Biochemical effects of inherited MMR gene mutations and diet on colon cancer risk

Colorectal cancer (CRC) is one of the leading causes of death in developed countries. Although, a small fraction of cancers are caused by inherited genetic predisposition most of the CRCs are sporadic. In CRC, cancer risk is associated with lifestyle factors and aging. Even in dominantly inherited CRC predisposition such as in Lynch syndrome (LS), which is linked to germline mu- tations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2, cancer develops as a result of accumulation of genetic and epigenetic changes. After diagnosing an LS family, to be able to offer contiguous pre-symptomatic surveillance and predictive gene counseling to mutation carriers in a family, the pathogenicity assessment of a mutation is needed. Dependent on the type and the site of a germline mutation, inherited cancer risk may vary from high to low and especially in the latter case cancer risk may be strongly affected by lifestyle factors such as diet. Epidemiological studies on humans and previous studies on mice have shown that especially a Western-style diet (WD) may predispose colon mucosa to CRC. However, the mechanisms, which mediate the effects of diet on tumorigenesis are largely unknown. Since both genetic and lifestyle factors have been shown to predispose to cancer, this the- sis analyzed biochemical defects caused by inherited MMR gene mutations and Western diet exposure. Different MMR gene mutations may compromise MMR function through various biochemical defects. Here, we studied 18 inherited non-truncating mutations in MSH2, the second most frequently mutated gene among Lynch syndrome patients. We assessed protein stability, DNA binding, and ATP mediated DNA release abilities of the MSH2 variants. The majority of variants in the amino terminal region including the connector and lever domains p.V161D, p.G162R, p.G164R, p.L173P, p.L187P, p.C333Y, p.D603N) affected protein stabil- ity. Variations in the ATPase domain (p.A636P, p.G674A, p.C697F, p.I745-I746del, p.E749K) totally abolished either mismatch binding or release. Four protein variants (p.T33P, p.A272 V, p.G322D, p.V923E) expressed slightly reduced mismatch binding and/or release efficiencies compared to wild-type (WT) MSH2 protein, while two variants (p.N127S, p.A834T) were in- distinguishable from WT. To define the effects of Western-style diet, we analyzed protein expression changes in histolog- ically normal colon mucosa of wild type (Mlh1+/+) and CRC predisposed mice (Mlh1+/-) after a long-term feeding experiment with WD and AIN-93G control diet. Using network analysis and data mining we also determined which of the affected proteins might be putative play- ers in early CRC development. Our results pinpoint changes in a complex protein interaction network involved in ATP synthesis coupled proton transport, oxidoreduction coenzyme and nicotinamide nucleotide metabolic processes, which are important in the generation of reactive oxygen species (ROS) and cellular protection against ROS toxicity. Additionally, we detected SELENBP1 and LGALS4, which are implied in neoplastic processes. Our studies show that mutations in the MMR gene affect the biochemistry of MMR, can have an effect on the phenotype of the mutation carriers and in the latest study suggest that the high sensitivity to Western diet may be linked to haplo-insufficiency caused by a loss of function mutation in the Mlh1+/- mice.Colorectal cancer (CRC) is one of the leading causes of death in developed countries. Although, a small fraction of cancers are caused by inherited genetic predisposition most of the CRCs are sporadic. In CRC, cancer risk is associated with lifestyle factors and aging. Even in dominantly inherited CRC predisposition such as in Lynch syndrome (LS), which is linked to germline mu- tations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2, cancer develops as a result of accumulation of genetic and epigenetic changes. After diagnosing an LS family, to be able to offer contiguous pre-symptomatic surveillance and predictive gene counseling to mutation carriers in a family, the pathogenicity assessment of a mutation is needed. Dependent on the type and the site of a germline mutation, inherited cancer risk may vary from high to low and especially in the latter case cancer risk may be strongly affected by lifestyle factors such as diet. Epidemiological studies on humans and previous studies on mice have shown that especially a Western-style diet (WD) may predispose colon mucosa to CRC. However, the mechanisms, which mediate the effects of diet on tumorigenesis are largely unknown. Since both genetic and lifestyle factors have been shown to predispose to cancer, this the- sis analyzed biochemical defects caused by inherited MMR gene mutations and Western diet exposure. Different MMR gene mutations may compromise MMR function through various biochemical defects. Here, we studied 18 inherited non-truncating mutations in MSH2, the second most frequently mutated gene among Lynch syndrome patients. We assessed protein stability, DNA binding, and ATP mediated DNA release abilities of the MSH2 variants. The majority of variants in the amino terminal region including the connector and lever domains p.V161D, p.G162R, p.G164R, p.L173P, p.L187P, p.C333Y, p.D603N) affected protein stabil- ity. Variations in the ATPase domain (p.A636P, p.G674A, p.C697F, p.I745-I746del, p.E749K) totally abolished either mismatch binding or release. Four protein variants (p.T33P, p.A272 V, p.G322D, p.V923E) expressed slightly reduced mismatch binding and/or release efficiencies compared to wild-type (WT) MSH2 protein, while two variants (p.N127S, p.A834T) were in- distinguishable from WT. To define the effects of Western-style diet, we analyzed protein expression changes in histolog- ically normal colon mucosa of wild type (Mlh1+/+) and CRC predisposed mice (Mlh1+/-) after a long-term feeding experiment with WD and AIN-93G control diet. Using network analysis and data mining we also determined which of the affected proteins might be putative play- ers in early CRC development. Our results pinpoint changes in a complex protein interaction network involved in ATP synthesis coupled proton transport, oxidoreduction coenzyme and nicotinamide nucleotide metabolic processes, which are important in the generation of reactive oxygen species (ROS) and cellular protection against ROS toxicity. Additionally, we detected SELENBP1 and LGALS4, which are implied in neoplastic processes. Our studies show that mutations in the MMR gene affect the biochemistry of MMR, can have an effect on the phenotype of the mutation carriers and in the latest study suggest that the high sensitivity to Western diet may be linked to haplo-insufficiency caused by a loss of function mutation in the Mlh1+/- mice

