PolyQ repeat expansions in ATXN2 associated with ALS are CAA interrupted repeats

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive disease leading to paralysis and death. Recently, intermediate length polyglutamine (polyQ) repeats of 27-33 in ATAXIN-2 (ATXN2), encoding the ATXN2 protein, were found to increase risk for ALS. In ATXN2, polyQ expansions of ≥ 34, which are pure CAG repeat expansions, cause spinocerebellar ataxia type 2. However, similar length expansions that are interrupted with other codons, can present atypically with parkinsonism, suggesting that configuration of the repeat sequence plays an important role in disease manifestation in ATXN2 polyQ expansion diseases. Here we determined whether the expansions in ATXN2 associated with ALS were pure or interrupted CAG repeats, and defined single nucleotide polymorphisms (SNPs) rs695871 and rs695872 in exon 1 of the gene, to assess haplotype association. We found that the expanded repeat alleles of 40 ALS patients and 9 long-repeat length controls were all interrupted, bearing 1-3 CAA codons within the CAG repeat. 21/21 expanded ALS chromosomes with 3CAA interruptions arose from one haplotype (GT), while 18/19 expanded ALS chromosomes with <3CAA interruptions arose from a different haplotype (CC). Moreover, age of disease onset was significantly earlier in patients bearing 3 interruptions vs fewer, and was distinct between haplotypes. These results indicate that CAG repeat expansions in ATXN2 associated with ALS are uniformly interrupted repeats and that the nature of the repeat sequence and haplotype, as well as length of polyQ repeat, may play a role in the neurological effect conferred by expansions in ATXN2.     

TATA Box-Binding Protein gene is associated with risk for schizophrenia, age at onset and prefrontal function

Schizophrenia is a common polygenic disease in distinct populations, while spinocerebellar ataxia type 17 (SCA17) is a rare autosomal dominant neurodegenerative disorder. Both diseases involve psychotic symptoms. SCA17 is caused by an expanded polyglutamine tract in the TATA box-binding protein ( TBP ) gene. In the present study, we investigated the association between schizophrenia and CAG repeat length in common TBP alleles with fewer than 42 CAG repeats in a Japanese population (326 patients with schizophrenia and 116 healthy controls). We found that higher frequency of alleles with greater than 35 CAG repeats in patients with schizophrenia compared with that in controls ( p = 0.042). We also examined the correlation between CAG repeats length and age at onset of schizophrenia. We observed a negative correlation between the number of CAG repeats in the chromosome with longer CAG repeats out of two chromosomes and age at onset of schizophrenia ( p = 0.020). We further provided evidence that TBP genotypes with greater than 35 CAG repeats, which were enriched in patients with schizophrenia, were significantly associated with hypoactivation of the prefrontal cortex measured by near-infrared spectroscopy during the tower of Hanoi, a task of executive function (right PFC; p = 0.015, left PFC; p = 0.010). These findings suggest possible associations of the genetic variations of the TBP gene with risk for schizophrenia, age at onset and prefrontal function.     

Correlation between CAG Repeat Length and Clinical Features in Machado-Joseph Disease

Machado-Joseph disease (MJD) is associated with the expansion of a CAG trinucleotide repeat in a novel gene on 14q32.1. We confirmed the presence of this expansion in 156 MJD patients from 33 families of different geographic origins: 15 Portuguese Azorean, 2 Brazilian, and 16 North American of Portuguese Azorean descent. Normal chromosomes contain between 12 and 37 CAG repeats in the MJD gene, whereas MJD gene carriers have alleles within the expanded range of 62–84 CAG units. The distribution of expanded alleles and the gap between normal and expanded allele sizes is either inconsistent with a premutation hypothesis or most (if not all) of the alleles we studied descend from a common ancestor. There is a strong correlation between the expanded repeat size and the age at onset of the disease as well as the clinical presentation. There is mild instability of the CAG tract length with transmission of the expanded alleles; both increase and decrease in size between parents and progeny occur, with larger variations in male than in female transmissions. Together, these effects can partly explain the variability of age at onset and of phenotypic features in MJD; however, other modifying factors must exist.     

