Spinocerebellar ataxia type 1

seminars in CELL

BIOLOGY,

Spinocerebellar

Vol 6, 1995: pp 29-35

ataxia type 1

Huda It Zoghbi” and Harry T. Or?

based on its genetic localization to the short arm of chromosome 6.s Identification of families with SCAl allowed the analysis and documentation of the clinical features seen in this subtype of hereditary ataxia. The clinical findings in the early stages of SCAl include gait ataxia, dysarthria and nystagmus. As the disease progresses, the ataxia worsens and other cerebellar signs such as dysmetria and hypotonia become apparent. Optic atrophy and ophthalmoparesis may be detected in some patients. Muscle atrophy, decreased deep tendon reflexes, and loss of proprioception and vibration sense are common in the later stages of the disease. Cognitive function typically remains intact with the exception of emotional lability in some patients. In the final stages of the disease, degeneration of several cranial nerves in the brain stem results in facial weakness and bulbar signs including severe dysarthria and dysphagia. This results in frequent choking spells, poor cough reflex and aspiration of food particles, which leads to recurrent pneumonia and eventually death. The typical age of onset for SC41 is in the third or fourth decade, but early onset in the first decade has been documented in two large families.4*5 Increase in the severity of the phenotype in later generations has been observed in these two kindreds suggesting that anticipation occurs in SCAl. The disease typically progresses over 10-15 years, but a more rapidly progressive course has been described in juvenile onset cases5 Neuroimaging findings in SCAl include cerebellar and brain stem atrophy as well as enlargement of the fourth ventricle secondary to volume loss in the posterior fossa. Neuropathologic studies reveal reduction in the size of the cerebellum with severe loss of Purkinje cells and dentate nucleus neurons. Additional fmdings include neuronal degeneration in the inferior olive and cranial nerve nuclei, III, IV, IX, X and XII, and demyelination of the spinocerebelk tracts and dorsal columns. The clinical features observed in SCAl families are not unique to this type of ataxia and have been observed in patients known to have a genetically distinct form of SCA such as type 2 spinocerebellar ataxia (SCA2) which maps to chromosome 12q2.3-q246 or type 3 spinocerebellar ataxia (%A$

Spinocerebellar ataxia type 1 (SGAl) is a dominantly inherited neurookgenerative disorder characterized 4 ataxia, dysarthria and progressive bulbar dysfunction. The SGA 1 gene which maps to the short arm of chromosome 6 has been isolated using a positional cloning approach. The XX1 transcript is 10660 bases and encodes a novel protein, ataxin-1, with a predicted molecular weight of 87 kDa. Expansion of a GAG repeat localized near the amino terminus of ataxin-1 has been found to be the mutational mechanism in XXI. This GAG repeat is highly polymorphic with normal alleles containing 6-39 repeats. Individuals affected with SC41 have one normal allele and one expanded allele containing 40-81 r@eats. The size of the r@eat correlates inversely with the age of onset of symptoms and the severity of disease. The repeat is a continuous GAG repeat tract on XX1 chromosomes whereas in 2 98% of normal alleles one or more GAT interruptions break the GAG repeat tracts into two tracts containing less than 18 repeats each. This suggests that loss of CAT interruptions within the SGAl GAG repeat on normal chromosomes leads to triplet instability

Key words: anticipation / glutamine / neurodegeneration / spinocerebellar ataxia / trinucleotide repeat .

Clinical

features

The inherited spinocerebek ataxias (SC&) are a group of neurodegenerative disorders characterized by variable degrees of dysfunction of the cerebellum, spinal tracts, and brain stern.‘*’ The overlap in the clinical and pathological features among the various subtypes of SCAs precludes their classifications based on clinical findings. Spinocerebellar ataxia type 1 (SCAl) is one subtype of the hereditary ataxias which has been classified From the *Departments of Pediatrics and Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza - 61OE Houston, TX 77030, USA and the tDepartments of Laboratory Medicine and Pathology, and Biochemist, and Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA 01995 Academic Press Ltd 1043-4682/95/010029 + 07$8:00/O

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H.Y Zaghbi and H.T Orr which maps to chromosome 14q31q24.3.’ Furthermore, these clinical features are observed in several families which segregate an ataxia that does not map to any of the three known loci on 6p, 12q or 14qs (H. Zoghhi, L. Ranum, unpublished data). These findings emphasize the heterogeneity of this group of diseases and the need for a classification which relies on identifying the relevant genes and gene products.

