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An Interstitial Deletion at 3P21.3 Results in the Genetic Fusion of MLH1 and ITGA9 in a Lynch Syndrome Family

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An Interstitial Deletion at 3P21.3 Results in the Genetic Fusion of MLH1 and ITGA9 in a Lynch Syndrome Family Human Cancer Biology An Interstitial Deletion at 3p21.3 Results in the Genetic Fusion of MLH1 and ITGA9 in a Lynch Syndrome Family Claus Meyer,1Angela Brieger,2 Guido Plotz,2 Nicole Weber,2 Sandra Passmann,2 Theo Dingermann,1 Stefan Zeuzem,2 Joerg Trojan,2 and Rolf Marschalek1 Abstract Purpose: Germline mutations in DNA mismatch repair genes, mainly MLH1 or MSH2, have been shown to predispose with high penetrance for the development of the clinical phenotype of hereditary nonpolyposis colorectal cancer (Lynch syndrome). Here, we describe the discovery and first functional characterization of a novel germline MLH1 mutant allele. Experimental Design: A large kindred including 54 potential carriers was investigated at the molecular level by using different types of PCR experiments, gene cloning, transfection studies, Western blot experiments, and mismatch repair assays to identify and characterize a novel MLH1 mutant allele.Twenty-two of 54 putative carriers developed colon cancer or other tumors, includ- ing breast cancer. Results:The identified MLH1 mutant allele emerged from an interstitial deletion on chromosome 3p21.3, leading to an in-frame fusion of MLH1 (exons 1-11) with ITGA9 (integrin a 9; exons 17-28).The deleted area has a size of about 400 kb; codes for LRRFIP2 (leucine-rich repeat in flightless interaction protein 2), GOLGA4 (Golgi autoantigen, golgin subfamily a, 4), and C3orf35/APRG1 (chromosome 3 open reading frame 35/AP20 region protein 1); and partly dis- rupts the AP20 region implicated in major epithelial malignancies. Tumor cells lost their second MLH1 allele. The MLH1.ITGA9 fusion protein provides no capability for DNA mismatch repair. Murine fibroblasts, expressing a doxycycline-inducible MLH1.ITGA9 fusion gene, exhibit a loss ^ of ^ contact inhibition phenotype. Conclusions: This is the first description of a functional gene fusion of the human MLH1 gene, resulting in the loss of mismatch repair capabilities.The MLH1.ITGA9 fusion allele, together with deletions of the AP20 region, presumably defines a novel subclass of Lynch syndrome patients, which results in an extended tumor spectrum known from hereditary nonpolyposis colorectal cancer and Muir-Torre syndrome patients. About 3% to 5% of all colorectal cancer cases in humans can dominant disorder, is caused by heterozygous defects in be classified as hereditary nonpolyposis colorectal cancer mismatch repair genes, mainly MLH1 (f50%) and MSH2 (Lynch syndrome). The Lynch syndrome, an autosomal- (f40%), also MSH6 or PMS2, and responsible for the early onset of colorectal and endometrial cancer (1). In addition, patients with Lynch syndrome are at increased risk to develop urinary tract, stomach, and small intestine cancer (2). Muir- Authors’ Affiliations: 1Institute of Pharmaceutical Biology/Diagnostic Center for Acute Leukemia/Zentrum fu« r Arzneimittelforschung, Entwicklung und Sicherheit/ Torre syndrome, a phenotypic subvariant of Lynch syndrome, is Cluster of Excellence Frankfurt, Biocenter, and 2Department of Internal Medicine I, characterized by the association between one or more visceral Goethe-University Frankfurt, Frankfurt/Main, Germany malignancies and sebaceous tumors or keratoacanthomas. The Received 7/24/08; revised 10/16/08; accepted 10/26/08. Muir-Torre phenotype is not linked to particular mismatch Grant support: Gisela Stadelmann-Stiftung (A. Brieger and J. Trojan); grants 2007.030.1 (G. Plotz and S. Zeuzem) and 2005.085.1 (A. Brieger and J.Trojan) of repair gene alterations; hence, the Muir-Torre syndrome the Wilhelm Sander-Stiftung; and Deutsche Forschungsgemeinschaft grant families present a wide phenotypic and genotypic variability MA1876/7-1, Bundesministerium fu« r Bildung und Forschung grant N1KR-S12T13, (3, 4). and Deutsche Krebshilfe grant 107819 (R. Marschalek, also a principal investigator Mismatch repair deficiencies are either caused by point within the Cluster of Excellence Frankfurt on Macromolecular Complexes funded by Deutsche Forschungsgemeinschaft grant EXC115). mutations, deletions (5, 6), or epigenetic processes, for The costs of publication of this article were defrayed in part by the payment of page example, silencing of specific mismatch repair promoters due charges. This article must therefore be hereby marked advertisement in accordance to DNA methylation (7). Absence of key proteins or the with 18 U.S.C. Section 1734 solely to indicate this fact. presence of mutated repair proteins impairs the formation of Note: C. Meyer and A. Brieger contributed equally to this work. J.Trojan and R. Marschalek shared senior