Hearing impairment risk and interaction of folate metabolism related gene polymorphisms in an aging study

Presbycusis is one of the most common sensory impairments affecting the elderly and is a multifactorial process that involves a multitude of intrinsic and extrinsic factors. Recent investigations demonstrated many genetic contributions to human age-related hearing impairment (ARHI) [1–7].

Folate metabolism is essential for cellular functioning because it provides one-carbon donors for the de novo synthesis of purines and pyrimidines, which are necessary for RNA and DNA synthesis, and provides methyl groups, which are necessary for the remethylation of homocysteine and for methylation reactions such as DNA methylation [8, 9]. The enzyme methylenetetrahydrofolate reductase (MTHFR) irreversibly catalyzes the conversion of 5,10-methylenetetrahydrofolate (5,10-methyleneTHF) to 5-methyltetrahydrofolate (5-methylTHF), a methyl donor. Methionine synthase (MTR) catalyzes the remethylation of homocysteine to methionine. Folate in the form of 5,10-methyleneTHF donates a methyl group to uracil, converting it to thymidine, which is used for DNA synthesis and repair [10]. (See Additional file 1, Figure S1.)

Folates cannot be synthesized de novo by mammals. Polymorphisms in genes encoding critical enzymes in folate metabolism play important and interrelated roles in the pathophysiology of cancer [9–13], cardio-cerebrovascular disease, atherosclerosis [14], pregnancy loss, renal failure [15], diabetic retinopathy [16], and the progression of Alzheimer's disease [17].

The MTHFR C677T polymorphism (rs1801133), which results in an alanine-to-valine substitution, is one of the most extensively investigated functional polymorphisms of genes encoding one-carbon metabolism enzymes and occurs frequently in Caucasian and Asian populations. This substitution results in a 30% decrease in MTHFR activity in heterozygotes and a 60% decrease in MTHFR activity in homozygotes [18].

Although reports are not uniformly consistent, this polymorphism has been shown to modify the risk of developing several cancers in a site-specific manner [13, 19, 20]. It decreases the risk of colorectal cancer, hepatocellular carcinoma, cervical cancer, and certain leukemias and lymphomas. In contrast, it increases the risk of cancer of the breast, endometrium, esophagus, stomach, pancreas, and bladder [13]. Epidemiological evidence indicates that this polymorphism has a protective effect against colorectal cancer in individuals with an adequate or high status of folate and nutrients involved in one-carbon metabolism [21–23].

The most common MTR gene polymorphism is MTR A2756G (rs1805087), which results in a substitution of aspartic acid for glycine and decreases methionine synthase activity [24]. This polymorphism increases the cellular homocysteine level [25], resulting in DNA hypomethylation [26].

The MTHFR and MTR polymorphisms have attracted interest as causes of sudden sensorineural hearing loss (SSNHL) [27–29]. We reported results from a nested case-control study using data from the National Institute for Longevity Sciences - Longitudinal Study of Aging (NILS-LSA) that showed that the T allele of MTHFR C677T is associated with susceptibility to SSNHL. However, little has been reported on the association between folate pathway gene polymorphisms and ARHI. Durga et al. investigated the effects of folate status and the MTHFR C677T polymorphism on hearing in middle-aged and elderly subjects [30].

In this study, we investigated whether the MTHFR C677T (rs1801133) and MTR A2756G (rs1805087) polymorphisms are associated with hearing impairment in middle-aged and elderly people in a Japanese community.

The main findings of our study were that the risk of developing hearing impairment in middle-aged and elderly subjects is influenced by an interaction between the MTR A2756G and MTHFR C677T gene polymorphisms and that the MTHFR 677T allele is associated with a reduced risk of hearing impairment, but only when the MTR A2756G genotype is a wild-type homozygote (AA).

We recently reported that the T allele of the MTHFR C677T gene polymorphism may be associated with an increased risk of SSNHL [29]. In the present analysis, the MTHFR 677 T allele decreased the risk of hearing impairment among the middle-aged and elderly, provided that the MTR A2756G genotype was AA. These apparently contradictory effects of the MTHFR C677T polymorphism on hearing are consistent with the results of Durga et al [30]. They examined the association between elevated plasma homocysteine level and its determinants with hearing levels in middle-aged and elderly subjects. Their hypothesis was that an elevated concentration of homocysteine and its determinants, including the MTHFR 677TT genotype, would be associated with poor hearing. Contrary to their hypothesis, the results demonstrated that MTHFR 677TT homozygotes had 5 dB (p = 0.037) and 2.6 dB (p = 0.021) lower PTA-high and PTA-low hearing thresholds, respectively, than subjects with a 677C allele when their folate status was above the median. They found that high serum folate concentrations were associated with better hearing in MTHFR 677TT homozygotes, whereas high serum folate concentrations were associated with poorer hearing in CC homozygotes. In the present study, the MTHFR variant allele conferred an advantage with respect to hearing when the MTR A2756G genotype was the wild type, which probably maintains MTR enzymatic activity at a higher level than in the variant [24]. Studies of carcinogenesis showed that an impaired MTR reaction, as observed in vitamin B₁₂ deficiency or in cases of chronic ethanol ingestion, results in the continuous conversion of 5,10-methylene-THF to 5-methyl-THF. Thus, a substantial proportion of cellular folate is converted into a metabolically unavailable form, which results in a functional folate deficiency [34]. The MTR 2756A allele may prevent functional folate deficiency via MTR activity.

