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Keywords:

  • Deafness;
  • EVA;
  • hearing loss;
  • MLPA;
  • Pendred;
  • SLC26A4

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Mutations in SLC26A4 cause Pendred syndrome (PS) – hearing loss with goitre – or DFNB4 – non-syndromic hearing loss (NSHL) with inner ear abnormalities such as Enlarged Vestibular Aqueduct (EVA) or Mondini Dysplasia (MD). We tested 303 unrelated Czech patients with early hearing loss (298 with NSHL and 5 with PS), all GJB2-negative, for SLC26A4 mutations and evaluated their clinical and radiological phenotype. Among 115 available HRCT/MRI scans we detected three MD (2.6%), three Mondini-like affections (2.6%), 16 EVA (13 bilateral – 19.2% and 15.6% respectively) and 61 EVA/MD-negative scans (73.4%). We found mutation(s) in 26 patients (8.6%) and biallelic mutations in eight patients (2.7%) out of 303 tested. In 18 of 26 (69%) patients, no second mutation could be detected even using MLPA. The spectrum of SLC26A4 mutations in Czech patients is broad without any prevalent mutation. We detected 21 different mutations (four novel). The most frequent mutations were p.Val138Phe and p.Leu445Trp (18% and 8.9% of pathogenic alleles respectively). Among 13 patients with bilateral EVA, six patients (50%) carry biallelic mutations. In EVA -negative patients no biallelic mutations were found but 4.9% had monoallelic mutations. SLC26A4 mutations are present mostly in patients with EVA/MD and/or progressive HL and those with affected siblings.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Hearing loss is the most common sensory deficit in humans with congenital or prelingual deafness affecting about 1 in 1000 newborns and infants (Morton, 1991). The majority of cases are expected to be of genetic origin with significant dominance of autosomal recessive (AR) inheritance (up to 75%) and non-syndromic phenotype (Schrijver, 2004). The most important role in non-syndromic hearing loss (NSHL) is played by biallelic mutations in the GJB2 gene (Snoeckx et al., 2005), being responsible for almost 40% of early NSHL cases in the Czech population (Seeman et al., 2004, 2005; Seeman & Sakmaryova, 2006).

Mutations in the SLC26A4 gene are probably the second most common cause of inherited hearing loss (after GJB2 mutations) and are responsible for Pendred syndrome (PS) (Everett et al., 1997) as well as DFNB4 (non-syndromic hearing loss with inner ear abnormalities – Enlarged Vestibular Aqueduct (EVA) and/or Mondini Dysplasia (MD)) (Pryor et al., 2005). Pendred syndrome comprises hearing loss with inner ear malformations (EVA and/or MD) (Reardon et al., 2000) and thyreopathy manifested as an eu/hypothyroid goitre and defective iodine organification (verifiable by perchlorate discharge test) (Pendred, 1896; Fraser, 1965). Thyroid impairment in PS usually appears during puberty or in early adulthood (Fraser, 1965; Reardon et al., 1999). PS is probably the most common cause of syndromic hearing loss with an estimated incidence about 7.5:100,000 inhabitants in the UK (Fraser, 1965; Pryor et al., 2005). Hearing loss in DFNB4/PS is typically progressive and/or fluctuant (Blons et al., 2004; Pryor et al., 2005). Vestibular impairment is an inconsistent part of the clinical picture (Goldfeld et al., 2005).

Biallelic SLC26A4 mutations were found in 30% (Azaiez et al., 2007) and 60% (Pera et al., 2008b) of PS patients. In cases of non-syndromic EVA, environmental and other genetic factors (like transcription control and mutations in other genes (Yang et al., 2007, 2009)) seem to play an important role. Monoallelic mutations in SLC26A4 are typical for non-syndromic EVA and biallelic mutations were frequently found in PS patients (Pryor et al., 2005; Azaiez et al., 2007).