Japanese families with autosomal dominant pure cerebellar ataxia map to chromosome 19p13.1-p13.2 and are strongly associated with mild CAG expansions in the spinocerebellar ataxia type 6 gene in chromosome 19p13.1.

Autosomal dominant cerebellar ataxia is a group of clinically and genetically heterogeneous disorders. We carried out genomewide linkage analysis in 15 families with autosomal dominant pure cerebellar ataxia (ADPCA). Evidence for linkage to chromosome 19p markers was found in nine families, and combined multipoint analysis refined the candidate region to a 13.3-cM interval in 19p13.1-p13.2. The remaining six families were excluded for this region. Analysis of CAG-repeat expansion in the alpha1A-voltage-dependent calcium channel (CACNL1A4) gene lying in 19p13.1, recently identified among 8 small American kindreds with ADPCA (spinocerebellar ataxia type 6 [SCA6]), revealed that 8 of the 15 families studied had similar, very small expansion in this gene: all affected individuals had larger alleles (range of CAG repeats 21-25), compared with alleles observed in neurologically normal Japanese (range 5-20 repeats). Inverse correlation between the CAG-repeat number and the age at onset was found in affected individuals with expansion. The number of CAG repeats in expanded chromosomes was completely stable within each family, which was consistent with the fact that anticipation was not statistically proved in the SCA6 families that we studied. We conclude that more than half of Japanese cases of ADPCA map to 19p13.1-p13.2 and are strongly associated with the mild CAG expansion in the SCA6/CACNL1A4 gene.     

Analysis of CAG repeats in SCA1, SCA2, SCA3, SCA6, SCA7 and DRPLA loci in spinocerebellar ataxia patients and distribution of CAG repeats at the SCA1, SCA2 and SCA6 loci in nine ethnic populations of eastern India

To identify various subtypes of spinocerebellar ataxias (SCAs) among 57 unrelated individuals clinically diagnosed as ataxia patients we analysed the SCA1, SCA2, SCA3, SCA6, SCA7 and DRPLA loci for expansion of CAG repeats. We detected CAG repeat expansion in 6 patients (10.5%) at the SCA1 locus. Ten of the 57 patients (17.5%) had CAG repeat expansion at the SCA2 locus, while four had CAG expansion at the SCA3/MJD locus (7%). At the SCA6 locus there was a single patient (1.8%) with 21 CAG repeats. We have not detected any patient with expansion in the SCA7 and DRPLA loci. To test whether the frequencies of the large normal alleles in SCA1, SCA2 and SCA6 loci can reflect some light on prevalence of the subtypes of SCAs we studied the CAG repeat variation in these loci in nine ethnic sub-populations of eastern India from which the patients originated. We report here that the frequency of large normal alleles (>31 CAG repeats) in SCA1 locus to be 0.211 of 394 chromosomes studied. We also report that the frequency of large normal alleles (>22 CAG repeats) at the SCA2 locus is 0.038 while at the SCA6 locus frequency of large normal alleles (>13 repeats) is 0.032. We discussed our data in light of the distribution of normal alleles and prevalence of SCAs in the Japanese and white populations.     

Role of histidine interruption in mitigating the pathological effects of long polyglutamine stretches in SCA1: A molecular approach

Polyglutamine expansions, leading to aggregation, have been implicated in various neurodegenerative disorders. The range of repeats observed in normal individuals in most of these diseases is 19-36, whereas mutant proteins carry 40-81 repeats. In one such disorder, spinocerebellar ataxia (SCA1), it has been reported that certain individuals with expanded polyglutamine repeats in the disease range (Q(12)HQHQ(12)HQHQ(14/15)) but with histidine interruptions were found to be phenotypically normal. To establish the role of histidine, a comparative study of conformational properties of model peptide sequences with (Q(12)HQHQ(12)HQHQ(12)) and without (Q(42)) interruptions is presented here. Q(12)HQHQ(12)HQHQ(12) displays greater solubility and lesser aggregation propensity compared to uninterrupted Q(42) as well as much shorter Q(22). The solvent and temperature-driven conformational transitions (beta structure <--> random coil --> alpha helix) displayed by these model polyQ stretches is also discussed in the present report. The study strengthens our earlier hypothesis of the importance of histidine interruptions in mitigating the pathogenicity of expanded polyglutamine tract at the SCA1 locus. The relatively lower propensity for aggregation observed in case of histidine interrupted stretches even in the disease range suggests that at a very low concentration, the protein aggregation in normal cells, is possibly not initiated at all or the disease onset is significantly delayed. Our present study also reveals that besides histidine interruption, proline interruption in polyglutamine stretches can lower their aggregation propensity.     

