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The genetically isolated yet heterogeneous and highly consanguineous Indian population has shown a higher prevalence of rare genetic disorders. However, there is a significant socioeconomic burden for genetic testing to be accessible to the general population. In the current study, we analyzed next-generation sequencing data generated through focused exome sequencing from individuals with different phenotypic manifestations referred for genetic testing to achieve a molecular diagnosis. Pathogenic or likely pathogenic variants are reported in 280 of 833 cases with a diagnostic yield of 33.6%. Homozygous sequence and copy number variants were found as positive diagnostic findings in 131 cases (15.7%) because of the high consanguinity in the Indian population. No relevant findings related to reported phenotype were identified in 6.2% of the cases. Patients referred for testing due to metabolic disorder and neuromuscular disorder had higher diagnostic yields. Carrier testing of asymptomatic individuals with a family history of the disease, through focused exome sequencing, achieved positive diagnosis in 54 of 118 cases tested. Copy number variants were also found in trans with single-nucleotide variants and mitochondrial variants in a few of the cases. The diagnostic yield and the findings from this study signify that a focused exome test is a good lower-cost alternative for whole-exome and whole-genome sequencing and as a first-tier approach to genetic testing., Competing Interests: Disclosure Statement All authors are current or past employees of Revvity Omics (previously PerkinElmer Genomics, Waltham, MA, and Chennai, India)., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Mitochondria are dynamic in structure, and undergo continuous fusion-fission to maintain their homeostasis. In diabetes, retinal mitochondria are swollen, their membrane is damaged and mitochondrial fusion protein, mitofusin 2 (Mfn2), is decreased. DNA methylation machinery is also activated and methylation status of genes implicated in mitochondrial damage and biogenesis is altered. This study aims to investigate the role of mitochondrial fusion in the development of diabetic retinopathy, and to illustrate the molecular mechanism responsible for Mfn2 suppression. Using human retinal endothelial cells, manipulated for Mfn2, we investigated the role of fusion in mitochondrial structural and functional damage in diabetes. The molecular mechanism of its suppression in diabetic milieu was determined by investigating Mfn2 promoter DNA methylation, and confirmed using molecular and pharmacological inhibitors of DNA methylation. Similar studies were performed in the retinal microvasculature (prepared by hypotonic shock method) of diabetic rats, and human donors with documented diabetic retinopathy. Overexpression of Mfn2 prevented glucose-induced increase in mitochondrial fragmentation, decrease in complex III activity and increase in membrane permeability, mtDNA damage and apoptosis. High glucose hypermethylated Mfn2 promoter and decreased transcription factor (SP1) binding, and Dnmt inhibition protected Mfn2 promoter from these changes. In streptozotocin-induced diabetic rats, intravitreal administration of Dnmt1-siRNA attenuated Mfn2 promoter hypermethylation and restored its expression. Human donors with diabetic retinopathy confirmed Mfn2 promoter DNA hypermethylation. Thus, regulating Mfn2 and its epigenetic modifications by molecular/pharmacological means will protect mitochondrial homeostasis in diabetes, and could attenuate the development of retinopathy in diabetic patients., (Copyright © 2019. Published by Elsevier B.V.)