Counting CAG repeats in the Huntington's disease gene by restriction endonuclease EcoP15I cleavage

Huntington's disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant inheritance. The disease is caused by a CAG trinucleotide repeat expansion located in the first exon of the HD gene. The CAG repeat is highly polymorphic and varies from 6 to 37 repeats on chromosomes of unaffected individuals and from more than 30 to 180 repeats on chromosomes of HD patients. In this study, we show that the number of CAG repeats in the HD gene can be determined by restriction of the DNA with the endonuclease EcoP15I and subsequent analysis of the restriction fragment pattern by electrophoresis through non-denaturing polyacrylamide gels using the ALFexpress DNA Analysis System. CAG repeat numbers in the normal (30 and 35 repeats) as well as in the pathological range (81 repeats) could be accurately counted using this assay. Our results suggest that this high-resolution method can be used for the exact length determination of CAG repeats in HD genes as well as in genes affected in related CAG repeat disorders.     

Counting CAG repeats in the Huntington’s disease gene by restriction endonuclease EcoP15I cleavage

Huntington’s disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant inheritance. The disease is caused by a CAG trinucleotide repeat expansion located in the first exon of the HD gene. The CAG repeat is highly polymorphic and varies from 6 to 37 repeats on chromosomes of unaffected individuals and from more than 30 to 180 repeats on chromosomes of HD patients. In this study, we show that the number of CAG repeats in the HD gene can be determined by restriction of the DNA with the endonuclease EcoP15I and subsequent analysis of the restriction fragment pattern by electrophoresis through non-denaturing polyacrylamide gels using the ALFexpress DNA Analysis System. CAG repeat numbers in the normal (30 and 35 repeats) as well as in the pathological range (81 repeats) could be accurately counted using this assay. Our results suggest that this high-resolution method can be used for the exact length determination of CAG repeats in HD genes as well as in genes affected in related CAG repeat disorders.     

Small-molecule ligand induces nucleotide flipping in (CAG)n trinucleotide repeats

DNA trinucleotide repeats, particularly CXG, are common within the human genome. However, expansion of trinucleotide repeats is associated with a number of disorders, including Huntington disease, spinobulbar muscular atrophy and spinocerebellar ataxia. In these cases, the repeat length is known to correlate with decreased age of onset and disease severity. Repeat expansion of (CAG)n, (CTG)n and (CGG)n trinucleotides may be related to the increased stability of alternative DNA hairpin structures consisting of CXG-CXG triads with X-X mismatches. Small-molecule ligands that selectively bound to CAG repeats could provide an important probe for determining repeat length and an important tool for investigating the in vivo repeat extension mechanism. Here we report that napthyridine-azaquinolone (NA, 1) is a ligand for CAG repeats and can be used as a diagnostic tool for determining repeat length. We show by NMR spectroscopy that binding of NA to CAG repeats induces the extrusion of a cytidine nucleotide from the DNA helix.     

A novel primer extension method to detect the number of CAG repeats in the androgen receptor gene in families with X-linked spinal and bulbar muscular atrophy.

X-linked spinal and bulbar muscular atrophy (SBMA), an adult-onset form of motor neuron disease, was recently reported to be caused by amplification of the CAG repeats in the androgen receptor gene. We report here a simple and rapid strategy to detect the precise number of the CAGs. After the DNA fragment containing the CAG repeats is amplified by the polymerase chain reaction, a primer extension is carried out; the extension of the end-labelled reverse primer adjacent to 3' end of CAG repeats stops at the first T after CAG repeats with the incorporation of dideoxy ATP in the reaction mixture. The resultant primer products are analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. This method could be quite useful to detect not only CAG repeats in SBMA but also other polymorphic dinucleotide and trinucleotide repeats.     

Expanded CAG/CTG repeat DNA induces a checkpoint response that impacts cell proliferation in Saccharomyces cerevisiae

Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases.     

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.     

Effects of temperature, Mg2+ concentration and mismatches on triplet-repeat expansion during DNA replication in vitro.

The human genome contains many simple tandem repeats that are widely dispersed and highly polymorphic. At least one group of simple tandem repeats, the DNA trinucleotide repeats, can dramaticallyexpand in size during transmission from one generation to the next to cause disease by a process known as dynamic mutation. We investigated the ability of trinucleotide repeats AAT and CAG to expand in size during DNA replication using a minimal in vitro system composed of the repeat tract, with and without unique flanking sequences, and DNA polymerase. Varying Mg2+concentration and temperature gave dramatic expansions of repeat size during DNA replication in vitro. Expansions of up to 1000-fold were observed. Mismatches partially stabilized the repeat tracts against expansion. Expansions were only detected when the primer was complementary to the repeat tract rather than the flanking sequence. The results imply that cellular environment and whether the growing strand contains a nick or gap are important factors for the expansion process in vivo.     

