TP53, a gene for colorectal cancer predisposition in the absence of Li-Fraumeni-associated phenotypes.

OBJECTIVE: Germline TP53 pathogenic (P) variants cause Li-Fraumeni syndrome (LFS), an aggressive multitumor-predisposing condition. Due to the implementation of multigene panel testing, TP53 variants have been detected in individuals without LFS suspicion, for example, patients with colorectal cancer (CRC). We aimed to decipher whether these findings are the result of detecting the background population prevalence or the aetiological basis of CRC. DESIGN: We analysed TP53 in 473 familial/early-onset CRC cases and evaluated the results together with five additional studies performed in patients with CRC (total n=6200). Control population and LFS data were obtained from Genome Aggregation Database (gnomAD V.2.1.1) and the International Agency for Research on Cancer (IARC) TP53 database, respectively. All variants were reclassified according to the guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP), following the ClinGen TP53 Expert Panel specifications. RESULTS: P or likely pathogenic (LP) variants were identified in 0.05% of controls (n=27/59 095) and 0.26% of patients with CRC (n=16/6200) (p<0.0001) (OR=5.7, 95% CI 2.8 to 10.9), none of whom fulfilled the clinical criteria established for TP53 testing. This association was still detected when patients with CRC diagnosed at more advanced ages (>50 and>60 years) were excluded from the analysis to minimise the inclusion of variants caused by clonal haematopoiesis. Loss-of-function and missense variants were strongly associated with CRC as compared with controls (OR=25.44, 95% CI 6.10 to 149.03, for loss of function and splice-site alleles, and OR=3.58, 95% CI 1.46 to 7.98, for missense P or LP variants). CONCLUSION: TP53 P variants should not be unequivocally associated with LFS. Prospective follow-up of carriers of germline TP53 P variants in the absence of LFS phenotypes will define how surveillance and clinical management of these individuals should be performed.

Candidate genes for hereditary colorectal cancer: Mutational screening and systematic review.

Genome-wide approaches applied for the identification of new hereditary colorectal cancer (CRC) genes, identified several potential causal genes, including RPS20, IL12RB1, LIMK2, POLE2, MRE11, POT1, FAN1, WIF1, HNRNPA0, SEMA4A, FOCAD, PTPN12, LRP6, POLQ, BLM, MCM9, and the epigenetic inactivation of PTPRJ. Here we attempted to validate the association between variants in these genes and nonpolyposis CRC by performing a mutational screening of the genes and PTPRJ promoter methylation analysis in 473 familial/early-onset CRC cases, a systematic review of the published cases, and assessment of allele frequencies in control population. In the studied cohort, 24 (5%) carriers of (predicted) deleterious variants in the studied genes and no constitutional PTPRJ epimutations were identified. Assessment of allele frequencies in controls compared with familial/early-onset patients with CRC showed association with increased nonpolyposis CRC risk of disruptive variants in RPS20, IL12RB1, POLE2, MRE11 and POT1, and of FAN1 c.149T>G (p.Met50Arg). Lack of association was demonstrated for LIMK2, PTPN12, LRP6, PTPRJ, POLQ, BLM, MCM9 and FOCAD variants. Additional studies are required to provide conclusive evidence for SEMA4A, WIF1, HNRNPA0 c.-110G>C, and FOCAD large deletions.

Role of POLE and POLD1 in familial cancer.

PURPOSE: Germline pathogenic variants in the exonuclease domain (ED) of polymerases POLE and POLD1 predispose to adenomatous polyps, colorectal cancer (CRC), endometrial tumors, and other malignancies, and exhibit increased mutation rate and highly specific associated mutational signatures. The tumor spectrum and prevalence of POLE and POLD1 variants in hereditary cancer are evaluated in this study. METHODS: POLE and POLD1 were sequenced in 2813 unrelated probands referred for genetic counseling (2309 hereditary cancer patients subjected to a multigene panel, and 504 patients selected based on phenotypic characteristics). Cosegregation and case-control studies, yeast-based functional assays, and tumor mutational analyses were performed for variant interpretation. RESULTS: Twelve ED missense variants, 6 loss-of-function, and 23 outside-ED predicted-deleterious missense variants, all with population allele frequencies <1%, were identified. One ED variant (POLE p.Met294Arg) was classified as likely pathogenic, four as likely benign, and seven as variants of unknown significance. The most commonly associated tumor types were colorectal, endometrial and ovarian cancers. Loss-of-function and outside-ED variants are likely not pathogenic for this syndrome. CONCLUSIONS: Polymerase proofreading-associated syndrome constitutes 0.1-0.4% of familial cancer cases, reaching 0.3-0.7% when only CRC and polyposis are considered. ED variant interpretation is challenging and should include multiple pieces of evidence.

Contribution to colonic polyposis of recently proposed predisposing genes and assessment of the prevalence of NTHL1- and MSH3-associated polyposes.

Technological advances have allowed the identification of new adenomatous and serrated polyposis genes, and of several candidate genes that require additional supporting evidence of causality. Through an exhaustive literature review and mutational screening of 177 unrelated polyposis patients, we assessed the involvement of MCM9, FOCAD, POLQ, and RNF43 in the predisposition to (nonserrated) colonic polyposis, as well as the prevalence of NTHL1 and MSH3 mutations among genetically unexplained polyposis patients. Our results, together with previously reported data and mutation frequency in controls, indicate that: MCM9 and POLQ mutations are not associated with polyposis; germline RNF43 mutations, with a prevalence of 1.5-2.5% among serrated polyposis patients, do not cause nonserrated polyposis; MSH3 biallelic mutations are highly infrequent among European polyposis patients, and the prevalence of NTHL1 biallelic mutations among unexplained polyposes is ~2%. Although nonsignificant, FOCAD predicted deleterious variants are overrepresented in polyposis patients compared to controls, warranting larger studies to provide definite evidence in favor or against their causal association with polyposis predisposition.

