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, including 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 intact open reading frames were verified by integrin receptor family. Integrins form heterodimeric integral sequence analyses. a membrane glycoproteins and are composed of an - and a Cell culture, transfection, and Western blot experiments. HEK 293T h-chain that mediate cell-cell and cell-matrix adhesion (11). cells, obtained from Dr. Kurt Ballmer (Paul Scherer Institute, Villingen, Integrin a 9 protein binds to integrin h 1 and thus forms a Switzerland), were grown in DMEM with 10% FCS. Transient trans- receptor for VCAM1, cytotactin, and osteopontin (12), also for fections were carried out using Gene Juice Transfection Reagent [Merck vascular endothelial growth factors (13). Very high expression (Novagene) Schwalbach]. Total cell lysates (50 Ag) were used for of ITGA9 has been shown in small cell lung cancer, and Western blot experiments along with 200 Ag total protein isolated from recently, ITGA9 has been described as a candidate cancer gene peripheral mononuclear cells. For detection the following antisera were in breast and colorectal cancer (14, 15). used: anti-MLH1 (clone LS-C566) raised against amino acids 387 to 403 of human MLH1 (Lifespan Biosciences), anti-ITGA9 (clone 3E4) Here, we report on an in-frame gene fusion of MLH1 and raised against amino acids 785 to 887 from human ITGA9 (Abnova), ITGA9 in a Lynch syndrome family. This novel allele is unable and anti–h Actin (Sigma). to confer mismatch repair activity. Mismatch repair assay. Because MutLa is not expressed in HEK 293T cells (18), these cells were transiently transfected according to standard Materials and Methods procedures. Cells were harvested after 48 h; whole cell extracts, as well as nuclear extracts, were prepared; and mismatch repair reactions were Family pedigree. The family pedigree and all data related to familiar done in vitro as described (19). Briefly, a substrate plasmid bearing a cancer histories were collected by patient 25. After informed consent, G-T mismatch within an AseI restriction site and a 3¶-single-strand nick peripheral blood samples of family members 9, 15, 18, 20, 21, 23, 25, in 83-bp distance to the mismatch was used for in vitro DNA repair and 28 were collected and used to isolate genomic DNA for further assays. Subsequent digestion with AseI was used to assess mismatch analysis. repair efficiency. Restriction digests were separated on 2% agarose gels Long-distance inverse PCR and genomic PCR experiments. Long- and stained with ethidium bromide, and bands were quantified using distance inverse PCR experiments were done as recently described (16). Quantity One Software v4.6.1 (Bio-Rad). Genomic DNA was isolated from peripheral blood mononuclear cells. Focus formation experiments. Focus formation experiments have About 2 Ag genomic DNA was digested with SpeI and NheI. An aliquot been carried out using commercially available MEF/tTA cells (BD of 500 ng was religated in 150 AL and subjected to long-distance inverse Clontech) as published recently (20, 21). Stably transfected cell lines PCR analysis. Different oligonucleotides were used to identify expressing either empty vector, mutant RAS* (G6V mutation), or the

www.aacrjournals.org 763 Clin Cancer Res 2009;15(3) February 1,2009 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Human Cancer Biology

