Scalable noninvasive amplicon-based precision sequencing (SNAPseq) for genetic diagnosis and screening of β-thalassemia and sickle cell disease using a next-generation sequencing platform

β-hemoglobinopathies such as β-thalassemia (BT) and Sickle cell disease (SCD) are inherited monogenic blood disorders with significant global burden. Hence, early and affordable diagnosis can alleviate morbidity and reduce mortality given the lack of effective cure. Currently, Sanger sequencing is considered to be the gold standard genetic test for BT and SCD, but it has a very low throughput requiring multiple amplicons and more sequencing reactions to cover the entire HBB gene. To address this, we have demonstrated an extraction-free single amplicon-based approach for screening the entire β-globin gene with clinical samples using Scalable noninvasive amplicon-based precision sequencing (SNAPseq) assay catalyzing with next-generation sequencing (NGS). We optimized the assay using noninvasive buccal swab samples and simple finger prick blood for direct amplification with crude lysates. SNAPseq demonstrates high sensitivity and specificity, having a 100% agreement with Sanger sequencing. Furthermore, to facilitate seamless reporting, we have created a much simpler automated pipeline with comprehensive resources for pathogenic mutations in BT and SCD through data integration after systematic classification of variants according to ACMG and AMP guidelines. To the best of our knowledge, this is the first report of the NGS-based high throughput SNAPseq approach for the detection of both BT and SCD in a single assay with high sensitivity in an automated pipeline

Development of pathophysiologically relevant models of sickle cell disease and β-thalassemia for therapeutic studies

Abstract Ex vivo cellular system that accurately replicates sickle cell disease and β-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study, we present the generation of erythroid progenitor lines with sickle cell disease and β-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles, globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally, these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably, we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype, which reactivates fetal hemoglobin levels and rescues the disease phenotypes, thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether, we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling, drug screenings and cell and gene therapy-based applications

Genomic analysis of early SARS-CoV-2 breakthrough infections from the state of Kerala suggest a preponderance of variants of concern

The SARS-CoV-2 Variant of Concern, Delta (B.1.617.2) was first reported in December 2020 in India and has spread colossally throughout the globe. Owing to factors like increased transmissibility, immune escape, and virulence, the delta variant has been considered as a potential public health threat apart from other variants of concern like alpha, beta and gamma. Kerala was one of the first states in India to enroll in the systematic genomic surveillance. In the present report, vaccine breakthrough infections were followed up in 147 patients including 55 healthcare workers who had been vaccinated with ChAdOx1 nCoV- 19/BBV152 across eleven districts from the state of Kerala. The timeline of samples analysed were from April 2021 till June 2021. Severity of the infections reported in the enrolled patients found to be mildly symptomatic, majorly with only 0.7% (n=1) of the cohort to be asymptomatic. Genomic analysis of the samples revealed the Delta variant (B.1.617.2) to constitute about 81.6% (n=120) in the studied cohort. This was followed by the Kappa variant B.1.617.1 (8.35%, n=9), AY.1 (0.6%, n= 1), AY.12 (0.6%, n= 1), AY.4 (1.2%, n= 2), AY.9 (1.2%, n= 2) and Eta variant, B.1.525 (0.6%, n= 1). 11 samples were not assigned any lineage. Evidence from this study suggests the preponderance of the Delta variant in the samples analysed