The frequency of biallelic SLC26A4 mutations in deaf patients among different populations seems to vary, appearing to be responsible for 3.5–12.5% of congenital hearing loss (Hutchin et al., 2005; Azaiez et al., 2007) vs. (Dai et al., 2008; Kahrizi et al., 2009). Frequencies range from 3.5 to 5% mainly in Caucasian populations (Hutchin et al., 2005; Azaiez et al., 2007; Madden et al., 2007) and around 8.8% to 12.5% in Middle East and Asian patients (Dai et al., 2008; Wu et al., 2008; Kahrizi et al., 2009).

Since EVA was revealed as a common feature of PS (Cremers et al., 1998), it was later recognised as a constant finding also in DFNB4 (Pryor et al., 2005). The presence of EVA was established as a crucial criterion for SLC26A4 analysis (Pryor et al., 2005). No study on a representative sample of deaf patients with EVA-negative findings on High Resolution Computed Tomography (HRCT) has yet been published. The spectrum and frequency of SLC26A4 mutations in neither the Czech population, nor any other in Central European region or in any Slavic population has been studied yet and was fully unknown before our study. To complete the information in this field and to establish the frequency and spectrum of SLC26A4 mutations among Czech deaf patients we performed SLC26A4 analysis in a large cohort of patients mostly from the Paediatric Cochlear Implant Centre in Prague, Czech Republic. We also aimed to evaluate and establish clinical selection criteria for effective diagnostic SLC26A4 testing in the future.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Patients

Three hundred and three unrelated Czech patients, children as well as young adults, diagnosed with prelingual severe-to-profound sensorineural hearing loss were included in the study. Two hundred and ninety eight were non-syndromic and five were diagnosed with PS. Twenty seven of the patients were from families with a similarly affected sibling. Severe to profound HL cases were selected since it is the typical phenotypic manifestation of AR early hearing impairment. All patients, including five with PS, had previously been shown to be negative for the most common cause of genetic hearing loss – biallelic mutations in the GJB2 gene. GJB2 mutations were excluded by direct sequencing of exon 2 followed by sequencing of exon 1 in patients with only one mutation in exon 2. All 303 patients were tested for the presence of mutations in the coding sequence of the SLC26A4 gene. Most patients were referred from the ENT clinics of University Hospital Motol and from various phoniatric and clinical genetics departments in the Czech Republic. Comprehensive family history with attention to hearing loss in the relatives was obtained from all investigated patients. Inclusion criteria for this study were: pre- or perilingual hearing loss, autosomal recessive inheritance or sporadic case and no presence of any following exclusion criteria. Exclusion criteria were: clearly acquired aetiology of hearing loss such as meningitis, asphyxia, neonatal ventilation, head trauma, craniofacial malformations, use of ototoxic drugs or very low birth weight, clearly dominant inheritance, otosclerosis and known genetic syndrome (except Pendred syndrome).

One hundred and twenty two patients in our cohort at the time of the study were cochlear implant users and had already been tested by a battery of tests related to this treatment.

All patients were originally evaluated and diagnosed by an ENT specialist before inclusion into our cohort. They were again evaluated and counselled by a clinical geneticist to rule out a common syndromic cause of deafness such as Waardenburg, Townes-Brock, Alport or Usher syndromes and to explain to the patients or their parents the purpose of SLC26A4 gene testing.

Retrospective re-evaluation of clinical data was focused on family and personal history, audiograms, hearing loss development records and eventual cochlear implantation protocol and radiological findings (HRCT or MRI of the temporal bone). The criterion for the presence of EVA was a diameter of vestibular aqueduct larger than 1.5 mm at the midpoint of its length. MD was diagnosed when aplasia of the last turn of the modiolus was clear.

Informed consent was obtained from all subjects or from their parents during genetic consultation before DNA testing. The project was approved by the Ethical committees of Charles University in Prague, Second Medical School and University Hospital Motol.

