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Biology subjects

Nakagawa, H.

Publications and source records attributed to Nakagawa, H..

At least 19 recordsLinked to original sources

MARiO: predicting cancer variant pathogenicity by integrating in silico evaluation and patient-level mutational contexts

Comprehensive genomic profiling (CGP) supports precision medicine in cancer care, but accurate assessment of missense variant pathogenicity, especially for variants without established consensus, remains challenging. Various computational tools have been developed for variant functional prediction, but most current tools rely solely on variant-level features and do not capture the clinical context of individual patients. To address this limitation, we developed MARiO (Missense Alteration Risk for Oncogenicity), a machine-learning model that integrates variant-level features and patient-level clinical and genomic contexts to effectively predict the pathogenicity of missense variants in cancer. We collected a total of 10,642 missense variants from 1271 patients, and evaluated candidate features for their association with variant pathogenicity, identifying informative features including in silico functional predictions, population allele frequency, variant allele frequency, and tumor mutational burden. Using these selected features, MARiO was developed with extreme gradient boosting. The model integrates multiple in silico prediction tools and patient-specific genomic contexts while accommodating missing values frequently observed in real-world CGP datasets. MARiO outperformed existing tools, achieving an area under the receiver operating characteristic curve of 0.942. The model demonstrated strong generalizability across multiple external datasets and showed consistency with real-world molecular treatment proposals. MARiO offers a robust and clinically relevant approach for missense variant pathogenicity assessment by integrating variant- and patient-level features and serves as a valuable tool to support clinical decision-making.

bioinformatics↗

Early detection of pancreatic ductal adenocarcinoma by single-molecule profiling of pancreatic enzyme activities

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related mortality, largely due to diagnosis at advanced stages. Early detection through minimally invasive liquid biopsy holds promise for improving patient outcomes. Here, we report a blood-based liquid biopsy platform based on single-molecule enzyme activity profiling (SEAP), which detects proteoform-level alterations in circulating pancreatic enzymes at single-molecule resolution. Using a tissue-centric biomarker discovery strategy, we identified activity signatures of pancreas-specific digestive enzymes associated with PDAC. A combinatorial classifier detected PDAC (stage I-IV) with 95.5% specificity and 75.0% sensitivity (74.2% for stage I-II) across 690 blood samples collected from multiple hospitals and biobanks. Performance was further validated in an independent cohort enriched for early-stage disease, where 54.2% (13/24) of stage IA, 64.3% (9/14) of stage IB, and 21.4% (3/14) of stage 0 lesions were classified as positive. These findings support the clinical potential of SEAP for early detection of PDAC.

biochemistry↗

Extrinsic MMPs drive epithelial shape change via basal ECM disassembly in the Drosophila wing disc

Epithelial morphogenesis generates complex tissue architectures with remarkable reproducibility, yet how such self-organized shape transformations are triggered in vivo remains poorly understood. Here, we show that epithelial morphogenesis during early pupal development of the Drosophila wing disc is regulated non-autonomously through remodeling of the basal extracellular matrix (ECM) by neighboring tissues. Live imaging analyses reveal a previously unrecognized epithelial shape transition at the larval-to-pupal stage, in which the wing disc epithelium transforms from a concave to a convex configuration. Rather than being driven autonomously by the epithelium, this morphogenetic transition depends on adjacent non-epithelial cell populations, including myoblasts and tracheal cells. Mechanistically, systemic ecdysone signaling activates Mmp1 and Mmp2 in these neighboring tissues, leading to spatially restricted basal ECM disassembly that permits collective epithelial shape reorganization. Together, our findings establish ECM remodeling at tissue interfaces as a non-autonomous regulatory layer that enables epithelial self-organization during development.

developmental biology↗

MitoDelta: identifying mitochondrial DNA deletions at cell-type resolution from single-cell RNA sequencing data

