Genetic Mapping of DNA Segments Relative to the Locus for the Fragile-X Syndrome at Xq27.3 L

Am J Hum Genet 37:463-472, 1985

Genetic Mapping of DNA Segments Relative to the Locus for the Fragile-X Syndrome at Xq27.3 L. M. MULLIGAN,' M. A. PHILLIPS,' C. J. FORSTER-GIBSON," 2 J. BECKETT,"12 M. W. PARTINGTON,2 N. E. SIMPSON,"12 J. J. A. HOLDEN,"12 AND B. N. WHITE" 2

SUMMARY We have tested linkage between the locus for the fragile-X [fra(X)] syndrome at Xq27.3 and five polymorphic restriction sites identified by four DNA probes mapping distal to Xq26. 1. A maximum distance of approximately 15 centimorgans (cM) between Xq27.3 and the marker loci mapping to this region was predicted based on the physical chromosome length. Close linkage between the disease and marker loci was excluded for probes DXSl9 and DXS37 (0 = .05, Z = - 2.94 and Z = - 4.17, respectively). These marker loci were estimated to be less than five cM apart but approximately 40 cM proximal to the fragile site, indicating that there is a significantly greater frequency of recombination in this region of the X chromosome than expected from the physical length. Linkage results for the other marker loci and the fra(X) syndrome were inconclusive. However, the pX45d probe locus appears very closely linked to the factor IX locus (Z = 1.94 at 0 = 0) and is approximately 20 cM proximal to Xq27.3. A relative map of the polymorphic restriction sites, fra(X) syndrome locus, and factor IX locus was constructed by maximizing lod scores over the Xq26. 1-*q27.3 region.

INTRODUCTION The fragile-X [fra(X)] syndrome is an X-linked form of mental retardation characterized by a heritable fragile site at Xq27.3 [1, 2]. Fragility is expressed

Received November 5, 1984; revised January 4, 1985. This study was supported by grants from the Scottish Rite Charitable Foundation of Canada, the Ontario Mental Health Foundation, the Hospital for Sick Children Foundation, the Canadian MRC MT 5783 (to N.E.S.), the Principal's Development Fund of Queen's University, and a research studentship from the Natural Sciences and Engineering Research Council (to L. M. M.). lDepartment of Biology, Queen's University, Kingston, Ontario, Canada. 2 Division of Medical Genetics, Department of Paediatrics, Queen's University, Kingston, On- tario, Canada. C 1985 by the American Society of Human Genetics. All rights reserved. 0002-9297/85/3703-0003$02.00 463 464 MULLIGAN ET AL. under appropriate culture conditions [3] in a variable proportion of cells from individuals who have inherited the mutation [4]. The mutation is partially penetrant with variable expressivity. It is estimated that 56% of heterozygous females and 79% of males with the mutation can be detected on the basis of identification of the fragile site at Xq27.3, with or without mental impairment [4]. Cytogenetic studies and mental status fail to confirm or exclude the presence of the mutation in approximately 70% of female and 60% of male offspring of mothers heterozygous for the mutation [4]. A reliable method of identifying both males and females who carry the fra(X) syndrome mutation is required. The use of restriction fragment length polymorphisms (RFLPs) as genetic markers for linkage analysis has proven successful in identifying a number of genetic disorders [5-10]. A polymorphic TaqI restriction site identified in the gene for coagulation factor IX has been shown to be linked to the fra(X) syndrome locus [11]. Subsequent investigation suggests that approximately 20% recombination occurs between these two loci [12]. The G6PD locus [13] and the 52A probe locus [14] have also been linked to the fra(X) syndrome locus. Marker loci with tighter linkage to the disease locus are still required. Only a proportion of fra(X) syndrome families will be informative for any one marker, and, therefore, a panel of probes identifying RFLPs at loci linked to the fra(X) syndrome locus is needed for classification of genetic status in the majority of families. We have isolated four DNA segments regionally localized near the fragile site at Xq27.3 that identify RFLPs [15]. Linkage of these polymorphic loci to the fra(X) syndrome locus has been tested in four families (fig. 1) [16, 17] using the LIPED computer program [18].

