[Show abstract][Hide abstract] ABSTRACT: Whilst common genetic variation in many non-coding genomic regulatory regions are known to impart risk of colorectal cancer (CRC), much of the heritability of CRC remains unexplained. To examine the role of recurrent coding sequence variation in CRC aetiology, we genotyped 12,638 CRCs cases and 29,045 controls from six European populations. Single-variant analysis identified a coding variant (rs3184504) in SH2B3 (12q24) associated with CRC risk (OR = 1.08, P = 3.9 × 10-7), and novel damaging coding variants in 3 genes previously tagged by GWAS efforts; rs16888728 (8q24) in UTP23 (OR = 1.15, P = 1.4 × 10-7); rs6580742 and rs12303082 (12q13) in FAM186A (OR = 1.11, P = 1.2 × 10-7 and OR = 1.09, P = 7.4 × 10-8); rs1129406 (12q13) in ATF1 (OR = 1.11, P = 8.3 × 10-9), all reaching exome-wide significance levels. Gene based tests identified associations between CRC and PCDHGA genes (P < 2.90 × 10-6). We found an excess of rare, damaging variants in base-excision (P = 2.4 × 10-4) and DNA mismatch repair genes (P = 6.1 × 10-4) consistent with a recessive mode of inheritance. This study comprehensively explores the contribution of coding sequence variation to CRC risk, identifying associations with coding variation in 4 genes and PCDHG gene cluster and several candidate recessive alleles. However, these findings suggest that recurrent, low-frequency coding variants account for a minority of the unexplained heritability of CRC.
Full-text · Article · Nov 2015 · Scientific Reports
[Show abstract][Hide abstract] ABSTRACT: Background:
The risk to develop colorectal and endometrial cancers among subjects testing positive for a pathogenic Lynch syndrome mutation varies, making the risk prediction difficult. Genetic risk modifiers alter the risk conferred by inherited Lynch syndrome mutations, and their identification can improve genetic counseling. We aimed at identifying rare genetic modifiers of the risk of Lynch syndrome endometrial cancer.
A family based approach was used to assess the presence of genetic risk modifiers among 35 Lynch syndrome mutation carriers having either a poor clinical phenotype (early age of endometrial cancer diagnosis or multiple cancers) or a neutral clinical phenotype. Putative genetic risk modifiers were identified by Next Generation Sequencing among a panel of 154 genes involved in endometrial physiology and carcinogenesis.
A simple pipeline, based on an allele frequency lower than 0.001 and on predicted non-conservative amino-acid substitutions returned 54 variants that were considered putative risk modifiers. The presence of two or more risk modifying variants in women carrying a pathogenic Lynch syndrome mutation was associated with a poor clinical phenotype.
A gene-panel is proposed that comprehends genes that can carry variants with putative modifying effects on the risk of Lynch syndrome endometrial cancer. Validation in further studies is warranted before considering the possible use of this tool in genetic counseling.
[Show abstract][Hide abstract] ABSTRACT: Lynch syndrome (LS), a heritable disorder with an increased risk of primarily colorectal cancer (CRC) and endometrial cancer (EC), can be caused by mutations in the PMS2 gene. We wished to establish whether genotype and/or parent-of-origin effects (POE) explain (part of) the reported variability in severity of the phenotype.
European PMS2 mutation carriers (n = 381) were grouped and compared based on RNA expression and whether the mutation was inherited paternally or maternally.
Mutation carriers with loss of RNA expression (group 1) had a significantly lower age at CRC diagnosis (51.1 years vs. 60.0 years, P = 0.035) and a lower age at EC diagnosis (55.8 years vs. 61.0 years, P = 0.2, nonsignificant) compared with group 2 (retention of RNA expression). Furthermore, group 1 showed slightly higher, but nonsignificant, hazard ratios (HRs) for both CRC (HR: 1.31, P = 0.38) and EC (HR: 1.22, P = 0.72). No evidence for a significant parent-of-origin effect was found for either CRC or EC.
PMS2 mutation carriers with retention of RNA expression developed CRC 9 years later than those with loss of RNA expression. If confirmed, this finding would justify a delay in surveillance for these cases. Cancer risk was not influenced by a parent-of-origin effect.Genet Med advance online publication 25 June 2015Genetics in Medicine (2015); doi:10.1038/gim.2015.83.
