BackgroundThe human KALRN gene, which encodes a complex, multifunctional Rho GDP/GTP exchange factor, has been linked to cardiovascular disease, psychiatric disorders and neurodegeneration. Examination of existing Kalrn knockout mouse models has focused only on neuronal phenotypes. However, Kalirin was first identified through its interaction with an enzyme involved in the synthesis and secretion of multiple bioactive peptides, and studies in C.elegans revealed roles for its orthologue in neurosecretion.ResultsWe used a broad array of tests to evaluate the effects of ablating a single exon in the spectrin repeat region of Kalrn (KalSRKO/KO); transcripts encoding Kalrn isoforms containing only the second GEF domain can still be produced from the single remaining functional Kalrn promoter. As expected, KalSRKO/KO mice showed a decrease in anxiety-like behavior and a passive avoidance deficit. No changes were observed in prepulse inhibition of acoustic startle or tests of depression-like behavior. Growth rate, parturition and pituitary secretion of growth hormone and prolactin were deficient in the KalSRKO/KO mice. Based on the fact that a subset of Kalrn isoforms is expressed in mouse skeletal muscle and the observation that muscle function in C.elegans requires its Kalrn orthologue, KalSRKO/KO mice were evaluated in the rotarod and wire hang tests. KalSRKO/KO mice showed a profound decrease in neuromuscular function, with deficits apparent in KalSR+/KO mice; these deficits were not as marked when loss of Kalrn expression was restricted to the nervous system. Pre- and postsynaptic deficits in the neuromuscular junction were observed, along with alterations in sarcomere length.ConclusionsMany of the widespread and diverse deficits observed both within and outside of the nervous system when expression of Kalrn is eliminated may reflect its role in secretory granule function and its expression outside of the nervous system.

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This study showed that knot security depends on suture material, tying technique, and number of throws, but is independent of suture size. Surgeon's knot security was greater than that for square and sliding knots when using sutures commonly used in the oral cavity. Vicryl had the greatest knot security and silk had the least. For surgeon's and square knots, at least 4 throws were generally indicated to achieve knot security; for sliding knots, at least 5 throws were generally indicated. Knot security did not increase after 5 throws and 2 throws are never indicated.

Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in‐cellulo functional assay in which we engineered site‐specific MSH2 VUS using clustered regularly interspaced short palindromic repeats‐Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild‐type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell‐based assay system for functional testing of MMR gene VUS will facilitate the identification of high‐risk LS patients.

Lynch syndrome (LS) is a hereditary cancer predisposition condition caused by inactivating germline mutations in one of the DNA mismatch repair (MMR) genes. Identifying a deleterious germline mutation by DNA sequencing is important for confirming an LS diagnosis. Frameshift and nonsense mutations significantly alter the protein product and likely impair MMR function. However, the implication of a missense mutation is often difficult to interpret. Referred to as variants of uncertain significance (VUS), their discovery hampers the definitive LS diagnosis. To determine the pathogenic significance of a VUS it is helpful to know its impact on protein function. Functional studies in the test tube and in cellular models have been performed for some VUS, however, these studies have been limited by the artificial nature of the assays. We report here an improved functional assay in which we engineered site-specific MSH2 VUS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We then characterized the impact of the variants on cellular MMR functions including DNA damage response signaling upon challenge with a DNA alkylating agent and the repair of DNA microsatellites. We classified the MMR functional capability of 8 of 10 VUS under study providing valuable information for determining their likelihood of being bona fide LS mutations. This improved human cell-based assay system for functionally testing MMR gene VUS will facilitate the identification of high risk LS patients. Significance StatementUnderstanding how cancer-associated missense variants in MMR genes affect function helps determine whether they truly contribute to disease. Laboratory assays previously utilized are limited by their artificial nature. To improve this, we introduced variants directly into the endogenous MMR loci in hESCs using CRISPR-Cas9 gene editing. This approach allows us to assess each variant while being expressed by its normal regulatory elements in a cellular environment. Our results will help guide the management of patients world-wide who carry these variants. At the same time, this study provides a technical road map for assessing the functional effects of all LS-associated variants, as well as variants linked to other genetic diseases where a cell-based functional assay is available.