Mitochondria in health and disease.

Mitochondrial biology has become an area of intense research owing to the unique physiology of the organelle and its role in several types of cancers and other disorders. It has been found that mitochondria-encoded proteins, mitochondrial DNA and even RNA influence the functioning of the cell in more ways than were previously imagined. This may contribute to disease phenotypes which require detailed investigation and communication to the community health care providers. Additionally, this provides several novel avenues in drug designing against various cancers, neurodegenerative diseases and other metabolic disorders. The sixth annual conference of the Society for Mitochondrial Research and Medicine - India (SMRM) titled, 'Mitochondria in Health and Disease' was organized by Rana P. Singh at the School of Life Sciences, Jawaharlal Nehru University in New Delhi, India from 10th to 11th February 2017. The underlying objective of the conference was to provide a platform to discuss the recent advances in basic and translational research in mitochondrial biology and diseases. The conference aimed to translate academic research into clinical practice by providing a forum for basic researchers and clinicians to share their knowledge and build collaborations towards development of advanced therapeutic in mitochondrial diseases. To facilitate the knowledge-sharing, six major themes for the scientific sessions were (1) understanding of mitochondrial biology in disease progression, (2) advances in basic and translational mitochondrial research, (3) mitochondria in evolution and development, (4) targeting mitochondria for cancer prevention and treatment, (5) mitochondria in metabolic and neurological disorders and (6) mitochondria in stem cell and regeneration biology. This report summarizes the major outcomes of the discussions at the conference.

Ionizing radiation effects on sex steroid hormone levels in serum and milt of freshwater fish Oreochromis mossambicus.

Effects of gamma rays on the sex steroid hormone levels [testosterone (T), 11-ketotestosterone (11-KT) and 17ß-estradiol (E2)] were studied in the freshwater fish Oreochromis mossambicus. Gamma radiation induced effects on hormone levels reported here for the first time in the fish. Since radionuclides released accidentally or during a nuclear disaster can contaminate inland water bodies, biomonitoring methods are required for assessing the impacts of certain dose levels of radiation that may ultimately result in ionizing radiation exposure to both humans and non-human biota. Three groups of (n=15 in each group) fishes were irradiated with a single dose of (60)Co 10Gy, 15Gy and 20Gy with a duration of .33, .50 and .66min. Significant decrease of the hormone levels was seen at higher doses of 15Gy and 20Gy. The sex steroid hormone levels in the fishes are vital for sperm production, development, differential functions related to the physiology and reproductive behavior. This study serves as biomonitoring tool to assess the ionizing radiation effects on reproductive behavior of aquatic biota.

Clinical and magnetic resonance imaging findings in patients with Leigh syndrome and SURF1 mutations.

BACKGROUND: Mutation in the SURF1 is one of the most common nuclear mutations associated with Leigh syndrome and cytochrome c oxidase deficiency. This study aims to describe the phenotypic and imaging features in four patients with Leigh syndrome and novel SURF1 mutation. METHODS: The study included four patients with Leigh syndrome and SURF1 mutations identified from a cohort of 25 children with Leigh syndrome seen over a period of six years (2006-2012). All the patients underwent a detailed neurological assessment, muscle biopsy, and sequencing of the complete mitochondrial genome and SURF1. RESULTS: Three patients had classical presentation of Leigh syndrome. The fourth patient had a later age of onset with ataxia as the presenting manifestation and a stable course. Hypertrichosis, facial dysmorphism and hypopigmentation were the additional phenotypic features noted. On magnetic resonance imaging all patients had brainstem and cerebellar involvement and two had basal ganglia involvement in addition. The bilateral symmetrical hypertrophic olivary degeneration in these patients was striking. The SURF1 analysis identified previously unreported mutations in all the patients. On follow-up three patients expired and one had a stable course. CONCLUSIONS: Patients with Leigh syndrome and SURF1 mutation often have skin and hair abnormalities. Bilateral symmetrical hypertrophic olivary degeneration was a consistent finding on magnetic resonance imaging in these patients.

