Detection of PMS2 Mutations by Screening Hereditary Nonpolyposis Colon Cancer Families from Denmark and Sweden.

Background and Aims: Hereditary nonpolyposis colon cancer (HNPCC) and Lynch syndrome (LS) are characterized by defects in the mismatch repair (MMR) system, which protects the integrity of the genome. Pathogenic variants in four MMR genes (MLH1, MSH2, MSH6, and PMS2) are responsible for LS, an autosomal, dominant hereditary disease that occurs with a frequency of 2-5% among all colorectal cancer cases. It has been estimated that ∼2-5% of all pathogenic variants found in the four MMR genes in LS cases are detected in the PMS2 gene. An overview of detected variants is presented here. Materials and Methods: Long-range (LR) PMS2 polymerase chain reaction (PCR) and PMS2 multiplex ligation probe amplification (MLPA) assays were used to detect PMS2 variants in ∼1500 probands. In a subset of the probands, pathogenic PMS2 variants were detected by next-generation sequencing, and all detected variants were confirmed by LR-PCR combined with an MLPA assay. Results: A summary of PMS2 mutation analyses performed on colon cancer patients from molecular diagnostic laboratories in Denmark and Sweden is presented. By screening ∼1500 HNPCC probands, a total of 40 different PMS2 variants were detected in 71 probands (5%); 20 variants were classified as pathogenic (C5), 2 variants as likely pathogenic (C4), 15 variants as variants of unknown significance (VUSs) (C3), 1 variant as likely benign (C2), and 2 variants as benign (C1). In total, 22/71 (31%) of the probands carried a pathogenic sequence variant. Among the probands with isolated loss of pPMS2 expression, the fraction of pathogenic variants was 20/35 (55%). Conclusions: Approximately 5% of the probands found in the Danish and Swedish populations presented here carried a PMS2 variant. In this study, six novel pathogenic variants and seven VUSs are reported.

A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment.

Nonsyndromic hearing impairment (NSHI) is a highly heterogeneous condition with more than eighty known causative genes. However, in the clinical setting, a large number of NSHI families have unexplained etiology, suggesting that there are many more genes to be identified. In this study we used SNP-based linkage analysis and follow up microsatellite markers to identify a novel locus (DFNA66) on chromosome 6q15-21 (LOD 5.1) in a large Danish family with dominantly inherited NSHI. By locus specific capture and next-generation sequencing, we identified a c.574C>T heterozygous nonsense mutation (p.R192*) in CD164. This gene encodes a 197 amino acid transmembrane sialomucin (known as endolyn, MUC-24 or CD164), which is widely expressed and involved in cell adhesion and migration. The mutation segregated with the phenotype and was absent in 1200 Danish control individuals and in databases with whole-genome and exome sequence data. The predicted effect of the mutation was a truncation of the last six C-terminal residues of the cytoplasmic tail of CD164, including a highly conserved canonical sorting motif (YXXФ). In whole blood from an affected individual, we found by RT-PCR both the wild-type and the mutated transcript suggesting that the mutant transcript escapes nonsense mediated decay. Functional studies in HEK cells demonstrated that the truncated protein was almost completely retained on the plasma cell membrane in contrast to the wild-type protein, which targeted primarily to the endo-lysosomal compartments, implicating failed endocytosis as a possible disease mechanism. In the mouse ear, we found CD164 expressed in the inner and outer hair cells of the organ of Corti, as well as in other locations in the cochlear duct. In conclusion, we have identified a new DFNA locus located on chromosome 6q15-21 and implicated CD164 as a novel gene for hearing impairment.

Leiden Open Variation Database of the MUTYH gene.

The MUTYH gene encodes a DNA glycosylase involved in base excision repair (BER). Biallelic pathogenic MUTYH variants have been associated with colorectal polyposis and cancer. The pathogenicity of a few variants is beyond doubt, including c.536A4G/p.Tyr179Cys and c.1187G4A/p.Gly396Asp (previously c.494A4G/p.Tyr165Cys and c.1145G4A/p.Gly382Asp).However, for a substantial fraction of the detected variants, the clinical significance remains uncertain,compromising molecular diagnostics and thereby genetic counseling. We have established an interactive MUTYH gene sequence variant database (www.lovd.nl/MUTYH) with the aim of collecting and sharing MUTYH genotype and phenotype data worldwide. To support standard variant description, we chose NM_001128425.1 as the reference sequence. The database includes records with variants per individual, linked to available phenotype and geographic origin data as well as records with in vitro functional and in silico test data. As of April 2010, the database contains 1968 published and 423 unpublished submitted entries, and 230 and 61 unique variants,respectively. This open-access repository allows all involved to quickly share all variants encountered and communicate potential consequences, which will be especially useful to classify variants of uncertain significance.

