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Identification of SLC26A4 Mutations in Patients
with Hearing Loss and Enlarged Vestibular Aqueduct
Using High-Resolution Melting Curve Analysis
Stephen Mercer,
1
Patricia Mutton,
2
and Hans-Henrik M. Dahl
1,3
Mutations in the SLC26A4 gene can cause both Pendred syndrome and nonsyndromic enlargement of the ves-
tibular aqueduct, two conditions associated with sensorineural hearing loss. We analyzed the SLC26A4 gene in 44
hearing-impaired patients by nested polymerase chain reaction followed by high-resolution melt analysis. We also
used this approach to scan for mutations in KCNJ10 and FOXI1, two genes reported to play a role in the patho-
genesis of Pendred syndrome and enlarged vestibular aqueduct. Seven patients with known SLC26A4 mutations
were included as controls. All previously identified mutations were detected by high-resolution melt analysis. Of
the patients with no known mutations, we detected two SLC26A4 mutations in 5 probands (12%), one mutation in
9 probands (21%), and no mutations in 29 probands (67%). We identified two novel SLC26A4 mutations, p.T485M
and p.F718S, and found no evidence of a digenic contribution of KCNJ10 and FOXI1 mutations.
Introduction
Mutations in SLC26A4 are the second most common
cause of prelingual inherited hearing loss, accounting
for up to 10% of all hereditary deafness cases (Marazita et al.,
1993; Everett et al., 1997). Mutations in SLC26A4 can cause
Pendred syndrome (MIM 274600), an autosomal recessive
disorder associated with enlarged vestibular aqueduct (EVA,
MIM 600709), sensorineural hearing loss, goiter, and/or
Mondini dysplasia, as well as nonsyndromic EVA (MIM
600791), in which mild to severe hearing loss is inherited
without evidence of thyroid dysfunction (Azaiez et al., 2007).
A single mono-allelic SLC26A4 mutation is often detected in
patients with nonsyndromic EVA, whereas biallelic SLC26A4
mutations are common in Pendred syndrome (Pryor et al.,
2005b; Azaiez et al., 2007; Pera et al., 2008; King et al., 2010). No
SLC26A4 mutations can be detected in approximately one
third of EVA patients (Coyle et al., 1998; Campbell et al., 2001;
Pryor et al., 2005b; Albert et al., 2006), suggesting that addi-
tional genetic or environmental factors contribute to this type
of sensorineural hearing loss. Two recent studies have sug-
gested that mutations in the SLC26A4 interacting transcrip-
tion factor FOXI1 and the inward rectifying potassium
channel encoding gene KCNJ10 can contribute to the devel-
opment of EVA (Azaiez et al., 2007; Yang et al., 2007, 2009).
The coding region of SLC26A4 contains 21 exons (Everett
et al., 1997). High-resolution melting curve (HRM) analysis is a
highly sensitive, inexpensive alternative to other methods of
DNA sequence variant detection (Montgomery et al., 2007;
Reed et al., 2007; Millat et al., 2009). In this study, we have used
nested polymerase chain reaction (PCR) followed by HRM
analysis to screen a cohort of 44 EVA patients from 43 families
for DNA sequence changes in the coding regions and intron/
exon boundaries of the SLC26A4,KCNJ10, and FOXI1 genes.
Materials and Methods
Patient DNA samples
A total of 51 hearing-impaired individuals from 46 families
were recruited. Seven individuals from three families had
previously identified SLC26A4 mutations and were included
as controls for HRM analysis. The remaining 44 patients from
43 families had clinically confirmed EVA, based on computed
tomography scans done as part of the clinical investigations
on hearing loss. The vestibular aqueduct was considered
‘‘large’’ when it measured greater than 1.5 mm in width at the
midpoint between the common crus and the external aperture
(Swartz and Loevner, 2009). DNA was extracted from blood,
buccal swabs, or Guthrie cards.
Primary and nested PCR amplification
of SLC26A4, KCNJ10, and FOXI1 gene exons
All exons and exon/intron boundaries in the SLC26A4
(Ref. Seq. NG_008489.1), KCNJ10 (Ref. Seq. NM_002241),
and FOXI1 (Ref. Seq. NG_012068.1) genes were amplified in
1
Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Australia.
2
Deafness Centre Unit, The Children’s Hospital, Westmead, Australia.
3
Department of Paediatrics, University of Melbourne, Melbourne, Australia.