MLH1. ITGA9 fusion protein were established. Briefly, MEF/tTA cells gene, missing exons 12 to 19. The same deletion was verified by were grown in media containing 100 Ag/ml G418. Transfection of multiplex ligation-dependent probe amplification also in family . pTRE2puro, pTRE2puro::RAS*, and pTRE2puro::MLH1 ITGA9 was member 25, the daughter of family member 9. The latter family done using the FUGENE 6 Transfection Reagent (Roche). Selection member presented with a breast tumor at the age of 53, after she was started 48 h after transfection with 3 Ag/mL puromycin, and cells had already developed a sebaceous carcinoma at the age of 38. In were kept under selective conditions for a median time of 2 wks before single clones were isolated. Cell clones were tested by reverse addition, family member 25 was diagnosed to be positive for transcription-PCR for their ability to induce transgene expression microsatellite instability. The available pedigree suggested that upon doxycycline withdrawal using the following oligonucleotides: this MLH1 mutation represents a germline mutation that was RAS*. 3(5¶-ATGACAGAATACAAGCTTGTGG-3¶), RAS*. 5 transmitted over 5 generations (Fig. 1A). Twenty-two of 54 (5¶-GCCAGGTCACACTTGTTGCCCA-3¶), MLH1/ITGA9.3(5¶- potential allele carriers were affected from different tumors GAATGGTTACATATCCAATG-3¶), and MLH1/ITGA9.5(5¶-CCCAAAGC- involving endometrium, colon, liver, kidney, stomach, small TAGATACAGGGTTA-3¶). Transcriptional suppression of all transgenes intestine, pancreas, breast, brain, and sebaceous gland, or A was achieved by adding 10 g/mL doxycycline to the media. developed leukemia (Fig. 1B). . Mock-, MEF/tTA::RAS*-, and MLH1 ITGA9-transfected cell clones To characterize the genomic deletion in more detail, the were seeded in small Petri dishes (1 Â 104 cells) and grown in the MLH1 gene of family member 20was characterized at the presence or absence of doxycycline for at least 21 d. All cells were washed once with PBS and then fixed with a 2% formaldehyde molecular level using a long-distance inverse PCR strategy. solution for 2 mins. The dishes were rinsed with water and stained Because the genomic deletion was expected in vicinity of MLH1 with a 1 mg/mL methylene blue solution for 15 mins. Photographs exon 11, several oligonucleotides were designed to identify were taken from sections of selected Petri dishes (magnification, rearranged restriction fragments deriving from this genomic Â100). All experiments have been repeated in triplicates. region. As shown in Fig. 1C, three restriction polymorphic PCR amplimers (lanes a-c) were obtained in four different PCRs. The Results smallest PCR amplimer (lane a)wassubjectedtoDNA sequencing and revealed a genomic fusion between MLH1 Identification of the MLH1.ITGA9 fusion gene. A large family intron 11 (at nt 5099 of the 5173-nt-long intron) and intron 16 originating from French Guyana suffers from a variety of (at nt 4334 of the 30074-nt-long intron) of ITGA9 (Fig. 1D). different tumors. This family was diagnosed for the Lynch The fusion sequence contained a filler-DNA fragment of 13 nts syndrome after the detection of an MLH1 mutation in family that neither derived from MLH1 nor from ITGA9. Filler-DNA member 9 by multiplex ligation-dependent probe amplification. fragments at chromosomal fusion sites are a typical hallmark The mutation was characterized as a deletion within the MLH1 for nonhomologous end-joining DNA repair processes.

Fig. 1. Analysis of a Lynch syndrome family revealed a novel MLH1 mutation. A, family pedigree. Black symbols, tumor patients; white or gray symbols, all others (tested family members that carry the novel fusion allele). All family members with black symbols have already developed cancer, whereas family members 9,15,18, 20, 21,22, 23, 25, and 28 were genetically tested (asterisks, tested positive; open circle, tested negative). B, overview of all available cancer data. Numbers, family members in the family pedigree. Cancer type and age at diagnosis. C, long-distance inverse PCR identified the genomic MLH1.ITGA9 fusion. The MLH1.ITGA9 fusion allele (asterisks)was successfully amplified in three of four PCRs (lanes a-c).The sizes of PCR amplimers deriving from the mutant allele were3945, 5031,and 6171bp, respectively. M, size marker; D, sequence of the fusion site between MLH1 intron 11and ITGA9 intron 16. The underlined sequence in small letters represents a filler-DNA fragment that is neither encoded by the MLH1 nor by the ITGA9 gene.

