Am. J. Hum. Genet. 49:1-6, 1991
Invited Editorial: The End in Sight for Huntington Disease? Catrin Pritchard,* David R. Cox,t't and Richard M. Myers*,t Departments of *Physiology, Biochemistry and Biophysics, and $Psychiatry, University of California, San Francisco
These are exciting times for human geneticists. For many years our work has been directed toward developing better and faster techniques for understanding the human genome. Now, at last, we are seeing the fruits of this work with the cloning of many important disease genes. The approach for cloning such genes has become straightforward for diseases associated with a chromosomal rearrangement. However, the task is much more difficult for disease genes that are not as readily marked. In these cases, the general strategy is first to map the disease locus by performing genetic linkage analysis. Then, once an approximate location is known, a combination of chromosome walking, gene searching, and mutation detection techniques is used to home in gradually on the locus. The work involved is time-consuming and labor-intensive. Nevertheless, the approach has been successful for identifying the cystic fibrosis (CF) locus and holds promise for the cloning of other disease loci that are also not marked by a rearrangement. One such disease gene, that responsible for Huntington disease (HD), is the focus of considerable attention but has not yet succumbed to cloning. Many scientists following the human genetics field must be wondering why it is taking so long to clone this gene. After all, the HD mutation exhibits a simple mode of inheritance and was the first to be mapped by genetic linkage to a DNA marker. HD scientists clearly have some explaining to do. It is hoped that the Bates et al. (1991) paper in this issue of the Journal will help to satisfy the critics. The paper describes the localization of the gene to a 2.5Mb region, a result that may soon allow the cloning of the gene. HD has attracted genetic research for several years because it shows many unusual characteristics (Laird Received April 22, 1991. Address for correspondence and reprints: Catrin Pritchard, Ph.D., Department of Physiology, University of California, 513 Parnassus Avenue, San Francisco, CA 94143-0444. This article represents the opinion of the author and has not been peer reviewed. X 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4901-0001$02.00
1990). The disease is a neurodegenerative disorder characterized by dementia, chorea, and psychiatric disturbances such as depression (Martin and Gusella 1986). The most notable feature of HD is its low mutation rate. No proved case of a new mutation has been documented, and thus it is generally believed that the mutation arose only once in history. This view is supported both by studies showing that the disease lacks genetic heterogeneity (Conneally et al. 1989) and by recent linkage disequilibrium data (Snell et al. 1989; Theilmann et al. 1989). However, it remains to be proved that all affected individuals carry the same mutation. Another unusual feature of HD is its inheritance as an autosomal dominant trait with full penetrance. This is demonstrated by the finding that likely homozygotes show no more severe symptoms than do heterozygotes for the disease (Wexler et al. 1987; Myers et al. 1989). In addition, HD has a variable age at onset. The disease usually occurs in adulthood, at an average age of 38 years (Myers et al. 1982; Ridley et al. 1988). However, unlike other diseases that also show variable ages at onset, a class of "juvenile-onset" cases have been described for HD that show symptoms at an earlier age, before 21 years. It is interesting that it is much more common for such juvenile-onset cases to inherit the disease from their father than from their mother (Myers et al. 1982; Ridley et al. 1988). This has been taken to imply that either the HD gene itself or a modifier of the gene undergoes chromosomal imprinting (Erickson 1985; Reik 1988). Cloning of the HD gene should unravel many of these interesting facets of the disease. When the gene is in hand, it will also be possible to investigate how the mutation is manifested at the cellular level. The biochemical basis of HD is not known, and most attempts to clone the HD gene have relied on a knowledge of its chromosomal location. These genetic studies have yielded some valuable information. However, until recently, the studies failed to define a precise location for the gene. Genetic linkage between HD and the D4S1 0 marker was found in 1983 (Gusella et al. 1983), soon after DNA markers were first used for 1
Pritchard et al.
2 RAFI P1 centromere /, I
D4S62 D4S10 I approx. 6Mb I 17 cM
2 cM
m
p telomere
Are --4
HD *'--" 4cM
Figure I Localization of HD locus between D4S10 and 4p telomere. Linkage analysis places HD approximately 4 cM from D4S1 0 (Gilliam et al. 1987b; Conneally et al. 1989). RAFiP1 and D4S62 have a more centromeric location and are less tightly linked to HD than D4S10 (Gilliam et al. 1987b). The region between D4S10 and the telomere is estimated to span 6 Mb (Gilliam et al. 1987b; Bucan et al. 1990).
