Association of oxidative stress gene polymorphisms with presbycusis

Gene 593 (2016) 277–283

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Research paper

Association of oxidative stress gene polymorphisms with presbycusis Santoshi Kumari Manche a,b, Madhavi Jangala a,b, Padmavathi Putta a, Raja Meganadh Koralla a, Jyothy Akka b,⁎ a b

MAA Research Foundation, Somajiguda, Hyderabad, Telangana State, India Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Hyderabad, Telangana State, India

a r t i c l e

i n f o

Article history: Received 17 June 2016 Received in revised form 18 July 2016 Accepted 17 August 2016 Available online 22 August 2016 Keywords: Presbycusis Cytochrome P450 Glutathione S transferase N-acetyl transferase Uncoupled proteins

a b s t r a c t Introduction: Presbycusis is characterised by etiopathological changes in the cochlea of the inner ear due to genetic and environmental factors and has a serious impact on quality of life. The present study was aimed to evaluate the role of oxidant stress gene polymorphisms in the development of presbycusis. Subjects and methods: 220 subjects with confirmed presbycusis from ENT specialists of MAA ENT hospital, Hyderabad, India from 2012 to 2014 were considered for the study. 270 age and sex matched controls were included in the study. Analysis of gene polymorphisms of SNPs cytochrome P450 1A1 (CYP1A1) 3801 TN C, 2455 ANG and 2453 A NC; glutathione S transferase (GST) T1 and M1; N-acetyl transferase (NAT2) 282 C N T and 857 G N A; uncoupled proteins (UCP1) (−3826) ANG and (UCP2) (866)GN A was carried out. Variations in the allelic and genotypic frequencies obtained were computed and analysed using appropriate statistical methods. Results: The results of the study indicated that CYP1A1 gene polymorphism at 2453 CN A (adjusted OR: 1.59, 95% CI: 1.01–2.87) and 2455 ANG (adjusted OR: 1.87, 95% CI: 1.07–3.37), double null genotype of GSTM1 and GSTT1 (adjusted OR: 8.88, 95% CI: 4.10–19.19), NAT2 gene at C282T (adjusted OR: 1.77, 95% CI: 1.02–3.11) and G590 A (adjusted OR: 1.83, 95% CI 1.20–3.63) and UCP2 (−866) GN A (adjusted OR: 12.39; 95% CI: 6.51–23.56) showed increased risk for presbycusis while CYP1A1 at 3801 TNC and UCP1 (−3286) ANG exhibited no association. The haplotype combinations of T-G-A of CYP1A1 at 3801, 2455 and 2453 positions as well as T-A of NAT2*6 at 282 and 590 positions were found to contribute significant risk for the onset of presbycusis. Conclusions: Gene polymorphisms of CYP1A1 (A2455G, C2453A), NAT2*6 (C282T, G590 A), GST T1/M1 (double null genotype) and UCP2 (G-866 A) were found to contribute significant risk to presbycusis. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Presbycusis is a complex auditory disorder caused by degenerative changes due to aging in the cochlea of inner ear and central auditory pathways that lead to high-frequency hearing loss and loss of speech discrimination (Cruickshanks et al., 1998; Staecker et al., 2001). The cumulative effect of intrinsic and extrinsic factors like anatomical degeneration, noise exposure, medical disorders and their treatment, as well as hereditary susceptibility are reported to affect cochlea function (Bared et al., 2010). Especially, reductions in oxygen and an increase in the production of free radicals (Reactive oxygen species: ROS) leads to increase in the risk for age related hearing loss (Seidman et al., 2002; Coling et al., 2003; Ohlemiller, 2009). Gene polymorphisms of several detoxification and antioxidant phase I and II enzymes such as Abbreviations: ROS, Reactive oxygen species; CYP, cytochrome P450; GST, glutathione S-transferases; NAT, N-acetyltransferase; UCP, Uncoupled proteins; Ala, Alanine; Val, Valine; Thr, Threonine; PCR, Polymerase chain reaction; RFLP, Restriction Fragment Length Polymorphism. ⁎ Corresponding author at: Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Hyderabad, Telangana, India. E-mail address: [email protected] (J. Akka).

http://dx.doi.org/10.1016/j.gene.2016.08.029 0378-1119/© 2016 Elsevier B.V. All rights reserved.

cytochrome P450 (CYP1A1), glutathione peroxidase (GSTP1), glutathione S-transferases (GST M1 and T1), N-acetyltransferase (NATs) increase ROS levels and thereby induce cellular injury (Staecker et al., 2001; Seidman et al., 2002; Hein, 2002). In addition, mitochondrial mutations (UCP1, UCP2) acquired due to aging may also contribute to the degeneration process in the cochlea of inner ear and thereby contribute to hearing loss (Gates et al., 1999). CYP1A1 gene located on chromosome 15q22–24 spans 5810 bp has 7 exons and 6 introns codes for an enzyme aryl hydrocarbon hydroxylase responsible for the metabolism of polycyclic aromatic hydrocarbons (Hayashi et al., 1991). Studies have reported that substitution at position 3801 involves the transition of thymidine to cytosine in the 3′ non-coding region. The other well-known polymorphism CYP1A1 2455 A to G is downstream of exon 7 region involves replacement of isoleucine with valine in the catalytic region of the protein that leads to an increase in enzyme activity while 2453 substitution of C to A in exon 7 leads to changes in threonine to aspartate at codon 461 (Hayashi et al., 1991; Landi et al., 1994). Many phase II drug metabolizing enzymes such as the N-acetyltransferases (NAT1 and NAT2) and the glutathione S-transferases (GSTM and GSTT) were found to play an important role in the antioxidant protection of adult cochlea (Parl, 2005; Liu and Yan,

