Clinical, hematologic and molecular variability of sickle cell- β thalassemia in western India

BACKGROUND: Sickle cell- β thalassemia (HbS- β thalassemia) is a sickling disorder of varying severity, which results from compound heterozygosity for sickle cell trait and β thalassemia trait. The present study was undertaken to determine the genetic factors responsible for the clinical variability of HbS- β thalassemia patients from western India. MATERIALS AND METHODS: Twenty-one HbS- β thalassemia cases with variable clinical manifestations were investigated. The α and β globin gene clusters were studied by molecular analysis. RESULTS: Thirteen patients showed milder clinical presentation as against eight patients who had severe clinical manifestations. Four β thalassemia mutations were identified: IVS 1-5 (G  C), codon 15 (G  A), codon 30 (G  C) and codon 8/9 (+G). α thalassemia and XmnI polymorphism in homozygous condition (+/+) were found to be common among the milder cases. The β S chromosomes were linked to the typical Arab-Indian haplotype (#31). Framework (FW) linkage studies showed that four β thalassemia mutations were associated with different β globin gene frameworks. Linkage of codon 15 (G  A) mutation to FW2 is being observed for the first time. CONCLUSION: The phenotypic expression of HbS- β thalassemia is not uniformly mild and α thalassemia and XmnI polymorphism in homozygous condition (+/+) are additional genetic factors modulating the severity of the disease in the Indian subcontinent.


Introduction
The β S mutation is one of the most common single gene mutations in man and has a widespread geographic year, liver and spleen size were recorded. Ten milliliters of blood was collected in ethylenediaminetetraacetic acid (EDTA) after informed consent was obtained from all subjects. Blood samples for hematologic evaluation were collected at least 30 days after the last transfusion.
RBC indices were measured on an automated blood cell counter (Syxmex K-1000). Hemoglobin electrophoresis and quantitation of HbA 2 was done using alkaline cellulose acetate electrophoresis at pH 8.9 and elution. [6] The HbF level was quantified by the alkali denaturation method of Singer et al. [7] Molecular analysis was carried out in 19 HbS-β thalassemia cases. DNA was isolated from peripheral blood leukocytes using the standard phenol-chloroform method. The HbS and β thalassemia mutations were characterized by reverse dot blot hybridization, [8] while α globin genotype, β S haplotype and the presence of a CT mutation at position -158 of the G γ gene (XmnI polymorphism) were determined as described earlier. [9] The framework (FW) analysis was done by Denaturing gradient gel electrophoresis (DGGE) analysis. [10]

Results
The clinical, hematologic and molecular data of these cases are summarized in Table 1. The age at presentation varied from 6 months to 18 years.
Seven HbSβ thalassemia cases in the tribal group had a milder clinical presentation, whereas 8 of the 14 cases (57.2%) in the nontribal group had severe clinical manifestations with a history of vaso-occlusive crisis (3-8 per year) in the form of acute pain in the joints, abdomen, bones and chest and they were also dependent on regular blood transfusions. Some of these patients had infections, usually in the form of high grade fever and also required hospitalization for their painful crisis. Although hepatosplenomegaly was observed in both mild and severe cases, however, splenomegaly was more common in severe cases (87.5%) as compared to milder cases (53.8%).
Most of the patients had microcytic, hypocromic anemia with low Hb, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) levels.
However, the mean Hb levels among the tribals were    for α thalassemia.

Discussion
Silvestroni and Bianco [11] were the first to describe the compound heterozygosity (β S /β thal ) for the sickle gene and a β thalassemia gene, and since then, HbS-β thalassemia has been reported in different ethnic groups. [4] In India, the incidence of the β S gene varies from 0 to 40% and the relatively high frequency of β course. [12] The four different β thalassemia mutations found in the present study were broadly similar to the distribution observed in Indians. [13] Among these, IVS 1-5 (GC), a severe β + thalassemia allele was found thalassemia involving an AT transition at codon 132 of the β-globin gene had severe clinical manifestations. [16] The effects of β thalassemia mutations on the clinical severity of HbS-β thalassemia are well documented. Perseu et al. [17] have reported that patients with HbS-β + thalassemia had a mild to moderate presentation, while those with HbS-β 0 thalassemia showed more heterogeneity in clinical manifestations. Similarly, Schiliro et al. [18] and Mirabile et al. [19] have also shown that the type of β thalassemia alleles had an influence on the phenotypic expression of the disease. On the other hand, Nadkarni et al. [20] did not find any effect of β thalassemia mutations on the disease severity in the Indian thalassemic patients. Although our patients have inherited either severe β 0 or β + thalassemia mutations, the clinical presentation is quite variable and this could partially be explained by the association of α thalassemia (9/11) and XmnI polymorphism, either in homozygous or heterozygous state. Our earlier studies in sickle homozygous individuals from this region have also shown that α thalassemia is the major modulator of the severity of the disease, being more prevalent among tribals with a milder disease than among nontribals with more severe manifestations. [9] In the present study, the IVS 1-5 ( GC), codon 30 (GA) and codon 8/9 (+G) mutations were found to be associated with FW3a and 1, which is similar to the earlier observations from India. [10] This probably indicates a common origin of these mutations in Indian populations. The codon 15 (GA) mutation was earlier reported to be linked with FW1 and 3a; [10] however, we observed an association of this mutation with FW2 also in a tribal individual and this raises the possibility of a new independent origin of this mutation.
All the β S chromosomes in the present study were found to be linked to the typical Arab-Indian haplotype.
These results are consistent with earlier reports from India [5] and further confirm the earlier observations that the origin of the β S mutation in India is unicentric. [21] We conclude that the clinical manifestations of Indian HbS-β thalassemia patients are influenced by associated α thalassemia and XmnI polymorphism rather than β thalassemia mutations.