this purpose. D4S1 0 was subsequently localized both by in situ hybridization (Landegent et al. 1986) and by somatic cell genetics (MacDonald et al. 1987) to the short arm of chromosome 4, in the most terminal subband, 4pl 6.3. Progress toward localizing HD continued as multipoint linkage analysis was performed with D4S1 0 and two other markers known to be centromeric to D4S1 0 on 4p: D4S62 and RAFl P1 (fig. 1; Gilliam et al. 1 987b). HD was found to be more tightly linked to D4S1 0 than to the other markers (fig. 1). Such a result placed the disease locus between D4S1 0 and the 4p telomere, within a region estimated by cytogenetic measurement to contain less than 6 Mb of DNA (fig. 1; Gilliam et al. 1987b). Many new probes were isolated from the 6-Mb region (Pohl et al. 1988; Smith et al. 1988; Pritchard et al. 1989), and physical (Bucan et al. 1990) and genetic maps (MacDonald et al. 1989) were constructed with these probes. Genetic analysis was also performed on HD families by using polymorphisms identified with the new probes. The primary intention of this work was to identify crossover events between the new markers and HD so that the closest flanking markers for the disease locus could be identified. However, a major problem was encountered with the discovery of a hot spot for recombination very close to D4S1 0 (Skraastad et al. 1989; Allitto et al. 1991). Many of the recombination events occurring between D4S1 0 and HD were localized to this hot spot (Richards et al. 1988; Skraastad et al. 1989), thus reducing the number of events that could provide an exact location for HD. Four obligatory recombination events within the D4S10-4p telomere region but outside the hot spot remained known. These received considerable attention from several research groups. The results of the analyses produced conflicting locations for HD. In the case of three events (families 1-3 in fig. 2), HD was found to segregate with the wild-type allele at D4S1 0 (MacDonald et al. 1989; Robbins et al. 1989). All markers telomeric to D4S1 0 that were tested also originated from the wild-type chromosome. For families
1 and 2, the most telomeric informative marker was D4S1 11 (MacDonald et al. 1989), which maps 1,100 kb from the 4p telomere (Bucan et al. 1990). Family 3 was informative for D4S90 (Robbins et al. 1989), which resides approximately 300 kb from the telomere (Doggett et al. 1989; Bucan et al. 1990; Pritchard et al. 1990). As illustrated in figure 2, a telomeric location for HD was proposed on the basis of data on these families (MacDonald et al. 1989). Recombination events close to the telomere were thought to occur in each case to explain the observed segregation of HD. However, in family 4, the HD allele at D4S10 was inherited with HD. More telomeric markers such as D4S1 11 and D4S90 originated from the wild-type chromosome, indicating that a recombination event had occurred between these markers and D4S1 0. Such a result placed HD in a more centromeric region, at least 1 Mb proximal to the telomeric location. The two locations predicted for HD as defined by these recombination events clearly do not overlap. How can this be explained? Many different hypotheses were proposed, such as HD being a large gene and containing different mutations in the different families (MacDonald et al. 1989). However, at the time, it seemed most likely that the one recombination event predicting a more centromeric location (family 4 in fig. 2) was an exceptional case that could be explained as resulting from either misdiagnosis, mistyping, or, more likely, a second recombination event occurring very close to the telomere (MacDonald et al. 1989). With this double-crossover hypothesis in mind, attention was turned to the 300-kb region, defined by family 3, which placed HD between D4S90 and the 4p telomere. The decision to do this was risky, as no definitive interlocus recombination had been observed in family 3, even though recombination appeared to occur between the loci tested and HD. However, because of the region's small size, it was reasoned that it would be relatively quick and easy to analyze. In addition, by cloning more telomeric DNA, it would
3
Invited Editorial D4S10
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Possible locations for HD, based on four families displaying recombination events. The diagram (not drawn to scale) Figure 2 illustrates the genotypes of the region between D4S1 0 and the 4p telomere in the four recombinant chromosomes. Clear boxes represent regions originating from the wild-type chromosome. Hatched boxes signify regions whose genotype is unknown. Blackened boxes represent regions known to originate from the HD chromosome. In families 1-3, all of the tested polymorphic markers originate from the wild-type chromosome. To explain the inheritance of HD, the HD locus is placed close to the telomere (within the hatched boxes), and as yet unidentified recombination events are thought to occur telomeric to D4S1 11 (families 1 and 2; MacDonald et al. 1989) and to D4S90 (family 3; Robbins et al. 1989). For family 4 (MacDonald et al. 1989), markers in the more centromeric region originate from the HD chromosome. A recombination event has been identified between the markers D4S1 13 and D4S1 15, placing HD centromeric to D4S1 15. This location for HD is contradictory to that predicted by the data on families 1-3. Therefore a second recombination event, telomeric to D4S90, was initially thought to occur in family 4.
be possible to confirm the predicted crossovers for families 1-3 and to investigate whether the proposed second crossover in family 4 had indeed taken place. A telomeric location for HD also had romantic appeal. We became excited by the possibility of HD being a telomeric disorder, caused by a position effect of heterochromatic DNA (Laird 1990). Maybe this could explain the unusual genetic characteristics of the disease.
The DNA at the telomere of 4p was subsequently cloned. Bates et al. (1990) reported the isolation of a yeast artificial chromosome (YAC) which spans 11S kb and extends to the telomere of 4p. Other groups cloned DNA adjacent to the telomere, extending to D4S90 (Pritchard et al. 1990). However, a telomeric location for HD was brought into question on characterization of this DNA: few, if any, genes were found, and no DNA rearrangements associated with the disease were detectable (Pritchard et al. 1990). DNA at
the 4p telomere itself did not differ in size or structure in HD patients compared with control individuals when it was analyzed at a level of resolution that would detect changes of 5 kb or more (Pritchard et al. 1990). Doubts were also raised when it was realized that human telomeres carry homologous sequences (Brown et al. 1990) and can be polymorphic in length (Brown et al. 1990; Wilkie et al. 1991). If, as seems likely, this reflects DNA exchange at telomeres, then the HD mutation would no longer be linked to chromosome 4, if the mutation occurred prior to any exchange that involved 4p telomeric DNA. More direct evidence against a telomeric location was gathered from genetic studies. Polymorphisms were identified in two markers that mapped within 100 kb of the telomere: D4S142 (Bates et al. 1990) and D4S169 (Pritchard et al. 1990). When analysis with these markers was carried out, interlocus recombination was still not detected in family 3 (Pritchard,
Pritchard et al.
4 0 k 1 000 kb
D4S125 D4510
D45180
I
,1I
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D45113/114
D4595 D4543 I
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I
I
-ve NT
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/il -ve
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D4S90 D45142/169 I
-ve NT
1
4p telomere Linkage disequilibrium
Figure 3 Map of region between D4S1 0 and 4p telomere, indicating candidate location for HD as proposed by Bates et al. (1991; see also Whaley et al. 1991). The candidate region for HD is represented by the black bar and spans approximately 2.5 Mb. The double slashes indicate a gap in the map of unknown size. + ve = markers that show significant linkage disequilibrium; - ve = markers that do not show significant disequilibrium; NT = markers that have not been tested. At present, positive disequilibrium has only been observed at D4S95 (Snell et al. 1989; Theilmann et al. 1989; Adam et al. 1991). As no other markers close to D4S95 have yet been tested, the region of disequilibrium could be quite large, extending at most from D4S10 to D4S43 (approximately 2 Mb).
