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.     

Null alleles at the Huntington disease locus: implications for diagnostics and CAG repeat instability

PCR amplification of the CAG repeat in exon 1 of the IT15 gene is routinely undertaken to confirm a clinical diagnosis of Huntington disease (HD) and to provide predictive testing for at-risk relatives of affected individuals. Our studies have detected null alleles on the chromosome carrying the expanded repeat in three of 91 apparently unrelated HD families. Sequence analysis of these alleles has revealed the same mutation event, leading to the juxtaposition of uninterrupted CAG and CCG repeats. These data suggest that a mutation-prone region exists in the IT15 gene bounded by the CAG and CCG repeats and that caution should be exercised in designing primers that anneal to the region bounded by these repeats. Two of the HD families segregated null alleles with expanded uninterrupted CAG repeats at the lower end of the zone of reduced penetrance. The expanded repeats are meiotically unstable in these families, although this instability is within a small range of repeat lengths. The haplotypes of the disease-causing chromosomes in these two families differ, only one of which is similar to that reported previously as being specific for new HD mutations. Finally, no apparent mitotic instability of the uninterrupted CAG repeat was observed in the brain of one of the HD individuals.     

Phenotypic Characterization of Individuals with 30–40 CAG Repeats in the Huntington Disease (HD) Gene Reveals HD Cases with 36 Repeats and Apparently Normal Elderly Individuals with 36–39 Repeats

Abnormal CAG expansions in the IT-15 gene are associated with Huntington disease (HD). In the diagnostic setting it is necessary to define the limits of the CAG size ranges on normal and HD-associated chromosomes. Most large analyses that defined the limits of the normal and pathological size ranges employed PCR assays, which included the CAG repeats and a CCG repeat tract that was thought to be invariant. Many of these experiments found an overlap between the normal and disease size ranges. Subsequent findings that the CCG repeats vary by 8 trinucleotide lengths suggested that the limits of the normal and disease size ranges should be reevaluated with assays that exclude the CCG polymorphism. Since patients with between 30 and 40 repeats are rare, a consortium was assembled to collect such individuals.All 178 samples were reanalyzed in Cambridge by using assays specific for the CAG repeats. We have optimized methods for reliable sizing of CAG repeats and show cases that demonstrate the dangers of using PCR assays that include both the CAG and CCG polymorphisms. Seven HD patients had 36 repeats, which confirms that this allele is associated with disease. Individuals without apparent symptoms or signs of HD were found at 36 repeats (aged 74, 78, 79, and 87 years), 37 repeats (aged 69 years), 38 repeats (aged 69 and 90 years), and 39 repeats (aged 67, 90, and 95 years). The detailed case histories of an exceptional case from this series will be presented: a 95-year-old man with 39 repeats who did not have classical features of HD. The apparently healthy survival into old age of some individuals with 36–39 repeats suggests that the HD mutation may not always be fully penetrant.     

Sex-dependent mechanisms for expansions and contractions of the CAG repeat on affected Huntington disease chromosomes.

A total of 254 affected parent-child pairs with Huntington disease (HD) and 440 parent-child pairs with CAG size in the normal range were assessed to determine the nature and frequency of intergenerational CAG changes in the HD gene. Intergenerational CAG changes are extremely rare (3/440 [0.68%]) on normal chromosomes. In contrast, on HD chromosomes, changes in CAG size occur in approximately 70% of meioses on HD chromosomes, with expansions accounting for 73% of these changes. These intergenerational CAG changes make a significant but minor contribution to changes in age at onset (r2 = .19). The size of the CAG repeat influenced larger intergenerational expansions (> 7 CAG repeats), but the likelihood of smaller expansions or contractions was not influenced by CAG size. Large expansions (> 7 CAG repeats) occur almost exclusively through paternal transmission (0.96%; P < 10(-7)), while offspring of affected mothers are more likely to show no change (P = .01) or contractions in CAG size (P = .002). This study demonstrates that sex of the transmitting parent is the major determinant for CAG intergenerational changes in the HD gene. Similar paternal sex effects are seen in the evolution of new mutations for HD from intermediate alleles and for large expansions on affected chromosomes. Affected mothers almost never transmit a significantly expanded CAG repeat, despite the fact that many have similar large-sized alleles, compared with affected fathers. The sex-dependent effects of major expansion and contractions of the CAG repeat in the HD gene implicate different effects of gametogenesis, in males versus females, on intergenerational CAG repeat stability.     

