‘‘Second Hit’’ in Sebaceous Tumors from Muir–Torre Patients with Germline Mutations in MSH2: Allele Loss is Not the Preferred Mode of Inactivation
2001; Elsevier BV; Volume: 116; Issue: 3 Linguagem: Inglês
10.1046/j.1523-1747.2001.01265.x
ISSN1523-1747
AutoresRoland Kruse, Thomas Ruzicka, Arno Rütten, Hamid Reza Hosseiny–Malayeri, Michele Bisceglia, Waltraut Friedl, Peter Propping, Elisabeth Mangold,
Tópico(s)Cancer Diagnosis and Treatment
ResumoMuir–Torre syndrome is an autosomal-dominant inherited disorder predisposing to both sebaceous skin tumors and internal neoplasms. In a significant proportion of Muir–Torre syndrome patients skin tumors exhibit microsatellite instability as a hallmark of hereditary nonpolyposis colorectal cancer. Most individuals predisposed to hereditary nonpolyposis colorectal cancer harbor a germline mutation in the DNA mismatch repair genes MSH2 or MLH1. In Muir–Torre syndrome the vast majority of germline mutations have been identified in MSH2. Microsatellite instability in tumor tissue develops after somatic inactivation of the corresponding second mismatch repair allele (‘‘second hit’'). So far, the mechanisms of somatic inactivation of the second allele in microsatellite instability positive tumors from patients with known mismatch repair germline mutations are not well understood. We examined whether allele loss (loss of heterozygosity) is a frequent mechanism for inactivation of the second MSH2 allele in a sample of nine microsatellite instability positive skin tumors from eight unrelated Muir–Torre patients with known MSH2 germline mutations. Loss of heterozygosity was determined using microsatellite markers or heteroduplex analysis, respectively. Only one of the nine skin tumors exhibited loss of heterozygosity at the MSH2 locus. Thus, we could show in a sample of sebaceous tumors from patients with genetically proven Muir–Torre syndrome that loss of heterozygosity most probably is not the preferred mode of somatic inactivation of the second MSH2 allele. Muir–Torre syndrome is an autosomal-dominant inherited disorder predisposing to both sebaceous skin tumors and internal neoplasms. In a significant proportion of Muir–Torre syndrome patients skin tumors exhibit microsatellite instability as a hallmark of hereditary nonpolyposis colorectal cancer. Most individuals predisposed to hereditary nonpolyposis colorectal cancer harbor a germline mutation in the DNA mismatch repair genes MSH2 or MLH1. In Muir–Torre syndrome the vast majority of germline mutations have been identified in MSH2. Microsatellite instability in tumor tissue develops after somatic inactivation of the corresponding second mismatch repair allele (‘‘second hit’'). So far, the mechanisms of somatic inactivation of the second allele in microsatellite instability positive tumors from patients with known mismatch repair germline mutations are not well understood. We examined whether allele loss (loss of heterozygosity) is a frequent mechanism for inactivation of the second MSH2 allele in a sample of nine microsatellite instability positive skin tumors from eight unrelated Muir–Torre patients with known MSH2 germline mutations. Loss of heterozygosity was determined using microsatellite markers or heteroduplex analysis, respectively. Only one of the nine skin tumors exhibited loss of heterozygosity at the MSH2 locus. Thus, we could show in a sample of sebaceous tumors from patients with genetically proven Muir–Torre syndrome that loss of heterozygosity most probably is not the preferred mode of somatic inactivation of the second MSH2 allele. DNA mismatch repair microsatellite instability Muir–Torre syndrome Muir–Torre syndrome (MTS) (OMIM 158320) is a rare autosomal-dominant inherited disorder predisposing to both skin tumors (especially sebaceous gland tumors) and internal neoplasms (mostly colorectal carcinomas) (Schwartz and Torre, 1995Schwartz R.A. Torre D.P. The Muir–Torre syndrome: a 25-year retrospect.J Am Acad Dermatol. 