Genetic Epidemiology of Mitochondrial Pathogenic Variants Causing Nonsyndromic Hearing Loss in a Large Cohort of South Indian Hearing Impaired Individuals.

Mitochondria play a critical role in the generation of metabolic energy in the form of ATP. Tissues and organs that are highly dependent on aerobic metabolism are involved in mitochondrial disorders including nonsyndromic hearing loss (NSHL). Seven pathogenic variants leading to NSHL have so far been reported on two mitochondrial genes: MT-RNR1 encoding 12SrRNA and MT-TS1 encoding tRNA for Ser((UCN)) . We screened 729 prelingual NSHL subjects to determine the prevalence of MT-RNR1 variants at position m.961, m.1555A>G and m.1494C>T, and MT-TS1 m.7445A>G, m.7472insC m.7510T>C and m.7511T>C variants. Mitochondrial pathogenic variants were found in eight probands (1.1%). Five of them were found to have the m.1555A>G variant, two others had m.7472insC and one proband had m.7444G>A. The extended relatives of these probands showed variable degrees of hearing loss and age at onset. This study shows that mitochondrial pathogenic alleles contribute to about 1% prelingual hearing loss. This study will henceforth provide the reference for the prevalence of mitochondrial pathogenic alleles in the South Indian population, which to date has not been estimated. The m.1555A>G variant is a primary predisposing genetic factor for the development of hearing loss. Our study strongly suggests that mitochondrial genotyping should be considered for all hearing impaired individuals and particularly in families where transmission is compatible with maternal inheritance, after ruling out the most common variants.

Complete mitochondrial genome analysis and clinical documentation of a five-generational Indian family with mitochondrial 1555A>G mutation and postlingual hearing loss.

Hearing loss is the most common sensory disorder and is genetically heterogeneous. Apart from nuclear gene mutations, a number of inherited mitochondrial mutations have also been implicated. The m.1555A>G mutation in the mitochondrial MT-RNR1 gene is reported as the most common mutation causing nonsyndromic hearing loss in various ethnic populations. We report here for the first time the clinical, genetic and molecular characterisation of a single large five-generational Tamil-speaking South Indian family with maternally inherited nonsyndromic postlingual hearing loss. Molecular analysis led to identification of m.1555A>G in 28 maternal relatives with variable degree of phenotypic expression. The penetrance of hearing loss among the maternal relatives in this family was 55%. Sequence analysis of the complete mitochondrial genome in 36 members of this pedigree identified 25 known variants and one novel variant co-transmitted along with m.1555A>G mutation. The mtDNA haplotype analysis revealed that the maternal relatives carry the R*T2 haplotype similar to Europeans and South Asians. Sequencing of the coding exon of GJB2 nuclear gene did not show any pathogenic mutations. The results suggest that other nuclear or environmental modifying factors could have played a role in the differential expression of mutation m.1555A>G in postlingual hearing loss in this family.

Non-syndromic hearing impairment in India: high allelic heterogeneity among mutations in TMPRSS3, TMC1, USHIC, CDH23 and TMIE.

Mutations in the autosomal genes TMPRSS3, TMC1, USHIC, CDH23 and TMIE are known to cause hereditary hearing loss. To study the contribution of these genes to autosomal recessive, non-syndromic hearing loss (ARNSHL) in India, we examined 374 families with the disorder to identify potential mutations. We found four mutations in TMPRSS3, eight in TMC1, ten in USHIC, eight in CDH23 and three in TMIE. Of the 33 potentially pathogenic variants identified in these genes, 23 were new and the remaining have been previously reported. Collectively, mutations in these five genes contribute to about one-tenth of ARNSHL among the families examined. New mutations detected in this study extend the allelic heterogeneity of the genes and provide several additional variants for structure-function correlation studies. These findings have implications for early DNA-based detection of deafness and genetic counseling of affected families in the Indian subcontinent.

PARK2 gene mutations in early onset Parkinson's disease patients of South India.

With the etiology being unclear till date, a combination of age, genetic and environmental factors are known to play a significant role in the pathogenesis of Parkinson's disease. Mutations in PARK2 gene have been implicated to cause autosomal recessive early onset PD. We analyzed the 12 coding exons of PARK2 gene in 16 early onset PD patients of South Indian ethnicity. PARK2 mutations were present in 68% of the early onset cases. We report the presence of four PARK2 sequence variants c.1239G>C, c.171+25T>C, c.202A>G, c.601G>A, and a novel insertion mutation, c.798_799insA in the exon 7 of PARK2 gene. These results suggest that mutations in PARK2 gene may be a common cause of PD among South Indian early onset patients.

