The BRCA mutation spectrum among breast and ovarian cancers in India: highlighting the need to screen BRCA1 185delAG among South Indians.

Mutations in BRCA1 and BRCA2 significantly elevate the risk of developing breast and ovarian cancer. Limited data exists regarding the prevalence of BRCA mutations, and optimal, cost-effective testing strategies in developing countries like India. This study aimed to evaluate the utility of a Next Generation Sequencing (NGS) panel for BRCA1/2 mutation testing among women diagnosed with, or at risk of developing hereditary breast and ovarian cancers. We also aimed to identify population specific BRCA1/2 mutation hotspots, to enable the development of more affordable testing strategies. We identified 921 patients with breast and ovarian cancer who underwent mutation testing. The target enrichment was followed by targeted NGS in 772 patients and an allele-specific PCR (ASPCR) based genotyping for BRCA1:c.68_69delAG (or 185delAG), was carried out in 149 patients. We identified 188 (20.4%) patients with BRCA1/2 variants: 118 (62.8%) with pathogenic/likely pathogenic and 70 (37.2%) with VUS. The 185delAG was identified as a recurrent mutation in the Southern Indian population, accounting for 24.6% of the pathogenic variants. In addition, a family history of breast, ovary, pancreas, or prostate (BOPP) cancer was found to be associated with an increased risk of identifying a deleterious BRCA1/2 variant [OR = 2.11 (95% CI 1.45-3.07) p ≤ 0.001]. These results suggest that Targeted NGS is a sensitive and specific strategy for BRCA testing. For Southern Indian patients, a two-tiered approach can be considered: Initial screening with ASPCR for BRCA1 185delAG followed by NGS for those testing negative. Expanding the gene panel and identifying other population-specific mutation hot spots is a promising area with potential for improvements in testing and treatment strategies.

Genetic Heterogeneity and Challenges in the Management of Permanent Neonatal Diabetes Mellitus: A Single-Centre Study from South India.

Aim and Objectives: 1. To study the clinical outcome, growth and glycaemic control, 2. To study the frequency and type of genetic mutations. Methods: This is a retrospective study with a review of data of medical records from 2008 till date. Results: Twelve patients (six males) with neonatal diabetes mellitus (NDM) were identified. Median (interquartile range - (IQR)) age at diagnosis was 72 (31-95) days with a history of consanguinity in 75%. The median birth weight (range) was 2345 (900-3300) g. Follow-up data were available for eight patients with a median age at (IQR) follow-up of 3.3 (3-5.3) years. At follow-up, the mean annual HbA1c was 8.2% at a mean insulin dose of 1.1 U/kg/d. One patient with Wolcott-Rallison syndrome (WRS) and 21α-hydroxylase deficiency had poor growth and intellectual difficulty. The rest demonstrated satisfactory growth with an increase of mean weight centile from 2nd to 13th, height centile from 6.5th to 20th and normal neuro-cognitive development. Eleven patients underwent genetic testing with a molecular diagnosis in 54% (6/11): EIF2AK3 (n = 2) and one each in INS, PDX1, IL2RA and FOXP3. None had variants in ABCC8 or KCNJ11. One with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome underwent haematopoietic stem cell transplant (HSCT) and later succumbed. Conclusion: Our study demonstrates good clinical outcomes among NDM patients without immune dysfunction. Molecular diagnosis was attained only in around half of the patients (54%) with a great genetic heterogeneity.

WFS1 Gene-associated Diabetes Phenotypes and Identification of a Founder Mutation in Southern India.

CONTEXT: Wolfram syndrome (WFS) is a rare autosomal recessive disorder characterized by juvenile-onset diabetes, diabetes insipidus, optic atrophy, deafness, and progressive neurodegeneration. However, due to the progressive nature of the disease and a lack of complete clinical manifestations, a confirmed diagnosis of WFS at the time of onset of diabetes is a challenge. OBJECTIVE: With WFS1 rare heterozygous variants reported in diabetes, there is a need for comprehensive genetic screening strategies for the early diagnosis of WFS and delineating the phenotypic spectrum associated with the WFS1 gene variants in young-onset diabetes. METHODS: This case series of 11 patients who were positive for WFS1 variants were identified with next-generation sequencing (NGS)-based screening of 17 genemonogenic diabetes panel. These results were further confirmed with Sanger sequencing. RESULTS: 9 out of 11 patients were homozygous for pathogenic/likely pathogenic variants in the WFS1 gene. Interestingly, 3 of these probands were positive for the novel WFS1 (NM_006005.3): c.1107_1108insA (p.Ala370Serfs*173) variant, and haplotype analysis suggested a founder effect in 3 families from Southern India. Additionally, we identified 2 patients with young-onset diabetes who were heterozygous for a likely pathogenic variant or a variant of uncertain significance in the WFS1 gene. CONCLUSION: These results project the need for NGS-based parallel multigene testing as a tool for early diagnosis of WFS and identify heterozygous WFS1 variants implicated in young-onset diabetes.

Allele-specific PCR and Next-generation sequencing based genetic screening for Congenital Adrenal Hyperplasia in India.

Genetic screening of Congenital Adrenal Hyperplasia (CAH) is known to be challenging due to the complexities in CYP21A2 genotyping and has not been the first-tier diagnostic tool in routine clinical practice. Also, with the advent of massive parallel sequencing technology, there is a need for investigating its utility in screening extended panel of genes implicated in CAH. In this study, we have established and utilized an Allele-Specific Polymerase Chain Reaction (ASPCR) based approach for screening eight common mutations in CYP21A2 gene followed by targeted Next Generation Sequencing (NGS) of CYP21A2, CYP11B1, CYP17A1, POR, and CYP19A1 genes in 72 clinically diagnosed CAH subjects from India. Through these investigations, 88.7% of the subjects with 21 hydroxylase deficiency were positive for eight CYP21A2 mutations with ASPCR. The targeted NGS assay was sensitive to pick up all the mutations identified by ASPCR. Utilizing NGS in subjects negative for ASPCR, five study subjects were homozygous positive for other CYP21A2 variants: one with a novel c.1274G>T, three with c.1451G>C and one with c.143A>G variant. One subject was compound heterozygous for c.955C>T and c.1042G>A variants identified using ASPCR and NGS. One subject suspected for a Simple Virilizing (SV) 21 hydroxylase deficiency was positive for a CYP19A1:c.1142A>T variant. CYP11B1 variants (c.1201-1G>A, c.1200+1del, c.412C>T, c.1024C>T, c.1012dup, c.623G>A) were identified in all six subjects suspected for 11 beta-hydroxylase deficiency. The overall mutation positivity was 97.2%. Our results suggest that ASPCR followed by targeted NGS is a cost-effective and comprehensive strategy for screening common CYP21A2 mutations and the CAH panel of genes in a clinical setting.