1995; 33: 90-104Abstract Full Text PDF PubMed Scopus (323) Google Scholar). In a high proportion of MTS patients, skin tumors exhibit microsatellite instability (MSI) (Honchel et al., 1994Honchel R. Halling K.C. Schaid D.J. Pittelkow M. Thibodeau S.N. Microsatellite instability in Muir–Torre syndrome.Cancer Res. 1994; 54: 1159-1163PubMed Google Scholar), a characteristic feature of tumors related to hereditary nonpolyposis colorectal cancer (OMIM 120435 and 120436). Most hereditary nonpolyposis colorectal cancer patients harbor a germline mutation in one of the DNA mismatch repair (MMR) genes MSH2 or MLH1. In MTS the vast majority of germline mutations (19 of 22 published worldwide) have been identified in MSH2 (Kolodner et al. 1994;Liu et al., 1994Liu B. Parsons R.E. Hamilton S.R. et al.hMSH2 mutations in hereditary non-polyposis colorectal cancer kindreds.Cancer Res. 1994; 54: 4590-4594PubMed Google Scholar;Kruse et al., 1996Kruse R. Lamberti C. Wang Y. et al.Is the mismatch repair deficient type of Muir–Torre syndrome confined to mutations in the hMSH2 gene?.Hum Genet. 1996; 98: 747-750Crossref PubMed Scopus (57) Google Scholar,Kruse et al., 1998Kruse R. Rütten A. Lamberti C. et al.Muir–Torre phenotype has a frequency of DNA mismatch-repair-gene mutations similar to that in hereditary nonpolyposis colorectal cancer families defined by the Amsterdam criteria.Am J Hum Genet. 1998; 63: 63-70Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar;Esche et al., 1997Esche C. Kruse R. Lamberti C. Friedl W. Propping P. Lehmann P. Ruzicka T. Muir–Torre syndrome: Clinical features and molecular genetic analysis.Br J Dermatol. 1997; 136: 913-917Crossref PubMed Scopus (29) Google Scholar;Bapat et al., 1999Bapat B.V. Madlensky L. Temple L.K. et al.Family history characteristics, tumor microsatellite instability and germline MSH2 and MLH1 mutations in hereditary colorectal cancer.Hum Genet. 1999; 104: 167-176Crossref PubMed Scopus (86) Google Scholar;Doré et al., 1999Doré M.X. Dieumegard B. Grandjouan S. et al.Muir–Torre syndrome and familial colorectal cancer: two families with molecular genetic analysis.Ann Dermatol Venereol. 1999; 126: 582-586PubMed Google Scholar;Godard et al., 1999Godard V. Coulet F. Bernaudin J.F. Housset M. Soubrier F. MSH2 mutation in Muir–Torre syndrome.Ann Dermatol Venereol. 1999; 126: 600-603PubMed Google Scholar;Lynch et al., 1999Lynch H.T. Leibowitz R. Smyrk T. et al.Colorectal cancer and the Muir–Torre syndrome in a Gypsy family: a review.Am J Gastroenterol. 1999; 94: 575-580Crossref PubMed Google Scholar;Rütten et al., 1999Rütten A. Burgdorf W. Hügel H. et al.Cystic sebaceous tumors as marker lesions for the Muir–Torre syndrome. A histopathological and molecular genetic study.Am J Dermatopathol. 1999; 21: 405-413Crossref PubMed Scopus (78) Google Scholar;Caldes et al., 2000Caldes T. Godino J. Perez-Segura P. de la Hoya M. Diaz-Rubio E. Benito M. New mutation in the hMSH2 gene in a Spanish Muir–Torre syndrome.Am J Gastroenterol. 2000; 95: 2389-2390Crossref PubMed Google Scholar; this report), whereas only three mutations were found in MLH1 (Bapat et al., 1996Bapat B. Xia L. Madlensky L. Mitri A. Tonin P. Narod S.A. Gallinger S. The genetic basis of Muir–Torre syndrome includes the hMLH1 locus.Am J Hum Genet. 1996; 59: 736-739PubMed Google Scholar;Kruse et al., 1998Kruse R. Rütten A. Lamberti C. et al.Muir–Torre phenotype has a frequency of DNA mismatch-repair-gene mutations similar to that in hereditary nonpolyposis colorectal cancer families defined by the Amsterdam criteria.Am J Hum Genet. 1998; 63: 63-70Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar,Kruse et al., 1999Kruse R. Rütten A. Hosseiny Malayeri H.R. Günzl H.J. Friedl W. Propping P. A novel germline mutation in the hMLH1 DNA mismatch repair gene in a patient with an isolated cystic sebaceous tumor.J Invest Dermatol. 1999; 112: 117-118Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar;Rütten et al., 1999Rütten A. Burgdorf W. Hügel H. et al.Cystic sebaceous tumors as marker lesions for the Muir–Torre syndrome. A histopathological and molecular genetic study.Am J Dermatopathol. 