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|Year : 2013
: 15 | Issue : 65 | Page
|Lack of association between DNMT1 gene polymorphisms and noise-induced hearing loss in a Chinese population
Feifei Hu1, Xin Li1, Xiuting Li2, Meilin Wang1, Haiyan Chu1, Kai Liu3, Hengdong Zhang4, Zhengdong Zhang1, Baoli Zhu4
1 Department of Environmental Genomics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Cancer Center; Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing, China
2 Department of Environmental Genomics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Cancer Center; Institute of Occupational Disease Prevention, Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China
3 Center of Prevention and Health, Yizheng Hospital, Drum Tower Hospital Group of Nanjing, Yizheng, China
4 Institute of Occupational Disease Prevention, Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, China
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|Date of Web Publication||15-Jun-2013|
DNA methyltransferase 1 (DNMT1) plays a crucial role in maintaining of methylation and chromatin stability. And mutations in DNMT1 can induce one form of neurodegenerative diseases with dementia and sensorineural hearing loss. To assess whether single nucleotide polymorphisms (SNPs) or haplotypes of DNMT1 are related to noise-induced hearing loss (NIHL) in a Chinese population, we genotyped three functional polymorphisms (rs12984523, rs16999593, and rs2228612) in a case-control study involving 615 NIHL cases and 644 controls. However, no significant association was detected between these three SNPs and NIHL susceptibility in the Chinese population. Our data suggested that the DNMT1 polymorphisms may not contribute to risk of NIHL in the Chinese population.
Keywords: DNA methyltransferase 1, noise-induced hearing loss, polymorphism
|How to cite this article:|
Hu F, Li X, Li X, Wang M, Chu H, Liu K, Zhang H, Zhang Z, Zhu B. Lack of association between DNMT1 gene polymorphisms and noise-induced hearing loss in a Chinese population. Noise Health 2013;15:231-6
|How to cite this URL:|
Hu F, Li X, Li X, Wang M, Chu H, Liu K, Zhang H, Zhang Z, Zhu B. Lack of association between DNMT1 gene polymorphisms and noise-induced hearing loss in a Chinese population. Noise Health [serial online] 2013 [cited 2023 Mar 23];15:231-6. Available from: https://www.noiseandhealth.org/text.asp?2013/15/65/231/113517
| Introduction|| |
Noise-induced hearing loss (NIHL) is one of the leading occupational hazards around the world, which is most often caused by continuous and regular exposure to noise. Besides age-related hearing loss, NIHL is the major cause of adult sensorineural hearing loss.  Along with the industrialization process in the developing countries like China, Brazil, and India, the problem became more and more serious. In addition, NIHL is an irreversible disease, and there is no good treatment when it once happened.
NIHL is caused by continuous and regular exposure to noise, and it can also be modified by environment and genetic factors. The environmental risk factors like noise, organic solvents, smoking, high blood pressure, and cholesterol levels have been studied extensively. But little is known about the genetic factors. ,,,, As a formal heritability study has not yet been performed for NIHL in humans, several studies using knockout mice such as cadherin 23 (CDH23) +/-,  Cu/Zn superoxide dismutase 1 (SOD1) -/-,  and glutamate peroxidase 1 (GPX1) -/-  have demonstrated that they were more susceptible to NIHL. Up to now, only a few association studies with human subjects have identified potassium voltage-gated channel, isk-related family, member 1 (KCNE1),  glutathione s-transferase m1 (GSTM1)  and catalase (CAT)  as feasible NIHL susceptibility genes.
DNA methyltransferases (DNMTs) are the catalyst of the methylation, and play a key role in chromatin remodeling and regulation of gene expression.  DNA methyltransferase 1 (DNMT1) is the best-understood enzyme among three families of DNMTs: DNMT1, DNMT2, DNMT3a, and DNMT3b.  DNMT1 is mainly involved in the state of the maintaining of methylation, also is necessary for the extension of methylation status.  Recently, Christopher et al. found that mutations in DNMT1 caused one form of neurodegenerative diseases with dementia and sensorineural hearing loss by reducing the methyltransferase activity and impairing their combination ability to heterochromatin in the second gap (G2) phase.
