Am. J. Hum. Genet.

44:422-425, 1989

Evidence from Family Studies That the Gene Causing Huntington Disease Is Telomeric to D4S95 and D4S90
Carolyn Robbins, * Jane Theilmann, * Sandra Youngman, $ Jonathan Haines, Michael J. Altherr, § Peter S. Harper,: Cynthia Payne,# Ann Junkert John Wasmuth,§ and Michael R. Hayden*
Departments of *Medical Genetics and tPaediatrics, University of British Columbia, Vancouver; tMedical Genetics Division, University of Cardiff, Cardiff; §Department of Biological Chemistry, University of California, Irvine; IlDivision of Neurogenetics, Massachusetts General Hospital, Boston; and #Department of Neurology, Duke University, Durham, NC

Summary A DNA probe (D4S95) that detects a variable number of tandem repeats and a single-site-variation polymorphism after digestion with a single restriction enzyme, AccI, has previously been described. The order of this probe relative to the gene for Huntington disease (HD) and other previously described markers has not been established. Analysis of 24 affected families with HD has shown that D4S95 is in tight linkage with the gene causing HD, with a maximal Lod score of 12.489 at a 0 of .03. D4S90 is a probe which maps to 4pl6.3, telomeric to D4S95, and detects polymorphisms with HincII and other enzymes. In one affected person, recombination has occurred between D4S1O and HD, between D4S95 and HD, and in all likelihood also between D4S90 and HD, which strongly suggests that the gene for HD is telomeric to all these DNA probes. This suggests that the gene causing HD is located in the most distal region of the short arm of chromosome 4, flanked by D4S90 and the telomere, and supports the locus order D4S1O-D4S95-D4S90-HD-telomere. D4S95 is a most useful DNA marker for predictive testing programs, while D4S90 will serve as a useful starting point for identifying DNA fragments closer to the gene for HD.

Introduction

The basic defect underlying Huntington disease (HD) remains unknown. Because of this difficulty, considerable effort has been expended to approach the gene not by an understanding of its metabolic defect but rather as a result of refined knowledge of its map location. The first linked polymorphic marker for HD was described in 1983 (Gusella et al. 1983). Since then, numerous other markers closely linked to the gene for HD have been described (Gilliam et al. 1987; Hayden et al. 1988). We have recently described a DNA probe D4S95 (674) that detects a variable number of tandem repeats (VNTR) and numerous single-site polymorphisms on
Received October 20, 1988; revision received November 22, 1988. Address for correspondence and reprints: Dr. Michael R. Hayden, Department of Medical Genetics, Room F168, University Hospital, 2211 Wesbrook Mall, Vancouver, British Columbia V6T 2B5, Canada
i 1989 by The American Society of Human Genetics. All rights reserved. 0002-9297/89/4403-0016$02.00

the distal portion of chromosome 4. This probe is closely linked to the Huntington disease gene (Wasmuth et al. 1988). Another probe D4S90 (D5) has recently been identified which maps to 4pl6.3 when somatic cell hybrids are used and which is telomeric to D4S95 (Youngman et al. 1988). The likely order of D4S95 and D4S90 relative to the gene for HD and other newly described markers has not previously been established. In the present paper we provide evidence that supports the locus order D4S95-D4S90-HD-telomere.
Material and Methods
Isolation of Probes

A genomic library prepared from the hybrid cell line HHW693, which contains as its only human DNA the short arm of a translocated chromosome 5 in which the terminal one-third of 5p is replaced with the terminal one-third of 4p. This cell line was cloned into the lambda vector EMBL-4 and used for the selection of single-copy DNA inserts as described elsewhere (Was-

