Four Novel MSH2 and MLH1 Frameshift Mutations and Occurrence of a Breast Cancer Phenocopy in Hereditary Nonpolyposis Colorectal Cancer

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1 HUMAN MUTATION Mutation in Brief #420 (2001) Online MUTATION IN BRIEF Four Novel MSH2 and MLH1 Frameshift Mutations and Occurrence of a Breast Cancer Phenocopy in Hereditary Nonpolyposis Colorectal Cancer Oana Caluseriu 1, Emanuela Lucci Cordisco 1, Alessandra Viel 2, Silvia Majore 3, Riccardo Nascimbeni 4, Salvatore Pucciarelli 5, and Maurizio Genuardi 1* 1 Istituto di Genetica Medica, Università Cattolica del S. Cuore, Rome; 2 Divisione Oncologia Sperimentale 1, Centro di Riferimento Oncologico-IRCCS, Aviano; 3 Cattedra di Genetica Medica, Università degli Studi "La Sapienza"; 4 Cattedra di Chirurgia Generale, Università di Brescia, Italy; 5 Clinica Chirurgica II, Dipartimento di Scienze Oncologiche e Chirurgiche, Università degli Studi di Padova, Italy *Correspondence to: Maurizio Genuardi, Istituto di Genetica Medica, Facoltà di Medicina e Chirurgia "A. Gemelli", Università Cattolica del S. Cuore, Largo F. Vito 1, Rome, Italy; Tel: ; Fax: ; mgenuardi@rm.unicatt.it Contract grant sponsors: Ministero dell'università e della Ricerca Scientifica (MURST) COFIN99; Ministero della Sanità (Progetti Finalizzati) Communicated by Mark H. Paalman 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 delga, c inst; 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 Wiley-Liss, Inc. KEY WORDS: HNPCC; microsatellite instability; MSI; mismatch repair; MMR; colon cancer; breast cancer; MLH1; MSH2 Received 10 January 2001; accepted 28 March INTRODUCTION Mutations of the DNA mismatch repair (MMR) genes MLH1 (MIM# ; GenBank accession # AH003234) and MSH2 (MIM# ; GenBank accession # AH003235) account for the majority of hereditary nonpolyposis colorectal cancer (HNPCC) cases. The majority of HNPCC-associated defects are nonsense, frameshift, and splice-site changes, causing the synthesis of shorter, and therefore inactive, proteins (Peltomäki and 2001 WILEY-LISS, INC.

2 2 Caluseriu et al. Vasen 1997). Missense changes are also frequently identified, particularly in the MLH1 gene, but, in the absence of data from functional tests, their pathogenetic role is often questionable or undetermined (Genuardi et al. 1999). In a mutational survey of 7 Italian HNPCC families, we have identified 4 novel constitutional mutations in MLH1 and MSH2. These mutations consist of 1-2 bp insertions/deletions, which all involve removal or addition of nucleotides located within short tandem repeats. PATIENTS AND METHODS Informed consent was obtained from all subjects who underwent genetic testing. Mutation analysis was performed on probands from 7 colorectal cancer (CRC) families, for whom a diagnosis of HNPCC had been established according to the recently modified Amsterdam criteria (Vasen et al. 1999). In three of the families, molecular investigation was extended to further relatives following identification of the causative mutation. Tests were conducted on genomic DNA isolated from peripheral leukocytes. All MLH1 and MSH2 exons and flanking intron borders were directly sequenced using the Thermo Sequenase Dye Terminator kit (Amersham-Pharmacia Biotech, Buckinghamshire, England) and a Perkin Elmer-Applied Biosystems (Foster City, CA) 373 Stretch authomated sequencer. Putative DNA sequence variants identified in probands were confirmed by sequencing both DNA strands on 2 independent PCR products. Analysis of microsatellite instability (MSI) was performed on paired samples of leukocyte DNA and paraffinembedded tumor tissue, as previously described (Genuardi et al. 1998). The following microsatellite sequences were investigated for MSI status assessment: BAT25, BAT26, TGFßIIR, D2S123, and D17S250 (Boland et al. 1998). RESULTS AND DISCUSSION MLH1 or MSH2 DNA sequence variants were identified in 5 of the 7 HNPCC families investigated (Table 1). Four mutations were short (1-2 bp) insertions or deletions causing frameshifts, with consequent appearance of premature stop codons and production of non-functional, truncated proteins. Frameshifts within MLH1 and MSH2 are deemed to act as disease-causing mutations, since they cause inactivation of one allele. This is followed by complete functional disruption of one MMR locus by means of somatic mutations affecting the wild-type homologue before cancer development. On the other hand, the fifth mutation identified in this study is an MLH1 missense change which has been previously detected in HNPCC families (Liu et al. 1996; Maliaka et al. 1996), and whose clinical implications are not defined. Table 1. MLH1/MSH2 Mutations in HNPCC Families Family code Mutation Consequence Repeat involved 1 MSI status 2 RMS1 MLH1 exon 8 c delga (codons ) RMS2 MLH1 exon 13 c inst (codon 507) Premature stop at codon 204 Premature stop at codon 514 (GA) 3 n.d. (T) 4 + RMS3 None - n.a. + BS1 MSH2 exon 9 Premature stop at (A) + 2 c.1444dela codon 483 BS2 None - n.a. + PD7 MSH2 exon 1 Premature stop at (G) + 4 c.119delg codon 63 BO1 MLH1 exon 4 n.a. n.d. c.350c>t Thr117Met 1 n.a.: not applicable; 2 n.d.: not determined. Novel mutations in boldface type.

