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SUMMARYA rare case of thalassaemia-intermedia involving a non-deletion alpha thalassemia point mutation in the α1-globingene CD59 (GGC�GAC) and a deletion α+ (-α3.7) thalassaemiain which use of high performance liquid chromatography(HPLC) C-gram Hb subtype profile and DNA molecularanalysis helped establish the diagnosis.

KEY WORDS:Alpha-thalassaemia intermedia, Non-deletion α1 globin geneCD59, Deletion -α 3.7, HPLC, Molecular analysis.

INTRODUCTIONThe α-thalassemias are the most common single-gene diseasesin the world. They are characterized by a reduction orcomplete absence of α-globin gene expression. Normalindividuals have two α genes on each chromosome 16(αα/αα). The loss of one (-α) or both (--) of the cis-linkedgenes are the most common causes for α-thalassaemias.Patients with alpha-thalassaemia intermedia with only onefunctional α-globin gene (--/-α) develop chronic haemolyticanaemia of variable severity. HbH disease is the commoncause alpha-thalassaemia intermedia. This condition ischaracterized by a strongly positive H-inclusion test. Non-deletional HbH disease has been described to be more severethan the deletional type1. The most severe form of alphathalassaemia is Hb Barts hydrops foetalis where there are nofunctional α-globin genes (--/--) and results in the conditionwhere death occurs in utero or within a few hours of birth. Inthis case report, a rare case of alpha- thalassaemia intermediais presented where the H-inclusion test is negative.

CASE REPORTA 52-year old Malay male presented with pallor, generalizedmalaise, jaundice and hepatosplenomegaly. There was nofamily history of thalassaemia. The haematology work-upincluded a full blood picture, thalassaemia screen tests and aliver profile. Blood specimens were drawn into tubes (BectonDickinson Vacutainer Systems) containing dipotassiumethylene diamine tetraacetic acid (EDTA) for full bloodpicture and thalassaemia diagnosis (quantification of Hbsubtypes and DNA studies), into plain tubes for serumferrritin and liver profile studies.

Thalassaemia screen using the BHES protocol was done. Theacronym BHES refers to a multi-step process for screening ofthalassaemia where B is for blood counts and blood film, Hfor high performance liquid chromatography, E forelectrophoresis and S for stability. B: Blood counts weregenerated on an automated blood counter (Cell-Dyn, Abottlaboratories). He was noted to have severe anaemia with ahaemoglobin level of 5.6 gm/dl (normal range 13-18 gm/dl),hypochromic-microcytic red cell indices, reticulocytosis and athalassaemia peripheral blood film (anisopoikilocytosis,hypochromia, basophilic stippling, target cells, andfragments). H: a specimen collected in EDTA was analyzed onthe Bio-Rad Variant high performance liquid chromatography(HPLC) Hb analyzer (Bio-Rad Laboratories) to determine thedistribution of Hb subtypes with the use of the β-thalassaemiashort program recorder pack as described in the instructionmanual for the assay. The Hb subtypes noted were HbA, HbF,HbA and a pre-run peak of Hb Barts, shown Figure 1. Therewas no HbH seen. E: Hb electrophoresis by automatedagarose gel electrophoresis (Sebia) on alkaline pH8.5 showed

A Rare Case of Alpha-Thalassaemia Intermedia in a MalayPatient Double Heterozygous for α+-Thalassaemia and aMutation in α1 Globin Gene CD59 (GGC�GAC)

George E*, JAMA Tan**, A S Nor Azian***, Rahimah A****, Zubaidah Z***

*Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, **Department of Pathology, Hospital Malacca, ***UnitHaematology, Institute of Medical Research, ****Department of Molecular Genetics, Faculty of Medicine, Universiti of Malaya,50600 Kuala Lumpur, Malaysia

CASE REPORT

This article was accepted: 6 November 2009Corresponding Author: Elizabeth George, Haematology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia,43400 UPM Serdang, Selangor, Malaysia Email: elzageorge@hotmail.com

A=prerun peak of Hb Barts, RT=retention time in minutesY axis: percentage of Hb subtypes, X axis: retention time (RT)

Fig. 1: HPLC C-gram of alpha thalassaemia-intermedia (CD59/-α3.7)

similar findings of Hb subtypes seen with HPLC. S: the H-inclusion test was negative. The serum ferritin was 340µg/L(normal 150-300 µg/L). The liver enzymes were normal andthe unconjugated bilirubin level was raised. The antiglobulintest (Coombs test) was negative. Genomic DNA was extractedfrom peripheral blood using standard methods and multiplexpolymerase chain reaction (PCR)2 was done to detect thefollowing ·-thalassaemia mutations: single gene deletion (-α3.7,-α4.2) , two gene deletions (--SEA, --FIL, --MED, --THAI, --α20.5 and non-deletion α-thalassaemia (initiation codon (CD) (ATG�GTG),CD30, CD35 (TCC�CCC), Hb Quong Sze or CD125(CTG�CCG), Hb Constant Spring or CD 142 (TAA�CAA)and, CD59 (GGC�GAC) 3. The patient was found to beheterozygous for the single deletion (-α3.7) and the non-deletion mutation CD59 (GGC�GAC).

