Construction of a Detailed Physical and Transcript Map of the Candidate Region for Russell–Silver Syndrome on Chromosome 17q23–q24

Genomics 71, 174 –181 (2001) doi:10.1006/geno.2000.6413, available online at http://www.idealibrary.com on

Construction of a Detailed Physical and Transcript Map of the Candidate Region for Russell–Silver Syndrome on Chromosome 17q23– q24 Sylvia Do¨rr,* Alina T. Midro,† Claudia Fa¨rber,* ,1 Joannis Giannakudis,* and Ingo Hansmann* ,2 *Institut fu¨r Humangenetik und Medizinische Biologie, Universita¨t Halle-Wittenberg, 06097 Halle/Saale, Germany; and †Department of Clinical Genetics, Medical University Bialystok, 15-230 Bialystok, Poland Received June 29, 2000; accepted October 11, 2000

Russell–Silver syndrome (RSS) is a heterogeneous disorder characterized mainly by pre- and postnatal growth retardation and characteristic dysmorphic features. The genetic cause of this syndrome is unknown. However, two autosomal translocations involving chromosome 17q25 were reported in association with RSS. Molecular analysis of the breakpoint on chromosome 17 of the de novo translocation previously described as t(1;17)(q31;q25) enabled us to refine the localization of the chromosome 17 breakpoint to 17q23– q24. Since no detailed mapping data were available for this region, we established a contig of yeast artificial chromosomes, P1 artificial chromosomes, bacterial artificial chromosomes, and cosmid clones for a 17q segment flanked by the sequence-tagged site (STS) markers D17S1557 and D17S940. This contig covers a physical distance of 4 –5 Mb encompassing several novel markers. A transcript map was constructed by assigning genes and expressed sequence tags to the clone contig, and altogether 74 STS markers were mapped. Furthermore, the locus order and content provide insight into several duplication events that have occurred in the chromosomal region 17q23– q24. On the basis of our refined map, we have reduced the translocation breakpoint region to 65 kb between the newly derived markers 58T7 and CF20b. These data provide the molecular tools for the final identification of the RSS gene in 17q23– q24. © 2001 Academic Press

INTRODUCTION

Russell–Silver syndrome (RSS) (MIM 180860) is characterized mainly by intrauterine and postnatal growth retardation and dysmorphic features. The characteristic facial appearance includes a small triangular 1 Current address: Ingenium Pharmaceuticals AG, Lochhamer Strasse 29, 82152 Martinsried, Germany. 2 To whom correspondence should be addressed. Telephone: ⫹49/345/ 5574291. Fax: ⫹49/345/5574293. E-mail: ingo.hansmann@medizin. uni-halle.de.

0888-7543/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

face with frontal bossing, downturned corners of the mouth, a small chin, and low-set ears. Other features may include skeletal asymmetry, clinodactyly or brachydactyly of the little finger, cafe´-au-lait spots, syndactyly between the second and third toes, and muscular hypotrophy/-tony. Growth studies in 386 cases revealed a mean adult height of 151.2 cm in males and 139.9 cm in females (Wollmann et al., 1995). The genetic etiology of the syndrome is unknown and seems to be heterogeneous. Most cases occur sporadically, but in some cases familial occurrence has been described, indicating a genetic cause of the syndrome. In some families an autosomal recessive mode of inheritance was suggested (Rimoin, 1969; Fuleihan et al., 1971; Robichaux et al., 1981; Nair and Sabarinathan, 1984; Davies et al., 1988; Teebi, 1992), whereas other cases pointed to a dominant inheritance (Duncan et al., 1990; Zanchetta et al., 1990; Al-Fifi et al., 1996). About 350 RSS patients have been described, but the incidence of the syndrome is still unknown (Wollmann et al., 1995). Several chromosomal abnormalities have been reported in patients with features suggestive for RSS. These abnormalities include partial duplications of 1q (Kennerknecht and Rodens, 1991), deletions within 8q (Schinzel et al., 1994), deletions of distal 15q or ring chromosome 15 (Wilson et al., 1985; Francke et al., 1988; Roback et al., 1991; Dolon, 1993; Tamura et al., 1993; Rogan et al., 1996), and deletions within chromosome 18p (Christensen and Nielsen, 1978; Niklaus et al., 1992). Furthermore, maternal uniparental disomy of chromosome 7 (mat UPD7) in approximately 7% of RSS patients has been reported (Kotzot et al., 1995; Langlois et al., 1995; Shuman et al., 1996; Preece et al., 1997, 1999; Eggermann et al., 1997), suggesting that this chromosome harbors at least one locus for RSS. Recently reported duplications/inversions on this chromosome led to the supposition that the responsible region includes 7p13–p12 (Joyce et al., 1999; Monk et al., 2000). A candidate gene for RSS within this region is GRB10 (Yoshihashi et al., 2000).

