Nahid Akhtar Khan

Mitochondrial dysfunction and genetic heterogeneity in chronic periodontitis

We performed an extensive study on mitochondrial dysfunction in chronic periodontitis (CP). Electron microscopic analysis of gingival cells revealed abnormal mitochondria in 60% of the patients. Mitochondrial membrane potential and oxygen consumption of gingival cells were reduced by 4 fold and 5.8 fold, respectively; whereas ROS production was increased by 18%. The genetic analysis by complete mitochondrial DNA sequencing revealed the identification of 14 novel mutations only in periodontal tissues but not in the blood, suggesting a role of oxidative stress on periodontal tissues. Thus, our functional and genetic analysis provided an evidence for the mitochondrial dysfunction in CP.

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.

Mitochondrial DNA variations associated with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a primary disorder, characterized by unexplained hypertrophy of the left ventricle that frequently involved in the inter-ventricular septum. Mitochondrial DNA (mtDNA) mutations and haplogroups have been found to be associated with several diseases. Therefore, in the present study, we have sequenced the complete mtDNA of 114 clinically well-characterized HCM patients to look for the role of mtDNA variations and haplogroups in HCM phenotype among Indian patients. Complete mtDNA analysis revealed 28 novel variations, 25 disease-associated and 50 private mutations. We found 13 (11.40%) HCM patients having novel non-synonymous and/or MT-tRNA variations, of which two (m.4797C>M and m.8728T>Y) were in heteroplasmic condition. In silico prediction showed that a few mutations are pathogenic, which may affect the energy production in the heart. Unlike some of the other studies, we did not find association of mitochondrial haplogroup with HCM.

Haplogroup Heterogeneity of LHON Patients Carrying the m.14484T>C Mutation in India

PURPOSE To investigate the clinical and mitochondrial DNA (mtDNA) haplogroup background of Indian Leber hereditary optic neuropathy (LHON) patients carrying the m.14484T>C mutation. METHODS Detailed clinical investigation and complete mtDNA sequencing analysis was carried out for eight Indian LHON families with the m.14484T>C mutation. Haplogroup was constructed based on the evolutionarily important mtDNA variants. RESULTS In the present study, we characterized eight unrelated probands selected from 187 LHON cases. The overall penetrance of the disease was estimated to be 19.75% (16/81) in eight pedigrees with the m.14484T>C mutation and showed substantially higher sex bias (male: female = 13:3). The mtDNA haplogrouping revealed that they belong to diverse haplogroups; i.e., F1c1, M31a, U2a, M*, I1, M6, M3a1, and R30a. Interestingly, we did not find an association of the m.14484T>C mutation with any specific haplogroup within the Indian population. We also did not find any secondary mutation(s) in these pedigrees, which might affect the clinical expression of LHON. CONCLUSIONS Contrary to earlier reports showing preferential association of the m.14484T>C mutation with western Eurasian haplogroup J and increased clinical penetrance when present in J1 subhaplogroup background, the present study shows that m.14484T>C arose independently in a different mtDNA haplogroup and ethnic background in India, which may influence the clinical expression of the disease.

The “Double Panda” Sign in Leigh Disease

Although the “face of the giant panda” sign on magnetic resonance imaging (MRI) is traditionally considered to be characteristic of Wilson disease, it has also been reported in other metabolic disorders. This study describes the characteristic “giant panda” sign on MRI in a child with Leigh disease. The diagnosis was based on the history of neurological regression; examination findings of oculomotor abnormalities, hypotonia, and dystonia; raised serum lactate levels; and characteristic brain stem and basal ganglia signal changes on MRI. The midbrain and pontine tegmental signal changes were consistent with the “face of the giant panda and her cub” sign. In addition to Wilson disease, metabolic disorders such as Leigh disease should also be considered in the differential diagnosis of this rare imaging finding.

Mitochondrial DNA variations in Madras motor neuron disease

Although the Madras motor neuron disease (MMND) was found three decades ago, its genetic basis has not been elucidated, so far. The symptom at onset was impaired hearing, upper limb weakness and atrophy. Since some clinical features of MMND overlap with mitochondrial disorders, we analyzed the complete mitochondrial genome of 45 MMND patients and found 396 variations, including 13 disease-associated, 2 mt-tRNA and 33 non-synonymous (16 MT-ND, 10 MT-CO, 3 MT-CYB and 4 MT-ATPase). A rare variant (m.8302A>G) in mt-tRNA(Leu) was found in three patients. We predict that these variation(s) may influence the disease pathogenesis along with some unknown factor(s).

