Hemoglobin, 30 (3):393–396, (2006)Copyright © Taylor & Francis Group, LLCISSN: 0363-0269 print/1532-432X onlineDOI: 10.1080/03630260600755872

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LHEM0363-02691532-432XHemoglobin, Vol. 30, No. 3, May 2006: pp. 0–0Hemoglobin

SHORT COMMUNICATION

Hb SALLANCHES [a104(G11)Cys®Tyr, TGC®TAC (a2)]: AN

UNSTABLE HEMOGLOBIN VARIANT FOUND IN AN INDIAN CHILD

Hb Sallanches in an Indian ChildS. Dash et al.

Sumitra Dash,1 Keiko Harano,2 and Santosh Menon1

1Department of Haematology, Post Graduate Institute of Medical Education and Research, Chandigarh, India2Department of Clinical Nutrition, Kawasaki University of Medical Welfare, Kurashiki, Japan

� We report the fourth observation of Hb Sallanches [a104(G11)Cys →Tyr, TGC →TAC (a2)],an unstable a chain variant of intermediate severity in the homozygous state. Heterozygosity occa-sionally produces mild hypochromia and microcytosis in some patients. A balanced b/a ratio,found in previously reported cases, points to unstable ab dimers formed as a result of the Cys →Tyrsubstitution at the a1b1 contact site in this hemoglobin (Hb) variant. Our patient, and the previ-ous two of the three cases reported in patients of Pakistani origin, points to a common populationstock, separated by the mass population migration which occurred during the partition of Pakistanand India in 1947.

Keywords Hb Sallanches, Heinz body inclusions, Indian child

Case Report

When this 9-year-old Punjabi (north Indian) boy came to our attention,physical examination revealed pallor, jaundice, thalassemic bone alteration,an enlarged liver (2.5 cm below the costal margin) and spleen (5 cm belowthe costal margin). There was a history of jaundice episodes but no historyof any blood transfusions. The family history was not significant and he wasnot the product of a consanguineous marriage.

Received 9 December 2005, accepted 27 January 2006.Address correspondence to Professor Sumitra Dash, Department of Haematology, Post Graduate

Institute of Medical Education and Research (PGIMER), Sector-12, Chandigarh-160012, India; Tel.:+91-172-275-5126; Fax: +91-172-274-4401; E-mail: sumitradash2001@yahoo.com

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394 S. Dash et al.

Hematological analysis was carried out using standard techniques. DNAwas extracted by a chloroform-phenol method. DNA analysis was carriedout by Dr. Keiko Harano at the Department of Clinical Nutrition, KawasakiUniversity of Medical Welfare, Kurashiki, Japan. The β-globin gene wasamplified with Ex Taq DNA Polymerase (Takara Ex Taq, Start Version;Takara Bio Inc., Kyoto, Japan) and the α2 and α1 genes by the Hot StarTaqDNA Polymerase (Qiagen, Kyoto, Japan), using specific primer sets. Thepolymerase chain reaction (PCR) product was electrophoresed on agarosegel and the DNA was extracted by use of a spin column (Quantum PrepFreeze’N Squeeze DNA Gel Extraction Spin Column; Bio-Rad Laboratories,Hercules, CA, USA), Sequence analysis was performed using BigDye Termi-nator Cycle Sequencing Ready Reaction kits on an automatic ABI PRISM™3100 Genetic Analyzer (Applied BioSystems, Foster City, CA, USA). Com-mon deletional α-thalassemia (thal) genes were analyzed by the single tubemultiplex PCR method as described by Chong et al. (1).

