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Background: Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy, affecting approximately 1 in 4000 individuals worldwide. The most common form of syndromic RP is Usher syndrome (USH) accounting for approximately 20-30 % of RP cases. Mutations in the USH2A gene cause a significant proportion of recessive non-syndromic RP and USH type II (USH2). This study aimed to determine the causative role of the USH2A gene in autosomal recessive inherited ocular diseases and to establish genotype-phenotype correlation associated with USH2A variants., Methods: We performed direct Sanger sequencing and co-segregation analysis of the USH2A gene to identify disease causing variants in a non-syndromic RP family, two USH2 families and two Keratoconus (KC) families., Results: Disease causing variants in the USH2A gene were identified in two families displayed KC and USH2 phenotypes. A novel variant c.4029T > G, p.Asn1343Lys in the USH2A gene was detected in a Pakistani family with KC phenotype. In addition, a missense variant (c.7334 C > T, p. Ser2445Phe) in the USH2A gene was found segregating in another Pakistani family with USH2 phenotype. Homozygosity of identified missense USH2A variants was found associated with autosomal recessive inherited KC and USH2 phenotypes in investigated families. These variants were not detected in ethnically matched healthy controls. Moreover, the USH2A variants were predicted to be deleterious or potentially disease causing by PolyPhen-2, PROVEAN and SIFT., Conclusions: This study provided first evidence for association of a novel USH2A variant with KC phenotype in a Pakistani family as well as established the phenotype-genotype correlation of a USH2A variant (c.7334 C > T, p. Ser2445Phe) with USH2 phenotype in another Pakistani family. The phenotype-genotype correlations established in present study may improve clinical diagnosis of affected individuals for better management and counseling.

Hearing loss (HL) is clinically and genetically heterogeneous disorder and is the most frequent occurring sensory deficit in humans. This study was conducted to decipher the genetic cause of HL occurring in two large consanguineous Pakistani families (GCNF-01, GCNF-03). Family history and pure tone audiometry of both families suggested prelingual HL, while the affected individuals of GCNF-01 also had low vision and balance problems, consistent with cardinal features of Usher syndrome type I (USH1). Exome sequencing followed by segregating analysis revealed a novel splice site variant (c.877-1G > A) of USH1C occurring with USH1 phenotype in family GCNF01. While the affected individual of family GCNF-03 were homozygous for the c.716 T > A, p.(Val239Asp) previously reported pathogenic variant of SLC26A4. Both variants have very low frequencies in control database. In silico mutagenesis and 3-dimensional simulation analyses revealed that both variants have deleterious impact on the proteins folding and secondary structures. Our study expands the mutation spectrum of the HL genes and emphasizes the utility of exome sequencing coupled with bioinformatics tools for clinical genetic diagnosis, prognosis, and family counseling.

Background: Digenic inheritance is the simplest model of oligenic disease. It can be observed when there is a strong epistatic interaction between two loci. For both syndromic and non-syndromic hearing impairment, several forms of digenic inheritance have been reported., Methods: We performed exome sequencing in a Pakistani family with profound non-syndromic hereditary hearing impairment to identify the genetic cause of disease., Results: We found that this family displays digenic inheritance for two trans heterozygous missense mutations, one in PCDH15 [p.(Arg1034His)] and another in USH1G [p.(Asp365Asn)]. Both of these genes are known to cause autosomal recessive non-syndromic hearing impairment and Usher syndrome. The protein products of PCDH15 and USH1G function together at the stereocilia tips in the hair cells and are necessary for proper mechanotransduction. Epistasis between Pcdh15 and Ush1G has been previously reported in digenic heterozygous mice. The digenic mice displayed a significant decrease in hearing compared to age-matched heterozygous animals. Until now no human examples have been reported., Conclusions: The discovery of novel digenic inheritance mechanisms in hereditary hearing impairment will aid in understanding the interaction between defective proteins and further define inner ear function and its interactome.

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., 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 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., Conclusion: 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.

We ascertained two large Pakistani consanguineous families (PKDF231 and PKDF608) segregating profound hearing loss, vestibular dysfunction, and retinitis pigmentosa; the defining features of Usher syndrome type 1 (USH1). To date, seven USH1 loci have been reported. Here, we map a novel locus, USH1K, on chromosome 10p11.21-q21.1. In family PKDF231, we performed a genome-wide linkage screen and found a region of homozygosity shared among the affected individuals at chromosome 10p11.21-q21.1. Meiotic recombination events in family PKDF231 define a critical interval of 11.74 cM (20.20 Mb) bounded by markers D10S1780 (63.83 cM) and D10S546 (75.57 cM). Affected individuals of family PKDF608 were also homozygous for chromosome 10p11.21-q21.1-linked STR markers. Of the 85 genes within the linkage interval, PCDH15, GJD4, FZD4, RET and LRRC18 were sequenced in both families, but no potential pathogenic mutation was identified. The USH1K locus overlaps the non-syndromic deafness locus DFNB33 raising the possibility that the two disorders may be caused by allelic mutations.