The gene for autosomal dominant cerebellar ataxia with pigmentary macular dystrophy maps to chromosome 3p12-p21.1.

Autosomal dominant cerebellar ataxia with pigmentary macular dystrophy (ADCA type II) is a rare neurodegenerative disorder with marked anticipation. We have mapped the ADCA type II locus to chromosome 3 by linkage analysis in a genome-wide search and found no evidence for genetic heterogeneity among four families of different geographic origins. Haplotype reconstruction initially restricted the locus to the 33 cM interval flanked by D3S1300 and D3S1276 located at 3p12-p21.1. Combined multipoint analysis, using the Zmax-1 method, further reduced the candidate interval to an 8 cM region around D3S1285. Our results show that ADCA type II is a genetically homogenous disorder, independent of the heterogeneous group of type I cerebellar ataxias.

Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats.

Two forms of the neurodegenerative disorder spinocerebellar ataxia are known to be caused by the expansion of a CAG (polyglutamine) trinucleotide repeat. By screening cDNA expression libraries, using an antibody specific for polyglutamine repeats, we identified six novel genes containing CAG stretches. One of them is mutated in patients with spinocerebellar ataxia linked to chromosome 12q (SCA2). This gene shows ubiquitous expression and encodes a protein of unknown function. Normal SCA2 alleles (17 to 29 CAG repeats) contain one to three CAAs in the repeat. Mutated alleles (37 to 50 repeats) appear particularly unstable, upon both paternal and maternal transmissions. The sequence of three of them revealed pure CAG stretches. The steep inverse correlation between age of onset and CAG number suggests a higher sensitivity to polyglutamine length than in the other polyglutamine expansion diseases.

Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion.

The gene for spinocerebellar ataxia 7 (SCA7) has been mapped to chromosome 3p12-13. By positional cloning, we have identified a new gene of unknown function containing a CAG repeat that is expanded in SCA7 patients. On mutated alleles, CAG repeat size is highly variable, ranging from 38 to 130 repeats, whereas on normal alleles it ranges from 7 to 17 repeats. Gonadal instability in SCA7 is greater than that observed in any of the seven known neuro-degenerative diseases caused by translated CAG repeat expansions, and is markedly associated with paternal transmissions. SCA7 is the first such disorder in which the degenerative process also affects the retina.

Identification of the flavin monooxygenase responsible for ipso substitution of alkyl and alkoxyphenols in Sphingomonas sp. TTNP3 and Sphingobium xenophagum Bayram.

We previously showed that opdA from Sphingomonas sp. PWE1 encodes a putative flavin monooxygenase capable of transforming octylphenol (OP) via type II ipso substitution. Here, we demonstrate that an opdA homolog is responsible for OP and related alkyl/alkoxyphenol degradation in the nonylphenol degrader Sphingomonas sp. TTNP3. PCR and Southern blot analyses revealed that TTNP3 contained an opdA homolog, while a TTNP3 derivative unable to grow on nonylphenol (TTNP3d) did not. OpdA expression was confirmed in wild-type TTNP3 via two dimensional gel electrophoresis. Activity was restored to TTNP3d following complementation with opdA. Sequence analysis of an opdA homolog from another nonylphenol degrader, Sphingobium xenophagum Bayram, revealed that the predicted protein sequences from PWE1 and Bayram were identical, but differed from TTNP3 by four amino acids. In order to assess differences, we heterologously expressed the two unique opdA homologs and compared their effect on the disappearance of five alkyl/alkoxyphenol substrates and subsequent appearance of hydroquinone. For all substrates, except OP, the levels of substrate disappearance and hydroquinone appearance were significantly lower in cultures expressing odpA (TTNP3) than those expressing opdA (PWE1/Bayram). These differences in substrate specificity were consistent with an in silico model which predicted that two of the amino acid differences between odpA (TTNP3) and opdA (PWE1/Bayram) lay in a putative substrate binding pocket. While these strains are known to use the same type II ipso substitution mechanism for alkylphenol degradation, this work provides the first preliminary evidence that opdA homologs also encode the type I ipso substitution activity responsible for the degradation of alkoxyphenols.

