Proc. Natl. Acad. Sci. USA Vol. 91, pp. 6669-6673, July 1994 Genetics Bloom syndrome: An analysis of consanguineous families assigns the locus mutated to chromosome band 15q26.1 (homozygosity mapping/nka analysls/genomic instability/chromosome-breakge syndrome) JAMES GERMAN*, ANNE MARIE ROE*, MARK F. LEPPERTt, AND NATHAN A. ELLIS* *Laboratory of Human Genetics, New York Blood Center, New York, NY 10021; and tDepartment of Human Genetics, University of Utah, Salt Lake City, UT 84132 Communicated by Stanley M. Gartler, March 28, 1994

ABSTRACT By the principle of identity by descent, pa- tured BS cell lines exhibit hypersensitivity to certain DNA- rental consanguinity in individuals with rare recessively trans- damaging agents-e.g., mitomycin C, N-nitroso-N-ethyl- mitted disorders dictates homozygosity notjust at the mutated urea, and ethyl methanesulfonate (7-9). (iv) Altered activity disease-assoiated locus but also at sequences that flank that of several enzymes employed in DNA replication, DNA locus closely. In 25 of 26 individuals with Bloom syndrome repair, or both have been detected in some BS cell lines, examined whose parents were related, a polymorphic tetranu- including DNA ligase I, uracil DNA glycosylase, topoisom- cleotide repeat in an intron of the protooncogene FES was erase, 06-methylguanine methyltransferase, N-methylpurine homozygous, far more often than expected (P < 0.0001 by x2). DNA glycosylase, thymidylate synthetase, and superoxide Therefore, BLM, the gene that when mutated gives rise to dismutase (10-18). Therefore, elucidation of the primary Bloom syndrome, is tightly linked to FES, a gene whose defect probably will identify a factor ofcentral importance in chromosome position Is known to be l5q26.1. This successful the maintenance ofgenomic stability. The factor defective in approach to the aignment ofthe Bloom syndrome locus to one BS should have the potential ofaltering the activities ofmany short segment of the human genome simultaneously (i) dem- enzymes whose coordinated action ensures that stability. onstrates the power ofhomozygosity mapping and (it) becomes The investigation reported here permits the assignment of the first step in a "reverse" genetics defmition of the primary BLM to a specific sub-band-15q26.1-by the analysis of defect in Bloom syndrome. DNA from a small population ofpersons with BS who are the progeny ofconsanguineous marriages. This regional mapping Bloom syndrome (BS) (1, 2) is a rare autosomal recessive of BLM was carried out as part of a larger effort aimed at disorder the predominating clinical feature of which is a identifying BS's primary defect. In addition, it serves as a test well-proportioned smallness ofthe body. The locus mutated, of the value of an until-now-little-used method of detecting here named BLM, has long been recognized to be of central linkage in man, homozygosity mapping.* importance in the maintenance of genomic stability because persons with BS, blm/blm, feature a remarkable degree of genomic instability. In somatic cells having such a mutator MATERIALS AND METHODS genotype, increased numbers of different types of spontane- Families. Between 1960 and 1991, genetic and clinical ous mutations arise at various sites, mutations that qualita- information from essentially all families throughout the world tively are similar to those that arise spontaneously in normal affected with BS has been accumulated in files maintained in individuals. In addition to locus-specific mutations, in- the Laboratory of Human Genetics at the New York Blood creased numbers of microscopically visible chromosome Center-the Bloom's Syndrome Registry (20-23). From the mutations at randomly distributed sites are present in meta- outset, stringent clinical and cytogenetic diagnostic criteria phase chromosomes-gaps, breaks, and rearrangements. In for accessioning persons to the Registry have been utilized. particular, a striking excess ofchromatid exchanges occurs in For the present