Clin Cancer Res 2009;15(3) February 1, 2009 764 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. MLH1.ITGA9 Fusion Gene

Fig. 2. Schematic drawing of the interstitial deletion and genetic testing. A, interstitial deletion on 3p21.3. The deleted area codes for LRRFIP2, GOLGA4 ,and C3orf35/APRG1. MLH1 intron 11was fused to ITGA9 intron 16, leading to an in-frame gene fusion. Black arrows, test primers. B, genetic testing of available family members 9, 15, 18, 20, 21, 22, 23, 25, and 28. Genomic DNA was isolated from peripheral mononuclear cells; 7 of 9 tested members are carriers of the MLH1.ITGA9 fusion allele (left lanes). FA, fusion allele. Quality control of isolated genomic DNA was done by PCR on a portion of the human MLL gene (right lanes). CA, control allele.

Verification of the MLH1.ITGA9 fusion allele in other family Expression of the MLH1.ITGA9 fusion protein in peripheral members. The observed genetic recombination resulted in the mononuclear cells of affected family member 25. The genetic fusion of MLH1 exons 1 to 11 and ITGA9 exons 17 to MLH1.ITGA9 fusion allele is driven by the MLH1 promoter, 28. The gene fusion was indicative of an interstitial deletion at able to produce an MLH1.ITGA9 fusion mRNA, which can then 3p21.3 and encompasses an area of about 400 kb. The deleted be translated into the MLH1.ITGA9 fusion protein (Fig. 4A). area codes for three additional genes, LRRFIP2, GOLGA4, and The molecular weight of the fusion protein (85 kDa) is nearly C3orf35/APRG1, respectively (Fig. 2A). To verify these initial identical to that one of the wildtype MLH1 protein (84 kDa) but data, several members of the affected family were tested after differs from the ITGA9 protein (114 kDa). To verify the informed consent to confirm the presence of the MLH1.ITGA9 expression of the MLH1.ITGA9 fusion protein in somatic patient fusion allele. Genomic DNA from family members 9, 15, cells, MLH1, ITGA9, and the novel MLH1.ITGA9 fusion cDNA 18, 20, 21, 23, 25, and 28 was isolated from peripheral were cloned into an eukaryotic expression vector and transiently mononuclear cells and tested in genomic PCR experiments transfected into HEK 293T cells. All transfected cells (50 Ag total using two oligonucleotides specifically detecting the genomic protein) were analyzed together with peripheral mononuclear fusion between MLH1 intron 11 and ITGA9 intron 16. Seven of cells of family member 20and a healthy volunteer (200 Ag total 9 tested family members were positively diagnosed for the protein). As shown in Fig. 4B, Western blot experiments presence of the MLH1.ITGA9 fusion allele (Fig. 2B, leftlanes ). A confirmed the expression of recombinant ITGA9 (lane 2, 114 control PCR confirmed sufficient amounts of isolated genomic kDa), recombinant MLH1 (lane 3, 84 kDa), and the recombi- DNA (Fig. 2B, rightlanes ). Family members 9, 20, and 25 had nant MLH1.ITGA9 fusion protein (lane 4, 85 kDa). Peripheral already developed tumors (colon, endometrium, sebaceous mononuclear cells of patient 25 also revealed a weak expression gland, breast; marked with black symbols in Fig. 1A), whereas of the MLH1.ITGA9 fusion protein (lane 5, 85 kDa), also the family member 28 exhibits a benign keratoacanthoma since expression of MLH1 (lane 5, 84 kDa), whereas a healthy birth. Except family members 21 and 24 (marked with open volunteer showed only the expression of endogenous MLH1 circles in Fig. 1A), all other investigated family members bear protein but no expression of the ITGA9 protein in analyzed the identical MLH1.ITGA9 gene fusion (marked with asterisks peripheral mononuclear cells. Therefore, we concluded that the in Fig. 1A). This confirmed that the novel MLH1.ITGA9 allele MLH1.ITGA9 fusion gene is indeed expressed in somatic cells of is germline transmitted in this Lynch syndrome family. affected individuals. Minor differences in the migration of Deletion of the second MLH1 allele during tumor development. transfected and endogenous MLH1.ITGA9 fusion protein may For family member 25, a breast tumor biopsy sample (90% be due to (a) different amounts of protein used for the different tumor cells) was analyzed and compared with normal tissue lanes and (b) the expression of MLH1 and MHL1.ITGA9 that was of the same patient. Genomic PCR experiments revealed a simultaneously monitored in lane 5 when probed with an anti- complete loss of MLH1 exons 12 to 19 in the tumor biopsy MLH1 antibody. sample (Fig. 3A). Thus, we concluded that the second MLH1 MLH1.ITGA9 fusion protein is associated with mismatch repair allele was deleted in the tumor cells. To analyze this secondary deficiency. To analyze the MLH1.ITGA9 fusion protein for its event in more detail, another biopsy sample of the same tumor, mismatch repair capability, HEK 293T cells were transiently containing about 80% tumor cells, was analyzed for the transfected with expression vectors coding for PMS2 in homozygous deletion of LRRFIP2, GOLGA4,andC3orf35/ combination with either MLH1 or MLH1.ITGA9. Extracts of APRG1, respectively. As shown in Fig. 3B, MLH1 exon 11 these transfected cells were prepared and used for an in vitro (present in two copies in normal and tumor cells) served as single-bp mismatch repair assay. As shown in Fig. 5A, cells positive control. The other investigated genes were hemizygous expressing PMS2 and MLH1 are able to repair the single mispair in somatic cells (left), whereas tumor cells displayed the loss of of the substrate plasmid (faint band below linearized vector), all investigated genes (right). The observed faint PCR amplimers whereas cells expressing PMS2 and the MLH1.ITGA9 fusion presumably derived from contaminating normal cells (contain- protein were not able to repair the substrate accordingly. ing still a hemizygous set of all investigated genes). Therefore, we concluded that the MLH1.ITGA9 fusion protein

