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GDAŃSKI UNIWERSYTET MEDYCZNY
Marek Niedoszytko
Wykorzystanie badania ekspresji genów metodą
mikromacierzy RNA w ocenie efektywności immunoterapii
swoistej jadem owadów, rozpoznaniu mastocytozy i ocenie
zagrożenia alergią na jady owadów u chorych na mastocytozę
Klinika Alergologii Katedry Pneumonologii i Alergologii
Gdańskiego Uniwersytetu Medycznego
Kierownik: prof. dr hab. med. Ewa Jassem
Gdańsk 2011
Wydano za zgodą
Senackiej Komisji Wydawnictw
Gdańskiego Uniwersytetu Medycznego
Wydawca: Gdański Uniwersytet Medyczny
Druk: Dział Wydawnictw GUMed
Gdańsk, ul. Marii Skłodowskiej-Curie 3a
Zlecenie KW/42/11
SPIS TREŚCI
WYKAZ PRAC BĘDĄCYCH PRZEDMIOTEM ROZPRAWY ............................................. 7
WYKAZ SKRÓTÓW ................................................................................................................ 9
1. WSTĘP ................................................................................................................................ 11
1.1. Epidemiologia, rozpoznanie i leczenie alergii na jady owadów ............................... 11
1.1.1. Epidemiologia i patofizjologia alergii na jady owadów................................. 11
1.1.2. Rola szlaku renina angiotensyna aldosteron .................................................. 13
1.1.3. Leczenie alergii na jad owadów błonkoskrzydłych ....................................... 14
1.1.4. Ocena efektywności immunoterapii jadem owadów błonkoskrzydłych............ 16
1.2. Epidemiologia, rozpoznanie i leczenie mastocytozy ................................................ 17
1.2.1. Immunoterapia swoista w alergii na jady owadów u chorych na
mastocytozę .................................................................................................... 19
1.3. Farmakogenetyka w medycynie i alergologii ........................................................... 20
2. CELE PRACY..................................................................................................................... 21
3. MATERIAŁ I METODY .................................................................................................... 22
3.1. Badanie roli polimorfizmu AGT M235T w alergii na jady owadów........................ 22
3.2. Ocena skuteczności immunoterapii jadem owadów błonkoskrzydłych za pomocą
profilu ekspresji genów ............................................................................................. 23
3.3. Bezpieczeństwo i skuteczność immunoterapii jadem owadów w mastocytozie ..... 24
3.4. Ocena ryzyka alergii na jady owadów u chorych na mastocytozę za pomocą
profilu ekspresji genów ............................................................................................. 24
3.5. Profil ekspresji genów i system regulacji transkrypcji genów w mastocytozie
układowej .................................................................................................................. 25
3.6. Analiza ekspresji całego genomu za pomocą mikromacierzy RNA......................... 25
3.7. Analiza statystyczna wyników mikromacierzy RNA ............................................... 26
4. OMÓWIENIE WYNIKÓW ................................................................................................ 28
4.1. Rola polimorfizmu angiotensynogenu AGT M235T w alergii na jady owadów...... 28
4.2. Ocena skuteczności immunoterapii jadem owadów błonkoskrzydłych za pomocą
profilu ekspresji genów ............................................................................................. 29
4.3. Bezpieczeństwo i skuteczność immunoterapii jadem owadów w mastocytozie ...... 30
4.4. Ocena ryzyka alergii na jady owadów u chorych na mastocytozę za pomocą
profilu ekspresji genów ............................................................................................. 32
4.5. Profil ekspresji genów i system regulacji transkrypcji genów w mastocytozie
układowej .................................................................................................................. 33
5. WNIOSKI............................................................................................................................ 34
6. PIŚMIENNICTWO ............................................................................................................. 35
PRACE BĘDĄCE PRZEDMIOTEM ROZPRAWY............................................................... 45
– 7 –
WYKAZ PRAC BĘDĄCYCH PRZEDMIOTEM ROZPRAWY
1. Niedoszytko M. Mastocytoza - rozrostowa choroba komórek tucznych związana z
ryzykiem reakcji anafilaktycznej. Pol. Merk. Lek. 2006;21,126:570-572. MEiN 5
2. Niedoszytko M., Ratajska M., Chełmińska M., Makowiecki M., Malek E., Siemiń-
ska A., Limon J., Jassem E.: The angiotensinogen AGT p. M235T gene polymor-
phism may be responsible for the development of severe anaphylactic reactions to
insect venom allergens. Int. Arch. Allergy Immunol.2010;153:166-172.IF 2,542
3. Niedoszytko M., Bruinenberg M., de Monchy J., Wijmenga C., Platteel M., Jas-
sem E., Oude Elberink J.N.G.: Gene expression analysis in predicting the effec-
tiveness of insect venom immunotherapy. J. Allergy Clin. Immunol.
2010;125,5:1092-1097. IF 9,165
4. Niedoszytko M., de Monchy J., van Doormaal J., Jassem E., Oude Elberink
J.N.G.: Mastocytosis and insect venom allergy : diagnosis, safety and efficacy of
venom immunotherapy. Allergy 2009;64:1237-1245.IF 6,38
5. Niedoszytko M., Bruinenberg M., van Doormaal J., de Monchy J., Nedoszytko B.,
Koppelman G., Nawijn M., Wijmenga C., Jassem E. , Oude Elberink J. Gene
expression analysis predicts insect venom anaphylaxis in indolent systemic masto-
cytosis. Allergy 2011 doi:10.1111/j.1398-9995.2010.02521.x. IF 6,38
6. Niedoszytko M., Oude Elberink J.N.G., Bruinenberg M., Nedoszytko B., de Mon-
chy J., te Meerman G., Weersma R.K., Mulder A., Jassem E., van Doormaal J.J.
MD PHD. Gene expression profile, pathways and transcriptional system regula-
tion in indolent systemic mastocytosis. Allergy 2011;66,2:229-237. IF 6,38
(Łączny IF 30,847)
– 8 –
Finansowanie
Grant Ministerstwa Nauki i Szkolnictwa Wyższego 2008-2010
Numery: N402085934 i N40201031
Stypendium Kolumb Fundacji na Rzecz Nauki Polskiej
– 9 –
WYKAZ SKRÓTÓW
ACE – angiotensin converting enzyme / enzym konwertaza angiotensyny
AGT –angiotensinogen / angiotensynogen
GRADE – Grading of Recommendations Assessment, Development and Evaluation / system
oceny jakości danych i klasyfikacji siły zaleceń
GO – gene ontology / baza funkcji genów
ISM – indolent systemic mastocytosis / mastocytoza układowa o powolnym przebiegu
IVA – insect venom allergy / alergia na jady owadów
sIgE – specific immunoglobuline E / swoista immunoglobulina E
KEGG – Kyoto encyclopedia of genes and genomes / baza danych genów i genomów Kyoto.
MAPK – mitogen activated protein kinase / kinaza aktywowana mitogenami
NB – Naïve Bayes prediction model /.model predykcyjny Naïve Bayes
PCR - polymerase chain reaction / reakcja łańcuchowej polimerazy
RAS – renin angiotensin system / układ renina angiotensyna aldosteron
SPT - skin prick test / punktowe testy skórne
TSR – Transcriptional System Regulators / regulatory systemu transkrypcji
UMCG – Univeristy Medical Center Groningen – Centrum Medyczne Uniwersytetu w
Groningen
VIT –venom immunotherapy / immunoterapia swoista alergen jadów owadów
Wnt – wingless int pathway / szlak sygnałowy wnt
– 11 –
1. WSTĘP
1.1. Epidemiologia, rozpoznanie i leczenie alergii na jady owadów
1.1.1. Epidemiologia i patofizjologia alergii na jady owadów
Alergia na jady owadów, definiowana jako wystąpienie przynajmniej jednej reakcji
układowej w ciągu życia po użądleniu przez owada, występuje u 1 do 3% populacji [16]. Do
grup ryzyka reakcji układowej po użądleniu należą pszczelarze (reakcja układowa po użądle-
niu występuje u 13 do 43% z nich) [6,28] oraz chorzy na mastocytozę (wstrząs anafilaktycz-
ny, często o ciężkim przebiegu, dotyka 30% chorych, w tym 50% chorych na mastocytozę
układową) [12, 29,72,75]. Obecność swoistych IgE na jady owadów w teście skórnym lub w
badaniu sIgE stwierdza się u 20% osób w populacji ogólnej [32,34]. Odczyny miejscowe po
użądleniu (nawet o dużym nasileniu) występują u 26% osób. Nie stanowią one zagrożenia
życia i nie są wskazaniem do leczenia [32,34]. Do gatunków owadów najczęściej wywołują-
cych reakcje anafilaktyczne w Polsce należą osy, pszczoły, szerszenie (jad wykazuje reak-
tywność krzyżową z jadem osy) i trzmiele (jad wykazuje reaktywność krzyżową z jadem
pszczoły) [93]. W większości krajów europejskich opisywana jest większa częstość alergii na
jad osy w regionach nadmorskich, natomiast częstość alergii na jad pszczoły wzrasta wraz z
wysokością nad poziomem morza. W obszarze śródziemnomorskim Europy występują częste
reakcje anafilaktyczne po użądleniu przez klecanki. Doniesienia z Niemiec wskazują na po-
jawienie się tych owadów w południowej części kraju oraz przy granicy z Francją. Prawdo-
podobnie jest to związane z ocieplaniem się klimatu. Podobna sytuacja epidemiologiczna wy-
stępuje w USA [63]. Natomiast w krajach tropikalnych opisywane są reakcje na wiele gatun-
ków owadów, wśród których jedynie alergia na mrówki w Australii może być leczona za po-
mocą VIT [6].
Mechanizm immunologiczny alergii na jady owadów, jak i immunoterapii swoistej, nie
został w pełni poznany. Nadwrażliwość na jad może przebiegać w mechanizmie nadwrażli-
wości alergicznej I typu w klasyfikacji Gella i Coombsa (dominująca forma) jak i nieimmuno-
logicznej. Spotykane są również reakcje nietypowe, które pojawiają się zwykle w kilka dni po
użądleniu jak choroba posurowicza, zapalenie stawów, alergiczne zapalenie naczyń, zespół
– 12 –
nerczycowy, objawy neurologiczne (zapalenie nerwów obwodowych, zapalenia wielonerwo-
we, napady drgawek, zaburzenia koncentracji, zespół psychoorganiczny, zespół pozapirami-
dowy, zapalenie kłębuszków nerkowych) [6,10,93]. Alergia na jady owadów występuje z po-
dobną częstością u atopików jak i chorych bez atopii w wywiadzie. Osoby z wysokimi warto-
ściami sIgE na jady owadów często tolerują użądlenia, natomiast ciężkie reakcji poużądle-
niowe mogą występować u chorych o niskich wartościach sIgE w surowicy krwi
[6,10,28,58,60]. Niektóre osoby tolerujące użądlenia, nawet dużą ich liczbę (jak pszczelarze)
z niewiadomych przyczyn rozwijają objawy alergii. Nie jest również znana przyczyna znacz-
nie częstszego występowania IVA u chorych na mastocytozę (30%) w porównaniu z ogólną
populacją. Pierwsze doniesienia Muellera i wsp. [70] wskazywały na niskie bądź nieozna-
czalne stężenia IgE u wielu chorych. Dało to podstawę do teorii o farmakologicznym mecha-
nizmie nadwrażliwości. Badania wskazują na degranulację mastocytów pod wpływem alerge-
nu jadu owadów, której nie stwierdza się w kontakcie z alergenami wziewnymi i pokarmo-
wymi. Dotyczy to jednak stężeń alergenu, które nie występują w czasie użądlenia owada. Ba-
dania z użyciem obecnie stosowanych, czułych metod, pozwalają na potwierdzenie mechani-
zmu IgE zależnego u większości chorych [3]. Wyniki oznaczenia sIgE oraz SPT u chorych na
mastocytozę są przeważnie słabiej wyrażone niż u pozostałych chorych. Prawdopodobnie jest
to związane z adsorpcją krążących IgE na powierzchni mastocytów tkankowych [86]. Wpro-
wadzenie do diagnostyki testu aktywacji bazofilów umożliwiło stwierdzenie reakcji IgE za-
leżnej u prawie wszystkich chorych [9]. Alternatywny mechanizm aktywacji opisany został
na modelu zwierzęcym, gdzie kompleksy IgG antygen mogą aktywować makrofagi poprzez
łączenie z receptorem dla IgG (FcγRIII). Nie ma na razie danych potwierdzających znaczenie
tego mechanizmu u ludzi. Kluczowym elementem anafilaksji jest aktywacja mastocytów,
mediowana przez szlaki sygnałowe zależne od wewnątrzkomórkowych kinaz tyrozynowych
(Kit, Lyn, Syk and Fyn) [75]. Obecność mutacji KIT D816V może świadczyć o aktywacji i
proliferacji komórek tucznych, jakkolwiek nie wpływa na wzrost ryzyka anafilaksji
[1,12,95,96]. Natomiast zaburzenia czynności kanałów wapniowych, związane ze zwiększo-
nym napływem wapnia do komórek i łatwiejszą de granulacją, mogą odgrywać rolę w zwięk-
szeniu ryzyka anafilaksji [2,99]. Opisywana jest również rola kanałów TRMP (transient re-
ceptor potential membrane proteins), które odgrywają rolę w hamowaniu aktywacji komórek
tucznych [98]. Białko TRPM-4 jest zaangażowane w reakcjach nadwrażliwości. Substancje
aktywujące kanał jonowy, który tworzą białka TRMP, mogą służyć jako leki hamujące reak-
cje alergiczne [98].
– 13 –
Kluczowym elementem alergii na jady owadów jest zaburzenie stosunku pomiędzy spe-
cyficznymi alergenowo limfocytami T regulatorowymi i limfocytami Th2 [48]. Komórki pre-
zentujące antygen pod wpływem IL 4 wpływają na różnicowanie „naiwnych” limfocytów T w
komórki Th2. Aktywowane limfocyty Th2 wytwarzają IL-4, IL-5 i IL-13, które z kolei
zwiększają produkcję IgE, napływ i aktywację eozynofilów oraz skurcz mięśni gładkich [48].
Innym postulowanym mechanizmem alergii na jady owadów i efektywności immunote-
rapii jest szlak sygnałowy osteopontyny, którego aktywacja występuje głównie w monocytach
[55]. W literaturze są również doniesienia dotyczące udziału aktywacji dopełniacza [51] oraz
genów związanych z kalcytoniną [99].
1.1.2. Rola szlaku renina angiotensyna aldosteron
Ważnym mechanizmem, który może brać udział w nadwrażliwości na jady owadów jest
upośledzenie funkcji układu renina angiotensyna, aldosteron [43]. Angiotensyna II jest silną
substancją wazokonstrykcyjną [80,88]. Stężenia białka zależą od produkcji jej prekursorów:
angiotensynogenu i angiotensyny I, aktywności enzymu konwertującego angiotensynę I do II
oraz aktywności receptora dla angiotensyny II [81,88]. Angiotensynogen jest nieaktywnym
białkiem produkowanym w wątrobie. Renina, enzym obecny w nerkach, przekształca angio-
tensynogen w angiotensynę I, która z kolei po wpływem konwertazy angiotensyny zamienia-
na jest do angiotensyny II [81,88].
U większości ludzi użądlenie owada prowadzi do reakcji miejscowej charakteryzującej
się typowymi cechami stanu zapalnego: zaczerwienieniem, wzrostem temperatury, obrzę-
kiem, bólem [5,32]. Skurcz naczyń wywołany aktywacją angiotensyny II może ograniczyć
uogólnienie się reakcji [5,32]. Obserwacje kliniczne wskazują, że wielu chorych, którzy prze-
żyli reakcję anafilaktyczną po użądleniu przez owada, nie miało reakcji miejscowej po użą-
dleniu. W badaniach Hermanna i wsp. wykazano mniejsze stężenia angiotensynogenu, angio-
tensyny I, II reniny u chorych z alergią na jady owadów, w porównaniu z osobami zdrowymi
[40-44,55,]. Niskie stężenia białek tego układu stwierdzono również u chorych, u których
występowały niepożądane objawy leczenia, nawracające reakcje anafilaktyczne pomimo le-
czenia, z dodatnim wynikiem próby prowokacji z żywym owadem [40,41,42,44]. Niskie stę-
żenia białek układu RAS korelowały z ciężkością objawów klinicznych [40,41,42,44]. U cho-
rych, którzy osiągnęli tolerancję jadu owadów, stężenia angiotensyny I i II są podobne jak u
– 14 –
osób zdrowych, natomiast stężenie angiotensynogenu, pierwszego białka układu, nadal pozo-
stało istotnie niższe niż u osób zdrowych. Dotychczas nie udało się wykazać przyczyn niskie-
go stężenia białek układu RAA chorych leczonych z powodu alergii na jad owadów błonko-
skrzydłych.
1.1.3. Leczenie alergii na jad owadów błonkoskrzydłych
Reakcję kliniczną po użądleniu klasyfikuje się według kilku skal, z których najpopular-
niejsza jest klasyfikacja wg Muellera [67].
• stopień I: pokrzywka, świąd, nudności
• stopień II: obrzęk naczynioruchowy, świąd gardła, wymioty, biegunka, ból brzucha,
mdłości
• stopień III: duszność, świsty, trudności w mówieniu, zaburzenia połykania, lęk, hypo-
dynamia
• stopień IV: spadek ciśnienia tętniczego, utrata przytomności, nietrzymanie moczu i
stolca, sinica
Leczeniem z wyboru chorych z III i IV stopniem ciężkości reakcji według Muellera [67]
jest immunoterapia swoista (ang. VIT) [6,10,11,92,101]. Ryzyko reakcji układowej po użą-
dleniu wynosi u tych chorych około 70%, jest większe u chorych na mastocytozę, u których
dochodzi do 100%. W niektórych sytuacjach po użądleniu przez osę nie dochodzi do wnik-
nięcia jadu do ciała chorego, dlatego ryzyko reakcji nie jest 100% [33,34].
VIT można również stosować u chorych z mniej nasiloną reakcją o zwiększonym ryzyku
ciężkiej reakcji spowodowanym wykonywanym zawodem (pszczelarze, cukiernicy), choro-
bami współistniejącymi (np. mastocytozą) oraz znacznym upośledzeniem jakości życia [10].
Kwalifikacja do leczenia składa się z badania podmiotowego, w trakcie którego należy ocenić
(1) ciężkość reakcji, (2) sytuację, w której do niej doszło, (2) prawdopodobny gatunek bądź
gatunki owadów odpowiedzialnych za wystąpienie objawów, (3) ryzyko powtórzenia się re-
akcji w przyszłości, (4) występowanie chorób współistniejących (np. ciężka astma, niewydol-
ność krążenia, mastocytoza) oraz stosowanego leczenia (stosowanie B-blokerów, inhibitorów
enzymu konwertującego angiotensynę), które mogą wpłynąć na ryzyko lub przebieg reakcji
poużadleniowej. Ważnym elementem kwalifikacji do leczenia jest ocena jakości życia i nasi-
lenia lęku u chorych z reakcją o mniejszym stopniu nasilenia. Kolejnym etapem diagnostyki
– 15 –
jest wykonanie punktowych testów skórnych i testów śródskórnych, ich przeprowadzenie i
interpretację opisują standardy Europejskiej Akademii Alergologii [6,10]. Badaniami labora-
toryjnymi wykonywanymi u wszystkich chorych jest ocena stężenia swoistych IgE z jadem
osy, pszczoły i szerszenia. Zaleca się również ocenę stężenia tryptazy mastocytarnej w suro-
wicy, która może być markerem mastocytozy, jak i ryzyka działań niepożądanych oraz cięż-
kiej reakcji poużądleniowej u chorych bez tej choroby [60,83,84,86]. Wszyscy chorzy, u któ-
rych wystąpiła reakcja anafilaktyczna na jady owadów powinni być wyposażeni w zestaw
ratunkowy, którego najważniejszym elementem jest ampułkostrzykawka z adrenaliną oraz
leki przeciwhistaminowe i glikokortykoidy. Chorych należy poinstruować o sposobach uni-
kania narażenia na użądlenia przez owada. Jedyną przyczynową metodą leczenia IVA jest
immunoterapia swoista. W przeciwieństwie do chorych leczonych z powodu alergii na aler-
geny wziewne, VIT prowadzony jest jedynie w formie iniekcji podskórnych, badania klinicz-
ne z alergenem podjęzykowym nie wykazały różnic w skuteczności w porównaniu z placebo.
Faza wstępna VIT może być prowadzona schematem konwencjonalnym, przyspieszonym
(rush) i ultraszybkim (ulrarush). Po osiągnięciu dawki podtrzymującej ryzyko wystąpienia
reakcji poużądleniowej zmniejsza się do 2-3% i jest podobne do ryzyka takich reakcji w po-
pulacji ogólnej. U chorych, którzy nie osiągnęli tolerancji w wyniku leczenia ocenionej na
podstawie próby prowokacji alergenowej [30], użądlenia w warunkach naturalnych, bądź u
których w trakcie terapii podtrzymującej wystąpiły działania niepożądane leczenia, dawkę
leku można zwiększyć do 200 μg jadu [87]. Taką dawkę stosuje się również u chorych pracu-
jących jako pszczelarze [6,10]. Częstość występowania działań niepożądanych w trakcie le-
czenia zależy od stosowanego jadu owada (większe u chorych leczonych z powodu alergii na
jad pszczoły 25% w porównaniu z 11% leczonych jadem osy) [6,10]. U większości chorych
leczenie powinno być prowadzone przez 5 lat, a u osób ze współistniejącą mastocytozą praw-
dopodobnie do końca życia [10]. Stężenie tryptazy mastocytarnej w surowicy krwi wykazuje
liniową zależność z ciężkością reakcji anafilaktycznej oraz występowaniem działań niepożą-
danych podczas leczenia [83,84]. W tej grupie chorych stwierdzano przypadki śmiertelnej
anafilaksji po użądleniu, które nastąpiło po zakończeniu VIT [74,79]. Zalecenia amerykańskie
mówią o leczeniu trwającym do czasu negatywizacji testów skórnych, jednak u większości
chorych wydłuża to czas leczenia do 7-10 lat, nie wpływając na jego skuteczność [34]. Stan-
dardy Europejskiej Akademii Alergologii i Immunologii Klinicznej zalecają pięcioletni czas
leczenia. Może ono trwać trzy lata jeżeli stwierdza się negatywizację wyników testów skór-
nych i stężenia sIgE. Leczenie pięcioletnie umożliwia osiągnięcie tolerancji alergenu u więk-
szości chorych. Dłuższe leczenie wskazane jest u chorych zagrożonych wyższym ryzykiem
– 16 –
reakcji (1) leczonych z powodu mastocytozy, większym stężeniem tryptazy mastocytarnej, z
wywiadem ciężkiej reakcji poużądleniowej (2) osób, które doświadczyły reakcji niepożąda-
nych podczas leczenia podtrzymującego lub nie osiągnęły tolerancji użądlenia, (3) osób o
dużym ryzyku użądlenia jak pszczelarze i ich rodziny [10].
1.1.4. Ocena efektywności immunoterapii jadem owadów błonkoskrzydłych
Dotychczas nie ma wskaźników pozwalających ocenić skuteczność leczenia i reakcję
chorego po użądleniu. Ponad 90% chorych leczonych z powodu alergii na jad osy i 80% na
jad pszczoły osiąga tolerancję kolejnych użądleń po zakończeniu VIT. Gorsze efekty leczenia
stwierdza się u osób z cięższą reakcją przed leczeniem, chorych z działaniami niepożądanymi
w trakcie immunoterapii, współistniejącymi chorobami serca, zwiększonym stężeniem trypta-
zy mastocytarnej oraz u chorych na mastocytozę [62,65,66,68,69,83,84]. Bardziej efektywna
ochrona przed kolejnym użądleniem związana jest z dłuższym czasem leczenia i większym
dawką alergenu stosowanego w trakcie VIT [6,10]. Negatywizacja testów skórnych w wyniku
leczenia wskazuje prawdopodobnie na mniejsze ryzyko powtórnej reakcji, jednak występuje
ona jedynie u 20-30% leczonych chorych [33,34]. Ponadto u części osób (np. chorych na ma-
stocytozę) wyniki testów skórnych bywają negatywne bądź graniczne przed VIT, co nie kore-
luje z ciężkością reakcji anafilaktycznej [86]. Do badań laboratoryjnych stosowanych w oce-
nie skuteczności leczenia należą badanie swoistych IgE, test aktywacji bazofilów, ocena stę-
żenia IgG4, IL10, IL4. Zmniejszenie stężenia IgE, podobnie jak negatywizacja testów skór-
nych może u pewnej części chorych świadczyć o mniejszym ryzyku reakcji. Podobne zna-
czenie ma zmniejszenie reaktywności bazofilów [24,61]. Wykazanie wzrostu stężenia IL10 i
zmniejszenie stężenia IL4 może świadczyć o zwiększeniu puli limfocytów Th2 i zmniejszeniu
liczby limfocytów Th1. Dodatkowo wykazać można zwieszenie Foxp3 - białka świadczącego
o zwiększeniu puli limfocytów T regulatorowych [48]. U chorych leczonych z powodu alergii
na jad pszczoły wykazano zwiększenie stężenia osteopontyny w wyniku skutecznej immuno-
terapii [55]. Dotychczas żadne z powyższych badań nie weszło jednak do praktyki klinicznej i
ocena ryzyka reakcji po ponownym użądleniu z zastosowaniem metod in vitro nie jest możli-
wa.
– 17 –
1.2. Epidemiologia, rozpoznanie i leczenie mastocytozy
Mastocytoza to zespół chorobowy, w którym dochodzi do patologicznego rozrostu ko-
mórek tucznych w szpiku oraz innych narządach. U większości chorych występują zmiany
skórne, nacieki narządów wewnętrznych takich jak śledziona, wątroba, kości, przewód po-
karmowy, układ oddechowy, serce. Nacieki te mogą doprowadzić do upośledzenia funkcji
zajętych narządów. Pierwsze objawy choroby mogą pojawić się w każdym wieku. U dzieci
dominuje postać skórna choroby, rzadko występuje postać układowa. Dorośli chorują przede
wszystkim na mastocytozę układową. W najcięższych postaciach choroby często nie ma
zmian na skórze [1,27,47,95,96].
Klasyfikacja choroby według WHO obejmuje 7 postaci tego zespołu (Tabela 1).
Tabela 1. Klasyfikacja mastocytozy wg WHO [95]
1. Postać skórna (CM) a) pokrzywka barwnikowa (łac. urticaria pigmentosa) b) mastocytoma skóry
2. Systemowa mastocytoza o powolnym przebiegu (ISM) a) izolowana mastocytoza szpiku kostnego
3. Mastocytoza układowa z klonalnym rozrostem linii komórkowych nie- mastocytarnych (SM-AHNMD)
4. Agresywna układowa mastocytoza (ASM) 5. Białaczka mastocytarna (MCL) 6. Chłoniak mastocytarny 7. Mastocytoma w narządach poza skórą
Postacie agresywne choroby są bardzo rzadkie, dotyczą mniej niż 5% chorych dorosłych
i wyjątkowo występują u dzieci. Wymagają zastosowania chemioterapii z powodu występo-
wania nacieków proliferujących mastocytów upośledzających funkcję zajętych narządów
[47,94,95].
Mechanizm niekontrolowanej proliferacji mastocytów, jak i naciekania narządów w ma-
stocytozie, nie jest jasny. Dominującą zmianą genetyczną u chorych jest mutacja genu KIT
kodującego przezbłonowy receptor o aktywności kinazy tyrozynowej dla czynnika wzrostu
komórek pnia. Mutacja punktowa D816V stwierdzana jest u większości chorych, u części
spotykane są mutacje w innych miejscach genu. Obecność mutacji prowadzi do niekontrolo-
– 18 –
wanej autofosforylacji receptora i proliferacji mastocytów [1,36,95,96]. Wykazano jej obec-
ność w innych, poza mastocytarną, liniach komórkowych, co jest niekorzystnym czynnikiem
rokowniczym rozwoju agresywnych postaci mastocytozy [31]. Obecność samej mutacji genu
KIT nie jest wystarczająca do wystąpienia mastocytozy. Badania polimorfizmu genów wyka-
zały rolę polimorfizmu Q576R genu receptora IL4 [18] w rozwoju pokrzywki barwnikowej,
polimorfizmu, występowania allelu T w miejscu -1112 promotora genu IL-13 jako czynnika
ryzyka mastocytozy układowej [71]. Analiza ekspresji genów w szpiku chorych na mastocy-
tozę wykazała duże różnice w ekpresji genów u chorych na mastocytozę, w porównaniu z
osobami zdrowymi. Zidentyfikowano grupę 10 genów, których ekspresja znacznie różniła się
u chorych na mastocytozę, w tym największe różnice stwierdzono w odniesieniu do ekspresji
genu α-tryptazy [19]. Trwają obecnie badania nad zaburzeniem regulacji apoptozy w masto-
cytozie. Prawdopodobnie umożliwią one wykorzystanie nowych leków w leczeniu agresyw-
nych postaci choroby. Mastocytoza jest rzadką chorobą. ECNM (Europejska Sieć Mastocyto-
zy) podaje różne dane dotyczące epidemiologii choroby, zależne częściowo od zaawansowa-
nia badań nad chorobą i wielkości kraju. Wydaje się, że częstość występowania choroby
można określić na 7/100 000 mieszańców w tym 4/100 000 to mastocytoza układowa (tabela
2) [47].
Szacuje się, że w Polsce liczba chorych na mastocytozę może wynosić około 800 [72].
Pod opieką Polskiej Sieci Mastocytozy znajduje się obecnie 300 chorych (rejestr ośrodka
gdańskiego), w tym około 60% dorosłych i 40% dzieci [47].
Tabela 2. Epidemiologia mastocytozy w wybranych europejskich krajach i USA (dane ECNM) [47]
Kraj/liczba lud-ności
Liczba chorych na mastocytozę
N (n*)
Pokrzywka barw-nikowa N (n*)
Mastocytoza układowa
N (n*)
Agresywna mastocytoza
N (n*)
Austria/8 mln1 2000 (25) 1600 (20)
400 (5)
16 (0,2)
Holandia/16.5 mln2 1220 (7,4) 600
(3,6) 600 (3,6)
20 (0.12)
Niemcy/82 mln3 5000 (6)
USA5 3500 (1,13) Polska/38 mln5 300 (0,8) 200 (0,6) 100 (0,2) 5 (0,013)
* n – liczba chorych na 100 000
Autor uzyskał dane od kierowników ośrodków mastocytozy: 1 Peter Valent, 2 Jaap van Doormaal, 3 Knut Brockow, 4 USA Mastocytosis Group, 5 Gdański Ośrodek Mastocytozy
– 19 –
Rozpoznanie postaci układowej mastocytozy opiera się na kryteriach WHO [95]. Głów-
nym badaniem jest trepanobiopsja szpiku kostnego. Kryterium większym rozpoznania jest
stwierdzenie w badaniu histopatologicznym szpiku nacieków powyżej 15 atypowych masto-
cytów w skupisku, o atypowym kształcie. Do kryteriów mniejszych zalicza się stwierdzenie w
badaniu cytologicznym ponad 25% mastocytów o atypowym kształcie, obecność mutacji
D816V genu KIT, ekspresję CD2 i CD25 na mastocytach, stwierdzenie stężenia tryptazy po-
wyżej 20 ng/ml w surowicy krwi obwodowej [95]. Mastocytozę układową rozpoznaje się po
stwierdzeniu 1 dużego i 1 małego bądź 3 małych kryteriów WHO [95,96]. Wykonanie bada-
nia szpiku zlecane jest u wszystkich dorosłych, u których podejrzewa się mastocytozę tj. u
chorych na pokrzywkę barwnikową, anafilaksję ze współistniejącym zwiększonym stężeniem
tryptazy mastocytarnej, osteoporozę bez czynników ryzyka i zwiększonym stężeniem trypta-
zy, a także u chorych na choroby hematologiczne, u których wykazano obecność zwiększonej
liczby lub linii atypowych mastocytów w badaniu szpiku. Badania u dzieci wykonywane są w
przypadku podejrzenia agresywnej postaci choroby (upośledzenie funkcji narządów – szpik,
wątroba, śledziona, układ pokarmowy, osteoporoza) bądź w przypadku stężenia tryptazy ma-
stocytarnej powyżej 20 ng/ml [94,95,96]. Jedynie 1 z 5 kryteriów wg WHO opiera się na ba-
daniu krwi obwodowej, pozostałe wymagają biopsji szpiku kostnego. Wprowadzenie do prak-
tyki klinicznej narzędzia umożliwiającego rozpoznanie choroby w sposób mniej inwazyjny
mogłoby zwiększyć możliwości rozpoznania choroby.
1.2.1. Immunoterapia swoista w alergii na jady owadów u chorych na ma-stocytozę
Objawy degranulacji komórek tucznych występują u większości chorych na mastocyto-
zę, Ich nasilenie jest różne - od świądu skóry po hipotensję i wstrząs anafilaktyczny. Reakcje
anafilaktyczne występują u 50% chorych na mastocytozę układową, w tym u 30% chorych
reakcje anafilaktyczne występują po użądleniu przez owada. U większości chorych są to reak-
cje bardzo ciężkie, zagrażające życiu [8,12,15,35,36,59,96]. Uważa się, że większość zgonów
w wyniku anafilaksji na jady owadów dotyczy chorych na mastocytozę. Dotychczas opisano
co najmniej 6 zgonów po użądleniu przez owada u chorych na mastocytozę. Trzech chorych
nie było odczulanych [22,24,25]. U trzech kolejnych wstrząs nastąpił po użądleniu, do które-
go doszło po zakończeniu leczenia [74,90]. W przeciwieństwie do populacji ogólnej chorych
– 20 –
na alergię na jady owadów, w której immunoterapia jest leczeniem z wyboru, opinie na temat
odczulania chorych na mastocytozę znacznie różniły się. Część ośrodków uważała mastocy-
tozę za przeciwwskazanie do leczenia, głównie z powodu częstszych działań niepożądanych i
mniejszej skuteczności leczenia [22]. W innych klinikach współistnienie mastocytozy i alergii
na jady owadów uważano za jedno z najważniejszych wskazań do leczenia [8-10,82-87]. Po-
stulowano również profilaktyczne leczenie chorych na mastocytozę, u których do reakcji ana-
filaktycznej po użądleniu jeszcze nie doszło [86,100]. Stąd pojawiła się konieczność analizy
dostępnych danych z uwzględnieniem stosunku ryzyka do korzyści leczenia.
Metody diagnostyczne dostępne obecnie nie pozwalają na ocenę ryzyka anafilaksji u
chorych na mastocytozę. Wprowadzenie takiej metody miałoby duże znaczenie praktyczne i
pozwoliło na indywidualizację leczenia chorych.
1.3. Farmakogenetyka w medycynie i alergologii
Wyniki badań genetycznych stosowane są już szeroko w medycynie, zwłaszcza w hema-
tologii, onkologii, pediatrii [17,64,76,81,97], gdzie znacząco poprawiły wyniki leczenia,
zmniejszyły liczbę działań niepożądanych, koszty leczenia. Analiza obecności mutacji D816V
genu KIT jest też standardowym elementem rozpoznania mastocytozy układowej, gdzie jest
nie tylko małym kryterium rozpoznania wg WHO, ale pozwala uniknąć nieefektywnego le-
czenia imatinibem w przypadku występowania linii komórkowej D816V dodatniej, która jest
oporna na imatinib [1,94-96]. Z kolei leczenie chorych na astmę może być efektywniejsze po
uwzględnieniu oceny odpowiedzi na leki z grupy β2 agonistów za pomocą badania polimorfi-
zmu genu receptora β2 adrenergicznego (ARDB2), receptora kortykotropiny (CRHR1) i od-
powiedzi na glikokortykosteroidy, czy genu syntazy lekotrienu C4 i 5-lipooksygenazy w od-
powiedzi na inhibitory leukotrienów [53]. Wprowadzenie farmakogenetyki jako metody po-
mocniczej w badaniu chorych leczonych z powodu alergii na jad owadów błonkoskrzydłych
mogłoby poprawić wyniki leczenia i zwiększyć bezpieczeństwo chorych.
– 21 –
2. CELE PRACY
1. Ocena częstości występowania wariantów polimorficznych genu AGT (M235T) i ACE
(I/D, I/I, D/D) u chorych leczonych z powodu alergii na jady owadów, ocena ich związ-
ku z ciężkością reakcji anafilaktycznej i działaniami niepożądanymi podczas leczenia.
2. Ocena zastosowania badania ekspresji genów w ocenie skuteczności immunoterapii
swoistej jadem owadów błonkoskrzydłych.