investigation, those families from the Reg- BS, including homologous chromatid interchange which is istry were studied (i) in which the parents of the affected cytogenetic evidence of somatic crossing-over (3); therefore, person(s) were known to be related and (ii) from whom cells a mutation that does arise at any particular site in a BS of some type from the affected person(s) had been stored or somatic cell also is at an increased risk ofbecoming homozy- could now be obtained. Fig. 1 depicts the 21 families exam- gous. Other than the generalized growth deficiency ofclinical ined. The relatedness ofthe parents varies from first to fourth BS, which itself may be the consequence of an excessive cousins-e.g., the 52(PaDu) and 30(MaKa) families, respec- number oflethal mutations, the most important manifestation tively. The geographic or ethnic origins ofthe families are the of the genomic instability of the somatic cells is neoplasia; following: non-Jewish American with mixed Western Euro- cancers ofa wide variety of sites and types emerge unusually pean (six) and West African (two) ancestry; non-Jewish early and frequently in persons with BS. Italian (three), German (two), Turkish (two), Japanese (two), The primary defect in BS is unknown, and this has limited French Canadian (one), and Spanish (one); and Ashkenazi its usefulness as a model for studying neoplastic transforma- (one) and Sephardi (one) Jewish. tion and progression. Results of many studies do point to a Sources of DNA. Lymphoblastoid cell lines and blood disturbance of DNA replication. (i) The cytogenetic abnor- leukocytes stored in liquid nitrogen in the Bloom's Syndrome malities (4) are best explained on the basis of excessive Registry were the major source ofDNA. In some cases fresh exchange of chromatids via an error-prone mechanism acti- whole blood samples were obtained specifically for this vated during S-phase replication. (ii) Replication-fork pro- study, and in one case, archival (pathology) material of a gression is retarded (5), and an unusual size distribution of deceased person was employed. DNA replication intermediates is found (6). (iii) Some cul- Abbreviations: BS, Bloom syndrome; lod, logarithm of odds. The publication costs ofthis article were defrayed in part by page charge *The data reported here were presented as a poster at the 42nd payment. This article must therefore be hereby marked "advertisement" Annual Meeting of the American Society of Human Genetics, San in accordance with 18 U.S.C. §1734 solely to indicate this fact. Francisco, November 9-13, 1992 (19). 6669 Downloaded by guest on September 28, 2021 6670 Genetics: German et al. Proc. Natl. Acad. Sci. USA 91 (1994)

5(JaOa) " 7(RoTa) * 17(ChSm) * 21(RaRe) 22(El~a) 3*M~)*' 51(KeMc) M

00 ao ba rd0 *0 0 pi. 60(AnDa) 61(DoHop) ' 74(OmAy) 81(MaGrou) * 92(VaBia) FIG. 1. Abbreviated pedigrees of the families studied. Each fam- * ** ** ** ily is identified by the index case's designation in the Bloom's Syn- 6T , drome Registry (20-23). For ho-

th0 * l mozygosity mapping, the geno- type of every individual with BS * 4 was examined, save from the sec- 96(HiOk) 11O(MaKu) 1IMDaDern) - 122(RoHer) -* 127(TaLAI) 14K) - 149(Se~ - ond affected siblings in the fami- D lies of 17(ChSm) and 149(SeSat). 0 Dots indicate individuals from [uT aT) . 0~ whom genotype information was q analyzed by the program LINK- * 0 * AGE. Filled symbols, individuals 0 with BS; asterisk, a family in the * 0 initial phase of the study (see text).