No preview · Article · Jun 2015 · Genetics in medicine: official journal of the American College of Medical Genetics
[Show abstract][Hide abstract] ABSTRACT: A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT-PCR analyses of patient RNA or functional splicing reporter minigene assays, are required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch-repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants.
[Show abstract][Hide abstract] ABSTRACT: Familial adenomatous polyposis is most frequently caused by pathogenic variants in either the APC gene or the MUTYH gene. The detection rate of pathogenic variants depends on the severity of the phenotype and sensitivity of the screening method, including sensitivity for mosaic variants. For 171 patients with multiple colorectal polyps without previously detectable pathogenic variant, APC was reanalyzed in leukocyte DNA by one uniform technique: high-resolution melting (HRM) analysis. Serial dilution of heterozygous DNA resulted in a lowest detectable allelic fraction of 6 % for the majority of variants. HRM analysis and subsequent sequencing detected pathogenic fully heterozygous APC variants in 10 (6 %) of the patients and pathogenic mosaic variants in 2 (1 %). All these variants were previously missed by various conventional scanning methods. In parallel, HRM APC scanning was applied to DNA isolated from polyp tissue of two additional patients with apparently sporadic polyposis and without detectable pathogenic APC variant in leukocyte DNA. In both patients a pathogenic mosaic APC variant was present in multiple polyps. The detection of pathogenic APC variants in 7 % of the patients, including mosaics, illustrates the usefulness of a complete APC gene reanalysis of previously tested patients, by a supplementary scanning method. HRM is a sensitive and fast pre-screening method for reliable detection of heterozygous and mosaic variants, which can be applied to leukocyte and polyp derived DNA.
Electronic supplementary material
The online version of this article (doi:10.1007/s10689-015-9780-5) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: Germline variants affecting the exonuclease domains of POLE and POLD1 predispose to multiple colorectal adenomas and/or colorectal cancer (CRC). The aim of this study was to estimate the prevalence of previously described heterozygous germline variants POLE c.1270C>G, p.(Leu424Val) and POLD1 c.1433G>A, p.(Ser478Asn) in a Dutch series of unexplained familial, early onset CRC and polyposis index cases. We examined 1188 familial CRC and polyposis index patients for POLE p.(Leu424Val) and POLD1 p.(Ser478Asn) variants using competitive allele-specific PCR. In addition, protein expression of the POLE and DNA mismatch repair genes was studied by immunohistochemistry in tumours from POLE carriers. Somatic mutations were screened using semiconductor sequencing. We detected three index patients (0.25%) with a POLE p.(Leu424Val) variant. In one patient, the variant was found to be de-novo. Tumours from three patients from two families were microsatellite instable, and immunohistochemistry showed MSH6/MSH2 deficiency suggestive of Lynch syndrome. Somatic mutations but no germline MSH6 and MSH2 variants were subsequently found, and one tumour displayed a hypermutator phenotype. None of the 1188 patients carried the POLD1 p.(Ser478Asn) variant. POLE germline variant carriers are also associated with a microsatellite instable CRC. POLE DNA analysis now seems warranted in microsatellite instable CRC, especially in the absence of a causative DNA mismatch repair gene germline variant.European Journal of Human Genetics advance online publication, 5 November 2014; doi:10.1038/ejhg.2014.242.
