Germline Mutations In MSH2 Research Articles

Prospective Determination of Prevalence of Lynch Syndrome in Young Women With Endometrial Cancer

Age younger than 50 years at the time of colon cancer diagnosis is often used as a screening criterion for Lynch syndrome (hereditary nonpolyposis colorectal cancer syndrome). The purpose of this study was to determine the prevalence of MLH1, MSH2, and MSH6 mutations in an unselected cohort of women diagnosed with endometrial cancer at age younger than 50 years. A prospective, multicenter study was performed at three institutions. After written consent was obtained, germline mutation testing by full sequencing and large deletion analysis of the MLH1, MSH2, and MSH6 genes was performed. Tumor studies included immunohistochemistry of MLH1, MSH2, and MSH6; microsatellite instability analysis; and hypermethylation of the MLH1 promoter. Of the 100 women, nine (9%; 95% CI, 4.2 to 16.4) carried a deleterious germline mutation: seven women with mutations in MSH2, one woman with a mutation in MLH1, and one woman with a mutation in MSH6. Two additional women had molecular studies consistent with the diagnosis of Lynch syndrome. The mean body mass index (BMI) for the entire cohort was 34.4, which is significantly higher than 29.2, the mean BMI for the mutation carriers. Predictors of finding a germline mutation included having a first-degree relative with a Lynch syndrome-associated cancer, endometrial tumor with loss of MSH2 expression, tumors with high microsatellite instability, and lower BMI. In this prospective study of endometrial cancer patients younger than age 50 years, 9% were found to carry germline Lynch syndrome-associated mutations. In addition to young age of onset, family history, BMI, and molecular tumor studies can improve the likelihood of identifying a Lynch syndrome-associated germline mutation in MLH1, MSH2, and MSH6.

Gene-related cancer spectrum in families with hereditary non-polyposis colorectal cancer (HNPCC)

The family histories of 130 individuals with documented hereditary non-polyposis colorectal cancer (HNPCC) (caused by mutations in mismatch-repair (MMR) genes MSH2 (n = 64), MLH1 (n = 62) or MSH6 (n = 4)) were obtained, and incidence of cancers in those families was compared to that in the general population. There were a total of 982 cancers in 723 individuals. Colorectal cancer (CRC) was the commonest type (64% and 55% in individuals from families with germline MLH1 and MSH2 mutations respectively). Median age at diagnosis of first CRC in MSH6 mutation families was 59 years compared to 45 years in both MLH1 and MSH2 mutation families. The relative risk (RR) of endometrial cancer was 55 in MSH2 mutation families, compared with 27 in MLH1 mutation families, and 37 in MSH6 mutation families; median age at diagnosis 49 years. Even within MSH2 families, endometrial cancer tended to cluster, with 28 of the 58 cases coming from families with three or more cases (P < 0.001). Absolute risk of endometrial cancer in MLH1 families was still greater than any other cancer (other than CRC). 5% of cancers in both MLH1 and MSH2 mutation families were gastric (RR = 12); 53% of these were diagnosed before 50 years. Seven cases of small intestinal cancer occurred in MSH2 and MLH1 mutation families (RR = 26). There were 13 cases of cancer of the ureter; all were in MSH2 families. These cancers tended to cluster within families (P < 0.001); three of seven families with urothelial cancer had such cases in two or more individuals; two others had kidney cancer. Nineteen of 27 ovarian cancers (70%) were in MSH2 mutation families and 70% of these were diagnosed before age 50 years. There were 9 cases of sebaceous skin cancer, 3 in two MLH1 and 6 in four MSH2 mutation families. Of 22 pancreatic cancers, 14 were known to be diagnosed before 60 years. Breast cancer RR was 1.7 overall. The type of mutation (truncating or other type, and site of mutation) showed no obvious correlation with the presence or absence of extra-colonic cancers in families.