Genotype/phenotype correlation in a SCA1 family: anticipation without CAG expansion

We report on a family with spinocerebellar ataxia type 1 (SCA1), in which the age at onset and the severity of the disease do not correlate with the number of CAG repeat units. Although a marked anticipation was observed in the proband, it was not a consequence of an expansion of the CAG tract. None of the expanded alleles contained CAT interruptions. The pathologic expansion in this family was stable during the paternal but not maternal transmission, where it expanded by one trinucleotide and unexpectedly did not lead to anticipation. Our observations suggest that factors other than the length of the CAG repeat play a considerable role in determination of the disease course.     

Spinocerebellar ataxia 7 (SCA7)

Spinocerebellar ataxia 7 (SCA7) is a progressive autosomal dominant neurodegenerative disorder characterized clinically by cerebellar ataxia associated with progressive macular dystrophy. The disease affects primarily the cerebellum and the retina, but also many other CNS structures as the disease progresses. SCA7 is caused by expansion of an unstable trinucleotide CAG repeat encoding a polyglutamine tract in the corresponding protein, ataxin-7. Normal SCA7 alleles contain 4-35 CAG repeats, whereas pathological alleles contain from 36-306 CAG repeats. SCA7 has a number of features in common with other diseases with polyglutamine expansions: (i) the appearance of clinical symptoms above a threshold number of CAG repeats (>35); (ii) a correlation between the size of the expansion and the rate of progression of the disease: the larger the repeat, the faster the progression; (iii) instability of the repeat sequence (approximately 12 CAG/transmission) that accounts for the marked anticipation of approximately 20 years/generation. The CAG repeat sequence is particularly unstable and de novo mutations can occur during paternal transmissions of intermediate size alleles (28-35 CAG repeats). This can explain the persistence of the disease in spite of the anticipation that should have resulted in its extinction.     

Dentatorubral-pallidoluysian atrophy (DRPLA): Close correlation of CAG repeat expansions with the wide spectrum of clinical presentations and prominent anticipation

Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare autosomal dominant neurodegenerative disease characterized by various combinations of ataxia, choreoathetosis, myoclonus, epilepsy and dementia as well as various ages of onset. We have identified a specific unstable trinucleotide repeat expansion in a gene on the short arm of chromosome 12 as the pathogenic mutation for DRPLA. We investigated how the degree of the expansion of the CAG repeat affects the clinical manifestations of DRPLA. The sizes of the expanded alleles were well correlated with the ages of onset (r = −0.6955, P < 0.001). Patients with progressive myoclonus epilepsy (PME) phenotype had larger expansions (62–79 repeats) and earlier ages of onset (onset before age 20). Furthermore, most of the patients with PME phenotype inherited their expanded alleles from their affected fathers. On the other hand, patients with non-PME phenotype showed later ages of onset (onset after age 20) and smaller expansions (54–67 repeats). When ages of onset of each clinical symptom are compared with sizes of the CAG repeat, there is again a remarkably high correlation of the sizes of CAG repeat with each of the clinical symptoms. Thus the wide variation in clinical manifestations of DRPLA can now be clearly explained based on the degree of CAG repeat expansion, which strongly indicates that the expanded alleles are intimately involved in the neuronal degeneration in dentatofugal and pallidofugal systems.     

