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In studies of genetics of deafness, families with inherited hearing loss have been extremely valuable, providing paths to discovery of genes essential to mechanisms of hearing. Until now, such studies have depended on Sanger sequencing of genes in candidate genomic linkage regions. Constraints of PCR-based sequencing have largely limited gene discovery to conventional mutations in known genes in relatively small regions of linkage. The advent of next generation sequencing technology has the potential to dramatically accelerate the pace of gene discovery.
We have undertaken three approaches with next generation sequencing to discover novel genes responsible for the deafness phenotype in families with dominant or recessive inherited hearing loss. (1) For families in which hearing loss maps to linkage regions of less than 3 megabases, we tile the entire genomic interval on capture arrays. DNA fragment libraries from the hearing impaired probands are hybridized to the arrays and the enriched portion of the genome is sequenced to approximate 30 fold coverage to reveal the critical allele. (2) For small nuclear families in which hearing loss maps to one or more very large linkage regions, we capture the entire coding region or 'exome' and approximately 400 miRNAs with in-solution oligonucleotides, sequence to approximately 30 fold coverage, and evaluate all functional variants co-inherited with the phenotype. (3) In order to identify all classes of structural mutations - inversions, translocations, duplications, or deletions - in families unresolved by other methods, we construct large-insert mate-pair libraries from the proband and sequence the entire genome to high clone coverage. Point mutations and small insertions or deletions can be resolved directly. In addition, structural mutations can be resolved from discordant mate-pairs, in which orientation, strand location, or distance apart differs from expected values. The results emerging from these studies indicate that many more novel genes for hearing loss will be discovered in the near future.
Supported by NIH grant R01DC005641. |
