两个亨廷顿舞蹈病家系IT15基因的研究

为了探讨亨廷顿舞蹈病家系患者的临床特征与IT15基因中(CAG)_n重复拷贝数之间的相互关系, 对两家系患者的临床、影像学特征、发病年龄及遗传方式等进行分析; 用聚合酶链反应技术、6%聚丙烯酰胺凝胶电泳及直接测序等方法, 对42名家系成员的IT15基因的(CAG)_n三核苷酸重复序列进行分析。结果显示家系1患者无典型的临床“三联症”及尾状核的萎缩, 18名家系成员中9名患者IT15基因的(CAG)_n拷贝数介于40~50次之间,拷贝数与发病年龄无明显相关; 而家系2患者具有典型的“三联症”和尾状核的萎缩, 24名家系成员中5例患者(CAG)_n拷贝数大于等于50次, 发病年龄与(CAG)_n拷贝数相关。因此亨廷顿舞蹈病患者的临床特征在一定程度上受IT15基因的(CAG)_n三核苷酸重复拷贝数的影响, 拷贝数大于50次, 发病年龄与(CAG)_n拷贝数相关, 并有经父系遗传的(CAG)_n拷贝数的扩展, 且存在遗传早现现象。     

Analysis of the IT15 Gene in Huntington's Disease Families

Direct molecular genetic testing carried out in 59 Huntington's disease patients belonging to 46 families from Bashkortostan revealed the (CAG)_n repeat expansion in exon 1 of the IT15 gene in 57 of them. By use of this analysis the disease status was not confirmed in two patients with atypical form of the disease and negative family history. The (CAG)_n repeat expansion was identified in 27 out of 127 asymptomatic at-risk individuals. Analysis of the mutant (CAG)_n allele inheritance demonstrated extremely high instability and high mutation rate predominantly leading to the appearance of the alleles with increasing number of (CAG)_n repeats in subsequent generations. The instability was mostly observed in cases of paternal transmission. Almost complete linkage disequilibrium between the (CCG)₇ mutant alleles and the del2642 deletion was demonstrated. Three major haplotypes revealed, (CCG)₇/del–, (CCG)₇/del+, and (CCG)₁₀/del–, implied the existence of at least three sources of the origin of Huntington's disease in Bashkortostan. The identified haplotype frequency distribution patterns displayed similarities with those in European populations. The contribution of a number of genetic factors to the age of onset of Huntington's disease was analyzed.     

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.     

Geographical distribution of haplotypes in Swedish families with Huntington's disease

This study was planned to determine the number of origins of the mutation underlying Huntington's disease (HD) in Sweden. Haplotypes were constructed for 23 different HD families, using six different polymorphisms [(CCG) _n , GT70, 674, BS1, E2 and 4.2], including two within the gene. In addition, extensive genealogical investigations were performed, and the geographical origin of the haplotypes was studied. Ten different haplotypes were observed suggesting multiple origins for the HD mutation in Sweden. Analysis of the two polymorphic markers within the HD gene (the CCG repeat and GT70) indicates that there are at least three origins for the HD mutation in Sweden. One of these haplotypes (7/A) accounts for 89% of the families, suggesting that the majority of the Swedish HD families are related through a single HD mutation of ancient origin. Furthermore, three of the families that were previously considered to be unrelated could be traced to a common ancestor in the 15th century, a finding that is consistent with this hypothesis.     

An anthropometric assessment of Huntington's disease patients and families

An anthropometric investigation was designed to evaluate patterns of physical deterioration in Huntington's disease (HD). In this study a comprehensive set of measurements was taken including height, weight, body circumferences, skinfold thickness, and craniofacial, linear, and breadth components of the body, on 44 normal, 26 affected, and 70 at-risk individuals between 14 and 88 years of age. The anthropometric data were converted to z-scores using standards to adjust for age and sex differences. These scores were then adjusted for inter-family variation. There were significant differences among normal and affected individuals for all dimensions of body mass, as well as for several craniofacial and linear components of the body. Several significant differences were also found between normals and particular age cohorts of at-risk persons. HD gene carrier status was further assessed by factor analysis of the adjusted scores.     