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2007). The genetic polymorphisms of GSTM1 and GSTT1, involves deletion of 20 base pairs respectively which leads to loss in functional activity towards conjugation specific metabolites and enzymes (Duell et al., 2002). The genes encoding cytosolic N-acetyltransferase consist of an introns less protein coding exon with an open reading frame of 870 base pairs are located at 8p22 (Grant et al., 1989). Genetic variations in N-acetyltransferase genes leads to altered functional activity level of isoniazid acetylation, that has been classified into rapid, intermediate, and slow acetylator phenotypes (Butcher et al., 2002; Sabbagh et al., 2013). Eight different point mutations in NAT2, of which five cause amino acid changes at 191 G to A (Arg to Glu);341 T to C (Ile to Thr); 590 G to A (Arg to Gln); 803 A to G (Lys to Arg); 857 G o A (Gly to Glu), and three of them leading to silent amino acid changes at 111 T to C, 282 C to T and 481 C to T were commonly reported (Butcher et al., 2002). Several allelic variants of NAT2 result from certain combinations of the eight point mutations and the nomenclature of the alleles is based on the classification system of Vatsis et al. (1995). The slow acetylator phenotype for NAT2 cannot conjugate metabolites or toxins specific to these enzymes and can lead to an increase in the susceptibility to environmental toxins and oxidative free radical cellular damage. Uncoupling proteins (UCPs) are the important carrier proteins which facilitate the transfer of anions, thereby control the mitochondria-derived reactive oxygen species (ROS) (Giardina et al., 2008). UCP consists of 1, 2, 3, 4, and 5 members of an anion-carrier protein family which are located in the inner mitochondrial membrane (Souza et al., 2011). The UCP1 covers a 9 kb region on chromosome 4 (region 4q28-q31), and consists of 6 exons and 5 introns which is mainly expressed in brown adipose tissue (Yu et al., 2005). As UCP1 has been found to be involved in energy expenditure, several polymorphism especially at − 3826 A to G, −1766 A to G, and −112 A to C positions in the promoter region; Ala64Thr polymorphism in exon 2; and Met299Leu polymorphism in exon 5 were found to be associated with pathogenesis (Brondani et al., 2012). UCP2 is widely distributed and the gene consisting of eight exons and seven introns covers a 6.3 kb region on chromosome 11q13. Polymorphisms contribute to biological variation in insulin secretion but the main function of UCP2 is the control of mitochondria-derived ROS (Arsenijevic et al., 2000; Krempler et al., 2002). In Japanese population, it was recently reported that UCP2 gene polymorphism at Ala55Val in exon 4 is significantly associated with age related hearing loss but not with UCP1 A (− 3826) G polymorphism (Sugiura et al., 2010). Therefore, the damage caused by oxidative stress to auditory system might be causing susceptibility to presbycusis. Hence, the present study is the first of its kind in Indian population to evaluate the role of genes involved in oxidative stress and mitochondrial dysfunction leading to the etiopathogenesis of presbycusis.

analysis. Genomic DNA was isolated from whole blood samples by salting out method of Lahiri and Nurnberger (1991) and stored in −80 °C for molecular analysis. 2.2. CYP1A1 genotyping The CYP1A1 3081 T NC polymorphism was detected by PCR based Restriction Fragment Length Polymorphism (PCR-RFLP) (Cascorbi et al., 1996). The PCR was carried out with specific forward primer (5′-GGCTGAGCAATCTGACCCTA-3′) and reverse primer (5′TAGGAGTCTTGTCTCATGCCT-3′). After mixing all the contents, PCR tubes were kept in thermal cycler for a 3 step PCR with an initial denaturation at 95 °C for 5 min followed by cycling at 95 °C for 30 s, annealing for 60 °C at 60 s and extension at 72 °C for 60 s and a final extension at 72 °C for 5 min was carried out for about 30 cycles. The PCR product 899 bp was digested with MSPI restriction enzyme at 37 0 C for 5 min and electrophoresis was carried out on 1.5% agarose gel with ethidium bromide staining. The RFLP products showed homozygous ʹTTʹ genotype with 899 bp, heterozygous ʹTCʹ genotype with 899, 693, 206 bp and homozygous ʹCCʹ genotype with 693, 206 bp fragments. CYP1A1 was checked by amplifying a 204 bp fragment using forward (5′-CTGTCTCCCTCTGGTTACAGGAAGC-3′) and reverse (5′-TTCCACCCGTTGCAGCAGGATAGCC-3′) primers and digested with BsrDI while CYP1A1 (m4) was identified from the same 204 bp fragment by using forward (5′-CTGTCTCCCTCTGGTTACAGGAAGC-3′) and reverse (5′-TTCCACCCGTTGCAGCAGGATAGCC-3′) primers using BsaI restriction enzyme by 3% agarose gel electrophoresis. 2.3. GSTM1 and GSTT1 genotyping (multiplex - PCR) GSTM1 and GSTT1 gene polymorphisms were determined simultaneously using primers (5′-TTCCTTACTGGTCCTCACATCTC-3′ and 5′TCACCGGATCATGGCCAGCA-3′ as well as 5′-ACACAACTGTGTTCACTAGC3′ and 5′-CAACTTCATCCACGTTCACC-3′) and also using internal band forward 5′-ACACAACTGTGTTCACTAGC-3′ and reverse 5′CAACTTCATCCACGTTCACC-3′ primers by multiplex PCR approach method of Arand et al. (1996). The PCR conditions consisted of an initial single cycle of 10 min at 95 °C followed by 35 cycles of 30 s at 94 °C, 20 s at 59 °C and 5 s at 72 °C and was performed by using the primers of above mentioned. The PCR produced three DNA fragments of 215 bp (GSTM1), 480 bp (GSTT1) and 350 bp (albumin) used as internal positive control for the PCR efficiency. In both GSTM1 and GSTT1 polymorphisms, gene deletions were detected by the existence of null alleles and individuals homozygous with respect to a given null allele lacked the respective PCR amplified DNA fragment. 2.4. NAT2 genotyping

