Mitochondria in biology and medicine - 2023.

Mitochondria are an indispensable part of the cell that plays a crucial role in regulating various signaling pathways, energy metabolism, cell differentiation, proliferation, and cell death. Since mitochondria have their own genetic material, they differ from their nuclear counterparts, and dysregulation is responsible for a broad spectrum of diseases. Mitochondrial dysfunction is associated with several disorders, including neuro-muscular disorders, cancer, and premature aging, among others. The intricacy of the field is due to the cross-talk between nuclear and mitochondrial genes, which has also improved our knowledge of mitochondrial functions and their pathogenesis. Therefore, interdisciplinary research and communication are crucial for mitochondrial biology and medicine due to the challenges they pose for diagnosis and treatment. The ninth annual conference of the Society for Mitochondria Research and Medicine (SMRM)- India, titled "Mitochondria in Biology and Medicine" was organized at the Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad, India, on June 21-23, 2023. The latest advancements in the field of mitochondrial biology and medicine were discussed at the conference. In this article, we summarize the entire event for the benefit of researchers working in the field of mitochondrial biology and medicine.

Characterisation of Patients with SH3TC2 Associated Neuropathy in an Indian Cohort.

Background: SH3TC2 variations lead to demyelinating recessive Charcot-Marie-Tooth (CMT) disease, which is commonly associated with early-onset scoliosis and cranial neuropathy. Data from Indian ethnicity is limited. Objective: We aim to report the characteristics of patients with SH3TC2-associated neuropathy from an Indian cohort. Patients and Methods: Data of five unrelated subjects with SH3TC2 variations were analyzed. Results: Clinical features included female predominance (n = 4), early-onset neuropathy (n = 2), pes cavus and hammer toes (n = 4), kyphoscoliosis (n = 1), impaired vision and hearing (n = 1), facial muscle weakness (n = 1), impaired kinaesthetic sense (n = 3), tremor (n = 2), and ataxia (n = 1). Four patients had the "CMT" phenotype, while one patient had Roussy-Levy syndrome. All had demyelinating electrophysiology with conduction velocities being "very slow" in one, "slow" in one, "mildly slow" in two, and "intermediate" in one patient. Brain stem auditory evoked potentials were universally abnormal though only one patient had symptomatic deafness. Seven variants were identified in SH3TC2 [homozygous = 3 (c.1412del, c.69del, c.3152G>A), heterozygous = 4 (c.1105C>T, c.3511C>T, c.2028G>C, c.254A>T)]. Except for c.3511C>T variant, the rest were novel. Three patients had additional variations in genes having pathobiological relevance in other CMTs or amyotrophic lateral sclerosis. Conclusion: We provide data on a cohort of patients of Indian origin with SH3TC2 variations and highlight differences from other cohorts. Though the majority were not symptomatic for hearing impairment, evoked potentials disclosed abnormalities in all. Further studies are required to establish the functional consequences of novel variants and their interacting molecular partners identified in the present study to strengthen their association with the phenotype.

Neuromuscular disease genetics in under-represented populations: increasing data diversity.

Neuromuscular diseases (NMDs) affect ∼15 million people globally. In high income settings DNA-based diagnosis has transformed care pathways and led to gene-specific therapies. However, most affected families are in low-to-middle income countries (LMICs) with limited access to DNA-based diagnosis. Most (86%) published genetic data is derived from European ancestry. This marked genetic data inequality hampers understanding of genetic diversity and hinders accurate genetic diagnosis in all income settings. We developed a cloud-based transcontinental partnership to build diverse, deeply-phenotyped and genetically characterized cohorts to improve genetic architecture knowledge, and potentially advance diagnosis and clinical management. We connected 18 centres in Brazil, India, South Africa, Turkey, Zambia, Netherlands and the UK. We co-developed a cloud-based data solution and trained 17 international neurology fellows in clinical genomic data interpretation. Single gene and whole exome data were analysed via a bespoke bioinformatics pipeline and reviewed alongside clinical and phenotypic data in global webinars to inform genetic outcome decisions. We recruited 6001 participants in the first 43 months. Initial genetic analyses 'solved' or 'possibly solved' ∼56% probands overall. In-depth genetic data review of the four commonest clinical categories (limb girdle muscular dystrophy, inherited peripheral neuropathies, congenital myopathy/muscular dystrophies and Duchenne/Becker muscular dystrophy) delivered a ∼59% 'solved' and ∼13% 'possibly solved' outcome. Almost 29% of disease causing variants were novel, increasing diverse pathogenic variant knowledge. Unsolved participants represent a new discovery cohort. The dataset provides a large resource from under-represented populations for genetic and translational research. In conclusion, we established a remote transcontinental partnership to assess genetic architecture of NMDs across diverse populations. It supported DNA-based diagnosis, potentially enabling genetic counselling, care pathways and eligibility for gene-specific trials. Similar virtual partnerships could be adopted by other areas of global genomic neurological practice to reduce genetic data inequality and benefit patients globally.

