Exploring the evidence for mitochondrial dysfunction and genetic abnormalities in the etiopathogenesis of tropical ataxic neuropathy.

Tropical ataxic neuropathy (TAN) is characterised by ataxic polyneuropathy, degeneration of the posterior columns and pyramidal tracts, optic atrophy, and sensorineural hearing loss. It has been attributed to nutritional/toxic etiologies, but evidence for the same has been equivocal. TAN shares common clinical features with inherited neuropathies and mitochondrial disorders, it may be hypothesised that genetic abnormalities may underlie the pathophysiology of TAN. This study aimed to establish evidence for mitochondrial dysfunction by adopting an integrated biochemical and multipronged genetic analysis. Patients (n = 65) with chronic progressive ataxic neuropathy with involvement of visual and/or auditory pathways underwent deep phenotyping, genetic studies including mitochondrial DNA (mtDNA) deletion analysis, mtDNA and clinical exome sequencing (CES), and respiratory chain complex (RCC) assay. The phenotypic characteristics included dysfunction of visual (n = 14), auditory (n = 12) and visual + auditory pathways (n = 29). Reduced RCC activity was present in 13 patients. Mitochondrial DNA deletions were noted in five patients. Sequencing of mtDNA (n = 45) identified a homoplasmic variant (MT-ND6) and a heteroplasmic variant (MT-COI) in one patient each. CES (n = 45) revealed 55 variants in nuclear genes that are associated with neuropathy (n = 27), deafness (n = 7), ataxia (n = 4), and mitochondrial phenotypes (n = 5) in 36 patients. This study provides preliminary evidence that TAN is associated with a spectrum of genetic abnormalities, including those associated with mitochondrial dysfunction, which is in contradistinction from the prevailing hypothesis that TAN is related to dietary toxins. Analysing the functional relevance of these genetic variants may improve the understanding of the pathogenesis of TAN.

A unique case of hyperammonemia due to CA5A deficiency: Impact of coexisting gene mutations, pseudogene, and microdeletion.

Carbonic anhydrase 5A (CA5A) belongs to a family of carbonic anhydrases which are zinc metalloenzymes involved in the reversible hydration of CO2 to bicarbonate. Mutations in CA5A are very rare and known to cause Carbonic anhydrase 5A deficiency (CA5AD), an autosomal recessive inborn error of metabolism characterized clinically by acute onset of encephalopathy in infancy or early childhood. CA5A also has two very identical pseudogenes whose interference may result in compromised accuracy in targeted sequencing. We report a unique case of CA5AD caused by compound heterozygous variant (NM_001739.2: c.721G>A: p.Glu241Lys & NM_001739.2: c.619-3420_c.774 + 502del4078bp) in an infant in order to expand the phenotypic spectrum and underscore the impact of pseudogenes, which can introduce complexities in molecular genetic analysis.

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.

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.

Reconstructing the demographic history of the Himalayan and adjoining populations.

The rugged topography of the Himalayan region has hindered large-scale human migrations, population admixture and assimilation. Such complexity in geographical structure might have facilitated the existence of several small isolated communities in this region. We have genotyped about 850,000 autosomal markers among 35 individuals belonging to the four major populations inhabiting the Himalaya and adjoining regions. In addition, we have genotyped 794 individuals belonging to 16 ethnic groups from the same region, for uniparental (mitochondrial and Y chromosomal DNA) markers. Our results in the light of various statistical analyses suggest a closer link of the Himalayan and adjoining populations to East Asia than their immediate geographical neighbours in South Asia. Allele frequency-based analyses likely support the existence of a specific ancestry component in the Himalayan and adjoining populations. The admixture time estimate suggests a recent westward migration of populations living to the East of the Himalaya. Furthermore, the uniparental marker analysis among the Himalayan and adjoining populations reveal the presence of East, Southeast and South Asian genetic signatures. Interestingly, we observed an antagonistic association of Y chromosomal haplogroups O3 and D clines with the longitudinal distance. Thus, we summarise that studying the Himalayan and adjoining populations is essential for a comprehensive reconstruction of the human evolutionary and ethnolinguistic history of eastern Eurasia.

Novel magnetic resonance imaging findings in a patient with short chain acyl CoA dehydrogenase deficiency.