Mitochondrial oxidative phosphorylation disorders in children: Phenotypic, genotypic and biochemical correlations in 85 patients from South India

Mitochondrial oxidative phosphorylation (OXPHOS) disorders account for a variety of neuromuscular disorders in children. In this study mitochondrial respiratory chain enzymes were assayed in muscle tissue in a large cohort of children with varied neuromuscular presentations from June 2011 to December 2013. The biochemical enzyme deficiencies were correlated with the phenotypes, magnetic resonance imaging, histopathology and genetic findings to reach a final diagnosis. There were 85 children (mean age: 6.9±4.7years, M:F:2:1) with respiratory chain enzyme deficiency which included: isolated complex I (n=50, 60%), multiple complexes (n=24, 27%), complex IV (n=8, 9%) and complex III deficiencies (n=3, 4%). The most common neurological findings were ataxia (59%), hypotonia (59%) and involuntary movements (49%). A known mitochondrial syndrome was diagnosed in 27 (29%) and non-syndromic presentations in 57 (71%). Genetic analysis included complete sequencing of mitochondrial genome, SURF1, POLG1&2. It revealed variations in mitochondrial DNA (n=8), SURF1 (n=5), and POLG1 (n=3). This study, the first of its kind from India, highlights the wide range of clinical and imaging phenotypes and genetic heterogeneity in children with mitochondrial oxidative phosphorylation disorders.

Response to Letter to the Editor “Mitochondrial haplogroups are associated with hypertrophic cardiomyopathy in the Indian population”

Complete mitochondrial DNA (mtDNA) analysis of 114 HCM patients along with 300 ethnically matched controls from India revealed several novel and disease-associated mutations (Govindaraj et al., 2014). However, in contrast to studies on other populations (Castro et al., 2006; Hagen et al., 2013), none of the mitochondrial haplogroup was found to be associated with HCM in India (Govindaraj et al., 2014). Christiansen et al. (in press) have reanalyzed this data by combining M, N, I and W haplogroups (as early cluster) and R, U and H haplogroups (as late cluster), and suggested that these two clusters of haplogroups were significantly different between HCM patients and controls. Hence, they proposed that mitochondrial haplogroups are associated with HCM in the Indian population. This analysis has the following issues: 1. samples with N, I, W (early cluster) and H (late cluster) haplogroups are very less in number (please refer to Table 1 of Christiansen et al., 2014); hence inclusion of such a low sample numbers would introduce bias; 2. p-values were not subjected to Bonferroni correction. Since more than one haplogroup was considered for the analysis, it is appropriate to correct the p-values with Bonferroni correction. Even with the inclusion of early and late cluster haplogroups, when Bonferroni corrections were applied to the p-values, obtained by Christiansen et al. (2014), we found that both early (p = 0.092) and late (p = 0.069) haplogroups are not significantly associated with HCM in the Indian population (Table 1). We once again reiterate that mitochondrial haplogroup, either independently or in combination, is not associated with HCM in India.

Author Response: Penetrance of the LHON Mutation m.11778G>A May Depend on Factors Other Than Haplotype or Heteroplasmy Rate