The DNMT1 gene locates at chromosome 19p13.3-p13.2 including two parts, one is a large N-terminal regulatory region and the other is a smaller C-terminal catalytic region.  Several studies have investigated the associations between the DNMT1 polymorphisms and risk of disease, including breast cancer,  hepatitis b virus (HBV) infection,  systemic lupus erythematosus.  To the best of our knowledge, no published study has investigated the associations between the DNMT1 polymorphisms and the susceptibility to NIHL.
In this study, we hypothesized that the three SNPs (rs12984523, rs16999593, and rs2228612) in DNMT1 are associated with susceptibility to NIHL. To test this hypothesis, we genotyped the polymorphisms and assessed the associations with risk of NIHL in the Chinese population.
| Methods|| |
The study group was recruited from the factories where they were all exposed to at least 1 year of exposure to the continuous and steady noise in the cities of Xuzhou, Yizheng, and Nanjing in Jiangsu province, China, during the period from April 2010 to May 2011. Included workers had to meet the following criteria: Han Chinese, no history of middle ear disease, no family history of hearing loss, no history of using used potentially ototoxic drugs (e.g., aspirin, quinolones, and aminoglycosides), no history of fever or common infections (e.g., influenza, diarrhea, and hepatitis) within 1 month before medical examination.  A questionnaire was used to obtain general information, occupational history, lifestyle (smoking and drinking status), history of explosive noise exposure, and history of disease. Based on previous research,  those who had smoked >100 cigarettes in their life-times were defined as long-term smokers and the others were considered as non-smokers and those who drank at least 3 times per week for more than 1 year were defined as long-term drinkers and the rest were considered as non-drinkers. The questionnaire was administered through face-to-face interviews by trained interviewer to obtain information. After the interview, a 5 ml venous blood sample was collected from each subject. Informed consent was obtained from all subjects and the research protocol was approved by the Institutional Review Board of Nanjing Medical University.
Environmental noise monitoring and audio logical assessment
According to the Chinese national criteria for noise in the workplace (GBZ43-2002, http://www.zybw.net ), we used a sound pressure noise meter (Noise-Pro, Quest, Oconomowoc, WI) at 10 AM, 3 PM, and 5 PM for 3 consecutive days, twice per year to assess noise exposure levels at the workplaces. To evaluate the actual noise exposure level for workers, normalization of equivalent continuous A-weighted sound pressure to a nominal 8 h a day (Lex. 8 h) were recorded.
Pure-tone audiometry was performed for both ears of every subject at 0.5, 1.0, 2.0, 3.0, 4.0, and 6.0 kHz by a trained technician according to the standards set in ''Diagnostic Criteria of Occupational NIHL" (Chinese Occupational Health Standard, (GBZ49-2002). An ascending method in 5-dB (A) steps was used to ascertain the hearing threshold levels by the technician. The lowest signal intensity detected in the subject with a minimum of 3 tries was defined as the final threshold value for each ear. If the subject had a threshold value that differed by 40 dB or more between both ears, masking was performed.  We also performed the otoscopic examination of the external acoustic meatus and tympanic membrane to exclude any ear diseases. As far as we know, hearing loss can occur either in the low-frequency range (0.5-2.0 kHz) or high-frequency range (3.0-6.0 kHz). And the mean threshold of 0.5, 1.0, and 2.0 kHz was considered as low-frequency hearing status and the mean threshold of 3.0, 4.0, and 6.0 kHz as high-frequency hearing status. Briefly, hearing threshold worse than 25 dB in high-frequency or in both high-frequency and low-frequency was defined as hearing loss. Controls were matched to the cases on age, exposure level, and exposure time. In our study, due to the requirement of the Diagnostic Criteria of Occupational NIHL, the workers with low-frequency hearing loss should be transferred from original noise-exposed environment immediately. So we only accepted subjects with high-frequency hearing loss. A total of 615 cases and 644 controls were recruited.