Contribution of muscle biopsy and genetics to the diagnosis of chronic progressive external opthalmoplegia of mitochondrial origin.

Chronic progressive external opthalmoplegia (CPEO) is the most common phenotypic syndrome of the mitochondrial myopathies. Muscle biopsy, which provides important morphological clues for the diagnosis of mitochondrial disorders, is normal in approximately 25% of patients with CPEO, thus necessitating molecular genetic analysis for more accurate diagnosis. We aimed to study the utility of various histochemical stains in the diagnosis of CPEO on muscle biopsy and to correlate these results with genetic studies. Between May 2005 and November 2007 all 45 patients diagnosed with CPEO were included in the study (23 males; mean age at presentation, 35 years). Thirty-nine patients had CPEO only and six had CPEO plus; two had a positive family history but the remaining 39 patients had sporadic CPEO. Muscle biopsy samples were stained with hematoxylin and eosin, modified Gomori's trichrome stain, succinic dehydrogenase (SDH), cytochrome C oxidase (COX) and combined COX-SDH. Ragged red fibers were seen in 27 biopsies; seven showed characteristics of neurogenic atrophy only, and 11 were normal. The abnormal fibers were best identified on COX-SDH stain. A complete mitochondrial genome was amplified in muscle and blood samples of all patients. Mutations were found in transfer RNA, ribosomal RNA, ND, CYTB, COX I, II and III genes. Mitochondrial gene mutations were found in ten of the 11 patients with a normal muscle biopsy. The genetic mutations were classified according to their significance. The observed muscle biopsy findings were correlated with genetic mutations noted. Histological studies should be combined with genetic studies for the definitive diagnosis of CPEO syndrome.

Estrogen receptor beta gene mutations in Indian infertile men.

Recent studies suggest that estrogens play an important role in male fertility. Estrogen signaling is mediated by Estrogen Receptors (ERalpha and ERbeta). Association of ERbeta with male infertility has not been analyzed to date except for genotyping of known polymorphisms in two different studies, which yielded controversial interpretation. Hence, we performed sequencing of all the exons and untranslated regions of ERbeta gene in 300 infertile and 255 fertile control Indian men. We identified eight novel mutations and four known single nucleotide polymorphisms (SNPs). Of the eight novel mutations, four were non-synonymous, of which one was detected only in infertile men, whereas the other three mutations were detected only in fertile men. Using different bioinformatics tools, we predicted that non-synonymous mutations were benign and they neither altered the structure nor the function of the protein. Among synonymous novel mutations, one was detected in both fertile and infertile men, two were exclusive to infertile men and one was exclusive to fertile men. None of the known SNPs or novel mutations showed statistically significant difference between infertile and fertile men. Moreover, infertile men having ERbeta mutations had normal reproductive tract and serum hormone levels. Our results suggest that the SNPs and mutations in ERbeta gene are not a common cause of spermatogenesis failure in Indian men, although mutations specifically found in infertile men can affect transcription, translation or have synergic effect with other variants in causing infertility.

CA repeat and RsaI polymorphisms in ERbeta gene are not associated with infertility in Indian men.

Oestrogen Receptor beta (ERbeta) gene plays an important role in the regulation of fertility in both males and females. Polymorphism in CA repeat located in the flanking region of ERbeta has been shown to be associated with several diseases, but its association with male infertility has not been analysed so far. However, RsaI polymorphism (rs1256049) in exon 5 of ERbeta has been shown to be associated with male infertility in Caucasian patients. Hence, we have analysed 695 Indian men, including 443 infertile and 252 fertile men to evaluate the association of CA repeat length and RsaI polymorphisms in male infertility. Our results revealed no significant difference in the distribution of CA repeat length between infertile (mean +/- SD 23.24 +/- 2.06, median 24) and fertile men (mean +/- SD 23.16 +/- 2.27, median 24). The analysis of dosage effect by classifying samples into SS (short/short), SL (short/long) and LL (long/long) groups also did not show any significant difference between infertile and fertile men. Similarly, RsaI polymorphism also did not show any significant difference between infertile and fertile men. Furthermore, the combined analysis of CA repeat and RsaI polymorphisms by haplotyping showed that the distribution of haplotypes was not significantly different between fertile and infertile men. Our results suggest that CA repeat length and RsaI polymorphisms in ERbeta are not associated with infertility in Indian men.

