Am. J. Hum. Genet. 48:595-603, 1991

Linkage Disequilibrium and Modification of Risk for Huntington Disease Shelin Adam, * Jane Theilmann, * Ken Buetow, t Amy Hedrick, * Colin Collins, * Bernard Weber, * Marlene Huggins,* and Michael Hayden* *Department of Medical Genetics, University of British Columbia, Vancouver; and tFox Chase Cancer Center, Philadelphia

Summary

The major limitation in performing predictive testing for Huntington disease (HD) is the unavailability of DNA from crucial family members. In our program approximately 20% (36/183) of persons have been excluded from predictive testing because of this reason. The major aim of this study was to examine whether data derived from linkage disequilibrium could modify risk analysis for persons at risk for HD. As a first step, we assessed whether the previously reported linkage disequilibrium between alleles recognized by probe pBS674E-D at locus D4S95 remained significant in a much larger data set. A total of 1,150 chromosomes from 622 individuals-200 affected and 422 unaffected-from 118 families were assessed. Significant haplotype association was detected with AccI and MboI RFLPs at the locus D4S95, with all the families (P = .00003), as well as for a subset from the United Kingdom (P = .0037). Data derived from linkage disequilibrium studies using D4S95 modifies the risk for HD, especially in persons of U.K. descent. Utilization of this approach for risk modification of HD awaits both validation of these data and additional information concerning ethnic-specific alleles at the D4S95 locus.

Introduction The gene causing Huntington disease (HD) has been localized to a region close to the telomere of chromosome 4 (Gilliam et al. 1987; Whaley et al. 1988; Robbins et al. 1989). While the precise location is still unknown, the discovery of DNA markers linked to the HD gene has resulted in the establishment of predictive testing programs in many parts of the world. Additional markers located closer to the gene have im-

proved the informativeness of predictive testing results (Wasmuth et al. 1988; MacDonald et al. 1989a). Approximately 20% (36/183) of persons participating in our predictive testing program have been unable to receive results because of the unavailability of DNA from crucial family relatives (Hayden et al. Received June 14, 1990; final revision received November 8, 1990. Address for correspondence and reprints: Michael R. Hayden, M.D., Department of Medical Genetics, F168 University Hospital, 2211 Wesbrook Mall, Vancouver, British Columbia V6T 2B5, Canada. i 1991 by The American Society of Human Genetics. All rights reserved. 0002-9297/91 /4803-0019$02.00

1988; S. Adam andJ. Thielmann, unpublished data). This has been the major limitation in performing predictive testing. Estimates of uninformativeness in other centers range from 22% (16/74) in Edinburgh (Brock et al. 1989) to 24% (13/55) in Baltimore (Brandt et al. 1989) and 31% (5/ 16) in Boston (Meissen et al. 1988). Analogous limitations in carrier detection and prenatal diagnosis occurred for cystic fibrosis (CF) prior to the cloning of the gene. In one study, 19% (32/167) of CF families were unable to have prenatal diagnostic studies, because of unavailability of DNA from the affected child (Beaudet et al. 1989). However, the use of linkage disequilibrium data has allowed substantial risk modification for some of these families (Beaudet et al. 1989). We (Theilmann et al. 1989a) and others (Snell et al. 1989) have previously reported significant linkage disequilibrium between two DNA markers (D4S95 and D4S98) and the HD gene. In the present paper we present haplotype data for an increased number of families confirming the finding of significant nonrandom association between the HD gene and both AccI and MboI RFLPs recognized by the probe pBS674E-D 595

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at the locus D4S95. We have utilized these data to determine whether this information might be used to alter an individual's risk of having inherited the HD gene. We show that, in some instances, significant risk modification can be made using data derived from linkage disequilibrium studies. This approach, if validated in independent studies, might have application for predictive testing for persons who do not have a sufficient number of relatives to currently allow for an informative test. Material and Methods Family Studies