DNA Genotyping. Selected DNA probes for loci known to one parent in each pedigree was duplicated, arbitrarily the map to chromosome 15 were obtained (Fig. 2) for use in mother of the affected. hybridization studies. For Southern analysis, 1-10 pug of To estimate the recombination fraction, the total numberof DNA was digested with restriction enzymes according to the meioses (the denominator in the fraction) was estimated by supplier's recommendations, and the DNA fragments were counting each ancestor in the loop ofa pedigree (Fig. 1) as one separated by electrophoresis through 0.8% agarose (FMC) meiotic event, except for the ancestor at the top of the loop gels in 89 mM Tris/89 mM boric acid/2 mM EDTA. DNA was who was counted as two, and summing over all pedigrees. transferred to Hybond N+ nylon membrane (Amersham) as Affected siblings for which genotypic information was ob- described (27). Hybridization was 1 M NaCl/1% SDS/10%o tained were counted as two additional meioses. The total was dextran sulfate containing sheared and denatured salmon then multiplied by the frequency of homozygosity of the testis DNA (0.25 mg/ml) and denatured labeled probe (2-10 polymorphisms under study; this adjustment was made be- ng/ml). Probes were labeled by the random hexamer method cause recombination between BLM and any test locus would using [a-32P]dCTP (28). When necessary, the probe was not be detected in homozygotes. The number of heterozy- prehybridized with Cotl DNA (BRL) in 1 ml ofhybridization gotes at any test locus was taken to be the number of solution according to the manufacturer's instructions. recombinants and became the numerator of the fraction. PCR was carried out in 10-25 A4 of 50 mM KCI/0.5-2.5 mM MgCl2/10 mM Tris HCl, pH 8.3/0.1% gelatin/100 nM THEORY tetramethylammonium chloride/200 jAM dNTPs (Pharmacia) with 1.25 units of Taq polymerase (Cetus or Boehringer The principle behind the approach to detecting linkage by Mannheim) and 4 pmol of each oligonucleotide primer. homozygosity mapping is the following. An individual with Oligonucleotide sequences specific for selected loci on chro- BS who is the offspring of a consanguineous union is ex- mosome 15 (Fig. 2) were obtained from Human Gene Map- pected to be homozygous for the mutation at BLM- ping 11 (29); sequence information concerning loci ACTC, specifically, not to be a compound heterozygote should more CYP, FBN, and D15S87 was generously provided by A. M. than one disease-associated mutant allele at that locus be Bowcock (University of Texas Southwestern Medical segregating in the population. Similarly, the base sequences School). Two hundred femtomoles of oligonucleotide end- that flank the mutant locus closely usually will be homozy- labeled with [-t32P]ATP or [y-33P]ATP (NEN) was intro- gous, including any polymorphic sequences contained in duced into the PCR mixture. Cycle times and temperatures those segments of DNA. Accordingly, the goal here was to were taken from the literature and modified as necessary. identify polymorphic DNA sequences that were homozygous The PCR products were separated in a 6.8 M urea/6% more often than expected in a small group of "consanguin- acrylamide (Long Ranger, AT Biochem, Malvern, PA) se- eous" individuals with BS. quencing gel. Gels and blots were exposed to film (Kodak This approach to the detection of linkage was considered XAR5) with intensifying screens. particularly suitable for mapping BLM for seven reasons. (i) Pedigree Analyses. The genotypes of 26 of the 28 individ- The syndrome is rare and is recessively transmitted (32), and uals with BS (H and * in Fig. 1) were determined at one locus the penetrance and expressivity of homozygosity for the BS after another. The raw data periodically were examined mutation are effectively complete. (ii) The clinical diagnosis visually for possible excesses of homozygosity. The data can be confirmed by demonstrating a greatly elevated sister- finally were analyzed "by hand" with the x2 test. The chromatid exchange frequency in somatic cells, a finding that rationale for this approach is presented below, under Theory. is unique to BS (4). (iii) Parental cons ty is increased In addition, conventional linkage analysis was carried out in BS except among Ashkenazi Jews, where the mutation is with the program LINKAGE (30, 31). Genotype information relatively common, so that cumulation of an adequate num- was obtained for this analysis from DNA samples from ber of informative