Supplementary Notes. This document includes description of studies that they have been used for the analysis.

Supplementary Figure 2. Absolute Risk (With no family history of prostate cancer.)

Supplementary Tables 1-8. Supplementary table 1: Total number of cases and controls in PRACTICAL III and GWAS stage 3 by population and study. Supplementary Table 2: Data information for family history and age at diagnosis/observation. Supplementary Table 3: Grade-specific and family history-specific odds ratios. Supplementary Table 4: PSA levels by genotype in controls. Supplementary Table 5: Age-specific odds ratios. Supplementary Table 6: The results of 29 pair wise interaction of 25 SNPs significant at P <0.05 (29 out of 300 interactions; Bonferroni correction 0.05/300=1.67x10-4). Supplementary Table 7: Estimated odds ratios of PRS percentiles adjusted for age at diagnosis of PrCa (five categories) in a model allowing for an interaction between PRS and Age. Supplementary Table 8: Estimated odds ratios used for estimating absolute risk.

Background: Germline pathogenic variants in the breast cancer type 1 susceptibility gene BRCA1 are associated with a 60% lifetime risk for breast and ovarian cancer. This overall risk estimate is for all BRCA1 variants; obviously, not all variants confer the same risk of developing a disease. In cancer patients, loss of BRCA1 function in tumor tissue has been associated with an increased sensitivity to platinum agents and to poly-(ADP- ribose) polymerase (PARP) inhibitors. For clinical management of both at- risk individuals and cancer patients, it would be important that each identified genetic variant be associated with clinical significance. Unfortunately for the vast majority of variants, the clinical impact is unknown. The availability of results from studies assessing the impact of variants on protein function may provide insight of crucial importance. Results and conclusion: We have collected, curated, and structured the molecular and cellular phenotypic impact of 3654 distinct BRCA1 variants. The data was modeled in triple format, using the variant as a subject, the studied function as the object, and a predicate describing the relation between the two. Each annotation is supported by a fully traceable evidence. The data was captured using standard ontologies to ensure consistency, and enhance searchability and interoperability. We have assessed the extent to which functional defects at the molecular and cellular levels correlate with the clinical interpretation of variants by ClinVar submitters. Approximately 30% of the ClinVar BRCA1 missense variants have some molecular or cellular assay available in the literature. Pathogenic variants (as assigned by ClinVar) have at least some significant functional defect in 94% of testable cases. For benign variants, 77% of ClinVar benign variants, for which neXtProt Cancer variant portal has data, shows either no or mild experimental functional defects. While this does not provide evidence for clinical interpretation of variants, it may provide some guidance for variants of unknown significance, in the absence of more reliable data. The neXtProt Cancer variant portal (https://www. nextprot. org/portals/breast- cancer) contains over 6300 observations at the molecular and/or cellular level for BRCA1 variants.

Although most of the pertinent data on the sequence-directed processes leading to genome rearrangements (GRs) have come from studies on somatic tissues, little is known about GRs in the germ line of patients with hereditary disorders. This study aims at identifying DNA motifs and higher order structures of genome architecture, which can result in losses and gains of genetic material in the germ line. We first identified candidate motifs by studying 112 pathogenic germ-line GRs in hereditary colorectal cancer patients, and subsequently created an algorithm, termed recombination type ratio, which correctly predicts the propensity of rearrangements with respect to homologous versus nonhomologous recombination events.