Melatonin does not suppress the pituitary luteinizing hormone response to luteinizing hormone-releasing hormone in men.

The effect of an iv melatonin infusion on the pituitary LH response to LHRH was studied in five young men. Melatonin (30 micrograms/min; total dose, 7.2 mg) was infused for a 4-h period, 2 h before and 2 h after a LRH stimulation (single iv 150-microgram dose). Each subject's control response to LRH was obtained previously. During the melatonin infusion, supraphysiological concentrations of melatonin (20 times) were documented using a specific RIA. All five subjects had a LH rise after LRH stimulation, and this response was not affected by the melatonin infusion. These results indicate that an acute constant infusion of a pharmacological amount of melatonin does not suppress LRH-induced LH release from the pituitary in men. In addition, no change in sleepiness and behavior was found.

Clinical and genetic analysis of a Tunisian family with autosomal dominant cerebellar ataxia type 1 linked to the SCA2 locus.

Autosomal dominant cerebellar ataxias (ADCA) type 1 are a clinically and genetically heterogeneous group of neurodegenerative disorders. We report a large Tunisian ADCA type 1 family in which 17 patients (mean age at onset +/- SD = 35.6 +/- 15.3 years) were examined. There was mean anticipation of 10.3 +/- 15.4 years in this family; anticipation was greater in paternal (28 +/- 8.2 years) than in maternal (2.7 +/- 10.9 years) transmission. Linkage analysis performed with microsatellite markers linked to the spinal cerebellar ataxia 1 (SCA1) locus on chromosome 6p and the SCA2 locus on chromosome 12q excluded linkage to SCA1, but there was close linkage with marker D12S105 (Zmax = 2.51 at theta = 0.00). This result was confirmed by multipoint analysis, which generated a maximal lod score of 3.46 at this locus. Multipoint analysis and haplotype reconstruction reduced the interval containing the SCA2 locus to 6.4 cM, a narrowing of the 35-cM interval in a previously described Cuban SCA2 family with a clinical picture similar to that of our family, including a high frequency of postural and action tremor.

Clinical and molecular features of spinocerebellar ataxia type 6.

The mutation involved in spinocerebellar ataxia type 6 (SCA6) is a small CAG expansion in the alpha-1A subunit of the voltage-dependent calcium channel gene. We looked for this mutation in 91 families with autosomal-dominant cerebellar ataxias and found that SCA6 is a minor locus in our series (2%) and is rare in France (1%). Furthermore, we did not detect the SCA6 mutation on 146 sporadic cases with isolated cerebellar ataxia or olivopontocerebellar atrophy. The normal and expanded alleles ranged from 4 to 15 and 22 to 28 CAG repeats, respectively, and age at onset was correlated to CAG repeat length (r = -0.87). In contrast with other SCA, the expanded allele was stable during transmission. Clinically, SCA6 patients (n = 12) presented with moderate to severe cerebellar ataxia with a lower frequency of associated signs compared with other SCA and a mean age at onset of 45 +/- 14 years (range, 24 to 67). MRI showed extensive cerebellar atrophy but not of the brainstem or cerebral cortex.

SCA2 is not a major locus for ADCA type I in French families.

Autosomal dominant cerebellar ataxias (ADCA) of type I, a group of clinically heterogeneous neurodegenerative disorders, are known to be genetically heterogeneous since a second locus for ADCA type I (SCA2) has been identified on the long arm of chromosome 12. Linkage analysis was performed in 7 French ADCA type I families in order to estimate its frequency. We analysed 121 individuals, 39 of whom were affected. In 6 families, the SCA2 candidate interval, spanning 12.8 cM, was excluded by bi- and multipoint analysis. In one family (SAL-315), however, the maximal positive lod score reached 2.03 at the D12S79 locus. A posterior probability of 94% in favor of linkage to SCA2 was calculated by homogeneity analysis. The clinical profile of this family was similar to that of previously described SCA1 and non-SCA1 families, except that dementia was observed in 2 out of 6 patients. This may be a clinical idiosyncrasy in this family and was insufficient for a genotype-phenotype correlation.

