Screening for proteins with polyglutamine expansions in autosomal dominant cerebellar ataxias
Screening for proteins with polyglutamine expansions in autosomal dominant cerebellar ataxiasGiovanni Stevanin1, Yvon Trottier2, Géraldine Cancel1, Alexandra Dürr1, Gilles David1, Olivier Didierjean1, Katrin Bürk3, Georges Imbert2, Frederic Saudou2, Myriem Abada-Bendib1,4, Isabelle Gourfinkel-An1, Ali Benomar5, Nacer Abbas1, Thomas Klockgether3, Djamel Grid6,+, Yves Agid1, Jean-Louis Mandel2 and Alexis Brice1,*
1INSERM U289 and Fédération de Neurologie, Hôpital de la Salpêtrière, Paris, France, 2Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, CU de Strasbourg, France, 3Department of Neurology, University of Tübingen, Tübingen, Germany, 4Service de Neurologie, Hôpital de Ben-Aknoun, CHU Alger-ouert, Algiers, Algeriaand 5Service de Neurologie, Hôpital des Spécialités, Rabat, Morocco, 6Service de Neurologie, CHU Mustapha, Algiers, Algeria
Received July 30, 1996;Revised and Accepted September 16, 1996
Expansion of trinucleotide CAG repeats coding for polyglutamine has been implicated in five neurodegenerative disorders, including spinocerebellar ataxia (SCA) 1 and SCA3 or Machado-Joseph disease (SCA3/MJD), two forms of type I autosomal dominant cerebellar ataxias (ADCA). Using the 1C2 antibody which specifically recognizes large polyglutamine tracts, particularly those that are expanded, we recently reported the detection of proteins with pathological glutamine expansions in lymphoblasts from another form of ADCA type I, SCA2, as well as from patients presenting with the distinct phenotype of ADCA type II. We now have screened a large series of patients with ADCA or isolated cases with cerebellar ataxia, for the presence of proteins with polyglutamine expansions. A 150 kDa SCA2 protein was detected in 16 out of 40 families with ADCA type I. This corresponds to 24% of all ADCA type I families, which is much more frequent than SCA1 in this series of patients (13%). The signal intensity of the SCA2 protein was negatively correlated to age at onset, as expected for an expanded and unstable trinucleotide repeat mutation. The disease segregated with markers closely linked to the SCA2 locus in all identified SCA2 families. In addition, a specific 130 kDa protein, which segregated with the disease, was detected in lymphoblasts of patients from nine families with ADCA type II. It was also visualized in the cerebral cortex of one of the patients, demonstrating its translation in the nervous system. Finally, no new disease-related proteins containing expanded polyglutamine tracts could be detected in lymphoblasts from the remaining patients with ADCA or isolated cases with cerebellar ataxia.
The autosomal dominant cerebellar ataxias (ADCA) are a group of neurological disorders characterized clinically by progressive and unremitting cerebellar ataxia variably associated with other neurological signs. A classification, established on the basis of these associated signs (1 ,2 ), now includes molecular criteria (3 ,4 ). ADCA type I is the most common subtype and variably combines cerebellar ataxia and dysarthria, ophthalmoplegia, pyramidal and extrapyramidal signs, deep sensory loss, amyotrophy and dementia. The genes for two forms of ADCA type I have been identified: spinocerebellar ataxia (SCA) 1 on chromosome 6p and SCA3, which is also responsible for Machado-Joseph disease (MJD), on chromosome 14q. Two other genes have been localized on chromosomes 12q (SCA2) and 16q (SCA4), and the existence of yet another is likely. The association of macular degeneration with cerebellar ataxia is characteristic of ADCA type II, the gene for which has been mapped to the SCA7 locus on chromosome 3p. Finally, ADCA type III denotes a `pure' late onset cerebellar syndrome and has been mapped to chromosome 11cen (SCA5).
SCA1 and SCA3/MJD share common molecular features with Huntington's disease (HD), dentatorubropallidoluysian atrophy (DRPLA) and spinobulbar muscular atrophy (SBMA), three other neurodegenerative disorders [reviewed in (3 ,5 )]: (i) the mutation is the expansion of a translated trinucleotide CAG repeat sequence; (ii) there is a strong negative correlation between the number of CAG repeats and the age at onset; (iii) the CAG repeat is unstable particularly during paternal transmission, partially accounting for the anticipation phenomenon; and (iv) the normal, as well as the pathological protein, which contain an expanded polyglutamine tract, are expressed in the nervous system (6 -14 ).
