Huntington disease in Finland: a molecular and genealogical study

Summary Huntington disease (HD) is found at exceptionally low frequency in the Finnish population. In this population, linkage disequilibrium was earlier established with markers from the D4S10 and D4S43 loci. We now report a continuation to the restriction fragment length polymorphism haplotype analysis, in combination with a genealogical study of all the Finnish HD families. When the HD pedigrees were systematically traced to the 18th century, only one consanguinity was found, and a high percentage (28%) of the families had foreign ancestors. The majority of the Finnish ancestors were localized to border regions or trade centers of the country following the old postal routes. The observed high risk haplotypes formed with markers from the D4S10 and D4S43 loci were evenly distributed among the HD families in different geographical locations. Consequently, the HD gene(s) has most probably arrived in Finland on several occasions via foreign immigrants during the last few centuries.     

Complex patterns of linkage disequilibrium in the Huntington disease region.

The genetic defect causing Huntington disease (HD) has been mapped to 4p16.3 by linkage analysis using DNA markers. Two apparently contradictory classes of recombination events in HD kindreds preclude precise targeting of efforts to clone the disease gene. Here, we report a new recombination event that increases support for an internal candidate region of 2.5 Mb between D4S10 and D4S168. Analysis of 23 DNA polymorphisms in 4p16.3 revealed a complex pattern of association with the disease gene that failed to narrow the size of the candidate region. The degree of linkage disequilibrium did not show a continuous increase across the physical map, nor was a region of extreme disequilibrium identified. Markers displaying no association with the disorder were interspersed with and, in many cases, close to markers displaying significant disequilibrium. Comparison of closely spaced marker pairs on normal and HD chromosomes, as well as analysis of haplotypes across the HD region, suggest that simple recombination subsequent to a single original HD mutation cannot easily explain the pool of HD chromosomes seen today. A number of different mechanisms could contribute to the diversity of haplotypes observed on HD chromosomes, but it is likely that there has been more than one and possibly several independent origins of the HD mutation.     

Recombination of 4p16 DNA markers in an unusual family with Huntington disease

The Huntington disease (HD) mutation has been localized to human chromosome 4p16, in a 6-Mb region between the D4S10 locus and the 4p telomere. In a report by Robbins et al., a family was identified in which an affected individual failed to inherit three alleles within the 6-Mb region originating from the parental HD chromosome. To explain these results, it was suggested that the HD locus (HD) lies close to the telomere and that a recombination event took place between HD and the most telomeric marker examined, D4S90. As a test of this telomere hypothesis, we examined six members of this family, five of whom are affected with HD, for the segregation of 12 polymorphic markers from 4p16, including D4S169, which lies within 80 kb of the 4p telomere. We separated, in somatic cell hybrids, the chromosomes 4 from each family member, to determine the phase of marker alleles on each chromosome. We excluded nonpaternity by performing DNA fingerprint analyses on all six family members, and we found no evidence for chromosomal rearrangements when we used high-resolution karyotype analysis. We found that two affected siblings, including one of the patients originally described by Robbins et al., inherited alleles from the non-HD chromosome 4 of their affected parents, throughout the 6-Mb region. We found that a third affected sibling, also studied by Robbins et al., inherited alleles from the HD chromosome 4 of the affected parent, throughout the 6-Mb region. Finally, we found that a fourth sibling, who is likely affected with HD, has both a recombination event within the 6-Mb region and an additional recombination event in a more centromeric region of the short arm of chromosome 4. Our results argue against a telomeric location for HD and suggest that the HD mutation in this family is either associated with DNA predisposed to double recombination and/or gene conversion within the 6-Mb region or is in a gene that is outside this region and that is different from that mutated in most other families with HD.     

Batten disease gene, CLN3: linkage disequilibrium mapping in the Finnish population, and analysis of European haplotypes.