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.     

Quantification of age-dependent somatic CAG repeat instability in Hdh CAG knock-in mice reveals different expansion dynamics in striatum and liver

Background

Age at onset of Huntington''s disease (HD) is largely determined by the CAG trinucleotide repeat length in the HTT gene. Importantly, the CAG repeat undergoes tissue-specific somatic instability, prevalent in brain regions that are disease targets, suggesting a potential role for somatic CAG repeat instability in modifying HD pathogenesis. Thus, understanding underlying mechanisms of somatic CAG repeat instability may lead to discoveries of novel therapeutics for HD. Investigation of the dynamics of the CAG repeat size changes over time may provide insights into the mechanisms underlying CAG repeat instability.

Methodology/Principal Findings

To understand how the HTT CAG repeat length changes over time, we quantified somatic instability of the CAG repeat in Huntington''s disease CAG knock-in mice from 2–16 months of age in liver, striatum, spleen and tail. The HTT CAG repeat in spleen and tail was very stable, but that in liver and striatum expanded over time at an average rate of one CAG per month. Interestingly, the patterns of repeat instability were different between liver and striatum. Unstable CAG repeats in liver repeatedly gained similar sizes of additional CAG repeats (approximately two CAGs per month), maintaining a distinct population of unstable repeats. In contrast, unstable CAG repeats in striatum gained additional repeats with different sizes resulting in broadly distributed unstable CAG repeats. Expanded CAG repeats in the liver were highly enriched in polyploid hepatocytes, suggesting that the pattern of liver instability may reflect the restriction of the unstable repeats to a unique cell type.

Conclusions/Significance

Our results are consistent with repeat expansion occurring as a consequence of recurrent small repeat insertions that differ in different tissues. Investigation of the specific mechanisms that underlie liver and striatal instability will contribute to our understanding of the relationship between instability and disease and the means to intervene in this process.     

Two polymorphic microsatellites in a coding segment of the canine androgen receptor gene

A 0.6-kb segment of exon 1 of the canine androgen receptor gene contains two polymorphic CAG tandem repeats which encode strings of glutamine homopolymers. The number of CAGs in each tandem repeat was determined by (1) polymerase chain reaction (PCR) amplification of a gene segment containing both repeats, (2) cleavage between repeats with restriction enzyme EcoO109I and (3) fractionation of the restriction fragments containing individual CAG repeats by denaturing polyacrylamide gel electrophoresis (PAGE). Individual genomic DNA samples from 80 unrelated dogs (53 males plus 27 females for a total of 107 X chromosomes) contained 10–12 CAGs in the 5′ repeats and 10–13 CAGs in the 3′ repeats. Thirteen distinct androgen receptor genotypes were identified. Eleven (or 41%) of the 27 unrelated females were heterozygous in one or both repeat regions, whereas all male samples produced single bands as expected for X chromosome markers. A total of seven distinct haplotypes contributed to the 13 genotypes. The ‘polymorphism information content’ or PIC for this seven-allele X chromosome marker was 0.67.     

亨廷顿病的基因诊断

为了简单高效检测HD基因开放阅读框5’端(CAG)ｎ三核苷酸重复序列,建立快速准确的亨廷顿病(Huntington disease, HD)基因诊断方法,应用TaKaRa LA Taq DNA聚合酶配合GC buffer扩增HD基因包含(CAG)ｎ重复序列的目的片段,非变性聚丙烯酰胺凝胶电泳检测后回收(CAG)ｎ拷贝数异常增多的目的片段,再次PCR扩增后将产物连接至T载体,进行DNA测序确定CAG的拷贝数。应用该方法对一个HD家系的3名成员以及20名正常人进行基因诊断,结果显示该HD家系3名成员的一条染色体上的(CAG)ｎ拷贝数在正常范围内,而另一条染色体上的(CAG)ｎ拷贝数异常增多,分别为39、40、41,而20例正常人(CAG)ｎ拷贝数均在正常范围内,正常和HD等位基因之间的(CAG)ｎ拷贝数不相重叠。因此,应用该方法可以对HD进行准确的基因诊断,结果同时也证明HD基因的动态突变是导致中国人亨廷顿病的遗传基础。     

