The progress made in research on hereditary hearing loss indicates the importance of molecular diagnosis using the SLC26A4 gene in patients with hearing loss
. The molecular diagnosis of hearing impairment associated with the SLC26A4 gene is reliable because deafness is caused by homozygous or compound heterozygous SLC26A4 mutations
[6, 23]. Previous studies
[13, 24, 25]. suggested that SLC26A4 c.919-2A>G is the most frequent mutation in Taiwan and mainland China, and that less-frequent mutations of other exons may be detected by direct DNA sequence analysis
. In the present study, 58.8% of patients carrying a heterozygous c.919-2A>G mutation (47/80) had a definite form of SNHL caused by a SLC26A4 mutation. We also found that compound heterozygosity accounted for nearly 60% of patients with two mutated alleles, which highlights the importance of screening for SLC26A4 mutations other than c.919-2A>G.
In this study, 19 mutations were identified, seven of which were novel. Of these mutations, 16 were found only in the Asiatic deaf population, whereas three were identified in both Asians and Caucasians. It seems that the spectrum of SLC26A4 mutations in the Chinese population is different from that of European populations. The most common allele variant was p.H723R, situated within exon 19, which accounted for 34% of all mutant alleles. P.H723R appeared to be highly correlated with nonsyndromic recessive deafness with EVA among Chinese populations
. Park et al.
 reported that the p.H273R mutation is prevalent; this allele exists in the majority of SLC26A4 mutations in Korean and Japanese populations. The second-most-frequent mutations (i.e., p.T410M, 15+5G>A (c.1705+5G>A), and p.L676Q) occur at exon 10, exon 15 flanking sequences, and exon 17, respectively. Exons 19, 10, 17 and 15 (flanking sequences) have highly variable region(s) in which multiple mutations were detected. Therefore, this study showed that mutations other than c.919-2A>G exist mostly in four exons (i.e., exons 19, 10, 17 and 15). The mutation frequency in SLC26A4 exons 19, 17, 10, and 15 was consistent with the results of previous studies
. However, our study was performed on patients with severe to profound hearing impairment or deafness (general deaf population), whereas Wang investigated patients with hearing impairment and EVA/MD
. The distribution of mutant SLC26A4 alleles revealed by our study suggests that mutation screening of four exons (i.e., exons 19, 10, 17 and 15) following c.919 A>G identification should be the priority for NSHL genetic testing, especially in EVA/MD patients.
We found seven SLC26A4 mutations that have not been described previously. These included two missense mutations, four splice-site mutations, and one frameshift mutation. The missense mutations were probably pathogenic, based on both their presence in affected individuals and their predicted biological consequences. That is, a change in an amino acid that is located in a functional domain or that is conserved in related genes or species is likely to be pathogenic. The missense mutations p.S93R and p.G222V are most likely to be pathogenic based on the changes in evolutionarily conserved amino acids, Serine to Arginine and Glycine to Valine, respectively. However, as the SLC26A4 gene is expressed only in thyroid, kidney, and brain
[7, 12, 28], and since none of these tissues was available from patients, we were unable to evaluate the effect of the mutation at the mRNA level. The splice site mutation is frequently found in compound heterozygotes with a c.919-2A>G mutation
. The pathogenic potential of splice site mutations is unknown since their effect on splicing has not been determined. We presume that these splice site mutations could cause exon deletion and affect SLC26A4 mRNA integrity and pendrin function. The SLC26A4 frameshift mutation (c.1825 delG) was a nonsense mutation, resulting in an unstable mRNA or truncated protein.
A second mutant allele was not detected among 33 patients in our study. It is unlikely that a single SLC26A4 mutant allele is sufficient to cause EVA, as there are no published reports of vertical co-segregation of EVA with a single SLC26A4 mutant allele or sporadic cases associated with a single de novo SLC26A4 mutant allele
. In a previous study, we performed single mutation screening of deaf individuals from Chifeng and Nantong Cities, and found one mutant allele mutation in SLC26A4 was identified in 8.45%(24/284)
It still remains unclear whether or why the heterozygous c.919-2A>G mutation affects the inner ear. It can be assumed that patients with a single c.919-2A>G mutation are more likely to develop hearing loss and EVA in the presence of additional genes or environmental factors. On the basis of current knowledge, we speculate that other regulatory factors or genes are involved in the production or activity of the inner ear proton pump or that environmental factors affect pump function in some patients. First, a subset of individuals with a monoallelic SLC26A4 gene mutation might harbor a second unidentified mutation in the promoter or intronic regions of this gene. It has been demonstrated that Foxil-null mice have both enlarged endolymphatic ducts/sacs and hearing loss, and Foxil is a possible upstream regulator of SLC26A4
. Yang et al. described several EVA patients with a heterozygous SLC26A4 mutation in combination with a heterozygous hypo-functional variant of FOXI1 or KCNJ10
[33, 34]. Mutations in introns that may activate cryptic splice sites might be present in human populations. Second, since deafness is associated with GJB2 and GJB3
, it should be noted that the deafness associated with the SLC26A4 gene might be a consequence of digenic inheritance. Last, environmental or unknown factors may explain the phenotype of EVA patients with a single heterozygous c.919-2A>G mutation. Alternatively, SLC26A4 epigenetic modifications, such as DNA methylation, might repress gene transcription and account for the observed co-segregation of deafness and SLC26A4 monoallelic mutations
We propose a new algorithm for genetic screening in association with SLC26A4 mutations, based on the frequency of mutations in different exons. In SNHL patients, the SLC26A4 gene should be examined by preferential exon screening. If results are inconclusive, then we propose mutation screening in sequence, since the four most frequent types of mutations account for the majority mutant alleles (66%) in our experience (Table
1). If no mutations are detected in exons 19, 10, 17 or 15, then analysis of other SLC26A4 exons should be undertaken. This algorithm should detect almost two thirds of mutant alleles and is particularly useful for genetic screening in hearing loss patients.
EVA is associated with characteristic clinical findings, including fluctuating and sometimes progressive sensorineural hearing loss. Hearing loss in some EVA patients is mild to moderate. This study was limited by the moderate-to-profound hearing loss requirement, which decreased EVA patient enrollment. However, our findings show that biallelic SLC26A4 mutations are a common cause of moderate-to-profound hearing loss. Our results underscore the importance of a genetic screening program that is capable of detecting different SLC26A4 mutations. The screening of SLC26A4 gene mutations will assist us with genetic counseling, and screening for SLC26A4 gene mutations in patients with moderate or profound hearing loss should be routine. Furthermore, our data provide information that will foster genetic approaches to early diagnosis in the Chinese population.