Am. J. Hum. Genet. 51:357-362, 1992
A Recombination Event That Redefines the Huntington Disease Region Russell G. Snell,* Leslie M. Thompson,T Danilo A. Tagle4 Tracey L. Holloway,* Glenn Barnes,§ Helen G. Harley,* Lodewijk A. Sandkuijl,* Marcy E. MacDonald,§ Francis S. Collinst James F. Gusella,§ Peter S. Harper, * and Duncan J. Shaw* *Department of Medical Genetics, University of Wales College of Medicine, Cardiff; tDepartment of Biological Chemistry, College of Medicine, University of California, Irvine; $Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor; and §Molecular Neurogenetics Laboratory Neuroscience Center, Massachusetts General Hospital East, Charlestown
Summary We report both a recombination event that places the Huntington disease gene proximal to the marker D4S98 and an extended linkage-disequilibrium study that uses this marker and confirms the existence of disequilibrium between it and the HD locus. We also report the cloning of other sequences in the region around D4S98, including a new polymorphic marker R10 and conserved sequences that identify a gene in the region of interest.
Introduction Huntington disease (HD) is a neurodegenerative disorder with an autosomal dominant mode of inheritance (Harper et al. 1991). Symptoms generally first appear in middle age, becoming more severe until death 1015 years later. The gene has been localized to the region 4p16.3-4pter, by linkage studies, in situ hybridization, somatic cell genetics, and more recently, linkage-disequilibrium studies (Gusella et al. 1983; Landegent et al. 1986; Magenis et al. 1986; Youngman et al. 1986; MacDonald et al. 1987; Snell et al. 1989a; Theilman et al. 1989). There is now a complete physical map of the region expected to contain the gene, extending from the locus D4S10 3 Mb to the locus D4S168 (Bates et al. 1991). The interpretation of recombinants in the region, which encouraged the search for the gene toward the telomere, now suggests the existence of two distinct alternative locations for the gene (MacDonald et al. 1989b; Robbins et al. 1989; Bates et al. 1991). Of the published recombinants, three suggest that the gene is located close to Received January 14, 1992; revision received March 30, 1992. Address for correspondence and reprints: Dr. Russell G. Snell, Department of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, United Kingdom. © 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5102-0015$02.00
the telomere. We and others have previously reported linkage disequilibrium with the markers D4S95 and D4S98, which supports the nontelomeric location of the HD gene (Snell et al. 1989a; Theilman et al. 1989; Barron et al. 1991; MacDonald et al. 1991). It has been suggested that the recombinants indicating a distal location may be either the result of a double recombination in a small region or, possibly, a result of gene conversion (MacDonald et al. 1991). We have studied our HD families for further recombinants, to define the candidate region more precisely. We have also extended our disequilibrium study around D4S98 and have cloned some of that region, identifying a new polymorphism that has been used in our disequilibrium and recombinant study. Material and Methods Restriction-digested DNA was separated by agarose gel electrophoresis and was transferred to nylon membrane (Hybond N + Amersham) by alkali vacuum
blotting (Snell and Shaw 1991). Hybridization and autoradiography were performed according to standard techniques (Gusella et al. 1983). The conditions for amplification and resolution of the C-A repeat were as described by Tagle et al. (in press). Probes used in the present study are shown in table 1. 357
4
Cl n m
H
-i
m 0
400Q
H tSn r-i H U H 0 Cl0 CO ( H * CA U) r0 rH U i) U1t : lP. U) U) U) >4:m Q G Q Q
O O -4U
C
.
