Jayalakshmi Srinidhi

Structure and expression of the Huntington's disease gene: Evidence against simple inactivation due to an expanded CAG repeat

Huntingtons disease, a neurodegenerative disorder characterized by loss of striatal neurons, is caused by an expanded, unstable trinucleotide repeat in a novel 4p16.3 gene. To lay the foundation for exploring the pathogenic mechanism in HD, we have determined the structure of the disease gene and examined its expression. TheHD locus spans 180 kb and consists of 67 exons ranging in size from 48 bp to 341 bp with an average of 138 bp. Scanning of theHD transcript failed to reveal any additional sequence alterations characteristic of HD chromosomes. A codon loss polymorphism in linkage disequilibrium with the disorder revealed that both normal and HD alleles are represented in the mRNA population in HD heterozygotes, indicating that the defect does not eliminate transcription. The gene is ubiquitously expressed as two alternatively polyadenylated forms displaying different relative abundance in various fetal and adult tissues, suggesting the operation of interacting factors in determining specificity of cell loss. TheHD gene was disrupted in a female carrying a balanced translocation with a breakpoint between exons 40 and 41. The absence of any abnormal phenotype in this individual argues against simple inactivation of the gene as the mechanism by which the expanded trinucleotide repeat causes HD. Taken together, these observations suggest that the dominant HD mutation either confers a new property on the mRNA or, more likely, alters an interaction at the protein level.

Gametic but not somatic instability of CAG repeat length in Huntington's disease.

Instability of a CAG repeat in 4p16.3 has been found in Huntingtons disease (HD) chromosomes. Unlike a similar repeat in the fragile X syndrome, the expanded HD repeat showed no evidence of somatic instability in a comparison of blood, lymphoblast, and brain DNA from the same persons. Four pairs of monozygotic HD twins displayed identical CAG repeat lengths suggesting that repeat size is determined in gametogenesis. In contrast with the fragile X syndrome and with HD somatic tissue, mosaicism was readily detected as a diffuse spread of repeat lengths in DNA from HD sperm samples. Typically, the modal repeat size was larger in the sperm DNA than in corresponding lymphoblast DNA, with the greatest degree of gametic mosaicism coinciding with the longest somatic CAG repeats. These data indicate that the developmental timing of repeat instability appears to differ between HD and fragile X syndrome, and that the fundamental mechanisms leading to repeat expansion may therefore be distinct.

Quantitative neuropathological changes in presymptomatic Huntington's disease

Morphometric studies of the tail of the caudate nucleus, the site where the pathology is first seen, were performed on 16 brain specimens collected from individuals at risk for inheriting Huntingtons disease (HD). Medical records and information obtained from immediate family members indicated that all had died without symptoms of HD. Six individuals had 37 or more CAG repeats and were designated HD gene carriers, whereas 10 were determined to be non‐carriers. Cell counts of the tail of the caudate nucleus revealed an increased density of oligodendrocytes among the presymptomatic HD gene carriers (mean cells/field: carriers = 40.0, noncarrier = 21.3; age, sex, repeated measure adjusted F[126] = 11.7, p = 0.0008). No statistically significant differences were found between HD carriers and noncarriers in the density of neurons (carriers = 16.9, noncarriers = 15.5), astrocytes (carriers = 27.8, noncarriers = 21.3) or microglial cells (carriers = 7.9, noncarriers = 5.6). Ubiquitin immunostaining performed in 3 gene carriers revealed intranuclear inclusions in all 3 cases, including 1, with 37 repeats, who died 3 decades before the expected age for onset of the clinical syndrome. Normal densities of other cell types and careful macroscopic examination suggest that the increase in oligodendroglial density is not a consequence of atrophy and may instead reflect a developmental effect of the HD gene. Ann Neurol 2001;49:29–34

Trinucleotide repeat length and progression of illness in Huntington's disease.