3 MSH2 and MLH1 Frameshift Mutations 3 Figure 1. Pedigree of family RMS1. Co: colorectal cancer; En: endometrial cancer; OS: osteosarcoma; Ad: colonic adenoma; Bl: bladder carcinoma; Br: breast carcinoma; Co40: colorectal cancer diagnosed at 40 years of age; d96: dead at 96 years; *: tested for MLH1 mutation; c: carrier; nc: not carrier; the arrow indicates the proband through whom the family was ascertained. Tumor tissue for MSI analysis was available from 5 of the 7 HNPCC probands. Instability at >2 microsatellite loci was found in all 5 CRC samples examined. MSI analysis was also performed on a CRC and an infiltrating ductal breast carcinoma (BC) specimen from individual IV-3 in family RMS2 (Fig. 1), who is a carrier of the c inst mutation. MSI was present in the CRC, but not in the BC from this patient. These results further confirm that small deletions/insertions represent a very frequent mutational mechanism within the MLH1 and MSH2 genes underlying cancer predisposition in the context of HNPCC. The overrepresentation of specific types of constitutional defects, such as frameshift and nonsense changes, among all mutations identified within MMR genes is explained by selection for their inactivating effects on protein function. An additional reason for such high frequency could lie in the relatively high likelihood of occurrence of specific mutational events. In fact, it has been shown that the mutational mechanism most often underlying nonsense mutations is represented by C>T (or, conversely, G>A) transitions at CpG dinucleotides, which likely result from deamination of methylated cytosine residues (Maliaka et al. 1996). Moreover, large MLH1 or MSH2 genomic deletions, which account for a relevant fraction of constitutional defects in some populations, are driven by Alumediated recombination (Nyström-Lahti et al. 1995; Mauillon et al. 1996; Wijnen et al. 1998). In this regard, it is interesting to note that all of the novel small deletions/insertions identified in this study occur within small stretches of repeated mono- or dinucleotides (Table 1). Likewise, in reviewing the HNPCC mutation database ( nfdht.nl/database), we observed that >50% of MLH1/MSH2 1-2 bp insertions/deletions involve simple coding repetitive tracts. Thus, the nature and location of such small frameshift mutations indicate that they are the consequence of spontaneous DNA polymerase slippage during replication. On the other hand, larger (>4 bp) insertions/deletions must be caused by different mechanisms, since they are usually not located within repetitive tracts, as inferred from the sequence context of mutations reported in the International Collaborative Group on Hereditary NonPolyposis Colorectal Cancer database ( nfdht.nl/database).

