American Journal of Medical Genetics 40374-376 (1991)
Non-Random Association Between DNA Markers and Huntington Disease Locus in the Italian Population Andrea Novelletto, Paola Mandich, Emilia Bellone, Patrizia Malaspina, Giuseppa Vivona, F’ranco Ajmar, and Marina F’rontali Dipartimento di Biologia, Uniuersita Tor Vergata, Roma, Italy (A.N.,P.Ma1.); Cattedra di Genetica Medica, ISMI, Universita di Genova, Italy (P.Man., E.B., F.A.); Dipartimento di Sanita e Biologia Cellulare, Uniuersita Tor Vergata, Roma, Italy (G.V.); Istituto di Medicina Sperimentale, CNR, Roma, Italy (M.F.) A group of Huntington disease (HD) families of Italian ancestry was analyzed for 11 RFLPs from genetic loci mapped in 4p16 and genetically linked to the HD gene. We found a statistically significant difference of allele distributions in HD vs normal chromosomes for loci D4S10, D4S127, and D4S43. This observation increases the number of loci in linkage disequilibriumwith HD. However,the amount of disequilibrium does not allow either a finer localization of the HD gene or a substantial improvement in risk calculations.
KEY WORDS: linkage disequilibrium, chromosome 4p markers. INTRODUCTION Huntington disease (HD) is an autosomal dominant neurodegenerative disorder with late onset. The gene responsible for this condition has been localized on the short arm of chromosome 4, distally to the genetic locus D4S10 [Gusella et al., 1983; Gilliam et al., 1987al. However, subsequent efforts to further localize the gene with respect to newly discovered markers have led to contrasting results. In fact, the analysis of a set of recombination events locates the HD gene close to the chromosome telomere, whereas other events locate it more proximally [Robbinset al., 1989;MacDonald et al., 19891. Recently, Theilmann et al. [1989] and Snell et al. [1989]reported the finding of linkage disequilibrium, or the non-random association of specific alleles a t D4S98 and D4S95 marker loci with the HD mutation, in a sampleof families with different ethnic origin [Theilmann et al., 19891and in England and Wales [Snell et al., 19891. Received for publication May 16,1990;revision received September 26, 1990. Address reprint requests to A. Novelletto, Dipartimento di Biologia, Universita Tor Vergata, Via 0. Raimondo 00173 &ma, Italy.
0 1991 Wiley-Liss, Inc.
This is taken as evidence for a location of the HD gene in the proximity of these loci. Here, we report evidence that linkage disequilibrium also exists in the Italian population, concerning the D4S43 and D4S127 loci that are physically very close to D4S98 and D4S95, and spans proximally to at least a part of D4S10.
MATERIALS AND METHODS Families The DNA polymorphisms described below were examined in 46 families (348 subjects) collected either in Rome or Genoa. These include families taking part in a cooperative study to assess the feasibility of testing programs, and families that later entered the testing protocol. All of the pedigrees showed a positive family history of HD and were of Italian ancestry. The analysis of birth places of the pedigree members showed that this sample is representative of the whole of Italy. DNA Analysis Southern blot analysis was carried out on genomic DNA obtained from peripheral blood leukocytes or lymphoblastoid cell lines. The following 2-allele polymorphisms were analyzed: Hind111 (sites 1 and 2) and TaqI revealed by pK082, BglI by R7 [Gusella et al., 19861and EcoRI by F5.53 [Bakker et al., 19881from D4S10 locus; PvuII detected by BJ56PS18 from locus D4S127 [MacDonald et al., 19891; TaqI, PstI, and AccI detected by BS674E-D [Smith et al., 19881 from the D4S95 locus; TaqI revealed by S1.5 [Gilliam et al., 1987151 from the D4S43 locus; PvuII revealed by D5 [Youngman et al., 19881from the locus D4S90. For all the polymorphisms the larger fragment is referred to as A and the smaller fragment as B. These markers have been shown [MacDonald et al., 19891 to be arranged in 4p16.3 in the following order: D4SlO-D4S127-D4S95-D4S43-D4S90telomere. Molecular analysis was performed either in Rome or in Genoa. This is the reason why the 2 subsets of families were not all typed for the same RFLPs. Haplotype and phase reconstruction was by visual inspection. Non-random association of marker alleles in
HD Locus in the Italian Population HD vs. non-HD chromosomes was assayed by Fisher’s exact test.