Transmission distortion of the mutant alleles in spinocerebellar ataxia

Spinocerebellar ataxia type 1 and type 3 (SCA1, SCA3) are autosomal dominant neurodegenerative disorders caused by expanded CAG trinucleotide repeats in novel genes. In our collective of SCA1 and SCA3 families, we observed distortion of the Mendelian 1:1 segregation of the disease. The mutated alleles were preferentially transmitted by female carriers in SCA3, whereas a gender effect on clinical features such as age of onset was not obvious. The mechanism underlying segregation distortion remains to be established. Received: 9 September 1996 / Revised: 7 October 1996     

Evolution and function of CAG/polyglutamine repeats in protein-protein interaction networks

Expanded runs of consecutive trinucleotide CAG repeats encoding polyglutamine (polyQ) stretches are observed in the genes of a large number of patients with different genetic diseases such as Huntington's and several Ataxias. Protein aggregation, which is a key feature of most of these diseases, is thought to be triggered by these expanded polyQ sequences in disease-related proteins. However, polyQ tracts are a normal feature of many human proteins, suggesting that they have an important cellular function. To clarify the potential function of polyQ repeats in biological systems, we systematically analyzed available information stored in sequence and protein interaction databases. By integrating genomic, phylogenetic, protein interaction network and functional information, we obtained evidence that polyQ tracts in proteins stabilize protein interactions. This happens most likely through structural changes whereby the polyQ sequence extends a neighboring coiled-coil region to facilitate its interaction with a coiled-coil region in another protein. Alteration of this important biological function due to polyQ expansion results in gain of abnormal interactions, leading to pathological effects like protein aggregation. Our analyses suggest that research on polyQ proteins should shift focus from expanded polyQ proteins into the characterization of the influence of the wild-type polyQ on protein interactions.     

Molecular analysis of CAG repeats at five different spinocerebellar ataxia loci: correlation and alternative explanations for disease pathogenesis

Spinocerebellar ataxias (SCAs) are caused by expansion of (CAG)n triplet repeats. These repeats occur as polymorphic forms in general population; however, beyond a threshold size they become pathogenic. The sizes and distributions of repeats at the SCA1, SCA2, SCA3, SCA7 and DRPLA loci were assessed by molecular analysis of 124 unrelated ataxia patients and 44 controls, and the association of larger normal (LN) alleles with disease prevalence was evaluated. Triplet repeat expansions in the disease range were detected in 8% (10/124) of the cases, with the majority having expansion at the SCA1 locus. Normal allele ranges in the cohort studied were similar to the Caucasian and North Indian populations but differed from the Korean and Japanese populations at various loci. The percentage of individuals with LN alleles at the SCA1 and SCA2 loci was higher than reported in Indians, Japanese and Caucasians. LN alleles showed a good correlation with the incidence of SCA1, indicating that SCA1 is the most prevalent ataxia in our population. The majority of cases with clinical symptoms of SCA could not be diagnosed by established CAG repeat criteria, suggesting that there may be an alternative basis for disease pathogenesis: (i) Repeats lower than the normal range may also result in abnormal phenotypes (ii) LN alleles at different loci in the same individual may contribute to symptoms (iii) Exogenous factors may play a role in triggering disease symptoms in individuals with LN alleles (iv) Triplet repeats may reach the disease range in the brain but not in the blood.     

Disruption of the toxic conformation of the expanded polyglutamine stretch leads to suppression of aggregate formation and cytotoxicity

The polyglutamine (polyQ) diseases are a class of inherited neurodegenerative diseases including Huntington's disease, caused by the expansion of a polyQ stretch within each disease protein. This expansion is thought to cause a conformational change in the protein leading to aggregation of the protein, resulting in cytotoxicity. To analyze whether disrupting the toxic conformation of the polyQ protein can alter its aggregation propensity and cytotoxicity, we examined the effect of interruption of the expanded polyQ stretch by proline insertion, since prolines cause great alterations in protein conformation. Here, we show that insertion of prolines into the expanded polyQ stretch indeed disrupts its ordered secondary structure, leading to suppression of polyQ protein aggregation both in vitro and in cell culture, and reduction of cytotoxicity in correlation with the number of proline interruptions. Furthermore, we found that a short polyQ stretch with a proline interruption is able to inhibit aggregation of the expanded polyQ protein in trans. These results show that a gain in toxic conformation of the expanded polyQ protein is essential for aggregation and cytotoxicity, providing insight into establishing therapies against the polyQ diseases.     