Recently, Trottier and co-workers (13 ) reported that the monoclonal antibody 1C2 (mAb1C2) specifically recognizes long polyglutamine stretches, with a detection threshold that varies according to the protein. Above the threshold, the intensity of the signal increases with the number of glutamines. This antibody allowed the identification of expanded polyglutamine containing proteins of 150 and 130 kDa in lymphoblasts of patients with SCA2 and SCA7, respectively, suggesting that two additional subtypes of ADCA are caused by trinucleotide repeat expansion (13 ).
In order to determine whether other proteins with polyglutamine expansions are involved in ADCAs, we have used mAb1C2 to analyze proteins extracted from lymphoblastoid cell lines of a large subset of patients. We were able to evaluate the frequency of SCA2, characterized by the segregation of a 150 kDa protein. A specific 130 kDa protein, expressed in the cerebral cortex, was observed in the majority of the ADCA type II kindreds. No evidence for new proteins with polyglutamine expansions was found either in the remaining ADCA families or in isolated cases with cerebellar ataxia (15 ).
The initial screening for proteins with expanded polyglutamine tracts using the mAb1C2 was performed only on the patients with the earliest ages at onset in each of 40 families with ADCA type I, 13 with ADCA type II and four with ADCA type III, in addition to seven isolated cases with cerebellar ataxia. Expansions of trinucleotide CAG repeats at the SCA1, SCA3/MJD and DRPLA loci had been excluded previously in all probands by PCR analysis (16 -18 ).
Detection of the 150 kDa SCA2 protein. A 150 kDa protein was strongly detected in the selected patients from 16 of the 40 ADCA type I families (Fig. 1 A). In six of them, linkage to the SCA2 locus has been established or suspected previously (19 -22 ). This strong 150 kDa contrasts with a much weaker band at the same size that was frequently observed in controls (Fig. 1 B, lane 1; Fig. 1 C, lane 6 and Fig. 1 D, lane 8). The presence of the SCA2 protein and its segregation with the disease was confirmed in 29 other patients from 10 of these 16 families, with ages at onset ranging from 13 to 57 years (Fig. 1 B). However, only the weak 150 kDa band was visible in two patients, with ages at onset of 46 (Fig. 1 C, lane 2) and 63 years (Table 1 1). In addition, a strong signal was detected in an asymptomatic at risk individual, who was 33 years old and carried the haplotype segregating with the disease in his family (Fig. 1 B, lane 2).
Thirteen families with the specific ADCA type II phenotype were analyzed. Their clinical features were similar to those found in previous reports (23 ,24 ). The mean age at onset was 27.3 +- 15.2 years (range 1.5-70; n = 49) and was similar in men (24.01 +- 15.9, n = 26) and women (31.0 +- 13.9, n = 23). A mean 18.2 +- 13.1 years (range -10 to +45) anticipation was observed in 22 parent-child couples, and was significantly more marked in paternal (23.1 +- 15.6 years, n = 8) compared with maternal (15.3 +- 11.1 years, n = 14) transmissions (P<0.05).
In six of the 13 ADCA type II families, a specific 130 kDa band was observed in 12 patients with ages at onset ranging from 5 to 43 years (Table 1 and Fig. 2 , individuals SAL-306-001, Rbt-002-029 and Rbt-002-027). The co-segregation of the protein and the disease could be observed in two of these kindreds. In some patients from SCA7-linked families (24 ), a 150 kDa band (n = 3) or a 180 kDa (n = 1) were detected in addition to the 130 kDa protein (Fig. 2 ). Their presence was not correlated with an early age at onset (range 5-43 years). The 130 kDa protein was not detected in eight patients, with ages at onset from 27 to 55 years. Interestingly, in one of these negative patients with an age at onset of 34 years (Rbt-007-016), a strong signal of higher molecular weight was present at 150 kDa (Fig. 2 ).
There were no detectable proteins with expanded polyglutamine stretches in the lymphoblastoid cell lines from 21 of the 24 remaining ADCA type I families, nor in those with ADCA type III or isolated cases with cerebellar ataxia. In three patients with ADCA type I, no conclusion could be drawn since the intensity of the 150 kDa band was intermediate between the normal control signal and the more intense signal corresponding to the SCA2 protein. A 190 and a strong 230 kDa protein, as well as the TATA-binding protein (TBP) at ~50 kDa, were detected in lymphoblasts of all patients and controls, as already reported (13 ).
