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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

GDAŃSKI UNIWERSYTET MEDYCZNY - pbc.gda.plpbc.gda.pl/Content/11406/habilitacja_NIEDOSZYTKO_Marek.pdf · zagrożenia alergi ą na jady owadów u ... Wnt – wingless int pathway

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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

Marcie, Józiowi, Rodzicom i Rodzeństwu

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 –

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Fax +41 61 306 12 34E-Mail [email protected]

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

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8 Hermann K, Ring J: Association between the renin angiotensin system and anaphylaxis. Adv Exp Med Biol 1995; 377: 299–309.

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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.

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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.

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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.

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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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VOLUME 125, NUMBER 5

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.

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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

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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.

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Risk assessment in anaphylaxis: current and future approaches. J Allergy Clin

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8. Kazmierska J, Malicki J. Application of the naıve Bayesian classifier to optimize

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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.

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16. Nogales-Cadenas R, Carmona-Saez P, Vazquez M, Vicente C, Yang X, Tirado F,

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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

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therapy—T-cell tolerance and more. Allergy 2006;61:796-807.

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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.

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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.

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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

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