Homozygous PMS2 germline mutations in two families with early-onset haematological malignancy, brain tumours, HNPCC-associated tumours, and signs of neurofibromatosis type 1

Heterozygous germline mutations in mismatch repair (MMR) genes MLH1, PMS2, MSH2, and MSH6 cause Lynch syndrome. New studies have indicated that biallelic mutations lead to a distinctive syndrome, childhood cancer syndrome (CCS), with haematological malignancies and tumours of brain and bowel early in childhood, often associated with signs of neurofibromatosis type 1. We provide further evidence for CCS reporting on six children from two consanguineous families carrying homozygous PMS2 germline mutations. In family 1, all four children had the homozygous p.I590Xfs mutation. Two had a glioblastoma at the age of 6 years and one of them had three additional Lynch-syndrome associated tumours at 15. Another sibling suffered from a glioblastoma at age 9, and the fourth sibling had infantile myofibromatosis at 1. In family 2, two of four siblings were homozygous for the p.G271V mutation. One had two colorectal cancers diagnosed at ages 13 and 14, the other had a Non-Hodgkin's lymphoma and a colorectal cancer at ages 10 and 11, respectively. All children with malignancies had multiple café-au-lait spots. After reviewing published cases of biallelic MMR gene mutations, we provide a concise description of CCS, revealing similarities in age distribution with carriers of heterozygous MMR gene mutations.

A novel MSH2 germline mutation in a Druze HNPCC family

Germline mutations in DNA mismatch repair (DNA-MMR) genes, mainly MLH1, MSH2, and MSH6, underlie Hereditary non-polyposis colorectal cancer (HNPCC) and are mostly family-specific, with few reported founder mutations in MSH2 (Ashkenazim) MLH1 (Finnish). No mutations in colon cancer susceptibility genes have ever been reported in Druze individuals, a Moslem related faith encompassing approximately 1,000,000 individuals worldwide. A novel MSH2 mutation is described in a Druze HNPCC family: a multigenerational family with 10 members in 4 generations affected with colorectal cancer (mean age of diagnosis 46.5 years), two with gastric cancer and one--endometrial cancer. Mutational analysis of the MSH2 gene using denaturing gradient gel electrophoresis (DGGE) and direct sequencing revealed the c.702delA mutation in codon 234 of exon 4 of the MSH2 gene leading to a premature early stop in codon 245, p.Thr234fsX245. Analysis of mutation-carrying or presumed carriers individuals' offspring, revealed 11/42 asymptomatic mutation carriers, age range 17-50 years. The mutation was not present in two additional Druze HNPCC families and 20 Druze sporadic colon cancer patients. This is the first mutation ever reported in a colon cancer susceptibility gene in a Druze family and it appears not to be a founder mutation in Druze individuals with HNPCC.

A de novo deletional germline mutation in the MLH1 gene resulting in early onset colorectal cancer in a woman with a negative family history: A case report

21067 Background: Hereditary non-polyposis colorectal cancer (HNPCC) is a genetic disorder that results in an increased risk of early onset colorectal cancer (CRC). HNPCC is caused by germline mutations in DNA mismatch repair (MMR) genes, including MLH1 and MSH2, and is transmitted in an autosomal dominant manner. De novo germline mutations in MMR genes are exceedingly rare. We now describe a case of a de novo germline mutation in MLH1 associated with early onset CRC in a young woman with a negative family history. Methods: We present a case of a 31-year-old Caucasian female who presented with abdominal pain. Colonoscopy revealed a moderately differentiated adenocarcinoma with focal mucin production involving the sigmoid colon, with a final staging of T4N0M0. Genetic testing was offered because of her young age at diagnosis, having fulfilled the Bethesda guidelines. Microsatellite instability (MSI) testing using a panel of six microsatellite loci was performed on the tumor sample from the affected patient. Immunohistochemistry (IHC) testing for MLH1 and MSH2 was performed. Following counseling and with informed consent, DNA was isolated and sequenced using bi-directional PCR of the MLH1 gene. All exons of MLH1 and MSH2 were analyzed by standard Southern blot methods. Paternity was established using eight genetic loci. Results: The patient's tumor revealed high MSI and complete absence of MLH1 immunoreactivity. MSH2 IHC staining was normal. A large deletional mutation involving exons 5–12 of MLH1 was identified by Southern blot analysis. The patient's parents and siblings were tested and found to have wild type MLH1. Paternity was confirmed with greater than 99.9% certainty. Conclusions: De novo mutations in MMR genes are a rare cause of HNPCC. We report the first case of a large de novo deletion in the MLH1 gene accounting for early onset CRC. Such de novo mutations, albeit rare, must be considered in patients who present with early onset CRC and a negative family history. These results support the use of the Bethesda guidelines to identify individuals who may carry mutations in the MMR genes. No significant financial relationships to disclose.