unpublished data), and the putative second recombination event in family 4 was not observed (Bates et al. 1990). Final proof against a telomeric location was provided by the detection of nonrandom allelic association (linkage disequilibrium) between HD and two DNA markers in the more centromeric region: D4S95 and D4S98 (Snell et al. 1989; Theilmann et al. 1989; fig. 3). Significant allele association was not observed for markers in the telomeric region, such as D4S90 (Snell et al. 1989; Theilmann et al. 1989). The realization that the HD gene was not in the telomeric region was a setback for many. It precipitated a slowing up of research and a reevaluation of the approaches needed to find the real location for the gene. The past year has been spent collecting data, with careful consideration of which recombination events are best to evaluate. Bates et al. (1991), in this issue of the Journal, and Whaley et al. (1991), in a recent issue of Somatic Cell and Molecular Genetics, have now published the first results of this reevaluation. Whaley et al. analyze family 4 in detail and provide a very precise location for the recombination event: between the markers D4S168 and D4S1 13/114 (see fig. 3). Bates et al. focus on defining a proximal boundary for the gene. This boundary is provided by the recombination hot spot which lies close to D4S1 0. Of the recombination events that fall within this hot spot, Bates et al. identify one which appears to be the most telomeric, occurring between D4S125 and D4S1 80 (fig. 3). The conclusion from these studies is that HD lies proximal to D4S1 68 but distal to D4S1 25 (fig. 3). Physical mapping of this region shows that the two flanking markers are separated by approximately
2.5 Mb. It would be prudent to exercise some caution in accepting this location for HD, given that it depends on only two recombination events. However, perhaps we can afford to be more confident this time, as the new location is strongly supported by the linkage disequilibrium data. It is pertinent at this point to draw lessons from the successful cloning of the CF gene (Kerem et al. 1989; Riordan et al. 1989; Rommens et al. 1989). As with HD, some false starts were also made in localizing CF. Why, then, was it possible to clone this gene so rapidly? The answer lies partly with the fact that CF is encoded by a large gene and that few other candidate genes were detectable in the CF region. However, in addition, the CF researchers made some wise strategic moves that greatly aided cloning of the gene. The most intuitive was the decision to use linkage disequilibrium as a measure of genetic distance from the disease locus. Despite the analysis of many families, CF could only be narrowed to a 900-kb region based on the available recombination events (Kerem et al. 1989). Additional genetic methods were required to locate the gene. The CF researchers looked for linkage disequilibrium over the 900-kb region. Although fluctuations occurred at several locations, one very strong peak of disequilibrium was detected in a region that subsequently revealed the presence of the CF gene (Kerem et al. 1989). It was not known at the time whether this strategy would identify the gene, as previous studies had shown that other factors besides genetic distance - such as mutation rate, selection, and an uneven distribution of recombination events - can influence the strength of disequilibrium (Chakravarti et al. 1984). In the case
Invited Editorial of CF, other indicators such as the flanking crossovers and the presence of candidate genes were considered before effort was focused on the region of strong disequilibrium (Riordan et al. 1989; Rommens et al. 1989). The use of linkage disequilibrium for this purpose is instructive for the cloning of other disease loci for which allele association can be detected. For HD, several research groups are now actively involved in cloning the 2.5-Mb region. Although the size of this region is three times greater than that which existed for CF, YAC technology is now available to speed up the cloning. The linkage disequilibrium studies are also being extended. Indeed, a recent study has shown that the allele association is strong at D4S95 but no longer significant at D4S98 (fig. 3; Adam et al. 1991). Negative results have already been reported for D4S10 (Youngman et al. 1986; Theilmann et al. 1989), D4S43 (Gilliam et al. 1987a), and D4S96 (Theilmann et al. 1989). Therefore, most attention is currently being given to the region near D4S95. Once a smaller candidate region is identified and cloned, searches will be performed for candidate genes for HD. This may take a long time, as there maybe as many as 50 genes in the HD region. To identify which candidate gene is the HD gene, screens will need to be undertaken to determine whether there are any quantitative or qualitative differences in their expression in HD patients as compared with normal controls. Searches will also be performed for a genomic DNA rearrangement, by using the candidate gene sequences as probes against DNA isolated from both HD and non-HD individuals. If HD is caused by a small DNA change that is not detectable by these other approaches, then the candidate genes will be sequenced with the aim of identifying sequence differences between the genes of HD individuals and those of nonHD individuals. Although a lot of work lies ahead before the gene is available, we have reason to be optimistic, now that a more definitive location for HD has been achieved. The search for HD has been arduous, compounded by limited resources and bad luck. However, we remain intrigued by this disease and eagerly await knowing what type of mutation causes such unusual genetic features. We also anticipate an explanation for why we were misled by the three families showing a telomeric location for HD (families 1-3 in fig. 2). Are there really different mutations in the different families, or can they be explained by other events, such as rearrangements of chromosome 4p or gene conversion? Finally, cloning of the gene promises to be a major
relief for the families afflicted by HD. They, more than any of us, must look forward to the news that a more reliable predictive test is available and that potential treatments are in sight.