3. Ocena danych dotyczących występowania, rozpoznania, bezpieczeństwa i skuteczności
immunoterapii swoistej jadem owadów błonkoskrzydłych u chorych na mastocytozę
układową.
4. Ocena różnic w ekspresji genów u chorych na mastocytozę układową i alergię na jady
owadów w porównaniu z chorymi, u których nigdy nie wystąpiła reakcja anafilaktycz-
na.
5. Ocena różnic w ekspresji genów we krwi obwodowej u chorych na mastocytozę ukła-
dową i określenie profilu genów charakterystycznego dla chorych na mastocytozę.
– 22 –
3. MATERIAŁ I METODY
3.1. Badanie roli polimorfizmu AGT M235T w alergii na jady owadów
Materiał i metodę badań opublikowano w [pracy 2]. Badanie wykonane we współpracy
z Katedrą i Zakładem Genetyki GUMed, kierownik Katedry Prof. dr hab. med. Janusz Limon.
Grupę badaną stanowiło 107 chorych leczonych z powodu alergii na jady owadów błon-
koskrzydłych w Klinice Alergologii Gdańskiego Uniwersytetu Medycznego, średnia wieku
41 lat (zakres 18-75), w tym 59 (55%) kobiet i 48 (45%) mężczyzn. Rozpoznanie alergii na
jady owadów ustalono zgodnie z zaleceniami EAACI. Grupę kontrolną stanowiło 113 zdro-
wych dawców krwi o średniej wieku 41 lat (zakres 21-74), w tym 48 (42%) kobiet i 65 (58%)
mężczyzn. Badanie uzyskało zgodę komisji etycznej Gdańskiego Uniwersytetu Medycznego
(NKEBN/811/2004).
Badanie polimorfizmu genu (p.M235T) wykonano metodą ASO-PCR (allele-specific
oligonucleotide polymerase chain reaction) [45,54]. W celu potwierdzenia otrzymanych wy-
ników co dziesiąta próbka analizowana była za pomocą sekwencjonowania analizatorem ABI
PRISM 310.
Badanie polimorfizmu ACE I/D, I/I, D/D (rs1799752) wykonano metodą PCR [54,80].
W celu potwierdzenia otrzymanych wyników co dziesiątą próbkę sekwencjonowano analiza-
torem ABI PRISM 310.
Pomiar stężenia angiotensyny I wykonano za pomocą metody ELISA (Phoenix Pharma-
ceuticals, CA, USA).
– 23 –
3.2. Ocena skuteczności immunoterapii jadem owadów błonko-skrzydłych za pomocą profilu ekspresji genów
Materiał i metodę badań opublikowano w [pracy 3]. Badanie wykonane we współpracy
z Katedrą Genetyki UMCG (Groningen, Holandia), kierownik Katedry Prof. Cisca Wijmenga.
Grupa badana składała się z 46 chorych leczonych z powodu alergii na jady owadów w
Klinice Alergologii Uniwersyteckiego Centrum Klinicznego w Groningen (Holandia). Wszy-
scy chorzy zakwalifikowani zostali do leczenia z powodu reakcji anafilaktycznej po użądleniu
przez owada ocenionej jako stopień III lub IV wg Muellera [67], dodatnich wyników testów
skórnych i/lub sIgE według zaleceń EAACI [10]. Kryteriami wyłączenia z badania był brak
zgody chorego, ciąża, choroby przewlekłe o ciężkim przebiegu, choroby nowotworowe i ma-
stocytoza.
Leczenie rozpoczęto według schematu semi-rush, pierwszego dnia leczenia chory osią-
gnął dawkę 10 µg leku. Wzrastające dawki leku do osiągnięcia dawki 100 µg podawane były
w odstępach tygodniowych. Dawki podtrzymujące podawane były w odstępach 6 tygodnio-
wych przez 3 do 5 lat. Badanie zaakceptowane było przez komisje etyczną UMCG (METc
2008/340).
Chorzy biorący udział w badaniu podzieleni zostali na 3 grupy:
Grupa 1. Osoby, które były leczone z powodu alergii na jady owadów, po zakończeniu
leczenia były użądlone co najmniej 3 razy przez owada i nie doszło u nich do reakcji anafi-
laktycznej (n = 17, średnia wieku 53 lata (zakres 28-70). W tym 9 mężczyzn (53%) i 8 kobiet
(47%))
Grupa 2. Osoby, które były leczone z powodu alergii na jady owadów, po jego zakoń-
czeniu byli użądleni przez owada przynajmniej 2 razy, pomimo leczenia doszło u nich do
reakcji anafilaktycznej (n=12, średnia wieku 56 (zakres 42-75) w tym 4 mężczyzn (33%) i 8
kobiet (67%))
Grupa 3. Osoby, które nadal leczone są w schemacie terapii podtrzymującej VIT, które
nie były użądlone w czasie odczulania (n=17, średnia wieku 55 (zakres 21-75) w tym 6 męż-
czyzn (35%) i 11 kobiet (65%)).
– 24 –
3.3. Bezpieczeństwo i skuteczność immunoterapii jadem owadów w mastocytozie
Materiał i metodę badań opublikowano w [pracy 4].
Analiza danych wykonana została wspólnie przez lekarzy z Kliniki w Groningen (Ho-
landia), gdzie po publikacjach Dubois [22] immunoterapia u chorych na mastocytozę nie była
wykonywana oraz przez lekarzy z Kliniki Alergologii w Gdańsku, gdzie leczenie było stoso-
wane na podstawie zaleceń EAACI [10] i publikacji Rueff [86]. W celu zebrania jak najwięk-
szej liczby danych przeanalizowano publikacje zawarte w bazie Pubmed, streszczenia z kon-
gresów alergologicznych w latach 2003-2008. W razie wątpliwości, co do interpretacji wyni-
ków z autorami kontaktowano się osobiście. Jakość dowodów naukowych oceniano za pomo-
cą systemu GRADE (Grading of Recommendations Assessment, Development and Evalua-
tion) [13,38]. Jakość dowodów oceniano w skali czterostopniowej (A wysoka, B średnia, C
niska, D bardzo niska), siłę rekomendacji określono jako: 1 – silną, 2 – słabą.
3.4. Ocena ryzyka alergii na jady owadów u chorych na mastocyto-zę za pomocą profilu ekspresji genów
Materiał i metodę badań opublikowano w [pracy 5]. Badanie wykonane we współpracy
z Katedrą Genetyki UMCG (Groningen, Holandia), kierownik Katedry Prof. Cisca Wijmenga.
Grupę 22 chorych na mastocytozę układową o powolnym przebiegu (ISM), leczonych w
Klinice Alergologii UMCG (średnia wieku 53 (zakres 35-73), w tym 7 mężczyzn (31%) i 15
kobiet (68%)) podzielono na dwie podgrupy w zależności od reakcji po użądleniu przez owa-
dy błonkoskrzydłe:
Grupa 1: Chorzy, u których w przeszłości wystąpiła reakcja anafilaktyczna IV stopnia
wg Muellera po użądleniu przez owady błonkoskrzydłe. Żaden z chorych nie otrzymywał
immunoterapii swoistej. Rozpoznanie alergii na jad owadów błonkoskrzydłych potwierdzono
dodatnim wynikiem SPT i/lub sIgE wg zaleceń EAACI.
– 25 –
Grupa 2: Chorzy, którzy byli użądleni przynajmniej raz przez owada błonkoskrzydłego
po rozpoznaniu mastocytozy układowej i nie wystąpiła u nich reakcja anafilaktyczna. Nie
wystąpiła u nich dotychczas reakcja anafilaktyczna ani reakcja hipotensyjna w żadnej innej
sytuacji po rozpoznaniu mastocytozy lub w ciągu ostatnich 10 lat. Badanie zaakceptowane
było przez komisje etyczną UMCG (METc 2008/340).
3.5. Profil ekspresji genów i system regulacji transkrypcji genów w mastocytozie układowej
Materiał i metodę badań opublikowano w [pracy 6]. Badanie wykonane we współpracy
z Katedrą Genetyki UMCG (Groningen, Holandia), kierownik Katedry Prof. Cisca Wijmenga.
Grupa badana składała się z 22 chorych na mastocytozę układową o powolnym przebie-
gu (ISM), leczonych w Klinice Alergologii UMCG (średnia wieku 53 (zakres 35-73), w tym 7
mężczyzn (31%) i 15 kobiet (68%)). Rozpoznanie choroby ustalone było zgodnie z zalece-
niami WHO i obejmowało badanie histopatologiczne szpiku kostnego, immunofenotypizację,
badanie cytologiczne, oznaczenie stężenia tryptazy mastocytarnej w surowicy krwi obwodo-
wej. Dodatkowo badano stężenie metabolitów histaminy w moczu. Grupa kontrolna składała
się z 43 zdrowych osób (średnia wieku 47,7 (zakres 19-73), w tym 22 mężczyzn (51%) i 21
kobiet (49%)). Badanie zaakceptowane było przez komisję etyczną UMCG (METc
2008/340).
3.6. Analiza ekspresji całego genomu za pomocą mikromacierzy RNA
Izolacja RNA
Próbki RNA krwi obwodowej zebrano za pomocą probówek PAXgene blood RNA tubes
(Qiagen, USA). Wszystkie probówki zamrożono w temperaturze -20 °C do czasu izolacji
(maksymalnie 2 miesiące od pobrania materiału). RNA izolowano za pomocą zestawu
– 26 –
PAXgene blood RNA Kit CE (Qiagen, Venlo, The Netherlands). Próbki RNA przechowywa-
no w temperaturze -80 °C do czasu znakowania i hybrydyzacji.
Jakość RNA oznaczano za pomocą analizatora 2100 Bioanalyzer (Agilent, Amstelveen,
The Netherlands) i Agilent RNA 6000 Nano Kit. Próbki krwi o wskaźniku integralności > 7,5
używane były do dalszej analizy.
Analiza ekspresji genomu
Znakowanie i amplifikacja RNA wykonana została zestawem Illumina TotalPrep 96
RNA Amplification Kit (Applied Biosystems, Nieuwerkerk ad IJssel, The Netherlands). Do
oznaczenia użyto 200 ng RNA z każdej próbki. Ludzkie tablice ekspresji całego genomu HT-
12_V3_expression arrays (Illumina, San Diego, USA) opracowano zgodnie z zaleceniami
producenta. Slajdy z wynikiem badania skanowano bezpośrednio po badaniu za pomocą Illu-
mina BeadStation iScan (Illumina, USA).
3.7. Analiza statystyczna wyników mikromacierzy RNA
Pierwszym etapem analizy statystycznej była korekcja sygnału tła i normalizacja kwan-
tylowa uzyskanych danych za pomocą programu Genomestudio Gene Expression Analysis
module v 1.0.6 Statistics. Geny, które w przynajmniej 75% próbek miały wartość sygnału
powyżej 20 percentyla całości sygnału porównywanych grup, włączano do dalszej analizy.
Analiza danych wykonana została za pomocą program GeneSpring package version
8.0.0 (Agilent Technologies Santa Clara CA, USA). Geny, których ekspresja różniła się w
porównywanych grupach, wybrane były na podstawie dwukrotnej różnicy ekspresji, istotności
statystycznej w teście t-Studenta i korekcji wyniku testem dla porównań wielokrotnych
Benjamini Hochberga. Model predykcyjny Naïve Bayes został stworzony w celu określenia
zestawu genów, który może być użyty w dalszych badaniach i w diagnostyce klinicznej
[52,56] . Metoda Naïve Bayes zakłada, że wpływ ekspresji pojedynczego genu nie jest zwią-
zany z wpływem ekspresji pozostałych genów na wartość wyniku predykcji. Metoda ta nie
bierze pod uwagę interakcji pomiędzy genami wchodzącymi w skład modelu predykcyjnego,
ani wpływu czynników środowiska.
– 27 –
Znaczenie funkcjonalne genów zostało zbadane za pomocą programu Genecodis
[14,73], bazy danych genów KEGG [49,50] i analizy GoSlim.
Różnice w ekspresji genów pomiędzy chorymi na mastocytozę i osobami zdrowymi
przeanalizowano również za pomocą analizy TSR (systemów regulacji transkrypcji) i analizy
wieloczynnikowej opisanej przez Fehrmana i wsp. [26]. Metoda ta stworzona została na pod-
stawie analizy 17550 eksperymentów badających ekspresję całego genu w różnych tkankach i
procesach chorobowych, przeprowadzonych metodą analizy ekspresji całego genomu zesta-
wem firmy Affimetrix. Zaobserwowano duże podobieństwo ekspresji genów w badanych
doświadczeniach. Na ich podstawie wyznaczono komponenty główne – TSR (systemy regu-
lacji transkrypcji) opierając się na korelacji pomiędzy genami ulegającymi wspólnej regulacji
ekspresji. Wprowadzona analiza pozwala wytłumaczyć 64% wszystkich różnic w ekspresji
genomu [26]. Interpretacja biologiczna i medyczna znaczenia różnic w ekspresji każdego z
TSR zależy od funkcji genów, które mają największy wpływ na jego ostateczną wartość. Ce-
lem analizy jest wykorzystanie podobieństwa w ekspresji genów i stworzenia TSR, które mają
większą powtarzalność i ich wartość jest pochodną ekspresji wielu genów współdziałających
ze sobą w różnych procesach biologicznych. W wyniku TSR utracie ulega wartość poje-
dynczego genu, ale rozwiązaniu ulega problem braku powtarzalności wyników i wpływu wa-
runków doświadczenia na ostateczny wynik badania.
Wartości ekspresji genów z bieżącego doświadczenia poddano logarytmicznej normali-
zacji, używając średnich wartości ekspresji genu. Następnie wartości ekspresji genów trans-
formowano do 50 głównych systemów regulacji transkrypcji, opisanych wcześniej przez Feh-
rmanna i wsp [26]. Następnie przeprowadzono analizę wieloczynnikową, redukując liczbę
parametrów opisujących ekspresję genów do 8 czynników.
Analizę statystyczną wykonano oprogramowaniem Systat 12.0 i programem napisanym
w języku Delphi 5.0.
– 28 –
4. OMÓWIENIE WYNIKÓW
4.1. Rola polimorfizmu angiotensynogenu AGT M235T w alergii na jady owadów
Wyniki badań opublikowano w [pracy 2]
Wyniki badania roli polimorfizmu AGT M235T genu angiotensynogenu potwierdzają
rolę układu RAS w alergii na jady owadów. Dodatkowo wskazują na czynnik genetyczny,
odpowiedzialny za niższe stężenia angiotensynogenu u chorych na cięższą postać nadwrażli-
wości. Wykazano, że wariant MM polimorfizmu genu AGT M235T jest znacznie częstszy u
chorych z ciężką reakcja anafilaktyczną po użądleniu przez owada. Związany jest on z niż-
szym stężeniem angiotensyniogenu w surowicy. Nie stwierdzono natomiast różnic w wystę-
powaniu wariantów polimorficznych I/D ACE genu konwertazy angiotensyny I.
Częstość występowania wariantu polimorficznego MM 235 genu AGT była niższa u
chorych leczonych z powodu alergii na jady owadów (30%) w porównaniu z osobami zdro-
wymi (17%). Obecność allela MM 235 była również czynnikiem ryzyka reakcji IV typu po
użądleniu przez owada (OR=2,5 CI 1,04-6,08). Dodatkowo, u homozygot MM stwierdzano
mniejsze stężenia angiotensyny I. Mniejsze stężenia białek układu RAS u chorych na ciężkie
postaci alergii na jady owadów opisano już poprzednio [40-44]. Mogą one wynikać z niższe-
go stężenia angiotensynogenu, pierwszego białka układu RAS. Podobne wyniki do stwierdza-
nych w tym badaniu wykazano u chorych na astmę i współistniejące inne choroby alergiczne
(alergiczny nieżyt nosa, atopowe zapalenie skóry) [45].
W badanej grupie nie stwierdzono różnic w występowaniu alleli I/D genu ACE zarówno
u chorych leczonych z powodu alergii na jady owadów i grupie kontrolnej, jak i porównując
chorych o różnych stopniach ciężkości reakcji anafilaktycznej. Różnic w aktywności tego
enzymu nie wykazano również w badaniach Hermanna i wsp. [40-44]. Wyniki badania po-
twierdzają również znaczenie mechanizmów innych niż reakcja alergiczna typu I wg Gella i
Coombsa.
Znaczenie układu RAS w alergii na jady owadów potwierdzają również wyniki badań, w
których wykazano znaczne zwiększenie ryzyka ciężkiej reakcji anafilaktycznej, u chorych
leczonych inhibitorami konwertazy angiotensyny [83,84,91].
– 29 –
Wyniki badania pozwalają przypuszczać, że leczenie oparte na farmakogenetyce może w
przyszłości pozwolić na indywidualizację farmakoterapii.
4.2. Ocena skuteczności immunoterapii jadem owadów błonko-skrzydłych za pomocą profilu ekspresji genów
Wyniki badań opublikowano w [pracy 3]
Wyniki badania wskazują na możliwość oceny efektywności VIT za pomocą badania
profilu ekspresji genów we krwi obwodowej. Różnice w ekspresji dotyczą znanych mechani-
zmów różnicowania limfocytów T, aktywacji komórek tucznych, jak i wskazują na nowe pro-
cesy pamięci immunologicznej. Użyta w badaniu metoda oceny ekspresji całego genomu we
krwi obwodowej, bez wcześniejszego sortowania komórek, jest wystandaryzowaną i prosta
metodą, która może stanowić podstawę do stworzenia narzędzia stosowanego w praktyce kli-
nicznej. Model predykcyjny, który może być użyty w dalszej praktyce klinicznej, oparty jest
na 18 genach. Profil ekspresji genów charakterystyczny dla tolerancji alergenu wykazano u
100% chorych uznanych za wyleczonych w wyniku stosowania VIT, nie znaleziono go u
żadnego chorego, który nie osiągnął tolerancji alergenu. Obecność tego profilu genów po-
twierdzono również u 88% chorych będących w trakcie immunoterapii podtrzymującej. Wy-
niki tego badania stanowią podstawę do dalszych badań, konieczna jest walidacja wyników w
innej populacji chorych, obserwacje prospektywne chorych i badania nad funkcją transkryp-
tów genów o nieznanej dotychczas roli w mechanizmie tolerancji alergenu. Analiza funkcjo-
nalna genów, których ekspresja różniła się w badanych grupach, wskazuje na udział znanych
mechanizmów VIT jak szlak związany z przekazaniem sygnału przez receptor FcγR1, JAK-
STAT, MAPK, Wnt, kanały wapniowe, przekazanie sygnału pomiędzy komórkami i regulację
transkrypcji genów. Funkcja wielu transkryptów nie jest jeszcze znana. Do genów o najwięk-
szym znaczeniu w identyfikacji chorych, którzy osiągnęli efekt leczenia zaliczono TWIST-2,
PRLR, CLDN1. TWIST 2 jest czynnikiem transkrypcyjnym stymulującym ekspresję IL10 i
zmniejszającym produkcję IL4 [89]. Po leczeniu zwiększeniu uległa również ekspresja genu
CLDN1, którego produkt klaudyna 1 jest cząsteczką adhezyjną odpowiedzialną za adhezję i
migrację komórek dendrytycznych. Jego stężenie wzrasta po interakcji komórek z TGF-β,
cytokiną zwiększającą liczbę komórek T regulatorowych [102]. Zmniejszenie ekspresji PRLR
– 30 –
(receptora dla prolaktyny) po VIT może wskazywać na zwiększenie puli limfocytów Th1.
Prolaktyna stymuluje syntezę receptora γ/δ TCR, który zwiększa zależną od IL4 produkcję
IgE i IgG1, zwiększa również pulę limfocytów Th2 [46]. Zmiany w ekspresji wymienionych
genów pozwalają połączyć wyniki badania z doniesieniami dotyczącymi zmiany stężeń cyto-
kin w trakcie immunoterapii [48].
Wyniki badania pozwalają przypuszczać, że możliwa jest ocena skuteczności immunote-
rapii za pomocą badania ekspresji genów, które może dostarczyć nowych informacji na temat
mechanizmu tolerancji alergenu.
4.3. Bezpieczeństwo i skuteczność immunoterapii jadem owadów w mastocytozie
Wyniki badań opublikowano w [pracy 4]
Bezpieczeństwo immunoterapii swoistej alergenem jadów owadów oceniono na podsta-
wie analizy 117 chorych leczonych w 6 badaniach retrospektywnych [25,70,74,77] i 4 bada-
niach opisujących pojedyncze przypadki leczonych chorych [25,70,74,77]. Działania niepo-
żądane opisano u 28 (23,9%) chorych na mastocytozę leczonych VIT, w tym objawy układo-
we u 20,5% chorych. W populacji chorych bez mastocytozy takie porównywalne reakcje wy-
stępowały u 20,3% chorych (11,1 – 36%). Najsilniejsze działania niepożądane, w wyniku
których leczenie zostało przerwane, bądź objawy układowe wymagały w leczeniu podania
adrenaliny, występowały u 7,6% chorych. W populacji chorych bez mastocytozy takie dzia-
łania niepożądane występowały rzadziej, u 3 do 7% chorych. Porównanie działań niepożąda-
nych, w zależności od gatunku owada, który wywołał reakcje wykazało, że występowały one
częściej u chorych leczonych alergenem jadu osy, w porównaniu z populacją ogólną (11,2%
vs. 35%).
Efektywność VIT u chorych na mastocytozę opisano w 6 badaniach [9,20,21,22,39,86,]
i jednym opisie przypadku [25], oceniając ją na podstawie wyniku próby prowokacji z ży-
wym owadem, bądź reakcji na użądlenie przez owada w środowisku naturalnym. Reakcja
ogólnoustrojowa po użądleniu wystąpiła w 23,9% opisywanych prób prowokacji i 33,3%
użądleń w środowisku naturalnym. Najcięższą reakcję opisywano u chorego, który nie osią-
gnął dawki podtrzymującej VIT. Sumarycznie efektywność leczenia oceniono na 72%. Jest to
– 31 –
odsetek niższy niż w populacji ogólnej, który wynosi 80% dla chorych leczonych z powodu
alergii na jad pszczoły i 95% u chorych leczonych z powodu alergii na jad osy [32]. Wyniki
badania de Olano wskazują, że brak tolerancji użądlenia wystąpił zwłaszcza u chorych, u któ-
rych wystąpiły działania niepożądane leczenia. Badania Rueff [33,85,86,87] wskazują, że
podwyższenie dawki jadu stosowanej w czasie fazy podtrzymującej VIT może zwiększyć
efektywność leczenia. Brak jest danych oceniających długotrwałą efektywność VIT, zwłasz-
cza po zakończeniu leczenia. Opisano jednak przypadki chorych na mastocytozę, którzy
zmarli po użądleniu pomimo wcześniejszej VIT [74]. Na tej podstawie zalecenia EAACI
wskazują na długotrwały, być może trwający całe życie czas leczenie [10].
Ze względu na występujące działania niepożądane opisywane są metody premedykacji,
obejmujące stosowanie leków antyhistaminowych, sterydów, kromoglikanów, monitorowania
chorego [21,25], zmianę preparatu na formę depot, a także stosowanie omalizumabu
[21,22,25,37,57,74,87,101].
Ze względu na mniejszą efektywność leczenia w porównaniu z populacją ogólną chorzy
na mastocytozę powinni stale być zabezpieczenia w zestaw ratunkowy zawierający ampułko-
strzykawkę z adrenaliną [1,22,33,35,36,95,96].
Podsumowując wyniki analizy dostępnych danych na temat VIT u chorych na mastocy-
tozę, pomimo niskiej jakości zebranych danych (B – D wg system GRADE), można wysunąć
następujące wnioski: (1) chorzy na mastocytozę mają wyższe ryzyko reakcji po użądleniu
owadów w porównaniu z populacją ogólną, zwłaszcza po użądleniu przez osę, (2) VIT może
być zalecana u chorych na mastocytozę, (3) prawdopodobnie powinna być prowadzona przez
całe życie, (4) VIT zmniejsza ryzyko reakcji anafilaktycznej u chorych na mastocytozę, choć
jest mniej efektywny niż w populacji ogólnej, (5) u części chorych można rozważyć stosowa-
nie wyższych dawkę alergenu jadu, (6) VIT u chorych na mastocytozę związany jest z więk-
szą liczbą działań niepożądanych, (7) premedykacja i środki bezpieczeństwa powinny być
brane pod uwagę w czasie VIT, (8) pomimo leczenia chorzy powinni być wyposażeni w ze-
staw ratunkowy z adrenaliną. Ze względu na jakość danych siłę zaleceń ocenić można jako
słabą. Mimo tego zmieniły one pogląd wielu ośrodków zajmujących się leczeniem alergii na
jady owadów u chorych na mastocytozę i IVA jest obecnie szerzej stosowana w tej grupie
chorych.
– 32 –
4.4. Ocena ryzyka alergii na jady owadów u chorych na mastocyto-zę za pomocą profilu ekspresji genów
Wyniki badań opublikowano w [pracy 5]
Porównanie profilów ekspresji genów u chorych na mastocytozę układową, którzy cho-
rują dodatkowo na alergię na jady owadów, z chorymi, którzy nie doświadczyli nigdy reakcji
anafilaktycznej, pozwoliło na określenie genów różnicujących badane grupy. Analiza ich
funkcji sugeruje, że nadwrażliwość na jady owadów może być związana ze stopniem zróżni-
cowania komórek. Profil ekspresji genów chorych, którzy nie doświadczyli reakcji anafilak-
tycznej po użądleniu, wskazuje na pobudzenie szlaków biorących udział w powstawaniu no-
wotworów, adhezji komórek, przekazywaniu sygnału szlakiem MAPK, oddziaływaniu białek
międzykomórkowych. Tak więc nie sama liczba mastocytów ale ich stopień zróżnicowania
może decydować o reakcji na alergen. Stężenia tryptazy i metabolitów histaminy w moczu
(świadczące o całkowitej liczbie komórek tucznych) u chorych na mastocytozę i anafilaksję
były niższe w porównaniu z chorymi na mastocytozę bez reakcji anafilaktycznych w wywia-
dzie. Dodatkowym potwierdzeniem tej tezy jest obserwacja kliniczna chorych na agresywne
postaci choroby leczonych w Klinikach Alergologii w Groningen i Gdańsku, z których żaden
nie podawał objawów alergii. Dodatkowo wyniki badania wskazują na możliwe wykorzysta-
nie ich w praktyce klinicznej w celu różnicowania i modyfikacji leczenia chorych zagrożo-
nych reakcją anafilaktyczną. Zaproponowany profil ekspresji 104 genów umożliwił różnico-
wanie chorych zagrożonych anafilaksją z wysoką 100% czułością i swoistością, co nie było
możliwe przy użyciu dotychczas dostępnych metod. Wyniki tego badania wymagają jeszcze
potwierdzenia w innych populacjach, wskazują jednak na możliwość stworzenia nowego na-
rzędzia diagnostycznego. Być może możliwe będzie określenie ryzyka anafilaksji u chorego
na mastocytozę, który dotychczas nie odczuwał objawów alergii i wprowadzenie leczenia
profilaktycznego postulowanego przez Rueff i wsp. [86].
– 33 –
4.5. Profil ekspresji genów i system regulacji transkrypcji genów w mastocytozie układowej
Wyniki badań opublikowano w [pracy 6]
Porównanie profilów ekspresji genów u chorych na mastocytozę układową i u osób
zdrowych wykazało bardzo duże różnice w ekspresji pomiędzy porównywanymi grupami. W
oparciu o grupę genów o największej różnicy w ekspresji zaproponowano panel 29 transkryp-
tów, która może stać się podstawą do stworzenia nowego narzędzia diagnostycznego w ma-
stocytozie układowej.
Liczba mastocytów we krwi obwodowej, w przeciwieństwie do szpiku kostnego i tka-
nek, jest niewielka. Różnice w ekspresji określone w niniejszej pracy, wynikają prawdopo-
dobnie z różnicy w ekspresji w innych liniach komórkowych krwi obwodowej. Badania Gar-
cii Montero i wsp. [31] wskazują na występowanie mutacji KIT w innych liniach komórko-
wych poza mastocytarną, nasze badania wskazują dodatkowo na zmianę ekspresji innych ge-
nów poza KIT.
Uzyskane wyniki poddano niezależnej analizie za pomocą analizy systemów regulacji
transkrypcji (TSR), która potwierdziła znaczne różnice w ekspresji genów we krwi obwodo-
wej u chorych na mastocytozę układową. Analiza funkcji genów o odmiennej transkrypcji
wskazuje na odmienną transkrypcję genów zaangażowanych w szlaki biorące udział w nowo-
tworzeniu, szlaki MAPK, Jak-STAT, p53, cykl komórkowy i apoptozę. Wskazują one rów-
nież na nowe szlaki, które mogą stać się podstawą do stworzenia nowych leków na mastocy-
tozę. Wyniki analizy ekspresji genów pozwalają na dalsze badania nad zastosowaniem tej
techniki u chorych na mastocytozę, zarówno w celu rozpoznania choroby jak i rozpoznania
zagrożenia anafilaksją, a być może w przyszłości również innymi chorobami mieloproifera-
cyjnymi. Konieczne są jednak dalsze badania nad walidacją profilu w innych populacjach i
większej grupie chorych.
– 34 –
5. WNIOSKI
1. Wariant MM polimorfizmu genu AGT M235T występuje istotnie częściej u chorych le-
czonych z powodu alergii na jady owadów, związany jest z większym ryzykiem cięż-
kiej reakcji po użądleniu, mniejszym stężeniem angiotensyny I. Obserwowane wcze-
śniej upośledzenie działania układu RAS u chorych leczonych z powodu alergii na jady
owadów może mieć podłoże dziedziczne związane z mniejszą syntezą angiotensynoge-
nu.
2. Zastosowanie badania polimorfizmu genów może pozwolić na określenie efektywności
immunoterapii swoistej jadem owadów.
3. Różnice w ekspresji genów powalają na identyfikację chorych na mastocytozę układo-
wą zagrożonych reakcją anafilaktyczną po użądleniu przez owada. Dalsze badania mo-
gą pozwolić na stworzenie nowego narzędzia diagnostycznego stosowanego w praktyce
klinicznej.
4. Badanie profilu ekspresji genów wykazało istotne różnice w ekspresji genów w krwi
obwodowej u chorych na mastocytozę w porównaniu z osobami zdrowymi, które wska-
zują na nowe możliwości diagnostyczne i terapeutyczne.
– 35 –
6. PIŚMIENNICTWO
1. Akin C., Metcalfe D.: The biology of Kit in disease and the application of pharmacogenetics. J. Allergy Clin. Immunol. 2004, 114, 1, 13-19.
2. Baba Y., Nishida K., Fujii Y., Hirano T., Hikida M., Kurosaki T.: Essential function for the calcium sensor STIM1 in mast cell activation and anaphylactic responses. Nat. Immunol. 2008, 9,1,81-88.
3. Bernstein IL., Li JT., Bernstein DI., Hamilton R., Spector SL., Tan R., Golden DB., Khan DA., Nicklas RA., Portnoy JM., Blessing-Moore J., Cox L., Lang DM., Oppenheimer J., Randolph CC., Schuller DE., Tilles SA., Wallace DV., Levetin E., Weber R.: American Academy of Allergy, Asthma and Immunol-ogy; American College of Allergy, Asthma and Immunology. Allergy diagnos-tic testing: an updated practice parameter. Ann. Allergy Asthma Immunol. 2008,100,3 Suppl.3,1-148.
4. Biedermann T., Ruëff F., Sander CA., Przybilla B.: Mastocytosis associated with severe wasp sting anaphylaxis detected by elevated serum mast cell tryp-tase levels. Br. J. Dermatol. 1999,141,6,1110-1112.
5. Bilò MB., Brianzoni F., Cinti B., Napoli G., Bonifazi F. The dilemma of the negative skin test reactors with a history of venom anaphylaxis: will this always be the case? Eur. Ann. Allergy Clin. Immunol. 2005,37,9,341-342.
6. Biló BM., Rueff F., Mosbech H., Bonifazi F., Oude-Elberink JN.; the EAACI Interest Group on Insect Venom Hypersensitivity*. Diagnosis of Hymenoptera venom allergy. Allergy. 2005,60,11,1339-1349.
7. Birnbaum J., Ramadour M., Magnan A., Vervloet D.: Hymenoptera ultra-rush venom immunotherapy (210 min): a safety study and risk factors. Clin. Exp. Allergy. 2003,33,1,58-64.
8. Bonadonna P., Perbellini O., Passalacqua G., Caruso B., Colarossi S., Dal Fior D., Castellani L., Bonetto C., Frattini F., Dama A., Martinelli G., Chilosi M., Senna G., Pizzolo G., Zanotti R.: Clonal mast cell disorders in patients with systemic reactions to Hymenoptera stings and increased serum tryptase levels. J. Allergy Clin. Immunol. 2009,123,3,680-686.
9. Bonadonna P., Zanotti R., Caruso B., Castellani L., Perbellini O., Colarossi S., Chilosi M., Dama A., Schiappoli M., Pizzolo G., Senna G., Passalacqua G.: Al-lergen specific immunotherapy is safe and effective in patients with systemic mastocytosis and Hymenoptera allergy. J. Allergy Clin. Immunol. 2008,121,1,256-257.
– 36 –
10. Bonifazi F., Jutel M., Biló BM., Birnbaum J., Muller U.; EAACI Interest Group on Insect Venom Hypersensitivity. Prevention and treatment of hymenoptera venom allergy: guidelines for clinical practice. Allergy 2005,60,12,1459-1470.
11. Brehler R., Wolf H., Kütting B., Schnitker J., Luger T. Safety of a two-day ul-trarush insect venom immunotherapy protocol in comparison with protocols of longer duration and involving a larger number of injections. J Allergy Clin Im-munol. 2000,105,6 Pt 1,1231-1235.
12. Brockow K., Jofer C., Behrendt H., Ring J.: Anaphylaxis in patients with mas-tocytosis: a study on history, clinical features and risk factors in 120 patients. Allergy.2008,63,2,226-232.
13. Brożek JL., Akl EA., Alonso-Coello P., Lang D., Jaeschke R., Williams JW., Phillips B., Horvath AR., Bousquet J., Guyatt GH., Schünemann HJ.; GRADE Working Group. Grading quality of evidence and strength of recommendations in clinical practice guidelines. Allergy. 2009,64,8,1109-1116.
14. Carmona-Saez P., Chagoyen M., Tirado F., Carazo JM., Pascual-Montano A. GENECODIS: a web-based tool for finding significant concurrent annotations in gene lists. Genome Biol. 2007,8,1,R3.
15. Carter MC., Robyn JA., Bressler PB., Walker JC., Shapiro GG., Metcalfe DD.: Omalizumab for the treatment of unprovoked anaphylaxis in patients with sys-temic mastocytosis. J. Allergy Clin. Immunol. 2007,119,6,1550-1551.
16. Charpin D., Birnbaum J., Vervloet D.: Epidemiology of hymenoptera allergy. Clin. Exp. Allergy. 1994,24,11,1010-1015.
17. Crijns AP., Fehrmann RS., de Jong S., Gerbens F., Meersma GJ., Klip HG., Hollema H., Hofstra RM., te Meerman GJ., de Vries EG., van der Zee AG. Sur-vival-related profile, pathways, and transcription factors in ovarian cancer. PLoS Med. 2009,6,2,e24.
18. Daley T., Metcalfe D., Akin C.: Association of the Q576R polymorphism in the interleukin-4 receptor alpha chain with indolent mastocytosis limited to the skin. Blood 2001, 98,3:880-882.
19. D'ambrosio C., Akin C., Wu Y., Magnusson MK., Metcalfe DD. Gene expres-sion analysis in mastocytosis reveals a highly consistent profile with candidate molecular markers. J. Allergy Clin. Immunol. 2003,112,6,1162-1170.
20. Fricker M., Helbling A., Schwartz L., Müller U.: Hymenoptera sting anaphy-laxis and urticaria pigmentosa: clinical findings and results of venom immuno-therapy in ten patients. J. Allergy Clin. Immunol. 1997,100,1,11-15.
– 37 –
21. González de Olano D., Alvarez-Twose I., Esteban-López MI., Sánchez-Muñoz L., de Durana MD., Vega A. García-Montero A., González-Mancebo E., Belver T., Herrero-Gil MD., Fernández-Rivas M., Orfao A., de la Hoz B., Castells MC., Escribano L. Safety and effectiveness of immunotherapy in patients with indolent systemic mastocytosis presenting with Hymenoptera venom anaphy-laxis. J. Allergy Clin. Immunol. 2008,121,2,519-526.
22. Dubois AE.: Mastocytosis and Hymenoptera allergy. Curr. Opin. Allergy Clin. Immunol. 2004,4,4,291-295.
23. Dugas-Breit S., Przybilla B., Schöpf P., Ruëff F.: Possible circadian variation of serum mast cell tryptase concentration. Allergy. 2005,60,5,689-692.
