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.     

Association between CAG repeat length in the PPP2R2B gene and Alzheimer disease in the Japanese population

We analyzed the association between PPP2R2B gene CAG repeat length and Alzheimer disease (AD) susceptibility in the Japanese population. Blood samples were collected from 218 late-onset AD patients and 86 controls. DNA fragments containing the target CAG repeat region were amplified using polymerase chain reaction (PCR). PCR products were sequenced using ABI PRISM 310 genetic analyzer. The mean CAG repeat length did not differ significantly between the control and AD groups. In contrast, the frequency of CAG repeats shorter than 15 was significantly higher in AD group, specifically in the AD with APOE4 subgroup, than in the control group. The results suggest that CAG repeat lengths in the PPP2R2B gene may be potential genetic markers for AD susceptibility in the Japanese population.     

Polymorphisms in the CAG repeat--a source of error in Huntington disease DNA testing

Five of 400 patients (1.3%), referred for Huntington disease DNA testing, demonstrated a single allele on CAG alone, but two alleles when the CAG + CCG repeats were measured. The PCR assay failed to detect one allele in the CAG alone assay because of single-base silent polymorphisms in the penultimate or the last CAG repeat. The region around and within the CAG repeat sequence in the Huntington disease gene is a hot-spot for DNA polymorphisms, which can occur in up to 1% of subjects tested for Huntington disease. These polymorphisms may interfere with amplification by PCR, and so have the potential to produce a diagnostic error.     

Clinicopathology of spinocerebellar degeneration: Its correlation to the unstable CAG repeat of the affected gene

The recent advances in gene analysis have greatly facilitated the classification of autosomal dominant spinocerebellar ataxia (SCA). Analyses of linkage in large families with SCA have assigned gene foci to at least 8 chromosomes. One gene is located in the short arm of chromosome 6 (6p22-p23) and causes spinocerebellar ataxia type 1 (SCA1). A gene in the long arm of chromosome 14 (14q24.3-q32) underlies Machado-Joseph disease (MJD). A third gene locus is assigned to the short arm of chromosome 12 (12p2-pter) causing dentatorubropallidoluysian atrophy (DRPLA). The gene for spinocerebellar ataxia type 2 (SCA2) is located in the 12q23–24. Subsequently, a sporadic counterpart of hereditary olivopontocerebellar atrophy of the Menzel type is clearly defined, and all the syndromes (non-hereditary olivopontocerebellar atrophy, striatonigral degeneration and Shy-Drager syndrome) are now lumped under the term of multiple system atrophy (MSA). Oligodendroglial cytoplas-mic inclusions appear to be specific for and diagnostic of MSA. As the clinical features in SCA are variable and often appear to overlap with one another, which makes accurate classification difficult if not possible, the genotype is required for their unequivocal classification. However, major neuropathological features clearly distinguish SCA1 from SCA3/ MJD cases; the medial segment of the globus pallidus and intermediolateral column lesions in SCA3/MJD, and inferior olive and cerebellar cortical degeneration in SCA1. It has been stated that neurodegeneration in SCA3/MJD is more homogeneous than in SCA1 or SCA2 and that degeneration of the pallidoluysian system is not present in the latter. The pertinent pathology in each of the three types of SCA is illustrated. The background of clinicopathology and genetic analysis of dentatorubropallidoluysian atrophy is also reviewed.     

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.     

Dynamics of CAG repeat loci revealed by the analysis of their variability

In order to understand the dynamics of the expressed single tandem repeat trinucleotides (most of them involved in pathological expansion), the diversity in 10 different loci (SCA1, SCA2, SCA3, SCA6, SCA8, SCA12, DRPLA, HD, KCNN3, and NCOA3) was analyzed in four major human groups (Africans, Europeans, Indians, and East Asians). The present analysis intends to disentangle population-based from genetic-based factors having shaped STR (trinucleotide) variation and to recognize, for each locus, the specific rate and pattern of mutation (bias toward expansion or contraction, constraints on allele size), and the footprints of selection. Population differences account for a very small part of the total variation, but a clear footprint appears of population growth after a bottleneck in all non-African populations, giving support to the out-of-Africa model of modern humans. Most of the diversity is found among loci, and different dynamics are inferred for each of them. SCA2 and SCA3 follow an unrestricted stepwise mutation model, while the rest of loci are found under allele size constrictions and a bias to expansion (SCA1, SCA6, HD, and KCNN3), contraction (SCA12, DRPLA, and NCOA3), or unbiased (SCA8).     