Beta2‐microglobulin mutations in microsatellite unstable colorectal tumors

Defects of DNA mismatch repair (MMR) cause the high level microsatellite instability (MSI-H) phenotype. MSI-H cancers may develop either sporadically or in the context of the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome that is caused by germline mutations of MMR genes. In colorectal cancer (CRC), MSI-H is characterized by a dense lymphocytic infiltration, reflecting a high immunogenicity of these cancers. As a consequence of immunoselection, MSI-H CRCs frequently display a loss of human leukocyte antigen (HLA) class I antigen presentation caused by mutations of the beta2-microglobulin (beta2m) gene. To examine the implications of beta2m mutations during MSI-H colorectal tumor development, we analyzed the prevalence of beta2m mutations in MSI-H colorectal adenomas (n=38) and carcinomas (n=104) of different stages. Mutations were observed in 6/38 (15.8%) MSI-H adenomas and 29/104 (27.9%) MSI-H CRCs. A higher frequency of beta2m mutations was observed in MSI-H CRC patients with germline mutations of MMR genes MLH1 or MSH2 (36.4%) compared with patients without germline mutations (15.4%). The high frequency of beta2m mutations in HNPCC-associated MSI-H CRCs is in line with the hypothesis that immunoselection may be particularly pronounced in HNPCC patients with inherited predisposition to develop MSI-H cancers. beta2m mutations were positively related to stage in tumors without distant metastases (UICC I-III), suggesting that loss of beta2m expression may promote local progression of colorectal MSI-H tumors. However, no beta2m mutations were observed in metastasized CRCs (UICC stage IV, p=0.04). These results suggest that functional beta2m may be necessary for distant metastasis formation in CRC patients.

Microsatellite Instability Markers for Identifying Early-Onset Colorectal Cancers Caused by Germ-Line Mutations in DNA Mismatch Repair Genes

Microsatellite instability (MSI) testing of colorectal cancer tumors is used as a screening tool to identify patients most likely to be mismatch repair (MMR) gene mutation carriers. We wanted to examine which microsatellite markers currently used to detect MSI best predict early-onset colorectal cancer caused by germ-line mutations in MMR genes. Invasive primary tumors from a population-based sample of 107 cases of colorectal cancer diagnosed before age 45 years and tested for germ-line mutations in MLH1, MSH2, MSH6, and PMS2 and MMR protein expression were screened for MSI using the National Cancer Institute panel and an expanded 10-microsatellite marker panel. The National Cancer Institute five-marker panel system scored 31 (29%) as (NCI)MSI-High, 13 (12%) as (NCI)MSI-Low, and 63 (59%) as (NCI)MS-Stable. The 10-marker panel classified 18 (17%) as (10)MSI-High, 17 (16%) as (10)MSI-Low, and 72 (67%) as (10)MS-Stable. Of the 26 cancers that lacked the expression of at least one MMR gene, 24 (92%) were positive for some level of MSI (using either microsatellite panel). The mononucleotide repeats Bat26, Bat40, and Myb were unstable in all (10)MSI-High cancers and all MLH1 and MSH2 mutation carriers (100% sensitive). Bat40 and Bat25 were unstable in all tumors of MSH6 mutation carriers (100% sensitive). Bat40 was unstable in all MMR gene mutation carriers (100% sensitive). By incorporating seven mononucleotide repeats markers into the 10-marker panel, we were able to distinguish the carriers of MSH6 mutations (all scored (10)MSI-Low) from the MLH1 and MSH2 mutation carriers (all scored (10)MSI-High). In early-onset colorectal cancer, a microsatellite panel containing a high proportion of mononuclear repeats can distinguish between tumors caused by MLH1 and MSH2 mutations from those caused by MSH6 mutations.

