Journal of Medical Genetics 1989, 26, 676-681 Non-random association between alleles detected at D4S95 and D4S98 and the Huntington's disease gene J THEILMANN*, S KANANI*, R SHIANGt, C ROBBINS*, 0 QUARRELL*, M HUGGINS*, A HEDRICK*, B WEBER*, C COLLINS*, J J WASMUTHt, K H BUETOW§, J C MURRAYt, AND M R HAYDEN* From *the Department of Medical Genetics, University of British Columbia, Vancouver, Canada V6T 2B5; tDepartment of Pediatrics, University of Iowa; *Department of Biological Chemistry, University of California, Irvine; and §Division of Population Science, Fox Chase Cancer Centre, Philadelphia, USA. SUMMARY Analysis of many families with linked DNA markers has provided support for the Huntington's disease (HD) gene being close to the telomere on the short arm of chromosome 4. However, analysis of recombination events in particular families has provided conflicting results about the precise location of the HD gene relative to these closely linked DNA markers. Here we report an investigation of linkage disequilibrium between six DNA markers and the HD gene in 75 separate families of varied ancestry. We show significant non-random association between alleles detected at D4S95 and D4S98 and the mutant gene. These data suggest that it may be pos- sible to construct high and low risk haplotypes, which may be helpful in DNA analysis and genetic counselling for HD, and represent independent evidence that the gene for HD is centro- meric to more distally located DNA markers such as D4S90. This information may be helpful in defining a strategy to clone the gene for HD based on its location in the human genome. Directed attempts to clone the gene for Hunting- ton's disease (HD) depend on an accurate know- ledge of its location in the human genome. Over the last few years different DNA markers closely linked to the HD gene have been identified. 1-4 Analysis of many HD families with these markers has provided evidence that the gene causing HD lies close to the telomere of the short arm of chromosome 4.5 6 Cur- rently, attempts are being made to define physically the minimal genomic fragment which contains the HD gene. Towards this goal and in an effort to iden- tify clearly a flanking marker for the gene, recent efforts have been directed towards cloning the telo- mere of chromosome 4p.7 Analysis of families in which recombination between a closely linked DNA marker and the mutant gene has occurred can be extremely helpful in determining the order of the mutant gene relative to these linked DNA markers. We recently reported evidence suggesting the gene for HD was telomeric to the most distal marker D4S908 based on an analy- sis of a recombinant in one large family. More recently, contradictory evidence has been pre- sented. MacDonald et at9 have shown by analysis of Received for publication 3 August 1989. Accepted for publication 7 August 1989. recombinants in three families that the gene for HD may be flanked by the most distal markers in one family while analysis of two others favours a more telomeric location for the gene. How can these apparent contradictions be resolved? While analysis of recombination events in selected families is useful in providing evidence in favour of a certain locus order, such studies may also be misinterpreted. One source of error in such analyses may include misdiagnosis of the disease in a crucial family member. In addition, the simplest interpretation of the data is to assume a single recombination event. However, in the absence of a clearly defined distal marker, double recombination events cannot be excluded. This may be particularly relevant as one uses DNA markers close to the telo- mere where enhanced recombination has already been postulated to occur. 