Effect of deletions in the α-globin gene on the phenotype severity of ß-thalassemia.

Thalassemia is the most common inherited hemoglobinopathy worldwide. Variation of clinical symptoms in this hemoglobinopathy entails differences in disease-onset and transfusion requirements. The aim of this study was to investigate the role of α-globin gene deletions in modulating the clinical heterogeneity of ß-thalassemia (ß-thal) syndromes. A total number 270 ß-thal subjects were enrolled. Hematological parameters were recorded. ß-Globin mutations were determined by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR), gap-PCR and Sanger sequencing. α-Globin gene deletions were determined by multiplex PCR. Out of 270 ß-thal subjects, 19 carried ß+/ß+, 74 had ß0/ß0 and 177 had the ß0/ß+ genotype. When we determined the severity of the different ß-thal subjects in coinherited with the α gene deletion, it was revealed that, 84.2% ß+/ß+ subjects carried a non severe phenotype and did not have an α gene deletion. Of the ß0/ß0 individuals, 95.9% presented a severe phenotype, irrespective of α-globin gene deletions. In cases with the ß0/ß+ genotype, 19.2% subjects also carried a deletion on the α gene. Of these, 61.8% presented a non severe phenotype and 38.2% were severely affected. Only in the ß0/ß+ category did α gene deletions make a significant contribution (p < 0.001) toward alleviation of clinical severity. Therefore, it can be stated that α-globin gene deletions play a role in ameliorating the phenotype in patients with a ß+/ß0 genotype.

Post-transfusion hepatitis C virus infection among ß-thalassaemic individuals with associated clinical parameters.

Background & objectives: Multiple transfusions in ß-thalassaemia patients undergoing regular transfusion regimen are at a risk of developing transfusion transmitted infections, including hepatitis C virus (HCV). The present study was conducted to investigate the association of HCV viraemia and genotype with clinical parameters in HCV seroreactive ß-thalassaemic individuals. Methods: A total of 172 HCV seroreactive ß-thalassaemic individuals aged between 2-35 yr with at least 25 units of blood transfusion were catagorized into four groups (2-12 yr, group 1; 13-19 yr, group 2; 20-29 yr, group 3; 30-35 yr, group 4). Aged matched control samples (n=87; ß-thalassaemics without HCV infection) were also included. HCV RNA was detected by nested reverse transcriptase polymerase chain reaction (RT-PCR) based on 5' UTR of HCV genome, viral load was determined by real-time RT-PCR. Nested RT-PCR amplified partial core region was used for DNA sequencing. Liver function parameters [serum total bilirubin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] were also determined. Results: Of the 172 HCV seroreactive individuals, 59.30 per cent (n=102) were HCV RNA positive. HCV viral load ranged from 173 to 32.04×10[5] IU/ml; 87.65 per cent were infected with HCV genotype 3. Liver enzymes, such as ALT, AST and serum total bilirubin were significantly elevated in all age groups compared to control groups. Serum ferritin levels were found to be high in all individuals, but 16.27 per cent of HCV-infected individuals with >10,000 IU/ml viral load also showed high ferritin levels (>1500 µg/l) where the majority of them were infected with HCV genotype 3. Interpretation & conclusions: HCV genotype 3 was the major circulating genotype among ß-thalassaemia patients in this region. Our findings indicated an association between HCV replication and hepatic iron load and also highlighted the need for sensitive quantitative RT-PCR-based detection of HCV RNA in the high risk population.

Association of TNF-alpha (-308 A/G) and IFN-gamma (+874 A/T) gene polymorphisms in response to spontaneous and treatment induced viral clearance in HCV infected multitransfused thalassemic patients.

BACKGROUND: Multitransfused thalassemic individuals are at high risk of developing transfusion transmitted Hepatitis C virus (HCV) infection. The aim of the study was to correlate the effects of host cytokine single nucleotide polymorphisms of TNF-α (-308 A/G) and IFN-γ (+874 A/T) in spontaneous or IFN induced treatment response in the HCV infected thalassemic individuals. METHODS: A total of 427 HCV sero-reactive thalassemic individuals were processed for HCV viral genomic diversity and host gene polymorphisms analysis of TNF-α (-308 A/G) and IFN-γ (+874 A/T). RESULTS: Out of 427 HCV sero-reactive individuals, 69.09% were found to be HCV RNA positive with genotype 3 as the predominant infecting strain (94.29%). Study highlighted that, A allele was significantly associated with (p < .05) spontaneous clearance of HCV infection and G allele was correlated with viral persistence at TNF-α (-308) gene polymorphism. Whereas in case of IFN-γ (+874) SNPs, A allele was significantly responsible (p < .05) for spontaneous clearance than T allele. Our study also indicated that in relapsed cases, IFN-γ (+874) T allele is more responsible than A allele. Though no significant correlation was found at both TNF-α (-308) and IFN-γ (+874) gene polymorphism among SVR and relapsed thalassemic patients. CONCLUSION: A allele at both TNF-α (-308) and IFN-γ (+874) were strongly associated with spontaneous clearance among this population. But in case of SVR and relapsed cases no significant association was found. This cytokine gene polymorphisms pattern will help clinicians to take an informed decision about therapeutic management of HCV infected thalassemic individuals.