The gene for Batten disease (juvenile-onset neuronal ceroid lipofuscinosis, or Spielmeyer-Sjögren disease), CLN3, maps to 16p11.2-12.1. Four microsatellite markers--D16S288, D16S299, D16S298, and SPN--are in strong linkage disequilibrium with CLN3 in 142 families from 16 different countries. These markers span a candidate region of approximately 2.1 cM. CLN3 is most prevalent in northern European populations and is especially enriched in the isolated Finnish population, with an incidence of 1:21,000. Linkage disequilibrium mapping was applied to further refine the localization of CLN3 in 27 Finnish families by using linkage disequilibrium data and information about the population history of Finland to estimate the distance of the closest markers from CLN3. CLN3 is predicted to lie 8.8 kb (range 6.3-13.8 kb) from D16S298 and 165.4 kb (132.4-218.1 kb) from D16S299. Enrichment of allele "6" at D16S298 (on 96% of Finnish and 92% of European CLN3 chromosomes) provides strong evidence that the same major mutation is responsible for Batten disease in Finland as in most other European countries and that it is therefore not a Finnish mutation. Genealogical studies show that Batten disease is widespread throughout the densely populated regions of Finland. The ancestors of two Finnish patients carrying rare alleles "3" and "5" at D16S298 in heterozygous form originate from the southwestern coast of Finland, and these probably represent other foreign mutations. Analysis of the number and distribution of CLN3 haplotypes from 12 European countries provides evidence that more than one mutation has arisen in Europe.     

Epidemiological and linkage studies on Huntington's disease in Italy

Summary The results of an epidemiological survey on Huntington's disease in the Lazio Region, Central Italy, and of linkage studies in a subset of families are reported. From a total of 99 ascertained families and 491 patients, a prevalence of 25.6 × 10–6 was obtained, with distributions of age at onset and age at death similar to those described in the literature. No relationship with the sex of the transmitting parent was observed. Analysis of 10 chromosome 4 restriction fragment length polymorphisms in 11 families showed consistent linkage between the genetic loci D4S10, D4S43 and D4S95, and the disease. A recombination rate of 0.08 for D4S10 markers was obtained in this sample. Allelic frequencies of DNA markers in the general population are also reported.     

Cystic fibrosis in Finland: a molecular and genealogical study

Summary The incidence of cystic fibrosis (CF) in Finland is one tenth that in other Caucasian populations. To study the genetics of CF in Finland, we used a combined molecular and genealogical approach. Out of the 20 Finnish families with a living CF patient, 19 were typed for eight closely linked restriction fragment length polymorphisms (RFLP) at the MET, D7S8, and D7S23 loci. The birthplaces of the parents and grandparents were traced using population registries. Allele and haplotype frequencies in Finland are similar to those of other European and North American populations, but are modified by sampling: two regional CF gene clusters, evidently the results of a founder effect, were identified. Generally, the gene was evenly distributed over the population, carrier frequency being estimated at approximately 1.3%. We conclude that CF in Finland is caused by the common Caucasian mutation(s), and that the low frequency of the gene can be explained by a negative sampling effect and genetic drift.     

Synteny on mouse chromosome 5 of homologs for human DNA loci linked to the Huntington disease gene

Comparative mapping in man and mouse has revealed frequent conservation of chromosomal segments, offering a potential approach to human disease genes via their murine homologs. Using DNA markers near the Huntington disease gene on the short arm of chromosome 4, we defined a conserved linkage group on mouse chromosome 5. Linkage analyses using recombinant inbred strains, a standard outcross, and an interspecific backcross were used to assign homologs for five human loci, D4S43, D4S62, QDPR, D4S76, and D4S80, to chromosome 5 and to determine their relationships with previously mapped markers for this autosome. The relative order of the conserved loci was preserved in a linkage group that spanned 13% recombination in the interspecific backcross analysis. The most proximal of the conserved markers on the mouse map, D4S43h, showed no recombination with Emv-1, an endogenous ecotropic virus, in 84 outcross progeny and 19 recombinant inbred strains. Hx, a dominant mutation that causes deformities in limb development, maps approximately 2 cM proximal to Emv-1. Since the human D4S43 locus is less than 1 cM proximal to HD near the telomere of chromosome 4, the murine counterpart of the HD gene might lie between Hx and Emv-1 or D4S43h. Cloning of the region between these markers could generate new probes for conserved human sequences in the vicinity of the HD gene or possibly candidates for the murine counterpart of this human disease locus.     

