Exome sequencing of early-onset patients supports genetic heterogeneity in colorectal cancer.

Colorectal cancer (CRC) is a complex disease that can be caused by a spectrum of genetic variants ranging from low to high penetrance changes, that interact with the environment to determine which individuals will develop the disease. In this study, we sequenced 20 early-onset CRC patients to discover novel genetic variants that could be linked to the prompt disease development. Eight genes, CHAD, CHD1L, ERCC6, IGTB7, PTPN13, SPATA20, TDG and TGS1, were selected and re-sequenced in a further 304 early onset CRC patients to search for rare, high-impact variants. Although we found a recurring truncating variant in the TDG gene shared by two independent patients, the results obtained did not help consolidate any of the candidates as promising CRC predisposing genes. However, we found that potential risk alleles in our extended list of candidate variants have a tendency to appear at higher numbers in younger cases. This supports the idea that CRC onset may be oligogenic in nature and may show molecular heterogeneity. Further, larger and robust studies are thus needed to unravel the genetics behind early-onset CRC development, coupled with novel functional analyses and omic approaches that may offer complementary insight.

Candidate predisposing germline copy number variants in early onset colorectal cancer patients

Purpose. A great proportion of the heritability of colorectal cancer (CRC) still remains unexplained, and rare variants, as well as copy number changes, have been proposed as potential candidates to explain the so-called ‘missing heritability’. We aimed to identify rare high-to-moderately penetrant copy number variants (CNVs) in patients suspected of having hereditary CRC due to an early onset. Methods/patients. We have selected for genome-wide copy number analysis, 27 MMR-proficient early onset CRC patients (<50 years) without identifiable germline mutations in Mendelian genes related to this phenotype. Rare CNVs were selected by removing all CNVs detected at MAF >1% in the in-house control CNV database (n = 629 healthy controls). Copy number assignment was checked by duplex real-time quantitative PCR or multiplex ligation probe amplification. Somatic mutation analysis in candidate genes included: loss of heterozygosity studies, point mutation screening, and methylation status of the promoter. Results. We have identified two rare germline deletions involving the AK3 and SLIT2 genes in two patients. The search for a second somatic mutational event in the corresponding CRC tumors showed loss of heterozygosity in AK3, and promoter hypermethylation in SLIT2. Both genes have been previously related to colorectal carcinogenesis. Conclusions. These findings suggest that AK3 and SLIT2 may be potential candidates involved in genetic susceptibility to CRC (AU)

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Candidate predisposing germline copy number variants in early onset colorectal cancer patients.

PURPOSE: A great proportion of the heritability of colorectal cancer (CRC) still remains unexplained, and rare variants, as well as copy number changes, have been proposed as potential candidates to explain the so-called 'missing heritability'. We aimed to identify rare high-to-moderately penetrant copy number variants (CNVs) in patients suspected of having hereditary CRC due to an early onset. METHODS/PATIENTS: We have selected for genome-wide copy number analysis, 27 MMR-proficient early onset CRC patients (<50 years) without identifiable germline mutations in Mendelian genes related to this phenotype. Rare CNVs were selected by removing all CNVs detected at MAF >1% in the in-house control CNV database (n = 629 healthy controls). Copy number assignment was checked by duplex real-time quantitative PCR or multiplex ligation probe amplification. Somatic mutation analysis in candidate genes included: loss of heterozygosity studies, point mutation screening, and methylation status of the promoter. RESULTS: We have identified two rare germline deletions involving the AK3 and SLIT2 genes in two patients. The search for a second somatic mutational event in the corresponding CRC tumors showed loss of heterozygosity in AK3, and promoter hypermethylation in SLIT2. Both genes have been previously related to colorectal carcinogenesis. CONCLUSIONS: These findings suggest that AK3 and SLIT2 may be potential candidates involved in genetic susceptibility to CRC.

High incidence of large deletions in the PMS2 gene in Spanish Lynch syndrome families.

