Mutation sharing,predominant involvement of the MLH1 gene and description of four novel mutations in hereditary nonpolyposis colorectal cancer

Worldwide, the DNA mismatch repair genes MSH2 and MLH1 account for a major share and almost equal proportions of hereditary nonpolyposis colorectal cancer (HNPCC). Furthermore, the predisposing mutation usually varies from kindred to kindred. In this study, we screened 29 verified or putative HNPCC kindreds from Finland for mutations in these two genes and found 8 different mutations, 7 in MLH1 and 1 in MSH2, occurring in 13 families. Four of these mutations were novel. Altogether, we have to date studied 81 kindreds for mutations and 12 different mutations in 52 families have been identified, 10 in MLH1 and 2 in MSH2. These data show that Finnish HNPCC kindreds are characterized by the predominant involvement of MLH1 (49/52, 94% of the families) and a high rate of shared mutations (5/12, 42%) offering unique possibilities for mutation screening for both research and diagnostic purposes. Hum Mutat 11:482–483, 1998. © 1998 Wiley-Liss, Inc.     

hMLH1 mutations in hereditary nonpolyposis colorectal cancer kindreds. Mutations in brief no. 182. Online

Hereditary nonpolyposis colorectral cancer (HNPCC), an autosomal dominantly inherited predisposition for early onset colorectal cancer, accounts for at least 6% of all colorectal malignancies. HNPCC results from germ-line mutations in DNA mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1 and hPMS2) and is associated with a high rate of replication errors in tumor cells. Using PCR-SSCP, the protein truncation test and DNA sequencing we have analyzed the hMSH2 and hMLH1 genes in 10 Italian families that met the standard diagnostic criteria for HNPCC. We have identified three new mutations in the hMLH1 gene. One mutation consists in a deletion of one base pair at nucleotide 954 (954delC) in exon 11 that creates an early stop at codon 366 and is predicted to abolish normal protein function. The other two are missense mutations. Cys77Arg and Ser193Pro, that cause dramatic amino acid substitutions in two highly conserved MLH domains. The Cys77Arg mutation occurs within a domain (1-114 residues) that is very critical for MMR function. The Ser193Pro mutation occurs in a highly conserved central region of the MLH1 protein. No functional domains have yet been identified in this region. All mutant alleles cosegregate with the cancer phenotype.     

遗传性非息肉性结直肠癌家系的MLH1基因两个胚系新突变

目的初步评价遗传性非息肉性结直肠癌（HNPCC）胚系MLH1基因突变中新突变的病理性。方法收集符合AmsterdamⅡ标准的12个不同家系的12例患者外周血,用特异引物和耐热性逆转录酶特异地逆转录MLH1的mRNA;利用长模板PCR扩增酶扩增逆转录产物（cDNA）;测序分析扩增产物;利用PCR-Genescan技术和免疫组织化学染色分别检测有新突变患者肿瘤组织的5个微卫星位点（BAT26,BAT25,D5S346,D2S123和Mfd15）和MLH1蛋白的表达。结果在4例患者中检出4个MLH1突变,其中2个突变为第12外显子的第384密码子（1151bp处）GTT→GAT的突变,该突变是已报道的病理性突变;另外2个突变分别是第8外显子的第217密码子（649bp处）CGC→TGC突变和第16外显子的第581密码子（1742bp处）CC→CTG突变;后两者为尚未报道的新突变。2个新突变的患者肿瘤组织均呈高度微卫星不稳定性,两者的瘤组织MLH1蛋白均失表达。结论MLH1第8外显子的第217密码子突变和第16外显子的第581密码子的两个新突变很可能为病理性突变。     

Seven novel MLH1 and MSH2 germline mutations in hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most frequent hereditary form of colorectal cancer and is caused by germline mutations in mismatch repair (MMR) genes. The majority of mutations occur in MLH1 and MSH2. We report hereby seven novel germline mutations in these two genes (five in MLH1 and two in MSH2). All mutations have been found in families fulfilling criteria of the Bethesda guidelines and four of which also fulfilled the Amsterdam criteria. We identified three insertions or deletions of 1 bp leading to premature stop codons (MLH1: c.341delC, c.1413‐1414insA; MSH2: c.1119delG) and three nonsense mutations (MLH1: c.67G>T [E23X], c.436C>T [Q146X]; MSH2: c.1857T>G [Y619X]). The corresponding tumors showed a high level of microsatellite instability (MSI‐H) and a complete loss of expression of the affected protein. In addition, a missense mutation in MLH1 was identified (c.1984A>C [T662P]). The respective tumor also showed a high level of microsatellite instability but a reduced, rather then lost, expression of the MLH1‐protein. This missense mutation was not found in 107 healthy control individuals and in 54 HNPCC patients. © 2001 Wiley‐Liss, Inc.     