References Adam S, Theilmann J, Buetow K, Hedrick A, Collins C, Weber B, Huggins M, et al (1991) Linkage disequilibrium and modification of risk for Huntington disease. Am J Hum Genet 48:595-603 Allitto BA, MacDonald ME, Bucan M, RichardsJ, Romano D, Whaley WL, Falcone B, et al (1991) Increased recombination adjacent to the Huntington disease-linked D4S10 marker. Genomics 9:104-112 Bates GP, MacDonald ME, Baxendale S, Sedlacek Z, Youngman S, Romano D, Whaley WL, et al (1990) A yeast artificial chromosome telomere clone spanning a possible location of the Huntington disease gene. Am J Hum Genet 46:762-775 Bates GP, MacDonald ME, Baxendale S, Youngman S, Lin C, Whaley WL, WasmuthJJ, et al (1991) Defined physical limits of the Huntington disease gene candidate region. Am J Hum Genet 49:7-16 Brown WRA, MacKinnon PJ, Villasante A, Spurr N, Buckle VJ, Dobson M (1990) Structure and polymorphism of human telomere-associated DNA. Cell 63:119-132 Bucan M, Zimmer M, Whaley WL, Poustka A, Youngman S, Allitto BA, Ormondroyd E, et al (1990) Physical maps of 4pl6.3, the area expected to contain the Huntington disease mutation. Genomics 6:1-15 Chakravarti A, Buetow KH, Antonarakis SE, Waber PG, Boehm CD, Kazazian HH (1984) Nonuniform recombination within the human 0-globin gene cluster. Am J Hum Genet 36:1236-1258 Conneally PM, Haines JL, Tanzi RE, Wexler NS, Penchaszadeh GK, Harper PS, Folstein SE, et al (1989) Huntington disease: no evidence for locus heterogeneity. Genomics 5:304-308 Doggett NA, ChengJ-F, Smith CL, Cantor CR (1989) The Huntington disease locus is most likely within 325 kilobases of the chromosome 4p telomere. Proc Natl Acad Sci USA 86:10011-10014 Erickson RP (1985) Chromosomal imprinting and the parent transmission specific variation in expressivity of Huntington disease. Am J Hum Genet 37:827-829 Gilliam TC, Bucan M, MacDonald ME, Zimmer M, Haines JL, Cheng SV, Pohl TM, et al (1987a) A DNA segment encoding two genes very tightly linked to Huntington's disease. Science 238:950-952 Gilliam TC, Tanzi RE, HainesJL, Bonner TI, Faryniarz AG, Hobbs WJ, MacDonald ME, et al (1987b) Localization of the Huntington's disease gene to a small segment of chromosome 4 flanked by D4S10 and the telomere. Cell 50:565-571
6 Gusella JF, Wexler NS, Conneally PM, Naylor SL, Anderson MA, Tanzi RE, Watkins PC, et al (1983) A polymorphic marker genetically linked to Huntington's disease. Nature 306:234-238 Kerem B-S, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, et al (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073-1080 Laird C (1990) Proposed genetic basis of Huntington's disease. Trends Genet 6:242-247 Landegent JE, Jansen in de Wal N, Fisser-Groen YM, Bakker E, van der Ploeg M, Pearson PL (1986) Fine mapping of the Huntington disease linked D4S10 locus by nonradioactive in situ hybridization. Hum Genet 73:354-357 MacDonald ME, Anderson MA, Gilliam TC, Tranebjaerg L, Carpenter NJ, Magenis E, Hayden MR, et al (1987) A somatic cell hybrid panel for localizing DNA segments near the Huntington's disease gene. Genomics 1:29-34 MacDonald ME, HainesJL, Zimmer M, Cheng SV, Youngman S, Whaley WL, Wexler N, et al (1989) Recombination events suggest potential sites for the Huntington's disease gene. Neuron 3:183-190 Martin JB, Gusella JF (1986) Huntington's disease: pathogenesis and management. N Engl J Med 3 15: 1267-1276 Myers RH, Leavitt J, Farrer LA, Jagadeesh J, McFarlane H, Mastromauro CA, Mark RJ, et al (1989) Homozygote for Huntington disease. Am J Hum Genet 45:615-618 Myers RH, Madden JJ, Tiague JL, Falek A (1982) Factors related to onset age of Huntington disease. Am J Hum Genet 34:481-488 Pohl TM, Zimmer M, MacDonald ME, Smith B, Bucan M, Poustka A, Volinia S, et al (1988) Construction of a NotI linking library and isolation of new markers close to the Huntington's disease gene. Nucleic Acids Res 16:91859198 Pritchard C, Casher D, Bull L, Cox DR, Myers RM (1990) A cloned DNA segment from the telomeric region of human chromosome 4p is not detectably rearranged in Huntington disease patients. Proc Natl Acad Sci USA 87:73097313 Pritchard CA, Casher D, Uglum E, Cox DR, Myers RM (1989) Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to the Huntington disease gene. Genomics 4:408-418 Reik W (1988) Genomic imprinting: a possible mechanism for the parental origin effect in Huntington's chorea. J Med Genet 25:805-808 Richards JE, Gilliam TC, Cole JL, Drumm ML, Wasmuth JJ, Gusella JF, Collins FS (1988) Chromosome jumping from D4S10 (G8) toward the Huntington disease gene. Proc Natl Acad Sci USA 85:6437-6441
Pritchard et al. Ridley RM, Frith CD, Crow TJ, Conneally PM (1988) Anticipation in Huntington's disease is inherited through the male line but may originate in the female. J Med Genet 25:589-595 Riordan JR, Rommens JM, Kerem B-S, Alon N, Rozmahel R, Grzaelczak Z, Zielenski J, et al (1989) Identification of the cystic fibrosis gene: cloning and characteization of complementary DNA. Science 245:1066-1073 Robbins C, Theilmann J, Youngman S, Haines J, Altherr MJ, Harper PS, Payne C, et al (1989) Evidence from family studies that the gene causing Huntington disease is telomeric to D4S95 and D4S90. Am J Hum Genet 44: 422-425 Rommens JM, Iannuzzi MC, Kerem B-S, Drumm ML, Melmer G, Dean M, Rozmahel R, et al (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245:1059-1065 Skraastad MI, Bakker E, de Lange LF, Vegter-van der Vhs M, Klein-Breteler EG, van Ommen GJB, Pearson PL (1989) Mapping of recombinants near the Huntington disease locus by using G8 (D4S10) and newly isolated markers in the D4S10 region. Am J Hum Genet 44:560566 Smith B, Skarecky D, Bengtsson U, Magenis EM, Carpenter N, Wasmuth JJ (1988) Isolation of DNA markers in the direction of the Huntington disease gene from the G8 