Heterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair-deficiency syndrome patients. Mutation detection is... Show moreHeterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair-deficiency syndrome patients. Mutation detection is complicated by the occurrence of sequence exchange events between the duplicated regions of PMS2 and PMS2CL. We investigated the frequency of such events with a nonspecific polymerase chain reaction (PCR) strategy, coamplifying both PMS2 and PMS2CL sequences. This allowed us to score ratios between gene and pseudogene-specific nucleotides at 29 PSV sites from exon 11 to the end of the gene. We found sequence transfer at all investigated PSVs from intron 12 to the 3' end of the gene in 4 to 52% of DNA samples. Overall, sequence exchange between PMS2 and PMS2CL was observed in 69% (83/120) of individuals. We demonstrate that mutation scanning with PMS2-specific PCR primers and MLPA probes, designed on PSVs, in the 3' duplicated region is unreliable, and present an RNA-based mutation detection strategy to improve reliability. Using this strategy, we found 19 different putative pathogenic PMS2 mutations. Four of these (21%) are lying in the region with frequent sequence transfer and are missed or called incorrectly as homozygous with several PSV-based mutation detection methods. Hum Mutat 31:578-587, 2010. (C) 2010 Wiley-Liss, Inc. Show less
Purpose: Risk assessment for locoregional disease recurrence would be highly valuable in preoperative treatment planning for patients undergoing primary rectal tumor resection. Epigenetic... Show morePurpose: Risk assessment for locoregional disease recurrence would be highly valuable in preoperative treatment planning for patients undergoing primary rectal tumor resection. Epigenetic aberrations such as DNA methylation have been shown to be significant prognostic biomarkers of disease outcome. In this study, we evaluated the significance of a quantitative epigenetic multimarker panel analysis of primary tumors to predict local recurrence in rectal cancer patients from a retrospective multicenter clinical trial. Experimental Design: Primary tumors were studied from patients enrolled in the trial who underwent total mesorectal excision for rectal cancer (n = 325). Methylation levels of seven methylated-in-tumor (MINT) loci were assessed by absolute quantitative assessment of methylated alleles. Unsupervised random forest clustering of quantitative MINT methylation data was used to show subclassification into groups with matching methylation profiles. Results: Variable importance parameters [Gini-Index (GI)] of the clustering algorithm indicated MINT3 and MINT17 (GI, 20.2 and 20.7, respectively) to be informative for patient grouping compared with the other MINT loci (highest GI, 12.2). When using this two-biomarker panel, four different patient clusters were identified. One cluster containing 73% (184 of 251) of the patients was at significantly increased risk of local recurrence (hazard ratio, 10.23; 95% confidence interval, 1.38-75.91) in multivariate analysis, corrected for standard prognostic factors of rectal cancer. This group showed a significantly higher local recurrence probability than patients receiving preoperative radiation (P < 0.0001). Conclusion: Quantitative epigenetic subclassification of rectal cancers has clinical utility in distinguishing tumors with increased risk for local recurrence and may help tailor treatment regimens for locoregional control. Clin Cancer Res; 16(10); 2811-8. (C) 2010 AACR. Show less
Roon, E.H.J. van; Puijenbroek, M. van; Middeldorp, A.; Eijk, R. van; Meijer, E.J. de; Erasmus, D.; ... ; Morreau, H. 2010
Background: To investigate the etiology of MLH1 promoter methylation in mismatch repair (MMR) mutation-negative early onset MSI-H colon cancer. As this type of colon cancer is associated with high... Show moreBackground: To investigate the etiology of MLH1 promoter methylation in mismatch repair (MMR) mutation-negative early onset MSI-H colon cancer. As this type of colon cancer is associated with high ages, young patients bearing this type of malignancy are rare and could provide additional insight into the etiology of sporadic MSI-H colon cancer. Methods: We studied a set of 46 MSI-H colon tumors cases with MLH1 promoter methylation which was enriched for patients with an age of onset below 50 years (n = 13). Tumors were tested for CIMP marker methylation and mutations linked to methylation: BRAF, KRAS, GADD45A and the MLH1 -93G>A