RESULTS In this study only single-site polymorphisms were analyzed in order to make a comparison between alleles segregating in different families immediate. The 2 HindIII polymorphisms detected by pK082 were considered separately. Allele frequencies in the control group were obtained from the chromosomes of unrelated family members and from non-HD chromosomes of affected persons. The 2 subgroups did not show significant differences for any of the tested polymorphisms and were therefore pooled. The allele frequencies are in good agreement with those reported previously [Gusella et al., 1983; Youngman et al., 1986; Gilliam et al., 1987b; Quarrel1 et al., 1987; Wasmuth et al., 1988; Skraastad et al., 19891. Allele frequencies in HD chromosomes were obtained by considering a single cosegregating haplotype per family. Not all the families were typed for all polymorphisms. Cases where allele phase with respect to HD was not deducible were excluded. Table I shows absolute allele frequencies and percentages in HD and non-HD chromosomes. The examined polymorphisms are arranged accordingto their physical order. Non-random association is measured by the standardized disequilibrium statistics [Morton, 19821. The exact significance value is obtained by Fisher’s exact test. Among the examined polymorphisms, 3 [D4S43-TaqI, D4S127-PvuI1, D4SlO-HindII1, sitel] showed a significant nonrandom association between their alleles and
375
HD. Under the null hypothesis of no association, 0.55 -+ 0.72 comparisons out of the 11 tests performed were expected to be significant when a 0.05 threshold is considered (see, for example, Chakravarti et al. El9841 for the analysis of 15 RFLPs in the p-globin gene clusterl. At D4S43 locus the disease segregates preferentially with the smaller TaqI allele, whereas a t D4S127 and D4S10 it segregates preferentially with the larger allele. The non-random association at D4S10 is also statistically significant when the 2 HindIII polymorphisms are considered jointly (haplotypes A, B, C, D).
DISCUSSION In this study, which extends and confirmspreliminary results [Malaspina et al., 19891, we analyzed the presence of linkage disequilibrium between HD and chromosome 4 marker loci in the Italian population. The study of such a population is particularly meaningful since the genetic background in Italy is somewhat different from that in other areas studied so far. We have already reported [F’rontali et al., 19901 that HD in the Italian population shows the same linkage relationships found elsewhere. This study contributes another population to the analysis of linkage disequilibrium in HD. Our results confirm this finding and increase the overall number of genetic loci that exhibit linkage disequilibrium with HD, since we obtained a significant excess of non-random associations. The linkage disequilibrium with alleles at D4S43 and D4S127 loci is in agreement with the previous observation [Theilmann et al., 1989; Snell et al., 19891relative
TABLE I. RFLP Allele Freauencies and Percentages in HD and Non-HD Chromosomes in Italv
Locus D4S10
R.E. EcoRI
Probe F5.53
D4S10
HindIII (site2)
pK082
D4S10
TaqI
pK082
D4S10
BglI
R7
D4S10
HindIII (sitel)
pK082
D4S127
PVUII
BJ56
D4S95
TaqI
BS674E-D
D4S95
PstI
BS674E-D
D4S95
AccI
BS674E-D
D4S43
Taql
51.5
D4S90
PVUII
D5
-
Allele” A B A B A B A B A B A B A B A B A B A B A B
HD Chromosomes Abs % 50 12b 12 50 7 18 82 31 85 23 15 4 69 11 31 5 90 35 10 4 17 89 2 11 31 8 69 18 100 19 0 0 10 83 2 17 1 5 20 95 4 14 25 86
Non-HD Chromosomes Abs % 57 43 77 57 37 21 140 79 71 80 18 20 53 63 31 37 123 71 51 29 80 62 49 38 58 32 122 68 33 97 1 3 57 85 10 15 32 26 89 74 44 27 122 73
r
__-
P
+ .054
,323
- ,024
.463
+ .058 + .043
,375 ,449
+ .168
.009
+ .193
,014
.010
.538
+ .lo4
.642
.017
.582
- ,182
.021
- .lo5
,105
-
-
A and B refer to the larger and the smaller allele of each polymorphism, respectively. The values of the standardized disequilibrium statistics (r) and of the corresponding probability obtained by Fisher’s exact test (P)are reported. Includes 17Kb variant allele found in one HD family.
