Am. J. Hum. Genet. 46:5-11, 1990

Huntington Disease in Finland: Linkage Disequilibrium of Chromosome 4 RFLP Haplotypes and Exclusion of a Tight Linkage between the Disease and D4S43 Locus Elina Ikonen,* Jorma Palot Jurg Ott,$ James Gusella,§ Haninu Somerj Leena Karila,* Aarno Palotie, * and Leena Peltonen * Laboratory of Molecular Genetics, National Public Health Institute; and 1Department of Neurology, University of Helsinki, Helsinki; tDepartment of Psychiatry, Columbia University, New York; and §Neurogenetics Laboratory, Massachusetts General Hospital, and Department of Genetics, Harvard University, Boston

Summary The question about heterogeneity of Huntington disease (HD) at the DNA level can be approached by analyzing the RFLP haplotypes formed by several RFLP loci of the diseased chromosome in different populations. In genetically isolated populations such as Finland, it is further possible to use this approach to test the hypothesis of a single mutation enriched in this population demonstrating an exceptionally low prevalence of HD. In this study covering 70% of all diagnosed HD cases in Finland, linkage disequilibrium of RFLP haplotypes of D4S10 and D4S43 loci polymorphisms was found. This phenomenon, not so far reported in any other population, could support the hypothesis of one ancestor HD mutation in the Finnish population. Despite the lower heterozygosity obtained with some RFLP markers, the proportion of individuals receiving informative DNA test results did not significantly differ from that reported in more mixed populations. In one HD family we established a recombination event between HD and the D4S43 locus, an event which can be highly useful in the more precise mapping of the HD gene. Introduction

Huntington disease (HD) is a severe neurodegenerative condition with a relatively late age at onset, the first psychomotoric symptoms usually appearing in early middle age. Although the clinical findings are highly variable at an early stage, all the present knowledge suggests a single, dominantly inherited gene defect causing the disease (Hayden 1981; Conneally 1984; Martin and Gusella 1986). The precise mutation remains unknown, although tight linkage between the disease and the gene areas in the distal end of the short arm of chromosome 4 was reported several years ago (Gusella et al. 1983). Subsequently, several informative RFLP loci physically closer to the HD gene have been characterized in this area of several million base pairs (Gilliam Received February 15, 1989; final revision received June 21, 1989. Address for correspondence and reprints: Dr. Leena Peltonen, Laboratory of Molecular Genetics, National Public Health Institute, Mannerheimintie 166, SF-00300 Helsinki, Finland. i 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4601-0002$02.00

et al. 1987; Hayden et al. 1988; Wasmuth et al. 1988; Whaley et al. 1988; Robbins et al. 1989). To study the possible heterogeneity of a multisymptomatic disorder such as HD at the DNA level, it is important to analyze RFLP haplotypes formed by the combination of several RFLP loci of this chromosomal area coinherited with the disease in different populations. In genetically isolated populations it is also relevant to evaluate possible linkage disequilibrium between

the disease and marker loci, linkage disequilibrium which could support the presence of a single ancestral HD mutation enriched in this particular group of people. Further, as the DNA diagnosis of HD is still based on the coinheritance of certain RFLP haplotypes within the family, the evaluation of these haplotypes both in the general population and in the diseased persons is essential for reliable diagnosis of the presymptomatic cases in the given population. We have therefore studied the Finnish population, which has been genetically isolated for centuries. The isolation has been due to both geographic and linguistic factors. As a consequence, some rare diseases, such 5

Ikonen et al.

6

as aspartyl-glucosaminuria and infantile type of neuronal ceroid-lipofuscinosis, have been enriched while some rather common inherited diseases, such as phenylketonuria and cystic fibrosis, are virtually nonexistent (Nevanlinna 1980). HD is also very rare, the prevalence being only "-'5 cases/1 million population (Palo et al. 1987). We analyzed the polymorphisms of the HD region by using probes from the anonymous DNA area D4S10 reported to demonstrate a linkage at 4 cM from the disease (Gusella et al. 1983). In addition to these probes we also used a probe from another anonymous DNA locus, D4S43, which has been tentatively mapped significantly closer to the hypothetical HD gene, within 1.5 cM of the defect (Gilliam et al. 1987). This particular locus is in close proximity of D4S95 (Wasmuth et al. 1988), which is still considered the most useful DNA marker for predictive testing (Robbins et al. 1989). In one family an unequivocal recombination between the D4S43 locus and HD could be demonstrated. Furthermore, it seems that linkage disequilibrium of RFLP haplotypes exists in the Finnish HD patients, a phenomenon not found in any other population tested so far (Gilliam et al. 1987). Material and Methods All HD patients diagnosed in Finland since 1970 were collected in 1985 by systematically searching the archives of all university, central (regional), and central