GENETIC TESTING AND MOLECULAR BIOMARKERS
Volume 15, Number 5, 2011
ªMary Ann Liebert, Inc.
Pp. 365–368
DOI: 10.1089/gtmb.2010.0177
365
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nested PCR reactions following standard PCR procedures to
produce *150–350-bp amplicons.
Primary PCR amplification products of SLC26A4,KCNJ10,
and FOXI1 exons were diluted 1/10,000 and used in the
nested pre-HRM amplification.
Primers for nested PCR were designed *20–30 bp inside of
the first primer. The reaction mixtures consisted of 1
SensiMixHRM and 1EvaGreendye (Quantace; Bioline),
0.4 mL of each primer (10 mM), and 2 mL DNA template, with
the final volume made up to 12 mL with PCR-grade water. All
HRM reactions were performed in duplicate in a 100-well
gene disk (Corbett Life Science). PCR cycling and subsequent
HRM analysis was performed using the Rotor-GeneTM 6000
(Corbett Life Science). Primers and reaction conditions are
available on request.
HRM analysis
Following nested PCR amplification, dsDNA was sub-
jected to HRM analysis, using rising temperature increments
of 0.058C per second between 658C and 958C. Relative fluo-
rescence was recorded at each temperature increase. All
HRM-associated analyses were conducted using Rotor-Gene
1.7.34 software.
Post-HRM analysis DNA sequencing
PCR fragments identified as variants based on analysis of
their HRM normalization and derivative graphs were
sequenced using the BigDye
Terminator v3.1 Cycle Se-
quencing Kit (Applied Biosystems) according to the manu-
facturer’s protocol. The data were examined using the
Mutation Surveyor
Software (SoftGenetics).
Results
HRM and DNA sequence analyses
of the SLC26A gene
Forty-four patients with EVA from 43 families were
screened for mutations in the SLC26A4,KCNJ10, and FOXI1
genes. In addition, we included seven patients from three
families with previously identified SLC26A4 mutations
(Table 1) as positive controls for HRM analysis.
All known mutations in the seven individuals were suc-
cessfully detected in the HRM analysis as variant melt profiles
and again verified by post-HRM sequencing (data not shown).
Monoallelic and biallelic SLC26A4 mutations
Monoallelic or biallelic SLC26A4 mutations were identified
in 14 of the 43 probands with no previously known mutations.
Two SLC26A4 mutations were found in 5 probands from the
43 families (Table 1, Fig. 1). Single monoallelic SLC26A4 mu-
tations were identified in 9 probands (Table 1), with the re-
maining 29 EVA patients having no SLC26A4 mutations.
A total of 16 different SLC26A4 mutations were identified
by HRM analysis in this study (Table 1). Two of the SLC26A4
changes, p.T485M and p.F718S, have not been previously
reported.
HRM and DNA sequence analyses
of KCNJ10 and FOXI1
It was recently shown that a subgroup of EVA patients with
a monoallelic SLC26A4 mutation carry changes in the KCNJ10
or FOXI1 gene, suggesting the existence of digenic inheri-
tance (Yang et al., 2007, 2009). We screened our cohort of 51
Table 1. Genotypes of 19 Nonsyndromic Enlarged Vestibular Aqueduct Patients
with One or Two SLC26A4 Mutations
SLC26A4 genotype
Patient ID Family ID Allele 1 Allele 2
8 8 c.2153T>C (p.F718S)
a
14142 14142 c.1226G>A (p.R409H)
15626 15626 c.1001 þ1G>A (IVS 8 þ1G>A) c.1246A>C (p.T416P)
15941 15941 c.626G>T (p.G209V)
15942 15941 c.626G>T (p.G209V)
16498 16498 c.485C>T (p.T485M)
a
c.485C>T (p.T485M)
a
18936 18936 c.1790T>C (p.L597S)
20721 20721 c.1229C>T (p.T410M)
20767 20767 c.1181_1183delTCT (p.F394del)
28140 28140 c.919-2A>G (IVS8-2 A>G) c.2015G>A (p.G672E)
28759 28759 c.1790T>C (p.L597S)
28765 28765 c.626G>T (p.G209V)
28768 28768 c.916_917insG (p.V306GfsX24) c.1975G>C (p.V659L)
31587 31587 919-2A>G (IVS8-2 A>G) 919-2A>G (IVS8-2 A>G)
39397 39397 c.626G>T (p.G209V) c.1284_1286delTGC (A429del)
39398 39397 c.626G>T (p.G209V) c.1284_1286delTGC (A429del)
40677 39397 c.626G>T (p.G209V) c.1284_1286delTGC (A429del)
08V1964 08V1964 165-1G>A (IVS2-1 G>A)
08V3059 08V3059 c.1001 þ1G>A (IVS 8 þ1G>A) c.1246 A>C (p.T416P)
GHR-1 GHR-1 c.2048T>C (F683S)
Numbering is based on reference sequences NM000441.1 (cDNA) and NP_000432.1. Data of patients with known mutations from DNA
sequence analysis done previous to high-resolution melting curve analysis are indicated in boldface.
