American Journal of Medical Genetics 39:211-214 (1991)
Genetic Analysis of Huntington Disease in Italy F. Ajmar, P. Mandich, E. Bellone, and G. Abbruzzese Section of Medical Genetics. ISMI (F.A..P.M., E.B.) and Neurology Institute (G.A.), University of Genoua, Genoua, Italy
Twelve Italian families with Huntington disease were tested with 10 probes known to be linked to the disease locus and able to detect polymorphisms at the following loci on chromosome 4: D4S10, D45127, D4S95, D4S43, D4S115, D4Sll1, D4S90. The results confirmed the applicability of the linkage approach for presymptomatic diagnosis in Italian families. Positive lod scores were found between D4S10, D4S95, D4S43 and the disease, whereas D4S90 did not indicate significant linkage values. With the limitations due to the small size of the tested sample, no genetic heterogeneity was detected in the families examined for loci D4S10, D4S95iS127, D4S43. KEY WORDS: HD, chromosome 4, D4S10, D4S127, D4S95, D4S43, D45175, D4Sll1, D4S90 INTRODUCTION Huntington disease (HD) is a neurodegenerative disorder characterized by progressive loss of motor control, impaired cognition, and psychiatric disturbances [Martin and Gusella, 19861. Neuropathological examination of affected brains shows atrophy of basal ganglia, particularly in the caudatum nucleus. Usually psychiatric and cognition symptoms precede the appearance of abnormal movements by many years. Many emotional disturbances are present in HD patients such as irritability, depression, character, and mood changes and sometimes delusions and hallucinations. Progressively chorea or involuntary movements of the body are the typical and early motor symptoms. Dystonia, bradychinesia, and dysarthria can develop gradually. Patients also loose short-term memory, organizational ability, and other cognitive abilities. The average age of onset is approximately 38 years ranging from less than 5
Received for publication January 2,1990; revision received May 14, 1990.
Address reprint request to F’ranco AJMAR, MD, PhD, Section on Medical Genetics, ISMI, University of Genova, Viale Benedetto XV, 6, 16132 Genova, Italy.
0 1991 Wiley-Liss, Inc.
years of age to more than 70 years and in about 10% of cases the disease begins before age 20 years. Patients die 15-20 years after the onset of symptoms [Conneally, 19841. The biochemical basis for neuronal cell loss in this disorder is not yet known, and a t present there is no treatment for delaying or preventing the manifestations of this disease. HD is caused by a n autosomal dominant mutation, whose locus has been assigned by linkage analysis and somatic cell hybrids to the short arm of chromosome 4, a t 4 ~ 1 6 . 3between , the anonymous DNA marker D4S10 and the telomere [Gilliam et al., 1987133. The characterization of linked markers for HD has also introduced the possibility of diagnosis in presymptomatic individuals. For presymptomatic and prenatal tests, it is critical to increase the number and informativeness of linked DNA markers to ensure a n accurate test. The discovery of new tightly linked DNA markers with the characteristics of VNTR markers [Macdonald et al., 19891 has improved the informativeness and the accuracy of the test. The approach of linked markers in presymptomatic diagnosis of HD requires also that different populations be tested to confirm general applicability. Analysis of different populations can also give informations about the origin of the disease (unique or multiple), about genetic heterogeneity, and about linkage disequilibrium. The populations so far tested include, aside from the large Venezuelan pedigrees [Gusella et al., 19831,North Americans and northern Europeans [Conneally et al., 1989; Skraastad et al., 19891. No reports have demonstrated heterogeneity for this disease [Conneally et al., 19891, and the linkage between the available markers and the disease was always confirmed, except for among a Finnish population [Ikonen et al., 19901. No data on linkage analysis or heterogeneity in Italy have been described. The present report describes linkage analysis and heterogeneity evaluation in the Italian population using some recently available polymorphic markers.
