Cloning of rat MLH1 and expression analysis of MSH2, MSH3, MSH6, and MLH1 during spermatogenesis

The mismatch repair system has been highly conserved in various species. In eukaryotic cells, the Mut S and Mut L homologues play crucial roles in both DNA mismatch repair and meiotic recombination. A full-length rat cDNA clone for rat MLH1 has been constructed using the RT-PCR method. The cDNA has an open reading frame of 2274 nucleotides for a protein of 757 amino acids. We have also obtained partial cDNA clones for MSH3 and MSH6. Northern blot analysis of rat MLH1, MSH2, MSH3, and MSH6 in the testes of rats of different ages showed differential expression of these genes as a function of developmental maturation of the testes. The expression analysis suggests that MSH3 may have a more predominant role in the meiotic recombination process.     

Nine novel germline mutations of STK11 in ten families with Peutz-Jeghers syndrome

Peutz-Jeghers Syndrome (PJS) is an autosomal dominant hereditary disease characterized by hamartomatous polyposis involving the entire bowel. Recently STK11, a gene bearing a mutation responsible for PJS, was isolated. We investigated the entire coding region of STK11 in 15 unrelated PJS families by the PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) method and PCR-direct sequence analysis, and found nine different, novel mutations among ten of those families. One nonsense mutation and five different frameshift mutations (two families carried the same mutation), all of which would cause truncation of the gene product, were found in seven families; mutations found in five families were clustered within exon 6. Among these five mutations, three occurred at the mononucleotide-repeat region (CCCCCC) of codons 279–281, suggesting that this region is likely to be a mutational hotspot of this gene. One of the remaining three families carried a 3-bp in-frame deletion that would eliminate an asparagine residue within a kinase domain of the product; the other two carried intronic mutations at or adjacent to the consensus dinucleotide sequences of splice-acceptor or -donor sites, which were likely to lead to aberrant splicing. Received: 9 March 1998 / Accepted: 1 May 1998     

Distinct mutations in MLH1 and MSH2 genes in hereditary non-polyposis colorectal cancer (HNPCC) families from China

Hereditary non-polyposis Colorectal Cancer (HNPCC) is an autosomal dominant inheritance syndrome. HNPCC is the most common hereditary variant of colorectal cancer (CRC), which accounts for 2-5% CRCs, mainly due to hMLH1 and hMSH2 mutations that impair DNA repair functions. Our study aimed to identify the patterns of hMSH2 and hMLH1 mutations in Chinese HNPCC patients. Ninety-eight unrelated families from China meeting Amsterdam or Bethesda criteria were included in our study. Germline mutations in MLH1 and MSH2 genes, located in the exons and the splice-site junctions, were screened in the 98 probands by direct sequencing. Eleven mutations were found in ten patients (11%), with six in MLH1 (54.5%) and five in MSH2 (45.5%) genes. One patient had mutations in both MLH1 and MSH2 genes. Three novel mutations in MLH1 gene (c.157_160delGAGG, c.2157dupT and c.-64G>T) were found for the first time, and one suspected hotspot in MSH2 (c.1168C>T) was revealed.     

Deletions account for 17% of pathogenic germline alterations in MLH1 and MSH2 in hereditary nonpolyposis colorectal cancer (HNPCC) families

Hereditary nonpolyposis colorectal cancer (HNPCC) is due to defects in DNA mismatch repair (MMR) genes MSH2, MLH1, MSH6, and to a lesser extent PMS2. Of 466 suspected HNPCC families, we defined 54 index patients with either tumors of high microsatellite instability (MSI-H) and/or loss of expression for either MLH1, MSH2, and/or MSH6, but without a detectable pathogenic point mutation in these genes. This study cohort was augmented to 64 patients by 10 mutation-negative index patients from Amsterdam families where no tumors were available. Deletion/duplication screening using the multiplex ligation-dependent probe amplification (MLPA) revealed 12 deletions in MSH2 and two deletions in MLH1. These deletions constitute 17% of pathogenic germline alterations but elucidate the susceptibility to HNPCC in only 22% of the mutation-negative study cohort, pointing towards other mutation mechanisms for an inherited inactivation of MLH1 or MSH2. We describe here four novel deletions. One novel and one known type of deletion were found for three and two unrelated families, respectively. MLPA analysis proved a reliable method for the detection of genomic deletions in MLH1 and MSH2; however, sequence variations in the ligation-probe binding site can mimic single exon deletions.     

