PMS2-Related Genes Flank the Rearrangement ... - CiteSeerX

SHORT COMMUNICATION PMS2-Related Genes Flank the Rearrangement Breakpoints Associated with Williams Syndrome and Other Diseases on Human Chromosome 7 Lucy R. Osborne,* Jo-Anne Herbrick,* Tom Greavette,* Henry H. Q. Heng,* Lap-Chee Tsui,*,† and Stephen W. Scherer*,1 *Department of Genetics, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada; and †Department of Molecular and Medical Genetics, The University of Toronto, Toronto, Ontario M5S 1A1 Canada Received June 2, 1997; accepted July 23, 1997

The human PMS2 mismatch repair gene and a family of at least 17 other related genes (named human PMSR or PMS2L genes) have been localized to human chromosome 7. Human PMS2 has been mapped previously to 7p22 and shown to be causative in hereditary nonpolyposis colon cancer (HNPCC), but the human PMS2L genes have not been positioned in the context of the physical or genetic map of chromosome 7. In this study we have used various mapping methodologies to determine the precise location of the human PMS2L genes at 7q11.22, 7q11.23, and 7q22. Within 7q11.23, human PMS2L genes were found to be present at at least three sites as part of duplicated genomic segments that flank the most common rearrangement breakpoints in Williams syndrome. q 1997 Academic Press

The PMS genes belong to a family of mismatch repair genes originally identified in bacteria and yeast (13, 33). The human PMS proteins have greatest homology to the yeast PMS proteins but also share conserved sequences with mutL from Escherichia coli and its other human homologues hMSH2 and hMLH1 (8). Defects in the DNA mismatch repair system are associated with an increased risk of accumulation of mutations in genes involved in tumorigenesis (5, 23), leading to an increased risk of cancer. Germline mutations in the mutL homologues hMSH2 (15), hMLH1 (22), hPMS1, and hPMS2 (17) have been identified in families with hereditary nonpolyposis colorectal cancer (HNPCC), indicating that some of these genes play important and distinct roles in tumor suppression. The hPMS1 and hPMS2 genes were previously mapped to chromosome regions 2q31–q33 and 7p22, respectively (17). At least 17 other human PMS-related (hPMSR or PMS2L) genes have been identified (8, 19). These PMS2L genes are highly conserved with the The HGMW-approved symbols for the genes described in this paper are PMS2LI–PMS2LII, PMS2LP1 and PMS2LP2 1 To whom correspondence should be addressed at Department of Genetics, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. GENOMICS

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MutL genes, suggesting a potential functional role in mismatch repair. Horii et al. (8) used degenerate oligonucleotide primed reverse transcription PCR of total human stomach RNA followed by screening of human fetal brain libraries and identified at least six PMS2L transcribed sequences that were named hPMS3– hPMS8. These cDNA clones were then used to identify cosmid clones, and DNA sequence analysis resulted in the identification of at least 3 additional PMS2L genes, suggesting that these genes may occur in clusters. Simultaneously, Nicolaides et al. (19) used a similar approach to identify from a small intestine cDNA library and a genomic P1 library at least 8 different PMS2L genes named hPMSR1–hPMSR8 (19). All of the genes mapped to chromosome 7 but they contained a high degree of DNA sequence identity in both the exons and the introns, making regional localization experiments difficult. As a result, only hPMSR5 (PMS2LP1) and hPMSR2 (PMS2L8) could be mapped to a broad region at 7q11 and 7q22, respectively (19). Moreover, cosmids containing the hPMS genes always hybridized to multiple locations on chromosome 7, which revealed only the approximate cytogenetic positions of the genes (Refs. 8 and 19; also see Fig. 1). In our effort to construct a fully integrated map of human chromosome 7 (30), we have used several methodologies to determine a more precise location of the PMS2L genes on the chromosome. First, for the hPMS2 gene, PCR primers specific for the 3*UTR (5*-CAACCATGAGACACATCGC-3* and 5*-AGGTTAGTGAAGACTCTGTC-3*) (19), which was shown previously to map to 7p22, were used to screen the G4 radiation hybrid (RH) panel (from Research Genetics) and a collection of chromosome 7-specific yeast artificial chromosome (YAC) clones (27). No YACs were identified but the gene could be located using RH hybrids between the microsatellite markers D7S517 and D7S481, thereby integrating the cytogenetic, genetic, and physical positions of the hPMS2 gene on 7p. Second, all of the hPMS cDNA probes (8) were used to screen the Lawrence Livermore National Laboratory chromosome 7-specific cosmid library, the collection of

