Am. J. Hum. Genet. 49:723-734, 1991
Complex Patterns of Linkage Disequilibrium in the Huntington Disease Region Marcy E. MacDonald, * Carol Lin, * Lakshmi Srinidhi, * Gillian Bates, t Michael Altherr,t W. Lance Whaley,* Hans LehrachT John Wasmuth,$ and James F. Gusella* Neurogenetics Laboratory, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Boston; tImperial Cancer Research Fund, Lincoln's Inn Fields, London; and tDepartment of Biological Chemistry, University of California, Irvine
Summary The genetic defect causing Huntington disease (HD) has been mapped to 4pl6.3 by linkage analysis using DNA markers. Two apparently contradictory classes of recombination events in HD kindreds preclude precise targeting of efforts to clone the disease gene. Here, we report a new recombination event that increases support for an internal candidate region of 2.5 Mb between D4S10 and D4S168. Analysis of 23 DNA polymorphisms in 4pl6.3 revealed a complex pattern of association with the disease gene that failed to narrow the size of the candidate region. The degree of linkage disequilibrium did not show a continuous increase across the physical map, nor was a region of extreme disequilibrium identified. Markers displaying no association with the disorder were interspersed with and, in many cases, close to markers displaying significant disequilibrium. Comparison of closely spaced marker pairs on normal and HD chromosomes, as well as analysis of haplotypes across the HD region, suggest that simple recombination subsequent to a single original HD mutation cannot easily explain the pool of HD chromosomes seen today. A number of different mechanisms could contribute to the diversity of haplotypes observed on HD chromosomes, but it is likely that there has been more than one and possibly several independent origins of the HD mutation.
Introduction
Huntington disease (HD) is a progressive late-onset neurologic disorder resulting from a dominant defect (HD) on chromosome 4 (Martin and Gusella 1986). The disease gene was first localized by genetic linkage analysis using the DNA marker D4S1 0 in two HD pedigrees, an extremely large Venezuelan family and a smaller American kindred (Gusella et al. 1983, 1984). Subsequent family studies have provided no evidence for nonallelic heterogeneity (Conneally et al. 1989). Identification of recombination events between HD and D4S1 0 placed the disease gene between the DNA marker and the telomere in 4p16.3 (Gilliam et al. Received March 14, 1991; revision received June 6, 1991. Address for correspondence and reprints: James F. Gusella, Ph.D., Molecular Neurogenetics Laboratory, Building 149, 6th Floor, Massachusetts General Hospital, 13th Street, Charlestown, MA 02129. © 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4904-0005$02.00
1987b). More detailed analysis using additional DNA markers from this region has not yielded a single candidate region for the defect (MacDonald et al. 1989b). Three distinct classes of crossover event have been observed. The majority of recombinations between D4S10 and HD occurred in a 200-300-kb segment immediately distal to the DNA marker. This region has been shown to display a much higher frequency of recombination than would be expected on the basis of its size (Allitto et al. 1991). In three families, events have been detected in which HD displays recombination with every informative marker between D4S10 and the telomere, suggesting a location for the disease gene in the terminal 250 kb of the chromosome (Whaley et al. 1988; Robbins et al. 1989). Finally, a single event has been reported in which markers from 100 kb and 1,500 kb from the telomere have recombined with HD while more proximal markers, including D4S10, have not (MacDonald et al. 1989b). The conflicting predictions provided by the latter two classes of recombination event require that two 723
MacDonald et al.