Una'
rl H
C
P4
r-
U)
r-4H
UE
U)
>nQa
Recombination Defining HD Region
359
Table I Loci, Probes, and Enzymes Used in Present Study
Locus
Probe
Enzyme detecting RFLP
HindIII D4S10 .... pKO82 D4S43 .... C-A dinucleotide repeat SstI D4S98 .. BS731 BamHI R10 Y1P18 SstI PstI D4S111 ... 157.9 D4S115 ... 252.3 PstI D4S141 ... 2R3 HindIII
Reference Gusella et al. 1983 Tagle et al., in press Wasmuth et al. 1988 Present paper Lin et al. 1991 MacDonald et al. 1989a MacDonald et al. 1989a Snell et al. 1989b
Clone Isolation
A preparative pulsed field gel containing NruIdigested DNA from the hybrid HHW693 (Wasmuth et al. 1986) was cut into 17 4-mm slices perpendicular to the direction of mobility (Michiels et al. 1987). The DNA, of size 50-2,000 kb, was extracted by agarase digestion. Samples from four fractions expected to contain fragments of "'150 kb were digested with SstI, were separated on a standard gel, and hybridized with the probe BS731 (D4S98). The positive fraction was partially digested with Sau3A and was cloned into lambda EMBL3A. The library was plated out and screened by hybridization with labeled human DNA. Hybridizing plaques were screened a second time and were localized to chromosome 4 by using a hybrid (JS4A9. 1) with 4 as its only human chromosome (Saxon et al. 1985). The positive clones were then mapped by pulsed-field electrophoresis using HHW693 DNA digested with the enzymes Nod, MluI, and NruI. Results Analysis of the Recombinant Family
For analysis, the family (fig. 1) was divided into four sections; the three branches that resulted from the
transmission of the gene by individuals 3, 5, and 7 three sections, and their three sibs-9, 10, and 11-constituted the fourth. The disease status of affected individuals only was used to establish phase. Derived haplotypes are shown in brackets, and typings where phase cannot be assigned are displayed in parentheses. A minimum number of recombination events were assumed. Paternity tests have been applied in the form of four probes for hypervariable loci (DNA profiling kit; Amersham), and, where possible, critical patients have been resampled. This analysis suggests that three recombination events have occurred in this family - two in the first generation, resulting in haplotypes 5 and 6, and the other in either the first or the second generation, producing haplotype 7. Haplotype 1 is assumed to represent the original mutation-bearing chromosome, because it segregates with the HD phenotype in the progeny of two of the three affected sibs in the second generation. This chromosome is evident in patients 12, 14, 17, 18, and 22, but it is not present in the apparently unaffected individuals 9 and 10, who were last examined at ages 84 years and 68 years, respectively. The recombination event that resulted in haplotype 5 has reset the distal location boundary of the HD gene. Haplotype 5 segregates with the disease in individual 21 and probably arose in person 1 by a recombination between the loci D4S98 and D4S43 (for the physical locations of the markers, see fig. 2). Patient 21 therefore inherited the proximal region of the HD chromosome, from a point somewhere between D4S98 and D4S43. The gene, therefore, must be proximal to D4S98. Haplotype 6 is also probably a product of a recombination in person 1 and is present in individual 11. In this case the recombination took place between Y1P1 8 and D4S1 11, with the distal portion of the HD chromosome being retained. This patient, whose agewere
Figure I Pedigree of an HD family, which sets the distal boundary for the HD gene. Phase was established and typings were inferred for deceased individuals, by assuming the least number of recombinants. The family was divided into four sections for analysis. Three sections were the branches originating from persons 3, 5, and 7; the fourth included persons 9, 10, and 11. Where possible, sections were analyzed separately. Large brackets denote derived haplotypes, and parentheses contain real or derived typings for cases where phase cannot be established. The locations between markers of recombination events are shown by arrows, although the events did not necessarily first occur in the people shown. Haplotypes 1 and 5 segregate with the disease. A recombination event between loci D4S43 and D4S98 in person 1 would result in haplotype 5, which is present in person 21. This implies that the gene is proximal to D4S98, since this region is retained in the patient. Haplotype 6 in person 11 also resulted from a recombination in person 1. The original mutation-bearing chromosome (1) was retained distal to probe Y1P18, although the patient was unaffected at age 79 years. The third recombination occurred in either person 2 or person 3, resulting in haplotype 7 being apparent in person 13. This recombination took place either between loci D4S1 15 and D4S141, if in person 2, or somewhere between D4S98 and D4S141 in person 3.
360
Snell et al. BS731
M RN
(M)
_3 S 5
R (R) N
S37 180kb
1
R
160kb
cen D4S1 10 D4S125 D4S95
_s-
RlO/S31
D4S43
I
R10 Y1P18 D4S98
tel D4S111 D4S115
I
I
;I
//
*
D4S141
7
HD gene location
region in which flanking crossovers occured
Figure 2 Physical map of the HD candidate region. The lower map shows the approximate locations of the markers used in the analysis of the family in fig. 1. The enlarged section includes new clones isolated from the region around BS731 (D4S98). A phage library was constructed from NruI-digested hybrid DNA, size selected by pulsed-field electrophoresis for the 160-kb fragments. Clones S37, S35, and R10/S31 mapped back to the region by hybridization to pulsed-field filters of MluI (M)- , NotI (N)- , and Nrul (R)-digested HHW693 DNA.
