MSH2 in Contrast to MLH1 and MSH6 Is Frequently Inactivated by Exonic and Promoter Rearrangements in Hereditary Nonpolyposis Colorectal Cancer1

[CANCER RESEARCH 62, 848–853, February 1, 2002] MSH2 in Contrast to MLH1 and MSH6 Is Frequently Inactivated by Exonic and Promoter Rearrangements in Hereditary Nonpolyposis Colorectal Cancer1

Françoise Charbonnier, Sylviane Olschwang, Qing Wang, Cećile Boisson, Cosette Martin, Marie-Pierre Buisine, Alain Puisieux, and Thierry Frebourg2 Laboratoire de Geńe´tique Molećulaire, CHU de Rouen et Institut National de la Santeét de la Recherche Me´dicale EMI 9906, Faculte´deMe´decine et de Pharmacie, IFRMP, 76183 Rouen [F. C., C. M., T. F.]; Unite´ d’Oncologie Molećulaire, Institut National de la Sante´ et de la Recherche Me´dicale U453, Centre León Be´rard, 69373 Lyon [Q. W., A. P.]; Fondation Jean Dausset-Centre d’Etude du Polymorphisme Humain, Paris [S. O., C. B.]; and Laboratoire de Biochimie et Biologie Molećulaire, CHU de Lille 59037 Lille [M-P. B.], France

