Background
Pendred syndrome (PDS) (OMIM 274600) is one of the most common autosomal recessive disorders of hearing [
1,
2], characterised by mild to severe sensorineural hearing impairment (SNHL) [
2‐
5] goitre, and inner ear malformations, typically an enlargement of the vestibular aqueduct (EVA) [
6]. Some PDS cases can also exhibit Mondini dysplasia which involves the bilateral enlargement of the vestibular aqueducts (EVA) with cochlear hypoplasia [
7]. EVA can also be associated with other forms of syndromic and non-syndromic deafness (DFBN4 (OMIM 600791)) [
8,
9], particularly non-syndromic enlarged vestibular aqueducts.
Pendred syndrome is the result of malfunction in pendrin, an apical protein anion transporter which mediates chloride (Cl
-), hydroxide (OH
-), bicarbonate (HCO3
-) and iodide (I
-) exchange [
10,
11]. Pendrin is expressed in the thyroid and importantly, in the inner ear to maintain the endocochlear potential [
5] as well as in the kidney. Dysfunction of ion transport affects iodide organification for thyroid hormone biosynthesis [
4] resulting in goitre [
1,
3,
12], which distinguishes it from non-syndromic EVA. However, the majority of patients remain euthyroid [
13]. It is now widely acknowledged that mutations in the gene
SLC26A4 located on chromosome 7q21-34 [
14,
15] are involved in the presentation of an EVA/PDS phenotype [
14,
16,
17]. Biallelic mutations in the gene
SLC26A4 have been shown to cause Pendred syndrome, accounting for its autosomal recessive inheritance [
9,
14,
18]. However, there is evidence to show that a cohort of patients affected by hearing loss and EVA, have only one mutant allele of
SCL26A4; Yang
et al. showed that in patients with hearing loss and EVA, in whom thyroid disorder was not used as a clinical criterion, 19% of siblings had a single mutation in
SLC26A4 and 42% had zero mutations, suggesting that other genetic factors may be involved [
19] in the aetiology of their hearing loss. Rendtorff
et al., in a clinically well-phenotyped cohort of 109 Scandinavian patients, showed that 10% had monoallelic mutations. Likely heterogeneity of PDS and non-syndromic EVA has been proposed and investigated, with some suggesting a complex relationship between heterozygosity and the severity of the EVA/PDS phenotype [
20].
Two genes,
KCNJ10 and
FOXI1, have been investigated for their role in the PDS disease spectrum and it has been proposed that digenic mutations in both
SLC26A4 and either
FOXI1 or
KCNJ10 may cause Pendred syndrome [
19,
21]. The gene
KCNJ10 encodes an inwardly rectifying potassium (K
+) channel, namely the Kir4.1 channel, expressed in a wide variety of tissues but most importantly in the case of Pendred syndrome, in the cochlear stria vascularis which maintains the endocochlear potential and K + homeostasis [
22‐
24]. It has been shown that mice models that lacked the
Slc26a4 gene (
Slc26a4
−/−) did not express
Kcnj10 and that lack of this protein led to the loss of the endocochlear potential via endolymphatic acidification and Ca
2+ absorption inhibition [
25,
26] which may be the direct cause of deafness in Pendred syndrome. Variants of
KCNJ10 have also been considered as a risk factor for seizure susceptibility in genetic association studies [
27] and biallelic mutations in
KCNJ10 in humans are known to cause a syndromic form of hearing loss, EAST syndrome (epilepsy, ataxia, sensorineural deafness and renal tubulopathy) [
28].
FOXI1 is a transcription factor gene and an upstream regulator of
SCL26A4[
19,
29]. It is also involved in the regulation of vascular H
+-ATPase proton pumps in the inner ear, epididymis and kidney [
30]. It was proposed that digenic inheritance of mutations in both
FOXI1 and
SLC26A4 were involved in the genetic basis of Pendred syndrome, as mutations in
FOX1 were shown to reduce the transcription of
SLC26A4[
19]. Moreover,
Foxi1 null mice showed phenotypic features of sensorineural deafness due to the defective pendrin-chloride mediated reabsorption in which FOXI1 was most likely a key regulator [
29].
Only a few studies have found evidence that KCNJ10 or FOXI1 gene mutations may influence the disease phenotype shown in patients with monoallelic mutations in the SLC26A4 gene. The present study investigates whether variants in KCNJ10 and FOXI1 in combination with heterozygosity for SLC26A4 mutations are likely to be associated with the disease phenotype seen in Pendred syndrome/EVA patients (who already have one mutation in the SLC26A4 gene). Because of the suggested role of KCNJ10 in both epilepsy and PDS/EVA, we also screened 3 unrelated patients with the combination of Pendred syndrome and seizures.