Linkage disequilibrium analysis in Machado-Joseph disease patients of different ethnic origins

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration originally described in families of Portuguese-Azorean ancestry. The hypothesis that its present world distribution could result from the spread of an original founder mutation has been raised. To test this possibility we have conducted a linkage disequilibrium study of markers segregating with the MJD1 locus in a total of 64 unrelated families of different geographical origins. Significant association was detected between the MJD1 locus and marker alleles at loci D14S280, D14S1050 and D14S81. All affected individuals, except one Chinese family, had allele 3 (237 bp) at D14S280. This finding is consistent with a founder effect in our MJD population. However, distinct haplotypes were observed in patients originating from the two Azorean islands showing the highest disease prevalence; therefore, the possible existence of more than one founder mutation can not be excluded with the markers currently available. Received: 27 February 1996 / Revised: 4 June 1996     

Studies of cystic fibrosis in Hutterite families by using linked DNA probes.

Several multigenerational S-leut Hutterite families with cystic fibrosis (CF) were ascertained. Linkage studies with DNA marker loci MET and pJ3.11 (D7S8) were performed to determine whether (1) the CF gene in this inbred population is linked to DNA markers on chromosome 7, as it is in outbred populations of European origins, and (2) ancestral origin(s) of the CF gene could be determined. Our results indicate that the CF gene in Hutterite families segregates with chromosome 7 markers, identified by probes metH and pJ3.11. Thus, the CF mutation in Hutterites is likely to be either at the same locus as or at one closely linked to that reported in outbred populations. Heterozygous carriers could be distinguished from normal homozygous sibs of affected individuals. In the families studied, three different chromosome 7 haplotypes carried the CF mutation, raising the possibility that the CF gene may have been introduced into this population by as many as three different ancestors.     

Significant linkage disequilibrium between the Huntington disease gene and the loci D4S10 and D4S95 in the Dutch population.

Significant linkage disequilibrium has been found between the Huntington disease (HD) gene and DNA markers located around D4S95 and D4S98. The linkage-disequilibrium studies favor the proximal location of the HD gene, in contrast to the conflicting results of recombination analyses. We have analyzed 45 Dutch HD families with 19 DNA markers and have calculated the strength of linkage disequilibrium. Highly significant linkage disequilibrium has been detected with D4S95, consistent with the studies in other populations. In contrast with most other studies, however, the area of linkage disequilibrium extends from D4S10 proximally to D4S95, covering 1,100 kb. These results confirm that the HD gene most likely maps near D4S95.     

Defined physical limits of the Huntington disease gene candidate region.

The Huntington disease (HD) gene has been mapped 4 cM distal to D4S10 within the telomeric chromosome band, 4p16.3. The published physical map of this region extends from D4S10 to the telomere but contains two gaps of unknown size. Recombination events have been used to position the HD mutation with respect to genetic markers within this region, and one such event places the gene proximal to D4S168, excluding the distal gap as a possible location for the defect. One previously published recombination event appeared to have excluded the proximal gap. We have reassessed this event and have moved the proximal boundary for the HD candidate region centromeric to the gap within a "hot spot" for recombination between D4S10 and D4S125. We have closed the proximal gap and report here the complete physical map spanning the HD candidate region from D4S10 to D4S168, the maximum size of which can now be placed accurately at 2.5 Mb.     

Ancestral origins of the Machado-Joseph disease mutation: a worldwide haplotype study

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder originally described in families of Portuguese-Azorean ancestry. The cloning of the MJD1 gene allowed identification of the disease in many other populations, and MJD is now known to be the most common cause of dominant spinocerebellar ataxia. The hypothesis that its present world distribution could result from the spread of an original founder mutation has been raised, both at historical and molecular levels. In the present study, we tested this hypothesis by linkage-disequilibrium analysis of tightly linked polymorphisms and by haplotype comparison, in 249 families from different countries. We typed five microsatellite markers surrounding the MJD1 locus (D14S1015, D14S995, D14S973, D14S1016, and D14S977), and three intragenic single-base-pair polymorphisms (A(669)TG/G(669)TG, C(987)GG/G(987)GG, and TAA(1118)/TAC(1118)). The results show two different haplotypes, specific to the island of origin, in families of Azorean extraction. In families from mainland Portugal, both Azorean haplotypes can be found. The majority of the non-Portuguese families also share the same intragenic haplotype seen in the families coming from the island of Flores, but at least three other haplotypes were seen. These findings suggest two introductions of the mutation into the Portuguese population. Worldwide, the sharing of one intragenic haplotype by the majority of the families studied implies a founder mutation in MJD.     