In order to determine whether an unstable mutation could be excluded in the 21 remaining ADCA type I families, anticipation was evaluated. Mean age at onset was 33.3 +- 12.9 years (n = 61), and differed significantly between those who inherited the disease from their father (28.8 +- 10.3 years, n = 26) and those who received it from their mother (34.8 +- 13.2 years, n = 27) (P<0.05). The mean anticipation in 28 parent-child couples was 9.9 +- 13.1 years and differed, although not significantly (P = 0.055), according to the gender of the transmitting parent (13.7 +- 14.2 years, n = 15 and 5.4 +- 10.6 years, n = 13 for paternal and maternal inheritance, respectively). In two of these negative kindreds, linkage to SCA4 and SCA5 could be excluded (data not shown). The remaining families were not informative for linkage analysis.
Systematic screening for proteins with expanded polyglutamine tracts in lymphoblastoid cell lines from a large series of families with various forms of ADCA or isolated cerebellar ataxia permitted the detection of new SCA2 families in which the abnormal protein segregated, and the analysis of kindreds with the ADCA type II phenotype.
An intensely labeled 150 kDa protein was detected in 16 families for which linkage to markers D12S1339 and D12S1340 was then established. These results demonstrate that the 150 kDa signal corresponds to the SCA2 protein which carries an expanded polyglutamine tract. Taking into account the relative frequencies of SCA1 (13%) and SCA3 (28%) in this sample (18 ), the frequency of the SCA2 mutation was evaluated at ~24%. In this series, SCA2 was more frequent than SCA1, the first mapped and identified triplet repeat responsible for an ADCA. As previously reported in SCA2 kindreds (18 ,19 ,21 ), the group of SCA2 patients differed from SCA1 and SCA3/MJD groups by higher frequencies of decreased or abolished reflexes of the lower limbs, fasciculations, slow eye movements and tremor, and by a lower frequency of extensor plantar responses (data not shown).
The SCA2 protein was detected in families with various geographical origins: France (n = 7), Germany (n = 3), French West Indies (n = 3), as well as in Belgium (n = 1), Algeria (n = 1) and Yugoslavia (n = 1) where this mutation had not been previously reported. Recombination events were detected in two patients with the marker D12S1339, confirming that this marker represents the centromeric boundary of the SCA2 candidate interval. Marker D12S1340 is closely linked to the SCA2 locus since no recombination events were detected and linkage disequilibrium was observed at this locus. The 147 bp allele of D12S1340 segregated with the disease in eight of 15 SCA2 families tested (data not shown), as well as in a Tunisian SCA2 family (25 ,26 ), but was found in only 26 of 96 control chromosomes (P<0.05). More SCA2 families are needed to determine whether a limited number of founder events or several different mutations are involved.
The mAb1C2 is a sensitive tool for the detection of the SCA2 protein. Only two out of 47 patients tested did not present detectable SCA2 protein. This might be due to a smaller expansion, that would be compatible with their rather late ages at onset (46 and 63 years). In addition, the 150 kDa protein was detected in a still asymptomatic at risk individual who carried the disease-associated haplotype.
Trottier and co-workers have reported previously that the signal intensity with the mAb1C2 increases with the size of the polyglutamine tract in huntingtin, ataxin-1 and TBP (13 ). The presence of anticipation in the 16 SCA2 families, and the tendency for the signal corresponding to the 150 kDa protein to increase as age at onset decreases, suggest an unstable mutation due to a polyglutamine-coding trinucleotide expansion. The imperfect correlation between age at onset and the intensity of the 150 kDa band, observed in some instances, may be explained, as in other neurodegenerative disorders, by the contribution of other genetic or environmental factors (18 ,27 ,28 ). In these diseases, the number of CAG repeats only accounts for ~50-70% of the variability in age at onset.
