Molecular characterization of variants in mitochondrial DNA encoded genes using next generation sequencing analysis and mitochondrial dysfunction in women with PCOS

Emerging studies indicates mitochondrial dysfunction and involvement of mitochondrial DNA (mtDNA) variants in the pathogenesis of polycystic ovary syndrome (PCOS). Cumulative effect of mtDNA rare variants are now gaining considerable interest apart from common variants in the pathogenesis of complex diseases. Rare variants may modify the effect of polymorphism or in combination with the common variants may affect the risk of disease. With the evolution of high throughput sequencing techniques, which can be utilized to identify common as well as rare variants along with heteroplasmy levels, comprehensive characterization of the mtDNA variants is possible. Till date, few studies reported common mtDNA variants using traditional sequencing techniques but rare variants in mtDNA encoding genes remain unexplored in women with PCOS. These mtDNA variants may be responsible for mitochondrial dysfunction and may contribute in PCOS pathogenesis. In this study we determined mtDNA copy number, a biomarker of mitochondrial dysfunction and first time analysed variants in mtDNA encoded genes in women with PCOS using mitochondrial Next Generation sequencing (NGS) approach and compared allele fre-quency from mitochondrial 1000 genome dataset. Variant annotation and prioritization was done using highly automated pipeline, MToolBox that excludes reads mapped from nuclear mitochondrial DNA sequences (NumtS) to identify unique mtDNA reads. The present study identified significant reduction in mtDNA copy number in women with PCOS compared to non-PCOS women. A total of unique 214 prioritized common to rare variants were iden-tified in mtDNA encoded genes, 183 variants in OXPHOS complexes, 14 variants in MT-tRNA and 17 variants in MT-rRNA genes that may be involved in mitochondrial dysfunction in PCOS. Numerous variants were heteroplasmic, pathogenic in nature and occurred in evolutionary conserved region. Heteroplasmic variants were more frequently occurred in MT-CO3 gene. Non-synonymous variants were more than synonymous variants and mainly occurred in OXPHOS complex I and IV. Few variants were found to be associated with diseases in MITOMAP database. The study provides a better understanding towards pathogenesis of PCOS from novel aspects focusing on mitochondrial ge-netic defects as underlying cause for contributing mitochondrial dysfunction in women with PCOS.

Automated summary

Emerging studies have found that mitochondrial dysfunction and mtDNA variants play a role in the development of PCOS. Rare variants in mtDNA, in addition to common variants, may modify the risk of disease. High throughput sequencing techniques allow for comprehensive characterization of mtDNA variants, but rare variants in mtDNA encoding genes have not been extensively explored in women with PCOS. This study analyzed mtDNA copy number and variants in mtDNA encoded genes in PCOS patients for the first time using NGS, and identified significant reduction in mtDNA copy number and numerous prioritized variants that may contribute to mitochondrial dysfunction in PCOS. This study provides novel insights into the pathogenesis of PCOS by focusing on mitochondrial genetic defects as an underlying cause of mitochondrial dysfunction in women with PCOS.