Signatures of high altitude adaptation in Tibeto-Burman tribes of the Darjeeling Hill Region.

OBJECTIVES: The long-term isolation, endogamy practices, and environmental adaptations have shaped the enormous human diversity in India. The genetic and morphological variations in mainland Indians are well studied. However, the data on the Indian Himalayan populations are scattered. Thus, the present study attempts to understand variations in the selected parameter among four Tibeto-Burman speaking ethnic tribal populations from the Darjeeling Hill Region (DHR) in the Eastern Himalaya Biodiversity Hotspot region of India. METHODS: A total of 178 healthy male individuals (Lepcha 98, Sherpa 31, Bhutia 27, and Tibetans 22) living at an altitudinal range of 1467-2258 m above the sea level were studied for the 10 parameters namely, weight (kg), height (cm), body mass index (BMI) (kg/m2 ) systolic and diastolic pressure (mm of Hg), pulse rate (per minute), saturation of peripheral oxygen (SPO2 ) (%), hemoglobin (g/dl), hematocrit (HCT) (%), and blood glucose (mg/dl). The data was statistically analyzed using analysis of variance and multiple linear regression methods. RESULTS: Our analysis revealed comparatively lower hemoglobin and HCT levels, and higher systolic and diastolic blood pressure in the Sherpas followed by the Tibetans. This may be reflecting the persistence of high-altitude adaptation signatures even in lowlands. Interestingly, the Tibetans differed significantly from other populations in terms of their higher body weight, height, and BMI. CONCLUSION: Thus, our study showed the persistence of high altitude signatures in Tibetans and Sherpa inhabited the DHR. Additionally, we also observed significant differences in the anthropometric and physiological parameters among the Tibeto-Burman populations of the DHR.

A proteomic study to unveil lead toxicity-induced memory impairments invoked by synaptic dysregulation.

Lead (Pb2+), a ubiquitously present heavy metal toxin, has various detrimental effects on memory and cognition. However, the molecular processes affected by Pb2+ causing structural and functional anomalies are still unclear. To explore this, we employed behavioral and proteomic approaches using rat pups exposed to lead acetate through maternal lactation from postnatal day 0 (P0) until weaning. Behavioral results from three-month-old rats clearly emphasized the early life Pb2+ exposure induced impairments in spatial cognition. Further, proteomic analysis of synaptosomal fractions revealed differential alteration of 289 proteins, which shows functional significance in elucidating Pb2+ induced physiological changes. Focusing on the association of Small Ubiquitin-like MOdifier (SUMO), a post-translational modification, with Pb2+ induced cognitive abnormalities, we identified 45 key SUMO target proteins. The significant downregulation of SUMO target proteins such as metabotropic glutamate receptor 3 (GRM3), glutamate receptor isoforms 2 and 3 (GRIA 2 and GRIA3) and flotilin-1 (FLOT1) indicates SUMOylation at the synapses could contribute to and drive Pb2+ induced physiological imbalance. These findings identify SUMOylation as a vital protein modifier with potential roles in hippocampal memory consolidation and regulation of cognition. Data availbility: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034212".

Genetic Spectrum of Inherited Neuropathies in India.

