Contribution of SLC26A4 to the molecular diagnosis of nonsyndromic prelingual sensorineural hearing loss in a Brazilian cohort
Carvalho et al. BMC Res Notes
Contribution of SLC26A4 to the molecular diagnosis of nonsyndromic prelingual sensorineural hearing loss in a Brazilian cohort
Simone da Costa e Silva Carvalho 0 3
Carlos Henrique Paiva Grangeiro 0 2 3
Clarissa Gondim Picanço‑Albuquerque 0 2 3
Thaís Oliveira dos Anjos 1
Greice Andreotti De Molfetta 0 1 3
Wilson Araujo Silva Jr 0 1 3 4
Victor Evangelista de Faria Ferraz 0 1 2 3
0 Department of Genetics, Ribeirão Preto Medical School, University of São Paulo , Ribeirão Preto, SP , Brazil
1 Center for Medical Genomics at University Hospital of the Ribeirão Preto Medical School, University of São Paulo , Ribeirão Preto, SP , Brazil
2 Medical Genetics Service of the Univer‐ sity Hospital of the Ribeirão Preto Medical School, University of São Paulo , Ribeirão Preto, SP , Brazil
3 Department of Genetics, Ribeirão Preto Medical School, University of São Paulo , Ribeirão Preto, SP , Brazil
4 Regional Blood Center of Ribeirão Preto (FUNDHERP) of the Ribeirão Preto Medical School, University of São Paulo , Ribeirão Preto, SP , Brazil
Objective: Hereditary hearing loss (HL) is the most common sensorineural disorder in humans. Besides mutations in GJB2 and GJB6 genes, pathogenic variants in the SLC26A4 gene have been reported as a cause of hereditary HL due to its role in the physiology of the inner ear. In this research we wanted to investigate the prevalence of mutations in SLC26A4 in Brazilian patients with nonsyndromic prelingual sensorineural HL. We applied the high‑ resolution melting technique to screen 88 DNA samples from unrelated deaf individuals that were previously screened for GJB2, GJB6 and MT‑ RNR1 mutations. Results: The frequency of mutations in the SLC26A4 gene was 28.4%. Two novel mutations were found: p.Ile254Val and p.Asn382Lys. The mutation c.‑ 66C>G (rs17154282) in the promoter region of SLC26A4, was the most frequent mutation found and was significantly associated with nonsyndromic prelingual sensorineural HL. After mutations in the GJB2, GJB6 and mitochondrial genes, SLC26A4 mutations are considered the next most common cause of hereditary HL in Brazilian as well as in other populations, which corroborates with our data. Furthermore, we suggest the inclusion of the SCL26A4 gene in the investigation of hereditary HL since there was an increase in the frequency of the mutations found, up to 22.7%.
Nonsyndromic hearing loss; SLC26A4; Mutation screening; DFNB4
Sensorineural hearing loss (SNHL) is the most common
sensorineural impairment in humans, and it is
associated with abnormalities of inner ear structures. In most
cases, SNHL occurs before speech development
(prelingual), and approximately 80% of these cases are affected
by mutations in genes related to the hearing process [
]. Many proteins have been associated with hereditary
SNHL by affecting the hair-cell structure, extracellular
matrix, ion homeostasis, and transcription factors [
]. Mutations in two proteins encoded by the GJB2 and
GJB6 genes (DFNB1 locus) expressed in cochlea cells,
constitute the primary cause of genetic deafness,
especially in Caucasian populations [
studies with the Brazilian population have shown that the
screening of the GJB2 and GJB6 genes explains the
etiology of hearing loss (HL) in only 1–24.7% of the subjects
analyzed, being the c.35delG (rs80338939) mutation in
GJB2 (NM_004004.5) gene, the most frequent
pathogenic variant [
]. Mutations in the SLC26A4 gene are
associated with both syndromic (Pendred Syndrome)
and nonsyndromic (DFNB4) cases of SNHL. This gene
encodes pendrin, a transmembrane ion transporter
which exchanges chloride for iodide and bicarbonate, in
the thyroid gland and inner ear. In the cochlea, pendrin
is expressed in the epithelial and supporting cells, and it
is related to the regulation of pH homeostasis and the ion
composition of the endolymph [
]. After mutations
in the GJB2 and GJB6 genes, mutations in SLC26A4 are
considered the major cause of hereditary hearing loss in
the Brazilian population and in many other populations
], contributing to up to 14% of cases of moderate,
profound or severe deafness [
]. Thus, this study aimed
to investigate and describe the prevalence of mutations in
the SLC26A4 gene in nonsyndromic prelingual
sensorineural hearing loss (SNHL) patients of a cohort from the
Southeast of Brazil who had been previously tested for
GJB2, GJB6, and MT-RNR1.