ABSTRACT MLH1 mutations may be explained by: (a) alterations of MSH2 or MLH1 missed by conventional screening methods based on PCR To estimate the relative frequency of mismatch repair genes, rear- amplification of each exon; (b) the involvement of other MMR genes; rangements in hereditary nonpolyposis colorectal cancer (HNPCC) fam- or (c) the existence of HNPCC not related to a defect within the MMR ilies without detectable mutations in MSH2 or MLH1, we have analyzed by multiplex PCR of short fluorescent fragments MSH2, MLH1, and MSH6 pathway. The first case of a MLH1 genomic deletion, involving exon in 61 families, either fulfilling Amsterdam criteria or including cases of 16, was reported in 1995 by Albert de la Chapelle’s group in Finnish multiple primary cancers belonging to the HNPCC spectrum. We detected families. This deletion, detected by RT-PCR analysis, was shown to 13 different genomic rearrangements of MSH2 in 14 families (23%), result from an Alu-mediated recombination (13). Using also RT-PCR whereas we found no rearrangement of MLH1 and MSH6. Analysis of 31 analysis, we subsequently detected two distinct Alu-mediated dele- other families, partially meeting Amsterdam criteria, revealed no addi- tions removing exons 13–16 and exon 2 of MLH1 in two French tional rearrangement of MSH2. All of the MSH2 rearrangements, except HNPCC families (14, 15). An extensive screening by Southern blot one, corresponded to genomic deletions involving one or several exons. In analysis of 137 HNPCC families, without detectable point mutations 8 of 13 families with a MSH2 genomic deletion, the MSH2 promoter was within the MMR genes, led Wijnen et al. (16) to identify, in 8 also deleted, and the 5؅ breakpoint was located either within or upstream families, four distinct genomic deletions of MSH2, removing exon 1, the MSH2 gene. This study demonstrates the heterogeneity of MSH2 exonic and promoter rearrangements and shows that, in HNPCC families exon 2, exon 3, and exon 6. Two other genomic deletions of MSH2, without detectable MSH2 or MLH1 point mutation, one must consider the involving exons 1–6 and exons 1–7, were identified using the con- presence of MSH2 genomic rearrangements before the involvement of version approach, based on the cell fusion strategy (17). other mismatch repair genes. The simplicity and rapidity of their detec- To facilitate the detection of genomic rearrangements, we recently tion, using fluorescent multiplex PCR, led us to recommend to begin the developed a simple and rapid screening method, based on the multi- molecular analysis in HNPCC by screening for MSH2 rearrangements. plex PCR amplification of short fluorescent fragments. This method allowed us to identify two additional deletions of MSH2, removing exon 5 and exons 1–15, and the first case of a partial duplication of INTRODUCTION this gene affecting exons 9–10 (15). However, the frequency of the HNPCC3 is the most common form of inherited colorectal cancer rearrangements in the MMR genes has not been estimated thus far, and (for review, see Ref. 1). In HNPCC, detection of the causal alteration this information is essential to determine the best strategy of the of the MMR gene is essential for a proper management of the families, molecular analyses in HNPCC families. Therefore, we have system- because it allows to identify relatives with high risk for colorectal or atically screened for genomic rearrangements a series of 92 families endometrial cancer, who require the appropriate screening (2, 3) and, without detectable point mutations. conversely, to avoid useless surveillance in noncarrier relatives. The efficiency of the colonoscopy screening, especially in terms of mor- MATERIALS AND METHODS tality, has been demonstrated recently in HNPCC families (4). Mo- lecular diagnosis of HNPCC is complicated by the genetic heteroge- Families. This study included a total of 92 families corresponding to: (a) neity of the syndrome because of the involvement of the different 49 families fulfilling the Amsterdam criteria I or II for HNPCC (at least three MutS and MutL homologues, MSH2 (2p22-p21), MSH6 (2p16), and relatives with CRC, cancer of the endometrium, small bowel, ureter, or renal MLH1 (3p21) and PMS2 (7p22), respectively (5–11). Mutation stud- pelvis, one of whom is a first-degree relative of the other two; at least two ies (12) have shown that MSH2 and MLH1 are involved in approxi- successive generations affected; and at least one diagnosed before age 50; mately half of the families fulfilling the Amsterdam criteria I (at least Refs. 18 and 19); (b) 12 families, partially fulfilling the Amsterdam criteria, but including cases with MPCs belonging to the HNPCC spectrum (CRC, three relatives with CRC, one of whom is a first-degree relative of the cancer of the endometrium, stomach, ovary, small bowel, ureter, or renal other two; at least two successive generations affected; and at least pelvis), the first of which developed before 50 years of age; and (c) 31 families one of the cases of CRC diagnosed before age 50; tumors verified by which met only partial Amsterdam criteria and without cases of MPC. For each pathological examination). family, previous screening of MSH2 and MLH1 by Denaturating Gradient Gel The large number of HNPCC families without detectable MSH2 or Electrophoresis, heteroduplex, and/or direct sequencing analysis, from genomic DNA of the index case, had revealed no point mutation. RT-PCR Received 10/8/01; accepted 12/3/01. analysis had also been performed in 43 families, for which high quality mRNA The costs of publication of this article were defrayed in part by the payment of page was available, and had revealed no alteration. charges. This article must therefore be hereby marked advertisement in accordance with Fluorescent Multiplex PCR. We used a slightly modified version of the 18 U.S.C. Section 1734 solely to indicate this fact. protocol described previously (15). Briefly, short fragments (Ͻ300 bp) of the 1 Supported by l’Association pour la Recherche sur le Cancer and La Fondation pour la Recherche Me´dicale. 16 MSH2, 19 MLH1, and 10 MSH6 coding exons (Table 1) or of the MSH2 2 To whom requests for reprints should be addressed, at Institut National de la Sante´ upstream region (Table 2) were simultaneously PCR amplified, using 6-Fam et de la Recherche Me´dicale EMI 9906, Faculte´deMe´decine et de Pharmacie, 22 labeled primers. For MSH2 and MLH1, two multiplex PCRs were necessary to Boulevard de Gambetta, 76183 Rouen, France. cover all of the exons (Table 1). An additional fragment, corresponding to 3 The abbreviations used are: HNPCC, hereditary nonpolyposis colorectal cancer; MMR, mismatch repair; RT-PCR, reverse transcription-PCR; CRC, colorectal cancer; another gene used as a control, was systematically amplified in each multiplex MPC, multiple primary cancers. PCR. PCR was performed in a final volume of 25 ␮l containing 100 ng of 848