MATERIALS AND METHODS Pedigrees Families with a documented history of HD were ascertained. They were referred to our genetic counselling department either directly or from the neurology clinics. A protocol for informed consent was used. Paternity testing was performed with VNTR markers in doubtful
212
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Table I presents the different level of informativeness obtained with each probe. This allowed us to decide which probe to use first in each new family. Informativeness increased with the use of VNTR probes, which were DNA Analysis the latest available. Therefore, several tests with previSamples of venous blood (20 ml) were used for DNA ously available probes were also reported. We do not isolation. Genomic DNA was extracted according to the systematically test the whole set of probes, but we begin methods described by Maniatis et al. 119821. DNA ali- with the most informative and closest to the gene and quots of 5 p.g were digested to completion with the appro- proceed to other probes if the initial ones are noninforpriate enzymes, then separated by agarose gel electro- mative. This procedure explains why the linkage analphoresis and transferred to nylon membrane by ysis does not include all loci in all families. Southern blotting. The probes were labeled with [ C X ~ ~ P ]The results of linkage analysis are reported in Table dCTP following the procedure of Feinberg and Vogel- 11. The lod score values obtained between HD and loci stein [1984]. Filters were hybridized in 0.25 M Na phos- known to be closely linked (e.g., D4S95 and 049127 or phate buffer, 7% sulfate sodium dodecyl (SDS) a t 65"C, D4S111 and D4S115)or detected by overlapping probes then washed a t 65°C in 0.0125 M Na phosphate 1%SDS, (e.g.,G8 pK082 and pK083, D5, and 2R3) were combined as indicated in Table 11. and autoradiographed. Although the lod score values are rather modest, they DNA Probes confirm the close genetic association between the tested For the D4S10 locus [Gusella et al., 19831, two single markers and the disease in a small sample of Italian copy probes were used: 1)pK082 is a 5.5 kb EcoRI frag- families. Lower values of lod scores are found for ments subcloned from G8 phage clone, and i t detects D4S111lD4S115 loci, with Z = 1.031 a t 0=0.10 and are polymorphic fragment with HindIII; 2) pK083 is a 2.4 kb even lower for D4S90.Few families were tested a t the EcoRI fragment subcloned from G8 phage clone, and it locus, however, and one recombinant was found between detects EcoRI polymorphism. For the D4S95 locus the locus and HD. Five recombinants altogether were [Wasmuth et al., 19881, a 1 kb single copy fragment found: 2 were nonaffected relatives, 2 affected patients, BS674 E-D, which reveals polymorphisms with TaqI, and one a n at risk individual. The locations of recomHindIII, and AccI, was used. For the 04843 locus [Gill- bination events were, respectively, one between D4S10 iam et al., 1987a1, two probes were used: S1.5, a 1.2 kb and 048127,2 between 04843 and D4S115, one besingle copy fragment polymorphic in TaqI, and pKPl.65, tween D4S115 and D4S90, and one between 048125 a 1.65 kb PstI fragment that detects a multiallelic poly- and D4S95.Due to the relevance of some of these recommorphism with BglI. Probe 252.3 is a PstI-Sau3A frag- binations to HD gene mapping, they are presently being ment from a cosmid that overlaps the NotI-linking clone tested with other probes (D4S98 and D4S96), and 252 a t the D4S115 locus [MacDonald et al., 19891 poly- the results will be reported separately (manuscript in morphic for many enzymes. Probe p157.9 is a 450 bp preparation). Homogeneity testing indicates no significant heteroPstI-Sau3A fragment subcloned into pGEM3 from a cosmid that overlaps the NotI-linking clone 151 defining geneity among the families tested for loci 04510, D4S111 locus [MacDonald et al., 19891. For the D4S90 D4S95lS127,and D4S43 (Table 11). Values for D4Sllll locus [Youngman et al., 19891, two single copy probes S115 and for D4S90 are also included. were used: D5 is a 1.5 kb EcoRI insert polymorphic for PVUII and 2R3 is a 1.3 kb XbaI fragment polymorphic for HindIII. cases. Since the results of presymptomatic testing were still on an experimental basis, no result of testing was given to the patients.
Linkage Analysis Two point linkage analysis was carried out using the LINKAGE 5.03 program [Lathrop and Lalouel, 19841; the homogeneity test was carried out with the HOMOG program [Ott, 19851.
RESULTS Twelve HD families from different regions of Italy were examined, consisting of 115 individuals. Four families were not included in the lod score calculations because of family structure; one was excluded because of incompatibility in paternity testing of the affected individual. Of the tested subjects, 20 were affected, 55 were unaffected relatives, and 40 were a t risk of developing HD. With DNA testing, 14 resulted positive, 19 negative, and 7 were uninformative due to family structure or because of recombination events occurring in the family and not allowing the localization of the disease gene. A total of 115 meioses (108 informative) were counted.
