Homology modeling and global computational mutagenesis of human myosin VIIa

Usher syndrome type 1B (USH1B) is a genetic disorder caused by mutations in the unconventional Myosin VIIa (MYO7A) protein. USH1B is characterized by hearing loss due to abnormalities in the inner ear and vision loss due to retinitis pigmentosa. Here, we present the model of human MYO7A homodimer, built using homology modeling, and refined using 5 ns molecular dynamics in water. Global computational mutagenesis was applied to evaluate the effect of missense mutations that are critical for maintaining protein structure and stability of MYO7A in inherited eye disease. We found that 43.26% (77 out of 178 in HGMD) and 41.9% (221 out of 528 in ClinVar) of the disease-related missense mutations were associated with higher protein structure destabilizing effects. Overall, most mutations destabilizing the MYO7A protein were found to associate with USH1 and USH1B. Particularly, motor domain and MyTH4 domains were found to be most susceptible to mutations causing the USH1B phenotype. Our work contributes to the understanding of inherited disease from the atomic level of protein structure and analysis of the impact of genetic mutations on protein stability and genotype-to-phenotype relationships in human disease.

Spectrum of MYO7A Mutations in an Indigenous South African Population Further Elucidates the Nonsyndromic Autosomal Recessive Phenotype of DFNB2 to Include Both Homozygous and Compound Heterozygous Mutations

MYO7A gene encodes unconventional myosin VIIA, which, when mutated, causes a phenotypic spectrum ranging from recessive hearing loss DFNB2 to deaf-blindness, Usher Type 1B (USH1B). MYO7A mutations are reported in nine DFNB2 families to date, none from sub-Saharan Africa.In DNA, from a cohort of 94 individuals representing 92 families from the Limpopo province of South Africa, eight MYO7A variations were detected among 10 individuals. Family studies identified homozygous and compound heterozygous mutations in 17 individuals out of 32 available family members. Four mutations were novel, p.Gly329Asp, p.Arg373His, p.Tyr1780Ser, and p.Pro2126Leufs*5. Two variations, p.Ser617Pro and p.Thr381Met, previously listed as of uncertain significance (ClinVar), were confirmed to be pathogenic. The identified mutations are predicted to interfere with the conformational properties of myosin VIIA through interruption or abrogation of multiple interactions between the mutant and neighbouring residues. Specifically, p.Pro2126Leufs*5, is predicted to abolish the critical site for the interactions between the tail and the motor domain essential for the autoregulation, leaving a non-functional, unregulated protein that causes hearing loss. We have identified MYO7A as a possible key deafness gene among indigenous sub-Saharan Africans. The spectrum of MYO7A mutations in this South African population points to DFNB2 as a specific entity that may occur in a homozygous or in a compound heterozygous state.

Regenerating hair cells in vestibular sensory epithelia from humans

Human vestibular sensory epithelia in explant culture were incubated in gentamicin to ablate hair cells. Subsequent transduction of supporting cells with ATOH1 using an Ad-2 viral vector resulted in generation of highly significant numbers of cells expressing the hair cell marker protein myosin VIIa. Cells expressing myosin VIIa were also generated after blocking the Notch signalling pathway with TAPI-1 but less efficiently. Transcriptomic analysis following ATOH1 transduction confirmed up-regulation of 335 putative hair cell marker genes, including several downstream targets of ATOH1. Morphological analysis revealed numerous cells bearing dense clusters of microvilli at the apical surfaces which showed some hair cell-like characteristics confirming a degree of conversion of supporting cells. However, no cells bore organised hair bundles and several expected hair cell markers genes were not expressed suggesting incomplete differentiation. Nevertheless, the results show a potential to induce conversion of supporting cells in the vestibular sensory tissues of humans.