Independent origin of 185delAG BRCA1 mutation in an Indian family.

BRCA1 and BRCA2 are the two major breast cancer susceptibility genes. We tested members of a family with multiple cases of breast cancer, for mutations in the BRCA1 gene. Analysis of the PCR amplicons of all the exons (22) of the BRCA1 gene using conformation sensitive gel electrophoresis (CSGE) revealed a heteroduplex band pattern in exon 2 of the proband (III-3) in this family. The amplicon was further sequenced to assess the nature of the mutation, which revealed a deletion of AG nucleotides at the 185th position (185delAG). The two base pair deletion introduces a stop codon at the 39th amino acid residue. A similar analysis was carried out on other extended family members to evaluate their allelic status. We detected the same deletion in 7 of the 19 members tested. Two of them are males. Haplotype analysis suggested an independent origin for this mutation. Our study highlights the importance of testing hereditary cases of breast/ovarian cancer for BRCA1 mutations in extended families in order to identify high-risk individuals at a pre-clinical stage and provide genetic counseling.

Is there an inter-relationship between prostate specific antigen, kallikrein-2 and androgen receptor gene polymorphisms with risk of prostate cancer in north Indian population?

Androgen receptor (AR) and kallikrein (KLK-2) regulates the PSA (prostate specific antigen) transcription and activation, respectively. We investigated the individual and combined risk of KLK-2, PSA and AR gene polymorphism in histologically confirmed CaP patients and healthy controls from north India. DNA was extracted from peripheral blood leucocytes pellet of 277 subjects. AR repeats analysis was done by PCR-Genscan method. PSA and KLK-2 were genotyped by PCR-RFLP method. Kruskal-Wallis test and logistic regression was applied for mean comparison and risk determination. A significant association for CaP risk was observed with short AR-CAG repeats (OR=3.36, p<0.001) and CC genotype of KLK-2 (OR=2.78, p=0.031), however, no association was found with PSA and AR-GGN repeat polymorphism. PSA/GG genotype was significantly associated with higher Gleason score (> or =7) of tumor (OR=6.23, p<0.01). No association was observed with other confounding variables such as PSA and age with any of these polymorphisms. Thus, we hypothesize that these polymorphisms may influence the etiology of CaP and may have the probability to become appropriate marker either independently or in combination. The combined information on serum PSA level, PSA (G/A), KLK-2 (C/T) genotypes and AR (CAG; GGN repeat) may assist in the deterrence of unnecessary biopsies.

A novel BRCA1 mutation in an Indian family with hereditary breast/ovarian cancer.

Germ-line mutations in BRCA1 gene contribute to a majority of familial breast and ovarian cancers. A group of 23 Tamil Nadu (south India) patients with positive family history for breast and ovarian cancer were screened for BRCA1 mutations by conformation sensitive gel electrophoresis (CSGE) followed by sequencing. In the present study, we report a novel 1307delT mutation in exon 11 of BRCA1 gene in a 43-year-old woman of Indian origin with breast cancer. This mutation gives rise to a premature stop codon at amino acid residue 409 and also creates a novel DdeI restriction site. The same mutation was also detected in the patient's maternal uncle and his son through extended family analysis. The 1307delT is a novel mutation that has not been documented in any population or published report to the best of our knowledge. Identification of this novel mutation stresses the need for developing a database of BRCA1 mutations, which will aid in breast cancer screening in this population.

Nonprogressive juvenile-onset spinal muscular atrophy: A clinico-radiological and CAG repeat study of androgen receptor gene.