422

03 (table 1) is revealed with 95% confidence limits of . the haplotype AA is segregating with HD when either D4S10 or D4S95 is used. N N £O N N N m 0% N C . who was recombinant between D4S10 and the gene for HD was also recombinant with D4S95 (fig. . had inherited i DNA haplotypes with D4S10 and D4S95 that were similar to those of a person with an onset age of 35.01-. 1987). Analysis of family members reveals that HD is segregating with the B haplotype after digestion of DNA with EcoRI and hybridization with pko83 and is segregating with the B haplotype after digestion with TaqI and hybrid- Nof o 00 Vo 0 ON T. A peak Lod score of 12. 1). In one large family. and the gene for HD. NO 0 C) No .w 0 z . IGQ-"N irL--izoi0). and MboI and hybridized with D4S95 as described elsewhere (Southern 1975. in close proximity to the HD gene. one affected individual. Feinberg and Vogelstein 1983.. Table 1 shows the two-point linkage analysis for HD versus D4S95. -1 mutn et ai. J o ization with D4S95. However. The frequency of heterozygosity when D4S90 is used is only . Probe D4S90 was isolated from a flow-sorted chromosome 4 library. 1988).q . A subclone of D4S95 which mapped distal to D4S10 was used to identify RFLPs. however. SN ro e ON r< t- N 00 . - 4-j '- o =) 0 :.489 at a 0 of . in 111-1.. All these families were ascertained as part of the predictive testing program for HD in British Colombia. Lod scores were calculated using an age-at-onset correction for at-risk persons who had not manifested signs and symptoms (Conneally et al. TaqI.--A usea u w u z < -E W4 > . u 'A 7. Hayden et al. D4S95.0 z r-. In six large families . Thirty-six of these families were similarly analyzed with D4S100. The data generated were analyzed for linkage to HD by using the computer program LIPED.- ON 0. suggesting that this person also was recombinant between D4S10..- < To . In another ° 1n 0 large family an unaffected sibling. who is clearly affected with HD.14 (Youngman et al.09. wi 9:4 N Linkage Analysis DNA from persons in 44 families with HD was digested with AccI.Huntington Disease Gene Location -4-L 423 4-- to seiect for markers on the distal portion of the short arm of chromosome 4. C) < a 0 0 . 111-1. -0 ". Seven persons who have been previously shown to be recombinant between D4S10 (G8) and/or D4S62 (P8) and HD are all nonrecombinant for D4S95 and HD (Wasmuth et al.N Results 0. a 0 . A polymorphic DNA fragment (D4S90) which is telomeric to D4S95 was also used for analysis in seven of these families in Southern blot experiments with DNA digested with HincII. -*. 1988).D a Y. 1984). aged 62. nese prooes were .

D4S95. M. In view of the fact that the recombination estimate based on genetic analysis may not be directly related to the proximity of the gene. and. D4S90 has a low heterozygous frequency of around 14% and will therefore be not very useful for predictive testing. and HD is strong evidence that the gene for HD is telomeric to D4S95. Huntington disease: estimation of heterozy- . after digestion with HincII and hybridization with D4S90. Affected person Ill-1 is a recombinant between the loci D4S1O. M. is segregating with HD. In the absence of double recombination. it is important to develop a physical map of 4pl6 so that the limits of the region which must contain the HD gene can be defined. Discussion We have elsewhere demonstrated by both somatic hybrid cell mapping and genetic linkage analysis. strongly suggests that the gene for HD is located in 4p16. D4S90 and the HD gene. J. in all likelihood. studied. in an effort to eventually isolate the mutant gene. 1988). 1988). 1984. D4S95 is a highly polymorphic probe that is extremely useful for predictive testing in HD. D4S95. the Hereditary Disease Foundation. I~~~~~~ [B II BB| A + oI Taq 1 Hinc II AB AB AB AB AB BA BB BB BB BB BA BA AB AB BA BB BB BB BB BB AB BB BB AB 2 AB AB AB AB BA BB D4S1O EcoRi D4S95 D4S90 AB AB BB BB AB AB III <& 6 D4S1O EcoRs AA AB AB BB AB BB D4S95 Taq 1 AA AB BB BA AB BB D4S90 Hinc II AB AA AB BA AA AB Figure I A large family with HD. Acknowledgments This study was supported by grants from the Medical Research Council of Canada.. However. and it favors the order D4S1O-D4S95HD. F. the affected person 111-1 in the large pedigree in figure 1 who was recombinant between D4S1O. A double recombinant in 111-1 between D4S95 and HD and separately between D4S90 and HD cannot. and HD is also likely to be recombinant between D4S90 and HD. the Huntington Society of Canada. Despite the possible increased frequency of recombination in this region of chromosome 4 (Richards et al. on the one hand. Wallace. 1988). however. this marker was uninformative. that D4S95 is closer to the HD gene (Smith et al. D4S95. the National Health Research Development Program. D4S90 will serve as a useful starting point for identifying additional cloned DNA fragments closer to the gene for HD. D4S90 has elsewhere been shown to be telomeric to D4S95 by using somatic hybrid cell mapping which separates the two loci (Youngman et al. R.424 Robbins et al. S. this currently would represent the described probe closest to the gene for HD. In this family the A haplotype. and N. However. this places D4S95 and D4S90 centromeric to the gene for HD. The demonstration of a clear recombinant event between D4S10. The order of DNA markers in this region is therefore D4S62-D4S1O-D4S95-HD. be completely excluded. and the gene for HD. Wasmuth et al. References Conneally. On the other hand. this would. and D4S90. Gusella. D4S62 has been shown to be centromeric to D4S10 (Hayden et al. the Woodward Foundation. the affected person 111-1 has inherited the B haplotype from the affected parent. however. 1988. be a highly unlikely occurrence. The demonstration of an affected person who is recombinant between D4S10. 1988). D4S95. Wexler. which at this distance from the HD gene is extremely unlikely.3 distal to all these three loci. However. P. and the NIH. on the other.