Molecular Genetic Analysis

DNA was isolated from whole blood by standard techniques. We performed direct sequencing of all 21 exons of the SLC26A4 gene and its exon/intron boundaries using previously published primers (Prasad et al., 2004). In brief: After PCR, fragments were purified with AMPure set (Beckman Coulter, Beverly, MA, USA) and sequenced with the Big Dye Terminator 3.1 kit (Applied Biosystems, Foster City, CA, USA). Sequencing reaction products were purified with CleanSeq (Beckman Coulter) and subsequently run and analysed on an automated Genetic Analyzer ABI 3130. The resulting sequences were edited and compared against the published SLC26A4 sequence from NCBI – gene ID 5172.

All 18 patients with only one detected SLC26A4 pathogenic mutation were analysed with MLPA KIT SALSA R280-A1 Pendred-SLC26A4 (MRC-Holland, Amsterdam, Netherlands) to detect any possible larger deletion or duplication affecting the SLC26A4 gene which would be missed by sequencing.

The carrier frequency for p.Val138Phe and p.Leu597Ser in the Czech normal hearing population was established by screening of 503 anonymous DNA samples using restriction endonucleases DdeI and Tsp509I (New England Biolabs, Ipswich, MA, USA). This sample comprised of 400 anonymous blood donors and 103 anonymised healthy relatives of patients with peripheral neuropathies (individuals with evident hearing loss were excluded). To define p.Glu6Val carrier frequency, exon 2 was sequenced in 190 healthy controls (randomly selected from the group of 400 anonymous blood donors).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Clinical and Radiological Findings

Evaluating the clinical data of 303 Czech patients with early hearing loss, we found 19 (6.5%) patients with clinical progression of hearing loss (all single cases) but no patient with fluctuant hearing loss.

Thyroid dysfunction was noticed in 12 patients (seven sporadic and five familial). Neonatal thyroid screening was positive in three patients.

Detailed re-evaluation of all 115 available CT/MRI scans revealed MD in 3 patients and in three patients a Mondini-like malformation was observed (2.6% of all available CT each). In this collection, 83 HRCT/MRI scans valuable for EVA (vestibular aqueduct level was manifest) were present. Among these 83 HRCT/MRI scans we detected EVA in 16 patients (in 13 of them the EVA was bilateral) (19.2% respectively 15.6%) and in 61 patients EVA/MD was not present (73.4% of all valuable CT scans).

On the basis of these findings we divided our patients into 3 groups:

  • Group A (highest expected SLC26A4 mutation probability) – 22 patients with EVA and/or MD (two of them familial)

  • Group B – 220 patients with no valuable CT/MRI scans (23 of them familial)

  • Group C – 61 patients with EVA/MD-negative result from HRCT/MRI scan of the temporal bone (two of them familial)

Mutational frequencies in each group are summarised in Table 1.

Table 1.  Frequency of SLC26A4 mutations among Czech NSHL patients.
group# patientsPatients with monoallelic mutations (% in the group)Patients with biallelic mutations (% in the group)Patients with ≥1 mutation (% in the group)
A223 (13.6%)6 (27.3%)9 (40.9%)
B22012 (5.5%)2 (0.9%)14 (6.4%)
C613 (4.9%)03 (4.9%)
All30318 (5.9%)8 (2.7%)26 (8.6%)

The highest mutational frequency (40.9% in group) was in group A, as we assumed.

In familial cases mutations in SLC26A4 were three times more frequent than in sporadic – see Table 2.

Table 2.  Frequency of SLC26A4 mutations in sporadic and familial Czech NSHL patients.
 # patientsPatients with monoallelic mutations (% in the group)Patients with biallelic mutations (% in the group)Patients with ≥1 mutation (% in the group)
Familial cases27 (8.9%)4 (14.8%)2 (7.4%)6 (22.2%)
Sporadic cases276 (91.1%)14 (5.1%)6 (2.2%)20 (7.3%)
All cases303 (100%)18 (5.9%)8 (2.7%)26 (8.6%)