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A PHYSICAL AND TRANSCRIPT MAP OF CHROMOSOME 17q23– q24

Furthermore, two probands with the typical RSS phenotype and a balanced autosomal translocation involving chromosome 17q were reported in association with RSS (Ramirez-Duen˜as et al., 1992; Midro et al., 1993). The proband described by Midro et al. (1993) has a de novo 1q;17q translocation, whereas the proband described by Ramirez-Duen˜as et al. (1992) inherited her 17q;20q translocation from the phenotypically normal father. Because both translocation breakpoints on chromosome 17 were cytogenetically determined in band 17q25, we suggested that sequences around the breakpoints on chromosome 17 are related to RSS. To identify a gene for RSS in this region by positional cloning, a contig containing yeast artificial chromosomes (YACs) and several types of bacterial clones [P1 artificial chromosomes (PACs), bacterial artificial chromosomes (BACs), and cosmids] was developed for the translocation breakpoint segment. Here, we report on the physical and transcript mapping of the critical region for the RSS translocation breakpoint on chromosome 17q23– q24. Our data provide the basic tool for the final identification of the presumed RSS gene in this region. The construction of the physical map allowed us to isolate several novel markers and to refine significantly the localization of the supposed RSS locus. The high-resolution physical mapping also led to the localization of the gene for karyopherin ␣-2 (KPNA2) in close proximity to the analyzed translocation breakpoint. Therefore, this gene is a positional candidate gene for RSS. MATERIALS AND METHODS Isolation of genomic clones. YAC clones positive for the previously mapped sequence-tagged sites (STSs) and expressed sequence tags (ESTs) (Dib et al., 1996; http://www.genome.wi.mit.edu/) in the region 17q21– qter between markers D17S790 and D17S785 were provided by the Centre d’Etude du Polymorphisme Humain (CEPH) in Paris. Total yeast DNA from individual clones was extracted and stored in agarose plugs. For PCR and fluorescence in situ hybridization (FISH), YAC DNA was isolated from agarose plugs by agarase treatment according to the manufacturer’s recommendation (Roche Molecular Biochemicals). The chimerism of YAC clones was analyzed by the metaphase FISH technique. Clones mapping to 17q23– q24 were used for further mapping. The presence of STSs and ESTs (Dib et al., 1996; http://www.genome.wi.mit.edu; http://www.ncbi.nlm. nih.gov/genemap/) in the YACs was tested by PCR. PCR pools of the RPCI1 PAC library (Ioannou et al., 1994) were obtained from the UK HGMP Resource Centre, and several STSs and ESTs were used for library screening. High-density gridded membranes containing cosmid clones from chromosome 17 (library No. 105) were provided by the Resource Center/Primary Database (Berlin, Germany) and screened with different 32P-radiolabeled PCR products. DNA of PACs and cosmids was prepared using standard procedures. FISH. Metaphase chromosomes were obtained from phytohemagglutinin-stimulated human peripheral blood lymphocytes and EBV cells by standard techniques. Probes derived from YAC/PAC/ cosmid clones were labeled with biotin-16 – dUTP (Roche Molecular Biochemicals) by nick-translation. Hybridization and immunofluorescence detection of biotin in FITC were performed using standard procedures (Pinkel et al., 1986; Lichter et al., 1988). Finally, the slides were mounted in Vectashield containing DAPI (0.2 ␮g/ml) and propidium iodide (1 ␮g/ml). Digital images were taken with a CCD