Abstract Background: Genome-wide association studies have identified multiple genetic variants associated with prostate cancer risk which explain a substantial proportion of familial relative risk. These variants can be used to stratify individuals by their risk of prostate cancer. Methods: We genotyped 25 prostate cancer susceptibility loci in 40,414 individuals and derived a polygenic risk score (PRS). We estimated empirical odds ratios (OR) for prostate cancer associated with different risk strata defined by PRS and derived age-specific absolute risks of developing prostate cancer by PRS stratum and family history. Results: The prostate cancer risk for men in the top 1% of the PRS distribution was 30.6 (95% CI, 16.4–57.3) fold compared with men in the bottom 1%, and 4.2 (95% CI, 3.2–5.5) fold compared with the median risk. The absolute risk of prostate cancer by age of 85 years was 65.8% for a man with family history in the top 1% of the PRS distribution, compared with 3.7% for a man in the bottom 1%. The PRS was only weakly correlated with serum PSA level (correlation = 0.09). Conclusions: Risk profiling can identify men at substantially increased or reduced risk of prostate cancer. The effect size, measured by OR per unit PRS, was higher in men at younger ages and in men with family history of prostate cancer. Incorporating additional newly identified loci into a PRS should improve the predictive value of risk profiles. Impact: We demonstrate that the risk profiling based on SNPs can identify men at substantially increased or reduced risk that could have useful implications for targeted prevention and screening programs. Cancer Epidemiol Biomarkers Prev; 24(7); 1121–9. ©2015 AACR.

Advanced donor age adversely affects allograft outcome following renal transplantation.Candidate genes involved in cellcycle regulation and telomere shortening have been already described as markers for cellular senescence.There is still a lack of biomarkers for defining older and marginal organs in order to adopt immunosuppression for improved long-term outcome.To characterize senescence in the donor organ, we first studied gene expression for selected candidate markers(CM) in a small sample cohort of 57 zero hour kidney biopsies derived from deceased donors.Among them 26 biopsies were derived from donors>55 yrs(mean66.5±7.4) and 31 specimens were derived from donors<55 yrs(mean 41.35±8.4).Compared with younger donors,elderly donors revealed a significant de novo mRNA expression of CM including immunoproteasome subunits PSMB8,9,10;(p<0.001 respectively), MHC classII transcripts including HLA-DRB(p<0.01)or transcripts of the receptor NKG2D(p=0.0036).Next, we confirmed gene expression of CM in an independent patient cohort consisting of 139 biopsy samples.Based on sample size,3 groups were defined according to donor age:0-30yrs(group I,21±4.9yrs,n=30),31-54yrs(group II, 46.5±6.6yrs,n=50)and>55 yrs(group III,64.07±7yrs,n=59).Whereas no differences were observed between group I and group II, aged kidneys(group III) revealed a significant gene expression of PSMB9(p=0.0405) and PSMB10(p=0.0223) compared with middle aged kidneys(group II).In addition,HLA-DRB(p=0.0215) and PSMB9(p=0.0129) were significantly induced in group III in comparison with group I.Strikingly,transcripts of the activating NK cell receptor NKG2D revealed the highest gene induction in group III versus group II and group I(p<0.001,respectively) indicating enhanced infiltration of NKG2D+ CD8+ T cells or NK cells in elderly kidneys. Linear restriction analysis revealed a strong correlation especially for pre-transplant NKG2D mRNA expression with serum creatinine levels at hospital discharge(p=0.0003), at 3months(p=0.004) and at 6months post transplantation(p=0.0031).Our results reveal novel candidate markers in aged renal allografts providing help in the assessment of organ quality and implicating the potential of pretreatment strategies.

CHARACTERISTICS 1.1 Name of the disease (synonyms) Lynch syndrome/HNPCC.1.2 OMIM# of the disease 276300, 613244.1.3 Name of the analysed genes or DNA/chromosome segments MLH1, MSH2, MSH6, PMS2, and EPCAM.1.4 OMIM# of the gene(s) MLH1 (120436), MSH2 (609309), MSH6 (600678), PMS2 (600259), EPCAM (185535). Mutational spectrumPoint mutations, large deletions and duplications, large genomic insertions and promoter methylation. Analytical methodsStepwise analyses:

Recently, we identified 3' end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele-specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch-like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2-deficient families from multiple geographical origins with varying deletions all encompassing the 3' end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu-repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3' end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics.

Lynch syndrome is an autosomal dominant disease associated with an important risk of cancer, mainly endometrial and colorectal-cancer. This risk can be efficiently lessen by an appropriate screening as far as the mutations carriers are identified. As current clinicopathological recommendations lack sensitivity, a systematic pre-screening of every patient with a colorectal or endometrial cancer can be proposed. Oncogenetic units of the HUG in Geneva and ICHV in Valais have set up a population-based study to evaluate the efficacy of such a strategy. Whatever the approach, the pathologist is directly implicated as Lynch syndrome harbors specific histological aspects that can help to its identification, but also as pre-screening tests are directly realized on tumor-tissue.