www.aacrjournals.org 765 Clin Cancer Res 2009;15(3) February 1,2009 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Human Cancer Biology

Fig. 3. Comparison of normal and tumor tissue. A, normal versus tumor tissue. Available DNA material of affected family member 25 was used to analyze the second MLH1 allele. Top, genomic DNA prepared from paraffin-embedded normal tissue; bottom, DNA extracted from breast tumor material. B, normal versus tumor tissue. Available DNA material of affected family member 25 was used to analyze for the presence of LRRFIP2, GOLGA4 ,andC3orf35/APRG1, respectively. PCR analysis of MLH1 exon 11 (300 bp), LRRFIP2 exon 28 (195 bp), GOLGA4 exon 14 (201bp), APRG1 exon 6 (202 bp), and ITGA9 exon16(192bp)fornormal(left) and tumor cells (right).

does not confer any mismatch repair capability. This is most seeded in small Petri dishes and grown for at least 21 days. likely due to its inability to bind to PMS1/2 (Fig. 4A). Medium was exchanged every 3 days. Mock- and untransfected Focus formation experiments. Stable cell lines transfected cells did not show any growth-transformed phenotype, whereas with empty vector, a mutant RAS* and MLH1.ITGA9 were loss of contact inhibition was observed in a doxycycline- grown either in the presence (no transgene expression) or dependent manner for RAS*- and MLH1.ITGA9-expressing absence of 10 Ag/ml doxycycline (transgene expression). As cells (right). The number of foci observed for MLH1.ITGA9 shown in Fig. 5B, both transgenes were induced upon overexpressing cells was roughly half of the amount of foci doxycycline withdrawal (left, lane f). A total of 104 cells were observed for RAS* expressing cells.

Fig. 4. Recombination sites within MLH1and ITGA9 and expression of the resulting fusion protein. A, precise localization of the chromosomal fusion sites within MLH1 and ITGA1 at 3p21.3(vertical line). Nt positions and certain exons of MLH1 (top)and ITGA9 (bottom) on chromosome 3.Wildtype MLH1, ITGA9,andtheMLH1.ITGA9 fusion alleles are in the central part. Protein domains and lengths. BD, binding domain. B, Western blot analysis of HEK 293Tcells, transiently transfected HEK 293Tcells, and peripheral mononuclear cells. Lane 1, HEK 293T cells; lane 2, HEK 293Tcells transfected with ITGA9 (114 kDa); lane 3, HEK 293Tcells transfected with MLH1 (84 kDa); lane 4, HEK 293Tcells transfected with MLH1.IITGA9 (85 kDa); lane 5, peripheral mononuclear cells of 25; lane 6, peripheral mononuclear cells of a healthy volunteer. Loading controls, h-actin. Left, molecular weight of comigrated marker proteins.