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camera placed on an Axioplan 2 fluorescence microscope equipped with multibandpass filters. Estimation of clone insert sizes by pulsed-field gel electrophoresis. High-molecular-weight YAC DNA was prepared in low-meltingpoint agarose and separated by standard pulsed-field gel electrophoresis (PFGE). Gels containing YAC clones were blotted and hybridized with radiolabeled Cot-1 DNA. PAC DNA was digested with NotI to release the insert prior to PFGE, and the fragments were visualized by ethidium bromide staining. Generation of new STSs. To establish STSs from the termini of YACs, PACs, and cosmids, clone insert end-fragments were isolated by an Alu vector PCR-based method (Nelson et al., 1989) and sequenced. New STSs were designed from the nonrepetitive end sequences using Primer software (version 0.5, Whitehead Institute/ MIT). The sequences used to generate novel STSs and primer sequences have been submitted to the EMBL/GenBank Data Libraries (for accession numbers see Table 1). Isolation of PCR products. PCR products generated from single clones were isolated from 8% polyacrylamide gels by incubating crushed DNA bands for 1 h at 65°C. After a centrifugation step, the DNA was precipitated from the supernatant, dissolved in a smaller volume, and directly sequenced. Alternatively, PCR products were isolated from agarose gels by centrifugation of the DNA through a filter. Southern analysis. DNA from clones and genomic DNA were digested with several enzymes and electrophoresed on 0.8% agarose gels. Southern blotting and hybridization were carried out according to standard protocols. Filters were probed with radiolabeled PCR products, with clone fragments isolated from agarose gels and with whole clone DNAs. Filters were stripped and reprobed. Mapping of candidate genes. Radiolabeled probes used for Southern hybridization on blots with EcoRI-digested YAC DNA were a human PRKCA cDNA insert (GenBank Accession No. X52479) and a PCR product containing the coding region of human CYB561 (GenBank Accession No. U06715). For genes associated with an STS, PCR screening of genomic clones was performed with primers that are available upon request from the authors.

RESULTS

Construction of the Physical Map To assign the chromosome 17 breakpoint of the proband with the 1;17 translocation (Midro et al., 1993) to a specific chromosomal area, nine cytogenetically mapped cosmids (Inazawa et al., 1993) were used for FISH analyses on metaphases of this proband. Two cosmids, which were previously mapped to 17q23.3 (cCI17-696 and cCI17-743), yielded signals on the der(17), indicating their localization proximal to the breakpoint. Seven cosmids from segment 17q24 – qter (cCI17-509, cCI17-591, cCI17-815, cCI17-464, cCI17546, cCI17-469, and cCI17-519) showed signals on the der(1) only, thus reflecting a localization distal to the breakpoint. Therefore, the breakpoint is enclosed in the region 17q23.3– q24. This result deviates from the cytogenetic analysis carried out by Midro et al. (1993), who localized the breakpoint within 17q25. YACs were retrieved from the CEPH (http://www. cephb.fr/infoclone.html) and the Whitehead Institute for Medical Research (http://www-genome.wi.mit.edu/ cgi-bin/contig/phys_map) databases. Using FISH analysis, a total of 33 YACs from the region 17q22– q25 were localized with regard to the translocation break-

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176 TABLE 1 Newly Developed STSs STS name

Accession No.

83t7 83sp6 696t3 696t7 901ura 306sp6 875trp 188sp6 120t7 2A3t3 58t7 112t7 KPNA2-E1 KPNA2-E2 CF20bt7 KPNA2-3⬘ 81t7 293t7 241t7 CF58t7 7t7 293sp6 241sp6