Background and Objectives: Charcot-Marie-Tooth (CMT) disease is the commonest inherited neuromuscular disorder and has heterogeneous manifestations. Data regarding genetic basis of CMT from India is limited. This study aims to report the variations by using high throughput sequencing in Indian CMT cohort. Methods: Fifty-five probands (M:F 29:26) with suspected inherited neuropathy underwent genetic testing (whole exome: 31, clinical exome: 17 and targeted panel: 7). Their clinical and genetic data were analysed. Results: Age at onset ranged from infancy to 54 years. Clinical features included early-onset neuropathy (n=23), skeletal deformities (n=45), impaired vision (n=8), impaired hearing (n=6), facial palsy (n=8), thickened nerves (n=4), impaired cognition (n=5), seizures (n=5), pyramidal signs (n=7), ataxia (n=8) and vocal cord palsy, slow tongue movements and psychosis in one patient each. Twenty-eight patients had demyelinating electrophysiology. Abnormal visual and auditory evoked potentials were noted in 60.60% and 37.5% respectively. Sixty two variants were identified in 37 genes including variants of uncertain significance (n=34) and novel variants (n=45). Eleven patients had additional variations in genes implicated in CMTs/ other neurological disorders. Ten patients did not have variations in neuropathy associated genes, but had variations in genes implicated in other neurological disorders. In seven patients, no variations were detected. Conclusion: In this single centre cohort study from India, genetic diagnosis could be established in 87% of patients with inherited neuropathy. The identified spectrum of genetic variations adds to the pool of existing data and provides a platform for validation studies in cell culture or animal model systems.

Disrupted structural connectome and neurocognitive functions in Duchenne muscular dystrophy: classifying and subtyping based on Dp140 dystrophin isoform.

OBJECTIVE: Neurocognitive disabilities in Duchenne muscular dystrophy (DMD) children beginning in early childhood and distal DMD gene deletions involving disruption of Dp140 isoform are more likely to manifest significant neurocognitive impairments. MRI data analysis techniques like brain-network metrics can provide information on microstructural integrity and underlying pathophysiology. METHODS: A prospective study on 95 participants [DMD = 57, and healthy controls (HC) = 38]. The muscular dystrophy functional rating scale (MDFRS) scores, neuropsychology batteries, and multiplex ligand-dependent probe amplification (MLPA) testing were used for clinical assessment, IQ estimation, and genotypic classification. Diffusion MRI and network-based statistics were used to analyze structural connectomes at various levels and correlate with clinical markers. RESULTS: Motor and executive sub-networks were extracted and analyzed. Out of 57 DMD children, 23 belong to Dp140 + and 34 to Dp140- subgroup. Motor disabilities are pronounced in Dp140- subgroup as reflected by lower MDFRS scores. IQ parameters are significantly low in all-DMD cases; however, the Dp140- has specifically lowest scores. Significant differences were observed in global efficiency, transitivity, and characteristic path length between HC and DMD. Subgroup analysis demonstrates that the significance is mainly driven by participants with Dp140- than Dp140 + isoform. Finally, a random forest classifier model illustrated an accuracy of 79% between HC and DMD and 90% between DMD- subgroups. CONCLUSIONS: Current findings demonstrate structural network-based characterization of abnormalities in DMD, especially prominent in Dp140-. Our observations suggest that participants with Dp140 + have relatively intact connectivity while Dp140- show widespread connectivity alterations at global, nodal, and edge levels. This study provides valuable insights supporting the genotype-phenotype correlation of brain-behavior involvement in DMD children.

Leukodystrophy Due to eIF2B Mutations in Adults.

Vanishing white matter disease (VWMD) due to eIF2B mutations is a common leukodystrophy characterised by childhood onset, autosomal recessive inheritance, and progressive clinical course with episodic worsening. There are no reports of genetically confirmed adult patients from India. We describe the phenotype of two adults with genetically confirmed VWMD and typical radiological findings. Both had spastic ataxia and cognitive and behavioural disturbances. Other neurological features included myoclonic jerks and parkinsonism. At the last follow-up (duration: 2-9 years), one patient was wheelchair-bound. VWMD is rare in adults but should be suspected based on radiological findings and confirmed by eIF2B mutation.

Serum fibroblast growth factor 21 and growth differentiation factor 15: Two sensitive biomarkers in the diagnosis of mitochondrial disorders.

Mitochondrial disorders are often difficult to diagnose because of diverse clinical phenotypes. FGF-21 and GDF-15 are metabolic hormones and promising biomarkers for the diagnosis of these disorders. This study has systematically evaluated serum FGF-21 and GDF-15 levels by ELISA in a well-defined cohort of patients with definite mitochondrial disorders (n = 30), neuromuscular disease controls (n = 36) and healthy controls (n = 36) and aimed to ascertain their utility in the diagnosis of mitochondrial disorders. Both serum FGF-21 and GDF-15 were significantly elevated in patients with mitochondrial disorders, especially in those with muscle involvement. The levels were higher in patients with mitochondrial deletions (both single and multiple) and translation disorders compared to respiratory chain subunit or assembly factor defects.

