Introduction
Sudden sensorineural hearing loss (SNHL) is a relatively frequent otological disease of unknown aetiology with an incidence of approximately 10 in 100 000 per year in South Korea. It is characterised by the sudden onset of hearing loss that occurs in minutes, hours or even over a few days and is usually unilateral.Reference Lazarini and Cameargo1
Various aetiologies have been hypothesised for this disease, including vascular disorders, viral infections of the labyrinth or cochlear nerve, and autoimmune causes.Reference Stokroos, Albers and Schirm2–Reference Cadoni, Agostino, Manna, De Santis, Fetoni and Vulpiani5 However, classifying sudden SNHL by aetiology is insufficient, as there is still a lack of knowledge concerning its pathogenetic mechanisms. Whatever the underlying cause of the hearing loss, impaired cochlear perfusion seems to be the most important pathogenetic event. As the cochlea is highly sensitive to minimal changes in localised blood flow and to anoxia, cochlear ischaemia is a likely cause of sudden SNHL.Reference Schweinfurth and Cacace6
The vascular hypothesis is plausible, since the cochlea is a highly vascular organ supplied by the labyrinthine artery, a functional end artery.Reference Guo, Zhang, Du, Nair and Yoo7 The possibility of impaired labyrinthine circulation in patients with sudden SNHL has been discussed previously. In 1973, Schuknecht et al. reported that alterations in the microcirculation of the cochlea may be a cause of sudden SNHL, while Nagahara et al. reported a 30 per cent reduction in perilymphatic oxygen tension in their sudden SNHL group.Reference Schuknecht, Kimura and Naufal8, Reference Nagahara, Fisch and Yagi9
Recently, a considerable number of studies have investigated the relationship between sudden SNHL epidemiological and vascular risk factors and treatment interventions. The incidence of sudden SNHL in western countries has increased.Reference Suckfull10 One recent study suggested that an exaggerated western diet rich in saturated fatty acids could be a risk factor for sudden SNHL. This is consistent with the hypothesis that vascular factors can cause sudden SNHL.Reference Nakamura, Whitlock, Aoki, Nakashima, Hoshino and Yokoyama11
Several inherited vascular risk factors for sudden SNHL have been detected in recent years, including genetic variations in folate-dependent homocysteine metabolism (Figure 1).Reference Menachem, Gideon, Ron, Nira, Neta and Iman12 Homocysteine is an amino acid that is related to folate through the active 5-methyltetrahydrofolate form of folic acid. Its concentration depends upon three enzymes: methylenetetrahydrofolate reductase, methionine synthase and cystathionine-B synthase. A deficiency in any one of these enzymes causes an accumulation of unmetabolised intracellular homocysteine, which is exported from the cells into the plasma. This favours functional impairment of the endothelial and vascular smooth muscle cells and the extracellular matrix. In this way, increased plasma homocysteine concentration promotes coagulation and impairs fibrinolysis. The associated endothelial damage may favour the formation of atheromatous cholesterol plaques, which are known to have an additive negative effect on endothelial function.Reference Van Guldenet and Stehouwer13
Folate-dependent homocysteine metabolism. MTHF = methylenetetrahydrofolate
Methylenetetrahydrofolate reductase, the key homocysteine metabolic enzyme, catalyses the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which acts as a cofactor in the methylation of homocysteine to form methionine. Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with increased plasma homocysteine concentration, a recognised risk factor for thrombosis and atherosclerosis, which can induce occlusive vascular disease.Reference Friedman, Goldschmidt, Friedlander, Ben-Yehuda, Selhub and Babaey14 Specific mutations in the methylenetetrahydrofolate reductase gene have been associated with increased total plasma homocysteine concentration. The cytosine (C) → thymine (T) substitution mutation at nucleotide 677 of the coding region (i.e. C677T) results in the substitution of valine for alanine at position 226 of the amino acid sequence, and is linked to elevated total plasma homocysteine levels in homozygotes (i.e. TT) compared with heterozygotes (i.e. CT) or normal individuals (i.e. CC).Reference Leclerc, Campeau, Goyette, Adjalla, Christensen and Ross15 The methylenetetrahydrofolate reductase C677T gene mutation reduces methylenetetrahydrofolate reductase enzymatic activity, and subsequently leads to increased plasma homocysteine and decreased plasma folate levels.Reference Lievers, Boers, Verhoef, den Heijer, Kluijtmans and van der Put16
Based on these considerations, and in order to further elucidate the mechanisms of inner ear vascular impairment and to investigate the possible role of genetic factors, we investigated the role of the methylenetetrahydrofolate reductase C677T mutation as a predisposing vascular factor for sudden SNHL, and its effect on homocysteine, folate and cholesterol concentrations.