Allele-specific silencing of mutant Huntington's disease gene

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a poly-glutamine expansion in huntingtin, the protein encoded by the HD gene. PolyQ-expanded huntingtin is toxic to neurons, especially the medium spiny neurons of the striatum. At the same time, wild-type huntingtin has important – indeed essential – protective functions. Any effective molecular therapy must preserve the expression of wild-type huntingtin, while silencing the mutant allele. We hypothesized that an appropriate siRNA molecule would display the requisite specificity and efficacy. As RNA interference is incapable of distinguishing among alleles with varying numbers of CAG (glutamine) codons, another strategy is needed. We used HD fibroblasts in which the pathogenic mutation is linked to a polymorphic site: the Δ2642 deletion of one of four tandem GAG triplets. We silenced expression of the harmful Δ2642-marked polyQ-expanded huntingtin without compromising synthesis of its wild-type counterpart. Following this success in HD fibroblasts, we obtained similar results with neuroblastoma cells expressing both wild-type and mutant HD genes. As opposed to the effect of depleting wild-type huntingtin, specifically silencing the mutant species actually lowered caspase-3 activation and protected HD cells under stress conditions. These findings have therapeutic implications not only for HD, but also for other autosomal dominant diseases. This approach has great promise: it may lead to personalized genetic therapy, a holy grail in contemporary medicine.     

Variation within the Huntington's disease gene influences normal brain structure

Genetics of the variability of normal and diseased brain structure largely remains to be elucidated. Expansions of certain trinucleotide repeats cause neurodegenerative disorders of which Huntington's disease constitutes the most common example. Here, we test the hypothesis that variation within the IT15 gene on chromosome 4, whose expansion causes Huntington's disease, influences normal human brain structure. In 278 normal subjects, we determined CAG repeat length within the IT15 gene on chromosome 4 and analyzed high-resolution T1-weighted magnetic resonance images by the use of voxel-based morphometry. We found an increase of GM with increasing long CAG repeat and its interaction with age within the pallidum, which is involved in Huntington's disease. Our study demonstrates that a certain trinucleotide repeat influences normal brain structure in humans. This result may have important implications for the understanding of both the healthy and diseased brain.     

Recombination events suggest potential sites for the Huntington's disease gene

The Huntington's disease gene (HD) maps distal to the D4S10 marker in the terminal 4p16.3 subband of chromosome 4. Directed cloning has provided several DNA segments that have been grouped into three clusters on a physical map of approximately 5 X 10(6) bp in 4p16.3. We have typed RFLPs in both reference and HD pedigrees to produce a fine-structure genetic map that establishes the relative order of the clusters and further narrows the target area containing the HD gene. Despite the large number of meiotic events examined, the HD gene cannot be positioned relative to the most distal cluster. One recombination event with HD suggests that the terminal-most markers flank the disease gene; two others favor a telomeric location for the defect. Efforts to isolate the HD gene must be divided between these two distinct intervals until additional genetic data resolve the apparent contradiction in localization.     

Mapping of the DNA locus D4S10 and the linked Huntington's disease gene to 4p16----p15

An anonymous DNA fragment (G8) detects two restriction fragment length polymorphic alleles (RFLPs) called D4S10 in HindIII-digested human genomic DNA. This segment had been assigned to chromosome 4 and shows close linkage to the Huntington's disease gene. With in situ hybridization, we mapped D4S10 to the terminal region of the short arm of chromosome 4, localizing the Huntington's disease gene to bands 4p16----p15. This information may prove useful for the development of strategies to clone the Huntington's disease gene.     

Neural expression of the Huntington's disease gene as a chordate evolutionary novelty

Huntington's disease is a progressive neuro-degenerative disorder in humans, which is scharacterized by onset of dementia, muscular ataxia, and death. Huntington's disease is caused by the expansion of the polyglutamine (polyQ) tract in the N-terminus of the HD protein (Huntingtin). CAG expansion is a dominant gain of function mutation that affects striated neurons in the brain (Cattaneo, 2003, News Physiol Sci 18:34). The evolutionary origins of the vertebrate Hd gene are not well understood. In order to address the evolutionary history of the Hd gene, we have cloned and characterized the expression of the Hd gene in two invertebrate deuterostomes, an echinoderm and an ascidian, and have examined the expression patterns in a phylogenetic context. Echinoderms are basal deuterostomes and ascidians are basal chordates; both are useful for understanding the origins of and evolutionary trends in genes important in vertebrates such as the Huntigton's disease gene. Expression of Hd RNA is detected at all stages of development in both the echinoderm and ascidian studied. In the echinoderm Heliocidaris erythrogramma, Hd is expressed in coelomic mesodermal tissue derivatives, but not in the central nervous system. In the ascidian Halocynthia roretzi expression is located in both mesoderm and nervous tissue. We suggest that the primitive deuterostome expression pattern is not neural. Thus, neural expression of the Hd gene in deuterostomes may be a novel feature of the chordate lineage, and the original role(s) of HD in deuterostomes may have been non-neural.     