Whole exome sequencing reveals a homozygous C1QBP deletion as the cause of progressive external ophthalmoplegia and multiple mtDNA deletions.

Whole exome sequencing (WES), analyzed with GENESIS and WeGET, revealed a homozygous deletion in the C1QBP gene in a patient with progressive external ophthalmoplegia (PEO) and multiple mtDNA deletions. The gene encodes the mitochondria-located complementary 1 Q subcomponent-binding protein, involved in mitochondrial homeostasis. Biallelic mutations in C1QBP cause mitochondrial cardiomyopathy and/or PEO with variable age of onset. Our patient showed only late-onset PEO-plus syndrome without overt cardiac involvement. Available data suggest that early-onset cardiomyopathy variants localize in important structural domains and PEO-plus variants in the coiled-coil region. Our patient demonstrates that C1QBP mutations should be considered in individuals with PEO with or without cardiomyopathy.

Ethylmalonic encephalopathy ETHE1 p. D165H mutation alters the mitochondrial function in human skeletal muscle proteome.

Ethylmalonic encephalopathy (EE) is a rare autosomal recessive inborn error of metabolism. To study the molecular effects of ETHE1 p. D165H mutation, we employed mass spectrometry-based mitochondrial proteome and phosphoproteome profiling in the human skeletal muscle. Eighty-six differentially altered proteins were identified, of which thirty-seven mitochondrial proteins were differentially expressed, and most of the proteins (37%) were down-regulated in the OXPHOS complex-IV. Also, nine phosphopeptides that correspond to eight mitochondrial proteins were significantly affected in EE patient. These altered proteins recognized are involved in several pathways and molecular functions, predominantly in oxidoreductase activity. This is the first study that has integrated proteome and phosphoproteome of skeletal muscle and identified multiple proteins associated in the pathogenesis of EE.

Clinico-pathological and Molecular Spectrum of Mitochondrial Polymerase γ Mutations in a Cohort from India.

Polymerase γ catalytic subunit (POLG), a nuclear gene, encodes the enzyme responsible for mitochondrial DNA (mtDNA) replication. POLG mutations are a major cause of inherited mitochondrial diseases. They present with varied phenotypes, age of onset, and severity. Reports on POLG mutations from India are limited. Hence, this study aimed to describe the clinico-pathological and molecular observations of POLG mutations. A total of 446 patients with clinical diagnosis of mitochondrial disorders were sequenced for all exons and intron-exon boundaries of POLG. Of these, 19 (4.26%) patients (M:F: 10:9) had POLG mutations. The age of onset ranged from 5 to 55 years with an overlapping phenotypic spectrum. Ptosis, peripheral neuropathy, seizures, and ataxia were the common neurological features observed. The most common clinical phenotype was chronic progressive external ophthalmoplegia (CPEO) and CPEO plus (n = 14). Muscle biopsy showed characteristic features of mitochondrial myopathy in fourteen patients (14/19) and respiratory chain enzyme deficiency in eleven patients (11/19). Multiple mtDNA deletions were seen in 47.36% (9/19) patients. Eight pathogenic POLG variations including two novel variations (p.G132R and p.V1106A) were identified. The common pathogenic mutation identified was p.L304R, being present in eight patients (42.1%) predominantly in the younger age group followed by p.W748S in four patients (21%). To the best of our knowledge, this is the first extensive study from India, highlights the clinico-pathological and molecular spectrum of POLG mutations.

Contribution of nuclear and mitochondrial gene mutations in mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome.