Patients with an unexplained microsatellite instable tumour have a low risk of familial cancer

The cancer risk is unknown for those families in which a microsatellite instable tumour is neither explained by MLH1 promoter methylation nor by a germline mutation in a mismatch repair (MMR) gene. Such information is essential for genetic counselling. Families suspected of Lynch syndrome (n=614) were analysed for microsatellite instability, MLH1 promoter methylation and/or germline mutations in MLH1, MSH2, MSH6, and PMS2. Characteristics of the 76 families with a germline mutation (24 MLH1, 2 PMS2, 32 MSH2, and 18 MSH6) were compared with those of 18 families with an unexplained microsatellite instable tumour. The mean age at diagnosis of the index patients in both groups was comparable at 44 years. Immunohistochemistry confirmed the loss of an MMR protein. Together this suggests germline inactivation of a known gene. The Amsterdam II criteria were fulfilled in 50/75 families (66%) that carried a germline mutation in an MMR gene and in only 2/18 families (11%) with an unexplained microsatellite instable tumour (P<0.0001). Current diagnostic strategies can detect almost all highly penetrant MMR gene mutations. Patients with an as yet unexplained microsatellite instable tumour likely carry a different type of mutation that confers a lower risk of cancer for relatives.

The additive effect of p53 Arg72Pro and RNASEL Arg462Gln genotypes on age of disease onset in Lynch syndrome patients with pathogenic germline mutations in MSH2 or MLH1

p53 and the prostate-cancer-susceptibility gene RNASEL are tumour suppressor genes involved in apoptosis. We have previously reported that the common, functionally different variants Arg72Pro in p53 and Arg462Gln in RNASEL are associated with the age of disease onset of colorectal cancer in Lynch syndrome patients. To assess the combined effect of both variants, we screened 246 unrelated Lynch syndrome patients with a pathogenic germline mutation either in MSH2 ( n = 138) or in MLH1 ( n = 108) and colorectal cancer as first tumour, and 245 healthy controls. The global log rank test revealed significant differences in the age of disease onset for the genotypes of each variant ( p = 0.0176 for p53 and p = 0.0358 for RNASEL) and for the combined genotypes of both variants ( p = 0.0174). The highest difference in median age of disease onset was seen between homozygotes for the wild-types in both genes (42 years [range 22–75]) and homozygotes for the variant alleles in both genes (30 years [range 26–47]). A multivariate Cox regression model indicated that only the p53 and RNASEL genotypes had a significant influence on age of disease onset ( p = 0.016 for p53 and p = 0.014 for RNASEL) in an additive mode of inheritance, and that the effects of both variants are purely additive, which supports the notion that the p53 and RNaseL pathways do not interact. These findings may be relevant for preventive strategies in Lynch syndrome.

Effectiveness of the crcapro program in identifying patients suspected for HNPCC

Subjects affected by hereditary non-polyposis colorectal cancer exhibit a high susceptibility to colon and extracolonic tumours, due to MMR gene defects. Revised Bethesda criteria are used to select patients as candidates for genetic tests. Recently, the CRCAPRO model has been developed, based on family history of colorectal and endometrial cancers. Our study aims to evaluate the reliability of CRCAPRO in identifying mutation carriers. We used the CRCAPRO program to evaluate carrier probability risk in 99 patients fulfilling Amsterdam or Bethesda guidelines. MLH1 and MSH2 were studied by direct sequencing in all the 99 patients, and the study of microsatellite instability and of MMR proteins expression was performed. Nine MLH1 and nine MSH2 germline mutations were identified. Five out of the nine patients with MLH1 mutation showed a CRCAPRO risk evaluation of less than 20%. The same happened for four out of nine patients with MSH2 mutation. Of the 17 patients with an estimated risk of more than 80%, only four harboured a mutation, all in the MSH2 gene. The highest risk calculated by the CRCAPRO system in the nine carriers of a MLH1 mutation has been 31.7%. In our experience, the CRCAPRO program sensitivity and specificity appears to be low but needs to be further evaluated in larger samples.

