Isolation of CAG/CTG repeats from within the chromosome 2p21-p24 locus for autosomal dominant spastic paraplegia (SPG4) by YAC fragmentation

Pure autosomal dominant spastic paraplegia (SPG) is a genetically heterogeneous neurodegenerative disorder of the central nervous system clinically characterized by progressive spasticity mainly affecting the lower limbs. Three distinct loci have been mapped to chromosomes 14q (SPG3), 2p (SPG4) and 15q (SPG6). In particular, SPG4 families show striking intrafamilial variability suggestive of anticipation and evidence has been provided that CAG/CTG repeat expansions may be involved. To isolate CAG/CTG repeat containing sequences from within the SPG4 candidate region, a novel approach was developed. Fragmentation vectors were assembled allowing direct fragmentation of yeast artificial chromosomes (YACs) with a short (> or = 21 bp) CAG/CTG sequence as the target site for homologous recombination. We used the CAG/CTG YAC fragmentation vectors to isolate CAG/CTG containing sequences from four YACs spanning the SPG4 candidate region between D2S400 and D2S367. A total of four CAG/CTG containing sequences were isolated of which three were novel. However, none of the four CAG/CTG repeats showed expanded alleles in two Belgian SPG4 families. In addition, we showed that the CAG/CTG alleles detected by the repeat expansion detection (RED) method could be fully explained by two polymorphic nonpathogenic CAG/CTG repeats on chromosomes 17 and 18, respectively. Also, the RED expansions in six SPG families could not be explained by amplification of the CAG/CTG repeats at the SPG4 locus. Together, our data do not support the hypothesis of a CAG/CTG repeat expansion as the molecular mechanism underlying SPG4 pathology.     

Expansion of 50 CAG/CTG repeats excluded in schizophrenia by application of a highly efficient approach using repeat expansion detection and a PCR screening set.

Studies of the transmission of schizophrenia in families with affected members in several generations have suggested that an expanded trinucleotide repeat mechanism may contribute to the genetic inheritance of this disorder. Using repeat expansion detection (RED), we and others have previously found that the distribution of CAG/CTG repeat size is larger in patients with schizophrenia than in controls. In an attempt to identify the specific expanded CAG/CTG locus or loci associated with schizophrenia, we have now used an approach based on a CAG/CTG PCR screening set combined with RED data. This has allowed us to minimize genotyping while excluding 43 polymorphic autosomal loci and 7 X-chromosomal loci from the screening set as candidates for expansion in schizophrenia with a very high degree of confidence.     

Search for unstable DNA in schizophrenia families with evidence for genetic anticipation.

Evidence for genetic anticipation has recently become an important subject of research in clinical psychiatric genetics. Renewed interest in anticipation was evoked by molecular genetic findings of a novel type of mutation termed "unstable DNA." The unstable DNA model can be construed as the "best fit" for schizophrenia twin and family epidemiological data. We have performed a large-scale Southern blot hybridization, asymmetrical PCR-based, and repeat expansion-detection screening for (CAG)n/(CTG)n and (CCG)n/(CGG)n expansions in eastern Canadian schizophrenia multiplex families demonstrating genetic anticipation. There were no differences in (CAG)n/(CTG)n and (CCG)n/(CGG)n pattern distribution either between affected and unaffected individuals or across generations. Our findings do not support the hypothesis that large (CAG)n/(CTG)n or (CCG)n/(CGG)n expansions are the major etiologic factor in schizophrenia. A separate set of experiments directed to the analysis of small (30-130 trinucleotides), Huntington disease-type expansions in individual genes is required in order to fully exclude the presence of (CAG)n/(CTG)n- or (CCG)n/(CGG)n-type unstable mutation.     

