Background: Identification of genes responsible for medically important traits is a major challenge in human genetics. Due to the genetic heterogeneity of hearing loss, targeted DNA capture and massively parallel sequencing are ideal tools to address this challenge. Our subjects for genome analysis are Israeli Jewish and Palestinian Arab families with hearing loss that varies in mode of inheritance and severity. Results: A custom 1.46 MB design of cRNA oligonucleotides was constructed containing 246 genes responsible for either human or mouse deafness. Paired-end libraries were prepared from 11 probands and bar-coded multiplexed samples were sequenced to high depth of coverage. Rare single base pair and indel variants were identified by filtering sequence reads against polymorphisms in dbSNP132 and the 1000 Genomes Project. We identified deleterious mutations in CDH23, MYO15A, TECTA, TMC1, and WFS1. Critical mutations of the probands co-segregated with hearing loss. Screening of additional families in a relevant population was performed. TMC1 p.S647P proved to be a founder allele, contributing to 34% of genetic hearing loss in the Moroccan Jewish population. Conclusions: Critical mutations were identified in 6 of the 11 original probands and their families, leading to the identification of causative alleles in 20 additional probands and their families. The integration of genomic analysis into early clinical diagnosis of hearing loss will enable prediction of related phenotypes and enhance rehabilitation. Characterization of the proteins encoded by these genes will enable an understanding of the biological mechanisms involved in hearing loss.

POU3F4 is a POU domain transcription factor that is required for hearing. In the ear, POU3F4 is essential for mesenchymal remodeling of the bony labyrinth and is the causative gene for DFNX2 human nonsyndromic deafness. Ear abnormalities underlie this form of deafness, characterized previously in multiple spontaneous, radiation‐induced and transgenic mouse mutants. Here, we report three novel mutations in the POU3F4 gene that result in profound hearing loss in both humans and mice. A p.Gln79* mutation was identified in a child from an Israeli family, revealed by massively parallel sequencing (MPS). This strategy demonstrates the strength of MPS for diagnosis with only one affected individual. A second mutation, p.Ile285Argfs*43, was identified by Sanger sequencing. A p.Cys300* mutation was found in an ENU‐induced mutant mouse, schwindel (sdl), by positional cloning. The mutation leads to a predicted truncated protein, similar to the human mutations, providing a relevant mouse model. The p.Ile285Argfs*43 and p.Cys300* mutations lead to a shift of Pou3f4 nuclear localization to the cytoplasm, demonstrated in cellular localization studies and in the inner ears of the mutant mice. The discovery of these mutations facilitates a deeper comprehension of the molecular basis of inner ear defects due to mutations in the POU3F4 transcription factor.

Oral tongue squamous cell carcinomas (OTSCCs) have an increasing incidence in young patients, and many have an aggressive course of disease. The objective of this study was to identify candidate prognostic protein markers associated with early-onset OTSCC. We performed an exploratory screening for differential protein expression in younger (≤45 years) versus older (>45 years) OTSCC patients in The Cancer Genome Atlas (TCGA) cohort (n = 97). Expression of candidate markers was then validated in an independent Austrian OTSCC patient group (n = 34) by immunohistochemistry. Kaplan–Meier survival estimates were computed, and genomic and mRNA enrichment in silico analyses were performed. Overexpression of protein kinase C alpha (PRKCA) was significantly more frequent among young patients of both the TCGA (p = 0.0001) and the Austrian cohort (p = 0.02), associated with a negative anamnesis for alcohol consumption (p = 0.009) and tobacco smoking (p = 0.02) and poorer overall survival (univariate p = 0.02, multivariate p< 0.01). Within the young subgroup, both overall and disease-free survival were significantly decreased in patients with PRKCA overexpression (both p < 0.001). TCGA mRNA enrichment analysis revealed 332 mRNAs with significant differential expression in PRKCA-upregulated versus PRKCA-downregulated OTSCC (all FDR ≤ 0.01). Our findings suggest that PRKCA overexpression may be a hallmark of a novel molecular subtype of early-onset alcohol- and tobacco-negative high-risk OTSCC. Further analysis of the molecular PRKCA interactome may decipher the underlying mechanisms of carcinogenesis and clinicopathological behavior of PRKCA-overexpressing OTSCC.