42 Med J Malaysia Vol 69 No 1 February 2014

SUMMARYHaemoglobin S D-Punjab is a rare compound heterozygoushaemoglobinopathy characterised by the presence of two βglobin gene variants: β6(GAG→GTG) and β121(GAA→CAA).These patients’ clinical and haematological features mimichaemoglobin S disease. We describe the first case of doublyheterozygous HbSD-Punjab from Malaysia managed withregular blood transfusion at the age of one. This casehighlights the propensity for occurrence of rare phenotypeswithin our multi-ethnic population and emphasises theimportance of accurate genotyping to avoid erroneouscounselling, and to plan an effective patient managementstrategy before complication evolves.

KeY WoRDS:Haemoglobinopathies; Hb SD-Punjab; Malaysia

INTRoDUCTIoNHaemoglobinopathies are characterised by structurallyabnormal haemoglobin variants of the normal adulthaemoglobin (HbA). Haemoglobin S, C, D and E are classicalexamples of β chain variants. Hb S (β6(A3), Glu→Val)variant has a worldwide distribution with highest genefrequencies seen in equatorial Africa, Quatif osases of easternSaudi Arabia and parts of India. In Malaysia, Hb S variant isseen amongst the Malaysian Indians at a low frequency 1. HbS heterozygotes (βS/βA) are usually asymptomatic but rarelymay be associated with clinical and haematologicmanifestation of significance. Homozygotes (βS/βS), however,has enormous clinical importance. Haemoglobin D Punjab(also known as D-Los Angeles) is characterized bysubstitution of glutamic acid by glutamine at position 121 ofthe β-globin chain and is most commonly encountered innorthwest India, Pakistan, and Iran; and is asymptomaticboth in heterozygous and homozygous states. Co-inheritanceof both haemoglobin S and haemoglobin D-Punjab, termedHbSD disease, may manifest with mild to moderatehaemolytic anaemia resembling those of sickle cell anaemia.

CASe RePoRT An Indian boy presented with a history of upper respiratorytract infection at the age of 4 months and was noted by hisGP to be very pale (Hb of 5.5 gm/dL). He was then referred toa district hospital for further investigation and management.Red cell indices at the hospital revealed the following: Hb 6.5gm/dL, RBC 2.3 X106/dL, MCV 86.0fL, MCV 27.5pg and

MCHC 32%. Blood smear showed marked anisopoikilocytosiswith microcytic hypochromic red cells, dacrocytes, targetcells, fragmented and polychromatic cells. A few sickle andboat shaped cells were also seen. Sickling test done waspositive. The Hb electrophoresis done at pH 8.5 showed avariant band at haemoglobin S position along with a thickband at HbF position. A presumptive diagnosis of sickle celldisease (SCD) was then made. Further analysis on HPLCshowed presence of Hb F and variant Hb peaks at the D(20.8%) and S (19.2%) windows. Hb A peak was virtually notvisible. Based on this result the diagnosis of sicklecell/haemoglobin D-Punjab compound heterozygosity wasmade.

At the age of one, the patient became transfusion dependentrequiring two monthly blood transfusions. To establish adefinitive molecular diagnosis, patient’s blood specimen wassent to our laboratory at Institute for Medical Research. Hisgenomic DNA was tested for β-gene mutations prevalent inour multiethnic population. Having excluded the commonpoint mutations by allele specific primer extension, β-globingene sequencing was done to detect the presence of othermutations. Sequencing of the β-globin gene read from -100bp to the 3’UTR showed the co-inheritance of a heterozygouschange at codon 6 (GAG→GTG) and also a heterozygousmutation at codon 121 (GAA→CAA). Thus, the diagnosis ofβS/βD-Punjab compound heterozygosity was confirmed. Theparent’s blood samples were not sent to our institute fortesting, therefore we were unable to fully determine thepattern of inheritance. The mother’s Hb analysis report wasconsistent with Hb S trait. However the father’s Hb analysiswas to have been reanalysed.

DISCUSSIoNSeveral variants of HbD such as Hb D Punjab, Hb D Iran, HbD Ibadan and Hb D Bushman have been noted to co-inheritwith HbS. With the exception of Hb D Punjab, compoundheterozygous states of HbS with HbD variants were clinicallyinnocuous. The substituted glutamine residue in Hb DPunjab interacts with Hb S to facilitate polymerisation duringdeoxygenation, and thus is presented perniciously withclinical and haematological features of variable degree ofhaemolytic anaemia resembling sickle cell anaemia 2. Studiesfrom India, Pakistan, Iran, UAE and Mexico have shownsimilar clinical presentations for HbSD-Punjab mimicingsevere form of sickle cell anaemia 3.