4 4 Caluseriu et al. Although BC is occasionally described in HNPCC pedigrees, it has yet to be established whether and how frequently its development is attributable to MMR gene defects segregating in these families. Anedoctical evidence for a causal relationship between BC and MLH1 constitutional mutations has been provided for both female and male BC, based on the observation of MSI in tumor specimens from 2 mutation carriers (Risinger et al. 1996; Boyd et al. 1999). However, the low frequency of MSI usually detected in BC (Jonsson et al. 1995; Anbazhagan et al. 1999), combined with data showing that the overall incidence of BC is not significantly increased in HNPCC (Aarnio et al. 1999; Vasen et al. 1999), suggests that the majority of BCs recorded in HNPCC pedigrees may represent phenocopies unrelated to the presence of MMR gene mutations. Furthermore, analysis of an HNPCC pedigree showing an excess of both male and female BCs has shown the presence of both a BRCA1 and an MLH1 mutation (Borg et al. 2000). In agreement with these findings, the BC sample from an MLH1 mutation carrier analyzed in this study did not show MSI. However, in order to better define the role played by MMR defects in the pathogenesis of HNPCC-associated BCs, it will be necessary to perform MSI and/or immunohistochemical investigations on larger sets of samples. REFERENCES Aarnio M, Sankila R, Pukkhala E, Salovaara R, Aaltonen LA, de la Chapelle A, Peltomäki P, Mecklin JP, Järvinen HJ Cancer risk in mutation carriers of DNA-mismatch repair genes. Int J Cancer 12: Anbazhagan R, Fujii H, Gabrielson E Microsatellite instability is uncommon in breast cancer. Clin Cancer Res 5: Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, Meltzer SJ, Rodriguez-Bigas MA, Fodde R, Ranzani GN, Srivastava S A National Cancer Institute Workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 58: Borg Å, Isola J, Chen, J, Rubio C, Johansson U, Werelius B, Lindblom A Germline BRCA1 and HMLH1 mutations in a family with male and female breast carcinoma. Int J Cancer 85: Boyd J, Rhei E, Federici MG, Borgen PI, Watson P, Franklin B, Karr B, Lynch J, Lemon SJ, Lynch HT Male breast cancer in the hereditary nonpolyposis colorectal cancer syndrome. Breast Cancer Res Treat 53:87-91 Genuardi M, Anti M, Capozzi E, Leonardi F, Fornasarig M, Novella E,,Bellacosa A, Valenti A, Gasbarrini GB, Roncucci L, Benatti P, Percesepe A, Ponz de Leòn M, Coco C, De Paoli A, Valentini M, Boiocchi M, Neri G, Viel A MLH1 and MSH2 constitutional mutations in colorectal cancer families not meeting the standard criteria for hereditary nonpolyposis colorectal cancer. Int J Cancer 75: Genuardi M, Carrara S, Anti M, Ponz de Leòn M, Viel A Assessment of pathogenicity criteria for constitutional missense mutations of the hereditary nonpolyposis colorectal cancer genes MLH1 and MSH2. Eur J Hum Genet 7: Jonsson M, Johansson O, Borg A Infrequent occurrence of microsatellite instability in sporadic and familial breast cancer. Eur J Cancer 31A : Liu B, Parsons R, Papadopoulos N, Nicolaides NC, Lynch HT, Watson P, Jass J., Dunlop M, Wyllie A, Peltomäki P, de la Chapelle A, Hamilton SR, Vogelstein B Kinzler KW Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nature Med 2: Maliaka YK, Chudina AP, Belev NF, Alday P, Bochkov NP, Buerstedde JM CpG dinucleotides in the hmsh2 and hmlh1 genes are hotspots for HNPCC mutations. Hum Genet 97:251-5 Mauillon JL, Michel P, Limacher J-M, Latouche J-B, Dechelotte P, Charbonnier F, Martin C, Moreau V, Metayer J, Paillot B, Frebourg T Identification of novel germline hmlh1 mutations including a 22 kb Alu-mediated deletion in patients with familial colorectal cancer. Cancer Res 56: Nyström-Lahti M, Kristo P, Nicolaides NC, Chang SY, Aaltonen LA, Moisio AL, Jarvinen HJ, Mecklin JP, Kinzler KW, Vogelstein B, de la Chapelle A, Peltomäki P Founding mutations and Alu-mediated recombination in hereditary colon cancer. Nature Med 1:1203

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