Distribution of RET proto-oncogene variants in children with appendicitis.

BACKGROUND: In addition to patient-related systemic factors directing the immune response, the pathomechanisms of appendicitis (AP) might also include insufficient drainage leading to inflammation caused by decreased peristalsis. Genetic predisposition accounts for 30%-50% of AP. M. Hirschsprung (HSCR), also characterized by disturbed peristalsis, is associated with variants in the RET proto-oncogene. We thus hypothesized that RET variants contribute to the etiology of AP. METHODS: DNA from paraffin-embedded appendices and clinical data of 264 children were analyzed for the RET c.135A>G variant (rs1800858, NC_000010.11:g.43100520A>G). In 46 patients with gangrenous or perforated AP (GAP), peripheral blood DNA was used for RET sequencing. RESULTS: Germline mutations were found in 13% of GAP, whereas no RET mutations were found in controls besides the benign variant p.Tyr791Phe (NC_000010.11:g.43118460A>T). In GAP, the polymorphic G-allele in rs2435352 (NC_000010.11:g.43105241A>G) in intron 4 was underrepresented (p = 0.0317). CONCLUSION: Our results suggest an impact of the RET proto-oncogene in the etiology of AP. Mutations were similar to patients with HSCR but no clinical features of HSCR were observed. The pathological phenotypes in both populations might thus represent a multigenic etiology including RET germline mutations with phenotypic heterogeneity and incomplete penetrance.

Adenoma and colorectal cancer risks in Lynch syndrome, Lynch-like syndrome and familial colorectal cancer type X.

Lynch syndrome (LS), Lynch-like syndrome (LLS) and familial colorectal cancer type X (FCCX) are different entities of familial cancer predisposition leading to an increased risk of colorectal cancer (CRC). The aim of this prospective study was to characterise and to compare the risks for adenoma and CRC in these three risk groups. Data was taken from the registry of the German Consortium for Familial Intestinal Cancer. Patients were prospectively followed up in an intensified colonoscopic surveillance programme that included annual examinations. Cumulative risks for adenoma and CRC were calculated separately for LS, LLS and FCCX, and then for males and females. Multivariate Cox regression was used to analyse the independent contributions of risk group, mismatch repair gene (within LS), sex and previous adenoma. The study population comprised 1448 individuals (103 FCCX, 481 LLS and 864 LS). The risks were similar for colorectal adenomas, but different for first and metachronous CRC between the three risk groups. CRC risk was highest in LS, followed by LLS and lowest in FCCX. Male sex and a prevalent adenoma in the index colonoscopy were associated with a higher risk for incident adenoma and CRC. In patients with LS, CRC risks were particularly higher in female MSH2 than MLH1 carriers. Our study may support the development of risk-adapted surveillance policies in LS, LLS and FCCX.

No Difference in Penetrance between Truncating and Missense/Aberrant Splicing Pathogenic Variants in MLH1 and MSH2: A Prospective Lynch Syndrome Database Study.

BACKGROUND: Lynch syndrome is the most common genetic predisposition for hereditary cancer. Carriers of pathogenic changes in mismatch repair (MMR) genes have an increased risk of developing colorectal (CRC), endometrial, ovarian, urinary tract, prostate, and other cancers, depending on which gene is malfunctioning. In Lynch syndrome, differences in cancer incidence (penetrance) according to the gene involved have led to the stratification of cancer surveillance. By contrast, any differences in penetrance determined by the type of pathogenic variant remain unknown. OBJECTIVE: To determine cumulative incidences of cancer in carriers of truncating and missense or aberrant splicing pathogenic variants of the MLH1 and MSH2 genes. METHODS: Carriers of pathogenic variants of MLH1 (path_MLH1) and MSH2 (path_MSH2) genes filed in the Prospective Lynch Syndrome Database (PLSD) were categorized as truncating or missense/aberrant splicing according to the InSiGHT criteria for pathogenicity. RESULTS: Among 5199 carriers, 1045 had missense or aberrant splicing variants, and 3930 had truncating variants. Prospective observation years for the two groups were 8205 and 34,141 years, respectively, after which there were no significant differences in incidences for cancer overall or for colorectal cancer or endometrial cancers separately. CONCLUSION: Truncating and missense or aberrant splicing pathogenic variants were associated with similar average cumulative incidences of cancer in carriers of path MLH1 and path_MSH2.