Neuronal distribution of intranuclear inclusions in Huntington's disease with adult onset.

Neuronal intranuclear inclusions were recently found in the brain of patients with inherited neurodegenerative disorders characterized by the expansion of a polyglutamine stretch in the mutated protein. These inclusions are ubiquitinated and, for some of these diseases, the presence of the mutated protein could be also identified. Using immunohistochemistry, we show here that ubiquitinated intranuclear inclusions are also observed postmortem in the brain of patients suffering from Huntington's disease characterized by small polyglutamine expansions and adult onset. We were, however, unable to detect the mutated form of huntingtin in these inclusions. These intranuclear inclusions were detected only in the affected cerebral regions, suggesting that their presence is probably linked to the neurodegenerative process.

Clinical and genetic analysis of three German kindreds with autosomal dominant cerebellar ataxia type I linked to the SCA2 locus.

The detailed clinical, electrophysiological and imaging data of three German autosomal dominant cerebellar ataxia (ADCA) families are reported. Linkage to SCA2 was established using microsatellite markers D12S105, D12S1339(1328), D12S1304(1329) yielding a lod score exceeding +3.0 for the combined data. Analysis of the pedigree data provided evidence of anticipation as observed in other neurodegenerative disorders due to polyglutamine expansion encoded by a CAG repeat. This hypothesis was confirmed by the detection of the SCA2-specific pathological protein using the 1C2 monoclonal antibody which selectively recognizes large polyglutamine expansions and the characterization of a CAG expansion in the patients. Clinically, the families were characterized by progressive ataxia of stance, gait and limbs. Saccade velocity was markedly reduced in SCA2. Further oculomotor findings were gaze palsy, impaired smooth pursuit and reduced optokinetic reflex. Dementia and pyramidal tract signs were rather rare, while peripheral involvement (reduced or absent ankle reflexes, fasciculation-like movements, amyotrophy) was a prominent feature. Electrophysiological investigations provided evidence of sensory neuropathy of the axonal type and degeneration of the posterior columns. Imaging studies demonstrated severe shrinkage of brain-stem structures even in early stages of the disease.

[Does the ataxo-choreic form of DRPLA exist in Europe? Search of mutation in 120 families]. / La forme ataxo-choréique de DRPLA existe-t-elle en Europe? Recherche de la mutation dans 120 familles.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by a degeneration of cerebellar and pallidal efferents, more frequent in Japan. Isolated cases are also encountered. Patients present with variable combination of signs including myoclonus, ataxia, epilepsy, choreoathetosis and dementia, with onset from childhood to the seventh decade. Clinically, DRPLA may be undistinguishable from other genetic disorders, in particular Huntington's disease or the spinocerebellar ataxias. The genetic basis of the inherited form of DRPLA is an expansion to more than 49 repeats of an unstable trinucleotide (CAG) in the DRPLA gene on the short arm of chromosome 12. We determined the frequency of this mutation in patients with the DRPLA phenotype. One hundred and seventeen patients with cerebellar ataxia, from 94 families and 23 isolated cases, as well as 3 patients from families with undiagnosed autosomal dominant neurodegenerative disorders were investigated for the presence of the expanded sequence. None of the patients carried this mutation. This finding suggests that DRPLA is rare in the French population. The search for the DRPLA mutation is justified in patients with the DRPLA phenotype, however, since genetic counselling is often requested and neither clinical, nor neuropathological examinations permit a definite diagnosis of the underlying disease.