Reports on magnetic resonance imaging findings in patients with short chain acyl -Coenzyme A dehydrogenase (SCAD) deficiency, an autosomal recessive disorder caused by mutations in the acyl-Coenzyme A dehydrogenase (ACADS), are limited. Many asymptomatic carriers of ACAD variants have also been described necessitating careful evaluation of clinical and biochemical findings for an accurate diagnosis. Here we report a an infant with short chain acyl -Coenzyme A dehydrogenase (SCAD) deficiency diagnosed based on the characteristic biochemical findings and confirmed by genetic testing. He presented with refractory seizures and neuro regression at 4 months of age. His metabolic work up revealed elevated butyryl carnitine in plasma and ethyl malonic acid in urine. Magnetic resonance imaging of the brain showed cortical and basal ganglia signal changes with cortical swelling. Serial scans showed progression of the lesions resulting in cystic leukomalacia with brain atrophy. Exome sequencing revealed a novel homozygous nonsense variation, c.1146C > G (p.Y382Ter) in exon ten of ACADS which was further validated by Sanger sequencing. Both parents were heterozygous carriers. Follow up at 15 months showed gross psychomotor retardation and refractory seizures despite being on optimal doses of anti-epileptic medications, carnitine and multivitamin supplementation. This report expands the phenotypic and genotypic spectrum of SCAD deficiency.

Genetic evidence for recent population mixture in India.

Most Indian groups descend from a mixture of two genetically divergent populations: Ancestral North Indians (ANI) related to Central Asians, Middle Easterners, Caucasians, and Europeans; and Ancestral South Indians (ASI) not closely related to groups outside the subcontinent. The date of mixture is unknown but has implications for understanding Indian history. We report genome-wide data from 73 groups from the Indian subcontinent and analyze linkage disequilibrium to estimate ANI-ASI mixture dates ranging from about 1,900 to 4,200 years ago. In a subset of groups, 100% of the mixture is consistent with having occurred during this period. These results show that India experienced a demographic transformation several thousand years ago, from a region in which major population mixture was common to one in which mixture even between closely related groups became rare because of a shift to endogamy.

Huppke-Brendel syndrome in a seven months old boy with a novel 2-bp deletion in SLC33A1.

Huppke -Brendel syndrome is a new addition to the evolving spectrum of copper metabolism defects. It is an autosomal recessive disorder characterized by congenital cataract, impaired hearing, and developmental delay with low copper and ceruloplasmin. It is caused by defects in SLC33A1 that codes for acetyl CoA transporter protein. Reports on variation in this gene causing human disease is extremely scarce and the metabolic link between this gene and copper metabolism is yet to be identified. Here we report a seven months old infant with Huppke-Brendel Syndrome. In addition to the already reported features, he also had hypo pigmented hair and hypogonadism. His magnetic resonance imaging revealed hypo myelination and cerebellar hypoplasia. Clinical exome sequencing revealed a homozygous two base pair deletion, c.542_543delTG (p.Val181GlyfsTer6) in exon 1 of the SLC33A1. This report expands the phenotypic and genotypic spectrum of Huppke Brendel syndrome.

DNA methylation analysis of phenotype specific stratified Indian population.

BACKGROUND: DNA methylation and its perturbations are an established attribute to a wide spectrum of phenotypic variations and disease conditions. Indian traditional system practices personalized medicine through indigenous concept of distinctly descriptive physiological, psychological and anatomical features known as prakriti. Here we attempted to establish DNA methylation differences in these three prakriti phenotypes. METHODS: Following structured and objective measurement of 3416 subjects, whole blood DNA of 147 healthy male individuals belonging to defined prakriti (Vata, Pitta and Kapha) between the age group of 20-30years were subjected to methylated DNA immunoprecipitation (MeDIP) and microarray analysis. After data analysis, prakriti specific signatures were validated through bisulfite DNA sequencing. RESULTS: Differentially methylated regions in CpG islands and shores were significantly enriched in promoters/UTRs and gene body regions. Phenotypes characterized by higher metabolism (Pitta prakriti) in individuals showed distinct promoter (34) and gene body methylation (204), followed by Vata prakriti which correlates to motion showed DNA methylation in 52 promoters and 139 CpG islands and finally individuals with structural attributes (Kapha prakriti) with 23 and 19 promoters and CpG islands respectively. Bisulfite DNA sequencing of prakriti specific multiple CpG sites in promoters and 5'-UTR such as; LHX1 (Vata prakriti), SOX11 (Pitta prakriti) and CDH22 (Kapha prakriti) were validated. Kapha prakriti specific CDH22 5'-UTR CpG methylation was also found to be associated with higher body mass index (BMI). CONCLUSION: Differential DNA methylation signatures in three distinct prakriti phenotypes demonstrate the epigenetic basis of Indian traditional human classification which may have relevance to personalized medicine.

Clinical and Neuroimaging Features in Two Children with Mutations in the Mitochondrial ND5 Gene.

Mutations in the mitochondrial-encoded nicotinamide adenine dinucleotide dehydrogenase 5 gene (MT-ND5) has been implicated as an important genetic cause of childhood mitochondrial encephalomyopathies. This study reports the clinical and magnetic resonance imaging findings in two pediatric patients with mutations in the ND5 gene of mitochondrial DNA. The 8-month-old boy with m.13513 G>A mutation presented with infantile basal ganglia stroke syndrome secondary to mineralizing angiopathy. The 7-year-old girl with the m.13514A>G mutation had episodic regression, progressive ataxia, optic atrophy, and hyperactivity. Magnetic resonance imaging of the brain showed bilateral symmetrical signal intensity changes in the thalamus, tectal plate, and inferior olivary nucleus, which subsided on follow-up image. Both the patients had a stable course. Familiarity with the various phenotypic and magnetic resonance imaging findings and the clinical course in childhood mitochondrial encephalomyopathies may help the physician in targeted metabolic-genetic testing and prognostication.

