Susanne Stemmler

Novel strategy for optimal sequential application of clinical criteria, immunohistochemistry and microsatellite analysis in the diagnosis of hereditary nonpolyposis colorectal cancer.

Clinical criteria, microsatellite analysis (MSA) and immunohistochemistry (IHC) are important diagnostic tools for identification of hereditary nonpolyposis colorectal cancer (HNPCC) patients who are likely to carry pathogenic germline mutations in mismatch repair genes. Based on MSA and IHC results and subsequent mutation analyses of 1,119 unrelated index patients meeting the Amsterdam II criteria or the classical Bethesda guidelines, we analyzed the value of these tools to predict MLH1 and MSH2 mutations with the aim of establishing optimal strategies for their most efficient sequential use. The overall prevalence of pathogenic germline mutations in our cohort was 20.6% (95% CI = 18.3–23.0%) and 61.8% (95% CI = 56.8–66.6%), respectively, after MSA/IHC‐based preselection. IHC was highly predictive (99.1%) and specific (99.6%) with regard to MSA. However, 14 out of 230 mutations (6%) escaped detection by IHC. Thus, IHC cannot be recommended to substitute MSA fully. Nonetheless, IHC is important to indicate the gene that is likely to be affected. To combine both methods efficiently, we propose a novel screening strategy that provides 2 alternative ways of sequential IHC and MSA application, either using IHC or MSA in the first place. A logistic regression model based on the age of the index patient at first tumor diagnosis and the number of fulfilled HNPCC criteria is used to allocate individual patients to that alternative pathway that is expected to be least expensive. A cost analysis reveals that about 25% of the costs can be saved using this strategy.

Recurrence and variability of germline EPCAM deletions in Lynch syndrome

Recently, we identified 3′ end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele‐specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch‐like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2‐deficient families from multiple geographical origins with varying deletions all encompassing the 3′ end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu‐repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3′ end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics. Hum Mutat 32:1–8, 2011.

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; Pvalue = 0.058) and statistically significant in the German population (OR = 1.36, 95% CI 1.06–1.75; Pvalue = 0.016) and in the joint analysis (OR = 1.34, 95% CI 1.12–1.61; Pvalue = 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.

Arg462Gln sequence variation in the prostate-cancer- susceptibility gene RNASEL and age of onset of hereditary non-polyposis colorectal cancer: a case-control study

BACKGROUND RNASEL is thought to be a susceptibility gene for hereditary prostate cancer and encodes the endoribonuclease RNase L, which has a role in apoptosis and is a candidate tumour-suppressor protein. A common sequence variation in RNASEL, Arg462Gln, has been associated with hereditary and sporadic prostate cancer, and the Gln variant has about three-fold reduced RNase activity in vitro. In view of the association between the age of onset of hereditary non-polyposis colorectal cancer and functionally different variants of P53, which play a key part in the apoptotic pathway, we aimed to assess whether the Arg462Gln variation of RNASEL affects the age of onset of hereditary non-polyposis colorectal cancer. METHODS We screened 251 patients with hereditary non-polyposis colorectal cancer who were unrelated, had pathogenic germline mutations in MSH2 (n=141) or MLH1 (n=110), and had colorectal carcinoma as the first tumour, for variation at codon 462 of RNASEL and compared them with 439 healthy controls. FINDINGS The median age of onset was 40 years (range 17-75) for patients with an Arg/Arg genotype at codon 462, 37 years (13-69) for patients with an Arg/Gln genotype, and 34 years (20-49) for those with a Gln/Gln genotype (p=0.0198). Only the RNASEL genotype had a significant effect on age of onset (p=0.0062) in an additive mode of inheritance. Pair-wise comparisons between genotype groups showed that the two homozygous groups (ie, Arg/Arg vs Gln/Gln) differed significantly in age of disease onset (mean age difference 4.8 years [SD 1.7], p=0.0044). INTERPRETATION A sequence variation in the prostate-cancer-susceptibility gene RNASEL has a role in a different, unassociated malignant disease. Genotypes at RNASEL codon 462 are associated with age of onset of hereditary non-polyposis colorectal cancer in a dose-dependent way, and might have a role in preventive strategies for this disease.

Association of interleukin-8 receptor |[alpha]| polymorphisms with chronic obstructive pulmonary disease and asthma

Chronic obstructive pulmonary disease (COPD) and asthma are common complex diseases characterized by airflow obstruction and inflammatory processes in the small airways. lnterleukin 8 (IL-8) is a potent proinflammatory cytokine which interacts with the IL-8 receptor α (IL8RA, CXCR1) and β (IL8RB, CXCR2), leading to activation and migration of leukocytes. In order to evaluate the role of the IL8RA gene in the pathogenesis of COPD and asthma, we screened the coding region of IL8RA for mutations by means of single-strand conformation polymorphism analysis in 50 COPD patients and identified three exchanges (M31R, S276T and R335C). These three polymorphisms were subsequently genotyped in 182 adult patients with COPD, 68 adult patients and 130 children with asthma as well as 454 healthy controls. The frequencies of the IL8RA 31R and 335C alleles were significantly increased in patients with COPD and in children with asthma compared to healthy controls (P=0.0073 and 0.023, respectively). Thus, these polymorphisms may play a role in the pathogenesis of COPD and asthma.

Truncating mutations in Peutz-Jeghers syndrome are associated with more polyps, surgical interventions and cancers.

