Efficient molecular genetic diagnosis of enlarged vestibular aqueducts in East Asians

Abstract

Context: Enlargement of the vestibular aqueduct (EVA) is a commonly detected inner ear anomaly related to hearing loss and often associated with mutations of SLC26A4 encoding pendrin, a transmembrane exchanger of Cl(-), I(-), and HCO(3)(-). Here we describe the phenotypes of 27 Korean EVA subjects and their SLC26A4 genotypes determined by bidirectional nucleotide sequencing.

Results: The detected variants include two novel missense substitutions (p.V138L and p.P542R). We characterized the ability of p.V138L and p.P542R pendrin products to traffic to the plasma membrane in COS-7 cells and to transport Cl(-), I(-), and HCO(3)(-) in Xenopus oocytes. The results indicate that p.P542R is a benign polymorphic variant, whereas p.V138L is a pathogenic mutation. Since this and other studies of East Asian EVA cohorts show that the majority of SLC26A4 mutations affect either or both of two amplicons (exons 7-8 and 19), we developed a hierarchical protocol that integrates direct sequencing with denaturing high-performance liquid chromatography analyses for detection of SLC26A4 mutations in these populations. We validated the cost efficiency of the integrated protocol by a simulated screen of published East Asian EVA cohorts with known SLC26A4 genotypes.

Conclusions: Our study further defines the spectrum of SLC26A4 mutations among East Asians and demonstrates a rapid and efficient protocol for their detection.

FIG. 1.

Temporal bone computed tomography scans. A coronal section of subject 7 (p.P542R/+) shows an enlarged vestibule (black arrowhead) and absent superior semicircular canal. An axial section demonstrates severe cochlear hypoplasia (black arrow) and poor mastoid pneumatization (white circle) in addition to enlargement of the vestibular aqueduct (EVA) (white arrow). Examination of adjacent sections confirmed this latter structure as the vestibular aqueduct and not the jugular bulb. Another subject (p.H723R/+) with a typical EVA has a superior semicircular canal (black dotted arrow), relatively normal cochlea, a well-pneumatized and aerated mastoid, and EVA (white arrow).

FIG. 2.

(a) Intracellular trafficking of pendrin variants in COS-7 cells. Merged representative images show green fluorescent protein–tagged wild-type or missense allele products (green) and concanavalin A staining (red) of plasma membrane. Colocalization (yellow) demonstrates targeting of pendrin to the plasma membrane. Scale bars = 20 μm. (b) Time course of ³⁶Cl⁻ efflux from representative oocytes and (c) rate constants (mean ± SE) for each group of (n) oocytes expressing wild-type or variant pendrin in extracellular medium containing 96 mM Cl⁻, 0 Cl⁻/5 mM I⁻, or 0 Cl⁻/0 I⁻, with gluconate as equimolar substituent anion. (d) Time course of ³⁶Cl⁻ efflux from representative oocytes and (e) rate constants (mean ± SE) for each group of (n) oocytes expressing wild-type or variant pendrin in extracellular medium containing 96 mM Cl⁻, 72 mM Cl⁻/24 mM HCO₃⁻, or 0 Cl⁻/24 mM HCO₃⁻/72 mM gluconate. Color images available online at www.liebertonline.com/gtmb.

FIG. 3.

(a) Electropherograms and denaturing high-performance liquid chromatography (DHPLC) elution profiles (EP) for novel SLC26A4 sequence variants (red arrowheads). (b, c) DHPLC EP of normal control, heterozygous, or homozygous c.919-2A>G (b) or p.H723R (c) variants with or without sequence-verified wild-type DNA added at a ratio of 2:1. EP differences for homozygotes are less prominent than for heterozygotes.

FIG. 4.

Exonic distribution of SLC26A4 mutations in East Asian EVA subjects from this and seven other studies (Tsukamoto et al., ; Park et al., ; Wu et al., ; Hu et al., ; Wang et al., ; Guo et al., ; Lee et al., 2008) and Caucasian (western) EVA subjects from four studies (Prasad et al., ; Pryor et al., ; Albert et al., ; Pera et al., 2008).

FIG. 5.

Schematic diagrams illustrating (a) nonhierarchical sequencing, (b) hierarchical integrated sequencing-DHPLC, and (c) nonhierarchical DHPLC simulated screens of a composite cohort of 247 East Asian EVA subjects from this and six other studies (Tsukamoto et al., ; Park et al., ; Wu et al., ; Hu et al., ; Wang et al., ; Lee et al., 2008). Numbers of subjects are in circles. Asterisks indicate numbers of DHPLC analysis temperatures.