BMPR2 as a Novel Predisposition Gene for Hereditary Colorectal Polyposis.

BACKGROUND & AIMS: Colorectal cancer (CRC) is one of the most prevalent tumors worldwide, with incidence quickly increasing (particularly in the context of early-onset cases), despite important prevention efforts, mainly in the form of population-wide screening programs. Although many cases present a clear familial component, the current list of hereditary CRC genes leaves a considerable proportion of the cases unexplained. METHODS: In this work, we used whole-exome sequencing approaches on 19 unrelated patients with unexplained colonic polyposis to identify candidate CRC predisposition genes. The candidate genes were then validated in an additional series of 365 patients. CRISPR-Cas9 models were used to validate BMPR2 as a potential candidate for CRC risk. RESULTS: We found 8 individuals carrying 6 different variants in the BMPR2 gene (approximately 2% of our cohort of patients with unexplained colonic polyposis). CRISPR-Cas9 models of 3 of these variants showed that the p.(Asn442Thrfs∗32) truncating variant completely abrogated BMP pathway function in a similar way to the BMPR2 knockout. Missense variants p.(Asn565Ser), p.(Ser967Pro) had varying effects on cell proliferation levels, with the former impairing cell control inhibition via noncanonical pathways. CONCLUSIONS: Collectively, these results support loss-of-function BMPR2 variants as candidates to be involved in CRC germline predisposition.

CTNND1 is involved in germline predisposition to early-onset gastric cancer by affecting cell-to-cell interactions.

BACKGROUND: CDH1 and CTNNA1 remain as the main genes for hereditary gastric cancer. However, they only explain a small fraction of gastric cancer cases with suspected inherited basis. In this study, we aimed to identify new hereditary genes for early-onset gastric cancer patients (EOGC; < 50 years old). METHODS: After germline exome sequencing in 20 EOGC patients and replication of relevant findings by gene-panel sequencing in an independent cohort of 152 patients, CTNND1 stood out as an interesting candidate gene, since its protein product (p120ctn) directly interacts with E-cadherin. We proceeded with functional characterization by generating two knockout CTNND1 cellular models by gene editing and introducing the detected genetic variants using a lentiviral delivery system. We assessed ß-catenin and E-cadherin levels, cell detachment, as well as E-cadherin localization and cell-to-cell interaction by spheroid modeling. RESULTS: Three CTNND1 germline variants [c.28_29delinsCT, p.(Ala10Leu); c.1105C > T, p.(Pro369Ser); c.1537A > G, p.(Asn513Asp)] were identified in our EOGC cohorts. Cells encoding CTNND1 variants displayed altered E-cadherin levels and intercellular interactions. In addition, the p.(Pro369Ser) variant, located in a key region in the E-cadherin/p120ctn binding domain, showed E-cadherin mislocalization. CONCLUSIONS: Defects in CTNND1 could be involved in germline predisposition to gastric cancer by altering E-cadherin and, consequently, cell-to-cell interactions. In the present study, CTNND1 germline variants explained 2% (3/172) of the cases, although further studies in larger external cohorts are needed.

Germline mutations in WNK2 could be associated with serrated polyposis syndrome.

BACKGROUND: Patients with serrated polyposis syndrome (SPS) have multiple and/or large serrated colonic polyps and higher risk for colorectal cancer. SPS inherited genetic basis is mostly unknown. We aimed to identify new germline predisposition factors for SPS by functionally evaluating a candidate gene and replicating it in additional SPS cohorts. METHODS: After a previous whole-exome sequencing in 39 SPS patients from 16 families (discovery cohort), we sequenced specific genes in an independent validation cohort of 211 unrelated SPS cases. Additional external replication was also available in 297 SPS cases. The WNK2 gene was disrupted in HT-29 cells by gene editing, and WNK2 variants were transfected using a lentiviral delivery system. Cells were analysed by immunoblots, real-time PCR and functional assays monitoring the mitogen-activated protein kinase (MAPK) pathway, cell cycle progression, survival and adhesion. RESULTS: We identified 2 rare germline variants in the WNK2 gene in the discovery cohort, 3 additional variants in the validation cohort and 10 other variants in the external cohorts. Variants c.2105C>T (p.Pro702Leu), c.4820C>T (p.Ala1607Val) and c.6157G>A (p.Val2053Ile) were functionally characterised, displaying higher levels of phospho-PAK1/2, phospho-ERK1/2, CCND1, clonogenic capacity and MMP2. CONCLUSION: After whole-exome sequencing in SPS cases with familial aggregation and replication of results in additional cohorts, we identified rare germline variants in the WNK2 gene. Functional studies suggested germline WNK2 variants affect protein function in the context of the MAPK pathway, a molecular hallmark in this disease.

