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.

Association of plasma homocysteine level with vaso-occlusive crisis in sickle cell anemia patients of Odisha, India.

Vascular complications of sickle cell anemia (SCA) are influenced by many factors. Elevated plasma homocysteine (Hcy) is supposed to be an independent risk factor and is either genetic or nutritional origin. The present study evaluated the plasma Hcy level, MTHFR C677T gene polymorphism, effect of folic acid (FA) supplementation' and hemato-biochemical parameters in SCA and their effect on the vaso-occlusive crisis (VOC) in SCA patients of an Asian-Indian haplotype population. One hundred twenty cases of SCA (HbSS) and 50 controls with normal hemoglobin(HbAA) were studied. It was found that the plasma Hcy level is significantly higher (p < 0.0001) in patients with SCA (22.41 ± 7.8 µmol/L) compared to controls (13.2 ± 4.4 µmol/L). Moreover, patients without FA supplementation had a significantly (p < 0.001) higher Hcy level (27 ± 7 µmol/L) compared to those with supplementation (17.75 ± 5.7 µmol/L). Turkey-Kramer multiple comparison tests show that there is a significant difference (p < 0.05) in HbF percent, hemoglobin (Hb), platelet count, serum bilirubin (direct:Bil-D and total:Bil-T), aspartate transaminase (AST), lactate dehydrogenase (LDH), and plasma Hcy levels between mild and severe VOC. Between moderate VOC and severe VOC, there was a significant difference (p < 0.05) in HbF%, Bil-D, AST, Hcy. Pearson correlation revealed that plasma Hcy had a significantly (p < 0.05) positive correlation with AST, serum bilirubin (indirect and total), LDH, jaundice, stroke, VOC per year, and hospitalization per year whereas it was inversely correlated with HbF percentage, Hb level, and FA treatment. In the study population, increased plasma Hcy level, hemolysis, and platelet activation were found to influence VOC in SCA.

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.

Hb Tianshui (HBB: C.119A > G) in Compound Heterozygosity with Hb S (HBB: C.20A > T) from Odisha, India.

We describe here a rare ß-globin gene variant, Hb Tianshui [ß39(C5)Glu→Arg; HBB: c.119A > G], detected during routine screening in Odisha, India. This is the second report of Hb Tianshui and the first to describe the cation exchange high performance liquid chromatography (HPLC) and DNA studies of two cases of this variant. Both cases had coinherited Hb S (HBB: c.20A > T) but none presented with typical symptoms of sickle cell disease. One of the cases was heterozygous for a common α-thalassemia (α-thal) allele (-α(3.7)) (rightward) (NG_000006.1: g.34164_37967del3804) and marginally raised Hb F percentage, while the other Hb S/Hb Tianshui case was completely benign and healthy. An atypical Asian Indian haplotype [+ - + - +] could be assigned to the Hb Tianshui variant. Hb Tianshui seems to mimic a few other Hb variants in cation exchange HPLC. However, we report two specific patterns in the chromatograms that are characteristic to Hb Tianshui. Combining an alkaline electrophoresis result with cation exchange HPLC at screening would be preferred to detect this rare variant, especially in regions with considerable frequency of Hb E [ß26(B8)Glu→Lys; HBB: c.79G > A] or Hb S.

First Report of Hb Limassol [ß8(A5)Lys→Asn; HBB: c.27G>C] from Odisha, India.

In this short report, we describe the clinical presentation of a rare hemoglobin (Hb) variant, Hb Limassol [ß8(A5)Lys→Asn; HBB: c.27G>C] with a faster electrophoretic mobility than Hb A and that elutes in the P3 window on cation exchange high performance liquid chromatography (HPLC). This sequence variation at codon 8 (AAG>AAC) of the HBB gene was found in the four heterozygous cases, all of whom were clinically asymptomatic.

Inheritance of Hereditary Persistence of Fetal Haemoglobin (HPFH) in a Family of Western Odisha, India.

Hereditary persistence of foetal haemoglobin (HPFH) is a rare inherited haemoglobin disorders in India. We encountered five cases of HPFH-3 in heterozygous condition in a single family of western Odisha, India. All the cases had raised % HbF (26.1±3.23%) with pancellular distribution of HbF in erythrocytes. There were no abnormalities found in the red cell indices. All the cases were asymptomatic till date with normal growth and development. Molecular confirmation of this haemoglobin disorders is important for control and prevention of haemoglobinopathies in this region.

Influence of Sickle Cell Gene on the Allelic Diversity at the msp-1 locus of Plasmodium falciparum in Adult Patients with Severe Malaria.

Although several studies have supported that sickle cell trait (HbAS) protects against falciparum malaria, the exact mechanism by which sickle gene confers protection is unclear. Further, there is no information on the influence of the sickle gene on the parasitic diversity of P. falciparum population in severe symptomatic malaria. This study was undertaken to assess the effect of the sickle gene on the parasite densities and diversities in hospitalized adult patients with severe falciparum malaria. The study was carried out in 166 adults hospitalized subjects with severe falciparum malaria at Sickle Cell Clinic and Molecular Biology Laboratory, Veer Surendra Sai Institute of Medical Sciences and Research, Burla, Odisha, India. They were divided into three groups on the basis of hemoglobin variants HbAA (n=104), HbAS (n=30) and HbSS (n=32). The msp-1 loci were genotyped using a PCR-based methodology. The parasite densities were significantly high in HbAA compared to HbAS and HbSS. The multiplicity of infection (MOI) and multi-clonality for msp-1 were significantly low in HbSS and HbAS compared to HbAA. The prevalence of K1 (p<0 .0001) and MAD20 (p=0.0003) alleles were significantly high in HbAA. The RO33 allele was detected at a higher frequency in HbSS and HbAS, compared to K1 and MAD20. Sickle gene was found to reduce both the parasite densities and diversity of P. falciparum in adults with severe malaria.