An unstable trinucleotide-repeat region on chromosome 13 implicated in spinocerebellar ataxia: a common expansion locus

Larger CAG/CTG trinucleotide-repeat tracts in individuals affected with schizophrenia (SCZ) and bipolar affective disorder (BPAD) in comparison with control individuals have previously been reported, implying a possible etiological role for trinucleotide repeats in these diseases. Two unstable CAG/CTG repeats, SEF2-1B and ERDA1, have recently been cloned, and studies indicate that the majority of individuals with large repeats as detected by repeat-expansion detection (RED) have large repeat alleles at these loci. These repeats do not show association of large alleles with either BPAD or SCZ. Using RED, we have identified a BPAD individual with a very large CAG/CTG repeat that is not due to expansion at SEF2-1B or ERDA1. From this individual's DNA, we have cloned a highly polymorphic trinucleotide repeat consisting of (CTA)n (CTG)n, which is very long ( approximately 1,800 bp) in this patient. The repeat region localizes to chromosome 13q21, within 1.2 cM of fragile site FRA13C. Repeat alleles in our sample were unstable in 13 (5.6%) of 231 meioses. Large alleles (>100 repeats) were observed in 14 (1. 25%) of 1,120 patients with psychosis, borderline personality disorder, or juvenile-onset depression and in 5 (.7%) of 710 healthy controls. Very large alleles were also detected for Centre d'Etude Polymorphisme Humaine (CEPH) reference family 1334. This triplet expansion has recently been reported to be the cause of spinocerebellar ataxia type 8 (SCA8); however, none of our large alleles above the disease threshold occurred in individuals either affected by SCA or with known family history of SCA. The high frequency of large alleles at this locus is inconsistent with the much rarer occurrence of SCA8. Thus, it seems unlikely that expansion alone causes SCA8; other genetic mechanisms may be necessary to explain SCA8 etiology.     

Haploinsufficiency of yeast FEN1 causes instability of expanded CAG/CTG tracts in a length-dependent manner

Trinucleotide repeat diseases, such as Huntington's disease, are caused by the expansion of trinucleotide repeats above a threshold of about 35 repeats. Once expanded, the repeats are unstable and tend to expand further both in somatic cells and during transmission, resulting in a more severe disease phenotype. Flap endonuclease 1 (Fen1), has an endonuclease activity specific for 5' flap structures and is involved in Okazaki fragment processing and base excision repair. Fen1 also plays an important role in preventing instability of CAG/CTG trinucleotide repeat sequences, as the expansion frequency of CAG/CTG repeats is increased in FEN1 mutants in vitro and in yeast cells defective for the yeast homolog, RAD27. Here we have tested whether one copy of yeast FEN1 is enough to maintain CAG/CTG tract stability in diploid yeast cells. We found that CAG/CTG repeats are stable in RAD27 +/- cells if the tract is 70 repeats long and exhibit a slightly increased expansion frequency if the tract is 85 or 130 repeats long. However for CAG-155 tracts, the repeat expansion frequency in RAD27 +/- cells is significantly higher than in RAD27 +/+ cells. This data indicates that cells containing longer CAG/CTG repeats need more Fen1 protein to maintain tract stability and that maintenance of long CAG/CTG repeats is particularly sensitive to Fen1 levels. Our results may explain the relatively small effects seen in the Huntington's disease (HD) FEN1 +/- heterozygous mice and myotonic dystrophy type 1 (DM1) FEN1 +/- heterozygous mice, and suggest that inefficient flap processing by Fen1 could play a role in the continued expansions seen in humans with trinucleotide repeat expansion diseases.     

Polymorphisms at 13 expressed human sequences containing CAG/CTG repeats and analysis in autosomal dominant cerebellar ataxia (ADCA) patients