Mitochondrial disorders: challenges in diagnosis & treatment.

Mitochondrial dysfunctions are known to be responsible for a number of heterogenous clinical presentations with multi-systemic involvement. Impaired oxidative phosphorylation leading to a decrease in cellular energy (ATP) production is the most important cause underlying these disorders. Despite significant progress made in the field of mitochondrial medicine during the last two decades, the molecular mechanisms underlying these disorders are not fully understood. Since the identification of first mitochondrial DNA (mtDNA) mutation in 1988, there has been an exponential rise in the identification of mtDNA and nuclear DNA mutations that are responsible for mitochondrial dysfunction and disease. Genetic complexity together with ever widening clinical spectrum associated with mitochondrial dysfunction poses a major challenge in diagnosis and treatment. Effective therapy has remained elusive till date and is mostly efficient in relieving symptoms. In this review, we discuss the important clinical and genetic features of mitochondrials disorders with special emphasis on diagnosis and treatment.

Indian Siddis: African descendants with Indian admixture.

The Siddis (Afro-Indians) are a tribal population whose members live in coastal Karnataka, Gujarat, and in some parts of Andhra Pradesh. Historical records indicate that the Portuguese brought the Siddis to India from Africa about 300-500 years ago; however, there is little information about their more precise ancestral origins. Here, we perform a genome-wide survey to understand the population history of the Siddis. Using hundreds of thousands of autosomal markers, we show that they have inherited ancestry from Africans, Indians, and possibly Europeans (Portuguese). Additionally, analyses of the uniparental (Y-chromosomal and mitochondrial DNA) markers indicate that the Siddis trace their ancestry to Bantu speakers from sub-Saharan Africa. We estimate that the admixture between the African ancestors of the Siddis and neighboring South Asian groups probably occurred in the past eight generations (∼200 years ago), consistent with historical records.

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.

Co-occurrence of m.1555A>G and m.11778G>A mitochondrial DNA mutations in two Indian families with strikingly different clinical penetrance of Leber hereditary optic neuropathy.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations are known to cause Leber hereditary optic neuropathy (LHON). However, the co-occurrence of double pathogenic mutations with different pathological significance in pedigrees is a rare event. METHODS: Detailed clinical investigation and complete mtDNA sequencing analysis was performed for two Indian families with LHON. The haplogroup was constructed based on evolutionarily important mtDNA variants. RESULTS: We observed the existence of double pathogenic mutations (m.11778G>A and m.1555A>G) in two Indian LHON families, who are from different haplogroup backgrounds (M5a and U2e1), with different clinical penetrance of the disease (visual impairment). The m.11778G>A mutation in the MT-ND4 gene is associated primarily with LHON; whereas, m.1555A>G in the 12S rRNA gene has been reported with aminoglycoside-induced non-syndromic hearing loss. CONCLUSIONS: The absence of hearing abnormality and widely varying clinical expression of LHON suggest additional nuclear modifier genes, environmental factors, and population heterogeneity might play an important role in the expression of visual impairment in these families.

Mitochondrial DNA variations in myelodysplastic syndrome.

There have been significant advances in the understanding of mitochondrial function and their contribution to human disease in the last few years. The myelodysplastic syndromes (MDSs) are heterogeneous group of disorders characterized by clonal proliferation of multipotent hematopoietic cells with ineffective hematopoiesis. They are often associated with evidence of mitochondrial dysfunction. Studies have shown mitochondrial DNA (mtDNA) mutations in different MDS subtypes; however, their pattern and their role in etiopathogenesis and disease progression are not yet clear. This study was undertaken to determine the frequency and spectrum of mutations of mtDNA in patients with MDS from Indian subcontinent. The entire mitochondrial genome was systematically analyzed in 21 patients and 21 age- and sex-matched controls by gene amplification and direct sequencing using 24 overlapping polymerase chain reactions. A total 37 variations were detected in the entire mitochondrial genome. Thirty-three were reported polymorphisms, one was novel polymorphism, also seen in the normal controls, and three were variations (mutations), not seen in normal controls. Three mutations were detected in four patients (20%).These were point mutations scattered over the entire mitochondrial genome including tRNAs, rRNAs, and protein genes. COI and COII are not the only spots in mtDNA where mutation may occur in MDS but tRNA, ND1, ND5, and 16S ribosomal DNA are all vulnerable to such mutations. These mutations seem to be an important component of molecular pathology of MDS. However, its role in severity and disease progression needs to be elucidated.

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.

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.

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".