Henrik Okkels

Neurofibromatosis von Recklinghausen Type I Phenotype and Early Onset of Cancers in Siblings Compound Heterozygous for Mutations in MSH6

We report on a nonconsanguineous family in which two siblings with cutaneous manifestations leading to a diagnosis of neurofibromatosis type 1 (NF1) developed CNS tumors at an early age. In addition, one of them developed a T‐cell lymphoma. Neither parent had NF1. The mother was known to be heterozygous for a MSH6 mutation, and the father was found to be heterozygous for a different MSH6 mutation. Screening of MSH2, MLH1, MSH6, PMS1, PMS2, and MLH3 in the affected children disclosed that they both were compound heterozygote for the MSH6 mutations of their parents. Most recently, about a dozen other cases of inherited bi‐allelic deficiency of mismatch repair (MMR) genes associated with early onset CNS tumors, hematologic malignancy, gastrointestinal neoplasia, café‐au‐lait spots, and other NF1 features have been reported. In the present study, we summarize the clinical findings of 27 individuals homozygous or compound heterozygous for an MMR gene mutation reported in the medical literature. We suggest that biparentally inherited mutations of one of the MMR genes should be considered in children with multiple café‐au‐lait spots who have early‐onset CNS tumors, hematologic malignancies, or early onset gastrointestinal neoplasia.

Exposure to urban and rural air pollution: DNA and protein adducts and effect of glutathione-S-transferase genotype on adduct levels

Ambient air in urban areas is polluted by agents suspected of causing cancer in humans. A number of epidemiological studies have revealed an increased cancer risk in urban communities, especially in lung cancer. The relative risk have been estimated to be in the order of 1.5. The objective of this study was to evaluate differences in genotoxic exposure through air pollution in urban and rural areas using DNA and protein adducts as biomarkers. Another objective was to investigate whether the GSTM1 genotype has any effect on adduct level. The analyses included 32P postlabelling of DNA adducts in lymphocytes, enzyme-linked immunosorbent assay for measuring benzo[a]pyrene protein adducts and polymerase chain reaction amplification of the GSTM1 genotype. The study was a cross-sectional study of non-smoking, healthy males from rural and urban Danish areas and from Athens, Greece. All individuals in the study were healthy, non-smoking males. The Danish urban group included 74 university students, the rural group 29 students from agricultural colleges and the Greek group 17 individuals. Adduct levels differed significantly in the three groups with median levels of 0.152 fmol/μg DNA (rural), 0.205 fmol/μg (urban) and 0.285 fmol/μg (Athens). The adduct patterns showed some identical spots, but also specific adducts. Here we report increasing DNA adduct levels comparing residents in rural, small urban and large urban residential areas; we found no influence of GSTM1 genotype on DNA or protein adduct levels in non-smokers exposed to low levels of air pollution.

Benign course of long-standing hepatitis B virus infection among Greenland Inuit?

Objective. Chronic hepatitis B virus (HBV) infection can present in different ways, from inactive carrier to liver failure or cancer. The role of the virus subtype is controversial. The purpose of this study was to characterize HBV infection in detail and its impact on general health, body-build and liver biochemistry. Material and methods. The study comprised a population-based cohort of Inuit exposed to HBV 3–7 decades ago in the capital in West Greenland, a coastal town and four settlements in rural East Greenland. Participants included 95% of the invited Inuit: 229 men, 205 women, aged 50–69 years. Results. Only 25% of the participants had never had HBV infection. HBsAg was positive in 86 participants (20.0%), more being found positive in rural East Greenland than in the city in West Greenland (28.9% versus 2.7%; p < 0.001). HBV-DNA was positive in 61 of those with median HBV-DNA 40,000 copies/ml. HBV genotype could be determined in 52: 47 participants had genotype B, 4 genotype D, and 1 had both B and D. At sequencing, genotype B resembled subtype Bj, but with more than 5% diversity in the C-gene it could be a new subtype B. Pre-core mutation was found in 55 of 56 participants investigated. None of the participants had signs of liver disease, and HBV infection did not influence body-build or liver biochemistry. Conclusions. More than 75% of participants had a marker of present or previous HBV infection but the infection seemed dormant. The majority harbored a special variant of genotype B that might be a new subtype giving a relatively benign disease. The role of detailed subtyping of HBV for prognostic evaluation should be investigated in more detail.

