Every year in China, about 30,000 children, compared to 840 in UK and one of every one thousand infants in US, are born with congenital hearing impairment123. Hearing impairment is the most common neurosensory disorder in human that has an incidence of approximately 1 in 1000 children worldwide4. About 50–60% of these cases have a genetic cause. The most common molecular defects for nonsyndromic autosomal recessive deafness lie on Connexin 26, a gap junction protein encoded by the GJB256789101112. More than 150 mutations, polymorphisms and unclassified variants have been described in GJB2 to account for about 8–40% of molecular etiology of the patients with nonsyndromic hearing impairment http://davinci.crg.es/deafness3. However, about 80% of the patients with nonsyndromic hereditary deafness in China do not have mutations in GJB213.

Pendred syndrome (PS) is the most common form of syndromic deafness that accounts for about 10% of hereditary hearing impairment14. It is an autosomal recessive disorder caused by biallelic mutations in SLC26A4 resulting in hearing loss, enlargement of the vestibular aqueduct (EVA) and iodine organification defect in the thyroid gland15. EVA is always detected in the ears of patients with PS by computed tomography (CT) and magnetic resonance imaging (MRI)16. EVA is the most common form of the inner ear malformation associated with prelingual or postlingual sensorineural hearing loss and is an important feature of PS1718. EVA may occur alone or in combination with an incomplete partition of the apical turn of the cochlea as part of Mondini deformity. PS is differentiated from nonsyndromic hearing loss with EVA by the presence of goiter, which usually develops later at around the time of puberty. Since environmental and other genetic factors may modulate the effects of SLC26A4 mutations on the development of goiter, the expression of goiter in PS patients is variable and may have incomplete penetrance19. SLC26A4 encodes an anion (chloride/iodide) transporter transmembrane protein, pendrin, which is expressed in the thyroid, kidney, and cochlea2021. DNA sequence analysis identified more than 100 different mutations in SLC26A41015222324252627. The mutation spectrum varies widely among different ethnic groups101519232627282930. Park and Pryor observed that patients with PS were always associated with two mutant alleles in SLC26A4 consistent with autosomal recessive disorder, whereas patients with nonsyndromic hearing loss and EVA might have one or zero mutant allele1519. In Caucasian nonsyndromic EVA cohort, about one third of the patients had two mutant alleles, one third had one mutant allele and one third had zero19. In Japanese and Korean EVA patients, the proportion of patients having two identified mutant alleles in SLC26A4 is much higher, 57% and 81%, respectively2429. Whereas in China, 97.9% EVA patients in simplex families were detected with either biallelic or monoallelic mutations, of which 88.4% were carrying biallelic variants and 9.5% with monoallelic mutation. Only 2.1% Chinese EVA patients had no mutant SLC26A4 allele detected27. In addition, the prevalent mutations in different ethnic groups are very different. Campbell et al. reported T416P and IVS8+1G>A as the two most frequent mutations in northern European population 22, while Blons et al. showed a completely different mutation spectrum that was extremely heterogeneous23. In Japanese, H723R accounted for 53% of the mutant alleles, and in Korean, the H723R and the IVS7-2A>G mutation was the most prevalent mutation accounting for 45.5% of patients with PS or EVA1929. In China, IVS7-2A>G mutation was the most common form accounting for 57.63% of the mutant alleles27. All of the above studies focused on the EVA or Pendred syndrome patients.

In order to investigate the ratio of EVA or Pendred syndrome in Chinese hearing impairment patients and provide effective genetic testing and accurate counseling for hearing loss patients and families in China, we performed SLC26A4 sequence analysis in hearing impairment patients in Chifeng City from Inner Mongolia and then made a genotype-phenotype correlation analysis.

All patients showed severe to profound bilateral sensorineural hearing impairment on audiograms except Patient 9 in Table 1 whose right ear pure tone average (PTA) is 55 dB.

Diagnosis of Pendred syndrome EVA requires the evaluation of inner ear malformation by temporal bone CT scan. Unfortunately, in Chifeng City, Inner Mongolia, China, the temporal bone CT scan was too expensive to perform and there was lack of expertise for temporal bone evaluation. Under these circumstances, SLC26A4 mutation analysis may be the only alternative way for the diagnosis of EVA, since blood samples can be collected locally and sent elsewhere for DNA analysis. In this study, 100% patients (17/17) with bi-allelic mutation were confirmed to have EVA by CT scan performed in Chifeng Second Hospital with the help of a specialist from Beijing. Perchlorate discharge testing, a routine testing for thyroid function, is not available in most area of China. We use thyroid hormone testing and ultrasound scan of thyroid to examine the function and structure of thyroid instead. Our results indicate that none of patients have PS. These may be explained by a). testing methods were different, b). the age of patients undertaking thyroid ultrasound and thyroid hormone assays, 3 to 20, average 13.24 ± 3.92, in this study may be too young to have symptoms, c). phenotypic diversity due to different genetic background.