A recurrent expansion of a maternal allele with 36 CAG repeats causes Huntington disease in two sisters

Large intergenerational repeat expansions of the CAG trinucleotide repeat in the HD gene have been well documented for the male germline. We describe a recurrent large expansion of a maternal allele with 36 CAG repeats (to 66 and 57 repeats, respectively, in two daughters) associated with onset of Huntington disease (HD) in the second and third decade in a family without history of HD. Our findings give evidence of a gonadal mosaicism in the unaffected mother. We hypothesize that large expansions also occur in the female germline and that a negative selection of oocytes with long repeats might explain the different instability behavior of the male and the female germlines.     

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.     

The nucleotide sequence, DNA damage location, and protein stoichiometry influence the base excision repair outcome at CAG/CTG repeats

Expansion of CAG/CTG repeats is the underlying cause of >14 genetic disorders, including Huntington's disease (HD) and myotonic dystrophy. The mutational process is ongoing, with increases in repeat size enhancing the toxicity of the expansion in specific tissues. In many repeat diseases, the repeats exhibit high instability in the striatum, whereas instability is minimal in the cerebellum. We provide molecular insights into how base excision repair (BER) protein stoichiometry may contribute to the tissue-selective instability of CAG/CTG repeats by using specific repair assays. Oligonucleotide substrates with an abasic site were mixed with either reconstituted BER protein stoichiometries mimicking the levels present in HD mouse striatum or cerebellum, or with protein extracts prepared from HD mouse striatum or cerebellum. In both cases, the repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely because of the lower level of APE1, FEN1, and LIG1. Damage located toward the 5' end of the repeat tract was poorly repaired, with the accumulation of incompletely processed intermediates as compared to an AP lesion in the center or at the 3' end of the repeats or within control sequences. Moreover, repair of lesions at the 5' end of CAG or CTG repeats involved multinucleotide synthesis, particularly at the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that the BER stoichiometry, nucleotide sequence, and DNA damage position modulate repair outcome and suggest that a suboptimal long-patch BER activity promotes CAG/CTG repeat instability.     

Huntington disease phenocopy is a familial prion disease

Huntington disease (HD) is a common autosomal dominant neurodegenerative disease with early adult-onset motor abnormalities and dementia. Many studies of HD show that huntingtin (CAG)n repeat-expansion length is a sensitive and specific marker for HD. However, there are a significant number of examples of HD in the absence of a huntingtin (CAG)n expansion, suggesting that mutations in other genes can provoke HD-like disorders. The identification of genes responsible for these "phenocopies" may greatly improve the reliability of genetic screens for HD and may provide further insight into neurodegenerative disease. We have examined an HD phenocopy pedigree with linkage to chromosome 20p12 for mutations in the prion protein (PrP) gene (PRNP). This reveals that affected individuals are heterozygous for a 192-nucleotide (nt) insertion within the PrP coding region, which encodes an expanded PrP with eight extra octapeptide repeats. This reveals that this HD phenocopy is, in fact, a familial prion disease and that PrP repeat-expansion mutations can provoke an HD "genocopy." PrP repeat expansions are well characterized and provoke early-onset, slowly progressive atypical prion diseases with an autosomal dominant pattern of inheritance and a remarkable range of clinical features, many of which overlap with those of HD. This observation raises the possibility that an unknown number of HD phenocopies are, in fact, familial prion diseases and argues that clinicians should consider screening for PrP mutations in individuals with HD-like diseases in which the characteristic HD (CAG)n repeat expansions are absent.     

CAG trinucleotide RNA repeats interact with RNA-binding proteins.

