Importance of the number of trinucleotide repeat expansions in the clinical manifestations of Huntington's chorea

INTRODUCTION: In 1993 the gene responsible for Huntington's disease (IT15) was isolated [5]. It was mapped to the tip of the short arm of chromosome 4 and within its coding sequence, near the 5' end, it contained a certain number of trinicleotide (CAG)n (cytosine-adenine-guanine) repeats (Figure 1). This gene codes for a protein (348 kd) called "huntington" that is widely expressed, and its sequence is not related to any protein [6]. The normal range of (CAG)n repeat numbers within IT15 was reported to be between 6 and 37 [6]. Mutation responsible for Huntington's disease implied expansion of (CAG)n repeats: in patients with Huntington's disease the pathologic range was determined to be between 35 and 121 repeats [7-10]. PATIENTS AND METHODS: In this study we correlated the age at onset, rate of progression and initial symptoms of Huntington's disease with the number of trinucleotide (CAG)n repeats in IT15. DNA was isolated from peripheral blood leukocytes of patients fulfilling clinical criteria for definite and probable Huntington's disease [2]. Genetic verification of Huntington's disease was made by the previously described and modified PRC (polymerase chain reaction) technique [17, 18]. In our laboratory a gene with 40 or more repeats was considered as a marker of Huntington's disease. RESULTS: The study comprised 26 patients (11 women and 15 men). At the onset of Huntington's disease they were between 19 and 66 years old (36.6 12.8 years), with the duration of the disease between 1 and 15 years (5.8 4.3 years). The number of (CAG)n, repeats in IT15 ranged between 40 and 95 (49.9 14.1). The negative correlation between the (CAG)n, count in the expanded allele and the age at onset of the disease has been confirmed. Regression analysis showed the correlation coefficient of -0.54 (p = 0.012). The effect of trinucleotide (CAG)n, repeats on the initial clinical manifestations and rate of progression of Huntington's disease is only one of the growing group of "CAG-repeat" disorders that also include entities such as spinocerebellar ataxia-type 1 and 3, spinobulbar muscular atrophy and dentato-rubo-pallidoluysian atrophy [6].     

Huntington's chorea: clinical aspects, genetics and current diagnosis

Huntington's disease is a late manifesting autosomal dominant neurodegenerative disorder. It is characterized by motor disturbance, loss of cognitive functions and psychiatric manifestations. The disease causing mutation, an unstable DNA sequence in the coding region of the Huntington gene on chromosome 2p, has recently been identified. A trinucleotide [CAG] repeat is expanded over the normal range and can be easily detected by standard laboratory methods. Accurate genetic testing can now be offered in clinically questionable cases and to presymptomatic subjects at risk for Huntington's disease. Furthermore, there is a correlation between the size of the expanded CAG repeat and the age of onset in affected individuals. The predictive value of this correlation, however, is limited due to the range of onset ages found at a given repeat length in large series of patients. Expanded triplet repeats exhibit a marked instability especially in male meiosis with a tendency to further increase during transmission over the generations. This is likely to be the molecular mechanism explaining anticipation, as well as the occurrence of juvenile cases and new mutations.     

1H NMR spectroscopy studies of Huntington's disease: correlations with CAG repeat numbers

Huntington's disease (HD) is the result of an expanded (CAG) repeat in a gene on chromosome 4. A consequence of the gene defect may be progressive impairment of energy metabolism. We previously showed increased occipital cortex lactate in HD using localized 1H spectroscopy. We have now extended these studies to show an almost threefold elevation in occipital cortex lactate in 31 HD patients as compared with 17 normal control subjects (p < 10(-11)). The spectra in three presymptomatic gene-positive patients were identical to normal control subjects in cortical regions, but three in eight showed elevated lactate in the striatum. Similar to recently reported increases in task-related activation of the striatum in the dominant hemisphere, we found that striatal lactate levels in HD patients were markedly asymmetric (higher on the left side). Markers of neuronal degeneration, decreased N-acetylaspartate (NAA)/creatine and increased choline/creatine levels, were symmetric. Both decreased NAA and increased lactate in the striatum significantly correlated with duration of symptoms. When divided by his or her age, an individual's striatal NAA loss and lactate increase were found to directly correlate with the subject's CAG repeat number, with correlation coefficients of 0.8 and 0.7, respectively. Similar correlations were noted between postmortem cell loss and age versus CAG repeat length. Together, these data provide further evidence for an interaction between neuronal activation and a defect in energy metabolism in HD that may extend to presymptomatic subjects.     