BACKGROUND: Mitochondrial disorders are clinically complex and have highly variable phenotypes among all inherited disorders. Mutations in mitochon drial DNA (mtDNA) and nuclear genome or both have been reported in mitochondrial diseases suggesting common pathophysiological pathways. Considering the clinical heterogeneity of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) phenotype including focal neurological deficits, it is important to look beyond mitochondrial gene mutation. METHODS: The clinical, histopathological, biochemical analysis for OXPHOS enzyme activity, and electron microscopic, and neuroimaging analysis was performed to diagnose 11 patients with MELAS syndrome with a multisystem presentation. In addition, whole exome sequencing (WES) and whole mitochondrial genome sequencing were performed to identify nuclear and mitochondrial mutations. RESULTS: Analysis of whole mtDNA sequence identified classical pathogenic mutation m.3243A > G in seven out of 11 patients. Exome sequencing identified pathogenic mutation in several nuclear genes associated with mitochondrial encephalopathy, sensorineural hearing loss, diabetes, epilepsy, seizure and cardiomyopathy (POLG, DGUOK, SUCLG2, TRNT1, LOXHD1, KCNQ1, KCNQ2, NEUROD1, MYH7) that may contribute to classical mitochondrial disease phenotype alone or in combination with m.3243A > G mutation. CONCLUSION: Individuals with MELAS exhibit clinical phenotypes with varying degree of severity affecting multiple systems including auditory, visual, cardiovascular, endocrine, and nervous system. This is the first report to show that nuclear genetic factors influence the clinical outcomes/manifestations of MELAS subjects alone or in combination with m.3243A > G mutation.

Exposure to the neurotoxin 3-nitropropionic acid in neuronal cells induces unique histone acetylation pattern: Implications for neurodegeneration.

Mitochondrial dysfunction is critical for neurodegeneration in movement disorders. Neurotoxicological models recapitulating movement disorder involve mitochondrial damage including inhibition of mitochondrial complexes. Previously, we demonstrated that neurotoxic models of Parkinson's disease and Manganism showed distinct morphological, electrophysiological and molecular profile indicating disease-specific characteristics. In a recent study, we demonstrated that the transcriptomic changes triggered by the neurotoxic mitochondrial complex II inhibitor 3-nitropropionic acid (3-NPA), was significantly different from the profile induced by the neurotoxic mitochondrial complex I inhibitor 1-methyl-4- phenylpyridinium (MPP+) and mitochondrial toxin Manganese (Mn). Among the plausible pathways, we surmised that epigenetic mechanisms could contribute to 3-NPA specific transcriptomic profile. To address this, we assessed global and individual lys-specific acetylation profile of Histone H3 and H4 in the 3-NPA neuronal cell model. Our data revealed histone acetylation profile unique to the 3-NPA model that was not noted in the MPP+ and Mn models. Among the individual lys, Histone H3K56 showed robust dose and time-dependent hyperacetylation in the 3-NPA model. Chromatin Immunoprecipitation-sequencing (ChIP-seq) revealed that acetylated H3K56 was associated with 13072 chromatin sites, which showed increased occupancy in the transcription start site-promoter site. Acetylated histone H3K56 was associated with 1747 up-regulated and 263 down-regulated genes in the 3-NPA model, which included many up-regulated autophagy and mitophagy genes. Western analysis validated the involvement of PINK1-Parkin dependent mitophagy in the 3-NPA model. We propose that 3-NPA specific chromatin dynamics could contribute to the unique transcriptomic profile with implications for movement disorders.

Leukodystrophies and Genetic Leukoencephalopathies in Children Specified by Exome Sequencing in an Expanded Gene Panel.

The overlapping clinical and neuroimaging phenotypes of leukodystrophies pose a diagnostic challenge to both clinicians and researchers alike. Studies on the application of exome sequencing in the diagnosis of leukodystrophies are emerging. We used targeted gene panel sequencing of 6440 genes to investigate the genetic etiology in a cohort of 50 children with neuroimaging diagnosis of leukodystrophy/genetic leukoencephalopathy of unknown etiology. These 50 patients without a definite biochemical or genetic diagnosis were derived from a cohort of 88 patients seen during a 2.5-year period (2015 January-2017 June). Patients who had diagnosis by biochemical or biopsy confirmation (n = 17) and patients with incomplete data or lack of follow-up (n = 21) were excluded. Exome sequencing identified variants in 30 (60%) patients, which included pathogenic or likely pathogenic variants in 28 and variants of unknown significance in 2. Among the patients with pathogenic or likely pathogenic variants, classic leukodystrophies constituted 13 (26%) and genetic leukoencephalopathies 15 (30%). The clinical and magnetic resonance imaging (MRI) findings and genetic features of the identified disorders are discussed.

PMP22 Gene-Associated Neuropathies: Phenotypic Spectrum in a Cohort from India.