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.

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.

The association between genetic variants in hMLH1 and hMSH2 and the development of sporadic colorectal cancer in the Danish population.

BACKGROUND: Mutations in the mismatch repair genes hMLH1 and hMSH2 predispose to hereditary non-polyposis colorectal cancer (HNPCC). Genetic screening of more than 350 Danish patients with colorectal cancer (CRC) has led to the identification of several new genetic variants (e.g. missense, silent and non-coding) in hMLH1 and hMSH2. The aim of the present study was to investigate the frequency of these variants in hMLH1 and hMSH2 in Danish patients with sporadic colorectal cancer and in the healthy background population. The purpose was to reveal if any of the common variants lead to increased susceptibility to colorectal cancer. METHODS: Associations between genetic variants in hMLH1 and hMSH2 and sporadic colorectal cancer were evaluated using a case-cohort design. The genotyping was performed on DNA isolated from blood from the 380 cases with sporadic colorectal cancer and a sub-cohort of 770 individuals. The DNA samples were analyzed using Single Base Extension (SBE) Tag-arrays. A Bonferroni corrected Fisher exact test was used to test for association between the genotypes of each variant and colorectal cancer. Linkage disequilibrium (LD) was investigated using HaploView (v3.31). RESULTS: Heterozygous and homozygous changes were detected in 13 of 35 analyzed variants. Two variants showed a borderline association with colorectal cancer, whereas the remaining variants demonstrated no association. Furthermore, the genomic regions covering hMLH1 and hMSH2 displayed high linkage disequilibrium in the Danish population. Twenty-two variants were neither detected in the cases with sporadic colorectal cancer nor in the sub-cohort. Some of these rare variants have been classified either as pathogenic mutations or as neutral variants in other populations and some are unclassified Danish variants. CONCLUSION: None of the variants in hMLH1 and hMSH2 analyzed in the present study were highly associated with colorectal cancer in the Danish population. High linkage disequilibrium in the genomic regions covering hMLH1 and hMSH2, indicate that common genetic variants in the two genes in general are not involved in the development of sporadic colorectal cancer. Nevertheless, some of the rare unclassified variants in hMLH1 and hMSH2 might be involved in the development of colorectal cancer in the families where they were originally identified.

Genotyping and annotation of Affymetrix SNP arrays.

In this paper we develop a new method for genotyping Affymetrix single nucleotide polymorphism (SNP) array. The method is based on (i) using multiple arrays at the same time to determine the genotypes and (ii) a model that relates intensities of individual SNPs to each other. The latter point allows us to annotate SNPs that have poor performance, either because of poor experimental conditions or because for one of the alleles the probes do not behave in a dose-response manner. Generally, our method agrees well with a method developed by Affymetrix. When both methods make a call they agree in 99.25% (using standard settings) of the cases, using a sample of 113 Affymetrix 10k SNP arrays. In the majority of cases where the two methods disagree, our method makes a genotype call, whereas the method by Affymetrix makes a no call, i.e. the genotype of the SNP is not determined. By visualization it is indicated that our method is likely to be correct in majority of these cases. In addition, we demonstrate that our method produces more SNPs that are in concordance with Hardy-Weinberg equilibrium than the method by Affymetrix. Finally, we have validated our method on HapMap data and shown that the performance of our method is comparable to other methods.

Presymptomatic diagnosis using a deletion of a single codon in families with hereditary non-polyposis colorectal cancer.