Clin Cancer Res 2009;15(3) February 1, 2009 766 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. MLH1.ITGA9 Fusion Gene

Fig. 5. Mismatch repair assay. A, mismatch repair assay. A substrate plasmid was incubated with nuclear extracts isolated from HEK 293Tcells, transiently transfected either with PMS2/MLH1 or PMS2/MLH1.ITGA9. Faint bands below the linearized vector, successful mismatch repair of the substrate plasmid. B, focus formation experiments. MEF/tTA cells transfected with empty vector (mock), the RAS G6V mutant (RAS*), or MLH1.ITGA9. Left, reverse transcription-PCR of untransfected cells (lanes a and b), mock-transfected cells (lanes c and d), and transgene-transfected cells (lanes e and f ). Lane g, water control. Doxycycline treatment. A, MLH1.ITGA9 transgene; B, RAS* (endogenous RAS expression was observed in all investigated cells; lanes a-f); C, GAPDH control. Right, focus formation was observed for RAS* and MLH1.ITGA9, whereas neither mock-transfected cells nor the absence of transgene transcription (+ Dox) did result in focus formation.

Discussion C3orf35/APRG1, and ITGA9). LOH of MLH1 will result in reduced mismatch repair activity. LRRFIP2 encodes a protein We have investigated a Lynch syndrome family that suffers that has been identified as an activator of the canonical WNT from a broad spectrum of tumors. The molecular analysis signaling pathway and, in association with DVL3, exerts its revealed an interstitial deletion of about 400 kb that fuses activity upstream of CTNNB1/h-CATENIN (24). The GOLGA4 portions of the MLH1 (exons 1-11) with the ITGA9 gene (exons gene encodes a protein that has been postulated to play a role 17-28), partly disrupting the AP20region at chromosome in Rab6-regulated membrane-tethering events in the Golgi 3p21.3. The AP20region is a genetic hotspot of homozygous apparatus (25). The C3orf35/APRG1 gene has been shown to deletions in renal, lung, and breast carcinomas (22), and has exhibit a tumor suppressive function in breast cancer and is been implicated in major epithelial malignancies (23). Lynch significantly down-regulated or lost in breast cancer and syndrome patients diagnosed for a 3¶-deletion of the MLH1 cervical carcinoma (26, 27). ITGA9 has been recently described gene may represent individuals that carry interstitial deletions as a candidate cancer gene that is overexpressed in human lung, involving the AP20region. Therefore, Lynch syndrome patients breast, and colorectal cancer (14, 15). Noteworthy, ITGA9 is that exhibit an unusual tumor spectrum may be candidates for normally not expressed in peripheral mononuclear cells (28). a molecular investigation of interstitial deletions at the AP20 Unfortunately, we had no access to detailed clinical records region with the presented long-distance inverse PCR–based of affected family members who belong to the first three strategy. The established primer set (MLH1I11.3and generations. Nevertheless, some family members of the last two ITGA9I16.5) is highly specific for the described MLH1.ITGA9 generations kindly provided their peripheral blood for further fusion. They will be useful in the future to screen Lynch analyses. These analyses revealed that seven of nine tested syndrome patients for the presence of that particular allele to family members were carriers of this particular fusion allele validate the prognostic value of our finding. (family members 9, 15, 18, 20, 22, 25, 28). Four of them were Because of this germline deletion, loss of heterozygosity already affected by tumors (endometrium, colon, small (LOH) was created for five genes (MLH1, LRRFIP2, GOLGA4, intestine, sebaceous adenoma, sebaceous carcinoma, breast