1999; 21: 405-413Crossref PubMed Scopus (78) Google Scholar). Fifteen of 19 MSH2 germline mutations and all three MLH1 germline mutations are either frameshift or non-sense mutations, leading to truncated MMR gene products. It was considered that in tumors MSI develops after somatic inactivation of the corresponding second MMR allele (‘‘second hit’'), analogous to inactivation of tumor suppressor function in hereditary tumor predisposition syndromes, e.g., neurofibromatosis or tuberous sclerosis (Green et al., 1994Green A.J. Smith M. Yates J.R. Loss of heterozygosity on chromosome 16p13.3 in hamartomas from tuberous sclerosis patients.Nat Genet. 1994; 6: 193-196Crossref PubMed Scopus (319) Google Scholar;Colman et al., 1995Colman S.D. Williams C.A. Wallace M.R. Benign neurofibromas in type 1 neurofibromatosis (NF1) show somatic deletions of the NF1 gene.Nat Genet. 1995; 11: 90-92Crossref PubMed Scopus (191) Google Scholar;Kluwe et al., 2000Kluwe L. Friedrich R.E. Hagel C. Lindenau M. Mautner V.F. Mutations and allelic loss of the NF2 gene in neurofibromatosis 2-associated skin tumors.J Invest Dermatol. 2000; 114: 1017-1021Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). According to Knudson's ‘‘two-hit’' hypothesis (Knudson, 1971Knudson A.G. Mutation and cancer. Statistical study of retinoblastoma.Proc Natl Acad Sci USA. 1971; 68: 820-823Crossref PubMed Scopus (5201) Google Scholar) in hereditary tumors one allele of a tumor suppressor gene is constitutionally inactivated whereas the second allele is subsequently inactivated (‘‘second hit’') at the somatic level. In tumor suppressor genes the ‘‘second hit’' is frequently accomplished by a large deletion encompassing the wild-type allele [loss of heterozygosity (LOH)]. So far, there are only few studies about mechanisms of somatic inactivation of the second allele in MSI positive tumors from patients with known MMR germline mutations. This study was performed in a sample of MSI positive skin tumors from MTS patients with known MSH2 germline mutations. The aim of this study was to examine whether LOH is the preferred mechanism for inactivation of the second MSH2 allele in MTS related skin tumors. We selected nine skin tumors with high MSI from eight unrelated MTS patients with known germline mutations in MSH2 (see Table 1). Patient 62 is a 34 y old otherwise healthy female from an hereditary nonpolyposis colorectal cancer kindred, diagnosed with a sebaceous carcinoma of the skull. Clinical data of the other seven patients are described in detail elsewhere (Kruse et al., 1998Kruse R. Rütten A. Lamberti C. et al.Muir–Torre phenotype has a frequency of DNA mismatch-repair-gene mutations similar to that in hereditary nonpolyposis colorectal cancer families defined by the Amsterdam criteria.Am J Hum Genet. 1998; 63: 63-70Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar).Table IExamination of nine MSI positive skin tumors from eight MTS patients with known MSH2 germline mutations for loss of heterozygosity at the MSH2 locusaMSI, Microsatellite instability (unstable markers/all examined markers); MM, Amplification of microsatellite markers; HD, heteroduplex analysis; –, no loss of heterozygosity; +, loss of heterozygosity.PatientMSH2 germline mutationSkin tumorMSIMethod for LOH assessmentLOH at MSH2130Exon 2: 289ins22bpSebaceous adenoma4/6HD–130Exon 2: 289ins22bpSebaceous epithelioma4/4HD–MTS-K10Exon 3: 380delATSebaceous epithelioma2/6MM+MTS-K2Exon 10: 1576delASebaceous epithelioma5/6HD–132Exon 11: 1676delTCystic sebaceous adenoma6/7HD–167Exon 11: 1699delAAACASquamous cell carcinoma (keratoacanthoma type)4/7HD–133Exon 12: 1809delTCystic sebaceous adenoma4/5HD–MTS-K8Exon 13: 2015delTSebaceous adenoma7/9MM–62Exon 13: C697FSebaceous carcinoma5/6MM–a MSI, Microsatellite instability (unstable markers/all examined markers); MM, Amplification of microsatellite markers; HD, heteroduplex analysis; –, no