The N-ethyl-N-nitrosourea-induced repro42 mutation, identified by a forward genetics strategy, causes both male and female infertility, with no other apparent phenotypes. Positional cloning led to the discovery of a nonsense mutation in Spata22, a hitherto uncharacterized gene conserved among bony vertebrates. Expression of both transcript and protein is restricted predominantly to germ cells of both sexes. Germ cells of repro42 mutant mice express Spata22 transcript, but not SPATA22 protein. Gametogenesis is profoundly affected by the mutation, and germ cells in repro42 mutant mice do not progress beyond early meiotic prophase, with subsequent germ cell loss in both males and females. The Spata22 gene is essential for one or more key events of early meiotic prophase, as homologous chromosomes of mutant germ cells do not achieve normal synapsis or repair meiotic DNA double-strand breaks. The repro42 mutation thus identifies a novel mammalian germ cell-specific gene required for meiotic progression.

The ENU-induced repro8 mutation was identified in a screen to uncover genes that control mouse gametogenesis. repro8 causes male-limited infertility, with failure of spermatocytes to exit meiotic prophase via the G2/MI transition. The repro8 mutation is in the Eif4g3 gene, encoding eukaryotic translation initiation factor 4, gamma 3. Mutant germ cells appear to execute events of meiotic prophase normally, and many proteins characteristic of the prophase-to-metaphase transition are not obviously depleted. However, activity of CDC2A (CDK1) kinase is dramatically reduced in mutant spermatocytes. Strikingly, HSPA2, a chaperone protein for CDC2A kinase, is absent in mutant spermatocytes in spite of the presence of Hspa2 transcript, consistent with the observation that the repro8 phenotype is markedly similar to the phenotype of the Hspa2 knockout. Thus, EIF4G3 is required for HSPA2 translation in spermatocytes, a finding that provides the first genetic evidence for selective translational control of meiotic exit in mammalian spermatocytes.

Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data. Using these new tools, putative pathogenic mutations were identified for 91 (53%) of the strains in our study. Despite the increased power offered by potentially unlimited pedigrees and controlled breeding, about half of our exome cases remained unsolved. Using a combination of manual analyses of exome alignments and whole-genome sequencing, we provide evidence that a large fraction of unsolved exome cases have underlying structural mutations. This result directly informs efforts to investigate the similar proportion of apparently Mendelian human phenotypes that are recalcitrant to exome sequencing.

Nonsense-mediated decay (NMD) is an important process that is best known for degrading transcripts that contain premature stop codons (PTCs) to mitigate their potentially harmful consequences, although its regulatory role encompasses other classes of transcripts as well. Despite the critical role of NMD at the cellular level, our knowledge about the consequences of deficiency of its components at the organismal level is largely limited to model organisms. In this study, we report two consanguineous families in which a similar pattern of congenital anomalies was found to be most likely caused by homozygous loss-of-function mutations in SMG9, encoding an essential component of the SURF complex that generates phospho-UPF1, the single most important step in NMD. By knocking out Smg9 in mice via CRISPR/Cas9, we were able to recapitulate the major features of the SMG9-related multiple congenital anomaly syndrome we observed in humans. Surprisingly, human cells devoid of SMG9 do not appear to have reduction of PTC-containing transcripts but do display global transcriptional dysregulation. We conclude that SMG9 is required for normal human and murine development, most likely through a transcriptional regulatory role, the precise nature of which remains to be determined.

Craniofacial abnormalities are among the most common features of human genetic syndromes and disorders. The etiology of these conditions is often complex, influenced by both genetic context and the environment. Frequently, craniofacial abnormalities present as part of a syndrome with clear comorbid phenotypes, providing additional insight into mechanisms of the causative gene or pathway. The mouse has been a key tool in our understanding of the genetic mechanisms of craniofacial development and disease, and can provide excellent models for human craniofacial abnormalities. While powerful genetic engineering tools in the mouse have contributed significantly our understanding of craniofacial development and dysmorphology, forward genetic approaches provide an unbiased means to identify new genes and pathways. Moreover, spontaneous mutations can occur on any number of genetic backgrounds, potentially revealing critical genes that require a specific genetic context. Here we report discovery and phenotyping of 43 craniofacial mouse models, derived primarily from a screen for spontaneous mutations in production colonies at the Jackson Laboratory. We identify the causative gene for 33 lines, including novel genes in pathways not previously connected to craniofacial development, and novel alleles of known genes that present with unique phenotypes. Together with our detailed characterization, this work provides a valuable gene discovery resource for the craniofacial community, and a rich source of mouse models for further investigation.