Heterogeneous attitudes in genetic testing practice exist among countries in terms of access, acceptance, use, and rules. The European Molecular Genetics Quality Network (EMQN) is trying to harmonize genetic testing services across Europe by developing External Quality Assessment (EQA) schemes for different inherited disorders. One that EMQN focussed on early is Lynch syndrome (HNPCC) associated with inherited mutations in mismatch repair (MMR) genes. In addition to early-onset colorectal cancer, such mutations confer an increased risk of gynaecological, urinary tract, and gastrointestinal adenocarcinomas. Genetic testing results guide screening programs for patients and families as specific clinical guidelines have been proven to reduce morbidity and mortality [Grover and Syngal, 2010]. Unclear or misinformed laboratory reports have major clinical implications because the presence or absence of a pathogenic MMR mutation is often instrumental to decisions made about patient management. Since the first pilot scheme on the MMR genes organized in 2003, annual EQAs have enabled the identification of the most frequent difficulties encountered by the participating labs, addressing disease/gene-specific points to improve the standard of testing strategies (Table 1). Globally, the performance of laboratories in this scheme increased each year as well as the number of participants and a high technical standard of genotyping has been reached with the decreasing use of home-made reagents and manual procedures. This result is very encouraging for labs that aim to become referent with long-term agreements. The feedback to the labs integrated the genotyping assessment and key points to improve the quality of the reports themselves. For example, in 2009, two separate reports were requested in one of the mock clinical questions—one for the index case (confirmation of the screening analysis) and one for a predictive test: 11 labs did not reanalyze the index case and 16 labs mixed the information from both cases in a single report, that is, 25% of the participants failed to write a reliable report for case 1. Best practice guidelines on reporting are available, which give clear guidance on the appropriate reporting procedures (http://www.sgmg.ch/view_page_professional.php?view=page&page_id=19). Specific to Lynch syndrome, the conclusion should have restated the genetic status in the clinical context with discussion on the patient's main risks (Lynch-related cancers, dominant inheritance) or the suitable prospective analyses. Before concluding the report, the reader should be convinced of the biological consequences of the genotype [Tavtigian et al., 2008]. In the 2008 scheme, 80% of participants provided consistent information because no case referred to missense variants. In contrast, the 2009 scheme (the same case was met in 2005) included such a situation and only 25% of them reached the maximum score [Adzhubei et al., 2010; Needham et al., 2006]. When analyzing separately the 78 labs having participated to both 2008 and 2009 schemes and the 34 participants newly registered in 2009, scores were slightly worse in the first group, decreasing from 42 to 22%, and being 29% for the new participants. This observation points out the difficulty linked to the interpretation of missense mutations itself rather than the lack of experience in quality controls. As observed in the other complex diseases such as breast cancer, approached through EMQN schemes, the EQA scheme results emphasise that is it very important to have good background knowledge of the genes being tested to guarantee the reliability of genetic testing as part of the medical evaluation, especially as relevant and validated information is now available on free access international and national mutation databases (http://www.mmrmissense.net/). We thank Shirley McQuaid, Kathleen Claes, Anna Krepelova, and Manuel Teixeira for their participation to the scheme assessments. We are also grateful to Outi Kamarainen and Rob Elles for their constant help in improving the management of the schemes.