24. Eberlein-König B., Varga R., Mempel M., Darsow U., Behrendt H., Ring J.: Comparison of basophil activation tests using CD63 or CD203c expression in patients with insect venom allergy. Allergy. 2006,61,9,1084-5.
25. Engler RJ., Davis WS.: Rush Hymenoptera venom immunotherapy: successful treatment in a patient with systemic mast cell disease. J. Allergy Clin. Immunol. 1994,94,3 Pt 1,556-559.
26. Fehrmann RS., de Jonge HJ., Ter Elst A., de Vries A., Crijns AG., Weidenaar AC., Gerbens F., de Jong S., van der Zee AG., de Vries EG., Kamps WA., Hof-stra RM., Te Meerman GJ., de Bont ES. A new perspective on transcriptional system regulation (TSR): towards TSR profiling. PLoS One. 2008,3,2,e1656.
27. Fine J. Mastocytosis. Int. J. Dermatol. 1980,19,3,117-123.
28. Finegold I. Issues in stinging insect allergy immunotherapy: a review. Curr. Opin. Allergy Clin. Immunol. 2008,8,4,343-347.
29. Florian S., Krauth MT., Simonitsch-Klupp I., Sperr WR., Fritsche-Polanz R., Sonneck K., Födinger M., Agis H., Böhm A., Wimazal F., Horny HP., Valent P.: Indolent systemic mastocytosis with elevated serum tryptase, absence of skin lesions, and recurrent anaphylactoid episodes. Int. Arch. Allergy Immunol. 2005,136,3,273-280.
30. Franken HH., Dubois AE., Minkema HJ., van der Heide S., de Monchy JG.: Lack of reproducibility of a single negative sting challenge response in the as-sessment of anaphylactic risk in patients with suspected yellow jacket hypersen-sitivity. J. Allergy Clin. Immunol. 1994,93,2:431-436.
31. Garcia-Montero AC., Jara-Acevedo M., Teodosio C., Sanchez ML., Nunez R., Prados A., Aldanondo I., Sanchez L., Dominguez M., Botana LM., Sanchez-Jimenez F., Sotlar K., Almeida J., Escribano L., Orfao A. KIT mutation in mast cells and other bone marrow hematopoietic cell lineages in systemic mast cell disorders: a prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients. Blood. 2006,108,7,2366-2372.
– 38 –
32. Golden DB. Insect sting anaphylaxis. Immunol. Allergy. Clin. North. Am. 2007,27,2,261-272.
33. Golden D., Kwiterovich K., Kagey-Sobotka A., Lichtenstein L.: Discontinuing venom immunotherapy: Extended observations. J. Allergy Clin. Immunol. 1998,101,3,298-305.
34. Golden DB., Marsh DG., Freidhoff LR., Kwiterovich KA., Addison B., Kagey-Sobotka A., Lichtenstein LM.: Natural history of Hymenoptera venom sensitiv-ity in adults. J. Allergy Clin. Immunol. 1997,100,P Pt 1,760-766.
35. Golkar L., Bernhard J.: Mastocytosis. Lancet.1997,349,9062,1379-1385.
36. Gonera R., Oranje W., Wolffenbuttel B.: Shock of unknown origin-think of mastocytosis! Neth. J. Med.1997;50,4,165-169.
37. Gorska L., Chelminska M., Kuziemski K., Skrzypski M., Niedoszytko M., Damps-Konstanska I. Szymanowska A., Siemińska A., Wajda B., Drozdowska A., Jutel M., Jassem E.: Analysis of safety, risk factors and pretreatment meth-ods during rush hymenoptera venom immunotherapy. Int Arch Allergy Immu-nol. 2008,147,3,241-245.
38. Guyatt GH., Oxman AD., Kunz R., Falck-Ytter Y., Vist GE., Liberati A., Schünemann HJ.;GRADE Working Group. Going from evidence to recommen-dations. BMJ. 2008,336,7652,1049-1051.
39. Haeberli G., Brönnimann M., Hunziker T., Müller U. Elevated basal serum tryp-tase and hymenoptera venom allergy: relation to severity of sting reactions and to safety and efficacy of venom immunotherapy. Clin. Exp. Allergy. 2003,33,9,1216-1220.
40.
Hermann K., Donhauser S., Ring J.: Angiotensin in Human Leukocytes of Pa-tients with Insect Venom Anaphylaxis and Healthy Volunteers. Int. Arch. Al-lergy Immunol.1995,107,1-3,385-86.
41. Hermann K., Ring J.: Association between the renin angiotensis system and anaphylaxis. Adv. Exp. Med. Biol. 1995, 377, 299-309.
42. Hermann J., Ring J.: Human leukocytes contain angiotensin I, angiotensin II and angiotensin metabolites. Int. Arch. Allergy Immunol. 1994,103,2,152-59.
43. Hermann K., Ring J.: The renin-angiotensin system in patients with treated ana-phylactic reactions during Hymenoptera venom hyposensitization and sting challenge. Int, Arch, Allergy Immunol. 1997,112,3,251-256.
44. Hermann K., Ring J.: The renin angiotensin system and hymenoptera venom anaphylaxis. Clin. Exp. Allergy. 1993,23,9,762-769.
– 39 –
45. Holla L., Vask A., Znojil V., Sisková L., Vácha J.: Association of 3 gene poly-morphisms with atopic diseases. J. Allergy Clin. Immunol. 1999,103,4,702-8.
46. Ippoliti F., De Santis W., Volterrani A., Lenti L., Canitano N., Lucarelli S., Frediani T.: Immunomodulation during sublingual therapy in allergic children. Pediatr. Allergy Immunol. 2003,14,3,216-221.
47. Jassem E., Niedoszytko M. Mastocytoza rozpoznanie i leczenie. W Fal A.(red): Alergia, choroby alergiczne, astma. T.2. Medycyna Praktyczna 2011 (w druku)
48. Jutel M., Akdis M., Blaser K., Akdis CA.: Mechanisms of allergen specific im-munotherapy-T-cell tolerance and more. Allergy. 2006,61,7,796-807.
49. Kanehisa M., Araki M., Goto S., Hattori M., Hirakawa M., Itoh M., Kawashima S., Okuda S., Tokimatsu T., Yamanishi Y.: KEGG for linking genomes to life and the environment. Nucleic Acids Res. 2008,36,480-484.
50. Kanehisa M., Goto S., Hattori M., Aoki-Kinoshita KF., Itoh M., Kawashima S., Katayama T., Araki M., Hirakawa M.: From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res. 2006,34,354-357.
51. Kawabata Y., Yan T., Yokochi T, Matsushita M, Fujita T., Shibazaki M., No-ikura T., Endo TY., Takada H.:. Complement system is involved in anaphylac-toid rections induced by lipopolisacharides in muramyldipeptide-treated mice. Shock 2000,14,5,572-577.
52. Kazmierska J., Malicki J. Application of the Naïve Bayesian Classifier to opti-mize treatment decisions. Radiother. Oncol. 2008,86,2,211-216.
53. Kazani S., Wechsler ME., Israel E. The role of pharmacogenomics in improving the management of asthma. J Allergy Clin Immunol. 2010,125,2,295-302.
54. Kim JJ., Kim HJ., Lee IK., Chung HT., Lee JH. Association between polymor-phisms of the angiotensin-converting enzyme and angiotensinogen genes and allergic rhinitis in a Korean population. Ann. Otol. Rhinol. Laryngol. 2004,113,4,297-302.
55. Konno S., Hizawa N., Nishimura M., Huang SK.: Osteopontin: a potential bio-marker for successful bee venom immunotherapy and a potential molecule for inhibiting IgE-mediated allergic responses. Allergol. Int. 2006,55,4,355-359.
56. Kononenko I.: Machine learning for medical diagnosis: history, state of the art and perspective. Artif. Intell. Med. 2001,23,1,89-109.
57. Kontou-Fili K. High omalizumab dose controls recurrent reactions to venom immunotherapy in indolent systemic mastocytosis. Allergy. 2008,63,3,376-378.
58. Kontou-Fili K. Patients with negative skin tests. Curr. Opin. Allergy Clin. Im-munol. 2002,2,4,353-357.
– 40 –
59. Kors JW., van Doormaal JJ., de Monchy JG.: Anaphylactoid shock following Hymenoptera sting as a presenting symptom of systemic mastocytosis. J. Intern. Med. 1993,233,3,255-258.
60. Kränke B., Sturm G., Aberer W.: Negative venom skin test results and mastocy-tosis. J. Allergy Clin. Immunol. 2004,113,1,180-181.
61. Kucera P., Cvackova M., Hulikova K., Juzova O., Pachl J.: Basophil activation can predict clinical sensitivity in patients after venom immunotherapy. J. Inves-tig. Allergol. Clin. Immunol. 2010,20,2,110-116.
62. Ludolph-Hauser D., Ruëff F., Fries C., Schöpf P., Przybilla B. Constitutively raised serum concentrations of mast-cell tryptase and severe anaphylactic reac-tions to Hymenoptera stings. Lancet. 2001,357,9253,361-362.
63. Mauss V. Potential effects of global warming on hymenoptera biology and the risk of stings. Allergy 2009,64,579.
64. Meeker ND., Yang JJ., Schiffman JD.: Pharmacogenomics of pediatric acute lymphoblastic leukemia. Expert Opin. Pharmacother. 2010,11,10,1621-1632.
65. Mosbech H., Müller U.: Side effects of insect venom immunotherapy: results from an EAACI multicenter study. Allergy 2000,55,11,1005-1010.
66. Mueller UR.: Cardiovascular disease and anaphylaxis. Curr. Opin. Allergy Clin. Immunol. 2007,7,4,337-341.
67. Mueller HL. Diagnosis and treatment of insect sensitivity. J. Asthma Res.1966,3,4,331-333.
68. Müller UR. New developments in the diagnosis and treatment of hymenoptera venom allergy. Int Arch Allergy Immunol. 2001,124,4:447-453.
69. Müller U., Helbling A., Berchtold E.: Immunotherapy with honeybee venom and yellow jacket venom is different regarding efficacy and safety. J. Allergy Clin. Immunol. 1992,89,2,529-535.
70. Müller UR., Horat W., Wüthrich B., Conroy M., Reisman RE.: Anaphylaxis after Hymenoptera stings in three patients with urticaria pigmentosa. J. Allergy Clin. Immunol. 1983,72,6,685-689.
71. Nedoszytko B., Niedoszytko M., Lange M., van Doormaal J., Gleń J., Zabłotna M., Renke J., Vales A., Buljubasic F., Jassem E., Roszkiewicz J., Valent P.: Interleukin-13 promoter gene polymorphism -1112C/T is associated with the systemic form of mastocytosis. Allergy. 2009,64,2,287-294.
– 41 –
72.
Niedoszytko M., Lange M., Chelminska M., Jaśkiewicz K., Piskosz A., Wasag B., Lewandowski K., Mital A., Renke J., Gruchała-Niedoszytko M., Woźniak M., Babińska A., Jassem E.: [Systemic mastocytosis] Pneumonol. Alergol. Pol. 2005,73,3,239-244.
73. Nogales-Cadenas R., Carmona-Saez P., Vazquez M., Vicente C., Yang X., Ti-rado F., Carazo JM., Pascual-Montano A.:GeneCodis: interpreting gene lists through enrichment analysis and integration of diverse biological information. Nucleic Acids Res. 2009,37,317-322.
74. Oude Elberink JN., de Monchy JG., Kors JW., van Doormaal JJ., Dubois AE.: Fatal anaphylaxis after a yellow jacket sting, despite venom immunotherapy, in two patients with mastocytosis. J. Allergy Clin. Immunol. 1997,99,1 Pt 1,153-154.
75.
Peavy R., Metcalfe D. Understanding the mechanisms of anaphylaxis. Curr Opin Allergy Clin Immunol. 2008,8,4,310-315.
76. Piekutowska-Abramczuk D., Olsen RK., Wierzba J., Popowska E., Jurkiewicz D., Ciara E., Ołtarzewski M., Gradowska W., Sykut-Cegielska J., Krajewska-Walasek M., Andresen BS., Gregersen N., Pronicka E. A comprehensive HADHA c.1528G>C frequency study reveals high prevalence of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency in Poland. J. Inherit. Metab. Dis. 2010 Sep 3. [Epub ahead of print]
77. Price LA., Safko M.: Bee venom allergy in a patient with urticaria pigmentosa. J. Allergy Clin. Immunol. 1987,79,2,407-409.
78. Pumphrey R., Roberts I.: Postmortem findings after fatal anaphylactic reaction. J. Clin. Pathol. 2000,53,4,273-276.
79. Reimers A., Müller U.: Fatal outcome of a Vespula sting in a patient with mas-tocytosis after specific immunotherapy with honey bee venom. Allergy Clin. Immunol. Int. J. WAO Org. 2005,17,68-70.
80. Rigat B., Hubert C., Alhenc-Gelas F., Cambien F., Corvol P., Soubrier F.: An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J. Clin. Invest. 1990,86,4,1343-46.
81. Roepman P., Jassem J., Smit EF., Muley T., Niklinski J., van de Velde T., Wit-teveen AT., Rzyman W., Floore A., Burgers S., Giaccone G., Meister M., Die-nemann H., Skrzypski M., Kozlowski M., Mooi WJ., van Zandwijk N.: An im-mune response enriched 72-gene prognostic profile for early-stage non-small-cell lung cancer. Clin. Cancer Res. 2009,15,1,284-290.
82. Ruëff F, Bilò MB, Jutel M, Mosbech H, Müller U, Przybilla B: Sublingual im-munotherapy with venom is not recommended for patients with Hymenoptera venom allergy. J. Allergy Clin. Immunol. 2009,123,1,272-273.
– 42 –
83. Ruëff F., Przybilla B., Biló MB., Müller U., Scheipl F., Aberer W., Birnbaum J., Bodzenta-Lukaszyk A., Bonifazi F., Bucher C., Campi P., Darsow U., Egger C., Haeberli G., Hawranek T., Körner M., Kucharewicz I., Küchenhoff H., Lang R., Quercia O., Reider N., Severino M., Sticherling M., Sturm GJ., Wüthrich B.: Predictors of severe systemic anaphylactic reactions in patients with Hymenop-tera venom allergy: importance of baseline serum tryptase-a study of the Euro-pean Academy of Allergology and Clinical Immunology Interest Group on In-sect Venom Hypersensitivity. J. Allergy Clin. Immunol. 2009,124,5,1047-1054.
84. Ruëff F., Przybilla B., Biló MB., Müller U., Scheipl F., Aberer W., Birnbaum J., Bodzenta-Lukaszyk A., Bonifazi F., Bucher C., Campi P., Darsow U., Egger C., Haeberli G., Hawranek T., Kucharewicz I., Küchenhoff H., Lang R., Quercia O., Reider N., Severino M., Sticherling M., Sturm GJ., Wüthrich B.: European Academy of Allergy and Clinical Immunology Interest Group. Predictors of side effects during the buildup phase of venom immunotherapy for Hymenoptera venom allergy: the importance of baseline serum tryptase. J. Allergy Clin. Im-munol. 2010;126,1,105-111.
85.
Rernick H., Przybilla B., Rueff F.: Venom immunotherapy (VIT) in patients with systemic mastocytosis (SM) and hymenoptera anaphylaxis (HVA): safety and efficacy of different maintenance doeses. Abstract no 936, AAAAI 2009 annual meeting.
86. Ruëff F., Placzek M., Przybilla B.: Mastocytosis and Hymenoptera venom al-lergy. Curr Opin Allergy Clin. Immunol. 2006,6,4,284-288.
87. Ruëff F., Wenderoth A., Przybilla B.: Patients still reacting to a sting challenge while receiving conventional Hymenoptera venom immunotherapy are protected by increased venom doses. J. Allergy Clin. Immunol. 2001,108,6,1027-1032.
88. Rush JW., Aultman CD.: Vascular biology of angiotensin and the impact of physical activity. Appl. Physiol. Nutr. Metab. 2008,33,1,162-72.
89. Sharabi AB., Aldrich M., Sosic D., Olson EN., Friedman AD., Lee SH., Chen SY.: Twist-2 controls myeloid lineage development and function. PLoS Biol. 2008,6,12,e316.
90. Soriano Gomis V., Gonzalez Delgado P., Niveiro Hernandez E.: Failure of omalizumab treatment after recurrent systemic reactions to bee-venom immuno-therapy. J. Investig. Allergol. Clin. Immunol. 2008,18,3,225-256.
91. Stumpf JL., Shehab N., Patel AC.: Safety of Angiotensin-converting enzyme inhibitors in patients with insect venom allergies. Ann. Pharmacother. 2006,40,4,699-703.
92. Sturm G., Kränke B., Rudolph C., Aberer W.: Rush Hymenoptera venom im-munotherapy: a safe and practical protocol for high-risk patients. J. Allergy Clin. Immunol. 2002,110,6,928-933.
– 43 –
93. Szymański W., Bodzenta-Łukaszyk A. Obraz kliniczny alergii na jady owadów. W red. Nitter-Marszalska M.: Alergia na jad owadów błonkoskrzydłych. Medi-ton. Łódź 2003.
94. Valent P., Akin C., Escribano L., Födinger M., Hartmann K., Brockow K., Ca-stells M., Sperr WR., Kluin-Nelemans HC., Hamdy NA., Lortholary O., Robyn J., van Doormaal J., Sotlar K., Hauswirth AW., Arock M., Hermine O., Hell-mann A., Triggiani M., Niedoszytko M., Schwartz LB., Orfao A., Horny HP., Metcalfe DD.: Standards and standardization in mastocytosis: consensus state-ments on diagnostics, treatment recommendations and response criteria. Eur. J. Clin. Invest. 2007, 37, 6,435-453.
95. Valent P., Horny HP., Escribano L., Longley BJ., Li CY., Schwartz LB., Ma-rone G., Nuñez R., Akin C., Sotlar K., Sperr WR., Wolff K., Brunning RD., Parwaresch RM., Austen KF., Lennert K., Metcalfe DD., Vardiman JW., Ben-nett JM.: Diagnostic criteria and classification of mastocytosis: a consensus pro-posal. Leuk. Res. 2001,25,7,603-625.
96. Valent P., Sperr W., Schwartz L., Horny H.: Diagnosis and classification of mast cell proliferative disorders: delineation from immunologic diseases and non-mast cell hematopoietic neoplasms. J. Allergy Clin. Immunol.2004,114,1,3-11.
97. van 't Veer LJ., Dai H., van de Vijver MJ., He YD., Hart AA., Mao M., Peterse HL., van der Kooy K., Marton MJ., Witteveen AT., Schreiber GJ., Kerkhoven RM., Roberts C., Linsley PS., Bernards R., Friend SH. Gene expression profil-ing predicts clinical outcome of breast cancer. Nature. 2002,415,6871,530-536.
98. Vennekens R., Olausson J., Meissner M., Bloch W., Mathar I., Philipp SE., Schmitz F., Weissgerber P., Nilius B., Flockerzi V., Freichel M.: Increased IgE dependent mast cell activation and anaphylactic responses in mice lacking the calcium activated nonselective cation channel TRPM4. Nat. Immunol. 2007,8,3,312-320.
99. Volcheck G., Butterfield J., Yunginger J., Klee G.: Elevated serum levels of calcitonin gene-related peptide in Hymenoptera venom anaphylaxis. J. Allergy Clin. Immunol. 1998,102,1,149-151.
100.
Wagner N., Fritze D., Przybilla B., Hagedorn M., Ruëff F.: Fatal anaphylactic sting reaction in a patient with mastocytosis. Int. Arch. Allergy Immunol. 2008,146,2,162-163.
101. Wenzel J., Meissner-Kraemer M., Bauer R., Bieber T., Gerdsen R.: Safety of rush insect venom immunotherapy. The results of a retrospective study in 178 patients. Allergy. 2003,58,11,1176-1179.
102. Zimmerli SC., Hauser C. Langerhans cells and lymph node dendritic cells ex-press the tight junction component claudin-1. J. Invest. Dermatol. 2007,127,10,2381-2390.
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Original Paper
Int Arch Allergy Immunol 2010;153:166–172 DOI: 10.1159/000312634
The Angiotensinogen AGT p.M235T Gene Polymorphism May Be Responsible forthe Development of Severe Anaphylactic Reactions to Insect Venom Allergens
Marek Niedoszytko a Magdalena Ratajska b Marta Chełmińska a Michał Makowiecki c Ewelina Malek b Alicja Siemińska a Janusz Limon b Ewa Jassem a
Departments of a Allergology, b Biology and Genetics, and c Laboratory Medicine, Medical University of Gdansk, Gdansk , Poland
prevalence of the ACE I/D polymorphism and angiotensin I levels between control groups and patients with different grades of anaphylactic reactions or patients with side effects of venom immunotherapy. Conclusion: The AGT M235T MM variant may be responsible for severe anaphylactic reactions to insect venom allergens in some patients.
Copyright © 2010 S. Karger AG, Basel
Introduction
Insect venom allergy (IVA), defined as at least one sys-temic IgE-mediated reaction (during an entire lifetime) following an insect sting, is present in approximately 1–3% of the population [1] . It is estimated that approxi-mately 2.4% of the patients with IVA have experienced at least one life-threatening reaction graded as III or IV on the Mueller scale during their lifetime [1–5] . The treat-ment of choice for IVA patients is venom immunotherapy (VIT) [2, 3] .
A large group of patients with high concentrations of specific IgE do not react to stings, whereas a proportion of patients with severe reactions have low specific IgE lev-els [1–4] . Consequently, factors other than the serum IgE level may also contribute to the allergic reaction to insect
Key Words
Genetics � I/D ACE � Insect venom allergy � M235T AGT
Abstract
Background: Insect venom allergy (IVA) is present in 1–3% of the population. A group of patients with high specific IgE do not react to stings. In contrast, a proportion of patients with IVA have low specific IgE levels. These findings indicate that factors other than specific IgE may also be involved in IVA. Dysfunction of the renin-angiotensin system (RAS) has been described as a potential factor in IVA. The objective of this study was to determine the prevalence of angiotensin AGT p.M235T and angiotensin-converting enzyme ACE I/D, I/I, D/D gene polymorphisms in patients with IVA and to re-late the presence of these gene variants to the course of IVA and the safety of treatment. Methods: A total of 107 patients with IVA and 113 controls were studied. AGT p.M235T and ACE (ID, I/I, D/D) gene polymorphisms were examined, and angiotensin I levels were measured by immunoassay. Re-
sults: The frequency of the AGT MM M235T variant was sig-nificantly higher in IVA patients (29.9%) than in controls (17%, p = 0.02). The presence of the MM M235T genotype increased the risk of grade IV reactions (odds ratio = 2.5 and 95% con-fidence interval 1.04–6.08). There were no differences in the
Received: June 16, 2009 Accepted after revision: November 26, 2009 Published online: April 22, 2010
Correspondence to: Dr. Marek Niedoszytko Department of Allergology Medical University of Gdansk Debinki 7, PL–80-952 Gdansk (Poland) Tel. +48 58 349 1626, Fax +48 58 349 1625, E-Mail mnied @ amg.gda.pl
© 2010 S. Karger AG, Basel1018–2438/10/1532–0166$26.00/0
Accessible online at:www.karger.com/iaa
AGT , ACE Polymorphisms in Insect Venom Allergy
Int Arch Allergy Immunol 2010;153:166–172 167
venom. In previous studies, dysfunction of the renin-an-giotensin system (RAS), for example, resulted in stronger allergic responses to Hymenoptera venom [6–11] .
Angiotensin II belongs to a family of agents exerting strong vasoconstrictive effects [12] . The level of angioten-sin II is regulated by the level of its precursors (angioten-sinogen and angiotensin I), the activity of the enzyme converting angiotensin I into angiotensin II, and angio-tensin II receptor activity [12] .
Angiotensinogen is an inactive plasma protein pro-duced by hepatocytes. Renin, an enzyme released by granular juxtaglomerular cells of the kidney, cleaves an-giotensinogen into angiotensin I, while the angiotensin-converting enzyme (ACE) cleaves angiotensin I into an-giotensin II, the main RAS effector [12] .
In most cases, a Hymenoptera sting leads to a local re-action characterized by redness, swelling and local ede-ma [1–4] . Constriction of the vessels mediated by angio-tensin may prevent a generalized reaction. Furthermore, many patients show symptoms of arrhythmia and/or hy-potension, which may be related to RAS activation [6–11] . Results of the study by Hermann and Ring [6–10] and Hermann et al. [11] showed that patients who experienced anaphylaxis to insect venom had significantly lower lev-els of angiotensin I, angiotensin II, renin and angioten-sinogen, i.e. molecules involved in RAS, in comparison to non-allergic controls. Patients who repeatedly experi-enced anaphylactic reactions during VIT had significant-ly lower levels of these molecules compared with patients without side effects of VIT [6–11] . Interestingly, patients with good VIT tolerance had RAS molecules within nor-mal ranges [6–11] . The low levels of these molecules also correlated with the severity of clinical symptoms, where-as no association with serum concentrations of aldoste-rone was observed [7] . Significant differences in RAS molecule levels were found in patients that reacted posi-tively in a provocation test to a living insect. They had lower levels of angiotensinogen and angiotensin I/II, whereas in patients with a negative reaction, who may be regarded as successfully treated, the levels of angioten-sinogen were even lower than in healthy subjects [6] .
The AGT gene, encoding for angiotensinogen, is lo-cated on the long arm of chromosome 1 (1q42). Enzy-matic activity is related to polymorphic variants (methio-nine ] threonine) at codon 235, where a thymine is sub-stituted by a cytosine [12–14] . The MM genotype of AGT is related to low levels of angiotensinogen [12–15] .
The ACE gene is located on the long arm of chromo-some 17, and several polymorphisms of this gene have been found so far. An insertion (I)/deletion (D) polymor-
phism of a 287-bp fragment within intron 16 was recent-ly identified. The presence of the D allele is related to a higher plasma ACE concentration and cardiovascular diseases [12–18] .
Both AGT and ACE gene polymorphisms are associ-ated with cardiovascular disorders [12–18] . However, lit-tle is known about the association of the ACE and AGT gene variants to allergic diseases. Studies by Benessiano et al. [19] and Holla et al. [20] demonstrated a relationship between the DD genotype of the ACE gene and the prev-alence of asthma in a Caucasian population, whereas the M235 allele of the AGT gene was more frequent among patients with asthma, allergic rhinitis and atopic derma-titis [19, 20] . Interestingly, such a relationship was absent in Korean populations [21–23] . ACE also inactivates ki-nins, substance P, bradykinin and prostaglandins (mol-ecules involved in the pathogenesis of asthma) [23] .Decreased ACE activity was noted in patients with aspi-rin-intolerant asthma, which leads to bronchial hyper-reactivity and eosinophilic inflammation [23] . Although the results might explain differences in the concentration of molecules involved in RAS, no studies have defined an association between gene polymorphisms and IVA pa-tients. It is likely that a pharmacogenetic approach to the diagnosis of IVA might predict the incidence of anaphy-lactic reactions during VIT and the efficacy of treatment.
The aim of this study was to determine the prevalence rates of AGT (p.M235T) and ACE (I/D, I/I and D/D ) gene polymorphisms in patients with IVA and to relate the presence of particular gene variants to the course of the disease (severity of anaphylactic reactions prior to treat-ment) and the safety of treatment.
Patients and Methods
Patients A total of 107 consecutive patients with Hymenoptera allergy
treated at the Department of Allergology, Medical University of Gdansk, were studied. Their mean age was 41 years (range 18–75). There were 59 (55%) women (mean age 41 years; range 18–75) and 48 (45%) men (mean age 40 years; range 18–73). IVA was diag-nosed according to the guidelines of the European Academy of Allergy and Clinical Immunology [1] .
The control group consisted of 113 healthy blood donors re-cruited from the Regional Center for Blood Donation in Gdansk. There were 65 (58%) men and 48 (42%) women (mean age 41 years; range 21–74). The age and gender differences were not significant, and the genes in the current study were not dependent on the X chromosome.
Clinical data on age, sex, personal and family history of IVA, previous hospitalizations, home and work characteristics, envi-ronmental exposures and physical examination were obtained af-
Niedoszytko et al. Int Arch Allergy Immunol 2010;153:166–172168
ter written informed consent. Exclusion criteria included malig-nancy, tuberculosis, pregnancy, and heart, renal or liver failure. None of the patients reported treatment with ACE inhibitors or � -blockers.
The study was approved by the Ethical Committee of the Med-ical University of Gdansk (NKEBN/811/2004).
Skin Test Skin prick tests (SPTs; concentrations of 10 and 100 � g/ml)
and intracutaneous tests (concentrations of 0.01 and 0.1 � g/ml) with allergens of yellow jacket and bee (HAL Allergy, Haarlem, The Netherlands) were carried out according to the recommenda-tions of the European Academy of Allergy and Clinical Immunol-ogy [1] . Positive SPTs were defined as a mean wheal diameter3 mm larger than that of the negative control. Intracutaneous tests were regarded positive when the mean wheal diameter was 5 mm larger than that of the negative control. Glycerol-buffered saline was the negative control, and histamine (1 mg/ml) was the posi-tive control. Allergopharma lancets and 0.7-mm needles were used for SPT and intracutaneous tests, respectively.
Specific IgE Specific IgE levels were measured with UniCAP (Pharmacia,
Uppsala, Sweden) according to the manufacturer’s instructions. Results 6 class I (IgE level of 0.35 kU/l) were considered positive.
Immunotherapy All patients with IVA (either grade III or IV reactions accord-
ing to the Mueller [5] scale) were treated with insect venom aller-gens. Bee allergens (Venomenhal Biene, HAL Allergy) were used in 26 (24%) cases, and yellow jacket allergens (Venomenhal Wespe, HAL Allergy) were used in 81 cases (76%). The initial phase was performed with the rush procedure according to the manufacturer’s instructions. Maintenance doses (100 � g) were administered every 4–6 weeks.
Collection of Blood Samples For genetic analysis, peripheral blood samples (10 ml) were
collected into EDTA-containing tubes and stored at –80 ° C. Ge-nomic DNA was isolated from peripheral blood leukocytes using Blood DNA Prep Plus according to the manufacturer’s instruc-
tions (A&A Biotechnology, Gdynia, Poland). DNA concentration was assessed using the Beckman DU-600 spectrophotometer. Pa-tient sera were collected and stored at –20 ° C. All samples were taken when patients were on the maintenance doses of VIT.
AGT M235T (rs699) Gene Polymorphism Analysis To detect AGT (p.M235T) gene polymorphisms, allele-specif-
ic oligonucleotide polymerase chain reaction (PCR) was used [20–21] . In order to confirm the results of the analysis, every 10th sample was sequenced using the ABI PRISM 310 genetic analyzer. More information on the protocols used, including the choice of primers, can be obtained from the corresponding author upon request.
ACE I/D, I/I, D/D (rs1799752) Gene Polymorphism Analysis To detect ACE (ID, I/I and D/D) gene polymorphisms, PCR
was applied [13] . Due to the possibility of misclassification of ID as DD, all DD homozygotes were reanalyzed with primers spe-cific for the insert allele ( fig. 1 ). In order to confirm the results of the analysis, every 10th sample was sequenced using the ABI PRISM 310 genetic analyzer. More information on the protocols used, including the choice of primers, can be obtained from the corresponding author upon request.
Measurement of Serum Angiotensin I Levels Serum angiotensin I levels were measured using a commercial
ELISA (Phoenix Pharmaceuticals, Burlingame, Calif., USA). The samples were taken during the maintenance phase of VIT, and the 3rd or 4th year of the treatment. The assay was performed accord-ing to the manufacturer’s instructions. The detection rate of the kit ranges from 0 to 25 ng/ml.
Statistical Analysis Results were expressed as a percentage of the patients, while
differences in percentages between groups were assessed by � 2 test. Data on continuous variables (angiotensinogen level) were presented as medians and standard deviations. The differences in angiotensinogen levels between groups were analyzed using the Mann-Whitney U test. Hardy-Weinberg equilibrium was tested by � 2 analysis using Tufts University calculator. Statistica 8.0 PL (StatSoft, Tulsa, Okla., USA) software was applied.
MT TT DD ID IIMM
AGT ACE
Fig. 1. AGT 235 MT and ACE I/D gene polymorphism analysis in IVA patients.
AGT , ACE Polymorphisms in Insect Venom Allergy
Int Arch Allergy Immunol 2010;153:166–172 169
Results
Insect Venom Allergy VIT was administered to 50 (47%) patients who expe-
rienced grade III anaphylactic reactions to insect venom and to 57 (53%) patients with grade IV reactions. During the rush phase of VIT, systemic reaction occurred in 11 (10%) cases. In 8 cases, the adverse reaction was graded as life-threatening (III or IV according to the Mueller [5] scale). Coexisting diseases diagnosed are described in ta-ble 1 . All patients with mastocytosis suffered from grade IV anaphylactic reactions before treatment, other co-morbidities did not relate to the severity of the preceding reaction. No relationship was found between co-morbid-ities present in the study group and the prevalence of side effects. Re-stings were reported in � 25% of the patients during VIT, but did not result in a systemic reaction.
High Frequency of the AGT M235T MM Polymorphism in Patients with Grade IV Anaphylactic Reactions The AGT gene polymorphism, in both the study and
the control groups, did not exhibit significant deviation from Hardy-Weinberg expectations. The frequency of the MM 235 AGT variant was significantly higher (p = 0.02) in patients with IVA (n = 32; 29.9%) in comparison with controls (n = 19; 17%). This genotype was significantly more prevalent (p = 0.03) in patients with grade IV ana-phylactic reactions prior to treatment (n = 22; 39%) com-pared with subjects with III grade reactions (n = 10; 20%). The difference was significant for patients with grade IV
reactions vs. controls (p = 0.001) but not for patients with grade III reactions. Presence of the MM variant increased the risk of grade IV reactions (odds ratio: 2.5; 95% confi-dence interval: 1.04–6.08; table 2 ). No differences were found in the presence of the MM variant between patients with and without side effects during the rush phase of immunotherapy.
ACE I/D, D/D and I/I Polymorphisms In the study and control groups, the ACE gene poly-
morphism did not exhibit significant deviation from Hardy-Weinberg expectations. The prevalence of the genotypes studied did not differ between the control group and patients with different grades of anaphylactic reactions or with patients that experienced side effects during immunotherapy ( table 2 ).
Angiotensin I Level The angiotensin I level was measured in 37 patients
with IVA (35% of the study group) without coexisting dis-eases influencing the level of angiotensin I. The serum level of angiotensin in patients possessing the MM geno-type of the AGT (1.04 8 0.7 ng/ml) gene was not signifi-
Table 1. C linical data of the patients and controls
IVA patients(n = 107)
Controls(n = 106)
Age, years (range) 41 (18–75) 41 (21–74)Males/females, % 45/55 58/42Yellow jacket/bee allergy, % 75/25Mueller class III/IV, % 47/53Hypertension, n (%) 34 (36)Hypercholesterolemia, n (%) 6 (6)Diabetes, n (%) 7 (6)Thyroid diseases, n (%) 13 (12)Coronary artery disease, n (%) 5 (5)Asthma, n (%) 18 (16)Allergic rhinitis, n (%) 22 (20)Chronic obstructive pulmonary
disease, n (%) 3 (3)Mastocytosis, n (%) 4 (4)
Table 2. AGT p.M235T and ACE I/D polymorphisms in the IVA patients and in the control group
MMII
MTID
TTDD
HWEp value
Control groupMT235 AGT 19 (17%)* 57 (51%) 37 (32%) 0.70I/D ACE 29 (25%) 64 (57%) 20 (18%) 0.13
IVA patients All
MT235 AGT 32 (30%)* 52 (48%) 23 (22%) 0.82I/D ACE 23 (21%) 51 (48%) 33 (31%) 0.69
Grade III anaphylactic reactionsMT235 AGT 10 (20%)** 25 (50%) 15 (30%) 0.94I/D ACE 11 (22%) 26 (52%) 13 (26%) 0.76
Grade IV anaphylactic reactionsMT235 AGT 22 (39%)** 27 (47%) 8 (14%) 0.95I/D ACE 12 (21%) 25 (44%) 20 (35%) 0.42
The frequency of the MM235 AGT genotype was significantly higher in the patients (30%) than in the controls (30%; * p = 0.02) and in the IVA patients with grade IV (39%) vs. grade III anaphy-lactic reactions (20%; ** p = 0.03). The presence of the MM geno-type increased the risk of grade IV reaction (odds ratio: 2.5; 95% confidence interval: 1.04–6.08). HWE = Hardy-Weinberg equi-librium.
