Rasheeda Bashir

Prioritized sequencing of the second exon of MYO15A reveals a new mutation segregating in a Pakistani family with moderate to severe hearing loss

Mutations in MYO15A are associated with deafness in humans, and shaker 2 mice also exhibit a hearing loss due to defects of unconventional myosin 15a. We ascertained a consanguineous Pakistani family with recessively inherited moderate to severe hearing loss, which putatively segregated with markers linked to the DFNB3 locus. Prioritized sequencing of the second exon of MYO15A from the DNA of all affected individuals of family revealed a duplication of Cytosine in a stretch of seven repetitive C nucleotides (c.1185dupC). This mutation results in a frameshift and incorporates a stop codon in the open reading frame of MYO15A (p.E396fsX431). The findings of less severe hearing loss in families with linkage to DFNB3 are only reported for some individuals with mutations in exon 2 of MYO15A, which are further supported by this study. Therefore, on basis of linkage data and the presence of a less severe hearing loss phenotype, sequencing of a single exon of MYO15A can efficiently identify the causative mutations in patients from these families.

A frameshift mutation in SANS results in atypical Usher syndrome

Usher syndrome is an autosomal, recessively inherited disorder involving progressive retinitis pigmentosa and hearing loss with or without vestibular dysfunction. Usher type I (USH1) is the severest form (1). It involves profound deafness, vestibular areflexia and onset of retinitis pigmentosa in early childhood (1). To date seven genetic loci for USH1 have been mapped (USH1B-USH1H) and five of the genes have been identified. USH1G (MIM #606943) is associated with mutations in SANS (2). The encoded 461 amino acid protein, SANS, is predicted to have three ankyrin-like domains, a PDZ binding motif and a Sterile alpha motif (SAM) domain (2). SANS is expressed in many tissues including the inner ear and retina (2, 3). Jackson shaker (js) mice have a mutation in Sans. They exhibit disorganized stereocilia and are profoundly deaf, but do not exhibit a retinal phenotype (3). USH1G appears to be a rare cause of USH1 as only five mutations in SANS have been reported to cause Usher syndrome in four families (2, 4, 5) (Table 1). All mutations, except one, cause classic symptoms of USH1. A mutation in exon 2 substituting p.V458D in SANS is associated with atypical Usher syndrome. It is therefore hypothesized that missense mutations in SANS may result in hypomorphic alleles and cause a less severe phenotype as compared to frameshift mutations (4). Table 1 List of all mutations in SANS We report a consanguineous family with four affected individuals with moderate to severe hearing loss, mild retinitis pigmentosa and normal vestibular function, a phenotype which resembles an USH2 diagnosis (1). Family HLRB12 (Fig. 1a) was recruited from Sheikhupura, Pakistan with Institutional Review Board approval at University of the Punjab, and written informed consent was obtained from all participants. Three affected children were subjected to audiometric examinations at the age of 12, 14 and 21 years, in ambient noise conditions. Clinical examination revealed moderate to severe hearing loss (Fig. 1b). Vestibular function was evaluated by Romberg and tandem gait tests. Additionally, inquiries were made about whether affected children felt insecure while walking in darkness or had motion sickness. There was no delay in independent ambulation and none of the affected individuals had difficulty with balance suggesting vestibular function was normal. Additionally, none of the patients reported problems with eyesight including night vision. However, funduscopy revealed mild symptoms of retinitis pigmentosa in three of the older affected individuals examined at 5, 13, 15, and 22 years respectively. No bone spicules were observed in the retinal epithelium (Fig. 1d). The optic discs were pale as compared to those in normal individuals. Electroretinography could not be performed. Optical testing revealed a mild loss of near sight vision, which was not noted by the patients. Figure 1 (a) Pedigree of family HLRB12. Haplotypes for two closest markers to SANS are shown for the genotyped members. Alleles of the two markers were homozygous for all affected individuals. The ancestral chromosome with the SANS mutation is shaded in gray (b) ... Genotyping with fluorescently labeled markers for linkage analyses excluded all Usher syndrome loci except USH1G. Markers D17S1807 and D17S1301 lie close to SANS and showed homozygosity by descent in all affected individuals of family HLRB12 (Fig. 1a). Using a disease allele frequency of 0.001 and coding the phenotype as a fully penetrant autosomal recessive disorder, maximum two-point LOD scores of 4.2 and 3.9 were obtained at recombination fraction θ = 0 with the markers D17S1807 and D17S1301 respectively. We PCR amplified and sequenced the three exons and flanking intronic regions of SANS. In the DNA of affected individuals of family HLRB12 we identified a homozygous 15bp deletion (c.163_164+13del15) involving nucleotides in the first exon and intron of SANS (Fig. 1c, e). We did not detect this mutation in 200 chromosomes from ethnically matched controls assayed by Tetra primers ARMS PCR (6). In order to identify the effect of c.163_164+13del15 on the SANS transcript, we obtained RNA from whole blood and generated cDNA. However we found that SANS is not expressed sufficiently in blood samples (data not shown). Usually mutations that delete donor splice sites in first exons result in retention of the following introns or use of a cryptic splice site within the affected exons or introns (7, 8) . Indeed, in silico analysis for cryptic splice sites in wild type and mutant genomic sequences of SANS with GeneSplicer (http://cbcb.umd.edu/software/GeneSplicer/gene_spl.shtml) predicted retention of the first intron of in the RNA resulting from mutant, but not from the normal, SANS genomic sequence (data not shown). The retention of the first intron will introduce a frameshift and a premature stop codon in the SANS open reading frame. The presence of premature stop codons are known to mark some mRNAs for nonsense mediated decay (9) and it is possible that no mutant mRNA will be produced in patients with the deletion mutation. However, if the mRNA escapes this surveillance mechanism, the frameshift will result in a truncated nonfunctional protein of 58 amino acids. Our work indicates that both missense and deletion mutations in SANS can result in atypical Usher syndrome. Thus the location or type of mutation does not predict the severity of the disorder and the phenotypic course can be modified by unknown genetic or epigenetic factors. Furthermore, some patients with no mutations in genes which cause USH2 may have mutations in SANS.

