Background: HCV infection is very common in multi-transfused β-thalassemia patients who need regular blood transfusions. Aim: The study was conducted to determine the epidemiology of HCV in multi-transfused β-thalassemia patients in West Bengal, India. Methods: Over a span of six years, blood samples were collected from HCV sero-reactive β-thalassemia patients and processed for viral RNA isolation followed by nested RT-PCR for qualitative viremia detection. The HCV genotype was determined by amplifying the partial HCV core gene by nested RT-PCR followed by DNA sequencing and NCBI genotyping tools. Phylogenetic and phylogeographic studies were performed with MEGA-X and BEAST software, respectively. Results: Out of 917 multi-transfused HCV sero-reactive β-thalassemia patients, 598 (65.21%) were HCV RNA positive while 250 (41.80%) had spontaneously cleared the virus. A significant percentage of male patients from rural areas (p = 0.042) and economically backward class (p = 0.002) were at higher risk of HCV infection. Female thalassemia patients and individuals belonging to ages 11–15 years had higher chances of spontaneous clearance. The most prevalent circulatory HCV genotype was 3a (78.26%) followed by 1b (12.04%). Phylogeographic analyses revealed that the 3a strains share genomic similarities with strains from Pakistan, Sri Lanka, and Thailand, whereas the 1b strains share similarities with strains from Thailand, Vietnam, Russia, and China. Uncommon HCV subtypes 3g and 3i were also detected. Conclusion: The high prevalence of HCV infection among β-thalassemia patients of West Bengal, India indicates NAT-based assays should be implemented for HCV screening in donor blood to eliminate HCV by 2030.

Hemoglobinopathies are the most common monogenic genetic disorders, primarily managed through blood transfusions or bone marrow transplantation. Clinical severity other than mutational effect is not well investigated and still unknown. This study aimed to identify dysregulated molecular pathways in red blood cells (RBCs) contributing to thalassemia severity. From a cohort of 285 patients with hemoglobinopathy, 10 age-matched individuals with identical compound heterozygous mutations (IVS 1-5 G>C and CD 26 G>A) were screened. Five had severe thalassemia requiring regular transfusions, whereas 5 had a nonsevere form requiring fewer transfusions. RNA sequencing and proteome analysis were conducted on isolated RBCs using NovaSeq and Orbitrap mass spectrometry platforms, respectively. Bioconductor R and different bioinformatics tools were used subsequently. Integrated transcriptome-proteome analysis revealed a global loss of messenger RNA–protein concordance. CDK11A and MCTS1 lost positive correlation, whereas RLP38 and H3C1 showed compensatory overtranslation, linked to transcription factors regulating erythropoiesis. In transfusion-dependent thalassemia (TDT), WNK3, HNRNPUL1, COPS7A, and HTATSF1 displayed discordant expression, indicating posttranscriptional aberrations. Protein-to-transcript ratio analysis showed reduced cytoskeletal (ankyrin, spectrin) expression, elevated chaperone activity, elevated ferroptosis markers (ferritin heavy chain 1, ferritin light chain, heme oxygenase-1), and suppressed autophagy. Collectively, these multilayered alterations, including splicing dysfunction, posttranscriptional deregulation, ferroptosis, autophagy suppression, oxidative stress, and cytoskeletal fragility, underlie the greater disease severity observed in TDT than non–transfusion-dependent thalassemia.

Background: Post transfusion acquired HCV infection is common in high-risk group individuals such as multi-transfused β-thalassemia patients who depend on regular blood transfusions. This study was conducted to determine epidemiology and distribution of HCV in multi-transfused β-thalassemia patients, in West Bengal, India. Methods: Over a span of six years blood samples were collected from HCV sero-reactive β-thalassemic patients and processed for viral RNA isolation followed by nested RT-PCR for qualitative viremia detection. HCV genotype was determined by amplifying partial HCV core gene by nested RT-PCR, DNA sequencing and using NCBI genotyping tools. Phylogenetic and phylogeographic studies were performed with online MEGA-X and BEAST 1.10.0 software respectively. Results: Out of 917 multi-transfused HCV sero-reactive β-thalassemic patients, 598 (65.21%) were positive for HCV RNA while 250 (41.80%) had spontaneously cleared the virus. Female thalassemic patients and individuals belonging to ages 10-14 years had higher chances of spontaneous clearance. The most prevalent circulatory HCV genotype was 3a (78.11%) followed by 1b (12.20%). Phylogeographic analyses revealed that the 3a strains share similarity with Pakistan, Sri Lanka and Thailand whereas the 1b strains share similarity with Thailand, Vietnam, Russia and China. Conclusion: The prevalence of HCV infection is very high among Indian β-thalassemic patients, necessitates a critical look into the prevailing transfusion practices and requires implementation of more rigid donor screening criteria to decrease the rate of transfusion transmitted HCV infection, especially in multi-transfused thalassemic patients. The use of more sensitive NAT based assays for HCV detection in donor blood is a compressing need of the hour.