TABLE I. Heterozygosity of HD Markers in Italy* Heterozygosity Locus D4S90 D4S90 04943 D4S95 D4S95 049127 D4SlO 04510 D4S43 D4S95 D4S115 D4Slll 049125 D4S95
Probe 2R3 D5 S1.5 674 674 BJ56 KO82 KO83 KPI.65 674 252.3 157.9 YNZ32 674
Polymorphism HindIII PVUII TaqI TaqI AccI PVUII HindIII EcoRI BglI HindIII PstI PstI TaqI AccI
(%I RFLP 135) RFLP (28) RFLP (18) RFLP (33) RFLP (22) RFLP (30) RFLP (28) RFLP (27) Multiallelic (33) Multiallelic (30) VNTR (73) VNTR (59) VNTR (57) VNTR (59)
*The heterozygosity of each probe was measured in unrelated individuals a s percentage of heterozygous meioses.
Genetic Analysis of HD in Italy
213
TABLE 11. Lod Scores for Linkage of HD Polvmoruhic Sites of D4* Locus 8
0.005
0.01
0.02
0.04
0.06
0.08
0.10
0.20
D4S10 1.353 1.347 1.338 1.304 1.270 1.224 1.181 0.879 (n = 11) D4S95/S127 2.149 2.117 2.052 1.923 1.796 1.671 1.547 0.964 ( n = 11) D4S43 1.480 1.467 1.442 1.381 1.334 1.276 1.218 0.897 (n = 7) D4SllllS115 0.029 0.320 0.598 0.843 0.957 1.012 1.031 0.885 (n = 9) D4S90 -1.676 -1.091 -1.037 -0.700 -0.503 -0.367 -0.264 -0.008 (n = 5)
0.30 0.491 0.478 0.561 0.575 0.058
*n =number ofinformative pedigrees combinedper locus: probes pK082 and pK082 (HindIII and EcoRI) for B4S10; BS 674 (TaqI, HindIII, and AccI) and BJ 56 (PvuII) for loci D4S9.5 and B4S127, respectively; probe 51.5 and pKP1.65 (TaqI and BglI) forD4S43; probe p157.9 and 252.3 (PstI)for D 4 S l l l and D4S115; probe D5 and 2R3 (PvuII and HindIII) for D4S90. Lod scores for closelv linked loci were mouDed a s indicated. The homogeneity test (HOMOG program) indicated the following*values: for D4SYO: 14L=3.115, a = 1.0 at 0 = 0.005; xz = 6.231, P = 0.006; for D4S956127: 1nL = 4.948, a = 1.0 at 0 = 0.005; x’ = 9.897, P = 0,0008; for D4S43: lnL=3.408, a=1.0 a t 0=0.005; x2=6.816, P=O.O045; for D4SllliS115: lnL=2.415, a=0.55 at 0=0.005; ~‘4.748,P=O.O15 (lnL=2.398 at a=0.7 and 0=0.05); for D4S90: lnL=0.133 at a = 1 . 0 and 0 = 0.005, xz = 0.267, P = 0.303) (see Discussion).
DISCUSSION The use of probes linked to the HD gene in the presymptomatic diagnosis requires a preliminary survey to verify that this approach is applicable in the population being tested. Although the sample is limited, our results confirm that polymorphic probes of loci D4910, D4S95, 049127, and 0 4 9 4 3 can be used in presymptomatic testing of Italian families. Our results on the informativeness of different probes suggest also the order in which the analysis for presymptomatic diagnosis should be performed, starting with the closest and most informative probes; however, since the recombinational events have a relatively high frequency, it is appropriate to test systematically several loci even if informativeness is reached with a single probe. The use of multiple probes a t different loci allows, in most cases, detection of the risk aplotype. The small sample studied does not permit us yet to attribute a significance to the relatively lower values of linkage between HD and loci previously described as tightly linked: In particular, D4S11 llD4S115 and D4990 show higher genetic distance than D4S10, D4S95, D4S127, or 0 4 9 4 3 . This is likely due to the fact that one recombinant is found between D4S90 and HD, and the number of informative families for this locus is only 5. The homogeneity test confirmed that no significant genetic heterogeneity is present in our population. It is known that significance for heterogeneity test requires a considerably higher number of families [CavalliSforza and King, 1986; Ott 19851. With these limitations, our results further support the suggestion that the same gene is responsible for HD in different populations [Conneally et al., 19891. ACKNOWLEDGMENTS We thank Drs. J. Gusella, M. Hayden, P. Harper, J. Wasmuth, and Y. Nakamura for providing us with the probes and Drs. M. Lathrop and J. Ott for the LINKAGE
program. This work was supported by grants from Regione Liguria, MPI and CNR (International Projects).