BackgroundDeletion variants in mitochondrial DNA (mtDNA) are associated with various diseases, such as mitochondrial disorders and neurodegenerative diseases. Traditionally, mtDNA deletions have been studied using bulk DNA sequencing, but bulk methods average signals across cells, thereby masking the cell-type-specific mutational landscapes. Resolving mtDNA deletions at single-cell resolution is beneficial for understanding how these mutations affect distinct cell populations. To date, no specialized method exists for detecting cell-type-specific mtDNA deletions from single-cell RNA sequencing data. Notably, mtDNA possesses unique molecular features: a high copy number, stable transcription, and compact structure of the mitochondrial genome. This results in a relatively high abundance of mtDNA-derived reads even in single-cell RNA sequencing data, suggesting the possibility of detecting mtDNA deletion variants directly from transcriptomic data. ResultsHere, we present MitoDelta, a computational pipeline that enables the detection of mtDNA deletions at cell-type resolution solely from single-cell RNA sequencing data. MitoDelta combines a sensitive alignment strategy with robust statistical filtering based on a beta-binomial model, allowing accurate identification of deletion events even from noisy single-cell transcriptomes. To capture cell-type-specific deletion patterns, MitoDelta analyzes reads pooled by annotated cell types, enabling quantification of deletion burden across distinct cellular populations. We benchmarked MitoDelta against existing mtDNA deletion detection tools and demonstrated superior overall performance. As a practical application, we applied MitoDelta to a published single-nucleus RNA sequencing dataset for Parkinsons disease and revealed distinct mtDNA deletion burdens across neuronal subtypes. ConclusionsMitoDelta enables the transcriptome-integrated, cell-type-specific detection of mtDNA deletions from single-cell RNA sequencing data alone, offering a valuable framework for reanalyzing public datasets and studying mitochondrial genome alterations at cell-type resolution. This integrated approach enables insights into how mtDNA deletions are distributed across specific cell types and cellular states, providing new opportunities to investigate the role of mtDNA deletions in cell-type-specific disease mechanisms. The tool is available at https://github.com/NikaidoLaboratory/mitodelta.

bioinformatics↗

Multi-color fluorescence live-cell imaging in Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum, a member of the Amoebozoa, has been extensively studied in cell and developmental biology. D. discoideum is unique in that they are genetically tractable, with a wealth of data accumulated over half a century of research. Fluorescence live-cell imaging of D. discoideum has greatly facilitated studies on fundamental topics, including cytokinesis, phagocytosis, and cell migration. Additionally, its unique life cycle places Dictyostelium at the forefront of understanding aggregative multicellularity, a recurring evolutionary trait found across the Opisthokonta and Amoebozoa clades. The use of multiple fluorescent proteins (FP) and labels with separable spectral properties is critical for tracking cells in aggregates and identifying co-occurring biomolecular events and factors that underlie the dynamics of the cytoskeleton, membrane lipids, second messengers, and gene expression. However, in D. discoideum, the number of frequently used FP species is limited to two or three. In this study, we explored the use of new-generation FP for practical 4- to 5-color fluorescence imaging of D. discoideum. We showed that the yellow fluorescent protein Achilles and the red fluorescent protein mScarlet-I both yield high signals and allow sensitive detection of rapid gene induction. The color palette was further expanded to include blue (mTagBFP2 and mTurquosie2), large Stoke-shift LSSmGFP, and near-infrared (miRFP670nano3) FPs, in addition to the HaloTag ligand SaraFluor 650T. Thus, we demonstrated the feasibility of deploying 4- and 5- color imaging of D. discoideum using conventional confocal microscopy.

cell biology↗

EPHA2 Regulates SOX2 during Esophageal Development

The human esophagus, derived from the anterior foregut endoderm, requires proper dorsal-ventral patterning for development. The transcription factor SOX2, crucial in this process, when dysregulated, leads to congenital esophageal abnormalities. EPHA2, a receptor tyrosine kinase, is vital in various developmental processes and cancer models, where it activates SOX2. This study demonstrates that EPHA2 regulates SOX2 expression during esophageal development using human iPSCs and iPSC-derived human esophageal organoids (HEO). Inhibition of EPHA2 decreased iPSC-derived HEO formation and SOX2 expression. These findings provide evidence of EPHA2 as being a key regulator of SOX2 signaling in early esophageal development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/617209v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@16b8a8aorg.highwire.dtl.DTLVardef@1548672org.highwire.dtl.DTLVardef@46e28corg.highwire.dtl.DTLVardef@1c35578_HPS_FORMAT_FIGEXP M_FIG SFD: Serum-Free Differentiation media; EPC: esophageal progenitor cells; HEO: human esophageal organoids Created with BioRender.com C_FIG HighlightsO_LISOX2 is crucial for proper esophageal development. C_LIO_LIEPHA2 is a receptor tyrosine kinase involved in various developmental processes. C_LIO_LIEPHA2 activates SOX2. C_LIO_LIInhibition of EPHA2 decreased SOX2 expression and human esophageal organoid formation. C_LI

developmental biology↗

Modeling epithelial homeostasis and perturbation in three-dimensional human esophageal organoids

BackgroundEsophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbeccos Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. MethodsWe have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-{beta} receptor-mediated signaling, both key regulators of proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. ResultsThe formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGF{beta} receptor-mediated signaling. In optimized HOME0, normal human esophageal organoid formation was improved, whereas IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.

cell biology↗

Adaptive evolution of sesquiterpene deoxyphomenone in mycoparasitism by Hansfordia pulvinata associated with horizontal gene transfer from Aspergillus species

Leaf mold caused by the ascomycete fungus Cladosporium fulvum is a devastating disease of tomato plants. The mycoparasitic fungus Hansfordia pulvinata is an effective biocontrol agent that parasitizes C. fulvum hyphae on leaves and secretes 13-deoxyphomenone, an eremophilane-type sesquiterpene, which was also identified as a sporulation-inducing factor in Aspergillus oryzae. Here, we identified deoxyphomenone biosynthesis (DPH) gene clusters conserved in both H. pulvinata and Aspergillus section Flavi including A. oryzae and A. flavus. Functional disruption of DPH1 orthologous genes encoding sesquiterpene cyclase in H. pulvinata, A. oryzae and its close relative A. flavus revealed that deoxyphomenone in H. pulvinata had exogenic antifungal activity against the host fungus C. fulvum and controlled endogenic sporulation in Aspergillus species. Deoxyphomenone also inhibited mycelial growth of C. fulvum and the non-host tomato pathogen Pseudocercospora fuligena. Complete DPH clusters, highly similar to those in H. pulvinata, were exclusive to Aspergillus section Flavi, while species in other Aspergillus sections contained fragmented DPH clusters. A comparative genomics analysis revealed that these DPH gene clusters share a common origin and are horizontally transferred across large taxonomic distances from an ancestor of Aspergillus to H. pulvinata. Our results suggest that, after horizontal transfer, H. pulvinata maintained the DPH cluster as the inhibitory effect of deoxyphomenone on spore germination and mycelial growth contributed to its mycoparasitism on the host fungus C. fulvum.

microbiology↗

Cancer-associated DNA Hypermethylation of Polycomb Targets Requires DNMT3A Dual Recognition of Histone H2AK119 Ubiquitination and the Nucleosome Acidic Patch

During tumor development, promoter CpG islands (CGIs) that are normally silenced by Polycomb repressive complexes (PRCs) become DNA hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) catalyze CpG methylation at PRC-regulated regions remains unclear. Here we report a cryo-EM structure of the DNMT3A long isoform (DNMT3A1) N-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine 119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 N-terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Furthermore, aberrant redistribution of DNMT3A1 to Polycomb target genes inhibits their transcriptional activation during cell differentiation and recapitulates the cancer-associated DNA hypermethylation signature. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for countering promoter CGI DNA hypermethylation, a major molecular hallmark of cancer.

cancer biology↗

ALDH2 dysfunction accelerates ESCC pathogenesis

The alcohol metabolite acetaldehyde is a potent human carcinogen. Aldehyde dehydrogenase 2 (ALDH2) is the primary enzyme that detoxifies acetaldehyde in the mitochondria. Acetaldehyde accumulates and causes genotoxic stress in cells expressing the dysfunctional ALDH2E487K mutant protein linked to ALDH2*2, the single nucleotide polymorphism highly prevalent amongst East Asians. Chronic alcohol users with heterozygous ALDH2*2 display an increased risk for the development of esophageal squamous cell carcinoma (ESCC) and other alcohol-related cancers. However, how ALDH2 influences ESCC pathobiology is incompletely understood. Herein, we characterize how ESCC and preneoplastic cells respond to alcohol exposure using cell lines, three dimensional organoids, and xenograft models. We find that alcohol exposure results in increased organoid formation and tumor growth concurrent with increased reactive oxygen species (ROS), increased DNA damage, and the enrichment of putative cancer stem cells (CSCs) characterized by high CD44 expression. Pharmacological activation of ALDH2 function by Alda-1 inhibits this phenotype, indicating that acetaldehyde is the primary driver of these changes. ALDH2 dysfunction also affects response to a commonly used chemotherapy for the treatment of ESCC. We find that Aldh2 dysfunction facilitated enrichment of CSCs following cisplatin-induced cell death and oxidative stress in murine organoids. Together, these data provide evidence that alcohol exposure, results in more aggressive tumors through enrichment of CSCs, which is augmented by ALDH2 dysfunction.