The genetic defect causing Huntingtons disease (HD) has been identified as an unstable expansion of a trinucleotide (CAG) repeat sequence within the coding region of the IT15 gene on chromosome 4. In 50 patients with manifest HD who were evaluated prospectively and uniformly, we examined the relationship between the extent of the DNA expansion and the rate of illness progression. Although the length of CAG repeats showed a strong inverse correlation with the age at onset of HD, there was no such relationship between the number of CAG repeats and the rate of clinical decline. These findings suggest that the CAG repeat length may influence or trigger the onset of HD, but other genetic, neurobiological, or environmental factors contribute to the progression of illness and the underlying pace of neuronal degeneration.

Differential expression of normal and mutant Huntington's disease gene alleles.

Huntingtin expression was examined by Western blot and immunoprecipitation studies of lymphoblastoid cell lines from Huntingtons disease (HD) homozygotes, heterozygotes, and a phenotypically normal individual with a t(4p16.3;12p13.3) breakpoint in the HD gene. The latter produced a reduced level of normal huntingtin without evidence of an altered protein, indicating that simple loss of huntingtin activity does not cause HD. In juvenile onset HD heterozygotes, NH2- and COOH-terminal antisera revealed reduced relative expression from the mutant allele. Pulse-chase studies indicated that huntingtin is a stable protein whose differential allelic expression is not due to destabilization of the mutant isoform. No stable breakdown products specific to mutant huntingtin were detected in either HD homozygotes or heterozygotes. These data are consistent with HD involving either a gain of function or a dominant negative loss of function that operates within severe constraints and suggest that in either case the pathogenic process is usually saturated by the amount of abnormal huntingtin produced from a single mutant allele.

HUNTINGTON'S DISEASE

Early in 1993, an unstable, expanded trinucleotide repeat in a novel gene of unknown function was identified on HD chromosomes. This discovery unleased a flurry of experimentation that has established the expanded CAG repeat the almost universal cause of the characteristic neurologic symptoms and pathology of this neurodegenerative disorder of midlife onset. The biochemical basis for the specific neuronal loss of HD remains uncertain, but the genetic lesion probably acts via its consequent polyglutamine segment in the protein product, huntingtin. This review will describe the basic parameters of the HD repeats behavior and the knowledge that has accumulated concerning its potential mechanisms of action.

Mouse Huntington's disease gene homolog (Hdh)

The incurable neurodegenerative disorder, Huntingtons disease (HD), is caused by an expanded, unstable CAG repeat encoding a stretch of polyglutamine in a 4p16.3 gene (HD) of unknown function. Near the CAG repeat is a polyproline-encoding CCG repeat that shows more limited allelic variation. The mouse homologue,Hdh, has been mapped to chromosome 5, in a region devoid of mutations causing any comparable phenotype. We have isolated overlapping cDNAs from theHdh gene and compared their sequences with the human transcript. The consensus mouse coding sequence is 86% identical to the human at the DNA level and 91% identical at the protein level. Despite the overall high level of conservation,Hdh possesses an imperfect CAG repeat encoding only seven consecutive glutamines, compared to the 13–36 residues that are normal in man. Although no evidence for polymorphic variation of the CAG repeat was seen, a nearby CCG repeat differed in length by one unit between several strains of laboratory mouse andMus spretus. The absence of a long CAG repeat in the mouse is consistent with the lack of a spontaneous mouse model of HD. The information presented concerning the sequence of the mouse gene should facilitate attempts to create such a model.

A genetic linkage map of the chromosome 4 short arm

We have generated an 18-interval contiguous genetic linkage map of human chromosome 4 spanning the entire short arm and proximal long arm. Fifty-seven polymorphisms, representing 42 loci, were analyzed in the Venezuelan reference pedigree. The markers included seven genes (ADRA2C, ALB, GABRB1, GC, HOX7, IDUA, QDPR), one pseudogene (RAF1P1), and 34 anonymous DNA loci. Four loci were represented by microsatellite polymorphisms and one (GC) was expressed as a protein polymorphism. The remainder were genotyped based on restriction fragment length polymorphism. The sex-averaged map covered 123 cM. Significant differences in sex-specific rates of recombination were observed only in the pericentromeric and proximal long arm regions, but these contributed to different overall map lengths of 115 cM in males and 138 cM in females. This map provides 19 reference points along chromosome 4 that will be particularly useful in anchoring and seeding physical mapping studies and in aiding in disease studies.