REFERENCES Cavalli-Sforza LL, King MC (1986):Detecting linkage for genetically heterogeneous diseases and detecting heterogeneity with linkage data. Am J Hum Genet 38:599-616. Conneally PM (1984):Huntington disease: Genetics and epidemiology. Am J Hum Genet 36506-526. Conneally PM, Haines JL, Tanzi RE, Wexler NS, Penchaszadeh GK, Harper PS, Folstein SE, Cassiman J J , Myers RH, Young AB, Hayden MR, Falek A, Tolosa ES, Crespi s, Di Maio L, Holmgren G, Anvret M, Kanazawa I, Gusella JF (1989):Huntington disease: No evidence for locus heterogeneity. Genomics 5304-308. Feinberg AP, Vogelstein B (1984):A technique for radio labelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 123:6-13. Gilliam TC, Bucan M, MacDonald ME, Zimmer M, Haines JL, Cheng SV, Pohl TM, Whaley WL, Allitto BA, Faryniarz A, Wasmuth JJ, hischauf AM, Conneally PM, Lehrach H, Gusella J F (1987a): A DNA segment encoding two genes very tightly linked to Huntington’s disease. Science 238:950-952. Gilliam TC, Tanzi RE, Haines JL, Bonner TI, Faryniarz AG, Hobbs, WJ, MacDonald ME, Cheng SV, Folstein SE, Conneally PM, Wexler NS, Gusella JF (1987b): Localization of the Huntington’s diease gene to a small segment of chromosome 4 flanked by D4S10 and the telomere. Call 50:565-571. Gusella JF, Wexler NS, Conneally PM, Naylor SL, Anderson MA, Tanzi RE, 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. Ikonen E, Palo J , Ott J , Gusella J , Somer H, Karila L, Palotie A , Peltonen L (1990):Huntington disease in Finland: Linkage disequilibrium of chromosome 4 RFLP haplotypes and exclusion of a tight linkage between the disease and D4S43 locus. Am J Hum Genet 465-1 1. Lathrop GM, Lalouel JM (1984): Easy calculations of lad scores and genetic risk on small computers. Am J Hum Genet 36:460-465. MacDonald ME, Cheng SV, Zimmer N, Haines JL, Poutska AM, Allitto B, Smith B, Whaley WL, Romano DM, Jagadeesh J, Myers RH, Lehrach H, Wasmuth JJ, FrischaufAM, Gusella JF (1989):Clustering of multi-allele DNA markers near the Huntington’s disease gene. J Clin Invest 84:1013-1016. Maniatis T, Fritsch EF, Sanbrock J (1982): “Molecular Cloning. A Laboratory manual.” Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
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Martin JB, Gusella JF (1986):Huntington’s disease: Pathogenesis and management. N Engl J Med 315:1267-1276. Ott J (1985):“Analysis of Human Linkage.” Baltimore: Johns Hopkins University Press. Skraastad MI, Bakkar 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. Am J Hum Genet 44560-566.
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. Nature 332:734-736. Youngman S, Sarfarazi M, Bucan M, MacDonald ME, Smith B, Zimmer M, Gilliam C, Frischauf AM, Wasmuth JJ, Gusella J F , Lehrach H, Harper PS, Shaw DJ (1989):A new DNA marker (D4S90)is located terminally on the short arm of chromosome 4, close to the Huntington disease gene. Genomics 5:802-809.