Germline Mutations in FAF1 Are Associated With Hereditary Colorectal Cancer.

BACKGROUND & AIMS: A significant proportion of colorectal cancer (CRC) cases have familial aggregation but little is known about the genetic factors that contribute to these cases. We performed an exhaustive functional characterization of genetic variants associated with familial CRC. METHODS: We performed whole-exome sequencing analyses of 75 patients from 40 families with a history of CRC (including early-onset cases) of an unknown germline basis (discovery cohort). We also sequenced specific genes in DNA from an external replication cohort of 473 families, including 488 patients with colorectal tumors that had normal expression of mismatch repair proteins (validation cohort). We disrupted the Fas-associated factor 1 gene (FAF1) in DLD-1 CRC cells using CRISPR/Cas9 gene editing; some cells were transfected with plasmids that express FAF1 missense variants. Cells were analyzed by immunoblots, quantitative real-time polymerase chain reaction, and functional assays monitoring apoptosis, proliferation, and assays for Wnt signaling or nuclear factor (NF)-kappa-B activity. RESULTS: We identified predicted pathogenic variant in the FAF1 gene (c.1111G>A; p.Asp371Asn) in the discovery cohort; it was present in 4 patients of the same family. We identified a second variant in FAF1 in the validation cohort (c.254G>C; p.Arg85Pro). Both variants encoded unstable FAF1 proteins. Expression of these variants in CRC cells caused them to become resistant to apoptosis, accumulate beta-catenin in the cytoplasm, and translocate NF-kappa-B to the nucleus. CONCLUSIONS: In whole-exome sequencing analyses of patients from families with a history of CRC, we identified variants in FAF1 that associate with development of CRC. These variants encode unstable forms of FAF1 that increase resistance of CRC cells to apoptosis and increase activity of beta-catenin and NF-kappa-B.

Colorectal cancer genetic variants are also associated with serrated polyposis syndrome susceptibility.

BACKGROUND: Serrated polyposis syndrome (SPS) is a clinical entity characterised by large and/ormultiple serrated polyps throughout the colon and increased risk for colorectal cancer (CRC). The basis for SPS genetic predisposition is largely unknown. Common, low-penetrance genetic variants have been consistently associated with CRC susceptibility, however, their role in SPS genetic predisposition has not been yet explored. OBJECTIVE: The aim of this study was to evaluate if common, low-penetrance genetic variants for CRC risk are also implicated in SPS genetic susceptibility. METHODS: A case-control study was performed in 219 SPS patients and 548 asymptomatic controls analysing 65 CRC susceptibility variants. A risk prediction model for SPS predisposition was developed. RESULTS: Statistically significant associations with SPS were found for seven genetic variants (rs4779584-GREM1, rs16892766-EIF3H, rs3217810-CCND2, rs992157-PNKD1/TMBIM1, rs704017-ZMIZ1, rs11196172-TCF7L2, rs6061231-LAMA5). The GREM1 risk allele was remarkably over-represented in SPS cases compared with controls (OR=1.573, 1.21-2.04, p value=0.0006). A fourfold increase in SPS risk was observed when comparing subjects within the highest decile of variants (≥65) with those in the first decile (≤50). CONCLUSIONS: Genetic variants for CRC risk are also involved in SPS susceptibility, being the most relevant ones rs4779584-GREM1, rs16892766-EIF3H and rs3217810-CCND2.