Table 1 Primers used for the fluorescent multiplex PCR analysis of the MMR gene exonsa Size of the Exon Sense primer Antisense primer ampliconb MSH2c 15Ј TTCGTGCGCTTCTTTCAG 3Јd 5Ј TGGGTCTTGAACACCTCCCG 3Ј 127 25Ј GGTTAAGGAGCAAAGAATCTGC 3Јd 5Ј ATGCCAAATACCAATCATTC 3Ј 158 35Ј GGATATGTCAGCTTCCATTGGT 3Јd 5Ј GGTAAAACACATTCCTTTGG 3Ј 190 45Ј TCATATCAGTGTCTTGCACA 3Јd 5Ј TTTTGTGGAAAAGTCAGCTT 3Ј 200 55Ј GGACTGTCTGCGGTAATCAAGT 3Јd 5Ј AAAGGTTAAGGGCTCTGACT 3Ј 133 65Ј TAATGAGCTTGCCATTCTTT 3Јd 5Ј GAGAGGCTGCTTAATCCAC 3Ј 152 75Ј CAAGCAGCAAACTTACAAGA 3Јd 5Ј ACCACCACCAACTTTATGAG 3Ј 176 85Ј GGGGAAGCTTTGAGTGCTACAT 3Јd 5Ј AGAGGAGTCACAAAAACTGC 3Ј 265 95Ј GGTGACTTGGAAAAGAAGATGC 3Јd 5Ј GGGCTTGTTTAAATGACATC 3Ј 229 10 5Ј GGTTTCGTGTAACCTGTAAGGAA 3Јd 5Ј GGGCTATTTAACAAATGGTG 3Ј 262 11 5Ј GATACTTTGGATATGTTTCA 3Јd 5Ј CCAGGTGACATTCAGAACA 3Ј 225 12 5Ј GGTTAGGAAATGGGTTTTGAAT 3Јd 5Ј ATGCCTGGATGCTTTTAAT 3Ј 275 13 5Ј ATTGTGGACTGCATCTTAGC 3Јd 5Ј AAAGTCCACAGGAAAACAAC 3Ј 242 14 5Ј GGCTACGATGGATTTGGGTTAG 3Јd 5Ј TTTCCCATTACCAAGTTCTG 3Ј 242 15 5Ј GGGCTGTCTCTTCTCATGCTGT 3Јd 5Ј GATAGCACTTCTTTGCTGCT 3Ј 217 16 5Ј GGCTGTCCAAGGTGAAACAAAT 3Јd 5Ј CCATTACTGGGATTTTTCAC 3Ј 162 MSH6e 15Ј GGGTACAGCTTCTTCCCCAAGT 3Јd 5Ј CCTCCGTTGAGGTTCTTC 3Ј 207 25Ј GGCTGCCTTTAAGGAAACTTGA 3Јd 5Ј ACATGAACACGGACTGATTT 3Ј 195 35Ј GGGGAGGTCATTTTTACAGTGC 3Јd 5Ј CCTCCATCTCTTCTTCCTCT 3Ј 147 45Ј GGGGAGGAAGGAAGCAGTGATG 3Јd 5Ј GGGCTTGGGATTCAGAATTT 3Ј 246 55Ј GGTTTACTGTGCCTGGCTAACT 3Јd 5Ј ACAAGCACACAATAGGCTTT 3Ј 223 65Ј GGATTCACAGGCTGGCTTATTA 3Јd 5Ј TGAGAACTTAAGTGGGAAACA 3Ј 160 75Ј GGTTGTTGAATTAAGTGAAACTGC 3Јd 5Ј TCTTCAAATGAGAAGTTTAATGTC 3Ј 105 85Ј GGAACAGGAAGAGGTACTGCAA 3Јd 5Ј TGAATGGTAGTGAGTTGAAAA 3Ј 113 95Ј GGCATGCATGGTAGAAAATGAA 3Јd 5Ј ACTTCCTCTGGGAGATTAGC 3Ј 134 10 5Ј GGCTGGCTAGTGAAAGGTCAAC 3Јd 5Ј AGAAAATGGAAAAATGGTCA 3Ј 185 a Sequences of the MLH1 primers have been published previously (15). b In bp. c For MSH2, the two multiplex PCRs corresponded to exons 2, 3, 5, 9, 10, 12, 14, and 15 and exons 1, 4, 6–8, 11, 13, and 16, respectively. d 5Ј (6-FAM) labelled. e For MSH6, the multiplex PCR was performed in the presence of 10% of DMSO to facilitate amplification of exon 1.