Major contribution from recurrent alterations and MSH6 mutations in the Danish Lynch syndrome population

An increasing number of mismatch-repair (MMR) gene mutations have been identified in hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. This study presents the population-based Danish MMR gene mutation profile, which contains 138 different MMR gene alterations. Among these, 88 mutations in 164 families are considered pathogenic and an additional 50 variants from 76 families are considered to represent variants of unknown pathogenicity. The different MMR genes contribute to 40% (MSH2), 29% (MLH1), and 22% (MSH6) of the mutations and the Danish population thus shows a considerably higher frequency of MSH6 mutations than previously described. Although 69/88 (78%) pathogenic mutations were present in a single family, previously recognized recurrent/founder mutations were causative in 75/137 (55%) MLH1/MSH2 mutant families. In addition, the Danish MLH1 founder mutation c.1667+2_1667_+8TAAATCAdelinsATTT was identified in 14/58 (24%) MLH1 mutant families. The Danish Lynch syndrome population thus demonstrates that MSH6 mutations and recurrent/founder mutations have a larger contribution than previously recognized, which implies that the MSH6 gene should be included in routine diagnostics and suggests that directed analysis of recurrent/founder mutations may be feasible e.g. in families were diagnostic material is restricted to archival tissue.

Mutations in CARD15 and smoking confer susceptibility to Crohn`s disease in the Danish population

Objective. Three CAspase Recruitment Domain (CARD15) mutations have shown to predispose to Crohns disease in Caucasian populations. The aim of this study was to investigate the mutation frequency in patients with inflammatory bowel disease and in healthy controls in Denmark. Material and methods. Genotyping of the three common CARD15 mutations was carried out on 388 patients with Crohns disease, 565 patients with ulcerative colitis and 796 healthy controls using real-time PCR. Allele and genotype frequencies in the three groups were compared. A possible additive effect of smoking on CARD15 mutations was also examined. Results. Carrying at least one CARD15 mutation was significantly more common in patients with Crohns disease compared with healthy controls (21% versus 10%; p <0.001). A gene–dosage effect was observed (ORadj.smoking 22.2; p<0.001 for carrying two CARD15 mutations versus ORadj.smoking 1.8; p=0.01 for carrying one CARD15 mutation). The 1007insC protein truncating mutation was the major contributing mutation. Ileal involvement was more common in Crohns disease patients with CARD15 mutations as opposed to patients without CARD15 mutations (ORadj.smoking 3.6; p<0.001). Smoking was independently associated with Crohns disease (OR 1.8; p<0.001), but no multiplicative effect of smoking on CARD15 genotypes was found. Conclusions. In the Danish population, CARD15 mutations were found to be associated with Crohns disease, hence supporting the hypothesis of a genetic component contributing to the disease. Further research for other genes possibly involved in Crohns disease may result in the use of genetic testing for diagnosis or treatment of Crohns disease in the future.

Glutathione S-transferase mu as a risk factor in bladder tumours.

Glutathione transferases are involved in the detoxification of many zenobiotica involved in the etiology of cancer. To investigate the role of the glutathione S-transferase M1 deletion (GSTM1*0/0) in bladder carcinogenesis, the polymerase chain reaction was used to determine the GSTM1 genotypes of cancer patients (n = 234) and hospital controls (n = 202). Overall, the proportion of GSTM1*0/0 in the case group was 57%, compared to 50% in the control group giving an odds ratio (OR) of 1.33, (0.91-1.94; 95% confidence interval (CI)). Dividing the bladder cancer group into incident (n = 87) and surviving case groups (n = 147), a modest association between the GSTM1*0/0 genotype and bladder cancer was found in the surviving group, whereas, in the incident group no association was found. Logistic regression analysis of the incident cases, adjusting for age, gender, and cigarette smoking, revealed ORs of 1.12 (0.61-2.08) and 0.74 (0.33-1.73) for the malignant and benign tumours, respectively. The corresponding adjusted ORs for the surviving cases were 1.81 (1.04-3.13) for benign and 1.43 (0.80-2.56) for malignant tumours. Thus, in this study, the GSTM1 deletion is not a risk factor for the development of bladder cancer, but may be related to the survival of the bladder cancer patients. This finding is very important for the design of case-control studies in general, and for the interpretation of existing data.

Challenges in the identification of MSH6-associated colorectal cancer: rectal location, less typical histology, and a subset with retained mismatch repair function.

Identification of Lynch syndrome tumors is challenging. This relates particularly to MSH6-associated cases, which show reduced penetrance of colorectal cancer and a higher age at diagnosis. We recorded the clinical and morphologic features of 52 MSH6-associated colorectal cancers in comparison with MLH1/MSH2-mutant tumors and sporadic mismatch repair-deficient cancers. In the MSH6 subset, we confirmed a higher age (median, 56 y) at diagnosis and found a significantly larger proportion (25%) of rectal cancers. Presence of dirty necrosis was the sole histologic component that significantly differed between MSH6 and MLH1/MSH2 tumors. Compared with the sporadic mismatch repair-defective cohort, MSH6 cases had a lower prevalence of tumor-infiltrating lymphocytes and Crohn-like reactions. Mismatch repair defects were identified in 92% of MSH6 tumors, with high concordance between microsatellite instability and loss of immunohistochemical MSH6 expression. The remaining 8% showed a mismatch repair-stable phenotype, which suggests that analysis of additional tumors might be considered in families suspected of Lynch syndrome.