Identification of New Genes Involved in Germline Predisposition to Early-Onset Gastric Cancer.

The genetic cause for several families with gastric cancer (GC) aggregation is unclear, with marked relevance in early-onset patients. We aimed to identify new candidate genes involved in GC germline predisposition. Whole-exome sequencing (WES) of germline samples was performed in 20 early-onset GC patients without previous germline mutation identified. WES was also performed in nine tumor samples to analyze the somatic profile using SigProfilerExtractor tool. Sequencing germline data were filtered to select those variants with plausible pathogenicity, rare frequency and previously involved in cancer. Then, a manual filtering was performed to prioritize genes according to current knowledge and function. These genetic variants were prevalidated with Integrative Genomics Viewer 2.8.2 (IGV). Subsequently, a further selection step was carried out according to function and information obtained from tumor samples. After IGV and selection step, 58 genetic variants in 52 different candidate genes were validated by Sanger sequencing. Among them, APC, FAT4, CTNND1 and TLR2 seem to be the most promising genes because of their role in hereditary cancer syndromes, tumor suppression, cell adhesion and Helicobacter pylori recognition, respectively. These encouraging results represent the open door to the identification of new genes involved in GC germline predisposition.

Using linkage studies combined with whole-exome sequencing to identify novel candidate genes for familial colorectal cancer.

Colorectal cancer (CRC) is a complex disorder for which the majority of the underlying germline predisposition factors remain still unidentified. Here, we combined whole-exome sequencing (WES) and linkage analysis in families with multiple relatives affected by CRC to identify candidate genes harboring rare variants with potential high-penetrance effects. Forty-seven affected subjects from 18 extended CRC families underwent WES. Genome-wide linkage analysis was performed under linear and exponential models. Suggestive linkage peaks were identified on chromosomes 1q22-q24.2 (maxSNP = rs2134095; LODlinear = 2.38, LODexp = 2.196), 7q31.2-q34 (maxSNP = rs6953296; LODlinear = 2.197, LODexp = 2.149) and 10q21.2-q23.1 (maxSNP = rs1904589; LODlinear = 1.445, LODexp = 2.195). These linkage signals were replicated in 10 independent sets of random markers from each of these regions. To assess the contribution of rare variants predicted to be pathogenic, we performed a family-based segregation test with 89 rare variants predicted to be deleterious from 78 genes under the linkage intervals. This analysis showed significant segregation of rare variants with CRC in 18 genes (weighted p-value > 0.0028). Protein network analysis and functional evaluation were used to suggest a plausible candidate gene for germline CRC predisposition. Etiologic rare variants implicated in cancer germline predisposition may be identified by combining traditional linkage with WES data. This approach can be used with already available NGS data from families with several sequenced members to further identify candidate genes involved germline predisposition to disease. This approach resulted in one candidate gene associated with increased risk of CRC but needs evidence from further studies.

Involvement of exosomes in lung inflammation associated with experimental acute pancreatitis.

A frequent complication of acute pancreatitis is the lung damage associated with the systemic inflammatory response. Although various pro-inflammatory mediators generated at both local and systemic levels have been identified, the pathogenic mechanisms of the disease are still poorly understood. In recent years, exosomes have emerged as a new intercellular communication system able to transfer encapsulated proteins and small RNAs and protect them from degradation. Using an experimental model of taurocholate-induced acute pancreatitis in rats, we aimed to evaluate the role of exosomes in the extent of the systemic inflammatory response. Induction of pancreatitis increased the concentration of circulating exosomes, which showed a different proteomic profile to those obtained from control animals. A series of tracking experiments using PKH26-stained exosomes revealed that circulating exosomes effectively reached the alveolar compartment and were internalized by macrophages. In vitro experiments revealed that exosomes obtained under inflammatory conditions activate and polarize these alveolar macrophages towards a pro-inflammatory phenotype. Interestingly, the proteomic analysis of circulating exosomes during acute pancreatitis suggested a multi-organ origin with a relevant role for the liver as a source of these vesicles. Tracking experiments also revealed that the liver retains the majority of exosomes from the peritoneal cavity. We conclude that exosomes are involved in the lung damage associated with experimental acute pancreatitis and could be relevant mediators in the systemic effects of pancreatitis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Lipids generated during acute pancreatitis increase inflammatory status of macrophages by interfering with their M2 polarization.