www.aacrjournals.org 767 Clin Cancer Res 2009;15(3) February 1,2009 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. Human Cancer Biology cancer, and a benign keratoacanthoma), whereas family presence of this particular fusion protein may provide complex members 15, 18, and 22, although carriers, have no tumor effects on the biology of affected cells. Tumor cells without history thus far. Tested family members 21 and 23 were wildtype MLH1 alleles but at least one copy of the negative and thus are not at risk to develop a Lynch syndrome MLH1.ITGA9 fusion allele are unable to provide mismatch because they do not carry the mutated MLH1 allele. repair capabilities due to the missing PMS1/2 binding sites. Although the family history fulfills the Amsterdam criteria II Therefore, cells expressing this particular fusion protein have (29), the spectrum of tumors caused by the novel MLH1.ITGA9 reduced ability to cope with genotoxic stress situations. Such a gene fusion is not fully compatible with the spectrum of tumors genetic situation will result in microsatellite instability and normally observed in Lynch syndrome patients (1). The global genetic instability. occurrence of sebaceous gland tumors is a typical feature of Because this is the first time that an MLH1 fusion protein has Muir-Torre syndrome patients (30). Breast (n = 4), liver (n = 2), been identified in a Lynch syndrome family, we tested the and kidney carcinomas (n = 1) are generally not associated with hypothesis whether the fusion protein may provide a biological the Lynch syndrome. Presumably, an impaired mismatch repair function. Focus formation experiments show that the doxycy- system in combination with either the deletion of AP20-region cline-dependent overexpression of the MLH1.ITGA9 fusion genes or a yet to show gain of function due to the presence of protein resulted in a loss–of–contact inhibition phenotype in the MLH1.ITGA9 fusion protein may account for the unusual murine embryonic fibroblasts. These preliminary data should tumor spectrum in the investigated Lynch syndrome family. be handled with care because the ectopic overexpression of Loss of the remaining MLH1 wildtype allele during tumor many proteins displays similar phenotypes and is not development was not unexpected. An experimental comparison necessarily representing a true biological phenotype. As a of the genomic DNA isolated from paraffin embedded normal matter of fact, we used 200 Ag of total cellular protein for the and tumor cells revealed also the loss of LRRFIP2, GOLGA4, detection of the MLH1.ITGA9 fusion protein in peripheral and C3orf35/APRG1 at the second 3p21.3 allele. There are two mononuclear cells of one investigated family member. Thus, possible explanations for this finding: it may (a) argue for an the fusion protein is only weakly expressed in somatic cells of interallelic recombination event resulting in uniparental affected patients. This may account for hyperplastic growth, disomy (31, 32) or (b) be explained by a complete loss of which is presumably always a first step for any type of tumor the second chromosome 3. This should be investigated in more developing along to a multistep carcinogenic pathway. There- detail in the future, for example, by single-nucleotide polymor- fore, studies are currently being done, aiming to dissect the phism array analysis, because neither genomic PCR of genes biological properties of this novel MLH1.ITGA9 fusion protein, surrounding the genomic fusion site nor microsatellite PCR will which may provide clues to understand the clinical observa- be informative. Genes surrounding the fusion site are still tions in more detail. hemizygously on the derivative chromosomes, and microsatellite PCR experiments will not be of value in tumor cells that were positively tested to exhibit microsatellite instability Disclosure of Potential Conflicts of Interest (10). Other important issues are potential functions of the No potential conflicts of interest were disclosed. MLH1.ITGA9 fusion protein. Because the MLH1.ITGA9 fusion protein does not exhibit a leader peptide, neither a membrane insertion nor the typical ITGA9-specific N-glycosylation can be Acknowledgments expected. The remaining transmembrane domain and cytosolic We thank Prof. Michael Karas for critically reading the manuscript, Dr. Jochen . tail present in the 3¶-terminal portion of the MLH1 ITGA9 Raedle and Dr. Brigitte Tandonnet for the medical help, and all family members who fusion protein may attract other cellular proteins, and thus, the provided peripheral blood after informed consent.