Genetic locus

and physical

mapping

were found in the region; the EcoRl fragments containing these repeats where hybridized to BstNl- or Taql- digested DNAs from individuals with juvenile onset of disease in search of expansions. A 3.36kb EcoRl fragment containing a CAG repeat detected expanded fragments in all SCAl individuals but in none of the unaffected individuals. Sequence analysis of a 500 bp (GAG),,-containing subclone of the 3.36 kb EcoRl fragment revealed a repeat configuration of (CAG),, CAT CAG CAT (CAG),,. I4 Using oligonucleotide primers immediately flanking the repeat, PCR analysis confirmed the expansion of the GAG repeat in DNAs from SC41 individuals. Normal individuals displayed alleles containing 6 to 39 repeat units, whereas individuals with SC41 had two alleles, one normal allele with less than 39 repeats and one expanded allele with 40-81 repeat units (Figure 1). Analysis of the SCAl CAG repeat in 20 families known to have SCAl based on linkage data revealed an expanded allele in all affected individuals. Expansion of the SCAl CAG repeat was found in 24 out of 173 additional ataxia kindreds that were too small for linkage analysis.15-18 These data indicate that CAG repeat expansion is the mutational mechanism in SCAl and that other mutations are less likely. Furthermore, these data suggest that SCAl accounts for 1423% of dominantly inherited ataxias depending on whether one does or does not include families known to have SC41 based on linkage analysis. The number of repeat units on SC41 chromosomes correlates inversely with the age of onset and disease severity. In a study by Ranum and colleagues,” the relationships between the age of onset and the number of repeat units on normal and SCAl alleles were evaluated in 113 individuals. A correlation coefficient of -0.815 was obtained for age of onset versus number of repeat units on expanded alleles indicating that 66% of the variability in the age of onset can be accounted for by the number of CAG repeat units (Figure 2). The correlation between the severity of the disease and the number of the repeats on SCAl chromosome was evaluated by using age of death minus the age of onset as a measure of severity in 29 individuals. A linear correlation of -0.58 was obtained suggesting that 34% of the variation in the duration of the disease is due to the number of the repeat units. ” The size of the repeat on normal chromosomes had no significant effect on age of onset or severity of disease. Similar correlations between age of onset and number of repeats were observed in the study of 55 patients by Jodice et aZi6 and in a study of 42 patients by Dubourg et aZ18where

of the SCAl

The SCAl gene was first localized to the short arm of chromosome 6 based on linkage to the human leukocyte antigen (HLA) loci.sV” In 1991, the genetic mapping of the SCAl gene was refined by demonstrating very close linkage to the marker D6S89 which maps telomeric to HLA in 6p22-p23.*‘*” Linkage analysis using D6S89 and approximately 120 affected individuals from nine SCAl kindreds revealed a single recombinational event between SCAl and D6S89 identifying D6S89 as a telomeric flanking marker to the SCAl locus at a genetic distance s 1 CM.‘* D6S274 was subsequently identified as a centromeric flanking marker and the region between the two flanking markers was estimated to be 1.2 mega base pairs based on long range restriction analysis of yeast artificial chromosome (YAC) clones in the region.‘s The SCAl critical region was represented in a minimum of four overlapping YAC clones which were subsequently cloned into cosmids. These cosmid clones were used in experiments aimed at identifying genes from the candidate region.

Molecular

basis of SCAl

The finding of anticipation in some SCAl families led to the hypothesis that expansion of unstable trinucleotide repeats could be the mutational mechanism in SCAl given that amplification of unstable trinucleotide repeats was the underlying mechanism for three ,d.isorders which display anticipation, fragile X syndrome, myotonic. dystrophy and Huntingdon’s disease. To test this hypothesis, cosmid clones from the candidate SW region were screened for the presence of trinucleotide repeat sequences by hybridi&g, ,a probe containing oligonucleotides representingall &e. ,permutations. of trinucleotide repeats to Southern blots .of EcoWdigested DNAs from these oii&r@l~.~~ Seven, independent trinucleotide repeats 30

GAG rqbeats and sjkocereDella~

25-+

I

I

I

TX-SCA

LA-SCA

Figure 1. analysis of the PCR products using primers flanking the SCAl CAG repeat from unaffected and affected members of the TX- and LA- SCAI kindreds. The range for nonnal (LAG),, is s 34 and expanded is zz 46 on this autoradiograph. From ref 25, with permission of McGraw-Hill, New York.