locus. Am J Hum Genet 42:335-344 Snell RG, Lazarou L, Youngman S, Quarrell OWJ, Wasmuth JJ, Shaw DJ, Harper PS (1989) Linkage disequilibrium in Huntington's disease: an improved localization for the gene. J Med Genet 26:673-675 Theilmann J, Kanani S, Shiang R, Robbins C, Quarrell 0, Huggins M, Hedrick A, et al (1989) Non-random association between alleles detected at D4S95 and D4S98 and the Huntington's disease gene. J Med Genet 26:676-681 Wexler NS, Young AB, Tanzi RE, Travers H, StarostaRubinstein S, Penney JB, Snodgrass SR, et al (1987) Homozygotes for Huntington's disease. Nature 326:194197 Whaley WL, Bates GP, Novelletto A, Sedlacek Z, Cheng S, Romano D, Ormondroyd E, et al (1991) Mapping of cosmid clones in Huntington's disease region of chromosome 4. Somatic Cell Mol Genet 17:83-91 Wilkie AOM, Higgs DR, Rack KA, Buckle VJ, Spurr NK, Fischel-Ghodsian N, Ceccherini I, et al (1991) Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16. Cell 64:595-606 Youngman S, Sarfarazi M, Quarrell OWJ, Conneally PM, Gibbons K, Harper PS, Shaw DJ, et al (1986) Studies of a DNA marker (G8) genetically linked to Huntington's disease in British families. Hum Genet 73:333-339
Invited Editorial: The End in Sight for Huntington Disease? Catrin Pritchard,* David R. Cox,t't and Richard M. Myers*,t Departments of *Physiology, Biochemistry and Biophysics, and $Psychiatry, University of California, San Francisco
These are exciting times for human geneticists. For many years our work has been directed toward developing better and faster techniques for understanding the human genome. Now, at last, we are seeing the fruits of this work with the cloning of many important disease genes. The approach for cloning such genes has become straightforward for diseases associated with a chromosomal rearrangement. However, the task is much more difficult for disease genes that are not as readily marked. In these cases, the general strategy is first to map the disease locus by performing genetic linkage analysis. Then, once an approximate location is known, a combination of chromosome walking, gene searching, and mutation detection techniques is used to home in gradually on the locus. The work involved is time-consuming and labor-intensive. Nevertheless, the approach has been successful for identifying the cystic fibrosis (CF) locus and holds promise for the cloning of other disease loci that are also not marked by a rearrangement. One such disease gene, that responsible for Huntington disease (HD), is the focus of considerable attention but has not yet succumbed to cloning. Many scientists following the human genetics field must be wondering why it is taking so long to clone this gene. After all, the HD mutation exhibits a simple mode of inheritance and was the first to be mapped by genetic linkage to a DNA marker. HD scientists clearly have some explaining to do. It is hoped that the Bates et al. (1991) paper in this issue of the Journal will help to satisfy the critics. The paper describes the localization of the gene to a 2.5Mb region, a result that may soon allow the cloning of the gene. HD has attracted genetic research for several years because it shows many unusual characteristics (Laird Received April 22, 1991. Address for correspondence and reprints: Catrin Pritchard, Ph.D., Department of Physiology, University of California, 513 Parnassus Avenue, San Francisco, CA 94143-0444. This article represents the opinion of the author and has not been peer reviewed. X 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4901-0001$02.00
1990). The disease is a neurodegenerative disorder characterized by dementia, chorea, and psychiatric disturbances such as depression (Martin and Gusella 1986). The most notable feature of HD is its low mutation rate. No proved case of a new mutation has been documented, and thus it is generally believed that the mutation arose only once in history. This view is supported both by studies showing that the disease lacks genetic heterogeneity (Conneally et al. 1989) and by recent linkage disequilibrium data (Snell et al. 1989; Theilmann et al. 1989). However, it remains to be proved that all affected individuals carry the same mutation. Another unusual feature of HD is its inheritance as an autosomal dominant trait with full penetrance. This is demonstrated by the finding that likely homozygotes show no more severe symptoms than do heterozygotes for the disease (Wexler et al. 1987; Myers et al. 1989). In addition, HD has a variable age at onset. The disease usually occurs in adulthood, at an average age of 38 years (Myers et al. 1982; Ridley et al. 1988). However, unlike other diseases that also show variable ages at onset, a class of "juvenile-onset" cases have been described for HD that show symptoms at an earlier age, before 21 years. It is interesting that it is much more common for such juvenile-onset cases to inherit the disease from their father than from their mother (Myers et al. 1982; Ridley et al. 1988). This has been taken to imply that either the HD gene itself or a modifier of the gene undergoes chromosomal imprinting (Erickson 1985; Reik 1988). Cloning of the HD gene should unravel many of these interesting facets of the disease. When the gene is in hand, it will also be possible to investigate how the mutation is manifested at the cellular level. The biochemical basis of HD is not known, and most attempts to clone the HD gene have relied on a knowledge of its chromosomal location. These genetic studies have yielded some valuable information. However, until recently, the studies failed to define a precise location for the gene. Genetic linkage between HD and the D4S1 0 marker was found in 1983 (Gusella et al. 1983), soon after DNA markers were first used for 1
Pritchard et al.
