CAG repeat instability, cryptic sequence variation and pathogeneticity: evidence from different loci.

Different aspects of expanded polyglutamine tracts and of their pathogenetic role are taken into consideration here. (i) The (CAG)n length of wild-type alleles of the Huntington disease gene was analysed in instability-prone tumour tissue from colon cancer patients to test whether the process leading to the elongation of alleles towards the expansion range involves single-unit stepwise mutations or larger jumps. The analysis showed that length changes of a single unit had a relatively low frequency. (ii) The observation of an expanded spinocerebellar ataxia (SCA)1 allele with an unusual pattern of multiple CAT interruptions showed that cryptic sequence variations are critical not only for sequence length stability but also for the expression of the disease phenotype. (iii) Small expansions of the (CAG)n sequence at the CACNA1A gene have been reported as causing SCA6. The analysis of families with SCA6 and episodic ataxia type 2 showed that these phenotypes are, in fact, expressions of the same disorder caused either by point mutations or by small (CAG)n expansions. A gain of function has been hypothesized for all proteins containing an expanded polyglutamine stretch, including the alpha 1A subunit of the voltage-gated calcium channel type P/Q coded by the CACNA1A gene. Because point mutations at the same gene with similar phenotypic consequences are highly unlikely to have this effect, an alternative common pathogenetic mechanism for all these mutations, including small expansions, can be hypothesized.     

Orientation dependence of trinucleotide CAG repeat instability in Saccharomyces cerevisiae.

To examine the chromosomal stability of repetitions of the trinucleotide CAG, we have cloned CAG repeat tracts onto the 3' end of the Saccharomyces cerevisiae ADE2 gene and placed the appended gene into the ARO2 locus of chromosome VII. Examination of chromosomal DNA from sibling colonies arising from clonal expansion of strains harboring repeat tracts showed that repeat tracts often change in length. Most changes in tract length are decreases, but rare increases also occur. Longer tracts are more unstable than smaller tracts. The most unstable tracts, of 80 to 90 repeats, undergo changes at rates as high as 3 x 10(-2) changes per cell per generation. To examine whether repeat orientation or adjacent sequences alter repeat stability, we constructed strains with repeat tracts in both orientations, either with or without sequences 5' to ADE2 harboring an autonomously replicating sequence (ARS; replication origin). When CAG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively stable regardless of the orientation of ADE2. When CTG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively unstable regardless of the orientation of ADE2. Removal of the ARS as well as other sequences adjacent to the 5' end of ADE2 alters the orientation dependence such that stability now depends on the orientation of ADE2 in the chromosome. These results suggest that the proximity of an ARS or another sequence has a profound effect on repeat stability.     

Mitotic and meiotic instability of the CAG trinucleotide repeat in spinocerebellar ataxia type 1

Spinocerebellar ataxia type 1 (SCA1) is an autosomal, dominantly inherited neurodegenerative disease caused by an unstable CAG trinucleotide repeat expansion in the ataxin-1 gene located on chromosome 6p22-p23. The expanded CAG repeat is unstable during transmission, and a variation in the CAG repeat length has been found in different tissues, including sperm samples from affected males. In order further to examine the mitotic and meiotic instability of the (CAG)_n stretch we have performed single sperm and low-copy genome analysis in SCA1 patients and asymptomatic carriers. A pronounced variation in the size of the expanded allele was found in sperm cells and peripheral blood leucocytes, with a higher degree of instability seen in the sperm cells, where an allele with 50 repeat units was contracted in 11.8%, further expanded in 63.5% and unchanged in 24.6% of the single sperm analysed. We found a low instability of the normal alleles; the normal alleles from the individuals carrying a CAG repeat expansion were significantly more unstable than the normal alleles from the control individuals (P<0.001), indicating an interallelic interaction between the expanded and the normal alleles. Received: 8 June 1998 / Accepted: 10 September 1998     