:

60

80 Number of repeats

Figure 2. Relationship between alleles of individuals affected obtained indicating that 66% size of the CAG repeat on the Chicago.

the age of with SCAl. (R* = 0.66) expanded

onset and the number of CAG repeats on the expanded A correlation coefficient R of -0.815 (PsO.0001) was of the variation in the age of onset is attributable to the allele. From ref 17, with permission of the University of

31

ataxia

H. I! Zoghbi and H.T Orr Table

1. Comparison Normal

of the number

of CAG repeats on normal

chromosomes

SCAl

Number of chromosomes

GW.

and SCAl

chromosomes

chromosomes Number of chromosomes

GW.

19-36

304

40-81

114

6-39

3:: 116

41-57 46-66 42-67

10 it

the correlation respectively.

Features

coefficients

of the SCM

were -0.74 and -0.81

Ref 1’7; unpublished

data

:z 18

repeats). lg The difference in intergenerational change between maternal and paternal transmissions is statistically significant (P
GAG repeat

The SCA1 CAG repeat is highly polymorphic with a heterozygosity rate of 84%. The distribution of the number of repeat units on normal chromosomes has been studied in a large number of individuals from diverse ethnic backgrounds. Ranum et al found the distribution to be 19-36 repeats based on analysis of 304 chromosomes. ” Matilla and coworkers found the range in the number of repeat units on normal chromosomes to be 6-39 based on analysis of 68 chromosomes.i5 Jodice et al reported a distribution of 2637 based on 365 normal chromosomes.i6 Dubourg et al found the normal alleles to contain 27-35 repeats. i* In summary, normal individuals displayed 24 alleles ranging from 6-39 repeat units. Recently an SCAI affected individual has been identified with a perfect CAG tract of 40 triplets (L. Ranum, unpublished data). Thus, the size of expanded alleles found so far ranges from 40 to 81 triplets. Based on these data it appears that, to date, there is no overlap between the number of repeat units on normal versus SCAl chromosomes (Table 1). The intergenerational stability of the SCAl CAG repeat was examined in normal and SCAl meioses. No instances of instability were observed for the CAG repeat on normal alleles in over 1000 meioses.16*” In contrast, the CAG repeat on SC41 alleles was unstable and variations which included both increases and decreases were noted. Chung et al examined 28 maternal and 16 paternal transmissions in five SC41 kindreds; they found a significant difference in the type or size of variation in maternal versus paternal transmissions with 69% of maternal transmissions having no change or a decrease in the number of CAG repeats (average change of -0.4 repeats) and 63% of paternal transmissions having an increase in the number of CAG repeats (average of +3.3 32

CAG repeats and spinocerebellur ataxia Table 2. Summary of repeat sequence configurations on normal chromosomes Number of repeats 19 &I 23-36 32-33

Sequence configuration (UG) I9 (CA%, (UG) I,-I,Q=(~G) (~G),.,,(CATM~G),,, (MG) ,sa(~T)s(QW