Characterization of a germline mosaicism in families with Lowe syndrome, and identification of seven novel mutations in the OCRL1 gene.

The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked disorder characterized by major abnormalities of eyes, nervous system, and kidneys. Mutations in the OCRL1 gene have been associated with the disease. OCRL1 encodes a phosphatidylinositol 4, 5-biphosphate (PtdIns[4,5]P2) 5-phosphatase. We have examined the OCRL1 gene in eight unrelated patients with OCRL and have found seven new mutations and one recurrent in-frame deletion. Among the new mutations, two nonsense mutations (R317X and E558X) and three other frameshift mutations caused premature termination of the protein. A missense mutation, R483G, was located in the highly conserved PtdIns(4,5)P2 5-phosphatase domain. Finally, one frameshift mutation, 2799delC, modifies the C-terminal part of OCRL1, with an extension of six amino acids. Altogether, 70% of missense mutations are located in exon 15, and 52% of all mutations cluster in exons 11-15. We also identified two new microsatellite markers for the OCRL1 locus, and we detected a germline mosaicism in one family. This observation has direct implications for genetic counseling of Lowe syndrome families.     

Three novel KCNA1 mutations in episodic ataxia type I families

Hereditary paroxysmal ataxia, or episodic ataxia (EA), is a rare, genetically heterogeneous neurological disorder characterized by attacks of generalized ataxia. By direct sequence analysis, a different missense mutation of the potassium channel gene (KCNA1) has been identified in three families with EA. Received: 20 November 1997 / Accepted: 12 January 1998     

Three synchronous primary carcinomas in a patient with HNPCC associated with a novel germline mutation in MLH1: Case report

Background

Adnexal masses are not uncommon in patients with breast cancer. Breast cancer and ovarian malignancies are known to be associated. In patients with breast cancer and co-existing pleural effusions, ascites and adnexal masses, the probability of disseminated disease is high. Nevertheless, benign ovarian masses can mimic this clinical picture when they are associated with Meigs' syndrome making the work-up and management of these patients challenging. To our knowledge, there are no similar reports in the literature and therefore we present this case to highlight this entity.

Case presentation

A 56-year old woman presented with a 4 cm, grade 2, invasive ductal carcinoma of her left breast. Pre-treatment staging investigations showed a 13.5 cm mass in her left ovary, a small amount of ascites and a large right pleural effusion. Serum tumour markers showed a raised CA125 supporting the malignant nature of the ovarian mass. The cytology from the pleural effusion was indeterminate but thoracoscopic biopsy failed to show malignancy. The patient was strongly against mastectomy and she was commenced on neo-adjuvant Letrozole 2.5 mg daily with a view to perform breast conserving surgery. After a good response to the hormone manipulation, the patient had breast conserving surgery, axillary sampling and laparoscopic excision of the ovarian mass which was eventually found to be a benign ovarian fibroma.

Conclusion

Despite the high probability of disseminated malignancy when an ovarian mass associated with ascites if found in a patient with a breast cancer and pleural effusion, clinicians should be aware about rare benign syndromes, like Meigs', which may mimic a similar picture and mislead the diagnosis and management plan.     