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.     

No evidence of expansion of CAG or GAA repeats in schizophrenia families and monozygotic twins

Many diseases caused by trinucleotide expansion exhibit increased severity and decreased age of onset (genetic anticipation) in successive generations. Apparent evidence of genetic anticipation in schizophrenia has led to a search for trinucleotide repeat expansions. We have used several techniques, including Southern blot hybridization, repeat expansion detection (RED) and locus-specific PCR to search for expanded CAG/CTG repeats in 12 families from the United Kingdom and 11 from Iceland that are multiplex for schizophrenia and demonstrate anticipation. The unstable DNA theory could also explain discordance of phenotype for schizophrenia in pairs of monozygotic twins, where the affected twin has a greater number of repeats than the unaffected twin. We used these techniques to look for evidence of different CAG/CTG repeat size in 27 pairs of monozygotic twins who are either concordant or discordant for schizophrenia. We have found no evidence of an increase in CAG/CTG repeat size for affected members in the families, or for the affected twins in the MZ twin sample. Southern hybridization and RED analysis were also performed for the twin and family samples to look for evidence of expansion of GAA/TTC repeats. However, no evidence of expansion was found in either sample. Whilst these results suggest that these repeats are not involved in the etiology of schizophrenia, the techniques used for detecting repeat expansions have limits to their sensitivity. The involvement of other trinucleotide repeats or other expandable repeat sequences cannot be ruled out. Received: 8 September 1997 / Accepted: 13 March 1998     

Premutation huntingtin allele adopts a non-B conformation and contains a hot spot for DNA damage

The expansion of a CAG trinucleotide repeat (TNR) sequence has been linked to several neurological disorders, for example, Huntington's disease (HD). In HD, healthy individuals have 5-35 CAG repeats. Those with 36-39 repeats have the premutation allele, which is known to be prone to expansion. In the disease state, greater than 40 repeats are present. Interestingly, the formation of non-B DNA conformations by the TNR sequence is proposed to contribute to the expansion. Here we provide the first structural and thermodynamic analysis of a premutation length TNR sequence. Using chemical probes of nucleobase accessibility, we found that similar to (CAG)(10), the premutation length sequence (CAG)(36) forms a stem-loop hairpin and contains a hot spot for DNA damage. Additionally, calorimetric analysis of a series of (CAG)(n) sequences, that includes repeat tracts in both the healthy and premutation ranges, reveal that thermodynamic stability increases linearly with the number of repeats. Based on these data, we propose that while non-B conformations can be formed by TNR tracts found in both the healthy and premutation allele, only sequences containing at least 36 repeats have sufficient thermodynamic stability to contribute to expansion.     

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.     

Repair in haploid male germ cells occurs late in differentiation as chromatin is condensing

Huntington's Disease (HD) is one of eight progressive neurodegenerative disorders in which the underlying mutation is a CAG expansion encoding a polyglutamine tract. The mechanism of trinucleotide expansion remains poorly understood. We have followed heritable changes in CAG length in male transgenic mice. In germ cells, expansion is limited to the post-meiotic, haploid cell and therefore cannot involve mitotic replication or recombination between a homologous chromosome and a sister chromatid. Expansion occurs by gap filling synthesis when DNA loops comprising the CAG trinucleotide repeats are sealed into the DNA strand. Our data support a model in which expansion occurs late in male germ cell development as spermatids are entering the epididymis at a time when chromatin is condensing. These data indicate that repair can be carried out in germ cells as long as the DNA is accessible. The capacity for repair of germ cells may have important implications for future gene therapy.     

PCR片段测序和基因分型两种方法对Kennedy遗传病的诊断

X连锁脊延髓肌萎缩症（SBMA）或肯尼迪病是一种成年人发病的神经变性疾病,以肌无力与慢性、进行性肌萎缩为特征. 通过PCR片段测序和基因分型法准确检测雄激素受体（AR）基因CAG复制数目,兄弟俩（来自同一个中国家庭）被确诊为隐性遗传性SBMA. 为了得到该中国家庭SMBA家系人员AR基因的CAG复制数目,我们采用了PCR片段测序和基因分型两种方法. 在该SMBA家系中有两个已发病的成年男性、未发病的年轻男性,及女性基因携带者. 两个已发病男性患者AR基因中CAG三核苷酸串重复数目分别是48和45. 以前的研究表明特定三核苷酸串重复数目的扩增可导致人类遗传性神经障碍疾病发病。我们的研究结果完全支持这一观点,SMBA中国家系的三核苷酸CAG拷贝数目检测结果表明,AR基因CAG扩增数目与SMBA发病相关. 关键词雄性激素受体; CAG多重三核苷酸重复; 肯尼迪病; 脊延髓肌萎缩症; X连锁     

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.     