Analysis of the (CAG)n repeat causing Huntington's disease in a Mexican population

We investigated the allele distribution of the polymorphic (CAG)n repeat in the IT15 gene in 96 normal subjects from the Mexican population and 83 unrelated patients with Huntington's disease. Our results show that the size distributions of normal and affected alleles do not overlap. Normal alleles range from 13 to 32 triplets, with 18 being the most frequent allele, while HD alleles contain 37 to 76 repeats with 42 being the most frequent. One allele in the range of intermediate alleles was found (32 repeats) in a normal subject. The juvenile onset cases in this study are associated with an expansion greater than 49 repeats. In the available parent-offspring pairs, paternal alleles show instability with an expansion of 28 repeats in one case.     

Mutation screening and CAG repeat length analysis of the androgen receptor gene in Klinefelter's syndrome patients with and without spermatogenesis

BACKGROUND: Mutations of the androgen receptor (AR) gene give rise to a wide array of phenotypic abnormalities. A systematic analysis of the AR gene in patients with 47,XXY has not previously been performed. METHODS: Mutations of the AR gene and expansion of the CAG repeats in exon 1 of the AR gene were studied in 13 patients with Klinefelter's syndrome either with (n = 1) or without (n = 12) spermatogenesis. RESULTS: No abnormalities in the AR gene were detected by single strand conformational polymorphism analysis. The CAG lengths ranged from 17 to 27 (mean +/- SD 22.8 +/- 3.3, median 23) for Klinefelter patients or from 17 to 28 (mean +/- SD 23.2 +/- 2.6, median 23) for control subjects. X-inactivation analysis for the methylation status of the AR gene was performed in seven patients who were heterozygous for CAG repeats of different length, showing that the longer CAG repeat alleles underwent random but more frequent inactivation in five patients and skewed inactivation in two. CONCLUSIONS: An AR gene abnormality does not constitute an important factor for impaired spermatogenesis in patients with Klinefelter's syndrome.     

Accurate determination of the number of CAG repeats in the Huntington disease gene using a sequence-specific internal DNA standard

We have developed a sequence-specific internal DNA size standard for the accurate determination of the number of CAG repeats in the Huntington disease (HD) gene by cloning key fragments (between 15 and 64 CAG repeats) of the HD gene. These fragments, pooled to produce a sequence-specific DNA ladder, enabled us to observe the true number of CAG repeats directly, with no need for calculations. Comparison of the calculated numbers of CAG repeats in the HD gene using this sequence-specific DNA standard with a commercially available standard (GENESCAN-500 TAMRA) showed that the latter underestimated the number of CAG repeats by three when analyzed by capillary electrophoresis on the ABI 310 Genetic Analyzer (POP4 polymer). In contrast, the use of the same standard overestimated the number of CAG repeats by one when the samples were analyzed by denaturing polyacrylamide electrophoresis on ABI 377 DNA Sequencer (6% denaturing polyacrylamide gel). This suggests that our sequence-specific standard provides greater accuracy for the determination of the true number of CAG repeats in the HD gene than commercially available standards. The sequence-specific standard can be radioactively labeled and successfully replace conventional DNA size standards when analyzing polymerase chain reaction (PCR)-amplified HD alleles by denaturing polyacrylamide electrophoresis.     

Somatic stability of the expanded CAG trinucleotide repeat in X-linked spinal and bulbar muscular atrophy

Expansion of trinucleotide repeats has now been associated with eight inherited diseases: X-linked spinal and bulbar muscular atrophy, two fragile X syndromes, myotonic dystrophy, Huntington's disease, spinocerebellar ataxia type I, dentatorubral pallidoluysian atrophy and Machado-Joseph disease. It has been shown that these expanded DNA repeats are unstable in number when transmitted from parents to offspring (“meiotic instability”), while somatic variation in repeat number has also been found in the fragile X syndrome and myotonic dystrophy. Moderate meiotic instability has been demonstrated in X-linked spinal and bulbar muscular atrophy (SBMA, Kennedy's disease). In order to determine if the expanded CAG repeat in SBMA also shows somatic instability, we compared different tissues from two patients with SBMA. We then examined the in vitro stability of the CAG repeat expansion by analyzing fibroblast cell cultures. Length comparison of expanded CAG repeats from all these materials clearly demonstrates that the CAG trinucleotide repeat in SBMA does not exhibit somatic variation. © 1996 Wiley-Liss, Inc.     