According to an approach combining potentially functional polymorphisms of the DNMT1 gene, polymorphisms were selected from the NCBI database ( http://www.ncbi.nlm.cih.gov/ ) with a minor allele frequency (MAF) ≥0.05 in Han Chinese Beijing. Potentially functional polymorphisms were identified to meet the following criteria: Located in 5'-untranslated regions (UTR), 5'near gene, exon, 3'- UTR. According to the criteria, 3 SNPs were identified which were listed in [Table 1]. DNA was isolated from all samples (5 ml) using a Tiangen DNA extraction kit according to the manufacturer's instructions (Tiangen BiotechCompany, Beijing, China). And the genotypes for the three DNMT1 polymorphisms were performed with the MGB TaqMan probe assay using the 384-well Applied Biosystems Inc (ABI) 7900 high throughput (HT) Real Time polymerase chain reaction (PCR) System and the SDS 2.4 software for allelic discrimination from Applied Biosystems Inc. (Foster City, CA). The sequences of primer and probe for each SNP are available on request. The reaction mixture of 10 μl contained 20 ng genomic DNA, 3.5 μl of 2 × TaqMan Genotyping Master Mix, 0.25 μl of the primers and probes mix and 6.25 μl of double distilled water. The amplification was performed under the following conditions: 50°C for 2 min, 95°C for 10 min followed by 45 cycles of 95°C for 15 s, and 60°C for 1 min. The polymorphism analysis was performed by two persons independently in a blinded manner. About 10% of the samples were randomly selected for repeated genotyping for validation, and the results were 100% concordant.
The Hardy-Weinberg equilibrium (HWE) of the control's genotype distributions was tested for all individual SNPs using a goodness-of-fit χ2 test. Differences in the distributions of demographic characteristics, selected variables, and frequencies of genotypes of DNMT1 polymorphisms between the NIHL cases and controls were evaluated by using the Student's t-test (for continuous variables) or χ2 test (for categorical variables). Unconditional logistic regression analysis with the adjustment for age, sex, smoking status, drinking status, and exposure time were used to estimate the associations between the DNMT1 polymorphisms and risk of NIHL by calculating crude and adjusted odds ratios (ORs) and 95% confidence intervals. Because of the median noise expose year of the cases and controls, an exposure time cutoff of 20 years was used for stratification. According to the international reference standard ISO1999:1990 and our noise measurement data, noise exposure levels of the sample set were divided into three categories (<85 dB, 85-92 dB, and > 92 dB, [Lex. 8 h]). Statistics analysis system (SAS) 9.1 PROC HAPLOTYPE was used to infer haplotype frequencies based on our genotypes. P < 0.05 was considered statistically significant. All statistical tests were two-sided and performed with the software SAS 9.1 (SAS Institute, Inc., Cary, NC, USA).
| Results|| |
Characteristics of subjects and SNP genotyping
The frequency distributions of selected characteristics of the cases and controls are presented in [Table 2]. The cases and controls appeared to be adequately matched on age and sex (P = 0.973 for age and P = 0.700 for sex). In addition, we did not observe the statistically significant difference in smoking status, drinking status, exposure time, and exposure level [Table 2]. As expected, the average threshold value of NIHL workers was more than two times greater than that of the normal hearing workers (37.2 dB [A] vs. 14.1 dB [A]). The primary information and allele frequencies are summarized in [Table 1]. All the three polymorphism loci were in agreement with the HWE (P > 0.05 for all). And the MAF of all the three polymorphisms was consistent with that reported in the HapMap database.
|Table 2: Demographic and occupational characteristics of cases and controls|
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Association between DNMT1 polymorphisms and NIHL risk
The distribution of genotype of DNMT1 polymorphisms is shown in [Table 3]. For the SNP rs12984523, the frequencies of the CC, CT, and TT genotypes were 51.6%, 41.6% and 6.8%, respectively, among the cases and 52.6%, 38.7%, and 8.7%, respectively, among the controls. For the SNP rs16999593, the frequencies of the TT, CT, and CC genotypes were 62.6%, 33.7%, and 3.7%, respectively, among the cases and 64.5%, 31.8%, and 3.7%, respectively, among the controls. And for the SNP rs2228612 the frequencies of the TT, CT, and CC genotypes were 32.8%, 48.5% and 18.7%, respectively, among the cases and 31.1%, 47.8%, and 21.1%, respectively, among the controls. The distributions of these three SNPs were not significantly different between cases and controls (P = 0.338 for SNP rs12984523, P = 0.783 for SNP rs16999593, and P = 0.531 for SNP rs2228612).