Background and GoalsPeutz-Jeghers syndrome (PJS) is a rare autosomal dominant polyposis syndrome caused by STK11 germline mutations. PJS is associated with an increased risk of cancer. In our cohort, clinical and phenotypic parameters were correlated with genotypic findings and patients were prospectively followed by surveillance.StudyThirty-one patients treated between 2000 and 2006, were evaluated. STK11 genotyping was performed and phenotypes of patients with truncating (TM) and nontruncating mutations (NTM) were compared.ResultsMedian age at first symptoms was 11 years and complications occurred before the age of ten in 42% of patients. STK11 mutations were detected in 16 of 22 families (12 TM; four NTM). Patients with TM had more surgical gastrointestinal (GI) interventions (p = 0.021), and female patients in the TM group had an increased risk of undergoing gynecological surgery (p = 0.016). Also, there was a trend towards a higher polyp count (p = 0.11) and earlier age at first polypectomy (p = 0.13) in the TM group. Ten carcinomas were detected in six patients resulting in a cancer risk of 65% up to the age of 65 years. Patients with TM tended to develop more cancers (p = 0.10). Importantly, our surveillance strategy used detected 50% of cancers (n = 5) at an early potentially curable stage.ConclusionsOur study shows that almost half of PJ patients have complications early in life independent of mutational status. Patients with TM require more surgical GI interventions and tend to develop more polyps and cancers. Furthermore, close surveillance detects early stage cancers in patients. We propose that surveillance should be started as early as 8 years in all patients to avoid complications. Moreover, patients with TM may benefit from surveillance at shorter intervals.

A novel MSH2 germline mutation in homozygous state in two brothers with colorectal cancers diagnosed at the age of 11 and 12 years

Hereditary non‐polyposis colorectal cancer (HNPCC) syndrome is caused by heterozygous germline mutations in DNA mismatch repair genes (MMR), (MSH2, MLH1, MSH6, and PMS2) and it is inherited in an autosomal dominant pattern with high penetrance. Several patients have been reported carrying bi‐allelic MMR gene mutations and whose phenotype resembled a syndrome with childhood malignancies including hematological malignancies, brain, and colorectal tumors. This phenotype is similar to the tumor spectrum of MMR knockout mice. Herein we describe two brothers of healthy consanguineous parents from Pakistan, who had developed two and three colorectal cancers at the ages of 11 and 12 years, respectively, and less than 30 polyps. Tumor specimens were microsatellite instable (MSI‐H), and expression of MSH2 and MSH6 was lost. Mutation analyses of DNA samples from both patients revealed a novel homozygous c.2006‐5T > A mutation in intron 12 of the MSH2 gene. This phenotype of the brothers is unusual as they neither develop hematological malignancies nor brain tumors at an older age of presentation than other patients with homozygous MSH2 mutations. The milder phenotype may be due to the expression of low amounts of MSH2 protein with reduced activity.

Variation in the BDNF and NGFB genes in German atopic dermatitis patients

In humans, atopic dermatitis (AD) is believed to result from a complex interaction between genetic and environmental factors. Based on recent evidence, it has been proposed that neurotrophins play an important role in allergic inflammation. Levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in blood have been found to be elevated and correlated positively with disease in AD patients. Therefore, we sought to evaluate the role of nucleotide variation in the NGFB and BDNF genes in relation to the pathogenesis of AD. A functional polymorphism within the BDNF gene (Val66Met) and six selected polymorphisms in the NGFB gene were examined in 361 German AD patients and 325 non-atopic controls. In this cohort, no significant association with AD was detected, refuting the hypothesis that variation in these two neurotrophin genes contributes substantially to AD.

Unravelling the complex genetic background of atopic dermatitis: from genetic association results towards novel therapeutic strategies

Atopic dermatitis (AD) is a chronic inflammatory skin disease arising from complex interaction between genetic and environmental factors. As the starting point of the so-called “atopic march”, e.g. the progression towards allergic asthma in some but not all affected children, AD has come into focus for potential disease-modifying strategies. To elucidate the genetic factors influencing AD development, linkage, association as well as genome-wide association studies have been performed over the last two decades. The results suggest that besides variation in immune-mediated pathways, an intact skin barrier function plays a key role in AD development. Mutations in the gene encoding filaggrin, a major structural protein in the epidermis, have been consistently associated with AD, especially the early-onset persistent form of disease, and are regarded as the most significant known risk factor for AD development to date. Additionally, variation in some other genes involved in skin integrity and barrier function have shown association with AD. However, the known genetic risk factors can only explain a small part of the heritability at the moment. Whole-exome or whole-genome sequencing studies have not been reported yet, but will probably soon evaluate the influence of rare variations for AD development. Additionally, large multi-centre studies comprehensively incorporating gene–gene and gene–environment interactions as well as epigenetic mechanisms might further elucidate the genetic factors underlying AD pathogenesis and, thus, open the way for a more individualized treatment in the future.

Variation in genes of the epidermal differentiation complex in German atopic dermatitis patients

The filaggrin (FLG) gene is one of the most widely replicated susceptibility genes for atopic dermatitis (AD) so far. Yet, FLG mutations cannot fully account for the original linkage peak on chromosome 1q21, a region comprising the so‐called epidermal differentiation complex (EDC). Since the EDC contains numerous genes relevant for epidermal differentiation, we sought to evaluate variation in other genes located in this region in a German AD case–control cohort. Thirty‐two single nucleotide polymorphisms (SNPs) in 21 genes across the EDC were genotyped in 402 unrelated AD patients and 325 non‐atopic controls by means of restriction enzyme digestion or TaqMan assays. Allele and genotype frequencies were tested for differences between patients and controls by logistic regression. Haplotype frequencies were evaluated using the famhap software. Except for the already known association with FLG, we did not identify any additional significant associations of EDC genes with AD.