Genetic Analysis of Huntington Disease in Italy F. Ajmar, P. Mandich, E. Bellone, and G. Abbruzzese Section of Medical Genetics. ISMI (F.A..P.M., E.B.) and Neurology Institute (G.A.), University of Genoua, Genoua, Italy
Twelve Italian families with Huntington disease were tested with 10 probes known to be linked to the disease locus and able to detect polymorphisms at the following loci on chromosome 4: D4S10, D45127, D4S95, D4S43, D4S115, D4Sll1, D4S90. The results confirmed the applicability of the linkage approach for presymptomatic diagnosis in Italian families. Positive lod scores were found between D4S10, D4S95, D4S43 and the disease, whereas D4S90 did not indicate significant linkage values. With the limitations due to the small size of the tested sample, no genetic heterogeneity was detected in the families examined for loci D4S10, D4S95iS127, D4S43. KEY WORDS: HD, chromosome 4, D4S10, D4S127, D4S95, D4S43, D45175, D4Sll1, D4S90 INTRODUCTION Huntington disease (HD) is a neurodegenerative disorder characterized by progressive loss of motor control, impaired cognition, and psychiatric disturbances [Martin and Gusella, 19861. Neuropathological examination of affected brains shows atrophy of basal ganglia, particularly in the caudatum nucleus. Usually psychiatric and cognition symptoms precede the appearance of abnormal movements by many years. Many emotional disturbances are present in HD patients such as irritability, depression, character, and mood changes and sometimes delusions and hallucinations. Progressively chorea or involuntary movements of the body are the typical and early motor symptoms. Dystonia, bradychinesia, and dysarthria can develop gradually. Patients also loose short-term memory, organizational ability, and other cognitive abilities. The average age of onset is approximately 38 years ranging from less than 5
Received for publication January 2,1990; revision received May 14, 1990.
Address reprint request to F’ranco AJMAR, MD, PhD, Section on Medical Genetics, ISMI, University of Genova, Viale Benedetto XV, 6, 16132 Genova, Italy.
0 1991 Wiley-Liss, Inc.
years of age to more than 70 years and in about 10% of cases the disease begins before age 20 years. Patients die 15-20 years after the onset of symptoms [Conneally, 19841. The biochemical basis for neuronal cell loss in this disorder is not yet known, and a t present there is no treatment for delaying or preventing the manifestations of this disease. HD is caused by a n autosomal dominant mutation, whose locus has been assigned by linkage analysis and somatic cell hybrids to the short arm of chromosome 4, a t 4 ~ 1 6 . 3between , the anonymous DNA marker D4S10 and the telomere [Gilliam et al., 1987133. The characterization of linked markers for HD has also introduced the possibility of diagnosis in presymptomatic individuals. For presymptomatic and prenatal tests, it is critical to increase the number and informativeness of linked DNA markers to ensure a n accurate test. The discovery of new tightly linked DNA markers with the characteristics of VNTR markers [Macdonald et al., 19891 has improved the informativeness and the accuracy of the test. The approach of linked markers in presymptomatic diagnosis of HD requires also that different populations be tested to confirm general applicability. Analysis of different populations can also give informations about the origin of the disease (unique or multiple), about genetic heterogeneity, and about linkage disequilibrium. The populations so far tested include, aside from the large Venezuelan pedigrees [Gusella et al., 19831,North Americans and northern Europeans [Conneally et al., 1989; Skraastad et al., 19891. No reports have demonstrated heterogeneity for this disease [Conneally et al., 19891, and the linkage between the available markers and the disease was always confirmed, except for among a Finnish population [Ikonen et al., 19901. No data on linkage analysis or heterogeneity in Italy have been described. The present report describes linkage analysis and heterogeneity evaluation in the Italian population using some recently available polymorphic markers.
MATERIALS AND METHODS Pedigrees Families with a documented history of HD were ascertained. They were referred to our genetic counselling department either directly or from the neurology clinics. A protocol for informed consent was used. Paternity testing was performed with VNTR markers in doubtful
212
Ajmar et al.