families for analysis (i.e., those with individuals marked with dots on Fig. 1. To analyze consan- parental consanguinity) was feasible (and see vii below). (iv) guineous families with LINKAGE, the loops between the Because genetic complementation studies indicate that a parents and their common ancestors had to be broken, and single locus is mutated in BS (33), the genotypes of every Downloaded by guest on September 28, 2021 Genetics: German et al. Proc. Natl. Acad. Sci. USA 91 (1994) 6671 PROBE LOCUS human chromosome 15 into BS cells had been reported to 3-21 D1SSJO * bring toward normal the elevated sister-chromatid exchange frequency (34). This observation, reported after our study 13 a IR4-3R DISSI _ 12 was underway-confirmed in the present work-suggested 189-1 D15JS3 * 18.5 11.2 that the search for linkage of BLM and some polymorphic 11.1 CMWI D15S24 - 17.6 DNA sequences might be directed to chromosome 15 and, 11.1- 635/636 ACTC - 11.2 8.3 fortunately, limited considerably the scope of the project as THBS 1 .PCR THBS1 - originally designed. (Vii) Finally, contact had been estab- 12 4.9 13 THH 114 D1SS25 - lished and maintained with the majority of families in which 14 BS was diagnosed (20-23), so that reliable clinical, genetic, FIB15 FBN t 3.6 and cytogenetic information existed about affected persons 15 MSI1-14 DJSSJ t throughout the world, as did cultured cells and access to fresh CYP 1 9. PCR CYPI9 blood or archival cellular material. 21.1 DPI51 D15S2 t 21.2 15.2 HLIP LPC2A * RESULTS 21.3 EFZ33 D15S45 22.1 Fig. 1 depicts the relatedness of the parents of persons with 22.2 EFD49.3 D15S29 BS in the 21 families that were studied. In the initial phase of 22.3 EFD52. 1 D1SS36 the study, 16 affected individuals from 15 of the 21 families EKZ104 D15S30 (those families marked by asterisks in Fig. 1) were analyzed 23 by using the polymorphic loci shown in Fig. 2. Fifteen of the YNM18.1 D1SS35 16 individuals proved to be homozygous at one, and only one, YNZ90.1 D1SS28 24 ofthese loci, a polymorphic tetranucleotide repeat positioned MCT149.2 D1SS34 in intron V of the protooncogene FES (35). 25 MCT46.2 D15S26 t With this initial evidence that BLM and FES were tena- THH55 D15S27 t ciously cosegregating in BS families, the FES polymorphism was examined in 10 additional individuals with BS, bringing 26.1 FES.PCR FES to 26 the total number ofaffected progeny ofconsanguineous 26.2 EFD85.7 D15S37 unions genotyped at that one locus. The frequency of ho- 26.3 i MFD49 D15S87 mozygosity at this efficiently polymorphic, six-allele locus JU201 D15S3 was significantly greater than the homozygosity detected in the general population (Fig. 3; Table 1). In the entire group FIG. 2. Idiogram of a G-banded chromosome 15 and the poly- of 26, only 1 individual was heterozygous for the FES morphic genes and anonymous DNA segments genotyped in this polymorphism, 30(MaKa) in Fig. 1. study, identified by locus and probe names. The polymorphic loci Thus, by homozygosity mapping, BLM and FES were include 17 restriction fragment length polymorphisms, 1 variable- shown to be tightly linked. Then, employing the program number tandem repeat, and 7 PCR-based polymorphisms. Physical LINKAGE (30, 31), we calculated pairwise logarithm-of-odds positions of regionally mapped loci are indicated by vertical lines. (lod) scores for each of the Order ofloci along the chromosome corresponds to their order on the polymorphic markers and BLM sex-averaged genetic map. Map distances are indicated (24-26). (Table 2). A lod score of 11.37 at a recombination frequency Asterisks identify loci that have not yet been placed on the genetic of 0.001 confirmed tight linkage between FES and BLM. map, and daggers identify loci that have been placed in confidence Positive lod scores supporting linkage were found in every intervals. family except one, the Ka family, indicating strongly that a single locus is mutated in BS. If we assume that 30(MaKa), family should provide linkage information. (v) At the time the who is a heterozygote at FES, is a recombinant, then the study was initiated, DNA-based molecular markers com- genetic distance between BLM and FES is 0.8%. posed a 5- to lO-centimorgan genetic map that covered much FES has been mapped by in situ hybridization to 15q26.1 of the human genome. (vi) Microcell-mediated transfer of (36-38). Given the tight linkage of BLM and FES, the

