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 var iants by NGS platforms is now essential. Available approaches nowadays include bioinformatics, cell and mo lecular 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 ana lysis. 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

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

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

SUPPLEMENTARY MATERIAL accompanies this paper athttp://links.lww.com/CTG/A114OBJECTIVES: 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: 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 Pvalue > 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.M.D.-G. was supported by a contract from Agencia de Gestio d'Ajuts Universitaris i de Recerca (AGAUR) (Generalitat de Catalunya, 2018FI_B1_00213). S.F.-E., C.A.-C. and J.M. were supported by a contract from CIBEREHD. Y.S.L. was supported by a fellowship (LCF/BQ/DI18/11660058) from "la Caixa" Foundation (ID 100010434) funded EU Horizon 2020 Programme (Marie Sklodowska-Curie grant agreement no. 713673). LB was supported by a Juan de la Cierva postdoctoral contract (FJCI-2017-32593). CIBEREHD and CIBERONC are funded by the Instituto de Salud Carlos III. CT, BJO, and JMF were supported by Australian National Health and Medical Research (NHMRC) Project Grants 1063960 and 1066177. This research was supported by grants from Fondo de Investigacion Sanitaria/FEDER (16/00766, 17/00878), Fundacion Cientifica de la Asociacion Espanola contra el Cancer (GCB13131592CAST), PERIS (SLT002/16/00398, Generalitat de Catalunya), CERCA Programme (Generalitat de Catalunya), and Agencia de Gestio d'Ajuts Universitaris i de Recerca (Generalitat de Catalunya, GRPRE 2017SGR21, GRC 2017SGR653). This article is based on work from COST Action CA17118, supported by COST (European Cooperation in Science and Technology). www.cost.eu.Potential competing interests: None to report

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

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

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

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

Background: Colorectal cancer has high incidence and associ-ated 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 geno-typed. 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 = 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

Discovery of common and rare genetic risk variants for colorectal cancer.

To further dissect the genetic architecture of colorectal cancer (CRC), we performed whole-genome sequencing of 1,439 cases and 720 controls, imputed discovered sequence variants and Haplotype Reference Consortium panel variants into genome-wide association study data, and tested for association in 34,869 cases and 29,051 controls. Findings were followed up in an additional 23,262 cases and 38,296 controls. We discovered a strongly protective 0.3% frequency variant signal at CHD1. In a combined meta-analysis of 125,478 individuals, we identified 40 new independent signals at P < 5 × 10-8, bringing the number of known independent signals for CRC to ~100. New signals implicate lower-frequency variants, Krüppel-like factors, Hedgehog signaling, Hippo-YAP signaling, long noncoding RNAs and somatic drivers, and support a role for immune function. Heritability analyses suggest that CRC risk is highly polygenic, and larger, more comprehensive studies enabling rare variant analysis will improve understanding of biology underlying this risk and influence personalized screening strategies and drug development.Goncalo R Abecasis has received compensation from 23andMe and Helix. He is currently an employee of Regeneron Pharmaceuticals. Heather Hampel performs collaborative research with Ambry Genetics, InVitae Genetics, and Myriad Genetic Laboratories, Inc., is on the scientific advisory board for InVitae Genetics and Genome Medical, and has stock in Genome Medical. Rachel Pearlman has participated in collaborative funded research with Myriad Genetics Laboratories and Invitae Genetics but has no financial competitive interest

Genetic architectures of proximal and distal colorectal cancer are partly distinct.