Materials and methods
Subjects
We enrolled 33 consecutive patients with sudden SNHL (17 men and 16 women; mean age ± standard deviation (SD), 48.24 ± 14.50 years), from August 2008 to January 2009. The exclusion criteria were: diabetes mellitus; malignancy; anticoagulant therapy; oral contraception; a history of acute myocardial infarction, stroke, venous thrombosis, other cardiovascular diseases or autoimmune diseases; and pathological magnetic resonance imaging results.
Control subjects comprised 68 randomly selected, healthy individuals (26 men and 42 women; mean age ± SD, 43.91 ± 16.96 years).
Methods
All subjects underwent a general physical examination, audiological tests (i.e. pure tone audiometry, brainstem evoked response audiometry and otoacoustic emission testing), magnetic resonance imaging and haematologic examination (including methylenetetrahydrofolate reductase genotyping, and measurement of homocysteine, folate and cholesterol concentrations). All haematological examinations were performed via venepuncture. The methylenetetrahydrofolate reductase C677T mutation was analysed by polymerase chain reaction amplification, and by DNA fragment separation via electrophoresis with a SeeplexTM methylenetetrahydrofolate reductase genotyping kit (Seegen, Seoul, Korea). Plasma homocysteine concentration was measured by fluorescent polarising immunoassay, using the AxSYM automated immunoassay instrument system (Abbott, Abbott park, USA). Plasma folate concentration was determined using a radioassay kit.
All patients diagnosed with sudden SNHL were admitted immediately for seven days. On the day of admission, they received intravenous steroids, antiviral agents, vasodilators, antibiotics, blood volume expanders and fluid mixed with multivitamins. All patients were placed on a low salt diet with stellate ganglion block every day from admission.
Statistical analysis
The concordance of genotype frequencies and allele frequencies was tested with the chi-square test to determine whether the genotype distribution differed between sudden SNHL patients and controls. Student's t-test was used to compare cholesterol, homocysteine and folate concentrations for patients versus controls. Statistical significance was accepted at a level of p < 0.05. The relationship between the sudden SNHL patients' various mutation genotypes and their cholesterol, homocysteine and folate concentrations was tested using the Kruskal–Wallis H statistic, because of the small sample size for each genotype. Statistical tests were performed using the Statistical Package for the Social Sciences version 12.0 software.
Results and analysis
A total of 101 subjects participated in the study, comprising 33 patients with a clinical diagnosis of sudden SNHL (age range, 17–75 years) and 68 normal controls (age range, 11–73 years). There was no statistically significant difference in age or gender, comparing patients and controls (p = 0.2) (Table I).
Characteristics of sudden SNHL patients and controls
p = 0.2, comparing patients and controls for both age and gender (t-test). *n = 33; †n = 68. SNHL = sensorineural hearing loss; SD = standard deviation; yr = years
The wild-type methylenetetrahydrofolate reductase nucleotide 677 genotype (i.e. CC) was observed in 13 patients (39.4 per cent) and 24 controls (35.3 per cent). The heterozygous mutation genotype (i.e. CT) was observed in 16 patients (48.5 per cent) and 35 controls (51.5 per cent). The homozygous mutation genotype (i.e. TT) was observed in four patients (12.1 per cent) and nine controls (13.2 per cent). The prevalence of the homozygous methylenetetrahydrofolate reductase C677T mutation genotype in sudden SNHL patients (12.1 per cent) was not statistically significantly different to that in controls (13.2 per cent) (p = 0.922). The prevalence of the T allele of the methylenetetrahydrofolate reductase C677T polymorphism in sudden SNHL patients (23.5 per cent) was also not significantly different to that in controls (27.3 per cent) (p = 0.775). No statistically significant association was found between sudden SNHL and the presence of the methylenetetrahydrofolate reductase C677T gene mutation (Table II).