Accuracy of MSI testing in predicting germline mutations of MSH2 and MLH1: a case study in Bayesian meta-analysis of diagnostic tests without a gold standard

Microsatellite instability (MSI) testing is a common screening procedure used to identify families that may harbor mutations of a mismatch repair (MMR) gene and therefore may be at high risk for hereditary colorectal cancer. A reliable estimate of sensitivity and specificity of MSI for detecting germline mutations of MMR genes is critical in genetic counseling and colorectal cancer prevention. Several studies published results of both MSI and mutation analysis on the same subjects. In this article we perform a meta-analysis of these studies and obtain estimates that can be directly used in counseling and screening. In particular, we estimate the sensitivity of MSI for detecting mutations of MSH2 and MLH1 to be 0.81 (0.73-0.89). Statistically, challenges arise from the following: (a) traditional mutation analysis methods used in these studies cannot be considered a gold standard for the identification of mutations; (b) studies are heterogeneous in both the design and the populations considered; and (c) studies may include different patterns of missing data resulting from partial testing of the populations sampled. We address these challenges in the context of a Bayesian meta-analytic implementation of the Hui-Walter design, tailored to account for various forms of incomplete data. Posterior inference is handled via a Gibbs sampler.     

Three novel BRCA1/BRCA2 mutations in breast/ovarian cancer families in Croatia

BRCA1 and BRCA2 genes from 167 candidates (145 families) were scanned for mutations. We identified 14 pathogenic point mutations in 17 candidates, 9 in BRCA1 and 5 in BRCA2. Of those, 11 have been previously described and 3 were novel (c.5335C>T in BRCA1 and c.4139_4140dupTT and c.8175G>A in BRCA2). No large deletions or duplications involving BRCA1 and BRCA2 genes were identified. No founder mutations were detected for the Croatian population. Croatia shares most of the mutations with neighboring Slovenia and also with Germany, Austria and Poland. Two common sequence variants in BRCA1, c.2077G>A and c.4956G>A, were found more frequently in mutation carriers compared to healthy controls. No difference in BRCA2 variants was detected between the groups. Haplotype inference showed no difference in haplotype distributions between deleterious mutation carriers and non-carriers in neither BRCA1 nor BRCA2. In silico analyses identified one BRCA1 sequence variant (c.4039A>G) and two BRCA2 variants (c.5986G>A and c.6884G>C) as harmful with high probability, and inconclusive results were obtained for our novel BRCA2 variant c.3864_3866delTAA. Combination of QMPSF and HRMA methods provides high detection rate and complete coverage of BRCA1/2 genes. Benefit of BRCA1/2 mutation testing is clear, since we detected mutations in young unaffected women, who will be closely monitored for breast and ovarian cancer.     

Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants

Single base substitutions in DNA mismatch repair genes which are predicted to lead either to missense or silent mutations, or to intronic variants outside the highly conserved splicing region are often found in hereditary nonpolyposis colorectal cancer (HNPCC) families. In order to use the variants for predictive testing in persons at risk, their pathogenicity has to be evaluated. There is growing evidence that some substitutions have a detrimental influence on splicing. We examined 19 unclassified variants (UVs) detected in MSH2 or MLH1 genes in patients suspected of HNPCC for expression at RNA level. We demonstrate that 10 of the 19 UVs analyzed affect splicing. For example, the substitution MLH1,c.2103G>C in the last position of exon 18 does not result in a missense mutation as theoretically predicted (p.Gln701His), but leads to a complete loss of exon 18. The substitution MLH1,c.1038G>C (predicted effect p.Gln346His) leads to complete inactivation of the mutant allele by skipping of exons 10 and 11, and by activation of a cryptic intronic splice site. Similarly, the intronic variant MLH1,c.306+2dupT results in loss of exon 3 and a frameshift mutation due to a new splice donor site 5 bp upstream. Furthermore, we confirmed complete exon skipping for the mutations MLH1,c.1731G>A and MLH1,c.677G>A. Partial exon skipping was demonstrated for the mutations MSH2,c.1275A>G, MLH1,c.588+5G>A, MLH1,c.790+4A>G and MLH1,c.1984A>C. In contrast, five missense mutations (MSH2,c.4G>A, MSH2,c.2123T>A, MLH1,c.464T>G, MLH1,c.875T>C and MLH1,c.2210A>T) were found in similar proportions in the mRNA as in the genomic DNA. We conclude that the mRNA examination should precede functional tests at protein level. Databases: HNPCC – OMIM 114500, MSH2 – OMIM: 120435; GenBank: NM_000251.1, MLH1 – OMIM: 120436; GenBank: NM_000249.2, InSiGHT mutation database: , Programs: BDGP: , ESEfinder program:     

