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Purpose: Accurate diagnosis is required for management of Congenital adrenal hyperplasia (CAH). The conventional method for detection of mutations in the CYP21A2gene is targeted capillary sequencing which is labor intensive and has limited multiplexing capability. Next generation sequencing (NGS) provides data with high sequence coverage and depth. Our objective was to develop an accurate NGS-based assay to characterize the mutation spectrum in CYP21A2gene in Indian patients suspected to have 21-OH CAH. Methods: Cases with 21-OH CAH from 12 endocrine units across India were studied. DNA was extracted from proband’s and parent’s(subset) blood. Locus-specific long-range PCR and gel electrophoresis of amplicons was followed by NGS where no visible 30?kb homozygous/whole gene deletion was observed. Orthogonal confirmation was performed by capillary sequencing (ABI 3500) and Multiplex Ligation-dependent Probe Amplification (MLPA, MRC-Holland). PCR products were purified and individual libraries were pooled and sequenced (Illumina). Results: Of the 310 CAH cases, biallelic mutations (pathogenic/ likely pathogenic variants involving both CYP21A2gene copies) were detected in 256 (82.6%), heterozygous mutations in 13 (4.2 %), and none in 41 (13.2%). Most common mutation was c.293-13A/C>G (29.03%), followed by 30?kb deletion (18.24%). Thirty samples tested orthogonally (by capillary sequencing or MLPA) showed 100% concordance with NGS assay. Nine novel variants were identified. Conclusions: We have developed and validated a comprehensive NGS-based assay for detection of variants in CYP21A2gene in patients with 21-OH CAH. We describe CYP21A2mutation spectrum and novel variants in a large cohort of Indian patients with CAH.

Beta thalassemia major is a common genetic disorder characterized by the reduced production or absence of beta globin, a product of the haemoglobin subunit beta (HBB) gene. Every year, approximately 10,000–12,000 children with thalassemia major are born in India. Molecular methodologies like ARMS (amplification-refractory mutation system)-PCR (polymerase chain reaction) and capillary sequencing are used to detect HBBgene mutations. It is, however, challenging to achieve comprehensive coverage of the HBBgene by these methods. Next-generation sequencing (NGS) can be used to circumvent these problems. Commercial NGS panels are prohibitively expensive and hence are not routinely implemented in most laboratories. We have developed a cost-effective, highly sensitive and specific, indigenous targeted NGS assay for detecting mutations in the HBBgene. Using this custom NGS assay, we processed 1530 samples (3017 alleles), in which we detected a spectrum of 48 pathogenic/likely pathogenic variants (mutations); IVS-I-5 (c.92+5G>C) was the most common mutation detected (allele frequency (AF) 44.55%), followed by the 619-bp deletion (AF 10.74%), c.92+1G>T (AF 6.99%), c.27_28insG (AF 6.23%), c.47G>A (AF 5.77%) and c.126_129delCTTT (AF 4.71%). Additionally, we discovered a novel mutation (c.7delC) that was submitted to the HbVar database (HbVar ID 3193) as a variant of unknown significance (VUS), probably pathogenic. The targeted NGS assay developed during this study was validated using orthogonal methods and showed excellent correlation with currently available molecular methods. Additionally, this targeted NGS assay was used to analyse the mutation spectrum of the largest beta thalassemia cohort from India.