In this study, we found that SLC26A4 mutations were detected in nearly 20% of our patients with hearing impairment with IVS7-2A>G being the most prevalent mutation. Among the novel variants, Y375C, R470H, I491T, L597S and H723D were considered pathogenic based on a) they are located in evolutionarily conserved regions (Figure 2), b) substituted amino acids are structurally and functionally different from amino acids of the wild type, c) Y375C, R470H, I491T, L597S and H723D have been found in patients with EVA or other forms of inner ear malformation, and d) they were not present in our normal controls.

It's interesting to note that patient 18 with inner ear malformation carry one missense mutation only, whether the missense mutation causes dominant negative effect and/or specifies a different phenotype is not clear. Three patients (18 to 20) with EVA or other inner ear malformation harbored only one mutant allele. It's possible that the second mutant allele has not yet been identified due to a) mutations deep in introns or promoter regions that are not sequenced, b) intragenic exon deletions, c) mutations in genes other than SLC26A4 may involve in the pathogenesis (digenic). Thus, the mutations in the SLC26A4 gene account for at least 12.6% (17/135) of the patients with nonsyndromic hearing loss, making it as equally commonly mutated gene as GJB2 (23/135 no significant difference found after statistical analysis, P > 0.05) in patients from Inner Mongolia.

Unlike GJB2 which is a small gene with a lot of missence variants, SLC26A4 is a relatively large gene with rare missense benign polymorphisms or variants. Thus, novel missense variant in SLC26A4 is possibly pathogenic. Two questions were raised: can the autosomal recessive SLC26A4 mutations cause hearing impairment without EVA or other inner ear malformation, and are there other genes involved in the pathogenesis of hearing loss with SLC26A4 (digenic). To answer the first question, screening of the SLC26A4 mutations in a large NSHI population without EVA is necessary. For the second question, Malin Hulander reported that the lack of pendrin expression led to deafness and expansion of the endolymphatic compartment in inner ears of Foxi1 null mutant mice 34. His observation provides the direct evidence that other genes may modulate the expression of SLC26A4. Alternatively there may be dominant negative effect.

The SLC26A4 mutation spectrum in ChiFeng City, Inner Mongolia is similar to that reported in Chinese population but different from that of Japanese. There is a gradient shift of the most prevalent mutation from IVS7-2A>G to H723R, respectively, from Chinese to Japanese with both mutations being equally prevalent in Korean. This observation suggests that IVS7-2A>G and H723R mutations may be the ancient mutations in China and Japan respectively. The unique rare mutations evolved more recently. A recent study of 100 unrelated patients with EVA in European Caucasians by Albert et al. revealed a diverse mutation spectrum without prevalent mutations and only 40 patients carried SLC26A4 mutations26. Our previous study on the prevalence of GJB2 mutations in Chinese patients with hearing impairment demonstrated that GJB2 mutations were detected in 30.4% of the patients in ChiFeng city. Together, approximately 49.63% (41+26/135) of patients with NSHI in ChiFeng city carried mutations in GJB2 or SLC26A4 gene. Whereas about 33.1% and 3.5% of European patients with NSHI carried mutations in GJB2 and SLC26A4 respectively, with a total of 36.6%, comparable to that in our patient group 35. It is not clear why the mutations in SLC26A4 account for much lower percentage of patients with EVA in Caucasian patients. Presumably, other genetic factors and environmental factors are involved in the pathogenesis of EVA in Caucasians.

The striking spot of this study is that a new strategy that detects SLC26A4 mutations prior to the temporal bone CT scan to find EVA patients are established. In China, the cost of temporal CT scan is 200 to 300 RMB, because of the relatively high cost, it is not possible to perform CT scan in every hearing loss patient in molecular epidemiologic study to diagnose EVA. Since 97.9% of Chinese EVA patients carry SLC26A4 mutation 27, SLC26A4 mutation in hearing loss patients indicates a high possibility of EVA. This model presents unique advantage in epidemiologic study in large-scale deaf population to find EVA.

In Inner Mongolia, China, mutations in SLC26A4 gene account for at least 12.6% (17/135) of the patients with nonsyndromic hearing loss. Pendred syndrome is not detected in the Inner Mongolia deaf population. We established a new strategy that detects SLC26A4 mutations prior to the temporal bone CT scan to find EVA and inner ear malformation patients. This model has a unique advantage in epidemiologic study of large deaf population.

The authors declare that they have no competing interests.

Pu Dai, Yongyi Yuan and Deliang Huang carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. Xiuhui Zhu carried out temporal CT scan and thyroid hormone assays. Dongyang Kang participated in the sequence alignment. Fei Yu and Huijun Yuan participated in the design of the study and performed the statistical analysis. Dongyi Han and Bailin Wu conceived of the study, and participated in its design and coordination and helped to draft the manuscript. Lee-Jun Wong reviewed and interpreted the results, drafted and revised the manuscript. All authors read and approved the final manuscript.