Genetic anticipation – increasing severity and a decrease in the age of onset with successive generations of a pedigree – is clearly present in autosomal dominant cerebellar ataxia (ADCA). Anticipation is correlated with expansion of the CAG/CTG repeat sequence to sizes above those in the normal range through the generations of a pedigree. Genetic heterogeneity has been demonstrated for ADCA, with four cloned genes (SCA1, SCA2, SCA3/MJD, and SCA6) and three mapped loci (SCA4, SCA5 and SCA7). Another related dominant ataxia, dentatorubral-pallidoluysian atrophy (DRPLA), presents anticipation with CAG/CTG repeat expansions. We had previously analysed ADCA patients who had not shown repeat expansions in cloned genes for CAG/CTG repeat expansions by the repeat expansion detection method (RED) and had detected expansions of between 48 and 88 units in 17 unrelated familial cases. We present here an analysis of 13 genes and expressed sequence tags (ESTs) containing 10 or more CAG/ CTG repeat sequences selected from public databases in the 17 unrelated ADCA patients. Of the 13 selected genes and ESTs, 9 were found to be polymorphic with heterozygosities ranging between 0.09 and 0.80 and 2 to 17 alleles. In ADCA patients none of the loci showed expansions above the normal range of the CAG/CTG repeat sequences, excluding them as the mutation causing ADCA. Received: 28 May 1997 / Accepted: 30 June 1997     

A novel trinucleotide repeat expansion at chromosome 3q26.2 identified by a CAG/CTG repeat expansion detection array

CAG/CTG repeat expansions cause at least 12 different neurological disorders, and additional disorders of this type probably exist. Using the repeat expansion detection (RED) assay, we identified an expanded CAG/CTG repeat in a 50-year-old woman with an autosomal dominant syndrome with prominent progressive sensory neuropathy. The expansion could not be accounted for by any of the CAG/CTG repeats known to undergo expansion. To identify the locus of the expansion, we created a PCR array to assess the repeat length of all repeats of eight or more CAG or CTG triplets in the human genome. The expansion was localized to a repeat contained in an intron of a Genscan-predicted gene, 185 nt downstream of a predicted exon that is conserved through mouse. The closest experimentally verified gene in the region (TNIK, encoding a serine/threonine kinase) occurs approximately 63 Kb downstream from the repeat. The length of the expansion in the proband is 98 triplets. This repeat is not expanded in the proband’s cousin (the only other affected family member for whom DNA is currently available) and no expansions were detected in a set of 230 patients with movement disorders of unknown cause. An expanded allele containing 58 triplets was detected in a single control individual, and no other expansions were detected in a set of 255 controls. The normal repeat length ranges from 5 to 30 triplets, with 8 triplets the most common allele. Our results suggest that this new repeat expansion is probably not the direct cause of the phenotype in the proband. Whether the repeat contributes to the patient’s phenotype, or is associated with another phenotype, remains to be determined.Electronic Supplementary Material Supplementary material is available for this article at .     

Detection of triplet repeat expansion in the human genome by use of hybridization signal intensity

Triplet repeat disease is a group of hereditary neurodegenerative disorders caused by expansion of trinucleotide repeats such as CAG/CTG, CGG/CCG, and GAA/TTC. Direct detection of the expansion in the patient's genome shortcuts the tedious process needed for identification of disease genes by conventional approaches. Here we describe a method to detect triplet repeat expansion from the hybridization signal intensity. Using a digoxigenin-labeled (CTG)9 probe, the hybridization intensity and number of repeats showed a good linear correlation. The technique detected expansion in genomic DNA in all cases with moderate or large expansion. Even in the case of a small expansion, this method could detect the mutant fragment. The technique has advantages over related techniques because it is more sensitive and can be applied to cases where a small repeat expansion is involved.     

In vitro (CTG)*(CAG) expansions and deletions by human cell extracts

The mechanism of disease-associated (CTG)*(CAG) expansion may involve DNA replication slippage, replication direction, Okazaki fragment processing, recombination, or repair. A length-dependent bias for expansions is observed in humans affected by a trinucleotide repeat-associated disease. We developed an assay to test the effect of replication direction on (CTG)*(CAG) instabilities incurred during in vitro (SV40) DNA replication mediated by human cell extracts. This system recapitulates the bias for expansions observed in humans. Replication by HeLa cell extracts generated expansions and deletions that depended upon repeat tract length and the direction of replication. Templates with 79 repeats yielded predominantly expansions (CAG as lagging strand template) or predominantly deletions (CTG as lagging strand template). Templates containing 17 repeats were stable. Thus, replication direction determined the type of mutation. These results provide new insights into the orientation of replication effect upon repeat stability. This system will be useful in determining the contribution of specific human proteins to (CTG)*(CAG) expansions.     