a
376
Novelletto et al.
to D4S95 and D4S98.In fact the 4 loci are all clustered in the order cen-S127-S95-S43-S98-ter in a 2200 Kb segment that is candidate for HD location [Cox et al., 19891. We confirmed that the TaqI and PstI sites of D4S95 are randomly associated with HD,while our data as to the AccI site are still inconclusive. Moreover, we confirmed that the PvuII site in D4S90 is in linkage equilibrium with HD. As already pointed out [Theilmann et al., 19891 this finding does not necessarily imply absence of disequilibrium. In fact, it is not uncommon to observe linkage disequilibrium for only a subset of the polymorphic sites at a given locus. In contrast with previous reports, we found linkage disequilibrium with a single polymorphism at locus D4S10,located centromerically to D4S95,3.7cM apart. This, as the other disequilibria, cannot be attributed to inter-ethnic variations of allelic frequencies in normal chromosomes, but may reflect a peculiarity of the Italian population for HD,with a possible “founder effect” and a still incomplete randomization. A similar situation has been suggested for Finnish HD families that showed linkage disequilibrium between HD and D4S10 markers [Ikonen et al., 19901. In this context, it is tempting to speculate that the finding of disequilibrium with the Hind111site 1 alone is not purely coincidental, since this is in the direction of the HD gene [Gilliam et al., 1987al and it has been shown that the recombination within D4S10 can account for one third of the overall recombination between this locus and HD [Skraastad et al., 19891. In conclusion, our data strongly point towards substantial homogeneity of HD in Italy, and between families of Italian and northern European ancestry. However, at the moment, a finer localization of the HD gene through linkage disequilibrium data seems difficult to attain, due to lack of resolutive power. Finally, in view of its quantitative rather than qualitative nature, linkage disequilibrium in the Italian population will only add a little information in risk calculations, leaving classic family studies still unavoidable.
ACKNOWLEDGMENTS This work was supported by Minister0 Pubblica Istruzione (fondi 40% e 60%), CNR (progetti bilaterali e contratto 89.02715.041,and by a grant from Regione Liguria. REFERENCES Bakker E, Skraastad KI, Fisser-Groen YM, van Ommen GJB, Pearson PL (1988): Two additional RFLPs a t the D4S10 locus, useful for Huntington’s disease (HD)-family studies. Nucleic Acids Res 15:9100. Chakravarti A, Buetow KH, Antonarakis SE, Waber PG, Boehm CD, Kazazian HH (1984): Non-uniform recombination within the human p-globin gene cluster. Am J Hum Genet 36:1239-1258. Cox DR, Murray JC, Buetow KH (1989):Report ofthe committee on the genetic constitution of chromosome 4. Cytogenet Cell Genet 51: 121-136. Frontali M, Malaspina P, Rossi C, Jacopini AG, Vivona G, Pergola MS, Palena A, Novelletto A (1990): Epidemiological and linkage studies on Huntington’s disease in Italy. Hum Genet 85165-170.