2. Subjects and methods 2.1. Subjects In the present case–control study, 220 presbycusis cases referred to MAA ENT HOSPITALS, Hyderabad, Telangana State, from 2011 to 2014 and 270 age and sex matched controls were included as study subjects. All patients underwent a detailed medical otoscopic examination that includes tympanometry and pure tone audiometric test for evaluating hearing loss at 0.5, 1, 2, 4 and 8 kHz frequencies. The patients with sensorineural form of hearing loss (bilateral as well as symmetrical) whose age was equal or greater than 40 years and with no vestibular or any history of otological surgery were included in the study. Outer and middle ear diseases that include infections as well as patients residing/working in noisy environment were excluded from the study. Individuals without any disease were considered as healthy controls. Informed written consent was taken from all participants, and the study was carried out with the Institutional Ethics committee approval. Blood samples were collected from all study subjects and were kept in EDTA vials for

Detection of mutations in NAT2 gene at 590 GNA and 282 CN T. First the whole exon region containing variation in the NAT2 gene was amplified using forward (5'-GTCACACGAGGAAATCAAATGC-3') and reverse (5'-GTTTTCTAGCATGAATCACTC.TGC-3′) primers with specific reagents (Cascorbi et al., 1995). Thermal cycling conditions were 5 min denaturation at 95 °C, then 34 cycles at 95 °C for 30 s, 56.5 °C for 1 min and finally 72 °C for 2 min. This was followed by 5 min extension at 72 °C. The amplified product 1211 bp was determined by using 1% agarose gel electrophoresis. PCR evaluation of coding region containing nucleotide position at 282 C NT was amplified using 1211 bp fragment as template by forward (5'-GTCACACGAGGAAATCAAATGC-3') and reverse (5'ACCCAGCATCGACAATGTAATTCCTGCCCTCA-3') primers using suitable reagents (Cascorbi et al., 1995). 1 μl of the diluted PCR product of 1211 bp was added to the template. The thermal cycling conditions used for the amplification was denaturation at 94 °C for 5 min followed by 14 cycles at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 45 s. The extension was carried out at 72 °C for 5 min which resulted in the 442 bp

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fragment. Evaluation of nucleotide position 282 with FokI at 37 °C produced 'CC' genotype with 337, 105 bp, 'CT' genotype with 442, 337, 105 bp and 'TT' genotype with 442 bp fragment. For determining the variation at nucleotide position 590 in NAT2 gene, amplification was carried out on 1211 bp fragment by using specific forward (5'-CCTGGACCAAATCAGGAGAG-3') and reverse (5′ACACAAGGGTTTATTTTGTTCC-3') primers with specific reagents. The thermal cycling conditions used for the amplification was denaturation at 94 °C for 5 min then 14 cycles at 94 °C for 5 s, 60 °C for 1 min and 72 °C for 1 min with the final extension of 72 °C for 5 min (Cascorbi et al., 1995). The PCR product of 421 bp obtained was visualised by using 3% agarose gel electrophoresis. Evaluation of 590 position in NAT2 gene using TaqI at 65 °C produced 'GG' genotype with 170,142,109 bp, 'GA' genotype with 279, 170,142 bp and 'AA' genotype with 279,142 bp. 2.5. UCP1 (−3286) AN G genotyping The amplification for the determination of polymorphism in UCP1 at (− 3286) A N G was carried out by using forward (5′-CTTGGGTAGTGACAAAGTAT-3′) and reverse (5′-CCAAAGGGTCAGA TTTCTAC-3′) primers with specific reagents (Cassard-Doulcier et al., 1996). The PCR reaction was incubated at 94⁰C for 5 min, 55⁰C for 4 min, followed by 30 cycles at 94⁰C for 1 min and 55⁰C for 1 min. 470 bp PCR product obtained was subjected for digestion by BclI enzyme revealed UCP1 A-3826G homozygote ʹAAʹ genotype of 470 bp, heterozygote UCP1 (−3826) ʹAGʹ genotype of 470, 310 and 160 bp and homozygous UCP1 (−3826) ʹGGʹ genotype of 310 and 160 bp. 2.6. UCP2 (−866) GNA genotyping The amplification of UCP2 at (− 866) G NA was carried out by PCR RFLP using forward (5′-CACGCTGCTTCTGCCAGGAC-3′) and reverse primers (5′-AGGCGTCAGGAGATGGACCG-3′) with specific reagents (Sesti et al., 2003). The amplification was carried out by using initial condition of 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 68 °C for 30 s and extension at 72 °C for 30 s. A 363 bp PCR product was obtained. RFLP was performed by using MluI digestion that resulted in 363 bp of 'GG' genotype, 363, 295, 68 bp was of 'GA' genotype type and 295, 68 bp is of 'AA' genotype. 2.7. Statistical analysis The data obtained was coded for statistical evaluations. Appropriate statistical analysis was performed using the Statistical Package for Social Sciences, PASW STATISTICS 18.0 software (SPSS Inc., Chicago, IL, USA). Continuous data is represented as mean and standard deviations whereas categorical data as proportions. Statistical significance of the differences in frequency of genotypes was carried out by using binary and multinomial logistic regression analysis. And odds ratio was adjusted with age and gender. HardyWeinberg equilibrium was tested using goodness-of-fit. Chi-square test with one degree of freedom was used to compare the observed genotype frequencies among the subjects with the expected genotype frequencies. Haplotype frequencies were inferred using Haploview software.