ADCA type II, characteristically associated with progressive pigmentary macular dystrophy, has been considered to be a distinct clinical entity, that differs from other ADCAs (1 ,23 ,29 -31 ). This was confirmed recently by the demonstration of linkage homogeneity at the SCA7 locus in kindreds from different geographical origins (24 ,32 ,33 ). The detection of a 130 kDa protein in nine families with the ADCA type II phenotype strongly supports the hypothesis of genetic homogeneity. However, no abnormal protein was detected in eight patients from these families, with ages at onset ranging from 27 to 55 years, and in four other kindreds. This suggests that mAb1C2 does not detect the SCA7 protein with the same sensitivity as for SCA2. This is reminiscent of SCA1, where the mutated protein is only detected when there are at least 55 repeats (13 ). A low level of expression in some lymphoblastoid cell lines might also explain the absence of detectable protein. Consistent with this hypothesis, the SCA7 protein was detected in the nuclear fraction but not in whole cell extracts of patients with onset at 20, 22 and 40 years. The use of nuclear extracts, therefore, increases the sensitivity of the SCA7 protein assay. The absence of the 130 kDa protein in four ADCA type II kindreds does not rule out the homogeneity of linkage of this disease to the SCA7 locus.
Additional proteins containing expanded polyglutamine tracts of 150 or 180 kDa were also observed in lymphoblastoid cell lines from ADCA type II patients. They were not found in the brain, however, of one patient. The significance of these additional proteins is not clear. Dimer or trimerization (34 ), as observed with the TBP (Y. Trottier, unpublished data) or non-covalent association with other proteins cannot account for the apparent molecular weight of these proteins. These additional proteins may originate, however, from somatic mosaicism of the glutamine-coding trinucleotide repeat expansion, alternative splicing, post-translational modifications or from covalent linkage to other proteins. In HD, migration as a smear or as multiple bands of mutated, but not normal, proteins, has not yet been explained (9 ,12 ,35 ).
In a number of families, despite the analysis of patients with the earliest age at onset in 24 with ADCA type I, three with ADCA type III as well as in seven isolated cases of cerebellar ataxia, no proteins containing long polyglutamine tracts were detected. This result does not exclude the hypothesis that glutamine-encoding trinucleotide repeat expansions are involved in some of these families, particularly in the ADCA type I kindreds in which significant anticipation was found. Low levels of expression in the lymphoblastoid cell lines, or size of the polyglutamine expansion below the detection threshold of mAb1C2, might preclude detection.
In conclusion, mAb1C2 has proven to be a sensitive tool for identifying expanded polyglutamine tracts, particularly in patients carrying the SCA2 mutation. It also facilitated detailed analysis of SCA2 families, that were not informative in linkage studies, before the corresponding gene could be cloned. While different proteins with polyglutamine expansions were detected in lymphoblasts of ADCA type II patients, only the 130 kDa protein was detected in the cerebral cortex. The use of brain, instead of lymphoblasts, extracts is probably a more accurate and sensitive method. Although no new proteins with expanded polyglutamine tracts have been identified in the course of this study, mAb1C2 is a promising tool for the screening of other disorders with anticipation, such as familial Parkinson's disease (36 ) or autosomal dominant spastic paraplegia (37 -39 ).
Fifty seven families and seven isolated cases with cerebellar ataxia (15 ), in which the SCA1, SCA3/MJD and DRPLA mutations were excluded (16 -18 ), were analyzed in an ongoing clinical and molecular study of ADCA. Forty index cases fulfilled the diagnostic criteria of ADCA type I: progressive and unremitting cerebellar ataxia of autosomal dominant inheritance, and dysarthria associated with at least one other symptom (ophthalmoplegia, optic atrophy, pyramidal or extra pyramidal signs, deep sensory loss or dementia) (1 ,2 ). Six of these families were reported to be linked (19 ,21 ,22 ) and another possibly linked (20 ), to the SCA2 locus. Four families presented with a `pure' cerebellar ataxia, which was suggestive of ADCA type III. In the 13 other index cases, the cerebellar ataxia segregated with progressive macular degeneration (ADCA type II) (23 ,24 ). Linkage to the SCA7 locus was already demonstrated in three of these families (24 ). Proteins from 18 individuals without known pathology and seven with proven SCA1 or SCA3/MJD mutation were used as controls for the screening.