Localization of the Huntington's disease gene to a small segment of chromosome 4 flanked by D4S10 and the telomere

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder of late onset, characterized by progressive motor disturbance, psychological manifestations, and intellectual deterioration. The HD gene has been genetically mapped by linkage to the DNA marker D4S10, but the exact physical location of the HD defect has remained uncertain. To delineate critical recombination events revealing the physical position of the HD gene, we have identified restriction fragment length polymorphisms for two recently mapped chromosome 4 loci, RAF2 and D4S62, and determined the pattern of segregation of these markers in both reference and HD pedigrees. Multipoint linkage analysis of the new markers with D4S10 and HD establishes that the HD gene is located in a very small physical region at the tip of the chromosome, bordered by D4S10 and the telomere. A crossover within the D4S10 locus orients this segment on the chromosome, providing the necessary information for efficient application of directional cloning strategies for progressing toward, and eventually isolating, the HD gene.     

Evidence from family studies that the gene causing Huntington disease is telomeric to D4S95 and D4S90.

A DNA probe (D4S95) that detects a variable number of tandem repeats and a single-site-variation polymorphism after digestion with a single restriction enzyme, AccI, has previously been described. The order of this probe relative to the gene for Huntington disease (HD) and other previously described markers has not been established. Analysis of 24 affected families with HD has shown that D4S95 is in tight linkage with the gene causing HD, with a maximal Lod score of 12.489 at a theta of .03. D4S90 is a probe which maps to 4p16.3, telomeric to D4S95, and detects polymorphisms with HincII and other enzymes. In one affected person, recombination has occurred between D4S10 and HD, between D4S95 and HD, and in all likelihood also between D4S90 and HD, which strongly suggests that the gene for HD is telomeric to all these DNA probes. This suggests that the gene causing HD is located in the most distal region of the short arm of chromosome 4, flanked by D4S90 and the telomere, and supports the locus order D4S10-D4S95-D4S90-HD-telomere. D4S95 is a most useful DNA marker for predictive testing programs, while D4S90 will serve as a useful starting point for identifying DNA fragments closer to the gene for HD.     

DNA polymorphism of human HLA-linked complement C4 allotypes, including C4 null alleles, in the Finnish population

Human HLA-linked complement C4 gene products, C4A and C4B, show extensive genetic polymorphism. In both loci, an allele without a gene product, C4 null, is also observed. We have performed a restriction enzyme analysis of genomic DNA samples from individuals having all common (frequency over 1%) C4 protein allotypes observed in the Finnish population. Only one allotype-specific RFLP marker was observed. With some enzymes a DNA polymorphism was observed, which was not detectable by C4 protein typing. Analysis of 10 different C4B null haplotypes and 4 C4A null haplotypes suggested that only one haplotype, HLA-B8 C4A0 B1, carried a C4A gene deletion. This was observed in all 4 unrelated individuals homozygous for this haplotype.     

Nonrandom association between Huntington disease and two loci separated by about 3 Mb on 4p16.3.

The gene for Huntington disease (HD) has been localized close to the telomere on the short arm of chromosome 4. However, refined mapping using recombinant HD chromosomes has resulted in conflicting findings and mutually exclusive candidate regions. Previously reported significant nonrandom allelic association between D4S95 and HD provided support for a more proximal location for the defective gene. In this paper, we have analyzed 17 markers, spanning approximately 6 Mb of DNA distal to locus D4S62, for nonrandom association to HD. We confirm the previous findings of nonrandom allelic association between D4S95 and HD. In addition, we provide new data showing significant nonrandom association between HD and 3 markers at D4S133 and D4S228, which are approximately 3 Mb telomeric to D4S95.     