MSH6 mutations are frequent in hereditary nonpolyposis colorectal cancer families with normal pMSH6 expression as detected by immunohistochemistry.

Introduction:Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant condition accounting for 2% to 4% of all colorectal cancer cases worldwide. Families with germ line mutations in 1 of 6 mismatch repair genes are known as Lynch syndrome families. The largest number of mutations has been detected in the mismatch repair genes MLH1 and MSH2, but several mutations in MSH6 have also been demonstrated. Aim:Whether HNPCC families are screened for mutations in mismatch repair genes often relies on their immunohistochemical profile. The aim of the present study was to evaluate this approach in Lynch families carrying mutations in MSH6. Materials and Methods:Results of the screening of the MSH6 gene in HNPCC families were compared with those obtained on immunohistochemical protein analysis. Results:In 56 (7%) of 815 families, at least 1 MSH6 mutation, 23 definitively pathogenic mutations and 38 missense mutations or unclassified variants, and several polymorphisms in the MSH6 gene were detected. In families carrying a pathogenic MSH6 mutation, 69.6% of 23 colon adenocarcinomas showed absence of pMSH6 in tumor tissue by immunohistochemical analysis. In 34.5%, all proteins could be detected, whereas in 34.5% pMSH6 was present and pMLH1/pPMS2 was absent. Conclusions:If genetic screening of HNPCC families depended on immunohistochemical results, a substantial number of families harboring a pathogenic mutation in MSH6 and the vast majority of families harboring an MSH6 unclassified variant would not be detected.

Genetic variants of glutathione S-transferases mu, theta, and pi display no susceptibility to inflammatory bowel disease in the Danish population.

Abstract Introduction. A combination of genetic predisposition and interactions with environmental factors are believed to be responsible for disease phenotype and disease progression in inflammatory bowel diseases. The harmful effect of smoking and other environmental factors is believed to be highly dependent on the activity of detoxification enzymes. The aims of the study were to examine possible associations between the detoxifying glutathione S-transferases (GSTs) family μ, θ and π gene variants and inflammatory bowel disease, and secondly to examine a potential genotype–genotype interaction between these variants. Genotype–disease phenotype associations and a possible interaction between genotype and cigarette smoking were also assessed. Methods. Three hundred and eighty-eight patients with Crohns disease (CD), 565 patients with ulcerative colitis (UC) and 796 healthy Danish controls were included in the study. Genomic DNA was used for genotyping of the GST genes using PCR or real-time PCR. Results. No associations were found between GST genotypes and inflammatory bowel diseases. Neither did a combination of the GST genotypes reveal any associations. No genotype–disease phenotype associations were found. Smoking was positively associated with CD and negatively associated with UC. An interaction between smoking and GSTM1*0 genotype was found for UC, where the GSTM1*0 genotype appear to strengthen the protective effect of smoking on disease susceptibility. Conclusion. The GST genotypes do not seem to be important in susceptibility of inflammatory bowel disease in the Danish population. Nor did we find convincing evidence of associations between GST genotype and phenotypic features of inflammatory bowel diseases.

Functional characterization of rare missense mutations in MLH1 and MSH2 identified in Danish colorectal cancer patients

Recently, we have performed a population based study to analyse the frequency of colorectal cancer related MLH1 and MSH2 missense mutations in the Danish population. Half of the analyzed mutations were rare and most likely only present in the families where they were identified originally. Some of the missense mutations were located in conserved regions in the MLH1 and MSH2 proteins indicating a relation to disease development. In the present study, we functionally characterized 10 rare missense mutations in MLH1 and MSH2 identified in 13 Danish CRC families. To elucidate the pathogenicity of the missense mutations, we carried out in vitro functional analyses. The missense mutations were analyzed for their effect on protein expression and repair efficiency. The results of the functional analysis were correlated with clinical data on the families carrying these mutations. Eight missense mutations resulted in proteins with expression and repair efficiency similar to the wild type. One missense mutation (MSH2 p.Met688Val) caused reduced protein expression and one (MSH2 p.Leu187Arg) caused both reduced protein expression and repair deficiency. The MSH2 p.Leu187Arg mutation was found in an Amsterdam II family presenting with high microsatellite instability and loss of MSH2 and MSH6 proteins in tumours. In conclusion, only 1/10 missense mutations displayed repair deficiency and could be classified as pathogenic. No final conclusion can be drawn on the MSH2 p.Met688Val mutation, which caused reduced protein expression. Although, no deficiencies have been identified in the proteins harbouring the other missense mutations, pathogenicity of these variants cannot be unambiguously excluded.