Single-Cell Sequencing of Mouse Thymocytes Reveals Mutational Landscape Shaped by Replication Errors, Mismatch Repair, and H3K36me3

DNA mismatch repair (MMR) corrects replication errors and is recruited by the histone mark H3K36me3, enriched in exons of transcriptionally active genes. To dissect in vivo the mutational landscape shaped by these processes, we employed single-cell exome sequencing on T cells of wild-type andMMR-deficient (Mlh1(-/-)) mice. Within active genes, we uncovered a spatial bias in MMR efficiency: 3' exons, often H3K36me3-enriched, acquire significantly fewer MMR-dependent mutations compared with 5' exons. Huwe1 and Mcm7 genes, both active during lymphocyte development, stood out as mutational hotspots in MMR-deficient cells, demonstrating their intrinsic vulnerability to replication error in this cell type. Both genes are H3K36me3-enriched, which can explain MMR-mediated elimination of replication errors in wild-type cells. Thus, H3K36me3 can boost MMR in transcriptionally active regions, both locally and globally. This offers an attractive concept of thriftyMMR targeting, where critical genes in each cell type enjoy preferential shielding against de novo mutations.Peer reviewe

Western Diet Deregulates Bile Acid Homeostasis, Cell Proliferation, and Tumorigenesis in Colon

Western-style diets (WD) high in fat and scarce in fiber and vitamin D increase risks of colorectal cancer. Here, we performed a long-term diet study in mice to follow tumorigenesis and characterize structural and metabolic changes in colon mucosa associated with WD and predisposition to colorectal cancer. WD increased colon tumor numbers, and mucosa proteomic analysis indicated severe deregulation of intracellular bile acid (BA) homeostasis and activation of cell proliferation. WD also in-creased crypt depth and colon cell proliferation. Despite increased luminal BA, colonocytes from WD-fed mice exhibited decreased expression of the BA transporters FABP6, OST beta, and ASBT and decreased concentrations of secondary BA deoxycholic acid and lithocholic acid, indicating reduced activity of the nuclear BA receptor FXR. Overall, our results suggest that WD increases cancer risk by FXR inactivation, leading to BA deregulation and increased colon cell proliferation. (C) 2017 AACR.Peer reviewe

A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature

Single-cell transcriptomics promise to revolutionize our understanding of the vasculature. Emerging computational methods applied to high-dimensional single-cell data allow integration of results between samples and species and illuminate the diversity and underlying developmental and architectural organization of cell populations. Here, we illustrate these methods in the analysis of mouse lymph node (LN) lymphatic endothelial cells (LEC) at single-cell resolution. Clustering identifies five well-delineated subsets, including two medullary sinus subsets not previously recognized as distinct. Nearest neighbor alignments in trajectory space position the major subsets in a sequence that recapitulates the known features and suggests novel features of LN lymphatic organization, providing a transcriptional map of the lymphatic endothelial niches and of the transitions between them. Differences in gene expression reveal specialized programs for (1) subcapsular ceiling endothelial interactions with the capsule connective tissue and cells; (2) subcapsular floor regulation of lymph borne cell entry into the LN parenchyma and antigen presentation; and (3) pathogen interactions and (4) LN remodeling in distinct medullary subsets. LEC of the subcapsular sinus floor and medulla, which represent major sites of cell entry and exit from the LN parenchyma respectively, respond robustly to oxazolone inflammation challenge with enriched signaling pathways that converge on both innate and adaptive immune responses. Integration of mouse and human single-cell profiles reveals a conserved cross-species pattern of lymphatic vascular niches and gene expression, as well as specialized human subsets and genes unique to each species. The examples provided demonstrate the power of single-cell analysis in elucidating endothelial cell heterogeneity, vascular organization, and endothelial cell responses. We discuss the findings from the perspective of LEC functions in relation to niche formations in the unique stromal and highly immunological environment of the LN