Somatic expansion of the (CAG) n repeat in Huntington disease brains

The mutation causing Huntington disease (HD) has been identified as an expansion of a polymorphic (CAG) _n repeat in the 5 part of the huntingtin gene. The specific neuropathology of HD, viz. selective neuronal loss in the caudate nucleus and putamen, cannot be explained by the widespread expression of the gene. Since somatic expansion is observed in affected tissue in myotonic dystrophy, we have studied the length of the (CAG) _n repeat in various regions of the brain. Although we have not found clear differences when comparing severely and mildly affected regions, we have observed a minor increase in repeat length upon comparison of affected brain samples with cerebellum or peripheral blood. Hence, although further somatic amplification seems to occur in affected areas of the brain, the differences between affected and unaffected regions are too small to make this mechanism an obvious candidate for the cause of differential neuronal degeneration in HD.     

Fen1 does not control somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat in a knock-in mouse model for DM1

The mechanism of trinucleotide repeat expansion, an important cause of neuromuscular and neurodegenerative diseases, is poorly understood. We report here on the study of the role of flap endonuclease 1 (Fen1), a structure-specific nuclease with both 5' flap endonuclease and 5'-3' exonuclease activity, in the somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat of the DMPK gene in a mouse model for myotonic dystrophy type 1 (DM1). By intercrossing mice with Fen1 deficiency with transgenics with a DM1 (CTG)(n)*(CAG)(n) repeat (where 104n110), we demonstrate that Fen1 is not essential for faithful maintenance of this repeat in early embryonic cleavage divisions until the blastocyst stage. Additionally, we found that the frequency of somatic DM1 (CTG)(n)*(CAG)(n) repeat instability was essentially unaltered in mice with Fen1 haploinsufficiency up to 1.5 years of age. Based on these findings, we propose that Fen1, despite its role in DNA repair and replication, is not primarily involved in maintaining stability at the DM1 locus.     

Frequent germline mutations and somatic repeat instability in DNA mismatch-repair-deficient Caenorhabditis elegans

Mismatch-repair-deficient mutants were initially recognized as mutation-prone derivatives of bacteria, and later mismatch repair deficiency was found to predispose humans to colon cancers (HNPCC). We generated mismatch-repair-deficient Caenorhabditis elegans by deleting the msh-6 gene and analyzed the fidelity of transmission of genetic information to subsequent generations. msh-6-defective animals show an elevated level of spontaneous mutants in both the male and female germline; also repeated DNA tracts are unstable. To monitor DNA repeat instability in somatic tissue, we developed a sensitive system, making use of heat-shock promoter-driven lacZ transgenes, but with a repeat that puts this reporter gene out of frame. In genetic msh-6-deficient animals lacZ+ patches are observed as a result of somatic repeat instability. RNA interference by feeding wild-type animals dsRNA homologous to msh-2 or msh-6 also resulted in somatic DNA instability, as well as in germline mutagenesis, indicating that one can use C. elegans as a model system to discover genes involved in maintaining DNA stability by large-scale RNAi screens.     

Trinucleotide (CAG) repeat expansion in chromosomes of Spanish patients with Huntington's disease

Huntington's disease (HD) is a neurodegenerative and hereditary disease characterized by progressive movement disorders and mental and behavioral abnormalities. The HD gene is an expanding and unstable trinucleotide repeat (CAG repeat sequences). We studied 77 individuals from 38 families with HD in an attempt to obtain information for genetic counselling and differential diagnosis. Our results indicate that individuals with more than 40 repeats will be affected by the disease, whereas those with fewer than 30 will be healthy. There can be some overlap between 30 and 40 repeats, and one should be careful when interpreting these results.     

Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability

Mammalian prion diseases are fatal neurodegenerative disorders dependent on the prion protein PrP. Expansion of the oligopeptide repeats (ORE) found in PrP is associated with inherited prion diseases. Patients with ORE frequently harbor PrP aggregates, but other factors may contribute to pathology, as they often present with unexplained phenotypic variability. We created chimeric yeast-mammalian prion proteins to examine the influence of the PrP ORE on prion properties in yeast. Remarkably, all chimeric proteins maintained prion characteristics. The largest repeat expansion chimera displayed a higher propensity to maintain a self-propagating aggregated state. Strikingly, the repeat expansion conferred increased conformational flexibility, as observed by enhanced phenotypic variation. Furthermore, the repeat expansion chimera displayed an increased rate of prion conversion, but only in the presence of another aggregate, the [RNQ+] prion. We suggest that the PrP ORE increases the conformational flexibility of the prion protein, thereby enhancing the formation of multiple distinct aggregate structures and allowing more frequent prion conversion. Both of these characteristics may contribute to the phenotypic variability associated with PrP repeat expansion diseases.     

Structure and dynamics in DNA looped domains: CAG triplet repeat sequence dynamics probed by 2-aminopurine fluorescence

The triplet repeat sequence (CAG)n and related triplet repeats are associated with dynamic DNA mutations implicated in a number of debilitating human diseases. To gain insight into the dynamics of the (CAG)n repeat, we have substituted a single 2-aminopurine (2AP) fluorescent base for adenine at select positions within the 18 base looped domain of a (GC)3(CAG)6(GC)3 hairpin oligonucleotide. Using temperature-dependent steady-state fluorescence measurements in combination with time correlated photon counting spectroscopy, we show the conformation and dynamics of the C2APG domains to be strongly dependent on the position of the probe in the looped region. In other words, rather than being a uniform, single stranded loop, the (CAG)6 triplet repeat looped domain exhibits order and dynamics that are position dependent. The 2AP fluorescence dynamics within the C2APG repeat are well described by a 4 component exponential decay model, with lifetimes ranging from 5 ps to 4 ns. Differences in global DNA conformation (duplex, hairpin, single strand), as well as the local position of the probe within the loop of a given hairpin, predominantly are reflected in the relative amplitude rather than the lifetime of the probe. The time dependent 2AP anisotropy in the hairpin (CAG)n loops is sensitive to the position of the fluorescent base, with the fluorescence depolarization of a centrally located 2AP probe within the loop proceeding significantly more slowly than 2AP positioned at the 5'- or 3'-end of the repeat sequence near the loop-stem junction. These results are consistent with segmental motions of the CAG repeat, while also suggesting that the 2AP probe is significantly stacked, possibly even hydrogen bonded, within the partially structured CAG looped domain. Our results characterize the position-dependent and conformation-dependent dynamics and order within (CAG)n triplet repeat DNAs, properties of relevance to the biological mechanisms by which such domains can lead to disease states.     

Differential somatic CAG repeat instability in variable brain cell lineage in dentatorubral pallidoluysian atrophy (DRPLA): a laser-captured microdissection (LCM)-based analysis

Employing a laser-captured microdissection (LCM), we have investigated the somatic instability of CAG repeats in the variable brain cell lineage in three patients with dentatorubral pallidoluysian atrophy (DRPLA). LCM enables the isolation of single lineage brain cells for subsequent molecular analysis. We have found that CAG repeat size and the range of CAG repeats in the cerebellar granular cells is smaller than those in cerebellar glial cells. Similarly, those in the cerebral neuronal cells are significantly shorter than those in cerebral glial cells. These data directly indicate that the CAG repeat is relatively more stable in neuronal cells than in glial cells. Furthermore, cerebellar granular cells show significantly smaller main CAG repeat size and CAG repeat range than either Purkinje cells or cerebral neuronal cells, suggesting that somatic instability in the CAG repeat is markedly variable even among the different types of neuronal populations. The cell-specific CAG repeat instability may thus be more complex than has previously been considered. LCM is a powerful tool for elucidating the mechanism of the triplet repeat instability of each cell type.     