10 Additional independent sources of evidence in favour of a particular locus order relative to the HD gene are needed. Linkage disequilibrium shown by a non-random association between an allele at one locus with the mutant gene would provide strong evidence that two loci are physically in close proximity. Here we report on the assessment of linkage disequilibrium between six DNA markers [D4S62 (P8), D4S10 676 co p yrig h t. o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y h ttp ://jm g .b m j.co m / J M e d G e n e t: first p u b lish e d a s 1 0 .1 1 3 6 /jm g .2 6 .1 1 .6 7 6 o n 1 N o ve m b e r 1 9 8 9 . D o w n lo a d e d fro m http://jmg.bmj.com/ Non-random association between alleles detected at D4S95198 and the Huntington's disease gene 677 (G8), D4S95 (674), D4S98 (731), D4S96 (678), and D4S141 (2R3)] and the HD gene. We show that the gene for HD is in significant linkage disequilibrium with D4S95 and D4S98. This provides independent evidence that the gene for HD is proximal to D4S90 and distal to D4S10. The most distally located DNA markers including D4S90 are therefore likely to flank the. gene for HD. Materials and methods FAMILIES A total of 75 unrelated families with a documented history of HD was used in the analysis. All of these families were part of the Canadian Predictive Test- ing programme for HD. The ancestry of the families was determined during interviews with appropriate family members and is presented in table 1. For con- trols the alleles in unrelated family members and the alleles segregating with the non-HD chromo- some in affected persons were used. In all instances, the ancestry of the family members was assumed to be similar to that of the affected persons. Haplo- types for HD and marker restriction fragment length polymorphisms (RFLPs) were constructed using family studies where phase with HD was un- equivocal. DNA ANALYSIS A total of 495 persons, 148 affected and 347 TABLE 1 Ancestries of affected persons in 75 separate families with HD. No United Kingdom England Ireland Scotland Wales British (exact ancestry unknown) Total Non-United Kingdom Egypt French Canadian Germany Holland Pennsylvania Dutch Hungary Indian (Asian) Metis (North American Indian) Italy Mennonite Dutch Mennonite Russian Norway Romania Russia Sweden Grand total Ancestry unknown GCrand total 21 6 10 0 4 41 4 5 2 2 2 2 227 75 unaffected, from these 75 families was included in this analysis. DNA was extracted from leucocytes of each person and digested with restriction enzymes HincII, EcoRI, BglI, HindIII, AccI, TaqI, MboI, SstI, and MspI. Electrophoresis, transfer, and hybridisation conditions were performed as des- cribed previously.11-13 Ten polymorphisms detected by six DNA probes D4S62, D4SJO, D4S95, D4S98, D4S96, and D4S141 were examined. A HincII poly- morphism was detected by D4S62,2 EcoRI, BglI, and HindIII polymorphisms recognised by D4S10,1 AccI, TaqI, and MboI polymorhisms by D4S95,3 a SstI polymorphism by D4S98,1 and an MspI poly- morphism by D4S96. 14 Two polymorphisms are detected by AccI.3 The AccI polymorphism assessed in this analysis was the result of a single site varia- tion and resulted in fragments of 6-8 and/or 1-5 kb. D4S141 was isolated from the same cosmid as recognised by D4S90.15 A frequent HindIII poly- morphism was used in this analysis. The order of these DNA markers has been pre- viously established as D4S62-D4SIO-D4S95-D4S98- D4S96-D4S90-telomere.5 6 8 9 A diagramatic map with approximate genetic distances between these DNA markers is shown in fig 1. By convention, for single site variation polymorphisms, the larger band is termed A and the smaller band B (fig 2). STATISTICAL ANALYSIS Non-random association was evaluated by x2 analy- sis of allelic counts for locus pairs. Significant asso- ciation for biallelic pairs was determined by Fisher's exact probability test (one tailed). The linkage dise- quilibrium metric, r, was used to measure the mag- nitude of non-random association where r=(g-plp2) (pjq1P2q2)½ and pj and qj are the frequencies of the alternative alleles at site j, and g is the observed frequency of the P1P2 haplotype. Results The non-HD chromosome allelic frequencies for the 10 polymorphisms assessed are in general agreement with past studiesl and are shown in table 2. The frequencies of the alleles for the HD chromosomes are similar to their unrelated family member Centromere p ololo>>oY 4V .. Telomere ± 1cM + 4cM + 1cM + 1cM FIG 1 A diagramatic map showing approximate genetic distances between DNA markers used in this study. (Based on data presented in references 5, 6, 8, and 9). co p yrig h t. o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y h ttp ://jm g .b m j.co m / J M e d G e n e t: first p u b lish e d a s 1 0 .1 1 3 6 /jm g .2 6 .1 1 .6 