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FIG. 1. Cytogenetic localization of PMS2L loci on chromosome 7. (Left) Cosmid 43a11, which was known to originate from 7q11.23, was examined by FISH against human metaphase spreads. The clone hybridized to at least three different sites on chromosome 7 with the most intense signal originating from 7q11 and other less intense signals from 7p22 and 7q22. All other genomic clones tested by FISH gave identical results; (Right) A hPMS cDNA clone (hPMS4) was analyzed by FISH, and the difference in signal intensities was compared with that observed with the genomic clones, indicating that other DNA sequences are likely contributing to the cross-hybridization results observed in the left panel (a 1.3-kb hPMS4 cDNA, Ref. 8, was used in this case but the other cDNA probes gave the same result). The signal at 7p22 was difficult to observe when the cDNA probe was used. We failed to observe a specific signal above background at 7p13 using the cosmids, PACs, or cDNA probes as was observed in a previous study (18). Moreover, genomic library screening did not detect clones from this region.

YAC clones, and the RPCI-1 human genomic P1-derived artificial chromosome (PAC) library (10). A total of 200 cosmids, 17 YACs, and 23 PACs were identified. The identity of each clone was confirmed by restriction enzyme digestion and blot hybridization experiments. The genomic clones gave distinctive fingerprints upon blot hybridization with each hPMS probe, which allowed the cloned DNA segments to be grouped (data not shown). Due to the high degree of similarity of DNA sequence between each of the PMS2L genes, it was not possible using this methodology to distinguish which gene corresponded to which genomic clone. However, positive hybridization to genomic clones that were previously characterized and positioned on the map allowed the identification of, in addition to hPMS2 at 7p22, at least five regions of 7q containing PMS2L genes (Fig. 2). The PMS2L genes hybridized to representative genomic clones from contigs that contained the following reference markers (see Fig. 2); (a) YACs HSC7E295 and CGM2c1 containing the POM/POM-ZP3 genes at 7q11.2; (b) YACs HSC7E280 and HSC7E891, which contain NCF1, IB291, D7S1778, and D7S2479 at 7q11.23; (c) YAC HSC7E512 containing D7S489-B and D7S2518 at 7q11.23; (d) YACs HSC7E640, HSC7E902, and PAC286c17 containing the D7S489-A marker at 7q11.23; and (e) YAC HSC7E782 and PACs 76h2 and 770p8, which contain CUTL1 at 7q22. PCR primers specific for PMS2L8 and PMS2LP1 have been

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established (19), so the precise position of these genes is now determined within 7q22 and 7q11, respectively (Fig. 2). Previous studies have demonstrated that D7S489 is duplicated in three places in 7q11.23 (the three loci are all within 2 Mb of one another and have been named D7S489-A, D7S489-B, and D7S489-C) (25, 26). Our observations indicate that PMS2L genes are located in close proximity to each of the D7S489 loci (Fig. 2). Cosmids 43a11, 165g7, and PAC 76h2 were examined by fluorescence in situ hybridization (FISH) to human metaphase spreads. Cosmid 165g7 and PAC 76h2 were chosen since they were known to contain the D7S489-C marker and the CUTL1 gene and therefore must originate from 7q11.23 and 7q22, respectively. In each experiment, however, an identical pattern of hybridization was determined with the strongest signal observed at 7q11.22–q11.23 and less intense but distinct signals present at 7p22 and 7q22. An example of one of the FISH results is shown in Fig. 1. The more intense signal at 7q11.22–q11.23 can be explained, at least in part, by the presence of at least four nearby hPMSR loci in this region that are indistinguishable cytogenetically at this resolution of analysis (Fig. 2). But as is also shown in Fig. 1, hybridization with the hPMS4 cDNA (8) does not produce the same ratio of intensities as is observed when using the genomic clones, suggesting that larger cosmids, PACs, and YACs contain additional DNA sequences other than PMS2L that contribute to the cross-hybridization. In support of this we have observed that in addition to D7S489, at least three other markers (IB291, D7S1490, and D7S1778,) are also located in at least three places at 7q11.23 (Fig. 2) near the PMS2L loci. The location of PMS2L genes near the D7S489-B and D7S489-A loci is of particular interest in the study of Williams syndrome (WS) since the former two loci reside near the centromeric and telomeric boundaries, respectively, of the common deletion breakpoints in this contiguous deletion syndrome (25). D7S489-C appears not to be deleted in WS patients (26). Determining the exact site of the deletion breakpoints has been complicated because of the presence of the three duplicated stretches of DNA in the region. Previous studies have shown that most of the distal deletion breakpoints occur between D7S1870 and D7S489-A (7, 25). Therefore, it is possible to position the majority of the telomeric WS rearrangement breakpoints somewhere within YAC clones HSC7E640 and HSC7E902, which contain the PMS2LP1, D7S489-A, IB291, D7S1490, and D7S1778 loci. Two studies have now shown that a high percentage of WS deletions occur by a mechanism of unequal meiotic crossover (4, 31). Based on the observations reported in the present study, one might hypothesize that an unequal recombination event involving the duplicated regions could generate a deletion (and with equal frequency a chromosomal duplication) in a manner analagous to that observed in hereditary neuropathy with liability to pressure palsies (HNPP)

