Whole mitochondrial genome sequencing in individuals with Leber hereditary optic neuropathy negative for the common pathogenic mitochondrial DNA variants

Sundaramurthy Srilekha^1,2*Selvakumar Ambika³Nagarajan Hemavathy⁴Dharani Vidhya³Patrick Yu-Wai-Man^5,6,7,8

• ¹Sankara Nethralaya, Chennai, India
• ²SNONGC Department of Genetics & Molecular Biology, Medical Research Foundation, Chennai, India, Chennai, India
• ³Department of Neuro-Ophthalmology, Medical Research Foundation, Chennai, India, Chennai, India
• ⁴Centre for Bioinformatics, Vision Research Foundation, Chennai, India, Chennai, India
• ⁵John van Geest Centre for Brain Repair and MRC Mitochondrial Biology Unit, Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom, CAMBRIDGE, United Kingdom
• ⁶Cambridge Eye Unit, Addenbrooke’s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom, CAMBRIDGE, United Kingdom
• ⁷Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom, LONDON, United Kingdom
• ⁸Institute of Ophthalmology, University College London, London, United Kingdom, LONDON, United Kingdom

Purpose: This study aimed to explore the role of additional mitochondrial DNA (mtDNA) variants in the development of Leber hereditary optic neuropathy (LHON) by screening the entire mitochondrial genome in individuals who had previously tested negative for the three common mtDNA variants: m.3460G>A (MT-ND1), m.11778G>A (MT-ND4), and m.14484T>C (MT-ND6), by conventional Sanger sequencing.Methods: Forty-one individuals with a suspected clinical diagnosis of LHON were recruited from the neuro-ophthalmology clinic. Each participant had undergone a comprehensive neuro-ophthalmic examination, including slit lamp examination, indirect ophthalmoscopy, visual field perimetry, optical coherence tomography, and MRI of the brain and orbits.Targeted re-sequencing was conducted using next-generation sequencing (NGS) on the HiSeqX Ten platform (Illumina, San Diego, California) with a 2×150bp paired-end configuration. The sequencing reads were aligned to the human mitochondrial genome sequence (hg19). Variants were filtered with the VariMAT tool (v.2.3.9). Haplogroup analysis was performed using Haplogrep 2 (v2.0). To assess the deleteriousness of nonsynonymous variations, bioinformatics prediction tools such as PolyPhen2, SIFT, CADD, and Mutation Assessor were utilized. In addition, while tools like Consurf, PredictSNP, DynaMut, ENCoM, DUET, SDM, mCSM, were employed to evaluate evolutionary conservation, pathogenicity, structural stability, and functional impact.Results: Whole mitochondrial genome sequencing of 41 clinically suspected LHON cases identified a total of 1,518 mtDNA variants. Of these, 822 were located in the coding regions, including 555 synonymous and 273 non-synonymous variants. Two heteroplasmic diseasecausing variants (m.11778G>A and m.3460G>A) were identified in one individual each (90.0% and 63.6%, respectively). Additionally, rare mtDNA variants listed in Mitomap were found in five individuals (5/41, 12.1%), with variants in MT-ND1 being the most prevalent (3/41, 7.3%). MT-ND1 (m.3335T>C, m.3394T>C, m.3395A>G), MT-ND4L (m.10680G>A),MT-ND6 (m.14502T>C).Our study of a well-characterized Indian LHON cohort uncovered rare mtDNA variants that should be considered assessing of undiagnosed optic neuropathy cases.Additionally, it underscores the effectiveness of NGS in identifying heteroplasmic mtDNA variants. This indicates that whole mitochondrial genome sequencing via NGS is a more efficient and preferred approach for routine molecular genetic testing.