Reports of spectrum of clinical manifestations in PMP22 gene-associated neuropathies (duplication/mutations) are scarce. To identify the frequency of PMP22 gene variations and establish their genotype-phenotype correlation. Patients with suspected genetic demyelinating neuropathy (n = 128) underwent evaluation for copy number variations and point mutations in PMP22 gene by multiplex ligation-dependent probe amplification (MLPA) and direct sequencing respectively. Of these, only 27 patients (M:F:19:8) from 18 families had PMP22 gene-associated neuropathy; they were subsequently analyzed for genotype-phenotype correlation. Twenty-five patients had PMP22 duplication while two patients had PMP22 missense mutations (p.A114V and p.L80P). Age at onset of neuropathy ranged from infancy to 63 years and symptom duration ranged from 2 to 32 years. Cranial nerve dysfunction in the form of ptosis, ophthalmoplegia, bifacial weakness, and sensorineural hearing loss was observed in addition to a number of systemic features. Three patients were asymptomatic. All except one patient were ambulant. Velocity of median nerve and amplitude of evoked motor responses from common peroneal nerve were significantly reduced in male patients. There was significantly worse disability in the late-onset group as compared with the early-onset group. Otherwise, the mean age at onset, frequency of skeletal deformities, patterns of motor weakness, muscle stretch reflexes, sensory impairment, disability rating scales, and electrophysiological parameters were comparable irrespective of gender, onset age, family history and ulnar nerve conduction velocities. The relatively low frequency of PMP22 duplication in the present cohort warrants a more comprehensive search to establish the genetic etiology. Further research into the role of other genetic variants as well as modifier genes and their effect on phenotypic heterogeneity is indicated.

A Novel Splice Site Mutation in IFNGR2 in Patients With Primary Immunodeficiency Exhibiting Susceptibility to Mycobacterial Diseases.

Primary immunodeficiency (PID) refers to a group of heterogeneous genetic disorders with a weakened immune system. Mendelian susceptibility to mycobacterial disease (MSMD) is a subset of PID in which patients exhibit defects in intrinsic and innate immunity. It is a rare congenital disorder characterized by severe and recurrent infections caused by weakly virulent mycobacteria or other environmental mycobacteria. Any delay in definitive diagnosis poses a major concern due to the confounding nature of infections and immune deficiencies. Here, we report the clinical, immunological, and genetic characteristics of two siblings (infants) with recurrent infections. There was a history of death of two other siblings in the family after BCG vaccination. Whole exome sequencing of the two affected surviving infants along with their consanguineous parents identified a novel, homozygous single nucleotide splice acceptor site variant in intron 2 of the interferon gamma receptor 2 (IFNGR2) gene. Sanger sequencing of DNA obtained from blood and fibroblasts confirmed the variant. The patients underwent bone marrow transplantation from their father as a donor. RT-PCR and Sanger sequencing of the cDNA of patients from blood samples after transplantation showed the expression of both wild type and mutant transcript expression of IFNGR2. To assess partial or complete expression of IFNGR2 mutant transcripts, fibroblasts were cultured from skin biopsies. RT-PCR and Sanger sequencing of cDNA obtained from patient fibroblasts revealed complete expression of mutant allele and acquisition of a cryptic splice acceptor site in exon 3 that resulted in deletion of 9 nucleotides in exon 3. This led to an in-frame deletion of three amino acids p.(Thr70-Ser72) located in a fibronectin type III (FN3) domain in the extracellular region of IFNGR2. This illustrates individualized medicine enabled by next generation sequencing as identification of this mutation helped in the clinical diagnosis of MSMD in the infants as well as in choosing the most appropriate therapeutic option.

Mitochondrial genome variations in idiopathic dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (DCM) is a structural heart disease with strong genetic background. The aim of this study was to assess the role of mitochondrial DNA (mtDNA) variations and haplogroups in Indian DCM patients. Whole mtDNA analysis of 221 DCM patients revealed 48 novel, 42 disease-associated and 97 private variations. The frequency of reported variations associated with hearing impairment, DEAF, SNHL and LHON are significantly high in DCM patients than controls. Haplogroups H and HV were over represented in DCM than controls. Functional analysis of two private variations (m.8812A>G & m.10320G>A) showed decrease in mitochondrial functions, suggesting the role of mtDNA variations in DCM.