Niedoszytko et al. Int Arch Allergy Immunol 2010;153:166–172170
cantly lower than in patients having either the MT or TT genotype (1.16 8 0.61 ng/ml; fig. 2 ). Patients with grade IV reactions also had a lower level of angiotensin (1.0 8 0.6 ng/ml) in comparison to patients with grade III (1.12 8 0.6 ng/ml), although not significantly different (p = 0.1). The ACE I/D polymorphism was also not related to the level of angiotensin I. An association between the lev-el of angiotensin I and co-morbidities, including masto-cytosis, was not detected.
Discussion
The results of this study confirmed the relevant role of RAS in IVA. The presence of the AGT p.M235T MM vari-ant was found to be more prevalent in patients with IVA, particularly in those who suffered from the most severe (grade IV) reaction. No differences were found in the prevalence of the ACE I/D polymorphism and IVA. Sig-nificant differences between the serum level of angioten-sin I and either polymorphic variants of the ACE gene or the grade of anaphylactic reactions were absent.
In the present study, the frequency of the AGT gene MM235 variant, which is related to lower angiotensino-gen levels, was significantly higher in IVA patients (30%) compared with control subjects (17%). Levels of angioten-sinogen and its metabolites, angiotensin I and angioten-sin II, were reduced in IVA and in IVA patients with re-peated anaphylactic reactions during Hymenoptera ven-om hyposensitization and sting challenge [6–11] . These findings might be partially explained by a trend to de-creased angiotensin levels in carriers of the MM variant. Indeed, an inverse correlation of angiotensinogen and angiotensin I and II with the severity of anaphylaxis was reported [6–11] .
The presence of the MM235 variant (39% of patients with grade IV reactions) was highly associated with an increased risk of grade IV reactions (odds ratio = 2.5; 95% confidence interval 1.04–6.08) in this study. Additional-ly, angiotensin I was decreased in patients with grade IV reactions. Our results suggest that the observed differ-ences may have a genetic background. In the present study, plasma levels of angiotensin I were measured dur-ing the maintenance phase of VIT, thus this may explain why differences between the subgroups of patients with grades III and IV anaphylactic reactions were not signif-icant. However, the lack of significance may also be due to the low number of subjects included in the analysis. Previous studies showed that successful VIT induced a 9-fold increase in angiotensin I and II levels [6, 10] .
The association of the angiotensinogen gene p.M235T polymorphism was found in patients with asthma and atopic diseases (allergic rhinitis and atopic dermatitis) [20] . Similar to our study, the MM variant in association with the lowest angiotensin levels was more prevalent in patients with atopic disease (p = 0.02) [20] . This finding was not confirmed in Korean patients with allergic rhi-nitis [21, 22] . The differences in the frequencies of gene polymorphisms found in the above studies [20–22] may be related to different patient groups. Angiotensinogen is produced by hepatocytes and other cells, providing a constant level of the substrate for RAS [24] . Transcription of angiotensinogen might also be induced by acute-phase mediators, such as IL-1 � and TNF- � [20, 24] . Systemic inflammation, which affects acute-phase proteins, plays a more important role in asthma and atopic dermatitis than in IVA. This may explain the relevance of the AGT M235T gene variant in those patient groups.
The frequency of the ACE I/D polymorphism did not differ in the groups studied. ACE is a membrane-bound enzyme found in epithelial and endothelial cells of blood vessels, lungs and other organs [13, 15, 25] . The functions of the ACE enzyme (the cleavage of angiotensin I and in-activation of active substances like kinins, substance P and prostaglandins) are important in the pathology of al-lergic diseases. The role of the ACE I/D polymorphism in atopic diseases [20] and aspirin-intolerant asthma [23] was discussed previously. In contrast, no difference in the frequency of this polymorphism was found in the IVA patients studied, confirming the observation that there is no difference in plasma ACE activity between patients with IVA allergy and controls [6–8] . These results suggest that a low level of angiotensinogen is the most important factor responsible for the diminished function of the RAS pathway and severe insect venom anaphylaxis [6–11] .
00.20.40.60.81.01.21.41.61.82.0
An
gio
ten
sin
I(n
g/m
l)
MM
1.01
MT TT
1.051.18
Fig. 2. Angiotensin I levels (means 8 SD) in patients with differ-ent genotypes of the M235T AGT gene. The differences were not significant.
AGT , ACE Polymorphisms in Insect Venom Allergy
Int Arch Allergy Immunol 2010;153:166–172 171
In IVA patients, treatment of hypertension with ACE inhibitors has been the subject of previous discussions [25–27] . A review of papers published from 1966 to 2006 suggested that ACE inhibitors may exacerbate the re-sponse to insect venom, resulting in potentially life-threatening allergic reactions to insect stings or VIT [25] . Consequently, avoidance of ACE inhibitors has been ad-vocated in IVA patients [25, 27] . On the other hand, this group of drugs has proven protective effects in cardiovas-cular diseases and diabetes [12–14] . In a recently pub-lished retrospective analysis, these drugs were not found to be associated with an increased frequency of side ef-fects to IVA [26] . The results of the present study indicate that 39% of the IVA patients possess the M235T polymor-phism, which is related to the low level of angiotensino-gen. No difference was evidenced between the prevalence of side effects of VIT and the polymorphisms studied. However, such a correlation cannot be excluded. Further studies are required to determine the risk-benefit ratio of ACE inhibitors in IVA patients.
IVA is classified as an IgE-mediated non-atopic reac-tion. The most important pathway involved is IgE-medi-ated degranulation of mast cells associated with previous sensitization and a shift in the T-regulatory/T-helper 2 cell balance [28] . Alternative mechanisms of systemic re-action were studied, including complement activation, calcitonin-gene-related peptide overproduction, osteo-pontine pathway involvement and diminished RAS func-tion [6–11, 29–32] . In the present study, coexisting atopic diseases were found in 31% of patients. This result is sim-ilar to the prevalence of allergic diseases in the general
population [4] . This clinical observation may lead to the hypothesis that genes involved in insect venom anaphy-laxis may be responsible for factors different from those involved in allergic reactions graded according to the Gell-Coombs classification.
In summary, pharmacogenetics is one of the most promising fields of allergy management. There is hope that, in the future, diagnosis based on genetic assessment may facilitate tailoring of therapy for particular patients.
Conclusion
The AGT MM235 gene polymorphism may be respon-sible for severe anaphylactic reactions to insect venom al-lergens and should probably be considered in the diagno-sis of IVA. It is likely that a pharmacogenetic approach to the diagnosis of IVA may lead to safer and more effective treatment options.
Acknowledgments
This study was supported by the Foundation for Polish Science and a grant from the Polish Ministry of Science and Higher Edu-cation (No. N402085934).
The authors would like to thank Mrs. Henryka Murawska (RN; Department of Allergology) and Drs. Ewa Roik, Jolanta Jus-cinska and Malgorzata Szafran (Regional Center for Blood Dona-tion in Gdansk) for their help in blood collection, and Dr. Adam Burkiewicz (A&A Biotechnology) for his help with DNA isola-tion.
References
1 Bilo BM, Rueff F, Mosbech H, Bonifazi F, Oude-Elberink JN: Diagnosis of Hymenop-tera venom allergy. Allergy 2005; 60: 1339–1349.
2 Bonifazi F, Jutel M, Bilo BM, Birnbaum J, Muller U: Prevention and treatment of Hy-menoptera venom allergy: guidelines for clinical practice. Allergy 2005; 60: 1459–1470.
3 Finegold I: Issues in stinging insect allergy immunotherapy: a review. Curr Opin Aller-gy Clin Immunol 2008; 8: 343–347.
4 Lieberman P: Epidemiology of anaphylaxis. Curr Opin Allergy Clin Immunol 2008; 8: 316–320.
5 Mueller HL: Diagnosis and treatment of in-sect sensitivity. J Asthma Res 1966; 3: 331–333.
6 Hermann K, Ring J: The renin-angiotensin system in patients with repeated anaphylac-tic reactions during Hymenoptera venom hyposensitization and sting challenge. Int Arch Allergy Immunol 1997; 112: 251–256.
7 Hermann K, Ring J: Histamine, tryptase, norepinephrine, angiotensinogen, angioten-sin-converting enzyme, angiotensin I and II in plasma of patients with Hymenoptera ven-om anaphylaxis. Int Arch Allergy Immunol 1994; 104: 379–384.
8 Hermann K, Ring J: Association between the renin angiotensin system and anaphylaxis. Adv Exp Med Biol 1995; 377: 299–309.
9 Hermann K, Ring J: Human leukocytes con-tain angiotensin I, angiotensin II and angio-tensin metabolites. Int Arch Allergy Immu-nol 1994; 103: 152–159.
10 Hermann K, Ring J: The renin angiotensin system and Hymenoptera venom anaphylax-is. Clin Exp Allergy 1993; 23: 762–769.
11 Hermann K, Donhauser S, Ring J: Angioten-sin in human leukocytes of patients with in-sect venom anaphylaxis and healthy volun-teers. Int Arch Allergy Immunol 1995; 107: 385–386.
12 Rush JW, Aultman CD: Vascular biology of angiotensin and the impact of physical activ-ity. Appl Physiol Nutr Metab 2008; 33: 162–172.
13 Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F: An insertion/dele-tion polymorphism in the angiotensin I-con-verting enzyme gene accounting for half the variance of serum enzyme levels. J Clin In-vest 1990; 86: 1343–1346.
Niedoszytko et al. Int Arch Allergy Immunol 2010;153:166–172172
14 Azizi M, Hallouin MC, Jeunemaitre X, Guy-ene T, Menard J: Influence of the M235T polymorphism of human angiotensinogen (AGT) on plasma AGT and renin concentra-tions after ethinylestradiol administration. J Clin Endocr Metab 2000; 85: 4331–4337.
15 Mondorf UF, Russ A, Wiesemann A, Herre-ro M, Oremek G, Lenz T: Contribution ofangiotensin I converting enzyme gene poly-morphism and angiotensinogen gene poly-morphism to blood pressure regulationin essential hypertension. Am J Hypertens 1998; 11: 174–183.
16 Lindpaintner K, Pfeffer MA, Kreutz R, Stampfer MJ, Grodstein F, LaMotte F, Buring J, Hennekens CH: A prospective evaluation of an angiotensin-converting-enzyme gene polymorphism and the risk of ischemic heart disease. N Engl J Med 1995; 332: 706–711.
17 Prasad A, Narayanan S, Waclawiw MA, Ep-stein N, Quyyumi AA: The insertion/dele-tion polymorphism of the angiotensin-con-verting enzyme gene determines coronary vascular tone and nitric oxide activity. J Am Coll Cardiol 2000; 36: 1579–1586.
18 Villard E, Tiret L, Visvikis S, Rakotovao R, Cambien F, Soubrier F: Identification of new polymorphisms of the angiotensin I-con-verting enzyme (ACE) gene, and study of their relationship to plasma ACE levels by two-QTL segregation-linkage analysis. Am J Hum Genet 1996; 58: 1268–1278.
19 Benessiano J, Crestani B, Mestari F, Klouche W, Neukirch F, Hacein-Bey S, Durand G, Aubier M: High frequency of a deletion poly-morphism of the angiotensin-converting en-zyme gene in asthma. J Allergy Clin Immu-nol 1997; 99: 53–57.
20 Holla L, Vask A, Znojil V, Sisková L, Vácha J: Association of 3 gene polymorphisms with atopic diseases. J Allergy Clin Immunol 1999; 103: 702–708.
21 Kim JJ, Kim HJ, Lee IK, Chung HT, Lee JH: Association between polymorphisms of the angiotensin-converting enzyme and angio-tensinogen genes and allergic rhinitis in a Korean population. Ann Otol Rhinol Laryn-gol 2004; 113: 297–302.
22 Lee YC, Cheon KT, Lee HB, Kim W, Rhee YK, Kim DS: Gene polymorphism of endo-thelial nitric oxide synthase and angioten-sin-converting enzyme in patients with asth-ma. Allergy 2000; 55: 959–963.
23 Kim TH, Chang HS, Park SM, Nam BY, Park JS, Rhim T, Park HS, Kim MK, Choi IS, Cho SH, Chung IY, Park BL, Park CS, Shin HD: Association of angiotensin I-converting en-zyme gene polymorphisms with aspirin in-tolerance in asthmatics. Clin Exp Allergy 2008; 38: 1727–1737.
24 Brasier AR, Ron D, Tate JE, Habener JF: A family of constitutive C/EBP-like DNA binding proteins attenuate the IL-1 alpha in-duced, NF kappa B mediated trans-activa-tion of the angiotensinogen gene acute-phase response element. EMBO J 1990; 9: 3933–3944.
25 Stumpf JL, Shehab N, Patel AC: Safety of an-giotensin-converting enzyme inhibitors in patients with insect venom allergies. Ann Pharmacother 2006; 40: 699–703.
26 White KM, England RW: Safety of angioten-sin-converting enzyme inhibitors while re-ceiving venom immunotherapy. Ann Aller-gy Asthma Immunol 2008; 101: 426–430.
27 Lieberman P: Anaphylaxis. Med Clin North Am 2006; 90: 77–95.
28 Jutel M, Akdis M, Blaser K, Akdis CA: Mech-anisms of allergen specific immunotherapy – T-cell tolerance and more. Allergy 2006; 61: 796–807.
29 Kawabata Y, Yang TS, Yokochi TT, Matsu-shita M, Fujita T, Shibazaki M, Noikura T, Endo TY, Takada H: Complement system is involved in anaphylactoid reaction induced by lipopolysaccharides in muramyldipep-tide-treated mice. Shock 2000; 14: 572–577.
30 Volcheck G, Butterfield J, Yunginger J, Klee G: Elevated serum levels of calcitonin gene-related peptide in Hymenoptera venom ana-phylaxis. J Allergy Clin Immunol 1998; 102: 149–151.
31 Konno S, Hizawa N, Nishimura M, Huang SK: Osteopontin: a potential biomarker for successful bee venom immunotherapy and a potential molecule for inhibiting IgE-medi-ated allergic responses. Allergol Int 2006; 55: 355–359.
32 Lawley H, Hird H, Mallinder P: Detection of an activating c-kit mutation by real-time PCR in patients with anaphylaxis. Mutat Res 2005; 572: 1–13.
Gene expression analysis in predicting the effectivenessof insect venom immunotherapy
Marek Niedoszytko, MD, PhD,a,b Marcel Bruinenberg, PhD,c,d Jan de Monchy, MD, PhD,b Cisca Wijmenga, PhD,c
Mathieu Platteel, BSc,c Ewa Jassem, MD, PhD,a and Joanne N. G. Oude Elberink, MD, PhDb Gdansk, Poland, and Groningen,
The Netherlands
Abbreviations used
CLDN1: Claudin 1
IVA: Insect venom allergy
MAPK: Mitogen-activated protein kinase
PRLR: Prolactin receptor
SLC16A4: Solute carrier family 16
SNX33: sh3 and px domain containing 3
STAT: Signal transducer and activator of transcription
TWIST2: Transcription factor twist homolog 2
VIT: Venom immunotherapy
Background: Venom immunotherapy (VIT) enables longtimeprevention of insect venom allergy in the majority of patients.However, in some, the risk of a resystemic reaction increasesafter completion of treatment. No reliable factors predictingindividual lack of efficacy of VIT are currently available.Objective: To determine the use of gene expression profiles topredict the long-term effect of VIT.Methods: Whole genome gene expression analysis wasperformed on RNA samples from 46 patients treated with VITdivided into 3 groups: (1) patients who achieved and maintainedlong-term protection after VIT, (2) patients in whom insectvenom allergy relapsed, and (3) patients still in the maintenancephase of VIT.Results: Among the 48.071 transcripts analyzed, 1401 showeda >2 fold difference in gene expression (P < .05); 658 genes(47%) were upregulated and 743 (53%) downregulated. Forty-three transcripts still show significant differences in expressionafter correction for multiple testing; 12 of 43 genes (28%) wereupregulated and 31 of 43 genes (72%) downregulated. A naiveBayes prediction model demonstrated a gene expression patterncharacteristic of effective VIT that was present in all patientswith successful VIT but absent in all subjects with failure ofVIT. The same gene expression profile was present in 88% ofpatients in the maintenance phase of VIT.Conclusion: Gene expression profiling might be a useful tool toassess the long-term effectiveness of VIT. The analysis ofdifferently expressed genes confirms the involvement ofimmunologic pathways described previously but also indicatesnovel factors that might be relevant for allergen tolerance.(J Allergy Clin Immunol 2010;125:1092-7.)
Key words: Insect venom allergy, venom immunotherapy, geneexpression, microarray assessment, prediction of treatment efficacy
From athe Department of Allergology, Medical University of Gdansk; and bthe Depart-
ment of Allergology, cthe Department of Genetics, and dLifelines, University Medical
Center Groningen, University of Groningen.
Supported by the Foundation for Polish Science and a grant from the Polish Ministry of
Science and Higher Education, no. N402085934.
Disclosure of potential conflict of interest: J. de Monchy has received research support
from ALK-Abello and Novartis. The rest of the authors have declared that they have no
conflict of interest.
Received for publication September 3, 2009; revised December 29, 2009; accepted for
publication January 6, 2010.
Available online March 24, 2010.
Reprint requests: Marek Niedoszytko, MD, PhD, Department of Allergology, Medical
University of Gdansk, Debinki 7 80-952 Gdansk, Poland. E-mail: [email protected].
0091-6749/$36.00
� 2010 American Academy of Allergy, Asthma & Immunology
doi:10.1016/j.jaci.2010.01.021
1092
Insect venom allergy (defined as at least 1 systemic IgEmediated reaction in a lifetime after an insect sting) is presentin approximately 1% to 3% of general population.1
Venom immunotherapy (VIT) with bee, yellow jacket, orPolistes venom is the treatment of choice in patients with insectvenom allergy (IVA). At reaching maintenance dose, the risk ofa systemic reaction to a subsequent sting is reduced from 70%(ie, before the start of VIT) to 3% to 15%.2 To reach long-termprotection, the maintenance phase has to be continued for at least3 years in patients with mild systemic reactions and at least 5 yearsin patients with severe systemic reactions.3 This procedure prob-ably enables lifelong prevention of anaphylactic reactions in themajority of patients.3
However, in some patients, the risk of a systemic reaction to are-sting reappears and increases after stopping the treatment.Currently there is no certain way to predict the individual efficacyof VITexcept for deliberate sting challenges, but it is known that anumber of factors are associated with a worse outcome ofimmunotherapy. First is the duration of treatment. The risk of aresystemic reaction after 2 years of VIT is higher than in patientswho stopped after 3 to 5 years (30% vs 3%).1,2,4 Second, it isknown that patients with side effects during treatment are moreprone to a lower degree of protection.1,2 Hence, prolongation ofVIT may reduce the risk for resystemic reaction in thesepatients.1,2 Third, the amount of allergen routinely administeredmight not be sufficient to stimulate full protection in all individ-uals. It has been shown that continuation of VIT with higherdose (eg, 200 ug) is able to reduce this risk.5 Fourth, it was dem-onstrated that the risk at a systemic reaction after completing thetreatment is related to the culprit insect. In patients with yellowjacket venom allergy, the long-term effectiveness of therapy isassessed to be 85% to 95%, whereas in patients allergic to beevenom, this is 75% to 85%.1 Fifth, coexistence of mastocytosisand even elevated serum tryptase level might increase the riskof inefficacy of VIT.6,7 The current guidelines of European Acad-emy of Allergy and Clinical Immunology indicate that patientswith negative skin tests and undetectable specific IgE to insectvenom have a diminished risk of relapse after stopping VIT.2-4
TABLE I. Demographic and clinical patient data
Long-term
protection
Group 1
Failure of
treatment
Group 2
Maintenance
phase of VIT
Group 3
No. of subjects 17 12 17
Age (y), (range) 53 (28-70) 56 (42-75) 54 (21-75)
Sex male/female (%) 50/50 36/64 31/69
Years of VIT, no. (SD) 3.15 (0.6) 3.3 (0.7) 4 ( 0.8)
Yellow jacket/bee allergy (%) 94/6 84/16 100/0
Mueller class III/IV (%) 64/36 58/42 0/100
sIgE yellow jacket
(kU/L), mean (SD)
5.7 (7) 9.5 (19) 4.2 (4.7)
sIgE honeybee
(kU/L), mean (SD)
0.2 (0.5) 0.9 (1.7) 0.3 (0.5)
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NIEDOSZYTKO ET AL 1093
Finally, it is known that less severe sting reactions are associatedwith better protection after completing the treatment.4
Overall, this means that 10% to 20% of subjects remainvulnerable to the culprit insect venom in spite of completing thetreatment.1-3,6,7
The aim of this study was to determine whether gene expres-sion profiles may predict the efficacy or inefficacy of VIT. We de-termined whole genome gene expression profiles of patients whosuccessfully completed treatment and compared their gene ex-pression profiles with patients who had repeated systemic stingreactions in spite of VIT. On the basis of these results, we builta naive Bayes prediction model that subsequently was evaluatedin a group of patients still on a maintenance dose of VIT.8,9
Tryptase
(ng/mL), mean (SD)
— — 2.2 (4.3)
Methylhistamine in urine
(mm/mkrea), mean (SD)
94 (38) 101 (29) 109.6 (41)
Asthma, no. (%) 1 (7) 1 (9) 4 (25)
Hypertension, no. (%) 1 (7) 3 (27) 2 (12.5)
No. of re-stings after VIT,
mean (range)
5 (2-30) 2 (1-3) —
Reaction to re-sting Mueller
class III/IV (%)
0 80/20 —
Time interval between end of
VIT and re-sting (y), (range)
3.5 (2-12) 4.2 (2-8) —
METHODS
PatientsA total of 46 patients treated with VIT were included. All patients
experienced 1 or more severe systemic reactions before starting VIT. Inclusion
criteria were the diagnosis of IVA on the basis of medical history (grade III or
IV systemic reaction according to Mueller10 before VIT) and positive skin
tests or specific immunoglobulin E. Exclusion criteria were lack of consent,
pregnancy, severe chronic or/and malignant disease, or mastocytosis. Patients
started immunotherapy at the day ward, reaching 1/10 of the maintenance
dose, and continued in the outpatient clinic with 1 injection weekly, increasing
the amount of venom during approximately 6 weeks. Subsequently all patients
received a maintenance dose of 100 mg every 6 weeks for 3 to 5 years. The
study was approved by the Medical Ethical Committee of the University Med-
ical Center Groningen (METc 2008/340).
The following 3 groups of patients were included (Table I):
Group 1 included patients who did not experience a systemic reaction in
spite of being stung at least 3 times with the relevant insect after stopping VIT
(n 5 17). There were 9 (53%) men and 8 (47%) women, with a mean age of 53
years (range, 28-70) in this group.
Group 2 included patients who experienced at least 2 systemic reactions
after field re-stings with the relevant insect (n 5 12). There were 4 (33%) men
and 8 (67%) women, with a mean age of 56 years (range, 42-75) in this group.
The severity of the reaction to the re-sting was assessed as grade III in 80%
(before VIT, 58%) and grade IV in 20% (before VIT, 42%) of patients
according to the Mueller10 scale. The restart of venom immunotherapy was
offered to all patients from this group.
Group 3 included patients who were still in the maintenance phase of VIT
(3-5 years) and had not been stung since the start of the therapy (n 5 17). There
were 6 (35%) men and 11 (65%) women, with a mean age of 55 years (range,
21-75) in this group.
Collection of blood samplesFrom all patients, RNA was isolated from the whole blood by using the
PAXgene Blood RNA Tubes (Qiagen, Valencia, Calif). All tubes were imme-
diately frozen and stored at –208C until RNA isolation (maximum period, 2
months). RNA was isolated by using the PAXgene Blood RNA Kit CE (Qia-
gen, Venlo, The Netherlands). All RNA samples were stored at –808C until la-
beling and hybridization.
The quality and concentration of RNA were determined by using the 2100
Bioanalyzer (Agilent, Amstelveen, The Netherlands) with the Agilent RNA
6000 Nano Kit. Samples with a RNA integrity number >7.5 were used for
further analysis on expression arrays.
Gene expressionFor amplification and labeling of RNA the Illumina TotalPrep 96 RNA Am-
plification Kit was used (Applied Biosystems, Nieuwerkerk ad IJssel, The
Netherlands). For each sample, we used 200 ng RNA. The Human
HT-12_V3_expression arrays (Illumina, San Diego, Calif) were processed ac-
cording to the manufacturer’s protocol. Slides were scanned immediately by
using an Illumina BeadStation iScan (Illumina).
Image and data analysisFirst line check, background correction and quantile normalization of the
data were performed with Genomestudio Gene Expression Analysis module
v 1.0.6 Statistics. Entities containing at least 75% of samples with a signal
intensity value above the 20the percentile in 100% of the samples in at least
2 groups were included for the further analysis.
Data analysis was performed by using the GeneSpring package version
8.0.0 (Agilent Technologies, Santa Clara, Calif). Genes for which expres-
sion was significantly different between compared groups were chosen
based on a log2 fold change >2 in gene expression, t test P value <.05 and
Benjamin-Hochberg false discovery rates <.01.11,12 The naive Bayes
prediction model was used to build a prediction model assessing the
effectiveness of VIT.8,9 The naive Bayesian classifier is a mathematical pro-
cess computing the probability of classifying the patient from group 3 as a
treatment success or treatment failure based on the results of gene expres-
sion.8,9 The selection of genes and their influence on classification in a par-
ticular group is based on results obtained in groups 1 and 2. The naive
Bayesian classifier assumes that the impact of single gene expression is un-
related to other genes in the prediction model. The method does not take
into account the interactions of the genes composing the model or gene-
environmental interactions.
Functional annotation of genes was described by using the Go Process anal-
ysis and Kyoto encyclopedia of genes and genomes pathways13-15 with the
Genecodis functional annotation web based tool.16,17
Clinical data for this study were analyzed with Statistica 8.0 (StatSoft,
Tulsa, Okla).
RESULTSWhole genome gene expression analysis was performed on
RNA samples isolated from all blood cells in whole blood of 46patients with IVA treated with VIT. From all 48.804 probespresent on the array, 48.071 transcripts had sufficient data forfurther analysis.
TABLE II. The list of 18 genes composing the naive Bayes
prediction model of successful VIT
Corrected
P value
P
value
Ratio*
group 1/
2
Ratio*
group 2/
1
Gene
symbol Gene name
.0014 .0002 3.51 0.28 AFAP1L1 Hypothetical
protein flj36748
.0048 .0012 0.38 2.63 C16ORF13 Hypothetical
protein mgc13114
.0014 .0001 5.07 0.20 CLDN1 Claudin 1
.0049 .0013 0.27 3.76 COMMD8 comm domain
containing 8
.0049 .0014 0.37 2.70 HS.129800
.0033 .0007 0.44 2.27 HS.205446
.0049 .0014 0.39 2.57 HS.21177
.0014 .0001 2.57 0.39 HS.428102
.0014 .0002 0.34 2.95 HS.532515
.0033 .0008 2.39 0.42 HS.581554
.0004 .0000 0.20 5.06 HS.583392
.0014 .0002 0.24 4.23 LOC644019Similar to cobw
domain containing 3
.0031 .0006 0.22 4.48 PCDHB10 Protocadherin b 10
.0031 .0006 0.28 3.58 PRLR Prolactin receptor
.0014 .0002 2.57 0.39 SLC16A4 Solute carrier
family 16
(monocarboxylic
acid transporters),
member 4
.0027 .0004 0.24 4.12 SLC47A2 Hypothetical protein
flj31196
.0014 .0001 2.25 0.44 SNX33 sh3 and px domain
containing 3
.0014 .0002 4.43 0.23 TWIST2 twist homolog
2 (Drosophila)
*Ratio of the expression levels for each individual gene comparing patients with
success of VIT (group 1) with patients with failure of treatment (group 2).
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Comparison of gene expression profiles between
patients with long-term protection of VIT versus
failure of treatmentOf the analyzed transcripts, 1401 showed at log2 >2 fold differ-
ence in gene expression (P < .05), of which 658 genes (47%) wereupregulated and 743 genes (53%) were downregulated. Signifi-cant differences (P < .05) in single gene expression were foundfor 978 transcripts. Correction for multiple testing reduced thenumber of significantly expressed genes to 43, of which12 (28%) were upregulated and 31 (72%) downregulated. Weidentified a group of 18 genes with the most discriminative changein gene expression that fulfilled the following conditions: (1) log2
>3 fold change in gene expression, (2) P <.0015, (3) confirmed bycorrection for multiple testing with a P <.005. These genes wereused to build the prediction model (Table II). A hierarchical den-drogram of those genes is presented in Fig 1.
Functional annotation of genes differentially
expressedFunctional annotation of 978 genes with log2 >2 fold change
and a significant difference (P < .05) was assigned by Geneco-dis16,17 (Table III). The main functions of the differently ex-pressed genes were signal transduction, ion transport,
multicellular organism development, transcription, cell prolifera-tion, cell-cell signaling, and cytoskeletal organization. The mostimportant signaling transduction pathways identified were theFceRI signaling pathway, the mitogen-activated protein kinase(MAPK) signaling pathway, the Wnt signaling pathway, theJak–signal transducer and activator of transcription (STAT) sig-naling pathway, and the calcium signaling pathway.
Among the 18 most differentially expressed transcripts used forthe prediction model (Table II) were actin filament associated pro-tein 1-like 1 (AFAP1L1), which is involved in intracellular signal-ing and is a constituent of the cytoskeleton; claudin 1 (CLDN1)and protocadherin b 10 (PCDHB10), which are involved in celladhesion; the prolactin receptor (PRLR), which is involved in sig-nal transduction; and transcription factor twist homolog 2(TWIST2), which increases the expression of the anti-inflammatory cytokine IL-10, which in turn is related to the suc-cess of immunotherapy.18 For a majority of the transcripts, thefunction is unknown.
We also analyzed the expression profiles of leukocyte-specificgenes expressed in dendritic cells, B cells, effector memoryT cells, mast cells, and basophils as described by Liu at al.19 A sta-tistically significant difference in expression between patientswith long-term protection compared with the group of patientswith failure of VIT was found for the mast cell–specific genefollistatin (FST; P 5 .003), the memory T-cell gene galactokinase(GALK; P 5 .008), and B-cell specific Fc receptor-like 5 (FCRL5;P 5 .04).
Comparing our data set with the set of genes as described byKonno et al20 in a similar group of patients treated with bee venomallergy demonstrated statistically significant differences(P 5 .04) in gene expression for IL-1 receptor (IL1R1) and IL-1 -receptor antagonist (IL1RN).
Prediction of the outcome of treatment in a group
of patients in the maintenance phase of VITWe subsequently predicted the potential outcome of treatment
in patients still treated with VIT (group 3). We built 3 predictionmodels by using a naive Bayes8,9 classifier based on (1) the 978genes differentially expressed between the groups with failureand success of VIT (log2 fc > 2; P < .05), (2) the 56 genes withlog2 fc >3 and P <.05 withstanding multiple test correction, and(3) the most discriminative 18 genes with P <.0015 withstandingmultiple testing P <.005 (Table II). Because the 3 predictionmodels gave the same results, the one based on the lowest numberof genes was used for further analysis. We were interested howthis model would predict the percentage of failure of VIT inpatients still in the maintenance phase of VIT. Of this patientgroup, according to this model, 2 (12%) would have treatmentfailure of VIT, whereas 15 (88%) would be protected. These per-centages are in accordance with the known data of protection,1-3
which might substantiate the usefulness of this model in thefuture.
DISCUSSIONIn this study, we have shown that there is a gene expression
profile that may help differentiate patients with success fromthose with failure after VIT. The differences in gene expressionare related to known mechanisms of T-lymphocyte differentiationand mast cell activation, but probably also to other, yet unknownmechanisms. For this study, we used the RNA isolated from the
FIG 1. Hierarchical clustering dendrogram of the most differentially expressed genes (FC > 3; P < .0015; Ben-
jamin Hochberg test < .005) from patients with long-term protection of VIT and the group with failure of VIT.
Each column represents a patient sample, each row an individual gene. For each gene, green represents
underexpression; red, overexpression; and black, missing data.
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whole blood. This not only is a simple and standardized methodthat can be used in a routine setting but also reduces the effect ofsample handling, thereby making it an easy tool for clinicaldiagnosis.
Clinical relevance of the resultsWe were able to identify a gene expression pattern that is
characteristic for the success of VIT in 100% of the patients withsuccess of VIT, whereas it was not found in the patients withfailure of VIT. We built 3 prediction models based on 978, 56, and18 genes with the same results in all models. Therefore, weconcluded that the number of genes in the prediction model maybe reduced to 18. Subsequently we used the final prediction modelto see whether this model gives realistic percentages in the groupof patients who are yet in the maintenance phase of VIT. The geneprofile characteristic for the success of treatment was present in88% of patients on maintenance treatment of VIT, which is inagreement with the epidemiologic data on the risk of a reactionto a re-sting in this group.4 It will be necessary to follow these pa-tients over time to test the true predictive value of our model, be-cause this is necessary before applying such a model in clinicalpractice. It is planned to perform sting challenges before theend of VIT to ensure the final outcome of VIT.
A potential selection bias has to be taken into account, becausethe patient group with failure of treatment was selected on thebasis of the medical history given by the patient and available
medical records. In cases in which the data were not clear, thegeneral physicians were contacted, and the data in medicalrecords were compared with the anamnesis. In spite of theseefforts, it is possible that the severity of the reaction in somepatients in fact was different from the classified one. Theanamnesis of patients with successful treatment is more reliable,because at least 3 re-stings were observed without side effects,although they could have been stung by a not relevant insect. Thecurrent data suggest that patients with the reaction assessed as IIIand IV in the Mueller scale should be treated for 5 years, whichmay increase the effectiveness of VIT. The duration of treatmentof the patients described in this study was shorter (3 years) but didnot differ between the groups. Therefore, our results should berepeated in independent patient groups to evaluate the validity ofthe model.
The main question for further studies is whether we can use thegene expression analysis in daily practice. The severity of thereaction to a re-sting not only may depend on intrinsic patientfactors but also may be related to the stinging insect, patientcomorbidities and condition, and used medication. It is likely thatan optimal diagnostic tool should include these factors as well asgene expression profiling. The definitive prediction of theoutcome will always be difficult. Prospective studies in largergroups of patients treated in different centers should be performedto evaluate the accuracy of this gene profile. In the future, theanalysis of gene expression profiles might also be used for the
TABLE III. Gene co-occurence annotation found by Genecodis14,15 (GO Process) for the genes differentially expressed (FC > 2; P < .05)
between groups with success and failure of VIT
No. of genes NGR NG Hyp Hyp* Annotations
52 1700(37435) 52(434) 2.30e-10 4.37e-09 GO:0007165 :signal transduction
FceRI signaling pathway (MS4A2, IL5, PLA2G12A) Hyp* 5 0.04
MAPK signaling pathway (EGFR, FLNA, NR4A1, IL1R1, MINK1, MAP3K12, PLA2G12A, FGF17, MAPK8IP1) Hyp* 5 0.03
Wnt signaling pathway (DVL1, DKK2, TCF7L2, WNT1, WNT10B) Hyp* 5 0.04
Jak-STAT signaling pathway (IL29, IL5, IL21R, PRLR) Hyp* 5 0.02
Calcium signaling pathway (CHRNA7, EGFR, ERBB2, ERBB4) Hyp* 5 0.05
21 503(37435) 21(434) 5.76e-07 5.47e-06 GO:0006811: ion transport
KCNG1, SLC12A5, KCNJ14, SLC22A7, ATP1B1, PKDREJ, MLC1, TTYH2, KCNMA1,CHRNA7, FXYD2 SLC17A1, SLC22A12, KCNK4, KCNA1,
KCNC2, ACCN4, CLCNKA, CLCA1, HTR3D, SCN1B
28 874(37435) 28(434) 1.64e-06 1.04e-05 GO:0007275: multicellular organismal
development
SNAI2, IFRD1, PPP1R9B, TBX3, HEY1, SCMH1, GTF2IRD1, PLXNA1, HOXD4, ERBB4, FST, VDR, DLX1
ISL2, WNT1, MINK1, WNT10B, DKK2, FOXL1, PAX8, DVL1, MGP, OSGIN1, GRHL1, THEG, HYDIN
NANOS3, TRAF4
35 1516(37435) 35(434) 0.000100479 0.000477 GO:0006350 :transcription
RRN3, SNAI2, RARA, ASH1L, SPZ1, MCM4, MTERFD3, TCF7L2, ZNF10, TAF1, GTF2IRD1, SOX18,
RBM4, ZNF740, RBL1, ZNF37A, ZNF322A, ZFP14, HIPK1, FOXL2, MED21, ZNF322B, ZNF404, FOXL1
TFDP2, TFAP2E, RNF2, SCRT1, GRHL1, BRMS1L, PATZ1, ZNF197, ZNF192, ZNF492, NFKBIB
11 258(37435) 11(434) 0.000237206 0.000901 GO:0008283: cell proliferation
PCNA, TPX2, TCF7L2, ERBB2, C6ORF108, ERBB4, CDV3, ELN, CENPF, OCA2, MS4A2
9 237(37435) 9(434) 0.00191438 0.00606221 GO:0007267: cell-cell signaling
ASH1L, SSTR3, CALCA, ADORA1, CXCL9, FGF17, INSL3, ISG15, CCL15
4 54(37435) 4(434) 0.00356701 0.00968189 GO:0007010: cytoskeleton organization
MARK1, SGCZ, ABLIM1, KRT86
13 471(37435) 13(434) 0.00372335 0.00884295 GO:0008152: metabolic process
C8ORF79, PNPLA4, ARSD, UGT2B17, GSTM4, ISOC2, MCAT, AGL, KCNMA1, ALPI, ATP12A
GALK2, ACSM2A
P values have been obtained through hypergeometric analysis (Hyp) corrected by the false discovery rate method (Hyp*). NGR, Number of annotated genes in the reference list of
NG, number of annotated genes in the input list. The most important signaling transduction pathways annotated by Kyoto encyclopedia of genes and genomes12,13 are shown.
