Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2.

The gene for spinocerebellar ataxia type 2 (SCA2) has been mapped to 12q24.1. A 1.1-megabase contig in the candidate region was assembled in P1 artificial chromosome and bacterial artificial chromosome clones. Using this contig, we identified a CAG trinucleotide repeat with CAA interruptions that was expanded in patients with SCA2. In contrast to other unstable trinucleotide repeats, this CAG repeat was not highly polymorphic in normal individuals. In SCA2 patients, the repeat was perfect and expanded to 36-52 repeats. The most common disease allele contained (CAG)37, one of the shortest expansions seen in a CAG expansion syndrome. The repeat occurs in the 5'-coding region of SCA2 which is a member of a novel gene family.

Cytotoxicity of capsaicin and its analogs in vitro.

Capsaicin (CAP) is the main compound responsible for the spicy flavor of Capsicum plants. However, its application can be inhibited due to its pungency and toxicity. This study aimed to evaluate and compare the cytotoxic effect of CAP and its analogs N-benzylbutanamide (AN1), N-(3-methoxybenzyl) butanamide (AN2), N-(4-hydroxy-3-methoxybenzyl) butanamide (AN3), N-(4-hydroxy-3-methoxybenzyl) hexanamide (AN4) and N-(4-hydroxy-3-methoxybenzyl) tetradecanamide (AN5) on the hepatoma cells of Rattus norvegicus using the MTT test. The results showed cytotoxicity of CAP at concentrations of 100, 150, 175, and 200 µM (24 hours), AN1 at 150 and 175 µM (48 hours), AN2 at 50 µM (24 hours) and 10, 25, 50, and 75 µM (48 hours), AN4 at 175 µM (24 hours), and AN5 at 50 µM (48 hours). Removing the hydroxyl radical from the vanillyl group of capsaicin, together with reducing the acyl chain to 3 carbons, which is the case of AN2, resulted in the best biological activity. Increasing the carbon chain in the acyl group of the capsaicin molecule, which is the case of AN5, also showed evident cytotoxic effects. The present study proves that the chemical modifications of capsaicin changed its biological activity.

Gal80 proteins of Kluyveromyces lactis and Saccharomyces cerevisiae are highly conserved but contribute differently to glucose repression of the galactose regulon.

We cloned the GAL80 gene encoding the negative regulator of the transcriptional activator Gal4 (Lac9) from the yeast Kluyveromyces lactis. The deduced amino acid sequence of K. lactis GAL80 revealed a strong structural conservation between K. lactis Gal80 and the homologous Saccharomyces cerevisiae protein, with an overall identity of 60% and two conserved blocks with over 80% identical residues. K. lactis gal80 disruption mutants show constitutive expression of the lactose/galactose metabolic genes, confirming that K. lactis Gal80 functions in essentially in the same way as does S. cerevisiae Gal80, blocking activation by the transcriptional activator Lac9 (K. lactis Gal4) in the absence of an inducing sugar. However, in contrast to S. cerevisiae, in which Gal4-dependent activation is strongly inhibited by glucose even in a gal80 mutant, glucose repressibility is almost completely lost in gal80 mutants of K. lactis. Indirect evidence suggests that this difference in phenotype is due to a higher activator concentration in K. lactis which is able to overcome glucose repression. Expression of the K. lactis GAL80 gene is controlled by Lac9. Two high-affinity binding sites in the GAL80 promoter mediate a 70-fold induction by galactose and hence negative autoregulation by Gal80. Gal80 in turn not only controls Lac9 activity but also has a moderate influence on its rate of synthesis. Thus, a feedback control mechanism exists between the positive and negative regulators. By mutating the Lac9 binding sites of the GAL80 promoter, we could show that induction of GAL80 is required to prevent activation of the lactose/galactose regulon in glycerol or glucose plus galactose, whereas the noninduced level of Gal80 is sufficient to completely block Lac9 function in glucose.

Pathological mechanisms in Huntington's disease and other polyglutamine expansion diseases.