Genotype distribution and allelic frequencies of sudden SNHL patients and controls
*Patients vs controls, chi-square test. †n = 33; ‡n = 68. SNHL = sensorineural hearing loss; MTHFR = methylenetetrahydrofolate reductase; C = cytosine; T = thymine; pts = patients; ctls = controls; CC = wild type; CT = mutation heterozygotes; TT = mutation homozygotes
Plasma concentrations of homocysteine, folate and cholesterol were measured in the 33 sudden SNHL patients and 68 controls, to detect any differences between the two groups. The mean homocysteine level was significantly higher in the sudden SNHL patients than in the controls (mean ± SD, 9.43 ± 6.1 vs 7.21 ± 4.2 µmol/l, respectively; reference limits, 5–20 µmol/l; p = 0.037). The mean folate level was significantly lower in the sudden SNHL patients than in the controls (mean ± SD, 10.74 ± 6.0 vs 13.01 ± 5.1 ng/ml, respectively; reference limits, 3–17 ng/ml; p = 0.049). The mean cholesterol level was significantly higher in the sudden SNHL patients than in the controls (mean ± SD, 176.45 ± 25.8 vs 158.75 ± 33.1 mg/dl, respectively; reference limits, below 200 mg/dl; p = 0.008) (Table III).
Homocysteine, folate and cholesterol plasma concentrations in sudden SNHL patients and controls
Data represent means ± standard deviations unless otherwise specified. *n = 33; †n = 68. ‡Student's t-test; §p < 0.05. Reference ranges: **5–20 µmol/l; #3–17 ng/ml; abelow 200 mg/dl. SNHL = sudden sensorineural hearing loss
Within the sudden SNHL patient group, no statistically significant difference was found in the mean homocysteine, folate and cholesterol levels, comparing patients with different methylenetetrahydrofolate reductase C677T genotypes (p = 0.852, 0.434 and 0.477, respectively) (Table IV).
Homocysteine, folate and cholesterol plasma concentrations in sudden SNHL patients*, by MTHFR C677T mutation genotype
Data represent mean values unless otherwise specified.
* n = 33. †Kruskal–Wallis H test. Reference ranges: ‡5–20 µmol/l; **3–17 ng/ml; §??–200 mg/dl. SNHL = sudden sensorineural hearing loss; MTHFR = methylenetetrahydrofolate reductase; C = cytosine; T = thymine; CC = wild type; CT = mutation heterozygotes; TT = mutation homozygotes
Discussion
Impaired cochlear perfusion is the most widely accepted hypothesis for the pathogenesis of sudden SNHL. However, the anatomy of the cochlea, and its location within the temporal bone, hinder adequate investigation of the pathophysiological mechanism of sudden SNHL.Reference Capaccio, Ottaviani, Cuccarini, Ambrosetti, Fagnani and Bottero17 Previous studies have contributed to the vascular theory of sudden SNHL by investigating thromboembolism, blood factor disturbances and cochlear microcirculation alteration.Reference Schuknecht, Kimura and Naufal8 The cochlear blood supply is maintained by the labyrinthine artery, which has no collateral vasculature.Reference Schweinfurth and Cacace6 Vascular derangement, caused by cochlear injury or dysfunction due to anoxia or hypoxia, may cause sudden SNHL.Reference Schweinfurth and Cacace6 Furthermore, endothelial dysfunction, high levels of haemostatic factors and blood flow disturbance – the fundamental features of thromboembolic disease – can impair cochlear microcirculation.Reference Yamasoba, Kikuchi, Higo, O'uchi and Tokumaru18 Although there are few data demonstrating impaired cochlear perfusion in sudden SNHL, therapeutic concepts are widely based on the assumption of such impairment.Reference Capaccio, Ottaviani, Cuccarini, Bottero, Schindler and Cesana19
Recent studies have assessed a number of inherited prothrombotic risk factors and their genetic alterations, and have correlated these with the occurrence of vascular disorders.Reference Friedman, Goldschmidt, Friedlander, Ben-Yehuda, Selhub and Babaey14–Reference Capaccio, Ottaviani, Cuccarini, Ambrosetti, Fagnani and Bottero17, Reference Capaccio, Ottaviani, Cuccarini, Bottero, Schindler and Cesana19 In particular, methylenetetrahydrofolate reductase gene mutation, which impairs enzymatic activity, has frequently been mentioned.Reference Capaccio, Ottaviani, Cuccarini, Ambrosetti, Fagnani and Bottero17–Reference Capaccio, Ottaviani, Cuccarini, Bottero, Schindler and Cesana19 The effects of methylenetetrahydrofolate reductase C677T gene mutation on cardiovascular disease, in association with plasma homocysteine concentrations, have been discussed.Reference Lievers, Boers, Verhoef, den Heijer, Kluijtmans and van der Put16 In 2001, Lievers et al. reported that the TT genotype of this mutation gave rise to higher homocysteine levels than the CT genotype, causing an increased risk of cardiovascular disease.Reference Lievers, Boers, Verhoef, den Heijer, Kluijtmans and van der Put16 In 2005, Capaccio et al. reported that the methylenetetrahydrofolate reductase C677T mutation genotypes TT and CT were more frequent in sudden SNHL patients.Reference Capaccio, Ottaviani, Cuccarini, Ambrosetti, Fagnani and Bottero17 On this basis, we too investigated the relationship between the methylenetetrahydrofolate reductase C677T mutation and sudden SNHL; however, unexpectedly, we did not find a statistically significant association.
