Approximately one third of all mammalian genes are essential for life. Phenotypes resulting from mouse knockouts of these genes have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5000 knockout mouse lines, we have identified 410 lethal genes during the production of the first 1751 unique gene knockouts. Using a standardised phenotyping platform that incorporates high-resolution 3D imaging, we identified novel phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes identified in our screen, thus providing a novel dataset that facilitates prioritization and validation of mutations identified in clinical sequencing efforts.

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Interphase chromosomes are organized into discrete chromosome territories (CTs) that may occupy preferred sub-nuclear positions. While chromosome size and gene density appear to influence positioning, the biophysical mechanisms behind CT localization, especially the relationship between morphology and positioning, remain obscure. One reason for this has been the difficulty in imaging, segmenting, and analyzing structures with variable or imprecise boundaries. This prompted us to develop a novel approach, based on the two-dimensional (2D) wavelet-transform modulus maxima (WTMM) method, adapted to perform objective and rigorous CT segmentation from nuclear background. The wavelet transform acts as a mathematical microscope to characterize spatial image information over a continuous range of size scales. This multiresolution nature, combined with full objectivity of the formalism, makes it more accurate than intensity-based segmentation algorithms and more appropriate than manual intervention. Using the WTMM method in combination with numerical simulation models, we show that CTs have a highly nonspherical 3D morphology, that CT positioning is nonrandom, and favors heterologous CT groupings. We discuss potential relationships between morphology, positioning, chromosomal function, and instability.

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.

Primary immunodeficiencies are rare but serious diseases with diverse genetic causes. Accumulating evidence suggests that defects in DNA double-strand break (DSB) repair can underlie many of these syndromes. In this context, the nonhomologous end joining pathway of DSB repair is absolutely required for lymphoid development, but possible roles for the homologous recombination (HR) pathway have remained more controversial. While recent evidence suggests that HR may indeed be important to suppress lymphoid transformation, the specific relationship of HR to normal lymphocyte development remains unclear. We have investigated roles of the X-ray cross-complementing 2 (Xrcc2) HR gene in lymphocyte development. We show that HR is critical for normal B-cell development, with Xrcc2 nullizygosity leading to p53-dependent early S-phase arrest. In the absence of p53 (encoded by Trp53), Xrcc2-null B cells can fully develop but show high rates of chromosome and chromatid fragmentation. We present a molecular model wherein Xrcc2 is important to preserve or restore replication forks during rapid clonal expansion of developing lymphocytes. Our findings demonstrate a key role for HR in lymphoid development and suggest that Xrcc2 defects could underlie some human primary immunodeficiencies.Human immunodeficiencies are complex, multivariate diseases, the underlying genetic causes of which remain mostly unknown (9,11,31,32,35). In the past decade, defects in DNA double-strand break (DSB) repair have been implicated in some lymphodeficiencies, such as radiosensitive severe combined immunodeficiency. To resist the deleterious effects of DSBs, cells must efficiently respond to, and repair, the damage and maintain genomic integrity. Most eukaryotes employ two predominant DSB repair pathways: nonhomologous end joining (NHEJ), which catalyzes the religation of cognate broken DNA ends irrespective of sequence homology; and homologous recombination (HR), which utilizes a homologous DNA template to effect error-free DSB repair. While all cells likely incur random DSBs as a result of endogenous metabolism or exposure to environmental agents, certain cell types also deliberately generate DSBs to effect specific developmental programs (for review, see references 18 and 20). In this context, NHEJ is required to resolve DNA breaks associated with V(D)J recombination in developing lymphocytes, failure of which can lead to lymphodeficiency (2,20). In contrast, possible roles for the HR pathway, in either normal lymphocyte development or in lymphodeficiency, remain controversial. Recently the controversy has been rekindled by evidence that HR may be important to suppress T-cell lymphomagenesis (21).Here we investigate the role of the X-ray cross-complementing 2 (Xrcc2) gene in B-cell development. Xrcc2 is a member of the Rad51 gene family and was initially identified by its ability to complement DNA damage sensitivity in mutant Chinese hamster ovary (CHO) cells (34). Subsequent studies have shown XRCC2 to participate in one or more unique, multiprotei...