References 1. de la Chapelle A. Genetic predisposition to colorectal 7. Herman JG, Umar A, Polyak K, et al. Incidence and beta5, alpha5beta1 and alpha9beta1 to osteopontin. cancer. Nat Rev Cancer 2004;4:769 ^80. functional consequences of hMLH1promoter hyper- Matrix Biol 2005;24:418^ 27. 2. UmarA, RisingerJI, Hawk ET, et al.Testing guidelines methylation in colorectal carcinoma. Proc Natl Acad 13. Vlahakis NE, Young BA, Atakilit A, et al. Integrin for hereditary non-polyposis colorectal cancer. Nat Sci U S A1998;95:6870^5. alpha9beta1 directly binds to vascular endothelial Rev Cancer 2004;4:153 ^ 58. 8. Iyer RR, Pluciennik A, Burdett V, Modrich PL. DNA growth factor (VEGF)-A and contributes to VEGF-A- 3. Lynch HT, Lynch PM, Pester J, Fusaro RM. The can- mismatch repair: functions and mechanisms. Chem induced angiogenesis. J Biol Chem 2007;282: cer family syndrome. Rare cutaneous phenotypic link- Rev 2006;106:302^23. 15187 ^ 9 6. age of Torre’s syndrome. Arch Intern Med 1981;141: 9. WangY, Cortez D,Yazdi P, et al. BASC, a super com- 14. Hibi K, Yamakawa K, Ueda R, et al. Aberrant upre- 607^11. plex of BRCA1-associated proteins involved in the rec- gulation of a novel integrin alpha subunit gene at 4. Mangold E, Pagenstecher C, Leister M, et al. A ognition and repair of aberrant DNA structures. Genes 3p21.3 in small cell lung cancer. Oncogene 1994;9: genotype-phenotype correlation in HNPCC: strong Dev 2000;14:927^ 39. 611^ 9. predominance of msh2 mutations in 41 patients 10. Aaltonen LA, Peltomaki P, Leach FS, et al. Clues to 15. Sjo« blomT, Jones S, Wood LD, et al. The consensus with Muir-Torre syndrome. J Med Genet 2004;41: the pathogenesis of familial colorectal cancer. Science coding sequences of human breast and colorectal 567^72. 1993;260:812^ 16. cancers. Science 2006;314:268 ^ 74. 5. Papadopoulos N, Lindblom A. Molecular basis of 11. Palmer EL, Ru« eggC,FerrandoR,PytelaR, 16. Meyer C, Schneider B, Jakob S, et al. A new diag- HNPCC: mutations of MMR genes. Hum Mutat 1997; Sheppard D. Sequence and tissue distribution of the nostic tool for the identification of MLL rearrange- 10 :89 ^ 99. integrin alpha 9 subunit, a novel partner of beta 1that ments including unknown partner genes. Proc Natl 6. Peltoma« ki P,Vasen HF. Mutations predisposing to he- is widely distributed in epithelia and muscle. JCell Biol Acad Sci U S A 2005;102:449 ^ 54. reditary nonpolyposis colorectal cancer: database 1994;123:1289 ^ 97. 17. Kolodner RD, Hall NR, Lipford J, et al. Structure of and results of a collaborative study. The International 12. Yokosaki Y,Tanaka K, Higashikawa F, Yamashita K, the human MLH1locus and analysis of a large heredi- Collaborative Group on Hereditary Nonpolyposis Co- Eboshida A. Distinct structural requirements for bind- tary nonpolyposis colorectal carcinoma kindred for lorectal Cancer. Gastroenterology 1997;113:1146 ^ 58. ing of the integrins alphavbeta6, alphavbeta3, alphav- mlh1mutations. Cancer Res 1995;55:242 ^ 8.