MATERIALS AND METHODS X-Chromosome Probes A human X-chromosome library, prepared from DNA of a human X/hamster somatic cell hybrid line, was the source of human X-chromosome unique sequence probes DXS37, DXSI9, pX58c, and pX45d [15]. Probes were regionally localized by in situ hybridization and hybridization to DNAs from cell lines containing deletions and duplications for parts of the X chromosome [15, 19]. The factor IX probe (VIII) [7, 20] was provided by Dr. G. G. Brownlee of Oxford University, Oxford, England. Lymphoblastoid Cell Lines Peripheral lymphocytes from family members were transformed using Epstein-Barr virus [21]. Cultured lymphoblastoid cells were collected at a density of 5-10 x 106 cells/ ml, lysed with 1% SDS, and digested with 50 Kg/ml proteinase K at 560C for 5-20 hrs. Samples were phenol and chloroform extracted, then incubated 1 hr at 370C with 200 Kug/ ml ribonuclease A and 3 hrs at 560C with 200 pug/ml proteinase K. After further phenol and chloroform extraction, the genomic DNA was extensively dialyzed against TNE2. DNA Extraction from Peripheral Lymphocytes Blood samples were collected in acid citrate dextrose anticoagulant, frozen and thawed to lyse red blood cells, and the white cell pellet collected and washed in Hank's buffer. DNA was extracted as from lymphoblastoid cells with the addition of a dialysis step against TNE2 after the first set of phenol and chloroform extractions. FRAGILE-X 465

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IV 2 3 4 5 6 FIG. 1.-Pedigrees of families 1-4. Only family members included in the LIPED linkage analyses are shown. Individuals not indicated were used to identify obligate heterozygotes in families I and 3 6; ii; = fra(X) chromosome expressed, = heterozygote by history, t = mentally retarded.

Genomic DNA Digests and Southern Blotting Genomic DNA was digested with restriction enzymes (Bethesda Research, Gaithers- burg, Md.) at 3-4 U/,pg DNA for 6-14 hrs. Fragments were electrophoretically separated on 1% agarose gels and transferred to Gene Screen Plus Membrane by the tech- nique of Southern [22] as modified by New England Nuclear, Boston, Mass. (brochure NEF-976). Hybridization of Probes DNA was nick-translated with 32P[dCTP] (NEN, Boston, Mass.) to a specific activity of 3-10 x 108 cpm/,lg [23]. Blots were hybridized with 32P-labeled probes and washed as described (NEN, brochure NEF-976). Blots were autoradiographed on Kodak XAR-5 film with Dupont Lightning Plus intensifying screens at - 70'C for 10-48 hrs. Linkage Analysis The LIPED computer program [18] was used to calculate lod scores for each pair of loci. Individuals were classified as affected if the fra(X) chromosome was expressed in 2% oftheir cultured lymphocytes. Obligate carrier females were defined as females with two 466 MULLIGAN ET AL. or more affected offspring including at least one affected son or with one affected son whose maternal relatives included at least one other affected individual. The fra(X) syndrome genotype of phenotypically normal offspring of females heterozygous for the fra(X) mutation could not be unambiguously determined, and therefore a correction for partial penetrance was required. This correction was based on the phenotypic frequencies for affected and normal individuals and the allele frequency of the fra(X) mutation estimated by Sherman et al. [4]. RESULTS Linkage between five polymorphic restriction sites and the locus for the fra(X) syndrome was tested in four kindreds (fig. 1). DNA from 46 individuals, including 34 offspring of 13 females heterozygous for the fra(X) chromosome by expression or history (fig. 1), was used in these studies. The four probes, which identified the five RFLPs, were previously localized distal to Xq26. 1 [15] (table 1). Each RFLP appears to result from a base substitution causing the presence or absence of a restriction site. Allele frequencies were calculated by screening a minimum of 50 X chromosomes of unrelated individuals (table 1). Segregation of Polymorphisms in fra(X) Syndrome Families DXS19. Four females in families 1 and 2 (table 2) with 20 offspring were heterozygous for the fra(X) mutation and the TaqI RFLP identified by DXS 19 (fig. 2). Analysis of these results produced a total lod score of - 2.94 at a recombination fraction (0) of .05 (table 3), excluding close linkage between DXS19 and the fra(X) syndrome locus. DXS37. Three females informative for the A alleles and four heterozygous for the B alleles of DXS37 (table 1) as well as at the fra(X) syndrome locus were identified (fig. 2; table 2). Because of the proximity of the two polymorphic

TABLE I CHARACTERIZATION OF X-CHROMOSOME PROBE SEQUENCES

X CHROMOSOME POLYMORPHISM PROBE Regional localization Enzyme Alleles* Band size (kb) Frequencyt DXS19 ...... Xq26. 1--qtert TaqI Al 3.7 .89 A2 2.6, 1.1 .11 DXS37 ...... Xq26.1-*q27t§ TaqI Al 9.4, 3.1, 1.3 .88 A2 9.4, 6.7, 1.3 .12 PvuII B1 7.5, 2.4, 2.1 .76 B2 4.5, 2.4, 2.1 .24 pX58c ...... Xq26.1-*q27t SstI A1 9.0 .51 A2 6.0 .49 pX45d ...... Xq26. l-qterf PstI A1 5.4 .84 A2 3.4 .16 Factor IX ...... Xq26--q27t TaqI Al 5.3, 1.8 .65 A2 5.3, 1.3 .35

