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Targeted High-Throughput Sequencing Used for Hereditary Hearing Loss Genetic Testing

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – A new study appearing online last night in the Proceedings of the National Academy of Sciences suggests target-enriched high-throughput sequencing can be used to help find variants behind some forms of hereditary hearing loss.

Researchers from the University of Iowa and Baylor College of Medicine explored the possibility of using targeted sequence capture in combination with massively parallel sequencing to test for mutations in dozens of genes in individuals with non-syndromic hearing loss — hearing loss that isn't part of a larger syndrome that includes other symptoms.

Based on their findings for nine individuals, they concluded that "[m]assively parallel sequencing technologies provide sensitivity, specificity, and reproducibility at levels sufficient to perform genetic diagnosis of hearing loss."

"Our sequencing results are consistent with other studies reporting utility of these technologies for diagnosis of other genetic diseases and suggest that massively parallel sequencing is suitable for genetic testing of [non-syndromic hearing loss]," corresponding author Richard Smith, a pediatric otolaryngology researcher at the University of Iowa, and co-authors wrote.

Although determining the genetic cause of deafness and hearing loss can be important to an individual's prognosis and treatment, the researchers explained, non-syndromic loss is notoriously difficult to diagnose, because it stems from diverse genetic causes.

Dozens of genes and more than 100 loci have been implicated in deafness, they added, making targeted Sanger sequencing of potentially mutated genes laborious. To try to overcome some problems associated with existing diagnosis method, the team turned to target capture combined with high-throughput massively parallel sequencing.

"Although genetic diagnoses can also be made by whole-genome sequencing or targeted sequence capture of the entire exome, these approaches are expensive, and time-consuming data analysis is required," they explained. "Therefore, our aim was to develop and test a streamlined, comprehensive genetic diagnostic platform that targets only the 0.014 [percent] of the genome currently associated with [non-syndromic hearing loss]."

For the current study, they focused on samples from nine individuals, including three positive control samples from two individuals with known mutations, a negative control sample, and six samples from individuals with unknown mutations.

As part of their custom-variant calling and annotation pipeline to find variants involved in hearing loss, which they dubbed "otologic sequence capture of pathogenic exons" or OtoSCOPE, the researchers first captured sequence from the exons of 54 genes using either an array-based target enrichment system from NimbleGen solid phase or a solution-based system from Agilent (SureSelect).

The genes selected had been previously associated with either hearing loss or Usher syndrome, a condition with symptoms similar to non-syndromic hearing loss in young children that can eventually lead to both hearing and vision problems.

The team then sequenced the samples using either the Roche 454 GS FLX or Illumina GAII platforms, comparing the capture efficiency, read depth, and variant detection characteristics of each target enrichment-sequencing combination.

For several hundred of the SNPs that appeared to be especially heterogeneous, the researchers also compared their initial findings with sequence generated using Sanger sequencing.

Based on the results for the first few samples, the team settled on a combination of SureSelect sequence capture and Illumina sequencing to assess the remaining samples, explaining that "the SureSelect-Illumina method is superior in terms of scalability, cost, and increased sensitivity."

Using this strategy, they reported that they could accurately root out hearing loss-related variants in the three positive control individuals and in five of the six individuals whose hearing loss had previously had no known cause.

"[W]e have demonstrated that OtoSCOPE has the potential to improve the efficiency of genetic testing for [non-syndromic hearing loss] and Usher syndrome," the team concluded. "Our results show that targeted capture plus massively parallel sequencing has a sensitivity and specificity comparable to Sanger sequencing."

The findings hint that such high-throughput methods could improve the ability to distinguish between different forms of inherited deafness, they argued, and provide an opportunity to find individuals with Usher syndrome-related hearing loss earlier. That, in turn, may allow for earlier treatments for other components of their condition, such as vision problems.

"Comprehensive genetic screening for deafness using platforms like OtoSCOPE would allow clinicians to improve patient care by providing prognostic information and genetic counseling," the authors explained, "and in cases like Usher syndrome, offer families preventative strategies to minimize the rate of progression of retinitis pigmentosa."

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