Gilliam TC, Tanzi RE, Haines JL, Bonner TI, Faryniarz AG, Hobbs WJ, MacDonald ME, Cheng SV, Folstein SE, Conneally PM, Wexler NS, Gusella JF (1987a):Localization ofthe Huntington’s disease gene to a small segment of chromosome 4 flanked by D4S10 and the telomere. Cell 50:565-571. Gilliam TC, Bucan M, MacDonald ME, Zimmer M, Haines J , Cheng SV, Pohl TM, Myers RH, Whaley WL, Allitto B, Faryniarz A, Wasmuth J J , Frischauf AM, Conneally PM, Gusella JF (1987b):A DNA segment encoding two genes very tightly linked to Huntington disease gene. Science 238:950-952. Gusella J F , Wexler NS, Conneally PM, Naylor SL. Anderson MA, Tanzi R, Watkins PC, Ottina K, Wallace MR, Sakaguchi AY, Young AB, Shoulson I, Bonilla E, Martin J B (1983): A polymorphic DNA marker genetically linked to Huntington’s disease. Nature 306: 234-238. Gusella JF, Gilliam TC, Tanzi RE, MacDonald ME, Chang SV, Wallace M, Haines J , Conneally PM, Wexler NS (1986): Molecular genetics of Huntington’s disease. Cold Spring Harbor Symp Quant Biol 51:359-364. Ikonen E, Palo J , Ott J, Gusella J, Somer H, Karila L, Palotie A, Peltonen L (1990): Huntington’s disease in Finland: Linkage disequilibrium on chromosome 4 RFLP haplotypes and exclusion of a tight linkage between the disease and D4S43 locus. Am J Hum Genet 465-11. MacDonald ME, Haines JL, Zimmer M, Cheng SV, Youngman S, Whaley WL, Wexler N, Bucan M, Allitto BA, Smith B, Leavitt J, Poutska A, Harper P, Lehrach H, Wasmuth JJ, Frischauf AM, Gusella JF (1989):Recombination events suggest potential sites for Huntington’s disease gene. Neuron 3:183-190. Malaspina P, Vivona G, Palena A, Novelletto A, Frontali M (1989): Non-random association of DNA markers in the proximity of Huntington’s disease locus. Atti Ass Genet Ital XXV:405. Morton NE (1982):“Outline of Genetic Epidemiology.” Basel, Switzerl a n d S. Karger. Quarrell OWJ, Tyler A, Upadhyaya M, Meredith AL, Youngman AL, Harper PS (1987): Exclusion testing for Huntington’s disease in pregnancy with a closely linked DNA marker. Lancet June 6:1281-1283. , Robbins C, Theilmann J, Youngman S, Haines J , Altherr MJ, Harper PS, Payne C, Junker A, Wasmuth J J , Hayden MR (1989):Evidence from family studies that the gene causing Huntington disease is telomeric to D4S95 and D4S90. Am J Hum Genet 44:422-425. Skraastad MI, Bakker E, de Lange LF, Vegter-van der Vlis M, KleinBreteler EG, van Ommen GJB, Pearson PL (1989): Mapping of recombinants near the Huntington disease locus by using G8 (D4S10) and newly isolated markers in the D4S10 region. Hum Genet 44:560-566. Smith B, Skarecky D, Bengtsson U, Magenis RE, Carpenter N , Wasmuth JJ (1988): Isolation of DNA marker in the direction of the Huntington disease gene from the G8 locus. Am J Hum Genet 42:335-344. Snell RG, Lazarou L, Youngman S, Quarrell OWJ, Wasmuth JJ, Shaw DJ, Harper PS (1989):Linkage disequilibrium in Huntington’s disease: An improved localization for the gene. J Med Genet 26:673-675. Theilmann J, Kanani S, Shiang R, Robbins C, Quarrell OWJ, Huggins M, Hedrick A, Weber B, Collins C, Wasmuth J J , Buetow KH, Murray JC, Hayden MR (1989): Non-random association between alleles detected by D4S95 and the Huntington’s disease gene. J Med Genet 26:676-681. Wasmuth JJ, Hewitt J, Smith B, Allard D, Haines JL, Skarecky D, Partlow E, Hayden MR (1988): A highly polymorphic locus very tightly linked to the Huntington’s disease gene. Nature 332: 734-736. Youngman S, Sarfarazi M, Quarrel OWJ, Conneally PM, Gibbons K, Harper PS, Shaw DJ, Tanzi RE, Wallace MR, Gusella JF (1986): Studies of a DNA marker (G8) genetically linked to Huntington’s disease in British families. Hum Genet 73:333-339. Youngman S, Shaw DJ, Gusella J F , MacDonald ME, Stanbridge EJ, Wasmuth J J , Harper PS (1988): A DNA probe D5 [D4S901mapping to human chromosome 4~16.3.Nucleic Acids Res 16:1648.