Low and fixed dose of hydroxyurea is effective and safe in patients with HbSß(+) thalassemia with IVS1-5(G→C) mutation.

BACKGROUND: Despite compelling evidence that hydroxyurea is safe and effective in sickle cell disease, it is prescribed sparingly due to several barriers like knowledge gaps in certain genotypes, apprehension about its safety and toxicity, and limited resources. We undertook this study to find out the efficacy and safety of HU in patients with HbSß(+) -thalassemia with IVS1-5(G→C) mutation. PROCEDURE: We registered 318 patients with HbSß(+) -thalassemia with IVS1-5(G→C) mutation. Of these, 203 were enrolled for hydroxyurea treatment at a low and fixed dose of 10 mg/kg/day. One hundred four patients (Group-I: 37 children and Group-II: 67 adults) with ≥2 years of hydroxyurea treatment were studied. RESULTS: The rate of vaso-occlusive crises, requirement of blood transfusion and rate of hospitalization reduced from 3 to 0.5, 1 to 0 and 1 to 0 in Group-I and 3 to 0, 1 to 0 and 0.5 to 0 in Group-II respectively after HU therapy (P < 0.0001). %HbF level, hemoglobin, MCV and MCH increased significantly, whereas HbS, WBC, platelet count, serum-bilirubin and LDH levels decreased significantly after HU therapy. It has been observed that along with fairly subtle hematological changes following HU therapy, there was a substantial clinical improvement occurred in these patients. Transient myelotoxicity was observed in 4.8%. There was minimal gonadal toxicity without affecting reproductive function. CONCLUSION: In view of easy affordability, better acceptability, minimal toxicity, the need of infrequent monitoring and its potential effectiveness, low and fixed dose of hydroxyurea is suitable for treatment of patients with HbSß(+) -thalassemia in resource poor setting.

Prevalence of deletional alpha thalassemia and sickle gene in a tribal dominated malaria endemic area of eastern India.

Inherited hemoglobin disorders like alpha thalassemia and sickle gene are common in the Indian subcontinent. These disorders in the heterozygous state act as malaria resistance genes and influence the susceptibility to Plasmodium falciparum malaria. There is inadequate knowledge about the epidemiology of these malaria resistance genes in the tribal dominated malaria endemic region of the state of Odisha in eastern India. A cross sectional prevalence study was undertaken in 594 subjects in five tribal populations in this region, namely, Sahara (42.4%), Kutia Kandha (30.0%), Kuda (15.8%), Gond (9.8%), and Oraon (2.0%). Sickling test, Hb electrophoresis, HPLC, and molecular studies were undertaken to diagnose the prevalence of sickle allele, ß -thalassemia allele, and deletional alpha thalassemia. Sickle and ß thalassemia alleles were found in 13.1% and 3.4% of subjects, respectively. Sickle allele was found both in heterozygous (10.1%) and homozygous state (3.03%). The prevalence of alpha thalassemia was 50.84% with an allelic frequency of 0.37. Both α (-3.7) and α (-4.2) alpha thalassemia were detected with an allele frequency of 0.33 and 0.04, respectively. The high prevalence of alpha thalassemia and sickle gene in this population is probably due to selection pressure of endemic malaria in this part of India.

The effect of hydroxyurea on compound heterozygotes for sickle cell-hemoglobin D-Punjab--a single centre experience in eastern India.

BACKGROUND: Although hydroxyurea is the only effective agent for the treatment of sickle cell disease, published experience with this drug is limited to treatment of homozygous sickle cell anemia and HbS/ß thalassemia. The role of hydroxyurea in the treatment of patients with HbSD-Punjab, a rare hemoglobinopathy with phenotypic expression similar to that of sickle cell anemia is unknown. PROCEDURE: Over a period of 10 years, we followed 42 patients with HbSD-Punjab, of which 20 presented with severe clinical manifestations (≥3 episodes of VOC and/or ≥2 units of blood transfusion in the previous 12 months). These 20 patients were enrolled for treatment with hydroxyurea at a dose of 10 mg/kg/day and followed prospectively for a period of 24 months. RESULTS: The frequency of VOC decreased significantly and none of them required blood transfusion while receiving hydroxyurea. The HbF, total hemoglobin, MCV, MCH, and MCHC levels increased significantly, whereas HbS, WBC, platelet count, total serum bilirubin, and LDH levels decreased significantly in all the patients. No short-term drug toxicity was observed. CONCLUSION: This study describes the use of hydroxyurea therapy in patients with HbSD-Punjab. Low dose hydroxyurea (10 mg/kg/day) was found to be effective in reducing the clinical severity in patients with HbSD-Punjab without any short-term toxicity. In view of easy affordability amongst poor patients, widespread acceptability by patients and doctors, the need of infrequent monitoring and its potential effectiveness, low dose hydroxyurea is suitable for treatment of patients with HbSD-Punjab.