One hundred eighteen unrelated families with a documented history of HD were used in the analysis. Some of the families were part of the Canadian Collaborative Study of Predictive Testing for Huntington Disease, while others participated for research purposes. The ancestries of the families were determined during interviews and are presented in table 1. We were unable to determine whether the families of Irish descent were from Northern Ireland or the Irish Republic (Eire). For this reason and because of their close geographical relationship, families from Ireland have been included with those from the United Kingdom. Haplotypes composed of two markers linked to HD were constructed in family studies where phase with HD was unequivocal. Control chromosomes consisted of the alleles in spouses and of the alleles segregating with the non-HD chromosome in affected persons. DNA Analysis

A total of 1,150 chromosomes from 200 affected and 422 unaffected individuals were analyzed. DNA analysis was performed using DNA that was extracted from leukocytes of each person and then digested with the restriction enzymes AccI, MboI, and SstI and hybridized with the probes pBS674E-D and pBS731B-C according to methods described elsewhere (Southern 1975; Feinberg and Vogelstein 1983; Hayden et al. 1987). AccI and MboI detect RFLPs identified by the DNA probes pBS674E-D at the locus D4S95 (Wasmuth et al. 1988; Theilmann et al. 1989b), and SstI detects an RFLP identified by pBS73 1 B-C at the locus D4S98 (Smith et al. 1988). The AccI polymorphism assessed in this analysis was the result of a single site variation and resulted in fragments of 6.8 and/or 1.5 kb. By convention, the large band of a single-sitevariation polymorphism is termed "A" and the smaller band is termed "B." The order of these markers has

Table I Ancestries of Affected Persons in II 8 Families with HD

Ancestry

United Kingdom: England .............................. Scotland .............................. . Ireland ................... Wales .............................. British (exact ancestry unknown) ...... Subtotal .............................. Non-United Kingdom:

Germany .............................. French-Canadian ........................... .............. Russia ................ Sweden .............................. Holland .................... .......... Indian (Asian) ..............................

Italy

..............................

Mennonite-Russian ........................ Norway .................... .......... ................ Egypt .............. Pennsylvania Dutch ........................ Hungary ...................... ........ Metis (North American Indian) ........ Mennonite-Dutch ........................... Romania .............................. Native Canadian ........................... Lebanon ...................... ........ .............. France ................ Subtotal .............................. Unknown ..............................

No. of Affected Persons 27 19 9 2 6 63 8 4 3 3 3 2 2 2 2 1 1 1 1 1 1 1 1 1

38 17 118

been previously established as D4S95-D4S98-telomere (MacDonald et al. 1989b). Statistical Analysis Nonrandom association of HD with marker RFLPs located in 4pl 6.3 was evaluated by contingency-table analysis. Linkage disequilibrium was measured using the method of Hedrick and Thomson (1986). In this method, the contingency x2 is divided by the product of the df and sample size, to obtain a standardized measure of nonrandom association, r2. The square root of this value, r, is the correlation between the locus of interest and the marker haplotypes. Given the range of sample sizes and structure of our data, statistical significance for biallelic systems was determined by Fisher's exact probability (one-tailed test).

Significance of comparisons with multiallelic systems

Linkage Disequilibrium and HD (i.e., those in which the number of alleles is greater than two) was evaluated by X2 tests. Homogeneity of linkage disequilibrium values estimated from groups with differing ancestries was determined by taking the weighted sum of squares of the Z transformed interlocus correlation coefficients (Sokal and Rohlf 1969). Risk estimates associated with given DNA haplotypes were determined by Bayesian methods standard in genetic counseling (Cox and Hinkley 1974). In other biostatistical applications, risk is assessed in the form of odds ratios. The odds ratio can be algebraically converted to express a Bayesian probability (Cox and Hinkley 1974). This algebraic identity was used to determine 95% confidence intervals for each Bayesian risk estimate. In practice this was accomplished by determining the interval estimate associated with the odds ratio for a given comparison (Cox and Hinkley 1974). The odds ratio interval estimates were then converted to Bayesian risk estimates. Results

Statistically significant nonrandom association of the HD gene and the RFLPs detected with AccI and MboI (D4S95) was seen in the group of families as a whole (table 2). The allele frequencies for these markers for the non-HD chromosome are in agreement with the results of past studies (Wasmuth et al. 1988; Theilmann et al. 1989a). When families of U.K. ancestry