polymorphism. When available, normal colon and leukocyte DNA was tested for GADD45A mutations and germline MLH1 methylation. SNP array analysis was performed on a subset of tumors. Results: We identified two cases (33 and 60 years) with MLH1 germline promoter methylation. BRAF mutations were less frequent in colon cancer patients below 50 years relative to patients above 50 years (p-value: 0.044). CIMP-high was infrequent and related to BRAF mutations in patients below 50 years. In comparison with published controls the G>A polymorphism was associated with our cohort. Although similar distribution of the pathogenic A allele was observed in the patients with an age of onset above and below 50 years, the significance for the association was lost for the group under 50 years. GADD45A sequencing yielded an unclassified variant. Tumors from both age groups showed infrequent copy number changes and loss-of-heterozygosity. Conclusion: Somatic or germline GADD45A mutations did not explain sporadic MSI-H colon cancer. Although germline MLH1 methylation was found in two individuals, locus-specific somatic MLH1 hypermethylation explained the majority of sporadic early onset MSI-H colon cancer cases. Our data do not suggest an intrinsic tendency for CpG island hypermethylation in these early onset MSI-H tumors other than through somatic mutation of BRAF. Show less
Drost, M.; Zonneveld, J.B.M.; Dijk, L. van; Morreau, H.; Tops, C.M.; Vasen, H.F.A.; ... ; Wind, N. de 2010
The hereditary colon and endometrium cancer predisposition Lynch Syndrome (also called HNPCC) is caused by a germ-line mutation in one of the DNA mismatch repair (MMR) genes. A significant fraction... Show moreThe hereditary colon and endometrium cancer predisposition Lynch Syndrome (also called HNPCC) is caused by a germ-line mutation in one of the DNA mismatch repair (MMR) genes. A significant fraction of the gene alterations detected in suspected Lynch Syndrome patients is comprised of amino acid substitutions. The relevance for cancer risk of these variants is difficult to assess, as currently no time- and cost-effective, validated, and widely applicable functional assays for the measurement of MMR activity are available. Here we describe a rapid, cell-free, and easily quantifiable MMR activity assay for the diagnostic assessment of variants of the MLH1 MMR protein. This assay allows the parallel generation and functional analysis of a series of variants of the MLH1 protein in vitro using readily available, or preprepared, reagents. Using this assay we have tested 26 MLH1 variants and of these, 15 had lost activity. These results are in concordance with those obtained from first-generation assays and with in silico and pathology data. After its multifocal technical and clinical validation this assay could have great impact for the diagnosis and counseling of carriers of an MLH1 variant and their relatives. Show less
Objective: Cure rate for primary hyperparathyroidism (PHPT) is reported to be 94-100% 1 year after surgery, but recent data suggest recurrence in 4% of the patients 1-5 years post-operatively. The... Show moreObjective: Cure rate for primary hyperparathyroidism (PHPT) is reported to be 94-100% 1 year after surgery, but recent data suggest recurrence in 4% of the patients 1-5 years post-operatively. The aim of our study was to establish the cure rate and its maintenance in the long-term after parathyroidectomy (PTx) in patients with sporadic PHPT. Design: Evaluation of recurrence in patients with sporadic hyperparathyroidism who underwent PTx 1-24 years prior to the study. Patients and methods: We identified 111 patients who underwent initial PTx between 1984 and 2008, and had no MEN-1, MEN-2, or CaR mutation; parathyroid carcinoma; a history of lithium use; or renal failure. Thirty-eight patients were lost to follow-up or were unwilling or unable to participate in the study. Cure was defined as maintenance of normal serum calcium and parathyroid hormone concentrations 6 months after PTx. Results: Cure was achieved in 68 of 73 patients studied (93%) and was sustained in all for 6+/-5 years. Conclusion: The cure rate of sporadic PHPT after initial surgery is 93%. When cure is achieved, this is sustained in 100% of the patients for up to 24 years post-operatively. Our data suggest that closer early follow-up is advocated in all patients undergoing PTx to definitively establish cure and to provide a safety net for those with residual gland pathology. The data do not support the need for long-term follow-up when cure is established 6 months after PTx. Show less
