Genomic Analysis News

Using Next Generation Sequencing to Discover Novel Associations in Bilateral Sensorineural Hearing Loss:

Successful adoption of next generation sequencing (NGS) technology for medical sequencing requires coordination between informaticists, geneticists, subject matter experts, and ethical/legal/regulatory domains. CBMi is collaborating with disease experts and clinical diagnostics groups to develop and promote best practices in the areas of robust data analysis and management, research study design, and clinical diagnostic workflows using this transformative technology. One early success has been in developing novel NGS-based methods and analyses to improve clinical diagnostics for hearing disorders seen at CHOP.

Bilateral Sensorineural Hearing Loss (BLSNHL)

BLSNHL is a common birth defect affecting 1 of every 500 newborns. About half of these cases involve a genetic component, but there is a complex, multifactorial basis in the family of hearing loss diseases that is not well understood. Research just in the past decade has implicated more than 40 genes associated with this disorder and future research will likely reveal many more. However, genetic screening panels currently available use standard molecular techniques and assess only a small number of genes for BLSNHL, such that no diagnosis is obtained in a substantial number of patients.

With the goal of using NGS to develop a more comprehensive BLSNHL screening panel, CBMi's NGS group has been working with CHOP’s Molecular Genetics Laboratory to discover potential molecular markers of BLSNHL. The team, led by Dr. Xiaowu Gai and including Juan Perin, Monica D’arcy, and Michael Grauer, has collaborated with Drs. Catherine Stolle and Avni Santani, Director and Assistant Director of the Molecular Genetics Laboratory, respectively. “We wanted to determine whether we could efficiently use NGS to capture and analyze all implicated hearing loss loci in one comprehensive diagnostic test", said Dr. Santani. “Ideally, we would be able to identify mutations with high sensitivity and specificity at a cost that would be comparable to a single gene disorder test."

Targeted Capture of BLSNHL Risk Loci

As a test, exons of 35 genes previously implicated in BLSNHL spanning a total of 139 kilobases were captured using febit’s HybSelect enrichment technology. Captured material was sequenced on the SOLiD platform for 13 BLSHNL patients with a previously confirmed molecular diagnosis, and 82 additional patients where a mutation was not known. These patients had previously been diagnosed for BLSNHL by a clinical group led by Dr. Ian Krantz. The capture experiments yielded a mean of 13X coverage for each sample (range: 2X-44X). Together, this experiment generated over 3.4 million sequence reads comprising 171 million base pairs of unique sequence.

Establishing an Analysis Workflow

Providing a customized workflow for analyzing the results of each NGS run required integrating a series of open-source and custom analytical software and resources. "Due to the current lack of maturity of NGS sequence analysis software, we find that combining several approaches and algorithms provides the best results", said Dr. Gai. For this experiment, the CBMi group primarily used the sequence alignment tools BWA and samtools, the analysis platform GATK, and the annotation tools PolyPhen-2, SIFT, and HGMD. Custom programs and databases provide the logic flow and automation that stitch together the set of algorithms and resources. Variations of this general workflow have worked well for the group using data from the major sequencing platforms (i.e., SOLiD, 454, Illumina) and via different sequencing strategies (i.e., pooled, targeted capture, whole exome). The process results in fully automated variant calls with high sensitivity and specificity. "For this project, our workflow was able to provide sequence annotation, assess functional significance, and filter results based on known polymorphisms and genetic models”, stated Dr. Gai. “By combining these tools together, we were ultimately able to identify and prioritize the most likely disease-causing variants."

Results of the BLSNHL Study

The study identified 41 potentially disease-causing variants in 28 individuals that were confirmed by Sanger sequencing. These included 36 missense variants, 4 nonsense mutations, and one deletion of three nucleotides. Among these, 18 were newly discovered while the other 23 were previously reported pathogenic variants. To date, a positive molecular diagnosis has been assigned for 8 of the 82 previously undiagnosed patients, and additional variants are being assessed. These findings highlight the potential for NGS utility in both the research and clinical diagnostic settings. "The initial success of our findings, combined with those of other laboratories, indicate that NGS will likely be a game changer for the clinical assessment of rare genetic disorders in the very near future", said Dr. Santani. Initial findings of this study were recently presented by Dr. Santani at the Hereditary Hearing Impairment Consortium during the 60th annual meeting of the American Society of Human Genetics (Washington, DC; November 2-6, 2010).

Attribution

Mutation screening of 95 patients with bilateral sensorineural hearing loss using HybSelect and massively parallel sequencing - experience of a clinical diagnostic laboratory. A. Santani, X. Gai, M. D'Arcy, M. Grauer, A. Caruso, H. Wu, T. Tischler, A. Keller, V. Boisguerin, L. Francey, D. Clark, I. Krantz, C. Stolle

Presented At: Hereditary Hearing Impairment Consortium during the 60th annual meeting of the American Society of Human Genetics (Washington, DC; November 2-6, 2010).