Patient samples and clinical data
This study was approved by the Ethics Research
Committee of the University Hospital of the Ribeirão Preto
Medical School-USP (n. 8736/2007). Genomic DNAs from
88 unrelated individuals diagnosed with nonsyndromic
prelingual SNHL were extracted from whole blood
samples, collected on EDTA, using Wizard® Genomic DNA
Purification Kit (Promega®, Wisconsin, EUA), according
to manufacturer’s instructions. To access clinical data,
we reviewed the medical archives, and some variables
were analyzed, such as type, degree, and progression of
hearing loss, familial history, and risk factors. The study
enrolled patients of both sexes at an age varying from
2–62 years old, who had been previously tested for GJB2
mutations by Sanger sequencing of the entire exon; for
del(GJB6-D13S1830) and del(GJB6-D13S1854) deletions
in GJB6 by multiplex PCR; as well as for m.1555A>G
(rs267606617) and m.961delT point mutations in the
mitochondrial gene MT-RNR1, by PCR and
Restriction Fragment Length Polymorphism (PCR-FRLP) using
HaeIII (Invitrogen®, Wisconsin, EUA) and MnlI (Fisher
Scientific, New Hampshire, EUA) restriction enzymes.
We used DNA samples from 96 individuals of the same
population as a control group. Individuals were all adults,
of both sexes and did not present a personal history of
Screening of mutations on SLC26A4 gene
The genomic DNA of the affected individuals was
examined for SLC26A4 mutations based on melting curve
analyses compared with wild-type samples using High
Resolution Melting (HRM) technique. A total of 26 HRM
primers were adapted from Chen et al. [
] to cover
all the 21 exons (including the non-coding Exon 1) of
SLC26A4 with a maximum amplicon size of 250pb. The
primers were optimized using In-Silico PCR (https://
genome.ucsc.edu/cgi-bin/) and checked by the Melting
Curve Prediction Software (https://www.dna.utah.edu/
umelt/umelt.html) to reach the best melting curve profile
PCR for HRM analysis was performed on the 7500
Real-Time PCR Systems (Applied Biosystems®,
California, EUA) using MeltDoctor™ HRM Master Mix
(fluorescent DNA intercalating dye) (Applied Biosystems®,
California, EUA). The reaction mixture in a final
volume of 20 μL was made using 10 μL of 1X
MeltDoctor™ HRM Master Mix, 2.4 μL of each primer (2.5 nM),
20 ng of genomic DNA and 1.2 μL of dH2O. The cycling
and melting conditions were as follows: 40 cycles at
95 °C for 15 s, the annealing temperature of each
amplicon for 1 min and one cycle at 95 °C for 10 s, 60 °C for
1 min, a melt at 95 °C for 15 s and 60 °C for 15 s. All
samples were tested in duplicate.
Samples with mutations identified by HRM
analysis were sequenced. Each sample with altered
melting curves was purified using the Wizard SV Gel and
PCR Clean-up System (Promega®, Wisconsin, EUA).
Next, the product of purification was sequenced using
Sanger sequencing on an automated 3500 XL Genetic
Analyzer (Applied Biosystems®, California, EUA),
using BigDye® Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems®, California, EUA) according to
the manufacturer´s instructions. Sequencing data were
analyzed with the Geneious R7 software v7.1 using the
NM_000441.1—GRCh37/hg19 sequence as reference.
In silico analysis of single nucleotide polymorphisms
was done using three online tools (Sift Score,
PolyPhen-2 and Combined Annotation Dependent
Depletion—CADD) to predict the pathogenicity of the
non-synonymous variants identified. Then, the
software FunSeq and RegulomeDB were used to evaluate
the effect of mutations on non-coding regions (exon
1). The Project HOPE Web Server tool was also used to
predict the protein structural effects.