Replication and expansion of trinucleotide repeats in yeast

The mechanisms of trinucleotide repeat expansions, underlying more than a dozen hereditary neurological disorders, are yet to be understood. Here we looked at the replication of (CGG)(n) x (CCG)(n) and (CAG)(n) x (CTG)(n) repeats and their propensity to expand in Saccharomyces cerevisiae. Using electrophoretic analysis of replication intermediates, we found that (CGG)(n) x (CCG)(n) repeats significantly attenuate replication fork progression. Replication inhibition for this sequence becomes evident at as few as approximately 10 repeats and reaches a maximal level at 30 to 40 repeats. This is the first direct demonstration of replication attenuation by a triplet repeat in a eukaryotic system in vivo. For (CAG)(n) x (CTG)(n) repeats, on the contrary, there is only a marginal replication inhibition even at 80 repeats. The propensity of trinucleotide repeats to expand was evaluated in a parallel genetic study. In wild-type cells, expansions of (CGG)(25) x (CCG)(25) and (CAG)(25) x (CTG)(25) repeat tracts occurred with similar low rates. A mutation in the large subunit of the replicative replication factor C complex (rfc1-1) increased the expansion rate for the (CGG)(25) repeat approximately 50-fold but had a much smaller effect on the expansion of the (CTG)(25) repeat. These data show dramatic sequence-specific expansion effects due to a mutation in the lagging strand DNA synthesis machinery. Together, the results of this study suggest that expansions are likely to result when the replication fork attempts to escape from the stall site.     

Genetic anticipation in Portuguese kindreds with familial amyloidotic polyneuropathy is unlikely to be caused by triplet repeat expansions

Familial amyloidotic polyneuropathy (FAP) is a lethal autosomal dominant type of amyloidosis resulting from the deposition of transthyretin (ATTR) variants in the peripheral and autonomic nervous systems. ATTR V30M-associated FAP exhibits marked genetic anticipation in some families, with clinical symptoms developing at an earlier age in successive generations. The genetic basis of this phenomenon in FAP is unknown. Anticipation has been associated with the dynamic expansion of trinucleotide repeats in several neurodegenerative disorders, such as Huntington disease, myotonic dystrophy, and fragile X syndrome. We have used the repeat expansion detection (RED) assay to screen affected members of Portuguese FAP kindreds for expansion of any of the ten possible trinucleotide repeats. Nine generational pairs with differences in their age of onset greater than 12 years and a control pair with identical ages of onset were tested. No major differences were found in the lengths of the ten trinucleotide repeats analyzed. The distribution of the maximal repeat sizes was consistent with reported studies in unrelated individuals with no known genetic disease. The present data do not support a role for trinucleotide repeat expansions as the molecular mechanism underlying anticipation in Portuguese FAP. Received: 13 December 1998 / Accepted: 23 March 1999     

A SCA7 CAG/CTG repeat expansion is stable in Drosophila melanogaster despite modulation of genomic context and gene dosage