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assessment of effectiveness of immunotherapy with other aller-gens to evaluate whether the gene pattern is specific for the IVAallergens or whether it is a more common predictor for themechanism of treatment with immunotherapy.
Immunological mechanisms that might be involved
in the long-term effectiveness or ineffectiveness of
VITFunctional annotation of the genes differentially expressed
between patient groups with success and failure of VIT includedgenes involved in well known mechanisms of immunotherapy,such as FceRI, JAK-STAT, MAPK, and Wnt, and calcium signal-ing pathways, cell signaling, or transcription. The function ofmany other differently expressed transcripts is yet unknown(Table II) or questionable, which is a common situation in wholegene expression studies.21,22 Interestingly, genes commonly re-lated to known mechanism of VIT like IL-10, IL-4, and osteopon-tin were not differentially expressed. This does not excludesignificant differences in protein levels or differences in RNA ex-pression in subpopulation of cells, like regulatory T lymphocytes,but they could not be demonstrated by the model chosen in thestudy examining the whole blood RNA. The description heretherefore is based on the genes with known function and on thehighest differences in expression composing the prediction modelused in the study.
TWIST2 was upregulated in patients who gained success ofVIT. It has been shown that this gene promotes the productionof the IL-10 and decreases the synthesis of IL-4.13 In spite of
the fact that no difference in expression of IL-10 and IL-4 was ob-served in our study, the upregulation in TWIST2 expression maybe responsible for the differences in cytokine levels and cell sub-types typical for immunotherapy.18 Further studies are needed toaddress this finding.
The downregulation of PRLR in successfully treated patientsmay also indicate a shift toward TH1. A decrease in serum levelsof prolactin is found in patients during sublingual immuno-therapy.23 Prolactin induces overexpression of g/d T-cell receptor,which increases the IL-4–dependent IgE and IgG1 responseessential for the development of TH2 lymphocytes.23 The down-regulation of PRLR is consistent with this finding.
CLDN1 expression was higher in patients who were protectedfrom re-sting reactions after VIT. This protein is a crucial struc-tural component of tight junctions and plays an important rolein adhesion and migration of dendritic cells.24 The expressionof CLDN1 is increased by TGF-b. Higher expression of CLDN1in dendritic cells may be related to the role of these cells in regu-latory T differentiation.18
The function of solute carrier family 16 (SLC16A4), sh3 and pxdomain containing 3 (SNX33), and MCT5 is known although it isdifficult to relate it to the mechanism of immunotherapy.SLC16A4 product monocarboxylic acid transporter 5 plays arole in monocarboxylic acid transport.25 SNX33 product—sortingnexin 33—modulates endocytosis trafficking.26 COMM domaincontaining 8 (COMMD8) gene may play a role in cell prolifera-tion.27 The function of all other transcripts (AFAP1L1,C16ORF13, HS.129800, HS.205446, HS.21177, HS.428102,HS.532515, HS.581554, HS.583392, LOC644019, SLC47A2)
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is unknown. The functional studies of the genes described andstudies indicating the change in gene expression during immuno-therapy may explain the mechanisms of venom immunotherapyin the future.
In conclusion, the use of gene expression profiles might be auseful tool to predict the effectiveness of VIT. The analysis of dif-ferentially expressed genes confirms the involvement of immuno-logic pathways described before but also indicates novelpathways potentially involved in induction of allergen tolerance.Further studies in larger groups of patients are required to confirmthis prediction model before it can be used in clinical practice.
Clinical implications: Gene expression profiles may help iden-tify patients who fail to achieve longtime protection by insectvenom immunotherapy. The results of this study may be a basisfor further studies.
REFERENCES
1. Golden D. Stinging insect allergy. Am Fam Physician 2003;67:2541-6.
2. Golden D, Kagey-Sobotka A, Lichtenstein L. Survey of patients after discontinu-
ing venom immunotherapy. J Allergy Clin Immunol 2000;105:385-90.
3. Bonifazi F. Prevention and treatment of hymenoptera venom allergy: guidelines for
clinical practice. Allergy 2005;60:1459-70.
4. Lerch E, M€uller UR. Long-term protection after stopping venom immunotherapy:
results of re-stings in 200 patients. J Allergy Clin Immunol 1998;101:606-12.
5. Ru€eff F, Wenderoth A, Przybilla B. Patients still reacting to a sting challenge while
receiving conventional Hymenoptera venom immunotherapy are protected by
increased venom doses. J Allergy Clin Immunol 2001;108:1027-32.
6. Oude Elberink JNG, de Monchy JGR, Kors JJ, van Doormaal JJ, Dubois AE. Fatal
anaphylaxis after a yellow jacket sting, despite venom immunotherapy, in two
patients with mastocytosis. J Allergy Clin Immunol 1997;99:153-4.
7. Simons FE, Frew AJ, Ansotegui IJ, Bochner BS, Golden DB, Finkelman FD, et al.
Risk assessment in anaphylaxis: current and future approaches. J Allergy Clin
Immunol 2007;120:2-24.
8. Kazmierska J, Malicki J. Application of the naıve Bayesian classifier to optimize
treatment decisions. Radiother Oncol 2008;86:211-6.
9. Kononenko I. Machine learning for medical diagnosis: history, state of the art and
perspective. Artif Intell Med 2001;23:89-109.
10. Mueller HL. Diagnosis and treatment of insect sensitivity. J Asthma Res 1966;3:
331-3.
11. Benjamini Y, Yekutieli D. The control of the false discovery rate in multiple testing
under dependency. Ann Stat 2001;29:1165-88.
12. Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I. Controlling the false discovery
rate in behavior genetics research. Behav Brain Res 2001;125:279-84.
13. Sharabi AB, Aldrich M, Sosic D, Olson EN, Friedman AD, Lee SH, et al. Twist-2
controls myeloid lineage development and function. PLoS Biol 2008;6:e316.
14. Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, et al. KEGG
for linking genomes to life and the environment. Nucleic Acids Res 2008;36:
480-4.
15. Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF, Itoh M, Kawashima S, et al.
From genomics to chemical genomics: new developments in KEGG. Nucleic Acids
Res 2006;34:354-7.
16. Nogales-Cadenas R, Carmona-Saez P, Vazquez M, Vicente C, Yang X, Tirado F,
et al. GeneCodis: interpreting gene lists through enrichment analysis and integra-
tion of diverse biological information. Nucleic Acids Res 2009;37:317-22.
17. Carmona-Saez P, Chagoyen M, Tirado F, Carazo JM, Pascual-Montano A. GENE-
CODIS: a web-based tool for finding significant concurrent annotations in gene
lists. Genome Biol 2007;8:R3.
18. Jutel M, Akdis M, Blaser K, Akdis CA. Mechanisms of allergen specific immuno-
therapy—T-cell tolerance and more. Allergy 2006;61:796-807.
19. Liu SM, Xavier R, Good KL, Chtanova T, Newton R, Sisavanh M, et al. Im-
mune cell transcriptome datasets reveal novel leukocyte subset-specific genes
and genes associated with allergic processes. J Allergy Clin Immunol 2006;
118:496-503.
20. Konno S, Golden DB, Schroeder J, Hamilton RG, Lichtenstein LM, Huang SK.
Increased expression of osteopontin is associated with long-term bee venom immu-
notherapy. J Allergy Clin Immunol 2005;115:1063-7.
21. Diosdado B, Wapenaar MC, Franke L, Duran KJ, Goerres MJ, Hadithi M, et al.
A microarray screen for novel candidate genes in coeliac disease pathogenesis.
Gut 2004;53:944-51.
22. Crijns AP, Fehrmann RS, de Jong S, Gerbens F, Meersma GJ, Klip HG, et al.
Survival-related profile, pathways, and transcription factors in ovarian cancer.
PLoS Med 2009;6:e24.
23. Ippoliti F, De Santis W, Volterrani A, Lenti L, Canitano N, Lucarelli S, et al.
Immunomodulation during sublingual therapy in allergic children. Pediatr Allergy
Immunol 2003;14:216-21.
24. Zimmerli SC, Hauser C. Langerhans cells and lymph node dendritic cells express
the tight junction component claudin-1. J Invest Dermatol 2007;127:2381-90.
25. SLC16A4 gene function. Available at: http://www.ncbi.nlm.nih.gov/IEB/Research/
Acembly/av.cgi?db5human&q5SLC16A4.
26. Sch€obel S, Neumann S, Hertweck M, Dislich B, Kuhn PH, Kremmer E, et al.
A novel sorting nexin modulates endocytic trafficking and alpha-secretase cleavage
of the amyloid precursor protein. J Biol Chem 2008;283:14257-68.
27. COMMD8 gene function. Available at: http://www.ncbi.nlm.nih.gov/IEB/
Research/Acembly/av.cgi?db5human&q5COMMD8.
Review article
Mastocytosis and insect venom allergy: diagnosis, safety and
efficacy of venom immunotherapy
Mastocytosis is an uncommon disease resulting from amonoclonal proliferation of pathological mast cells indifferent tissues including skin, bone marrow, liver,spleen, lymph nodes and gastrointestinal tract (1–5). Theclinical presentation of mastocytosis is heterogeneous,varying from a manifestation limited to skin in urticariapigmentosa (UP), diffuse cutaneous mastocytosis andmastocytoma, to different forms of systemic diseases(extradermal tissue involvement) including indolent sys-temic mastocytosis (ISM), smoldering systemic masto-cytosis, aggressive systemic mastocytosis and mast cellleukemia (3–5). In adult patients with systemic masto-cytosis, the large majority (approx. 90%) has theindolent form of the disease (3–5). Mast cell infiltrationleads to organ failure in rare forms of aggressivemastocytosis, whereas skin involvement and mast cellmediator release affect the majority of patients. Symp-toms of mast cell degranulation may vary from itchingand flushing to profound hypotension and anaphylaxis(1, 2, 4, 6). About half of the patients experienceanaphylactic reactions (6). The most important trigger isinsect venom. It is estimated that 30% of all mastocy-
tosis patients suffer from venom anaphylaxis (6), andthese reactions are often more severe than in the generalinsect venom allergic (IVA) population (7). The treat-ment of choice in IVA patients is venom immunother-apy (VIT) with the relevant venom (honey bee, yellowjacket and/or Polistes) (8, 9). The opinions concerningVIT in mastocytosis patients with IVA are conflictingand range from considering it as a contraindication, toseeing these patients as the group who needs VIT mostof all (8, 10, 11). These conflicting opinions were thereason to analyse the available data concerning diagno-sis, safety and effectiveness of VIT among mastocytosispatients.
We analysed data both published in the journals listedin the Pubmed database (search terms: mastocytosis IVA,immunotherapy), as data presented at allergologicalcongresses from 2003–2008. If data were unclear, authorswere contacted personally for further information. Qual-ity of evidence (A: high, B: moderate, C: low and D: verylow) and strength of recommendation (strong 1 andweak 2) concerning VIT in mastocytosis patients wasassessed according to the Grading of Recommendations
The most important causative factor for anaphylaxis in mastocytosis are insectstings. The purpose of this review is to analyse the available data concerningprevalence, diagnosis, safety and effectiveness of venom immunotherapy (VIT)in mastocytosis patients. If data were unclear, authors were contacted per-sonally for further information. Quality of evidence (A: high, B: moderate, C:low and D: very low) and strength of recommendation (strong 1 and weak 2)concerning VIT in mastocytosis patients are assessed according to the Gradingof Recommendations Assessment, Development and Evaluation and aremarked in square brackets. Results of VIT were described in 117 patients todate. The mean rate of side-effects during treatment in studies published so faris 23.9% (7.6% requiring adrenaline) with an overall protection rate of 72%.Based on the review we conclude that (1) mastocytosis patients have a highrisk of severe sting reactions in particular to yellow jacket, (2) VIT could besuggested [2] in mastocytosis, (3) probably should be done life long [2], (4) VITin mastocytosis is accompanied by a higher frequency of side-effects, so (5)special precautions should be taken into account notably during the builtup phase of the therapy [2], (6) VIT is able to reduce systemic reactions, butto a lesser extent compared to the general insect venom allergic population[2], so (7) patients should be warned that the efficacy of VIT might be lessthan optimal and they should continue carrying two adrenaline auto injectors[2].
M. Niedoszytko1,2, J. de Monchy2,J. J. van Doormaal2, E. Jassem1,J. N. G. Oude Elberink2
1Department of Allergology, Medical University ofGdansk, Gdansk, Poland; 2Department ofAllergology, University Medical Center Groningen,University of Groningen, Groningen, the Netherlands
Key words: insect venom allergy; mastocytosis; venomimmunotherapy.
Marek NiedoszytkoDepartment of AllergologyMedical University of GdanskDebinki 7 80-952 GdanskPoland
Accepted for publication 24 April 2009
Allergy 2009: 64: 1237–1245 � 2009 John Wiley & Sons A/S
DOI: 10.1111/j.1398-9995.2009.02118.x
1237
Assessment, Development and Evaluation (GRADE) (12,13) and are marked in square brackets.
Epidemiology
Insect venom allergic (defined as at least one systemicIgE-mediated reaction in lifetime after an insect sting) isreported in approx. 1–3% of population (14). It is higherin beekeepers and reaches 14–43% (9, 15). The epidemi-ology of mastocytosis has not been properly studied sofar. It is estimated that 1 in 1000 –1 in 8000 ofdermatological patients suffer from urticaria pigmentosa(16). The prevalence of mastocytosis is higher in patientswith IVA and varies from 0.9–2.6% (Table 1) (10, 11, 17,18). In many centers, serum tryptase levels are notroutinely measured which may lead to under diagnosis ofthe frequency of mastocytosis in IVA patients, especiallyin patients without skin involvement (19).Compared to patients with IVA without mastocytosis
and normal basal tryptase levels, the systemic stingreaction is usually more severe in patients with mastocy-tosis (7), as well as in those with increased levels oftryptase without diagnosed mastocytosis (20, 21).In mastocytosis patients, IVA is the most important
trigger factor for anaphylaxis (6, 22), the prevalence ofIVA in this group is estimated between 20% and 30% (6,19, 22, 23). In addition to Brockow and Florian weevaluated our own data and found about the sameprevalence (Table 2). In the study by Haeberli et al.(where in general more patients with a honey bee venomallergy are referred to) it was remarkable that yellowjacket venom allergy was more prevalent in patients with
elevated tryptase compared to the general population ofIVA patients (20).
Fatalities
At least six cases of patients with mastocytosis with afatal reaction after an insect sting have been described todate. Three patients (10, 24, 25) had not been treated withVIT, in three others, treatment was stopped before thefatal sting occurred (25, 26). No fatalities were describedin patients while on maintenance dose of VIT so far.
Concerning the patients who died despite VIT, OudeElberink et al. (26) described two cases. The first patientwas treated with VIT for 5 years without side-effects aftersuffering a severe systemic reaction. Four years aftercompletion of VIT, systemic mastocytosis was diagnosed.The patient died after an accidental sting 9 years after theend of VIT in spite of immediate emergency treatment.The second patient described was treated with VIT for4 years. The treatment was stopped because of side-effects at maintenance dose of VIT, after which thediagnosis of mastocytosis was established. This patientdied 1 year later, after an insect sting. Both fatal reactionsraise the question whether continuation of VIT wouldhave been able to prevent these fatal reactions. A thirdpatient described by Reimers and Muller (27) was treatedwith honey bee VIT after honey bee venom anaphylaxis,but died 10 years later due to a Vespula sting anaphylaxis.Both Rueff et al. (11) and Reimers and Muller (27) raisethe question whether VIT should be performed both withhoney bee and yellow jacket in mastocytosis patients evenif there is a clinical relevant allergy to only one species.The natural history of IVA in mastocytosis patients isunknown, as is the natural history after stopping VIT (seeEfficacy of treatment during venom immunotherapy).Despite this uncertainty, the guidelines of the EuropeanAcademy of Allergology and Clinical Immunology(EAACI) advice life long treatment with VIT (8), butonly with venom of the culprit insect.
Concerning the patients who were not treated withVIT, three patients with mastocytosis were described whodied after an insect sting in whom this was probably thefirst systemic reaction in their life (10, 24, 26). In onepatient, the diagnosis of mastocytosis was made post-mortem (28, 29). In two others, the diagnosis of masto-cytosis was made before the fatal sting (24, 26).Therefore, Wagner et al. (24) suggested prophylacticVIT in all mastocytosis patients. However, the naturalhistory of IVA in mastocytosis patients is presently notknown sufficiently well to draw such a conclusion.
Pathogenesis of insect venom anaphylaxis in mastocytosispatients
The mechanism of IVA in mastocytosis is only partiallyunderstood. The first publications of coexistence of
Table 1. Epidemiology of mastocytosis in insect venom allergic (IVA) patients
Author (ref.)Number of patients
evaluatedn (%) of patients suffering
from mastocytosis
Rueff F (11) 1102 2.6%Dubois A (10) 2375 0.9%Bonadonna P (17) 552 2.9%Bonadonna P (18) 379 5.5%
Table 2. Epidemiology of insect venom allergy (IVA) among mastocytosis patients(disease diagnosed according to World Health Organization (WHO) criteria [3])
Author (ref.)Studied mastocytosis
patientsn (%) of patients
suffering from IVA
Brockow K. (6) 46 children, 74 adults(59 ISM, 1 ASM, 1 SMAHD)
Children 0%,adults 27%
Florian S. (22) 40 adults (40 ISM) 20%Van Doormaal J* 160 adults with ISM 30%Niedoszytko M* 60 adults (35 ISM) 30%
ISM: indolent systemic mastocytosis; ASM aggressive systemic mastocytosis;SM-AHNMD: systemic mastocytosis associated hematological nonmast cell lineagedisease.*Unpublished data.
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both diseases came from Muller et al. (30). Theyreported three patients with UP suffering from anaphy-laxis following a Hymenoptera sting. Two had detect-able sIgE and positive skin prick tests (SPT), whereas inone patient no specific IgE mediated allergy could befound. This raised questions concerning the IgE mech-anisms. The authors concluded that the mediatorrelease by a pharmacologic mechanism may be analternative way of induction of anaphylaxis afterHymenoptera stings in mastocytosis (30). However, theintroduction of the basophil histamine release test andthe basophil activation test in the diagnostic work-upallows nowadays detecting IgE in most, if not allpatients (11). Therefore, the non-IgE mediated IVA isregarded as a very rare reaction (20, 31), although theresults of sIgE and skin test in mastocytosis patients areless pronounced compared to patients suffering reac-tions with a similar grade without mastocytosis (20). Asuggested explanation for this phenomenon is theadsorption of circulating IgE on the abundant massof tissue mast cells (30).Also, alternative pathways of mast cell activation have
been demonstrated, although none of them has beenproven so far to play a crucial role in the mechanism ofanaphylaxis in mastocytosis. In mouse models, activa-tion of macrophages by IgG-antigen complexes cross-linking low-affinity IgG receptor (FccRIII) has beendemonstrated to induce anaphylaxis. No data so farhave confirmed the relevance of this model to humananaphylaxis (23). Another element of anaphylaxis is theactivation of mast cells by intracellular tyrosine kinase-mediated cascades especially via the well known Kit, butalso Lyn, Syk and Fyn pathways (23). The presence of aKIT mutation, especially the D816V, is found in thelarge majority of patients with systemic mastocytosis(3–5). The D816V mutation might induce exaggeratedmast cell stimulation, as is shown in in vitro studies (23).However, the presence of this mutation does notcorrelate with the severity and prevalence of anaphylaxis(6). Further, deregulation of the calcium influx for mastcell activation may also play a role in anaphylaxissusceptibility (23, 32, 33). Transient receptor potentialmembrane proteins (TRPM) channels are involved inthe mast cell degranulation, compounds activating thesechannels can serve as drugs inhibiting allergic reactions(32).There are also data indicating complement activation,
calcitonine gene-related peptide overproduction, osteo-pontine pathway involvement and diminished function ofangiotensinogen and renin–angiotensin system in IVA(34–39). It is not known to which extent these pathwayscould be relevant for mastocytosis and effectiveness ofVIT in this disease.Unfortunately, in spite of the findings mentioned
above, still it is not known why one out of two patientswith mastocytosis suffer from anaphylaxis and one out ofthree from IVA.
Diagnostic tests of insect venom allergy in mastocytosispatients
An elevated level of tryptase is an indicator of mastocy-tosis and should be measured in IVA patients, especiallysubjects with severe reactions after an insect sting andthose who react abnormally to treatment (9, 40–44). In allpatients with a history of a systemic allergic reaction to aninsect sting before VIT specific IgE has to be demon-strated (9, 15, 31, 40), by SPT, intracutaneous tests and/or serological specific IgE in order to detect specific IgEand to identify the culprit insect (9, 15, 31, 40). It must betaken into account that skin tests in mastocytosis patientsmay provoke systemic reactions (10, 26). In the majorityof patients, this set of tests is sufficient for a diagnosis (9,21). In individual patients with negative results, the testsshould be repeated after 1 or 2 months. If still no specificIgE can be demonstrated for all possibly relevant insects,additional examinations as basophil histamine releasetest, leukotriene release test or basophil activation testmay be considered (9, 11, 31, 40, 41). With these noveldiagnostic methods the diagnosis of IgE mediated IVAcan be established in 99% patients (31, 40, 41, 43). Theamount of specific IgE does not correlate with the severityof the preceding reaction.
In nonmastocytosis patients, sting challenges may alsobe considered, however in mastocytosis patients diagnos-tic challenges are considered contra-indicated [2] (10). Itcan not be excluded that in rare occasions patients withmastocytosis may have non-IgE-mediated anaphylaxis toan insect sting, but this diagnosis can only be made afterall other methods described evaluating the existence ofIgE are negative.
Immunotherapy
The review of the articles describing VIT in patients withmastocytosis reveals conflicting opinions. Data availableso far come from case reports and observational studies,of which most are retrospective with a high risk ofselection bias. Additionally, only the studies by de Olano(45) and Bonadonna (17) provide a complete diagnosis ofmastocytosis in all patients. Therefore, the power ofevidence is moderate [B] to very low [D] according toevidence based medicine guidelines (12, 13). Below, wedescribe the most relevant studies. None of the studieswas a randomized trial.
De Olano et al. (45) examined 21 patients with IVAand mastocytosis treated in the centers cooperatingwithin the Spanish Network on Mastocytosis [B]. Thediagnosis of mastocytosis was made according to theWorld Health Organization (WHO) criteria (3). A totalof 21 patients were treated (seven with Polistes venom, sixwith Vespula, five with Apis venom, and three with twovenoms). Fourteen patients were treated according toconventional and seven according to cluster protocol. The
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maintenance dose was 100 lg in 20 patients and in theremaining patient 300 lg mixed vespid together with100 lg honey bee was used. Field stings in the studygroup were reported. There were no sting challengesperformed.Bonadonna et al. (17) retrospectively studied 16
patients treated in Verona (Italy) with IVA and masto-cytosis diagnosed according to the WHO guidelines [C](3). The patients were selected from 552 IVA cases treatedin the years 2004–2007. Seven patients were treated withPolistes, eight with Vespula and one with Apis venom. Aconventional protocol of VIT was used reaching mainte-nance dose of 100 lg of venom. The dose was increasedto 200 lg in two patients who reacted to a field sting.Similarly to the previous study sting challenges were notperformed, field stings occurred in 13 patients.Fricker et al. (46) studied 10 patients with IVA and
urticaria pigmentosa treated in the years 1980–1994 inBern (Switzerland) [C]. Three patients were treated withhoney bee, five with Vespula and two with both honey beeand Vespula venoms. The study was performed before theWHO criteria on the diagnosis of mastocytosis wereestablished; it is unknown how many patients could havehad a systemic mastocytosis. Seven patients were restung:three by a sting challenge and four experienced a fieldsting.Dubois (10) described seven patients with systemic
mastocytosis who were treated with VIT according to asemi rush protocol in the Netherlands [D]. The diagnosisof mastocytosis was made by histopathological examina-tion of the bone marrow. The reaction to field stings wasreported in six cases.Rueff et al. (11) described 55 patients with mastocyto-
sis and IVA. Honey bee venom allergy was diagnosed inseven (13%), yellow jacket venom allergy in 35 (66.7%)
and both yellow jacket and honey bee venom allergy in 11(20.3%) of the patients [C]. About 48 patients weretreated with VIT according to a rush protocol. Addition-ally, sting challenges were performed in 33 of them.
Haeberli et al. (20) treated 19 IVA patients with anelevated basal tryptase level (‡13.5 ng/ml), of whom 16underwent the histopatological examination of the skinbiopsies confirming urticaria pigmentosa [C]. Sevenpatients were treated with honey bee and 12 with Vespulavenom. The treatment was performed according to anultrarush, rush or conventional protocol. Sting challengeswere performed in 10 patients.
Safety of treatment
The safety of VIT in mastocytosis patients could beevaluated in a total of 117 patients described in sixretrospective analyses [C] (10, 11, 17, 20, 45, 46) and fourcase reports (26, 30, 47, 48) (Table 3). Side-effects werenot described in all studies. Side-effects during VIT weredocumented in 28 (23.9%) patients and systemicside-effects in 24 (20.5%).
We compared these percentages with side-effects in thegeneral population of IVA patients treated with VIT.Studies evaluating at least >100 patients were includedfor this analysis (Table 4). Both conventional, rush andultrarush schedules were used. Side-effects are describedin 20.3% (11.1–36%) of the population, which seems tobe comparable with the percentage of side-effects in themastocytosis patients. However, it is known that studiesevaluating one Hymenoptera venom cannot be extrapo-lated to the other Hymenoptera venoms (49). In thegeneral population side-effects due to honey bee venomoccurred more frequently compared to yellow jacketvenom allergic patients. In the studies we included side-
Table 3. Side-effects of venom immunotherapy (VIT) in mastocytosis patients
Author (ref.)
Number (%) of mastocytosis patients
VIT
Side-effects
All side-effects Systemic side-effectsAdrenaline1 was used or caused
discontinuation of treatment2
Rueff F (11) 48 mastocytosis 9 (18.8%) built up phase of VIT 9 (18.8%) built up phase of VIT 21*
Dubois A (10) 7 ISM 6 (85%) 6 (85%) 42
Bonadonna P (17) 16 ISM (2 UP+, 14 UP–) 2 (12.5%) 0 0Haeberli (20) 10 patients with tryptase >13.5 lg/L
and sting challenge performed1 (10%) 1 (10%) 0
Fricker (46) 3 ISM + UP, 3 ISM – UP +,4 UP no BM diagnosis
2 (20%) 1 (10%) 0
De Olano (45) 21 ISM (5 UP+, 16 UP–) 6 (29%) 3 built up, 3maintenance phase of VIT
5 (24%) 12
Engler, M�ller,Oude Elberink(26, 30, 47, 48)
4 UP 1 ISM 2 (33%) 2 (33%) 21,2
Total 117 28 (23.9%) 24 (20.5%) 9 (7.6%)
ISM: indolent systemic mastocytosis; UP: urticaria pigmentosa.*Unpublished data.
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effects occur in 26.6% (12–30%) of the honey bee venomtreated patients and in 11.2% (2–20.9%) of the yellowjacket treated patients. In the mastocytosis patients, VITwas performed with yellow jacket venom in 36, honey beevenom in 15, Polistes in 14, and with two venoms in foursubjects, while in the study by Rueff the specific venomwas not stated, but most were yellow jacket allergicpatients (11).Side-effects to yellow jacket venom VIT were present in
12 out of 34 (35%) patients, to honey bee venom VIT infive out of 13 (38%) and to Polistes venom VIT in two of17 (12%). Thus, there is no difference in side-effectsbetween yellow jacket and honey bee venom treatedpatients with mastocytosis. However, compared with thegeneral IVA population, side-effects are more frequent,especially in yellow jacket allergic patients.In addition, we evaluated the number of side-effects in
which adrenaline was used or the reaction was so severethat therapy has been stopped. In the population of
nonmastocytosis patients treated with VIT, this variedfrom 3–7% vs 7.6% in patients with mastocytosis(Tables 3 and 4).
These results suggest [2] that side-effects of VIT areprobably more frequent in mastocytosis patients,especially in patients with a yellow jacket allergy.
Efficacy of treatment during venom immunotherapy
The efficacy of VIT in mastocytosis was described in sixreview papers covering 81 patients [B, C, D] (10, 11, 17,20, 45, 46) and one case report including one patient (47)by evaluating the outcome of sting challenges or reactionto field stings (Table 5). A systemic reaction was observedin 11 of 46 (23 9%) sting challenges described, whereas asystemic reaction to a field sting occurred in 12 of 36 (333%) patients during VIT (Table 4). The most severereaction described (requiring resuscitation and intuba-tion) was found in a patient who was not yet on
Table 4. Side-effects of venom immunotherapy (VIT) in general population of insect venom allergy (IVA) patients (papers with >101 patients published in English)
Author (ref.) Characteristics of patients and VIT protocoln (%) of patientswith side-effects
Side-effects of Honey bee/yellow jacket/Polistesallergic patients (%)
n (%) of patients withsystemic side-effects
n (%) of patients wereadrenaline was used
Haeberli (20) 151 honey bee and yellow jacket allergicnormal basal tryptase, ultrarush, rush
conventional
21 (13.9%) 17 / 7 – –
G�rska L. (55) 118 honey bee and yellow jacket allergic5 day rush
18 (15.2%) 28.6/11.1 7 (5.9%) III or IVMueller (59)
6 (5%)
Mosbech H. (60) 840 honey bee and yellow jacket allergicultrarush, rush, conventional
20%(24%rush/ulrarush;12% conventional)
24/19/0 3% 3%
Wenzel J. (61) 178 honey bee and yellow jacket, rushprotocol
(32) 17,9% Not known III or IV Mueller (59)10 (5.6%)
0
Birnbaum (62) 325 patients 90 honey bee, 186 yellowjacket, 49 Polistes ultra-rush
33 (11.1%) 30/3.3/6.1 9 (2.7%) 2 (0.6%)
M�ller (49) 205 patients honey bee 148, yellow jacket 57rush and conventional VIT
74 (36%) Objective side-effects21/4
Objective side-effects33 (16%)
Not known
Brehler (63) 1055 ultrarush VIT in 966 patients yellowjacket VIT in 933 honey bee VIT in 122
patients
224 (21.4%) 23.8/20.9 160 (15.2%) 0
Sturm (64) 101 patients 52 honey bee VIT, 47 yellowjacket VIT rush VIT
7 (6.9%) 12/2 2 (2%) 0
Table 5. Effectiveness of venom immunotherapy (VIT) in mastocytosis
Author (ref.) Time of sting challengen (%) of patients with systemic
reaction in sting challengen (%) of patients with systemic
reaction on field stingCumulative number ofreactions to re-sting
Rueff F (11) 6–12 months after reachingmaintenance dose
7/33 (21.6%) – 7/33 (21.6%)
Dubois A (10) ND 6/7 (85%) 6/7 (85%)Bonadonna P (17) ND – 2/13 (15%) 2/13 (15%)Haeberli (20) 3–5 years of VIT 4/10 (40%) – 4/10 (40%)Fricker (46) maintenance dose 0/3 1/3 (33%) 1/6 (16%)De Olano (45) ND – 3/12 (25%) 3/12 (25%)Engler (47) ND 0/1 0/1Total 11/46 (23.9%) 12/36 (33.3%) 23/82 (28%)
ND: not done.
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maintenance phase of VIT (45). The cumulative (stingchallenge and field stings) number of reactions was 23 in82 (28%) patients, indicating a protection rate of 72%.However, individual studies show a large variation inprotection rate varying form 15–85%. A protection rateof 72% is lower compared to patients without mastocy-tosis, where the protection is about 80% in honey beeallergic patients and about 95% in yellow jacket allergicpatients during maintenance phase of VIT (41).The assessment of efficacy of VIT however may be
overstated due to the possibility of false negative stingreactions as the field sting could have been by anirrelevant insect or due to the relatively low predictivevalue of one single negative sting reaction in a patient (itis possible that patients with one negative sting reaction,still will react to a second sting) (50).All patients with a reaction to a later sting in study by
de Olano et al. had side-effects during build-up phase ofVIT (45). The same has been demonstrated in the generalpopulation of patients treated with VIT, where half of thepatients who experience sting reactions after stoppingVIT have a history of previous systemic reactions duringtreatment due to a VIT injection or to a field sting (51).This may suggest that at least these patients should bewarned that VIT might be less efficacious.Rueff et al. (11) strongly recommend performing sting
challenges to assess the efficacy of VIT in order to detectunprotected patients in whom the dose of venom shouldbe increased to gain protection [B] (11, 51). Theyincreased maintenance dose in 39 patients of whicheight had elevated tryptase levels (>13.5 ng/ml) after apositive challenge (52), and subsequently demonstratedthat seven of these eight patients were protected at asubsequent sting challenge, whereas in one patient thesting challenge was not repeated. In another set ofpatients, the comparison of different maintenance dosesof venom in mastocytosis patients revealed betterprotection of 200 lg (all 17 patients had no reactionto sting challenge) in comparison to 100 lg (five of 24subjects developed systemic symptoms during the stingchallenge) P = 0.05 (53). The �effect� of a highermaintenance dose was also demonstrated in anotherstudy in two mastocytosis patients reacting to a fieldsting who tolerated subsequent stings after increasingthe maintenance dose (17).No data are available to date evaluating the long-term
efficacy of VIT in mastocytosis patients. However, twofatalities have been described in two patients afterstopping VIT respective 5 and 2.5 years (54). Therefore,the guidelines of the EAACI advise lifelong treatment forthis group of patients [2] (8). However, it is presentlyunclear whether continuation of VIT will be able toprevent fatal anaphylactic reactions.Taking into consideration all available data, we con-
clude, that VIT seems to reduce the amount of systemicreactions in patients with mastocytosis although theefficacy is less compared to the general IVA population
treated with VIT, especially in yellow jacket allergicpatients [2].
Pretreatment and means of precautions
Pretreatment during the built-up phase of VIT in mast-ocytosis patients was described in two papers (45, 47).Engler et al. used prednisone, hydroxyzine, ranitidine andastemizole during the 5 days of rush therapy. A heparinelock and cardiovascular monitoring was applied (47). DeOlano et al. considered VIT in mastocytosis patients as arisky procedure, therefore they used premedication (oraldisodium cromoglicate) and intensive care unit or mon-itored setting with management for resuscitation in allpatients. In subjects who did not tolerate the maintenancedose in addition antihistamines, prednisone and antileu-kotrienes were given (45). In this last group they alsochanged Pharmalgen to Aguagen SQ – amine free extract(ALK, Denmark) allowing the continuation of thetherapy without further side-effects in most patients(45). Concerning the maintenance phase of VIT, systemicreactions also have been reported. Therefore, pretreat-ment may also be considered in this phase of treatment(10, 26). Antihistamines blocking H1 receptor in highdoses (i.e. 30 mg of cetirizine a day for adult patients)may be used in all patients (52, 55) [C], as steroids mightbe advised in patients with side-effects during treatment[2] (47, 52). Kontou Fili demonstrated that omalizumabdecreased the incidence and severity of side-effects inmastocytosis patient during VIT (56). However, failuresof pretreatment with omalizumab in prevention ofrecurrent anaphylaxis have also been reported (57), sothe efficacy of omalizumab needs further evaluation.
Patients with mastocytosis, who have experiencedsystemic reactions, should carry two or more epinephrineself-injectors [B] (1–5, 51). This is also advised for all VIT-treated mastocytosis patients in spite of having reachedmaintenance dose, because of the persistent risk of asystemic reaction (10) and the possibility that systemicreactions may also occur after a sting of an insect whosevenom was not used for VIT (27).