Crude and adjusted ORs for NIHL were used without and with adjustment for confounding factors including age, sex, smoking status, drinking status, and exposure time. No significant higher risk was found in any genotype of the three SNPS (P > 0.05), and the similar results were observed in the stratification analyses (data not shown).
|Table 3: Distribution of genotype of DNMT1 polymorphisms with the risk of noise-induced hearing loss|
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Associations between the DNMT1 haplotypes and NIHL risk
The distribution of haplotypes between NIHL cases and NIHL controls are presented in [Table 4]. Eight common haplotypes were found from the analyses of the combinations of the three SNPs. And the haplotypes with a frequency <0.05 were pooled into the others group. However, no significant difference was observed between the cases and controls and the haplotypes showed no apparent relationship with risk of NIHL.
|Table 4: Frequencies of the inferred haplotypes of DNMT1 among the cases and controls and the associations with risk of noise-induced hearing loss|
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| Discussion|| |
The present study was to explore the association between three polymorphisms (rs12984523, rs16999593, and rs2228612) and risk of NIHL in a Chinese population. The distribution of genotypic frequencies and haplotypes of the DNMT1 polymorphisms showed no significant association between the NIHL cases and healthy controls. These results suggested that the selected DNMT1 polymorphisms may not affect the susceptibility to NIHL in the Chinese population.
As we all know, NIHL is a complex disease caused by a gene-environment interaction. For the same noise exposure, some individuals are more susceptible to NIHL than others.  More and more attention around the world was put into the field of how to explain the differences in the susceptibility to NIHL. Currently, most studies on NIHL have focused on the three mainstream pathways which are oxidative stress, K-recycling pathway, and heat shock proteins. However, the exact mechanism of the NIHL is not clear until now. As far as we all know, NIHL is one form of sensorineural hearing loss accompanied by auditory nerve damage. Neural development, neural survival, and connectivity have been related to DNMT1 because it plays a key role for DNA methylation and chromatin stability. ,, Christopher et al. found that mutations in DNMT1 can cause hearing loss. Therefore, we performed this study and to the best of our knowledge, this is the first study to evaluate the association between the DNMT1 polymorphisms and NIHL risk. In this case-control study, we failed to find evidence for an association between the DNMT1 polymorphisms and NIHL risk. But DNMT1 may not be excluded from the candidate genes for NIHL. For the following reasons, the first is ethnic differences while we studied the Chinese Han population which may be the crucial factor leading to the lack of association. The second, in China, once identified by the government as an occupational disease, patients can enjoy a series of preferential policies. Considering the self-interest factors, there may be a certain subjective difference from audiological assessment. We expect that a more objective and accurate audiological assessment method will be applied in the near future. The third, our sample size is moderate and the statistical power of the study is limited.
The strength of our study is that the genotype distributions in the population are similar to the study which was reported to have been association with risk of breast carcinoma among Han Chinese women.  For example, the frequencies of the TT, CT, and CC genotypes of the rs16999593 among our 644 southern Chinese controls (64.5%, 31.8%, and 3.7%, respectively) were comparable to frequencies (70.6%, 28.4%, and 1.6%) reported for 314 northern Chinese women controls. This tiny difference may come from different gender of the two studies. The other two SNPs have not been reported yet in Chinese population.
| Conclusion|| |
In conclusion, we found that the three polymorphisms of DNMT1 were not associated with the susceptibility to NIHL. These findings suggested that the polymorphisms of DNMT1 may not contribute to the risk of NIHL in a Chinese population. Larger population-based studies with different races should be conducted to further confirm our findings.
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Department of Environmental Genomics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Cancer Center, Nanjing Medical University, 818 East Tianyuan Road, Nanjing 211166
Source of Support: This study was mainly supported by Projects of Jiangsu Society Development (BS2005661), Jiangsu Province’s Outstanding Medical Academic Leader program, the National Natural Science Foundation of China (81230068, and 30972444), the Key Program of Natural Science Foundation of Jiangsu Province (BK2010080, and BK2010575), the Qing Lan Project of Jiangsu Provincial Department of Education, and the Priority Academic Program development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine), Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]
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