Supplementary Material for: Phenotypic Analyses and Mutation Screening of the <i>SLC26A4</i> and <i>FOXI1</i> Genes in 101 Taiwanese Families with Bilateral Nonsyndromic Enlarged Vestibular Aqueduct (DFNB4) or Pendred Syndrome

Recessive mutations in the <i>SLC26A4</i> gene are responsible for nonsyndromic enlarged vestibular aqueduct (EVA) and Pendred syndrome. However, in some affected families, only 1 or 0 mutated allele can be identified, as well as no clear correlation between <i>SLC26A4</i> genotypes and clinical phenotypes, hampering the accuracy of genetic counseling. To elucidate the genetic composition of nonsyndromic EVA and Pendred syndrome, we screened related genomic fragments, including the <i>SLC26A4</i> coding regions, the <i>SLC26A4</i> promoter and the <i>FOXI1 </i>transcription factor gene, in 101 Taiwanese families, and analyzed their phenotypic and genotypic results. Mutation screening in the <i>SLC26A4</i> coding regions by direct sequencing and quantitative polymerase chain reaction detected 2 mutations in 63 (62%) families, 1 mutation in 24 (24%) families and no mutation in 14 (14%) families. The radiological findings, the presence of goiters and the audiological results were not different among probands (i.e. index cases of the families) with different <i>SLC26A4</i> genotypes. Specifically, probands heterozygous for <i>SLC26A4</i> mutations demonstrated clinical features indistinguishable from those of probands with 2 mutated alleles, implicating that there might be undetected mutations. However, except for a variant (c.–2554G>A of <i>SLC26A4</i>) with possible pathological consequences, no definite mutation was detected after extensive screening in the <i>SLC26A4</i> promoter and <i>FOXI1</i>. In other words, in most Taiwanese families nonsyndromic EVA or Pendred syndrome might not result from aberrance in the transcriptional control of <i>SLC26A4</i> by <i>FOXI1</i>. Meanwhile, exploration of undetected mutations in the <i>SLC26A4</i> noncoding regions revealed 9 divergent haplotypes among the 21 no-mutation-detected <i>SLC26A4</i> alleles of the c.919-2A>G heterozygotes, indicating that there might be no common and predominant mutations in the <i>SLC26A4</i> introns.