Results
Screening of
FOXI1 and
KCNJ10 was completed for the cohort of 68 patients all of whom had previously been found to have only a single mutation in the
SLC26A4 gene. Results are shown in Table
2. In
KCNJ10, three unrelated patients were identified with the variant p.Arg271Cys (c.811C > T) and one with p.Arg18Gln (c.52G > A). In
FOXI1, only one patient was observed to have a variant, p.Arg123Trp (c.367C > T). This variant was not found in the 95 pan-ethnic controls. No other mutations in either
KCNJ10 or
FOXI1 were found.
Table 2
KCNJ10
and
FOXI1
mutations identified in patients with monoallelic mutations in
SLC26A4
25215 | c.1001 + 1G > A | | N | N |
25278 | c.1790T > C | p.Leu597Ser | N | N |
34515 | c.716T > A | p. Val239Asp | N | N |
28979 | c.1061T > C | p.Phe354Ser | N | N |
35265 | c.707T > C | p.Leu236Pro | N | N |
40257 | c.1001 + 1G > A | | N | N |
37952 | c.1151A > G | p.Glu384Gly | N | N |
38201 | c.1790T > C | p.Leu597Ser | N | N |
42836 | c.707T > C | p.Leu236Pro | N | N |
42564 | (c.1234G > A | p.Val412Ile) | N | N |
41066 | c.1001 + 1G > A | | N | N |
40187 | c.412G > T | p.Val138Phe | N | N |
44595 | c.2127delT | p.Phe709Leufs*12 | N | c.811C > T (p.Arg271Cys)/N |
45381 | c.1790T > C | p.Leu597Ser | N | N |
13343 | c.1342-2_1343dup | p.Leu450Glyfs*19 | N | N |
45592 | c.340G > A | p.Gly114Arg | N | N |
48799 | c.1001 + 1G > A | | N | N |
46182 | c.707T > C | p.Leu236Pro | N | N |
50939 | c.2190G > T | p.Gln730His | N | N |
51079 | c.707T > C | p.Leu236Pro | N | N |
54165 | c.2T > C | p.Met1? | N | N |
59858 | c.2080T > C | p.Ser694Pro | N | N |
61452 | c.412G > T | p.Val138Phe | N | N |
54483 | c.1790T > C | p.Leu597Ser | N | N |
63420 | c.1151A > G | p.Glu384Gly | N | N |
65983 | c.1211C > T | p.Thr404Ile | N | N |
50886 | c.1151A > G | p.Glu384Gly | N | N |
66609 | c.-3-2A > G | | N | N |
66643 | c.[1001 + 1G > A(;) 2219C > T] | (p.Gly740Val) | N | N |
66830 | c.1790T > C | p.Leu597Ser | N | N |
69863 | c.113T > C | p.Phe335Leu | N | N |
47141 | c.1790T > C | p.Leu597Ser | N | N |
72446 | c.-3-2A > G | | N | N |
72770 | c.412G > T | p.Val138Phe | N | N |
72617 | c.1151A > G | p.Glu384Gly | N | N |
76349 | c.1790T > C | p.Leu597Ser | N | N |
76715 | c.1826T > G | p.Val609Gly | N | N |
78124 | c.1468A > C | p.Ile490Leu | N | N |
78231 | c.1001 + 1G > A | | N | N |
23853 | c.2T > C | p.Met1? | N | N |
71753 | c.1342-2_1343dup | p.Leu450Glyfs*19 | N | N |
72950 | c.1229C > T | p.Thr410Met | N | N |
79945 | (c.2219C > T) | (p.Gly740Val) | c.367C > T(p.Arg123Trp) | N |
80435 | c.707T > C | p.Leu236Pro | N | N |
10576 | c.[1343C > T]; [1991C > T] | p.[Ser448Leu]; [Ala664Ser] | N | N |
83112 | c.1790T > C | p.Leu597Ser | N | N |
84175 | c.119delT (c.918G > A) | p.Leu40ArgfsX26 | N | N |
86297 | c.2153G > T | p.Phe718Ser | N | c.53G > A (p.Arg18Gln)/N |
86482 | c.707T > C | p.Leu236Pro | N | N |
85020 | c.1234G > T | p.Gln421Arg | N | N |
83883 | c.1790T > C | p. Leu597Ser | N | N |
87823 | c.1790T > C | p. Leu597Ser | N | N |
84236 | (c.73C > T) | (p.Pro25Ser) | N | N |
88933 | c.1151A > G | p.Glu384Gly | N | N |
89770 | c.1001 + 1G > A | | N | N |
90473 | c.1790T > C | p.Leu597Ser | N | c.811C > T (p.Arg271Cys) /N |
90511 | c.1003T > C | p.Phe335Leu | N | c.811C > T (p.Arg271Cys) /N |
90643 | (c.970A > T) | (p.Asn324Tyr) | N | N |
91820 | c.1790T > C | p.Leu597Ser | N | N |
40013 | c.1000G > T | p.Gly334Trp | N | N |
89620 | c.[1790T > C(;) 412G > T] | p.[Val138Phe(;) (Leu597Ser)] | N | N |
94065 | c.1342-2_1343dup | p.Leu450Glyfs*19 | N | N |
89792 | c.-103T > C | | N | N |
94743 | c.1003T > C | p.Phe335Leu | N | N |
96669 | c.2015G > A | p.Gly672Glu | N | N |
95020 | c.1363A > T | p.Ile455Phe | N | N |
99311 | c.626G > T | p.Gly209Val | N | N |
99458 | c.1334T > G | p.Leu445Trp | N | N |