DISCUSSIONPatients with alpha thalassaemia-intermedia have moderateanaemia with Hb levels between 7-10gm/dl. Themanifestations include thalassaemia facies, jaundice andhepatosplenogaly that range from mild to moderate. Growthand development is normal. Gallstones and iron overloadingin the absence of blood transfusions have been seen. The H-inclusion test is strongly positive in these cases. In this casereport, the patient had α-thalassaemia intermedia phenotypewith severe anaemia (Hb 5.6 gm/dl) with thalassaemiafeatures in the peripheral blood film in the absence of apositive H-inclusion test.

Alpha-thalassaemia is caused by both deletion and non-deletion mutations in the alpha globin gene complex. Incontrast to β-thalassaemia, deletions are more common thannon-deletional defects. Commonly, α-thalassaemia-intermedia presents as HbH disease from the interaction ofdeletion of both the α-globin genes in cis (α0) (αthal 1) and α+

(αthal 2) phenotype. The molecular defects resulting in asingle deletion of the α globin gene are the rightward deletion-α3.7 and the leftward deletion -α4.2 with -α3.7 being the morecommon mutation. The double deletion defects (α0) of the αglobin genes in cis are --SEA, --FIL, --MED, --THAI and --20.5. The mostcommon non-deletion single gene defect (α+) is Hb ConstantSpring. This is a termination codon defect (TAA�CAA) resultsin a long mRNA with 31 extra amino acids being formed. Thelong mRNA is unstable resulting in reduced production of αglobin chains and a α+ phenotype. HbH-Constant Spring hasa more severe clinical phenotype than deletion HbH disease.The nondeletion molecular defect in the α-globin 1 gene

CD59 (GGC�GAC) results in the formation of hyperunstableα Hb variant. It is a rare cause for α-thalassaemia-intermediawhen the mutation combines with a α+ deletion defect. If themutation occurs in the α2-globin gene it results in hydropsfetalis. mRNA formation in α2:α1 is 3:1 and accounts for thecondition being more severe when the α2 gene is involved.The two cases first reported with α-thalassaemia-intermediawere from Turkey4. The true frequency of the moleculardefect in the α-globin 1 gene CD59 (GGC�GAC) is unknownas the defect can only be ascertained by DNA studies. This αHb variant is hyperunstable and has no product to bevisualized by routine haematology studies. Screening forthalassaemia by blood counts and Hb subtyping will miss thediagnosis in contrast to beta-thalassaemia wheredetermination of Hb subtypes presumptively identifies thepresence of thalassaemia.

This is the first report described in a Malay patient fromMalaysia. The presence of α1CD59 (GGC�GAC) may be theresult of migration from countries bordering theMediterranean sea as the Malays have links with Turkey, Iranand the Middle East. The true frequency of this alpha Hbvariant cannot be identified by screening methods such as theBHES protocol as it is hyper-unstable. This case reporthighlights the need to consider a hypersunstable α Hb variant[CD59 (GGC�GAC)] in a patient with alpha thalassaemia-intermedia in the absence of a strongly positive H-inclusiontest with normal Hb subtypes and presence of Hb Barts.Confirmation is required by DNA analysis as shown in thiscase.

ACKNOWLEDGEMENTThe study was supported by an e-Science grant 02-01-04-SF0876, Ministry of Science Technology and InnovationMalaysia (MOSTI).

REFERENCES1. George E, Ferguson V, Yakas J et al. A molecular marker associated with

mild hemoglobin H disease. Pathology 1989; 21: 27-30.2. Chong SC, Boehm CD, Higgs DR et al. Single-tube multiplex-PCR screen

for common deletional determinants for α-thalassemia. Blood 2000; 95:360-2.

3. Eng B, Patterson M, Walker L et al. Detection of severe nondeletional α-thalassemia mutations using a single-tube multiplex ARMS assay. Genetictesting 2001; 5: 327-9.

4. Douna V, Passotiriou I, Garoufi A et al. A rare thalassemia syndrome causedby interaction of Hb Adana [alpha59(E)Gly�Asp] with alpha+ deletionthalassemia: clinical aspects in two cases. Hemoglobin 2008; 32: 361-9.

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Case Report