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Table 1 Demographic and associated co-morbidities in presbycusis subjects. Cases (%) (n = 220)

Controls (%) (n = 270)

127 (57.7) 93 (42.3) 63.7 ± 9.98 36 (16.4) 80 (36.4) 104 (47.2) 52.9 ± 8.51 12 (5.5) 208 (94.5)

160 (59.3) 110 (40.7) 62.9 ± 9.86 44 (16.3) 101 (37.4) 125 (46.2) 12.1 ± 0.36 0 (0.0) 0 (0.0)

Tinnitus Vertigo Co-morbidities

42 (19.1) 9 (4.1)

0 (0.0) 0 (0.0)

NA NA

Diabetes Hypertension Hypothyroidism

48 (21.8) 30 (13.6) 3 (1.4)

0 (0.0) 0 (0.0) 0 (0.0)

NA NA NA

Parameters Gender Male Female Age (years)a 40–50 50–60 N60 Degree of hearing loss (Decibels-dB) b40 dB N40 dB Associated risk factors

a b

P-valuea 0.732 NAb 0.97

NA NA

χ2 test. NA-not applicable.

frequency (N40 dBHL). Diabetes was present in 21.8%, hypertension in 13.6% and tinnitus in 19.1% of the cases.

3.1. CYP1A1 genotyping 3.1.1. Distribution of allelic and genotypic frequency of 2453 C NA, 2455 AN G, 3801 TNC polymorphism in CYP1A1 gene In the CYP1A1, the polymorphism of C to A at 2453 revealed that the frequency of 'A' allele was found to be predominant and 1.43 fold risk in presbycusis subjects compared to controls (46.1% vs 37.4% respectively). 'AA' genotype was found to be predominant with 1.59 folds risk respectively for presbycusis when compared to controls. It was observed that 'CA + AA' genotype showed 2.03 folds risk when compared with 'CC' genotype of controls. And the CYP1A1 A2455G polymorphism, the frequency of 'G' allele was found to be more in presbycusis group compared to controls (47.7% vs 38.9% respectively), with a 1.44 fold increased risk for presbycusis. 'GG' genotype was found to be 25% predominant in presbycusis when compared to controls with 1.87 folds risk for presbycusis. ʹGGʹ genotype showed 1.65 folds risk when compared with ʹAA + GGʹ genotype for presbycusis when compared to controls. And CN T polymorphism at 3801 position of CYP1A1 showed that the frequency of 'CC' genotype (10.9%) and 'C′ allele (30%) did not show any association with presbycusis (Table 2).

3.1.2. Haplotype analysis of CYP1A1 gene_at 2453, 2455 and 3801 positions Haplotype analysis was performed in the present study for understanding the influence of genetic variations of CYP1A1 gene on the manifestation of presbycusis. The haplotypes were constructed based on the three polymorphisms and their association with onset of presbycusis (Table 3). The haplotype analysis has revealed that wild type allele at 3801, and recessive allele at 2455 & 2453 of CYP1A1 in T-G-A combination were found to be significantly predominant and contributing to 2.43 fold risk in presbycusis when compared to controls.

3. Results 3.2. GSTM1/GSTT1 polymorphism genotyping The frequency of cases with presbycusis was found to be increased with increasing age. The mean age of onset was 63.7 ± 9.98 years in the study subjects. The demographic, audiological and associated comorbidities of presbycusis subjects and controls are represented in Table 1. Male preponderance was observed in the study subjects of presbycusis and the disability of hearing loss was noticed at higher

The GSTM1 and GSTT1 polymorphism double null genotype (i.e., absence of both alleles) was 24.5% in cases while 3.3% was in controls (Table 2). There was a significant association of GSTM1 and GSTT1 double null genotype with presbycusis (adjusted OR 8.88, 95% CI 4.10– 19.19, p b 0.001).