7 6 o n 1 N o ve m b e r 1 9 8 9 . D o w n lo a d e d fro m http://jmg.bmj.com/ 678 J Theilmann et al TABLE 2 Allele frequencies and percentages for RFLPs on HD and non-HD chromosomes. HD chromosomes Non-HD chromosomes r p value No % No % D4S62 (P8) A 25 62-5 115 71-9 0-07 NS (HincII) B 9 22-5 30 18-8 C 6 15-0 14 8-7 D 0 1 0-6 Total 40 100-0 160 100-0 D4S10 (G8) A 17 44-7 73 49-0 -0-03 NS (EcoRI) B 21 55-3 76 51-0 Total 38 100-0 149 100-0 D4S10 (G8) A 18 60-0 93 65-5 -0-04 NS (Bgll) B 12 40 0 49 34-5 Total 30 100-0 142 100-0 D4S10 (G8) A 24 85 7 103 72-0 0-08 NS (HindIII) B 1 3 6 13 9 1 C 3 10-7 24 16-8 D 0 3 2-1 Total 28 100-0 143 1(0-( D4S95 (674) A 15 27-3 76 31-0 -0-03 0-35 (TaqI) B 40 72-7 169 69-0 Total 55 100.0 245 100 0 D4S9S (674) A 54 80-6 175 63-6 0-14 0-005 (AccI) B 13 19-4 100 36-4 Total 67 100-0 275 100-0 D4S95 (674) A 39 76-5 107 56-9 0-16 0-007 (MboI) B 12 23-5 81 43-1 Total 51 100-0 188 100-0 D4S98 (731) A 6 25-0 7 7-6 0-22 0-026 (SstI) B 18 75 0 85 92-4 Total 24 100.0 92 100-0 D4S96 (678) A 27 56-3 128 59-8 -0)03 NS (MspI) B 21 43-7 86 40-2 Total 48 10070 214 100 0 D45141 (2R3) A 5 22 7 28 39 4 -0 15 NS (HindIII) B 17 77.3 43 60-6 Total 22 100-0 71 100.0 NS=not significant at p_0-05. TABLE 3 AccI and MboI alleles of HD and non-HD chromosomes by ancestry. AccI: United Kingdom English Irish Scottish British Total Alieles HD non-HD HD non-HD HD non-HD HD non-HD HD non-HD r p A 14 43 5 14 7 30 4 11 30 98 0-16 0-02 B 4 26 0 10 2 11 0 8 6 55 AccI: Non-United Kingdom French Canadian German Other Total Alleles HD non-HD HD non-HD HD non-HD HD non-HD r p A 8 14 3 7 11 38 18 59 0-09 0-23 B 0 2 1 8 4 23 6 33 MboI: United Kingdom English Irish Scottish British Total Alleles HD non-HD HD non-HD HD non-HD HD non-HD HD non-HD r p A 9 24 4 7 5 21 4 9 22 62 0.19 0-02 B 4 21 0 8 2 2 0 8 6 50 MboI: Non-United Kingdom French Canadian German Other Total Alleles HD non-HD HD non-HD HD non-HD HD non-HD r p A 4 11 2 0 5 17 11 28 0.10 030 B 0 1 1 6 4 14 5 21 co p yrig h t. o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y h ttp ://jm g .b m j.co m / J M e d G e n e t: first p u b lish e d a s 1 0 .1 1 3 6 /jm g .2 6 .1 1 .6 7 6 o n 1 N o ve m b e r 1 9 8 9 . D o w n lo a d e d fro m http://jmg.bmj.com/ Non-random association between alleles detected at D4S95198 and the Huntington's disease gene 679 Acci 668 '- m mA VTlRL -si7:vbo .4 OM SW I' 3 "i$"r- 0*7 06 AB AB AA 1 5 - _ -- BB AB AB AB AA FIG 2 The polymorphisms of AccI and MboI detected by D4S95. A refers to the larger fragment (6&8, 1-3 kb) while B pertains to the smaller fragments (0 7, 0-6 kb). TABLE 4 AccI and MboI haplotype frequencies in persons affected with HD and their relatives. HD Non-HD r p chromosome chromosome No % No % A (AccI) A (Mbol) 39 76 91 55 0-15 001 AB 0 12 7 BA 0 1 1 BB 12 24 62 37 Total 51 100 166 100 controls for the polymorphisms detected by D4S62, D4SJO, D4S96, and D4S141. However, two of the three RFLPs (AccI and MboI) (fig 2) detected by D4S95 show statistically significant differences (p=0005 and p=0-007, respectively) in allelic fre- quencies between HD chromosomes and controls. The third RFLP (TaqI) detected by D4S95 shows no allelic frequency difference between HD and non- HD chromosomes (p=035). The polymorphism detected by D4S98 is also in linkage disequilibrium with HD (p=003). The families in this study represent many different ancestries (table 1). A significant difference for AccI (p=0-02) and MboI alleles (p=0-02) on HD and non-HD chromosomes was seen in families emanat- ing from the United Kingdom. However, for the smaller number of families outside the UK the trend in allele frequencies was similar but was not statisti- cally different (table 3). As noted previously,3 the single site variation polymorphism detected by AccI is in strong linkage disequilibrium with MboI. For these two RFLPs the A alleles cosegregate as do the B alleles. Thirty-nine of 51 HD chromosomes have an AA haplotype and 12 have a BB haplotype. No AB or BA haplotypes were detected in this group (table 4). It was possible to determine the phase of the AccI and MboI polymorphism segregating with HD in 67 and 51 of 75 families assessed respectively (table 4). There were significant differences in the haplotype fre- quencies between