* Alleles are designated according to Human Gene Mapping 7 [24]. t Allele frequencies were calculated from at least 50 X chromosomes of unrelated individuals. t Localized by hybridization to DNA from cell lines with deletions or duplications for parts of the X chromosome [6, 15]. § Localized by in situ hybridization to prometaphase chromosomes [19]. FRAGILE-X 467 TABLE 2 GENOTYPES AT MARKER LoCI FOR MOTHERS HETEROZYGOUS AT THE fra(X) SYNDROME Locus

GENOTYPE FAMILY PEDIGREE NO. DXSI9 DXS37 pX58c pX45d 1 111-2 ...... A1-2 A1-l/Bl-l B2-2 Al-i 111-4 ...... Al-i Al-l/Bl-1 B2-2 Al-2 IV-5 ...... Al-I Al-2/Bl-l B1-2 Al-I IV-8 ...... Al-I AI-2/Bl-l B1-2 Al-I

2 11-2 ...... A1-2 Al-l/BI-2 B1-2 Al-I 11-4 ...... A1-2 Al-1/B1-1 B1-2 A1-2 III-4 ...... Al-i A1-1/B1-2 B1-2 Al-I 111-8 ...... A1-2 Al-1/Bl-l B1-1 Al-I 111-10 ...... Al-I A1-1/B1-2 B1-2 Al-I 3 1-2 ...... Al-I Al-l/B1-l B1-2 Al-i 11-2 ...... Al-i Al-2/B1-l B1-1 Al-i 4 I I -2 ...... Al-1 Al-1/BI-2 B1-2 A1-2

NOTE: Additional genotype information is available on request. restriction sites detected by DXS37 (less than 8 kilobases [kb] apart as determined by mapping the probe sequence), results for the two sites were summed to determine linkage to the fra(X) syndrome locus. Seventeen offspring of the seven females were available. A total lod score of -2.73 at 0 = .10 (table 2) excludes linkage between these loci. Linkage was also excluded if only the B alleles of DXS37 were considered (Z = - 2.12 at 0 = .05). pX58c. Eight females with a total of 21 progeny were doubly heterozygous for the SstI RFLP and at the fra(X) syndrome locus. Insufficient data were obtained to exclude or confirm linkage between the pX58c and fra(X) syndrome

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Al A2 AIAI-2 BI B2 Al A2 Al A2 FIG. 2.-Southern blot analysis of RFLP alleles identified by four probes: DXS19, DXS37, pX58c, and pX45d. a, TaqI digestions probed with DXS19. b, TaqI, and c, PvuII digestions probed with DXS37. d, Sstl digestions probed with pX58c. e, PstI digestions probed with pX45d. All fragment sizes are given in kb. 468 MULLIGAN ET AL.

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:NN :r: FRAGILE-X 469 loci. A positive peak (Z = 1.01) was detected at 0 = .2, suggesting the possibil- ity of loose linkage between pX58c and the disease locus. pX45d. Three females, with eight progeny, were informative for linkage of the fra(X) syndrome locus and the variable PstI restriction site detected by pX45d. Data were insufficient for conclusions regarding linkage to be reached with a maximum lod score of 0.27 at 0 = .3. Relative Mapping of Marker Loci The positions of the probe loci relative to each other, the fra(X) syndrome locus, and the factor IX gene were determined where female parents heterozygous at two or more loci were observed. A relative map for these loci was established by maximizing the total lod scores over the Xq26 -' q27.3 region (fig. 3) [25]. The factor IX gene and pX45d locus appear closely linked, and lod scores were calculated for two additional kindreds in which the fra(X) syndrome did not occur (pedigrees not shown) (table 3). No recombinations were detected in 14 informative meioses (Z = 1.94 at 0 = 0). The DXS37 and DXSl9 loci also appear closely linked (Z = 2.18 at 0 = 0). No recombinations were detected between these loci in eight informative meioses.

