CAG Expansion Research Articles

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.

SCA6 is caused by moderate CAG expansion in the alpha1A-voltage-dependent calcium channel gene.

Recently, moderate (CAG)>20 repeat expansions in the alpha1A-voltage-dependent calcium channel gene (CACNL1A4) have been identified in a previously unmapped type of SCA which has been named SCA6. We investigated the (CAG)n repeat length of the CACNL1A4 gene in 733 patients with sporadic ataxia and in 46 German families with dominantly inherited SCA which do not harbor the SCA1, SCA2, or MJD1/SCA3 mutation, respectively. The SCA6 (CAG)n expansion was identified in 32 patients most frequently with late manifestation of the disease. The (CAG)n stretch of the affected allele varied between 22 and 28 trinucleotide units and is therefore the shortest trinucleotide repeat expansion causing spinocerebellar ataxia. The (CAG)n repeat length is inversely correlated with the age at onset. In 11 parental transmissions of the expanded allele no repeat instability has been observed. Repeat instability was also not found for the normal allele investigating 431 meioses in the CEPH families. Analyzing 248 apparently healthy octogenerians revealed one allele of 18 repeats which is the longest normal CAG repeat in the CACNL1A4 gene reported. The SCA6 mutation causes the disease in approximately 10% of autosomal dominant SCA in Germany. Most importantly, the trinucleotide expansion was observed in four ataxia patients without obvious family history of the disease which necessitates a search for the SCA6 (CAG)n expansion even in sporadic patients.

Toward understanding the molecular pathology of Huntington's disease.

Huntington's Disease (HD) is caused by expansion of a CAG trinucleotide beyond 35 repeats within the coding region of a novel gene. Recently, new insights into the relationship between CAG expansion in the HD gene and pathological mechanisms have emerged. Survival analysis of a large cohort of affected and at-risk individuals with CAG sizes between 39 and 50 repeats have yielded probability curves of developing HD symptoms and dying of HD by a certain age. Animals transgenic for the first exon of huntingtin with large CAG repeats lengths have been reported to have a complex neurological phenotype that bears interesting similarities and differences to HD. The repertoire of huntingtin-interacting proteins continues to expand with the identification of HIP1, a protein whose yeast homologues have known functions in regulating events associated with the cytoskeleton. The ability of huntingtin to interact with two of its four known protein partners appears to be influenced by CAG length. Caspase 3 (apopain), a key cysteine protease known to play a seminal role in neural apoptosis, has also been demonstrated to specifically cleave huntingtin in a CAG length-dependent manner. Many of these features are combined in a model suggesting mechanisms by which the pathogenesis of HD may be initiated. The development of appropriate in vitro and animal models for HD will allow the validity of these models to be tested.

Clinical aspects of CAG repeat diseases.

Seven neurodegenerative disorders are known to be caused by unstable expansions of the trinucleotide CAG within human genes, and more will be discovered in the coming years. These disorders share some clinical similarities, as well as some differences, which are summarized here. These diseases have unusual clinical genetic properties related to the dynamic nature of CAG repeat expansions, including instability of the repeat expansion in meiosis, particularly male meiosis; a strong correlation between onset age and size of the repeat expansion; anticipation (earlier disease onset in succeeding generations); new mutations arising from unstable, mutable alleles with a high-normal CAG repeat number; and reduced penetrance for alleles in the low-affected range. Much more remains to be learned about the molecular biology and clinical pathophysiology of this new class of genetic diseases.

Expanded CAG repeats in spinocerebellar ataxia (SCA1) segregate with distinct haplotypes in South african families.

The autosomal dominant late onset spinocerebellar ataxias (SCAs) are genetically heterogeneous. Three genes, SCA1 on 6p, SCA2 on 12q and MJD1 on 14q, have been isolated for SCA1, SCA2 and Machado-Joseph disease (MJD), respectively. In these three autosomal dominant disorders the mutation is an expanded CAG repeat. Evidence for heterogeneity in families not linked to the SCA1, SCA2 and MJD loci is provided by the mapping of SCA loci to chromosomes 16q, 11cen and 3p. A total of 14 South African kindreds and 22 sporadic individuals with SCA were investigated for the expanded SCA1 and MJD repeats. None of the families nor the sporadic individuals showed expansion of the MJD repeat. Expanded SCA1 and CAG repeats were found to cosegregate with the disorder in six of the families tested and were also observed in one sporadic individual with a negative family history of SCA. The use of the microsatellite markers D6S260, D6S89 and D6S274 provided evidence that the expanded SCA1 repeats segregated with three distinct haplotypes in the six families. Use of the highly polymorphic tightly linked microsatellite markers is still important as this stage, particularly where this coincides with the possibility of a homozygous genotype with the trinucleotide repeat marker. Importantly, our molecular findings indicate: (1) an absence of MJD expanded repeats underlying SCA; (2) the major disease in this group is due to mutations in the SCA1 gene; and (3) the familial disorder in the majority population group (i.e. mixed ancestry) in the Western Cape region of South Africa is most likely to be the result of two distinct founder events.