724 separate physical locations be considered as candidate HD gene regions: a terminal region of 100 kb from the most distal marker to the telomere and an internal region of 2.5 Mb located between D4S10 and D4S168 (Whaley et al. 1991). The terminal candidate region has been cloned in its entirety as a yeast artificial chromosome (Bates et al. 1990). For the internal candidate region, a detailed physical map has been completed (Bates et al. 1991) and a significant proportion of the DNA has been isolated (Whaley et al. 1991; Lin et al., in press). Two recent reports of linkage disequilibrium between HD and the DNA markers D4S95 and D4S98 have favored the proximal candidate region but have not significantly narrowed its size (Snell et al. 1989;
Theilmann
et
al. 1989). The discovery of
a
family
displaying an additional convincing recombination event that predicts the internal location for HD has reinforced the need for a more extensive correlation of linkage disequilibrium to the physical map of this candidate region. Material and Methods Pedigree DNAs and Marker Typing
DNA was prepared from fresh blood (Seizinger et al. 1986) or Epstein-Barr virus-transformed lymphoblastoid cell lines (Gusella et al. 1979; Anderson and Gusella 1984) from affected members of HD pedigrees and their unaffected relatives. Clinical diagnosis of HD, based on the characteristic movement disorder (Wexler et al. 1987), was made by neurologists familiar with HD who were blind to the specific genetic data. For the members of the family displaying recombination, diagnoses have been reconfirmed, and independent blood samples have been obtained and retyped. Agarose gel electrophoresis, DNA transfer, hybridization, and autoradiography were performed as described elsewhere (Gusella et al. 1983), except that probes were labeled by the oligonucleotide priming method of Feinberg and Vogelstein (1984). The following probe/enzyme combinations were used for the linkage disequilibrium studies: D4S90, D5 (StuI) (Youngman et al. 1989); D4S111, p157.9 (PstI and BclI) (MacDonald et al. 1989a); D4S11S, p252.3 (PstI) (MacDonald et al. 1989b); D4S96, pBS678 (MspI) (Smith et al. 1988); D4S168, E4PS18 (RsaI) (Whaley et al. 1991); D4S113, p337 (StuI) (Whaley et al. 1988); D4S186, Ylpl8 (SacI) (Lin et al., in press); D4S114, p312 (SacI) (Whaley et al. 1988);
D4S98, pBS731 (Sad) (Smith et al. 1988); D4S43, LCD150 (Sau96), C34Sac2.4 (HinclI), pKP1.65 (StuI) (Gilliam et al. 1987a; MacDonald et al. 1989a; Lin et al., in press); D4S183, pR4pl7 (RsaI) (Lin et al., in press); D4S182, Y12Eco2.3 (EcoT22) (Lin et al., in press); D4S95, pBS674E-D (TaqI and AccI) (Smith et al. 1988; Wasmuth et al. 1988); D4S127, p358 (PvuII), p363 (StuI) (Allitto et al. 1991); D4S180, L19ps11 (BamHI), L19ps14 (XmnI) (Lin et al., in press); D4S125, F4psl9 (BglII) (Lin et al., in press); and D4S126, p309 (Sad) (Allitto et al. 1991). Note that, although the probes for the Sad RFLPs at D4S1 14 and D4S98 lie within 3 kb of each other and were previously thought to detect the same polymorphic site (MacDonald et al. 1989b), the polymorphisms are in fact distinct. Statistical Analysis For individual markers, we typed single HD patients from part or all of a collection of 97 independent families and, by typing one or both parents, deduced the particular allele cosegregating with HD. Typing results from the unaffected parents were used to estimate allele frequencies in the normal population. Where phase could not be determined (usually
Complex Patterns of Linkage Disequilibrium in the Huntington Disease Region Marcy E. MacDonald, * Carol Lin, * Lakshmi Srinidhi, * Gillian Bates, t Michael Altherr,t W. Lance Whaley,* Hans LehrachT John Wasmuth,$ and James F. Gusella* Neurogenetics Laboratory, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Boston; tImperial Cancer Research Fund, Lincoln's Inn Fields, London; and tDepartment of Biological Chemistry, University of California, Irvine
Summary The genetic defect causing Huntington disease (HD) has been mapped to 4pl6.3 by linkage analysis using DNA markers. Two apparently contradictory classes of recombination events in HD kindreds preclude precise targeting of efforts to clone the disease gene. Here, we report a new recombination event that increases support for an internal candidate region of 2.5 Mb between D4S10 and D4S168. Analysis of 23 DNA polymorphisms in 4pl6.3 revealed a complex pattern of association with the disease gene that failed to narrow the size of the candidate region. The degree of linkage disequilibrium did not show a continuous increase across the physical map, nor was a region of extreme disequilibrium identified. Markers displaying no association with the disorder were interspersed with and, in many cases, close to markers displaying significant disequilibrium. Comparison of closely spaced marker pairs on normal and HD chromosomes, as well as analysis of haplotypes across the HD region, suggest that simple recombination subsequent to a single original HD mutation cannot easily explain the pool of HD chromosomes seen today. A number of different mechanisms could contribute to the diversity of haplotypes observed on HD chromosomes, but it is likely that there has been more than one and possibly several independent origins of the HD mutation.
