Genetic Analysis through OtoSeq of Pakistani Families Segregating Prelingual Hearing Loss

Objective. To identify the genetic cause of prelingual sensori- neural hearing loss in Pakistani families using a next- generation sequencing (NGS)-based mutation screening test named OtoSeq. Study Design. Prospective study. Setting. Research laboratory. Subjects and Methods. We used 3 fluorescently labeled short tandem repeat (STR) markers for each of the known auto- somal recessive nonsyndromic (DFNB) and Usher syndrome (USH) locus to perform a linkage analysis of 243 multige- nerational Pakistani families segregating prelingual hearing loss. After genotyping, we focused on 34 families with potential linkage to MYO7A, CDH23, and SLC26A4 .W e screened affected individuals from a subset of these families using the OtoSeq platform to identify underlying genetic var- iants. Sanger sequencing was performed to confirm and study the segregation of mutations in other family members. For novel mutations, normal hearing individuals from ethni- cally matched backgrounds were also tested. Results. Hearing loss was found to co-segregate with locus- specific STR markers for MYO7A in 32 families, CDH23 in 1 family, and SLC26A4 in 1 family. Using the OtoSeq platform, a microdroplet PCR-based enrichment followed by NGS, we identified mutations in 28 of the 34 families including 11 novel mutations. Sanger sequencing of these mutations showed 100% concordance with NGS data and co- segregation of the mutant alleles with the hearing loss phe- notype in the respective families.