Analysis of triplet repeats in the huntingtin gene in Japanese families affected with Huntington''s disease.

Huntington's disease (HD) is associated with the expansion of a CAG repeat in the huntingtin gene. Molecular analysis of the repeat in Japanese HD patients and normal controls was performed. The size of the CAG repeat ranged from 37 to 95 repeats in affected subjects and from seven to 29 in normal controls. A significant correlation was found between the age of onset and the CAG expansion. The length of the expanded repeat is unstable in meiotic transmission and large increases occur in paternal transmission. At the same time the CCG repeat polymorphism adjacent to the CAG repeat was analysed and haplotypes of HD chromosomes were identified. Strong linkage disequilibrium was found between the CAG repeat expansion and an allele of (CCG)10 in Japanese HD chromosomes. It is distinct from that described previously in western populations. Western HD chromosomes strongly associate with an allele of (CCG)7. Possible mechanisms underlying the disequilibrium in Japan are discussed.     

Sequence analysis of the CCG polymorphic region adjacent to the CAG triplet repeat of the HD gene in normal and HD chromosomes.

The CAG expansion responsible for Huntington's disease (HD) is followed by an adjacent polymorphic CCG repeat region which may interfere with a PCR based diagnosis. We have sequenced this region in 52 unrelated HD patients, from both normal and HD chromosomes. Fifty percent of the normal alleles were (CCG)7(CCT)2, 48% (CCG)10(CCT)2, and 2% (CCG)7(CCT)3. In contrast (CCG)7(CCT)2 was found in 85% of the HD alleles which represents significant linkage disequilibrium with the HD mutation.     

DNA haplotype analysis of Huntington disease reveals clues to the origins and mechanisms of CAG expansion and reasons for geographic variations of prevalence

This study of allelic association using three Intra- and twoextragenlc markers within 150 kb of the Huntlngton disease (HD)mutation has provided evidence for linkage disequilibrium forfour of five markers. Haplotype analysis of 67 HD families usingmarkers in strong linkage disequilibrium with HD Identifiedtwo haplotypes underlying 77.6% of HD chromosomes. Normal chromosomeswith these two haplotypes had a mean number of CAG repeats significantlylarger than and an altered distribution of CAG repeats comparedwith other normal chromosomes. Furthermore, haplotype analysisof five new mutation families reveals that HD has arisen onthese same two chromosomal haplotypes. These findings suggestthat HD arises more frequently on chromosomes with specificDNA haplotypes and higher CAG repeat lengths. We then studiedCAG and CCG repeat lengths In the HD gene on 896 control chromosomesfrom different ancestries to determine whether the markedlyreduced frequency of HD in Finland, Japan, China and AfricanBlacks Is associated with an altered frequency of DNA haplotypesand subsequently lower CAG lengths on control chromosomes comparedto populations of Western European descent. The results showa highly significant positive correlation between CAG size onnormal chromosomes and the frequency of HD and a significantinverse relationship between CAG and CCG repeat lengths. Inpopulations with lowered prevalence rates of HD, CAG repeatlengths are smaller and the distribution of CCG alleles Is markedlydifferent from Western European populations. These findingssuggest that, in addition to European emigration, new mutationsmake a contribution to geographical variation of prevalencerates and is consistent with a multistep model of HD developingfrom normal chromosomes with higher CAG repeat lengths.     

Analysis of CAG and CCG repeats in Huntingtin gene among HD patients and normal populations of India

We have analysed the distribution of CAG and adjacent polymorphic CCG repeats in the Huntingtin gene in 28 clinically diagnosed unrelated Huntington's disease (HD) patients and in normal individuals belonging to different ethnic groups of India. The range of expanded CAG repeats in HD patients varied from 41 to 56 repeats, whereas in normal individuals this number varied between 11 and 31 repeats. We identified six CCG alleles from a total of 380 normal chromosomes that were pooled across different ethnic populations of India. There were two predominant alleles: (CCG)7 (72.6%) and (CCG)10 (20%). We report here for the first time one four-repeat CCG allele which has not been found in any population so far. We found 30 haplotypes (two loci CAG-CCG) for 380 normal chromosomes. In the present study, no statistically significant preponderance of expanded HD alleles was found on either (CCG)7 or (CCG)10 backgrounds. Our studies suggest that the overall prevalence of HD in Indian populations may not be as high as in Western populations. Further studies are necessary to identify the origin of HD mutation in these populations.     