Introduction
Huntington disease (HD) is a progressive late-onset neurologic disorder resulting from a dominant defect (HD) on chromosome 4 (Martin and Gusella 1986). The disease gene was first localized by genetic linkage analysis using the DNA marker D4S1 0 in two HD pedigrees, an extremely large Venezuelan family and a smaller American kindred (Gusella et al. 1983, 1984). Subsequent family studies have provided no evidence for nonallelic heterogeneity (Conneally et al. 1989). Identification of recombination events between HD and D4S1 0 placed the disease gene between the DNA marker and the telomere in 4p16.3 (Gilliam et al. Received March 14, 1991; revision received June 6, 1991. Address for correspondence and reprints: James F. Gusella, Ph.D., Molecular Neurogenetics Laboratory, Building 149, 6th Floor, Massachusetts General Hospital, 13th Street, Charlestown, MA 02129. © 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4904-0005$02.00
1987b). More detailed analysis using additional DNA markers from this region has not yielded a single candidate region for the defect (MacDonald et al. 1989b). Three distinct classes of crossover event have been observed. The majority of recombinations between D4S10 and HD occurred in a 200-300-kb segment immediately distal to the DNA marker. This region has been shown to display a much higher frequency of recombination than would be expected on the basis of its size (Allitto et al. 1991). In three families, events have been detected in which HD displays recombination with every informative marker between D4S10 and the telomere, suggesting a location for the disease gene in the terminal 250 kb of the chromosome (Whaley et al. 1988; Robbins et al. 1989). Finally, a single event has been reported in which markers from 100 kb and 1,500 kb from the telomere have recombined with HD while more proximal markers, including D4S10, have not (MacDonald et al. 1989b). The conflicting predictions provided by the latter two classes of recombination event require that two 723
MacDonald et al.
724 separate physical locations be considered as candidate HD gene regions: a terminal region of 100 kb from the most distal marker to the telomere and an internal region of 2.5 Mb located between D4S10 and D4S168 (Whaley et al. 1991). The terminal candidate region has been cloned in its entirety as a yeast artificial chromosome (Bates et al. 1990). For the internal candidate region, a detailed physical map has been completed (Bates et al. 1991) and a significant proportion of the DNA has been isolated (Whaley et al. 1991; Lin et al., in press). Two recent reports of linkage disequilibrium between HD and the DNA markers D4S95 and D4S98 have favored the proximal candidate region but have not significantly narrowed its size (Snell et al. 1989;
Theilmann
et
al. 1989). The discovery of
a
family
displaying an additional convincing recombination event that predicts the internal location for HD has reinforced the need for a more extensive correlation of linkage disequilibrium to the physical map of this candidate region. Material and Methods Pedigree DNAs and Marker Typing
DNA was prepared from fresh blood (Seizinger et al. 1986) or Epstein-Barr virus-transformed lymphoblastoid cell lines (Gusella et al. 1979; Anderson and Gusella 1984) from affected members of HD pedigrees and their unaffected relatives. Clinical diagnosis of HD, based on the characteristic movement disorder (Wexler et al. 1987), was made by neurologists familiar with HD who were blind to the specific genetic data. For the members of the family displaying recombination, diagnoses have been reconfirmed, and independent blood samples have been obtained and retyped. Agarose gel electrophoresis, DNA transfer, hybridization, and autoradiography were performed as described elsewhere (Gusella et al. 1983), except that probes were labeled by the oligonucleotide priming method of Feinberg and Vogelstein (1984). The following probe/enzyme combinations were used for the linkage disequilibrium studies: D4S90, D5 (StuI) (Youngman et al. 1989); D4S111, p157.9 (PstI and BclI) (MacDonald et al. 1989a); D4S11S, p252.3 (PstI) (MacDonald et al. 1989b); D4S96, pBS678 (MspI) (Smith et al. 1988); D4S168, E4PS18 (RsaI) (Whaley et al. 1991); D4S113, p337 (StuI) (Whaley et al. 1988); D4S186, Ylpl8 (SacI) (Lin et al., in press); D4S114, p312 (SacI) (Whaley et al. 1988);
D4S98, pBS731 (Sad) (Smith et al. 1988); D4S43, LCD150 (Sau96), C34Sac2.4 (HinclI), pKP1.65 (StuI) (Gilliam et al. 1987a; MacDonald et al. 1989a; Lin et al., in press); D4S183, pR4pl7 (RsaI) (Lin et al., in press); D4S182, Y12Eco2.3 (EcoT22) (Lin et al., in press); D4S95, pBS674E-D (TaqI and AccI) (Smith et al. 1988; Wasmuth et al. 1988); D4S127, p358 (PvuII), p363 (StuI) (Allitto et al. 1991); D4S180, L19ps11 (BamHI), L19ps14 (XmnI) (Lin et al., in press); D4S125, F4psl9 (BglII) (Lin et al., in press); and D4S126, p309 (Sad) (Allitto et al. 1991). Note that, although the probes for the Sad RFLPs at D4S1 14 and D4S98 lie within 3 kb of each other and were previously thought to detect the same polymorphic site (MacDonald et al. 1989b), the polymorphisms are in fact distinct. Statistical Analysis For individual markers, we typed single HD patients from part or all of a collection of 97 independent families and, by typing one or both parents, deduced the particular allele cosegregating with HD. Typing results from the unaffected parents were used to estimate allele frequencies in the normal population. Where phase could not be determined (usually
