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.