title: Genetic analysis through OtoSeq of Pakistani families segregating prelingual hearing loss.
abstract: OBJECTIVE
  To identify the genetic cause of prelingual sensorineural hearing loss in Pakistani families using a next-generation sequencing (NGS)-based mutation screening test named OtoSeq.
  
  
  METHODS
  Prospective study.
  
  
  METHODS
  Research laboratory.
  
  
  METHODS
  We used 3 fluorescently labeled short tandem repeat (STR) markers for each of the known autosomal recessive nonsyndromic (DFNB) and Usher syndrome (USH) locus to perform a linkage analysis of 243 multigenerational Pakistani families segregating prelingual hearing loss. After genotyping, we focused on 34 families with potential linkage to MYO7A, CDH23, and SLC26A4. We screened affected individuals from a subset of these families using the OtoSeq platform to identify underlying genetic variants. Sanger sequencing was performed to confirm and study the segregation of mutations in other family members. For novel mutations, normal hearing individuals from ethnically 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 phenotype in the respective families.
  
  
  CONCLUSIONS
  Using NGS-based platforms like OtoSeq in families segregating hearing loss will contribute to the identification of common and population-specific mutations, early diagnosis, genetic counseling, and molecular epidemiology.
authors.affiliation: Divisions of Ophthalmology, Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio USA.
authors.forename: Mohsin
authors.initials: M
authors.lastname: Shahzad
authors.forename: Theru A
authors.initials: TA
authors.lastname: Sivakumaran
authors.forename: Tanveer A
authors.initials: TA
authors.lastname: Qaiser
authors.forename: Julie M
authors.initials: JM
authors.lastname: Schultz
authors.forename: Zawar
authors.initials: Z
authors.lastname: Hussain
authors.forename: Megan
authors.initials: M
authors.lastname: Flanagan
authors.forename: Munir A
authors.initials: MA
authors.lastname: Bhinder
authors.forename: Diane
authors.initials: D
authors.lastname: Kissell
authors.forename: John H
authors.initials: JH
authors.lastname: Greinwald
authors.forename: Shaheen N
authors.initials: SN
authors.lastname: Khan
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Friedman
authors.forename: Kejian
authors.initials: K
authors.lastname: Zhang
authors.forename: Saima
authors.initials: S
authors.lastname: Riazuddin
authors.forename: Sheikh
authors.initials: S
authors.lastname: Riazuddin
authors.forename: Zubair M
authors.initials: ZM
authors.lastname: Ahmed
mesh_terms: D000483:Alleles; D000070585:Cadherin Related Proteins; D015820:Cadherins / Q000235:genetics*; D005260:Female; D008040:Genetic Linkage; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D006319:Hearing Loss, Sensorineural / Q000235:genetics*; D006801:Humans; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics*; D018895:Microsatellite Repeats; D009154:Mutation; D000081412:Myosin VIIa; D009218:Myosins / Q000235:genetics*; D010154:Pakistan; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011446:Prospective Studies; D000076988:Sulfate Transporters; D052245:Usher Syndromes / Q000235:genetics
chemical_list: C442480:CDH23 protein, human; D000070585:Cadherin Related Proteins; D015820:Cadherins; C000631455:MYO7A protein, human; D026901:Membrane Transport Proteins; D000081412:Myosin VIIa; C085609:SLC26A4 protein, human; D000076988:Sulfate Transporters; D009218:Myosins
keywords: CDH23; MYO7A; PDS; Usher syndrome; clinical diagnosis; genetic etiology; hearing loss; microdroplet PCR; next-generation sequencing