The CAG repeat at the Huntington disease gene in the Portuguese population: insights into its dynamics and to the origin of the mutation

Huntington disease (HD) is caused by an expansion of a CAG repeat. This repeat is a dynamic mutation that tends to undergo intergenerational instability. We report the analysis of the CAG repeat in a large population sample (2,000 chromosomes) covering all regions of Portugal, and a haplotype study of (CAG)_n and (CCG)_n repeats in 140 HD Portuguese families. Intermediate class 2 alleles represented 3.0% of the population; and two expanded alleles (36 and 40 repeats, 0.11%) were found. There was no evidence for geographical clustering of the intermediate or expanded alleles. The Portuguese families showed three different HD founder haplotypes associated with 7-, 9- or 10-CCG repeats, suggesting the possibility of different origins for the HD mutation among this population. The haplotype carrying the 7-CCG repeat was the most frequent, both in normal and in expanded alleles. In general, we propose that three mechanisms, occurring at different times, may lead to the evolution from normal CAGs to full expansion: first, a mutation bias towards larger alleles; then, a stepwise process that could explain the CAG distributions observed in the more recent haplotypes; and, finally, a pool of intermediate (class 2) alleles more prone to give rise to expanded HD alleles.     

Dynamic mutation in Dutch Huntington''s disease patients: increased paternal repeat instability extending to within the normal size range.

Analysis of the distribution of normal and expanded alleles of the polymorphic (CAG)n repeat in the IT15 gene in the Dutch population confirmed the presence of an expanded repeat on all Huntington's disease (HD) chromosomes. Our results show that the size distributions of normal and affected alleles overlap. Normal alleles range from 11 to 37 repeats and HD alleles contain 37 to 84 repeats. A clear correlation is found between age at onset and repeat length, but the spread of the age at onset in the major repeat range producing characteristic HD is too wide to be of diagnostic value. In the available parent-offspring pairs, maternal HD alleles show a moderate instability with a slight preponderance of size increase over size decrease. Paternal alleles have a bimodal distribution: the majority (69%) behave similarly to the maternal alleles, while the remainder (31%) show a dramatic expansion, the degree of which appears proportional to the initial size. This is shown in three out of four juvenile patients, who have repeats of 71, 74, and 84 copies, respectively, originating from expanded paternal HD alleles in the previous generation. Two sporadic cases are caused by expansion of 'large' normal paternal alleles of 32 and 34 repeats, respectively, to 46 copies. This not only confirms the diagnosis of HD in two de novo cases, but it also underlines the increased paternal instability. In addition paternal repeat instability was once detected within the normal range in two sibs who inherited 21 and 22 repeats, respectively, on the same paternal chromosome. In two Dutch HD families the segregation of the expanded (CAG)n repeat was found. Analysis of the (CAG)n repeat in our previously reported recombinants confirmed their disease status.     

Genetic background of Huntington disease in Croatia: Molecular analysis of CAG,CCG, and Δ2642 (E2642del) polymorphisms

