Molecular diagnostics for congenital hearing loss including 15 deafness genes using a next generation sequencing platform

NXTGNT, Ghent University, Ghent, Belgium. .
BMC Medical Genomics (Impact Factor: 2.87). 05/2012; 5(1):17. DOI: 10.1186/1755-8794-5-17
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Hereditary hearing loss (HL) can originate from mutations in one of many genes involved in the complex process of hearing. Identification of the genetic defects in patients is currently labor intensive and expensive. While screening with Sanger sequencing for GJB2 mutations is common, this is not the case for the other known deafness genes (> 60). Next generation sequencing technology (NGS) has the potential to be much more cost efficient. Published methods mainly use hybridization based target enrichment procedures that are time saving and efficient, but lead to loss in sensitivity. In this study we used a semi-automated PCR amplification and NGS in order to combine high sensitivity, speed and cost efficiency.
In this proof of concept study, we screened 15 autosomal recessive deafness genes in 5 patients with congenital genetic deafness. 646 specific primer pairs for all exons and most of the UTR of the 15 selected genes were designed using primerXL. Using patient specific identifiers, all amplicons were pooled and analyzed using the Roche 454 NGS technology. Three of these patients are members of families in which a region of interest has previously been characterized by linkage studies. In these, we were able to identify two new mutations in CDH23 and OTOF. For another patient, the etiology of deafness was unclear, and no causal mutation was found. In a fifth patient, included as a positive control, we could confirm a known mutation in TMC1.
We have developed an assay that holds great promise as a tool for screening patients with familial autosomal recessive nonsyndromal hearing loss (ARNSHL). For the first time, an efficient, reliable and cost effective genetic test, based on PCR enrichment, for newborns with undiagnosed deafness is available.

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    • "In the past few years, next-generation sequencing (NGS; high-throughput massively parallel sequencing) has allowed an unprecedented increase in our ability to sequence large numbers of genes at an equivalent cost to traditional Sanger sequencing. Although there have been several reports using NGS in a diagnostic setting, including one pilot study of five patients with ataxia (Hoischen et al., 2010), most studies include patients with very homogeneous clinical features (Shanks et al., 2012) or a modest number of genes requiring analysis (Fokstuen et al., 2011; De Keulenaer et al., 2012; Schrauwen et al., 2013) and there are no reports examining the realities of introducing NGS for highly varied disorders into clinical practice. As obtaining a specific molecular diagnosis has significant implications for patient management including genetic counselling, prognosis, long-term investigations and development of therapeutic strategies, we decided to investigate a heterogeneous group of patients with ataxia who were a diagnostic challenge and are representative of the range of cases referred for serial genetic testing. "
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    ABSTRACT: Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the 'diagnostic odyssey' for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1-3, 6, 7 and Friedrich's ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3-35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US$620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.
    Brain 09/2013; 136(10). DOI:10.1093/brain/awt236 · 9.20 Impact Factor
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    • "They successfully confirmed the causative mutations in all positive controls and detected additional variants in the other selected genes [50]. De Keulenaer et al. screened 15 ARNSHL genes in 1 positive control and 3 ARNSHL families with MPS and identified a pathogenic mutation in 2 ARNSHL families of Iranian ancestry [51]. In the present study, we performed targeted capture of 80 known deafness genes in 10 ADNSHL and 2 ARNSHL families and identified causative variants in 4 ADNSHL families. "
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    ABSTRACT: Despite the clinical utility of genetic diagnosis to address idiopathic sensorineural hearing impairment (SNHI), the current strategy for screening mutations via Sanger sequencing suffers from the limitation that only a limited number of DNA fragments associated with common deafness mutations can be genotyped. Consequently, a definitive genetic diagnosis cannot be achieved in many families with discernible family history. To investigate the diagnostic utility of massively parallel sequencing (MPS), we applied the MPS technique to 12 multiplex families with idiopathic SNHI in which common deafness mutations had previously been ruled out. NimbleGen sequence capture array was designed to target all protein coding sequences (CDSs) and 100 bp of the flanking sequence of 80 common deafness genes. We performed MPS on the Illumina HiSeq2000, and applied BWA, SAMtools, Picard, GATK, Variant Tools, ANNOVAR, and IGV for bioinformatics analyses. Initial data filtering with allele frequencies (<5% in the 1000 Genomes Project and 5400 NHLBI exomes) and PolyPhen2/SIFT scores (>0.95) prioritized 5 indels (insertions/deletions) and 36 missense variants in the 12 multiplex families. After further validation by Sanger sequencing, segregation pattern, and evolutionary conservation of amino acid residues, we identified 4 variants in 4 different genes, which might lead to SNHI in 4 families compatible with autosomal dominant inheritance. These included p.R75Q, p.T381M, p.S680F, and p.E1256K. Among them, p.S680F and p.E1256K were novel. In conclusion, MPS allows genetic diagnosis in multiplex families with idiopathic SNHI by detecting mutations in relatively uncommon deafness genes.
    PLoS ONE 02/2013; 8(2):e57369. DOI:10.1371/journal.pone.0057369 · 3.23 Impact Factor
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    • "Another recent study utilized a PCR-based approach combined with massive parallel sequencing. However, due to the fact that this was done using semi-automated conventional PCR, this method is very time consuming [De Keulenaer et al., 2012]. "
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    ABSTRACT: Implementing DNA diagnostics in clinical practice for extremely heterogeneous diseases such as hearing loss is challenging, especially when attempting to reach high sensitivity and specificity in a cost-effective fashion. Next generation sequencing has enabled the development of such a test, but the most commonly used genomic target enrichment methods such as hybridization-based capture suffer from restrictions. In this study, we have adopted a new flexible approach using microdroplet PCR-based technology for target enrichment, in combination with massive parallel sequencing to develop a DNA diagnostic test for autosomal recessive hereditary hearing loss. This approach enabled us to identify the genetic basis of hearing loss in 9 of 24 patients, a success rate of 37.5%. Our method also proved to have high sensitivity and specificity. Currently, routine molecular genetic diagnostic testing for deafness is in most cases only performed for the GJB2 gene and a positive result is typically only obtained in 10-20% of deaf children. Individuals with mutations in GJB2 had already been excluded in our selected set of 24 patients. Therefore, we anticipate that our deafness test may lead to a genetic diagnosis in roughly 50% of unscreened autosomal recessive deafness cases. We propose that this diagnostic testing approach represents a significant improvement in clinical practice as a standard diagnostic tool for children with hearing loss. © 2012 Wiley Periodicals, Inc.
    American Journal of Medical Genetics Part A 01/2013; 161A(1). DOI:10.1002/ajmg.a.35737 · 2.16 Impact Factor
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