Hemoglobin, 2012; 36(4): 362–370Copyright © Informa Healthcare USA, Inc.ISSN: 0363-0269 print/1532-432X onlineDOI: 10.3109/03630269.2012.679717

ORIGINAL ARTICLE

SYMPTOMATIC ERYTHROCYTOSIS ASSOCIATEDWITH A COMPOUNDHETEROZYGOSITY FOR Hb LEPORE-BOSTON-WASHINGTON(δ87-β116) AND Hb JOHNSTOWN [β109(G11)Val!Leu, GTG>TTG]

Susumu Inoue,1,2 Jennifer L. Oliveira,3 James D. Hoyer,3 andMahesh Sharman1,21Department of Pediatrics, Hurley Medical Center, Flint, Michigan, USA2Department of Pediatrics/Human Development, Michigan State University, College of Human Medicine,East Lansing, Michigan, USA3Mayo Clinic, Metabolic Hematology Laboratory, Rochester, Minnesota, USA

� Hb Johnstown [β109(G11)Val!Leu, GTG>TTG] has previously been described as a highoxygen affinity variant in a heterozygous state and in combination with β0-thalassemia (β0-thal).Because the variant does not separate from Hb A by routine methods it may be easily missed unlessclinical suspicion is high. Hb Lepore-Boston-Washington (Hb LBW; δ87-β116) is a δβ hybrid variantthat clinically manifests similarly to a βþ-thal. Hb LBW is not detected by routine polymerase chainreaction (PCR) sequencing but is easily detected by electrophoretic methods. We describe a 19-year-oldAfrican American male with a compound heterozygosity for Hb Johnstown and Hb LBW. The patientpresented with progressively worsening chest pains, headaches and erythrocytosis. He was repeatedlyphlebotomized with symptomatic improvement and subsequently was confirmed to have the high oxygenaffinity hemoglobin (Hb) variant. The lowest Hb and hematocrit (packed cell volume, PCV) achievedby phlebotomy was 16.1 g/dL and 0.51 L/L, respectively. Currently, he is no longer being phleboto-mized, and is feeling relatively well except for minor chest pain. It is unclear to what degree thephlebotomies contributed to his subjective improvement. The combination of Hbs Johnstown and LBWhas not been heretofore described, and in this case, was associated with marked symptomatic erythro-cytosis. This unique combination results in a more pronounced phenotype, similar to or slightly moresevere than, compound Hb Johnstown/β0-thal. This compound hemoglobinopathy will likely not becorrectly classified using a single method of Hb detection and underscores the need for multiplecharacterization methods when indicated by the clinical picture.

Keywords Hb Johnstown, High oxygen affinity hemoglobin (Hb), Erythrocytosis, HbLepore-Boston-Washington (Hb LBW), Left-shifted oxygen dissociation,Decreased p50

Received 8 November 2011; Accepted 8 February 2012.Address correspondence to Susumu Inoue, M.D., Hurley Medical Center, One Hurley Plaza, Flint, MI

48503, USA; Tel.: þ810-626-6211; Fax: þ810-262-9972; E-mail: sinoue1@hurleymc.com

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INTRODUCTION

High oxygen affinity hemoglobins (Hbs) are commonly found in a simpleheterozygous state, a subset in a compound heterozygous state and only rarelyin a homozygous state. The paucity of homozygosity suggests a selective disad-vantage of increased variant percentages.When found in combinationwith a β0-thalassemia (β0-thal), a pseudohomozygous state is simulated due to increasedpercentage of the variant and the lack of normal Hb A. Hb Johnstown [β109(G11)Val!Leu, GTG>TTG] (HGVS HBB:C.[328G>C or 328G>T]) (1) is ahigh oxygen affinity variant that has been characterized in such a manner. Thesimple heterozygous state is associated withmild erythrocytosis and aHb variantlevel of approximately 45-50%. As a compound heterozygote with β0-thal, thevariant level is approximately 90% and is associated with more pronouncederythrocytosis. It is electrophoretically silent and will be missed by routine Hbelectrophoresis screening methods. In the course of investigating a young manwith extreme erythrocytosis, a previously undescribed combination of HbsJohnstown and Lepore-Boston-Washington (Hb LBW; δ87-β116) (HGVSNG_000007.3:g.63632_71046del) was found. He was repeatedly phleboto-mized, and interestingly, his subjective symptoms of headaches and chest painimproved. However, a review of the literature regarding the benefit of phlebot-omy in patients with high oxygen affinity Hb variants was generally negative.Therefore, we would like to describe our experience in this case.