Correction: Cancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: findings from the Prospective Lynch Syndrome Database.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cancer risks by gene, age, and gender in 6350 carriers of pathogenic mismatch repair variants: findings from the Prospective Lynch Syndrome Database.

PURPOSE: Pathogenic variants affecting MLH1, MSH2, MSH6, and PMS2 cause Lynch syndrome and result in different but imprecisely known cancer risks. This study aimed to provide age and organ-specific cancer risks according to gene and gender and to determine survival after cancer. METHODS: We conducted an international, multicenter prospective observational study using independent test and validation cohorts of carriers of class 4 or class 5 variants. After validation the cohorts were merged providing 6350 participants and 51,646 follow-up years. RESULTS: There were 1808 prospectively observed cancers. Pathogenic MLH1 and MSH2 variants caused high penetrance dominant cancer syndromes sharing similar colorectal, endometrial, and ovarian cancer risks, but older MSH2 carriers had higher risk of cancers of the upper urinary tract, upper gastrointestinal tract, brain, and particularly prostate. Pathogenic MSH6 variants caused a sex-limited trait with high endometrial cancer risk but only modestly increased colorectal cancer risk in both genders. We did not demonstrate a significantly increased cancer risk in carriers of pathogenic PMS2 variants. Ten-year crude survival was over 80% following colon, endometrial, or ovarian cancer. CONCLUSION: Management guidelines for Lynch syndrome may require revision in light of these different gene and gender-specific risks and the good prognosis for the most commonly associated cancers.

Analysis of gastrin-releasing peptide gene and gastrin-releasing peptide receptor gene in patients with agoraphobia.

A gastrin-releasing peptide receptor (GRPR) knock-out mouse model provided evidence that the gastrin-releasing peptide (GRP) and its neural circuitry operate as a negative feedback-loop regulating fear, suggesting a novel candidate mechanism contributing to individual differences in fear-conditioning and associated psychiatric disorders such as agoraphobia with/without panic disorder. Studies in humans, however, provided inconclusive evidence on the association of GRP and GRPR variations in agoraphobia with/without panic disorder. Based on these findings, we investigated whether GRP and GRPR variants are associated with agoraphobia. Mental disorders were assessed via the Munich-Composite International Diagnostic Interview (M-CIDI) in 95 patients with agoraphobia with/without panic disorder and 119 controls without any mental disorders. A complete sequence analysis of GRP and GRPR was performed in all participants. We found no association of 16 GRP and 7 GRPR variants with agoraphobia with/without panic disorder.

Evaluating the performance of clinical criteria for predicting mismatch repair gene mutations in Lynch syndrome: a comprehensive analysis of 3,671 families.

Carriers of mismatch repair (MMR) gene mutations have a high lifetime risk for colorectal and endometrial cancers, as well as other malignancies. As mutation analysis to detect these patients is expensive and time-consuming, clinical criteria and tumor-tissue analysis are widely used as pre-screening methods. The aim of our study was to evaluate the performance of commonly applied clinical criteria (the Amsterdam I and II Criteria, and the original and revised Bethesda Guidelines) and the results of tumor-tissue analysis in predicting MMR gene mutations. We analyzed 3,671 families from the German HNPCC Registry and divided them into nine mutually exclusive groups with different clinical criteria. A total of 680 families (18.5%) were found to have a pathogenic MMR gene mutation. Among all 1,284 families with microsatellite instability-high (MSI-H) colorectal cancer, the overall mutation detection rate was 53.0%. Mutation frequencies and their distribution between the four MMR genes differed significantly between clinical groups (p < 0.001). The highest frequencies were found in families fulfilling the Amsterdam Criteria (46.4%). Families with loss of MSH2 expression had higher mutation detection rates (69.5%) than families with loss of MLH1 expression (43.1%). MMR mutations were found significantly more often in families with at least one MSI-H small-bowel cancer (p < 0.001). No MMR mutations were found among patients under 40-years-old with only colorectal adenoma. Familial clustering of Lynch syndrome-related tumors, early age of onset, and familial occurrence of small-bowel cancer were clinically relevant predictors for Lynch syndrome.