<, t30i , te

C3*.;n~~~~~~~~~~~~~~~.---_ _ - C2 - C3 - C4

FIG. 3. Autoradiograph of 33P-end-labeled PCR products separated by polyacrylamide gel electrophoresis, showing homozygosity at FES in 24 of 25 of the affected progeny of consanguineous couples examined. [Not represented on this gel is one additional person, 21(RaRe) (Fig. 1), and he also proved to be homozygous C2/C2.] Only 30(MaKa) is heterozygous, C2/C3. The index cases of affected families are identified by their Bloom's Syndrome Registry designations, as in Fig. 1; siblings of index cases when examined are identifiable by having the same final (surname) initials. The FES polymorphism consists of six alleles, C1-C6, of which C2, C3, and C4 are the most common, represented in 93% ofindividuals of Western European ancestry (35). Our own (unpublished) genotyping of 107 grandparents from 31 unrelated Centre d'Atude du Polymorphisme Humain (CEPH) families yielded the following allele frequencies ofFES: C1, 0.056; C2, 0.168; C3, 0.416; C4, 0.346; C5, 0.005; C6, 0.009. Downloaded by guest on September 28, 2021 6,6712 Genetics: German et al. Proc. Natl. Acad. Sci. USA 91 (1994)

Table 1. x2 analysis of the observed vs. expected frequencies of homozygotes and heterozygotes for polymorphic DNA markers on chromosome 15 in individuals with BS whose parents are cousins Heterozygosity No. of heterozygotes in BS No. of homozygotes in BS in general Locus population Observed (0) Expected (E) (O - E)2/E Observed (0) Expected (E) (O - E)2/E X2 DISSJO 0.28 1 4.2 2.44 14 10.8 0.95 3.39 DJSSII 0.50 11 7.5 1.63 4 7.5 1.63 3.26 D15S13 0.43 8 6.5 0.35 7 8.5 0.26 0.61 D15S24 0.52 9 7.3 0.39 5 6.7 0.43 0.82 ACTC 0.75 10 11.3 0.15 5 3.7 0.46 0.61 THBSJ 0.56 6 8.4 0.69 9 6.6 0.87 1.56 D1SS2S 0.44 5 6.2 0.23 9 7.8 0.18 0.41 FBN 9 6 DJSS) 0.50 5 7.5 0.83 10 7.5 0.83 1.66 CYPI9 0.72 7 10.8 1.34 8 4.2 3.44 4.78* D15S2 0.42 2 5.9 2.58 12 8.1 1.88 4.46* LPC2A 0.52 10 7.8 0.62 5 7.2 0.67 1.29 D1SS4S 0.33 5 5.0 0.00 10 10.0 0.00 0.00 D15S29 0.37 5 5.6 0.06 10 9.4 0.04 0.10 D1SS36 0.31 4 4.3 0.02 10 9.7 0.01 0.03 DISS30 0.51 7 7.7 0.06 8 7.3 0.07 0.13 D1SS3S 0.36 5 5.0 0.00 9 9.0 0.00 0.00 D15S28 0.38 7 5.7 0.30 8 9.3 0.18 0.48 D1SS34 0.24 5 3.6 0.54 10 11.4 0.17 0.71 D15S26 0.25 4 3.5 0.07 10 10.5 0.02 0.09 DIS527 0.45 3 6.3 1.73 11 7.7 1.41 3.14 FES 0.67 1 14.1 12.17 20 6.9 24.87 37.04** D15S37 0.47 4 6.6 1.02 10 7.4 0.91 1.93 D15S87 0.86 13 12.0 0.08 1 2.0 0.50 0.58 D1S53 0.47 6 6.6 0.05 8 7.4 0.05 0.10 The value of heterozygosity of the loci that exists in the general population was obtained from the Welch Medical Library, except for the following: the heterozygosity ofACTC was obtained from A. M. Bowcock, and that ofFES was estimated from our own analysis of unrelated Centre d'Etude du Polymorphisme Humain (CEPH) families; population data are unavailable for the FBN polymorphism. Observations ofonly one sibling per family are included in the table, thus 15 "observeds" in the first phase of the study (see text). The number of observeds is sometimes <15 due to technical failures, whereas the observeds for FES is >15 as explained in the text. When the number of loci tested, 25, is taken into account, only the P value at FES is significant. *, 0.05 > P > 0.01; **, P < 10-8. chromosomal location of BLM is concluded also to be FES is considered improbable on biological grounds, and a 15q26.1. recently reported transfection experiment (42) may exclude this possibility. The analysis of BS reported here demonstrates that the DISCUSSION wedding ofclinical genetic observation-which includes ped- The mapping of