The number of triplet repeats in five brain-expressed loci with CAG repeats is not associated with schizophrenia

We have previously shown that large expansions of CAG (CTG) triplets are associated both with schizophrenia itself and with an early age-at-onset of the disease. However, the repeat expansion detection (RED) method used did not provide a chromosomal location for the expanded region(s) (Morris et al., 1995). In a further study of our schizophrenic and control patients, we have now examined the length of the repeated sequence in five loci that are expressed in brain and are known to contain CAG repeat regions (Li et al., 1993). No enlarged repeat regions were identified; it is unlikely therefore that expansions at any of these five loci can account for expansions of up to 136 triplets identified by the RED method.

Identification of an HD patient with a (CAG)180 repeat expansion and the propagation of highly expanded CAG repeats in lambda phage.

The Huntington's disease mutation has been identified as a CAG/polyglutamine repeat expansion in a large gene of unknown function. In order to develop the transgenic systems necessary to uncover the molecular pathology of this disorder, it is necessary to be able to manipulate highly expanded CAG repeats in a cloned form. We have identified a patient with an expanded allele of greater than 170 repeat units and have cloned the mutant allele in the lambda zap vector. The recovery of highly expanded repeats after clone propagation was more efficient when repeats were maintained as lambda phage clones rather than as the plasmid counterparts. Manipulation of the repeats as phage clones has enabled us to generate Huntington's disease transgenic mice that contain highly expanded (CAG)115-(CAG)150 repeats and that develop a progressive neurological phenotype.

Machado-Joseph disease: clinical, molecular, and metabolic characterization in Chinese kindreds.

Machado-Joseph disease, an autosomal dominant multisystem motor degeneration, has been described mainly in people of Portuguese descent. Our report documents the presence of Machado-Joseph disease in the Chinese population, based on the specific molecular marker of a CAG repeat array in the 3' end of the MJD gene. We screened 21 Chinese families with dominant spinocerebellar ataxia. The results showed that Machado-Joseph disease with CAG expansion accounted for 52% of families with autosomal dominant cerebellar ataxia in this series. The clinical characteristics, besides the well-documented cerebellar ataxia, dysarthria, nystagmus, corticospinal dysfunctions, a variable degree of facial muscle fasciculation, and proprioceptive loss, included loss of optokinetic nystagmus and autonomic nervous system dysfunction. The CAG repeat number in the MJD gene ranged from 14 to 39 among normal alleles, and from 63 to 81 among MJD alleles. There was a strong inverse correlation (gamma = -0.77) between number of CAG repeats and age at symptom onset, accounting for 60% of the variance of age at onset. A strong clinical anticipation of age at onset existed in successive generations. Mild instabilities of expanded CAG repeat numbers during meiotic transmission occurred, with no significant difference according to the gender of the transmitting parent. Finally, brain metabolism in Machado-Joseph disease, studied with positron emission tomography, was characterized by significant progressive regional hypometabolism in the occipital cortex, as well as the cerebellar hemispheres, vermis, and brainstem.

Clinical and genetic analysis of three German kindreds with autosomal dominant cerebellar ataxia type I linked to the SCA2 locus.

The detailed clinical, electrophysiological and imaging data of three German autosomal dominant cerebellar ataxia (ADCA) families are reported. Linkage to SCA2 was established using microsatellite markers D12S105, D12S1339(1328), D12S1304(1329) yielding a lod score exceeding +3.0 for the combined data. Analysis of the pedigree data provided evidence of anticipation as observed in other neurodegenerative disorders due to polyglutamine expansion encoded by a CAG repeat. This hypothesis was confirmed by the detection of the SCA2-specific pathological protein using the 1C2 monoclonal antibody which selectively recognizes large polyglutamine expansions and the characterization of a CAG expansion in the patients. Clinically, the families were characterized by progressive ataxia of stance, gait and limbs. Saccade velocity was markedly reduced in SCA2. Further oculomotor findings were gaze palsy, impaired smooth pursuit and reduced optokinetic reflex. Dementia and pyramidal tract signs were rather rare, while peripheral involvement (reduced or absent ankle reflexes, fasciculation-like movements, amyotrophy) was a prominent feature. Electrophysiological investigations provided evidence of sensory neuropathy of the axonal type and degeneration of the posterior columns. Imaging studies demonstrated severe shrinkage of brain-stem structures even in early stages of the disease.

Trinucleotide repeats in the human genome: size distributions for all possible triplets and detection of expanded disease alleles in a group of Huntington disease individuals by the repeat expansion detection method.