genomic DNA, 0.2–1 ␮M of each pair of primers, and 1 unit of Taq DNA RESULTS polymerase (Eurobio, Les Ulis, France). After a denaturation of 3 min at 95°C, the PCR consisted of 21 cycles of 15 s at 94°C,15sat55°C, and 15 s at 72°C, Using the fluorescent multiplex PCR assay, based on the simulta- followed by a final extension of 7 min at 72°C. neous amplification of short genomic fragments (15), we first Analysis of the Multiplex PCR. After electrophoresis for3honan screened for rearrangements of MSH2, MLH1, or MSH6 in 61 automated sequencer (Applied Biosystems), data were analyzed using the HNPCC families, either fulfilling the Amsterdam criteria (49 families) Gene scanner Model 672 Fluorescent Fragment Analyzer (Applied Biosys- or selected for the presence of cases with MPC belonging to the tems). Electropherograms were superposed to those generated from control HNPCC spectrum (12 families). In 14 families (23%), a genomic DNAs, and the areas of the corresponding peaks between the different samples were compared. In the fluorescent multiplex PCR assay, a 0.5 reduction of the rearrangement of MSH2 was detected (Fig. 1; Table 3). MSH2 peak(s) area indicates an heterozygote deletion of the corresponding exon(s), genomic rearrangements (Table 3) were detected in 10 of the 49 whereas exonic duplications result in a 1.5 increase (15). Each positive result families fulfilling Amsterdam criteria I or II (20%) and in 4 of the 12 was controlled on a second independent fluorescent multiplex PCR. families with MPC (33%). In contrast, the fluorescent multiplex PCR Sequence Analysis. After purification by electrophoresis on low-melt aga- assay revealed no MLH1 or MSH6 genomic alteration. We then rose gel, PCR products were directly sequenced on both strands using the Big extended the analysis of MSH2 to 31 other families partially meeting Dye Terminator Kit (Applied Biosystems) and a model 377 automated se- Amsterdam criteria, and we detected no additional rearrangement of quencer (Applied Biosystems). Long-range PCR. Long-range PCR was performed using the Expand MSH2. Long Template PCR system from Boehringer Mannheim according to the All of the MSH2 rearrangements, except one, corresponded to protocol of the supplier. genomic deletions, which involved in 6 families a single exon and in

Table 2 Primers used for the fluorescent multiplex PCR analysis of the MSH2 upstream region Primer Sequence Location Amplified region H1F 5Ј TATCAGAAATGGTAGTAGCTTCTCTAAAGG 3Јa MSH2 promoterb Ϫ3589-3386c (204)d H1R 5Ј CTAATTTTTGTAATTTTTGTGGAGATGAGT 3Ј ZF 5Ј TAAGTCTAGTTAAAGGTAACTGTCTTGTGC 3Јa MSH2 promoter Ϫ4242-4083c (160)d ZR 5Ј GAAAAATTCCTTAAGTCCAGGTGTTTA 3Ј YF 5Ј GGCCTGATCTACTTTGAATAAGAATTTATC 3Ј Intergenice Ϫ5110-4941c (170)d YR 5Ј ACGCCCCACTAATTTTTATATTTTTAG 3Јa LOCF 5Ј ATGGACCTGACAGTAAATGG 3Јa LOC 65359e,f Exon 7 (138)d LOCR 5Ј CACCACAACCACAATAACAG 3Ј a 5Ј (6-FAM) labelled. b Defined by Iwahashi et al. (1998); Genbank accession no.: AB006445. c Numbered from the MSH2 transcription initiation site (Ϫ68 bp from the ATG). d Size of the amplicon indicated in bp. e Sequences and location of primers were based on the chromosome 2 working draft sequence (build 25, contig NT_005400.5). f Sequence segment c659182-640641. 849

Fig. 1. Detection of exonic deletions of MSH2 by fluorescent multiplex PCR. In each panel, the electropherogram of the patient (in red) was superposed to that of a control (in blue). The Y axis displays fluorescence in arbitrary units, and the X axis indicates the size in bp. Exon numbers are indicated. Representative results are shown: a, deletion of exon 1; b, deletion of exon 7; c, deletion of exons 1–7; d, deletion of exons 1–8.