Comparative study of the methods of extracting mesenchymal stem cells from cryopreserved Wharton's Jelly.

The Wharton's Jelly (WJ) is an established source of mesenchymal stem cells (MSC). We compared 3 methods of extracting WJ-MSC from cryopreserved tissue and determined that enzymatic digestion of the WJ yielded the most viable MSC, compared to the explant and mechanical digestion methods. The enzymatically-released WJ-MSC conformed to the International Society for Cellular Therapy (ISCT) criteria: displayed plastic-adherence, co-expressed CD73, CD90, CD105 and were negative for hematopoietic lineage cell markers.

Interferon λ3 gene (IL28B) is associated with spontaneous or treatment-induced viral clearance in hepatitis C virus-infected multitransfused patients with thalassemia.

BACKGROUND: Hepatitis C virus (HCV) is the major posttransfusion infection in multitransfused individuals in India with thalassemia major. To our knowledge, this study is the first conducted to correlate and comprehend the effects of the host interleukin (IL)28B gene polymorphism at loci rs12979860 and rs8099917 in spontaneous or interferon (IFN)-induced treatment response in the HCV-seroreactive individuals with thalassemia major. STUDY DESIGN AND METHODS: A total of 557 HCV-seroreactive individuals with thalassemia were processed for HCV viral genotyping and host IL28B single-nucleotide polymorphism analysis at loci rs12979860 and rs8099917. RESULTS: Of 557 individuals, 70.92% were found to be HCV RNA positive with Genotype 3 (95.18%) as predominant strain. A favorable CC allele at locus rs2979860 and TT allele at rs8099917 were 75.31 and 77.16%, respectively, which was strongly associated with spontaneous clearance of infection (p < 0.05). Of 85 IFN-treated cases, 56 achieved sustained virologic response (SVR) whereas 27 were relapsed cases. Among these patients who achieved SVR, a favorable CC/TT allele at rs12979860/rs8099917 was found to be predominant with 76.79 and 66.07%, respectively, whereas in the case of relapsed patients, unfavorable CT (55.56%) and TG (59.26%) alleles were found to be predominant. Additionally, low serum ferritin level was significantly associated with SVR. CONCLUSION: CC at rs12979860 and TT at rs8099917 was strongly associated with spontaneous clearance and SVR in the population with thalassemia. Low age group and low serum ferritin level are important cofactors. This allelic pattern will aid clinicians in making an informed decision about prognosis and therapeutic management.

Cross-Sectional Study for the Detection of Mutations in the Beta-Globin Gene Among Patients with Hemoglobinopathies in the Bengali Population.

AIMS: Thalassemia is a common autosomal recessive blood disorder, which is most prevalent in South East Asian and Mediterranean populations. It is considered as a major health burden in the Indian population. The aims of the present study were to investigate the common, as well as uncommon, mutations responsible for thalassemia in the Bengali population. METHODS: The Bengali state was divided into four sampling zones. Mutation detection was done using Sanger sequencing of the HBB gene. RESULTS: A total of 14 different mutations were observed, including rare mutations IVS1-130(G>C), IVS1-129(A>C), -90(T>C), CD16(-C), -30(T>C), CD15(-T), and a novel mutation CD53(C>T). The frequencies of IVS1-5(G>C) and CD26(G>A) mutations were higher than other mutations. There were also some silent polymorphisms found in the studied group, CD3(T>C), CD10(C>A), IVSII-16(G>C), IVSII-74(T>G), -42(C>G). CONCLUSION: The present study is the first attempt to screen for ß-thalassemia-causing mutations by direct sequencing in different districts of West Bengal. The information obtained from the present study may be helpful for thalassemia management and prenatal mutation detection.

Hb Midnapore [ß53(D4)Ala→Val; HBB: c.161C>T]: A Novel Hemoglobin Variant with a Structural Abnormality Associated with IVS-I-5 (G>C) (HBB: c.92+5G>C) Found in a Bengali Indian Family.

We describe a novel C>T substitution at codon 53 of the HBB gene (HBB: c.161C>T). The proband was a transfusion-dependent ß-thalassemia major (ß-TM) patient. DNA was extracted and subsequently, DNA sequencing was done to detect the mutations on the HBB gene. Capillary zone electrophoresis (CZE) revealed the presence of an unknown peak. She inherited this mutation from her grandmother through her mother. This mutation exists in cis with the common ß0 mutation IVS-I-5 (G>C) (HBB: c.92+5G>C). The proband is homozygous for HBB: c.92+5G>C and needs monthly transfusions. On the other hand, her grandmother, mother and sister all possess this novel mutation cis with the heterozygous HBB: c.92+5G>C. They are carriers not thalassemic. This mutation produces the substitution ß53(D4)Ala→Val; HBB: c.161C>T, a new structural hemoglobin (Hb) variant. As this variant was identified in a Bengali family from Paschim Midnapore district of West Bengal, India, it has been designated as Hb Midnapore. This variant has now been reported to the HbVar database.