Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PAPERCLIP:

Background: The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms. Results: We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems. Conclusions: Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.

Adult-onset hearing loss is very common, but we know little about the underlying molecular pathogenesis impeding the development of therapies. We took a genetic approach to identify new molecules involved in hearing loss by screening a large cohort of newly generated mouse mutants using a sensitive electrophysiological test, the auditory brainstem response (ABR). We review here the findings from this screen. Thirty-eight unexpected genes associated with raised thresholds were detected from our unbiased sample of 1,211 genes tested, suggesting extreme genetic heterogeneity. A wide range of auditory pathophysiologies was found, and some mutant lines showed normal development followed by deterioration of responses, revealing new molecular pathways involved in progressive hearing loss. Several of the genes were associated with the range of hearing thresholds in the human population and one, SPNS2, was involved in childhood deafness. The new pathways required for maintenance of hearing discovered by this screen present new therapeutic opportunities. Progressive hearing loss with age is extremely common in the population, leading to difficulties in understanding speech, increased social isolation, and associated depression. We know it has a significant heritability, but so far we know very little about the molecular pathways leading to hearing loss, hampering the development of treatments. Here, we describe a large-scale screen of 1,211 new targeted mouse mutant lines, resulting in the identification of 38 genes underlying hearing loss that were not previously suspected of involvement in hearing. Some of these genes reveal molecular pathways that may be useful targets for drug development. Our further analysis of the genes identified and the varied pathological mechanisms within the ear resulting from the mutations suggests that hearing loss is an extremely heterogeneous disorder and may have as many as 1,000 genes involved.

Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing. Progressive hearing loss is common in the human population but we know very little about the molecular mechanisms involved. Mutant mice are useful for investigating these mechanisms and have revealed a wide range of different abnormalities that can all lead to the same outcome: deafness. We report here our findings of a new mouse line with a mutation in the Spns2 gene, affecting the release of a lipid called sphingosine-1-phosphate, which has an important role in several processes in the body. For the first time, we report that this molecular pathway is required for normal hearing through a role in generating a voltage difference that acts like a battery, allowing the sensory hair cells of the cochlea to detect sounds at extremely low levels. Without the normal function of the Spns2 gene and release of sphingosine-1-phosphate locally in the inner ear, the voltage in the cochlea declines, leading to rapid loss of sensitivity to sound and ultimately to complete deafness. The human version of this gene, SPNS2, may be involved in human deafness, and understanding the underlying mechanism presents an opportunity to develop potential treatments for this form of hearing loss.

Deafness is a highly heterogenous disorder with over 100 genes known to underlie human non-syndromic hearing impairment. However, many more remain undiscovered, particularly those involved in the most common form of deafness: adult-onset progressive hearing loss. Despite several genome-wide association studies of adult hearing status, it remains unclear whether the genetic architecture of this common sensory loss consists of multiple rare variants each with large effect size or many common susceptibility variants each with small to medium effects. As next generation sequencing is now being utilised in clinical diagnosis, our aim was to explore the viability of diagnosing the genetic cause of hearing loss using whole exome sequencing in individual subjects as in a clinical setting. We performed exome sequencing of thirty patients selected for distinct phenotypic sub-types from well-characterised cohorts of 1479 people with adult-onset hearing loss. Every individual carried predicted pathogenic variants in at least ten deafness-associated genes; similar findings were obtained from an analysis of the 1000 Genomes Project data unselected for hearing status. We have identified putative causal variants in known deafness genes and several novel candidate genes, including NEDD4 and NEFH that were mutated in multiple individuals. The high frequency of predicted-pathogenic variants detected in known deafness-associated genes was unexpected and has significant implications for current diagnostic sequencing in deafness. Our findings suggest that in a clinic setting, efforts should be made to a) confirm key sequence results by Sanger sequencing, b) assess segregations of variants and phenotypes within the family if at all possible, and c) use caution in applying current pathogenicity prediction algorithms for diagnostic purposes. We conclude that there may be a high number of pathogenic variants affecting hearing in the ageing population, including many in known deafness-associated genes. Our findings of frequent predicted-pathogenic variants in both our hearing-impaired sample and in the larger 1000 Genomes Project sample unselected for auditory function suggests that the reference population for interpreting variants for this very common disorder should be a population of people with good hearing for their age rather than an unselected population.

Early-onset hearing loss is mostly of genetic origin. The complexity of the hearing process is reflected by its extensive genetic heterogeneity, with probably many causative genes remaining to be identified. Here, we aimed at identifying the genetic basis for autosomal dominant non-syndromic hearing loss (ADNSHL) in a large German family. A panel of 66 known deafness genes was analyzed for mutations by next-generation sequencing (NGS) in the index patient. We then conducted genome-wide linkage analysis, and whole-exome sequencing was carried out with samples of two patients. Expression of Osbpl2 in the mouse cochlea was determined by immunohistochemistry. Because Osbpl2 has been proposed as a target of miR-96, we investigated homozygous Mir96 mutant mice for its upregulation. Onset of hearing loss in the investigated ADNSHL family is in childhood, initially affecting the high frequencies and progressing to profound deafness in adulthood. However, there is considerable intrafamilial variability. We mapped a novel ADNSHL locus, DFNA67, to chromosome 20q13.2-q13.33, and subsequently identified a co-segregating heterozygous frameshift mutation, c.141_142delTG (p.Arg50Alafs*103), in OSBPL2, encoding a protein known to interact with the DFNA1 protein, DIAPH1. In mice, Osbpl2 was prominently expressed in stereocilia of cochlear outer and inner hair cells. We found no significant Osbpl2 upregulation at the mRNA level in homozygous Mir96 mutant mice. The function of OSBPL2 in the hearing process remains to be determined. Our study and the recent description of another frameshift mutation in a Chinese ADNSHL family identify OSBPL2 as a novel gene for progressive deafness.