For association analyses, we used X2 Test and Fisher
Exact Test, conducted using R Commander package
“/wt” means the presence of a wild type allele, i.e. when mutations were found
ND no mutation detected
version 2.4-x with R software environment. p-values
lower than 0.05 were considered as a measurement of
In relation to the degree of hearing impairment, most
individuals presented profound (55.7%) or severe (20.4%)
hearing loss (HL). About 87.5% presented
non-progressive HL, 28.4% reported a positive familial history of
deafness and about 13.6% reported parental
consanguinity. The frequency of mutations in the GJB2, GJB6, and
mitochondrial genes, was 18.2%. The mutation c.35delG
(rs80338939) in the GJB2 gene was the most frequent,
being present in 14 individuals (15.9%) both in
homozygous and heterozygous genotypes (Table 2). Three
patients presented the del(GJB6-D13S1854) mutation in
double heterozygosity with c.35delG in GJB2, and two
patients presented the del(GJB6-D13S1854) in
heterozygosity. No mutation was found in mitochondrial genes
(Table 2). However, recessive genotypes and pathogenic
mutations that can be associated with the phenotype
were found in only 10.2% of the cases.
The inclusion of the SLC26A4 gene in the molecular
screening showed a higher frequency of mutations. Ten
non-synonymous mutations were identified in 25
individuals (28.4%). Two mutations were located in the promoter
region (exon 1); seven were missense mutations, and two
out of seven have not been described yet; as well as this,
one synonymous mutation was found. About 22.7% of
the mutations were found in heterozygosity or
compound heterozygosity and 5.7% in double heterozygosity
with the GJB2 and GJB6 genes (Table 3). For three of
those mutations, it was possible to screen the frequency
in the control group of 96 healthy individuals. The most
frequent mutation found was the c.-66C>G (rs17154282),
located in the non-coding exon 1 of the SLC26A4 gene
(NM_000441.1) and it was found in 14.8% of the cohort.
This mutation was significantly associated with
nonsyndromic prelingual SNHL patients (OR = 0.33, 95% CI
0.09–1.05, p = 0.03684) when compared to the control
group. The novel mutations p.Ile254Val and p.Asn382Lys
were found, and this study constitutes the first report on
both mutations in prelingual SNHL patients. Both
mutations were shown to be pathogenic by the in silico tools
PolyPhen-2 and CADD, but we found no significant
association of those mutations with SNHL (p = 0.3052)
when we compared to the mutation frequency in the
control group. Three missense mutations also presented
pathogenic scores with the in silico prediction tools
(rs111033304; rs36039758 and rs55638457), but they had
been previously reported as Benign/Likely benign by the
ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/).
No correlations were found between the clinical features
and the genotypes.
The SLC26A4 (OMIM 605646) gene encodes pendrin, a
protein associated with regulation of the ion composition
and pH of endolymph in cochlear cells [
Mutations in SLC26A4 gene may promote pendrin
downregulation or loss of function, and constitute the second most
frequent cause of autosomal recessive NSHL . In this
study, the frequency of mutations in GJB2, GJB6, and
mitochondrial genes was 18.2%, and the inclusion of the
SLC26A4 in the investigation increased the frequency of
mutations to up to 40.9%.
Hereditary NSHL is a disease characterized by an
autosomal recessive inheritance pattern [
], however, about
18.1% of SLC26A4 mutations found in our study were
monoallelic. These findings corroborate with many
studies with NSHL patients [
] that report up to 61% of
cases presenting heterozygous mutations in the SLC26A4
gene and suggest that these mutations might
contribute to phenotype due to the presence of other
mutations not detected (in genes or in regulatory regions not
investigated), which together affect pendrin expression in
the inner ear. The mutation c.-66C>G (rs17154282) was
the most frequent mutation found in our study. It was
present in 14.8% (13/88) of the individuals, mainly in a
heterozygous genotype. This mutation was also reported
by Choi et al. [
] in patients with NSHL and EVA. In the
Tunisian population [
], a high frequency of c.-66C>G
was also found in patients with autoimmune thyroid
diseases, but this was considered as a non-pathogenic
polymorphism. The c.-66C>G presents a high allele frequency
specially in the African population [
], which may
explain the high frequency of that mutation in both the
Tunisian and Brazilian populations, once both of whom
present a strong African ancestry. In our study, the in
silico analyses showed low pathogenicity scores (Table 3)
but also suggested that the mutation affects a chromatin
regulatory region that is also important for the ligation
of enhancers and transcriptional factors (ELF1, RAD21,
REST, ZEB1, CTCF). The screening of c.-66C>G in the
control group of the same population revealed a
significant association of that mutation with hereditary NSHL
(OR = 0.33, 95% CI 0.09–1.05, p = 0.03684).