Thalassemia is a common monogenic autosomal disorder prevalent in India. HbE beta thalassemia is a compound heterozygous state of two different beta globin mutations (HBB), predominant in the Eastern India. In HbE-beta thalassemia (β⁺/β°) patients, one HBB mutation does not produce any functional protein (β°); another mutation produces a structural altered haemoglobin, variant E (β⁺). It has been observed that in HbE-beta thalassemia patients with same type of HBB mutations, there exist diverse phenotypic presentation ranging from very severe, with regular transfusion-dependence to clinically non severe requiring very less transfusion or no transfusion. To explore this phenotypic mystery, we performed trio based expanded whole exome sequencing (WES) of two extreme phenotypes of HbE beta thalassemia subjects with similar HBB genotype and their parents. For WES library preparation, Agilent Sure select V6+UTR kit was used and sequencing was done through the Illumina HiSeq 2500 platform. This study aimed to search for de novo or inherited variants associated with the clinical severity. Six significant inherited variants were found in the genes, ATOH8, TTLL4, P2RX7, PRSS54, SLC9C2 and VRK2 in severe subject, however only one significant inherited variant in MTRR gene was found in non-severe subject. Bioinformatic analysis also confirms that each variant significantly changes in respective protein structure. The identified genes were either associated with iron absorption, oxidative stress, hematopoietic stem cell differentiation, structural organization of RBC cytoskeleton or regulating anemia. No significant de novo variant were identified associated with clinical severity. The finding of this study indicates that the combined effect of mutation load in major pathways may responsible for severe phenotype.

Thalassemia is the commonest inherited hemoglobinopathy worldwide. Variation of clinical symptoms entail differences in disease-onset and transfusion requirements. Our objective was to investigate the role of alpha gene deletions in modulating the clinical heterogeneity of thalassemia syndromes. A total of 214 individuals with diagnosed beta-thalassemia major/intermedia were included in the study. Beta globin mutations were determined and categorized as β ⁺ and β ⁰ . Eight common alpha globin gene deletions were detected by multiplex GAP-PCR. Out of the 17 individuals with β ⁺ /β ⁺ , 16 did not harbour alpha deletions (αα/αα), and most of them were non-severe. On the other hand, out of 46 individuals with β ⁰ /β ⁰ , 30 did not reveal alpha deletions, whereas 16 possessed one or more alpha deletion(s). Accordingly, most of them presented as clinically severe. Out of the 151 β ⁰ /β ⁺ individuals, 119 were negative for alpha deletion, whereas 32 possessed alpha deletions. It was observed that, only in this last category, alpha deletions made a significant contribution (P< 0.0001) in modulation of clinical non severity in this genotype. In conclusion, alpha globin gene deletions play a role to help in ameliorating the phenotype in the β ⁺ /β ⁰ genotype. However, they may have only minor/no role in patients with β ⁺ /β ⁺ or β ⁰ /β ⁰ genotype.

β-thalassemia patients often experience ocular abnormalities such as angioid streaks (ASs), retinal pigmented epithelium degradation, visual field defects, and in rare instances choroidal neovascularization (CNV). Although ASs are common in individuals with hemoglobinopathies, the occurrence of choroidal neovascularization without preceding ASs is exceptionally rare. In this report, we describe a β-thalassemia patient who had developed CNV at the age of 27 years and also had experience of renal stones at the age of 19 years. He had undergone splenectomy and was under conservative therapy of iron supplementation. We conducted whole-exome sequencing (WES) in search of CNV-associated variants. Through variant filtering and Phenolyzer analysis, we have identified a rare heterozygous missense variant in the ABCC6 gene, ABCC6:NM_001171:exon25:c.3524T>C (rs376062004). In silico analysis revealed that this variant is present in the highly conserved region and is likely to decrease the stability of the protein. Mutation in the ABCC6 gene leads to pseudoxanthoma elasticum (PXE). Previously, it was believed that ASs and subsequent CNV-like ocular complication may develop due to the pathophysiological condition of thalassemia. However, our study provides compelling evidence that rare mutations in the ABCC6 gene, in combination with oxygen insufficiency, may contribute to the development of CNV in β-thalassemia patients. This finding highlights the potential genetic basis of PXE-mediated CNV development in β-thalassemia.