This study presents the first molecular data on the basis and the origin of Huntington disease in Croatia and is the first such analysis performed among a Slavic population. We analyzed three trinucleotide polymorphisms in the HD gene: CAG, CCG and GAG Δ2642 (E2642del) triplets. Analysis of the CAG repeat size among 44 Huntington patients (39‐66 CAGs) and 51 normal individuals (9‐34 CAGs) showed that the range of the repeats was similar to previous findings. The frequency of the CCG and Δ2642 polymorphic alleles on N and HD chromosomes was found to correlate well with earlier reports for Western European populations. We found significance for both the CCG7 allele (p=0.004) and the Δ2642 allele (p<0.001) among HD chromosomes. The CCG7 allele was overpresented among affected chromosomes (94.6%), but was also the most frequent CCG allele among normal chromosomes (66.7%). Interestingly, the Δ2642 allele was present on 40.5% HD chromosomes compared to only 9.8% of control chromosomes. Our results indicate that HD mutations in Croatia could be of the same origin as in Western populations and also support the multi‐step hypothesis for generating new HD alleles. Similar frequencies and distributions of both the CCG and the Δ2642 polymorphisms in Croatia and Western European normal chromosomes indicate that the prevalence rate of HD in Croatia may be as high as in Western populations. Since we estimated a lower prevalence rate (1 : 100,000), we assume that there are still many misdiagnosed and/or unrecognized cases of Huntington disease in Croatia. © 2002 Wiley‐Liss, Inc.     

Genetic background of Huntington disease in Croatia: Molecular analysis of CAG,CCG, and Delta2642 (E2642del) polymorphisms

This study presents the first molecular data on the basis and the origin of Huntington disease in Croatia and is the first such analysis performed among a Slavic population. We analyzed three trinucleotide polymorphisms in the HD gene: CAG, CCG and GAG Delta2642 (E2642del) triplets. Analysis of the CAG repeat size among 44 Huntington patients (39-66 CAGs) and 51 normal individuals (9-34 CAGs) showed that the range of the repeats was similar to previous findings. The frequency of the CCG and Delta2642 polymorphic alleles on N and HD chromosomes was found to correlate well with earlier reports for Western European populations. We found significance for both the CCG7 allele (p=0.004) and the Delta2642 allele (p<0.001) among HD chromosomes. The CCG7 allele was overpresented among affected chromosomes (94.6%), but was also the most frequent CCG allele among normal chromosomes (66.7%). Interestingly, the Delta2642 allele was present on 40.5% HD chromosomes compared to only 9.8% of control chromosomes. Our results indicate that HD mutations in Croatia could be of the same origin as in Western populations and also support the multi-step hypothesis for generating new HD alleles. Similar frequencies and distributions of both the CCG and the Delta2642 polymorphisms in Croatia and Western European normal chromosomes indicate that the prevalence rate of HD in Croatia may be as high as in Western populations. Since we estimated a lower prevalence rate (1 : 100,000), we assume that there are still many misdiagnosed and/or unrecognized cases of Huntington disease in Croatia.     

Study of the Huntington''s disease (HD) gene CAG repeats in schizophrenic patients shows overlap of the normal and HD affected ranges but absence of correlation with schizophrenia.

The CAG repeats in the Huntington's disease gene were investigated in chromosomes from 71 unrelated schizophrenic persons and 18 patients with schizoaffective disorder in order to determine if any of these patients had abnormal expansions. All of the probands had repeat sizes in the normal range (< 35 repeats) and there was no significant difference between the allele distributions of these patients and the normal controls. The families of two patients with 32 repeats and one patient with 34 repeats were investigated further and showed no uniform segregation of the disease with the large repeat alleles. The proband with 34 repeats inherited a chromosome that originally had 36 repeats in her father. The presence of 36 CAG repeats in members of her family and in HD patients suggests that there is an overlap between the normal and Huntington's disease CAG repeat size ranges. The more recently described CCG polymorphism in this gene was also examined in the schizophrenic and schizoaffective persons. All patients had alleles in the normal range.     

Interaction of normal and expanded CAG repeat sizes influences age at onset of Huntington disease