HD is an autosomal dominant neurodegenerative disorder characterized by involuntary movements, cognitive impairment progressing to dementia, and mood disturbances. The brains of patients show extensive neuronal loss in the striatum, and the cerebral cortex is also affected. The genetic defect causing HD is an expansion of a CAG repeat encoding a polyglutamine stretch in the target protein, named huntingtin. The age of onset of HD is inversely correlated with the size of the expansion. Polyglutamine expansion represents a novel cause of neurodegeneration, which has been shown to be responsible for seven other inherited disorders. The polyglutamine expansion confers a gain of toxic property to the mutated target proteins. Molecular and cellular studies of the brains of patients and of mice models of polyglutamine expansion diseases have led to the identification of abnormal intracellular inclusions representing aggregation of the mutated protein. However, the mechanism whereby such polyglutamine expansion leads to selective neuronal dysfunction and death is still puzzling.

A cellular model that recapitulates major pathogenic steps of Huntington's disease.

To gain insight into the pathogenic mechanisms of Huntington's disease (HD), we have developed a stable cellular model, using a neuroblastoma cell line in which the expression of full-length or truncated forms of wild-type and mutant huntingtin can be induced. While the wild-type forms have the expected cytoplasmic localization, the expression of mutant proteins leads to the formation of cytoplasmic and nuclear inclusions in a time- and polyglutamine length-dependent manner. The inclusions are ubiquitinated, appear more rapidly in cells expressing truncated forms of mutant huntingtin and are correlated with enhanced apoptosis. In lines expressing mutant full-length huntingtin, major characteristics present in Huntington's patients could be modelled. Selective processing of the mutant, but not the wild-type, full-length huntingtin was observed at late time points, with appearance of a breakdown product corresponding to a predicted caspase-3 cleavage product. A more truncated N-terminal fragment of huntingtin is also produced, that appears involved in building up cytoplasmic inclusions at early time points, and later on also nuclear inclusions. This fits with the finding that inclusions in the brain of HD patients are detected only using antibodies directed against epitopes very close to the polyglutamine stretch. This unique model should thus be useful to study the processing mechanism of mutant huntingtin, its role in the formation of intracellular aggregates and the effect of the latter on cellular physiology.

Molecular heterogeneity of autosomal dominant cerebellar ataxia: analysis of flanking microsatellites of the spinocerebellar ataxia 1 locus in a northern European family unequivocally demonstrates non-linkage.

This study addresses the question whether the different forms of autosomal dominant cerebellar ataxia (ADCA) are related to different ethnic/geographical regions in Europe. One mutation in families originating from Holland, Prussia and Italy has previously been localized to chromosome 6p (SCA1 locus), whereas the mutation in families of Iberic origin has been excluded from chromosome 6p. In a Danish five-generation pedigree with ADCA and in which previous HLA-serotyping had shown inconclusive linkage results, the present study shows unequivocal exclusion from the SCA1 locus, firstly through the use of the new, highly informative microsatellites D6S89 and D6S109, which closely flank the SCA1 locus, and secondly through the manifestation of disease in four pedigree members previously scored as unaffected. Additional molecular genetic analysis of the HLA DRbeta and F13A polymorphisms also argue against a cluster of ADCA genes on chromosome 6p. Since this study demonstrates the existence of non-SCA1 families and therefore heterogeneity in the North-European population, molecular family counselling remains restricted to the few known SCA1 families.

Properties of polyglutamine expansion in vitro and in a cellular model for Huntington's disease.

Eight neurodegenerative diseases have been shown to be caused by the expansion of a polyglutamine stretch in specific target proteins that lead to a gain in toxic property. Most of these diseases have some features in common. A pathological threshold of 35-40 glutamine residues is observed in five of the diseases. The mutated proteins (or a polyglutamine-containing subfragment) form ubiquitinated aggregates in neurons of patients or mouse models, in most cases within the nucleus. We summarize the properties of a monoclonal antibody that recognizes specifically, in a Western blot, polyglutamine stretches longer than 35 glutamine residues with an affinity that increases with polyglutamine length. This indicates that the pathological threshold observed in five diseases corresponds to a conformational change creating a pathological epitope, most probably involved in the aggregation property of the carrier protein. We also show that a fragment of a normal protein carrying 38 glutamine residues is able to aggregate into regular fibrils in vitro. Finally, we present a cellular model in which the induced expression of a mutated full-length huntingtin protein leads to the formation of nuclear inclusions that share many characteristics with those observed in patients: those inclusions are ubiquitinated and contain only an N-terminal fragment of huntingtin. This model should thus be useful in studying a processing step that is likely to be important in the pathogenicity of mutated huntingtin.