Nine novel pathogenic germline mutations in MLH1, MSH2, MSH6 and PMS2 in families with Lynch syndrome

Many germline mutations in the DNA mismatch repair genes have been described so far leading to the clinical phenotype of Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC). Most mutations are private mutations. We report on nine novel pathogenic germline mutations that have been found in families meeting either the Amsterdam or the Bethesda criteria. These findings include the mutations MLH1,c.884+4A>G, MLH1,c.1377_1378insA;p.Glu460ArgfsX19, MLH1,c.1415_1416delGA;p.Arg472ThrfsX5, MSH2,c.301G>T;p.Glu101X, MSH2,c.638_639delTG;p.Leu213GlnfsX18, MSH2,c.842C>A;p.Ser281X, MSH2,c.859G>T;p.Gly287X, MSH6,c.2503C>T;p.Gln835X and a large genomic deletion of exons 1–10 of the PMS2 gene. The mutation MLH1,c.884+4A>G detected in two families results in a complete skipping of exon 10 on mRNA level and thus has been considered as pathogenic. In all cases the tumor tissue of the index patient revealed high microsatellite instability (MSI-H) and showed a complete loss of expression of the affected protein in the tumor cells by immunohistochemistry (IHC). The findings underline the importance of a pre-screening of tumor tissue for an efficient definition of conspicuous cases.

Very low prevalence of germline MSH6 mutations in hereditary non-polyposis colorectal cancer suspected patients with colorectal cancer without microsatellite instability

Hereditary non-polyposis colorectal cancer (HNPCC) is caused by mutations in one of the mismatch repair genes MLH1, MSH2, MSH6, or PMS2 and results in high-level microsatellite instability (MSI-high) in tumours of HNPCC patients. The MSI test is considered reliable for indicating mutations in MLH1 and MSH2, but is questioned for MSH6. Germline mutation analysis was performed in 19 patients with an MSI-high tumour and absence of MSH2 and/or MSH6 protein as determined by immunohistochemistry (IHC), without an MLH1 or MSH2 mutation, and in 76 out of 295 patients suspected of HNPCC, with a non-MSI-high colorectal cancer (CRC). All 295 non-MSI-high CRCs were analysed for presence of MSH6 protein by IHC. In 10 patients with an MSI-high tumour without MSH2 and/or MSH6 expression, a pathogenic MSH6 mutation was detected, whereas no pathogenic MSH6 mutation was detected in 76 patients with a non-MSI-high CRC and normal MSH6 protein expression. In none of the 295 CRCs loss of MSH6 protein expression was detected. The prevalence of a germline MSH6 mutation is very low in HNPCC suspected patients with non-MSI-high CRC. Microsatellite instability analysis in CRCs is highly sensitive to select patients for MSH6 germline mutation analysis.