rare recessive, disease-associated loci poses igree analysis-with recombinant DNA technology generates special problems, because of the paucity of affected siblings a powerful and efficient means of detecting linkage in rare in human families and because of the difficulty for any one autosomal recessive disorders, potentially ofparticular value geneticist to accumulate adequate numbers of affected fam- in the study of conditions in which the primary biochemical ilies for analysis. Four decades ago, Smith (39), in reviewing defects are unknown. Other reports support this conclusion the methods available for detecting linkage of rare autosomal (43-47). recessive loci associated with a disease, emphasized the An early reward ofthe assignment ofBLM to band 15q26.1 potential value of studying affected persons whose parents is the exclusion as candidates for the primary defect in BS of are consanguineous. Lander and Botstein (40, 41) reempha- all ofthe enzymes whose activities have been reported so far sized the potential value of the analysis of consanguineous to be abnormal in BS cells (mentioned above): the DNA families for homozygosity mapping of disease loci now that ligase I gene, LIGI, maps to band 19q13.3; uracil DNA many polymorphic DNA marker loci have been mapped in glycosylase, UNG, maps to chromosome 12; the topoisom- humans. Success in homozygosity mapping rare recessive erase genes TOP], TOP2A, and TOP2E map to 20q12-q13.1, disease genes depends on the density of polymorphic DNA 17q21-q22, and chromosome 3, respectively; 06-methylgua- loci on the genetic linkage map, the polymorphism informa- nine methyltransferase, MGMT, maps to 10q26; 3-methylad- tion content of those loci, the number of affected progeny of enine DNA glycosylase, AAG (or MPG), maps to chromo- consanguineous couples who are available for study, and the some 16; thymidylate synthetase, TYMS, maps to 18pll.31; degrees of relatedness of the parents of the affected individ- and, the superoxide dismutase genes SOD), SOD2, and uals. SOD3 map to 21q22.1, 6q21, and 4p16.3-q21, respectively. In the present study BLM and FES are concluded to be (Gene assignments were extracted from the Genome Data- tightly linked because recombination occurred between them base and On-line Mendelian Inheritance in Man in the Welch perhaps only once in the -120 meioses represented in the 21 Medical Library, Johns Hopkins University.) families that were studied. Identity by descent at BLM had Also, these results confirm genetic complementation stud- dictated homozygosity also at FES, a locus that displays ies (33) which showed that a single locus is mutated in families extensive heterozygosity in a random sample from the gen- that were ascertained through a person with clinical BS eral population. It is formally possible that BLM and FES are regardless of whether they were of Ashkenazi Jewish, non- one and the same gene; however, the identity of BLM and Jewish Western European, or Japanese ancestry. And, as Downloaded by guest on September 28, 2021 Genetics: German et al. Proc. Natl. Acad. Sci. USA 91 (1994) 6673

Table 2. Lod scores calculated by using the genotypes of those 8. Kurihara, T., Inoue, M. & Tatsumi, K. (1987) Mutat. Res. 184, members of the BS families denoted by a large dot in Fig. 1: 147-151. 9. Krepinsky, A. B., Heddle, J. A. & German, J. (1979) Hum. Genet. pairwise analysis of polymorphic markers on chromosome 50, 151-156. 15 and the disease locus, BLM 10. Willis, A. E. & Lindahl, T. (1987) Nature (London) 325, 355-357. 9 11. Chan, J. Y. H., Becker, F. F., German, J. & Ray, J. H. (1987) Nature (London) 325, 357-359. Locus 0.001 0.01 0.05 0.1 0.2 0.3 12. Kurihara, T., Teraoka, H., Inoue, M., Takebe, H. & Tatsumi, K. (1991) Jpn. J. Cancer Res. 82, 51-57. D1SSJO 0.65 1.16 1.38 1.17 0.65 0.3 13. Seal, G., Brech, K., Karp, S. J., Cool, B. L. & Sirover, M. A. D15JSS -11.65 -8.02 -3.6 -1.79 -0.53 -0.15 (1988) Proc. Nat!. Acad. Sci. USA 85, 2339-2343. D15S3 -8.36 -5.63 -2.29 -1.04 -0.27 -0.08 14. Heartlein, M. W., Tsuji, H. & Latt, S. A. (1987) Exp. CellRes. 