Gipc3 mutations associated with audiogenic seizures and sensorineural hearing loss in mouse and human.

Sensorineural hearing loss affects the quality of life and communication of millions of people, but the underlying molecular mechanisms remain elusive. Here, we identify mutations in Gipc3 underlying progressive sensorineural hearing loss (age-related hearing loss 5, ahl5) and audiogenic seizures (juvenile audiogenic monogenic seizure 1, jams1) in mice and autosomal recessive deafness DFNB15 and DFNB95 in humans. Gipc3 localizes to inner ear sensory hair cells and spiral ganglion. A missense mutation in the PDZ domain has an attenuating effect on mechanotransduction and the acquisition of mature inner hair cell potassium currents. Magnitude and temporal progression of wave I amplitude of afferent neurons correlate with susceptibility and resistance to audiogenic seizures. The Gipc3(343A) allele disrupts the structure of the stereocilia bundle and affects long-term function of auditory hair cells and spiral ganglion neurons. Our study suggests a pivotal role of Gipc3 in acoustic signal acquisition and propagation in cochlear hair cells.

In-vivo Antioxidant Effects of Ethyl Acetate Fraction of Mentha spicata L. on 4-Nitroquinoline-1-Oxide Injected Mice.

Antioxidant effects of ethyl acetate fraction of Mentha spicata (L.) were evaluated against 4-nitroquinoline-1-oxide injected mice. For this study, experiment setup consisted of 36 albino mice of either sex divided into 6 groups: Control (25% DMSO in water), ethyl acetate fraction (EAF) alone group (80, 160 mg/Kg body weight-bwt), 4-NQO (7.5 mg/Kg bwt-IP) alone and 4-NQO + EAF. EAF and vehicles were administered orally for five consecutive days. 4-NQO (7.5 mg/Kg bwt) was injected intraperitoneally on the 6(th) day. After 24 h, the animals were killed; liver sample was extracted and used for bio-assay. 4-NQO alone treated group decreased (27-60%) the antioxidant activities and promoted lipid peroxidation (LPO-60%) over their respective control values. Pretreatment with EAF, at the maximum dose (160 mg/Kg bwt) brought down the LPO up to 87% enhanced by 4-NQO. Among the enzymatic antioxidants, glutathione S-transferase (GST) was the most affected enzyme with 4-NQO and the least was catalase (CAT). Pretreatment with EAF (160 mg/Kg bwt), the restoration of antioxidants like glutathione peroxidase, superoxide dismutase, and CAT were found equal or less than 1.2 fold higher than that of the respective control values whereas, GST was observed to be the most restored antioxidant. Be reduced glutathione (GSH) and the least vitamin C over their control values. EAF restored the GSH and Vitamin E levels were found to be 1.2 fold higher than the respective control values.

Associations for lipoprotein lipase and peroxisome proliferator-activated receptor-gamma gene and coronary artery disease in an Indian population.

BACKGROUND AND AIMS: Peroxisome proliferator activated receptor-gamma (PPARgamma) and lipoprotein lipase (LPL) genes are important in pathways of triglyceride metabolism, insulin resistance and adipogenesis. We hypothesized that polymorphisms of PPARgamma Pro12Ala, LPL HindIII and LPL Ser447X influence severity of coronary artery disease (CAD) in an Indian population. METHODS: PPARgamma Pro12Ala, LPL HindIII and LPL Ser447X polymorphisms were genotyped in 414 patients with CAD and matched with 424 controls. The study subjects were inducted after standard diagnostic procedures and analyzed statistically for the association of polymorphisms with clinical characteristics. RESULTS: We found that PPARgamma alleles were not associated with CAD among Indians although proline carriers had significantly higher levels of HDL-cholesterol (p = 0.03) among CAD patients. The LPL HindIII also had no significant correlations for CAD or for any clinical characteristics. The Ser447X polymorphism (p = 0.015) influenced lower triglyceride levels among CAD patients with significant associations (OR = 0.66, 95% CI 0.483-0.915, p = 0.012). This protective effect of the 447X allele was more pronounced among the CAD patients without the risk factor of diabetes (OR = 0.60, 95% CI 0.403-0.907, p = 0.014) along with less progression of a severe atherosclerotic disease. CONCLUSIONS: PPARgamma and LPL have intractable roles in pathways that lead to CAD, but their gene polymorphisms associate differently. Our results imply a significant correlation of Ser447X polymorphism and its protective effect on Indians against severity of CAD modified by the risk of diabetes, than LPL HindIII and PPARgamma Pro12Ala.