Chemically induced increases and decreases in the rate of expansion of a CAG*CTG triplet repeat

Somatic mosaicism of repeat length is prominent in repeat expansion disorders such as Huntington disease and myotonic dystrophy. Somatic mosaicism is age-dependent, tissue-specific and expansion-biased, and likely contributes toward the tissue-specificity and progressive nature of the symptoms. We propose that therapies targeted at somatic repeat expansion may have general utility in these disorders. Specifically, suppression of somatic expansion would be expected to be therapeutic, whilst reversion of the expanded mutant repeat to within the normal range would be predicted to be curative. However, the effects of genotoxic agents on the mutational properties of specific nuclear genes are notoriously difficult to define. Nonetheless, we have determined that chronic exposure over a three month period to a number of genotoxic agents can alter the rate of triplet repeat expansion in whole populations of mammalian cells. Interestingly, high doses of caffeine increased the rate of expansion by ~60%. More importantly, cytosine arabinoside, ethidium bromide, 5-azacytidine and aspirin all significantly reduced the rate of expansion by from 35 to 75%. These data establish that drug induced suppression of somatic expansion is possible. These data also suggest that highly unstable expanded simple sequence repeats may act as sensitive reporters of genotoxic assault in the soma.     

Uninterrupted CAG repeat drives striatum-selective transcriptionopathy and nuclear pathogenesis in human Huntingtin BAC mice

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CTG repeat instability and size variation timing in DNA repair-deficient mice

Type 1 myotonic dystrophy is caused by the expansion of an unstable CTG repeat in the DMPK gene. We have investigated the molecular mechanisms underlying the CTG repeat instability by crossing transgenic mice carrying >300 unstable CTG repeats in their human chromatin environment with mice knockout for genes involved in various DNA repair pathways: Msh2 (mismatch repair), Rad52 and Rad54 (homologous recombination) and DNA-PKcs (non-homologous end-joining). Genes of the non-homologous end-joining and homologous recombination pathways did not seem to affect repeat instability. Only lack of Rad52 led to a slight decrease in expansion range. Unexpectedly, the absence of Msh2 did not result in stabilization of the CTG repeats in our model. Instead, it shifted the instability towards contractions rather than expansions, both in tissues and through generations. Furthermore, we carefully analyzed repeat transmissions with different Msh2 genotypes to determine the timing of intergenerational instability. We found that instability over generations depends not only on parental germinal instability, but also on a second event taking place after fertilization.     

Quantitative lipidomics reveals age-dependent perturbations of whole-body lipid metabolism in ACBP deficient mice

The acyl-CoA binding protein (ACBP) plays a key role in chaperoning long-chain acyl-CoAs into lipid metabolic processes and acts as an important regulatory hub in mammalian physiology. This is highlighted by the recent finding that mice devoid of ACBP suffer from a compromised epidermal barrier and delayed weaning, the physiological process where newborns transit from a fat-based milk diet to a carbohydrate-rich diet. To gain insights into how ACBP impinges on weaning and the concomitant remodeling of whole-body lipid metabolism we performed a comparative lipidomics analysis charting the absolute abundance of 613 lipid molecules in liver, muscle and plasma from weaning and adult Acbp knockout and wild type mice. Our results reveal that ACBP deficiency affects primarily lipid metabolism of liver and plasma during weaning. Specifically, we show that ACBP deficient mice have elevated levels of hepatic cholesteryl esters, and that lipids featuring an 18:1 fatty acid moiety are increased in Acbp depleted mice across all tissues investigated. Our results also show that the perturbation of systemic lipid metabolism in Acbp knockout mice is transient and becomes normalized and similar to that of wild type as mice grow older. These findings demonstrate that ACBP serves crucial functions in maintaining lipid metabolic homeostasis in mice during weaning.     