Detection of BRAF V600E Mutation in Colorectal Cancer: Comparison of Automatic Sequencing and Real-Time Chemistry Methodology

Mutation V600E of BRAF, a kinase-encoding gene from the RAS/RAF/MAPK pathway, in colorectal carcinoma (CRC) suggests a sporadic origin of the disease, providing an exclusion criterion for hereditary nonpolyposis colorectal cancer. Here we describe detection of this mutation by real-time chemistry TaqMan MGB probes, confirmed by direct DNA sequencing as the gold standard. DNA was extracted from paraffin-embedded tissue from 112 tumors obtained from the EPICOLON study. Seventy-two tumors were CRC with defective DNA mismatch repair (MMR; microsatellite instability and/or loss of protein expression by immunohistochemical analysis), and 40 were proficient MMR controls. BRAF mutation was detected in 20/72 (27.8%) CRC with defective MMR and in 3/40 (7.5%) proficient MMR controls (P = 0.011). BRAF mutation was detected in 19/51 (37.3%) tumors with loss of MLH1 expression and in none of the tumors with loss of MSH2 expression (0/13). BRAF mutation was not found in cases with germline mutation of MLH1 (4/112) or MSH2 (3/112) genes. The sensitivity and specificity of our real-time chemistry were both 100% for detecting the V600E mutation. Because real-time chemistry methodology has advantages in cost, time, and labor, we consider it a valuable alternative to automatic direct sequencing, particularly for serial measurements.

Germline mutations and polymorphic variants in MMR, E‐cadherin and MYH genes associated with familial gastric cancer in Jiangsu of China

Germline mutations in MSH2, MLH1, E-cadherin and MutY (MYH) genes have been implicated in the occurrence of gastric cancer (GC). Epidemiological investigation was performed by recruiting patients with GC onset during 2002 in Jiangsu province, China. We identified suspected hereditary GC patients based on either the GC family history or GC onset at early ages. We have screened germline variations in 101 suspected hereditary GC patients at the coding sequences of MSH2, MLH1, E-cadherin and MYH genes with polymerase chain reaction-denaturing high-performance liquid chromatography (PCR-DHPLC) analysis and DNA sequencing. The result showed that about 40% of patients carried germline variations, predominantly with missense mutations. Of the variations detected are 2 base pair substitutions, c.53C > T and c.74G > A, which is predicted to generate missense mutations of p.Pro18Leu and p.Gly25Asp, respectively, and occurred at the same allele of MYH gene. The frequency of variant haplotype T/A in patients was higher than that in the control group (p = 0.021, odds ratio [OR] = 4.43, 95% confidence interval [95% CI] = 1.33-14.72). Difference in the frequency of the silent mutation p.Asn751Asn in E-cadherin gene was also found between patients and controls (p = 0.009, OR = 2.54, 95% CI = 1.30-4.95). Moreover, 6 types of variations were detected in MSH2 and MLH1 genes in 14 of 101 patients. Most of them occurred at exon7 of MSH2, frequently c.1168C > T, resulting in mutation of p.Leu390Phe. In summary, germline mutation at MSH2, MLH1, E-cadherin and MYH genes is a frequent event in the familial GC. They may form a genetic basis for the familial GC susceptibility in Chinese population.

Microsatellite instability and novel mismatch repair gene mutations in northern Chinese population with Hereditary non‐polyposis colorectal cancer