DISCUSSION The genetic map of the X chromosome is evolving rapidly as new loci are assigned and the relative positions of loci are more accurately defined. Until

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_ 1 Fragile Site (Xq 27.3) FIG. 3.-The linkage relationships of four probe loci, the factor IX gene, and fra(X) syndrome locus. The fra(X) syndrome locus and the fragile site are assumed to be the same site for this map. Distance between loci is shown as a recombination fraction. 470 MULLIGAN ET AL. recently, a genetic length of 165 cM (1.65 x 105 kb DNA) was predicted for the X chromosome [26, 27]. Random recombination along the length of this chromosome would give the region Xq26. 1->qter a genetic length of approximately 22 cM, based on its physical proportion of the chromosome. No loci localized to this region should be further than 15 cM from the locus for the fra(X) syndrome. We isolated and localized 17 unique sequence X- chromosome probes, nine of these distal to Xq26. 1. DNAs isolated from family members were screened with 14 restriction enzymes and a total of five RFLPs identified by four of the nine probes. Although both the fra(X) syndrome and the DXS37 loci were mapped distal to Xq26. 1, linkage between these loci was excluded at 13 cM (table 3) with the maximum lod score at 47 cM. Similar results were found with DXS19, which appears to be less than five cM from DXS37. The two other probe loci studied (pX45d and pX58c) also appear to be further than expected from the fra(X) syndrome locus. Our data support the conclusion of increased recombination in the distal bands of the X-chromosome long arm [14, 25]. Based on the map presented by Keats [25], which gives the genetic length of the X chromosome as 267 cM, the Xq26 -- qter region has a genetic length of 72 cM. Therefore, loci localized to this region should be no further than 57 cM from the fragile site at Xq27.3. It is not surprising that the DXS19 and DXS37 loci are not closely linked to the fra(X) syndrome locus. The pX45d and factor IX loci appear tightly linked and are located 20 cM proximal to the locus for fra(X) syndrome [12]. The RFLP identified in the coagulation factor IX gene has already proved useful in screening families with factor IX deficiencies (hemophilia B) [6, 7, 28]. The PstI RFLP of pX45d may also have considerable value to families with hemophilia B, particularly in cases where factor IX polymorphisms are uninformative. A probe with close linkage but distal to the fragile site has been identified [14, 29]. However, the increased recombination in the distal bands of the long arm of the X chromosome makes it unlikely that a single probe will be isolated that is useful in genetic counseling of individuals at risk for fra(X) syndrome. The need for a bank of probes identifying RFLPs at loci linked to the fra(X) syndrome locus is apparent. A panel of probes for loci bracketing the disease locus has two distinct advantages over any single probe: (1) heterozygosity for at least one marker locus will be more frequently identifiable and (2) multiple markers and haplotype determination for these loci will increase the accuracy of the genetic classification.