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.     

Spinocerebellar ataxia 1 (SCA1) in the Japanese: Analysis of CAG trinucleitide repeat expansion and instability of the repeat for paternal transmission

Summary SCA1 is caused by expansion of an unstable CAG triplet repeat in a novel gene located on the short arm of chromosome 6. In 126 Japanese individuals from 12 pedigrees with SCA1, studies were done to determine if they carried this mutant gene. All the affected and presymptomatic individuals, determined by haplotype segregation analyses, carried an abnormally expanded allele with the range of 39–63 repeat units. This repeat size inversely correlated with the age at onset. However, contrary to reported results, size of the repeat did not correlate with gender of the transmitting parent. Therefore, the CAG triplet repeat instability on paternal transmission is not likely to be fundamental to SCA1.     

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.     

Correlation between magnitude of CAG repeat length alterations and length of the paternal repeat in paternally inherited Huntington's disease

An increasing number of diseases are being found to be due to elongation of specific trinucleotide repeat sequences. Inverse correlation between the age at onset and the length of the repeat has been found in most of these. The elongated CAG repeat causing Huntington's disease is highly unstable when inherited from an affected father. In this study we found an average parent-to-offspring difference of +0.08 repeat units in maternally inherited repeats, significantly less than the average difference of +2.92 repeat units with paternal transmission. Large repeat expansions, of more than 5 repeat units, were seen only in paternally inherited cases. With paternal transmission the magnitude of repeat length alterations was directly correlated to increasing paternal repeat length. Increasing variation in repeat length among siblings was correlated to increasing average repeat length in the sibship in both maternally and paternally inherited HD. Comparison of the magnitude of repeat length alterations to parental age at the time of birth of the offspring showed no correlation.     

Mitotic and meiotic stability of the CAG repeat in the X-linked spinal and bulbar muscular atrophy gene

X-linked spinal and bulbar muscular atrophy (SBMA) occurs due to an expansion of the trinucleotide repeat (CAG)_n in the androgen receptor gene. Anticipation is relatively rare in SBMA in contrast to spinocerebellar ataxia type 1 (SCA1), and dentatorubral and pallidoluysian atrophy (DRPLA) which show obvious paternal anticipation. The differences in the CAG repeat number were compared among sperm, leukocytes and skeletal muscles of SBMA patients. In SBMA, the sperm of most patients and the skeletal muscle of all patients showed the same repeat number as their leukocytes, whereas the increase in the repeat number from leukocytes to sperm was evident in SCA1 and DRPLA patients. The higher mosaicism level in sperm compared with leukocytes was common in SBMA, SCA1 and DRPLA, and the level of sperm was lower in SBMA than in SCA1 and DRPLA. Thus, spermatogenesis was suggested to be strongly associated with paternal anticipation. The mosaicism level was smaller in SBMA than in other (CAG)_n expanded disorders, and smallest in the SBMA carrier females. These findings demonstrate that the CAG repeat in SBMA is relatively stable in mitotic and meiotic processes, and there is a possibility that the lower mosaicism level of the carrier females compared with the SBMA patients is associated with X-linked recessive inheritance.     

Analysis of (CAG)n size heterogeneity in somatic and sperm cell DNA from intermediate and expanded Huntington disease gene carriers

The length of the CAG repeat responsible for Huntington disease has been analysed by two PCR methods in blood and sperm DNA of 13 expansion carriers, two carriers of intermediate alleles, and four normal subjects. The two methods consistently confirmed size heterogeneity, more pronounced in sperm and confined to the CAG stretch. Based on densitometric scanning of films, four indexes addressed to different features of the PCR pattern were used to quantitate mosaicism. These revealed strong correlations with CAG size and intergenerational instability. However, mosaicism did not show a greater similarity in sibs who shared the same HD chromosome, nor was correlated with instability in the proband's pedigree. Our data do not support the hypothesis that cis-acting factors play a major role in the instability and leave the CAG size per se as the major determinant of sperm cell CAG instability. Hum Mutat 10:458–464, 1997. © 1997 Wiley-Liss, Inc.