It is well known that reduced methylenetetrahydrofolate reductase enzyme activity leads to folate dysmetabolism and increased plasma homocysteine levels, both of which may play a role in microvascular disease.Reference Capaccio, Ottaviani, Cuccarini, Ambrosetti, Fagnani and Bottero17–Reference Capaccio, Ottaviani, Cuccarini, Bottero, Schindler and Cesana19 It has also been hypothesised that the increase in homocysteine levels associated with altered folate metabolism, although slight, may favour the impairment of endothelial function in small vessels such as the auditory artery and its branches.Reference Yamasoba, Kikuchi, Higo, O'uchi and Tokumaru18 Mild to moderate increases in homocysteine levels have been associated more often with peripheral vascular and cerebrovascular disease than with coronary artery disease.Reference Mattson, Kruman and Duan20 Furthermore, high blood cholesterol levels are known to be a major risk factor for endothelial dysfunction and vascular thrombosis.Reference Hirano, Ikeda, Kawase, Oshima, Kekehata and Takahashi21 In our study, no significant associations were found between sudden SNHL patients' homocysteine, folate and cholesterol concentrations and their methylenetetrahydrofolate reductase C677T mutation genotypes; however, statistically significant differences were observed between the homocysteine, folate, and cholesterol concentrations of patients versus controls. This finding could further support the theory of a relationship between methylenetetrahydrofolate reductase mutation, folate-dependent homocysteine metabolism and sudden SNHL.
In fact, it could be argued that there is a trend towards an increased risk of sudden SNHL even with homocysteine concentrations which are increased but still within reference limits, which may still lead to microvascular impairment, especially in such terminal vascular systems as that of the cochlea. In addition to this mechanism, a spasm or thromboembolic event in the cochlear arterial system may cause sudden SNHL because of cochlear ischaemia or anoxia.Reference Yamasoba, Kikuchi, Higo, O'uchi and Tokumaru18
Other enzymes involved in the folate-dependent homocysteine mechanism, such as methionine synthase, have been discussed as inherited vascular risk factors in sudden SNHL.Reference Menachem, Gideon, Ron, Nira, Neta and Iman12 Alterations of the methionine synthase and cystathionine-B synthase enzymes can affect homocysteine and folate plasma concentrations; however, neither enzyme was examined in our study. Other mutations of the methylenetetrahydrofolate reductase gene, such as A1298C, have also to be investigated. Such variable factors may affect the folate-dependent homocysteine mechanism, and require further investigation.
• Impaired cochlear perfusion appears to be the most important event in the development of sudden sensorineural hearing loss (SNHL)
• This study investigated the relationship between the methylenetetrahydrofolate reductase C677T mutation and sudden SNHL
• There was a statistically significant difference between the homocysteine, folate and cholesterol levels in sudden SNHL patients vs controls, although there was no significant difference in these levels among patients with various methylenetetrahydrofolate reductase C677T mutation genotypes
• Further studies are required to explore the relationship between folate-dependent homocysteine metabolism and sudden SNHL
Conclusion
Our data indicated no statistically significant association between the methylenetetrahydrofolate reductase C677T gene mutation and sudden SNHL. We did observe a statistically significant difference between the levels of homocysteine, folate and cholesterol in our sudden SNHL patients versus controls. However, there was no statistically significant difference in these levels, comparing sudden SNHL patients with the three methylenetetrahydrofolate reductase C677T mutation genotypes. These findings could further support the theory of a relationship between folate-dependent homocysteine metabolism and the development of sudden SNHL. Further studies should be performed on other methylenetetrahydrofolate reductase mutations, and on other homocysteine metabolic enzymes.
If performed on a larger series of patients and controls, genetic studies of homocysteine metabolism could be useful in detecting those patients at risk of developing sudden hearing loss or other microvascular disease. In the future, newly discovered genetic variations in folate-dependent homocysteine metabolism will probably have important implications for the prognosis of patients with sudden SNHL.