Results of the analysis of the three further patients referred for
SLC26A4 screening who also had a history of seizures are shown in Table
3. One patient was found to have p.Arg271Cys, and one had the variant p.Arg18Gln.
Table 3
Patients with biallelic mutations of
SLC26A4
tested for variants within the
KCNJ10
gene
66119 | c.1229C > T p.Thr410Met | c.1229C > T p.Thr410Met | c.811C > T p.Arg271Cys | rs1130183 |
5472 | c.707T > C p.Leu236Pro | c.626G > T p.Gly209Val | c.53G > A p.Arg18Gln | rs115466046 |
6401 | c.707T > C p.Leu236Pro | c.707T > C p.Leu236Pro | N | |
Discussion
The evidence that genetic factors in addition to mutations in the coding region of the
SLC26A4 gene contribute to Pendred syndrome and non-syndromic EVA has originated from both mouse and human studies. In humans the most compelling fact is that bi-allelic
SLC26A4 mutations are found in only a minority of patients, even those with the most severe phenotype of EVA-Mondini and thyroid dysfunction or goitre. Undiscovered non-coding region mutations of
SLC26A4 are unlikely to fully explain this observation as a number of groups have performed extensive analysis of non-coding regions with little yield, although a variant in the promoter region of
SLC26A4 which binds the transcription factor, FOXI1 was noted. Furthermore, siblings with discordant phenotypes, a mono-allelic
SLC26A4 mutation and concordant
SLC26A4 haplotypes, as well as siblings with concordant phenotypes but discordant haplotypes, indicate evidence for locus heterogeneity [
9].
Studies based on mouse models
Studies in mice have suggested other genes that might influence the PDS/EVA phenotype and may act in a digenic, or additive manner. Mice lacking FOXI1 are deaf, have inner ear malformations (EVA and cochlear dysplasia) as well as renal tubular acidosis and male infertility. Furthermore these mice show no expression of SLC26A4 in the inner ear, implying that as an upstream regulator,
FoxI1 expression is necessary for the transcription of
Slc26a4. Therefore the contribution of
FOXI1 to EVA/PDS was investigated in humans by Yang
et al., who found a variant in the promoter of
SLC26A4 in a consensus binding motif for FOXI1, c.-101T > C; they showed using EMSAs that binding affinity of FOXI1 to the promoter was reduced in the presence of c.-101T > C and used Luciferase reporter constructs to show that transcription from the promoter was completely abolished in the presence of this variant [
19]. Further screening of the coding region of
FOXI1 itself revealed five further non-synonymous variants, p. Asp161del, p.Arg267Gln, p.Gly335Val, p.Gly258Arg, p.Gly258Glu among 372 probands with EVA in whom one or zero
SLC26A4 mutations had previously been found. All five mutations showed reduced activation of the luciferase reporter construct. Finally mice which are double heterozygotes for variants in
Slc26a4 and
Foxi1 (
Slc26a4
+/−,
Foxi1
+/−) were reported to have EVA although hearing of these mice was not commented on [
19].
The potential role of the inwardly rectifying potassium channel
KCNJ10 in PDS/EVA was also suggested from studies in mice. The
Slc26a4
−/− mouse is deaf and has EVA and shows loss of endocochlear potential and no expression of KCNJ10 protein in the stria vascularis. KCNJ10 is thought to be crucial in the generation and maintenance of the endocochlear potential necessary for auditory transduction. In 2 out of 89 EVA/PDS patients who were heterozygous for a single pathogenic
SLC26A4 mutation, Yang
et al. found two heterozygous mutations in
KCNJ10 in unrelated probands, and suggested a probable digenic interaction between these two genes. In their hands, expression of mutant KCNJ10 in Xenopus oocytes showed markedly reduced K
+ conductance [
21].