CAG and CTG repeat expansions are the cause of at least a dozen inherited neurological disorders. In these so-called "dynamic mutation" diseases, the expanded repeats display dramatic genetic instability, changing in size when transmitted through the germline and within somatic tissues. As the molecular basis of the repeat instability process remains poorly understood, modeling of repeat instability in model organisms has provided some insights into potentially involved factors, implicating especially replication and repair pathways. Studies in mice have also shown that the genomic context of the repeat sequence is required for CAG/CTG repeat instability in the case of spinocerebellar ataxia type 7 (SCA7), one of the most unstable of all CAG/CTG repeat disease loci. While most studies of repeat instability have taken a candidate gene approach, unbiased screens for factors involved in trinucleotide repeat instability have been lacking. We therefore attempted to use Drosophila melanogaster to model expanded CAG repeat instability by creating transgenic flies carrying trinucleotide repeat expansions, deriving flies with SCA7 CAG90 repeats in cDNA and genomic context. We found that SCA7 CAG90 repeats are stable in Drosophila, regardless of context. To screen for genes whose reduced function might destabilize expanded CAG repeat tracts in Drosophila, we crossed the SCA7 CAG90 repeat flies with various deficiency stocks, including lines lacking genes encoding the orthologues of flap endonuclease-1, PCNA, and MutS. In all cases, perfect repeat stability was preserved, suggesting that Drosophila may not be a suitable system for determining the molecular basis of SCA7 CAG repeat instability.     

三核苷酸重复序列失稳定机制:重复序列形成non-B二级结构的必要性和细胞反式作用因子的作用

与三核苷酸重复序列CAG.CTG、CGG·CCG和GAA·TTC扩增和缺失有关的分子机制尚不能得到清楚的阐释.体外研究表明,上述疾病相关的重复序列可以在体外形成non-B二级结构,并介导重复序列扩增.然而,迄今为止,类似的观察尚未在体内研究过程中得以实现.利用模型生物大肠杆菌和酵母等进行的有关研究并不能模拟三核苷酸重复序列的扩增,这暗示三核苷酸重复序列的体内扩增可能与重复序列形成non-B二级结构关联性并不大.尽管理论上较长的三核苷酸重复序列可以在复制和后复制过程中较易形成non-B DNA二级结构,但这样的二级结构倾向于导致重复序列出现"脆性",而不是扩增.事实上,患者所具有的三核苷酸重复序列扩增并非一定需要通过non-B二级结构的介导,这些重复序列的扩增是可以通过一种RNA转录诱导的局部DNA重复序列的复制和其后的DNA重排得以发生.     

Highly Specific Contractions of a Single CAG/CTG Trinucleotide Repeat by TALEN in Yeast

Trinucleotide repeat expansions are responsible for more than two dozens severe neurological disorders in humans. A double-strand break between two short CAG/CTG trinucleotide repeats was formerly shown to induce a high frequency of repeat contractions in yeast. Here, using a dedicated TALEN, we show that induction of a double-strand break into a CAG/CTG trinucleotide repeat in heterozygous yeast diploid cells results in gene conversion of the repeat tract with near 100% efficacy, deleting the repeat tract. Induction of the same TALEN in homozygous yeast diploids leads to contractions of both repeats to a final length of 3–13 triplets, with 100% efficacy in cells that survived the double-strand breaks. Whole-genome sequencing of surviving yeast cells shows that the TALEN does not increase mutation rate. No other CAG/CTG repeat of the yeast genome showed any length alteration or mutation. No large genomic rearrangement such as aneuploidy, segmental duplication or translocation was detected. It is the first demonstration that induction of a TALEN in an eukaryotic cell leads to shortening of trinucleotide repeat tracts to lengths below pathological thresholds in humans, with 100% efficacy and very high specificity.     

CTG trinucleotide repeat "big jumps": large expansions, small mice

Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including fragile X mental retardation, Huntington disease, and myotonic dystrophy type 1. Disease severity and age of onset are critically linked to expansion size. Previous mouse models of repeat instability have not recreated large intergenerational expansions ("big jumps"), observed when the repeat is transmitted from one generation to the next, and have never attained the very large tract lengths possible in humans. Here, we describe dramatic intergenerational CTG*CAG repeat expansions of several hundred repeats in a transgenic mouse model of myotonic dystrophy type 1, resulting in increasingly severe phenotypic and molecular abnormalities. Homozygous mice carrying over 700 trinucleotide repeats on both alleles display severely reduced body size and splicing abnormalities, notably in the central nervous system. Our findings demonstrate that large intergenerational trinucleotide repeat expansions can be recreated in mice, and endorse the use of transgenic mouse models to refine our understanding of triplet repeat expansion and the resulting pathogenesis.     

