Targeted Exon Sequencing Successfully Discovers Rare Causative Genes and Clarifies the Molecular Epidemiology of Japanese Deafness Patients

<div><p>Target exon resequencing using Massively Parallel DNA Sequencing (MPS) is a new powerful strategy to discover causative genes in rare Mendelian disorders such as deafness. We attempted to identify genomic variations responsible for deafness by massive sequencing of the exons of 112 target candidate genes. By the analysis of 216randomly selected Japanese deafness patients (120 early-onset and 96 late-detected), who had already been evaluated for common genes/mutations by Invader assay and of which 48 had already been diagnosed, we efficiently identified causative mutations and/or mutation candidates in 57 genes. Approximately 86.6% (187/216) of the patients had at least one mutation. Of the 187 patients, in 69 the etiology of the hearing loss was completely explained. To determine which genes have the greatest impact on deafness etiology, the number of mutations was counted, showing that those in <i>GJB2</i> were exceptionally higher, followed by mutations in <i>SLC26A4, USH2A, GPR98, MYO15A, COL4A5</i> and <i>CDH23</i>. The present data suggested that targeted exon sequencing of selected genes using the MPS technology followed by the appropriate filtering algorithm will be able to identify rare responsible genes including new candidate genes for individual patients with deafness, and improve molecular diagnosis. In addition, using a large number of patients, the present study clarified the molecular epidemiology of deafness in Japanese. <i>GJB2</i> is the most prevalent causative gene, and the major (commonly found) gene mutations cause 30–40% of deafness while the remainder of hearing loss is the result of various rare genes/mutations that have been difficult to diagnose by the conventional one-by-one approach. In conclusion, target exon resequencing using MPS technology is a suitable method to discover common and rare causative genes for a highly heterogeneous monogenic disease like hearing loss.</p></div

Examples of the families and audiograms of the patients with missense mutations after confirmation by Sanger sequencing.

<p>Examples of the families and audiograms of the patients with missense mutations after confirmation by Sanger sequencing.</p

The pedigrees and audiograms of the patients with insertion-deletion mutations after confirmation by Sanger sequencing.

<p>The pedigrees and audiograms of the patients with insertion-deletion mutations after confirmation by Sanger sequencing.</p

Examples of the families and audiograms of the patients with missense mutations after confirmation by Sanger sequencing.

<p>Examples of the families and audiograms of the patients with missense mutations after confirmation by Sanger sequencing.</p

A: The number of mutations/mutation candidates indicating that the majority of the responsible gene mutations are accumulated in particular major causative genes.

<p>B: The number of mutations/mutation candidates in the early-onset group. C: The number of mutations/mutation candidates in the late-detected group.</p

The pedigrees and audiograms of the patients with nonsense mutations after confirmation by Sanger sequencing.

<p>The pedigrees and audiograms of the patients with nonsense mutations after confirmation by Sanger sequencing.</p

Algorithm applied in this study.

<p><b>Nonsense mutations, splice-site mutations, and missense mutations were chosen according to this algorithm.</b></p

Variation in degree of inflammation of rat paws after treatment with vehicle or IL-23 blockade

<p><b>Copyright information:</b></p><p>Taken from "IL-23 induces human osteoclastogenesis via IL-17 , and anti-IL-23 antibody attenuates collagen-induced arthritis in rats"</p><p>http://arthritis-research.com/content/9/5/R96</p><p>Arthritis Research & Therapy 2007;9(5):R96-R96.</p><p>Published online 23 Sep 2007</p><p>PMCID:PMC2212562.</p><p></p> Photographs showing paws of rats with collagen-induced arthritis (CIA) that were treated with vehicle or anti-IL-23 antibody on day 21. Control rats. Rats with CIA that were treated with vehicle. Rats with CIA that were treated with anti-IL-23 antibody (3.0 μg) from day 14. Slides stained with hematoxylin and eosin representing synovial tissues obtained from right paws on day 28. Control rats. Rats with CIA that were treated with vehicle. Rats with CIA that were treated with anti-IL-23 antibody (3.0 μg) from day 14. Original magnification ×100

Typical radiographic images obtained on day 28, showing differences in paws

<p><b>Copyright information:</b></p><p>Taken from "IL-23 induces human osteoclastogenesis via IL-17 , and anti-IL-23 antibody attenuates collagen-induced arthritis in rats"</p><p>http://arthritis-research.com/content/9/5/R96</p><p>Arthritis Research & Therapy 2007;9(5):R96-R96.</p><p>Published online 23 Sep 2007</p><p>PMCID:PMC2212562.</p><p></p> The groups were control rats, vehicle-treated rats with collagen-induced arthritis (CIA), and rats with CIA treated with anti-IL-23 antibody on day 28. Frontal views; lateral views. Control rats. Rats with CIA treated with vehicle. Rats with CIA treated with anti-IL-23 antibody (3.0 μg) from day 14. Note that rats wth CIA treated with vehicle showed radiographic changes characterized by bone erosion and joint space narrowing

Variation in paw volume in rats with CIA treated with vehicle or IL-23 blockade

<p><b>Copyright information:</b></p><p>Taken from "IL-23 induces human osteoclastogenesis via IL-17 , and anti-IL-23 antibody attenuates collagen-induced arthritis in rats"</p><p>http://arthritis-research.com/content/9/5/R96</p><p>Arthritis Research & Therapy 2007;9(5):R96-R96.</p><p>Published online 23 Sep 2007</p><p>PMCID:PMC2212562.</p><p></p> Paw volumes of control rats, vehicle-treated rats with collagen-induced arthritis (CIA), and rats with CIA that were treated with anti-IL-23 antibody, from day 0 to day 21. Open squares, rats with CIA treated with vehicle; open circles, controls; filled triangles, rats with CIA treated with 3.0 μg of anti-IL-23 antibody; filled diamonds, rats with CIA treated with 6.0 μg of anti-IL-23 antibody. Means ± SD are shown. = 0.021 versus controls, = 0.021 versus controls, = 0.014 versus vehicle, = 0.007 versus vehicle (Mann–Whitney test). Arthritis scores of control rats, vehicle-treated rats with CIA, and rats with CIA that were treated with anti-IL-23 antibody, from day 0 to day 28. Open squares, rats with CIA treated with vehicle; open circles, controls; filled triangles, rats with CIA treated with 3.0 μg of anti-IL-23 antibody; filled diamonds, rats with CIA treated with 6.0 μg of anti-IL-23 antibody. Means ± SD are shown. = 0.021 versus controls, = 0.021 versus controls (Mann–Whitney test)