cancer biology↗

Tumor Niche Network-Defined Subtypes Predict Immunotherapy Response of Esophageal Squamous Cell Cancer

Despite the promising outcomes of immune checkpoint blockade (ICB), resistance to ICB presents a new challenge. Therefore, selecting patients for specific ICB applications is crucial for maximizing therapeutic efficacy. Herein we curated 69 human esophageal squamous cell cancer (ESCC) patients tumor microenvironment (TME) single-cell transcriptomic datasets to subtype ESCC. Integrative analyses of the cellular network transcriptional signatures of T cells, myeloid cells, and fibroblasts define distinct ESCC subtypes characterized by T cell exhaustion, Interferon (IFN) a/b signaling, TIGIT enrichment, and specific marker genes. Furthermore, this approach classifies ESCC patients into ICB responders and non-responders, as validated by liquid biopsy single-cell transcriptomics. Our study stratifies ESCC patients based on TME transcriptional network, providing novel insights into tumor niche remodeling and predicting ICB responses in ESCC patients.

cancer biology↗

COOBoostR: an extreme gradient boosting-based tool for robust tissue or cell-of-origin prediction of tumors

We here present COOBoostR (https://github.com/SWJ9385/COOBoostR), a computational method designed for the putative prediction of tissue-or cell-of-origin of various cancer types. COOBoostR leverages regional somatic mutation density information and chromatin mark features to be applied to an extreme gradient boosting-based machine-learning algorithm. COOBoostR ranks chromatin marks from various tissue and cell types which best explain the somatic mutation density landscape of any sample of interest. Through integrating either ChIP-seq based chromatin data or bulk/single cell chromatin accessibility data along with regional somatic mutation density data derived from normal cells/tissue, precancerous lesions, and cancer types, we show that COOBoostR outperforms existing random forest-based methods in prediction speed with comparable or better tissue or cell-of-origin prediction performance. In addition, our results suggest a dynamic somatic mutation accumulation at the normal tissue or cell stage which could be intertwined with the changes in open chromatin marks and enhancer sites. These results further represent chromatin marks shaping the somatic mutation landscape at the early stage of mutation accumulation, possibly even before the initiation of precancerous lesions or neoplasia.

bioinformatics↗

Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion

Background and aimsDespite recent progress in identifying aberrant genetic and epigenetic alterations in esophageal squamous cell carcinoma (ESCC), the mechanism of ESCC initiation remains unknown. MethodsUsing CRISPR/Cas 9-based genetic ablation, we targeted 9 genes (TP53, CDKN2A, NOTCH1, NOTCH3, KMT2D, KMT2C, FAT1, FAT4, and AJUBA) in murine esophageal organoids (EOs). Transcriptomic phenotypes of organoids and chemokine released by organoids were analyzed by single-cell RNA sequencing (scRNA-seq). Tumorigenicity and immune evasion of organoids were monitored by allograft transplantation. Human ESCC scRNA-seq datasets were analyzed to classify patients and find subsets relevant to organoid models and immune evasion. ResultsWe established 32 genetically engineered EOs and identified key genetic determinants that drive ESCC initiation. A single-cell transcriptomic analysis uncovered that Trp53, Cdkn2a, and Notch1 (PCN) triple-knockout (KO) induces neoplastic features of ESCC by generating cell lineage heterogeneity and high cell plasticity. PCN KO also generates an immunosuppressive niche enriched with exhausted T cells and M2 macrophages via the CCL2-CCR2 axis. Mechanistically, CDKN2A inactivation transactivates CCL2 via NF-{kappa}B. Moreover, comparative single-cell transcriptomic analyses stratified ESCC patients and identified a specific subtype recapitulating the PCN-type ESCC signatures, including the high expression of CCL2 and CD274/PD-L1. ConclusionsOur study unveils that loss of TP53, CDKN2A, and NOTCH1 induces esophageal neoplasia and immune evasion for ESCC initiation and proposes the CCL2 blockade as a viable option for targeting PCN-type ESCC.

