High-throughput detection of mutations responsible for childhood hearing loss using resequencing microarrays

Prachi Kothiyal,John H Greinwald,Margaret A Kenna,Ammar Husami,Heidi L Rehm,Jonathan Ebert,Bruce J Aronow,Stephanie Cox

doi:10.1186/1472-6750-10-10

Abstract

BackgroundDespite current knowledge of mutations in 45 genes that can cause nonsyndromic sensorineural hearing loss (SNHL), no unified clinical test has been developed that can comprehensively detect mutations in multiple genes. We therefore designed Affymetrix resequencing microarrays capable of resequencing 13 genes mutated in SNHL (GJB2, GJB6, CDH23, KCNE1, KCNQ1, MYO7A, OTOF, PDS, MYO6, SLC26A5, TMIE, TMPRSS3, USH1C). We present results from hearing loss arrays developed in two different research facilities and highlight some of the approaches we adopted to enhance the applicability of resequencing arrays in a clinical setting.ResultsWe leveraged sequence and intensity pattern features responsible for diminished coverage and accuracy and developed a novel algorithm, sPROFILER, which resolved >80% of no-calls from GSEQ and allowed 99.6% (range: 99.2-99.8%) of sequence to be called, while maintaining overall accuracy at >99.8% based upon dideoxy sequencing comparison.ConclusionsTogether, these findings provide insight into critical issues for disease-centered resequencing protocols suitable for clinical application and support the use of array-based resequencing technology as a valuable molecular diagnostic tool for pediatric SNHL and other genetic diseases with substantial genetic heterogeneity.

Highlights

Despite current knowledge of mutations in 45 genes that can cause nonsyndromic sensorineural hearing loss (SNHL), no unified clinical test has been developed that can comprehensively detect mutations in multiple genes
Genetic testing of the GJB2 gene has been added to the diagnostic evaluation
We propose a novel algorithm for resolution of no-calls from GeneChip Sequence Analysis Software (GSEQ)

Summary

Introduction

Despite current knowledge of mutations in 45 genes that can cause nonsyndromic sensorineural hearing loss (SNHL), no unified clinical test has been developed that can comprehensively detect mutations in multiple genes. The medical evaluation of sensorineural hearing loss (SNHL) involves a combination of non-genetic laboratory and radiographic tests. The former provide little diagnostic or prognostic information [1]. Radiographic evaluations are helpful in diagnosing temporal bone anomalies, but are expensive and require sedation or general anaesthesia in children [2]. These tests are time-consuming and stressful for the child and family. Genetic testing of the GJB2 gene has been added to the diagnostic evaluation Mutations in this gene account for about 20% of children with nonsyndromic SNHL [3]. When the genetic etiology can be determined in a large cohort of patients, it will provide a better understanding of the genotype-phenotype correlations that exist for each of the genes examined, which could direct specific therapeutic interventions

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Conclusion