HD and non-HD chromosomes (p=0-01) (table 4) and the families from the UK when analysed as a sepa-rate group (p=002) (table 5). TABLE 5 AccI and MboI haplotypes of HD and non-HD United Kingdom Haplotypes AA AB BA BB Total English HD Non-HD 9 20 0 4 0 0 4 16 13 40 Non-United Kingdom French Canadian Haplotypes HD Non-HD AA 4 11 AB 0 0 BA 0 0 BB 0 1 Total 4 12 Ancestry unknown Haplotypes HD AA 6 AB 0 BA 0 BB I Total 7 % Non-HD 86 10 0 1 0 0 14 7 100 18 Irish HD 4 0 0 0 4 Non-HD 7 2 0 6 15 German HD Non-HD 2 0 0 0 0 0 1 6 3 6 56 5 0 39 100 Scottish HD Non-HD 5 20 0 2 0 0 2 9 7 31 Other HD 5 0 0 4 9 Non-HD 16 2 0 12 30 chromosomes by ancestry. British HD 4 0 0 0 4 Non-HD 7 1 1 5 14 Total HD 22 0 0 6 28 % Non-HD 79 54 0 9 0 1 21 36 100 100 Total HD % Non-HD % r 11 69 27 56 0-09 0 0 2 4 0 0 0 0 5 31 19 40 16 100 48 100 r p 0.30 0-21 B r p 0-18 0-0254 9 1 36 100 p 0-34 co p yrig h t. o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y h ttp ://jm g .b m j.co m / J M e d G e n e t: first p u b lish e d a s 1 0 .1 1 3 6 /jm g .2 6 .1 1 .6 7 6 o n 1 N o ve m b e r 1 9 8 9 . D o w n lo a d e d fro m http://jmg.bmj.com/ J Theilmann et al However, there is a similar trend but no signifi- cant difference in the haplotype distributions of HD and non-HD chromosomes for the families from outside the United Kingdom (p=034) and for fami- lies with unknown ancestry (p=0O21) (table 5). Discussion The major finding of this study is the non-random association between alleles for AccI and MboI detected by D4S95, SstI alleles for D4S98, and the mutant gene for HD. Linkage disequilibrium was detected with MboI and AccI and not with TaqI with D4S95. Possible explanations for this include the presence of a recombinational hot spot between the sites for TaqI on the one hand and MboI and AccI polymorphisms on the other. Alternatively it has already been shown that the recognition site for TaqI itself may act as a mutational hot spot'6 with the TaqI poly- morphism arising independently on several occa- sions. Finally it is possible that there are multiple origins of the HD gene on identical AccI and MboI haplotypes but different TaqI backgrounds. In all these instances linkage disequilibrium would not be apparent with TaqI. The 75 families in this study are of many different ancestries with the majority being of British descent (41/75). The other 34 families include at least 15 dif- ferent ancestries. The allele frequencies of AccI and MboI show significant differences between HD and non-HD chromosomes for the whole group and for families from the United Kingdom. A similar trend, but which with smaller numbers does not show sta- tistical significance, was seen with the non-UK fami- lies. It is particularly noteworthy that the haplotype distributions do not significantly differ between UK and non-UK families. HD is a disease with an extremely low mutation rate. The frequency of the disease in persons of Caucasian descent varies between 5 and 7/100 000, while there is a particularly low prevalence in per- sons of Black and Japanese descent.'7 These find- ings, together with accumulating genealogical evi- dence which traced families in Australia,18 South Africa,'9 and North America20' to Europe, impli- cates north western Europe as the major source of the gene and suggests that there may have been only a few separate mutations underlying the frequency of HD in the world. 17 The finding of similar haplotype distributions in both British families and other families of many different ancestries is sugges- tive evidence against many different mutations underlying HD. On the contrary this would suggest that there may be only a few HD mutations which have spread around the world during particular waves of immigration. These findings, however, must be viewed as preliminary and do not exclude the possibility that multiple mutations may have occurred by chance on the same chromosomal back- ground. Clearly, additional investigations in certain ethnic groups are needed to determine whether the same non-random association is found. The failure to find the same non-random association in other studies may occur because of separate origins for the HD gene in different ancestries, or may reflect recom- bination early in history in a particular