cancer biology↗

Cell-surface protein YwfG of Lactococcus lactis binds to α-1,2-linked mannose

Lactococcus lactis strains are used as starter cultures in the production of fermented dairy and vegetable foods, but the species also occurs in other niches such as plant material. Lactococcus lactis subsp. lactis G50 (G50) is a plant-derived strain and potential candidate probiotics. Western blotting of cell-wall proteins using antibodies generated against whole G50 cells detected a 120-kDa protein. MALDI-TOF MS analysis identified it as YwfG, a Leu-Pro-any-Thr-Gly cell-wall-anchor-domain-containing protein. Based on a predicted domain structure, a recombinant YwfG variant covering the N-terminal half (aa 28-511) of YwfG (YwfG28-511) was crystallized and the crystal structure was determined. The structure consisted of an L-type lectin domain, a mucin-binding protein domain, and a mucus-binding protein repeat. Recombinant YwfG variants containing combinations of these domains (YwfG28-270, YwfG28-311, YwfG28-511, MubR4) were prepared and their interactions with monosaccharides were examined by isothermal titration calorimetry; the only interaction observed was between YwfG28-270, which contained the L-type lectin domain, and D-mannose. Among four mannobioses, -1,2-mannobiose had the highest affinity for YwfG28-270 (dissociation constant = 34 M). YwfG28-270 also interacted with yeast mannoproteins and yeast mannan. Soaking of the crystals of YwfG28-511 with mannose or -1,2-mannobiose revealed that both sugars bound to the L-type lectin domain in a similar manner, although the presence of the mucin-binding protein domain and the mucus-binding protein repeat within the recombinant protein inhibited the interaction between the L-type lectin domain and mannose residues. Three of the YwfG variants (except MubR4) induced aggregation of yeast cells. Strain G50 also induced aggregation of yeast cells, which was abolished by deletion of ywfG from G50, suggesting that surface YwfG contributes to the interaction with yeast cells. These findings provide new structural and functional insights into the interaction between L. lactis and its ecological niche via binding of the cell-surface protein YwfG with mannose.

microbiology↗

Human papilloma virus E6 regulates therapy responses in oropharyngeal cancer by repressing the PGC-1α/ERRα axis

Therapy with radiation plus cisplatin kills human papilloma virus-related (HPV+) oropharyngeal squamous cell carcinomas (OPSCCs) by increasing reactive oxygen species beyond cellular antioxidant capacity. To explore why some patients fail these standard treatments, we evaluated whether the variation in HPV oncoprotein levels among HPV+ OPSCCs impacts mitochondrial metabolism, a source of antioxidant capacity. In cell line and patient-derived xenograft models, levels of HPV full-length E6 (fl-E6) inversely correlated with oxidative phosphorylation, antioxidant capacity, and therapy resistance, and fl-E6 was the only HPV oncoprotein to display such correlation. Ectopically expressing fl-E6 in models with low levels reduced mitochondrial mass, depleted antioxidant capacity, and sensitized to therapy. In this setting, fl-E6 repressed the PGC-1/ERR pathway for mitochondrial biogenesis by reducing p53-dependent PGC-1 transcription. Concordant observations were made in three clinical cohorts, where expression of mitochondrial components was higher in tumors of patients with reduced survival. These tumors contained the lowest fl-E6 levels, highest p53 target gene expression, and an activated PGC-1/ERR pathway. Our findings demonstrate that E6 can potentiate treatment responses by depleting mitochondrial antioxidant capacity and provide evidence for low E6 negatively impacting patient survival. E6s interaction with the PGC-1/ERR axis has implications for predicting and targeting treatment resistance in OPSCC.

cancer biology↗

Comprehensive analysis of full-length transcripts reveal aberrations of splicing variants in liver cancer