OBJECTIVE: An understanding of the etiologic heterogeneity of colorectal cancer (CRC) is critical for improving precision prevention, including individualized screening recommendations and the discovery of novel drug targets and repurposable drug candidates for chemoprevention. Known differences in molecular characteristics and environmental risk factors among tumors arising in different locations of the colorectum suggest partly distinct mechanisms of carcinogenesis. The extent to which the contribution of inherited genetic risk factors for CRC differs by anatomical subsite of the primary tumor has not been examined. DESIGN: To identify new anatomical subsite-specific risk loci, we performed genome-wide association study (GWAS) meta-analyses including data of 48 214 CRC cases and 64 159 controls of European ancestry. We characterised effect heterogeneity at CRC risk loci using multinomial modelling. RESULTS: We identified 13 loci that reached genome-wide significance (p<5×10-8) and that were not reported by previous GWASs for overall CRC risk. Multiple lines of evidence support candidate genes at several of these loci. We detected substantial heterogeneity between anatomical subsites. Just over half (61) of 109 known and new risk variants showed no evidence for heterogeneity. In contrast, 22 variants showed association with distal CRC (including rectal cancer), but no evidence for association or an attenuated association with proximal CRC. For two loci, there was strong evidence for effects confined to proximal colon cancer. CONCLUSION: Genetic architectures of proximal and distal CRC are partly distinct. Studies of risk factors and mechanisms of carcinogenesis, and precision prevention strategies should take into consideration the anatomical subsite of the tumour

Variants genètiques de baixa penetrància en la predisposició germinal al càncer colorectal i la síndrome de poliposi serrada