Full-text · Article · Nov 2014 · European journal of human genetics: EJHG
[Show abstract][Hide abstract] ABSTRACT: SDHB mutation carriers are predisposed to developing paragangliomas (PGLs). The objective of this study was to assess genotype-phenotype correlations of a Dutch cohort of SDHB mutation carriers and assess potential differences in clinical phenotypes related to specific SDHB founder mutations. Forty-seven consecutive SDHB mutation carriers were included. Initial screening consisted of measurement of 24 h urinary excretion of catecholamines and their metabolites in duplicate, repeated annually if initial biochemical screening was negative. Whole-body imaging studies with magnetic resonance imaging (MRI) or computed tomography (CT) and/or (123)I-MIBG scintigraphy were performed in case of catecholamine excess, and MRI or CT scans of thorax, abdomen and pelvis were performed every 2 years regardless of catecholamine levels. Repetitive head-and-neck MRI was performed at 2 year intervals. Mean follow-up was 3.6 ± 3.6 years. Twenty-seven persons (57 %) carried the SDHB c.423+1 G>A mutation and seven persons (15 %) the SDHB c.201-4429_287-933del (exon 3 deletion) mutation. No differences were found in the clinical phenotype of carriers of these two specific SDHB mutations. By end of follow-up, 49 % of SDHB mutation carriers displayed no biochemical or radiological evidence of manifest disease, i.e. they were unaffected carriers. Three persons (6 %) had been diagnosed with a pheochromocytoma (PCC), four with a sympathetic PGL (sPGL) (9 %), 18 with a HNPGL (38 %), and two persons (4 %) had developed a malignant paraganglioma, i.e. metastatic disease. In conclusion, the two main Dutch SDHB founder mutations do not differ in clinical expression and result in a relatively mild phenotype. Over one-third of SDHB mutation carriers develop HNPGL, with sPGL/PCC in only 15 % and malignancy in only 4 %.
[Show abstract][Hide abstract] ABSTRACT: Familial adenomatous polyposis (FAP) is usually characterised by the appearance of hundreds-to-thousands of adenomas throughout the colon and rectum and if left untreated the condition will develop into CRC with close to 100% penetrance. Germline mutations in the APC gene, which plays an integral role in the Wnt-signalling pathway, have been found to be responsible for 70-90% of FAP cases. Several studies suggest that modifier genes may play an important role in the development of CRC and possible modifiers for FAP have been suggested. Interestingly, a study has found that SNPs within ATP5A1 is associated with raised levels of ATP5A1 expression and high expression levels may facilitate CRC development. We aimed to determine if SNPs in ATP5A1 modify the risk of developing CRC/adenomas in FAP patients.
Genomic DNA from 139 Australian FAP patients with a germline APC mutation underwent genotyping at the Australian Genome Research Facility (AGRF) utilising iPLEX GOLD chemistry with Sequenom MassArray on an Autoflex Spectrometer for 16 SNPs in the ATP5A1 gene. Association between ages of diagnosis/risk of CRC/adenomas was tested with Kaplan-Meier estimator analysis, logistic regression and cox proportional hazard regression.
An association between age of diagnosis of CRC and genotypes was observed for SNP rs2578189 (p = 0.0014), with individuals harbouring the variant genotype developing CRC 29 years earlier than individuals harbouring the wildtype genotype. Individuals harbouring the variant genotype of SNP rs2578189 were also at increased risk of CRC (HR = 13.79, 95% CI = 2.36-80.64, p = 0.004). We used an independent Dutch FAP cohort (n = 427) to validate our results; no association between SNP rs2578189 and CRC was observed.
These results highlight the difficulties in studying a disease that has a high degree of intervention and also emphasize the importance of large sample sizes when searching for modifier genes in patients with an inherited predisposition to disease. To fully determine if there are genetic modifiers of disease in FAP we would encourage people that are interested in collaborating in future studies into the role of modifier genes in disease expression in FAP to join forces.
Full-text · Article · Dec 2013 · Hereditary Cancer in Clinical Practice
[Show abstract][Hide abstract] ABSTRACT: The clinical classification of hereditary sequence variants identified in disease-related genes directly affects clinical management of patients and their relatives. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) undertook a collaborative effort to develop, test and apply a standardized classification scheme to constitutional variants in the Lynch syndrome–associated genes MLH1, MSH2, MSH6 and PMS2. Unpublished data submission was encouraged to assist in variant classification and was recognized through microattribution. The scheme was refined by multidisciplinary expert committee review of the clinical and functional data available for variants, applied to 2,360 sequence alterations, and disseminated online. Assessment using validated criteria altered classifications for 66% of 12,006 database entries. Clinical recommendations based on transparent evaluation are now possible for 1,370 variants that were not obviously protein truncating from nomenclature. This large-scale endeavor will facilitate the consistent management of families suspected to have Lynch syndrome and demonstrates the value of multidisciplinary collaboration in the curation and classification of variants in public locus-specific databases.