Genes generate various transcripts by alternative splicing, and these transcripts can have diverse functions. However, in most transcriptome studies, short-reads sequencing technologies (next-generation sequencers) have been used and full-length transcripts have not been observed directly. Although long-reads sequencing technologies would enable us to sequence full-length transcripts, analysis of the data is a difficult task. In the present study, we developed an analysis pipeline named SPLICE to analyze full-length cDNA sequences. Using this method, we analyzed cDNA sequences from 42 pairs of hepatocellular carcinoma (HCC) and matched non-cancerous liver with Oxford Nanopore technology. Our analysis detected 46,663 transcripts from the protein-coding genes in the HCCs and the matched non-cancerous livers, of which 5,366 (11.5 %) were novel. Comparison of expression levels identified 9,933 differentially expressed transcripts (DETs) in 4,744 genes. Importantly, 746 genes with DET were not found by the gene-level analysis. We also identified novel exons derived from transposable elements (TEs). In the analysis of transcripts from hepatitis B virus (HBV), HBx-human TE fusions were found to be overexpressed in the HCCs. Furthermore, fusion gene detection showed novel recurrent fusion events. These results suggest that long-reads sequencing technologies allow us to analyze full-length transcripts, and show the importance of splicing variants in carcinogenesis.

cancer biology↗

Aberrant integration of Hepatitis B virus DNA promotes major restructuring of human hepatocellular carcinoma genome architecture

Most cancers are characterized by the somatic acquisition of genomic rearrangements during tumour evolution that eventually drive the oncogenesis. There are different mutational mechanisms causing structural variation, some of which are specific to particular cancer types. Here, using multiplatform sequencing technologies, we identify and characterize a remarkable mutational mechanism in human hepatocellular carcinoma caused by Hepatitis B virus, by which DNA molecules from the virus are inserted into the tumour genome causing dramatic changes in its configuration, including non-homologous chromosomal fusions and megabase-size telomeric deletions. This aberrant mutational process, present in at least 8% of all HCC tumours, is active early during liver cancer evolution and can provide the driver rearrangements that a cancer clone requires to survive and grow.

cancer biology↗

Population-wide Screening for Germline Variants of Hereditary Cancer Genes in 12K Unselected Japanese Colorectal Cancers and 27K Controls

Background & AimsColorectal cancer (CRC) is one of the most common cancers in Western countries and Japan. Currently, a few % of CRCs can be attributed to recognizable hereditary germline variants of known CRC susceptibility genes, predominantly the DNA mismatch repair genes. To establish a universal screening strategy for hereditary CRCs, it is necessary to explore the prevalence of hereditary CRC and pathogenic variants of multiple cancer-predisposing genes in non-European populations. MethodsWe analyzed the coding regions of 27 cancer-predisposing genes, including mismatch repair genes, APC, and BRCA1/2, in 12,503 unselected Japanese CRC patients and 23,705 controls aged [&ge;] 60 years without any personal or family history of cancer by target sequencing and genome-wide SNP chip data. Their clinical significance was assessed using ClinVar and the guidelines by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). ResultsWe identified 4,804 variants in the 27 genes and annotated them as 397 pathogenic variants, 941 benign variants, and 3,466 variants of uncertain significance, of which 43.6% were registered in neither ClinVar nor dbSNP. In total, 3.3% of the unselected CRC patients and 1.5% of the controls had a pathogenic variant of the 27 genes. The pathogenic variants of MSH2 (odds ratio (OR) =18.1), MLH1 (OR=8.6), MSH6 (OR=4.9), APC (OR=49.4), BRIP1 (OR=3.6), BRCA1 (OR=2.6), BRCA2 (OR=1.9), and TP53 (OR=1.7) were significantly associated with CRC development in the Japanese population (P-values < 0.01, FDR<0.05). Furthermore, we confirmed copy number variants (CNVs) of MSH2/EPCAM, MLH1, and APC by multiplex ligation-dependent probe amplification (MLPA) and quantitative PCR in this cohort (n = 23), including whole gene duplications of MSH2 and APC. These pathogenic variants were significantly associated with the diagnostic age and personal/family history of other types of cancer. In total, at least 3.5% of the Japanese CRC population had a pathogenic variant or CNV of the 27 cancer-predisposing genes. ConclusionsThis is the largest study of CRC heredity in the Asian population and would contribute to the development of guidelines for genetic testing and variant interpretation for heritable CRCs. Universal screening for CRC risk should be assessed in multiple genes, including BRCA1/2 and BRIP1. These data would facilitate risk assessment of cancer and optimize the screening strategy.

genetics↗