a
The two novel mutations.
366 MERCER ET AL.
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individuals for mutations in KCNJ10 and FOXI, but found no
causative mutations.
Discussion
A successful HRM analysis is dependent on a number
of factors, including purity of the PCR fragments to be ana-
lyzed, uniformity of the concentration of PCR fragments, as
well as equivocal salt and reagent conditions in all PCRs
(Montgomery et al., 2007; Reed et al., 2007). We employed a
nested PCR approach to generate PCR fragments that can be
used directly in the HRM analysis.
The five known SLC26A4 mutations in the seven control
individuals were all detected, suggesting that the nested PCR
HRM approach is proficient and detects most, if not all,
SLC26A4 sequence variants, including homozygous mutations.
We detected biallelic SLC26A4 mutations in 5 probands
from 43 families with no previously known mutations (Table
1, Fig. 1). A patient from a consanguineous marriage is ho-
mozygous for the novel p.T485M mutation. The p.T485M
missense mutation is located in the 11th transmembrane
section and Pfam protein domain of the pendrin transmem-
brane protein (Everett et al., 1997). Sequence alignment shows
that the Thr
485
residue is highly conserved in the orthologs
and paralogs of SLC26A4 across all species, supporting the
functional significance of this amino acid. We have also
screened DNA from 96 people with normal hearing and 370
people with hearing loss for mutations in SLC26A4 using
HRM. The p.T485M and p.F718S mutations were not present
in any of these 466 samples.
Seven different mutations were found in the nine non-
syndromic EVA probands with monoallelic SLC26A4 muta-
tions (Table 1). The p.F718S missense mutation is a novel
SLC26A4 mutation. It is located in the STAS domain of
the C-terminal extracellular domain (Everett et al., 1997).
Sequence alignment of the SLC26A4 polypeptide sequence
FIG. 1. Duplicate aberrant
melt curves detected in the
high-resolution melting curve
screen of exon 10 (A) and exon
17 (B) of the SLC26A4 gene.
Mutations were identified from
subsequent sequencing.
IDENTIFICATION OF SLC26A4 MUTATIONS 367
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shows that the Phe
718
residue is highly conserved, suggesting
that it plays an important role in pendrin function.
Previous studies have failed to identify SLC26A4 mutations
in one or both alleles in many patients. This has led to a
speculation that additional environmental or genetic factors
can be involved in these conditions (Pryor et al., 2005b). Two
such genetic factors have been suggested: FOXI1 and KCNJ10.
We found no indication that FOXI1 and KCNJ10 mutations
were involved in the hearing loss in any of our nine families
with a single SLC26A4 mutation and our data, therefore,
concur with results from other studies (Pryor et al., 2005a;
Adler et al., 2008; Pera et al., 2008). Although genetic studies
suggest that occult mutations in SLC26A4—or another auto-
somal gene—are likely to contribute to EVA in patients with
only one detectable mutation, the nature and location of such
mutations are not known (Choi et al., 2009).
Although we are aware that mutations might be missed by
this approach, we conclude that nested PCR followed by
HRM is an efficient and cost-effective approach to detect
known and unknown SLC26A4 coding sequence variants.
Acknowledgments
This study was supported by J.&J. Calvert Jones.
H.-H.M.D. is a NHMRC Principal Research Fellow (NHMRC
ID: 334313). The authors thank Dr. K. Prelog for diagnosing
EVA in the patients. Thanks to Drs. K. Miller, L. Williams, and
S. Manji for helpful discussions and to Mr. Thomas Dantoft for
assistance with the CAS-1200 workstation and HRM analysis.
Thanks to MCRI diagnostic service for preparing some of the
patient DNAs used in this study.