loss of heterozygosity; +, loss of heterozygosity. Open table in a new tab Ethylenediamine tetraacetic acid anticoagulated blood samples were taken from all MTS patients who had given informed consent. Normal DNA was extracted from the peripheral blood by salting out (Miller et al., 1988Miller S.A. Dykes D.D. Polesky H.F. A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res. 1988; 16: 1215Crossref PubMed Scopus (17177) Google Scholar). Tumor DNA was extracted from microdissected paraffin-embedded tumor tissue by use of the QIAamp tissue kit (Qiagen, Hilden, Germany). In one part of the tumor DNA we assessed LOH by examining the DNA from tumors and corresponding normal tissue with three microsatellite markers located within a region of about 4 cM, which includes the MSH2 gene on chromosome 2p16 (D2S2378 and D2S2240 proximal of MSH2, D2S123 distal of MSH2 according to the Marshfield map: http://research.marshfieldclinic.org/genetics). One primer of each primer pair was labeled with ABI fluorescence dye. Polymerase chain reaction (PCR) products were separated by electrophoresis on an ABI 377 automated sequencer (Applied Biosystems, Weiterstadt, Germany). Allele loss was scored comparing fluorescence signals of tumor DNA with DNA from normal tissue. Both PCR and electrophoresis steps were performed twice. As an alternative method for LOH assessment we used heteroduplex analysis in tumor DNA on PCR fragments harboring the known MSH2 germline mutation. In detail, for PCR amplification of MSH2 fragments from tumor DNA we used the primer pairs and PCR conditions described byKolodner et al., 1994Kolodner R.D. Hall N.R. Lipford J. et al.Structure of the human MSH 2 locus and analysis of two Muir–torre kindreds for MSH 2 mutations.Genomics. 1994; 24: 516-526Crossref PubMed Scopus (266) Google Scholar. Nonisotopic heteroduplex analyses on nondenaturing 10% polyacrylamide gels were performed as described elsewhere (Mandl et al., 1994Mandl M. Paffenholz R. Friedl W. Caspari R. Sengteller M. Propping P. Frequency of common and novel inactivating APC mutations in 202 families with familial adenomatous polyposis.Hum Mol Genet. 1994; 3: 181-184Crossref PubMed Scopus (59) Google Scholar). Each reaction was performed twice. We assessed LOH at the MSH2 locus in a sample of nine MSI positive skin tumors from eight unrelated MTS patients with known MSH2 germline mutations (Table 1). We examined LOH in three of the nine tumors using the highly polymorphic microsatellite markers D2S123, D2S2378, and D2S2240 closely flanking the MSH2 region (see Figure 1). All three patients were heterozygous for these markers. Only one of the tumors (a sebaceous epithelioma of patient MTS-K10) showed LOH with marker D2S123 (Figure 1). In six tumors from five MTS patients we used heteroduplex analysis as an alternative method for assessing LOH in MSH2. This method has been successfully used in hereditary breast cancer (Mansukhani et al., 1997Mansukhani M.M. Nastiuk K.L. Hibsboosh H. Kularatne P. Russo D. Krolewski J.J. Convenient, nonradioactive, heteroduplex-based methods for identifying recurrent mutations in the BRCA1 and BRCA2 genes.Diagn Mol Pathol. 1997; 6: 229-237Crossref PubMed Scopus (7) Google Scholar). We examined all six tumor DNA by heteroduplex analysis of the exons harboring the germline mutation and obtained the same strong heteroduplex bands that were seen in corresponding normal DNA (Figure 2). These observations demonstrate that two alleles are present in all six tumor tissues. Taken together, only one of the nine skin tumors showed loss of the second allele at the MSH2 locus whereas in eight of nine skin tumors from MSH2-deficient MTS patients the second MSH2 allele is still present (Table 1). In tumorigenesis the ‘‘second hit’' in tumor suppressor genes is often accomplished by a deletion of the second allele (LOH). The present results suggest that in sebaceous tumors from MTS patients with germline MSH2 mutations, somatic inactivation of the second MSH2 