Mitochondria in Huntington's disease

Huntington's disease (HD) is an inherited progressive neurodegenerative disorder associated with involuntary abnormal movements (chorea), cognitive deficits and psychiatric disturbances. The disease is caused by an abnormal expansion of a CAG repeat located in exon 1 of the gene encoding the huntingtin protein (Htt) that confers a toxic function to the protein. The most striking neuropathological change in HD is the preferential loss of medium spiny GABAergic neurons in the striatum. The mechanisms underlying striatal vulnerability in HD are unknown, but compelling evidence suggests that mitochondrial defects may play a central role. Here we review recent findings supporting this hypothesis. Studies investigating the toxic effects of mutant Htt in cell culture or animal models reveal mitochondrial changes including reduction of Ca²⁺ buffering capacity, loss of membrane potential, and decreased expression of oxidative phosphorylation (OXPHOS) enzymes. Striatal neurons may be particularly vulnerable to these defects. One hypothesis is that neurotransmission systems such as dopamine and glutamate exacerbate mitochondrial defects in the striatum. In particular, mitochondrial dysfunction facilitates impaired Ca²⁺ homeostasis linked to the glutamate receptor-mediated excitotoxicity. Also dopamine receptors modulate mutant Htt toxicity, at least in part through regulation of the expression of mitochondrial complex II. All these observations support the hypothesis that mitochondria, acting as “sensors” of the neurochemical environment, play a central role in striatal degeneration in HD.     

Neurodegenerative processes in Huntington's disease

Huntington''s disease (HD) is a complex and severe disorder characterized by the gradual and the progressive loss of neurons, predominantly in the striatum, which leads to the typical motor and cognitive impairments associated with this pathology. HD is caused by a highly polymorphic CAG trinucleotide repeat expansion in the exon-1 of the gene encoding for huntingtin protein. Since the first discovery of the huntingtin gene, investigations with a consistent number of in-vitro and in-vivo models have provided insights into the toxic events related to the expression of the mutant protein. In this review, we will summarize the progress made in characterizing the signaling pathways that contribute to neuronal degeneration in HD. We will highlight the age-dependent loss of proteostasis that is primarily responsible for the formation of aggregates observed in HD patients. The most promising molecular targets for the development of pharmacological interventions will also be discussed.     

Iron dysregulation in Huntington's disease

Huntington's disease (HD) is one of many neurodegenerative diseases with reported alterations in brain iron homeostasis that may contribute to neuropathogenesis. Iron accumulation in the specific brain areas of neurodegeneration in HD has been proposed based on observations in post‐mortem tissue and magnetic resonance imaging studies. Altered magnetic resonance imaging signal within specific brain regions undergoing neurodegeneration has been consistently reported and interpreted as altered levels of brain iron. Biochemical studies using various techniques to measure iron species in human samples, mouse tissue, or in vitro has generated equivocal data to support such an association. Whether elevated brain iron occurs in HD, plays a significant contributing role in HD pathogenesis, or is a secondary effect remains currently unclear.

     

Protein aggregation in Huntington's disease

The presence of an expanded polyglutamine produces a toxic gain of function in huntingtin. Protein aggregation resulting from this gain of function is likely to be the cause of neuronal death. Two main mechanisms of aggregation have been proposed: hydrogen bonding by polar-zipper formation and covalent bonding by transglutaminase-catalyzed cross-linking. In cell culture models of Huntington's disease, aggregates are mostly stabilized by hydrogen bonds, but covalent bonds are also likely to occur. Nothing is known about the nature of the bonds that stabilize the aggregates in the brain of patients with Huntington's disease. It seems that the nature of the bond stabilizing the aggregates is one of the most important questions, as the answer would condition the therapeutic approach to Huntington's disease.