Molecular analysis of the IT15 gene in 79 Spanish families with Huntington's disease: diagnostic confirmation and presymptomatic diagnosis

BACKGROUND: Huntington's disease (HD) is a neurodegenerative disorder with late age of onset, caused by (CAG), expansion in the IT15 gene. We present here the results of IT15 gene study in Spanish families in order to show the usefulness of diagnosis, genetic counseling and clinical-genetic correlation in Spanish population. PATIENTS AND METHODS: We have studied the number of (CAG)n repeats in the IT15 gene by PCR analysis in 137 individuals from 79 Spanish families with HD. RESULTS: The number of (CAG)n repeats in HD chromosomes varied from 35 to 85, while the range for the normal chromosomes was from 13 to 31. In four juvenile cases the number of (CAG)n repeats was above 50. In three of these cases the transmission was paternal. The (CAG)n expansion was demonstrated in 98.3% of the cases. We established the diagnosis in 15 uncertain clinical diagnosis. We made a presymptomatic diagnosis after psychological-psychiatric evaluation in 50 HD at risk individuals. We showed an inverse correlation between the number of (CAG)n repeats and the age at onset of the disease. CONCLUSIONS: The (CAG)n repeats study in the IT15 gene in Spanish populations allows the confirmation of diagnosis of HD as well as presymptomatic testing enabling the genetic counseling. There is an inverse correlation between the age of onset of the disease and the number of (CAG)n repeats in the IT15 gene.     

Molecular genetics of Huntington's disease]

Huntington's disease (HD) is a progressive neurodegenerative disorder which is clinically characterized by chorea, cognitive decline, and emotional disturbance; it is inherited in an autosomal dominant manner. The HD gene maps to chromosome 4p16.3. Our linkage analysis demonstrated a significant genetic linkage between Japanese HD families and the flanking markers, D4S127, D4S43. The molecular basis of the disease is an expansion of CAG repeat in the huntingtin gene. We performed molecular analysis of the repeat in Japanese HD patients and normal controls. The size of the CAG repeat ranged from 37 to 95 repeats in affected subjects and from seven to 29 in normal controls. A significant correlation was found between the age of onset and the CAG expansion. The length of the expanded repeat is unstable in meiotic transmission and large increases occur in paternal transmission. At the same time the CCG repeat polymorphism adjacent to the CAG repeat was analysed and haplotypes of HD chromosomes were identified. Striking linkage disequilibrium was found between the CAG repeat expansion and an allele of (CCG)10 in Japanese HD chromosome. It is distinct from that described previously in western populations. Western HD chromosomes strongly associate with an allele of (CCG)7.     

The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family)

Striatal grafts have been proposed as a potential strategy for striatal repair in Huntington's disease, but it is unknown whether the diseased brain will compromise graft survival. A transgenic mouse line has recently been described in which hemizygotes with an expanded CAG repeat in exon 1 of the HD gene exhibit a progressive neurological phenotype similar to the motor symptoms of Huntington's disease. We have therefore evaluated the effects of the transgenic brain environment on the survival, differentiation, and function of intrastriatal striatal grafts and undertaken a preliminary analysis of the effects of the grafts on the development of neurological deficits in the host mice. Hemizygote transgenic and wild-type littermate female mice received striatal grafts at 10 weeks of age and were allowed to survive 6 weeks. Normal healthy grafts were seen to survive and differentiate within the striatum of transgenic mice in a manner comparable to that seen in control mice. The transgenic mice exhibited a progressive decline in body weight from 9 weeks of age and a progressive hypoactivity in an open field test of general locomotor behavior. Although striatal grafts exerted a statistically significant influence on several indices of this impairment, all behavioral effects were small and did not exert any clinically relevant effect on the profound neurological deficiency of the transgenic mice.     

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.     

Spinocerebellar ataxia type 6. Molecular and clinical features of 35 Japanese patients including one homozygous for the CAG repeat expansion

Spinocerebellar ataxia type 6 (SCA6) is a newly classified autosomal-dominant cerebellar ataxia (ADCA) associated with CAG repeat expansion. We screened 111 patients with cerebellar ataxia for the SCA6 mutation. Of these, 35 patients were found to have expanded CAG repeats in the SCA6 gene, indicating that second to SCA3, SCA6 is the most common ADCA in Japan. Expanded alleles ranged from 21 to 29 repeats, whereas normal alleles had seven to 17 repeats. There was no change in the CAG repeat length during meiosis. The age at onset was inversely correlated with the repeat length. The main clinical feature of the 35 patients with SCA6 was slowly progressive cerebellar ataxia; multisystem involvement was not common. The 35 patients included nine cases without apparent family history of cerebellar ataxia. The sporadic cases had smaller CAG repeats (21 or 22 repeats) and a later age at onset (64.9 +/- 4.9 years) than the other cases with established family history. We also identified one patient who was homozygous for the SCA6 repeat expansion. The homozygote showed an earlier age of onset and more severe clinical manifestations than her sister, a heterozygote carrying an expanded allele with the same repeat length as the homozygote. This finding suggests that the dosage of the CAG repeat expansion plays an important role in phenotypic expression in SCA6.     