Spinocerebellar ataxia type 1 and Machado-Joseph disease: incidence of CAG expansions among adult-onset ataxia patients from 311 families with dominant, recessive, or sporadic ataxia.

The ataxias are a complex group of diseases with both environmental and genetic causes. Among the autosomal dominant forms of ataxia the genes for two, spinocerebellar ataxia type 1 (SCA1) and Machado-Joseph disease (MJD), have been isolated. In both of these disorders the molecular basis of disease is the expansion of an unstable CAG trinucleotide repeat. To assess the frequency of the SCA1 and MJD trinucleotide repeat expansions among individuals diagnosed with ataxia we have collected DNA from individuals representing 311 families with adult-onset ataxia of unknown etiology and screened these samples for trinucleotide repeat expansions within the SCA1 and MJD genes. Within this group there are 149 families with dominantly inherited ataxia. Of these, 3% had SCA1 trinucleotide repeat expansions, whereas 21% were positive for the MJD trinucleotide expansion. Thus, together SCA1 and MJD represent 24% of the autosomal dominant ataxias in our group, and the frequency of MJD is substantially greater than that of SCA1. For the 57 patients with MJD trinucleotide repeat expansions, a strong inverse correlation between CAG repeat size and age at onset was observed (r = -.838). Among the MJD patients, the normal and affected ranges of CAG repeat size are 14-40 and 68-82 repeats, respectively. For SCA1 the normal and affected ranges are much closer, containing 19-38 and 40-81 CAG repeats, respectively.     

Structure-Forming CAG/CTG Repeat Sequences are Sensitive to Breakage in the Absence of Mrc1 Checkpoint Function and S-Phase Checkpoint Signaling: Implications for Trinucleotide Repeat Expansion Diseases

Expansion of trinucleotide repeat sequences is the cause of multiple inherited human genetic diseases including Huntington’s disease and myotonic dystrophy. CTG and CAG repeats have been shown to form stable secondary structures that can impair Okazaki fragment processing and may impede replication fork progression. We recently showed that mutation of DNA damage checkpoint proteins results in increased chromosome breaks at expanded CAG/CTG repeats and in increased repeat instability (expansions and contractions).1 Here we report that long CAG~155 tracts are especially sensitive to absence of Mrc1 (Claspin) checkpoint function, implicating the S-phase checkpoint in maintenance of trinucleotide repeats and other secondary-structure forming sequences. Based on all of our results, we propose a model for the detection of different types of structures by different checkpoint signaling pathways.     

In vitro repair of DNA hairpins containing various numbers of CAG/CTG trinucleotide repeats

Expansion of CAG/CTG trinucleotide repeats (TNRs) in humans is associated with a number of neurological and neurodegenerative disorders including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism leads to TNR instability. However, the molecular mechanism by which cells recognize and repair CAG/CTG hairpins is largely unknown. Recent studies have identified a novel DNA repair pathway specifically removing (CAG)(n)/(CTG)(n) hairpins, which is considered a major mechanism responsible for TNR instability. The hairpin repair (HPR) system targets the repeat tracts for incisions in the nicked strand in an error-free manner. To determine the substrate spectrum of the HPR system and its ability to process smaller hairpins, which may be the intermediates for CAG/CTG expansions, we constructed a series of CAG/CTG hairpin heteroduplexes containing different numbers of repeats (from 5 to 25) and examined their repair in human nuclear extracts. We show here that although repair efficiencies differ slightly among these substrates, removal of the individual hairpin structures all involve endonucleolytic incisions within the repeat tracts in the nicked DNA strand. Analysis of the repair intermediates defined specific incision sites for each substrate, which were all located within the repeat regions. Mismatch repair proteins are not required for, nor do they inhibit, the processing of smaller hairpin structures. These results suggest that the HPR system ensures CAG/CTG stability primarily by removing various sizes of (CAG)(n)/(CTG)(n) hairpin structures during DNA metabolism.     

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.