Twelve novel RB1 gene mutations in patients with hereditary retinoblastoma. Mutations in brief no. 206. Online

Hereditary predisposition to retinoblastoma is caused by germline mutations in the RB1 gene. Mutation analysis in this gene is important because knowledge of the causative mutation is often required for accurate risk prediction in relatives. We have performed RB1 gene mutation analysis in 45 patients with hereditary retinoblastoma. Screening by heteroduplex and SSCP analysis resulted in the identification of small mutations in 28 (62%) patients. Recurrent mutations, mostly CpG-transitions, were found in 16 patients. Two patients with isolated bilateral retinoblastoma showed missense mutations, S567L and C712R, which have previously been reported in a patient with bilateral tumors and in a family with low penetrance, respectively. Twelve of the mutations identified here have not been reported to date. These include a novel missense mutation, L662P, which was identified in two bilaterally affected siblings and their mother with unilateral retinoma.     

Six novel heterozygous MLH1, MSH2, and MSH6 and one homozygous MLH1 germline mutations in hereditary nonpolyposis colorectal cancer

Most hereditary nonpolyposis colorectal cancer (HNPCC) cases are caused by germline mutations of mismatch repair (MMR) genes (i.e., MLH1, MSH2, or MSH6). Here we describe six novel mutations in patients referred for genetic assessment. All of these mutations lead to premature translation termination. Five single base pair deletions lead to frameshift (MLH1: g.38-39insCCCA, g.1971del.T; MSH2: g.163del.C, g.746del.A; MSH6: g.3320del.A) and one nonsense mutation in MSH2 g.1030C>T leads to a stop codon: p.Q344X. In one patient, the previously described MLH1 nonsense mutation g.806C>G was found in a homozygous state. In this patient, the familial histories of both the mother and father suggested HNPCC syndrome. This patient developed colon cancer at 22 years of age, suggesting a more aggressive phenotype. The results of our study provide further insight into the mutational spectrum of MMR genes in HNPCC families.     

Four new mutations in the DNA mismatch repair gene MLH1 in colorectal cancers with microsatellite instability. Mutations in brief no. 157. Online

Hereditary nonpolyposis colorectal cancer (HNPCC) is frequently associated with inherited mutation in one of four DNA mismatch repair genes. Somatic mutations in the same genes are also found in a subset of sporadic colorectal cancers. A defect in DNA mismatch repair results in a RER (replication error) tumor phenotype. We screened 110 archival and 11 prospectively acquired colorectal cancers for the RER phenotype. A total of 22 cancers were RER-positive. RER-positive tumors were investigated for mutations in the DNA mismatch repair gene MLH1 using single-strand-conformation-polymorphism (SSCP) analysis. We identified four previously undescribed mutations in four different samples. Three mutations were exonic: a point mutation at codon 69 (AGG-->AAG(arg-->lys]); a single base pair deletion at codon 42/43 (GCAAAATCC-->GCAAATCC) leading to a new stop codon downstream; and a point mutation at codon 757 (TAA-->TAT [termination-->tyr] which extend the MLH1 peptide by 36 ammino acids. The fourth mutation was a 1 base pair insertion six base pairs 5' to the start of exon 14 (tttgtttt-->tttggtttt). The mutations were not seen in the patients' constitutional DNA. The somatic MLHI mutations identified appear to be causally associated with the RER phenotype.     

Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer.