2 RAFI P1 centromere /, I
D4S62 D4S10 I approx. 6Mb I 17 cM
2 cM
m
p telomere
Are --4
HD *'--" 4cM
Figure I Localization of HD locus between D4S10 and 4p telomere. Linkage analysis places HD approximately 4 cM from D4S1 0 (Gilliam et al. 1987b; Conneally et al. 1989). RAFiP1 and D4S62 have a more centromeric location and are less tightly linked to HD than D4S10 (Gilliam et al. 1987b). The region between D4S10 and the telomere is estimated to span 6 Mb (Gilliam et al. 1987b; Bucan et al. 1990).
this purpose. D4S1 0 was subsequently localized both by in situ hybridization (Landegent et al. 1986) and by somatic cell genetics (MacDonald et al. 1987) to the short arm of chromosome 4, in the most terminal subband, 4pl 6.3. Progress toward localizing HD continued as multipoint linkage analysis was performed with D4S1 0 and two other markers known to be centromeric to D4S1 0 on 4p: D4S62 and RAFl P1 (fig. 1; Gilliam et al. 1 987b). HD was found to be more tightly linked to D4S1 0 than to the other markers (fig. 1). Such a result placed the disease locus between D4S1 0 and the 4p telomere, within a region estimated by cytogenetic measurement to contain less than 6 Mb of DNA (fig. 1; Gilliam et al. 1987b). Many new probes were isolated from the 6-Mb region (Pohl et al. 1988; Smith et al. 1988; Pritchard et al. 1989), and physical (Bucan et al. 1990) and genetic maps (MacDonald et al. 1989) were constructed with these probes. Genetic analysis was also performed on HD families by using polymorphisms identified with the new probes. The primary intention of this work was to identify crossover events between the new markers and HD so that the closest flanking markers for the disease locus could be identified. However, a major problem was encountered with the discovery of a hot spot for recombination very close to D4S1 0 (Skraastad et al. 1989; Allitto et al. 1991). Many of the recombination events occurring between D4S1 0 and HD were localized to this hot spot (Richards et al. 1988; Skraastad et al. 1989), thus reducing the number of events that could provide an exact location for HD. Four obligatory recombination events within the D4S10-4p telomere region but outside the hot spot remained known. These received considerable attention from several research groups. The results of the analyses produced conflicting locations for HD. In the case of three events (families 1-3 in fig. 2), HD was found to segregate with the wild-type allele at D4S1 0 (MacDonald et al. 1989; Robbins et al. 1989). All markers telomeric to D4S1 0 that were tested also originated from the wild-type chromosome. For families
1 and 2, the most telomeric informative marker was D4S1 11 (MacDonald et al. 1989), which maps 1,100 kb from the 4p telomere (Bucan et al. 1990). Family 3 was informative for D4S90 (Robbins et al. 1989), which resides approximately 300 kb from the telomere (Doggett et al. 1989; Bucan et al. 1990; Pritchard et al. 1990). As illustrated in figure 2, a telomeric location for HD was proposed on the basis of data on these families (MacDonald et al. 1989). Recombination events close to the telomere were thought to occur in each case to explain the observed segregation of HD. However, in family 4, the HD allele at D4S10 was inherited with HD. More telomeric markers such as D4S1 11 and D4S90 originated from the wild-type chromosome, indicating that a recombination event had occurred between these markers and D4S1 0. Such a result placed HD in a more centromeric region, at least 1 Mb proximal to the telomeric location. The two locations predicted for HD as defined by these recombination events clearly do not overlap. How can this be explained? Many different hypotheses were proposed, such as HD being a large gene and containing different mutations in the different families (MacDonald et al. 1989). However, at the time, it seemed most likely that the one recombination event predicting a more centromeric location (family 4 in fig. 2) was an exceptional case that could be explained as resulting from either misdiagnosis, mistyping, or, more likely, a second recombination event occurring very close to the telomere (MacDonald et al. 1989). With this double-crossover hypothesis in mind, attention was turned to the 300-kb region, defined by family 3, which placed HD between D4S90 and the 4p telomere. The decision to do this was risky, as no definitive interlocus recombination had been observed in family 3, even though recombination appeared to occur between the loci tested and HD. However, because of the region's small size, it was reasoned that it would be relatively quick and easy to analyze. In addition, by cloning more telomeric DNA, it would
3
Invited Editorial D4S10
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HD?
Possible locations for HD, based on four families displaying recombination events. The diagram (not drawn to scale) Figure 2 illustrates the genotypes of the region between D4S1 0 and the 4p telomere in the four recombinant chromosomes. Clear boxes represent regions originating from the wild-type chromosome. Hatched boxes signify regions whose genotype is unknown. Blackened boxes represent regions known to originate from the HD chromosome. In families 1-3, all of the tested polymorphic markers originate from the wild-type chromosome. To explain the inheritance of HD, the HD locus is placed close to the telomere (within the hatched boxes), and as yet unidentified recombination events are thought to occur telomeric to D4S1 11 (families 1 and 2; MacDonald et al. 1989) and to D4S90 (family 3; Robbins et al. 1989). For family 4 (MacDonald et al. 1989), markers in the more centromeric region originate from the HD chromosome. A recombination event has been identified between the markers D4S1 13 and D4S1 15, placing HD centromeric to D4S1 15. This location for HD is contradictory to that predicted by the data on families 1-3. Therefore a second recombination event, telomeric to D4S90, was initially thought to occur in family 4.
be possible to confirm the predicted crossovers for families 1-3 and to investigate whether the proposed second crossover in family 4 had indeed taken place. A telomeric location for HD also had romantic appeal. We became excited by the possibility of HD being a telomeric disorder, caused by a position effect of heterochromatic DNA (Laird 1990). Maybe this could explain the unusual genetic characteristics of the disease.
The DNA at the telomere of 4p was subsequently cloned. Bates et al. (1990) reported the isolation of a yeast artificial chromosome (YAC) which spans 11S kb and extends to the telomere of 4p. Other groups cloned DNA adjacent to the telomere, extending to D4S90 (Pritchard et al. 1990). However, a telomeric location for HD was brought into question on characterization of this DNA: few, if any, genes were found, and no DNA rearrangements associated with the disease were detectable (Pritchard et al. 1990). DNA at
the 4p telomere itself did not differ in size or structure in HD patients compared with control individuals when it was analyzed at a level of resolution that would detect changes of 5 kb or more (Pritchard et al. 1990). Doubts were also raised when it was realized that human telomeres carry homologous sequences (Brown et al. 1990) and can be polymorphic in length (Brown et al. 1990; Wilkie et al. 1991). If, as seems likely, this reflects DNA exchange at telomeres, then the HD mutation would no longer be linked to chromosome 4, if the mutation occurred prior to any exchange that involved 4p telomeric DNA. More direct evidence against a telomeric location was gathered from genetic studies. Polymorphisms were identified in two markers that mapped within 100 kb of the telomere: D4S142 (Bates et al. 1990) and D4S169 (Pritchard et al. 1990). When analysis with these markers was carried out, interlocus recombination was still not detected in family 3 (Pritchard,
Pritchard et al.