BACKGROUND: Occurrence of nonprogressive juvenile-onset spinal muscular atrophy (SMA) predominantly in males suggests a possibility of X-linked disorder but there is no such report addressing this problem. AIMS: To evaluate CAG repeat expansion of androgen receptor (AR) gene in patients with nonprogressive juvenile-onset SMA. SETTING: Tertiary medical teaching institute. SUBJECTS AND METHODS: Patients fulfilling the diagnostic criteria of nonprogressive juvenile-onset SMA were included. Detailed clinical evaluation and pedigree charting were done in all. Nerve conduction study, electromyography and cervical spinal MRI were carried out. From peripheral venous blood, DNA was separated and AR gene CAG repeat exon polymorphism was assayed using polymerase chain reaction (PCR) in conjugation with genotyping and Gene scan soft ware. Number of CAG repeats was compared with normal controls. RESULTS: 25 patients with nonprogressive juvenile-onset SMA from 24 families were included and their mean age was 22.2 years. Age at the time of disease onset ranged between 15 and 30 years with a mean duration of illness 2.6 years. None of the patients had testicular atrophy or gynecomastia. C7-T1 myotomal wasting and weakness although was unilateral to begin with but became bilateral in 16 and 4 more patients had evidences of subclinical involvement of the other side as revealed by EMG. Spinal MRI revealed cord atrophy at C6-8 vertebral level in 16 patients. CAG repeat study of AR gene was carried out in 16 patients. The number of CAG repeats in patients ranged between 15 and 39 (median 21) which were within the normal range. CONCLUSION: Abnormal CAG repeat expansion of AR gene is not found in patients with nonprogressive juvenile-onset SMA.

A novel androgen receptor mutation resulting in complete androgen insensitivity syndrome and bilateral Leydig cell hyperplasia.

Androgens drive male secondary sexual differentiation and maturation. Mutations in the androgen receptor (AR) gene cause a broad spectrum of abnormal phenotypes in humans, ranging from mild through partial to complete androgen insensitivity. We have analyzed the AR gene by using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing and have studied gonads histologically in a familial case of complete androgen insensitivity syndrome. Sequence analysis of the AR gene showed a novel C2578T missense mutation, resulting in the replacement of a highly conserved leucine residue with phenylalanine (L859F) in ligand-binding domain of the receptor. The residue L859, located in helix 10 of the androgen receptor, plays a significant role in overall architecture of ligand-binding pocket. The mutation was absent from the father, normal brother of the patients, and 100 normal males recruited in this study as controls. The inheritance of the mutation in the family clearly shows that C2578T is the underlying mutation for the eventual phenotype in the patients. Histology of patient's gonads showed Leydig cell hyperplasia, with a few or no spermatogonium. It is thought that AR gene mutations result in hormonal imbalance, resulting in the high levels of luteinizing hormone (LH) and ultimately Leydig cell hyperplasia or tumor formation. In the present study, we have reported a rare familial case of Leydig cell hyperplasia despite consistently normal LH levels. The finding will help in giving counseling to this family and prevent the transmission of the mutated X chromosome to the coming generations.

Is reduced CAG repeat length in androgen receptor gene associated with risk of prostate cancer in Indian population?

Shorter CAG repeats in androgen receptor (AR) gene have been found to be associated with an increased risk of prostate cancer (CaP). Ethnic variations in CAG repeat length may contribute to varying risks in different populations. To evaluate the prognostic significance of androgen receptor (AR) CAG repeats in Indian population for CaP, genomic DNA from 113 CaP, 57 benign prostate hyperplasia (BPH) patients and 133 normal healthy controls were examined by using a PCR-based GeneScan analysis. The mean number of CAG repeat in CaP was significantly lower as compared to the healthy controls (20.26 vs 22.98; p = 0.016). The odds ratio for CaP was 2.96 (p < 0.01), when individuals with short CAG repeat (< or =22) were compared with those having longer repeats (>22). A significant association was also observed between short CAG repeat and young age at diagnosis (OR 2.18; p = 0.04). The mean CAG repeat was not significantly different in BPH and healthy controls; however, BPH patients showed a tendency towards short CAG repeats. Thus, our results show that CAG repeat polymorphism in AR gene is significantly associated with CaP risk, suggesting that AR CAG polymorphism may act as a risk modifier to CaP in Indian population.