Hereditary nonpolyposis colon cancer (HNPCC) is associated with malfunction of postreplicative mismatch repair (MMR). While a majority of HNPCC-associated mutations in the MMR genes MLH1, MSH2, or MSH6 genes cause truncations-and thus loss of function--of the respective polypeptides, little is currently known about the biochemical defects associated with nontruncating mutations. We studied the interactions of six MLH1 variants, carrying either missense mutations or in-frame deletions, with normal PMS2 and tested the functionality of these heterodimers of MLH1 and PMS2 (MutL(alpha)) in an in vitro MMR assay. Three MLH1 carboxy-terminal mutations, consisting of internal deletions of exon 16 (amino acids 578-632) or exon 17 (amino acids 633-663), or a missense R659P mutation in exon 17, affected the formation of a functional MutL(alpha). Interestingly, mutations C77R and I107R in the amino-terminal part of MLH1 did not affect its heterodimerization with PMS2. The complexes MLH1(C77R)/PMS2 and MLH1(I107R)/PMS2, however, failed to complement a MMR-deficient extract lacking a functional MutL(alpha). As all these five mutations were identified in typical HNPCC families and produce nonfunctional proteins, they can be considered disease-causing. In contrast, the third amino-terminal mutation S93G did not affect the heterodimerization, and the MLH1(S93G)/PMS2 variant was functional in the in vitro MMR assay, given thus the nature of the HNPCC family in question. Although the missense mutation segregates with the disease, the mean age of onset in the family is unusually high (approximately 65 years).     

Four novel MSH2 and MLH1 frameshift mutations and occurrence of a breast cancer phenocopy in hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations of genes encoding for proteins of the mismatch repair (MMR) machinery. The majority of mutations occur in the MLH1 and MSH2 genes, and consist of splice-site, frameshift and nonsense changes, leading to loss of protein function. In this study, we screened 7 HNPCC families for MLH1/MSH2 mutations. Sequence changes were identified in 5 families. Four alterations were novel 1- or 2-bp deletions or insertions causing a frameshift and appearance of premature stop codons (MLH1: c.597-598delGA, c.1520-1521insT; MSH2: c.1444delA, c.119delG). The four small insertions/ deletions were located within stretches of simple repeated sequences. By reviewing the HNPCC mutation database, we found that the majority of 1-2 bp frameshift mutations similarly affects simple repetitive stretches, pointing to DNA polymerase slippage during replication as the most likely source of such errors. We also evaluated microsatellite instability (MSI) in a breast carcinoma (BC) from an MLH1 mutation carrier. While a colon cancer from the same individual showed MSI, the BC specimen was MSI-negative, indicating that development of the latter tumor was unrelated to MMR impairment, despite presence of a constitutional MLH1 mutation. Hum Mutat 17:521, 2001.     

Peutz-Jeghers syndrome: four novel inactivating germline mutations in the STK11 gene. Mutations in brief no. 227. Online

The diagnosis of Peutz-Jeghers syndrome is based on the occurrence of hamartomatous gastrointestinal polyps and perioral pigment spots. In view of the development of hamartomatous polyps in several syndromes and the variability of pigment spots in Peutz-Jeghers patients, identification of affected individuals is difficult. Recently, germline mutations in the STK11 gene have been reported as a molecular cause of Peutz-Jeghers syndrome. We present four novel inactivating mutations identified by direct sequencing of all 9 exons of the STK11 gene in 4 patients suggestive of Peutz-Jeghers syndrome: three frameshift mutations (125-137del; 474-480del; 516-517insT) and one nonsense mutation (Q220X). Our data obtained in these patients and in those reported previously emphasize the diagnostic value of histological discrimination between different types of hamartomatous polyps and of molecular analysis, particularly in cases with no family history of the disease.     

两个遗传性非患肉性结直肠癌家系中MLH1和MSH2基因的突变检测

目的 确定两个遗传性非息肉性结直肠癌(hereditary nonpolyposis colorectal cancer,HNPCC)家系的致病基因,选择MLH1基因和MSH2基因进行突变检测.方法 采用聚合酶链反应结合DNA直接测序法,对两个遗传性非息肉性结直肠癌家系的患者进行MLH1基因和MSH2基因的突变检测;发现变异后,采用PCR-限制性片段长度多态性或直接测序法鉴定此变异是否属于突变.结果 在家系A的患者中发现了位于MLH1基因第3外显子内的新突变c.243_244 insA;在家系B的患者中发现了MSH2基因第7外显子内的c.1215_1218dupCCGA突变,这两个突变都导致了编码蛋白的提前终止.结论 MLH1基因的c.243_244insA突变和MSH2基因的c.1215_1218dupCCGA突变分别是导致家系A和家系B发生遗传性非息肉性结直肠癌的致病突变.     