4 0 k 1 000 kb
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1
4p telomere Linkage disequilibrium
Figure 3 Map of region between D4S1 0 and 4p telomere, indicating candidate location for HD as proposed by Bates et al. (1991; see also Whaley et al. 1991). The candidate region for HD is represented by the black bar and spans approximately 2.5 Mb. The double slashes indicate a gap in the map of unknown size. + ve = markers that show significant linkage disequilibrium; - ve = markers that do not show significant disequilibrium; NT = markers that have not been tested. At present, positive disequilibrium has only been observed at D4S95 (Snell et al. 1989; Theilmann et al. 1989; Adam et al. 1991). As no other markers close to D4S95 have yet been tested, the region of disequilibrium could be quite large, extending at most from D4S10 to D4S43 (approximately 2 Mb).
unpublished data), and the putative second recombination event in family 4 was not observed (Bates et al. 1990). Final proof against a telomeric location was provided by the detection of nonrandom allelic association (linkage disequilibrium) between HD and two DNA markers in the more centromeric region: D4S95 and D4S98 (Snell et al. 1989; Theilmann et al. 1989; fig. 3). Significant allele association was not observed for markers in the telomeric region, such as D4S90 (Snell et al. 1989; Theilmann et al. 1989). The realization that the HD gene was not in the telomeric region was a setback for many. It precipitated a slowing up of research and a reevaluation of the approaches needed to find the real location for the gene. The past year has been spent collecting data, with careful consideration of which recombination events are best to evaluate. Bates et al. (1991), in this issue of the Journal, and Whaley et al. (1991), in a recent issue of Somatic Cell and Molecular Genetics, have now published the first results of this reevaluation. Whaley et al. analyze family 4 in detail and provide a very precise location for the recombination event: between the markers D4S168 and D4S1 13/114 (see fig. 3). Bates et al. focus on defining a proximal boundary for the gene. This boundary is provided by the recombination hot spot which lies close to D4S1 0. Of the recombination events that fall within this hot spot, Bates et al. identify one which appears to be the most telomeric, occurring between D4S125 and D4S1 80 (fig. 3). The conclusion from these studies is that HD lies proximal to D4S1 68 but distal to D4S1 25 (fig. 3). Physical mapping of this region shows that the two flanking markers are separated by approximately
2.5 Mb. It would be prudent to exercise some caution in accepting this location for HD, given that it depends on only two recombination events. However, perhaps we can afford to be more confident this time, as the new location is strongly supported by the linkage disequilibrium data. It is pertinent at this point to draw lessons from the successful cloning of the CF gene (Kerem et al. 1989; Riordan et al. 1989; Rommens et al. 1989). As with HD, some false starts were also made in localizing CF. Why, then, was it possible to clone this gene so rapidly? The answer lies partly with the fact that CF is encoded by a large gene and that few other candidate genes were detectable in the CF region. However, in addition, the CF researchers made some wise strategic moves that greatly aided cloning of the gene. The most intuitive was the decision to use linkage disequilibrium as a measure of genetic distance from the disease locus. Despite the analysis of many families, CF could only be narrowed to a 900-kb region based on the available recombination events (Kerem et al. 1989). Additional genetic methods were required to locate the gene. The CF researchers looked for linkage disequilibrium over the 900-kb region. Although fluctuations occurred at several locations, one very strong peak of disequilibrium was detected in a region that subsequently revealed the presence of the CF gene (Kerem et al. 1989). It was not known at the time whether this strategy would identify the gene, as previous studies had shown that other factors besides genetic distance - such as mutation rate, selection, and an uneven distribution of recombination events - can influence the strength of disequilibrium (Chakravarti et al. 1984). In the case
Invited Editorial of CF, other indicators such as the flanking crossovers and the presence of candidate genes were considered before effort was focused on the region of strong disequilibrium (Riordan et al. 1989; Rommens et al. 1989). The use of linkage disequilibrium for this purpose is instructive for the cloning of other disease loci for which allele association can be detected. For HD, several research groups are now actively involved in cloning the 2.5-Mb region. Although the size of this region is three times greater than that which existed for CF, YAC technology is now available to speed up the cloning. The linkage disequilibrium studies are also being extended. Indeed, a recent study has shown that the allele association is strong at D4S95 but no longer significant at D4S98 (fig. 3; Adam et al. 1991). Negative results have already been reported for D4S10 (Youngman et al. 1986; Theilmann et al. 1989), D4S43 (Gilliam et al. 1987a), and D4S96 (Theilmann et al. 1989). Therefore, most attention is currently being given to the region near D4S95. Once a smaller candidate region is identified and cloned, searches will be performed for candidate genes for HD. This may take a long time, as there maybe as many as 50 genes in the HD region. To identify which candidate gene is the HD gene, screens will need to be undertaken to determine whether there are any quantitative or qualitative differences in their expression in HD patients as compared with normal controls. Searches will also be performed for a genomic DNA rearrangement, by using the candidate gene sequences as probes against DNA isolated from both HD and non-HD individuals. If HD is caused by a small DNA change that is not detectable by these other approaches, then the candidate genes will be sequenced with the aim of identifying sequence differences between the genes of HD individuals and those of nonHD individuals. Although a lot of work lies ahead before the gene is available, we have reason to be optimistic, now that a more definitive location for HD has been achieved. The search for HD has been arduous, compounded by limited resources and bad luck. However, we remain intrigued by this disease and eagerly await knowing what type of mutation causes such unusual genetic features. We also anticipate an explanation for why we were misled by the three families showing a telomeric location for HD (families 1-3 in fig. 2). Are there really different mutations in the different families, or can they be explained by other events, such as rearrangements of chromosome 4p or gene conversion? Finally, cloning of the gene promises to be a major
relief for the families afflicted by HD. They, more than any of us, must look forward to the news that a more reliable predictive test is available and that potential treatments are in sight.