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PDGFA D7S517 hPMS2 D7S481

D7S2549 POM-ZP3 PMS2L GUSB D7S663

EPO PMS2L8 D7S518 CUTL1 D7S666

NCF1 PMS2L/IB291/D7S1778 D7S489-C/D7S1490 PMS2L/IB291/D7S1778 D7S489-B/D7S1490 FZD3 D7S2476 STX1A ELN LIMK1 WBSCR1 RFC2 D7S1870 D7S489-A /D7S1490 PMS2LP1/IB291/D7S1778

FIG. 2. Physical localization of PMS2L gene on chromosome 7. The position of the hPMS2 gene and five other PMS2L loci within the context of nearby chromosome 7 reference markers could be determined by radiation hybrid and cosmid–PAC–YAC analysis. The cytogenetic localization was based on the results from Fig. 1 and previous experiments that mapped genomic clones containing the reference markers to individual bands. The relative position of the three PMS2L genes at 7q11.23 and corresponding genomic clones in the Williams syndrome (WS) region is schematically shown. The location of STX1A, ELN, LIMK1, WBSCR1, RFC2, and D7S1870 within the WS region (20, 21) and D7S489-A (the 145-bp band), D7S489-B (the 175-bp band), and D7S489-C (the 165-bp band) (25) has been described. FZD3, which was also mapped in the critical region (32), could be positioned proximal to STX1A based on its hybridization to HSC7E797, which contained the D7S489-B locus. It is important to note that the clone contig currently covering the WS region is incomplete with at least one gap existing between D7S2476 and FZD3. In addition the YAC clones in this region are all inherently unstable (for example all cosmid and PAC mapping data indicate that a PMS2L locus must reside on HSC7E797, but upon repeated analysis the cDNA probe failed to identify the YAC). The order of the six duplicated markers highlighted by boxes is not certain, and we predict that the D7S1778 locus (underlined) near D7S489-B will be identified (it has yet to be found in the existing clones) based on the fact that it is present in the most centromeric and telomeric duplications. EST IB291 has now been identified to be part of the BAP-135 gene. It should be noted that STS content maps of this region of 7q11.23 (2, 9) are inaccurate. The region deleted in most WS patients is shown as a stippled box, but in some individuals a larger deletion has been observed, the boundaries of which extend to an interval covered by the open boxes. The complete list of 200 cosmids, 17 YACs, and 23 PACs specific for PMS2L and further details on the clone contigs are available at http://www.genet.sickkids.on.ca/chromosome7/.

and Charcot–Marie–Tooth disease type 1A (CMT1A) (3, 24). Other examples of repeated regions flanking genomic intervals prone to unequal exchange and intrachromosomal deletion have been described at the Emery–Dreifuss muscular dystrophy (28), spinal muscular atrophy (16), juvenile nephronophthisis (12), factor VIII (14), and distal Xp loci (1, 34). The exact mechanism and the DNA sequences involved in the rearrangements in WS remain to be elucidated. The PMS2L8 gene at 7q22 also appears to reside in a region near the CUTL1 gene that is frequently deleted or rearranged in leiomyomas (35) and in some myeloid leukemias (6, 11, 29). While it has been shown that some of the PMS2L genes on chromosome 7 are transcribed (8, 17), so far, only hPMS2 has been demonstrated to encode a functional protein. Therefore it is not clear if alterations causing homozygous inactiva-

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tion of the 7q22 PMS2L loci would lead to mismatch repair and malignancy. The q22 interval of chromosome 7 demonstrates a preponderance for chromosome breakage and in at least four cases of acute leukemia with constitutional inversions, [inv(7)(q11.2q22)] (Ref. 29 and our unpublished data), it is tempting to speculate that the rearrangement might arise by intrachromosomal recombination between hPMS gene clusters. The elucidation of the complete DNA sequence of the genomic clones containing PMS2L loci will resolve the exact number, organization, and evolution of this gene family on chromosome 7. ACKNOWLEDGMENTS The authors thank Jack Huizenga and Xiao-Mei Shi for technical assistance, Dr. Yusuke Nakamura for providing the hPMS probes,

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