Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting

Abstract

Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss and mutations in the SLC26A4 gene. Extensive allelic heterogeneity, however, necessitates analysis of all exons and splice sites to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously identified by sequencing. Subsequently, a 384-well plate format was designed for up to three patient samples per run. Blinded HRM testing on these plates of patient samples collected over 1 year in a clinical diagnostic laboratory accurately detected all variants identified by sequencing. In conclusion, HRM with targeted sequencing is a reliable, simple, and cost-effective method for SLC26A4 mutation screening and detection. Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss. Whereas Pendred syndrome is thought to be the most common cause of syndromic hearing loss, 1,2 DFNB4 is a nonsyndromic form of hearing loss caused by mutations in the same gene. 3 Clinically, both can be associated with temporal bone abnormalities. 4 In addition, an abnormal perchlorate discharge test result and/or euthyroid goiter can be present in patients with Pendred syndrome but not in those with DFNB4. 3,4 This may be due to allelic heterogeneity because differences in residual activity of the protein products render different phenotypes. 5

SLC26A4 mutation frequency and spectrum in 109 Danish Pendred syndrome/DFNB4 probands and a report of nine novel mutations

“…However, autoimmune thyroid disease could also be a possibility in some of these patients. At present, Sanger sequencing is the standard for SLC26A4 molecular diagnosis, but other efficient methods such as high-resolution melting are available (4). Development of next-generation sequencing for molecular diagnosis is expected to reduce the utility of methods such as APEX, which here (besides screening for common SLC26A4 mutations) was used to establish whether the HI, alternatively, could be explained by other genes.…”

Genetic Alterations in Pendrin (SLC26A4) Gene in Adult Hypothyroid Patients

“…Present study was undertaken for the first time to screen the genetical variants in SLC26A4 gene, if any, in association with nonautoimmune and noncongenital hypothyroidism, which was devoid of any characteristics of Pendred syndrome. The alterations in SL-C26A4 gene are mostly associated with Pendred syndrome or congenital hypothyroidism [24][25][26][27][28][29]32]. However, none of the patients enrolled in this study were registered with any possible sign of Pendred syndrome and/or congenital hypothyroidism, which specify that whatever the genetical alteration noted in this study, they are not associated with Pendred syndrome and/or congenital hypothyroidism; rather might be related with the hypothyroidism detected in adulthood.…”

Genotyping on ALDH2: Comparison of Four Different Technologies

“…And our results demonstrated that HRM is a reliable method for genotyping ALDH2 rs671. Besides genotyping, HRM was also used for gene scanning and methylation analysis [ 14 , 15 ]. One current limitation of HRM is the possibility that another unexpected SNP interferes in the target SNP.…”