7 families several exons (Table 3). We also analyzed families R3 and of intron 1, deleting 1297 bp of the promoter region (Fig. 4b). R4 (Fig. 2a), as well as families P5 and L14 (15), using long-range Alignment and analysis of the nucleotide sequences, using the Re- PCR. In each case, the rearrangement was confirmed. In 8 of 13 (62%) peatMasker program,4 revealed that the recombination had occurred families, exon 1 was deleted (Table 3; Fig. 4a), suggesting that the in Alu repeats and involved a 54-bp homologue sequence between the MSH2 promoter was affected by the rearrangement. To confirm the promoter region and intron 1. In family L7, the breakpoint within the promoter deletion within these families, we first performed long-range promoter region was mapped to the same position as in family R2, and Ј PCR using a sense primer (P1) corresponding to the 5 end of the the promoter sequence was fused to the last 148 bp of intron 4. The MSH2 promoter region (Fig. 4b). In only 2 families (R2 and L7), we breakpoint within intron 4 was located ϳ210 bp downstream an Alu detected an aberrant PCR product (Fig. 2, b and c), confirming the repeat. In the 6 other families with an exon 1 deletion (R1, Li8, R9, deletion. Sequencing analysis revealed that, in the R2 family, the first R10, R11, and P12), the absence of an abnormal PCR product de- 3041 bp of the MSH2 promoter region were fused to the last 2132 bp tected by long-range PCR (Fig. 2b) suggested that the 5Ј breakpoint was located upstream the MSH2 promoter region. We then designed Table 3 Detection of MSH2 exonic rearrangements a new multiplex PCR assay (Table 2; Fig. 3) to analyze ϳ24 kb of Family Genomic rearrangement Presentation genomic sequences upstream the transcription initiation site of MSH2 R1 Del exon 1 AMS Ia (Fig. 4b). The multiplex PCR assay revealed (Fig. 3) that the 5Ј R2 Del exon 1 MPCb (E, 47; C, 50)c breakpoint was located, in 2 families (R9 and R11), downstream the L7 Del exons 1–4 AMS I Li8 Del exons 1–6 AMS IId LOC65359 gene and in the 4 other families (R1, Li8, R10, and P12), R9 Del exons 1–7 AMS II at least 24 kb upstream the transcription initiation site of MSH2, R10 Del exons 1–8e AMS I R11 Del exons 1–8e MPC (CRC, 35; CRC, 35) because the LOC65359 gene was also deleted (Fig. 4b). In family R9, P12 Del exons 1–15f AMS I long-range PCR, using a sense primer corresponding to exon 9 (P9) of f R3 Del exon 3 MPC (CRC, 41; CRC, 44; CRC, 59) the LOC65359 gene (Fig. 4a) and an antisense primer corresponding R4 Del exon 3 MPC (CRC, 32; O, 53) P5 Del exon 5f AMS I to exon 8 of MSH2, confirmed the presence of a large genomic P13 Del exons 5–6 AMS I deletion of ϳ55 kb (Fig. 2d). Sequence analysis showed that the L6 Del exon 7 AMS I L14 Dup 9–10f AMS I breakpoint had occurred within an Alu repeat, 529 bp downstream the a Fulfilling Amsterdam criteria I (19). last exon of LOC65359, removing ϳ16 kb of genomic sequences b Including cases of multiple primary cancers belonging to the HNPCC spectrum. upstream the transcription initiation site of MSH2 (Fig. 4b). Within c E, endometrial cancer; O, cancer of the ovary. d Fulfilling Amsterdam criteria II (19). intron 7, the breakpoint was located 600 bp upstream the splice e Haplotype analysis, using the microsatellite markers D2S288, D2S2227, and D2S123, acceptor site also within an Alu repeat. had indicated that, in families R10 and R11, the rearrangements had been recurrently generated. f Previously reported in Charbonnier et al. (15). 4 Internet address: http://repeatmasker.genome. washington.edu. 850

arrangements are frequently found in MSH2 but not in MLH1 and MSH6. We observed that 20% of the HNPCC families fulfilling Amsterdam criteria had a genomic rearrangement of MSH2 (10 of 49), a value higher than the first estimate provided by Wijnen et al. (16) and obtained by Southern blot analysis (6 of 51, 12%). An additional finding of the present study is the high frequency of MSH2 rearrangements in patients with MPCs belonging to the HNPCC spectrum, which confirms that this criterion, independently of the familial his- tory, is highly suggestive of HNPCC. In 31 families, which only partially met Amsterdam criteria and without cases of MPC, we detected no rearrangement of MSH2. Wijnen et al. (16) had described, among 86 HNPCC families not fitting Amsterdam criteria, only 2

