Identification of SLC26A4 Mutations in Patients with Hearing Loss and Enlarged Vestibular Aqueduct Using High-Resolution Melting Curve Analysis

Abstract

Mutations in the SLC26A4 gene can cause both Pendred syndrome and nonsyndromic enlargement of the vestibular 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 pathogenesis 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 additional genetic or environmental factors contribute to this type of sensorineural hearing loss. Two recent studies have suggested that mutations in the SLC26A4 interacting transcription factor FOXI1 and the inward rectifying potassium channel encoding gene KCNJ10 can contribute to the development 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 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 × SensiMix™ HRM and 1 × EvaGreen™ dye (Quantace; Bioline), 0.4 μL of each primer (10 μM), and 2 μL DNA template, with the final volume made up to 12 μL 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 subjected to HRM analysis, using rising temperature increments of 0.05°C per second between 65°C and 95°C. Relative fluorescence 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 Sequencing Kit (Applied Biosystems) according to the manufacturer'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.

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 + 1G>A (IVS 8 + 1 G>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 + 1G>A (IVS 8 + 1 G>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.

The two novel mutations.

All known mutations in the seven individuals were successfully 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 mutations were identified in 9 probands (Table 1), with the remaining 29 EVA patients having no SLC26A4 mutations.

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.

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 inheritance (Yang et al., 2007, 2009). We screened our cohort of 51 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 analyzed, 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 homozygous 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 transmembrane protein (Everett et al., 1997). Sequence alignment shows that the Thr⁴⁸⁵ 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 nonsyndromic EVA probands with monoallelic SLC26A4 mutations (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 shows that the Phe⁷¹⁸ 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 autosomal 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.

Footnotes

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.

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