Genetic mapping of the spinocerebellar ataxia 2 (SCA2) locus on chromosome 12q23-q24.1.

A refined genetic map of the spinocerebellar ataxia 2 locus was constructed through linkage and haplotype analysis of 11 large pedigrees from the Holguín SCA2 family collective. Three-point analysis makes a localization of the SCA2 mutation in the 6-cM interval D12S84-D12S79 likely. This is consistent with haplotype results indicating a crossover event between two branches of the SCA2 family Rs and placing the mutation on the telomeric side of D12S84. The microsatellite D12S105 within this interval shows a peak two-point lod score of Z = 16.14 at theta = 0.00 recombination and complete linkage disequilibrium among affected individuals. These data together with the observation of a common disease haplotype among all family ancestors support the notion of an SCA2 founder effect in Holguín province.

Machado-Joseph disease is genetically different from Holguin dominant ataxia (SCA2).

Machado-Joseph disease (MJD) and Holguin ataxia (SCA2) are autosomal dominant multisystem degenerations with spinocerebellar involvement that are predominant among people of Portuguese-Azorean and of Cuban descent, respectively. Their clinical distinction may at times be difficult to make in individual patients, due to significant phenotypic overlapping (similar overall age-of-onset and duration of cerebellar ataxia, eye movement, and, often, other common problems. The recent mapping of SCA2 to chromosome 12q provided another candidate region for linkage studies of MJD. Original data on 10 families with Holguin ataxia show that the locus for phenylalanine hydroxylase (PAH) on chromosome 12q is linked to SCA2 at 4 cM and is thus far its closest marker. The exclusion of linkage 15 cM on each side of PAH in 16 families with MJD shows that these two forms of dominant ataxia are genetically distinct and at different chromosomal locations (nonallelic).

Unstable triplet repeat and phenotypic variability of spinocerebellar ataxia type 1.

A Siberian kindred with spinocerebellar ataxia genetically linked to the SCA1 locus on chromosome 6p has been screened for the CAG triplet expansion within the coding region of the SCA1 gene. The kindred includes 1,484 individuals, 225 affected and 656 at risk, making this collection the largest spinocerebellar ataxia type 1 (SCA1) pedigree known. Each of the studied 78 SCA1 patients carried an expanded allele containing a stretch of 39 to 72 uninterrupted CAG repeats. Normal alleles had 25 to 37 trinucleotide repeats. Expanded alleles containing 40 to 55 repeats were found in 26 at-risk relatives. The number of CAG repeats in the mutated allele was inversely correlated with age at disease onset. Cerebellar deficiency was present in each patient and its severity was moderately affected by the number of CAG repeats. In contrast, the associated signs, dysphagia, diffuse skeletal muscle atrophy with fasciculations, and tongue atrophy were absent or mild in patients with low CAG repeat numbers, but severely complicated the course of illness in patients with a larger number of repeat units. One female mutation carrier was asymptomatic at age 66, more than 2 standard deviations beyond the average age of risk, suggesting incomplete penetrance. In 2 symptomatic individuals who had an expanded number of CAG repeats on both chromosomes, age at onset, rate of progression, and clinical manifestation corresponded to the size of the larger allele.

Mutation detection in Machado-Joseph disease using repeat expansion detection.