mental hospitals (total number 44). The search revealed 23 families that had one or several living or deceased members with the disease. Since then, four more families have been found in the follow-up, bringing the total to 27. The diagnostic criteria included typical clinical course with movement disorder mostly of choreiformic type and progressive dementia often combined with psychiatric symptoms (Hayden 1981). Computer tomography (CT) of the head was carried out only in more recently diagnosed patients because the method became available for the first time in 1978. Positron emission tomography (PET) has not been used. One family was found with the juvenile type of HD and with patients known in 5 generations (also see fig. 1). The diagnosis was based on repeated neurologic examinations in the university central hospitals. The first index case of the family was a 58-year-old male who had suffered from the typical symptoms of HD for several years. The result of CT was also compatible with the diagnosis. He died 2 years after the initiation of the study, but no neuropathologic examination was

done. His son got first symptoms in early adolescence (between 15 and 18 years of age), and his 12-year-old granddaughter has suffered from HD-like symptoms since age 10 years. Repeated neurologic examinations in a university hospital also have confirmed her diagnosis.

Blood samples were collected from a total of 113 individuals belonging to nine HD families with at least 2 generations of affected members (see table 4). Ofthese, 18 were affected, representing 70% of all diagnosed HD cases in Finland (Palo et al. 1987). Four patients belonged to the family with the juvenile type of the disease. Fifty-three persons had a 50% risk of carrying the disease gene, according to calculations from the pedigree data. Control samples for the RFLP analyses of the general Finnish population were collected from volunteer individuals representing the Finnish family material collected in our laboratory as well as from those 23 individuals married into HD families whose RFLP haplotype it was possible to determine unequivocally. DNA was extracted and digested to completion by using the restriction enzymes HindIll, EcoRI, and MspI, and Southern blot analysis was performed according to a method described elsewhere (Southern 1975; Vandenplas et al. 1984). Three of the probes used to detect polymorphisms originate from the D4S10 area (G8 locus), detecting four RFLPs, and one originates from the D4S43 locus, which is genetically closer to the disease area. The probes were labeled radioactively to specific activity of 2-8 x 108 cpm/ gg by standard nick-translation reaction according to the protocol of the supplier (Amersham, United Kingdom). Multipoint linkage analysis was carried out by the MLINK computer program using four-point linkage analyses (Lathrop et al. 1984). The delayed onset of the disease was taken into account by accommodating 12 liability classes in the MLINK program. The probability of having the HD gene was both evaluated by following the haplotype cosegregating with the disease in each HD family and confirmed by computing it with the risk-calculation option of the MLINK program. Results

The usefulness of RFLP linkage analyses is, to a great extent, determined by the informativeness of the individual RFLP markers in the particular population. The allelic frequencies of the five RFLP loci in the general Finnish population are given in table 1. As expected from this isolated gene pool (Nevanlinna 1980), in the

Linkage Disequilibrium of HD-RFLPs in Finland

7

Table I Allelic Frequencies, Heterozygosity, and PIC of HD markers in Finland ALLELIC FREQUENCY, HETEROZYGOSITY, AND PIC PROBE

D4S10 locus: F5.52 ........

pK082

pK082

.......

.......

RESTRICTION ENZYME, ALLELES (kb)

52/48 75 .37

40/60

Frequency Heterozygosity

73/27 27 .32

65/35 48 .35

12/88 24 .19

26/74 36 .31

54/46 62 .37

40/60 49 .36

16/84 23 .23

n.d.

PIC HindIII, 4.9/3.7 PIC

........

D4S43 locus: C4H .........

Elsewhere

MspI, 3.5/2.8 Frequency Heterozygosity PIC HindIII, 13/11

Frequency Heterozygosity FS.53

Finland (100 chromosomes)