Huntington disease (HD) is a neurodegenerative disorder caused by the abnormal expansion of CAG repeats in the HD gene on chromosome 4p16.3. Past studies have shown that the size of expanded CAG repeat is inversely associated with age at onset (AO) of HD. It is not known whether the normal Huntington allele size influences the relation between the expanded repeat and AO of HD. Data collected from two independent cohorts were used to test the hypothesis that the unexpanded CAG repeat interacts with the expanded CAG repeat to influence AO of HD. In the New England Huntington Disease Center Without Walls (NEHD) cohort of 221 HD affected persons and in the HD-MAPS cohort of 533 HD affected persons, we found evidence supporting an interaction between the expanded and unexpanded CAG repeat sizes which influences AO of HD (P = 0.08 and 0.07, respectively). The association was statistically significant when both cohorts were combined (P = 0.012). The estimated heritability of the AO residual was 0.56 after adjustment for normal and expanded repeats and their interaction. An analysis of tertiles of repeats sizes revealed that the effect of the normal allele is seen among persons with large HD repeat sizes (47-83). These findings suggest that an increase in the size of the normal repeat may mitigate the expression of the disease among HD affected persons with large expanded CAG repeats.     

A study of the Huntington''s disease associated trinucleotide repeat in the Scottish population.

Accurate measurements of a specific CAG repeat sequence in the Huntington's disease (HD) gene in 337 HD patients and 229 normal controls from the Scottish population showed a range from 35 to 62 repeats in affected subjects and eight to 33 in normal subjects. A link between early onset of symptoms and very high repeat number was seen. For HD patients with the most common affected allele sizes (39 to 42 repeats) absolute repeat size was a poor index for the age at onset of symptoms. There was variability in the transmitted repeat size for both sexes in the HD size range. We observed a significant increase of repeat size for paternal transmission of the disease and greater instability for paternally transmitted CAG repeats in the HD size range.     

Analysis of the trinucleotide repeat expansion in Italian families affected with Huntington disease

150 subjects affected with HD and 45 at high risk for the diseasewere typed for the CAG trinucleotide repeat at the 5‘end of IT15. In all of them we find expanded segments showingmarked instability upon transmission. Their length distributionmatches those previously reported and inversely correlates (–0.686)with age at onset. Two at risk sibs from a large HD pedigreeshow expanded segments that overlap the normal distributionand can represent reductions from the HD to the normal range.A case of instability on a normal chromosome is also reported.Finally, an analysis of the CAG repeat as a function of threepolymorphic DNA markers at D4S127 and D4S95 loci shows no significantdifference in the average repeat length on HD chromosomes groupedaccording to the cosegregating allele of each marker or to thecorresponding haplotype. Despite the marked heterogeneity inrepeat length among HD families, haplotypes are not associatedwith different values around which the repeat length fluctuates.     

DNA analysis of Huntington''s disease in southern Chinese.

Allelic frequencies of RFLPs at loci closely linked to the HD gene, D4S95, D4S91, D4S141, and D4S90, were determined in 13 Huntington's disease (HD) patients from nine Chinese families and 129 normal subjects. These were similar for non-HD and HD chromosomes and the HD gene in Chinese is associated with multiple haplotypes. Hence the HD gene probably arose independently in the background haplotypes of the Chinese population. The heterozygosity rates for the two most useful RFLP sites are 0.659 for D4S95-AccI VNTR and 0.494 for D4S141-HindIII. (CAG)n repeat numbers ranged from 12 to 27 in 174 normal chromosomes. In 52 meiotic recombinations, the (CAG)n repeats were stably inherited in normal families. In HD families, 12 of 13 HD patients had expanded (CAG)n repeats of 40 to 58. Additionally, 10 asymptomatic family members had expanded (CAG)n repeats and the inheritance of the expanded repeat was unstable in these families.     

A novel PCR-based approach for the detection of the Huntington disease associated trinucleotide repeat expansion

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder associated with expansions of an unstable CAG trinucleotide repeat in exon 1 of the IT15 gene. In normal individuals, IT15 contains up to 35 CAG repeats, while in affected the repeat length is >36. Polymerase chain reaction (PCR) is used to estimate the number of CAG repeats but may be inefficient in long repeats because of the high C+G content of the HD locus. We present a novel PCR approach for the diagnosis of HD, which permits direct visualization of the amplified products on agarose gel, using ethidium bromide. It is based on the methylation-sensitive conversion of C residues to U by bisulfite treatment of single-stranded DNA and subsequent amplification of the sense strand with specific primers. The bisulfite treatment dramatically reduces the C + G content of the region; thus, the high Tm and stable secondary structures are no longer obstacles to PCR. In both normal and affected individuals, UAG repeats (5'- CAG-3', before bisulfite treatment) in the sense strand can easily be amplified and visualized on a gel by ethidium bromide staining. The method has considerable advantages compared with other described PCR-based diagnostic tests for HD.     