Promoter Methylation of MLH1, PMS2, MSH2 and p16 Is a Phenomenon of Advanced-Stage HCCs

Epigenetic silencing of tumour suppressor genes has been observed in various cancers. Looking at hepatocellular carcinoma (HCC) specific protein silencing was previously demonstrated to be associated with the Hepatitis C virus (HCV). However, the proposed HCV dependent promoter methylation of DNA mismatch repair (MMR) genes and thereby enhanced progression of hepatocarcinogenesis has been the subject of controversial discussion. We investigated promoter methylation pattern of the MMR genes MLH1, MSH2 and PMS2 as well as the cyclin-dependent kinase inhibitor 2A gene (p16) in 61 well characterized patients with HCCs associated with HCV, Hepatitis B virus infection or alcoholic liver disease. DNA was isolated from formalin-fixed, paraffin-embedded tumour and non-tumour adjacent tissue and analysed by methylation-specific PCR. Moreover, microsatellite analysis was performed in tissues showing methylation in MMR gene promoters. Our data demonstrated that promoter methylation of MLH1, MSH2, PMS2 and p16 is present among all considered HCCs. Hereby, promoter silencing was detectable more frequently in advanced-stage HCCs than in low-stage ones. However, there was no significant correlation between aberrant DNA methylation of MMR genes or p16 and HCV infection in related HCC specimens. In summary, we show that promoter methylation of essential MMR genes and p16 is detectable in HCCs most dominantly in pT3 stage tumour cases. Since loss of MMR proteins was previously described to be not only responsible for tumour development but also for chemotherapy resistance, the knowledge of mechanisms jointly responsible for HCC progression might enable significant improvement of individual HCC therapy in the future.     

Molecular characterization of germline mutations in the BRCA1 and BRCA2 genes from breast cancer families in Taiwan

A total of 18 families with multiple cases of breast cancer were identified from southern Taiwan, and 5 of these families were found to carry cancer-associated germline mutations in the BRCA1 and BRCA2 genes. One novel cryptic splicing mutation of the BRCA1 gene, found in two unrelated families, was shown to be a deletion of 10 bp near the branch site in intron 7. This mutation causes an insertion of 59 nucleotides derived from intron 7 and results in a frameshift, leading to premature translational termination of BRCA1 mRNA in exon 8. Deletions of 2670delC, 3073delT and 6696-7delTC in the BRCA2 gene were found in three other breast cancer families. All three deletions are predicted to generate frameshifts and to result in the premature termination of BRCA2 protein translation. Several genetic polymorphisms in both BRCA1 and BRCA2 genes were also detected in this investigation. Received: 28 September 1998 / Accepted: 20 November 1998     

Phenotypic variability in two families with novel splice-site and frameshift NF2 mutations

Neurofibromatosis 2 (NF2) is a clinically variable autosomal dominant disorder, caused by mutations in the NF2 tumor suppressor gene on chromosome 22q12, that predisposes to nervous system tumors and ocular abnormalities. To assess intrafamilial phenotypic variability, we performed mutation analysis and clinical assessment on two multigeneration NF2 families with five patients and seven asymptomatic first-degree relatives of patients. One family had a point mutation of agCC→ggCC at position 1447–2 at the exon 13/14 boundary predicted to lead to an altered splice acceptor sequence and exon deletion. The other family had an insertion of 2 base pairs (TC) at position 761 in exon 8, leading to a frameshift. Both mild and severe phenotypes occurred in each family, indicating that phenotypic variability in NF2 can be caused by factors other than NF2 mutations. Genetic counseling of NF2 families should include the possibility that presymptomatic NF2 mutation carriers can develop a different phenotype than previously diagnosed patients. Received: 4 January 1996 / Revised: 26 March 1996     