EcoRI, 13/9.0 Frequency Heterozygosity PIC

n.d. .36

MspI, 15/8.0 Frequency Heterozygosity PIC

Finnish population the degree of heterozygosity obtained with some markers was lower than that previously reported for more mixed populations, only 25% of individuals being heterozygous in the case of three markers. The degree of heterozygosity and the PIC of the markers used in the general population are also given in table 1. When RFLP haplotypes were originally formed from the five tested marker loci, in 73 meiotic events studied, obligatory recombinations were found even between the markers in the D4S10 locus. For the two markers at both ends of the D4S10 locus (F5.52 and F5.53) the two-point recombination estimate calculated from our families was .05, but the difference from the earlier observed value of .01 was not significant (J. Gusella, unpublished data) when maximal LOD scores at these recombination fractions were compared. Therefore the value .01 was used in linkage calculations. Linkage between the D4S10 and D4S43 loci was uninformative in our small families. Therefore, the value of .04 given by Gilliam et al. (1987) was applied. Multipoint linkage analyses were thus carried out with the locus order

8/92 .14

F5.52-F5.53-D4S43-HD and respective recombination fractions of .01, .04, and .01. In eight families, individual linkage analyses were almost uninformative, so their LOD scores were combined (table 2). In one family, the only one representing the juvenile type of the disease (fig. 1), a LOD score of -5.69 (0 = 0) between HD and the markers was found in multilocus analysis between the D4S10 and D4S43 markers and the disease. This suggests the presence of at least one known recombination. Inspection of the pedigree (fig. 1) reveals the following recombination event between HD and C4H (D4S43): when absence of homozygous affected genotypes is assumed, individual (1) identifies the HD gene as being coupled with the + allele of C4H. On the other hand, individual (2) received the - allele with the disease gene from her father. In the other eight families, no unequivocal recombinations either between D4S10 and HD or between D4S43 and HD occurred. The heterogeneity test was applied to analyze whether this juvenile-diseasetype family demonstrating a recombination event between C4H and the disease would possibly represent

Ikonen et al.

8 Table 2 LOD Scores for Linkage of HD versus Combined Polymorphic Sites of D4S10 and D4S43

RECOMBINATION FRACTIONS (0)

SAMPLE

0

.01

.03

.05

.10

.20

.30

.40 .50

Eight families ................. - 1.07 -.98 -.82 -.68 -.42 -.13 -.00 +.02 .00 One family (juvenile type HD)... . -5.69 -1.12 -.67 -.48 -.25 -.07 -.02 .00 .00 Total for all nine families ...... -6.76 -2.10 -1.49 -1.16 -.67 -.20 -.02 +.02 .00

NOTE. - Recombination fractions allowed between the markers were .01 for those within the D4S1 0 locus and .04 between the D4S10 and D4S43 markers.

a mutation in a locus other than that implicated in all the other eight HD families. No evidence for allelic heterogeneity could be found (P = .3 8). Since no evidence for heterogeneity could be demonstrated, we combined the information from all the families to determine the map distance excluded as a site for HD mutation in the vicinity of the used markers. The result of the anal-

++

++

yses performed using the LINKMAP program from the LINKAGE package is given in figure 2 and excludes a DNA region covering the distance starting from 2 cM from G8 locus toward the centromere and reaching 3 cM from C4H toward the 4 pter. Beyond increasing the informativeness in individual pedigrees, the RFLP haplotypes demonstrated the enrichment of certain chromosome 4 haplotypes in the Finnish HD patients. When RFLP haplotypes were formed using both the D4S1O and D4S43 polymorphisms in the other eight HD families and following the inheritance of marker combinations in pedigrees, only three different haplotypes could be found in HD patients (tables 3 and 4). In 62 individuals representing

+

+

Figure I Pedigree of the family with the juvenile type of HD, showing haplotypes detected after hybridization of restriction-digested DNA with FS.52 (MspI), F5.53 (EcoRI), and C4H (MspI). The least important parts of the pedigree have been slightly changed to maintain the anonymity of the family. The paternities have been ascertained with Jeffreys et al.'s (1985) method of DNA fingerprints. The earliest age at onset of symptoms was 10 years. Numbers in parentheses refer to the individuals discussed in Results.

Figure 2 Multipoint linkage analysis of HD, D4S10, and D4S43. The program LINKMAP from the LINKAGE package was used to calculate LOD scores for different locations of the HD gene relative to fixed positions of two marker loci. The centromeric end of the D4S10 locus (F5.52) was arbitrarily placed at .0. Recombination frequencies between the markers were based on reported values such as described in the text. The recombination fractions were transformed to physical map distances by Haldane's formula. Dashed lines indicate the limits of the chromosomal area excluded as a locus for the HD gene.