BACKGROUND: Necrosis of adipose tissue is a common complication of acute pancreatitis. The areas of steatonecrosis become a source of inflammatory mediators, including chemically modified fatty acids which could influence the progression of the systemic inflammation. In an experimental model of acute pancreatitis we analyzed the effects of lipids generated by two representative areas of adipose tissue on the switch to the M1 phenotype in macrophages. METHODS: Pancreatitis was induced in rats by intraductal administration of 5% taurocholate and after 6 h, lipids from retroperitoneal, mesenteric or epididymal adipose tissues were collected. Lipid uptake, phenotype polarization and the activation of PPARγ and NFκB were evaluated in macrophages treated with these lipids. RESULTS: After induction of pancreatitis, lipids from visceral adipose tissue promote the switch to an increased pro-inflammatory phenotype in macrophages. This effect is not related with a higher activation of NFκB but with an interfering effect on the activation of M2 phenotype. CONCLUSIONS: During acute pancreatitis, lipids generated by some areas of adipose tissue interfere on the M2 polarization of macrophages, thus resulting in a more intense pro-inflammatory M1 response.

HAP1, a new revolutionary cell model for gene editing using CRISPR-Cas9.

The use of next-generation sequencing (NGS) technologies has been instrumental in the characterization of the mutational landscape of complex human diseases like cancer. But despite the enormous rise in the identification of disease candidate genetic variants, their functionality is yet to be fully elucidated in order to have a clear implication in patient care. Haploid human cell models have become the tool of choice for functional gene studies, since they only contain one copy of the genome and can therefore show the unmasked phenotype of genetic variants. Over the past few years, the human near-haploid cell line HAP1 has widely been consolidated as one of the favorite cell line models for functional genetic studies. Its rapid turnover coupled with the fact that only one allele needs to be modified in order to express the subsequent desired phenotype has made this human cell line a valuable tool for gene editing by CRISPR-Cas9 technologies. This review examines the recent uses of the HAP1 cell line model in functional genetic studies and high-throughput genetic screens using the CRISPR-Cas9 system. It covers its use in an attempt to develop new and relevant disease models to further elucidate gene function, and create new ways to understand the genetic basis of human diseases. We will cover the advantages and potential of the use of CRISPR-Cas9 technology on HAP1 to easily and efficiently study the functional interpretation of gene function and human single-nucleotide genetic variants of unknown significance identified through NGS technologies, and its implications for changes in clinical practice and patient care.

Molecular functions of MCM8 and MCM9 and their associated pathologies.

Minichromosome Maintenance 8 Homologous Recombination Repair Factor (MCM8) and Minichromosome Maintenance 9 Homologous Recombination Repair Factor (MCM9) are recently discovered minichromosome maintenance proteins and are implicated in multiple DNA-related processes and pathologies, including DNA replication (initiation), meiosis, homologous recombination and mismatch repair. Consistent with these molecular functions, variants of MCM8/MCM9 may predispose carriers to disorders such as infertility and cancer and should therefore be included in relevant diagnostic testing. In this overview of the (patho)physiological functions of MCM8 and MCM9 and the phenotype of MCM8/MCM9 variant carriers, we explore the potential clinical implications of MCM8/MCM9 variant carriership and highlight important future directions of MCM8 and MCM9 research. With this review, we hope to contribute to better MCM8/MCM9 variant carrier management and the potential utilization of MCM8 and MCM9 in other facets of scientific research and medical care.