AJ296823 AJ296824 AJ296841 AJ296842 AJ296838 AJ296825 AJ296839 AJ296826 AJ296827 AJ296840 AJ296848 AJ296829 AJ296843 AJ296844 AJ296836 AJ296845 AJ296830 AJ296831 AJ296832 AJ296837 AJ296833 AJ296834 AJ296835

point. STS content mapping of 17 selected YACs from the breakpoint region was performed with the available chromosome 17q markers. YACs were assembled into a contig map accordingly. To build a more detailed map and to design additional markers to narrow the breakpoint region further, we screened a pooled PAC library (Ioannou et al., 1994). A total of 19 STSs known to map to our region of interest were used for library screening: 5 of them represent published markers (WI-9571, D17S1870, stSG3721, stSG2549, and WI-9461), whereas the remaining 14 STSs (306sp6, TRP875, 188sp6, 2A3t3, 58t7, 112t7, KPNA2-E1, KPNA2-E2, CF20bt7, KPNA2-3⬘, 81t7, 293t7, CF58t7, and 241sp6) were developed during this study. In addition, two PAC clones (120L21 and 250M4) previously isolated for the genes PRKCA and PRKAR1A (Ba¨rlund et al., 1998) were included in the map. Furthermore, 2 cosmids (ICRFc105D0337D1 and ICRFc105G1042D1) were derived by screening the ICRF chromosome 17 cosmid library with hybridization probes from the breakpoint region. Twenty novel STSs (Table 1) for the confirmation of clone overlaps and for further screening of clones were designed by sequencing of Alu-PCR end products starting from selected clones. Furthermore, three loci (KPNA2-E1, KPNA2-E2, and KPNA2-3⬘) were derived from the genomic sequence of KPNA2 determined in our laboratory (S. Do¨rr, unpublished results). The localization of all YAC and PAC clones and of selected cosmid clones with respect to the translocation breakpoint was determined by FISH. Surprisingly, some clones from the proximal end of our physical map were found to be positive for markers from the distal

end of the map and vice versa. These initial results suggested that regions on both sides of the breakpoint shared a high degree of sequence homology or identity. To examine directly whether sequences were repeated within our region of interest, we sequenced isolated PCR products of seven loci generated from clones located distal or proximal to the breakpoint. With the exception of one T/A polymorphism within the sequence of the locus (WI-9571), we detected no sequence variations between clones located on one side of the breakpoint but the respective sequences obtained from clones located on the other side of the breakpoint showed specific differences. By this analysis we were able to confirm that these loci are duplicated within our region of interest. The sequence identity between the duplicated loci located on both sides of the breakpoint ranges between 82 and 98% (Table 2). Because clones located proximal to the breakpoint give only weak PCR signals with primers for locus 293sp6, we were not able to sequence the respective PCR products. Given that this locus is also duplicated, we were able to distinguish between eight duplicated loci and to assign these to our map definitively. By sequence comparisons of the duplicated loci within genome databases, we found that the loci 293t7 and WI-9461 also show sequence homologies to the sequenced BAC clone RP11-271K11 (GenBank Accession No. AC005562), which is also located on chromosome 17 but outside of our contig. Therefore, chromosome 17 harbors at least one more region with homology to the duplicated sequences within 17q23– q24. To estimate the physical size of the contig, we determined the insert size of most YAC and PAC clones by PFGE (Fig. 1). The insert sizes of all clones containing the minimal tiling path are adding to a maximum size of approximately 4 –5 Mb. Eleven BAC clones that have been sequenced at the Whitehead Institute/MIT were found by performing BLAST comparisons with the STS sequences (Fig. 1). TABLE 2 Duplicated STSs Proximal STS/cases [Accession No.]

Distal STS/cases [Accession No.]

(KPNA2-E2)/2 [AJ296828] (KPNA2-3⬘)/3 [AJ296847] (293t7)/1 [AJ296846] (WI-9461)/3 [AJ296849] 2A3t3/2 [AJ296840] (7t7)/2 [AJ296850] WI-9571/3 [Z39152]

KPNA2-E2/6 [AJ296844] KPNA2-3⬘/4 AJ296845] 293t7/2 [AJ296831] WI-9461/4 [Z39035] (2A3t3)/2 [AJ296852] 7t7/4 [AJ296833] (WI-9571)/3 [AJ296851]

a

Primers were excluded.