Ancient origin of the CAG expansion causing Huntington disease in a Spanish population

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder characterized clinically by progressive motor impairment, cognitive decline, and emotional deterioration. The disease is caused by the abnormal expansion of a CAG trinucleotide repeat in the first exon of the huntingtin gene in chromosome 4p16.3. HD is spread worldwide and it is generally accepted that few mutational events account for the origin of the pathogenic CAG expansion in most populations. We have investigated the genetic history of HD mutation in 83 family probands from the Land of Valencia, in Eastern Spain. An analysis of the HD/CCG repeat in informative families suggested that at least two main chromosomes were associated in the Valencian population, one associated with allele 7 (77 mutant chromosomes) and one associated with allele 10 (two mutant chromosomes). Haplotype A-7-A (H1) was observed in 47 out of 48 phase-known mutant chromosomes, obtained by segregation analysis, through the haplotype analysis of rs1313770-HD/CCG-rs82334, as it also was in 120 out of 166 chromosomes constructed by means of the PHASE program. The genetic history and geographical distribution of the main haplotype H1 were both studied by constructing extended haplotypes with flanking short tandem repeats (STRs) D4S106 and D4S3034. We found that we were able to determine the age of the CAG expansion associated with the haplotype H1 as being between 4,700 and 10,000 years ago. Furthermore, we observed a nonhomogenous distribution in the different regions associated with the different extended haplotypes of the ancestral haplotype H1, suggesting that local founder effects have occurred.     

Mitotic stability and meiotic variability of the (CAG)n repeat in the Huntington disease gene

The gene causing Huntingtons's disease, an autosomal dominantlyInherited, neurodegenerative disorder, has been Identified recently.The corresponding mutation Is Involving an expansion In thenumber of (CAG)_n repeats In the coding region of the Huntington'sdisease gene on chromosome 4. In this report, we demonstratethe length variation of the repeat In 513 non-HD chromosomesfrom normal Individuals and HD patlents showing 23 alleles with11 to 33 repeats. Analyzing the Inherltance of the (CAG)_n stretchwe found melotic instability for HD alleles ([CAG]₄₀ to [CAG]₇₅)with a mutation frequency of approximately 0.7, while In 431meloses of normal alleles only two expanslons were Identified.The risk of expansion during spermatogenesis is enhanced comparedto oogenesls explaining juvenile onset by transmission fromaffected fathers. Further, the number of (CAG)_n copies In anaffected individual In relation to the sex of the transmittingparent was evaluated and no significant differences were found.No mosalcism or differences In the repeat lengths were observedIn the DNA from different tissues Including brain and lymphocytesof two HD patients indicating mltotic stability of the mutation.Therefore, the determination of the repeat number In the DNAof blood lymphocytes Is probably representative of all tissuesIn a patient.     

A single allele from the polymorphic CCG rich sequence immediately 3' to the unstable CAG trinucleotide in the IT15 cDNA shows almost complete disequilibrium with Huntington's disease chromosomes in the Scottish population

The CCG rich sequence immediately 3' to the CAG repeat thatis expanded in Huntington's disease (HD) has recently been shownto be polymorphic with at least 4 alleles differing by multiplesof 3 bp being found in the normal population. We have studiedthe allele distribution in 180 HD families resident in Scotlandand have found very strong evldence for disequilibrium in thispopulation. For the 131 families where phase was unambiguouslydetermined, 130 were shown to have a CCG repeat allele of 176bp cosegregating with the HD chromosome. This observation issignificantly different to the normal population distributionwhere 31% of people have an allele of 185 bp. The evidence forand against a possible founder effect in the Scottish HD populationis discussed. We propose the hypothesis that we may have identifieda region of the IT15 gene that is critical in Huntington's disease.