Unraveling the impact of a germline heterozygous POLD1 frameshift variant in serrated polyposis syndrome.

Serrated polyposis syndrome (SPS) is one of the most frequent polyposis syndromes characterized by an increased risk for developing colorectal cancer (CRC). Although SPS etiology has been mainly associated with environmental factors, germline predisposition to SPS could also be relevant for cases with familial aggregation or a family history of SPS/CRC. After whole-exome sequencing of 39 SPS patients from 16 families, we identified a heterozygous germline frameshift variant in the POLD1 gene (c.1941delG, p.(Lys648fs*46)) in a patient with SPS and CRC. Tumor presented an ultra-hypermutated phenotype and microsatellite instability. The POLD1 germline variant segregated in three additional SPS-affected family members. We attempted to create yeast and cellular models for this variant but were no viable. Alternatively, we generated patient-derived organoids (PDOs) from healthy rectal tissue of the index case, as well as from a control donor. Then, we challenged PDOs with a DNA-damaging agent to induce replication stress. No significant differences were observed in the DNA damage response between control and POLD1-Lys648fs PDOs, nor specific mutational signatures were observed. Our results do not support the pathogenicity of the analyzed POLD1 frameshift variant. One possible explanation is that haplosufficiency of the wild-type allele may be compensating for the absence of expression of the frameshift allele. Overall, future work is required to elucidate if functional consequences could be derived from POLD1 alterations different from missense variants in their proofreading domain. To our knowledge, our study presents the first organoid model for germline POLD1 variants and establishes the basis for its use as a model for disease in SPS, CRC and other malignancies.

Evaluating the Potential of Polygenic Risk Score to Improve Colorectal Cancer Screening.

BACKGROUND: Colorectal cancer has high incidence and associated mortality worldwide. Screening programs are recommended for men and women over 50. Intermediate screens such as fecal immunochemical testing (FIT) select patients for colonoscopy with suboptimal sensitivity. Additional biomarkers could improve the current scenario. METHODS: We included 2,893 individuals with a positive FIT test. They were classified as cases when a high-risk lesion for colorectal cancer was detected after colonoscopy, whereas the control group comprised individuals with low-risk or no lesions. 65 colorectal cancer risk genetic variants were genotyped. Polygenic risk score (PRS) and additive models for risk prediction incorporating sex, age, FIT value, and PRS were generated. RESULTS: Risk score was higher in cases compared with controls [per allele OR = 1.04; 95% confidence interval (CI), 1.02-1.06; P < 0.0001]. A 2-fold increase in colorectal cancer risk was observed for subjects in the highest decile of risk alleles (≥65), compared with those in the first decile (≤54; OR = 2.22; 95% CI, 1.59-3.12; P < 0.0001). The model combining sex, age, FIT value, and PRS reached the highest accuracy for identifying patients with a high-risk lesion [cross-validated area under the ROC curve (AUROC): 0.64; 95% CI, 0.62-0.66]. CONCLUSIONS: This is the first investigation analyzing PRS in a two-step colorectal cancer screening program. PRS could improve current colorectal cancer screening, most likely for higher at-risk subgroups. However, its capacity is limited to predict colorectal cancer risk status and should be complemented by additional biomarkers. IMPACT: PRS has capacity for risk stratification of colorectal cancer suggesting its potential for optimizing screening strategies alongside with other biomarkers.

Comprehensive Genomic Characterization of Fifteen Early-Onset Lynch-Like Syndrome Colorectal Cancers.