[Show abstract][Hide abstract] ABSTRACT: Colorectal adenomatous polyposis is associated with a high risk of colorectal cancer (CRC) and is frequently caused by germline mutations in APC or MUTYH. However, in about 20-30% of patients no underlying gene defect can be identified. In this study, we tested if recently identified CRC risk variants play a role in patients with >10 adenomas.
We analysed a total of 16 SNPs with a reported association with CRC in a cohort of 252 genetically unexplained index patients with >10 colorectal adenomas and 745 controls. In addition, we collected detailed clinical information from index patients and their first-degree relatives (FDRs).
We found a statistically significant association with two of the variants tested: rs3802842 (at chromosome 11q23, OR=1.60, 95% CI 1.3 to 2.0) and rs4779584 (at chromosome 15q13, OR=1.50, 95% CI 1.2 to 1.9). The majority of index patients (84%) had between 10 and 100 adenomas and 15% had >100 adenomas. Only two index patients (1%), both with >100 adenomas, had FDRs with polyposis. Forty-one per cent of the index patients had one or more FDRs with CRC.
These SNPs are the first common, low-penetrant variants reported to be associated with adenomatous polyposis not caused by a defect in the APC, MUTYH, POLD1 and POLE genes. Even though familial occurrence of polyposis was very rare, CRC was over-represented in FDRs of polyposis patients and, if confirmed, these relatives will therefore benefit from surveillance.
No preview · Article · Nov 2013 · Journal of Medical Genetics
[Show abstract][Hide abstract] ABSTRACT: Lynch syndrome (LS), one of the most frequent forms of hereditary colorectal cancer (CRC), is caused by a defect in one of the mismatch repair (MMR) genes. Carriers of MMR defects have a strongly increased risk of developing CRC and endometrial cancer. Over the last few years, value-based healthcare has been introduced as an approach to the cost-effective delivery of measurable patient value over complete cycles of care. This requires all involved stakeholders to formulate and validate 'patient value' for Lynch syndrome, as well as to identify targets and associated costs. The aim of this study was to develop a value-based care model for Lynch syndrome that can determine patient value and associated costs, and to design a coordinated care pathway from existing guidelines. All specialists in our hospital involved in the management of LS patients evaluated the care delivered to these patients at their department and formulated outcome measures relevant to patient value. Patients were then invited to complete a questionnaire that assessed the importance of these measures on a scale of 1-10. Six high-value outcomes were identified: (1) prevention of cancer or detection of early stage cancer (2) rapid results from MMR gene mutation testing (3) rapid investigation of the colon and uterus (4) no/little pain during colonoscopy and gynaecologic examination/biopsy (5) the offer of psychological help and (6) registration with the Dutch Lynch syndrome registry. A total of 38 (59 %) out of 62 patients completed the questionnaire. The relevance of all outcomes was confirmed by the patients and mean scores varied from 7.2 to 9.9. Patients underscored the relevance of both proper patient education and the efficiency of surveillance during their care cycle. Value-based care delivery for Lynch syndrome includes the implementation of six parameters related to prevention and early detection of cancer, a short cycle time and registration to ensure continuation of care. Estimated costs are 3320 for the first cycle of care ( 3550 including gynaecologic surveillance) and approximately 720 per subsequent annual cycle ( 950 including gynaecologic surveillance).
[Show abstract][Hide abstract] ABSTRACT: Two colorectal cancer (CRC) susceptibility loci have been found to be significantly associated with an increased risk of CRC in Dutch Lynch syndrome (LS) patients. Recently, in a combined study of Australian and Polish LS patients, only MLH1 mutation carriers were found to be at increased risk of disease. A combined analysis of the three data-sets was performed to better define this association. This cohort-study includes three sample populations combined totaling 1,352 individuals from 424 families with a molecular diagnosis of LS. Seven SNPs, from six different CRC susceptibility loci, were genotyped by both research groups and the data analyzed collectively. We identified associations at two of the six CRC susceptibility loci in MLH1 mutation carriers from the combined LS cohort: 11q23.1 (rs3802842, HR = 2.68, p ≤ 0.0001) increasing risk of CRC, and rs3802842 in a pair-wise combination with 8q23.3 (rs16892766) affecting age of diagnosis of CRC (log-rank test; p ≤ 0.0001). A significant difference in the age of diagnosis of CRC of 28 years was observed in individuals carrying three risk alleles compared to those with 0 risk alleles for the pair-wise SNP combination. A trend (due to significance threshold of p ≤ 0.0010) was observed in MLH1 mutation carriers towards an increased risk of CRC for the pair-wise combination (p = 0.002). This study confirms the role of modifier loci in LS. We consider that LS patients with MLH1 mutations would greatly benefit from additional genotyping of SNPs rs3802842 and rs16892766 for personalized risk assessment and a tailored surveillance program.