597 are examined separately, similar association is observed at the D4S95 locus (table 3). Families having ancestors from other countries demonstrate only marginally significant nonrandom association (table 4). However, the haplotype distribution does not differ significantly from that of the sample of families with U.K. ancestry (X2 = 1.33, df = 1, P = .25). Linkage disequilibrium previously reported with SstI (D4S98) is no longer significant for persons of U.K. or nonU.K. descent (tables 3 and 4). The magnitude of nonrandom association is critically influenced by allele frequency distributions on both disease-bearing and non-disease-bearing chromosomes. Under the assumption of a single origin of HD, the allele distribution of disease-bearing chromosomes is expected to be relatively homogeneous. However, allele distributions on non-disease-bearing chromosomes could vary widely, reflecting differing genetic backgrounds of various ancestries. The AccI and MboI (D4S95) polymorphisms on HD chromosomes and non-HD chromosomes for the various populations included in the study is shown in table 5. Examination of this table shows that, as expected, allele distributions on the HD chromosomes are very similar. Minor differences in allele frequency are observed among non-HD chromosomes. These differences do not, however, result in statistically significant differences in allelic association. The homogeneity x2 values for D4S95 (AccI) and D4S95 (MboI) are 2.29

Table 2 Allele Frequencies for RFLPs Associated with HD Chromosomes and Non-HD Chromosomes of Families Listed in Table I No. (%) OF ALLELES ON

MARKER (no. of individuals), ENZYME, AND ALLELE TYPE

D4S95 (674): AccI: A ..........

.....................................

B ..........

.....................................

Total ............... MboI: A .......... B ..........

Total ............... D4S98 (731): SstI: A .......... B .......... C ..........

Total ...............

................................

..................................... .....................................

................................

..................................... ..................................... ..................................... ................................

HD Chromosomes

Non-HD Chromosomes

P VALUE

86 (82.7) 18 (17.3) 104 (100.0)

315 (66.3) ) 160 (33.7) 475 (100.0)

.0016

82 (84.5)

214 (57.8)

15 (15.5) 97 (100.0)

156 (42.2) 370 (100.0)

18 (17.0) 88 (83.0) 0

46 (11.6) 346 (86.9) 6 (1.5) ) 398 (100.0)

106 (100.0)

.00000

0

.158

Adam et al.

598 Table 3 Allele Frequencies for RFLPs Associated with HD Chromosomes and Non-HD Chromosomes of Families from the United Kingdom

MARKER (no. of individuals), ENZYME, AND ALLELE TYPE

D4S95 (674): AccI: A ............................................. B ............................................. ................................ Total ............. Mbol: A ............................................. B ............................................. ................................ Total .............. D4S98 (731): SstI: A ............................................. B ............................................. C ............................................. ................................ Total ..............

No. (%) HD Chromosomes

OF

ALLELES ON Non-HD Chromosomes

47 (83.9) 9 (16.1) 56 (100.0)

163 (64.4) 90 (35.6) 3 253 (100.0)

43 (84.3) 8 (15.7) 51 (100.0)

118 (56.5) 10004 91 (43.5) 3 209 (100.0)

8 (14.5) 47 (85.5) 0

26 (11.5) 195 (85.9) 6 (2.6)

55 (100.0)

227 (100.0)

P VALUE

.008

.405

Table 4 Allele Frequencies for RFLPs Associated with HD Chromosomes and Non-HD Chromosomes in Families with Origins outside the United Kingdom

No. (%)

OF

ALLELES

ON

MARKER (no. of individuals), ENZYME, AND ALLELE TYPE

HD Chromosomes

Non-HD Chromosomes

P VALUE

D4S95 (674): AccI: A ............................................. B .............................................

26 (78.8) 7 (21.2)

102 (70.3) 43 (29.7) )

.448

33 (100.0)

145 (100.0)

26 (83.9) 5 (16.1)

71 (61.2) 45 (38.8)

31 (100.0)

116 (100.0)

8 (22.9) 27 (77.1)

86 (87.8)

Mbol: A ............................................. B ............................................. ................................ Total ............. D4S98 (731): SstI: A ............................................. B ............................................. C ...............................................0 0.

Total .............

................................