The molecular mechanisms of the instability of the CAG repeat

Expansion of CAG trinucleotide repeats coding for polyglutamine stretches has been identified for seven neurodegenerative diseases including Machado-Joseph disease (MJD). There are many common features shared among these disease such as genetic anticipation i.e. accelerated age at onset in successive generations, which is also a result of intergenerational increase in the size of expanded CAG repeats. To identify elements affecting the intergenerational instability of the CAG repeat, we investigated whether the CGG/GGG polymorphism at the 3' end of the CAG repeat in the MJD1 gene affects intergenerational instability. We suggested that an inter-allelic interaction is involved in the intergenerational instability of the CAG repeat and provide a clue to the molecular mechanisms of the instability of the CAG repeat.     

Analysis of CAG/CTG repeat size in Chinese subjects with schizophrenia and bipolar affective disorder using the repeat expansion detection method

BACKGROUND: Family studies of schizophrenia and bipolar affective disorder provide evidence for genetic anticipation, which (in common with a number of mendelian disorders), may be caused by triplet repeat expansion. This hypothesis is strengthened by evidence from repeat expansion detection (RED) analysis revealing association between the psychoses and long CAG/CTG trinucleotide repeats. METHODS: We performed RED on Han Chinese subjects with schizophrenia (82), bipolar affective disorder (43), and normal controls (61), using a CTG10 oligonucleotide. RESULTS: Comparison between cases and controls revealed no significant association between long repeats and affected status. We also found no detectable association with age at onset and repeat length in either bipolar affective disorder or schizophrenia. Overall, the size distribution of CAG/CTG repeats in Chinese subjects was not significantly different from those reported previously for Caucasian subjects. CONCLUSIONS: These findings indicate that CAG/CTG repeat expansion is not likely to be a major etiological factor for psychosis in Chinese populations.     

Spinocerebellar ataxia 3 and Machado-Joseph disease: clinical, molecular, and neuropathological features

Patients with spinocerebellar ataxia 3 (SCA3) and Machado-Joseph disease (MJD) carry an expanded CAG repeat in the MJD1 gene. One hundred twenty families of different geographic origin with autosomal dominant cerebellar ataxia (ADCA) type I were tested. Thirty-four families (126 patients) carried an expanded CAG repeat. The expanded and the normal allele did not overlap and the repeat was unstable during transmission, with variation in the size of the CAG length ranging from -8 to +5 and a mean expansion of 0.86 repeats without differences according to the parental sex. There was a combined effect of the number of CAG repeats of the expanded and normal allele on the age at onset, which accounted for 70% of its variability. The length of the CAG repeat influenced the frequency of clinical signs associated with cerebellar ataxia, such as abnormal tendon reflexes or decreased vibration sense, whereas the interindividual variation of supranuclear ophthalmoplegia, sphincter and swallowing difficulties, and amyotrophy was mostly determined by different disease durations. We compared the clinical profile of 91 SCA3/MJD patients with 51 SCA1 and 32 SCA2 patients. There were striking differences between the SCA3/MJD and SCA2 but not with SCA1 groups of patients. Despite their clinical similarities, distinct neuropathological features were observed in 2 SCA3/MJD and 2 SCA1 patients.     

Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation

Huntington's disease (HD) is one of an increasing number of human neurodegenerative disorders caused by a CAG/polyglutamine-repeat expansion. The mutation occurs in a gene of unknown function that is expressed in a wide range of tissues. The molecular mechanism responsible for the delayed onset, selective pattern of neuropathology, and cell death observed in HD has not been described. We have observed that mice transgenic for exon 1 of the human HD gene carrying (CAG)115 to (CAG)156 repeat expansions develop pronounced neuronal intranuclear inclusions, containing the proteins huntingtin and ubiquitin, prior to developing a neurological phenotype. The appearance in transgenic mice of these inclusions, followed by characteristic morphological change within neuronal nuclei, is strikingly similar to nuclear abnormalities observed in biopsy material from HD patients.     