Unsolved problems
Golden et al. (41, 58) evaluated the risk of a systemicreaction in the general population taking into consider-ation asymptomatic sensitization and the occurrence oflarge local reaction and/or systemic reactions in the past.Similar studies have not been performed for mastocytosispatients so far, so it is difficult to assess the long term riskof a systemic reaction based on the response to previousstings. We consider that all mastocytosis patients with asystemic reaction (grade 1–4) to a Hymenoptera sting,might be eligible to preventive treatment. Venom immu-notherapy lowers the response rate to the future stings,
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but the long term efficacy of VIT in mastocytosis patientsis less favorable compared to the general population ofIVA patients. It is probable that higher doses of venommay increase the efficacy. As mastocytosis patients areregarded as highly vulnerable to insect sting anaphylaxis(6, 7, 9, 45), the question is whether patients with anallergy for one venom have an increased risk for systemicreactions to other venoms. If so, the question is whetherprophylactic treatment with both honey bee and yellowjacket venom could be useful (24, 27).
Conclusions
In spite of the uncertainties listed above and the moderate[B] to very low quality [D] of data gathered so far, itseems that (1) mastocytosis patients have a high risk atsevere sting reactions in particular to yellow jacket, (2)VIT could be suggested [2] in mastocytosis patients, (3)
probably should be done life long [2]. (4) VIT is able toreduce systemic reactions, but to a lesser extent comparedwith the general IVA population [2], so (5) increased doseof venom might be considered [2]. (6) VIT in mastocytosisis accompanied by a higher frequency of side-effects,especially in yellow jacket allergic patients, thus (7)special precautions such as premedication and promptavailability of the emergency treatment should be takeninto account notably during the built up phase of thetherapy [2] (8). Patients should be warned that the efficacyof the treatment may be less than optimal and they shouldcontinue carrying two adrenaline auto injectors [2].
Acknowledgments
The publication was supported by the Foundation for Polish Sci-ence.
References
1. Golkar L, Bernhard J. Mastocytosis.Lancet 1997;349:1379–1385.
2. Gonera R, Oranje W, Wolffenbuttel B.Shock of unknown origin-think ofmastocytosis! Neth J Med 1997;50:165–169.
3. Valent P, Horny H, Escribano L.Diagnostic criteria and classification ofmastocytosis: a consensus proposal.Leuk Res 2001;25:603–625.
4. Valent P, Sperr W, Schwartz L, HornyH. Diagnosis and classification of mastcell proliferative disorders: delineationfrom immunologic diseases andnonmast cell hematopoietic neoplasms.J Allergy Clin Immunol 2004;114:3–11.
5. Akin C, Metcalfe D. The biology of kitin disease and the application of phar-macogenetics. J Allergy Clin Immunol2004;114:13–19.
6. Brockow K, Jofer C, Behrendt H,Ring J. Anaphylaxis in patients withmastocytosis: a study on history, clinicalfeatures and risk factors in 120 patients.Allergy 2008;63:226–232.
7. Ludolph-Hauser D, Rueff F, Fries C,Schopf P, Przybilla B. Constitutivelyraised serum concentrations of mast-celltryptase and severe anaphylactic reac-tions to Hymenoptera stings. Lancet2001;357:361–362.
8. Bonifazi F. Prevention and treatment ofhymenoptera venom allergy: guidelinesfor clinical practice. Allergy2005;60:1459–1470.
9. BiloBM,RueffF,MosbechH,BonifaziF,Oude-Elberink JN. The EAACI interestgroup on insect venom hypersensitivity.Diagnosis of Hymenoptera venomallergy. Allergy 2005;60:1339–1349.
10. Dubois AE. Mastocytosis and hyme-noptera allergy. Curr Opin Allergy ClinImmunol 2004;4:291–295.
11. Rueff F, Placzek M, Przybilla B. Masto-cytosis and hymenoptera venom allergy.Curr Opin Allergy Clin Immunol 2006;6:284–288.
12. Bro _zek JL, Akl EA, Alonso-Coello P,Lang D, Jaeschke R, Williams JW et al.Grading quality of evidence andstrength of recommendations in clinicalpractice guidelines. Allergy 2009;64:669–677.
13. Guyatt GH, Oxman AD, Kunz R,Falck-Ytter Y, Vist GE, Liberati Aet al. Going from evidence to recom-mendations. BMJ 2008;336:1049–1051.
14. Charpin D, Birnbaum J, Vervloet D.Epidemiology of hymenoptera allergy.Clin Exp Allergy 1994;24:1010–1015.
15. Finegold I. Issues in stinging insectallergy immunotherapy: a review. CurrOpin Allergy Clin Immunol 2008;8:343–347.
16. Fine J. Mastocytosis. In J Dermatol1980;19:117–123.
17. Bonadonna P, Zanotti R, Caruso B,Castellani L, Perbellini O, Colarossi Set al. Allergen specific immunotherapyis safe and effective in patients withsystemic mastocytosis and hymenopteraallergy. J Allergy Clin Immunol2008;121:256–257.
18. Bonadonna P, Perbellini O, PassalacquaG, Caruso B, Colarossi S, Dal Fior Det al. Clonal mast cell disorders inpatients with systemic reactions toHymenoptera stings and increasedserum tryptase levels. J Allergy ClinImmunol 2009;123:680–686.
19. Niedoszytko M, Lange M, ChelminskaM, Jaskiewicz K, Piskosz A, Wasag Bet al. Systemic mastocytosis. PneumonolAlergol Pol 2005;73:239–244.
20. Haeberli G, Bronnimann M, HunzikerT, Muller U. Elevated basal serum tryp-tase and hymenoptera venom allergy:relation to severity of sting reactions andto safety and efficacy of venom immu-notherapy. Clin Exp Allergy 2003;33:1216–1220.
21. Muller UR. New developments in thediagnosis and treatment of hymenopteravenom allergy. Int Arch Allergy Immu-nol 2001;124:447–453.
22. Florian S. Indolent systemic mastocy-tosis with elevated serum tryptase,absence of skin lesions, and recurrentanaphylactoid episodes. Int ArchAllergy Immunol 2005;136:273–280.
23. Peavy R, Metcalfe D. Understandingthe mechanisms of anaphylaxis. CurrOpin Allergy Clin Immunol 2008;8:310–315.
24. Wagner N, Fritze D, Przybilla B,Hagedorn M, Rueff F. Fatal anaphy-lactic sting reaction in a patient withmastocytosis. Int Arch Allergy Immunol2008;146:162–163.
Mastocytosis and insect venom allergy
� 2009 John Wiley & Sons A/S Allergy 2009: 64: 1237–1245 1243
25. Soriano Gomis V, Gonzalez Delgado P,Niveiro Hernandez E. Failure ofomalizumab treatment after recurrentsystemic reactions to bee-venom immu-notherapy. J Investig Allergol ClinImmunol 2008;18:225–256.
26. Oude Elberink JN, de Monchy JG, KorsJW, van Doormaal JJ, Dubois AE.Fatal anaphylaxis after a yellow jacketsting, despite venom immuno-therapy, in two patients withmastocytosis. Allergy Clin Immunol1997;99:153–154.
27. Reimers A, Muller U. Fatal outcome ofa vespula sting in a patient with masto-cytosis after specific immunotherapywith honey bee venom. Allergy ClinImmunol Int J WAO Org 2005;17:68–70.
28. Pumphrey R, Roberts I. Postmortemfindings after letal anaphylactic reaction.J Clin Pathol 2000;53:273–276.
29. Mueller UR. Cardiovascular disease andanaphylaxis. Curr Opin Allergy ClinImmunol 2007;7:337–341.
30. Muller UR, Horat W, Wuthrich B,Conroy M, Reisman RE. Anaphylaxisafter hymenoptera stings in threepatients with urticaria pigmentosa.J Allergy Clin Immunol 1983;72:685–689.
31. Bernstein IL, Li JT, Bernstein DI,Hamilton R, Spector SL, Tan R et al.American Academy of Allergy, Asthmaand Immunology; American College ofAllergy, Asthma and Immunology.Allergy diagnostic testing: an updatedpractice parameter. Ann AllergyAsthma Immunol 2008;100:1–148.
32. Vennekenens R, Olausson J, MeissnerM, Bloch W, Mathar I, Philipp S et al.Increased IgE dependent mast cell acti-vation and anaphylactic responses inmice lacking the calcium activated non-selective cation channel TRPM4. NatImmunol 2007;8:312–320.
33. Baba Y, Nishida K, Fujii Y et al.Essential function for the calcium sensorSTIM1 in mast cell activation andanaphylactic responses. Nat Immunol2008;9:81–88.
34. Jutel M, Akdis M, Blaser K, Akdis CA.Mechanisms of allergen specific immu-notherapy-T-cell tolerance and more.Allergy 2006;61:796–807.
35. Konno S, Hizawa N, Nishimura M,Huang SK. Osteopontin: a potentialbiomarker for successful bee venomimmunotherapy and a potential mole-cule for inhibiting IgE-mediatedallergic responses. Allergol Int 2006;55:355–359.
36. Kontou-Fili K. Patients with negativeskin tests. Curr Opin Allergy ClinImmunol 2002;2:353–357.
37. Kawabata Y, Yan T, Yokochi T et al.Complement system is involved in ana-phylactoid rections induced by lipopol-isacharides in muramyldipeptide-treatedmice. Shock 2000;14:572–577.
38. Hermann K, Ring J. The rennin-angio-tensin system in patients with treatedanaphylactic reactions during Hyme-noptera venom hyposensitization andsting challenge. Int Arch AllergyImmunol 1997;112:251–256.
39. Volcheck G, Butterfield J, Yunginger J,Klee G. Elevated serum levels of calci-tonin gene-related peptide in Hyme-noptera venom anaphylaxis. J AllergyClin Immunol 1998;102:149–151.
40. Bilo MB, Brianzoni F, Cinti B, NapoliG, Bonifazi F. The dilemma of thenegative skin test reactors with a historyof venom anaphylaxis: will this alwaysbe the case? Eur Ann Allergy ClinImmunol 2005;37:341–342.
41. Golden DB. Insect sting anaphylaxis.Immunol Allergy Clin North Am2007;27:261–272.
42. Dugas-Breit S, Przybilla B, Schopf P,Rueff F. Possible circadian variation ofserum mast cell tryptase concentration.Allergy 2005;60:689–692.
43. Kranke B, Sturm G, Aberer W. Nega-tive venom skin test results and masto-cytosis. J Allergy Clin Immunol 2004;113:180–181.
44. Kors JW, van Doormaal JJ, de MonchyJG. Anaphylactoid shock followinghymenoptera sting as a presentingsymptom of systemic mastocytosis.J Intern Med 1993;233:255–258.
45. Gonzalez de Olano D, Alvarez-Twose I,Esteban-Lopez MI, Sanchez-Munoz L,de Durana MD, Vega A et al. Safetyand effectiveness of immunotherapy inpatients with indolent systemic masto-cytosis presenting with Hymenopteravenom anaphylaxis. J Allergy ClinImmunol 2008;121:519–526.
46. Fricker M, Helbling A, Schwartz L,Muller U. Hymenoptera sting anaphy-laxis and urticaria pigmentosa: clinicalfindings and results of venom immuno-therapy in ten patients. Allergy ClinImmunol 1997;100:11–15.
47. Engler RJ, Davis WS. Rush hymenop-tera venom immunotherapy: successfultreatment in a patient with systemicmast cell disease. J Allergy Clin Immu-nol 1994;94:556–559.
48. Price LA, Safko M. Bee venom allergyin a patient with urticaria pigmentosa. JAllergy Clin Immunol 1987;79:407–409.
49. Muller U, Helbling A, Berchtold E.Immunotherapy with honeybee venomand yellow jacket venom is differentregarding efficacy and safety. J AllergyClin Immunol 1992;89:529–535.
50. Franken HH, Dubois AE, Minkema HJ,van der Heide S, de Monchy JG. Lackof reproducibility of a single negativesting challenge response in the assess-ment of anaphylactic risk in patientswith suspected yellow jacket hypersen-sitivity. J Allergy Clin Immunol 1994;93:431–436.
51. Golden D, Kwiterovich K, Kagey-Sobotka A, Lichtenstein L. Discontinu-ing venom immunotherapy:Extendedobservations. J Allery Clin Immunol1998;101:298–305.
52. Rueff F, Wenderoth A, Przybilla B.Patients still reacting to a sting challengewhile receiving conventional hymenop-tera venom immunotherapy are pro-tected by increased venom doses.J Allergy Clin Immunol 2001;108:1027–1032.
53. Rernick H, Przybilla B, Rueff F. Venomimmunotherapy (VIT) in patients withsystemic mastocytosis (SM) and hyme-noptera anaphylaxis (HVA): safety andefficacy of different maintenance doeses.Abstract no 936, AAAAI 2009 annualmeeting.
54. Biedermann T, Rueff F, Sander CA,Przybilla B. Mastocytosis associatedwith severe wasp sting anaphylaxisdetected by elevated serum mast celltryptase levels. Br J Dermatol1999;141:1110–1112.
55. Gorska L, Chelminska M, KuziemskiK, Skrzypski M, Niedoszytko M,Damps-Konstanska I et al. Analysis ofsafety, risk factors and pretreatmentmethods during rush hymenopteravenom immunotherapy. Int ArchAllergy Immunol 2008;147:241–245.
56. Kontou-Fili K. High omalizumabdose controls recurrent reactions tovenom immunotherapy in indolentsystemic mastocytosis. Allergy 2008;63: 376–378.
57. Carter MC, Robyn JA, Bressler PB,Walker JC, Shapiro GG, Metcalfe DD.Omalizumab for the treatment ofunprovoked anaphylaxis in patientswith systemic mastocytosis. J AllergyClin Immunol 2007;119:1550–1551.
58. Golden DB, Marsh DG, Freidhoff LR,Kwiterovich KA, Addison B, Kagey-Sobotka A et al. Natural history ofhymenoptera venom sensitivity inadults. J Allergy Clin Immunol 1997;100:760–766.
59. Mueller HL. Diagnosis and treatment ofinsect sensitivity. J Asthma Res 1966;3:331–333.
60. Mosbech H, Muller U. Side-effects ofinsect venom immunotherapy: resultsfrom an EAACI multicenter study.Allergy 2000;55:1005–1010.
Niedoszytko et al.
1244 � 2009 John Wiley & Sons A/S Allergy 2009: 64: 1237–1245
61. Wenzel J, Meissner-Kraemer M, BauerR, Bieber T, Gerdsen R. Safety of rushinsect venom immunotherapy. Theresults of a retrospective study in 178patients. Allergy 2003;58:1176–1179.
62. Birnbaum J, Ramadour M, Magnan A,Vervloet D. Hymenoptera ultra-rushvenom immunotherapy (210 min): asafety study and risk factors. Clin ExpAllergy 2003;33:58–64.
63. Brehler R, Wolf H, Kutting B, SchnitkerJ, Luger T. Safety of a two-day ultra-rush insect venom immunotherapy pro-tocol in comparison with protocols oflonger duration and involving a largernumber of injections. J Allergy ClinImmunol 2000;105:1231–1235.
64. Sturm G, Kranke B, Rudolph C, AbererW. Rush Hymenoptera venom immu-notherapy: a safe and practical protocolfor high-risk patients. J Allergy ClinImmunol 2002;110:928–933.
Mastocytosis and insect venom allergy
� 2009 John Wiley & Sons A/S Allergy 2009: 64: 1237–1245 1245
ORIGINAL ARTICLE EPIDEMIOLOGY AND GENETICS
Gene expression analysis predicts insect venom anaphylaxisin indolent systemic mastocytosisM. Niedoszytko1,2, M. Bruinenberg3,4, J. J. van Doormaal2, J. G. R. de Monchy2, B. Nedoszytko5,G. H. Koppelman6, M. C. Nawijn7, C. Wijmenga3, E. Jassem1 & J. N. G. Oude Elberink2
1Department of Allergology, Medical University of Gdansk, Gdansk, Poland; 2Department of Allergology, University Medical Center
Groningen, University of Groningen, Groningen, the Netherlands; 3Department of Genetics, University Medical Center Groningen, University
of Groningen, Groningen, the Netherlands; 4LifeLines, University Medical Center Groningen, University of Groningen, Groningen, the Nether-
lands; 5Department of Dermatology, Medical University of Gdansk, Gdansk, Poland; 6Department of Pediatric Pulmonology and Pediatric
Allergology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands; 7Laboratory of Allergology and
Pulmonary Diseases, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen,
Groningen, the Netherlands
To cite this article: Niedoszytko M, Bruinenberg M, van Doormaal JJ, de Monchy JGR, Nedoszytko B, Koppelman GH, Nawijn MC, Wijmenga C, Jassem E,
Oude Elberink JNG. Gene expression analysis predicts insect venom anaphylaxis in indolent systemic mastocytosis. Allergy 2010; DOI: 10.1111/
j.1398-9995.2010.02521.x.
Mastocytosis is an uncommon disease resulting from a path-
ological increase in mast cells in different tissues including
skin, bone marrow, liver, spleen, and lymph nodes (1). The
clinical presentation of mastocytosis is heterogeneous, vary-
ing from sole skin presentation found in urticaria pigmentosa
and mastocytoma, to different forms of systemic disease
including indolent systemic mastocytosis (ISM), smoldering
systemic mastocytosis, aggressive systemic mastocytosis and
mast cell leukemia (1, 2). Of the adult patients with systemic
mastocytosis, the large majority (ca. 90%) has the indolent
form of the disease.
Symptoms of the disease result from skin involvement,
mast cell mediator release and massive mast cell infiltration,
as found in aggressive variants of the disease. Symptoms of
mast cell degranulation may vary from pruritus and flushing
Keywords
anaphylaxis; gene expression; insect venom
allergy; mastocytosis; microarray
assessment; prediction of anaphylaxis.
Correspondence
Marek Niedoszytko, MD, PhD, Department
of Allergology, Medical University of
Gdansk, Debinki 7 80-952, Gdansk, Poland.
Tel.: +48583491626
Fax: +48583491625
E-mail: [email protected]
Accepted for publication 9 November 2010
DOI:10.1111/j.1398-9995.2010.02521.x
Edited by: Thomas Bieber
Abstract
Background: Anaphylaxis to insect venom (Hymenoptera) is most severe in patients
with mastocytosis and may even lead to death. However, not all patients with mastocy-
tosis suffer from anaphylaxis. The aim of the study was to analyze differences in gene
expression between patients with indolent systemic mastocytosis (ISM) and a history
of insect venom anaphylaxis (IVA) compared to those patients without a history of
anaphylaxis, and to determine the predictive use of gene expression profiling.
Methods: Whole-genome gene expression analysis was performed in peripheral
blood cells.
Results: Twenty-two adults with ISM were included: 12 with a history of IVA and
10 without a history of anaphylaxis of any kind. Significant differences in single gene
expression corrected for multiple testing were found for 104 transcripts (P < 0.05).
Gene ontology analysis revealed that the differentially expressed genes were involved
in pathways responsible for the development of cancer and focal and cell adhesion
suggesting that the expression of genes related to the differentiation state of cells is
higher in patients with a history of anaphylaxis. Based on the gene expression
profiles, a naıve Bayes prediction model was built identifying patients with IVA.
Conclusions: In ISM, gene expression profiles are different between patients with a
history of IVA and those without. These findings might reflect a more pronounced
mast cells dysfunction in patients without a history of anaphylaxis. Gene expression
profiling might be a useful tool to predict the risk of anaphylaxis on insect venom in
patients with ISM. Prospective studies are needed to substantiate any conclusions.
Abbreviations
Fc, fold change difference; GO, gene ontology; IgE,
immunoglobulin E; ISM, indolent systemic mastocytosis; IVA,
insect venom anaphylaxis; KEGG, Kyoto encyclopedia of genes and
genomes; MAPK, mitogen-activated protein kinase; SPT, skin-prick
test; VIT, venom immunotherapy; Wnt, wingless int pathway.
Allergy
ª 2010 John Wiley & Sons A/S
to anaphylaxis with profound hypotension and with occa-
sionally even fatal outcome (1–5). The cumulative prevalence
of anaphylaxis in patients with mastocytosis has been
reported to be as high as 50% (3). Anaphylaxis is thought to
be the most important burden for the majority of patients
with mastocytosis.
The most important eliciting factor of anaphylaxis in
patients with mastocytosis is an insect sting (3). It is estimated
that 30% of patients with mastocytosis suffer from insect
venom anaphylaxis (IVA) (3, 6). Thus, patients are advised to
carry epinephrine auto-injectors and often start with venom
immunotherapy (VIT), probably lifelong (6, 7). Because fatal
anaphylaxis also has been reported, even without a history of
previous anaphylactic sting reactions (4, 5), some authors pos-
tulate prophylactic treatment for all patients with mastocytosis
(7, 8). However, because less than half of the patients with
mastocytosis will develop anaphylaxis on insect stings during
their lifetime, such a strategy of prophylactic treatment would
inflict a significant burden on a large group of patients that is
in no need for such an intervention. Therefore, it is of great
interest to identify those patients at risk for IVA, ideally using
a minimally invasive and highly sensitive assay. Unfortunately,
it has not been possible so far to predict which patients with
mastocytosis are at risk for anaphylaxis to an insect sting (1–9).
The mechanism(s) responsible for development of anaphy-
laxis in some and the lack of such reactions in other patients
with mastocytosis are mainly unknown (2, 3, 6, 9–11). It has
been hypothesized that the predisposition to anaphylaxis is
related to activation of mast cell kinase pathways or calcium
ion channels (2, 3, 8–11). Although severe anaphylaxis may
occur in patients with all variants of mastocytosis (1–3), they
are most frequent in the indolent form of mastocytosis. The
mast cells in mastocytosis display several phenotypic abnor-
malities, among which the activating somatic mutation of the
KIT receptor that also affects histamine release (2). However,
the contribution of these intrinsic alterations to the sensitivity
for developing (insect venom) anaphylaxis is unknown.
Better understanding of the molecular differences between
mastocytosis patients with an IgE mediated anaphylaxis to
insect venom and mastocytosis patients without such reactions
might be helpful to predict the risk for anaphylaxis in those
patients with mastocytosis who have not been stung yet. Anal-
ysis of gene expression allows for the direct recognition of gene
activation. Whole-genome expression analysis has the potential
to reveal differences in activation of gene networks involved in
anaphylaxis between patients with and without IVA.
The aim of this study therefore was to analyze whether in
ISM differences in gene expression profiles can be identified
in peripheral blood cells between patients with a history of
anaphylaxis to insect venom who never received VIT and
those patients without anaphylaxis of any kind.
Materials and methods
Patients
Patients with ISM from the Department of Allergology, Uni-
versity Medical Center Groningen (UMCG) were included.
ISM has been diagnosed in all patients according to the
WHO criteria (1) by serum tryptase measurement and bone
marrow investigation including histopathological, cytological,
CD2 and CD25 flow cytometry and genetic examinations.
None of these patients had been treated with VIT in the past.
Subjects suffering from other severe chronic or/and malig-
nant diseases and pregnant women were excluded.
Patients were divided into two groups:
Group 1: patients with a history of anaphylaxis caused by
Hymenoptera venom. The diagnosis of IVA was based on
medical history (grade IV reaction according to Mueller (12)
and a positive skin tests and/or sIgE.
Group 2: patients who did not experience any allergic reac-
tions after insect stings (they were stung at least once after
ISM has been diagnosed) or by other known or unknown
factors in the past during at least 10 years of follow-up after
the diagnosis of ISM has been made.
The study was approved by the Medical Ethical Commit-
tee of the UMCG (METc 2008/340).
Collection of blood samples
From all patients, RNA was isolated from whole blood using
the PAXgene Blood RNA Tubes (Qiagen, Valencia, CA,
USA). All tubes were immediately frozen and stored in
)20�C till RNA isolation (maximal period 2 months). RNA
was isolated using PAXgene Blood RNA Kit CE (Qiagen,
Venlo, the Netherlands). All RNA samples were stored in
)80�C till labeling and hybridization.
The quality and concentration of RNA was determined
using the 2100 Bioanalyzer (Agilent, Amstelveen, the Nether-
lands) with the use of Agilent RNA 6000 Nano Kit. Samples
with a RNA integrity number >7.5 were used for further
analysis on expression arrays.
Gene expression
For amplification and labeling of RNA with Illumina, Total-
Prep 96 RNA Amplification kit was used (Applied Biosys-
tems, Nieuwerkerk ad IJssel, the Netherlands). For each
sample, we used 200 ng of RNA. The Human HT-12_V3_
expression arrays (Illumina, San Diego, CA, USA) were pro-
cessed according to the manufacturer’s protocol. Slides were
scanned immediately using an Illumina BeadStation iScan
(Illumina).
Image and data analysis
First-line check, background correction, and quantile normal-
ization of the data were performed with Genomestudio Gene
Expression Analysis module v 1.0.6 Statistics. Entities con-
taining at least 75% of samples with a signal intensity value
above the 20th percentile in 100% of the samples in at least
2 groups were included for the further analysis.
Data analysis was performed using genespring package
version 10.0.0 (Agilent Technologies, Santa Clara, CA,
USA). Genes for which expression was significantly different
between both groups were chosen based on a log2 fold
Gene expression profile predicting insect venom anaphylaxis in mastocytosis Niedoszytko et al.
ª 2010 John Wiley & Sons A/S
change >2 in gene expression, t-test P-value <0.05 and
a Benjamini–Hochberg false discovery rates <0.05. The
P-values presented in the text were corrected for multiple
testing. A naıve Bayes prediction model was used to build a
prediction model assessing the risk of anaphylaxis in patients
with ISM (13). A naıve Bayesian classifier is a mathematical
process computing the probability of classifying the patient
in a group of risk of anaphylaxis or without risk based on
the results of gene expression (13). The expression of each
particular gene is regarded as an independent predictor classi-
fying patient to the particular group.
Functional annotation of genes was described by the Go
Process Analysis and pathways of the Kyoto encyclopedia of
genes and genomes (KEGG) (14) with Genecodis functional
annotation web-based tool (15).
Clinical data of this study were analyzed with statistica
8.0 (StatSoft, Tulsa, OK, USA). The chi-square was used to
compare the number of patients with IVA in the studied
groups. The mean difference of serum tryptase and urinary
excretions of methylhistamine and methylimidazole acetic
acid was analyzed using the U-Mann–Whitney test. P-values
<0.05 were considered statistically significant.
Results
A total of 22 patients with ISM were included. Twelve
patients (two men) had a history of IVA (group 1) and 10
patients (five men) had no history of anaphylaxis caused by
insect venom or other factors (group 2). In group 2, one
patient was asymptomatically sensitized to wasp and bee
venom. No significant differences in clinical characteristics
were found between both groups, except for sensitization to
insect venom (P = 0.0001) (Table 1). Ages at diagnosis of
ISM and levels of serum tryptase and urinary excretions of
methylhistamine and methylimidazole acetic acid were in
medians and ranges in group 1: 49 (25-70) years, 29.4 (5.13-
112) lg/l, 335 (153-1024) lmol/mol creatinine, and 3.3 (1.4-
7.7) mmol/mol creatinine; and in group 2: 50 (34-64) years,
48.4 (4.62-155) lg/l, 452 (77-1046) lmol/mol creatinine, and
4.5 (1.4-10.9) mmol/mol creatinine, respectively.
Comparison of gene expression profiles
Whole-genome gene expression analysis was performed on
RNA samples isolated from peripheral blood cells. From all
48.804 probes present on the array, 48.676 transcripts passed
the quality control and were included for further analysis.
Of all analyzed transcripts, 1951 showed a log2 > 2-fold
difference in gene expression: 967 (49%) genes were increased
and 984 (51%) were decreased in patients with IVA.
To assess whether the differentially expressed genes were
enriched for a specific cellular function, we performed gen-
ome ontology (GO) analysis using the Genecodis website.
The main processes to which the differentially expressed
genes map are signal transduction, multicellular organism
development, transcription, cell differentiation, metabolic
process, and ion transport (Table 2). Using the KEGG data-
base (14), we found that the pathways that are most signifi-
cantly enriched for in our list of log2 > 2-fold differentially
expressed genes are pathways involved in cancer, focal adhe-
sion, cell adhesion, ubiquitin mediated proteolysis, Wnt sig-
naling, and calcium signaling (Table 3).
Subsequently, we analyzed the 104 transcripts that were
significantly different between the two patient groups in sin-
gle gene expression corrected for multiple testing (P < 0.05)
revealing a log2 > 3-fold difference in expression. In patients
with a history of IVA, 37 transcripts (36%) were increased,
whereas 67 transcripts (64%) were decreased in expression
(Table 4). A hierarchical clustering of the differentially
expressed genes is presented in Figure 1. The most significant
differences (P corrected for multiple testing <0.002) were
found in eight transcripts: HS 552770, HS 41192,
RBMY1A3P, DVL1, G0S2, HS 540329, HS 546027, and
LOC283487. Except for HS 552770, which was up-regulated
in patients with a history of IVA, the remaining seven tran-
scripts were up-regulated in patients without anaphylaxis. Of
these genes, it is known that DVL1 and G0S2 are related to
the development of cancer, the function of the remaining
transcripts is still unknown. GO analysis using the KEGG
database on this list of 104 transcripts revealed the enrich-
ment of genes mapping to two processes: pathways in cancer
and the mitogen-activated protein kinase (MAPK) signaling
pathway (Table 3). The function of 46 (39%) genes is
unknown yet.
Subsequently, we used a prediction model that uses a naıve
Bayes (NB) classifier, based on the 104 most significant dif-
ferentially expressed genes. This model was able to differenti-
ate ISM patients with IVA from those without anaphylaxis
with a sensitivity and specificity of 100%.
To relate the whole-genome expression results to specific
cell expression profiles, we analyzed the expression profiles of
leukocyte-specific genes expressed in dendritic cells, B cells,
memory T cells, mast cells, and basophils as described by
Liu et al. (16). A statistically significant difference in expres-
sion comparing both patient groups was found for the mast
cell–specific genes CTTNBP2 encoding cortactin-binding pro-
tein 2, a central regulator of cytoskeletal rearrangement in
mitosis (17), which was up-regulated in patients with IVA
(P = 0.03) and SIGLEC6 encoding sialic acid–binding Ig-like
lectin 6, playing a role in cell–cell interactions (18), which
was down-regulated in patients with IVA (P = 0.04).
Discussion
The results of our study demonstrate that we are able to
identify in ISM genes that are differentially expressed
between the peripheral blood cells from patients with IVA
and those from patients without a history of anaphylaxis.
GO analysis reveals that the differentially expressed genes are
enriched for genes that function in several pathways that
regulate the balance between proliferation versus terminal
differentiation: Wnt signaling pathway, focal and cell adhe-
sion, calcium signaling, extracellular matrix interactions,
pathways in cancer and MAPK signaling (Tables 3 and 4).
So, our data indicate that in spite of a high number of mast
cells in all patients, the sensitivity to develop anaphylaxis
Niedoszytko et al. Gene expression profile predicting insect venom anaphylaxis in mastocytosis
ª 2010 John Wiley & Sons A/S
Tab
le1
Clin
ical
chara
cte
ristics
of
the
patients
with
indole
nt
syste
mic
masto
cyto
sis
with
ahis
tory
of
insect
venom
anaphyla
xis
(gro
up
1)
and
without
such
ahis
tory
(gro
up
2)
Gro
up
Sex
Age
at
dia
gnosis
(years
)U
P
Seru
m
trypta
se
(lg/l)*
Urine
MH
(lm
ol/m
ol
cre
at)
*
Urine
MIM
A
(mm
ol/m
ol
cre
at)
*
MC
sin
bone
marr
ow
aspirate
(%)
CD
2
imm
uno-
phenoty
pe
CD
25
imm
uno-
phenoty
pe
D816V
KIT
muta
tion
inbone
marr
ow
cells
‡2 aggre
gate
s
of
‡15
MC
s
inbone
marr
ow
Abnorm
al
morp
holo
gy
of
‡25%
of
MC
sin
bone
marr
ow
His
tolo
gic
al
bone
marr
ow
cellu
larity
Sensitiz
ation
tow
asp
venom
�
Sensitiz
ation
tobee
venom
�
Gro
up
1M
50
neg
28.9
153
1.4
0.0
9neg
pos
neg
neg
pos
norm
al
pos
neg
F43
adult
UP
34.1
380
4.8
0.2
9neg
pos
pos
pos
pos
norm
al
pos
neg
F64
adult
UP
21.7
166
3.1
0.1
1pos
pos
pos
neg
pos
norm
al
pos
neg
F47
neg
48.3
604
6.5
0.1
7pos
pos
pos
pos
pos
norm
al
pos
neg
F25
adult
UP
27.4
1024
3.4
n.d
.n.d
.n.d
.n.d
.pos
pos
norm
al
pos
neg
F61
neg
15.2
266
2.3
0.3
0neg
pos
neg
pos
pos
norm
al
pos
neg
F51
neg
74.5
194
2.7
0.1
0pos
pos
pos
pos
pos
norm
al
pos
pos
F38
adult
UP
5.1
3190
1.7
0.1
3pos
pos
neg
pos
pos
norm
al
pos
neg
M37
adult
UP
112
404
7.7
n.d
.n.d
.n.d
.pos
pos
pos
norm
al
pos
neg
F36
neg
31.3
542
6.5
0.1
7pos
pos
pos
pos
pos
slig
htly
hyperp
lastic
pos
neg
F70
neg
19.7
289
2.7
0.1
0pos
pos
pos
neg
pos
norm
al
pos
neg
F54
adult
UP
29.8
593
4.1
n.d
.n.d
.n.d
.pos
pos
pos
norm
al
pos
neg
Gro
up
2F
64
adult
UP
155
1046
10.9
0.4
9pos
pos
pos
pos
pos
norm
al
neg
neg
F50
adult
UP
20.4
293
3.0
0.0
6pos
pos
pos
pos
pos
norm
al
neg
neg
M34
adult
UP
55.4
433
4.9
0.4
5pos
pos
pos
pos
pos
norm
al
neg
neg
M41
juvenile
UP
146
470
4.0
1.6
1neg
pos
pos
pos
pos
norm
al
neg
neg
F64
adult
UP
4.6
2102
1.6
0.1
0pos
pos
pos
neg
pos
norm
al
neg
neg
M54
neg
28.2
77
1.4
0.0
9pos
pos
pos
neg
pos
norm
al
pos
pos
M50
adult
UP
52.3
530
6.4
n.d
.n.d
.n.d
.n.d
.pos
pos
norm
al
neg
neg
F43
adult
UP
36.5
851
5.9
0.3
4pos
pos
pos
pos
pos
norm
al
neg
neg
M38
neg
44.4
310
3.4
n.d
.n.d
.n.d
.pos
pos
pos
norm
al
neg
neg
F63
adult
UP
109
823
6.3
n.d
.n.d
.n.d
.n.d
.pos
pos
norm
al
neg
neg
Abbre
via
tions:
M:
male
;F:
fem
ale
;n.d
.:not
done;
UP
:urt
icaria
pig
mento
sa;
juvenile
UP
:ju
venile
-onset
UP
;adult
UP
:adult-o
nset
UP
;M
H:
meth
ylh
ista
min
e;
MIM
A:
meth
ylim
idazo
leacetic
acid
;M
Cs:
mast
cells
.
*R
efe
rence
valu
es:
trypta
se
11.4
lg/l
(95
perc
entile
);M
H167
lm
ol/m
olcre
atinin
e(9
7.5
perc
entile
);M
IMA
1.9
mm
ol/m
olcre
atinin
e(9
7.5
mm
ol/m
olcre
atinin
e(9
7.5
perc
entile
).
�Sensitiz
ation
pos:
sIg
E>
0.3
5kU
/lor
positiv
eskin
-prick
test.
Gene expression profile predicting insect venom anaphylaxis in mastocytosis Niedoszytko et al.
ª 2010 John Wiley & Sons A/S
might be correlated to the differentiation state of the mast
cells. The functional annotation of the four most significantly
differentially expressed genes between the two patient groups
confirms this observation (Table 5). Together, these data
indicate that ISM patients with a more differentiated mast
cell phenotype are at risk for developing of IVA.
Further, our data indicate that gene expression profiling
on peripheral blood cells from patients with ISM enables to
differentiate patients with IVA from ISM patients without
anaphylaxis in their medical history. It indicates that this
procedure might be adapted to identify mastocytosis patients
at risk for anaphylaxis to benefit from prophylactic treat-
ment.