Identities a 185/205 (90%) 93/97 (95%) 60/62 (96%) 100/121 (82%) 71/72 (98%) 185/199 (92%) 221/235 (94%)

FIG. 1. The genomic clone contig of the region 17q23– q24. The location of the breakpoint region of our RSS proband is marked by a hatched bar. Markers used to construct the physical map are indicated above the line representing chromosome 17q23– q24. The orientation of the map is centromere to telomere, left to right. The polymorphic genetic markers are characterized by asterisks, whereas genes or ESTs are indicated by open circles. Duplicated loci are marked by boxes. Clones are positioned at the bottom; the YACs are indicated by black bars, the sequenced BACs (with GenBank accession numbers) are indicated by red bars, PACs are indicated by blue bars, and cosmids are indicated by green bars. Loci that were mapped to the clones by PCR or hybridization (PRKCA) are indicated by vertical bars. ESTs stSG35126, H59098, CACNG5, CACNG4 –5⬘, CACNG4 –3⬘, stSG48128, stSG26107, KIAA0054, R95427, stSG52950, stSG41045, stSG45498, stSG28454, stSG36055, sts-N33189, KIAA1001, and stSG22116 were assigned to the contig by sequence comparisons with the known sequences of the BAC clones. Solid bars indicate confirmed marker content, dashed lines represent the possible extent of the clones, and deletions of YACs are shown by open bars. Dots at the ends of BACs represent sequence overlap with another sequenced BAC clone. The final order of STSs, polymorphic markers, and transcripts was generated by combining data from mapping experiments and BLAST results from homology searches and clones sequenced by the Whitehead Institute. A caret above markers indicates that these markers could not be ordered relative to one another. The following YACs are chimeric by FISH: 765F4, 929D11, 955H4, 680F6, 742A4, 875C4, and 804H7. The map is not drawn to scale.

A PHYSICAL AND TRANSCRIPT MAP OF CHROMOSOME 17q23– q24

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The sequence information obtained from these BAC clones was used to refine and confirm our mapping data. The smallest clone showing FISH signals on both derivative chromosomes is PAC clone 81B9, with an insert size of 95 kb. Due to the duplicated regions on both sides of the translocation breakpoint, we intended to exclude the possibility that the FISH signal of this clone is due to a duplication of target sequences. Therefore, it was necessary to prove that clone 81B9 indeed overlaps with clone 112L5 located proximal to the translocation breakpoint and with clone ICRFc105D0337D1 located distal to this breakpoint. To address this question, we sequenced the PCR products of the loci 112t7 and CF20bt7 from PAC 81B9. The sequence of these loci generated from clone 81B9 is completely identical with the respective sequence of clones 112L5 and ICRFc105D0337D1 and consistent with clone overlap. To determine the size of the overlapping segments from these clones, we performed Southern analysis. DNA of the respective clones was digested with several restriction enzymes (single and double digestions), and PCR products of the loci 112t7, KPNA2-E1, and KPNA2-3⬘ as well as DNA from clone 81B9 and all five KpnI insert fragments from this clone were used as probes. According to this analysis, the overlap between clones 112L5 and 81B9 was determined to be approximately 10 kb, whereas the overlap between clones ICRFc105D0337D1 and 81B9 is approximately 20 kb (Fig. 2). Thus, the breakpoint region was narrowed to a 65-kb region of 81B9, which is not contained within clones 112L5 and ICRFc105D0337D1. Furthermore, we investigated whether the translocation generated an abnormal genomic fragment in comparison to controls. Therefore, we used the five KpnI fragments containing the insert of PAC clone 81B9 as probes on blots with differentially digested genomic DNA from the patient and controls. Using MspI for cleavage, two KpnI fragments of clone 81B9 detected one band in the lane with the patient’s DNA but that was not present in the lanes for the two controls (Fig. 2). Thus, the translocation generates an abnormal genomic fragment. However, abnormal fragments were not detectable by using the enzymes BamHI, PstI, XbaI, and SacI. Since the two fragments of clone 81B9 that detect the abnormal MspI fragment are not adjacent to each other on the basis of our restriction map of clone 81B9, the translocation seems to be more complex. It should be mentioned that the 5⬘ end of KPNA2 is located on one of the KpnI fragments that detects an abnormal MspI fragment in the patient’s DNA (Fig. 2). Further characterization of the translocation breakpoint is in progress and will be reported elsewhere. Integration of Genes and Transcripts Genes and ESTs were selected from genome databases (http://www.ncbi.nlm.nih.gov/genemap/; http://