adjusted risk of inheriting the disorder was
A Recombination Event That Redefines the Huntington Disease Region Russell G. Snell,* Leslie M. Thompson,T Danilo A. Tagle4 Tracey L. Holloway,* Glenn Barnes,§ Helen G. Harley,* Lodewijk A. Sandkuijl,* Marcy E. MacDonald,§ Francis S. Collinst James F. Gusella,§ Peter S. Harper, * and Duncan J. Shaw* *Department of Medical Genetics, University of Wales College of Medicine, Cardiff; tDepartment of Biological Chemistry, College of Medicine, University of California, Irvine; $Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor; and §Molecular Neurogenetics Laboratory Neuroscience Center, Massachusetts General Hospital East, Charlestown
Summary We report both a recombination event that places the Huntington disease gene proximal to the marker D4S98 and an extended linkage-disequilibrium study that uses this marker and confirms the existence of disequilibrium between it and the HD locus. We also report the cloning of other sequences in the region around D4S98, including a new polymorphic marker R10 and conserved sequences that identify a gene in the region of interest.
Introduction Huntington disease (HD) is a neurodegenerative disorder with an autosomal dominant mode of inheritance (Harper et al. 1991). Symptoms generally first appear in middle age, becoming more severe until death 1015 years later. The gene has been localized to the region 4p16.3-4pter, by linkage studies, in situ hybridization, somatic cell genetics, and more recently, linkage-disequilibrium studies (Gusella et al. 1983; Landegent et al. 1986; Magenis et al. 1986; Youngman et al. 1986; MacDonald et al. 1987; Snell et al. 1989a; Theilman et al. 1989). There is now a complete physical map of the region expected to contain the gene, extending from the locus D4S10 3 Mb to the locus D4S168 (Bates et al. 1991). The interpretation of recombinants in the region, which encouraged the search for the gene toward the telomere, now suggests the existence of two distinct alternative locations for the gene (MacDonald et al. 1989b; Robbins et al. 1989; Bates et al. 1991). Of the published recombinants, three suggest that the gene is located close to Received January 14, 1992; revision received March 30, 1992. Address for correspondence and reprints: Dr. Russell G. Snell, Department of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, United Kingdom. © 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5102-0015$02.00
the telomere. We and others have previously reported linkage disequilibrium with the markers D4S95 and D4S98, which supports the nontelomeric location of the HD gene (Snell et al. 1989a; Theilman et al. 1989; Barron et al. 1991; MacDonald et al. 1991). It has been suggested that the recombinants indicating a distal location may be either the result of a double recombination in a small region or, possibly, a result of gene conversion (MacDonald et al. 1991). We have studied our HD families for further recombinants, to define the candidate region more precisely. We have also extended our disequilibrium study around D4S98 and have cloned some of that region, identifying a new polymorphism that has been used in our disequilibrium and recombinant study. Material and Methods Restriction-digested DNA was separated by agarose gel electrophoresis and was transferred to nylon membrane (Hybond N + Amersham) by alkali vacuum
blotting (Snell and Shaw 1991). Hybridization and autoradiography were performed according to standard techniques (Gusella et al. 1983). The conditions for amplification and resolution of the C-A repeat were as described by Tagle et al. (in press). Probes used in the present study are shown in table 1. 357
4
Cl n m
H
-i
m 0
400Q
H tSn r-i H U H 0 Cl0 CO ( H * CA U) r0 rH U i) U1t : lP. U) U) U) >4:m Q G Q Q
O O -4U
C
.