Clin Cancer Res 2009;15(3) February 1, 2009 768 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. MLH1.ITGA9 Fusion Gene

18. Trojan J, Zeuzem S, Randolph A, et al. Functional integrated physical and gene map of the 3.5-Mb chro- the integrin alpha 9 subunit, a novel partner of beta analysis of hMLH1variants and HNPCC-related muta- mosome 3p21.3 (AP20) region implicated in major 1 that is widely distributed in epithelia and muscle. tions using a human expression system. Gastroenter- human epithelial malignancies. Cancer Res 2003;63: JCellBiol1993;123:1289^97. ology 2002;122:211^ 9. 404^12. 29. Syngal S, Fox EA, Eng C, Kolodner RD, Garber JE. 19. PlotzG,WelschC,Giron-MonzonL,etal.Mutations 24. Liu J, Bang AG, Kintner C, et al. Identification of Sensitivity and specificity of clinical criteria for heredi- in the MutSalpha interaction interface of MLH1 can the Wnt signaling activator leucine-rich repeat in tary non-polyposis colorectal cancer associated abolish DNA mismatch repair. Nucl Acids Res 2006; flightless interaction protein 2 by a genome-wide mutations in MSH2 and MLH1. J Med Genet 2000; 34:6574^86. functional analysis. Proc Natl Acad Sci U S A 2005; 37:641 ^ 5. 20. Bursen A, Moritz S, Gaussmann A, et al. Interaction 102:1927 ^32. 30. South CD, Hampel H, Comeras I, et al. The fre- of AF4 wild-type and AF4.MLL fusion protein with 25. Barr FA. A novel Rab6-interacting domain defines a quency of Muir-Torre syndrome among Lynch SIAH proteins: indication for t(4;11) pathobiology? family of Golgi-targeted coiled-coil proteins. Curr Biol syndrome families. J Natl Cancer Inst 2008;100: Oncogene 2004;23:6237 ^ 49. 19 9 9;9 :3 81 ^ 4. 277 ^ 81. 21. Gaussmann A, Wenger T, Eberle I, et al. Combined 26. SenchenkoV, Liu J, Braga E, et al. Deletion mapping 31. Raghavan M, Lillington DM, Skoulakis S, et al. effects of the two reciprocal t(4;11) fusion proteins using quantitative real-time PCR identifies two distinct Genome-wide single nucleotide polymorphism anal- MLL.AF4 and AF4.MLL confer resistance to apopto- 3p21.3 regions affected in most cervical carcinomas. ysis reveals frequent partial uniparental disomy due sis, cell cycling capacity and growth transformation. Oncogene 2003;22:2984 ^ 92. to somatic recombination in acute myeloid leuke- Oncogene 2007;26:3352^63. 27. Leris AC, Roberts TR, Jiang WG, Newbold RF, mias. Cancer Res 2005;65:375 ^ 8. 22. Senchenko V, Liu J, Loginov W, et al. Discovery of Mokbel K. Evidence for a tumour suppressive func- 32. Melcher R, Al-Taie O, KudlichT, et al. SNP-array gen- frequent homozygous deletions in chromosome tion of APRG1 in breast cancer. Breast Cancer Res otyping and spectral karyotyping reveal uniparental 3p21.3 LUCA and AP20 regions in renal, lung and Treat 2005;293:97 ^100. disomy as early mutational event in MSS- and MSI- breast carcinomas. Oncogene 2004;23:5719^ 28. 28. PalmerEL,RueeggC,FerrandoR,PytelaR, colorectal cancer cell lines. Cytogenet Genome Res 23. Protopopov A, Kashuba V, Zabarovska VI, et al. An Sheppard D. Sequence and tissue distribution of 2007;118:214^ 21.

www.aacrjournals.org 769 Clin Cancer Res 2009;15(3) February 1,2009 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2009 American Association for Cancer Research. An Interstitial Deletion at 3p21.3 Results in the Genetic Fusion of MLH1 and ITGA9 in a Lynch Syndrome Family

Claus Meyer, Angela Brieger, Guido Plotz, et al.

Clin Cancer Res 2009;15:762-769.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/15/3/762

Cited articles This article cites 32 articles, 13 of which you can access for free at: http://clincancerres.aacrjournals.org/content/15/3/762.full#ref-list-1

Citing articles This article has been cited by 2 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/15/3/762.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/15/3/762. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.