Rapid Genetic Diagnosis for Okinawan Patients with Enlarged Vestibular Aqueduct Using Single-Stranded Tag Hybridization Chromatographic Printed-Array Strip

Abstract

Both Pendred syndrome (PS) and nonsyndromic hearing loss with an enlarged vestibular aqueduct (EVA) are autosomal recessive disorders caused by SLC26A4 pathogenic variants. The spectrum of SLC26A4 pathogenic variants varies with the ethnic background. Among the patients with EVA in Okinawa, 94% had some combination of NM_000441.2(SLC26A4):c.1707+5G>A and NM_000441.2(SLC26A4):c.2168A>G(p.His723Arg), the two SLC26A4 pathogenic variants that are the most common in this population. We identified these two pathogenic variants using a novel genotyping method that employed an allele-specific polymerase chain reaction (PCR) from a gDNA and single-stranded tag hybridization chromatographic printed-array strip (STH-PAS) in DNA samples obtained from 48 samples in Okinawa, including 34 patients with EVA and 14 carriers of c.1707+5G>A or c.2168A>G. In addition, whole blood and saliva samples were used for analysis in this genotyping method with direct PCR. The results of STH-PAS genotyping were consistent with those obtained using standard Sanger sequencing for all samples. The accuracy of the STH-PAS method is 100% under the optimized conditions. STH-PAS genotyping provided a diagnosis in 30 out of 34 patients (88%) in Okinawan patients with EVA in under 3 h. The turn-around time for STH-PAS genotyping used with direct PCR was 2 h as a result of the omission of the DNA extraction and purification steps. Using information about the ethnic distribution of pathogenic variants in the SLC26A4 gene, STH-PAS genotyping performs a rapid genetic diagnosis that is simple and has a considerably improved efficiency.

Keywords: Okinawan patients; Pendred syndrome; SLC26A4; enlarged vestibular aqueduct; rapid diagnosis.

Figure 1

Geographic location of the Okinawa Islands: this string of small islands is situated between the Japanese island of Kyushu and Taiwan.

Figure 2

(a) Schematic diagram of the STH-PAS genetic analysis. (b) Results of PCR amplicon signals detected by STH-PAS for seven subjects that are representative of the entire group of patients. Red lines are positional markers. Blue lines indicate the presence of pathogenic variants. The genotype, represented by the pattern of blue lines, was summarized at the bottom of each lane. NC, negative control; −, template pathogenic variant absent; +, template pathogenic variant carrier.

Figure 3

Stability test of the STH-PAS genetic analysis for the c.1707+5G>A and p.H723R pathogenic variants in SLC26A4. Each panel summarizes results obtained at a different pair of conditions. Rows are organized by template DNA concentration: top row (a–c), high (50 ng/μL); bottom row (d–f), low (5 ng/μL). Similarly, columns are organized by annealing temperature: left (a,d), 59 °C; middle (b,e), 60 °C; and right (c,f), 61 °C. Red arrowheads (strips 4, 5, 6 in a) indicate false positives. NC, negative control; −, template pathogenic variant absent; +, template pathogenic variant carrier.

Figure 4

Results of the STH-PAS genetic analysis for c.1707+5G>A and p.H723R in SLC26A4 using direct PCR from samples of (a) whole blood and (b) saliva of a single patient (strip 1) and two controls (strips 2 and 3). The genotype of each lane was presented in the bottom graphics. NC, negative control; −, template pathogenic variant absent; +, template pathogenic variant carrier.