Nuclear inclusions in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 is due to a CAG repeat expansion in the gene encoding ataxin-1. In a case with an expansion of 56 repeats, intranuclear inclusions were found only in neurons, both in severely affected regions (such as the pons) and in areas where the lesions were inconspicuous (such as the cortex or the striatum). The inclusions were labelled by a monoclonal antibody directed against long polyglutamine stretches (1C2); they were also detected by the anti-ubiquitin antibody. They were faintly eosinophilic, Congo red negative and were not stained by thioflavin S or by ethidium bromide.

Phenotypic variability in autosomal dominant cerebellar ataxia type I is unrelated to genetic heterogeneity.

Families with autosomal dominant cerebellar ataxia (ADCA), a heterogeneous group of diseases, were investigated prior to and during genetic linkage analysis. We report here on the clinical features of 122 affected individuals from 36 unrelated families with ADCA type I, the most common type. Our results indicate an anticipation expressed in a mean 9.4 year earlier age at onset and more rapid clinical progression in successive generations. There was no imprinting, since age at onset, disease duration and severity of the disease were independent of parental transmission. Progressive cerebellar ataxia was variably associated with signs such as ophthalmoplegia, dysphagia, sphincter disturbances, briskness or loss of tendon reflexes, decreased vibration sense and amyotrophy, a variability correlated with disease duration. Linkage analysis of 10 informative families with microsatellite markers, located on the short arm of the chromosome 6, allowed the identification of four families showing positive linkage to the SCA1 (spinal cerebellar ataxia 1) locus and six non-SCA1 families for whom linkage to this locus was excluded. This reflects non-allelic genetic heterogeneity. Thus, the analysis of clinical signs associated with cerebellar ataxia in SCA1 versus non-SCA1 kindreds did not distinguish between the two groups. The clinical picture of ADCA type I did not reflect the genetic heterogeneity of the disease.

Autosomal dominant cerebellar ataxia type I linked to chromosome 12q (SCA2: spinocerebellar ataxia type 2).

Spinocerebellar ataxia 2 (SCA2) is one of the loci for the clinically and genetically heterogeneous group of autosomal dominant type I cerebellar ataxias. After initial linkage to chromosome 12q in Cuban families, SCA2 was shown to be the gene responsible for the disease in Italian, Tunisian, French-Canadian, Austrian-Canadian and Martinican kindreds with dominant ataxia, and the candidate interval was reduced to 6.4 cM between markers D12S84 and D12S79. Comparison of patients from families of different geographical origins clearly demonstrates the clinical interfamilial variability of the clinical signs which reaches statistical significance for the frequency of extrapyramidal rigidity, postural tremor and dementia. The most striking difference between the 29 Martinican SCA2 patients and those with SCA1 on chromosome 6p or SCA3/MJD on chromosome 14q is the greater frequency of hyporeflexia in the former. A mean 12.5 year anticipation is observed, with a more rapid clinical course of the disease in successive generations, indicating that an expanded trinucleotide repeat probably constitutes the underlying molecular mechanism.

Heterogeneous intracellular localization and expression of ataxin-3.

Spinocerebellar ataxia type 3 or Machado-Joseph disease (SCA3/MJD) is an autosomal dominant neurodegenerative disorder caused by an unstable and expanded CAG trinucleotide repeat that leads to the expansion of a polyglutamine tract in a protein of unknown function, ataxin-3. We have generated and characterized a panel of monoclonal and polyclonal antibodies raised against ataxin-3 and used them to analyze its expression and localization. In Hela cells, multiple isoforms are expressed besides the major 55-kDa form. While the majority of ataxin-3 is cytosolic, both immunocytofluorescence and subcellular fractionation studies indicate the presence of ataxin-3, in particular, of some of the minor isoforms, in the nuclear and mitochodrial compartments. We also show that ataxin-3 can be phosphorylated. In the brain, only one ataxin-3 isoform containing the polyglutamine stretch was detected, and normal and mutated proteins were found equally expressed in all patient brain regions analyzed. In most neurons, ataxin-3 had a cytoplasmic, dendritic, and axonal localization. Some neurons presented an additional nuclear localization. Ataxin-3 is widely expressed throughout the brain, with a variable intensity specific for subpopulations of neurons. Its expression is, however, not restricted to regions that show intranuclear inclusions and neurodegeneration in SCA3/MJD.

