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.