Haptoglobin Genotypes Associated with Vaso-Occlusive Crisis in Sickle Cell Anemia Patients of Eastern India.

Sickle cell anemia is hallmarked by hemolysis, which releases hemoglobin (Hb) into the plasma promoting vaso-occlusive crisis (VOC). Haptoglobin (Hp) clears free Hb and decreases Hb-related pathophysiology in sickle cell anemia. There are two alleles (HP1 and HP2) and three genotypes (HP1-1, HP1-2 and HP2-2) of Hp with different frequencies in different populations. This study involved Hp level and genotype among normal and sickle cell anemia patients with varying severity of VOC. A total of 297 sickle cell anemia patients and 98 healthy controls were selected for the study. The sickle cell anemia patients were categorized as 'mild-phenotype' with no pain episodes and 'severe-phenotype' as having three or more acute pain episodes in the preceding 12 months. The Hp level was significantly lower (p < 0.001) in sickle cell patients anemia than controls; HP1-1 genotype had a higher Hp level compared to HP1-2 and HP2-2 (p < 0.05). Turkey-Kramer multiple comparison tests showed that mild and severe phenotypes have significant differences (p < 0.05) in Hb F%, Hb, platelet count, aspartate aminotransferase (AST), alanine aminotransferase (ALT), direct-bilirubin (Bil-D), total-bilirubin (Bil-T), lactate dehydrogenase (LDH) and Hp level. Pearson correlation revealed that Hp level has a positive (p < 0.05) correlation with Hb F%, Hb, packed cell volume (PCV) and serum urea; in contrast its level is negatively correlated with AST, ALT, Bil-T and LDH. A significantly higher frequency of HP2 allele and HP2-2 genotypes was found in severe phenotypes. In the studied population, it was found that higher HP2 frequency, low Hp level and more hemolysis favors the onset of VOC in sickle cell anemia.

Profiling of 35 Cases of Hb S/Hb E (HBB: c.20A>T/HBB: c.79G>a), Disease and Association with α-Thalassemia and ß-Globin Gene Cluster Haplotypes from Odisha, India.

Hb S/Hb E (HBB: c.20A>T/HBB: c.79G>A) is an uncommon variant of sickle cell disease resulting from coinheritance of Hb S and Hb E. Clinico-hematological and biochemical parameters of 35 cases of Hb S/Hb E disease were studied and compared with 70 matched cases of homozygous sickle cell disease (Hb SS) and Hb S/ß-thalassemia (ß-thal) with IVS-I-5 (G>C) (HBB: c.92+5G>C). The influence of α-thal and that of of ß-globin gene cluster haplotypes among Hb S/Hb E disease was also studied. Statistical analysis was done using GraphPad InStat version 3.06. Of the 35 cases, 20 (57.14%) had a moderate clinical presentation. Mean lactate dehydrogenase (LDH) level, vaso-occlusive crises (VOCs) per year, and annual blood transfusion requirements were significantly lower in Hb S/Hb E cases than in the other two groups. The hemoglobin (Hb) and packed cell volume (PCV) levels were significantly high in Hb S/Hb E cases with α-thal and these cases were associated with microcytic-hypochromic anemia. α-Thalassemia did not influence clinical presentation in Hb S/Hb E cases. The ß-globin gene cluster haplotypes of 70 alleles of Hb S/Hb E revealed an association of five typical haplotypes [Arab-Indian (A-I), Benin, Bantu, Cameroon and Senegal] in 95.71% cases. Hb S/Hb E disease exhibit asymptomatic to moderate phenotypic expression. However, further in-depth studies on Hb S/Hb E will help in reducing the disease burden especially in high-risk countries like India.

Influence of rs1042713 and rs1042714 polymorphisms of ß2-adrenergic receptor gene with erythrocyte cAMP in sickle cell disease patients from Odisha State, India.

The vaso-occlusive crisis (VOCs) in sickle cell disease (SCD) is often associated with stress. Epinephrine released during stress acts via beta 2-adrenergic receptors (ß2-AR or ADRB2) to stimulate the synthesis of cyclic adenosine monophosphate (cAMP) in the red blood cells (RBCs). Higher cAMP levels promote adhesion of sickled RBCs to vascular endothelium, a major contributor for VOCs. Several single-nucleotide polymorphisms (SNPs) of the ß2-AR gene have been reported; two of them at codon 16 (rs1042713) and codon 27 (rs1042714) have been extensively studied for their clinical relevance. Therefore, we assessed the influence of polymorphism at these two sites of the ß2-AR gene on the RBC cAMP concentrations with and without epinephrine stimulation in SCD subjects. We determined the frequency distribution of different genotypes of codon 16 and codon 27 of the ß2-AR gene using the Sanger sequencing method in the SCD subjects. We measured the RBC-cAMP levels at baseline and after stimulation with epinephrine, to ascertain the influence of different genotypes in determining cAMP levels. There was no difference in the socio-demographic and hematological indicators in different genotypes of both codon 16 and 27. In the sham-treated erythrocytes, the cAMP levels were significantly different with three genotypes of codon 16 (F = 3.39, P = 0.036; one way ANOVA) but not with different genotypes of codon 27. A significant increase in cAMP levels was noticed with epinephrine treatment in all genotypes of codons 16 and 27 (P = 0.001; Wilcoxon signed-rank test). However, the extent of increase in the epinephrine-treated cAMP values from the sham-treated (baseline) cAMP values was significantly different between the three genotypes of codon 16 (H = 8.74; P = 0.012; Kruskal-Wallis test) but not in codon 27 genotypes. Polymorphism in codon 16 (rs1042713) of the ß2-AR gene influences cAMP concentrations in the RBC both before and after epinephrine treatment. Higher cAMP levels may lead to increased adhesion of sickle cell RBCs to vascular endothelium and may increase the frequency of VOCs.

Compound Heterozygote of Hb S (HBB: c.20A>T)/Hb Westdale (HBB: c.380_396delTGCAGGCTGCCTATCAG): Report of Four Cases from Odisha State, India.

We report four cases of compound heterozygotes for Hb S (HBB: c.20A>T) and a rare ß0-thalassemia (ß0-thal) mutation, Hb Westdale (HBB: c.380_396delTGCAGGCTGCCTATCAG), characterized by a 17 bp deletion between codons 126 to 131 in exon 3 of the ß-globin gene of human hemoglobin (Hb) confirmed by direct ß-globin gene sequencing. All four cases were from four unrelated families belonging to the Agharia caste, an endogamous ethnic community of the Sundargarh and Jharsuguda districts of Odisha State, India. Detailed observations indicated that all four cases of Hb S/Hb Westdale were clinically severe. On family screening, six family members were found to be heterozygous for Hb Westdale and were asymptomatic. Deletional α-thalassemia (α-thal) and XmnI polymorphism were studied for all the Hb Westdale cases. The Hb S/Hb Westdale cases had an early median age at onset of symptoms and presentation, more requirement of blood transfusions, splenomegaly and hepatomegaly and were found to be clinically more severe when compared with the Hb S-ß-thal with IVS-I-5 (G>C) (HBB: c.92 + 5G>C) cases. Overall, the findings indicate that this rare and hitherto unreported compound heterozygosity of Hb S/Hb Westdale is a clinically significant hemoglobinopathy and its finding in a large endogamous community of Odisha State, India will have important implication in the epidemiology and understanding of the clinical spectrum of sickle cell disease in Indian context and prenatal diagnosis.