Spectrum and Frequency of Mutations

Among 303 selected patients we detected 30 different sequence variants (nine of them novel), of which 21 were considered pathogenic (four of them novel) – see Table 3. At least one pathogenic mutation was detected in 26 patients (8.6% of all tested patients). Both pathogenic mutations in both gene alleles were found in eight patients (2.7% of all examined patients) – all as compound heterozygotes (see Tables 1 and 4). A single pathogenic mutation on one allele only was found in 18 patients (5.9% of all examined patients) – see Tables 1 and 5. This high number of patients with only monoallelic mutations (69.2% of patients with an identified mutation) led us to the assumption that there could be a defect in the SLC26A4 gene unidentifiable by sequencing, such as a larger deletion or duplication of several exons or even the whole gene. Thus we performed MLPA analysis in all 18 patients with only one pathogenic mutation identified, but we did not find any deletion or duplication of any part of the gene in these patients.

Table 3.  Spectrum of SLC26A4 mutations in Czech NSHL patients and their frequencies among pathogenic alleles.
Mutation No. and frequency of mutationFirst describedExon
p.Val138Phec.412 G>T6 (18% of all pathogenic alleles)van Hauwe et al. 19984
p.Leu445Trpc.1334 T>G3 (8.9%)van Hauwe et al. 199811
p.Glu29Glnc.85 C>A2 (5.9%)Campbell et al. 20012
p.Arg409Hisc.1226 G>A2 (5.9%)Coyle et al. 199810
p.Thr416Proc.1245 A>C2 (5.9%)van Hauwe et al. 199810
p.Asn457Lysc.1371 C>A2 (5.9%)Park et al. 200312
c.1001 + 1G>A2 (5.9%)Coyle et al. 1998Intron 8
c.1614 + 1 G>A2 (5.9%)Blons et al. 2004Intron 14
p.Thr99Metc.296 C>T1 (2.9%)Propst et al. 20063
p.Val144Alac.431 T>C1 (2.9%)this study5
p.Arg185Thrc.554 G>C1 (2.9%)this study5
p.Thr193Ilec.580 C>T1 (2.9%)Adato et al. 20005
p.Gly209Valc.626G>T1 (2.9%)van Hauwe et al. 19986
p.Leu236Proc.707 T>C1 (2.9%)van Hauwe et al. 19986
p.Val281Ilec.841 G>A1 (2.9%)this study7
p.Phe335Leuc.1003 T>C1 (2.9%)Campbell et al. 20019
p.Trp518Stopc.1554 G>A1 (2.9%)this study14
p.Tyr530Serc.1589 A>C1 (2.9%)Pryor et al. 200514
p.Thr721Metc.2162 C>T1 (2.9%)Usami et al. 199919
p.Gly740Valc.2219 G>T1 (2.9%)Prasad et al. 200419
p.Arg776Cysc.2326 C>T1 (2.9%)Pryor et al. 200521
Total # 34 (100%)  
Table 4.  Phenotypes of patients with biallelic pathogenic SLC26A4 mutations (new mutations in italics).
# DNAs/f-*Age**hearing lossHRCT/MRIThyroidAllele 1Allele 2
  1. *sporadic or familial (s = sporadic, fam = familial)

  2. **Age at examination (y = year/s)

1790s10yprogressivebil. EVAnormalp.Thr193Ilep.Asn457Lys
1928s9ycongenitally deafbil. EVANTHp.Leu236Prop.Thr721Met
2333s19yprogressivebil. EVAgoiterp.Glu29Glnp.Leu445Trp
3830fam55yprogressiven/agoiterp.Asn457Lysc.1001 + 1 G>A
3832s6yprogressivebil. EVAnormalp.Val138Phec.1001 + 1 G>A
4745s38yprogressiven/agoiterp.Val281Ilec.1614 + 1 G>A
4748s13yprogressivebil. EVAgoiterp.Val138Phep.Leu445Trp
4947fam5ycongenitally deafbil. EVAnormalp.Arg409Hisp.Leu445Trp
Table 5.  Phenotypes of patients with monoallelic pathogenic SLC26A4 mutations (new mutations in italics).
# DNAs/f-*Age**hearing lossCT/HRCTThyroidAllele 1Allele 2MLPA
  1. *sporadic or familial (s = sporadic, fam = familial)