Germline truncating-mutations in BRCA1 and MSH6 in a patient with early onset endometrial cancer.

BACKGROUND: Hereditary Breast and Ovarian Cancer Syndrome (HBOCS) and Hereditary Non-Polyposis Colorectal Cancer Syndrome (HNPCC, Lynch Syndrome) are two tumor predisposition syndromes responsible for the majority of hereditary breast and colorectal cancers. Carriers of both germline mutations in breast cancer genes BRCA1 or BRCA2 and in mismatch repair (MMR) genes MLH1, MSH2, MSH6 or PMS2 are very rare. CASE PRESENTATION: We identified germline mutations in BRCA1 and in MSH6 in a patient with increased risk for HBOC diagnosed with endometrial cancer at the age of 46 years. CONCLUSIONS: Although carriers of mutations in both MMR and BRCA genes are rare in Caucasian populations and anamnestical and histopathological findings may guide clinicians to identify these families, both syndromes can only be diagnosed through a complete gene analysis of the respective genes.

Mutation and association analyses of the candidate genes ESR1, ESR2, MAX, PCNA, and KAT2A in patients with unexplained MSH2-deficient tumors.

Lynch syndrome (Hereditary non-polyposis colorectal cancer/HNPCC) is a cancer susceptibility syndrome which is caused by germline mutations in DNA mismatch repair (MMR) genes, in particular MLH1 and MSH2. A pathogenic germline mutation in the respective MMR gene is suggested by the finding of a loss of a mismatch repair protein in tumor tissue on immunohistochemical staining combined with an early age of onset and/or the familial occurrence of colorectal cancer. Pathogenic germline mutations are identifiable in around 60% of patients suspected of Lynch syndrome, depending on the familial occurrence. The aim of the present study was to identify novel susceptibility genes for Lynch syndrome. 64 Healthy controls and 64 Lynch syndrome patients with no pathogenic MSH2 mutation but a loss of MSH2 expression in their tumor tissue were screened for rare and disease causing germline mutations in the functional candidate genes ESR1, ESR2, MAX, PCNA, and KAT2A. Thirty variants were identified, and these were then genotyped in an independent sample of 36 mutation negative Lynch syndrome patients and 234 controls. Since a trend towards association was observed for KAT2A, an additional set of 21 tagging SNPs was analyzed at this locus in a final case-control sample of 142 mutation negative Lynch syndrome patients and 298 controls. The mutation analysis failed to reveal any rare disease-causing mutations. No association was found at the single-marker or haplotypic level for any common disease-modifying variant. The present results suggest that neither rare nor common genetic variants in ESR1, ESR2, MAX, PCNA, or KAT2A contribute to the development of Lynch syndrome.

Association between TAS2R38 gene polymorphisms and colorectal cancer risk: a case-control study in two independent populations of Caucasian origin.