Linkage Disequilibrium of HD-RFLPs in Finland

9

Table 3 RFLP Haplotypes and Their Distribution in the General Finnish Population and in HD Families HAPLOTYPE

I II III IV V VI VII VIII

Restriction enzyme (probe):

Mspl (F5.52)

..................+ - + -

+ + - +

.................. + + + +

--

EcoRI (F5.53) .................+ +

Mspl (C4H)

-

-

Frequency (%) in general population (124 chromosomes)a ... 38 22 12 10 6 6 4 No. of HD families with the haplotypea ........ 3 3 2

+

2

NOTE. - + = restriction site present; - = restriction site absent. When compared in the two-tailed x2 test, P < .001.

a

the general population, at least eight different haplotypes could be identified. The haplotypes of HD patients did not represent the most common haplotypes of the general population. So far, no information or genetic counseling based on DNA analysis has been given to the members of the Finnish HD families. In the nine HD families analyzed here, there were a total of 53 nonsymptomatic individuals having one affected parent, and, for 50 of them, RFLP haplotypes could be formed and risk calculations could be performed. Their risk for HD was originally estimated on the basis of the current age of the individuals, and the final risks based on the performed DNA analyses were calculated with the MLINK program by four-point linkage analyses (disease vs. three markers) under the assumption of linkage equilibrium.

The change in the risk evaluation of each tested individual is shown in figure 3. When compared with the age-dependent risk without using marker information, the risk obtained from DNA analyses increased significantly in five individuals (risk r-90% or more), decreased in 14 cases, and did not change much for 31 individuals. However, seven of these 31 individuals showed informative results from DNA analyses. Their MLINK risk percentage was significantly low (r-15% or less), but they already belonged to the old-age group, which had a very low risk estimate. Thus their primary risk was only confirmed by the DNA analysis. Discussion

The gene defect resulting in HD seems to be exceptionally rare in the Finnish population, the incidence being r-1.6/1 million and the prevalence being r-5/1 million inhabitants (Palo et al. 1987). These numbers are only one-tenth of those reported in neighboring Sweden (Mattson and Ottosson 1985). The spectrum of other inherited diseases is also unique as a consequence of the founder effect (Nevanlinna 1980). It was therefore relevant to analyze the value of current DNA probes in the predictive testing of HD in this unique population and to test the hypothesis of one mutation in the HD families by analyzing the haplotypes formed with polymorphic markers. The present study does not provide evidence for linkage between HD and the chromosome 4 markers studied but, apart from one known recombination, does not

give significant evidence against

it either. There-

fore, on the basis of the present study, we have no rea-

Table 4 HD Families Studied with RFLP Analysis

1 2 3 4 5 6 7 8 9

Type of

No. of

Family

HD

Individuals Analyzed

No. of Diseased Individuals Analyzed

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

Adult Adult Adult Juvenile Adult Adult Adult Adult Adult

43 16 11 10 9 8 8 4 4 113

4 3 2 4 1 1 1 1 1 18

.........

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

a Haplotype numbers refer to the haplotypes presented in table 3. b Recombination exists between the disease and the RFLP haplotype.

Diseased Haplotypea IV IV V .

VI VI V V IV

b

Ikonen et al.

10

risk 100 90 80 70 60 50 40 30 20 10

0

0

.

.

I

0

*0

' 0

0

0

dp

~~0

0

0

.

0

0

0 4a

.

0

01-

~~0

0

000 00 1

20

l

30

40

el

O

50

age-dependent risk (%)

The change in age-dependent risk for HD, accordFigure 3 ing to the MLINK program. Each tested individual is presented as a point. The points below the oblique line correspond to a decrease in risk, and points above the line correspond to a risk increase.

son to postulate an HD gene at a location different from the one found in other populations. The informativeness of RFLP markers can be limited

in isolated populations because of the low degree of heterozygosity. This was the case when chromosome 4-specific markers linked to HD were analyzed in the Finnish population, the values for heterozygosity of some loci being lower than those found elsewhere (Gusella et al. 1985; Harper et al. 1985; Bakker et al. 1987; Quarrell et al. 1987), although the difference was not

striking.