[cat] El càncer colorectal (CCR) representa una de les neoplàsies amb major incidència i mortalitat a escala global. Per aquest motiu els sistemes nacionals de salut europeus han posat en marxa programes de cribratge poblacionals amb l’objectiu d’augmentar la detecció precoç de lesions precursores o del càncer, i així, poder evitar l’aparició de CCR o disminuir-ne la mortalitat. Tanmateix, les modalitats dels programes de cribratge amb prova intermèdia, com la prova FIT (prova immunohistoquímica fecal), tendeixen a tenir un elevat nombre de falsos positius a causa de l’especificitat subòptima d’aquesta prova, el que es tradueix en colonoscòpies innecessàries. Per aquest motiu, és fonamental establir noves estratègies que millorin el rendiment de les actuals. La síndrome de poliposi serrada (SPS) és una entitat clínica caracteritzada per la presència de grans i/o múltiples lesions/pòlips serrats en el còlon i un alt risc a CCR. La causa de la SPS és desconeguda. No obstant una petita proporció de casos podrien ser resultat de factors genètics heretats, sobretot per la multiplicitat de les lesions, el ràpid desenvolupament d’un CCR i les edats joves de diagnòstic. Tanmateix, gran part de la predisposició germinal a la SPS està per determinar. En aquest sentit, pel CCR es coneix que variants genètiques comunes i de baixa penetrància contribueixen a la predisposició germinal a CCR, no obstant, encara no s’ha explorat el paper d’aquest tipus de variants en la predisposició germinal a la SPS. En aquest context, el primer objectiu proposat de la present tesi doctoral va ser identificar quines variants genètiques comunes i de baixa penetrància associades a la predisposició germinal a CCR estan també implicades en la predisposició germinal a SPS. Per contra, el segon objectiu, va ser avaluar si les variants genètiques comunes i de baixa penetrància associades a la predisposició germinal a CCR es poden aplicar en el programa de cribratge actual per optimitzar-lo. Per una banda, per tal d’avaluar si les variants genètiques comunes i de baixa penetrància associades a la predisposició germinal a CCR es poden aplicar en el programa de cribratge actual per optimitzar-lo, es van genotipar 65 variants genètiques germinals associades consistentment a la predisposició germinal del CCR en una cohort de 2.893 participants del programa de detecció precoç del CCR de Barcelona, amb una prova de sang oculta en femta positiva. Posteriorment, es va calcular el PRS per a cada individu. Els resultats obtinguts al comparar la mitjana dels valors del PRS entre casos i controls van assenyalar que els casos tenien valors de PRS més elevats en comparació amb els controls (OR 1,4; IC 95% 1,02-1,06; p-valor < 0,0001). Al comparar el PRS dels individus de l’últim decil amb els del primer decil, s’observava que els de l’últim decil tenien gairebé 2,5 vegades més risc de tenir un CCR o una lesió associada a risc de CCR (OR 2,22; IC 95% 1,59-3,12; p-valor <0,0001). Amb aquests resultats semblava factible identificar un subgrup de participants amb un major risc de tenir lesions precanceroses o un CCR a conseqüència d’un PRS elevat. Seguidament, es van generar 4 models de predicció de risc amb les variables edat, sexe, valor del FIT i valor del PRS. Els resultats estimats directament del model predictiu generat a partir de l’edat, el sexe, el valor del FIT i el PRS (AUC 0,639; IC 95% 0,619-0,660) evidenciaven una millora en la discriminació en comparació amb el model predictiu actual utilitzat en el programa de cribratge de CCR el qual només es basa en el valor del FIT (AUC 0,626; IC 95% 0,605-0,647). Per aquest motiu, els resultats obtinguts a partir d’aquest estudi van permetre suggerir que el PRS podria ajudar a millorar els resultats actuals dels programes de cribratge de CCR. No obstant això, la seva capacitat és limitada i s'ha de complementar amb biomarcadors addicionals com el microbioma o factors ambientals. Per l’altra, per tal d’identificar quines variants genètiques comunes i de baixa penetrància associades a la predisposició germinal a CCR estan també implicades en la predisposició germinal a SPS, es va realitzar un estudi d’associació de cas-control. Per això, es van genotipar 65 variants genètiques germinals associades consistentment a la predisposició germinal del CCR en 219 pacients diagnosticats amb la SPS i 548 controls. Posteriorment, es va calcular un PRS per avaluar si les persones amb SPS tenien en general un nombre més elevat d’al·lels de risc que les persones del grup control. Els resultats de l’estudi d’associació van permetre identificar 7 variants associades amb la susceptibilitat germinal de la SPS en la nostra cohort (rs4779584-GREM1, rs16892766-EIF3H, rs3217810-CCND2, rs992157-PNKD1/TMBIM1, rs704017-ZMIZ1, rs11196172-TCF7L2, rs6061231-LAMA5). D’aquestes, la variant que va mostrar una associació més significativa amb la SPS va ser la variant rs4779584 (OR 1,573; IC 95% 1,21-2,04; p-valor 0,0006). Els resultats obtinguts a partir d’aquest estudi van permetre suggerir que algunes variants genètiques de risc de CCR també podrien estar involucrades en la susceptibilitat a l'SPS i que la variant rs4779584, prop de GREM1, semblaria ser la variant genètica comuna i de baixa penetrància amb una associació més important amb la susceptibilitat de la SPS identificada fins ara.[eng] Colorectal cancer (CRC) 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. In the other hand serrated polyposis syndrome (SPS) is a clinical entity characterised by large and/or multiple 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. Accordingly, this thesis has two main purposes. The first one was to evaluate the potential of polygenic risk score to improve colorectal cancer screening while the second one was to evaluate if common, low-penetrance genetic variants for CRC risk are also implicated in SPS genetic susceptibility. To assess whether common genetic variants and low penetrance associated with germinal predisposition in CCR can be applied in the current screening program to optimize it, 65 germinal genetic variants associated consistent with the germinal predisposition of the CCR were genotyped in a cohort of 2,893 participants of the Barcelona CCR early detection program with a positive FIT. Subsequently, the PRS was calculated for each individual. To achieve the first objective, all the individuals included in the study 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. To achieve the second objective 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. The results obtained by comparing the average PRS values between cases and controls noted that 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]. Moreover, 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 results obtained by comparing the different risk models showed that 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]. On the other hand, to identify which common genetic variants and low penetrance associated with germinal predisposition in CCR are also involved in germinal predisposition in SPS a case-control association study was conducted. Therefore, 65 germinal genetic variants associated consistent with germinal CCR predisposition in 219 patients diagnosed with SPS and 548 controls were genotyped. Subsequently, a PRS was calculated to assess whether people with SPS generally had a higher number of risk alleles than those in the control group. The results showed that seven genetic variants were significantly associated to SPS (rs4779584-GREM1, rs16892766-EIF3H, rs3217810-CCND2, rs992157-PNKD1/TMBIM1, rs704017-ZMIZ1, rs11196172-TCF7L2, rs6061231-LAMA5). Moreover, a 4-fold increase in SPS risk was observed when comparing subjects within the highest decile of variants (≥65) with those in the first decile (≤50). Consequently, we could conclude that PRS has capacity for risk stratification of colorectal cancer suggesting its potential for optimizing screening strategies alongside with other biomarkers and that genetic variants for CRC risk are also involved in SPS susceptibility