It is assumed that baseline serum tryptase concentrations
reflect the mast cell mass of the body. In patients without
mastocytosis, an increased tryptase concentration is associated
with an increased risk for the occurrence of insect venom
allergy (28), and in addition, it is also associated with the
more severe reactions (28). In patients with mastocytosis,
higher basal tryptase values were also associated with a
greater risk of anaphylaxis (3). In our ISM group, however,
we found in patients with IVA a trend toward lower levels of
tryptase in serum and lower urinary excretion of the histamine
metabolites methylhistamine and methylimidazole acetic acid,
which are also thought to reflect mast cell burden. This differ-
ence might be because of the fact that we only included
Table 2 Gene co-occurrence annotation found by Genecodis (19, 20) (GOSlim Process Function) for the genes differentially expressed
(log2fc>2) between patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis and without anaphylaxis of any
kind
Genes NGR NG Hyp Hyp* Annotations
88 genes 1700 (37435) 88 (1022) 8.37904e-09 2.5975e-07 GO:0007165: signal transduction
50 genes 874 (37435) 50 (1022) 9.52928e-07 1.47704e-05 GO:0007275: multicellular organismal
development
73 genes 1516 (37435) 73 (1022) 2.37117e-06 2.45021e-05 GO:0006350: transcription
8 genes 43 (37435) 8 (1022) 1.8644e-05 0.000144491 GO:0007165: signal transduction
GO:0030154: cell differentiation
29 genes 471 (37435) 29 (1022) 4.93153e-05 0.000305755 GO:0008152: metabolic process
30 genes 503 (37435) 30 (1022) 6.56447e-05 0.000339164 GO:0006811: ion transport
18 genes 232 (37435) 18 (1022) 7.74516e-05 0.000343 GO:0006629: lipid metabolic process
Hyp: P-values have been obtained through Hyper geometric analysis; Hyp*: P-values have been obtained through hypergeometric analysis,
corrected by false discovery rate method; NGR: number of annotated genes in the reference list; NG: number of annotated genes in the
input list.
Table 3 Gene co-occurrence annotation found by Genecodis (Kyoto encyclopedia of genes and genomes [KEGG] pathways) (17–20) for the
genes differentially expressed (log2fc>2) between patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis
and without anaphylaxis of any kind
Genes NGR NG Hyp Hyp* Annotations
25 genes 320 (37435) 25 (1022) 3.05203e-06 0.000378452 (KEGG) 05200: Pathways in cancer
17 genes 194 (37435) 17 (1022) 2.65238e-05 0.00164448 (KEGG) 04510: Focal adhesion
13 genes 131 (37435) 13 (1022) 6.47261e-05 0.00267535 (KEGG) 04514: Cell adhesion molecules (CAMs)
5 genes 19 (37435) 5 (1022) 0.000126934 0.00393495 (KEGG) 05200: Pathways in cancer (KEGG) 04120:
Ubiquitin mediated proteolysis
12 genes 129 (37435) 12 (1022) 0.000225735 0.00559822 (KEGG) 04120: Ubiquitin mediated proteolysis
13 genes 150 (37435) 13 (1022) 0.000254129 0.00525199 (KEGG) 04310: Wnt signaling pathway
14 genes 176 (37435) 14 (1022) 0.000362833 0.00642733 (KEGG) 04020: Calcium signaling pathway
9 genes 82 (37435) 9 (1022) 0.000400788 0.00621222 (KEGG) 04512: ECM-receptor interaction
15 genes 206 (37435) 15 (1022) 0.000580926 0.00800387 (KEGG) 04810: Regulation of actin cytoskeleton
9 genes 96 (37435) 9 (1022) 0.00126496 0.0130712 (KEGG) 04912: GnRH signaling pathway
9 genes 98 (37435) 9 (1022) 0.00146319 0.0139566 (KEGG) 04916: Melanogenesis
7 genes 62 (37435) 7 (1022) 0.00147513 0.0130655 (KEGG) 00980: Metabolism of xenobiotics by
cytochrome P450
16 genes 262 (37435) 16 (1022) 0.0024577 0.0190472 (KEGG) 04010: MAPK signaling pathway
Hyp: P-values have been obtained through hypergeometric analysis; Hyp*: P-values have been obtained through hypergeometric analysis,
corrected by false discovery rate method; NGR: number of annotated genes in the reference list; NG: number of annotated genes in the
input list.
Niedoszytko et al. Gene expression profile predicting insect venom anaphylaxis in mastocytosis
ª 2010 John Wiley & Sons A/S
Table 4 List of genes that were differentially expressed (log2 fc > 3, P < 0.05 corrected for multiple testing by Benjamini–Hochberg method
P < 0.05) between patients with indolent systemic mastocytosis with a history of insect venom anaphylaxis and without anaphylaxis of any
kind
Gene symbol Gene name P-value
Corrected
P-value R* 1/2 R* 2/1
ABI3BP Abi gene family, member 3 (nesh) binding protein 0.0018 0.0094 0.26 3.81
ANKRD6 Ankyrin repeat domain 6 0.0099 0.0158 0.46 2.15
B3GAT1 Beta-1,3-glucuronyltransferase 1 (glucuronosyltransferase p) 0.0102 0.0158 0.42 2.41
C14ORF115 Chromosome 14 open reading frame 115 0.0025 0.0094 0.25 4.05
C14ORF118 Chromosome 14 open reading frame 118 0.0034 0.0104 3.08 0.33
C14ORF165 Chromosome 14 open reading frame 165 0.0108 0.0159 0.34 2.96
C18ORF56 Chromosome 18 open reading frame 56 0.0212 0.0269 0.42 2.41
C20ORF106 Chromosome 20 open reading frame 106 0.0035 0.0104 3.13 0.32
C20ORF132 Chromosome 20 open reading frame 132 0.0073 0.0138 2.36 0.42
CCDC134 Hypothetical protein flj22349 0.0095 0.0153 0.42 2.36
CDC42BPA cdc42-binding protein kinase alpha (dmpk-like) 0.0044 0.0112 0.39 2.56
CEP57 Centrosomal protein 57 kda 0.0147 0.0204 1.98 0.51
COL9A1 Collagen, type ix, alpha 1 0.0089 0.0148 0.23 4.29
CYORF15A Chromosome y open reading frame 15a 0.0162 0.0217 0.19 5.31
CYORF15B Chromosome y open reading frame 15b 0.0080 0.0141 0.22 4.59
CYORF15B 0.0417 0.0477 0.11 9.07
DHX9 Deah (asp-glu-ala-his) box polypeptide 9 0.0068 0.0138 2.73 0.37
DVL1 Dishevelled, dsh homolog 1 (drosophila) 0.0001 0.0020 0.22 4.46
EIF1AY Eukaryotic translation initiation factor 1a, y-linked 0.0294 0.0347 0.41 2.42
EWSR1 Ewing sarcoma breakpoint region 1 0.0258 0.0317 0.36 2.79
FAM110A Chromosome 20 open reading frame 55 0.0088 0.0148 2.12 0.47
FBLN1 Fibulin 1 0.0081 0.0141 0.55 1.81
FBXL21 F-box and leucine-rich repeat protein 21 0.0035 0.0104 0.24 4.19
FLJ39660 Hypothetical protein dkfzp434p055 0.0038 0.0104 0.27 3.64
G0S2 G0/g1switch 2 0.0002 0.0023 0.13 7.74
GLYAT Glycine-n-acyltransferase 0.0014 0.0089 0.40 2.49
HGD Homogentisate 1,2-dioxygenase (homogentisate oxidase) 0.0012 0.0089 0.40 2.52
HLA-DRB4 Major histocompatibility complex, class ii, dr beta 1 0.0184 0.0238 0.46 2.20
HOXA6 Homeobox a6 0.0168 0.0221 2.65 0.38
HRB Hiv-1 rev-binding protein 0.0037 0.0104 5.05 0.20
HS,107801 0.0090 0.0148 0.36 2.80
HS,124514 0.0060 0.0138 0.28 3.55
HS,134088 0.0071 0.0138 0.33 3.04
HS,189987 0.0107 0.0159 3.23 0.31
HS,41192 0.0000 0.0009 0.20 4.90
HS,436654 0.0024 0.0094 0.37 2.68
HS,527174 0.0022 0.0094 2.61 0.38
HS,537553 0.0007 0.0055 0.24 4.20
HS,540329 0.0001 0.0023 0.21 4.87
HS,540415 0.0072 0.0138 0.23 4.31
HS,541520 0.0003 0.0036 4.50 0.22
HS,544017 0.0060 0.0138 2.35 0.43
HS,545032 0.0084 0.0143 2.22 0.45
HS,545163 0.0061 0.0138 0.24 4.15
HS,545866 0.0020 0.0094 3.43 0.29
HS,546019 0.0353 0.0409 3.31 0.30
HS,546027 0.0001 0.0023 0.27 3.68
HS,549742 0.0106 0.0159 0.24 4.15
HS,552354 0.0015 0.0089 0.39 2.54
HS,552770 0.0000 0.0006 4.81 0.21
HS,562039 0.0287 0.0346 2.80 0.36
HS,562265 0.0004 0.0044 4.82 0.21
HS,563189 0.0007 0.0055 2.48 0.40
Gene expression profile predicting insect venom anaphylaxis in mastocytosis Niedoszytko et al.
ª 2010 John Wiley & Sons A/S
Table 4 (Continued)
Gene symbol Gene name P-value
Corrected
P-value R* 1/2 R* 2/1
HS,563982 0.0114 0.0166 0.20 4.90
HS,565704 0.0018 0.0094 0.43 2.30
HS,566231 0.0133 0.0187 2.51 0.40
HS,572999 0.0014 0.0089 0.46 2.17
HS,576804 0.0038 0.0104 3.77 0.27
HS,580555 0.0002 0.0026 0.25 4.03
HS,581365 0.0041 0.0110 0.46 2.18
HS,581671 0.0015 0.0089 3.51 0.28
HS,581933 0.0027 0.0094 0.31 3.23
HS,583304 0.0023 0.0094 0.36 2.76
HS,583989 0.0076 0.0139 0.38 2.66
HS3ST4 Heparan sulfate (glucosamine) 3-o-sulfotransferase 4 0.0131 0.0186 0.26 3.91
IIP45 Invasion inhibitory protein 45 0.0064 0.0138 0.34 2.96
INOC1 Ino80 complex homolog 1 (s, cerevisiae) 0.0073 0.0138 0.46 2.17
JARID1D Smcy homolog, y-linked (mouse) 0.0216 0.0271 0.11 8.92
JUP Junction plakoglobin 0.0106 0.0159 2.46 0.41
KLRC1 Killer cell lectin-like receptor subfamily c, member 1 0.0054 0.0129 0.44 2.30
LBH Hypothetical protein dkfzp566j091 0.0004 0.0042 2.94 0.34
LBP Lipopolysaccharide-binding protein 0.0077 0.0140 0.22 4.48
LIPJ Lipase-like, ab-hydrolase domain containing 1 0.0027 0.0094 2.90 0.34
LOC283487 Hypothetical protein loc283487 0.0002 0.0023 0.32 3.13
LOC387885 Hypothetical loc387885 0.0020 0.0094 0.35 2.87
LOC388588 Hypothetical gene supported by bc035379; bc042129 0.0007 0.0055 3.57 0.28
LOC391025 Similar to protein tyrosine phosphatase, receptor type,
u isoform 2 precursor
0.0035 0.0104 5.55 0.18
LOC641742 Hypothetical protein loc641742 0.0063 0.0138 0.29 3.48
LOC648897 Similar to atp-binding cassette sub-family d member
1 (adrenoleukodystrophy protein) (aldp)
0.0064 0.0138 3.67 0.27
LOC650227 Similar to mucin 6, gastric 0.0291 0.0347 3.79 0.26
LOC652418 Similar to hypothetical protein flj36492 0.0033 0.0104 2.36 0.42
LOC653308 Similar to n-acylsphingosine amidohydrolase 2 0.0101 0.0158 0.39 2.57
LOC731682 0.0253 0.0314 0.10 9.90
LRTM1 Leucine-rich repeats and transmembrane domains 1 0.0069 0.0138 2.97 0.34
LTK Leukocyte tyrosine kinase 0.0160 0.0217 2.54 0.39
MAP2K3 Mitogen-activated protein kinase kinase 3 0.0025 0.0094 0.22 4.54
MAP7 Microtubule-associated protein 7 0.0433 0.0492 2.46 0.41
MEGF8 Egf-like domain, multiple 4 0.0036 0.0104 4.98 0.20
N4BP2L1 Hypothetical gene cg018 0.0156 0.0214 0.32 3.16
PDGFA Platelet-derived growth factor alpha polypeptide 0.0024 0.0094 0.26 3.84
PKIB Protein kinase (camp-dependent, catalytic) inhibitor beta 0.0165 0.0218 0.33 3.03
PRKY Protein kinase, y-linked 0.0276 0.0336 0.11 8.87
RBM3 Rna-binding motif (rnp1, rrm) protein 3 0.0189 0.0243 0.41 2.47
RBMY1A3P RNA-binding motif protein, Y-linked, family 1, member A3 pseudogene 0.00001 0.0017 0.24 4.20
RGMB rgm domain family, member b 0.0304 0.0356 2.43 0.41
RHD rh blood group, ccee antigens 0.0051 0.0128 3.00 0.33
RP11-45B20,2 Similar to hypothetical protein mgc48915 0.0016 0.0092 0.27 3.71
SPAG17 Sperm associated antigen 17 0.0069 0.0138 0.18 5.45
SPN Sialophorin (gpl115, leukosialin, cd43) 0.0116 0.0167 0.50 1.99
SUDS3 Suppressor of defective silencing 3 homolog (s, cerevisiae) 0.0071 0.0138 0.34 2.96
TBPL2 Tata box-binding protein like 2 0.0052 0.0128 0.37 2.69
TMSB4Y Thymosin, beta 4, y-linked 0.0026 0.0094 0.19 5.30
TNFRSF4 Tumor necrosis factor receptor superfamily, member 4 0.0081 0.0141 4.05 0.25
TRAF4 Tnf receptor-associated factor 4 0.0043 0.0112 0.40 2.51
*Ratio of the expression levels for each individual gene when comparing patients with indolent systemic mastocytosis with a history of
insect venom anaphylaxis and without anaphylaxis of any kind.
Niedoszytko et al. Gene expression profile predicting insect venom anaphylaxis in mastocytosis
ª 2010 John Wiley & Sons A/S
patients with ISM and excluded of patients with solely cutane-
ous mastocytosis (i.e. mastocytosis in the skin without sys-
temic involvement). This is in contrast to the findings of
Brockow who analyzed patients with both cutaneous and sys-
temic mastocytosis. Our data might indicate that in patients
with mastocytosis, the total mast cell load does not contribute
to the particular risk for IVA or anaphylaxis of any kind. In
fact, we postulate that the absence of anaphylaxis might be
because of a more pronounced mast cell dysfunction in the
patients with nonanaphylactic mastocytosis . This assumption
is supported by the observation that in our group of Dutch
and Polish patients with a more advanced (i.e. smoldering,
aggressive, or leukemic) form of mastocytosis (n = 10 + 13),
none of the subjects is suffering from IVA (Kluin-Nelemans
H., de Monchy J.G.R., van Doormaal J.J., Oude Elberink
J.N.G. and Niedoszytko M. unpublished observation). The
differences in expression of the described genes were not
found in patients with IVA without mastocytosis as described
previously by our group (29). Therefore, these effects seem to
be specific for patients with mastocytosis.
Patients withmastocytosis without
anaphylaxisPatients with
mastocytosis and IVA
Figure 1 Hierarchical clustering dendro-
gram of differentially expressed genes
that were differentially expressed (log2
fc>3, P < 0.05 corrected for multiple
testing by Benjamini–Hochberg method
P < 0.05) between patients with indolent
systemic mastocytosis with a history of
insect venom anaphylaxis and without
anaphylaxis of any kind. Each column
represents a patient sample, each row an
individual gene. For each gene green color
represents underexpression, red color
overexpression, and black signal missing
data.
Table 5 Function of the most differently expressed genes with P < 0.01
Gene Known function of the gene
DVL 1 Wnt signaling pathway (19, 20)
Up-regulation of DVL1 was found in inflammatory bowel disease mucosa and also in colon cancer cells (19)
DVL1 is a key molecule in Wnt/planar cell polarity signaling pathway controlling tissue polarity and cell movement (19, 20)
and is up-regulated in various types of human cancers like melanoma, gastric and lung cancer, and neuroblastoma (19, 20)
PDGFA Up-regulation of both PDGFA and PDGFRA was found in patients with gastrointestinal stromal tumors forming
autocrine-paracrine loop (21), and in prostate cancer, where the over expression was found not only in cancer but also in
stromal cells (22)
MAPK signaling pathway (http://www.genome.jp/kegg/pathway.html)
G0S2 Expressed i.e. in lymphocytes and monocytes during the switch from G0 to G1 phase of the cell cycle (23, 24)
Up-regulation was found also in patients with vasculitis, psoriasis, rheumatoid arthritis and systemic lupus erythematosus (23)
MAP2K3 Product of this gene MAP kinase kinase 3 activates p38 MAP kinase that induces IL1a, IL1b IL12 production (25), B-cell
proliferation (26), and probably also enhances murine mast cell survival (27)
These genes were over expressed in patients with indolent systemic mastocytosis without a history of anaphylaxis of any kind.
Gene expression profile predicting insect venom anaphylaxis in mastocytosis Niedoszytko et al.
ª 2010 John Wiley & Sons A/S
The observation that the risk of anaphylaxis is related to
more differentiated phenotype of mast cells may also fit very
well to the recently developed concept of a mast cell activa-
tion syndrome (MMAS) (30–32). Patients with mast cell acti-
vation syndrome suffer from repeated and severe anaphylaxis
and show some criteria of systemic mastocytosis, but fail to
meet sufficient criteria to be classified as mastocytosis. It
might be possible that the mast cell phenotype in these
patients is yet more differentiated compared to patients with
mastocytosis, and it would be interesting to evaluate whether
the findings of our study might also be of relevance in this
new patient group (30–32).
Using a prediction model that uses naıve Bayes (NB) clas-
sifier based on the 104 most significantly and most differen-
tially expressed genes, it was possible to differentiate ISM
patients with IVA from those without anaphylaxis. The value
of single gene expression was regarded as an independent
predictor of IVA. This differentiation was not possible using
well-known measurements, such as serum tryptase, urinary
histamine metabolites, and KIT mutation analysis.
It is known that the prevalence of Hymenoptera sensitiza-
tion in the general population is higher than the prevalence
of IVA. Sensitization to insect venom as evidenced by skin-
prick tests and specific IgE was also found in one ISM
patient without a history of IVA. In some patients with IVA,
especially with co-existing mastocytosis, it is difficult to detect
the presence of specific IgE (6, 29, 33). Here, it has been
speculated that other mechanisms, besides specific IgE, might
be involved in insect venom-associated activation of mast
cells. As we have found no differences in other factors that
could be related to the level of specific IgE (i.e. time elapsed
since the last sting, number of stings, atopic status), the
observed differences in gene expression may be related to the
regulation of the production, secretion, binding to cells, and/
or clearance of IgE.
Our data indicate that it might be possible to construct a
simple method based on expression values of several genes in
peripheral blood cells that can be used in clinical practice to
assess the risk of IVA by a minimally invasive technique in a
similar way to predicting the response to chemotherapy in
oncology (34, 35). Our results need to be replicated in inde-
pendent populations, especially in a prospective way evaluat-
ing the natural history of anaphylaxis in patients with ISM.
So far, it is not recommended to perform prophylactic
VIT in mastocytosis patients, although this has been recently
suggested by Rueff et al. (7, 8) who reported a patient with
ISM and urticaria pigmentosa who died of an anaphylaxis
after a yellow jacket sting without a history of previous ana-
phylactic sting reactions. Our study indicates that it might be
possible to identify patients with mastocytosis at risk for
anaphylaxis and might enable to define criteria for the selec-
tion of mastocytosis patients eligible for ‘prophylactic’ VIT
as has been suggested elsewhere (7, 8).
The analysis of peripheral whole-blood cells was chosen
because sampling is less invasive for patients compared to
bone marrow aspiration. Furthermore, the standardized
method of RNA isolation and analysis we used reduces
potentially deleterious effects of sample handling on the
result and allows reliable clinical application. Because of this
methodology, the observed differences in expression were
detected in other cell lines of peripheral blood than mast
cells. The analysis of whole-blood cells might be justified
because recent data show that KIT mutation can be present
not only in mast cells but also in myeloid and lymphoid cell
lineages (36).
We can only speculate whether the higher expression of
genes related to the neoplastic transformation of cells is
clinically relevant. At the one side, it is known that patients
with ISM may develop haematological malignancies, but at
the other side this phenomenon has proven to be very rare
in our patient population that is presenting mainly with
urticaria pigmentosa, osteoporosis, and/or anaphylaxis. The
presenting symptom in all our patients with mastocytosis
who have a haematological malignancy was that malig-
nancy.
In conclusion, we demonstrate that in ISM (1), differen-
tially expressed genes in patients with IVA are annotated to
act in pathways favouring cellular differentiation over prolif-
eration, indicating that the differentiation state of the mast
cell might be a critical determinant of the sensitivity to ana-
phylaxis, and (2) gene expression profiling might be a useful
tool in identifying patients who are at risk for IVA by a
minimally invasive technique. Further studies in larger groups
of patients are required to validate our approach for the
development of a predictive tool to be used in clinical prac-
tice.
Acknowledgment
The research was supported by the Foundation for Polish
Science and a grant of the Polish Ministry of Science and
Higher Education N40201031/0386 and N402085934.
References
1. Valent P, Sperr W, Schwartz L, Horny H.
Diagnosis and classification of mast cell
proliferative disorders: delineation from
immunologic diseases and non-mast cell
hematopoietic neoplasms. J Allergy Clin
Immunol 2004;114:3–11.
2. Akin C, Metcalfe D. The biology of Kit in
disease and the application of pharmaco-
genetics. J Allergy Clin Immunol 2004;114:
13–19.
3. Brockow K, Jofer C, Behrendt H, Ring J.
Anaphylaxis in patients with mastocytosis: a
study on history, clinical features and risk
factors in 120 patients. Allergy 2008;63:
226–232.
4. Oude Elberink JN, de Monchy JG, Kors
JW, van Doormaal JJ, Dubois AE. Fatal
anaphylaxis after a yellow jacket sting,
despite venom immunotherapy, in two
patients with mastocytosis. J Allergy Clin
Immunol 1997;99:153–154.
5. Reimers A, Muller U. Fatal outcome of a
Vespula sting in a patient with mastocytosis
after specific immunotherapy with honey bee
venom. Allergy Clin Immunol Int J WAO
Org 2005;17:68–70.
6. Niedoszytko M, De Monchy J, Van
Doormaal JJ, Jassem E, Oude Elberink
JNG. Mastocytosis and insect venom
Niedoszytko et al. Gene expression profile predicting insect venom anaphylaxis in mastocytosis
ª 2010 John Wiley & Sons A/S
allergy: diagnosis, safety and efficacy of
venom immunotherapy. Allergy 2009;64:
1237–1245.
7. Wagner N, Fritze D, Przybilla B, Hagedorn
M, Rueff F. Fatal anaphylactic sting
reaction in a patient with mastocytosis. Int
Arch Allergy Immunol 2008;146:162–163.
8. Rueff F, Placzek M, Przybilla B. Masto-
cytosis and Hymenoptera venom allergy.
Curr Opin Allergy Clin Immunol 2006;6:
284–288.
9. Simons FE, Frew AJ, Ansotegui IJ, Bochner
BS, Golden DB, Finkelman FD et al. Risk
assessment in anaphylaxis: current and
future approaches. J Allergy Clin Immunol
2007;120:S2–S24.
10. Jutel M, Akdis M, Blaser K, Akdis CA.
Mechanisms of allergen specific immuno-
therapy-T-cell tolerance and more. Allergy
2006;61:796–807.
11. Konno S, Hizawa N, Nishimura M, Huang
SK. Osteopontin: a potential biomarker for
successful bee venom immunotherapy and a
potential molecule for inhibiting IgE-medi-
ated allergic responses. Allergol Int 2006;
55:355–359.
12. Mueller HL. Diagnosis and treatment of
insect sensitivity. J Asthma Res 1966;3:
331–333.
13. Kazmierska J, Malicki J. Application of
the Naıve Bayesian Classifier to optimize
treatment decisions. Radiother Oncol 2008;
86:211–216.
14. Kanehisa M, Araki M, Goto S, Hattori M,
Hirakawa M, Itoh M et al. KEGG for
linking genomes to life and the environment.
Nucleic Acids Res 2008;36:480–484.
15. Nogales-Cadenas R, Carmona-Saez P,
Vazquez M, Vicente C, Yang X, Tirado F
et al. GeneCodis: interpreting gene lists
through enrichment analysis and integration
of diverse biological information. Nucleic
Acids Res 2009;37:317–322.
16. Liu SM, Xavier R, Good KL, Chtanova T,
Newton R, Sisavanh M et al. Immune cell
transcriptome datasets reveal novel leuko-
cyte subset-specific genes and genes associ-
ated with allergic processes. J Allergy Clin
Immunol 2006;118:496–503.
17. Cheung J, Petek E, Nakabayashi K, Tsui
LC, Vincent JB, Scherer SW. Identification
of the human cortactin-binding protein-2
gene from the autism candidate region at
7q31. Genomics 2001;78:7–11.
18. Patel N, Brinkman-Van der Linden ECM,
Altmann SW, Gish K, Balasubramanian S,
Timans JC et al. OB-BP1/Siglec-6: a leptin-
and sialic acid-binding protein of the
immunoglobulin superfamily. J Biol Chem
1999;274:22729–22738.
19. You XJ, Bryant PJ, Jurnak F, Holcombe
RF. Expression of Wnt pathway compo-
nents frizzled and disheveled in colon cancer
arising in patients with inflammatory bowel
disease. Oncol Rep 2007;18:691–694.
20. Liu X, Mazanek P, Dam V, Wang Q, Zhao
H, Guo R et al. Deregulated Wnt/beta-
catenin program in high-risk neuroblastomas
without MYCN amplification. Oncogene
2008;27:1478–1488.
21. Negri T, Bozzi F, Conca E, Brich S,
Gronchi A, Bertulli R et al. Oncogenic and
ligand-dependent activation of KIT/PDG-
FRA in surgical samples of imatinib-treated
gastrointestinal stromal tumours (GISTs).
J Pathol 2009;217:103–112.
22. van der Heul-Nieuwenhuijsen L, Dits N,
Van Ijcken W, de Lange D, Jenster G. The
FOXF2 pathway in the human prostate
stroma. Prostate 2009;69:1538–1547.
23. Kobayashi S, Ito A, Okuzaki D, Onda H,
Yabuta N, Nagamori I et al. Expression
profiling of PBMC-based diagnostic gene
markers isolated from vasculitis patients.
DNA Res 2008;15:253–265.
24. Kitareewan S, Blumen S, Sekula D, Bisson-
nette RP, Lamph WW, Cui Q et al. G0S2 is
an all-trans-retinoic acid target gene. Int J
Oncol 2008;33:397–404.
25. Dong C, Davis RJ, Flavell RA. MAP
kinases in the immune response. Annu Rev
Immunol 2002;20:55–72.
26. Ringshausen I, Dechow T, Schneller F,
Weick K, Oelsner M, Peschel C et al. Con-
stitutive activation of the MAPkinase p38 is
critical for MMP-9 production and survival
of B-CLL cells on bone marrow stromal
cells. Leukemia 2004;18:1964–1970.
27. Sly LM, Kalesnikoff J, Lam V, Wong D,
Song C, Omeis S et al. IgE-induced mast cell
survival requires the prolonged generation of
reactive oxygen species. J Immunol 2008;181:
3850–3860.
28. Rueff F, Przybilla B, Bilo MB, Muller U,
Scheipl F, Aberer W et al. Predictors of
severe systemic anaphylactic reactions in
patients with Hymenopteravenom allergy:
importance of baseline serum tryptase – a
study of the European Academy of Aller-
gology and Clinical Immunology Interest
Group on Insect Venom Hypersensitivity.
J Allergy Clin Immunol 2009;124:1047–
1054.
29. Niedoszytko M, Bruinenberg M, de Monchy
J, Wijmenga C, Platteel M, Jassem E et al.
Gene expression analysis in predicting the
effectiveness of insect venom immunother-
apy. J Allergy Clin Immunol 2010;125:
1092–1097.
30. Akin C, Scott LM, Kocabas CN, Kushnir-
Sukhov N, Brittain E, Noel P et al. Demon-
stration of an aberrant mast-cell population
with clonal markers in a subset of patients
with ‘‘idiopathic’’ anaphylaxis. Blood 2007;
110:2331–2333.
31. Alvarez-Twose I, Gonzalez de Olano D,
Sanchez-Munoz L, Matito A, Esteban-
Lopez MI, Vega A et al. Clinical, biological,
and molecular characteristics of clonal mast
cell disorders presenting with systemic mast
cell activation symptoms. J Allergy Clin
Immunol 2010;125:1269–1278.
32. Bonadonna P, Perbellini O, Passalacqua G,
Caruso B, Colarossi S, Dal Fior D et al.
Clonal mast cell disorders in patients with
systemic reactions to Hymenoptera stings
and increased serum tryptase levels.
J Allergy Clin Immunol 2009;123:
680–686.
33. Golden DB, Marsh DG, Freidhoff LR,
Kwiterovich KA, Addison B, Kagey-
Sobotka A et al. Natural history of
Hymenoptera venom sensitivity in adults.
J Allergy Clin Immunol 1997;100:760–766.
34. Crijns AP, Fehrmann RS, de Jong S,
Gerbens F, Meersma GJ, Klip HG et al.
Survival-related profile, pathways, and tran-
scription factors in ovarian cancer. PLoS
Med 2009;6:e24.
35. van ‘t Veer LJ, Dai H, van de Vijver MJ,
He YD, Hart AA, Mao M et al. Gene
expression profiling predicts clinical outcome
of breast cancer. Nature 2002;415:530–
536.
36. Garcia-Montero AC, Jara-Acevedo M,
Teodosio C, Sanchez ML, Nunez R, Prados
A et al. KIT mutation in mast cells and
other bone marrow hematopoietic cell lin-
eages in systemic mast cell disorders: a pro-
spective study of the Spanish Network on
Mastocytosis (REMA) in a series of 113
patients. Blood 2006;108:2366–2372.
Gene expression profile predicting insect venom anaphylaxis in mastocytosis Niedoszytko et al.
ª 2010 John Wiley & Sons A/S
ORIGINAL ARTICLE EPIDEMIOLOGY AND GENETICS
Gene expression profile, pathways, and transcriptionalsystem regulation in indolent systemic mastocytosisM. Niedoszytko1,2, J. N. G. Oude Elberink2, M. Bruinenberg3,4, B. Nedoszytko5, J. G. R. de Monchy2,G. J. te Meerman3, R. K. Weersma6, A. B. Mulder7, E. Jassem1 & J. J. van Doormaal2
1Department of Allergology Medical University of Gdansk, Gdansk, Poland; 2Department of Allergology, University Medical Center Gronin-
gen, University of Groningen; 3Department of Genetics University Medical Center Groningen, University of Groningen; 4Lifelines, University
Medical Center Groningen, University of Groningen, Groningen, the Netherlands; 5Department of Dermatology Medical University of Gdansk,
Gdansk, Poland; 6Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen;7Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
To cite this article: Niedoszytko M, Oude Elberink JNG, Bruinenberg M, Nedoszytko B, de Monchy JGR, te Meerman GJ, Weersma RK, Mulder AB, Jassem E,
van Doormaal JJ. Gene expression profile, pathways, and transcriptional system regulation in indolent systemic mastocytosis. Allergy 2011; 66: 229–237.
Mastocytosis is an uncommon disease resulting from prolifer-
ation of abnormal mast cells in different tissues including skin,
bone marrow, liver, spleen, and lymph nodes (1). One of the
key elements in the pathogenesis of the disease is the presence
of a specific KIT mutation in mastocytes but also in other
peripheral blood cells (1, 2). The clinical presentation of mast-
ocytosis is heterogenous, varying from solely skin presentation
found in urticaria pigmentosa and mastocytoma, to different
forms of systemic disease including indolent systemic masto-
cytosis, smouldering systemic mastocytosis, aggressive sys-
temic mastocytosis, and mast cell leukaemia (1). Of the adult
patients with systemic mastocytosis, the large majority
(ca. 90%) have the indolent form of the disease. Most symp-
toms (like anaphylaxis, hypotension, urticaria, and diarrhoea)
are related to mast cell infiltration and degranulation (1).
However, other symptoms such as osteoporosis, hypertension,
Keywords
gene expression; gene profiling;
mastocytosis.
Correspondence
Marek Niedoszytko, MD, PhD,
Department of Allergology Medical
University of Gdansk, Debinki 7 80-952
Gdansk, Poland.
Tel.: +48583491626
Fax: +48583491625
E-mail: [email protected]
Accepted for publication 26 July 2010
DOI:10.1111/j.1398-9995.2010.02477.x
Edited by: Stephan Weidinger
Abstract
Background: Mastocytosis is an uncommon disease resulting from proliferation of
abnormal mast cells infiltrating skin, bone marrow, liver, and other tissues. The aim
of this study was to find differences in gene expression in peripheral blood cells of
patients with indolent systemic mastocytosis compared to healthy controls. The
second aim was to define a specific gene expression profile in patients with
mastocytosis.
Methods: Twenty-two patients with indolent systemic mastocytosis and 43 healthy
controls were studied. Whole genome gene expression analysis was performed on
RNA samples isolated from the peripheral blood. For amplification and labelling of
the RNA, the Illumina TotalPrep 96 RNA Amplification Kit was used. Human
HT-12_V3_expression arrays were processed. Data analysis was performed using
GeneSpring, Genecodis, and Transcriptional System Regulators.
Results: Comparison of gene expression between patients and controls revealed a
significant difference (P < 0.05 corrected for multiple testing) and the fold change
difference >2 in gene expression in 2303 of the 48.794 analysed transcripts. Func-
tional annotation indicated that the main pathways in which the differently
expressed genes were involved are ubiquitin-mediated proteolysis, MAPK signalling
pathway, pathways in cancer, and Jak-STAT signalling. The expression distributions
for both groups did not overlap at all, indicating that many genes are highly differ-
entially expressed in both groups.
Conclusion: We were able to find abnormalities in gene expression in peripheral
blood cells of patients with indolent systemic mastocytosis and to construct a gene
expression profile which may be useful in clinical practice to predict the presence of
mastocytosis and in further research of novel drugs.
Abbreviations
TSR, transcriptional system regulators; FA, factor analysis; NB,
Naıve Bayes prediction; FC, fold change.
Allergy
Allergy 66 (2011) 229–237 ª 2010 John Wiley & Sons A/S 229
pain syndromes, and neurological symptoms are only partially
understood and may involve other mechanisms (1).
The mechanism(s) involved in the development of masto-
cytosis are mainly unknown (1). Activation of kinase path-
ways (i.e. D816V mutation of KIT), and IL-13 and rIL-4
polymorphisms have previously been shown to be relevant in
this respect (3, 4). It seems quite likely that more than one
pathway maybe located in other cells than mast cells alone
are involved in the development of the disease. The studies
by Garcia-Montero (2) showed that the KIT mutation is
present not only in mast cells but also in myeloid and lym-
phoid cell lineages. Furthermore, the presence of the KIT
mutation in various cell lineages was related to the prognosis
of mastocytosis (2).
The aim of this study was to find differences in gene
expression in peripheral blood cells of patients with indolent
systemic mastocytosis compared to healthy controls. The sec-
ond aim was to define a specific gene expression profile in
patients with mastocytosis.
Methods
Patients
A total of 22 Caucasian patients with indolent systemic mast-
ocytosis from the Department of Allergology, University
Medical Center Groningen (UMCG) were studied [median
age 50 range 35–73 years; 7 (31%) men and 15 (68%)
women]. All patients underwent standard diagnostic proce-
dures based on WHO guidelines for the workup of systemic
mastocytosis including bone marrow histopathological, cyto-
logical, and flow cytometric (CD2, CD25) examinations of
bone marrow. To detect the KIT D816V mutation, we used
two different techniques in time. Initially, RNA was isolated
from EDTA anti-coagulated bone marrow cells with the help
of the QIAamp�RNA Blood MINI Kit (Qiagen, Westburg,
Leusden, the Netherlands). The Promega Reverse Transcrip-
tase kit (Promega Benelux, Leiden, the Netherlands) was
used to synthesize c-DNA from approximately 500 ng RNA.