authorship. functional repair initiating complexes. MLH1 binds either to Requests for reprints: Rolf Marschalek, Institute of Pharmaceutical Biology, PMS1 or PMS2, and both heterodimers (MutLh and MutLa) Biocenter, Goethe-University Frankfurt, N230, 3.03, Max-von-Laue-Street 9, bind either to the heterodimers MSH2/MSH6 (MutSa; recog- D-60438 Frankfurt/Main, Germany. Phone: 49-69-798-29647; Fax: 49-69-798- nition of mispairs) or MSH2/MSH3 [MutSh; recognition of 29662; E-mail: [email protected]. F 2009 American Association for Cancer Research. DNA insertions/deletions up to 12 nucleotides (nt) in length]. doi:10.1158/1078-0432.CCR-08-1908 In mammalian cells, the association with proliferating cell Clin Cancer Res 2009;15(3) February 1, 2009 762 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. MLH1.ITGA9 Fusion Gene restriction polymorphic DNA fragments within the MLH1 exon 11/ Translational Relevance intron 11 region: MLH1.R4 (5¶-ACCTTAGATAGTGGGAGGGGGA- GAAAAAGC-3¶) was used in combination with either MLH1.F5 We have discovered a novel type of MLH1 mutation in a (5¶-AAGATTCATAAGTGGGTTCAGCTGGTGACA-3¶), MLH1.F4 large Lynch syndrome family: the human MLH1 mismatch (5¶-CCACACACTAAACAAGCAGTCAGTTCCAGA-3¶), MLH1.F3 (5¶- ¶ . ¶ repair gene (exons 1-11) is fused in-frame with the ITGA9 TGTTTGAGGATCTGAAAGTTGAGTGCAGTG-3 ), or MLH1 F2A (5 - ¶ (integrin a 9) gene (exon 17-28), located about 400 kb CAGTTAGGAAAGGGAGGGAAGAGACATGG-3 ). PCR amplimers were cut out from the gel and subjected to sequence analysis. downstream of MLH1 at 3p21. Established diagnostic The presence of the MLH1.ITGA9 allele was confirmed by using the methods for MLH1 mutations are not able to detect this following oligonucleotides: MLH1I11.3(5¶-GAGGAAGGGGAAGTGA- novel type of MLH1 mutation. Thus, all Lynch syndrome GAAATTGTTTAATGG-3¶) and ITGA9I16.5(5¶-CCAGTCAGTTATAG- patients that carry deletions of MLH1 extending beyond GATTAGGAGGGTAAAC-3¶). The amount of genomic DNA was the 5¶-or3¶-end of the gene might be diagnosed by confirmed using the MLL-specific oligonucleotides MLL.F7 (5- the method presented in this manuscript. Moreover, the TTGTGAGCCCTTCCACAAGTTTTGTTTAGAGG-3) and MLL.CR2 (5¶- established primer set to test for the presence of the GTCCCAGGCACTCAGGGTGATAGCTGTTTCGG-3¶). MLH1.ITGA9 fusion allele might be of prognostic value. The presence of all 19 MLH1 exons was analyzed using extracted We experimentally show that this genomic fusion is able DNA from paraffin embedded breast tumor and normal tissue of to produce the MLH1.ITGA9 fusion protein, the first mis- patient 25. PCRs for all 19 MLH1 exons were carried out using exon- specific primer combinations as previously published (17). match repair fusion protein ever described in the literature. The presence of MLH1 exon 11, LRRFIP2 (leucine-rich repeat in . Preliminary data show that the MLH1 ITGA9 fusion protein flightless interaction protein 2) exon 28, GOLGA4 (Golgi autoantigen, does not confer mismatch repair capability. golgin subfamily a, 4) exon 14, C3orf35/APRG1 (chromosome 3 open reading frame 35/AP20region protein 1) exon 6, and ITGA9 exon 16 was verified with the following oligonucleotides: MLH1ex11.3 . nuclear antigen (which interacts with MSH3 and 6), RPA, (5¶-GGGCTTTTTCTCCCCCTCCC-3¶), MLH1ex11 5(5¶- ¶ . ¶ exonuclease I, and DNA repair polymerases y and q is a crucial AAAATGTGGGCTCTCACG-3 ), LRRFIP2 F1 (5 -TACGAACAGCACTG- ¶ . ¶ ¶ step for subsequent repair processes (8). Moreover, the proteins GACAAGAT-3 ), LRRFIP2 R1 (5 -TAAAAGGGGTTTGTGTCAATGG-3 ), GOLGA4.F1 (5¶-CACCAGCAGCAAGTTGACAGTA-3¶), GOLGA4.R1 MLH1, MSH2, and MSH6 are part of the ‘‘surveillance and (5¶-TAGCAGATGCCTGCTGGAGTTC-3¶), APRG1.F1 (5¶-GACTGACT- repair complex’’ involving ATM, BLM helicase, BRCA1, BRCA2, CAATCCCAGCTGCT-3¶), APRG1.R1 (5¶-TGCCAGAGCAATGCTGACC- RAD50, MRE11, NBS1, Ku70, KU86, DNAPKcs, and RFC (9). CAG-3¶), ITGA9.F1 (5¶- ATCGTGTTTGAAGCAGCCTACA-3¶), and Therefore, many MLH1 and MSH2 mutations compromise the ITGA9.R1 (5¶- TTTGGGCCATTATTTCTGCTCT-3¶). assembly of different protein complexes and result in the Cloning and expression of MLH1, ITGA9, and MLH1.ITGA9. The inability to cope with genotoxic stress situations. Mismatch pcDNA3.1+ expression vector (Invitrogen) containing the entire open repair deficiency causes the accumulation of numerous reading frame of MLH1 was a gift of Dr. Hong Zhang (Huntsman mutations in repetitive DNA sequences, leading to micro- Cancer Institute, University of Utah, Salt Lake City, UT). cDNAs of . satellite instability, which is typically found in tumors of ITGA9 and MLH1 ITGA9 were amplified from peripheral mononuclear patients with Lynch syndrome (10). cells of a healthy volunteer or family member 25, respectively. The obtained cDNA amplimers were cloned into the pcDNA3.1+ expression The ITGA9 (integrin a 9) gene codes for a member of the plasmid. Orientation and
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