Molecular sensing in the lingual mucosa and in the gastro-intestinal tract play a role in the detection of ingested harmful drugs and toxins. Therefore, genetic polymorphisms affecting the capability of initiating these responses may be critical for the subsequent efficiency of avoiding and/or eliminating possible threats to the organism. By using a tagging approach in the region of Taste Receptor 2R38 (TAS2R38) gene, we investigated all the common genetic variation of this gene region in relation to colorectal cancer risk with a case-control study in a German population (709 controls and 602 cases) and in a Czech population (623 controls and 601 cases). We found that there were no significant associations between individual SNPs of the TAS2R38 gene and colorectal cancer in the Czech or in the German population, nor in the joint analysis. However, when we analyzed the diplotypes and the phenotypes we found that the non-taster group had an increased risk of colorectal cancer in comparison to the taster group. This association was borderline significant in the Czech population, (OR = 1.28, 95% CI 0.99-1.67; P(value) = 0.058) and statistically significant in the German population (OR = 1.36, 95% CI 1.06-1.75; P(value) = 0.016) and in the joint analysis (OR = 1.34, 95% CI 1.12-1.61; P(value) = 0.001). In conclusion, we found a suggestive association between the human bitter tasting phenotype and the risk of CRC in two different populations of Caucasian origin.

Polymorphisms in CTNNBL1 in relation to colorectal cancer with evolutionary implications.

Colorectal cancer (CRC) is a complex disease related to environmental and genetic risk factors. Several studies have shown that susceptibility to complex diseases can be mediated by ancestral alleles. Using RNAi screening, CTNNBL1 was identified as a putative regulator of the Wnt signaling pathway, which plays a key role in colorectal carcinogenesis. Recently, single nucleotide polymorphisms (SNPs) in CTNNBL1 have been associated with obesity, a known risk factor for CRC. We investigated whether genetic variation in CTNNBL1 affects susceptibility to CRC and tested for signals of recent selection. We applied a tagging SNP approach that cover all known common variation in CTNNBL1 (allele frequency >5%; r(2)>0.8). A case-control study was carried out using two well-characterized study populations: a hospital-based Czech population composed of 751 sporadic cases and 755 controls and a family/early onset-based German population (697 cases and 644 controls). Genotyping was performed using allele specific PCR based TaqMan® assays (Applied Biosystems, Weiterstadt, Germany). In the Czech cohort, containing sporadic cases, the ancestral alleles of three SNPs showed evidence of association with CRC: rs2344481 (OR 1.44, 95%CI 1.06-1.95, dominant model), rs2281148 (OR 0.59, 95%CI 0.36-0.96, dominant model) and rs2235460 (OR 1.38, 95%CI 1.01-1.89, AA vs. GG). The associations were less prominent in the family/early onset-based German cohort. Data derived from several databases and statistical tests consistently pointed to a likely shaping of CTNNBL1 by positive selection. Further studies are needed to identify the actual function of CTNNBL1 and to validate the association results in other populations.

Mutational status of KIT and PDGFRA and expression of PDGFRA are not associated with prognosis after curative resection of primary gastrointestinal stromal tumors (GISTs).

BACKGROUND: The aim of this study was to investigate if immunohistochemical expression and mutational status of KIT and PDGFRA in GISTs are associated with the clinical course and disease-free survival after curative resection of the primary tumor without adjuvant systemic therapy. METHODS: Paraffin-embedded tumor sections of 95 GISTs were analyzed for KIT and PDGFRA expression by immunohistochemistry. PDGFRA expression was judged using a scoring system subdividing tumors in negative/weak and strong immunoreactivity groups. For mutation analysis, exons 9, 10, 11, 13, and 17 of KIT and exons 10, 12, 14, and 18 of PDGFRA were sequenced. RESULTS: Of 95 R0-resected GISTs, 69% showed strong PDGFRA immunoreactivity. Gastric GISTs revealed a significantly higher rate of strong PDGFRA immunoreactivity (P = 0.01) and longer DFS (P = 0.015) than GISTs of the small intestine. KIT mutations were detected in 43 of 63 (68.3%) completely sequenced cases while PDGFRA mutations were identified in 6 cases (10%). In multivariate analysis, neither KIT/PDGFRA expression nor mutational status of KIT or PDGFRA were independent prognostic factors. Only mitotic rate predicted recurrence independently. CONCLUSION: Our data do not support the notion that expression of PDGFRA or mutations in KIT or PDGFRA are independent prognostic factors after curative resection of primary GIST.