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Table 2 Genotypic and Allelic frequencies of CYP1A1, GSTT1, GSTM1, NAT2, UCP1 and UCP2 gene polymorphisms in presbycusis subjects and controls.

CYP1A1 2453 CNA(rs1799814; Thr461Asn) Genotypes CC CA AA CC vs CA + AA CC + CA vs AA CC + AA vs CA Alleles C A

Subjects (%)

Controls (%)

OR (95 CI)a

p-Valuea

Adjusted OR (95% CI)b

57 (25.9) 123 (55.9) 40 (18.2) 163 (74.1) 40 (18.2) 123 (55.9)

112 (41.5) 114 (42.2) 44 (16.3) 158 (58.5) 44 (16.3) 114 (42.2)

1.00 (Reference) 2.12 (1.41–3.19) 1.79 (1.05–3.05) 2.03 (1.38–2.98) 1.14 (0.72–1.83) 1.74 (1.21–2.50)

– b0.001 0.033 b0.001 0.582 0.003

1.00 (Reference) 2.38 (1.52–2.74)⁎ 1.59 (1.01–2.87)⁎ 2.03 (1.36–3.03)⁎⁎ 0.97 (0.57–1.61) 2.03 (1.36–3.03)⁎⁎

237 (53.9) 203 (46.1)

330 (61.1) 210 (38.9)

1.00 (Reference) 1.43 (1.11–1.85)

0.006

100 (37.0) 120 (44.4) 50 (18.6) 170 (63.0) 50 (18.6) 120 (44.4)

1.00 (Reference) 1.18 (0.78–1.78) 2.12 (1.27–3.53) 1.40 (0.96–2.05) 1.92 (1.22–3.02) 0.89 (0.63–1.28)

– 0.417 0.004 0.05 0.448 0.552

330 (61.1) 210 (38.9)

1.00 (Reference) 1.44 (1.11–1.85)

0.005

137 (50.7) 103 (38.1) 30 (11.1) 133 (49.3) 30 (11.1) 103 (38.1)

1.00 (Reference) 0.99 (0.68–1.46) 0.98 (0.54–1.77) 1.01 (0.71–1.44) 1.07 (0.60–1.90) 0.99 (0.69–1.44)

– 0.99 0.943 0.97 0.816 0.994

377 (69.8) 163 (30.2)

1.00 (Reference) 0.99 (0.75–1.30)

0.95

74 (33.6) 47 (21.4) 45 (20.5) 54 (24.5)

142 (52.6) 59 (21.9) 60 (22.2) 9 (3.3)

1.00 (Reference) 1.53 (0.95–2.46) 1.44 (0.89–2.32) 11.51 (5.39–24.61)⁎⁎⁎

– 0.080 0.135 b0.001

1.00 (Reference) 1.37 (0.84–2.24) 1.50 (0.92–2.45) 8.88 (4.10–19.19)⁎⁎⁎

50 (22.7) 110 (50.0) 60 (27.3) 170 (77.3) 60 (27.3) 110 (50.0)

84 (31.1) 136 (50.4) 50 (18.5) 186 (68.9) 50 (18.5) 136 (49.8)

1.00 (Reference) 1.36 (0.88–2.09) 2.02 (1.21–3.37) 1.54 (1.02–2.31) 1.65 (1.08–2.53) 0.99 (0.69–1.41)

0.163 0.007 0.039 0.022 0.935

1.00 (Reference) 1.43 (0.98–2.29) 1.77 (1.02–3.11)⁎ 1.53 (0.98–2.39)⁎ 1.40 (1.02–2.24)⁎ 1.10 (0.75–1.63)

210 (47.7) 230 (52.3)

304 (56.3) 236 (43.7)

1.00 (Reference) 1.41 (1.10–1.82)

79 (35.9) 112 (50.9) 29 (13.2) 141 (64.1) 29 (13.2) 112 (50.9)

128 (47.4) 122 (45.2) 20 (7.4) 142 (52.6) 20 (7.4) 122 (45.2)

1.00 (Reference) 1.48 (1.02–2.18) 2.35 (1.25–4.43) 1.61 (1.12–2.32) 1.89 (1.04–3.46) 1.26 (0.88–1.79)

270 (61.4) 170 (38.6)

378 (70.0) 162 (30.0)

1.00 (Reference) 1.47 (1.13–1.92)

0.005

45 (20.5) 97 (44.1) 78 (35.5) 175 (79.5) 78 (35.5) 97 (44.1)

48 (17.8) 114 (42.2) 108 (40.0) 222 (82.2) 108 (40.0) 114 (42.2)

1.00 (Reference) 0.91 (0.56–1.48) 0.77 (0.47–1.27) 0.84 (0.54–1.32) 0.82 (0.57–1.19) 1.08 (0.75–1.55)

– 0.697 0.307 0.453 0.303 0.678

187 (42.5) 253 (57.5)

210 (38.9) 330 (61.1)

1.00 (Reference) 1.16 (0.89–1.50)

