Pathogenic substitution of IVS15 + 5G > A in SLC26A4 in patients of Okinawa Islands with enlarged vestibular aqueduct syndrome or Pendred syndrome

Abstract

Background: Pendred syndrome (PS) and nonsyndromic hearing loss associated with enlarged vestibular aqueduct (EVA) are caused by SLC26A4 mutations. The Okinawa Islands are the southwestern-most islands of the Japanese archipelago. And ancestral differences have been reported between people from Okinawa Island and those from the main islands of Japan. To confirm the ethnic variation of the spectrum of SLC26A4 mutations, we investigated the frequencies of SLC26A4 mutations and clinical manifestations of patients with EVA or PS living in the Okinawa Islands.

Methods: We examined 22 patients with EVA or PS from 21 unrelated families in Okinawa Islands. The patient's clinical history, findings of physical and otoscopic examinations, hearing test, and computed tomography (CT) scan of the temporal bones were recorded. To detect mutations, all 21 exons and the exon-intron junctions of SLC26A4 were sequenced for all subjects. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for SLC26A4 and calculations using the comparative CT (2(-ΔΔCT)) method were used to determine the pathogenicity associated with gene substitutions.

Results: SLC26A4 mutations were identified in 21 of the 22 patients. We found a compound heterozygous mutation for IVS15 + 5G > A/H723R in nine patients (41%), a homozygous substitution of IVS15 + 5G > A in six patients (27%), and homozygous mutation for H723R in five patients (23%). The most prevalent types of SLC26A4 alleles were IVS15 + 5G > A and H723R, which both accounted for 15/22 (68%) of the patients. There were no significant correlations between the types of SLC26A4 mutation and clinical manifestations. Based on qRT-PCR results, expression of SLC26A4 was not identified in patients with the homozygous substitution of IVS15 + 5G > A.

Conclusions: The substitution of IVS15 + 5G > A in SLC26A4 was the most common mutation in uniquely found in patients with PS and EVA in Okinawa Islands. This suggested that the spectrum of SLC26A4 mutation differed from main islands of Japan and other East Asian countries. The substitution of IVS15 + 5G > A leads to a loss of SLC26A expression and results in a phenotype of PS and EVA.

Figure 1

Computed tomography of the temporal bone showing an enlarged vestibular aqueduct. Circles show the vestibular aqueduct. The vestibular aqueduct is not identified in control subject (A). The enlarged vestibular aqueduct is identified in a patient with EVA (B).

Figure 2

Location of the Okinawa islands in relation to East Asia. The Okinawa islands are located between Taiwan and the Japanese island of Kyushu. The Japanese archipelago comprises Hokkaido, Honshu, Kyusyu, and the Okinawa islands, as well as some smaller islands.

Figure 3

Examples of direct sequence analysis of the SLC26A4 gene. Representative results of H723R and the IVS15 + 5G > A mutation analysis are shown. Genomic sequences of the SLC26A4 gene in normal individuals (A), (B). A compound heterozygous mutation for IVS15 + 5G > A/H723R (C), (D). A homozygous mutation for H723R (E). A homozygous substitution of IVS15 + 5G > A (F). The arrows indicate the variant nucleotide.

Figure 4

Expression of the SLC26A4 gene in patients with PS or EVA. The expected RT-nested PCR amplification product of SLC26A4 was 154 base pairs (bp) in length. Agarose gel electrophoresis shows the 154 bp band for the control subject (A) and the patient with IVS15 + 5G > A/H723R compound heterozygous mutation (B); however, there was no band for the patient with IVS15 + 5G > A homozygous substitution (C).

Figure 5

Relative expression of the SLC26A4 gene in control subjects and in patients with a homozygous mutation of IVS15 + 5G > A or compound heterozygous mutation of IVS15 + 5G > A/H723R. The ratio of SLC26A4 mRNA to GAPDH mRNA is shown in three control subjects (A, B, C), three patients with compound heterozygous mutation of IVS15 + 5G > A/H723R (D, E, F), and three patients with IVS15 + 5G > A homozygous substitution (G, H, I). No expression of SLC26A4 was observed in the three patients with the IVS15 + 5G > A homozygous substitution (G, H, I). All experiments were done in tripricate.