Disclosure Statement
No competing financial interests exist.
References
Adler L, Efrati E, Zelikovic I (2008) Molecular mechanisms of
epithelial cell-specific expression and regulation of the human
anion exchanger (pendrin) gene. Am J Physiol 294:C1261–
C1276.
Albert S, Blons H, Jonard L, et al. (2006) SLC26A4 gene is fre-
quently involved in nonsyndromic hearing impairment with
enlarged vestibular aqueduct in Caucasian populations. Eur J
Hum Genet 14:773–779.
Azaiez H, Yang T, Prasad S, et al. (2007) Genotype-phenotype
correlations for SLC26A4-related deafness. Hum Genet
122:451–457.
Campbell C, Cucci RA, Prasad S, et al. (2001) Pendred syndrome,
DFNB4, and PDS/SLC26A4 identification of eight novel mu-
tations and possible genotype-phenotype correlations. Hum
Mut 17:403–411.
Choi BY, Madeo AC, King KA, et al. (2009) Segregation of en-
larged vestibular aqueducts in families with non-diagnostic
SLC26A4 genotypes. J Med Genet 46:856–861.
Coyle B, Reardon W, Herbrick JA, et al. (1998) Molecular analysis
of the PDS gene in Pendred syndrome. Hum Mol Genet
7:1105–1112.
Everett LA, Glaser B, Beck JC, et al. (1997) Pendred syndrome is
caused by mutations in a putative sulphate transporter gene
(PDS). Nat Genet 17:411–422.
King KA, Choi BY, Zalewski C, et al. (2010) SLC26A4 genotype,
but not cochlear radiologic structure, is correlated with hear-
ing loss in ears with an enlarged vestibular aqueduct. Lar-
yngoscope 120:384–389.
Marazita ML, Ploughman LM, Rawlings B, et al. (1993) Genetic
epidemiological studies of early-onset deafness in the U.S.
school-age population. Am J Med Genet 46:486–491.
Millat G, Chanavat V, Rodriguez-Lafrasse C, et al. (2009) Rapid,
sensitive and inexpensive detection of SCN5A genetic variations
by high resolution melting analysis. Clin Biochem 42:491–499.
Montgomery J, Wittwer CT, Palais R, et al. (2007) Simultaneous
mutation scanning and genotyping by high-resolution DNA
melting analysis. Nat Protoc 2:59–66.
Pera A, Villamar M, Vinuela A, et al. (2008) A mutational analysis
of the SLC26A4 gene in Spanish hearing-impaired families
provides new insights into the genetic causes of Pendred syn-
drome and DFNB4 hearing loss. Eur J Hum Genet 16:888–896.
Pryor SP, Demmler GJ, Madeo AC, et al. (2005a) Investigation of
the role of congenital cytomegalovirus infection in the etiology
of enlarged vestibular aqueducts. Arch Otolaryngol Head
Neck Surg 131:388–392.
Pryor SP, Madeo AC, Reynolds JC, et al. (2005b) SLC26A4/PDS
genotype-phenotype correlation in hearing loss with enlarge-
ment of the vestibular aqueduct (EVA): evidence that Pendred
syndrome and non-syndromic EVA are distinct clinical and
genetic entities. J Med Genet 42:159–165.
Reed GH, Kent JO, Wittwer CT (2007) High-resolution DNA
melting analysis for simple and efficient molecular diagnos-
tics. Pharmacogenomics 8:597–608.
Swartz JD, Loevner LA (2009) Imaging of the Temporal Bone.
Thieme, New York.
Yang T, Gurrola JG, 2nd, Wu H, et al. (2009) Mutations of
KCNJ10 together with mutations of SLC26A4 cause digenic
nonsyndromic hearing loss associated with enlarged vestibu-
lar aqueduct syndrome. Am J Hum Genet 84:651–657.
Yang T, Vidarsson H, Rodrigo-Blomqvist S, et al. (2007) Tran-
scriptional control of SLC26A4 is involved in Pendred syn-
drome and nonsyndromic enlargement of vestibular aqueduct
(DFNB4). Am J Hum Genet 80:1055–1063.
Address correspondence to:
Hans-Henrik M. Dahl, Ph.D.
Murdoch Childrens Research Institute
The Royal Children’s Hospital
Flemington Road
Parkville
Melbourne 3052
Australia
E-mail: henrik.dahl@mcri.edu.au
368 MERCER ET AL.
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