Fig. 2. Confirmation by long-range PCR of the MSH2 genomic deletions revealed by the multiplex PCR. a, amplification of genomic DNA from the R3 family index case, using the sense primer corresponding to exon 2 and the antisense primer corresponding to exon 4 indicated in Table 1. Instead of the expected normal fragment of ϳ4 kb, observed in a control, an abnormal shorter PCR product of 1.8 kb was detected in the index case. The same pattern was observed in the family R4 index case; b, amplification of genomic DNA from the R2 family index case (Table 2), using the sense primer P1 (5Ј CCTTA- AACACCTGGACTTAAGGAATTTTTC 3Ј) corresponding to the 5Ј end of the MSH2 promoter region (Ref. 22; GenBank accession no. AB006445) and the antisense primer corresponding to exon 2 and indicated in Table 1. Instead of the expected normal fragment of 9 kb, an abnormal shorter PCR product of ϳ5 kb was detected in the index case. In contrast, in the R1 family index case, only the normal allele could be amplified. c, amplification of genomic DNA from the L7 family index case, using the P1 primer and the antisense primer corresponding to exon 5, indicated in Table 1. Note that the normal fragment (expected size: 14 kb) could not be amplified, but an abnormal 1.5-kb fragment was amplified in the L7 patient. d, amplification of genomic DNA from the R9 family index case, using the sense primer P9 corresponding to exon 9 of the LOC 65359 gene (5Ј CAGATAAAGGAGATGGGTGAG 3Ј) and the antisense primer corresponding to exon 8ofMSH2 and indicated in Table 1. Whereas the expected size of the normal fragment is ϳ57 kb, an aberrant fragment of 1.7 kb was detected in the R9 patient. In each panel, the arrow indicates the abnormal PCR product. The fragment sizes (in kb) of relevant markers are indicated.

DISCUSSION This study was designed to estimate the relative frequency of MMR gene rearrangements in HNPCC families without detectable point mutations within the MSH2 and MLH1 genes. Southern blot analysis, which is the method commonly used to detect genomic rearrangements, can miss small deletions or duplications, and restriction site polymorphisms may complicate the interpretation of the restriction patterns. Therefore, we took advantage of a simple semiquantitative method, based on the multiplex PCR of short fluorescent fragments (15). We had initially validated this method on MLH1 and MSH2 rearrangements, detected previously by RT-PCR and confirmed by long-range PCR (15). The fluorescent multiplex PCR assay was subsequently shown to be a powerful method for the detection of Fig. 3. Characterization of the deletions of the MSH2 upstream region by fluorescent multiplex PCR. In each panel, the electropherogram of the patient (in red) was superposed heterozygote deletions of SMN1 in spinal muscular atrophy (20) and to that of a control (in blue). The Y axis displays fluorescence in arbitrary units, and the for the detection of C1NH exonic deletions and duplications (21). In X axis indicates the size in bp. The amplicons, corresponding to the primers listed in Table 2, are indicated. Ex 1, exon 1 of MSH2. a, deletion detected in patient R2; b, deletion the present study, this method allowed us to show that, in HNPCC detected in patient R9. The same profile was obtained for patient R11; c, deletion detected families screened previously by conventional methods, genomic re- in patient Li8. The same profile was obtained for patients R1, R10, and P12. 851

Fig. 4. Distribution of the MSH2 genomic rearrangements detected in HNPCC families.a,exonic rearrangements. MSH2 exons are numbered below the solid boxes, and the size of the introns in kb is indicated. ⅐⅐⅐⅐, the sequence presently missing from the chromosome 2 working draft sequence. OOO, the rearrangements detected in the families. ----, the duplication. Arrows, the deletions whose intronic boundaries have been accurately determined, thus allowing an accurate estimate of the extent of the deletions (indicated in kb above the lines). b, mapping of the 5Ј boundaries of the promoter deletions. The map of this region was based on the contig NT_005400.5 (build 25). The MSH2 promoter region is indicated by the gray bar. The first exon of MSH2 and the last exons of the LOC65359 gene are shown as well as the size of introns and of the interval between the two genes. The size of the intergenic region sequence missing from the chromosome 2 working draft sequence was estimated by long-range PCR to ϳ4 kb. Am- plicons used in the upstream region fluorescent multiplex PCR (Fig. 3) are marked by vertical arrows and are designed Y, Z, and H1, respectively. Primers used to characterize the deletions by long- range PCR are shown by short horizontal arrows.