Using a modified Repeat Expansion Detection (RED) assay, that was optimized for individual oligonucleotides, unrelated individuals were systematically screened for maximal repeat sizes of each of the ten possible trinucleotide repeats. Cloned trinucleotide repeats were generated and used as standards for the detectability of single copy trinucleotide repeat fragments. When the size distributions of trinucleotide repeats were compared to previously reported data, significant differences were found for the CTT repeat, which corresponds to the expanded GAA repeat in Friedreich ataxia, as well as for ATT, CCT and GTT repeats. Since 30-35% of normal individuals have CTG/CAG trinucleotide repeat sizes of 180 bp or more, we investigated the question whether small-scale CTG/CAG repeat expansions are detectable on a population basis by using the RED technique. We blindly screened 20 HD probands with CAG expansions of the HD gene, ranging in size between 120 and 174 bp, and found that a shift to larger CAG size ranges is clearly detectable when comparing the distribution of maximal repeat sizes in the disease group to a control group. Our study, therefore, demonstrates that the application of the RED assay to a population of probands and a population of controls allows the detection of small-scale CTG/CAG repeat expansions in the size range of the expanded HD gene and present in a single allele. We also provide standards and control data for the detection of other trinucleotide repeat expansions.

Atypical Clinical Presentations of X-Linked Spinal and Bulbar Muscular Atrophy in Patients with Mild CAG Expansions in Androgen Receptor Gene

Atypical Clinical Presentations of X-Linked Spinal and Bulbar Muscular Atrophy in Patients with Mild CAG Expansions in Androgen Receptor Gene

Human huntingtin derived from YAC transgenes compensates for loss of murine huntingtin by rescue of the embryonic lethal phenotype.

Huntington disease (HD) is caused by expansion of a CAG trinucleotide repeat in exon 1 of a novel gene. The HD protein (huntingtin) plays a critical role in early embryonic development since homozygous targeted disruption of the murine HD gene results in embryonic lethality by day 7.5. To rescue this phenotype by transgene based huntingtin expression it is therefore essential to express the protein early enough in development in the appropriate cells. Since YAC based transgenes are known to be regulated in an appropriate temporal and tissue-specific manner, we sought to rescue the embryonic lethality by breeding YAC transgenic mice expressing human huntingtin with mice heterozygous for the targeted disruption. We generated viable offspring homozygous for the disrupted murine HD gene but expressing human huntingtin derived from the YAC. This result clearly shows that YAC transgene based expression of huntingtin occurs prior to 7.5 days gestation. Additionally, we show that human huntingtin expression in YAC transgenic mice follows an identical tissue distribution and subcellular localisation pattern as that of the murine endogenous protein and that expression levels of 2-3 times endogenous can be achieved. This shows that human huntingtin under the influence of its native promoter, despite differences to the murine protein, is functional in a murine background and can compensate for loss of the murine protein. These results show that YAC transgenic approaches are a particularly promising route to producing an animal model for disorders associated with CAG expansion.

Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2.

The gene for spinocerebellar ataxia type 2 (SCA2) has been mapped to 12q24.1. A 1.1-megabase contig in the candidate region was assembled in P1 artificial chromosome and bacterial artificial chromosome clones. Using this contig, we identified a CAG trinucleotide repeat with CAA interruptions that was expanded in patients with SCA2. In contrast to other unstable trinucleotide repeats, this CAG repeat was not highly polymorphic in normal individuals. In SCA2 patients, the repeat was perfect and expanded to 36-52 repeats. The most common disease allele contained (CAG)37, one of the shortest expansions seen in a CAG expansion syndrome. The repeat occurs in the 5'-coding region of SCA2 which is a member of a novel gene family.

An expanded CAG repeat sequence in spinocerebellar ataxia type 7.

Expanded CAG repeat sequences have been identified in the coding region of genes mutated in several neurodegenerative disorders, including spinocerebellar ataxia type 1 and Machado-Joseph disease. In all disorders described to date the CAG expansion codes for an elongated polyglutamine chain. An increased polyglutamine chain size leads to a more severe disease, thus correlating with the genetic anticipation seen in repeat expansion disorders. Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant spinocerebellar ataxia with anticipation and a progressive degeneration of the cerebellar cortex. Using repeat expansion detection (RED), a method in which a thermostable ligase is used to detect repeat expansions directly from genomic DNA, we have analyzed 8 SCA7 families for the presence of CAG repeat expansions. RED products of 150-240 bp were found in all affected individuals and found to cosegregate with the disease (P < 0.000001, n = 66), indicating strongly that a CAG expansion is the cause of SCA7. On the basis of a previously established correlation between RED product sizes and actual repeat sizes in Machado-Joseph disease, we were able to estimate the average expansion size in SCA7 to be 64 CAG copies.

Huntington disease: new insights into the relationship between CAG expansion and disease.