country between markers around the HD gene. However, if additional studies confirm and strengthen these initial findings, and continue to show particular linkage disequilibrium in certain ancestries, this would open the way to using linkage disequilibrium in DNA analysis and genetic counselling for HD in certain situations. It may be possible to calculate high and low risk haplotypes. Over 80% of persons requesting predictive testing for HD are able to receive a significant change in risk.2' Our major limitation in performing DNA analysis for such persons is the unavailability of DNA from crucial family members, which excludes up to 20% of persons from receiving a modification of risk. The ability to detect heterozygosity in an affected person is not now a problem if multiple highly polymorphic markers are used.2' Linkage dis- equilibrium data, if confirmed, may significantly reduce the need for DNA from numerous relatives and may allow predictive testing to be performed even in the absence of DNA from an affected person in certain instances. Such linkage disequilibrium data are now frequently used for risk calculation in some cystic fibrosis families where it has improved estimates of risk.22 These HD haplotype studies are similar to investi- gations in cystic fibrosis in that for both diseases the mutant gene is not cloned. In both instances linkage disequilibrium data can be used to provide clues as to the location of the mutant gene.23 In this study, non-random association was not seen with four other polymorphic DNA markers including D4S62, D4S10, D4S96, and D4S141. In particular no evidence for linkage disequilibrium was seen using distal markers D4S96 and D4S141. The absence of disequilibrium does not necessarily imply that it does not exist but rather that it was not detected. The sample sizes in this study are small compared with those that are absolutely necessary for a definite statement that disequilibrium does not exist. Nevertheless some conclusions based on the data presented can be made. In particular, these findings suggest that the gene for HD may be more proxi- 680 co p yrig h t. o n A p ril 5 , 2 0 2 1 b y g u e st. P ro te cte d b y h ttp ://jm g .b m j.co m / J M e d G e n e t: first p u b lish e d a s 1 0 .1 1 3 6 /jm g .2 6 .1 1 .6 7 6 o n 1 N o ve m b e r 1 9 8 9 . D o w n lo a d e d fro m http://jmg.bmj.com/ Non-random associationi between alleles detected at D4S95198 and the Huntington's disease gene 681 mally located than we,8 and others,6 have previously reported and may be centromeric to some of the more distally located DNA markers such as D4S90 or D4S141. A reasonable explanation for the appa- rent contradictory results concerning the location of the HD gene is that double recombination has occurred. Currently, in the absence of a polymor- phic sequence adjacent to the telomere, this hypoth- esis remains untested. Nevertheless these findings suggest that directed cloning strategies to identify the gene for HD should not be concentrated only on the most distal region close to the telomere on chro- mosome 4. We wish to thank Drs J Gusella, M Singer, R Snell, D Shaw, and S Youngman for gifts of the probes. We wish to thank the following people for their cooperation with this study; Ms M Klimek, Dr 0 Suchowersky, Calgary; Dr S Grover, Dr S Bam- forth, Edmonton; Mr B Lynch, Dr M Shokeir, Sas- katoon; Ms L Thompson, Dr C Greenberg, Winni- peg; Dr H Soltan, London; Ms D Eisenberg, Hamil- ton; Ms S Bobkin-Raz, North York; Mr M Cook, Dr A Hunter, Ottawa; Ms J Fleming, Dr P MacLeod, Dr P Bridges, Kingston; Dr P Welch, Ms A Fuller, Halifax; Dr E Ives, St John's, Canada. This study has been supported by grants from the MRC (MA9131) (MRH), The National Health Research Development Program, a Western Cana- dian private foundation, and NIH grants GM40864 (JCM, KHB) and GM07091 (RS). Oliver Quarrell is supported by the Royal Society (UK) and NSERC. Bernhard Weber is supported by a Fellowship from the Deutsche Forschungsgemeinschaft (We 1259/1-1) and Colin Collins is a predoctoral scholar of the Huntington Society of Canada. References lGusella JF, Wexler NS, Conneally PM, et al. 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D o w n lo a d e d fro m http://jmg.bmj.com/