(1) Clinical selection1: Lynch-related cancer (colon, rectum, endometrium, urinary tract, small bowel, biliary tract, ovary, stomach). Sporadic before 50 years of age, first-degree relative or prior Lynch-related cancer. (2) Study of MMR function in tumour cells2: microsatellite DNA analysis—genotyping of the consensus panel of five mononucleotidic repeats defined in 1998. Immunohistochemical study of the four mismatch repair proteins MLH1, MSH2, MSH6, and PMS2 (in case of MSI or in the absence of genotyping). BRAF codon 600 characterisation by pyrosequencing, sequencing, TaqMan, SNaPshot, and so on (in case of absence of the MLH1 protein). (3) Germline analysis3: MSH2 and/or MLH1 screening for point mutations by pre-screening (DHPLC) or direct sequencing, and for large genomic anomalies by MLPA including promoter regions and EPCAM gene. MSH6 or PMS2 screening for point mutations by pre-screening or direct sequencing, and for large genomic anomalies by MLPA (in case of negative results for MSH2/MLH1 screening, according to the tumour MMR status). MLH1 promoter methylation characterisation by MSP, bisulphite pyrosequencing (useful for diagnostic purpose, not for predictive testing). 1.7 Analytical validation Confirmation of mutation in an independent biological sample of the index case or an affected relative. In case of deletion/duplication of one exon, confirm with a second technique/kit based on different primers.

Lynch syndrome is one of the most common hereditary colorectal cancer (CRC) syndrome and is caused by germline mutations of MLH1 , MSH2 and more rarely MSH6 , PMS2 , MLH3 genes. Whereas the absence of MSH2 protein is predictive of Lynch syndrome, it is not the case for the absence of MLH1 protein. The purpose of this study was to develop a sensitive and cost effective algorithm to select Lynch syndrome cases among patients with MLH1 immunohistochemical silencing. Eleven sporadic CRC and 16 Lynch syndrome cases with MLH1 protein abnormalities were selected. The BRAF c.1799T> A mutation (p.Val600Glu) was analyzed by direct sequencing after PCR amplification of exon 15. Methylation of MLH1 promoter was determined by Methylation-Sensitive Single-Strand Conformation Analysis. In patients with Lynch syndrome, there was no BRAF mutation and only one case showed MLH1 methylation (6%). In sporadic CRC, all cases were MLH1 methylated (100%) and 8 out of 11 cases carried the above BRAF mutation (73%) whereas only 3 cases were BRAF wild type (27%). We propose the following algorithm: (1) no further molecular analysis should be performed for CRC exhibiting MLH1 methylation and BRAF mutation, and these cases should be considered as sporadic CRC; (2) CRC with unmethylated MLH1 and negative for BRAF mutation should be considered as Lynch syndrome; and (3) only a small fraction of CRC with MLH1 promoter methylation but negative for BRAF mutation should be true Lynch syndrome patients. These potentially Lynch syndrome patients should be offered genetic counselling before searching for MLH1 gene mutations.

BACKGROUND:Lynch syndrome is caused by germline mutations in MSH2, MLH1, MSH6, and PMS2 mismatch-repair genes and leads to a high risk of colorectal and endometrial cancer. We previously showed that constitutional 3' end deletions of EPCAM can cause Lynch syndrome through epigenetic silencing of MSH2 in EPCAM-expressing tissues, resulting in tissue-specific MSH2 deficiency. We aim to establish the risk of cancer associated with such EPCAM deletions. METHODS:We obtained clinical data for 194 carriers of a 3' end EPCAM deletion from 41 families known to us at the Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands and compared cancer risk with data from a previously described cohort of 473 carriers from 91 families with mutations in MLH1, MSH2, MSH6, or a combined EPCAM-MSH2 deletion. FINDINGS:93 of the 194 EPCAM deletion carriers were diagnosed with colorectal cancer; three of the 92 women with EPCAM deletions were diagnosed with endometrial cancer. Carriers of an EPCAM deletion had a 75% (95% CI 65-85) cumulative risk of colorectal cancer before the age of 70 years (mean age at diagnosis 43 years [SD 12]), which did not differ significantly from that of carriers of combined EPCAM-MSH2 deletion (69% [95% CI 47-91], p=0·8609) or mutations in MSH2 (77% [64-90], p=0·5892) or MLH1 (79% [68-90], p=0·5492), but was higher than noted for carriers of MSH6 mutation (50% [38-62], p<0·0001). By contrast, women with EPCAM deletions had a 12% [0-27] cumulative risk of endometrial cancer, which was lower than was that noted for carriers of a combined EPCAM-MSH2 deletion (55% [20-90], p<0·0001) or of a mutation in MSH2 (51% [33-69], p=0·0006) or MSH6 (34% [20-48], p=0·0309), but did not differ significantly from that noted for MLH1 (33% [15-51], p=0·1193) mutation carriers. This risk seems to be restricted to deletions that extend close to the MSH2 gene promoter. Of 194 carriers of an EPCAM deletion, three had duodenal cancer and four had pancreatic cancer. INTERPRETATION:EPCAM deletion carriers have a high risk of colorectal cancer; only those with deletions extending close to the MSH2 promoter have an increased risk of endometrial cancer. These results underscore the effect of mosaic MSH2 deficiency, leading to variable cancer risks, and could form the basis of an optimised protocol for the recognition and targeted prevention of cancer in EPCAM deletion carriers.