The mutation underlying Huntington disease (HD) is CAG expansion beyond 35 repeats within a novel gene. Recently, new insights into the role of the HD protein (huntingtin) in the pathogenesis of HD have emerged. The CAG is translated and expression of mutant huntingtin is essential for neuronal death. Huntingtin is crucial for normal development and may be regarded as a cell survival gene. Huntingtin is specifically cleaved during apoptosis by a key cysteine protease, apopain, known to play a pivotal role in apoptotic cell death. The rate of cleavage is enhanced by longer polyglutamine tracts, suggesting that inappropriate apoptosis underlies HD. Recently, three proteins have been identified and have been shown specifically to interact with huntingtin, two of these interactions being influenced by CAG length. Several different approaches to develop an animal model for HD include cDNA and YAC transgenics, as well as 'knock-in' strategies. Such a model will be critical for the understanding of the natural history of HD and for the testing of new therapeutic modalities.

Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract.

Apoptosis has recently been recognized as a mode of cell death in Huntington disease (HD). Apopain, a human counterpart of the nematode cysteine protease death-gene product, CED-3, has a key role in proteolytic events leading to apoptosis. Here we show that apoptotic extracts and apopain itself specifically cleave the HD gene product, huntingtin. The rate of cleavage increases with the length of the huntingtin polyglutamine tract, providing an explanation for the gain-of-function associated with CAG expansion. Our results show that huntingtin is cleaved by cysteine proteases and suggest that HD might be a disorder of inappropriate apoptosis.

Survey of CAG/CTG repeats in human cDNAs representing new genes: candidates for inherited neurological disorders

Expansion of polymorphic CAG and CTG repeats in transcripts is the cause of six inherited neurodegenerative or neuromuscular diseases and may be involved in several other genetic disorders of the central nervous system. To identify new candidate genes, we have undertaken a large-scale screening project for CAG and CTG repeats in human reference cDNAs. We screened 100 128 brain cDNAs by hybridization. We also scanned GenBank expressed sequence tags for the presence of long CAG/CTG repeats in the extremities of cDNAs from several human tissues. Of the selected clones, 286 were found to represent new genes, and 72 have thus far been shown to contain CAG/CTG repeats. Our data indicate that CAG/CTG repeated 10 or more times are more likely to be polymorphic, and that new 3'-directed cDNAs with such repeats are very rare (1/2862). Nine new cDNAs containing polymorphic (observed heterozygote frequency: 0.05-0.90) CAG/CTG repeats have been currently identified in cDNAs. All of the cDNAs have been assigned to chromosomes, and six of them could be mapped with YACs to 1q32-q41, 3p14, 4q28, 3p21 and 12q13.3, 13q13.1-q13.2, and 19q13.43. Three of these clones are highly polymorphic and represent the most likely candidate genes for inherited neurodegenerative diseases and, perhaps, neuropsychiatric disorders of multifactorial origin.

Expanded polyglutamine in the Machado–Joseph disease protein induces cell death in vitro and in vivo

Recently, we identified a novel gene, MJD1, which contains an expanded CAG triplet repeat in Machado-Joseph disease. Here we report the induction of apoptosis in cultured cells expressing a portion of the MJD1 gene that includes the expanded CAG repeats. Cell death occurs only when the CAG repeat is translated into polyglutamine residues, which apparently precipitate in large covalently modified forms. We also created ataxic transgenic mice by expressing the expanded polyglutamine stretch in Purkinje cells. Our results demonstrate the potential involvement of the expanded polyglutamine as the common aetiological agent for inherited neurodegenerative diseases with CAG expansions.

Psychiatric symptoms and CAG expansion in Huntington's disease

Psychiatric symptoms and CAG expansion in Huntington's disease

Psychiatric symptoms and CAG expansion in Huntington's disease.

The mutation responsible for Huntington's disease (HD) is an elongated CAG repeat in the coding region of the IT15 gene. A PCR-based test with high sensitivity and accuracy is now available to identify asymptomatic gene carriers and patients. An inverse correlation between CAG copy number and age at disease onset has been found in a large number of affected individuals. The influence of the CAG repeat expansion on other phenotypic manifestations, especially specific psychiatric symptoms has not been studied intensively. In order to elucidate this situation we investigated the relation between CAG copy number and distinct psychiatric phenotypes found in 79 HD-patients. None of the four differentiated categories (personality change, psychosis, depression, and nonspecific alterations) showed significant differences in respect to size of the CAG expansion. In addition, no influence of individual sex on psychiatric presentation could be found. On the other hand in patients with personality changes maternal transmission was significantly more frequent compared with all other groups. Therefore we suggest that clinical severity of psychiatric features in HD is not directly dependent on the size of the dynamic mutation involved. The complex pathogenetic mechanisms leading to psychiatric alterations are still unknown and thus genotyping does not provide information about expected psychiatric symptoms in HD gene carriers.