Nevertheless, despite the lack of functional evidences, this
mutation is reported as Benign/Likely benign on the ClinVar
database, probably due to its high allele frequency. Even
though there is conflicting data of pathogenicity, our
data suggests that c.-66C>G may be related to hereditary
NSHL. However, functional assays are necessary to
validate the contribution of this mutation to the phenotype.
One case showed c.-66C>G in trans with c.-103T>C
(rs60284988), which presents a nucleotide change in a
conserved region. It was functionally demonstrated [
that c.-103T>C integrates a critical binding site for
transcriptional regulatory elements (as described by the
prediction tools cited above), since c.-103T>C affects the
binding of FOXI1, and it completely abolishes the FOXI1
activation of SLC26A4 transcription. Given this case, we
suggest that the SLC26A4 5′UTR mutations contribute to
the phenotype due to the recessive genotype.
Two novel mutations were found in this cohort, but
only p.Asn382Lys presented pathogenic scores for all the
three in silico prediction tools analyzed (Table 3). None
of those mutations were found in the control group, and
no significant association with the phenotype was found
(p = 0.3052), probably due to the low number of
individuals analyzed. The mutations p.Asn324Tyr (rs36039758)
and p.Ile300Leu (rs111033304) also presented pathogenic
scores but ClinVar also characterizes them as benign.
In regard to p.Ile300Leu, this work constitutes the first
report in hereditary NSHL [
]. However, segregation
analyses were done with a first degree NSHL relative for
two of the three probands affected by p.Ile300Leu, and we
suggest that this mutation is not related to the phenotype
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since no segregation of p.Ile300Leu within the NSHL was
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19, 20, 28
The NSHL is a multigenic complex disease involving
many genetic and/or environmental factors [
]. It is
known that after mutations in the GJB2 and GJB6 genes,
SLC26A4 is considered the next most common cause of
hereditary HL in the Brazilian population and in many
other populations [
]. Our data showed that the
inclusion of a single gene in the investigation increased
the frequency of mutations from 18.2 to 40.9%,
reinforcing the importance of that gene to hereditary hearing loss
Many cases remained with an inconclusive molecular
diagnosis, which demonstrates the need for more studies
in order to characterize those mutations of unknown
significance as well as other non-coding regions and novel
genes associated with hereditary hearing loss.
CI: confidence interval; DFNB4: neurosensory nonsyndromic recessive deaf‑
ness 4; EVA: enlarged vestibular aqueduct; HL: hearing loss; NSHL: nonsyn‑
dromic hearing loss; OR: odds ratio; PCR: polymerase chain reaction; SNHL:
sensorineural hearing loss; SNP: single nucleotide polymorphism.
SCSC, CHPG, CGPA, TOA, and GAM performed the clinical characterization, PCR
standardization, HRM, Sanger sequencing and analyzed the data. VEFF and
WASJ provided DNA samples and/or clinical/genetic data. SCS, GAM, VEFF, and
WASJ wrote the manuscript. SCSC had full access to all the data in the study
and took responsibility for the integrity of the data and the accuracy of the
data analysis. All authors read and approved the final manuscript.
This work was possible due to the contribution of Prof. Dr. Wilson Araujo Silva
Jr and the Regional Blood Center of Ribeirão Preto (FUNDHERP). We are also
very thankful to all family members who participated in this study.
The authors declare that they have no competing interests.
Availability of data and materials
The publicly available datasets analyzed during the current study are available
in the 1000 genomes (http://www.internationalgenome.org/) repository. The
authors declare that all relevant data are included in the article and that it is
also available from the corresponding author by request.
Consent for publication
Ethical approval and consent to participate
A written informed consent was obtained from all patients, and the ethical
approval was granted by the Ethics Committee of the University Hospital of
the Ribeirão Preto Medical School‑USP (8736/2007).
This study was supported by the Molecular Genetics and Bioinformatics Labo‑
ratory, the Foundation for Education, Research and Assistance Support (FAEPA)
of the University Hospital of the Ribeirão Preto Medical School—USP, and
the National Council for Scientific and Technological Development (CNPq),
which supported the Master’s scholarship of the first author (process number
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.
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