Hearing loss is a genetically heterogeneous disorder affecting approximately 360 million people worldwide and is among the most common sensorineural disorders. Here, we report a genetic analysis of seven large consanguineous families segregating prelingual sensorineural hearing loss. Whole-exome sequencing (WES) revealed seven different pathogenic variants segregating with hearing loss in these families, three novel variants (c.1204G>A, c.322G>T, and c.5587C>T) in TMPRSS3, ESRRB, and OTOF, and four previously reported variants (c.208C>T, c.6371G>A, c.226G>A, and c.494C>T) in LRTOMT, MYO15A, KCNE1, and LHFPL5, respectively. All identified variants had very low frequencies in the control databases and were predicted to have pathogenic effects on the encoded proteins. In addition to being familial, we also found intersibship locus heterogeneity in the evaluated families. The known pathogenic c.226C>T variant identified in KCNE1 only segregates with the hearing loss phenotype in a subset of affected members of the family GCNF21. This study further highlights the challenges of identifying disease-causing variants for highly heterogeneous disorders and reports the identification of three novel and four previously reported variants in seven known deafness genes.identified in KCNE1 segregates only with the hearing loss phenotype in a subset of affected 132 members of the family GCNF21 ( Figure 1A). 133 134 135 Figure 1: Family pedigrees and hearing loss-causing variants. (A) Segregation of disease-causing alleles in 136 seven Pakistani families. Filled and empty symbols represent affected and unaffected individuals, respectively.137 Double lines indicate consanguineous marriages. The genotypes (wild type and heterozygous and homozygous 138 mutants) of the identified mutant alleles are also shown for each of the participating family members, (B) 139 Representative audiometric data from the affected individuals of seven Pakistani families revealed profound 140 hearing loss, (C) ClustalW multiple amino acid sequence alignments of orthologous proteins show 141 evolutionarily conserved mutated residues across species. 142To determine the effect of the identified missense variants on the encoded proteins, we 143 performed molecular modeling using Phyre2 software and a number of structures available online 144 at the Protein Database (https://www.ncbi.nlm.nih.gov/protein). The identified variant of TMPRSS3 145 [p.(Gly402Arg)] in family GCNF17 is present in very close vicinity to the substrate-binding site 146 p.Ser400. Our modeling data suggest that the p.Gly402Arg substitution might result in the loss of 147 correct protein folding, as it introduces a large residue and might add rigidity due to aberrant 148 hydrogen bonds and ionic interactions (Figure 2). Similarly, the p.(Asp108Tyr) variant identified in 149 ESRRB is located next to the zinc-binding site (p.Cys106), and this substitution might introduce new 150 ionic interactions and hydrogen bonding patterns (e.g., with p.Cys79) (Figure 2). 151 The KCNE1 variant p.(Asn76Asp...

In this study, prolactin gene polymorphism was investigated in Nili-Ravi buffaloes, Sahiwal and Achai cattle breeds, 100 per group, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Only genotype GG was observed in the case of Nili-Ravi buffaloes. In Sahiwal and Achai cattle, three genotypes were found, AA, AG and GG: the frequency of these genotypes were 72%, 18% and 10% in Sahiwal cattle and 44%, 34% and 22% in Achai cattle, respectively. The frequency of genotype AA was found to be higher in both cattle breeds. Results of chi-square test at P < 0.05 revealed that animals of Achai cattle were in Hardy-Weinberg equilibrium, whereas Sahiwal cattle were found to be deviating.

Kappa-casein (j-CN) is the subtype of casein protein, an important constituent of bovine milk protein. The current study was undertaken to investigate the genetic polymorphism in j-CN gene of Nili-ravi buffalo, Achai and Sahiwal cattle of Pakistan using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The Nili-ravi buffalo was found to be monomorphic (genotype BB only) for j-CN gene. Achai cattle were polymorphic for j-CN (having three genotypes AA, AB and BB) with a frequency of 0.70, 0.18 and 0.12, respectively, while in Sahiwal cattle, both the genotypes AA and AB were found with genotypic frequencies of 0.92 and 0.08, respectively. The presence of genotype BB in Achai cattle is surprising as it is absent in most of the cattle breeds worldwide.

We report the underlying genetic causes of prelingual hearing loss (HL) segregating in eight large consanguineous families, ascertained from the Punjab province of Pakistan. Exome sequencing followed by segregation analysis revealed seven potentially pathogenic variants, including four novel alleles c.257G>A, c.6083A>C, c.89A>G, and c.1249A>G of CLPP, CDH23, COL4A5, and LARS2, respectively. We also identified three previously reported HL-causing variants (c.4528C>T, c.35delG, and c.1219T>C) of MYO15A, GJB2, and TMPRSS3 segregating in four families. All identified variants were either absent or had very low frequencies in the control databases. Our in silico analyses and 3-dimensional (3D) molecular modeling support the deleterious impact of these variants on the encoded proteins. Variants identified in MYO15A, GJB2, TMPRSS3, and CDH23 were classified as “pathogenic” or “likely pathogenic”, while the variants in CLPP and LARS2 fall in the category of “uncertain significance” based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant pathogenicity guidelines. This paper highlights the genetic diversity of hearing disorders in the Pakistani population and reports the identification of four novel mutations in four HL families.