Studies in humans
However subsequent work by others has shown that recessive mutations in
KCNJ10 do indeed cause hearing loss, but as part of a complex syndrome, EAST syndrome, consisting of Epilepsy, Ataxia, Sensorineural deafness and renal Tubulopathy. Heterozygote parents are unaffected. In addition
KCNJ10 has also been considered as a ‘susceptibility gene’ for epilepsy in humans. However the variant p.Arg271Cys was thought to be a
protective allele against focal or generalised epilepsy [
22,
31,
32] and yet this was found in one of our patients with co-existing seizures and Pendred syndrome. As studies failed to show a functional or structural effect on channels, and because this variant is present in 563 of 8037 individuals (0.07) of European American descent in Exome Variant Server (
http://evs.gs.washington.edu/EVS/), p.Arg271Cys is most likely to be a polymorphism, unrelated to either EVA/PDS or even epilepsy. Similarly, the variant p.Arg18Gln has also been reported previously, but in a single patient with autistic spectrum disorder, seizures and intellectual disability but no deafness [
33]. This suggests that it may be unrelated to any of these phenotypes, consistent with its frequency in the population (present in 1 in 57 European Americans in EVS (148 in 8452 European Americans (0.0175)) (
http://evs.gs.washington.edu/EVS/). As the variant in
FOXI1, p.Arg123Trp (c.367C > T) has not been found at a high frequency previously or in controls screened here, we cannot be sure whether this is, or is not, a pathogenic mutation. However it appears that mutations in
FOXI1 are not a major contributor to digenic inheritance in these cases.
Other genetic studies of patients with EVA/PDS have failed to find convincing evidence that mutations of
FOXI1 and
KCNJ10 contribute to these phenotypes. Chen
et al.[
34] screened
SLC26A4,
FOXI1 and
KCNJ10 (as well as
GJB2) in patients with bilateral deafness and inner ear malformations and found no mutations in
FOXI1 or
KCNJ10 in the 15 who had one or zero
SLC26A4 mutations; Mercer
et al.[
35] screened 51 patients with EVA and found no mutations in
FOXI1 or
KCNJ10; Jonard
et al.[
36] screened 25 patients with unilateral deafness and unilateral EVA but found no mutations in either
KCNJ10 or
FOXI1 and Wu
et al. screened
FOXI1 in 100 patients with EVA and found no mutations, although
KCNJ10 was not screened at that time [
37]. Cirello
et al. did however find a novel missense variant in
FOXI1 p.Pro239Leu, and 3 patients with the variant p.Arg271Cys in
KCNJ10 out of 19 patients with PDS/EVA, but functional analysis of p.Pro239Leu showed no significant impairment in the transcriptional activation of
SLC26A4, and, p.Arg271Cys was classified as polymorphism [
38].
Our study is the largest study to screen both KCNJ10 and FOXI1 in patients who were heterozygous for a single SLC26A4 mutation in whom the promoter variant/FOXI1 (c.-101T > C) binding site had already been excluded. Our results indicate that heterozygosity for mutations in either of these genes in combination with a heterozygous SLC26A4 mutation is not a significant contributor to PDS/EVA in our cohort, even though previous in vitro work and mouse studies suggest their involvement in these phenotypes. The best evidence for a possible functional role of either KCNJ10 or FOXI1 in the PDS/EVA phenotype in humans would be genetic evidence - further families with mutations, which are shown to have a functional effect and which are present in a lower frequency in the general population than those with the PDS/EVA phenotype. Hopefully, NGS data from large sequencing efforts such as 1000 Genomes and EVS will be helpful in this respect.
It is possible that some of the cases presented here could be ‘co-incidental’ carriers for mutations in
SLC26A4 which do not contribute to their phenotype of hearing loss, or that we could have missed single or multiple exon deletions. However we think that this is unlikely to account for a significant proportion of these cases. Renorff
et al. found no deletions in their series of patients suggesting that deletions account for a very small number of mutations in
SLC26A4[
39]. Regarding carrier ship, we have recently performed an audit of all requests for
SLC26A4 screening performed in our laboratory from 2002 onwards. This shows that where testing of
SLC26A4 was requested, 66 out of a total of 538 patients tested, had monoallelic mutations (ie. 1 in 8). Even if those with variants of unknown significance are excluded, the heterozygote carriership rate for
SLC26A4 mutations would be almost 4 times that for
GJB2, the commonest form of inherited deafness [
40]. This suggests that heterozygosity for
SLC26A4 mutations is important in the aetiology of the hearing loss in these patients.
Competing interests
The authors declare no conflict of interest.
Authors’ contributions
MBG and AMD designed the study and chose the patient cohort. PL performed the laboratory work under the supervision of AMD. MBG, AMD and PL all participated in the analysis of the results. PL wrote the initial manuscript which was edited and revised by MBG and AMD. KR and LJ provided critical feedback and thoughts on the manuscript. All authors read and approved the final manuscript.