Hereditary non-polyposis colorectal cancer (HNPCC) syndrome is the most common cause of hereditary colorectal cancer with an early age of onset. Microsatellite instability (MSI) and germline mutation in one of the DNA mismatch repair (MMR) genes are found in the majority of HNPCC families and provide an opportunity for genetic diagnosis and prophylactic screening. The MMR gene mutation spectrum may vary across different populations and be influenced by founder mutations that prevail in specific ethnic groups. China is a big and ancient nation with enormous genetic diversity, which is especially notable between the northern and southern Chinese populations. A MMR gene mutation database for the southern Chinese population based in Hong Kong has been previously established. This study compares the MMR gene mutation spectrum and the MSI of HNPCC between the northern and southern Chinese populations. Twenty-five HNPCC families from northern China were systematically analyzed. The MSI analysis was performed using five loci in the USA National Cancer Institute (NCI) panel (D2S123, D5S346, BAT-25, BAT-26 and BAT-40) by PCR from the tumor and normal tissue. MSH2, MSH6 and MLH1 were performed using immunohistochemical staining. Two founder mutations of MSH2 and MLH1 were examined by PCR base analyses using primers flanking the two deletion sites (c.1452_1455delAATG in MSH2 and 1.8 kb deletion involving exon 11 of MLH1). Of the 25 families collected, 19 met Bethesda guideline (BG) 1 and six met BG3. Twenty-two (15.7%) were extra-colonic cancers with gastric cancer (in seven patients) being the most common cancer type. Of the 25 tumors analyzed, 21 (84%) were high level microsatellite instability (MSI-H) and four (16%) were microsatellite stable (MSS). Eighteen (86%) of the 21 MSI-H tumors showed loss of either the MLH1 or the MSH2 protein. Three MSI-H tumors and all four MSS tumors showed no loss of expression of the three MMR proteins. Out of the 21 patients with MSI-H tumors, 12 (57%) showed pathogenic germline mutations in either MLH1 (n = 8) or MSH2 (n = 4). Overall, three novel mutations (in patients H22, H17 and H29) have been identified. One of them, c.503_4insA, caused a frameshift mutation in the MLH1 gene. The other two were found in the MSH2 gene, including a frameshift (c.899_890insAT) and a splice junction (IVS7-1G-->A, SA of Exon 8) mutation. The results suggest a distinctly different mutation spectrum of MMR genes between northern and southern Chinese populations and call for a systematic, nationwide study to facilitate the design of a MMR gene mutation detection strategy tailored for individual populations in China.

The natural history of a combined defect in MSH6 and MUTYH in a HNPCC family

In the inherited syndromes, MUTYH-associated polyposis (MAP) and hereditary nonpolyposis colorectal cancer (HNPCC), somatic mutations occur due to loss of the caretaker function that base-repair (BER) and mismatch repair (MMR) genes have, respectively. Recently, we identified a large branch from a MSH6 HNPCC family in which 19 family members are heterozygous or compound heterozygous for MUTYH germ line mutations. MSH6/MUTYH heterozygote mutation carriers display a predominant HNPCC molecular tumour phenotype, with microsatellite instability and underrepresentation of G>T transversions. A single unique patient is carrier of the MSH6 germline mutation and is compound heterozygote for MUTYH. Unexpectedly, this patient has an extremely mild clinical phenotype with sofar only few adenomas at age 56. Four out of five adenomas show characteristic G>T transversions in APC and/or KRAS2, as seen in MUTYH associated polyposis. No second hit of MSH6 is apparent in any of the adenomas, due to retained MSH6 nuclear expression and a lack of microsatellite instability. Although this concerns only one case, we argue that the chance to find an additional one is extremely small and currently a mouse model with this genotype combination is not available. Moreover, the patients brother who is also compound heterozygous for MUTYH but lacks the MSH6 germline mutation presented with a full blown polyposis coli. In conclusion, these data would support the notion that abrogation of both MSH6 DNA mismatch repair and base repair might be mutually exclusive in humans.

No evidence for dual role of loss of heterozygosity in hereditary non-polyposis colorectal cancer

Hereditary non-polyposis colorectal cancer (HNPCC) is caused by germline mutations in mismatch repair (MMR) genes, mostly MLH1 and MSH2. Somatic inactivation of the wild-type allele of the respective MMR gene is required for tumor development. Unexpectedly, a recent study utilizing DNA from paraffin-embedded tissue material detected frequent loss of the mutant MMR gene allele in HNPCC tumors. Dual role for loss of heterozygosity (LOH) was proposed. If somatic loss of the wild-type MMR gene allele had occurred through point mutation or promoter hypermethylation, frequent somatic deletions at the region of the MMR gene locus, perhaps targeting other relevant cancer genes, could quite commonly lead to loss of the mutant allele. To test this hypothesis, we studied a population-based series of 25 fresh-frozen HNPCC tumors with a germline mutation in MLH1 or MSH2 for LOH. Fourteen of the 25 tumors (56%) showed LOH at the respective locus, and all 14 losses targeted the wild-type allele (P=0.00006). These results strongly support the traditional two-hit model of HNPCC gene inactivation.

