CASE REPORT Haemoglobin Lepore in a Malay family: a ... - MJPath

Malaysian J Pathol June 2005investigations revealed the diagnosis to behomozygous Haemoglobin Lepore. The clinical,haematological and laboratory features of thisdisorder are discussed in this paper.CASE REPORTA 2-year-old Malay boy was brought toUniversity Malaya Medical Centre forthalassemia/ haemoglobinopathy screening.Physical examination revealed thalassemiafacies, pallor, mild jaundice, hepatomegaly (3cm below the right subcostal margin) andsplenomegaly (3 cm below the left subcostalmargin). His height was at 3 rd percentile and hisweight was at 3-10 th percentile. Hishaematological profile showed haemoglobin 74g/L, red cell count 3.3 x 10 12 L, MCV 77 fl,MCH 22 pg and a reticulocyte count of 5.8%.The white blood cell count and platelet countwere normal. The patient’s blood smear revealedhypochromia, microcytosis, anisopoikilocytosis,polychromasia, basophilic stippling and a fewtarget cells (Fig.1).Haemoglobin electrophoresis on celluloseacetate at alkaline pH (pH 8.6) showed aprominent F band and an additional band in theS region (Fig. 2). There was no haemoglobin Aor haemoglobin A2 bands seen. Quantificationof haemoglobin F revealed a level of 90% andthe unknown haemoglobin constituted 9.3%.Electrophoresis at acid pH (pH 6.0) on citrateagar was carried out to identify the band in theS region. There was no Hb S detected. However,a prominent band was noted in the F region anda faint band in the A region. As the patient didnot have any A band on the cellulose acetatestrip, the faint band in the A region on citrateagar was suggestive of Hb D/G or Hb Lepore.Further testing to identify and quantify theabnormal haemoglobin variant was carried outby high performance liquid chromatography(HPLC) Bio-Rad Variant Haemoglobin testingsystem (Fig. 3). The unknown haemoglobin hada retention time (RT=3.59) similar to Hb A2 onthe “Beta Thal Short Program” and wasquantified as 10.7 %. There was no haemoglobinA synthesized and the rest of the haemoglobinpresent was hemoglobin F. Hb Lepore is knownto have the same retention time as Hb A2 on the“Beta Thal Short Program” and therefore apresumptive diagnosis of homozygous HbLepore was made for the patient.Screening was carried out for both parents.The father’s blood studies showed haemoglobin140 g/L, MCV 72 fl, and MCH 24 pg while themother’s haemoglobin was 111g/L, MCV 65.0fl,MCH 19 pg. The blood smear of both parentsrevealed hypochromia, microcytosis, few targetcells and occasional basophilic stippling (Fig.4). Haemoglobin electrophoresis on celluloseacetate (pH 8.6) of both parents (Fig. 5) showedbands at A, A2, and F regions and an additionalband in the S region while electrophoresis oncitrate agar at acid pH showed a prominent Aband and a faint band in the F region. There wasFIG. 1: Peripheral blood film of patient showinghypochromasia, microcytosis, anisopoikilocytosis,target cells and nucleated red bloodcell. Wright stain X 400FIG. 2: Haemoglobin electrophoresis on cellulose acetateat pH 8.6 Lane 1: normal control showingHb A and Hb A2. Lane 2: patient:absence of Hb A and Hb A2, increased Hb Fand an abnormal Hb (unknown band).34

HAEMOGLOBIN LEPOREno Hb S detected. The father’s Hb F level was1.6%, Hb A2 level 2.7% while the mother’s HbF level was 2.1%, Hb A2 level 3.0 %. Highperformance liquid chromatography (Fig. 6)revealed the unknown haemoglobin to be HbLepore (RT= 3.59) and the level in the fatherand mother was 13.1% and 13.8% respectively.FIG. 5: Haemoglobin electrophoresis on celluloseacetate at pH 8.6. Lane 1: normal controlshowing Hb A and Hb A2. Lane2: mother,heterozygous showing HbA, Hb A2 and anabnormal Hb (unknown). Lane 3: father,heterozygous showing similar findings.FIG. 3: High performance liquid chromatographyanalysis using the VARIANT HemoglobinTesting System (Bio-Rad Laboratories).Chromatogram of patient (homozygous). NoHb A, increased Hb F, Hb A2 + Leporeretention time 3.59FIG. 4: Peripheral blood film (mother) showinghypochromasia, microcytosis and target cells.Wright stain x 400FIG. 6: High performance liquid chromatographyanalysis using the VARIANT HemoglobinTesting System (Bio-Rad Laboratories) .Chromatogram of mother (heterozygous). HbA2 + Lepore retention time 3.5935