allele by LOH is a rare event. So far, there are only few studies on the mechanisms of somatic inactivation of the second allele in MSI positive visceral tumors from patients with known MMR germline mutations. These studies led to conflicting results.Lu et al., 1996Lu S.L. Akiyama Y. Nagasaki H. et al.Loss or somatic mutations of hMSH2 occur in hereditary nonpolyposis colorectal cancers with hMSH2 germline mutations.Jpn J Cancer Res. 1996; 87: 279-287Crossref PubMed Scopus (39) Google Scholar detected LOH of the MSH2 gene in four and somatic mutations in one among eight colorectal carcinomas of patients with MSH2 germline mutations.Tannergård et al., 1997Tannergård P. Liu T. Weger A. Nordenskjold M. Lindblom A. Tumorigenesis in colorectal tumors from patients with hereditary non-polyposis colorectal cancer.Hum Genet. 1997; 101: 51-55Crossref PubMed Scopus (69) Google Scholar showed that the inactivation of the wild-type allele of MLH1 in six of seven colorectal carcinomas from patients with known MLH1 germline mutations occurred through LOH and not through a somatic point mutation.Wang et al., 1997Wang Y. Friedl W. Lamberti C. Ruelfs C. Kruse R. Propping P. Hereditary nonpolyposis colorectal cancer: causative role of a germline missense mutation in the hMLH1 gene confirmed by independent occurrence of the same somatic mutation in tumour tissue.Hum Genet. 1997; 100: 362-364Crossref PubMed Scopus (13) Google Scholar identified the same somatic mis-sense MLH1 mutation in colorectal carcinomas from two hereditary nonpolyposis colorectal cancer patients with different MLH1 germline mutations. Analyses for LOH at the MSH2 and MLH1 loci in sporadic colorectal carcinomas with MSI were performed byTomlinson et al., 1996Tomlinson I.P. Ilyas M. Bodmer W.F. Allele loss occurs frequently at hMLH1, but rarely at hMSH2, in sporadic colorectal cancers with microsatellite instability.Br J Cancer. 1996; 74: 1514-1517Crossref PubMed Scopus (35) Google Scholar. They demonstrated that LOH at MSH2 is a rare event in sporadic colorectal carcinomas: only two of 22 (9%) tumors showed LOH at MSH2, whereas LOH at MLH1 was found in seven of 17 (41%) colorectal carcinomas. LOH studies on MSI positive sporadic colorectal carcinomas performed byBenachenhou et al., 1998Benachenhou N. Guiral S. Gorska-Flipot I. Michalski R. Labuda D. Sinnett D. Allelic losses and DNA methylation at DNA mismatch repair loci in sporadic colorectal cancer.Carcinogenesis. 1998; 19: 1925-1929Crossref PubMed Scopus (18) Google Scholar revealed hemizygous deletions at both MMR loci only rarely, with a similar proportion of MLH1 and MSH2: in six of 35 (17%) informative cases at MLH1 and in four of 27 (15%) at MSH2. In contrast to our study, however, including only patients with known MSH2 germline mutations, the frequency of MSH2 germline mutations had not been assessed in these studies. To date, only MSI positive sebaceous tumors from MTS patients with unknown MMR germline mutations have been examined for the presence of LOH (Peris et al., 1997Peris K. Onorati M.T. Keller G. et al.Widespread microsatellite instability in sebaceous tumours of patients with the Muir–Torre syndrome.Br J Dermatol. 1997; 137: 356-360Crossref PubMed Scopus (36) Google Scholar). These authors reported LOH at D2S119, a marker located in the vicinity of MSH2, in two of six sebaceous tumors from two MTS patients. In summary, we examined nine sebaceous tumors from eight unrelated MTS patients with known MSH2 germline mutations for LOH at the MSH2 locus. All the tumors exhibited MSI. Only one of nine sebaceous tumors showed LOH. Thus, we demonstrated, in a sample of sebaceous tumors from patients with genetically proven MTS, that LOH is not the preferred mode of somatic inactivation of the second MSH2 allele. We thank Marlies Sengteller and Nadine Schweiger for excellent technical assistance. The project was supported by grants of the Deutsche Forschungsgemeinschaft (KR 1620/3–1) and Deutsche Krebshilfe (70–2030-PR4).
Referência(s)