FIG. 2. (Top) KpnI restriction map of PAC clone 81B9, overlapping regions of clones 112L5 and ICRFc105D0337D1, and assignment of three loci. (Bottom) Southern blot of genomic DNA digested with MspI hybridized with two different KpnI fragments of PAC clone 81B9. DNA from the patient with a 1;17 translocation generated a 4.9-kb band (lane P), which was not present in two different controls (lanes C1 and C2).

gdb.org/; http://www.genome.wi.mit.edu) and from the literature by location within cytogenetic, physical, and genetic maps or by comparison with the syntenic region of mouse chromosome 11. Twenty-one genes, 1 pseudogene, and 10 ESTs were tested by PCR against the YAC clones. Of these, 4 known genes (APOH, PRKCA, CACNG1, and PRKAR1A), the pseudogene (G6PDL), and 7 ESTs (WI-9571, WI-9269, WI-8032, stSG2005, stSG3721, stSG2549, and WI-9461) could be mapped within our contig. Genes and ESTs that matched no clone were as follows: ACOX1, DCP1, FDXR, GAA, GALK1, GH1, ICAM2, ITGA2B, ITGB3, MPO, MYL4, PECAM1, RPL27, SCN4A, SSTR2, stSG9224, TBX2, TK1, WI-15726, WI-7618, and ZNF147. In addition, cDNA inserts of human PRKCA and CYB561 were used as probes for hybridization of blots with digested YAC DNA. Through this analysis, we were able to localize the gene encoding PRKCA within our map, whereas CYB561 was located on YAC 923H8 and therefore proximal to our region of interest. Furthermore, generation of new sequence data from cosmid ICRFc105D0337D1 and PAC 81B9 led to the localization of the gene encoding karyopherin ␣-2

A PHYSICAL AND TRANSCRIPT MAP OF CHROMOSOME 17q23– q24

(KPNA2) within our map. The PAC clone 81B9 overlaps with the translocation breakpoint and contains the entire KPNA2 gene, whereas the cosmid clone ICRFc105D0337D1 is localized distal to the breakpoint and harbors approximately 5 kb of the KPNA2 gene. Since more than 5 kb of the KPNA2 gene are localized within the narrowed breakpoint region of approximately 65 kb in size, this gene is located in close proximity to the breakpoint (data not shown). Therefore, KPNA2 is a positional candidate gene for Russell– Silver syndrome. Three loci derived from the genomic sequence of this gene (KPNA2-E1, KPNA2-E2, and KPNA2-3⬘) (derived in our laboratory) were integrated into the map. Moreover, by sequence comparisons with BAC clones sequenced from the Whitehead Institute, we were able to assign an additional 15 loci for genes/ ESTs to our map (Fig. 1). DISCUSSION

The cytogenetic and molecular analyses of diseaseassociated balanced chromosome rearrangements constitute an efficient strategy for mapping and cloning disease genes. We are investigating a RSS-specific balanced de novo translocation, cytogenetically defined as t(1;17)(q31;q25) (Midro et al., 1993), which by our molecular analysis can be refined as t(1;17)(q24;q23– q24). We assume that the gene responsible for the syndrome is disrupted by the translocation; i.e., it is localized on either chromosome 1 or chromosome 17. Since a second proband with a translocation involving chromosome 17q was reported by Ramirez-Duen˜as et al. (1992), we focused our search for a RSS gene to the breakpoint region on chromosome 17 by applying a positional candidate gene approach. This is made possible by the large amount of EST and gene sequence information provided by genome databases. However, this strategy relies heavily on the existence of a detailed physical map of the interval in question. Therefore, we have created a defined physical and EST-based transcript map for the chromosomal area 17q23– q24 harboring the translocation breakpoint of our proband. Furthermore, genomic clones arrayed in a contig also provide a resource from which novel candidate sequences can be recruited. Our clone contig consists of YACs, PACs, BACs, and cosmids and has been confirmed by STS content mapping using previously only roughly assigned STSs and ESTs as well as several novel PAC/YAC/cosmid endsequence-derived STSs (see Fig. 1). The contig assembly was further confirmed by FISH analyses on metaphases of the proband with 1;17 translocation. The length of the contig was estimated to be 4 –5 Mb. This estimation was based on the clone size determinations by PFGE. The contig is covered by at least two clones at most STSs. However, one gap remains between the markers 241SP6 and PRKAR1A. Evidence is provided for duplications of several loci within our region of interest (Fig. 1). The observation of