Haemoglobin Sickle D Punjab: - A Case Report

Rahimah Ahmad*, Syahira Lazira Omar*, Siti Hida H M Arif*, Faidatul Syazlin A Hamid*, Nur Aisyah Aziz*, NikHafidzah N Mustapha**, Zubaidah Zakaria*

*Haematology Unit, Institute for Medical Research (IMR), Jalan Pahang, 50588 Kuala Lumpur, **Pathology Unit, HospitalTuanku Jaafar, Negeri Sembilan Malaysia

CASE REPORT

This article was accepted: 29 October 2013Corresponding Author: Rahimah Ahmad, Haematology Unit, Institute for Medical Research (IMR), Jalan Pahang, 50588 Kuala Lumpur, Malaysia Email: ahmadrahimah@hotmail.com

Haemoglobin Sickle D Punjab

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Accurate delineation of these variants is very important tofacilitate an effective response to life threateningcomplication and to avoid erroneous counselling of theserare clinically important Hb S compound heterozygotepatients. As a rule of thumb, all samples showing a singleband at the haemoglobin S position on conservativeelectrophoresis at alkaline pH should be retested by analternative technique either by citrate agar/acid gel method,or by IEF. This step is crucial to exclude the possibility of acompound heterozygote. Alternatively, HPLC should bepreferred if available. Care must be taken if considering adiagnosis on HPLC chromatograph as it is possible for morethan one variant to overlap and co-elute within a givenretention time window. Haemoglobin eluting in the HbSwindow should have a sickle solubility test performed toconfirm the presence of sickling haemoglobin. It should benoted, however, that positive sickling test alone indicates thepresence of a sickling haemoglobin and does not providedefinitive diagnostic information on the identity of thehaemoglobin or which other haemoglobins may be present.Erythrocytes containing other variant haemoglobins inwhich β6(Glu→Val) is present, such as HbC-Geogtown and Hb S-Memphis also exhibit sickling. The sickle cell solubility testmay not be reliable at HbS level below 15-20%, reflecting thatthe test is unreliable in neonates, infants and in recenttransfusion with HbAA blood.

Hb D-Punjab can be readily distinguished from Hb S by itsnormal solubility, difference on electrophoretic mobility onagar gel at acidic pH and its failure to produce sickling.

Some factors affecting variability in clinical manifestation ofHb SD-Punjab may include co-inheritance of α-thalassaemia,enhanced HbF levels and the type of haplotype framework onwhich βS is inherited. The coexistence of α thalassaemia andsickle cell anaemia does have a proven affect on thephenotype reflected by less haemolysis. Moreover, it is knownthat Bantu and Benin haplotypes of βS are associated with theclinically severe sickle cell disease. However, unlike in HbSSsyndrome, elevated levels of HbF concentration do not seemto ameliorate the clinical phenotype of HbS D Punjab 4.

As the pathophysiology HbS D-Punjab is similar to HbSdisease, adopting the guidelines proposed for sickle cellanaemia to manage this clinically significant phenotype willbe critical to reduce morbidity and mortality 5.

CoNCLUSIoNAccording to our extensive literature review and to the best ofour knowledge, this is the first case of HbSD-Punjab reportedfrom Malaysia. Haemoglobin variant such as HbS and HbDare extremely rare in the multi-ethnic Malaysian population.However, demographic changes such as populationmigration, miscegenation causes new spectrum of inheritedhaemoglobin disorders to emerge. Therefore, it is importantto make a precise genotype diagnosis to facilitate error freecounselling and proper management HbS D disease. Acombined data from HPLC, electrophoresis at alkaline andacid pH, and the sickle solubility test enable definitiveidentification of HbA, HbF, HbS, HbC, and several others rarevariants.

ReFeReNCeS1. Lie-Injo LE, Hassan K, Joishy SK, Lim ML. Sickle cell anaemia associated

with alpha-thalassemia in Malaysian Indians. Am J Hematol 1986; 22:265-74.

2. Adachi K, Kim J, Ballas S, Surrey S, Asakura T. Facilitation of Hb Spolymerization by the substitution of Glu for Gln at beta 121. J Biol Chem1988; 263: 5607-10.

3. Mukherjee MB, Surve RR, Gangakhedhar RR, Mohanty D, Colah RB.Hemoglobin sickle D Punjab - a case report. Indian J Hum Genet 2005; 11:154-5.

4. Adekile A, Mullah AA, Akar NA. Does elevated hemoglobin F modulatethe phenotype in Hb SD-Los Angleles? Acta Haematol 2010; 123: 135-9.

5. Report by the Secretariat: Sickle cell anaemia.In Fifty-ninth world healthassembly World Health Organisation, 2006; A59/9: 1-5.

Fig. 1 : The sequence trace of the β globin gene above shows the codon 6 (GAG→GTG) and codon 121 (GAA →CAA) mutation inheritedby the patient.