BACKGROUND: Several neurological disorders have recently been explained through the discovery of expanded DNA repeat sequences. Among these is Machado-Joseph disease, one of the most common spinocerebellar ataxias (MJD/SCA3), caused by a CAG repeat expansion on chromosome 14. A useful way of detecting repeat sequence mutations is offered by the repeat expansion detection method (RED), in which a thermostable ligase is used to detect repeat expansions directly from genomic DNA. We have used RED to detect CAG expansions in families with either MJD/SCA3 or with previously uncharacterized spinocerebellar ataxia (SCA). MATERIALS AND METHODS: Five MJD/SCA3 families and one SCA family where linkage to SCA1-5 had been excluded were analyzed by RED and polymerase chain reaction (PCR). RESULTS: An expansion represented by RED products of 180-270 bp segregated with MJD/SCA3 (p < 0.00001) in five families (n = 60) and PCR products corresponding to 66-80 repeat copies were observed in all affected individuals. We also detected a 210-bp RED product segregating with disease (p < 0.01) in a non-SCA1-5 family (n = 16), suggesting involvement of a CAG expansion in the pathophysiology. PCR analysis subsequently revealed an elongated MJD/SCA3 allele in all affected family members. CONCLUSIONS: RED products detected in Machado-Joseph disease families correlated with elongated PCR products at the MJD/SCA3 locus. We demonstrate the added usefulness of RED in detecting repeat expansions in disorders where linkage is complicated by phenotyping problems in gradually developing adult-onset disorders, as in the non-SCA1-5 family examined. The RED method is informative without any knowledge of flanking sequences. This is particularly useful when studying diseases where the mutated gene is unknown. We conclude that RED is a reliable method for analyzing expanded repeat sequences in the genome.

Autosomal dominant spinocerebellar ataxia (SCA) in a Siberian founder population: assignment to the SCA1 locus.

In seven families from a Siberian founder population with autosomal dominant spinocerebellar ataxia (SCA) genetic analysis of the polymorphisms flanking the SCA1 locus on chromosome 6p showed allelic association with disease inheritance. While the association was absolute in the case of microsatellite D6S274, an allele switch was observed for D6S89 in two families, suggesting a historic recombinant. Further genetic and physical study of this recombinant event could be instrumental for the precise localization and identification of the SCA1 gene.

A gene for autosomal dominant paroxysmal choreoathetosis/spasticity (CSE) maps to the vicinity of a potassium channel gene cluster on chromosome 1p, probably within 2 cM between D1S443 and D1S197.

Paroxysmal choreoathetosis/episodic ataxia is a heterogeneous neurological syndrome usually inherited in an autosomal dominant manner. Recently, the association of one form of episodic ataxia (defined by the presence of additional myokymia) with point mutations in the potassium channel gene KCNA1 was described. This gene locus on chromosome 12p (HGMW-approved symbol CSE) was excluded in a large pedigree with paroxysmal choreoathetosis and additional spasticity. Linkage to chromosome 1p where a cluster of related potassium channel genes is located, was demonstrated. Genotyping of 18 affected and 11 unaffected family members with 28 microsatellites over a region of 45 cM proved linkage with a lod score of 7.2 at a recombination fraction theta = 0 to D1S451/421/447/GGAT4C11. Crossing-over events in 9 patients and 4 unaffected offspring suggested a probable assignment of the gene to a region of 2 cM between D1S443 and D1S197.

SCA2 trinucleotide expansion in German SCA patients.

Autosomal dominant spinocerebellar ataxias (SCA) are a group of clinically and genetically heterogeneous neurodegenerative disorders which lead to progressive cerebellar ataxia. A gene responsible for SCA type 2 has been mapped to human chromosome 12 and the disease causing mutation has been identified as an unstable and expanded (CAG)n trinucleotide repeat. We investigated the (CAG)n repeat length of the SCA2 gene in 842 patients with sporadic ataxia and in 96 German families with dominantly inherited SCA which do not harbor the SCA1 or MJD1/SCA3 mutation, respectively. The SCA2 (CAG)n expansion was identified in 71 patients from 54 families. The (CAG)n stretch of the affected allele varied between 36 and 64 trinucleotide units. Significant repeat expansions occurred most commonly during paternal transmission. Analysis of the (CAG)n repeat lengths with the age of onset in 41 patients revealed an inverse correlation. Two hundred and forty-one apparently healthy octogenerians carried alleles between 16 and 31 repeats. One 50-year old, healthy individual had 34 repeats; she had transmitted an expanded allele to her child. The small difference between 'normal' and disease alleles makes it necessary to define the extreme values of their ranges. With one exception, the trinucleotide expansion was not observed in 842 ataxia patients without a family history of the disease. The SCA2 mutation causes the disease in nearly 14% of autosomal dominant SCA in Germany.