  2. ** Age at examination (y = year/s)

  3. ***Patients with “non-MD” sign have only CT available with no valuable imaging of vestibular aqueduct level. MD however could have been excluded

1255s20yprogressivenon-MD***n/ap.Thr416Prowtnormal
2795s7ycongenitally deafbil. EVAnormalp.Val138Phewtnormal
3831s21yprogressivebil. EVAgoitrep.Arg185Thrwtnormal
4950s4ycongenitally deafbil. EVAnormalp.Arg409Hiswtnormal
1513fam29ycongenitally deafnon-MDn/ap.Val144Alawtnormal
1737s7ycongenitally deafnon -MDn/ap.Leu236Prowtnormal
2334s23ycongenitally deafnon-MDn/ap.Trp518Stopwtnormal
3163s9ycongenitally deafnon-MDn/ap.Gly740Valwtnormal
4771s44ycongenitally deafn/an/ap.Glu29Glnwtnormal
4776fam29yprogressivenon-MDgoiterp.Val138Phewtnormal
4784s16ycongenitally deafnon-MDn/ac.1614 + 1G>Awtnormal
4822s8ycongenitally deafn/an/ap.Thr99Metwtnormal
4825s33yprogressiven/an/ap.Thr416Prowtnormal
4837fam32ycongenitally deafnon-MDn/ap.Arg776Cyswtnormal
4840fam32yprogressivenon-MDgoiterp.Val138Phewtnormal
3836s5ycongenitally deafnon-EVA, non-MDnormalp.Val138Phewtnormal
4818s11ycongenitally deafnon-EVA, non-MDnormalp.Phe335Leuwtnormal
4954S4ycongenitally deafnon-EVA, non-MDnormalp.Tyr530Serwtnormal

There is no prevalent mutation in SLC26A4 in the Czech population. The most frequent mutations in Czech patients are p.Val138Phe and p.Leu445Trp, identified in six and three patients respectively (18% and 8.9% of all mutant alleles). Remaining mutations were found only once or twice each (representing 5.9% or 2.9% of all mutant alleles respectively) – see Table 3 (Coyle et al., 1998; Van Hauwe et al., 1998; Usami et al., 1999; Adato et al., 2000; Campbell et al., 2001; Park et al., 2003; Blons et al., 2004; Prasad et al., 2004; Pryor et al., 2005; Propst et al., 2006).

Parents (or descendants in cases 3830 and 4745) of all patients with two pathogenic mutations were subsequently tested and mutations were found in the heterozygous state in all of them, confirming their independent segregation. Mutations were found most commonly in exon 4 (6 alleles), exon 10 and exon 14 and its boundaries (4 alleles each). No pathogenic mutations were found in exons 1, 13, 15–18 and 20.

Although variants p.Leu597Ser and p.Glu6Val are non-synonymous changes of amino acids, we consider these to be non-pathogenic because firstly, their frequency in non-affected controls was very similar to that in NSHL patients and secondly, they were found only in the heterozygous state without any other pathogenic mutation found in “trans” in our patients (all in groups B and C).

Frequencies of all polymorphisms in our patients are shown in Table 6.

Table 6.  Polymorphisms and their frequencies in patients (and in a control population).
Allelic variantheterozygoteshomozygotes
c.607–17 C>T1%0
c.606 + 331 T>G41%33%
c.1437 + 243T>C3.5%0
c.1545-14 del ATT0.7%0
c.1708-18 T>A2.6%0
c.1-698 G>C0.3%0
c.1-697 T>G5.6%0.3%
c. 1790 T>C (p.Leu597Ser)2.1%(2.4%)0
c. 17 G>T (p.Gly6Val)1.7%(1.6%)0

In order to establish heterozygous frequency of the most commonly found mutation, p.Val138Phe, in the Czech hearing population we tested 503 samples from control subjects without evident HL, but no carrier was revealed. We conclude that the frequency of this mutation is lower than 1:500 (less than 0.2%).