Case Description

A 19-year-old African American male presented to the emergency depart-ment because of progressively worsening chest pain. The patient stated that hehad experienced headaches and chest pains “all his life.” The chest pain wasintermittent and worsened with deep breathing. He also stated that he hadrecently felt very weak. He smoked up to three cigarettes a day but did notsmoke marijuana. Past medical history is unremarkable. Physical examinationwas also unremarkable except a blood pressure of 144/82 mmHg. The work-up for cardiac causes of chest pain showed an EKGwith sinus rhythm and rightbundle brunch block, mild concentric left ventricular hypertrophy anddilated inferior vena cava (likely due to increased right atrial pressure). Thecardiac enzymes were normal. Evaluation for coronary artery disease wasnegative and he had no neurological deficits to suggest cerebral ischemia. ACBC showed an RBC value of 9.6 � 1012/L, Hb of 21.4 g/dL, hematocrit(packed cell volume, PCV) of 0.67 L/L, MCV of 68.3 fL, and platelet count of113,000/μL. The patient was not aware he had erythrocytosis. Upon question-ing, however, the patient recalled that his father and several members of hisfamily also had headaches and ruddy faces. An evaluation for erythrocytosisyielded the following results: normal TSH and T4, venous blood gas, pH 7.35,

Compound Heterozygosity for Hb Johnstown and Hb LBW 363

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pO2 164 mmHg, oxygen saturation 99%, pCO2 38 mmHg, bicarbonate 20.6mEq/L, normal renal function, no evidence of mass lesions by chest X-ray orultrasound study of abdomen and kidneys. A pulmonary function test wasnormal. Blood carboxyHb was 1.9%, serum erythropoietin assay was 21.4 milliinternational units (mIU) (reference range 3.7-31.5 mIU/mL), and the JAK2V617F mutation was not detected by polymerase chain reaction (PCR). Aninvestigation for a hemoglobinopathy was performed.

The patient was hospitalized, and placed on a regimen of 81 mg aspirin/day. While in the hospital, he underwent three phlebotomies every other daywith total withdrawal of 1400mL, after which the patient’s symptoms improved.At the end of hospitalization the Hb and PCV decreased to 18.9 g/dL and0.58 L/L, respectively. The patient was further phlebotomized for a total of ninetimes (400mL each) during a 55-day span following discharge from the hospitalreaching a nadirHb, PCV of 16.1 g/dL and 0.51 L/L, respectively. Interestingly,the patient maintained that phlebotomy continued to improve his symptoms.The phlebotomy was discontinued due to iron deficiency, and he has not beenphlebotomized for the last 4 months. Themost recent Hb and PCV values were18.4 g/dL and 0.61 L/L, respectively. He currently has no headaches or chestpains.

MATERIALS AND METHODS

Methods of Hb characterization included: p50 analysis, HEMOXAnalyzer(TCS Scientific Corporation, New Hope, PA, USA). The re-oxygenation curvewas measured at pH 7.6 and 37�C with in-house developed reagents. Highperformance liquid chromatography (HPLC) (VARIANT CLASSIC™,β-Thalassemia Short Program; Bio-Rad Laboratories, Hercules, CA, USA),capillary electrophoresis (CE) [CapillaryS Hemoglobin (E) Program, Sebia,Evry, France]. Additionally, intact globin chain mass spectrometry (MS) wasperformed using an electrospray ionization quadrupole-time-of-flight MS(Q-ToF Premier™; Waters Corporation, Milford, MA, USA) and results wereanalyzed using Waters BioPharmalynx software.