0.252

CYP1A1 2455 ANG (rs1048943; Ile462Val) Genotypes AA 65 (29.5) AG 100 (45.5) GG 55 (25.0) AA vs AG + GG 155 (70.5) AA +AG vs GG 55 (25.0) AA + GG vs AG 100 (45.5) Alleles A 230 (52.3) G 210 (47.7) CYP1A1 3801 TNC (rs4646903; 3′- non coding region) Genotypes TT 112 (50.9) TC 84 (38.2) CC 24 (10.9) TT vs TC + CC 108 (49.1) TT + TC vs CC 24 (10.9) TT + CC vs TC 84 (38.2) Alleles T 308 (70.0) C 132 (30.0) GSTM1 and GSTT1 GST M1/GST T1(Wild type) GST M1/GST_(Heterozygous null type) GST _/GST T1(Heterozygous null type) GST _/GST _(Double null type) NAT2 282 CNT(rs1041983; Tyr 94 Tyr) Genotypes CC CT TT CC vs CT + TT CC + CT vs TT CC + TT vs CT Alleles C T NAT2 590 GNA(rs 1799930; Arg197Gln) Genotypes GG GA AA GG vs GA + AA GG + GA vs AA GG + AA vs GA Alleles G A UCP1 A(−3826)G (rs1800592) Genotypes AA AG GG AA vs AG + GG AA + AG vs GG AA + GG vs AG Alleles A G

1.00 (Reference) 1.71 (1.10–2.66)⁎ 1.87 (1.07–3.37)⁎ 1.40 (0.93–2.13)⁎ 1.65 (1.01–2.71)⁎ 0.98 (0.67–1.46)

1.00 (Reference) 0.88 (0.58–1.35) 0.92 (0.48–1.76) 1.12 (0.76–1.66) 1.06 (0.57–1.99) 1.11 (0.74–1.67)

0.008

0.041 0.008 0.011 0.036 0.027

1.00 (Reference) 1.42 (1.00–2.14)⁎ 1.83 (1.20–3.63)⁎⁎ 1.48 (1.00–2.21)⁎ 1.52 (1.00–2.89)⁎ 1.25 (0.85–1.86)⁎

1.00 (Reference) 0.86 (0.52–1.43) 0.67 (0.39–1.13) 0.77 (0.48–1.23) 0.74 (0.50–1.09) 1.12 (0.77–1.63)

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Table 2 (continued)

UCP2 G(−866)A (rs659366) GG GA AA GG vs GA + AA GG + GA vs AA GG + AA vs GA Alleles G A

Subjects (%)

Controls (%)

OR (95 CI)a

p-Valuea

Adjusted OR (95% CI)b

42 (19.0) 100 (45.5) 78 (35.5) 178 (80.9) 78 (35.5) 100 (45.5)

156 (57.8) 93 (34.4) 21 (7.8) 114 (42.2) 21 (7.8) 93 (34.4)

1.00 (Reference) 3.99 (2.57–6.22) 13.79 (7.66–24.89) 5.79 (3.83–8.77) 6.51 (3.86–11.00) 1.59 (1.10–2.29)

– b0.001 b0.001 b0.001 b0.001 0.013

1.00 (Reference) 4.26 (2.62–6.92)⁎⁎⁎ 12.39 (6.51–23.56)⁎⁎⁎ 5.83 (3.69–9.21)⁎⁎⁎ 5.47 (3.12–9.61)⁎⁎⁎ 1.74 (1.16–2.60)⁎⁎

184 (41.8) 256 (58.2)

135 (25.0) 405 (75.0)

1.00 (Reference) 2.16 (1.64–2.83)

b0.001

a

-Binary logistic regression analysis; -Multinomial logistic Regression analysis; The distributions of all genotypes are in the Hardy-Weinberg equilibrium. Adjusted OR for gender and age. ⁎ p-Value b 0.05. ⁎⁎ p-Value b 0.01. ⁎⁎⁎ p-Value b 0.001. b

c

3.2.1. Distribution of allelic and genotypic frequency of 282 C NT and 590 GNA polymorphism in NAT2 gene At position 282 the polymorphism of C to T of NAT2 gene it was observed that the frequency of 'T' allele was found to be predominant in presbycusis group compared to controls (52.3% vs 43.7% respectively), with a 1.41 fold increased risk for presbycusis (Table 2). 'TT' genotype was found to be predominant in 27.3% of the presbycusis group compared to controls (19.4%) with 1.77 fold increased risk for presbycusis, which was statistically significant. It was also observed that polymorphism of G to A at position 590 of NAT2 the frequency of 'A' allele was found to be predominant in presbycusis group compared to controls (38.6% vs 30.0% respectively), with a 1.47 fold increased risk for presbycusis (Table 2). 'AA' genotype was found to be predominant in 13.2% of the presbycusis group compared to controls (7.4%) with 1.83 fold increased risk for presbycusis.

3.2.2. Haplotype combinations of NAT2 point mutations at 282 and 590 positions In this study, two point mutations allocated to allelic variants of NAT2 were tested and the frequencies obtained by haplotype analysis are presented in Table 3. The presence of a mutant genotype in the NAT2 gene at positions 282 C to T and 590 G to A is classified as NAT2*6A (haplotype T-A combination) was observed to be present in 38.6% of the study subjects and with 1.52 folds risk for presbycusis (Table 3).