Stanbridge. Hum. T. J. J. M. Anderson. Wallace. M. Langlois. Isolation of DNA markers in the direction of the Huntington disease gene from the G8 locus. Genet. C. and P. M. B. J. Macdonald. Hayden. J. Am. Meyers. U. Wasmuth. Nature 332:734-736. Allitto. Hewitt. Wasmuth. D. J. A. Frohlich. R. Rabkin. 40:421-430. B. Bengtsson. J. Faryniarz. L. Wasmuth. M. Ottina. B. Hum. Bucan. Collins. S. Wexler. 1988. J. B. J. J. E. Proc. J. 1987. M. J. Gusella. R. P. Frischauf. M. L. J. Gilliam. Hayden. Cole. Nat]. Haines. Smith.. A. J. J. J. 98:503-517. T. USA. M. J. 132:6-13. M. 1988. L. R. M.3. C. Lehrach. K. Sakaguchi. A DNA probe (D4S90) mapping to human chromosome 4pl6. Haines. Walkins. Young. Conneally. Feinberg. Cheng. Genet. Martin. Smith. M. Allard. A polymorphic DNA marker which represents a conserved gene in the region of the Huntington disease gene. 16:1648. L. U. and J.J. N. Harper. F. 425 Richards. McLeod. and J. and J. J. 1988. and F. Chromosome jumping from D4S10 (G8) toward the Huntington disease gene. Conneally. Southern. 39: 397-403. E. Carpenter.. Gusella. S. J. E. and D. A. H. Zimmer. C. Hum. 1983. Carlock. J. S. Hilbert... and L. 42:335-344. M. Am.. Epidemiol. P. Smith. Nucleic Acids Res. E. R. J. Tanzi. Conneally. Biol. M. R. R. Clark. DNA polymorphisms in and around the Apo-Al-CIII genes and genetic hyperlipidemias. Genet. Nature 306:234-239.. J. Acad. R. Gusella. D. Sci. Sharecky. A polymorphic DNA marker genetically linked to Huntington disease. J. Macdonald. L. J. Mol. Genet. B. A. J. Youngman. C. J. S. Kirk. Immken. 1983.. M. A polymorphic DNA marker tightly linked to the Huntington disease gene. A DNA segment encoding two genes very tightly linked to Huntington's disease. 85:6437-6441. Smith. P. Hewitt. J. Wasmuth. Anal. H. C. D. Bonilla.. 1988. 1987. Hayden. I. C. W. J. P. Am. A. L. H. Gilliam. and B. E. Hum. 1986. B. Wasmuth. 1:81-88. Gusella. P. R. J. M. 42:125-131. Biochem. J. Wasmuth. F. L.Huntington Disease Gene Location gote status using linked genetic markers. F.. Allard. A. Naylor. Vogelstein. Whaley. and J. Partlow. Wasmuth. B. A technique for labelling DNA restriction endonuclease fragments to high specific activity. Hewitt. S. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Pohl. Shaw. Y. 1975. Haines. and M. Am. D.. Skarecky. E. E. R. Drumin. . S. T. A cell hybrid and recombinant DNA library that facilitates identification of polymorphic loci in the vicinity of the Huntington disease gene. Shoulson. S. N. Science 238:950-952. A. Genet. L. 1988. Magenis. F. S.