Malaysian J Pathol June 2005A diagnosis of heterozygous haemoglobinLepore was made for both parents. The parentsare first degree relatives. The patient’s andparents’ samples were sent for DNA sequencingwhich confirmed this unknown haemoglobin tobe Hb Lepore Washington Boston. The patientis on regular follow-up and has received 3 bloodtransfusions at 4 -5 monthly intervals since thetime of diagnosis.DISCUSSIONHb Lepore is a common abnormal haemoglobinseen in the Mediterranean region. It is the mostcommon abnormal haemoglobin in Caucasiansin Central Portugal and in the Spanish AltaExtremadura. 1 It is a variant form of humanhaemoglobin that contains delta beta hybridchains or a fused globin chain, from the productof a hybrid gene: the unequal crossing overbetween the delta and beta globin genes resultingin a deletion of 7.4kb in the beta globin genecluster. 1,2Three different types of Hb Lepore have beendescribed so far, each with a different crossoverbreakpoint: Hb Lepore Washington Boston, HbLepore Baltimore and Hb Hollandia. 1,2,4 Thebasic defect in all the Hb Lepore disorders is theinefficient synthesis of the delta beta fusionchains of Hb Lepore, which leads to a variabledegree of globin imbalance, excess alpha chainproduction and the clinical phenotype of betathalassaemia. The ineffective erythropoiesis andthe shortened red cell survival results from thedeleterious effects of excess alpha chains. Theselection of cells which are synthesizingrelatively more gamma chains follow the samemechanism as occurs in beta thalassemia. 5 It hasbeen postulated that the deletion which resultedin the delta beta fusion is somehow responsiblefor increasing the absolute output of gammachains. 6 This is compatible with the observationthat Hb Lepore heterozygotes producesignificantly more Hb F than beta thalassaemiaheterozygotes.The Hb Lepore heterozygotes are usuallyasymptomatic. In a large Italian series noabnormal physical signs were reported, but afew of the Greek and Yugoslavian heterozygoteshad mild splenomegaly. 7,8 Heterozyogotes aretherefore healthy individuals with only a slightdecrease in haemoglobin levels, but with adistinct microcytosis and hypochromia of theirred blood cells. The mean cell haemoglobin(MCH) is thought to be the most reliableparameter that is altered in the Hb Leporeheterozygotes (range 20-25 pg). 9 In this series,the red cell indicies and morphologicalappearances of the blood film of betathalassaemic carriers were compared with thatof heterozygous Hb Lepore. It was found to besimilar in both conditions. Studies suggest thatthere are no major differences in thehaematological findings between carriers of thedifferent molecular varieties of Hb Lepore. 1 Redcell survival and ferrokinetic data available revealred cell survival is slightly shortened and Fe 59utilization is increased. 10The Hb Lepore homozygotes vary in theseverity of their clinical manifestations. 6,7,8 Atone end of the spectrum are patients who presentwithin the first 5 years of life with severe anaemiawith haemoglobin values ranging from 4 to 7 g/dl. Significant splenomegaly, hepatomegaly, andskeletal abnormalities indistinguishable fromthose of homozygous beta thalassaemia are seen.These patients require regular blood transfusionand exhibit shortened life span. At the other endof the spectrum are patients who have a milderdisorder indistinguishable from beta thalassemiaintermedia, who are anaemic throughoutchildhood and require only occasional bloodtransfusions. The homozygous state for HbLepore therefore varies from a transfusiondependent disease like beta thalassaemia majorthrough the milder spectrum of thalasaemiaintermedia that appears to be compatible withlongevity of life. 5 The haematological findingsin these patients are peripheral blood film featuresindistinguishable from the severe forms of betathalassaemia. Bone marrow smears show markederythroid hyperplasia due to high level oferythropoietin production in response to theanaemia. 