Cancer-Predicting Gene Expression Changes in Colonic Mucosa of Western Diet Fed Mlh1(+/-) Mice

Peer reviewe

A molecular map of murine lymph node blood vascular endothelium at single cell resolution

Blood vascular endothelial cells (BECs) control the immune response by regulating blood flow and immune cell recruitment in lymphoid tissues. However, the diversity of BEC and their origins during immune angiogenesis remain unclear. Here we profile transcriptomes of BEC from peripheral lymph nodes and map phenotypes to the vasculature. We identify multiple subsets, including a medullary venous population whose gene signature predicts a selective role in myeloid cell (vs lymphocyte) recruitment to the medulla, confirmed by videomicroscopy. We define five capillary subsets, including a capillary resident precursor (CRP) that displays stem cell and migratory gene signatures, and contributes to homeostatic BEC turnover and to neogenesis of high endothelium after immunization. Cell alignments show retention of developmental programs along trajectories from CRP to mature venous and arterial populations. Our single cell atlas provides a molecular roadmap of the lymph node blood vasculature and defines subset specialization for leukocyte recruitment and vascular homeostasis

Exploration of cell development pathways through high-dimensional single cell analysis in trajectory space

High-dimensional single cell profiling coupled with computational modeling is emerging as a powerful tool to elucidate developmental programs directing cell lineages. We introduce tSpace, an algorithm based on the concept of "trajectory space", in which cells are defined by their distance along nearest neighbor pathways to every other cell in a population. Graphical mapping of cells in trajectory space allows unsupervised reconstruction and exploration of complex developmental sequences. Applied to flow and mass cytometry data, the method faithfully reconstructs thymic T cell development and reveals development and trafficking regulation of tonsillar B cells. Applied to the single cell transcriptome of mouse intestine and C. elegans, the method recapitulates development from intestinal stem cells to specialized epithelial phenotypes more faithfully than existing algorithms and orders C. elegans cells concordantly to the associated embryonic time. tSpace profiling of complex populations is well suited for hypothesis generation in developing cell systems

Inferring direction of associations between histone modifications using a neural processes-based framework

Summary: Current technologies do not allow predicting interactions between histone post-translational modifications (HPTMs) at a system-level. We describe a computational framework, imputation-followed-by-inference, to predict directed association between two HPTMs using EpiTOF, a mass cytometry-based platform that allows profiling multiple HPTMs at a single-cell resolution. Using EpiTOF profiles of >55,000,000 peripheral mononuclear blood cells from 158 healthy human subjects, we show that neural processes (NP) have significantly higher accuracy than linear regression and k-nearest neighbors models to impute the abundance of an HPTM. Next, we infer the direction of association to show we recapitulate known HPTM associations and identify several previously unidentified ones in healthy individuals. Using this framework in an influenza vaccine cohort, we identify changes in associations between 6 pairs of HPTMs 30 days following vaccination, of which several have been shown to be involved in innate memory. These results demonstrate the utility of our framework in identifying directed interactions between HPTMs

A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature

Single-cell transcriptomics promise to revolutionize our understanding of the vasculature. Emerging computational methods applied to high-dimensional single-cell data allow integration of results between samples and species and illuminate the diversity and underlying developmental and architectural organization of cell populations. Here, we illustrate these methods in the analysis of mouse lymph node (LN) lymphatic endothelial cells (LEC) at single-cell resolution. Clustering identifies five well-delineated subsets, including two medullary sinus subsets not previously recognized as distinct. Nearest neighbor alignments in trajectory space position the major subsets in a sequence that recapitulates the known features and suggests novel features of LN lymphatic organization, providing a transcriptional map of the lymphatic endothelial niches and of the transitions between them. Differences in gene expression reveal specialized programs for (1) subcapsular ceiling endothelial interactions with the capsule connective tissue and cells; (2) subcapsular floor regulation of lymph borne cell entry into the LN parenchyma and antigen presentation; and (3) pathogen interactions and (4) LN remodeling in distinct medullary subsets. LEC of the subcapsular sinus floor and medulla, which represent major sites of cell entry and exit from the LN parenchyma respectively, respond robustly to oxazolone inflammation challenge with enriched signaling pathways that converge on both innate and adaptive immune responses. Integration of mouse and human single-cell profiles reveals a conserved cross-species pattern of lymphatic vascular niches and gene expression, as well as specialized human subsets and genes unique to each species. The examples provided demonstrate the power of single-cell analysis in elucidating endothelial cell heterogeneity, vascular organization, and endothelial cell responses. We discuss the findings from the perspective of LEC functions in relation to niche formations in the unique stromal and highly immunological environment of the LN