MSH6 germline mutations in early-onset colorectal cancer patients without family history of the disease

Germline MLH1 and MSH2 mutations are scarce in young colorectal cancer patients with negative family history of the disease. To evaluate the contribution of germline MSH6 mutations to early-onset colorectal cancer, we have analysed peripheral blood of 38 patients diagnosed with this disease before 45 years of age and who presented no family history of hereditary nonpolyposis colorectal cancer-related cancers. Blood samples from 108 healthy volunteers were analysed for those genetic alterations suspected to affect the function of MSH6. Of the seven (18.4%) MSH6 alterations found, we have identified three novel germline mutations, one 8 bp deletion leading to a truncated protein and two missense mutations resulting in the substitution of amino acids belonging to different polarity groups. High-frequency microsatellite instability was found in the patient with the MSH6 deletion, but not in the other 27 carcinomas analysed. No MLH1 promoter methylation was detected in tumour tissue. Our findings suggest that germline MSH6 mutations contribute to a subset of early-onset colorectal cancer. Further studies are warranted to understand the genetic and environmental factors responsible for the variable penetration of MSH6 germline mutations, as well as to identify other causes of early-onset colorectal cancer.

Genotype-Phenotype Comparison of German MLH1 and MSH2 Mutation Carriers Clinically Affected With Lynch Syndrome: A Report by the German HNPCC Consortium

Lynch syndrome is linked to germline mutations in mismatch repair genes. We analyzed the genotype-phenotype correlations in the largest cohort so far reported. Following standard algorithms, we identified 281 of 574 unrelated families with deleterious germline mutations in MLH1 (n = 124) or MSH2 (n = 157). A total of 988 patients with 1,381 cancers were included in this analysis. We identified 181 and 259 individuals with proven or obligatory and 254 and 294 with assumed MLH1 and MSH2 mutations, respectively. Age at diagnosis was younger both in regard to first cancer (40 v 43 years; P < .009) and to first colorectal cancer (CRC; 41 v 44 years; P = .004) in MLH1 (n = 435) versus MSH2 (n = 553) mutation carriers. In both groups, rectal cancers were remarkably frequent, and the time span between first and second CRC was smaller if the first primary occurred left sided. Gastric cancer was the third most frequent malignancy occurring without a similarly affected relative in most cases. All prostate cancers occurred in MSH2 mutation carriers. The proportion of rectal cancers and shorter time span to metachronous cancers indicates the need for a defined treatment strategy for primary rectal cancers in hereditary nonpolyposis colorectal cancer patients. Male MLH1 mutation carriers require earlier colonoscopy beginning at age 20 years. We propose regular gastric surveillance starting at age 35 years, regardless of the familial occurrence of this cancer. The association of prostate cancer with MSH2 mutations should be taken into consideration both for clinical and genetic counseling practice.

An Illustrative Case of Muir-Torre Syndrome

Muir-Torre syndrome (MTS) is an autosomal dominant genodermatosis characterized by the association of at least 1 cutaneous sebaceous tumor and 1 internal malignancy, often arising in the gastrointestinal tract. It is secondary to germline mutations in DNA mismatch repair genes, mainly MLH-1 and MSH-2. We report the case of a 54-year-old man with a 2-year history of skin-colored papules clinically reminiscent of large sebaceous hyperplasias on the nose and back, but histologically diagnosed as sebaceous adenomas and epitheliomas. His family history was positive for colon cancer in the mother and 2 brothers. A colonoscopy done during the hospitalization revealed 2 sessile polyps in the left colon, both showing a low-grade dysplasia on the biopsy specimen. Immunohistochemical staining performed on the cutaneous and colic biopsy specimens revealed a lack of expression of MSH-2 and MSH-6. Genetic testing revealed microsatellite instability in the colon and cutaneous tumors. The immunohistochemical testing for MSH-2, MSH-6, and MLH-1 is useful for rapid identification of an underlying mismatch repair defect and early diagnosis of MTS.