Table I presents the different level of informativeness obtained with each probe. This allowed us to decide which probe to use first in each new family. Informativeness increased with the use of VNTR probes, which were DNA Analysis the latest available. Therefore, several tests with previSamples of venous blood (20 ml) were used for DNA ously available probes were also reported. We do not isolation. Genomic DNA was extracted according to the systematically test the whole set of probes, but we begin methods described by Maniatis et al. 119821. DNA ali- with the most informative and closest to the gene and quots of 5 p.g were digested to completion with the appro- proceed to other probes if the initial ones are noninforpriate enzymes, then separated by agarose gel electro- mative. This procedure explains why the linkage analphoresis and transferred to nylon membrane by ysis does not include all loci in all families. Southern blotting. The probes were labeled with [ C X ~ ~ P ]The results of linkage analysis are reported in Table dCTP following the procedure of Feinberg and Vogel- 11. The lod score values obtained between HD and loci stein [1984]. Filters were hybridized in 0.25 M Na phos- known to be closely linked (e.g., D4S95 and 049127 or phate buffer, 7% sulfate sodium dodecyl (SDS) a t 65"C, D4S111 and D4S115)or detected by overlapping probes then washed a t 65°C in 0.0125 M Na phosphate 1%SDS, (e.g.,G8 pK082 and pK083, D5, and 2R3) were combined as indicated in Table 11. and autoradiographed. Although the lod score values are rather modest, they DNA Probes confirm the close genetic association between the tested For the D4S10 locus [Gusella et al., 19831, two single markers and the disease in a small sample of Italian copy probes were used: 1)pK082 is a 5.5 kb EcoRI frag- families. Lower values of lod scores are found for ments subcloned from G8 phage clone, and i t detects D4S111lD4S115 loci, with Z = 1.031 a t 0=0.10 and are polymorphic fragment with HindIII; 2) pK083 is a 2.4 kb even lower for D4S90.Few families were tested a t the EcoRI fragment subcloned from G8 phage clone, and it locus, however, and one recombinant was found between detects EcoRI polymorphism. For the D4S95 locus the locus and HD. Five recombinants altogether were [Wasmuth et al., 19881, a 1 kb single copy fragment found: 2 were nonaffected relatives, 2 affected patients, BS674 E-D, which reveals polymorphisms with TaqI, and one a n at risk individual. The locations of recomHindIII, and AccI, was used. For the 04843 locus [Gill- bination events were, respectively, one between D4S10 iam et al., 1987a1, two probes were used: S1.5, a 1.2 kb and 048127,2 between 04843 and D4S115, one besingle copy fragment polymorphic in TaqI, and pKPl.65, tween D4S115 and D4S90, and one between 048125 a 1.65 kb PstI fragment that detects a multiallelic poly- and D4S95.Due to the relevance of some of these recommorphism with BglI. Probe 252.3 is a PstI-Sau3A frag- binations to HD gene mapping, they are presently being ment from a cosmid that overlaps the NotI-linking clone tested with other probes (D4S98 and D4S96), and 252 a t the D4S115 locus [MacDonald et al., 19891 poly- the results will be reported separately (manuscript in morphic for many enzymes. Probe p157.9 is a 450 bp preparation). Homogeneity testing indicates no significant heteroPstI-Sau3A fragment subcloned into pGEM3 from a cosmid that overlaps the NotI-linking clone 151 defining geneity among the families tested for loci 04510, D4S111 locus [MacDonald et al., 19891. For the D4S90 D4S95lS127,and D4S43 (Table 11). Values for D4Sllll locus [Youngman et al., 19891, two single copy probes S115 and for D4S90 are also included. were used: D5 is a 1.5 kb EcoRI insert polymorphic for PVUII and 2R3 is a 1.3 kb XbaI fragment polymorphic for HindIII. cases. Since the results of presymptomatic testing were still on an experimental basis, no result of testing was given to the patients.
Linkage Analysis Two point linkage analysis was carried out using the LINKAGE 5.03 program [Lathrop and Lalouel, 19841; the homogeneity test was carried out with the HOMOG program [Ott, 19851.
RESULTS Twelve HD families from different regions of Italy were examined, consisting of 115 individuals. Four families were not included in the lod score calculations because of family structure; one was excluded because of incompatibility in paternity testing of the affected individual. Of the tested subjects, 20 were affected, 55 were unaffected relatives, and 40 were a t risk of developing HD. With DNA testing, 14 resulted positive, 19 negative, and 7 were uninformative due to family structure or because of recombination events occurring in the family and not allowing the localization of the disease gene. A total of 115 meioses (108 informative) were counted.