Lynch syndrome (LS) is caused by germline mutations in one of the four mismatch repair (MMR) genes. Defects in this pathway lead to microsatellite instability (MSI) in DNA tumors, which constitutes the molecular hallmark of this disease. Selection of patients for genetic testing in LS is usually based on fulfillment of diagnostic clinical criteria (i.e. Amsterdam criteria or the revised Bethesda guidelines). However, following these criteria PMS2 mutations have probably been underestimated as their penetrances appear to be lower than those of the other MMR genes. The use of universal MMR study-based strategies, using MSI testing and immunohistochemical (IHC) staining, is being one proposed alternative. Besides, germline mutation detection in PMS2 is complicated by the presence of highly homologous pseudogenes. Nevertheless, specific amplification of PMS2 by long-range polymerase chain reaction (PCR) and the improvement of the analysis of large deletions/duplications by multiplex ligation-dependent probe amplification (MLPA) overcome this difficulty. By using both approaches, we analyzed 19 PMS2-suspected carriers who have been selected by clinical or universal strategies and found five large deletions and one frameshift mutation in PMS2 in six patients (31%). Owing to the high incidence of large deletions found in our cohort, we recommend MLPA analysis as the first-line method for searching germline mutations in PMS2.

A genome-wide association study on copy-number variation identifies a 11q11 loss as a candidate susceptibility variant for colorectal cancer.

Colorectal cancer (CRC) is a complex disease, and therefore its development is determined by the combination of both environmental factors and genetic variants. Although genome-wide association studies (GWAS) of SNP variation have conveniently identified 20 genetic variants so far, a significant proportion of the observed heritability is yet to be explained. Common copy-number variants (CNVs) are one of the most important genomic sources of variability, and hence a potential source to explain part of this missing genetic fraction. Therefore, we have performed a GWAS on CNVs to explore the relationship between common structural variation and CRC development. Phase 1 of the GWAS consisted of 881 cases and 667 controls from a Spanish cohort. Copy-number status was validated by quantitative PCR for each of those common CNVs potentially associated with CRC in phase I. Subsequently, SNPs were chosen as proxies for the validated CNVs for phase II replication (1,342 Spanish cases and 1,874 Spanish controls). Four common CNVs were found to be associated with CRC and were further replicated in Phase II. Finally, we found that SNP rs1944682, tagging a 11q11 CNV, was nominally associated with CRC susceptibility (p value = 0.039; OR = 1.122). This locus has been previously related to extreme obesity phenotypes, which could suggest a relationship between body weight and CRC susceptibility.

Pharmacogenomics in colorectal cancer: a genome-wide association study to predict toxicity after 5-fluorouracil or FOLFOX administration.

The development of genotyping technologies has allowed for wider screening for inherited causes of variable outcomes following drug administration. We have performed a genome-wide association study (GWAS) on 221 colorectal cancer (CRC) patients that had been treated with 5-fluorouracil (5-FU), either alone or in combination with oxaliplatin (FOLFOX). A validation set of 791 patients was also studied. Seven SNPs (rs16857540, rs2465403, rs10876844, rs10784749, rs17626122, rs7325568 and rs4243761) showed evidence of association (pooled P-values 0.020, 9.426E-03, 0.010, 0.017, 0.042, 2.302E-04, 2.803E-03) with adverse drug reactions (ADRs). This is the first study to explore the genetic basis of inter-individual variation in toxicity responses to the administration of 5-FU or FOLFOX in CRC patients on a genome-wide scale.

A two-phase case-control study for colorectal cancer genetic susceptibility: candidate genes from chromosomal regions 9q22 and 3q22.

BACKGROUND: Colorectal cancer (CRC) is the second cause of cancer-related death in the Western world. Much of the CRC genetic risk remains unidentified and may be attributable to a large number of common, low-penetrance genetic variants. Genetic linkage studies in CRC families have reported additional association with regions 9q22-31, 3q21-24, 7q31, 11q, 14q and 22q. There are several plausible candidate genes for CRC susceptibility within the aforementioned linkage regions including PTCH1, XPA and TGFBR1 in 9q22-31, and EPHB1 and MRAS in 3q21-q24. METHODS: CRC cases and matched controls were from EPICOLON, a prospective, multicentre, nationwide Spanish initiative, composed of two independent phases. Phase 1 corresponded to 515 CRC cases and 515 controls, whereas phase 2 consisted of 901 CRC cases and 909 controls. Genotyping was performed for 172 single-nucleotide polymorphisms (SNPs) in 84 genes located within regions 9q22-31 and 3q21-q24. RESULTS: None of the 172 SNPs analysed in our study could be formally associated with CRC risk. However, rs1444601 (TOPBP1) and rs13088006 (CDV3) in region 3q22 showed interesting results and may have an effect on CRC risk. CONCLUSIONS: TOPBP1 and CDV3 genetic variants on region 3q22 may modulate CRC risk. Further validation and meta-analysis should be undertaken in larger CRC cohorts.