Correlation between the onset age of Huntington's disease and length of the trinucleotide repeat in IT-15

Huntington's disease (HD) is an autosomal dominant disorder with a variable age of onset that is influenced by the sex of the affected parent. The recent recognition that HD is caused by an expanded triplet repeat suggests the possibility that the onset age may be predicted by the length of the repeat. This hypothesis was tested by assaying the length of the repeat in 114 individuals who were clinically diagnosed with HD and had a known onset age. Every individual had an expanded allele. The range was from 36 to 82 repeats (mean = 48.4 +/- 9.51) and larger than the normal range (6 to 31). The size of the expanded allele was correlated with the age of onset (r = -0.65 p < .0001). Despite the highly significant correlation, the repeat size explains less than half of the variance in onset age. Furthermore, the age of onset cannot be predicted from the size of the triplet repeat, particularly if the number of repeats is in the smaller end of the expanded range. If the repeat is < or = 50 triplets, the amount of variation in the age of onset explained by the length of the triplet repeat is less than 10%. As an illustration, the onset age of individuals with 39 repeats ranges from 30 to 65 years old. The sex of the affected parent had no effect in our sample beyond the effect of the length of the repeat. Affected offspring of affected fathers had longer repeats and a larger variance in allele size than offspring of affected mothers, perhaps reflecting greater instability in paternal transmission.     

Direct genetic diagnosis in Huntington's chorea

Huntington's disease is a late manifesting autosomal dominant neurodegenerative disorder. It is characterized by motor disturbance, loss of cognitive functions and psychiatric manifestations. Recently, the disease causing mutation, an unstable DNA sequence in the coding region of the Huntington gene on chromosome 4p, has been identified. A trinucleotide (CAG) repeat is expanded over the normal range and can easily be detected by standard laboratory methods. Accurate genetic testing can now be offered in clinically questionable cases and to subjects at risk for Huntington's disease. Furthermore, there seems to be a correlation between the size of the expanded CAG repeat and the age of onset in affected individuals. We have investigated more than 130 individuals from different affected families and illustrate the advantages and the clinical application of the new method.     

Analysis of germline mutation spectra at the Huntington's disease locus supports a mitotic mutation mechanism

Trinucleotide repeat disease alleles can undergo 'dynamic' mutations in which repeat number may change when a gene is transmitted from parent to offspring. By typing >3500 sperm, we determined the size distribution of Huntington's disease (HD) germline mutations produced by 26 individuals from the Venezuelan cohort with CAG/CTG repeat numbers ranging from 37 to 62. Both the mutation frequency and mean change in allele size increased with increasing somatic repeat number. The mutation frequencies averaged 82% and, for individuals with at least 50 repeats, 98%. The extraordinarily high mutation frequency levels are most consistent with a mutation process that occurs throughout germline mitotic divisions, rather than resulting from a single meiotic event. In several cases, the mean change in repeat number differed significantly among individuals with similar somatic allele sizes. This individual variation could not be attributed to age in a simple way or to ' cis ' sequences, suggesting the influence of genetic background or other factors. A familial effect is suggested in one family where both the father and son gave highly unusual spectra compared with other individuals matched for age and repeat number. A statistical model based on incomplete processing of Okazaki fragments during DNA replication was found to provide an excellent fit to the data but variation in parameter values among individuals suggests that the molecular mechanism might be more complex.     

Twenty-four-hour heart preservation using continuous cold perfusion and copper (II) complexes

Spinocerebellar ataxia 7 (SCA7) is a neurodegenerative disorder characterized by degeneration of the cerebellum, brainstem and retina. The gene responsible for SCA7, located on chromosome 3p, recently was cloned and shown to contain a CAG repeat in the coding region of the gene, that is expanded in SCA7 patients of French origin. We examined the SCA7 repeat region in four Swedish SCA7 families as well as in 57 healthy controls. All Swedish SCA7 patients exhibited expanded CAG repeats with a strong negative correlation between repeat size and age of onset. The repeat length in SCA7 patients ranged from 40 to >200 repeats. The largest expansion was observed in a juvenile case with an age of onset of 3 months, and represents the longest polyglutamine stretch ever reported. In patients with 59 repeats or more, visual impairment was the most common initial symptom observed, while ataxia predominates in patients with <59 repeats. Two of the Swedish SCA7 families analysed in this study were shown to be related genealogically. The other two SCA7 families could not be traced back to a common ancestor. All four families shared the same allele on the disease chromosome at a locus closely linked to SCA7, suggesting the possibility of a founder effect in the Swedish population.     

