Supplementary Material for: Identification of Novel Functional Null Allele of <b><i>SLC26A4</i></b> Associated with Enlarged Vestibular Aqueduct and Its Possible Implication

Mutations in the <i>SLC26A4</i> gene, which encodes pendrin, cause congenital hearing loss as a manifestation of Pendred syndrome (PS) with an iodide organification defect or nonsyndromic enlarged vestibular aqueduct (NSEVA, DFNB4). There have been reports of differences between PS and NSEVA, including their auditory phenotypes and molecular genetic bases. For appropriate genetic diagnosis and counseling, it is important to functionally characterize <i>SLC26A4</i> variants. In this study, we identified and evaluated a novel null mutation of <i>SLC26A4</i> and report our method of assessing the pathogenic potential of mutations in <i>SLC26A4</i>, one of the most frequent causative genes of deafness in humans. A 3-year-old female with progressive sensorineural hearing loss and her parents were recruited. They underwent clinical, audiological, radiological and genetic evaluations, which revealed that the female patient had an enlarged vestibular aqueduct and an incomplete partition type II anomaly in the cochlea bilaterally. Sanger sequencing of the <i>SLC26A4</i> gene was also performed. For a confirmatory genetic diagnosis, we first characterized the anion/base exchange ability of mutant pendrin products in HEK 293 cells and, if necessary, evaluated whether the mutant pendrin traffics to the plasma membrane in COS-7 cells. We also expressed a null function mutant, p.H723R, and a previously documented polymorphism, p.P542R, as controls. The pure tone average was 66 dB HL in the right ear and 75 dB HL in the left ear. Sequencing of <i>SLC26A4</i> revealed a known pathogenic mutation (p.H723R) and a novel missense variant (p.V510D) as a compound heterozygote. When we expressed the p.V510D mutant pendrin in mammalian cells, the rate constants for Cl<sup>-</sup>/HCO<sub>3</sub><sup>-</sup> exchange were 10.96 ± 4.79% compared with those of wild-type pendrin. This figure was comparable to that of p.H723R, indicating p.V510D to be another pathogenic mutation with a null function. The p.V510D pendrin product was shown to be entrapped in the endoplasmic reticulum (ER) at 24-30 h after transfection, and not trafficked to the plasma membrane in COS-7 cells, suggesting retention in the ER and abnormal trafficking as the pathogenic mechanism. This was similar to p.H723R, which is a null function founder mutant in this population but is a candidate variant for future drug therapy to rescue the abnormal cell trafficking. Impaired cellular trafficking due to ER retention and abolished exchange activity of the newly detected p.V510D indicates the pathogenic potential of this variant. These missense variants may be good candidate variants for drug therapy if the intrinsic exchange activity is not damaged by the change.