Full-text · Article · Apr 2013 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: In the absence of a polyposis phenotype, colorectal cancer (CRC) patients referred for genetic testing because of early-onset disease and/or a positive family history, typically undergo testing for molecular signs of Lynch syndrome in their tumors. In the absence of these signs, DNA testing for germline mutations associated with other known tumor syndromes is usually not performed. However, a few studies in large series of CRC patients suggest that in a small percentage of CRC cases, bi-allelic MUTYH germline mutations can be found in the absence of the MUTYH-associated polyposis phenotype. This has not been studied in the Dutch population. Therefore, we analyzed the MUTYH gene for mutations in 89 patients with microsatellite-low or stable CRC cancer diagnosed before the age of 40 years or otherwise meeting the Bethesda criteria, all of them without a polyposis phenotype. In addition, we studied a series of 693 non-CRC patients with 1-13 adenomatous colorectal polyps for the MUTYH hotspot mutations Y179C, G396D and P405L. No bi-allelic MUTYH mutations were observed. Our data suggest that the contribution of bi-allelic MUTYH mutations to the development of CRC in Dutch non-polyposis patients that meet clinical genetic referral criteria, and to the development of low number of colorectal adenomas in non-CRC patients, is likely to be low.
[Show abstract][Hide abstract] ABSTRACT: Hereditary paraganglioma is a benign tumor syndrome with an age-dependent penetrance. Carriers of germline mutations in the SDHB or SDHD genes may develop parasympathetic paragangliomas in the head and neck region or sympathetic catecholamine-secreting abdominal and thoracic paragangliomas (pheochromocytomas). In this study, we aimed to establish paraganglioma risk in 101 asymptomatic germline mutation carriers and evaluate the results of our surveillance regimen. Asymptomatic carriers of an SDHD or SDHB mutation were included once disease status was established by MRI diagnosis. Clinical surveillance revealed a head and neck paraganglioma in 28 of the 47 (59.6%) asymptomatic SDHD mutation carriers. Risk of tumor development was significantly lower in SDHB mutation carriers: 2/17 (11.8%, P=0.001). Sympathetic paragangliomas were encountered in two SDHD mutation carriers and in one SDHB mutation carrier. In conclusion, asymptomatic carriers of an SDHD mutation are at a high risk for occult parasympathetic paraganglioma. SDHB carrier risk is considerably lower, consistent with lower penetrance of SDHB mutations. For both syndromes, the risk of symptomless sympathetic paragangliomas is small.European Journal of Human Genetics advance online publication, 5 September 2012; doi:10.1038/ejhg.2012.203.
No preview · Article · Sep 2012 · European journal of human genetics: EJHG
[Show abstract][Hide abstract] ABSTRACT: Heterozygous germline mutations in the mismatch repair gene PMS2 predispose carriers for Lynch syndrome, an autosomal dominant predisposition to cancer. Here, we present a LINE-1-mediated retrotranspositional insertion in PMS2 as a novel mutation type for Lynch syndrome. This insertion, detected with Southern blot analysis in the genomic DNA of the patient, is characterized as a 2.2 kb long 5' truncated SVA_F element. The insertion is not detectable by current diagnostic testing limited to MLPA and direct Sanger sequencing on genomic DNA. The molecular nature of this insertion could only be resolved in RNA from cultured lymphocytes in which nonsense-mediated RNA decay was inhibited. Our report illustrates the technical problems encountered in the detection of this mutation type. Especially large heterozygous insertions will remain unnoticed because of preferential amplification of the smaller wild-type allele in genomic DNA, and are probably underreported in the mutation spectra of autosomal dominant disorders.