RET-protooncogene variants in patients with sporadic neoplasms of the digestive tract and the central nervous system.

PURPOSE: The RET protooncogene plays a crucial role in neural crest development; accordingly, mutations of RET cause MEN2A and familial medullary thyroid carcinoma, while the expression deregulation of RET is involved in the pathophysiology of glioblastoma multiforme (GBM) and pancreatic cancer (PDAC). The aim of this study was to evaluate if germline variants of the RET protooncogene are associated with GBM, pancreatic cancer and gastric cancer (GC). METHODS: Genomic DNA from peripheral blood was isolated from 100 patients with GBM, 65 patients with GC and 54 patients with PDAC. The coding sequence of RET promoter, exon 2 and exon 13 was amplified. Sequence variations at -5 and -1 in the promotor and in exon 2 were determined through a LightCycler assay, and analysis of exon 13 was carried out by genomic sequencing. RESULTS: There was no significant association of the RET-promoter or exon 2 genotypes with the phenotype in the different populations, although there was an increase of the GG genotype of the -5G>A variant in all cancers compared to controls. Sequencing of exon 13 identified mutation c.2372A>T in codon 791 (Y791F) in heterozygous state in one of 100 GBM patients, in two of 65 patients with gastric cancer, in two of 54 PDAC patients and in none of the controls. CONCLUSIONS: Although our data did not reach significance in our small cohorts, we cannot rule out the involvement of the -5G promoter allele and the c.2372A>T mutation in the development of the aforementioned tumours.

Identification of candidate predisposing copy number variants in familial and early-onset colorectal cancer patients.

In the majority of colorectal cancers (CRCs) under clinical suspicion for a hereditary cause, the disease-causing genetic factors are still to be discovered. To identify such genetic factors we stringently selected a discovery cohort of 41 CRC index patients with microsatellite-stable tumors. All patients were below 40 years of age at diagnosis and/or exhibited an overt family history. We employed genome-wide copy number profiling using high-resolution SNP arrays on germline DNA, which resulted in the identification of novel copy number variants (CNVs) in six patients (15%) encompassing, among others, the cadherin gene CDH18, the bone morphogenetic protein antagonist family gene GREM1, and the breakpoint cluster region gene BCR. In addition, two genomic deletions were encountered encompassing two microRNA genes, hsa-mir-491/KIAA1797 and hsa-mir-646/AK309218. None of these CNVs has previously been reported in relation to CRC predisposition in humans, nor were they encountered in large control cohorts (>1,600 unaffected individuals). Since several of these newly identified candidate genes may be functionally linked to CRC development, our results illustrate the potential of this approach for the identification of novel candidate genes involved in CRC predisposition.

Polymorphisms of genes coding for ghrelin and its receptor in relation to colorectal cancer risk: a two-step gene-wide case-control study.

BACKGROUND: Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHSR), has two major functions: the stimulation of the growth hormone production and the stimulation of food intake. Accumulating evidence also indicates a role of ghrelin in cancer development. METHODS: We conducted a case-control study to examine the association of common genetic variants in the genes coding for ghrelin (GHRL) and its receptor (GHSR) with colorectal cancer risk. Pairwise tagging was used to select the 11 polymorphisms included in the study. The selected polymorphisms were genotyped in 680 cases and 593 controls from the Czech Republic. RESULTS: We found two SNPs associated with lower risk of colorectal cancer, namely SNPs rs27647 and rs35683. We replicated the two hits, in additional 569 cases and 726 controls from Germany. CONCLUSION: A joint analysis of the two populations indicated that the T allele of rs27647 SNP exerted a protective borderline effect (Ptrend = 0.004).