cases of MSH2 rearrangements. In both studies, this low frequency of umented by this study has important practical implications. In MSH2 genomic rearrangements in these families might be explained HNPCC families fulfilling Amsterdam criteria or with cases of MPC in part by the heterogeneity of the criteria used for the selection of the belonging to the HNPCC spectrum and without detectable MSH2 or kindreds. Nevertheless, it is also possible that genomic rearrange- MLH1 point mutations, one must consider the presence of MSH2 ments, in contrast to other types of germ-line alterations, such as genomic rearrangements. The frequency of these alterations is prob- missense or splice mutations, may result into a complete loss of ably higher than that of point mutations within the other MMR genes. function and, therefore, in a high penetrance. In 71 families fulfilling the original Amsterdam criteria and without Wijnen et al. (16) had identified by Southern blot analysis four detectable MSH2 or MLH1 point mutations, Wijnen et al. (23) have types of MSH2 rearrangements in 8 families. The present study detected a pathogenic MSH6 mutation only in 3 families. In a study highlights the remarkable allelic heterogeneity of MSH2 rearrange- including 90 HNPCC families, 23 of which meeting Amsterdam ments, because 13 distinct rearrangements were detected in 14 criteria, Huang et al. (24) recently identified only one germ-line HNPCC families (Fig. 4). Moreover, we showed that the MSH2 mutation of MSH6 and no mutation of MSH3, demonstrating that promoter was also deleted in 8 of 14 families. Furthermore, we these genes are rarely involved in HNPCC. The percentage of HNPCC demonstrated that the 5Ј breakpoint of the deletions removing the families fulfilling Amsterdam criteria and carrying a MSH2 genomic promoter is highly heterogeneous (Fig. 4b). The majority of the rearrangement can be estimated to ϳ10%, because the families ana- rearrangements that we have identified (Fig. 4), like those reported by lyzed in the present study had been selected on the basis of the Wijnen et al. (16) and Yan et al. (17), involve the region between the absence of point mutations in MSH2 or MLH1, a situation observed in 5Ј end of the gene and intron 8. This distribution could be explained approximately half of the kindreds. This significant percentage and by the distribution of the Alu sequences within the MSH2 gene. the simplicity and rapidity of the fluorescent multiplex PCR assay led Indeed, analysis of the genomic sequence, using the RepeatMasker us to suggest that it is probably efficient to begin the molecular program, revealed that among the 105 Alu sequences detected within screening of MMR genes in HNPCC families by searching for the MSH2 gene, the majority (88) was located between the promoter genomic rearrangements of MSH2. and exon 9. The involvement of Alu-mediated recombination events in the MSH2 rearrangements was confirmed by sequence analysis of the ACKNOWLEDGMENTS breakpoint in 3 families. The high frequency of genomic rearrangements of MSH2 contrasts We thank Mario Tosi for critical review of the manuscript, Gregory Raux with the absence of genomic rearrangements detected, in this study, for technical assistance, and the clinicians who referred HNPCC families. within MLH1 and MSH6. At the present time, only three Alu-mediated genomic rearrangements, involving respectively exon 2, exon 16, and REFERENCES exons 13–16, have been reported thus far within MLH1 (13–15). Nevertheless, in certain populations, as reported previously by Nys- 1. Lynch, H. T., and de la Chapelle, A. Genetic susceptibility to non-polyposis colorectal cancer. J. Med. Genet., 36: 801–818, 1999. tro¨m-Lahti et al. (13) for the deletion of exon 16, certain rearrange- 2. Dunlop, M. G., Farrington, S. M., Carothers, A. D., Wyllie, A. H., Sharp, L., Burn, ments of MLH1 might be associated with a founder effect, justifying J., Liu, B., Kinzler, K. W., and Vogelstein, B. Cancer risk associated with germline DNA mismatch repair gene mutations. Hum. Mol. Genet., 6: 105–110, 1997. their specific screening in the corresponding populations. The contrast 3. Burke, W., Petersen, G., Lynch, P., Botkin, J., Daly, M., Garber, J., Kahn, M. J., in the frequencies of MSH2, MLH1, and MSH6 rearrangements doc- McTiernan, A., Offit, K., Thomson, E., and Varricchio, C. Recommendations for 852

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Françoise Charbonnier, Sylviane Olschwang, Qing Wang, et al.

Cancer Res 2002;62:848-853.

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