Una'
rl H
C
P4
r-
U)
r-4H
UE
U)
>nQa
Recombination Defining HD Region
359
Table I Loci, Probes, and Enzymes Used in Present Study
Locus
Probe
Enzyme detecting RFLP
HindIII D4S10 .... pKO82 D4S43 .... C-A dinucleotide repeat SstI D4S98 .. BS731 BamHI R10 Y1P18 SstI PstI D4S111 ... 157.9 D4S115 ... 252.3 PstI D4S141 ... 2R3 HindIII
Reference Gusella et al. 1983 Tagle et al., in press Wasmuth et al. 1988 Present paper Lin et al. 1991 MacDonald et al. 1989a MacDonald et al. 1989a Snell et al. 1989b
Clone Isolation
A preparative pulsed field gel containing NruIdigested DNA from the hybrid HHW693 (Wasmuth et al. 1986) was cut into 17 4-mm slices perpendicular to the direction of mobility (Michiels et al. 1987). The DNA, of size 50-2,000 kb, was extracted by agarase digestion. Samples from four fractions expected to contain fragments of "'150 kb were digested with SstI, were separated on a standard gel, and hybridized with the probe BS731 (D4S98). The positive fraction was partially digested with Sau3A and was cloned into lambda EMBL3A. The library was plated out and screened by hybridization with labeled human DNA. Hybridizing plaques were screened a second time and were localized to chromosome 4 by using a hybrid (JS4A9. 1) with 4 as its only human chromosome (Saxon et al. 1985). The positive clones were then mapped by pulsed-field electrophoresis using HHW693 DNA digested with the enzymes Nod, MluI, and NruI. Results Analysis of the Recombinant Family
For analysis, the family (fig. 1) was divided into four sections; the three branches that resulted from the
transmission of the gene by individuals 3, 5, and 7 three sections, and their three sibs-9, 10, and 11-constituted the fourth. The disease status of affected individuals only was used to establish phase. Derived haplotypes are shown in brackets, and typings where phase cannot be assigned are displayed in parentheses. A minimum number of recombination events were assumed. Paternity tests have been applied in the form of four probes for hypervariable loci (DNA profiling kit; Amersham), and, where possible, critical patients have been resampled. This analysis suggests that three recombination events have occurred in this family - two in the first generation, resulting in haplotypes 5 and 6, and the other in either the first or the second generation, producing haplotype 7. Haplotype 1 is assumed to represent the original mutation-bearing chromosome, because it segregates with the HD phenotype in the progeny of two of the three affected sibs in the second generation. This chromosome is evident in patients 12, 14, 17, 18, and 22, but it is not present in the apparently unaffected individuals 9 and 10, who were last examined at ages 84 years and 68 years, respectively. The recombination event that resulted in haplotype 5 has reset the distal location boundary of the HD gene. Haplotype 5 segregates with the disease in individual 21 and probably arose in person 1 by a recombination between the loci D4S98 and D4S43 (for the physical locations of the markers, see fig. 2). Patient 21 therefore inherited the proximal region of the HD chromosome, from a point somewhere between D4S98 and D4S43. The gene, therefore, must be proximal to D4S98. Haplotype 6 is also probably a product of a recombination in person 1 and is present in individual 11. In this case the recombination took place between Y1P1 8 and D4S1 11, with the distal portion of the HD chromosome being retained. This patient, whose agewere
Figure I Pedigree of an HD family, which sets the distal boundary for the HD gene. Phase was established and typings were inferred for deceased individuals, by assuming the least number of recombinants. The family was divided into four sections for analysis. Three sections were the branches originating from persons 3, 5, and 7; the fourth included persons 9, 10, and 11. Where possible, sections were analyzed separately. Large brackets denote derived haplotypes, and parentheses contain real or derived typings for cases where phase cannot be established. The locations between markers of recombination events are shown by arrows, although the events did not necessarily first occur in the people shown. Haplotypes 1 and 5 segregate with the disease. A recombination event between loci D4S43 and D4S98 in person 1 would result in haplotype 5, which is present in person 21. This implies that the gene is proximal to D4S98, since this region is retained in the patient. Haplotype 6 in person 11 also resulted from a recombination in person 1. The original mutation-bearing chromosome (1) was retained distal to probe Y1P18, although the patient was unaffected at age 79 years. The third recombination occurred in either person 2 or person 3, resulting in haplotype 7 being apparent in person 13. This recombination took place either between loci D4S1 15 and D4S141, if in person 2, or somewhere between D4S98 and D4S141 in person 3.
360
Snell et al. BS731
M RN
(M)
_3 S 5
R (R) N
S37 180kb
1
R
160kb
cen D4S1 10 D4S125 D4S95
_s-
RlO/S31
D4S43
I
R10 Y1P18 D4S98
tel D4S111 D4S115
I
I
;I
//
*
D4S141
7
HD gene location
region in which flanking crossovers occured
Figure 2 Physical map of the HD candidate region. The lower map shows the approximate locations of the markers used in the analysis of the family in fig. 1. The enlarged section includes new clones isolated from the region around BS731 (D4S98). A phage library was constructed from NruI-digested hybrid DNA, size selected by pulsed-field electrophoresis for the 160-kb fragments. Clones S37, S35, and R10/S31 mapped back to the region by hybridization to pulsed-field filters of MluI (M)- , NotI (N)- , and Nrul (R)-digested HHW693 DNA.
adjusted risk of inheriting the disorder was