ACKNOWLEDGMENTS We gratefully acknowledge Mrs. L. Cooke who did much of the cell culturing and Marino Labinaz and Rob Mahon who helped with the probe isolation. We thank Dr. G. G. Brownlee (Oxford University) for providing the factor IX probe. FRAGILE-X 471 REFERENCES 1. DEARCE MA, KEARNS A: The fragile X syndrome: the patients and their chromosomes. J Med Genet 21:84-91, 1984 2. HARRISON CJ, JACK EM, ALLEN TD, HARRIS R: The fragile-X: a scanning electron microscope study. J Med Genet 20:280-285, 1983 3. SUTHERLAND GR: The fragile-X chromosome. Int Rev Cytol 81:107-142, 1983 4. SHERMAN SL, MORTON NE, JACOBS PA, TURNER G: The marker (X) syndrome: a cytogenetic analysis. Ann Hum Genet 48:21-37, 1984 5. GUSELLA JF, WEXLER NS, CONNEALLY PM, ET AL.: A polymorphic DNA marker genetically linked to Huntington's disease. Nature 306:234-238, 1983 6. CAMERINO G, GRZESCHIK KH, JAYE M, ET AL.: Regional localization on the human X chromosome and polymorphism of the coagulation factor IX gene (hemophilia B locus). Proc Natl Acad Sci USA 81:498-502, 1984 7. GIANNELLI F, CHoo KH, REES DJG, BOYD Y, RIZZA CR, BROWNLEE GG: Gene deletions in patients with haemophilia B and anti-factor IX antibodies. Nature 303:181-182, 1983 8. MURRAY JM, DAVIES KE, HARPER PS, MEREDITH L, MUELLER CR, WILLIAMSON R: Linkage relationship of a cloned DNA sequence on the short arm of the X- chromosome to Duchenne muscular dystrophy. Nature 300:69-71, 1982 9. KAN YW, DozY AM: Polymorphism of DNA sequence adjacent to human beta- globin structural gene: relationship to sickle mutation. Proc Natl Acad Sci USA 75:5631-5635, 1978 10. KAN Y, LEE KY, FURBETTA M, ANGIUS A, CAO A: Polymorphism of DNA sequence in the beta-globin gene region. N Engl J Med 302:185-188, 1980 11. CAMERINO G, MATTEI MG, MATTEI JF, JAYE M, MANDEL JL: Close linkage offragile- X mental retardation syndrome to haemophilia B and transmission through a normal male. Nature 306:701-703, 1983 12. FORSTER-GIBSON CJ, MULLIGAN LM, PARTINGTON MW, SIMPSON NE, HOLDEN JJA, WHITE BN: Recombination between the coagulation factor IX gene and the locus for the fragile X syndrome. Submitted for publication 13. FILIPPI G, RINALDI A, ARCHIDIACONO N, ROCCHI M, BALAZS I, SINISCALCO M: Linkage between G6PD and fragile X syndrome. Am J Med Genet 15:113-119, 1983 14. DRAYNA D, DAVIES K, HARTLEY D, ET AL.: Genetic map of the human X chromosome by using restriction fragment length polymorphisms. Proc Natl Acad Sci USA 81:1836-1839, 1984 15. HAMERTON JL, WANG HS, RIDDELL DC, ET AL.: Assignment and regional localization of a series of X chromosome specific DNA probes. Cytogenet Cell Genet 37:486, 1984 16. SOUDEK D, PARTINGTON MW, LAWSON JS: The fragile-X syndrome. I. Familial varia- tion in the proportion of lymphocytes with the fragile site in males. Am J Med Genet 17:241-252, 1984 17. PARTINGTON MW: The fragile-X syndrome. II. Preliminary data on growth and development in males. Am J Med Genet 17:175-194, 1984 18. OTT J: A computer program for linkage analysis of general human pedigrees. Am J Hum Genet 26:588-597, 1974 19. HOLDEN J, WANG HS, WHITE BN: The fragile-X syndrome. IV. Progress toward the identification of linked restriction fragment length variants (RFLVs). Am J Med Genet 17:259-274, 1984 20. GIANNELLI F, CHoo KH, WINSHIP PR, ET AL.: Characterization and use of an in- tragenic polymorphic marker for detection of carriers of haemophilia B (factor IX). Lancet i:239-241, 1984 21. ROBINSON J, MILLER G: Assay for Epstein-Barr virus based on stimulation of DNA synthesis in blood. J Virol 15:1065-1072, 1975 472 MULLIGAN ET AL. 22. SOUTHERN EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517, 1975 23. RIGBY PWJ, DICKMANN M, RHODES CR, BERG P: Labelling DNA to high specific activity in vitro by nick-translation with DNA polymerase I. J Mol Biol 113:237- 251, 1977 24. HUMAN GENE MAPPING WORKSHOP: Human Gene Mapping 7: Seventh International Workshop on Human Gene Mapping. Cytogenet Cell Genet 37:210-215, 1984 25. KEATS B: Genetic mapping: X-chromosome. Hum Genet 64:28-32, 1983 26. BOTSTEIN D, WHITE RL, SKOLNICK M, DAVIES RW: Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32:314-331, 1980 27. SOUTHERN EM: Application of DNA analysis to mapping the human genome. Cy- togenet Cell Genet 32:52-57, 1982 28. PEAKE IR, FURLONG BL, BLOOM AL: Carrier detection by direct gene analysis in a family with haemophilia B (factor IX deficiency). Lancet i:242-243, 1984 29. HARPER K, PEMBREY ME, DAVIES KE, WINTER RM, HARTLEY D, TUDDENHAM EGD: A clinically useful DNA probe closely linked to haemophilia A. Lancet ii:6-8, 1984

1985 NATIONAL SOCIETY OF GENETIC COUNSELORS EDUCATION CONFERENCE: RELIGIOUS, CULTURAL, AND ETHNIC INFLU- ENCES ON THE COUNSELING PROCESS. The Fifth Annual NSGS Edu- cation Conference will be held October 7-8, 1985, in Salt Lake City, Utah, preceding the meeting of the American Society of Human Genetics. Designed for genetic counselors and other health professionals providing or interested in genetic services, the program will focus on the effects of family background on the reception of genetic counseling. Cultural, religious, and sociological forces all shape a family's view of birth defects and genetic disease, creating possible barriers to the delivery of genetic services. For information regarding submis- sion of abstracts and registration forms, please contact: Barbara Biesecker, M.S., Chairperson 1985 NSGC Education Meeting, University of Wisconsin, Clinical Genetics Center, 1500 Highland Avenue, Madison, WI 53705. Tele- phone: (608)262-2507.