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. Received: 7 October 1996 / Revised: 5 December 1996     

Phenotypic characterization of Bbs4 null mice reveals age-dependent penetrance and variable expressivity

Bardet-Biedl syndrome (BBS) is a rare oligogenic disorder exhibiting both clinical and genetic heterogeneity. Although the BBS phenotype is variable both between and within families, the syndrome is characterized by the hallmarks of developmental and learning difficulties, post-axial polydactylia, obesity, hypogenitalism, renal abnormalities, retinal dystrophy, and several less frequently observed features. Eleven genes mutated in BBS patients have been identified, and more are expected to exist, since about 20–30% of all families cannot be explained by the known loci. To investigate the etiopathogenesis of BBS, we created a mouse null for one of the murine homologues, Bbs4, to assess the contribution of one gene to the pleiotropic murine Bbs phenotype. Bbs4 null mice, although initially runted compared to their littermates, ultimately become obese in a gender-dependent manner, females earlier and with more severity than males. Blood chemistry tests indicated abnormal lipid profiles, signs of liver dysfunction, and elevated insulin and leptin levels reminiscent of metabolic syndrome. As in patients with BBS, we found age-dependent retinal dystrophy. Behavioral assessment revealed that mutant mice displayed more anxiety-related responses and reduced social dominance. We noted the rare occurrence of birth defects, including neural tube defects and hydrometrocolpos, in the null mice. Evaluations of these null mice have uncovered phenotypic features with age-dependent penetrance and variable expressivity, partially recapitulating the human BBS phenotype.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Identification of RTG2 as a modifier gene for CTG*CAG repeat instability in Saccharomyces cerevisiae

Trinucleotide repeats (TNRs) undergo frequent mutations in families affected by TNR diseases and in model organisms. Much of the instability is conferred in cis by the sequence and length of the triplet tract. Trans-acting factors also modulate TNR instability risk, on the basis of such evidence as parent-of-origin effects. To help identify trans-acting modifiers, a screen was performed to find yeast mutants with altered CTG.CAG repeat mutation frequencies. The RTG2 gene was identified as one such modifier. In rtg2 mutants, expansions of CTG.CAG repeats show a modest increase in rate, depending on the starting tract length. Surprisingly, contractions were suppressed in an rtg2 background. This creates a situation in a model system where expansions outnumber contractions, as in humans. The rtg2 phenotype was apparently specific for CTG.CAG repeat instability, since no changes in mutation rate were observed for dinucleotide repeats or at the CAN1 reporter gene. This feature sets rtg2 mutants apart from most other mutants that affect genetic stability both for TNRs and at other DNA sequences. It was also found that RTG2 acts independently of its normal partners RTG1 and RTG3, suggesting a novel function of RTG2 that helps modify CTG.CAG repeat mutation risk.     

ATM and ATR protect the genome against two different types of tandem repeat instability in Fragile X premutation mice

Expansion of a tandem repeat tract is responsible for the Repeat Expansion diseases, a group of more than 20 human genetic disorders that includes those like Fragile X (FX) syndrome that result from repeat expansion in the FMR1 gene. We have previously shown that the ATM and Rad3-related (ATR) checkpoint kinase protects the genome against one type of repeat expansion in a FX premutation mouse model. By crossing the FX premutation mice to Ataxia Telangiectasia-Mutated (Atm) mutant mice, we show here that ATM also prevents repeat expansion. However, our data suggest that the ATM-sensitive mechanism is different from the ATR-sensitive one. Specifically, the effect of the ATM deficiency is more marked when the premutation allele is paternally transmitted and expansions occur more frequently in male offspring regardless of the Atm genotype of the offspring. The gender effect is most consistent with a repair event occurring in the early embryo that is more efficient in females, perhaps as a result of the action of an X-linked DNA repair gene. Our data thus support the hypothesis that two different mechanisms of FX repeat expansion exist, an ATR-sensitive mechanism seen on maternal transmission and an ATM-sensitive mechanism that shows a male expansion bias.