Non-Random Association Between DNA Markers and Huntington Disease Locus in the Italian Population Andrea Novelletto, Paola Mandich, Emilia Bellone, Patrizia Malaspina, Giuseppa Vivona, F’ranco Ajmar, and Marina F’rontali Dipartimento di Biologia, Uniuersita Tor Vergata, Roma, Italy (A.N.,P.Ma1.); Cattedra di Genetica Medica, ISMI, Universita di Genova, Italy (P.Man., E.B., F.A.); Dipartimento di Sanita e Biologia Cellulare, Uniuersita Tor Vergata, Roma, Italy (G.V.); Istituto di Medicina Sperimentale, CNR, Roma, Italy (M.F.) A group of Huntington disease (HD) families of Italian ancestry was analyzed for 11 RFLPs from genetic loci mapped in 4p16 and genetically linked to the HD gene. We found a statistically significant difference of allele distributions in HD vs normal chromosomes for loci D4S10, D4S127, and D4S43. This observation increases the number of loci in linkage disequilibriumwith HD. However,the amount of disequilibrium does not allow either a finer localization of the HD gene or a substantial improvement in risk calculations.
KEY WORDS: linkage disequilibrium, chromosome 4p markers. INTRODUCTION Huntington disease (HD) is an autosomal dominant neurodegenerative disorder with late onset. The gene responsible for this condition has been localized on the short arm of chromosome 4, distally to the genetic locus D4S10 [Gusella et al., 1983; Gilliam et al., 1987al. However, subsequent efforts to further localize the gene with respect to newly discovered markers have led to contrasting results. In fact, the analysis of a set of recombination events locates the HD gene close to the chromosome telomere, whereas other events locate it more proximally [Robbinset al., 1989;MacDonald et al., 19891. Recently, Theilmann et al. [1989] and Snell et al. [1989]reported the finding of linkage disequilibrium, or the non-random association of specific alleles a t D4S98 and D4S95 marker loci with the HD mutation, in a sampleof families with different ethnic origin [Theilmann et al., 19891and in England and Wales [Snell et al., 19891. Received for publication May 16,1990;revision received September 26, 1990. Address reprint requests to A. Novelletto, Dipartimento di Biologia, Universita Tor Vergata, Via 0. Raimondo 00173 &ma, Italy.
0 1991 Wiley-Liss, Inc.
This is taken as evidence for a location of the HD gene in the proximity of these loci. Here, we report evidence that linkage disequilibrium also exists in the Italian population, concerning the D4S43 and D4S127 loci that are physically very close to D4S98 and D4S95, and spans proximally to at least a part of D4S10.
MATERIALS AND METHODS Families The DNA polymorphisms described below were examined in 46 families (348 subjects) collected either in Rome or Genoa. These include families taking part in a cooperative study to assess the feasibility of testing programs, and families that later entered the testing protocol. All of the pedigrees showed a positive family history of HD and were of Italian ancestry. The analysis of birth places of the pedigree members showed that this sample is representative of the whole of Italy. DNA Analysis Southern blot analysis was carried out on genomic DNA obtained from peripheral blood leukocytes or lymphoblastoid cell lines. The following 2-allele polymorphisms were analyzed: Hind111 (sites 1 and 2) and TaqI revealed by pK082, BglI by R7 [Gusella et al., 19861and EcoRI by F5.53 [Bakker et al., 19881from D4S10 locus; PvuII detected by BJ56PS18 from locus D4S127 [MacDonald et al., 19891; TaqI, PstI, and AccI detected by BS674E-D [Smith et al., 19881 from the D4S95 locus; TaqI revealed by S1.5 [Gilliam et al., 1987151 from the D4S43 locus; PvuII revealed by D5 [Youngman et al., 19881from the locus D4S90. For all the polymorphisms the larger fragment is referred to as A and the smaller fragment as B. These markers have been shown [MacDonald et al., 19891 to be arranged in 4p16.3 in the following order: D4SlO-D4S127-D4S95-D4S43-D4S90telomere. Molecular analysis was performed either in Rome or in Genoa. This is the reason why the 2 subsets of families were not all typed for the same RFLPs. Haplotype and phase reconstruction was by visual inspection. Non-random association of marker alleles in
HD Locus in the Italian Population HD vs. non-HD chromosomes was assayed by Fisher’s exact test.