Structure of even/odd trinucleotide repeat sequences modulates persistence of non-B conformations and conversion to duplex

Expansion of trinucleotide repeats (TNR) has been implicated in the emergence of neurodegenerative diseases. Formation of non-B conformations such as hairpins by these repeat sequences during DNA replication and/or repair has been proposed as a contributing factor to expansion. In this work we employed a combination of fluorescence, chemical probing, optical melting, and gel shift assays to characterize the structure of a series of (CTG)(n) sequences and the kinetic parameters describing their interaction with a complementary sequence. Our structure-based experiments using chemical probing reveal that sequences containing an even or odd number of CTG repeats adopt stem-loop hairpins that differ from one another by the absence or presence of a stem overhang. Furthermore, we find that this structural difference dictates the rate at which the TNR hairpins convert to duplex with a complementary CAG sequence. Indeed, the rate constant describing conversion to (CAG)(10)/(CTG)(n) duplex is slower for sequences containing an even number of CTG repeats than for sequences containing an odd number of repeats. Thus, when both the CAG and CTG hairpins have an even number of the repeats, they display a longer lifetime relative to when the CTG hairpin has an odd number of repeats. The difference in lifetimes observed for these TNR hairpins has implications toward their persistence during DNA replication or repair events and could influence their predisposition toward expansion. Taken together, these results contribute to our understanding of trinucleotide repeats and the factors that regulate persistence of hairpins in these repetitive sequences and conversion to canonical duplex.     

PCR approach for detection of Fragile X syndrome and Huntington disease based on modified DNA: limits and utility

A group of mutations characterized by trinucleotide repeat expansion causes human diseases such as the Fragile X syndrome, Huntington disease (HD), and myotonic dystrophy. Methods based on PCR amplification of the CGG and CAG repeats region could facilitate the development of a rapid screening assay; unfortunately, amplification across CGG and CAG repeats can be inefficient and unreliable due to the G + C base composition. The utility of the PCR on modified DNA for amplification of the CGG and CAG repeats at the Fragile X syndrome and HD has been reported. In the present study, we analyzed the utility of PCR on modified DNA as a rapid screening method for diagnosis of patients with Fragile X syndrome and HD. A comparative analysis realized with 38 Fragile X and 29 HD patients showed that the molecular diagnosis by simple PCR on modified DNA has a sensitivity and specificity of 100% in Fragile X patients and 94.1% and 91.6% in HD patients. The results achieved from the statistical analysis allowed us to conclude that the amplification by simple PCR on modified DNA is a reliable and useful method for the molecular diagnosis of the Fragile X syndrome, but not for the HD.     

Twisting Right to Left: A…A Mismatch in a CAG Trinucleotide Repeat Overexpansion Provokes Left-Handed Z-DNA Conformation

Conformational polymorphism of DNA is a major causative factor behind several incurable trinucleotide repeat expansion disorders that arise from overexpansion of trinucleotide repeats located in coding/non-coding regions of specific genes. Hairpin DNA structures that are formed due to overexpansion of CAG repeat lead to Huntington’s disorder and spinocerebellar ataxias. Nonetheless, DNA hairpin stem structure that generally embraces B-form with canonical base pairs is poorly understood in the context of periodic noncanonical A…A mismatch as found in CAG repeat overexpansion. Molecular dynamics simulations on DNA hairpin stems containing A…A mismatches in a CAG repeat overexpansion show that A…A dictates local Z-form irrespective of starting glycosyl conformation, in sharp contrast to canonical DNA duplex. Transition from B-to-Z is due to the mechanistic effect that originates from its pronounced nonisostericity with flanking canonical base pairs facilitated by base extrusion, backbone and/or base flipping. Based on these structural insights we envisage that such an unusual DNA structure of the CAG hairpin stem may have a role in disease pathogenesis. As this is the first study that delineates the influence of a single A…A mismatch in reversing DNA helicity, it would further have an impact on understanding DNA mismatch repair.     

Trinucleotide repeat instability: a hairpin curve at the crossroads of replication, recombination, and repair

The trinucleotide repeats that expand to cause human disease form hairpin structures in vitro that are proposed to be the major source of their genetic instability in vivo. If a replication fork is a train speeding along a track of double-stranded DNA, the trinucleotide repeats are a hairpin curve in the track. Experiments have demonstrated that the train can become derailed at the hairpin curve, resulting in significant damage to the track. Repair of the track often results in contractions and expansions of track length. In this review we introduce the in vitro evidence for why CTG/CAG and CCG/CGG repeats are inherently unstable and discuss how experiments in model organisms have implicated the replication, recombination and repair machinery as contributors to trinucleotide repeat instability in vivo.