(df = 5, P = .81) and 1.84 (df = 5, P = .87), respectively. The results of haplotype analysis for the AccI and MboI (D4S95) polymorphisms are presented in table 6. As previously noted, the two polymorphisms are in almost complete linkage disequilibrium with each

.031

12 (12.2) .218

) 35 (100.0)

98 (100.0)

other on the HD chromosomes (Theilmann et al. 1989a). While this data set is larger than that previously published, the relative frequencies of the various haplotypes have remained stable. The most common haplotype in the HD chromosomes is AA (AccI and MboI). Haplotype AB is never seen in HD chro-

Linkage Disequilibrium and HD

599

Table 5 Accl and Mbol Alleles of HD Chromosomes and Non-HD Chromosomes, by Ancestry

ALLELE

AccI: A .... B MboI: A .....

....

B

.....

English HD non-HD

Irish HD non-HD

No. Scottish

OF

HD

non-HD

ALLELES

ON

British HD non-HD

Welsh HD non-HD

Other HD non-HD

19 6

62 42

9 0

27 17

13 3

51 24

4 0

17 5

2 0

6 2

26 7

102 43

16 6

41 40

8 0

20 15

13 2

39 27

4 0

12 9

2 0

1 2

26 5

71 45

Table 6 Accl and Mbol Haplotypes of HD Chromosomes and Non-HD Chromosomes

SAMPLE AND HAPLOTYPE

All familes: A(AccI) A(MboI) ................... AB....................0 BB ..........

..............

Total ......................... Families of U.K. descent: A(AccI) A(MboI) ................... AB ........... ............. BA ........... ............. BB .......... .............. Total ........................ Families of non-U.K. descent: A(AccI) A(MboI) ................... AB ........... ............. BA ........... BB ..........

Total

.............

..............

...............................

No. (%) HD Chromosomes

78 (82.1) BA .2 (2.1) 15 (15.8) 95 (100.0)

OF

HAPLOTYPES ON Non-HD Chromosomes 194 (57.7) 31 (9.2) 3 (1.0) 108 (32.1) 336 (100.0)

42 (82.3) 0 1 (2.0) 8 (15.7)

106 19 3 61

51 (100.0)

189 (100.0)

19 0 1 4 24

(79.1)

(56.1)' (10.1) (1.6)

(16.7)

(100.0)

83 (100.0)

mosomes but is found associated with about 8% of non-HD chromosomes. Differences in haplotypes in the HD chromosomes and non-HD chromosomes are highly significant (P = .00003). When the families are subdivided by ancestry, significant haplotype association is seen in those families originally from the United Kingdom (P = .0037) (table 6). Haplotype AA (Accl and MboI) remains the most common HD haplotype in both groups. The haplotype data can be utilized to modify risk estimates for at-risk individuals, on the basis of the four possible haplotypes (table 7). To be conservative in these estimates, it was assumed that each haplotype

.00003

.0037

(32.2))

52 (62.7) 10 (12.0) 0 21 (25.3)

(4.2)

P VALUE

.052

exists at least once in both the HD and non-HD sample, even if it was not observed. This was accomplished by assigning each missing haplotype a frequency of one over the total number of sampled haplotypes for that group. It is evident for the U.K. families that the calculated risks differ significantly from the prior probabilities. Furthermore, in some cases the risk estimate can be substantially altered if there is knowledge ofthe at-risk individual's DNA haplotype. With the exception of the risks associated with haplotype BA, which is derived from very few observations, each of the risk calculations has 95% confidence intervals that do not

Adam et al.

600 Table 7 Probability and Confidence Intervals That a D4S95 Haplotype Carries the HD Mutation

MODIFIED RISK (confidence interval) WHEN PRIOR PROBABILITY OF HD IS D4S95

AccI

MboI

A A B B Families of U.K. descent: A A B B Families of non-U.K. descent: A A B B B .........

A B A B

..59 ..10 ..70 ..33

A B A B

..59 ..16 ..55 ..32

.50 (birth)

.46 (age 30 years)

(.56-.62) (

Linkage disequilibrium and modification of risk for Huntington disease.

The major limitation in performing predictive testing for Huntington disease (HD) is the unavailability of DNA from crucial family members. In our pro...
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