Genetic variants on apolipoprotein gene cluster influence triglycerides with a risk of coronary artery disease among Indians.

Apolipoprotein C3 and apolipoprotien A5 are proteins coded from the APOA1/C3/A4/A5 gene cluster. Sst I polymorphism on apolipoprotein C3 and -1131C polymorphism of apolipoprotien A5 are key variants involved in triglyceride metabolism and cause a significant cardio-metabolic risk. Here, we have evaluated these two variants for their roles in coronary artery disease in patients of the Indian population. The apolipoprotein gene cluster variants were analysed in 416 angiographically determined coronary artery disease patients and matched 416 controls using polymerase chain reaction-restriction fragment length polymorphism. The characteristics of the study subjects were analyzed statistically for their association with the polymorphisms. The alleles were combined as haplotypes and their combined risks were evaluated. The minor allele genotypes of both apolipoprotein C3 (S2) and apolipoprotien A5 (C) had a significant risk for coronary artery disease. The S2 allele genotyped patients had a significantly increased triglyceride level (P < 0.001) and increased triglycerides were observed among both patient and control CC genotype carriers. We identified the haplotype S2/C with a significant increased risk (P < 0.001) to coronary artery disease with increased levels of circulating triglycerides compared to other haplotypes in patients. We conclude that the variants on apolipoprotein C3 and apolipoprotien A5 modulate serum triglyceride levels and increase the risk of coronary artery disease.

Antigenotoxic and antioxidant potential of aqueous fraction of ethanol extract of Mentha spicata (L.) against 4-nitroquinoline-1-oxide-induced chromosome damage in mice.

The antigenotoxic potential of an aqueous fraction of ethanol extract of Mentha spicata was evaluated by measuring the frequency of micronucleated polychromatic erythrocytes (MnPCEs) in mice bone marrow, using 4-nitroquinoline-1oxide (NQO) as the reference mutagen. In addition, lipid peroxidation (LPO) and antioxidant levels were also quantified with liver tissue of the same mice to assess their antioxidant potential. Swiss albino mice of either sex (25-30 g) were orally pretreated with an aqueous fraction (80, 160, and 320 mg/kg) for 5 consecutive days. NQO (7.5 mg/kg) was injected intraperitoneally after 2 hours until the final day (day 5) of treatment with aqueous fraction. Animals were sacrificed 24 hours later by cervical dislocation and processed for micronuclei and bioassays. A significant reduction (about 67%) of NQO-induced MnPCE frequency was observed at the dose of 320 mg/kg. The LPO was also suppressed effectively, with concomitant changes in both enzymatic and nonenzymatic antioxidants. The restoration level was dose dependent in LPO and glutathione-s-transferase, whereas it was dose independent in superoxide dismutase, glutathione peroxidase, catalase, and reduced glutathione. The results indicate that the aqueous fraction of M. spicata mediates their antigenotoxic effects by the modulation of LPO and antioxidant enzymes.

Functional consequences of novel connexin 26 mutations associated with hereditary hearing loss.

In a study of 530 individuals with non-syndromic, sensorineural hearing loss, we identified 18 mutations at connexin 26 (Cx26), four of which are novel (-23G>T, I33T, 377_383dupTCCGCAT, W172R) and the remaining 14 (ivs1+1G>A, M1V, 35delG, W24X, I35S, V37I, R75W, W77X, 312del14, E120del, Q124X, Y136X, R143W, R184P) being mutations previously described. To gain insight into functional consequences of these mutations, cellular localization of the mutant proteins and their ability to permit lucifer yellow transfer between cells was studied in seven of them (W24X, I33T, I35S, R75W, E120del, W172R and R184P). I35S and R184P showed impaired trafficking of the protein to the plasma membrane. I33T, R75W, E120del and W172R showed predominantly membrane localization but did not form functional gap junction channels. Surprisingly, W24X, a protein-truncating mutation, apparently permits formation of a full-length protein, perhaps due to a stop codon read-through mechanism. These results provide further evidence that Cx26 mutations affect gap junction activity by mis-regulation at multiple levels.

Protective effects of solvent fractions of Mentha spicata (L.) leaves evaluated on 4-nitroquinoline-1-oxide induced chromosome damage and apoptosis in mouse bone marrow cells.