Analysis of the SCA1 CAG repeat in a large number of families with dominant ataxia: clinical and molecular correlations.

Autosomal dominantly inherited ataxias are a clinically and genetically heterogeneous group of neurodegenerative disorders. The gene involved in one subtype, spinocerebellar ataxia 1 (SCA1), was first localized to chromosome 6p. An unstable CAG repeat has been identified as the responsible mutation. In this study, 88 families with various types of inherited ataxias and 16 individuals with sporadic cerebellar ataxia were investigated to determine the frequency of this mutation, the behavior of the SCA1 CAG repeat during transmission, and the clinical features specific to this form of disease. Only 12 of the families carried the SCA1 mutation; 10 of the 12 were of French origin. When transmitted paternally, the repeat was more unstable and larger in size. Age at onset was inversely correlated with the number of CAG repeats. Anticipation in age at onset of about 11 years was observed in offspring. Analysis of the clinical features did not distinguish SCA1 from other forms of dominantly inherited ataxias. In the absence of distinguishing clinical characteristics, the diagnosis of SCA1 in single affected patients or family members can only be made by direct detection of the mutation, opening the way for presymptomatic testing.

Somatic mosaicism of the CAG repeat expansion in spinocerebellar ataxia type 3/Machado-Joseph disease.

An expanded and unstable CAG repeat in the coding region of the MJD1 gene is the mutation responsible for spinocerebellar ataxia 3/Machado-Joseph disease. In order to determine whether there was a higher degree of instability in affected regions, the size of the expanded CAG repeat was analyzed in different regions of the central nervous system, in two unrelated SCA3/MJD patients. The degree of somatic mosaicism was quantified and compared to that in a SCA1 patient. Instability of the expanded CAG repeat was observed in peripheral tissues as well as in CNS of the three patients, but there was no correlation between the degree of mosaicism and the selective vulnerability of CNS structures. As in the other diseases caused by expanded CAG repeats, a lower degree of mosaicism was found in the cerebellar cortex of both SCA1 and SCA3/MJD patients, probably reflecting specific properties of this structure. In SCA3/MJD, the degree of mosaicism seemed to correlate with age at death rather than with the size of the expanded CAG repeat. Finally, somatic instability was more pronounced in SCA1 than in SCA3/MJD patients.

Autosomal dominant cerebellar ataxia type I in Martinique (French West Indies). Clinical and neuropathological analysis of 53 patients from three unrelated SCA2 families.

Autosomal dominant cerebellar ataxia type I was diagnosed in three unrelated families from Martinique (French West Indies), and linkage to the locus for spinocerebellar ataxia 2 (SCA2) was established. Neuropathological findings in two patients were those of olivopontocerebellar atrophy without oligodendroglial cytoplasmic inclusions. Cerebellar ataxia was associated with hyporeflexia in 68% of 31 examined patients, with slowed and/or limited eye movements in 65% and with dementia in 29%. No patients had optic atrophy, pigmentary retinal degeneration, spasticity or parkinsonism. Mean age at onset was 33 +/- 16 years, and onset before the age of 20 years was correlated with a more rapid and severe course of the disease. Movement disorders, oculomotor disturbances, sphincter disturbances and cognitive impairment were significantly more frequent in early than in late onset patients. This explains why the phenotype was strikingly different in one family, in which mean age at onset was much earlier. Comparison with previously described SCA2 families indicated similarities, such as reduced saccade velocity, supranuclear ophthalmoplegia and decreased reflexes, although phenotypic heterogeneity remains the outstanding feature of this disorder.