Genome-wide association study for colorectal cancer identifies risk polymorphisms in German familial cases and implicates MAPK signalling pathways in disease susceptibility.

Genetic susceptibility accounts for approximately 35% of all colorectal cancer (CRC). Ten common low-risk variants contributing to CRC risk have been identified through genome-wide association studies (GWASs). In our GWAS, 610 664 genotyped single-nucleotide polymorphisms (SNPs) passed the quality control filtering in 371 German familial CRC patients and 1263 controls, and replication studies were conducted in four additional case-control sets (4915 cases and 5607 controls). Known risk loci at 8q24.21 and 11q23 were confirmed, and a previously unreported association, rs12701937, located between the genes GLI3 (GLI family zinc finger 3) and INHBA (inhibin, beta A) [P = 1.1 x 10(-3), odds ratio (OR) 1.14, 95% confidence interval (CI) 1.05-1.23, dominant model in the combined cohort], was identified. The association was stronger in familial cases compared with unselected cases (P = 2.0 x 10(-4), OR 1.36, 95% CI 1.16-1.60, dominant model). Two other unreported SNPs, rs6038071, 40 kb upstream of CSNK2A1 (casein kinase 2, alpha 1 polypeptide) and an intronic marker in MYO3A (myosin IIIA), rs11014993, associated with CRC only in the familial CRC cases (P = 2.5 x 10(-3), recessive model, and P = 2.7 x 10(-4), dominant model). Three software tools successfully pointed to the overrepresentation of genes related to the mitogen-activated protein kinase (MAPK) signalling pathways among the 1340 most strongly associated markers from the GWAS (allelic P value < 10(-3)). The risk of CRC increased significantly with an increasing number of risk alleles in seven genes involved in MAPK signalling events (P(trend) = 2.2 x 10(-16), OR(per allele) = 1.34, 95% CI 1.11-1.61).

TCF-3, 4 protein expression correlates with beta-catenin expression in MSS and MSI-H colorectal cancer from HNPCC patients but not in sporadic colorectal cancers.

PURPOSE: The beta-catenin-T-cell factor-4 (TCF-4) complex is the main control switch of cell proliferation and differentiation of normal and malignant intestinal cells. The aim of our study was to analyze the protein expression of components of the Wnt pathway in microsatellite stable (MSS) and highly unstable (MSI-H) sporadic and hereditary nonpolyposis colorectal cancer (HNPCC) in human colorectal cancers. METHODS: Sixty seven colorectal tumors comprising of 15 sporadic MSS, 12 sporadic microsatellite instability colorectal tumors and 40 tumors from HNPCC patients, of which 20 were MSS and 20 MSI-H, were analyzed for the expression of APC, beta-catenin, and TCF-3, 4 proteins by immunohistochemistry. RESULTS: We found a significant difference in cytoplasmic APC expression frequency between sporadic MSS (52%) and HNPCC tumors (78%), whereas no difference was detected between MSI-H and MSS or HNPCC tumors. All tumor groups showed a similar pattern of decreased membranous staining and increased cytoplasmic and nuclear staining for beta-catenin compared to normal cells. Moreover, the TCF-3, 4 protein expression was higher (43%) in HNPCC-associated MSS tumors compared to sporadic tumors (14%; analysis of variance (ANOVA) p < 0.05). For HNPCC tumors, the subcellular beta-catenin expression (membranous, cytoplasmic, and nuclear) correlated with the nuclear TCF-3, 4 signal in MSS tumors (Spearman correlation p < 0.0007) and MSI-H tumors (Spearman correlation p < 0.0001). CONCLUSION: We have shown a previously unknown difference in TCF-3, 4 protein expression between sporadic and HNPCC MSS tumors. In addition, we found no difference in nuclear beta-catenin signal intensity, which may be caused by an alteration in Wnt pathway in MSS sporadic tumors by unknown mechanisms leading to lower TCF-3, 4 protein expression. This hypothesis has to be tested in future investigations.