Lymphoblastoid cell lines were established for 187 individuals by transformation with the Epstein-Barr virus (40 ). Two different protocols of extraction were used. Cells were washed in phosphate-buffered saline (PBS) and resuspended in either (i) 50 mM Tris-HCl pH 8.0, 10% (v/v) glycerol, 5 mM EDTA, 150 mM KCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 [mu]M Leu-peptine and 5 [mu]g/ml A-protonine, or (ii) 50 mM Tris-HCl pH 8, 1% Triton X-100, 0.5% NP-40, 10 mM 2-mercaptoethanol, 4% (v/v) glycerol, 1 mM PMSF, 20 [mu]M Leu-peptine and 5 [mu]g/ml A-protonine. Cells were left for 20 min at 4oC followed by a sonication, then centrifuged at 15 000 g for 20 min at 4oC. The supernatant was collected and the protein concentration determined by the Bradford assay (Biorad). Aliquots were stored at -80oC until further analysis.
According to Lee et al. (41 ), cells were washed in PBS and the pellet was resuspended in 2 vols of buffer A (1.5 mM MgCl2, 10 mM KCl, 10 mM HEPES pH 7.9, 1 mM PMSF, 0.5 mM dithiothreitol (DTT), 20 [mu]M Leu-peptine and 5 [mu]g/ml A-protonine). The cells were kept at 4oC for 20 min, then passed through a needle (0.25 gauge) at least 10 times to destroy the plasma membrane. After centrifugation at 13 000 g for 10 min at 4oC, the cytosolic fraction was removed and stored at -80oC. The pellet, containing the nuclear fraction, was washed twice with buffer A and finally resuspended in 2/3 volume of buffer C (25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.42 M NaCl, 20 mM HEPES pH 7.9, 1 mM PMSF, 0.5 mM DTT, 20 [mu]M Leu-peptine and 5 [mu]g/ml A-protonine). After 10 min at 4oC and centrifugation at 13 000 g for 10 min at 4oC, the supernatant containing the nuclear fraction was collected and stored at -80oC.
Protein samples (50 [mu]g/lane) were separated by SDS-PAGE, either using a 4-20% acrylamide/bisacrylamide (39/1) gradient gel (Biorad) for the screening, or a 6% acrylamide/bisacrylamide (39/1) gel. Proteins were then transferred at 4oC for 90 min onto nitrocellulose ( Hybond ECL, Amersham) which was blocked with 5% non-fat dry milk, 0.02% Tween for 1 h at room temperature. Membranes were incubated in 3% non-fat dry milk with the mAb1C2 (1:2000) overnight at 4oC, washed three times for 10 min each in PBS, and then treated with anti-mouse antisera conjugated to horseradish peroxidase (Amersham) at 1:10 000 in 3% non-fat dry milk for 45 min at room temperature. After washing three times for 10 min each in PBS, filters were processed for enhanced chemiluminescence according to the recommendations of the supplier (ECL kit, Amersham) and the blots were exposed to ECL films (Amersham) for 15 s-2 h. Due to a signal background caused by the presence of normal proteins containing polyglutamine stretches, the results of the Western blots were interpreted after comparison of the detection profiles with known SCA1, SCA2, SCA3 and SCA7 patients and healthy controls.
Two microsatellite markers (D12S1328 = D12S1339 = AFM240we1 and D12S1329 = D12S1340 = AFM291xe9), flanking the 1 cM SCA2 candidate interval were amplified by PCR and resolved in 6% acrylamide gels as previously described (22 ,26 ). Lod score analysis was performed using the LINKAGE package with the parameters set as in Gispert et al. (26 ) and Lezin et al. (22 ).
Comparisons of means were performed with the non-parametric Mann-Whitney U test, and comparison of frequencies with the [chi]2 test. Mean values are given with the standard deviations.
The authors are indebted to Drs M. Ruberg, S. Vyas, S. Fauré and E. Leguern for their help and technical advice. We especially thank Professors J. Julien, D. Laplane, G. Rancurel, B. Dubois, J-C. Vernant, and Drs H. Chneiweiss, M. Serdaru, D. Caparros-Lefevre, C. Vial, D. Smadja and A. Lezin for referring some of the probands. The contribution of J. Bou, A. Camuzat, C. Ponthieux and C. Penet, who prepared the cell lines of the patients, is greatly appreciated. We also thank Drs E. Hirsch and P. Damier for providing us with brain cortex from one ADCA type II patient. This work was supported financially by the Association Française contre les Myopathies (AFM), the VERUM foundation, the Groupement de Recherches et d'Etudes sur les Génomes (GREG) and the Association pour la Recherche sur les Maladies Génétiques Neurologiques et Psychiatriques (ADRMGNP). Y.T. and N.A. are held by a fellowship from the Hereditary Disease Foundation and AFM, respectively.
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