RESULTS In this study only single-site polymorphisms were analyzed in order to make a comparison between alleles segregating in different families immediate. The 2 HindIII polymorphisms detected by pK082 were considered separately. Allele frequencies in the control group were obtained from the chromosomes of unrelated family members and from non-HD chromosomes of affected persons. The 2 subgroups did not show significant differences for any of the tested polymorphisms and were therefore pooled. The allele frequencies are in good agreement with those reported previously [Gusella et al., 1983; Youngman et al., 1986; Gilliam et al., 1987b; Quarrel1 et al., 1987; Wasmuth et al., 1988; Skraastad et al., 19891. Allele frequencies in HD chromosomes were obtained by considering a single cosegregating haplotype per family. Not all the families were typed for all polymorphisms. Cases where allele phase with respect to HD was not deducible were excluded. Table I shows absolute allele frequencies and percentages in HD and non-HD chromosomes. The examined polymorphisms are arranged accordingto their physical order. Non-random association is measured by the standardized disequilibrium statistics [Morton, 19821. The exact significance value is obtained by Fisher’s exact test. Among the examined polymorphisms, 3 [D4S43-TaqI, D4S127-PvuI1, D4SlO-HindII1, sitel] showed a significant nonrandom association between their alleles and
375
HD. Under the null hypothesis of no association, 0.55 -+ 0.72 comparisons out of the 11 tests performed were expected to be significant when a 0.05 threshold is considered (see, for example, Chakravarti et al. El9841 for the analysis of 15 RFLPs in the p-globin gene clusterl. At D4S43 locus the disease segregates preferentially with the smaller TaqI allele, whereas a t D4S127 and D4S10 it segregates preferentially with the larger allele. The non-random association at D4S10 is also statistically significant when the 2 HindIII polymorphisms are considered jointly (haplotypes A, B, C, D).
DISCUSSION In this study, which extends and confirmspreliminary results [Malaspina et al., 19891, we analyzed the presence of linkage disequilibrium between HD and chromosome 4 marker loci in the Italian population. The study of such a population is particularly meaningful since the genetic background in Italy is somewhat different from that in other areas studied so far. We have already reported [F’rontali et al., 19901 that HD in the Italian population shows the same linkage relationships found elsewhere. This study contributes another population to the analysis of linkage disequilibrium in HD. Our results confirm this finding and increase the overall number of genetic loci that exhibit linkage disequilibrium with HD, since we obtained a significant excess of non-random associations. The linkage disequilibrium with alleles at D4S43 and D4S127 loci is in agreement with the previous observation [Theilmann et al., 1989; Snell et al., 19891relative
TABLE I. RFLP Allele Freauencies and Percentages in HD and Non-HD Chromosomes in Italv
Locus D4S10
R.E. EcoRI
Probe F5.53
D4S10
HindIII (site2)
pK082
D4S10
TaqI
pK082
D4S10
BglI
R7
D4S10
HindIII (sitel)
pK082
D4S127
PVUII
BJ56
D4S95
TaqI
BS674E-D
D4S95
PstI
BS674E-D
D4S95
AccI
BS674E-D
D4S43
Taql
51.5
D4S90
PVUII
D5
-
Allele” A B A B A B A B A B A B A B A B A B A B A B
HD Chromosomes Abs % 50 12b 12 50 7 18 82 31 85 23 15 4 69 11 31 5 90 35 10 4 17 89 2 11 31 8 69 18 100 19 0 0 10 83 2 17 1 5 20 95 4 14 25 86
Non-HD Chromosomes Abs % 57 43 77 57 37 21 140 79 71 80 18 20 53 63 31 37 123 71 51 29 80 62 49 38 58 32 122 68 33 97 1 3 57 85 10 15 32 26 89 74 44 27 122 73
r
__-
P
+ .054
,323
- ,024
.463
+ .058 + .043
,375 ,449
+ .168
.009
+ .193
,014
.010
.538
+ .lo4
.642
.017
.582
- ,182
.021
- .lo5
,105
-
-
A and B refer to the larger and the smaller allele of each polymorphism, respectively. The values of the standardized disequilibrium statistics (r) and of the corresponding probability obtained by Fisher’s exact test (P)are reported. Includes 17Kb variant allele found in one HD family.