Haplotype studies in Wilson disease.

In 51 families with Wilson disease, we have studied DNA haplotypes of dinucleotide repeat polymorphisms (CA repeats) in the 13q14.3 region, to examine these markers for association with the Wilson disease gene (WND). In addition to a marker (D13S133) described elsewhere, we have developed three new highly polymorphic markers (D13S314, D13S315, and D13S316) close to the WND locus. We have examined the distribution of marker alleles at the loci studied and have found that D13S314, D13S133, and D13S316 each show nonrandom distribution on chromosomes carrying the WND mutation. We have studied haplotypes of these three markers and have found that there are highly significant differences between WND and normal haplotypes in northern European families. These findings have important implications for mutation detection and molecular diagnosis in families with Wilson disease.     

Genetic linkage between Huntington disease and the D4S10 locus in South African families: further evidence against non-allelic heterogeneity

Summary A study of genetic linkage between Huntington disease (HD) and the D4S10 locus (G8) has been undertaken in 10 South African (SA) families originating from the black, white and mixed acestry population groups. Allele frequencies at the D4S10 locus have been established in the non-Caucasoid population groups. There are significant differences in the allele frequencies at the D4S10 locus between the various SA populations. Clearly, information about population-specific frequencies for all polymorphisms is essential prior to the implementation of predictive testing in different population groups. Linkage has been demonstrated within this mixed group of HD families in SA using the HindIII, EcoRI and MspI polymorphisms, detected by G8. A maximum lod score of 8.14 at a recombination fraction of 0.00 (confidence limit 0–0.058) has been calculated using a combined haplotype of the HindIII and MspI polymorphisms. Taking into account the diverse ethnic backgrounds of the different SA population groups in this investigation, the data obtained from the study provide further evidence that there is probably only a single HD locus.     

Mapping of recombinants near the Huntington disease locus by using G8 (D4S10) and newly isolated markers in the D4S10 region.

Genetic linkage between the marker G8 (D4S10) and Huntington disease (HD) was studied in six Dutch pedigrees. The informativeness of the D4S10 locus was increased by isolation of a cosmid, C5.5, with a G8 subclone used as probe. We present a restriction map of 70 kb in the D4S10 region. Two subclones of C5.5, H5.52 and F5.53, detect MspI and SinI RFLPs, respectively. These probes increase the informativeness of D4S10 in the Dutch HD population from 55% to 95%. Seven recombinations were found in 124 informative meioses in which multipoint segregation of D4S10 haplotypes and the HD locus was studied. Two of the recombinations occurred within the D4S10 region. The other five recombinations are highly valuable for the mapping of present and future markers relative to each other and to the HD gene. In addition, several recombinations between markers in meioses from unaffected parents were noted, which will also be useful in ordering new markers. On the basis of our three-point recombination data, the orientation of the D4S10 region relative to HD is HD-H5.52-G8-F5.53, which independently confirms the previously derived polarity for D4S10.     

Estimating European admixture in African Americans by using microsatellites and a microsatellite haplotype (CD4/Alu)

We have analyzed 10 unlinked microsatellites and a linked Alu deletion polymorphism at the CD4 locus in an African American population sample from Chicago (USA). Heterozygosity estimates at the microsatellite loci range from 0.727±0.025 (D3S1358) to 0.873±0.017 (D18S51), with an average of 0.794±0.016. These values are comparable to or higher than those reported for Europeans, with only one exception (D3S1358). The CD4/Alu haplotypic diversity (0.887±0.012) is comparable to diversity levels observed in sub-Saharan African populations and is higher than the diversity levels reported in European populations. No consistent pattern of within, between, or multi-locus deviations from Hardy-Weinberg expectations is observed, suggesting a low sub-heterogeneity within the sampled population. We have applied a maximum likelihood method and estimated the proportion of European admixture to the African American gene pool to be 0.26±0.02. The narrow confidence interval indicates that allele frequency data from multiple microsatellite loci, whether analyzed independently or as haplotypes, are particularly useful for estimating genetic admixture. Received: 9 September 1998 / Accepted: 3 November 1998