Marked Phenotypic Heterogeneity Associated with Expansion of a CAG Repeat Sequence at the Spinocerebellar Ataxia 3/Machado-Joseph Disease Locus

The spinocerebellar ataxia 3 locus (SCA3) for type I autosomal dominant cerebellar ataxia (ADCA type I), a clinically and genetically heterogeneous group of neuro-degenerative disorders, has been mapped to chromosome 14q32.1. ADCA type I patients from families segregating SCA3 share clinical features in common with those with Machado-Joseph disease (MJD), the gene of which maps to the same region. We show here that the disease gene segregating in each of three French ADCA type I kindreds and in a French family with neuropatho-logical findings suggesting the ataxochoreic form of dentatorubropallidoluysian atrophy carries an expanded CAG repeat sequence located at the same locus as that for MJD. Analysis of the mutation in these families shows a strong negative correlation between size of the expanded CAG repeat and age at onset of clinical disease. Instability of the expanded triplet repeat was not found to be affected by sex of the parent transmitting the mutation. Evidence was found for somatic and gonadal mosaicism for alleles carrying expanded trinucleotide repeats.     

Two Novel SNPs in ATXN3 3’ UTR May Decrease Age at Onset of SCA3/MJD in Chinese Patients

Spinocerebellar ataxia type 3 (SCA3), or Machado—Joseph disease (MJD), is an autosomal dominantly-inherited disease that produces progressive problems with movement. It is caused by the expansion of an area of CAG repeats in a coding region of ATXN3. The number of repeats is inversely associated with age at disease onset (AO) and is significantly associated with disease severity; however, the degree of CAG expansion only explains 50 to 70% of variance in AO. We tested two SNPs, rs709930 and rs910369, in the 3’ UTR of ATXN3 gene for association with SCA3/MJD risk and with SCA3/MJD AO in an independent cohort of 170 patients with SCA3/MJD and 200 healthy controls from mainland China. rs709930 genotype frequencies were statistically significantly different between patients and controls (p = 0.001, α = 0.05). SCA3/MJD patients carrying the rs709930 A allele and rs910369 T allele experienced an earlier onset, with a decrease in AO of approximately 2 to 4 years. The two novel SNPs found in this study might be genetic modifiers for AO in SCA3/MJD.     

Quantitative connection between polyglutamine aggregation kinetics and neurodegenerative process in patients with Huntington's disease

ABSTRACT: BACKGROUND: Despite enormous progress in elucidating the biophysics of aggregation, no cause-and-effect relationship between protein aggregation and neurodegenerative disease has been unequivocally established. Here, we derived several risk-based stochastic kinetic models that assess genotype/phenotype correlations in patients with Huntington's disease (HD) caused by the expansion of a CAG repeat. Fascinating disease-specific aspects of HD include the polyglutamine (polyQ)-length dependence of both age at symptoms onset and the propensity of the expanded polyQ protein to aggregate. In vitro, aggregation of polyQ peptides follows a simple nucleated growth polymerization pathway. Our models that reflect polyQ aggregation kinetics in a nucleated growth polymerization divided aggregate process into the lengthdependent nucleation and the nucleation-dependent elongation. In contrast to the repeatlength dependent variability of age at onset, recent studies have shown that the extent of expansion has only a subtle effect on the rate of disease progression, suggesting possible differences in the mechanisms underlying the neurodegenerative process. RESULTS: Using polyQ-length as an index, these procedures enabled us for the first time to establish a quantitative connection between aggregation kinetics and disease process, including onset and the rate of progression. Although the complexity of disease process in HD, the time course of striatal neurodegeneration can be precisely predicted by the mathematical model in which neurodegeneration occurs by different mechanisms for the initiation and progression of disease processes. Nucleation is sufficient to initiate neuronal loss as a series of random events in time. The stochastic appearance of nucleation in a cell population acts as the constant risk of neuronal cell damage over time, while elongation reduces the risk by nucleation in proportion to the increased extent of the aggregates during disease progression. CONCLUSIONS: Our findings suggest that nucleation is a critical step in gaining toxic effects to the cell, and provide a new insight into the relationship between polyQ aggregation and neurodegenerative process in HD.