TABLE I. Heterozygosity of HD Markers in Italy* Heterozygosity Locus D4S90 D4S90 04943 D4S95 D4S95 049127 D4SlO 04510 D4S43 D4S95 D4S115 D4Slll 049125 D4S95
Probe 2R3 D5 S1.5 674 674 BJ56 KO82 KO83 KPI.65 674 252.3 157.9 YNZ32 674
Polymorphism HindIII PVUII TaqI TaqI AccI PVUII HindIII EcoRI BglI HindIII PstI PstI TaqI AccI
(%I RFLP 135) RFLP (28) RFLP (18) RFLP (33) RFLP (22) RFLP (30) RFLP (28) RFLP (27) Multiallelic (33) Multiallelic (30) VNTR (73) VNTR (59) VNTR (57) VNTR (59)
*The heterozygosity of each probe was measured in unrelated individuals a s percentage of heterozygous meioses.
Genetic Analysis of HD in Italy
213
TABLE 11. Lod Scores for Linkage of HD Polvmoruhic Sites of D4* Locus 8
0.005
0.01
0.02
0.04
0.06
0.08
0.10
0.20
D4S10 1.353 1.347 1.338 1.304 1.270 1.224 1.181 0.879 (n = 11) D4S95/S127 2.149 2.117 2.052 1.923 1.796 1.671 1.547 0.964 ( n = 11) D4S43 1.480 1.467 1.442 1.381 1.334 1.276 1.218 0.897 (n = 7) D4SllllS115 0.029 0.320 0.598 0.843 0.957 1.012 1.031 0.885 (n = 9) D4S90 -1.676 -1.091 -1.037 -0.700 -0.503 -0.367 -0.264 -0.008 (n = 5)
0.30 0.491 0.478 0.561 0.575 0.058
*n =number ofinformative pedigrees combinedper locus: probes pK082 and pK082 (HindIII and EcoRI) for B4S10; BS 674 (TaqI, HindIII, and AccI) and BJ 56 (PvuII) for loci D4S9.5 and B4S127, respectively; probe 51.5 and pKP1.65 (TaqI and BglI) forD4S43; probe p157.9 and 252.3 (PstI)for D 4 S l l l and D4S115; probe D5 and 2R3 (PvuII and HindIII) for D4S90. Lod scores for closelv linked loci were mouDed a s indicated. The homogeneity test (HOMOG program) indicated the following*values: for D4SYO: 14L=3.115, a = 1.0 at 0 = 0.005; xz = 6.231, P = 0.006; for D4S956127: 1nL = 4.948, a = 1.0 at 0 = 0.005; x’ = 9.897, P = 0,0008; for D4S43: lnL=3.408, a=1.0 a t 0=0.005; x2=6.816, P=O.O045; for D4SllliS115: lnL=2.415, a=0.55 at 0=0.005; ~‘4.748,P=O.O15 (lnL=2.398 at a=0.7 and 0=0.05); for D4S90: lnL=0.133 at a = 1 . 0 and 0 = 0.005, xz = 0.267, P = 0.303) (see Discussion).
DISCUSSION The use of probes linked to the HD gene in the presymptomatic diagnosis requires a preliminary survey to verify that this approach is applicable in the population being tested. Although the sample is limited, our results confirm that polymorphic probes of loci D4910, D4S95, 049127, and 0 4 9 4 3 can be used in presymptomatic testing of Italian families. Our results on the informativeness of different probes suggest also the order in which the analysis for presymptomatic diagnosis should be performed, starting with the closest and most informative probes; however, since the recombinational events have a relatively high frequency, it is appropriate to test systematically several loci even if informativeness is reached with a single probe. The use of multiple probes a t different loci allows, in most cases, detection of the risk aplotype. The small sample studied does not permit us yet to attribute a significance to the relatively lower values of linkage between HD and loci previously described as tightly linked: In particular, D4S11 llD4S115 and D4990 show higher genetic distance than D4S10, D4S95, D4S127, or 0 4 9 4 3 . This is likely due to the fact that one recombinant is found between D4S90 and HD, and the number of informative families for this locus is only 5. The homogeneity test confirmed that no significant genetic heterogeneity is present in our population. It is known that significance for heterogeneity test requires a considerably higher number of families [CavalliSforza and King, 1986; Ott 19851. With these limitations, our results further support the suggestion that the same gene is responsible for HD in different populations [Conneally et al., 19891. ACKNOWLEDGMENTS We thank Drs. J. Gusella, M. Hayden, P. Harper, J. Wasmuth, and Y. Nakamura for providing us with the probes and Drs. M. Lathrop and J. Ott for the LINKAGE
program. This work was supported by grants from Regione Liguria, MPI and CNR (International Projects).
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