The combination of individual RFLP markers to haplotypes suggested the existence of linkage disequilibrium in the Finnish HD families. Only three of at least eight different haplotypes of the general population were found in HD patients. These HD haplotypes represented the more rare haplotypes of the general population. In other types of populations studied so far, no significant association between the defect and any specific RFLP allele has been observed (Gilliam et al. 1987). Although the number of HD patients (18 in nine families) was relatively small in the present study, it represented vv70% of all known cases in Finland. The hypothesis that most affected individuals in this particular gene pool could have inherited a gene defect

of common origin is supported by the linkage disequilibrium. The three different haplotypes cosegregating with HD can be explained as consequences of recent recombination events, especially since the recombination frequency in this region of chromosome 4 may be increased (Richards et al. 1988). However, the possibility of more than one HD mutation in the Finnish gene pool cannot be excluded. Combining the information on three RFLP markers, we analyzed the change in the risk evaluation after DNA analysis of 50 individuals at 50% risk in the families we studied. The proportion of individuals (52%) receiving informative test results agrees well with the numbers presented in similar studies in the United States (Hayden et al. 1988; Meissen et al. 1988). Since our screening included the majority (70%) of Finnish HD patients, it seems that with the available RFLP markers the proportion of informative cases is about the same irrespective of the population screened. Further, our relatively small family trees also demonstrate the fact that increase in the number of RFLP markers does not necessarily result in the increased information in individual families. The usefulness of predictive testing is rather dependent on both the pedigree structure and the availability of DNA samples, as suggested for HD families rising from more mixed populations (Myers et al. 1988; Robbins et al. 1989). Arrangements for voluntary pretest counseling and long-term follow-up of individuals at risk for HD are now in progress. In one HD family, a highly negative LOD score found in both two-point and multilocus linkage analyses excluded the DNA area covered by the probes of the D4S10 and D4S43 loci in the short arm of chromosome 4 as a cause of the disease. The negative LOD score of - 5.7 between the disease and the D4S43 locus clearly demonstrated at least one recombination event between HD and this locus tentatively placed within 0-1.5 cM of the genetic defect (Gilliam et al. 1987). We obtained this highly negative lod score at 0 = 0, instead of at 0 = -00, because in the linkage calculations we used the reduced age-dependent penetrance of the HD gene. During the submission of the manuscript of the present paper, we learned that recombination events between this area of chromosome 4 and HD also have been found in other families (Whaley et al. 1988; Robbins et al. 1989). The performed heterogeneity test did not provide evidence for allelic heterogeneity in the Finnish HD families. Thus one family with the found recombination must represent a rare chance event and can provide a highly useful starting point in the more precise mapping of the HD gene.

Linkage Disequilibrium of HD-RFLPs in Finland

Acknowledgments This study was supported by grants from the Paulo Foundation and the Klingendahl Foundation, Finland.

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11 Mattson B, OttossonJO (1985) Huntingtons sjukdom i uppdaterad register (in Swedish). Lakartidningen 82:1177 Meissen GJ, Myers RH, Mastromauro CA, Koroshetz WJ, Klinger KW, Farrer LA, Watkins PA, et al (1988) Predictive testing for Huntington's disease with use of a linked DNA marker. N Engl J Med 318:535-542 Myers RH, Farrer LA, Gusella JF, Martin JB (1988) Predictive testing for Huntington's disease using linked DNA markers. N Engi J Med 319:583 Nevanlinna HR (1980) Rare hereditary diseases and markers in Finland: an Introduction. In: Eriksson AW, Forssius H, Nevanlinna HR (eds) Population structure and genetic disorders, 10th ed. Academic Press, London, pp 569-576 Palo J, Somer H, Ikonen E, Karila L, Peltonen L (1987) Low prevalence of Huntington's disease in Finland. Lancet 2: 805-806 Quarrell OWJ, Meredith AL, Tyler A, Youngman S, Upadhyaya M, Harper PS (1987) Exclusion testing for Huntington's disease in pregnancy with a closely linked DNA marker. Lancet 1:1281-1283 Richards JE, Gilliam TC, Cole JL, Drumm ML, Wasmuth JJ, GusellaJF, Collins FS (1988) Chromosome jumping from D4S10 (G8) toward the Huntington disease gene. Proc Natd Acad Sci USA 85:6437-6441 Robbins C, TheilmannJ, Youngman S, HainesJ, 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 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517 Vandenplas S, Wud I, Grobler-Rabie A, Brebner K, Ricketts M, Wallis G, Bester A, et al (1984) Blot hybridisation analysis of genomic DNA. J Med Genet 21:164-172 WasmuthJJ, HewittJ, Smith B, Allard D, HainesJL, Skarecky D, Partlow E, et al (1988) A polymorphic DNA marker tightly linked to the Huntington disease gene. Nature 332: 734-736 Whaley WL, Michiels F, MacDonald ME, Romano D, Zimmer M, Smith B, LeavittJ, et al (1988) Mapping of D4S98/ S114/S113 confines the Huntington's defect to a reduced physical region at the telomere of chromosome 4. Nucleic Acids Res 16:11769-11780