We appreciate the effort shown by the authors and their comments on our article ‘‘Leber’s Hereditary Optic Neuropathy-Specific Mutation m.11778G>A Exists on Diverse Mitochondrial Haplogroups in India.’’ Leber’s hereditary optic neuropathy (LHON) is the most common, well-diagnosed, and maternally inherited mitochondrial disease. In this case, we would emphasize the statement that LHON is one of the most well characterized maternally inherited mitochondrial disease. We do agree with the statement that penetrance may depend on the age of individuals who carry the mutation. To date, our follow-up data do not reveal nonsymptomatic mutation carriers to have developed visual impairment and LHON. The data presented are to the best of our knowledge, till the time the manuscript was written. However, most of the families are in contact with the clinicians and have been informed about their genetic diagnosis. The families were followed up for the period of study (2005–2016), and patients were included if they developed visual disturbance. As discussed in the article, 13 out of 64 LHON families with m.11778G>A mutation were observed in heteroplasmic condition, and the remaining 51 were homoplasmic. In total (22 out of 145), 15% of manifesting mutation carriers was present with heteroplasmic form, while 31% of mutation carriers (124 out of 398) were present with homoplasmic condition. We do agree with the authors that knowledge of environmental factors (tobacco smoke) would greatly help in answering the penetrance of these LHON pedigrees. However, we realized during the study that quite a significant percentage of participants were not ready to share the details about their smoking and alcohol consumption history in an accurate way. At present, we lack proper details of the tobacco smoke and alcohol consumption for all the pedigrees and the amount of information, which we have, is not sufficient to make any specific conclusion about this issue. Keeping this in mind, we have decided to exclude this information from this study. During the initial analysis, 41 out of 64 index cases had positive family history of LHON. Seven affected individuals have mothers clinically manifesting LHON. During the course of this study, all the index cases were investigated for multiorgan involvement. We observed one patient from the index case had mild hepatosplenomegaly, while the other patients from index cases do not have any organ involvement. Hence, we would not be able to make conclusions about the multiorgan involvement and heteroplasmy level. None of the tRNA variants scored >11 on the Yarham score. One of the limitations was that we do not have all the information required for predicting the Yarham score for each patient due to absence of histology and transmitochondrial cybrid study for all the mutations. During initial clinical diagnosis of LHON-like symptoms for the patients, blood samples were collected for genetic diagnosis, which was confirmed by the presence of mutation. We, therefore, do not agree with collection of invasive samples like muscle biopsy in all the cases, but it was collected for a few cases where patients gave their consent. Hence, we were not able to perform all the analysis for predicting the Yarham score for additional variants observed together with m.11778G>A mutation. We also think it would be very difficult to segregate the effect of additional variant, since patients already have pathogenic mutation. We do agree with authors that pathogenicity of variants may be confirmed by using single fiber and cybrid analysis. However, it is not possible for us to make cybrids for all the variants; nevertheless, for the continuation of this project, we have a plan to select a few of the potential variants to study their role in different haplogroup backgrounds.

Outcome of epilepsy in patients with mitochondrial disorders: Phenotype genotype and magnetic resonance imaging correlations

OBJECTIVES Studies exploring the outcome of epilepsy in patients with mitochondrial disorders are limited. This study examined the outcome of epilepsy in patients with mitochondrial disorders and its relation with the clinical phenotype, genotype and magnetic resonance imaging findings. PATIENTS AND METHODS The cohort was derived from the database of 67 patients with definite genetic diagnosis of mitochondrial disorders evaluated over a period of 11years (2006-2016). Among this, 27 had epilepsy and were included in final analysis. Data were analyzed with special reference to clinical phenotypes, genotypes, epilepsy characteristics, EEG findings, anti epileptic drugs used, therapeutic response, and magnetic resonance imaging findings. Patients were divided into three groups according to the seizure frequency at the time of last follow up: Group I- Seizure free; Group II- Infrequent seizures; Group III- uncontrolled seizures. For each group the clinical phenotype, genotype, magnetic resonance imaging and duration of epilepsy were compared. RESULTS The phenotypes & genotypes included Mitochondrial Encephalopathy Lactic Acidosis and Stroke like episodes (MELAS) & m.3243A>G mutation (n = 10), Myoclonic Epilepsy Ragged Red Fiber syndrome (MERRF) & m.8344A>G mutation (n = 4), Chronic Progressive External Ophthalmoplegia plus &POLG1 mutation (CPEO, n = 6), episodic neuroregression due to nuclear mutations (n = 6; NDUFV1 (n = 3), NDUFA1, NDUFS2, MPV17-1 one each), and one patient with infantile basal ganglia stroke syndrome, mineralizing angiopathy &MT-ND5 mutations. Seven patients (25.9%) were seizure free; seven had infrequent seizures (25.9%), while thirteen (48.1%) had frequent uncontrolled seizures. Majority of the subjects in seizure free group had episodic neuroregression & leukoencephalopathy due to nuclear mutations (85.7%). Patients in group II with infrequent seizures had CPEO, POLG1 mutation and a normal MRI (71%) while 62% of the subjects in group III had MELAS, m.3243A>G mutation and stroke like lesions on MRI. CONCLUSIONS A fair correlation exists between the outcome of epilepsy, clinical phenotypes, genotypes and magnetic resonance imaging findings in patients with mitochondrial disorders. The recognition of these patterns is important clinically because of the therapeutic and prognostic implications.