Spearmint leaves (Mentha spicata L.) contain high levels of antioxidants that are known to protect against both exogenous and endogenous DNA damage. In this study, the protective effects of the hexane fraction (HF), chloroform fraction (CF) and ethyl acetate fraction (EAF) in an ethanol extract from M. spicata were evaluated against 4-nitroquinoline-1-oxide (4-NQO) induced chromosome damage and apoptosis in bone marrow cells of Swiss albino mice. Two (EAF; 80 and 160 mg/ kg body weight - bw) or three (HF and CF; 80, 160 and 320 mg/ kg bw) doses of solvent fractions or vehicle control (25% DMSO in water) were administered orally for five consecutive days. Upon the sixth day, 4-NQO was injected intraperitoneally. The animals were killed the following day. Other control groups were comprised of animals treated with either the vehicle control or the various doses of solvent fractions, but with no 4-NQO treatment. 4-NQO induced micro-nucleated polychromatic erythrocytes (MnPCEs) in all the test groups. However, pre-treatment of animals with the solvent fractions significantly reduced the 4-NQO-induced MnPCEs as well as the percentage of apoptotic cells. The reduction of both MnPCE and apoptosis was more evident following the pre-treatment of animals with 160 mg/kg bw EAF.

Protective effects of solvent fractions of Mentha spicata (L. ) leaves evaluated on 4-nitroquinoline-1-oxide induced chromosome damage and apoptosis in mouse bone marrow cells

Spearmint leaves (Mentha spicata L.) contain high levels of antioxidants that are known to protect against both exogenous and endogenous DNA damage. In this study, the protective effects of the hexane fraction (HF), chloroform fraction (CF) and ethyl acetate fraction (EAF) in an ethanol extract from M. spicata were evaluated against 4-nitroquinoline-1-oxide (4-NQO) induced chromosome damage and apoptosis in bone marrow cells of Swiss albino mice. Two (EAF; 80 and 160 mg/ kg body weight - bw) or three (HF and CF; 80, 160 and 320 mg/ kg bw) doses of solvent fractions or vehicle control (25 percent DMSO in water) were administered orally for five consecutive days. Upon the sixth day, 4-NQO was injected intraperitoneally. The animals were killed the following day. Other control groups were comprised of animals treated with either the vehicle control or the various doses of solvent fractions, but with no 4-NQO treatment. 4-NQO induced micro-nucleated polychromatic erythrocytes (MnPCEs) in all the test groups. However, pre-treatment of animals with the solvent fractions significantly reduced the 4-NQO-induced MnPCEs as well as the percentage of apoptotic cells. The reduction of both MnPCE and apoptosis was more evident following the pre-treatment of animals with 160 mg/kg bw EAF.

Mutations of the RDX gene cause nonsyndromic hearing loss at the DFNB24 locus.

Ezrin, radixin, and moesin are paralogous proteins that make up the ERM family and function as cross-linkers between integral membrane proteins and actin filaments of the cytoskeleton. In the mouse, a null allele of Rdx encoding radixin is associated with hearing loss as a result of the degeneration of inner ear hair cells as well as with hyperbilirubinemia due to hepatocyte dysfunction. Two mutant alleles of RDX [c.1732G>A (p.D578N) and c.1404_1405insG (p.A469fsX487)] segregating in two consanguineous Pakistani families are associated with neurosensory hearing loss. Both of these mutant alleles are predicted to affect the actin-binding motif of radixin. Sequence analysis of RDX in the DNA samples from the original DFNB24 family revealed a c.463C>T transition substitution that is predicted to truncate the protein in the FERM domain (F for 4.1, E for ezrin, R for radixin, and M for moesin) (p.Q155X). We also report a more complete gene and protein structure of RDX, including four additional exons and five new isoforms of RDX that are expressed in human retina and inner ear. Further, high-resolution confocal microscopy in mouse inner ear demonstrates that radixin is expressed along the length of stereocilia of hair cells from both the organ of Corti and the vestibular system.

Antioxidant activity measured in different solvent fractions obtained from Mentha spicata Linn.: an analysis by ABTS*+ decolorization assay.