Lynch-like syndrome (LLS) is an increasingly common clinical challenge with an underlying molecular basis mostly unknown. To shed light onto it, we focused on a very young LLS early-onset colorectal cancer (CRC) cohort (diagnosis ≤ 40 y.o.), performing germline and tumor whole-exome sequencing (WES) of 15 patients, and additionally analyzing their corresponding tumor mutational burden (TMB) and mutational signatures. We identified four cases (27%) with double somatic putative variants in mismatch repair (MMR) core genes, as well as three additional cases (20%) with double MSH3 somatic alterations in tumors with unexplained MSH2/MSH6 loss of expression, and two cases (13%) with POLD1 potential biallelic alterations. Average TMB was significantly higher for LLS cases with double somatic alterations. Lastly, nine predicted deleterious variants in genes involved in the DNA repair functions and/or previously associated with CRC were found in nine probands, four of which also showed MMR biallelic somatic inactivation. In conclusion, we contribute new insights into LLS CRC, postulating MSH3 and POLD1 double somatic alterations as an underlying cause of a microsatellite instability (MSI) phenotype, proposing intrinsic biological differences between LLS with and without somatic alterations, and suggesting new predisposing candidate genes in this scenario.

Germline and Somatic Whole-Exome Sequencing Identifies New Candidate Genes Involved in Familial Predisposition to Serrated Polyposis Syndrome.

The serrated polyposis syndrome (SPS) is the most common and yet underdiagnosed colorectal polyposis syndrome. It is characterized by multiple and/or large colonic serrated polyps and a higher associated risk for colorectal cancer (CRC). The main objective of this study was to identify new candidate genes involved in the germline predisposition to SPS/CRC. Thirty-nine SPS patients from 16 families (≥2 patients per family) were recruited without alterations in well-known hereditary CRC genes, and germline and somatic whole-exome sequencing were performed. Germline rare variants with plausible pathogenicity, located in genes involved in cancer development, senescence and epigenetic regulation were selected. Somatic mutational profiling and signature analysis was pursued in one sample per family, when possible. After data filtering, ANXA10, ASXL1, CFTR, DOT1L, HIC1, INO80, KLF3, MCM3AP, MCM8, PDLIM2, POLD1, TP53BP1, WNK2 and WRN were highlighted as the more promising candidate genes for SPS germline predisposition with potentially pathogenic variants shared within families. Somatic analysis characterized mutational profiles in advanced serrated polyps/tumors, revealing a high proportion of hypermutated samples, with a prevalence of clock-like mutational signatures in most samples and the presence of DNA mismatch repair-defective signatures in some cases. In conclusion, we identified new candidate genes to be involved in familial SPS. Further functional studies and replication in additional cohorts are required to confirm the selected candidates.

Germline biallelic Mcm8 variants are associated with early-onset Lynch-like syndrome.

Lynch syndrome is the most common cause of hereditary colorectal cancer (CRC), and it is characterized by DNA mismatch repair (MMR) deficiency. The term Lynch-like syndrome (LLS) is used for patients with MMR-deficient tumors and neither germline mutation in MLH1, MSH2, MSH6, PMS2, or EPCAM nor MLH1 somatic methylation. Biallelic somatic inactivation or cryptic germline MMR variants undetected during genetic testing have been proposed to be involved. Sixteen patients with early-onset LLS CRC were selected for germline and tumor whole-exome sequencing. Two potentially pathogenic germline MCM8 variants were detected in a male patient with LLS with fertility problems. A knockout cellular model for MCM8 was generated by CRISPR/Cas9 and detected genetic variants were produced by mutagenesis. DNA damage, microsatellite instability, and mutational signatures were monitored. DNA damage was evident for MCM8KO cells and the analyzed genetic variants. Microsatellite instability and mutational signatures in MCM8KO cells were compatible with the involvement of MCM8 in MMR. Replication in an independent familial cancer cohort detected additional carriers. Unexplained MMR-deficient CRC cases, even showing somatic biallelic MMR inactivation, may be caused by underlying germline defects in genes different than MMR genes. We suggest MCM8 as a gene involved in CRC germline predisposition with a recessive pattern of inheritance.

Approaches to functionally validate candidate genetic variants involved in colorectal cancer predisposition.