Eijk, R. van; Puijenbroek, M. van; Chhatta, A.R.; Gupta, N.; Vossen, R.H.A.M.; Lips, E.H.; ... ; Wezel, T. van 2010
Kirsten RAS (KRAS) is a small GTPase that plays a key role in Ras/mitogen-activated protein kinase signaling; somatic mutations in KRAS are frequently found in many cancers. The most common KRAS... Show moreKirsten RAS (KRAS) is a small GTPase that plays a key role in Ras/mitogen-activated protein kinase signaling; somatic mutations in KRAS are frequently found in many cancers. The most common KRAS mutations result in a constitutively active protein. Accurate detection of KRAS mutations is pivotal to the molecular diagnosis of cancer and may guide proper treatment selection. Here, we describe a two-step KRAS mutation screening protocol that combines whole-genome amplification (WGA), high-resolution melting analysis (HRM) as a prescreen method for mutation carrying samples, and direct Sanger sequencing of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue, from which limited amounts of DNA are available. We developed target-specific primers, thereby avoiding amplification of homologous KRAS sequences. The addition of herring sperm DNA facilitated WGA in DNA samples isolated from as few ass 100 cells. KRAS mutation screening using high-resolution melting analysis on wgaDNA from formalin-fixed, paraffin-embedded tissue is highly sensitive and specific; additionally, this method is feasible for screening of clinical specimens, as illustrated by our analysis of pancreatic cancers. Furthermore, PCR on wgaDNA does not introduce genotypic changes, as opposed to unamplified genomic DNA. This method can, after validation, be applied to virtually any potentially mutated region in the genome. (J Mol Diagn 2010, 12:27-34; DOI: 10.2353/jmoldx.2010.090028) Show less
Eijk, R. van; Eilers, P.H.C.; Natte, R.; Cleton-Jansen, A.M.; Morreau, H.; Wezel, T. van; Oosting, J. 2010
Background: Multiplex Ligation-Dependent Probe Amplification (MLPA) is an application that can be used for the detection of multiple chromosomal aberrations in a single experiment. In one reaction,... Show moreBackground: Multiplex Ligation-Dependent Probe Amplification (MLPA) is an application that can be used for the detection of multiple chromosomal aberrations in a single experiment. In one reaction, up to 50 different genomic sequences can be analysed. For a reliable work-flow, tools are needed for administrative support, data management, normalisation, visualisation, reporting and interpretation. Results: Here, we developed a data management system, MLPAInter for MLPA interpretation, that is windows executable and has a stand-alone database for monitoring and interpreting the MLPA data stream that is generated from the experimental setup to analysis, quality control and visualisation. A statistical approach is applied for the normalisation and analysis of large series of MLPA traces, making use of multiple control samples and internal controls. Conclusions: MLPAinter visualises MLPA data in plots with information about sample replicates, normalisation settings, and sample characteristics. This integrated approach helps in the automated handling of large series of MLPA data and guarantees a quick and streamlined dataflow from the beginning of an experiment to an authorised report. Show less
It is now recognised that a part of the inherited risk of colorectal cancer (CRC) can be explained by the co-inheritance of low-penetrance genetic variants. The accumulated experience to date in... Show moreIt is now recognised that a part of the inherited risk of colorectal cancer (CRC) can be explained by the co-inheritance of low-penetrance genetic variants. The accumulated experience to date in identifying these variants has served to highlight difficulties in conducting statistically and methodologically rigorous studies and follow-up analyses. The COGENT (COlorectal cancer GENeTics) consortium includes 20 research groups in Europe, Australia, the Americas, China and Japan. The overarching goal of COGENT is to identify and characterise low-penetrance susceptibility variants for CRC through association-based analyses. In this study, we review the rationale for identifying low-penetrance variants for CRC and our proposed strategy for establishing COGENT. British Journal of Cancer (2010) 102, 447-454. doi:10.1038/sj.bjc.6605338 www.bjcancer.com Published online 17 November 2009 (C) 2010 Cancer Research UK Show less
