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.     

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.     

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.     

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.     

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.     

A CCG repeat polymorphism adjacent to the CAG repeat in the Huntington disease gene: implications for diagnostic accuracy and predictive testing

The polymorphic CAG repeat that is expanded on Huntington disease(HD) chromosomes is flanked by a CCG repeat. Here we show thatthis CCG tract, previously assumed to be invariant at sevenCCG repeats, is also polymorphic. We have identified five CCGalleles from 205 normal chromosomes, with 137 (67%) having allelesof seven repeats, five (2%) with nine repeats, 61 (30%) with10 repeats, one (0.5%) with 11 repeats and one (0.5%) with 12repeats. In contrast, analysis of 113 HD chromosomes revealedthat the majority (105 chromosomes, 93%) contained seven CCGrepeats, while the remaining eight chromosomes (7%) had allelesizes of 10 CCG repeats. Despite evidence that both CAG andCCG are polymorphic on normal chromosomes, we have found thatit is only the CAG length that has a significant impact on ageof onset. The discovery of larger sized CCG alleles, however,has significant implications for the assessment of CAG repeatlength, particularly for persons with estimated CAG size of36–42 repeats, since an overestimation of CAG length inthis range could result in erroneous information being impartedto patients.     

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.     

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.     

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.     

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.     

Linkage disequilibrium between four intragenic polymorphic microsatellites of the NF1 gene and its implications for genetic counselling.

Four intragenic polymorphic microsatellite markers, AAAT Alu repeat, IVS27AC28.4, ACI27.2, and IVS38GT53.0, located along a 65 kb DNA region of the NF1 gene, were used to genotype 64 Spanish families with neurofibromatosis type 1 (NF1). Linkage disequilirium between each pair of markers was evaluated. Three of these markers, AAAT Alu repeat, ACI27.2, and IVS38GT53.0, exhibit linkage disequilibrium between each other. Analysis of extended haplotypes provides further evidence of the disequilibrium within this region since only 11 haplotypes account for 52% of the total chromosomes. Because of linkage disequilibrium, the informativeness of marker combinations for genotyping of NF1 families is diminished. There was no difference in the overall distribution of alleles between affected and normal chromosomes. An at risk haplotype was not found, as expected for a disease with at least 50% of cases being sporadic.     

Linkage disequilibrium at the SCA2 locus

Spinocerebellar ataxia type 2 (SCA2) is caused by the expansion of an unstable CAG repeat encoding a polyglutamine tract. Repeats with 32 to 200 CAGs are associated with the disease, whereas normal chromosomes contain 13 to 33 repeats. We tested 220 families of different geographical origins for the SCA2 mutation. Thirty three were positive (15%). Twenty three families with at least two affected subjects were tested for linkage disequilibium (LD) between the SCA2 mutation and three microsatellite markers, two of which (D12S1332-D12S1333) closely flanked the mutation; the other (D12S1672) was intragenic. Many different haplotypes were observed, indicating the occurrence of several ancestral mutations. However, the same haplotype, not observed in controls, was detected in the German, the Serbian, and some of the French families, suggesting a founder effect or recurrent mutations on an at risk haplotype.     

Instability of CAG repeats in Huntington''s disease: relation to parental transmission and age of onset.

Huntington's disease (HD) has recently been found to be caused by expansion of a trinucleotide (CAG) repeat within the putative coding region of a gene with an unknown function. We report here an analysis of HD mutation and the characteristics of its transmission in 36 HD families. CAG repeats on HD chromosomes were unstable when transmitted from parent to offspring. Instability appeared more frequent and stronger upon transmission from a male than from a female, with a clear tendency towards increased size. We have also found a significant inverse correlation (p = 0.0001) between the age of onset and the CAG repeat length. The observed scatter would, however, not allow an accurate individual prediction of age of onset. Three juvenile onset cases analysed had an HD mutation of paternal origin. In at least two of these cases a large expansion of the HD allele upon paternal transmission may explain the major anticipation observed. Our results suggest that several features of the expansion mutation in HD are similar to those previously observed for mutations of similar size in spinobulbar muscular atrophy and in myotonic dystrophy, and to those observed more recently in spinocerebellar ataxia type 1 and in dentatorubropallidoluysian atrophy, four diseases also caused by expansion of CAG repeats.     