TCR bias of in vivo expanded T cells in pancreatic islets and spleen at the onset in human type 1 diabetes.

Autoreactive T cells, responsible for the destruction of pancreatic ß cells in type 1 diabetes, are known to have a skewed TCR repertoire in the NOD mouse. To define the autoreactive T cell repertoire in human diabetes, we searched for intraislet monoclonal expansions from a recent onset in human pancreas to then trace them down to the patient's peripheral blood and spleen. Islet infiltration was diverse, but five monoclonal TCR ß-chain variable expansions were detected for Vß1, Vß7, Vß11, Vß17, and Vß22 families. To identify any sequence bias in the TCRs from intrapancreatic T cells, we analyzed 139 different CDR3 sequences. We observed amino acid preferences in the NDN region that suggested a skewed TCR repertoire within infiltrating T cells. The monoclonal expanded TCR sequences contained amino acid combinations that fit the observed bias. Using these CDR3 sequences as a marker, we traced some of these expansions in the spleen. There, we identified a Vß22 monoclonal expansion with identical CDR3 sequence to that found in the islets within a polyclonal TCR ß-chain variable repertoire. The same Vß22 TCR was detected in the patient's PBMCs, making a cross talk between the pancreas and spleen that was reflected in peripheral blood evident. No other pancreatic monoclonal expansions were found in peripheral blood or the spleen, suggesting that the Vß22 clone may have expanded or accumulated in situ by an autoantigen present in both the spleen and pancreas. Thus, the patient's spleen might be contributing to disease perpetuation by expanding or retaining some autoreactive T cells.

CDK6 is activated by the atypical cyclin I to promote E2F-mediated gene expression and cancer cell proliferation.

Cyclin-dependent kinases (CDKs), together with their cyclin partners, are the master cell cycle regulators. Remarkably, the cyclin family was extended to include atypical cyclins, characterized by distinctive structural features, but their partner CDKs remain elusive. Here, we conducted a yeast two-hybrid screen to identify new atypical cyclin-CDK complexes. We identified 10 new complexes, including a complex between CDK6 and cyclin I (CCNI), which was found to be active against retinoblastoma protein. CCNI upregulation increased the proliferation of breast cancer cells in vitro and in vivo, with a magnitude similar to that seen upon cyclin D upregulation, an effect that was abrogated by CDK6 silencing or palbociclib treatment. In line with these findings, CCNI downregulation led to a decrease in cell number and a reduction in the percentage of cells reaching S phase. Finally, CCNI upregulation correlated with the high expression of E2F target genes in large panels of cancer cell lines and tissue samples from breast cancer patients. In conclusion, we unveil CCNI as a new player in the pathways that activate CDK6, enriching the wiring of cell cycle control.

NTHL1 biallelic mutations seldom cause colorectal cancer, serrated polyposis or a multi-tumor phenotype, in absence of colorectal adenomas.

The cancer-predisposing syndrome caused by biallelic mutations in NTHL1 may not be a solely colorectal cancer (CRC) and polyposis syndrome but rather a multi-tumor recessive disease. The presence of ≤10 adenomas in several mutation carriers suggests a possible causal role of NTHL1 in hereditary or early-onset nonpolyposis CRC. The involvement of NTHL1 in serrated/hyperplastic polyposis remains unexplored. The aim of our study is to elucidate the role of NTHL1 in the predisposition to personal or familial history of multiple tumor types, familial/early-onset nonpolyposis CRC, and serrated polyposis. NTHL1 mutational screening was performed in 312 cancer patients with personal or family history of multiple tumor types, 488 with hereditary nonpolyposis CRC, and 96 with serrated/hyperplastic polyposis. While no biallelic mutation carriers were identified in patients with personal and/or family history of multiple tumor types or with serrated polyposis, one was identified among the 488 nonpolyposis CRC patients. The carrier of c.268C>T (p.Q90*) and 550-1G>A was diagnosed with CRC and meningioma at ages 37 and 45 respectively, being reclassified as attenuated adenomatous polyposis after the cumulative detection of 26 adenomas. Our findings suggest that biallelic mutations in NTHL1 rarely cause CRC, a personal/familial multi-tumor history, or serrated polyposis, in absence of adenomas.

msBiodat analysis tool, big data analysis for high-throughput experiments.

BACKGROUND: Mass spectrometry (MS) are a group of a high-throughput techniques used to increase knowledge about biomolecules. They produce a large amount of data which is presented as a list of hundreds or thousands of proteins. Filtering those data efficiently is the first step for extracting biologically relevant information. The filtering may increase interest by merging previous data with the data obtained from public databases, resulting in an accurate list of proteins which meet the predetermined conditions. RESULTS: In this article we present msBiodat Analysis Tool, a web-based application thought to approach proteomics to the big data analysis. With this tool, researchers can easily select the most relevant information from their MS experiments using an easy-to-use web interface. An interesting feature of msBiodat analysis tool is the possibility of selecting proteins by its annotation on Gene Ontology using its Gene Id, ensembl or UniProt codes. CONCLUSION: The msBiodat analysis tool is a web-based application that allows researchers with any programming experience to deal with efficient database querying advantages. Its versatility and user-friendly interface makes easy to perform fast and accurate data screening by using complex queries. Once the analysis is finished, the result is delivered by e-mail. msBiodat analysis tool is freely available at http://msbiodata.irb.hr.