Antioxidant compounds are abundantly available in plants and play an important role in scavenging free radicals, thus providing protection to humans against oxidative DNA damage. Mentha spicata Linn., commonly called spearmint, belongs to the family lamiaceae. It was selected in the present study because Mentha extracts have antioxidant properties due to the presence of eugenol, caffeic acid, rosmarinic acid and alpha-tocopherol. Four solvent fractions (hexane, chloroform, ethyl acetate and water) of ethanolic extract of dried leaves powder of M. spicata were analyzed for total antioxidant activity (TAA) and relative antioxidant activity (RAA) and compared with standard antioxidants such as Quercetin, beta-carotene, L-ascorbic acid and glutathione using ABTS*+ decolorization assay (ABTS/Potassium persulphate). The antioxidant activity was assumed to be from the total phenolic content of the ethanolic extract. Total phenolics are found to be highest in ethyl acetate fraction (54 mg/g) and least in hexane fraction (13 mg/g) and more or less similar in water and chloroform fractions (30-32 mg/g). TAA is found to be less in hexane and chloroform fractions (<53% at 50 microg/ml) and highest in ethyl acetate (95% at 20 microg/ml) and water (84% at 30 microg/ml) fractions. The RAA of ethyl acetate fraction is 1.1 compared to quercetin (at 5 microM/ml), but greater when compared to beta-carotene (15 microM/ml), L-ascorbic acid (15 microM/ml) and glutathione (15 microM/ml). The RAAs with these antioxidants are in the range of 1.31 -1.6. The values of RAAs for water fraction also show similar trend and are in the range of 1.0-1.4. The antioxidant activities of the solvent factions are closely related to the content of total phenolics present in them.

Interactive effects of saffron with garlic and curcumin against cyclophosphamide induced genotoxicity in mice.

Saffron is a well-known spice and food colorant commonly consumed in different parts of the world. Recently, much attention has been focused on the biological and medicinal properties of saffron. In the present study the interactive effects of saffron with two commonly consumed dietary agents, garlic and curcumin was evaluated for anti-genotoxic effects against cyclophosphamide (CPH) in the mouse bone marrow micronucleus test. Experimental animals were orally pretreated with saffron (100 mg/kg body weight), garlic (250 mg/kg body weight) and curcumin (10 mg/kg body weight), either alone or in combination for five consecutive days, 2h prior to the administration of CPH. Maximum reduction in the frequencies of micronucleated polychromatic erythrocytes (Mn PCEs) induced by CPH was observed when all the three test compounds were administered together. Furthermore, the protective effects were more pronounced in the garlic-administered groups compared to curcumin and/or saffron administered groups.

Refining the DFNB17 interval in consanguineous Indian families.

We previously mapped the DFNB17 locus to a 3-4 cM interval on human chromosome 7q31 in a large consanguineous Indian family with congenital profound sensorineural hearing loss. To further refine this interval, 30 new highly polymorphic markers and 8 SNPs were analyzed against the pedigree. Re-analysis in the original DFNB 17 family and additional data from a second unrelated consanguineous family with congenital deafness found to map to the interval, limited the area of shared homozygosity-by-descent (HBD) to approximately 4 megabase (Mb) between markers D7S2453 and D7S525. Nineteen known genes and over 20 other cDNAs have been identified in the refined DFNB 17 interval, including the SLC26A4 gene. We have analyzed 4 other cochlear-expressed genes that map to the DFNB17 interval as candidate genes. Analysis of coding and splice site regions of these cochlear expressed genes did not reveal any disease causing mutations. Further study of other candidate genes is currently underway.

Inhibitory effects of aqueous crude extract of Saffron (Crocus sativus L.) on chemical-induced genotoxicity in mice.

Saffron (dried stigmas of Crocus sativus L.), was evaluated in the mouse bone marrow micronucleus test for its possible protective effects against chromosomal damage induced by cisplatin (CIS), mitomycin-C (MMC) and urethane (URE). Three doses of saffron (25, 50 and 100 mg/kg body weight) were orally administered to mice for five consecutive days prior to administration of genotoxins under investigation. From the results obtained, it was evident that the administration of 50 and 100 mg saffron/kg body weight could significantly inhibit the in vivo genotoxicity of these genotoxins. However, all the three doses of saffron were effective in exerting a protective effect against urethane.

Mutations in a novel gene, TMIE, are associated with hearing loss linked to the DFNB6 locus.

We have identified five different homozygous recessive mutations in a novel gene, TMIE (transmembrane inner ear expressed gene), in affected members of consanguineous families segregating severe-to-profound prelingual deafness, consistent with linkage to DFNB6. The mutations include an insertion, a deletion, and three missense mutations, and they indicate that loss of function of TMIE causes hearing loss in humans. TMIE encodes a protein with 156 amino acids and exhibits no significant nucleotide or deduced amino acid sequence similarity to any other gene.