Most next generation sequencing (NGS) studies identified candidate genetic variants predisposing to colorectal cancer (CRC) but do not tackle its functional interpretation to unequivocally recognize a new hereditary CRC gene. Besides, germline variants in already established hereditary CRC-predisposing genes or somatic variants share the same need when trying to categorize those with relevant significance. Functional genomics approaches have an important role in identifying the causal links between genetic architecture and phenotypes, in order to decipher cellular function in health and disease. Therefore, functional interpretation of identified genetic variants by NGS platforms is now essential. Available approaches nowadays include bioinformatics, cell and molecular biology and animal models. Recent advances, such as the CRISPR-Cas9, ZFN and TALEN systems, have been already used as a powerful tool with this objective. However, the use of cell lines is of limited value due to the CRC heterogeneity and its close interaction with microenvironment. Access to tridimensional cultures or organoids and xenograft models that mimic the in vivo tissue architecture could revolutionize functional analysis. This review will focus on the application of state-of-the-art functional studies to better tackle new genes involved in germline predisposition to this neoplasm.

Identification of a Novel Candidate Gene for Serrated Polyposis Syndrome Germline Predisposition by Performing Linkage Analysis Combined With Whole-Exome Sequencing.

OBJECTIVES: Serrated polyposis syndrome (SPS) is a complex disorder with a high risk of colorectal cancer for which the germline factors remain largely unknown. Here, we combined whole-exome sequencing (WES) and linkage studies in families with multiple members affected by SPS to identify candidate genes harboring rare variants with higher penetrance effects. METHODS: Thirty-nine affected subjects from 16 extended SPS families underwent WES. Genome-wide linkage analysis was performed under linear and exponential models. The contribution of rare coding variants selected to be highly pathogenic was assessed using the gene-based segregation test. RESULTS: A significant linkage peak was identified on chromosome 3p25.2-p22.3 (maxSNP = rs2293787; LODlinear = 2.311, LODexp = 2.11), which logarithm of the odds (LOD) score increased after fine mapping for the same marker (maxSNP = rs2293787; LODlinear = 2.4, LODexp = 2.25). This linkage signal was replicated in 10 independent sets of random markers from this locus. To assess the contribution of rare variants predicted to be pathogenic, we performed a family-based segregation test with 11 rare variants predicted to be deleterious from 10 genes under the linkage intervals. This analysis showed significant segregation of rare variants with SPS in CAPT7, TMEM43, NGLY1, and FBLN2 genes (weighted P value > 0.007). DISCUSSION: Protein network analysis suggested FBLN2 as the most plausible candidate genes for germline SPS predisposition. Etiologic rare variants implicated in disease predisposition may be identified by combining traditional linkage with WES data. This powerful approach was effective for the identification of a new candidate gene for hereditary SPS.

Integrated Analysis of Germline and Tumor DNA Identifies New Candidate Genes Involved in Familial Colorectal Cancer.

Colorectal cancer (CRC) shows aggregation in some families but no alterations in the known hereditary CRC genes. We aimed to identify new candidate genes which are potentially involved in germline predisposition to familial CRC. An integrated analysis of germline and tumor whole-exome sequencing data was performed in 18 unrelated CRC families. Deleterious single nucleotide variants (SNV), short insertions and deletions (indels), copy number variants (CNVs) and loss of heterozygosity (LOH) were assessed as candidates for first germline or second somatic hits. Candidate tumor suppressor genes were selected when alterations were detected in both germline and somatic DNA, fulfilling Knudson's two-hit hypothesis. Somatic mutational profiling and signature analysis were also performed. A series of germline-somatic variant pairs were detected. In all cases, the first hit was presented as a rare SNV/indel, whereas the second hit was either a different SNV (3 genes) or LOH affecting the same gene (141 genes). BRCA2, BLM, ERCC2, RECQL, REV3L and RIF1 were among the most promising candidate genes for germline CRC predisposition. The identification of new candidate genes involved in familial CRC could be achieved by our integrated analysis. Further functional studies and replication in additional cohorts are required to confirm the selected candidates.