As referred to previously, the MTHFR 677T allele has favorable and unfavorable effects on human health. The conflicting effects of the MTHFR C677T polymorphism on carcinogenesis may be resolved as follows. When the dietary supply of folate and related nutrients is sufficient, individuals carrying variant MTHFR genotypes may be at reduced risk of cancer because high intracellular levels of 5,10-methyleneTHF may prevent imbalances in the nucleotide pool during DNA synthesis, thereby ensuring that DNA replication occurs with high fidelity [9, 13]. DNA methylation is crucial for epigenetic modification of the genome, which is involved in the regulation of many cellular processes. Some MTHFR variants are associated with DNA hypomethylation [35]. The degree of DNA methylation modulates the risks of various cancers as a consequence of gene-environment interactions [36]. The MTHFR 677T allele may affect disease susceptibility in a beneficial or an adverse manner.

Durga et al. explained the etiology of AHRI in terms of mitochondrial damage [37]. It was proposed that damage to mitochondrial DNA accumulates over time and that once a critical threshold is attained, the mitochondria are rendered bioenergetically inefficient and cells undergo apoptosis. Individuals with the MTHFR 677TT genotype may have increased levels of thymidylate synthesis, which may directly reduce the probability of DNA mutations by ensuring a smaller pool of uracil and, hence, a decreased likelihood of uracil misincorporation into DNA. In addition, MTHFR 677TT homozygotes may have high levels of 10-formylTHF, which may protect mitochondrial integrity by reducing cytochrome c [38] and, because of its structural lability, may exert antioxidant-like effects [39].

This study has certain limitations. A major weakness of this study is that homocysteine and folate levels were available for only 22% of the cumulative data because of cost restriction. Also lab tests for vitamin B12 and/or methylmalonic acid were not performed, which are additional metabolic parameters potentially influenced by MTR enzymatic action. Current opinion is that the 677 C-to-T variant exerts its effect via an elevated homocysteine level as described as 'the homocysteine hypothesis' [14]. On the other hand, the homocysteine hypothesis and homocysteine-lowering therapeutic approaches to vascular disease have been questioned in recent years. Secondary prevention trials have failed to confirm the benefit of homocysteine-lowering therapy on cardiovascular disease [40, 41]. Antoniades et al. proposed that folic acid and its circulating metabolite, 5-methylTHF, have direct effects on vascular function in humans, independent of their effects on plasma homocysteine level [42, 43]. Therefore, data on changes in metabolites of the folate and homocysteine pathways and the status of B vitamins would have strengthened this study.

Another issue that has created some confusion is the seemingly contradictory effects of the MTHFR 677T allele on SSNHL vs. ARHI in the NILS-LSA study population. We reported that the MTHFR 677T allele may be associated with an increased risk of SSNHL and that it may be associated with a reduced risk of hearing impairment later in life if the individuals are wild-type homozygotes for MTR A2756G. No plausible explanation for this contradiction can be offered at this stage. However, given that one of the many hypotheses on the etiology of SSNHL involves impaired perfusion and ischemic damage to the cochlea, the MTHFR 677T allele may contribute to risk of an acute vascular event in the cochlea. On the other hand, the MTHFR 677T allele has certain advantages for the better-hearing ear in homozygous MTR 2756AA individuals, which are independent of plasma total homocysteine level. Bilateral hearing impairment as defined by the World Health Organization requires the presence of a hearing impairment in the better-hearing ear. Therefore, the better-hearing ear was used in our study in order not to overestimate individuals with only one affected ear as subjects with ARHI. The contribution of the MTHFR 677T allele to the affected ear in SSNHL may differ from its contribution to the better-hearing ear in middle-aged and elderly people.

Although genetic contributions to ARHI have been increasingly reported, significant associations between gene polymorphisms and a reduced risk of ARHI are rare. The results of this study may help to elucidate the complex mechanisms responsible for ARHI. This is an observational epidemiological design, which helps us to understand the factors that determine the presence or absence of disorder. Hypotheses derived from our results need to be verified by further basic and applied researches.

The present study has found that the MTHFR 677T allele has a favorable effect on a risk of hearing impairment in the middle-aged and elderly population, only when the individuals were wild-type homozygotes for MTR A2756G. In addition, the favorable impact of the MTHFR 677T allele on the risk of developing hearing impairment in the MTR 2756AA genotype was independent of blood folate and homocysteine levels. These results may contribute to our understanding of the pathology of ARHI.