a
376
Novelletto et al.
to D4S95 and D4S98.In fact the 4 loci are all clustered in the order cen-S127-S95-S43-S98-ter in a 2200 Kb segment that is candidate for HD location [Cox et al., 19891. We confirmed that the TaqI and PstI sites of D4S95 are randomly associated with HD,while our data as to the AccI site are still inconclusive. Moreover, we confirmed that the PvuII site in D4S90 is in linkage equilibrium with HD. As already pointed out [Theilmann et al., 19891 this finding does not necessarily imply absence of disequilibrium. In fact, it is not uncommon to observe linkage disequilibrium for only a subset of the polymorphic sites at a given locus. In contrast with previous reports, we found linkage disequilibrium with a single polymorphism at locus D4S10,located centromerically to D4S95,3.7cM apart. This, as the other disequilibria, cannot be attributed to inter-ethnic variations of allelic frequencies in normal chromosomes, but may reflect a peculiarity of the Italian population for HD,with a possible “founder effect” and a still incomplete randomization. A similar situation has been suggested for Finnish HD families that showed linkage disequilibrium between HD and D4S10 markers [Ikonen et al., 19901. In this context, it is tempting to speculate that the finding of disequilibrium with the Hind111site 1 alone is not purely coincidental, since this is in the direction of the HD gene [Gilliam et al., 1987al and it has been shown that the recombination within D4S10 can account for one third of the overall recombination between this locus and HD [Skraastad et al., 19891. In conclusion, our data strongly point towards substantial homogeneity of HD in Italy, and between families of Italian and northern European ancestry. However, at the moment, a finer localization of the HD gene through linkage disequilibrium data seems difficult to attain, due to lack of resolutive power. Finally, in view of its quantitative rather than qualitative nature, linkage disequilibrium in the Italian population will only add a little information in risk calculations, leaving classic family studies still unavoidable.
ACKNOWLEDGMENTS This work was supported by Minister0 Pubblica Istruzione (fondi 40% e 60%), CNR (progetti bilaterali e contratto 89.02715.041,and by a grant from Regione Liguria. REFERENCES Bakker E, Skraastad KI, Fisser-Groen YM, van Ommen GJB, Pearson PL (1988): Two additional RFLPs a t the D4S10 locus, useful for Huntington’s disease (HD)-family studies. Nucleic Acids Res 15:9100. Chakravarti A, Buetow KH, Antonarakis SE, Waber PG, Boehm CD, Kazazian HH (1984): Non-uniform recombination within the human p-globin gene cluster. Am J Hum Genet 36:1239-1258. Cox DR, Murray JC, Buetow KH (1989):Report ofthe committee on the genetic constitution of chromosome 4. Cytogenet Cell Genet 51: 121-136. Frontali M, Malaspina P, Rossi C, Jacopini AG, Vivona G, Pergola MS, Palena A, Novelletto A (1990): Epidemiological and linkage studies on Huntington’s disease in Italy. Hum Genet 85165-170.
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