Mutations in CLDN14 are associated with different hearing thresholds

Mutations in CLDN14, encoding tight junction protein claudin 14, cause profound deafness in mice and humans. We identified a Pakistani family, in which the affected individuals were homozygous for a known pathogenic mutation c.254 T>A resulting in p.V85D substitution in CLDN14; however, in contrast to the previously reported families with mutations in CLDN14, most of the affected individuals in this family exhibit only a severe hearing loss (HL). In order to identify the contribution of CLDN14 to less than profound deafness, we screened for mutations of CLDN14 in 30 multiplex and 57 sporadic cases with moderately severe to severe HL from Pakistan. We identified one other affected individual homozygous for p.V85D substitution. Comparison of audiometric data from all patients indicates that mutations in CLND14 cause varying degrees of HL, which may be enhanced at high frequencies. This suggests that a modifier can reduce the severity of HL associated with mutations of CLDN14. Our data indicate that mutations in CLDN14 should be explored when considering the etiology of less severe HL.

Genetic causes of moderate to severe hearing loss point to modifiers

The genetic underpinnings of recessively inherited moderate to severe sensorineural hearing loss are not well understood, despite its higher prevalence in comparison to profound deafness. We recruited 92 consanguineous families segregating stable or progressive, recessively inherited moderate or severe hearing loss. We utilized homozygosity mapping, Sanger sequencing, targeted capture of known deafness genes with massively parallel sequencing and whole exome sequencing to identify the molecular basis of hearing loss in these families. Variants of the known deafness genes were found in 69% of the participating families with the SLC26A4, GJB2, MYO15A, TMC1, TMPRSS3, OTOF, MYO7A and CLDN14 genes together accounting for hearing loss in 54% of the families. We identified 20 reported and 21 novel variants in 21 known deafness genes; 16 of the 20 reported variants, previously associated with stable, profound deafness were associated with moderate to severe or progressive hearing loss in our families. These data point to a prominent role for genetic background, environmental factors or both as modifiers of human hearing loss severity.

SLC26A4 Mutations in Patients with Moderate to Severe Hearing Loss

Mutations in SLC26A4 cause either syndromic or nonsyndromic hearing loss. We identified a link between hearing loss and DFNB4 in 3 of the 50 families participating in this study. Sequencing analysis revealed two SLC26A4 mutations, p.V239D and p.S57X, in affected members of the 3 families. These mutations have been previously reported in deaf individuals from the subcontinent, all of whom manifested profound deafness. The patients investigated in our study exhibited moderate to severe hearing loss. Our results show that inactivating SLC26A4 mutations that cause profound deafness can also be involved in the etiology of moderate to severe hearing loss. The type of mutation cannot predict the severity of the hearing loss in all cases, and there may be additional epistatic interactions that could modify the phenotype.

CDC14A phosphatase is essential for hearing and male fertility in mouse and human

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.

In vitro evaluation of mutagenicity and genotoxicity of sitagliptin alone and in combination with artificial sweeteners

Purpose: To determine the in vitro genotoxicity and mutagenicity of sitagliptin alone and in combination with three commonly used artificial sweeteners (saccharin, aspartame and acesulfame-k). Methods: The in vitro genotoxicity and mutagenicity of Sitagliptin alone and in combination with three popular artificial sweeteners (saccharin, aspartame and acesulfame-k) were evaluated by Comet and Ames assays, respectively. Results: Sitagliptin demonstrated mutagenic potential only to TA 98 with S9 mix at a concentration of 3040 μg/plate. The mutagenicity of sitagliptin was enhanced when tested in combination with the artificial sweeteners. Furthermore, sitagliptin also caused pronounced DNA fragmentation at higher doses compared with negative control. Conclusion: At higher doses, sitagliptin showed both mutagenicity and genotoxicity. Thus, long-term use of artificial sweeteners with sitagliptin may lead to increase in both mutagenicity and genotoxicity. Keywords: Sitagliptin, Artificial sweeteners, Comet assay, DNA damage, Ames assay, Genotoxicity, Mutagenicity

The c.42_52del11 Mutation in TPRN and Progressive Hearing Loss in a Family from Pakistan

The DFNB79 locus harbors TPRN mutations in which have been reported in a few families with deafness. Four frameshift mutations in TPRN have been described to cause severe or severe-to-profound hearing loss in Moroccan and Pakistani families, and a single frameshift mutation was associated with progressive hearing loss in deaf individuals in a Dutch family. We identified a Pakistani family in which the affected individuals were homozygous for a pathogenic mutation, c.42_52del11, in TPRN (p.G15Afs150X). In contrast to the previously reported individuals affected by the same mutation, hearing loss is likely to be progressive in this family. Thus the same mutation of TPRN can be associated with different thresholds of hearing as well as differences in the stability of the phenotype.