Identification of four novel mutations of the XLRS1 gene in Japanese patients with X-linked juvenile retinoschisis. Mutation in brief no. 234. Online

The XLRS1 gene (HUGO-approved symbol, RS1) has been found to cause X-linked recessive retinoschisis (RS) which is characterized by splitting of the superficial layer of the retina. Recent mutation analysis of this gene revealed 82 different mutations in 214 patients with RS. We have now identified 10 mutations of the XLRS1 gene in 11 unrelated Japanese males with RS. Mutations found in these patients were; 1) a 20-kb deletion in exon 1 region; 2) mutations in the initiation sequence (M1V); 3) mutations in the splice donor site (IVS1 + 1 g-->a); 4) two nonsense mutations (Q88X, W163X); and 5) five missense mutations (E72K, Y89C, R182C, G109E, P203L). Four (M1V, Q88X, G109E, and W163X) of the 10 mutations were novel. The R182C mutation was identified in 2 unrelated patients. The 3 mutations found between exons 1 and 3 cause premature translation termination in the XLRS1 protein. The rest of the 7 mutations were clustered between exons 4 and 6. This region of the protein is homologous to the proteins implicated in cell-cell adhesion.     

Hereditary nonpolyposis coloretal cancer: identification of novel germline mutations in two kindreds not fulfulling the Amsterdam criteria. Mutations in brief no. 203. Online

Hereditary nonpolyposis colon cancer results from heritable defects in the MLH1, MSH2, PMS1 and PMS2 genes, which encode proteins involved in the mismatch repair process. In this work we report the identification of two novel germline mutations in the MLH1 gene from two unrelated HNPCC families. The two affected families do not fulfill the Amsterdam criteria. In family 1 we found a missense S93G mutation, which lies in a MLH1 domain critical for its MMR functions. In family 2 we found a two nucleotide insertion (AG) in position 523 from the AUG which determines an early stop codon at position 606 (codon 203). In both families the mutant alleles cosegregate with the cancer phenotype.     

Partial duplications of the MSH2 and MLH1 genes in hereditary nonpolyposis colorectal cancer

Numerous reports have highlighted the contribution of MSH2 and MLH1 genomic deletions to hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch's syndrome, but genomic duplications of these genes have been rarely reported. Using quantitative multiplex PCR of short fluorescent fragments (QMPSF), 962 and 611 index cases were, respectively, screened for MSH2 and MLH1 genomic rearrangements. This allowed us to detect, in 11 families, seven MSH2 duplications affecting exons 1-2-3, exons 4-5-6, exon 7, exons 7-8, exons 9-10, exon 11, and exon 15, and three MLH1 duplications affecting exons 2-3, exon 4 and exons 6-7-8. All duplications were confirmed by an independent method. The contribution of genomic duplications of MSH2 and MLH1 to HNPCC can therefore be estimated approximately to 1% of the HNPCC cases. Although this frequency is much lower than that of genomic deletions, the presence of MSH2 or MLH1 genomic duplications should be considered in HNPCC families without detectable point mutations.     

Novel missense and frameshift mutations in the activin receptor-like kinase-1 gene in hereditary hemorrhagic telangiectasia. Mutations in brief no. 164. Online

Hereditary hemmorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by multisystemic vascular dyplasia and recurrent hemorrhage. One of the causative genes is the activin receptor-like kinase-1 (ALK-1) gene located on chromosome 12q13. ALK-1 is an endothelial cell type I receptor for the TGF-beta superfamily of ligands. As a number of mutations have been identified in the kinase domain of ALK-1, we initiated a mutation analysis specifically targeting the first four coding exons of ALK-1 in order to determine if mutations in the extracellular and transmembrane domains are also present in HHT. Six new mutations have been identified. Three frameshift mutations were identified in exons encoding the extracellular and transmembrane domains. These mutations would grossly truncate the ALK-1 protein and are thus classic null alleles. Three new missense mutations within the exons encoding the extracellular domain, in addition to two previously described missense mutations, are located at or near highly conserved cysteines. These mutations may disrupt intra- or inter-molecular disulfide bridges required for ligand binding. The combined data suggest that both severe and subtle changes in the ALK-1 amino acid sequence can lead to receptor dysfunction and result in the HHT disease phenotype.