DNA was extracted from whole blood leukocytes using the Qiagen EZ1(Qiagen, Valencia, CA, USA) method. The β gene was amplified andsequenced using two sets of primers (β Hb gene exons 1 and 2 forward: 50-GTC CAA CTC CTA AGC CAG-30; reverse: 50-ATC ATT CGT CTG TTT CCCA-30 and β Hb gene β-thal rich region of intron 2 and exon 3 forward: 50-TTTCCC TAA TCT CTT TCT TTC-30 and reverse: 50-GCA GCC TCA CCT TCTTTC-30. The PC reaction conditions were as follows: 40 50 second cycles at54�C. After treatment with ExoSAP-IT, a direct sequencing reaction wasperformed utilizing the BigDye Terminator v1.1 reaction mixture (AppliedBiosystems, Foster City, CA, USA). The product was purified by the BigDye

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Xterminator Purification Kit. Results were read and analyzed using the ABIPRISM™ 3130xl instrument/SeqScape Software.

RESULTS

Hemoglobin variant evaluation by CE and HPLC showed 10%Hb Leporewith expected peak shapes and percentages in the Hbs A (83.4%), F (3.6%)and A2 (2.7%) regions (Figure 1a and 1b). A p50 evaluation was markedly left-shifted (12 mmHg, normal range 24–30) (Figure 2). Because Hb Lepore didnot explain the p50 results, a further search for an additional Hb variant wasundertaken. Mass spectrometry revealed two peaks with masses of 15881 and15865 Da, which are the calculated masses of Hb Johnstown and Hb LBW,respectively. No Hb A peak was present (Figure 3). β-Globin gene sequencingrevealed a substitution at codon 109 of GTG>TTG (Val!Leu) in a hetero-zygous state (Figure 4). As expected, Hb LBW was not detected by PCR.

DISCUSSION

Hb Johnstown was first described by Jones et al. (1) in 1990, in an asympto-matic patient with mild erythrocytosis. It is a high oxygen affinity Hb due to amutation causing an unstable configuration at the α1β1 or α1β2 contact sitewhen deoxygenated (tense state), thereby favoring the R (relaxed) (oxygen-ated) state of Hb. Currently, about 200 high oxygen affinity Hbs have beendescribed (2). Since the initial report, additional case reports of HbJohnstown have appeared in the literature (3–6). It is interesting to notethat one patient described by Feliu-Torres et al. (5), and two of four patients(in the same family) described by Ropero et al. (3), had β0-thal in addition toHb Johnstown. Two members of a family out of a total of 13 individuals (fivefamilies) with Hb Johnstown reported by Gonzalez Fernandez et al. (6) alsohad β0-thal. Hb Johnstown/β0-thal compound heterozygotes showed variantpercentages in the 90% range and manifested with higher PCV levels andlower p50 results than simple heterozygotes. Our case was in combination withHb LBW, a δβ hybrid variant that results from a crossover fusion of the δ and βgenes and phenotypically behaves like a β-thal trait. The microcytosis in ourpatient is associated with this abnormal variant. Our previously undescribedcombination has similar variant percentages as the Hb Johnstown/β0-thalcompound heterozygotes but appears to manifest more severe symptomsthan mentioned in the previous reports. Whether our patient’s smokinghistory exacerbated his symptoms is possible but presently unclear. Hb LBWis not typically associated with erythrocytosis and the oxygen affinity is similarin our case to those reported with β0-thal (3). Therefore, the contribution ofthe Hb LBW to the p50 may be negligible.

Compound Heterozygosity for Hb Johnstown and Hb LBW 365

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The patient subjectively improved with phlebotomy.His headaches becamemilder and less frequent, and the chest pains subsided. A literature reviewregarding the effect of phlebotomy on high oxygen affinity Hbs is generallynegative, indicating no benefit except in a few cases. A Thai child with

(a)

(b)

FIGURE 1 a) Capillary electrophoresis showsHb Lepore in zone 6 and a normal-appearingHb A peak. b)The HPLC tracing shows Hb Lepore in the Hb A2 window and normal-appearing Hb A peak.

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homozygous Hb Tak [β147 (þAC)] experienced frequent episodes of respira-tory difficulties that were temporarily reversed by exchange transfusions (7).A patient with Hb Yakima [β99(G1)Asp!His (GAT>CAT)] developed lethargy

FIGURE 2 The p50 test showed a markedly left-shifted oxygen dissociation curve; p50 ¼ 12 mm Hg(normal 24–30 mm Hg).