3.2.3. Distribution of allelic and genotypic frequency of (−3826)AN G and (−866)G N A polymorphisms in UCP1 gene The 'G' allelic and 'GGʹ genotypic frequency of A(−3826)G polymorphism of UCP1 was to be similar to controls indicating the difference being insignificant in presbycusis subjects. It was also observed that there was no difference in various genotypes combination of presbycusis with the controls (Table 2). And at the G(−866)A polymorphism of UCP2, the frequency of ‘A’ allele was found to be predominant in presbycusis group compared to controls (58.2% vs 75% respectively), with a 2.16 fold increased risk for presbycusis (Table 2). 'AA' genotype was found to be predominant in the presbycusis group compared to controls with 12.39 fold increased risk for presbycusis, which was statistically significant. It was observed that comparison of ʹGAʹ genotype with 'GG + AA' genotype combination and 'AA' genotype with 'GG + GA' genotype combination showed increased risk in presbycusis when compared to controls.

4. Discussion Presbycusis is one of the most common sensory disorder leading to progressive loss of hearing in elderly population (Gopinath et al., 2009; Someya and Prolla, 2010). It is associated with many factors, including environmental, medical and hereditary factors (Gopinath et al., 2009). Reactive oxygen species (ROS) and mitochondrial dysfunction affect the aging process and lead to the onset of various diseases.

Table 3 Haplotype combinations of CYP1A1 polymorphisms at positions of 3801, 2455 and 2453 and NAT2 at positions 282 and 590 in presbycusis and controls.

Haplotype combination CYP1A1 T3801C T C T T T

A2455G A G A G G

NAT2⁎6A C282T C T T

G590A G A G

a ‐Binary logistic regression analysis. ⁎ p-Value b 0.05. ⁎⁎ p-Value b 0.01. ⁎⁎⁎ p-Value b 0.001.

C2453 A C A A A C

Cases (%) (n = 440)

Controls (%) (n = 540)

OR (95% CI)a

p-Value

215 (48.9) 132 (30.0) 15 (3.4) 56 (12.7) 22 (5.0)

326 (60.4) 163 (30.2) 4 (0.7) 35 (6.5) 12 (2.2)

1.00 (Reference) 1.23 (0.92–1.64) 5.69 (1.86–17.36)⁎⁎ 2.43 (1.54–3.83)⁎⁎⁎ 2.78 (1.34–5.74)⁎⁎

– 0.161 0.002 b0.001 0.006

210 (47.7) 170 (38.6) 60 (13.7)

304 (58.3) 162(30.0) 74 (13.7)

1.00 (Reference) 1.52 (1.15–2.01)⁎⁎ 1.17 (0.80–1.72)

0.003 0.413

282

S.K. Manche et al. / Gene 593 (2016) 277–283

Abnormal levels of ROS production and mitochondrial dysfunction increase over age and thus lead to cochlear cell degeneration of inner ear (Balaban et al., 2005; Lin and Beal, 2006). The detoxification of reactive intermediates produced by ROS in phase I and II reactions are eliminated by antioxidant enzymatic scavengers such as cytochrome P450 (CYP), glutathione S-transferase (GST), glutathione peroxidase (GPX) and N-acetyl transferase (NAT) (Gutteridge and Halliwell, 1992; Beckman and Ames, 1998). Uncoupled proteins (UCPs) increase production of ROS over time and decrease of energy generated by mitochondria which leads to tissue damage in inner ear (Balaban et al., 2005; Lin and Beal, 2006). Therefore, the present study has been carried out to elucidate the role played by oxidative stress and mitochondrial dysfunction on cochlear senescence due to aging and in development of presbycusis. The prevalence of presbycusis in the present study was more likely to affect men than women which are in accordance with many of the studies (Sousa et al., 2009; Raynor et al., 2009). This is attributed to genetic, physiologic, environmental factors and occupational exposures as well as the presence of associated co-morbidities and other risk factors. Presbycusis tends to manifest in fifth decade of life and gradually progresses over years and increases over eighth decade (Sogebi et al., 2013). The prevalence of presbycusis in study subjects increased with advancing age. It may also be associated with symptoms of tinnitus and vertigo which indicate the pathophysiology of senile hearing loss (Wilson et al., 1999; Megighian et al., 2000). In the present study, 19.1% of the study subjects were found to be suffering from tinnitus. CYP1A1 is involved in detoxification of harmful components as it oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics (Masood and Kayani, 2011). To our knowledge no study has been reported so far on the association of CYP1A1 gene polymorphism with presbycusis. The present study analysed the association of CYP1A1 gene polymorphism at 2453, 2455 and 3801 positions in the onset of presbycusis. It was found that CYP1A1 A to G substitution at 2455 showed 1.87 folds risk for presbycusis indicating that change in enzyme levels may induces toxicity in cochlea of inner ear leading to hearing loss. Another variation of C to A at 2453 in the study population indicated AA genotype to posses 1.59 folds risk for presbycusis. However, there was no association of TNC polymorphism at position 3801 in CYP1A1 gene with presbycusis. Further, the haplo analysis has also revealed that T-G-A combination of CYP1A1 gene at positions 3801, 2455 and 2453 showed 2.43 fold increased risk for presbycusis. Anti-oxidant enzymes such as GSTT and GSTM are involved in conjugation of a wide range of electrophilic substances with glutathione thereby facilitate inactivation of toxic components and play an important role in antioxidant protection in adult cochlea (Bared et al., 2010). Specifically, glutathione S-transferase (GST) enzyme comprises of several genes including GSTM and GSTT especially GSTM1 and GSTT1 are the important subfamilies that exhibit deletion polymorphisms which have been reported to be associated with various disease susceptibilities (Mannervik et al., 1992; Cantlay et al., 1994). Previous studies have reported that double null genotype of GSTM1 and GSTT1 cannot conjugate specific metabolites and that may lead to oxidative stress which may damage the cochlea of the inner ear making the individual more susceptibility to presbycusis while others reported no association (Pemble et al., 1994; Ates et al., 2005). In the present study, it was observed that double null genotype of GSTM1 and GSTT1 were found to have 8.88 folds risk for the onset of presbycusis in when compared with controls of South Indian population. N-acetyltransferases (NATs) is one of the Phase II detoxification and antioxidant enzyme which is involved in causing damage to inner ear tissues with aging by ROS that induces cellular injury (Seidman et al., 2002). Insufficiently acetylated drugs accumulated in the body which are converted to reactive metabolites by oxidative enzymes are mostly eliminated by NAT enzymes (Dupret and Rodrigues-Lima, 2005). NAT2*6A(282 C NT and 590 G N A) encodes a slow acetylator, which