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6 Gusella JF, Wexler NS, Conneally PM, Naylor SL, Anderson MA, Tanzi RE, Watkins PC, et al (1983) A polymorphic marker genetically linked to Huntington's disease. Nature 306:234-238 Kerem B-S, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, et al (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073-1080 Laird C (1990) Proposed genetic basis of Huntington's disease. Trends Genet 6:242-247 Landegent JE, Jansen in de Wal N, Fisser-Groen YM, Bakker E, van der Ploeg M, Pearson PL (1986) Fine mapping of the Huntington disease linked D4S10 locus by nonradioactive in situ hybridization. Hum Genet 73:354-357 MacDonald ME, Anderson MA, Gilliam TC, Tranebjaerg L, Carpenter NJ, Magenis E, Hayden MR, et al (1987) A somatic cell hybrid panel for localizing DNA segments near the Huntington's disease gene. Genomics 1:29-34 MacDonald ME, HainesJL, Zimmer M, Cheng SV, Youngman S, Whaley WL, Wexler N, et al (1989) Recombination events suggest potential sites for the Huntington's disease gene. Neuron 3:183-190 Martin JB, Gusella JF (1986) Huntington's disease: pathogenesis and management. N Engl J Med 3 15: 1267-1276 Myers RH, Leavitt J, Farrer LA, Jagadeesh J, McFarlane H, Mastromauro CA, Mark RJ, et al (1989) Homozygote for Huntington disease. Am J Hum Genet 45:615-618 Myers RH, Madden JJ, Tiague JL, Falek A (1982) Factors related to onset age of Huntington disease. Am J Hum Genet 34:481-488 Pohl TM, Zimmer M, MacDonald ME, Smith B, Bucan M, Poustka A, Volinia S, et al (1988) Construction of a NotI linking library and isolation of new markers close to the Huntington's disease gene. Nucleic Acids Res 16:91859198 Pritchard C, Casher D, Bull L, Cox DR, Myers RM (1990) A cloned DNA segment from the telomeric region of human chromosome 4p is not detectably rearranged in Huntington disease patients. Proc Natl Acad Sci USA 87:73097313 Pritchard CA, Casher D, Uglum E, Cox DR, Myers RM (1989) Isolation and field-inversion gel electrophoresis analysis of DNA markers located close to the Huntington disease gene. Genomics 4:408-418 Reik W (1988) Genomic imprinting: a possible mechanism for the parental origin effect in Huntington's chorea. J Med Genet 25:805-808 Richards JE, Gilliam TC, Cole JL, Drumm ML, Wasmuth JJ, Gusella JF, Collins FS (1988) Chromosome jumping from D4S10 (G8) toward the Huntington disease gene. Proc Natl Acad Sci USA 85:6437-6441
Pritchard et al. Ridley RM, Frith CD, Crow TJ, Conneally PM (1988) Anticipation in Huntington's disease is inherited through the male line but may originate in the female. J Med Genet 25:589-595 Riordan JR, Rommens JM, Kerem B-S, Alon N, Rozmahel R, Grzaelczak Z, Zielenski J, et al (1989) Identification of the cystic fibrosis gene: cloning and characteization of complementary DNA. Science 245:1066-1073 Robbins C, Theilmann J, Youngman S, Haines J, Altherr MJ, Harper PS, Payne C, et al (1989) Evidence from family studies that the gene causing Huntington disease is telomeric to D4S95 and D4S90. Am J Hum Genet 44: 422-425 Rommens JM, Iannuzzi MC, Kerem B-S, Drumm ML, Melmer G, Dean M, Rozmahel R, et al (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245:1059-1065 Skraastad MI, Bakker E, de Lange LF, Vegter-van der Vhs M, Klein-Breteler EG, van Ommen GJB, Pearson PL (1989) Mapping of recombinants near the Huntington disease locus by using G8 (D4S10) and newly isolated markers in the D4S10 region. Am J Hum Genet 44:560566 Smith B, Skarecky D, Bengtsson U, Magenis EM, Carpenter N, Wasmuth JJ (1988) Isolation of DNA markers in the direction of the Huntington disease gene from the G8 locus. Am J Hum Genet 42:335-344 Snell RG, Lazarou L, Youngman S, Quarrell OWJ, Wasmuth JJ, Shaw DJ, Harper PS (1989) Linkage disequilibrium in Huntington's disease: an improved localization for the gene. J Med Genet 26:673-675 Theilmann J, Kanani S, Shiang R, Robbins C, Quarrell 0, Huggins M, Hedrick A, et al (1989) Non-random association between alleles detected at D4S95 and D4S98 and the Huntington's disease gene. J Med Genet 26:676-681 Wexler NS, Young AB, Tanzi RE, Travers H, StarostaRubinstein S, Penney JB, Snodgrass SR, et al (1987) Homozygotes for Huntington's disease. Nature 326:194197 Whaley WL, Bates GP, Novelletto A, Sedlacek Z, Cheng S, Romano D, Ormondroyd E, et al (1991) Mapping of cosmid clones in Huntington's disease region of chromosome 4. Somatic Cell Mol Genet 17:83-91 Wilkie AOM, Higgs DR, Rack KA, Buckle VJ, Spurr NK, Fischel-Ghodsian N, Ceccherini I, et al (1991) Stable length polymorphism of up to 260 kb at the tip of the short arm of human chromosome 16. Cell 64:595-606 Youngman S, Sarfarazi M, Quarrell OWJ, Conneally PM, Gibbons K, Harper PS, Shaw DJ, et al (1986) Studies of a DNA marker (G8) genetically linked to Huntington's disease in British families. Hum Genet 73:333-339