The resulting c-DNA was amplified using previously
described primers with the following PCR conditions: 30
cycles of denaturation (1 min at 95�C), annealing (1 min at
61�C), and extension (2 min at 72�C), followed by 7 min at
72�C and subsequent cooling (5). The resulting 346 -bp PCR
product was digested with the help of Hae III and Hinf I
(BioLabs, Westburg, Leusden, the Netherlands), resulting in
restriction fragments of 171, 127, and 48 (not detected) base
pairs to detect the wild type and 157, 127, 48 (not detected),
and 14 (not detected) base pairs to detect the Asp 816Val
mutation. The restriction fragments were separated on a 6%
agarose Multi purpose (Roche, Almere, the Netherlands) gel
and visualized using ethidium bromide (patient no. 1, 3, 5, 7,
9, 14, 15, 16, 17, 20, and 22). From December 2007, detec-
tion of the KIT D816V mutation was performed with a
real-time qPCR using previously published (6) primers
5¢-TTGTGATTTTGGTCTAGCCAGACT-3¢ and 5¢-GTGC-
CATCCACTTCACAGGTAG-3¢ (patient no. 2, 4, 8, 11, 12,
18, and 19). Urinary histamine metabolites and serum
tryptase measurements were also taken (Table 1) (1). A group
of 43 healthy Caucasian subjects [median age 50 range 19–
73 years, 22 (51%) men and 21 (49%) women] were used as
controls. They were nonrelated partners from patients with
inflammatory bowel disease visiting the outpatient depart-
ment of the inflammatory bowel disease unit of the UMCG.
The study was approved by the Medical Ethical Commit-
tee of the UMCG (METc 2008/340).
Collection of blood samples
PAXgene blood RNA tubes (Qiagen, Valencia, CA, USA)
were used for RNA sampling. All tubes were immediately
frozen and stored in )20�C till RNA isolation (maximal per-
iod 2 months). RNA was isolated using PAXgene blood
RNA Kit CE (Qiagen, Venlo, the Netherlands). All RNA
samples were stored in )80�C till labelling and hybridization.
The quality and concentration of RNA were determined
using 2100 Bioanalyzer (Agilent, Amstelveen, the Nether-
lands) and the Agilent RNA 6000 Nano Kit. Samples with
RNA integrity number >7.5 were used for further analysis
on expression arrays.
Gene expression
For amplification and labelling of the RNA with the Illumina
TotalPrep 96 RNA Amplification Kit (Applied Biosystems,
Nieuwerkerk ad IJssel, the Netherlands), 200 ng of RNA
from each sample was used. The human HT-12_V3_expres-
sion arrays (Illumina, San Diego, CA, USA) were processed
according to the manufacturer’s protocol. Slides were scanned
immediately using Illumina BeadStation iScan (Illumina).
Image and data analysis
First line check, background correction and quantile normali-
zation of the data were carried out with Genomestudio
Gene Expression Analysis module v 1.0.6 Statistics (San
Diego, CA, USA). Entities of which at least 75% of the sam-
ples had a signal intensity value above 20th percentile in
100% of the samples of at least two groups were included for
further analysis.
Data analysis was performed using GeneSpring package
version 8.0.0 (Agilent Technologies Santa Clara. CA, USA).
Genes of which expression was significantly different between
the compared groups were chosen based on a log2fold change
>2 in gene expression, t-test P-value <0.05 and corrected
for multiple testing by the Benjamin–Hochberg method. The
naıve Bayes prediction model was used to build a prediction
model which might be used in diagnosis of mastocytosis (7).
Naıve Bayesian classifier assumes that the impact of single
gene expression is unrelated to other genes in the prediction
model. The method does not take into account the interac-
tions of the genes composing the model or gene environmen-
tal interactions.
Functional annotation of genes was described using Gene-
codis (8), functional annotation web-based tool using KEGG
pathways (9) and GoSlim process analysis.
Gene expression in mastocytosis Niedoszytko et al.
230 Allergy 66 (2011) 229–237 ª 2010 John Wiley & Sons A/S
Tab
le1
Dem
ogra
phic
and
clin
icaldata
of
the
indiv
idualpatients
with
indole
nt
syste
mic
masto
cyto
sis
Patient
no.
Gender
Age
at
dia
gnosis
(years
)U
P
Seru
m
trypta
se
(lg/l)
Urine
MH
(lm
ol/m
ol
cre
at)
Urine
MIM
A
(mm
ol/m
ol
cre
at)
MC
s
inbone
marr
ow
aspirate
(%)
CD
2im
muno-
phenoty
pe
CD
25
imm
uno-
phenoty
pe
D816V
KIT
muta
tion
inbone
marr
ow
cells
‡2aggre
gate
s
of
‡15
MC
sin
bone
marr
ow
Abnorm
al
morp
holo
gy
of
‡25%
of
MC
sin
bone
marr
ow
His
tolo
gic
al
bone
marr
ow
cellu
larity
1M
50
)28.9
153
1.4
0.0
9)
+)
)+
Norm
al
2F
64
Adult
UP
155
1046
10.9
0.4
9+
++
++
Norm
al
3F
43
Adult
UP
34.1
380
4.8
0.2
9)
++
++
Norm
al
4F
64
Adult
UP
21.7
166
3.1
0.1
1+
++
)+
Norm
al
5F
47
)48.3
604
6.5
0.1
7+
++
++
Norm
al
6F
25
Adult
UP
27.4
1024
3.4
n.d
.n.d
.n.d
.n.d
.+
+N
orm
al
7F
50
Adult
UP
20.4
293
3.0
0.0
6+
++
++
Norm
al
8M
34
Adult
UP
55.4
433
4.9
0.4
5+
++
++
Norm
al
9F
61
)15.2
266
2.3
0.3
0)
+)
++
Norm
al
10
M41
Juvenile
UP
146
470
4.0
1.6
1)
++
++
norm
al
11
F64
Adult
UP
4.6
2102
1.6
0.1
0+
++
)+
Norm
al
12
M54
)28.2
77
1.4
0.0
9+
++
)+
Norm
al
13
M50
Adult
UP
52.3
530
6.4
n.d
.n.d
.n.d
.n.d
.+
+N
orm
al
14
F43
Adult
UP
36.5
851
5.9
0.3
4+
++
++
Norm
al
15
F51
)74.5
194
2.7
0.1
0+
++
++
Norm
al
16
F38
Adult
UP
5.1
3190
1.7
0.1
3+
+)
++
Norm
al
17
M38
)44.4
310
3.4
n.d
.n.d
.n.d
.+
++
Norm
al
18
M37
Adult
UP
112
404
7.7
n.d
.n.d
.n.d
.+
++
Norm
al
19
F36
)31.3
542
6.5
0.1
7+
++
++
Norm
al
20
F70
)19.7
289
2.7
0.1
0+
++
)+
Slig
htly
hyperp
lastic
21
F63
Adult
UP
109
823
6.3
n.d
.n.d
.n.d
.n.d
.+
+N
orm
al
22
F54
Adult
UP
29.8
593
4.1
n.d
.n.d
.n.d
.+
++
Norm
al
M,
male
;F,
fem
ale
;n.d
.,not
done;
UP
,urt
icaria
pig
mento
sa;
juvenile
UP
:ju
venile
-onset
UP
;adult
UP
,adult-o
nset
UP
;M
H,
meth
ylh
ista
min
e;
MIM
A,
meth
ylim
idazo
leacetic
acid
,M
Cs,
mast
cells
;)
,negative;
+,
positiv
e.
Niedoszytko et al. Gene expression in mastocytosis
Allergy 66 (2011) 229–237 ª 2010 John Wiley & Sons A/S 231
Differences in gene expression between patients with mast-
ocytosis and healthy controls were also analysed using tran-
scriptional system regulators (TSR) and factor analysis (FA)
described by Fehrmann et al. (10). This method uses princi-
pal components derived from the correlation between
expressed genes in 15.000 Affymetrix expression arrays.
The gene specific weights were applied to normalized log
transformed Illumina transcript data, using the average for
every gene, if more than one transcript was available. This
procedure was performed for the first 50 principal compo-
nents identified by Fehrmann et al. (10), resulting in a new
set of 50 data points for each person. Subsequently, FA was
performed on the component scores, further reducing the set
of data points per person to eight explaining 75% of the vari-
ance of the 50 original principal component scores. The
correlation between the factor scores is caused by the much
lower heterogeneity of the data in comparison with the
15.000 arrays used by Fehrmann et al. The aim of this
method is to use the correlation structure between genes to
find scores that have a higher reproducibility because the sig-
nal of many genes is added. The biological interpretation of
the factors is derived from those genes that have the stron-
gest contribution to the compound score. Gene related speci-
ficity is lost, but problems with overfitting and low reliability
of individual gene signals are strongly reduced. Factor analy-
sis was performed with Systat 12.0 (San Jose, CA, USA)
and component scores were computed with a computer
program written in Delphi 5.0 (Austin, TX, USA) available
on demand from GTM.
Power calculation to find differences in expression is diffi-
cult to compute a priori, as we have no knowledge of the
impact of systemic mastocytosis on expression. Considering
that the phenotype has a substantial impact on health, we
assumed that even with a small number of individuals com-
pared, significant differences between cases and controls
would be present even after correction for multiple testing.
The analysis based on metagenes is more sensitive than single
gene analysis as signals from many genes are combined and
errors cancelled out.
Clinical data were analysed with Statistica 8.0 (StatSoft,
Tulsa, OK, USA).
Results
Whole genome gene expression analysis was performed on
RNA samples isolated from all blood cells in whole blood.
From all 48.804 probes present in the array, 48.794 tran-
scripts had sufficient data for further analysis.
Comparison of gene expression profiles between patients
and controls revealed a significant difference in 5086 of the
analysed transcripts. A fold change difference >2 in gene
expression was found in 2330 of those transcripts among
which 1951 (84%) were upregulated and 379 (16%) down-
regulated. Functional annotation indicated that the main
pathways in which the differently expressed genes were
involved are ubiquitin-mediated proteolysis, MAPK signal-
ling pathway, pathways in cancer, Jak-STAT signalling, and
p53 signalling pathway (Table 2). The most important pro-
cesses influenced by mastocytosis are transcription, cell cycle,
protein transport, and signal transduction (Table 3).
We matched 13.032 transcripts with Affymetrix genes,
using official gene symbol agreement, and used in the factor
score analysis. Split-half correlations were computed for each
of the 50 factor scores as an indication of independence of
factor scores of individual genes.
Among the 50 TSRs described by Fehrmann et al. (10),
the TSRs 1, 2, 4, 5, 6, 7, 8, 10, 12, 13, 38, 46, 49, and 50 were
most different between patients and controls. In a second
FA, two uncorrelated (orthogonal) factors (nr 2 and 4) were
identified that both differentiated between cases and con-
trols). The factors 2 and 4 provided the best discriminative
properties of predicting the presence of mastocytosis. The
main function indicated by the TSRs and KEGG pathway
(9) analysis are MAPK signalling pathway, focal and cell
adhesion, calcium signalling pathway, neuroactive ligand–
receptor interaction, ribosome, cytokine–cytokine receptor
interactions, regulation of actin cytoskeleton, and oxidative
phosphorylation.
Using leucocyte-specific transcripts described by Liu et al.
(11), we analysed the expression profiles of the leucocyte-
specific genes characteristic for dendritic cells, B cells, effector
memory T cells, mast cells, and basophils. Statistically signifi-
cant differences in expression between patients and controls
Table 2 Gene co-occurence annotation found by Genecodis (KEGG pathways) for the genes differentially expressed (FC > 2, P < 0.05
corrected for multiple testing) between patients with indolent systemic mastocytosis and healthy controls. P-values have been obtained
through hypergeometric analysis (Hyp) corrected by FDR method (Hyp*) NGR – number of annotated genes in the reference list, NG – num-
ber of annotated genes in the input list (14, 15)
Genes NGR NG Hyp Hyp* Annotations
27 genes 129 (37435) 27 (1769) 5.93719e)11 1.82272e)08 (KEGG) 04120 :Ubiquitin-mediated proteolysis
35 genes 262 (37435) 35 (1769) 3.3285e)08 5.10924e)06 (KEGG) 04010 :MAPK signalling pathway
37 genes 320 (37435) 37 (1769) 5.6426e)07 5.77426e)05 (KEGG) 05200 :Pathways in cancer
22 genes 150 (37435) 22 (1769) 2.38024e)06 0.000182683 (KEGG) 04630 :Jak-STAT signalling pathway
14 genes 67 (37435) 14 (1769) 2.43652e)06 0.000149602 (KEGG) 04115 :p53 signalling pathway
18 genes 108 (37435) 18 (1769) 3.05782e)06 0.000156458 (KEGG) 04110 :Cell cycle
15 genes 87 (37435) 15 (1769) 1.3072e)05 0.000573302 (KEGG) 04210 :Apoptosis
20 genes 145 (37435) 20 (1769) 1.70886e)05 0.000655773 (KEGG) 00230 :Purine metabolism
20 genes 150 (37435) 20 (1769) 2.8221e)05 0.000962649 (KEGG) 04310 :Wnt signalling pathway
Gene expression in mastocytosis Niedoszytko et al.
232 Allergy 66 (2011) 229–237 ª 2010 John Wiley & Sons A/S
were found for the following genes expressed in mast cells:
ATP6VOA1, LOC348262, RFESD, OSBPL6, T cells: GALK2,
IL32, KLF12, IL12A, SOS1 dendritic cells: C2orf64, CD1B,
ZFP3 and B cells: MEF2C, and MS4A1. The genes identified
by D’Ambrosio et al. (12) studying gene expression analysis of
bone marrow mononuclear cells found similar changes in
expression in four of 10 described genes (CPA3, GATA2, KIT,
and MAF).
We subsequently went on to build the prediction model
which could be used in diagnosing indolent systemic mastocy-
tosis based on the gene expression in the peripheral blood
cells. We built the prediction model using a Naıve Bayes clas-
sifier based on the most discriminative 29 genes with
P < 10)10 corrected for multiple testing (Table 4). The sensi-
tivity of this predictive model was 100% with a specificity of
97%, a negative predictive value of 100%, and a positive pre-
dictive value of 96%. Clustering analysis divided those genes
into two clusters based on the similarities in gene expression
pattern (Fig. 1).
Discussion
The results of the present study show strong and very signifi-
cant differences in gene expression in peripheral blood cells
between patients with indolent systemic mastocytosis and
healthy controls. Additionally, 29 gene expression profile
differentiating patients from controls was created based on
the most differently expressed transcripts.
We were able to show that gene expression differences are
found in other cells than mast cells solely. It confirms the
finding that expression effects of the specific KIT mutation of
mastocytosis may be found in other cell lineages than mast
cells. In addition, it shows a difference in expression of 2330
other transcripts (3).
We also used TSR profiling in the data analysis. The
regulation of transcription is a complex mechanism;
however, the overlap between diseases and tissues analysed
led to the conclusion that a large part of differences in
transcription may be explained by a network of co-regulated
gene clusters. The studies by Fehrmann et al. (10) showed
that the number of orthogonal factors needed to explain
most of the variability in expression may be limited to 50
TSRs. Studies made on 17.550 human microarray experi-
ments led to identify 50 TSRs capturing 64% of the vari-
ability in gene expression (10). The TSR analysis in our
study showed profound transcriptosome abnormalities in
patients with mastocytosis. The results of the gene expres-
sion analysis made in GeneSpring and by the TSR method
indicate similar processes and pathways involved in masto-
cytosis. The biological complexities of these systems suggest
networks of co-regulated genes. The results of the TSR and
FA analysis suggest, that the abnormalities in gene expres-
sion in indolent systemic mastocytosis are related to biolo-
gical processes also found in other diseases. This approach,
which analyses transcriptional mechanisms common across
tissues and diseases, allows analysing of gene expression in
whole blood without cell sorting and reduces the probabi-
lity of finding gene expression patterns related to the exper-
imental conditions and sample studied. The functional
analysis reveals processes responsible for neoplastic cell
transformation (pathways in cancer, MAPK, Jak-STAT sig-
nalling, p53 signalling pathway, cell cycle, and apoptosis).
The finding of abnormally expressed genes and pathways
may also lead to the application of novel drugs in systemic
mastocytosis.
In the next step, we went on to create a gene expression
profile which could be of help in diagnosing patients suffering
from indolent systemic mastocytosis. We suggested a set of
29 most differently expressed genes divided in two clusters
according to the pattern of expression (Fig. 1).
The genes composing cluster 1 were described previously
in the pathogenesis of both solid tumours and haematological
malignancies. We observed both upregulation of proto-
oncogenes and downregulation of tumour suppressor genes.
Three of the genes were described in lung cancer, namely
ZMAT3 (p53 target zinc finger protein) (13), arp2 actin-
related protein 2 (ACTR2) (14), and cholinergic receptor,
nicotinic alpha 5 (CHRNA5) (15), and two others in breast
cancer, namely rho gtpase-activating protein 8 (PRR5) (16)
and plectin 1 (PLEC1) (17). Plectin 1 was also described in
ovarian cancer (17) and PRR5 (rho gtpase-activating protein
8) (16) in colorectal cancer. Four other genes were also
described in acute myeloid leukaemia, namely integrin beta 1
(ITGB1) (18), ataxia telangiectasia mutated (ATM) (19), and
Table 3 Gene co-occurence annotation found by Genecodis (14, 15) (GOSlim Process Function) for the genes differentially expressed
(FC > 2, P < 0.05 corrected for multiple testing) between patients with indolent systemic mastocytosis and healthy controls. P-values have
been obtained through hypergeometric analysis (Hyp) corrected by FDR method (Hyp*) NGR – number of annotated genes in the reference
list, NG – number of annotated genes in the input list
Genes NGR NG Hyp Hyp* Annotations
190 genes 1516 (37 435) 190 (1769) 3.22857e)35 1.51743e)33 GO:0006350 :transcription (BP)
61 genes 376 (37 435) 61 (1769) 3.51927e)17 8.27027e)16 GO:0007049 :cell cycle (BP)
53 genes 376 (37 435) 53 (1769) 1.34649e)12 2.10951e)11 GO:0015031 :protein transport (BP)
134 genes 1700 (37 435) 134 (1769) 4.64627e)09 5.45937e)08 GO:0007165 :signal transduction (BP)
51 genes 471 (37 435) 51 (1769) 3.90706e)08 3.67264e)07 GO:0008152 :metabolic process (BP)
18 genes 99 (37 435) 18 (1769) 8.26542e)07 6.47458e)06 GO:0006950 :response to stress (BP)
47 genes 505 (37 435) 47 (1769) 9.00404e)06 6.04557e)05 GO:0006810 :transport (BP)
21 genes 177 (37 435) 21 (1769) 0.00010213 0.000600016 GO:0006464 :protein modification process (BP)
Niedoszytko et al. Gene expression in mastocytosis
Allergy 66 (2011) 229–237 ª 2010 John Wiley & Sons A/S 233
v-ets erythroblastosis virus e26 oncogene homologue 1
(ETS1) (20) and seven in absentia homologue (SIAH1) (21).
Leucocyte-derived arginine aminopeptidase (LRAP) plays a
role in the development of lymphoma (22). Leucine-rich
repeat interacting protein (LRRFIP1) contributes to the
pathology of myelodysplastic syndrome (23) and glioblas-
toma (24). Multiple tumours including lymphomas and solid
tumours are related to overexpression of SERTAD2 (serta
domain containing) (25). RAB27A gene product is a protein
member of the ras oncogene family involved in neutrophil
secretion (26) and melanocyte shape (27).
All genes in cluster 2 were upregulated in mastocytosis.
Myeloid/lymphoid or mixed-lineage leukaemia (3MLL3) (28),
nuclear receptor coactivator 2 (NCOA2), and eosinophilic
leukaemia CCR2 [chemokine (c-c motif) receptor 2] (29) also
play a role in the pathology of myeloid leukaemia. Integrin
alpha v (ITGAV) involvement was described in laryngeal and
hypopharyngeal carcinomas (30). For the three genes lamin B
receptor (LBR), SGPP1, and MATR3 involvement in cancer
was not described, but their function may contribute to carci-
nogenesis. Lamin B receptor plays a role in the morpho-
logical maturation of neutrophils and granulopoiesis (31).
Sphingosine-1 phosphate phosphatase (SGPP1) is important
in the regulation of cell proliferation, angiogenesis and apop-
tosis (32). MATR3 plays a role in the regulation of transcrip-
tion (33).
The analysis of gene expression is becoming a popular
diagnostic method in neoplastic and inflammatory diseases
Table 4 The list of the 29 most significantly different expressed genes (FC > 2, P < 0.00000000001 corrected for multiple testing by Benja-
min Hochberg method) between patients with indolent systemic mastocytosis and healthy controls
Gene symbol Gene name
FC
M/C
FC
C/M Correlation P P Gene function
RAB27A rab27a, member ras oncogene family 0.39 2.58 3e)8 1.3e)11 Neutrophil secretion
and shape (26, 27)
ETS1 v-ets erythroblastosis virus e26 oncogene
homologue 1 (avian)
2.32 0.43 1.2e)8 3e)12 Carcinogenesis (20)
LOC730358 3.13 0.32 7.5e)8 6e)11 Unknown
ITGB1 Integrin, beta 1 (fibronectin receptor, beta
polypeptide, antigen cd29 includes mdf2, msk12)
2.69 0.37 5.2e)8 3.4e)11 Carcinogenesis (18)
ARL16 adp-ribosylation factor-like 16 0.38 2.66 1.4e)9 1.7e)11 Signal transduction (8, 9)
LRAP Leucocyte-derived arginine aminopeptidase 0.47 2.11 5.3e)11 3.3e)15 Carcinogenesis (22)
MLL3 Myeloid/lymphoid or
mixed-lineage leukaemia 3
5.07 0.20 1.9e)8 6.2e)12 Carcinogenesis (28)
PLEC1 Plectin 1, intermediate filament
binding protein 500 kDa
0.42 2.37 7.8e)8 6.6e)11 Carcinogenesis (17)
FAM39DP 0.48 2.09 1.2e)8 2.4e)12 Unknown
HSPC268 Hypothetical protein hspc268 0.37 2.69 5.2e)8 3.6e)11 Unknown
C3ORF34 Chromosome 3 open reading frame 34 0.41 2.44 3.4e)8 2e)11 Unknown
SERTAD2 Serta domain containing 2 2.02 0.49 1.2e)8 3.2e)12 Carcinogenesis (25)
ITGAV Integrin, alpha v (vitronectin receptor,
alpha polypeptide, antigen cd51)
4.29 0.23 9.2e)8 7.9e)11 Carcinogenesis (30)
SIAH1 Seven in absentia homologue 1 (drosophila) 2.06 0.49 1.2e)8 3.4e)12 Carcinogenesis (21)
CCR2 Chemokine (c-c motif) receptor 2 4.53 0.22 7.2e)8 5.6e)11 Carcinogenesis (29)
LRRFIP1 Leucine-rich repeat (in flii) interacting protein 1 0.41 2.42 2.9e)8 1.2e)11 Carcinogenesis (23)
C9ORF72 Hypothetical protein flj11109 5.23 0.19 5.2e)8 3.5e)11 Unknown
ATM Ataxia telangiectasia mutated (includes
complementation groups a, c and d)
2.74 0.37 9.7e)8 8.9e)11 Carcinogenesis (19)
PTP4A2 Protein tyrosine phosphatase type iva, member 2 0.47 2.14 2.6e)9 3.7e)13 Carcinogenesis (8, 9)
NCOA2 Nuclear receptor coactivator 2 7.36 0.14 6.6e)11 5.4e)15 Carcinogenesis (29)
LBR Lamin B receptor 4.36 0.23 3e)8 1.4e)11 Granulopoiesis maturation
of neutrophils (31)
MATR3 Matrin 3 4.80 0.21 9.7e)8 8.8e)11 Transcription (33)
ACTR2 arp2 actin-related protein 2 homologue (yeast) 2.14 0.47 6.8e)8 5.1e)11 Carcinogenesis (14)
PRR5 rho gtpase-activating protein 8 0.50 2.01 7.8e)8 6.6e)11 Carcinogenesis (16)
CHRNA5 Cholinergic receptor, nicotinic, alpha 5 0.41 2.42 3.9e)11 1.6e)15 Carcinogenesis (15)
ZMAT3 p53 target zinc finger protein 0.46 2.18 1e)11 7.2e)12 Carcinogenesis (13)
ZFAND5 Zinc finger, a20 domain containing 2 2.51 0.40 3.6e)8 2.2e)11 Carcinogenesis (8, 9)
SGPP1 Sphingosine-1-phosphate phosphatase 1 5.07 0.20 2.2e)8 8.8e)12 Cell proliferation,
apoptosis (32)
C14ORF153 Chromosome 14 open reading frame 153 0.38 2.65 4.4e)10 4.5e)14 Unknown
Gene expression in mastocytosis Niedoszytko et al.
234 Allergy 66 (2011) 229–237 ª 2010 John Wiley & Sons A/S
(34). Also, gene profiling is a standard procedure in assessing
the need for chemotherapy in breast cancer (35). To date, the
diagnosis of systemic mastocytosis is based on WHO criteria
(3–5). The large differences we observed between cases and
controls suggest that a gene expression based test could be
developed that would improve the reliability of current
diagnostic methods. A potential role for the described gene
profile may be in the differential diagnosis of patients with
myeloproliferative or myelodysplastic disorders co-existing or
masking mastocytosis, or in patients refusing bone marrow
examination.
Furthermore, in contrast to the study by D’Ambrosio
et al. (21), we analysed RNA isolated from whole blood
without prior cell separation. This approach (1) reduces the
effect of sample handling and (2) is a simple and standard-
ized method which may be used in the clinical practice in
the future, and furthermore (3) peripheral blood sampling is
less of a burden to patients in comparison with a bone mar-
row biopsy. Additionally, RNA isolation and gene expres-
sion analysis used in this protocol have become standardized
methods which avoid human laboratory errors and may be
further adapted to clinical practice in the future.
Some aspects of our study warrant comment. A necessary
next step is the validation of the abnormal gene profile in an
independent group of patients with indolent systemic masto-
cytosis to confirm our findings, and in cases with other
haematological diseases to see whether such RNA profiles
are specific for mastocytosis. Further studies may also answer
the questions whether analysis of gene expression profiles
may be used in clinical practice to (1) establish the diagnosis
of mastocytosis and its clinical variants, (2) assess the risk of
anaphylaxis in patients with mastocytosis and (3) assess the
risk of progressive disease or to develop a non–mast cell
haematological malignancy in these patients. The results of
the present study, although limited, may open a new area of
research.
In conclusion, we were able to find abnormalities in gene
expression in peripheral blood cells of patients with indolent
systemic mastocytosis and to construct a specific gene expres-
sion profile which may be useful in further research and
possibly in clinical practice.
Acknowledgments
The authors would like to thank Professor Cisca Wijmenga
Head of the Department of Genetics UMCG and Professor
Dirkje Postma from the Department of Pneumonology
UMCG for help in all steps of the scientific work, the nurses
from the Department of Allergology for help in collecting the
blood samples, Pieter van der Vlies and all colleagues from
the Department of Genetics UMCG for help in laboratory
work, Agata Somla from the Medical University of Gdansk
for help in financial logistics.
The research was supported by the Foundation for Polish
Science, and grant of the Polish Ministry of Science and
Higher Education, no. N402085934 and N40201031.
Controls Mastocytosis patients
Cluster 1
Cluster 2
Figure 1 Hierarchical clustering dendrogram of 29 most differen-
tially expressed genes between patients with mastocytosis and
controls. Each column represents a patient sample, each row an
individual gene. For each gene, green colour represents underex-
pression, red colour overexpression, and a black signal denotes
missing data.
Niedoszytko et al. Gene expression in mastocytosis
Allergy 66 (2011) 229–237 ª 2010 John Wiley & Sons A/S 235
References
1. Valent P, Sperr W, Schwartz L, Horny H.
Diagnosis and classification of mast cell pro-
liferative disorders: delineation from immu-
nologic diseases and non-mast cell
hematopoietic neoplasms. J Allergy Clin
Immunol 2004;114:3–11.
2. Garcia-Montero AC, Jara-Acevedo M,
Teodosio C, Sanchez ML, Nunez R, Prados
A et al. KIT mutation in mast cells and
other bone marrow hematopoietic cell lin-
eages in systemic mast cell disorders: a pro-
spective study of the Spanish Network on
Mastocytosis (REMA) in a series of 113
patients. Blood 2006;108:2366–2372.
3. Nedoszytko B, Niedoszytko M, Lange M,
van Doormaal J, Glen J, Zabotna M et al.
Interleukin-13 promoter gene polymorphism
-1112C/T is associated with the systemic
form of mastocytosis. Allergy 2009;64:287–
294.
4. Daley T, Metcalfe D, Akin C. Association
of the Q576R polymorphism in the interleu-
kin-4 receptor alpha chain with indolent
mastocytosis limited to the skin. Blood
2001;98:880–882.
5. Nagata H, Worobec AS, Oh CK, Chowdhu-
ry BA, Tannenbaum S, Suzuki Y et al.
Identification of a point mutation in the
catalytic domain of the protooncogene c-kit
in peripheral blood mononuclear cells of
patients who have mastocytosis with an
associated hematologic disorder. Proc
Natl Acad Sci USA 1995;92:10560–10564.
6. Lawley W, Hird H, Mallinder P, McKenna
S, Hargadon B, Murray A et al. Detection
of an activating c-kit mutation by real-time
PCR in patients with anaphylaxis. Mutat
Res 2005;572:1–13.
7. Kazmierska J, Malicki J. Application of the
Naıve Bayesian Classifier to optimize treat-
ment decisions. Radiother Oncol 2008;86:
211–216.
8. Nogales-Cadenas R, Carmona-Saez P, Vaz-
quez M, Vicente C, Yang X, Tirado F et al.
GeneCodis: interpreting gene lists through
enrichment analysis and integration of
diverse biological information. Nucleic Acids
Res 2009;124:514–521.
9. Kanehisa M, Araki M, Goto S, Hattori M,
Hirakawa M, Itoh M et al. KEGG for link-
ing genomes to life and the environment.
Nucleic Acids Res 2008;36:480–484.
10. Fehrmann RS, de Jonge HJ, Ter Elst A, de
Vries A, Crijns AG, Weidenaar AC et al. A
new perspective on transcriptional system
regulation (TSR): towards TSR profiling.
PLoS ONE 2008;3:e1656.
11. Liu SM, Xavier R, Good KL, Chtanova T,
Newton R, Sisavanh M et al. Immune cell
transcriptome datasets reveal novel leuko-
cyte subset-specific genes and genes associ-
ated with allergic processes. J Allergy Clin
Immunol 2006;118:496–503.
12. D’ambrosio C, Akin C, Wu Y, Magnusson
MK, Metcalfe DD. Gene expression analysis
in mastocytosis reveals a highly consistent
profile with candidate molecular markers.
J Allergy Clin Immunol 2003;112:1162–1170.
13. Varmeh-Ziaie S, Ichimura K, Yang F, Rab-
bits P, Collins VP. Cloning and chromo-
somal localization of human WIG-1/
PAG608 and demonstration of amplification
with increased expression in primary squa-
mous cell carcinoma of the lung. Cancer
Lett 2001;174:179–187.
14. Semba S, Iwaya K, Matsubayashi J, Seriza-
wa H, Kataba H, Hirano T et al. Coexpres-
sion of actin-related protein 2 and Wiskott-
Aldrich syndrome family verproline-homolo-
gous protein 2 in adenocarcinoma of the
lung. Clin Cancer Res 2006;12:2449–2454.
15. Wang JC, Cruchaga C, Saccone NL, Bertel-
sen S, Liu P, Budde JP et al. Risk for nico-
tine dependence and lung cancer is conferred
by mRNA expression levels and amino acid
change in CHRNA5. Hum Mol Genet
2009;18:3125–3135.
16. Johnstone CN, Castellvı-Bel S, Chang LM,
Sung RK, Bowser MJ, Pique JM et al.
PRR5 encodes a conserved proline-rich pro-
tein predominant in kidney: analysis of
genomic organization, expression, and muta-
tion status in breast and colorectal carcino-
mas. Genomics 2005;85:338–351.
17. Puiffe ML, Le Page C, Filali-Mouhim A,
Zietarska M, Ouellet V, Tonin PN et al.
Characterization of ovarian cancer ascites
on cell invasion, proliferation, spheroid for-
mation, and gene expression in an in vitro
model of epithelial ovarian cancer. Neoplasia
2007;9:820–829.
18. Pillozzi S, Brizzi MF, Bernabei PA, Bart-
olozzi B, Caporale R, Basile V et al. VEG-
FR-1 (FLT-1), beta1 integrin, and hERG
K+ channel for a macromolecular signaling
complex in acute myeloid leukemia: role in
cell migration and clinical outcome. Blood
2007;110:1238–1250.
19. Grosjean-Raillard J, Tailler M, Ades L,
Perfettini JL, Fabre C, Braun T et al. ATM
mediates constitutive NF-kappaB activation
in high-risk myelodysplastic syndrome and
acute myeloid leukemia. Oncogene 2009;
28:1099–1109.
20. Matsuo Y, Drexler HG, Harashima A,
Okochi A, Shimizu N, Orita K. Transcrip-
tion factor expression in cell lines derived
from natural killer-cell and natural killer-like
T-cell leukemia-lymphoma. Hum Cell 2004;
17:85–92.
21. Bursen A, Moritz S, Gaussmann A, Moritz
S, Dingermann T, Marschalek R. Interac-
tion of AF4 wild-type and AF4.MLL fusion
protein with SIAH proteins: indication for
t(4;11) pathobiology? Oncogene
2004;23:6237–6249.
22. Pinyol M, Bea S, Pla L, Ribrag V, Bosq J,
Rosenwald A et al. Inactivation of RB1 in
mantle-cell lymphoma detected by nonsense-
mediated mRNA decay pathway inhibition
and microarray analysis. Blood 2007;109:
5422–5429.
23. Soler G, Nusbaum S, Varet B, Macintyre
EA, Vekemans M, Romana SP et al.
LRRFIP1, a new FGFR1 partner gene asso-
ciated with 8p11 myeloproliferative syn-
drome. Leukemia 2009;23:1359–1361.
24. Li Y, Li W, Yang Y, Lu Y, He C, Hu G
et al. MicroRNA-21 targets LRRFIP1 and
contributes to VM-26 resistance in glioblas-
toma multiforme. Brain Res 2009;1286:
13–18.
25. Cheong JK, Gunaratnam L, Zang ZJ,
Yang CM, Sun X, Nasr SL et al. TRIP-Br2
promotes oncogenesis in nude mice and is
frequently overexpressed in multiple human
tumors. J Transl Med 2009;7:8.
26. Goishi K, Mizuno K, Nakanishi H, Sasaki
T. Involvement of Rab27 in antigen-induced
histamine release from rat basophilic leuke-
mia 2H3 cells. Biochem Biophys Res Commun
2004;324:294–301.
27. Kuroda TS, Fukuda M. Rab27A-binding
protein Slp2-a is required for peripheral
melanosome distribution and elongated cell
shape in melanocytes. Nat Cell Biol 2004;6:
1195–1203.
28. Ruault M, Brun ME, Ventura M, Roizes G,
De Sario A. MLL3, a new human member
of the TRX/MLL gene family, maps to
7q36, a chromosome region frequently
deleted in myeloid leukaemia. Gene 2002;
284:73–81.
29. Lee JS, Yang EJ, Kim IS. The roles of
MCP-1 and protein kinase Cdelta activation
in human eosinophilic leukemia EoL-1 cells.
Cytokine 2009;48:186–195.
30. Lu JG, Sun YN, Wang C, Jin de J, Liu M.
Role of the alpha v-integrin subunit in cell
proliferation, apoptosis and tumor metasta-
sis of laryngeal and hypopharyngeal squa-
mous cell carcinomas: a clinical and in vitro
investigation. Eur Arch Otorhinolaryngol
2009;266:89–96.
31. Zwerger M, Herrmann H, Gaines P, Olins
AL, Olins DE. Granulocytic nuclear differ-
entiation of lamin B receptor-deficient
mouse EPRO cells. Exp Hematol
2008;36:977–987.
32. Johnson KR, Johnson KY, Becker KP,
Bielawski J, Mao C, Obeid LM. Role of
human sphingosine-1-phosphate phosphatase
1 in the regulation of intra-and extracellular
Gene expression in mastocytosis Niedoszytko et al.
236 Allergy 66 (2011) 229–237 ª 2010 John Wiley & Sons A/S
sphingosine-1-phosphate levels and cell via-
bility. J Biol Chem 2003;278:34541–34547.
33. Valencia CA, Ju W, Liu R. Matrin 3 is a
Ca2+/calmodulin-binding protein cleaved
by caspases. Biochem Biophys Res Commun
2007;361:281–286.
34. Crijns AP, Fehrmann RS, de Jong S,
Gerbens F, Meersma GJ, Klip HG et al.
Survival-related profile, pathways, and
transcription factors in ovarian cancer.
PLoS Med 2009;6:e24.
35. van‘t Veer LJ, Dai H, van de Vijver MJ,
He YD, Hart AA, Mao M et al. Gene
expression profiling predicts clinical outcome
of breast cancer. Nature 2002;415:530–536.
Niedoszytko et al. Gene expression in mastocytosis
Allergy 66 (2011) 229–237 ª 2010 John Wiley & Sons A/S 237