slows down the detoxification mechanisms leading to an increase in higher concentration of xenobiotics in the inner ear (Van Eyken et al., 2007). Studies have reported that missense substitutions of the NAT2 alleles especially NAT2*6A were found to have an association with presbycusis (Unal et al., 2005; Van Eyken et al., 2007). The prevalence of NAT2 has been varying in different populations. Studies have reported that slow acetylator NAT2*6 had a prevalence of 1–7% in Turkey, 9% in European and 6% in the Finnish population (Unal et al., 2005; Van Eyken et al., 2007). In our study, the prevalence of NAT2*6A slow acetylator was 30.0% in controls which was in accordance with the reported literature. Similar observations regarding NAT2*6A have also been made by 31.7% in South and 31.5% in North India of the general population (Umamaheswaran et al., 2014). The prevalence of NAT2*6A was found to be higher in Indian population compared to other populations. To our knowledge the present study of NAT2 gene polymorphism and its association with presbycusis is the first to report the association of NAT2*6A with presbycusis. The NAT2*6A has shown significant association and was with 1.52 fold increased risk when compared to controls. Studies have reported that UCP2 Ala55Val polymorphisms at G(−866)A exhibited a significant association with presbycusis while UCP1 polymorphism at A(−3286)G showed no association in Japanese population (Arsenijevic et al., 2000; Sugiura et al., 2010). There have been no reports on the study of UCPs gene polymorphisms and hearing loss to date in Indian population. The UCP1 gene has a natural antioxidant effect and A to G substitution at −3826 position in the 5'flanking region leads to an increase in the pathology. The common polymorphism of G to A at − 866 position of UCP2 promoter was reported to show variation in different populations and ethnicities (Esterbauer et al., 2001). Similarly, the findings of the present study showed GA and AA genotype of UCP2(− 866) was found to be predominant in presbycusis when compared to controls with 4.26 and 12.39 fold increased risk respectively indicating that vasculature of blood vessels can lead to damage of cochlea in the inner ear. And the UCP1A (−3286)G polymorphism showed no association with presbycusis. The present study establishes the role of oxidative pathway gene polymorphisms especially involved in the activation of toxic components in cochlea of inner ear leading to aetio-pathology of presbycusis for the first time in South Indian population. Better understanding of these CYP1A1, GST M1/T1, NAT2 and UCP2 variants conferring the susceptibility to presbycusis may aid in diagnosis and development of personalized therapeutic strategies. Conflicts of interest There is no conflict of interest. Acknowledgements We would like to thank MAA Research Foundation for the funding and Ms. B. Sunita G Kumar, CMD, MAA ENT Hospitals for her support and cooperation in carrying out the work. I would also thank J.V. Ramakrishna and D. Dinesh in providing valuable support in carrying out the work. References Arand, M., Mühlbauer, R., Hengstler, J., et al., 1996. A multiplex polymerase chain reaction protocol for the simultaneous analysis of the glutathione S-transferase GSTM1 and GSTT1 polymorphisms. Anal. Biochem. 236, 184–186. Arsenijevic, D., Onuma, H., Pecqueur, C., et al., 2000. Disruption of the uncoupling protein2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat. Genet. 26, 435–439. Ates, N.A., Unal, M., Tamer, L., et al., 2005. Glutathione S-transferase gene polymorphisms in presbycusis. Otol. Neurotol. 26, 392–397. Balaban, R.S., Nemoto, S., Finkel, T., 2005. Mitochondria oxidants and aging. Cell 120, 483–495. Bared, A., Ouyang, X., Angeli, S., et al., 2010. Antioxidant enzymes, presbycusis., and ethnic variability. Otolaryngol. Head Neck Surg. 143, 263–268.

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