169, D15S24 -6.81 -3.56 0 1.19 1.41 0.89 245-254. 15. Kim, S., Vollberg, T. M., Ro, J. Y., Kim, M. & Sirover, M. A. THBES -2.65 0.36 2.65 2.87 2.04 1.05 (1986) Mutat. Res. 173, 141-145. D15S25 -3.73 -1.91 -0.14 0.29 0.29 0.14 16. Dehazya, P. & Sirover, M. A. (1986) Cancer Res. 46, 3756-3761. FBN -7.47 -4.55 -1.07 0.11 0.54 0.36 17. Shiraishi, Y., Taguchi, T., Ozama, M. & Bamezai, R. (1989) Mutat. DJSS) -4.47 -2.15 -0.02 0.46 0.43 0.23 Res. 211, 273-278. -2.03 0.3 2.54 2.82 2.01 18. Nicotera, T. M., Notaro, J., Notaro, S., Schumer, J. & Sandberg, CYPI9 1.04 A. A. (1989) Cancer Res. 49, 5239-5243. D15S2 -1.2 -0.33 0.37 0.41 0.21 0.07 19. Ellis, N. A., Roe, A. M., Otterud, B., Leppert, M. & German, J. LPC2A -8.71 -5.29 -1.44 -0.12 0.4 0.27 (1992) Am. J. Hum. Genet. 51, Suppl., A187 (abstr. 734). D15S45 -5.35 -3.2 -0.96 -0.27 0.04 0.05 20. German, J., Bloom, D. & Passarge, E. (1977) Clin. Genet. 12, D15S29 -1.9 -0.96 0.1 0.32 0.26 0.13 162-168. 21. German, J., Bloom, D. & Passarge, E. (1979) Clin. Genet. 15, D15S36 -3.56 -2.28 -0.79 -0.27 0 0.02 361-367. D15S30 -4.06 -2.59 -0.81 -0.2 0.07 0.06 22. German, J., Bloom, D. & Passarge, E. (1984) Clin. Genet. 25, D15S35 -4.01 -2.38 -0.5 -0.04 0.18 0.11 166-174. D15S28 -7.27 -5.11 -2.23 -1.06 -0.29 -0.08 23. German, J. & Passarge, E. (1989) Clin. Genet. 35, 57-69. D15S34 -2.89 -1.68 -0.1 0.34 0.36 0.19 24. National Institutes of Health/Centre d'Atude du Polymorphisme Humain Collaborative Mapping Group (1992) Science 258, 67-86. D15S26 -2.19 -1.06 0.3 0.67 0.61 0.35 25. Bowcock, A. M., Barnes, R. I., White, R. L., Kruse, T. A., Tsi- D15S27 -0.76 -0.05 0.58 0.6 0.37 0.17 pouras, P., Sarfarazi, M., Jenkins, T., Viljoen, C., Litt, M., FES 11.37 11.02 9.43 7.55 4.34 2.06 Kramer, P. L., Murray, J. C. & Vergnaud, G. (1992) Genomics 14, D15S37 -0.71 0.06 0.92 0.99 0.64 0.32 833-840. D1553 -5.39 -3.14 -0.71 0 0.21 0.13 26. Beckmann, J. S., Tomfohrde, J., Barnes, R. I., Williams, M., D15S87 -15.26 -10.06 -4.04 Broux, O., Richard, I., Weissenbach, J. & Bowcock, A. M. (1993) -1.74 -0.32 -0.03 Hum. Mol. Genet. 2, 2019-2030. Lod scores were not calculated for the ACTC locus. 27. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. 28. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132,6-13. pointed out above, the results confirm the assignment of 29. Williamson, R., Bowcock, A., Kidd, K., Pearson, P., Schmidtke, J., BLM to the distal half of chromosome 15 Ceverha, P., Chipperfield, M., Cooper, D. N., Coutelle, C., Hewitt, (34). J., Klinger, K., Langley, K., Beckmann, J., Tolley, M. & Maidak, Finally, the assignment of BLM to a specific, short chro- B. (1991) Cytogenet. Cell Genet. 58, 1190-1832. mosomal region via the demonstration of tight linkage to 30. Lathrop, G. M. & Lalouel, J. M. (1984) Am. J. Hum. Genet. 36, previously mapped FES represents the first step toward the 460-465. positional cloning of the BLM locus. The cloning itself then 31. Lathrop, G. M., Lalouel, J. M., Julier, C. & Ott, J. (1984) Proc. will constitute the second in a "reverse" Natl. Acad. Sci. USA 81, 3443-3446. step genetics 32. German, J. (1969) Am. J. Hum. Genet. 21, 196-227. approach that finally should permit the definition of the 33. Weksberg, R., Smith, C., Anson-Cartwright, L. & Moloney, K. primary biochemical defect of BS and thereby contribute to (1988) Am. J. Hum. Genet. 42, 816-824. an understanding of one ofthe most remarkable attributes of 34. McDaniel, L. D. & Schultz, R. A. (1992) Proc. Natl. Acad. Sci. the genetic material, its stability. USA 89, 7968-7972. 35. Polymeropoulos, M. H., Rath, D. S., Xiao, H. & Merril, C. R. (1991) Nucleic Acids Res. 19, 4018. We thank Brith Otterud and Dora Stouffer (Salt Lake City) for 36. Harper, M. E., Franchini, G., Love, J., Simon, M. I., Gallo, R. C. valuable advice and assistance in using LINKAGE and James Kozloski & Wong-Staal, F. 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