Three novel mutations causing a truncated protein within the RP2 gene in Italian families with X-linked retinitis pigmentosa

X-linked retinitis pigmentosa (XLRP) results from mutations in a number of loci, including RP2 at Xp11.3, and RP3 at Xp21.1. RP2 and RP3 genes have been identified by positional cloning. RP2 mutations are found in about 10% of XLRP patients. We performed a mutational screening of RP2 gene inpatients belonging to seven unrelated families in linkage with the RP2 locus. SSCP analysis detected three conformation variants, within exon 2 and 3. Direct sequencing of exon 2, disclosed a G-->A transition at nucleotide 449 (W150X), and a G-->T transversion in position 547 (E183X). Sequence analysis of exon 3 variant revealed an insertion (853/854insG), leading to a frameshift. In this patient, we detected an additional sequence alteration (A-->G at nucleotide 848, E283G). Each mutation was co-segregating with the disease in the affected family members available for the study. These mutations are expected to introduce a stop codon within the RP2 coding sequence probably resulting in a truncated or unstable protein.     

Identification and molecular characterization of two novel mutations in COL1A2 in two Chinese families with osteogenesis imperfecta

Osteogenesis imperfecta (OI, also known as brittle bone disease) is caused mostly by mutations in two type Ⅰ collagen genes, COL1A1 and COL1A2 encoding the pro-α1 (Ⅰ) and pro-α2 (Ⅰ) chains of type Ⅰ collagen, respectively. Two Chinese families with autosomal dominant OI were identified and characterized. Linkage analysis revealed linkage of both families to COL1A2 on chromosome 7q21.3-q22.1. Mutational analysis was carried out using direct DNA sequence analysis. Two novel missense mutations, c.3350A＞G and c.3305G＞C, were identified in exon 49 of COL1A2 in the two families, respectively. The c.3305G＞C mutation resulted in substitution of a glycine residue (G) by an alanine residue (A) at codon 1102 (p.G1102A), which was found to be mutated into serine (S), argine (R), aspartic acid (D), or valine (V) in other families. The c.3350A＞G variant may be a de novo mutation resulting in p.Y1117C. Both mutations co-segregated with OI in respective families, and were not found in 100 normal controls. The G1102 and Y1117 residues were evolutionarily highly conserved from zebrafish to humans. Mutational analysis did not identify any mutation in the COX-2 gene (a modifier gene of OI). This study identifies two novel mutations p.G1102A and p.Y1117C that cause OI, significantly expands the spectrum of COL1A2 mutations causing OI, and has a significant implication in prenatal diagnosis of OI.     

Predominant Ashkenazi BRCA1/2 mutations in families with pancreatic cancer

The present study aimed to identify pancreatic cancer patients who harbor a mutation in BRCA1/2 genes within hereditary breast-ovarian cancer families. History of cancer in 1014 families that attended our breast-ovarian oncogenetic clinic was evaluated. Twenty-three families with pancreatic cancer were studied. In nine families wherein the probands themselves presented with pancreatic cancer, two (22%) carried a BRCA mutation (185delAG in BRCA1 in one case and 6174delT in BRCA2 in the other). In 14 families, only a family history of pancreatic cancer was elicited. Of these, seven families segregated either the 185delAG (three families) or the 6174delT (three families) mutation; one family segregated both mutations, but the parental status was not studied. Pedigree analysis shows that four of the seven pancreatic cancer cases were obligatory carriers. In summary, from among 23 families with pancreatic cancer, 6 (26%) informative BRCA1/2 mutation carriers were identified, equally cosegregating the 185delAG or the 6174delT mutation. Yet, it is not fully elucidated whether the risk for pancreatic cancer attributed to BRCA1 is similar to the high risk conferred by BRCA2. In Ashkenazi Jews, mutations in BRCA1/2 may constitute a major cause for pancreatic cancer.