Single sperm analysis of the CAG repeats in the gene for dentatorubral-pallidoluysian atrophy (DRPLA): the instability of the CAG repeats in the DRPLA gene is prominent among the CAG repeat diseases

Dentatorubral-pallidoluysian atrophy (DRPLA) is known to show the most prominent genetic anticipation among CAG repeat diseases. To investigate the mechanism underlying the meiotic instability of expanded CAG repeats in the gene for DRPLA, we determined the CAG repeat sizes of 427 single sperm from two individuals with DRPLA. The mean variance of the change in the CAG repeat size in sperm from the DRPLA patients (288.0) was larger than any variances of the CAG repeat size in sperm from patients with Machado-Joseph disease (38. 5), Huntington's disease (69.0) and spinal and bulbar muscular atrophy (16.3), which is consistent with the clinical observation that the genetic anticipation on the paternal transmission of DRPLA is the most prominent among CAG repeat diseases. The variance of the change in CAG repeat size was significantly different between the two DRPLA patients (F-test, P < 0.0001). However, the segregation ratio of single sperm with an expanded allele to ones with a normal allele is not statistically different ( P = 0.161) from the expected 1:1 segregation ratio, and thus segregation distortion of expanded alleles in meiosis in male patients with DRPLA was not demonstrated.     

Reduction in enkephalin and substance P messenger RNA in the striatum of early grade Huntington's disease: a detailed cellular in situ hybridization study

The expression of enkephalin and substance P messenger RNAs was examined in the caudate-putamen of human post mortem tissue from control and Huntington's disease tissue using in situ hybridization techniques and human specific enkephalin and substance P [35S] oligonucleotides. Macroscopic and microscopic quantification of enkephalin and substance P gene expression was carried out using computer-assisted image analysis. Tissue was collected from six control cases with no sign of neurological disease and six Huntington's disease cases ranging from grades 0 to 3 as determined by neuropathological evaluation. The clinical and pathological diagnosis of Huntington's disease was confirmed unequivocally by genetic analysis of the CAG repeat length in both copies of IT15, the Huntington's disease gene. A marked reduction in both enkephalin and substance P messenger RNAs was detected in all regions of the caudate nucleus and putamen in Huntington's disease grades 2/3 when compared to controls; in the dorsal caudate few enkephalin or substance P messenger RNA-positive cells were detected. For the early grade (0/1) Huntington's disease cases, a heterogeneous reduction in both enkephalin and substance P messenger RNAs were noted; for enkephalin messenger RNA the striatal autoradiograms displayed a conspicuous patchy appearance. Detailed cellular analysis of the dorsal caudate revealed a striking reduction in the number of enkephalin and substance P messenger RNA-positive cells detected and in the intensity of hybridization signal/cell. These data suggest that both the "indirect" GABA/enkephalin and "direct" GABA/substance P pathways are perturbed very early in the course of the disease and that these early changes in chemical signalling may possibly underlie the onset of clinical symptoms.     

Codon repeats in genes associated with human diseases: fewer repeats in the genes of nonhuman primates and nucleotide substitutions concentrated at the sites of reiteration

Five human diseases are due to an excessive number of CAG repeats in the coding regions of five different genes. We have analyzed the repeat regions in four of these genes from nonhuman primates, which are not known to suffer from the diseases. These primates have CAG repeats at the same sites as in human alleles, and there is similar polymorphism of repeat number, but this number is smaller than in the human genes. In some of the genes, the segment of poly(CAG) has expanded in nonhuman primates, but the process has advanced further in the human lineage than in other primate lineages, thereby predisposing to diseases of CAG reiteration. Adjacent to stretches of homogeneous present-day codon repeats, previously existing codons of the same kind have undergone nucleotide substitutions with high frequency. Where these lead to amino acid substitutions, the effect will be to reduce the length of the original homopolymeric stretch in the protein.     

Inactivation of the mouse Huntington's disease gene homolog Hdh

Huntington's disease (HD) is a dominant neurodegenerative disorder caused by expansion of a CAG repeat in the gene encoding huntingtin, a protein of unknown function. To distinguish between "loss of function" and "gain of function" models of HD, the murine HD homolog Hdh was inactivated by gene targeting. Mice heterozygous for Hdh inactivation were phenotypically normal, whereas homozygosity resulted in embryonic death. Homozygotes displayed abnormal gastrulation at embryonic day 7.5 and were resorbing by day 8.5. Thus, huntingtin is critical early in embryonic development, before the emergence of the nervous system. That Hdh inactivation does not mimic adult HD neuropathology suggests that the human disease involves a gain of function.