6The multifaceted approach for thepresumptive identification of haemoglobinvariants includes a scrutiny of blood counts/redcell indices and haemoglobin analysis. Thisapproach easily identifies Hb Lepore. Oncellulose acetate electrophoresis at alkaline pH,Hb Lepore shows electrophoretic mobilitysimilar to Hb S. 5 The other haemoglobins thatrun in this position are Hb D and Hb G, whichare differentiated from Hb S by sickle solubilitytest and electrophoresis at acid pH. Hb Leporehas a similar retention time (RT) value as Hb A2on high performance liquid chromatography(HPLC) analysis with the Bio-Rad VariantHaemoglobin testing system. 2 Values greaterthan 10% suggest the presence of variant36

HAEMOGLOBIN LEPOREhaemoglobin. In Malaysia the most commonhaemoglobin variant seen is Hb E that isdifferentiated from Hb Lepore by celluloseacetate electrophoresis at alkaline pH. Hb Eruns in the same position as HbA2, whereas HbLepore runs at the position of Hb S. Therefore amultifaceted approach which includeshaemoglobin electrophoresis and highperformance liquid chromatography providessufficient information for the presumptiveidentification of Hb Lepore. The characterisationof the type of Hb Lepore requires analysis of theglobin chains by reversed phase HPLC andDNA studies would confirm the diagnosis ofthis variant haemoglobin. 4In the homozygous state, Hb A and Hb A2are absent and the haemoglobin is made up ofHbs F and Lepore only. The level of Hb Leporeranges from 8 to 30% with a mean value ofapproximately 15%, the remainder of thehaemoglobin being Hb F. In the heterozygousstate the haemoglobin contains Hbs A, Lepore,A2 and a variable amount of Hb F. The reportedlevel of Hb Lepore is between 5 to 15%, with amean level around 10%. The mean level of HbA2 is about 2% and the reported values for HbF range from between 1-14%. 5In conclusion, Hb Lepore is a structuralhaemoglobin variant coded for by a hybrid geneformed by the fusion of delta and beta genes.The homozygous state is clinically similar tobeta thalassaemia intermedia or major.Presumptive identification can be easily madein the laboratory by a multifaceted approach thatincludes the methods, scrutiny of blood counts/red cell indices, haemoglobin electrophoresis atalkaline and acid pH and haemoglobin analysisby high performance liquid chromatography.However, confirmation requires DNAcharacterization. A correct diagnosis is essentialfor proper clinical management and geneticcounseling.4. Ropero P, Gonzalez FA, Sanchez J et al. Identificationof the Hb Lepore phenotype by HPLC.Haematologica 1999; 84: 1081-45. The δ β and related thalassaemias. In: WeatherallDJ and Clegg JB, editors. The Thalassemia Syndromes.4 th ed. Blackwell Science Ltd; 2001. p.361-636. Olivieri NF, Rees DC, Ginder GD et al. Treatmentof thalassemia major with phenylbutyrate and hydroxyurea.Lancet 1997; 350: 491-27. Duma H, Efremov G, Sadikario A et al. Study ofnine families with haemoglobin Lepore. Br JHaematol. 1968; 15: 161-728. Quattrin N, Luzzatto L, Quattrin S. New clinicaland biochemical findings from 235 patients withhaemoglobin Lepore. Ann NY Acad Sci 1980;344: 364 -749. Huisman TH. Compound heterozygosity for HbSand the hybrid Hb S Lepore. Comparision ofhaematological and haemoglobin composition data.Hemoglobin 1997; 21: 249-5710. Pearson HA, Mc Farland W, King ER.Erythrokinetic studies in thalassemia trait. J LabClin Med 1960; 56: 866-73REFERENCES1. Ribeiro ML, Cunha E., Goncalves P et al. Hb.Lepore- Baltimore and Hb Lepore-Washington-Boston in Central Portugal and Spanish AltaExtremadura. Hum. Genet. 1997; 99: 669-732. Shaji RV, Edison ES, Krishamoorthy R, ChandyM, Srivastava A. Hb Lepore in the Indian population.Hemoglobin 2003; 27:7-143. Viprakasit V, Pung-Amritt P, Suwanthon L, ClarkK, Tanphaichitr VS. Complex interactions of [delta][beta] hybrid haemoglobin (Hb Lepore- Hollandia)Hb E ([beta] 26G>A ) and [alpha] + thalassaemia in aThai family. Eu J Haematol 2002; 68:107-12.37