Genotype – Phenotype Correlation

All six patients with both pathogenic mutations and HRCT documentation available, showed bilateral EVA associated with progressive and severe-to-profound hearing impairment.

Among 13 patients with bilateral EVA, biallelic mutations were detected in six patients (46%) and monoallelic in another three of them (23%). Interestingly we also detected monoallelic mutations in 4.9% of patients with EVA-negative HRCT.

In patients with unilateral EVA, MD and other malformations of the temporal bone, no SLC26A4 pathogenic mutation was found.

Neonatal transitory hypothyroidism (NTH) was observed in three patients, one showing two pathogenic SLC26A4 mutations (case no. 1928), but the others having wild type genotypes (cases no. 4743 and 4958).

All four post-pubertal patients with biallelic SLC26A4 mutations with initially normal thyroid function (no NTH) have shown hypo-functional goitre since adolescence.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Spectrum of the SLC26A4 Mutations

No prevalent mutation was detected in SLC26A4 in the Czech population as is the case in GJB2 with 35delG. The spectrum of mutations in SLC26A4 in the Czech population is broad with mutations affecting 14 of 21 exons and exon/intron boundaries. This finding will prevent a simplified routine DNA testing targeted to the prevalent mutation(s) in the future and sequencing of all 21 exons will be necessary in all patients. However the analysis could be started with exons 4, 10, 14 and 11 + 12, where we found the majority of pathogenic mutations. The most common mutation in our cohort, p.Val138Phe (18% of all pathogenic alleles), was also found in some familial cases in Germany (Borck et al., 2003) and was shown to be a founder mutation in the Central European area as was the mutation p.Thr416Pro (found twice – 5.9%– in our study) also described in Germany (Napiontek et al., 2004).

We detected all recurrent mutations commonly found in Caucasian patients in at least one of our patients, with the exclusion of p.Glu384Gly and p.Cys400fs. On the contrary we found two patients with mutation p.Asp457Lys which was detected previously only in one large consanguineous family in India (Park et al., 2003). We also detected four new sequence vatiants (p.Val144Ala, p.Arg185Thr, p.Val281Ile and p.Trp518Stop) each in the heterozygous state in one patient only. There is little doubt about the pathogenicity of the truncating mutation p.Trp518Stop and that of the mutation p.Val281Ile which was found in “trans” with the other pathogenic mutation (IVS14 + 1 G>A) in a phenotypically well-characterised patient (no. 4745), but the pathogenicity of mutations p.Val144Ala and p.Arg185Thr remains to be clarified.

Frequency of the SLC26A4 Mutations

Biallelic pathogenic mutations were found in 2.7% of all, and 7.4% of familial, AR cases among our GJB2-negative patients. This is a much lower frequency when compared to GJB2 mutations, which could be up to 42.4%, in an analogous group of Czech patients (Seeman et al., 2004, 2005). SLC26A4 was tested in several populations (France, Holland, Germany, Spain, UK, USA, China and Japan). However these cohorts are not comparable, since most of these publications refer to isolated cases (Borck et al., 2003; Napiontek et al., 2004– Germany; Van Hauwe et al., 1998– Holland; Usami et al., 1999– Japan) or the studies were performed on patients with EVA only (Blons et al., 2004; Albert et al., 2006– France; Pryor et al., 2005; Madden et al., 2007– USA; Pera et al., 2008a– Spain; Wu et al., 2005– China). There have been only four studies performed on non-selected NSHL patients (Azaiez et al., 2007– USA; Hutchin et al., 2005– UK; Dai et al., 2008– China, Kahrizi et al., 2009– Iran). The largest study of 1506, mostly Caucasian, NSHL patients (Azaiez et al., 2007) found biallelic SLC26A4 mutations in 4.38% of GJB2-negative patients, all of them having EVA. The other study, encompassing 142 AR familial NSHL Caucasian patients (Hutchin et al., 2005), revealed biallelic SLC26A4 mutations in 5.3% of all GJB2-negative patients.