The diagnosis of hereditary non-polyposis colorectal cancer (HNPCC) is often confirmed by a mutation in one of several mismatch-repair genes, in particular MLH1, MSH2 and MSH6. Presymptomatic diagnosis requires the identification of a mutation causing the disease. Three different deletions of a single amino acid codon have previously been published as assumed pathogenic. The objective of this study was to determine if an MSH2 3 base pair in-frame deletion (N596del) could be used in presymptomatic screening of at-risk individuals. We report on five HNPCC families with the N596del mutation, identified after mutation screening of MSH2 and MLH1. All patients in the families were haplotyped using markers flanking the MSH2 gene. The haplotypes revealed that the five families with high probability descended from only two founders. The N596del segregated with the HNPCC phenotype with lod scores of 3.2 and 2.0 at the recombination fraction of 0.0 in the two founder families. Sequencing of MSH2 and MLH1 did not reveal other pathogenic mutations, and N596del was not identified in 50 healthy controls. The mutation has previously been found expressed in mRNA, and is located in a conserved domain. The results support the hypothesis that N596del is the disease causing mutation and not a clinically silent variation. On this basis, the application of the MSH2 N596del mutation, in presymptomatic screening of HNPCC families, is recommended.

High-density single nucleotide polymorphism array defines novel stage and location-dependent allelic imbalances in human bladder tumors.

Bladder cancer is a common disease characterized by multiple recurrences and an invasive disease course in more than 10% of patients. It is of monoclonal or oligoclonal origin and genomic instability has been shown at certain loci. We used a 10,000 single nucleotide polymorphism (SNP) array with an average of 2,700 heterozygous SNPs to detect allelic imbalances (AI) in 37 microdissected bladder tumors from 17 patients. Eight tumors represented upstaging from Ta to T1, eight from T1 to T2+, and one from Ta to T2+. The AI was strongly stage-dependent as four chromosomal arms showed AI in > 50% of Ta samples, eight in T1, and twenty-two in T2+ samples. The tumors showed stage-dependent clonality as 61.3% of AIs were reconfirmed in later T1 tumors and 84.4% in muscle-invasive tumors. Novel unstable chromosomal areas were identified at chromosomes 6q, 10p, 16q, 20p, 20q, and 22q. The tumors separated into two distinct groups, highly stable tumors (all Ta tumors) and unstable tumors (2/3 muscle-invasive). All 11 unstable tumors had lost chromosome 17p areas and 90% chromosome 8 areas affecting Netrin-1/UNC5D/MAP2K4 genes as well as others. AI was present at the TP53 locus in 10 out of 11 unstable tumors, whereas 6 had homozygous TP53 mutations. Tumor distribution pattern reflected AI as seven out of eight patients with additional upper urinary tract tumors had genomic stable bladder tumors (P < 0.05). These data show the power of high-resolution SNP arrays for defining clinically relevant AIs.

Allelic imbalances in human bladder cancer: genome-wide detection with high-density single-nucleotide polymorphism arrays.

BACKGROUND: Bladder cancer is characterized by genomic instability. In this study, we investigated whether genome-wide screening using single-nucleotide polymorphism (SNP) arrays could detect allelic imbalance (loss or gain of at least one allele) in bladder cancers. METHODS: For microarray analysis, DNA was isolated from microdissected bladder tumors and leukocytes from 11 patients. The stage T1 tumor (connective tissue invasive) and the subsequent stage T2-4 tumor (muscle invasive) were available from eight of these patients, and only the first muscle-invasive stage T2-4 tumor was available from three of the 11 patients. The microarray contained 1494 biallelic polymorphic sequences. For microsatellite analyses, DNA was isolated from tumors and leukocytes of nine patients with primary T2-4 tumors and 13 patients with Ta (noninvasive) tumors. All statistical tests were two-sided. RESULTS: We assigned a genotype to 1204 loci, 343 of which were heterozygous. Allelic imbalance was detected in known areas of imbalance on chromosomes 6, 8, 9, 11, and 17, and a new area of imbalance was detected on the p arm of chromosome 6. Microsatellite analysis of nine other T2-4 tumors and 13 Ta tumors showed that allelic imbalance was more frequent in T2-4 tumors than in Ta tumors (P<.001). We detected 8.5 allelic imbalances (median) in 348 informative loci in T1 tumors and 28 allelic imbalances (median) in 329 informative loci in T2-4 tumors. When pairs of T1 and T2-4 tumors were analyzed from eight patients, 68% of imbalances detected in T1 tumors (146 imbalances) occurred in the subsequent T2-4 tumors (99 imbalances). Homozygous TP53 mutations were more often associated (P =.005) with high allelic imbalance than with low allelic imbalance. CONCLUSION: SNP arrays are feasible for high-throughput, genome-wide scanning for allelic imbalances in bladder cancer.