The investigations revealed the following parameters for the boy: hemo-globin (Hb) 7.8 g/dL, MCV 93.8 fL, MCH 25.5 pg, and reticulocytes 75%.Hemoglobin electrophoresis at pH 8.6 showed a normal pattern. The HbA2 value was 2.65% and for Hb F <1.0%. The peripheral blood smearshowed mild anisopoikilocytosis with a marked increase in polychromaticred cells, occasional target cells and nucleated red cells. His total serumbilirubin was 6.3 mg/dL (indirect 4.9 mg/dL). The red cell G6PD levelswere normal and the direct Coombs test was negative. The red cell osmoticfragility test was normal. The Heinz body inclusion test showed that almost90% of the peripheral red cells were loaded with both coarse Heinz bodiesand Hb H-like inclusions (Figure 1). The heat and isopropanol tests forunstable Hb gave ambiguous results. Investigation of the parents showedhypochromic microcytic red cells only in the mother.

Analysis of the α-thal genes failed to detect any deletional mutationssuch as −α3.7, −α4.2, −α20.5, – –SEA, – –MED and – –FIL. Direct sequencing ofthe α2 gene revealed a TGC→TAC single base substitution at codon 104,changing the cysteine residue to tyrosine that corresponds to Hb Sallanches[α104(G11)Cys→Tyr], a variant of the α2 chain. The patient did not haveany normal sequence, making him a homozygote for Hb Sallanches. TheDNA sequence of the β-globin genes was also determined and there was noabnormality except for a heterozygosity for the IVS-II-16 (G→C) mutation(AvaII site).

Unstable Hb variants are rarely discovered in different populations. Anoccasional case in the Indian population has been described (2). Hb Sal-lanches was first discovered in a family of French origin (3) in 1995, andsubsequently, in two patients and their families who were of Pakistani origin(4,5). This is the first case of Hb Sallanches to be reported in a patient ofIndian origin. In this Hb variant, biosynthetic studies have indicated that

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Hb Sallanches in an Indian Child 395

α-Sallanches globin chains are formed and detected biosynthetically, butare either unsuitable for αβ dimer formation or form highly unstable αβdimers that precipitate, giving rise to Hb H-like inclusions.

The TGC→TAC mutation at codon 104 of the α2-globin gene replacesthe normally present cysteine residue with a tyrosine. Normally, cysteine atα104(G11) is located at the α1β1 contact site that is important for the pack-ing of the normal Hb tetramer. At this position, cysteine is situated at inter-acting distances with Ala28(B9), Arg31(B12), Met32(B13), Phe36(C1),Leu109(G16) of the same α chain, and Gln127(H5) and Gln131(H9) of thepartner β chain. The substitution of tyrosine at position α104 in α-Sal-lanches most probably disturbs this network of interaction, which in turn isexpected to impair the formation and/or stability of the αβ dimer.

Hb Sallanches in this North Indian Punjabi patient and in two previ-ously reported Pakistani families is not surprising, considering the commonbackground of both the populations and the mass population migrationthat took place in 1947 following the partition of India and Pakistan.

REFERENCES

1. Chong SS, Boehm CD, Higgs DR, Cutting GR. Single-tube multiplex-PCR screen for common dele-tional determinants of α-thalassemia. Blood 2000; 95(1):360–362.

2. Dash S. Report of a case of unstable haemoglobin. Ind J Haematol 1990; 8(1):123–124.3. Morle F, Francina A, Ducrocq R, Wajcman H, Gonnet C, Philippe N, Souillet G, Godet J. A new α

chain variant Hb Sallanches [α104(G11)Cys→Tyr] associated with Hb H disease in one homozy-gous patient. Br J Haematol 1995; 91(3):608–611.

FIGURE 1 Peripheral blood smear showing a marked increase in reticulocytes and Heinz body inclu-sions in the red cells (brilliant cresyl blue stain).

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4. Waye JS, Walker L, Chui DHK, Lafferty J, Kirby M. Homozygous Hb Sallanches [α104(G11)Cys→Tyr] in a Pakistani child with Hb H disease. Hemoglobin 2000; 24(4):355–357.

5. Khan SN, Butt FI, Riazuddin S, Galanello R. Hb Sallanches [α104(G11)Cys→Tyr]: a rare α2-globinchain variant found in the homozygous state in three members of a Pakistani family. Hemoglobin2000; 24(1):31–35.

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