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several low-copy repeat sequences must be taken into account as a possible explanation for the genealogy of RSS. Such sequences may confer DNA instability at these regions by facilitating illegitimate recombination events during meiosis. The gene order and content of the region also provide insight into ancient duplication events within the chromosomal region 17q23– q24. Because gene duplication often generates paralogues that remain in close syntenic proximity (tandem duplication), Burgess et al. (1999) searched for Ca 2⫹ channel ␥-subunit genes in proximity to CACNG1 and identified the genes CACNG4 and CACNG5. This may reflect an ancient duplication event that occurred during evolution. It will be of interest to determine whether other genes within this region have also undergone duplication. Homology searches did not reveal extensive conserved synteny for gene loci of segment 17q23– q24 with the mouse. The mouse genome databases, The Jackson Laboratory (http://www.informatics.jax.org/) and the Whitehead Institute (http://www.genome.wi.mit.edu/), provide information about the mouse genomic region homologous to human 17q23– q24. The mouse genes Apoh, Prkar1a, and Pkca, homologous to genes within our candidate region, were mapped to mouse chromosome 11 (63– 68 cM, The Jackson Laboratory). Their order, however, differed somewhat, possibly due to rough mapping data or rearrangements during evolution. Furthermore, some human genes (ICAM2, SCN4A, DCP1, CYB561, GH1, MYL4, ITGA2B, and ITGB3) were mapped outside of our contig, whereas the homologous mouse genes were mapped to an area of 63– 68 cM on mouse chromosome 11 (The Jackson Laboratory). Moreover, the mouse homologue of human KPNA2, which is located in close proximity to the translocation breakpoint analyzed in this study, is located on mouse chromosome 3 (50.2 cM; The Jackson Laboratory). Our detailed physical map allowed us to isolate and precisely map several new markers from clone ends, which enabled the significant refinement of the translocation breakpoint of our RSS proband. This breakpoint was narrowed to a 65-kb region between the newly derived markers 58t7 and CF20bt7. Within this region, we identified part of the gene encoding KPNA2. This gene is involved in nuclear import of proteins (for review see Go¨rlich and Mattaj, 1996) and belongs to a multigene family with at least six members (Cortes et al., 1994; O’Neill and Palese, 1995; Cuomo et al., 1994; Weis et al., 1995; Takeda et al., 1997; Seki et al., 1997; Nachury et al., 1998; Ko¨hler et al., 1999). Transcription factors and many hormones involved in growth regulation depend on nuclear transport to exercise their function. KPNA2 may therefore play a role in the etiology of RSS. We are currently testing the positional candidate gene KPNA2 for mutations in RSS patients to analyze whether this gene is involved in the genealogy of RSS. Chromosome 17q23– q24 also plays an important role in a number of more common diseases. Sawcer et

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al. (1996) and Kuokkanen et al. (1997) provided suggestive evidence of a predisposing locus for multiple sclerosis within chromosomal region 17q22– q24, with the maximum lod score (Z max) occurring with D17S807 and D17S795. These markers are included in our physical map. Furthermore, comparative genomic hybridization analyses have defined a chromosomal site at 17q22– q24 that is often overrepresented in breast cancer, neuroblastoma, and several other tumor types (Ried et al., 1995, Kivipensas et al., 1996, Weber-Hall et al., 1996, Weber et al., 1997, Ba¨rlund et al., 1997, Tirkkonen et al., 1998). By means of FISH, Ba¨rlund et al. (1997) showed that the increased copy number in breast cancer is due to high-level amplification of two separate regions at 17q23. Our detailed physical map of the region 17q23– q24 would provide an important tool for the positional cloning of the genetic defects causing the disorders mentioned above. ACKNOWLEDGMENTS We thank the patient and his parents for contributing samples for this study. We also thank Dietmar Schlote for helpful comments and Daniel Barthelmes for assistance with preparation of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft.

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