Haplotype analysis of the 4p16.3 region in Portuguese families with Huntington's disease

Huntington's disease (HD) is a neurodegenerative disorder characterized by involuntary choreic movements, cognitive impairment, and behavioral changes, caused by the expansion of an unstable CAG repeat in HTT. We characterized the genetic diversity of the HD mutation by performing an extensive haplotype analysis of ～1Mb region flanking HTT in over 300 HD families of Portuguese origin. We observed that haplotype A, marked by HTT delta2642, was enriched in HD chromosomes and carried the two largest expansions reported in the Portuguese population. However, the most frequent HD haplotype B carried one of the largest (+12 CAGs) expansions, which resulted in an allele class change to full penetrance. Despite having a normal CAG distribution skewed to the higher end of the range, these two core haplotypes had similar expanded CAG repeat sizes compared to the other major core haplotypes (C and D) and there was no statistical difference in transmitted repeat instability across haplotypes. We observed a diversity of HTT region haplotypes in both normal and expanded chromosomes, representative of more than one ancestral chromosome underlying HD in Portugal, where multiple independent events on distinct chromosome 4 haplotypes have given rise to expansion into the pathogenic range. © 2015 Wiley Periodicals, Inc.     

Huntington disease mutation in Venezuela: age of onset,haplotype analyses and geographic aggregation

The aggregation of patients with Huntington's disease (HD) around Lake Maracaibo, Zulia State, Venezuela, is widely recognized, but the epidemiology of HD in the whole country is relatively unstudied. We have examined 279 individuals from 60 unrelated affected families residing in various areas of Venezuela for the presence of CAG repeats and other features associated with HD. The number of expanded repeats in 139 carriers varied from 35 to 112. Based on our examination of 71 symptomatic individuals, we developed a log-transformed regression equation, y = −0.0238x + 2.6616, to enable the prediction of age of onset in asymptomatic carriers. Intragenic haplotypes were constructed with two VNTRs (variable number of tandem repeats) and two SNPs (single nucleotide polymorphisms) in the promoter region as well as CCG repeat and Δ2642 polymorphisms to assess kinship between families. In 43 of 45 tested families, the haplotype on the mutated chromosome was 1;G;C;7;(A). The other haplotypes observed, 1;G;C;7;(B) and 4;G;C;7;(A), were of Peruvian and French origins, respectively. The geographic source of the first affected ancestor was assessed in 54 families from 15 different states. Residents of the states of Miranda, Lara and Táchira, excluding those of Zulia, had a mutated allele prevalence five- to ninefold higher than that of other areas. A low (approx. 1/200,000) prevalence, a wide-spread distribution with aggregation in some states and a likely remote European Caucasoid origin are defining epidemiologic features of HD in Venezuela. An erratum to this article can be found at     

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.     

Predictive testing for Huntington disease: interpretation and significance of intermediate alleles

Direct mutation analysis for Huntington disease (HD) became possible in 1993 with the identification of an expanded CAG trinucleotide repeat as the mutation underlying the disease. Expansion of CAG length beyond 35 repeats may be associated with the clinical presentation of HD. HD has never been seen in a person with a CAG size of <36 repeats. Intermediate alleles are defined as being below the affected CAG range but have the potential to expand to >35 CAG repeats within one generation. Thus, children of intermediate allele carriers have a low risk of developing HD. Currently, the intermediate allele range for HD is between 27 and 35 CAG repeats. In this study, we review the current knowledge on intermediate alleles for HD including the CAG repeat range, the intermediate allele frequency, and the clinical implications of an intermediate allele predictive test result. The factors influencing CAG repeat expansion, including the CAG size of the intermediate allele, the sex and age of the transmitting parent, the family history, and the HD gene sequence and haplotype, will also be reviewed.     

Gametic but not somatic instability of CAG repeat length in Huntington''s disease.

Instability of a CAG repeat in 4p16.3 has been found in Huntington's disease (HD) chromosomes. Unlike a similar repeat in the fragile X syndrome, the expanded HD repeat showed no evidence of somatic instability in a comparison of blood, lymphoblast, and brain DNA from the same persons. Four pairs of monozygotic HD twins displayed identical CAG repeat lengths suggesting that repeat size is determined in gametogenesis. In contrast with the fragile X syndrome and with HD somatic tissue, mosaicism was readily detected as a diffuse spread of repeat lengths in DNA from HD sperm samples. Typically, the modal repeat size was larger in the sperm DNA than in corresponding lymphoblast DNA, with the greatest degree of gametic mosaicism coinciding with the longest somatic CAG repeats. These data indicate that the developmental timing of repeat instability appears to differ between HD and fragile X syndrome, and that the fundamental mechanisms leading to repeat expansion may therefore be distinct.     

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.