FIGURE 3 Mass spectrometry showed Hb Johnstown (15881 Da) and Hb Lepore (15865 Da); no Hb A ispresent.

Compound Heterozygosity for Hb Johnstown and Hb LBW 367

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and swelling of hands and fingers. These symptoms subsided after phlebotomywhen the PCV level was reduced from 0.59 to 0.51 L/L. However, a treadmillstudy showed no difference in exercise tolerance in the patient before and afterreduction of PCV level (8). A patient with compound heterozygosity for HbRegina [β96(FG3)Leu!Val, CTG>GTG] and βþ-thal, developed hepatosple-nomegaly and renal vein thrombosis (9). This patient was treated with hydro-xyurea resulting in an increase in Hb F, shift of p50 and a decrease in red cellvolume, and the treatment led to an improvement in “general status.” A patientwith HbOsler [β145(HC2)Tyr!Asn, TAT>AAT] was studied as to the exercisetolerance before and after phlebotomy, with a reported conclusion of anabolictolerance being directly related to PCV levels, and the patient received nobenefit from phlebotomy (10). In another study, a patient with Hb Osler washemodiluted (PCV reduced from the initial 0.69 to 0.42 L/L), after which anexercise test was performed, and the result compared with a control subject whoalso had erythrocytosis of unknown etiology, also hemodiluted. The hemodilu-tion resulted in reduced maximal work and increased heart rate, increasedminute ventilation and increased lactic acid in the patient with the high oxygenaffinity Hb, whereas the control patient showed no changes in exercise perfor-mance after hemodilution (11). Länsimies et al. (12) measured the maximumwork capacity in 10 individuals with Hb Linköping [β36(C2)Pro!Thr,

FIGURE 4 DNA sequencing of the β-globin gene showed a mutation at codon 109 (GTG>TTG)(Val!Leu) confirming the Hb Johnstown variant. The δβ fusion variant Hb Lepore was not detected.

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CCT>ACT] using a bicycle ergometer and compared the results to that of 10normal controls and foundnodifference. Thus, in an overwhelmingmajority ofcases with high oxygen affinity Hbs, phlebotomy is not a recommended man-agement, mainly because the patients are asymptomatic (2,13). In our case, therelatively high erythropoietin level may indicate inadequate compensation ofhis tissue to poor oxygen unloading from the RBC to the tissues. But thisdeficiency most likely cannot be corrected by phlebotomy and may requireerythrocytapheresis. Thrombosis is another risk due to high viscosity, but thiscomplication has been very rare in high oxygen affinityHb variant patients. Theonly thrombotic episodes reported occurred in patients who had anotherthrombophilic condition, factor V Leiden in one (14), and inherited proteinS deficiency (15) in the other three of four family members who carried boththe protein S mutation and Hb Rainier [β145(HC2)Tyr!Cys (TAT>TGT)].There are a few exceptions including the patient with Hb Regina describedabove (9). Two of six patients reported by Percy et al. (16) were said to have hadthrombosis, but no details were given, thus it is unclear whether these patientshad additional thrombophilic risks. Hence, it may not be necessary to placethese patients on anti-thrombotic agents such as low-dose aspirin, unless thepatient has additional risk factor(s) for thrombophilia.

In conclusion, we report a patient with a symptomatic compound hetero-zygosity for Hb Johnstown/Hb LBW with marked erythrocytosis, microcytosisand a markedly left-shifted p50 curve. This combination may manifest in moresevere symptoms than the previously described Hb Johnstown/β0-thal cases.Importantly, multiple characterization methods were required to classify thisunique case correctly, as the only method that detected both Hb variants andconfirmed the absence of Hb A was MS. Without correlation with the p50results, Hb Johnstown would likely have beenmissed. The CE andHPLC resultsappeared like a typical Hb Lepore and PCR appeared as a simple heterozygousHb Johnstown. Further investigation is warranted when lifelong, hereditaryerythrocytosis is suspected. In the evaluation of erythrocytosis, Hb electrophor-esis results should be correlated with p50 and serum erythropoietin values toexclude electrophoretically silent high oxygen affinity Hb variants.

Declaration of Interest: The authors report no conflicts of interest. Theauthors alone are responsible for the content and writing of this article.

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Compound Heterozygosity for Hb Johnstown and Hb LBW 369

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