Genes associated with several neurological diseases are characterized by the presence of an abnormally long trinucleotide repeat sequence. By way of example, Huntington's disease (HD), is characterized by selective neuronal degeneration associated with the expansion of a polyglutamine-encoding CAG tract. Normally, this CAG tract is comprised of 11-34 repeats, but in HD it is expanded to > 37 repeats in affected individuals. The mechanism by which CAG repeats cause neuronal degeneration is unknown, but it has been speculated that the expansion primarily causes abnormal protein functioning, which in turn causes HD pathology. Other mechanisms, however, have not been ruled out. Interactions between RNA and RNA-binding proteins have previously been shown to play a role in the expression of several eukaryotic genes. Herein, we report the association of cytoplasmic proteins with normal length and extended CAG repeats, using gel shift and UV crosslinking assays. Cytoplasmic protein extracts from several rat brain regions, including the striatum and cortex, sites of neuronal degeneration in HD, contain a 63-kD RNA-binding protein that specifically interacts with these CAG-repeat sequences. These protein-RNA interactions are dependent on the length of the CAG repeat, with longer repeats binding substantially more protein. Two CAG repeat-binding proteins are present in human cortex and striatum; one comigrates with the rat protein at 63 kD, while the other migrates at 49 kD. These data suggest mechanisms by which RNA-binding proteins may be involved in the pathological course of trinucleotide repeat-associated neurological diseases.     

Investigation of tRNA<Superscript>Leu/Lys</Superscript> and ATPase 6 Genes Mutations in Huntington’s Disease

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats which code for glutamine in the HD gene product, huntingtin. Huntingtin is expressed in almost all tissues, so abnormalities outside the brain can also be expected. Involvement of nuclei and mitochondria in HD pathophysiology has been suggested. In fact mitochondrial dysfunction is reported in brains of patients suffering from HD. The tRNA gene mutations are one of hot spots that can cause mitochondrial disorders. In this study, possible mitochondrial DNA (mtDNA) damage was evaluated by screening for mutations in the tRNA^leu/lys and ATPase 6 genes of 20 patients with HD, using PCR and automated DNA sequencing. Mutations including an A8656G mutation in one patient were observed, which may be causal to the disease. Understanding the role of mitochondria in the pathogenesis of neurodegenerative diseases could potentially be important for the development of therapeutic strategies in HD.     

DNA polymerase θ promotes CAG•CTG repeat expansions in Huntington’s disease via insertion sequences of its catalytic domain

Huntington''s disease (HD), a neurodegenerative disease characterized by progressive dementia, psychiatric problems, and chorea, is known to be caused by CAG repeat expansions in the HD gene HTT. However, the mechanism of this pathology is not fully understood. The translesion DNA polymerase θ (Polθ) carries a large insertion sequence in its catalytic domain, which has been shown to allow DNA loop-outs in the primer strand. As a result of high levels of oxidative DNA damage in neural cells and Polθ''s subsequent involvement in base excision repair of oxidative DNA damage, we hypothesized that Polθ contributes to CAG repeat expansion while repairing oxidative damage within HTT. Here, we performed Polθ-catalyzed in vitro DNA synthesis using various CAG•CTG repeat DNA substrates that are similar to base excision repair intermediates. We show that Polθ efficiently extends (CAG)_n•(CTG)_n hairpin primers, resulting in hairpin retention and repeat expansion. Polθ also triggers repeat expansions to pass the threshold for HD when the DNA template contains 35 repeats upward. Strikingly, Polθ depleted of the catalytic insertion fails to induce repeat expansions regardless of primers and templates used, indicating that the insertion sequence is responsible for Polθ''s error-causing activity. In addition, the level of chromatin-bound Polθ in HD cells is significantly higher than in non-HD cells and exactly correlates with the degree of CAG repeat expansion, implying Polθ''s involvement in triplet repeat instability. Therefore, we have identified Polθ as a potent factor that promotes CAG•CTG repeat expansions in HD and other neurodegenerative disorders.     

Induced pluripotent stem cells from patients with Huntington's disease show CAG-repeat-expansion-associated phenotypes

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. Here, The HD Consortium reports the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Microarray profiling revealed CAG-repeat-expansion-associated gene expression patterns that distinguish patient lines from controls, and early onset versus late onset HD. Differentiated HD neural cells showed disease-associated changes in electrophysiology, metabolism, cell adhesion, and ultimately cell death for lines with both medium and longer CAG repeat expansions. The longer repeat lines were however the most vulnerable to cellular stressors and BDNF withdrawal, as assessed using a range of assays across consortium laboratories. The HD iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in HD and provides a human stem cell platform for screening new candidate therapeutics.