Prostate cancer (PrCa) is the most frequently diagnosed cancer in males in developed countries. To identify common PrCa susceptibility alleles, we previously conducted a genome-wide association study in which 541,129 SNPs were genotyped in 1,854 PrCa cases with clinically detected disease and in 1,894 controls. We have now extended the study to evaluate promising associations in a second stage in which we genotyped 43,671 SNPs in 3,650 PrCa cases and 3,940 controls and in a third stage involving an additional 16,229 cases and 14,821 controls from 21 studies. In addition to replicating previous associations, we identified seven new prostate cancer susceptibility loci on chromosomes 2, 4, 8, 11 and 22 (with P = 1.6 x 10(-8) to P = 2.7 x 10(-33)).

Lynch syndrome is mostly characterized by early-onset colorectal and endometrial adenocarcinomas. Over 90% of the causal mutations occur in two mismatch repair genes, MSH2 and MLH1 . The aim of this study was to evaluate the age-dependent cancer risk in MSH2 or MLH1 mutation carriers from data of DNA diagnostic laboratories. To avoid overestimation, evaluation was based on the age-dependent proportion of mutation carriers in asymptomatic first-degree relatives of identified mutation carriers. Data from 859 such eligible relatives were collected from 8 centers; 387 were found to have inherited the mutation from their relatives. Age-dependent risks were calculated either using a nonparametric approach for four discrete age groups or assuming a modified Weibull distribution for the dependence of risk on age. Cancer risk was estimated starting at 28 (25–32 0.68 confidence interval) and to reach near 0.70 at 70 years. The risks were very similar for MSH2 and MLH1 mutation carriers. Although not statistically significant, the risk in males appeared to precede that for females by ten years. This difference needs to be investigated on a larger dataset. If confirmed, this would indicate that the onset of the colonoscopic surveillance may be different in male and female mutation carriers.

Alterations at the X-linked Hmr gene of Drosophila melanogaster can fully restore viability and partially restore fertility in hybrid flies from crosses between D. melanogaster and any of its three most closely related species. Although more than one gene is expected to be involved in these barriers to reproduction, a single DNA-binding protein was recently identified as HMR. The Hmr gene was shown to evolve unusually fast, a feature that supports its role in causing genetic incompatibility in a hybrid genotype. The current treatment of hybrid genetics focuses not only on Hmr but also on the Rab9D gene, which lies only 1kb from Hmr. Rab9D is proposed also to influence hybrid viability. This gene has remained tightly linked to Hmr for about 10 million years, but it has diverged even more than Hmr with regard to D. melanogaster and its most closely related species. Furthermore, the 197-amino acid RAB9D protein contains four amino acid substitutions in the D. melanogaster-rescuing mutant Hmr1. Rab9D is shown to have evolved under very strong positive selection and to be the most recent member of a cluster of six paralogs that encode small RAB GTPases. Four of the six paralogs are unique to D. melanogaster in which they have diverged considerably, their encoded proteins sharing less than 50% amino acid identities with proteins from their orthologs in the closest species. Only two Rab orthologs are present in these sibling species and none is present in the genomes of more distantly related Drosophila species. Rapidly evolving Rab paralogs near the Hmr locus probably developed functional specialization of redundant proteins involved in trafficking macromolecules between cytoplasm and nucleus. Positive selection acting on duplicates of these Rab genes appears to participate in reproductive isolation.