In conclusion, our observed SLC26A4 mutational frequency in the Czech NSHL population is comparable with other Caucasian populations.

As there had been no systematic study of SLC26A4 mutations in EVA-negative patients, our observation of three patients with monoallelic mutations among 61 patients with EVA-negative HRCT scans (4.9% of patient in the group) is a new finding. This frequency is significantly higher than the expected heterozygote frequency in the general population which is estimated to be 1% (Pera et al., 2008a). Two explanations could account for this finding: 1) the heterozygote frequency in the general population is higher than expected or 2) a single mutation in the SLC26A4 gene can contribute to deafness by a mechanism other than by causing EVA. All sequence variants that can be considered non-pathogenic (p.Leu597Ser, p.Glu6Val and intronic variants) were excluded and all three mutations found in this group (p.Val138Phe, Phe335Leu and p.Thr416Pro) were repeatedly described (Campbell et al., 2001; Blons et al., 2004; Pryor et al., 2005), and have been functionally tested and proven to be pathogenic (Pera et al., 2008a). Additional surveys will be necessary to confirm or exclude one or both of the theories proposed above.

A large number of patients with monoallelic mutations were found in all groups of our cohort (69.2% of all patients with at least one mutation). In addition to the theory described above, that a single mutation in the SLC26A4 gene could play a pathogenic role, other explanations could be discussed: firstly, abundant mutations in the SLC26A4 gene may be simply rare non-pathogenic polymorphisms; secondly, there may be a prevalent mutation, presently unknown in these patients, which may be deeply intronic or not detectable by the techniques we used, and which could represent a majority or even all of the alleles found so far without any pathogenic mutation; thirdly the defect could be caused by a mutation in other genes such as FOXI1 or KCNJ10 (Yang et al., 2007, 2009). Therefore mutational screening in these two genes may be beneficial for diagnosing these patients in the future.

Genotype – Phenotype Correlation

As only one patient of three which had NTH was found to have biallelic SLC26A4 mutations we assume that, in agreement to a previous study (Banghova et al., 2008), this finding shows that NTH is rather a rare clinical manifestation of PS.

All four post-pubertal patients with biallelic pathogenic mutations and initially normal thyroid function (no NTH) have shown hypo-functional goitre since adolescence. This again raises the question of the existence of DFNB4 as an independent disorder, because there is still a high suspicion for this to be only a juvenile (incomplete) form of PS lacking goitre that will manifest post-pubertally. There is only a single published case of a patient of the age of 40 having EVA and 2 mutations in the SLC26A4 gene and no thyroid impairment (Wu et al., 2005). On the other hand there is evidence of thyroid impairment onset at the age of 37 years (Blons et al., 2004).

Conclusions

We conclude that SLC26A4 gene mutations are a much less common cause of inherited hearing loss than are GJB2 mutations, but they are still a relevant cause of NSHL in the Czech Republic, comprising 2.7 – 8.6% of all GJB2-negative Czech patients, which accounts for about 5% of all early non-syndromic hearing loss patients in the Czech Republic.

Thus we recommend performing SLC26A4 mutation analysis, following GJB2 analysis, in all HL patients with bilateral EVA and/or associated thyroid impairment, and also in patients with confirmed AR transmission of hearing loss even when they do not show any of the previously mentioned signs. On the other hand it is not reasonable to test the SLC26A4 gene in patients with sporadic prelingual deafness without knowledge of their temporal bone HRCT/MRI picture or even with its normal result.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References

Supported by IGA NS 9913/4.

We thank Dr. K. Huehne, O. Zwenger, V. Matejas, S. Uebe and Prof. B. Rautenstrauss from the Institute of Human Genetics in Erlangen for their kind support and cooperation.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of Interest
  9. References
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