does not share any homology with previously identified proteins. The SC41 transcript has 935 bp in the 5’untranslated region (UTR) and a large 3’ UTR of 7277 bp which currently is the longest sequenced 3’ UTR in Genbank (Figure 3A). The gene locus spans approximately 450 kb and is organized in nine exons (Figure 3B). The structure of the SC41 is unusual in that there are at least seven 5’ non coding exons, and that the coding region falls within two large exons measuring 2079 bp and 7805 bp, respectively. The seven non coding exons are small (49-157 bp) but are separated by large interns spanning 400 kb of genomic DNA.*’ Exons 24 undergo alternative splicing in a pattern which was similar in the five tissues which were examined. The gene structure appears to be conserved in the mouse including the large 5’ untranslated region (H. Zoghbi, unpublished data). The function of the large 5’ UTR is not clear at’ this point; one possibility is that it is involved in regulating the translation of ataxin-1. The pathogenesis of SC41 is not clear in spite of the identification of the mutational mechanism and the predicted protein sequence. It is quite likely that the mechanism which leads to neuronal loss in SCAl is similar to those involved in spinal-bulbar muscular atrophy (SBMA) ,*’ Huntington’s disease (I-ID),** and dentatorubral-pallidoluysian atrophy (DRPLA) ,s3V24 three neurodegenerative disorders caused by expansions of polyglutamine tracts. The expansion of the polyglutamine tracts in these disorders may lead to either gain of function or to a dominant negative effect given that deletions of the androgen receptor and hemizygosity at the I-ID locus do not result in SBMA and HD, respectively. Furthermore, expression analysis of the SCAl gene in lymphoblasts from SC41 patients revealed expression from both the normal and expanded alleles indicating that loss of transcription is not the mechanism involved.*’ The expansion of the polyglutamine tract in ataxin-1 may cause it to interact aberrantly with itself, with its normal target or with a protein that it does not normally interact with. The target protein may be cell specific explaining the selective neuronal loss in this disease. . In summary, using positional cloning the SC41 gene has been isolated and the mutational mechanism has been determined to be an expansion of a CAG repeat within ataxin-1, a novel protein whose function is still to be determined. The immediate implications of these findings include the feasibil@ of identifying families segregating this type of ataxia as well as the ability to make the diagnosis of SCAl in individual cases when family history may be lacking.

Number of chromosomes 1 141s 19-1s

3 36 2

revealed that all were cleaved with SfaNl indicating the presence of at least one CAT. These data indicate that 98% of normal chromosomes have an interrupted repeat configuration. In contrast analysis of the repeat configuration on 30 SCAl chromosomes (13 by sequence and 17 by restriction analysis) revealed a contiguous CAG repeat tract. The SC41 alleles were from seven unrelated SC41 kindreds which displayed at least four different haplotypes at D6S288 and D6S274 (markers within the SC41 gene) indicating that the contiguous CAG repeat on SC41 chromosomes is not the result of a single founder chromosome.‘g Jodice et al studied the repeat configuration of SC41 alleles from affected individuals representing 19 different SC41 families. All affected individuals had one allele with an uninterrupted CAG repeat and a normal allele with CAG interruption(s). SfaNl digestion and sequence analysis of the largest normal alleles, 37 and 39, observed by Jodice et al and by Matilla et al, respectively revealed CAT interruptions in both alleles l6 (E&ill, personal communication) . These data suggest that loss of CAT interruptions in alleles with 21 or more repeats renders the CAG repeat unstable and leads to subsequent expansions.‘g Furthermore, given that all 27 independent SCAl alleles analyzed to date have lost the CAT interruptions, SfaNl digestions of PCR products containing the repeat will be helpful for analysis of alleles containing 3840 repeats to determine if they are derived from normal or SCAl chromosomes.

The SCAl ,gene product model

and pathogenetic

The SC41 transcript is 10660 bp and is expressed in a variety of tissues based on Northern and RT-PCR analyses.*’ It has a coding region of 2448 bp encoding 816 amino acids. The CAG repeat falls within the coding region and encodes for glutamine. The SC41 protein, ataxin-1, appears to be a novel protein which 33

H. Y ZoghOi and H.T 6-r

A. eXon

4

1234567

157 115 129 136 49 126 61

6

9

2080

7805

500

B.

tel

ten !

8’‘Nr YAC

227Bl

L exon

SC

s

B

B

II

I

I

I

7%

a

M I I

5

Nr

N&M

I

I II I

B II

Nr

SM

IIII II

!!

EC

R

69

Figure 3. (A) Structure of the SCAl cDNA; the exons are drawn to scale and the shaded areas represent the coding region. (B) genomic structure of the SCAl gene. The nine exons were localized on a long range restriction map ofYAC clone 227Bl which encompasses the SCAl gene. The exons are shown as solid rectangles not drawn to scale. The centromere-telomere orientation is indicated by ‘ten’ and ‘tel’ L, left YAC end; R, right YAC end; B, Bss HII; C, Cspl; M, Mlul; N, Notl; Nr, Nrul; S, SacII. 2. Koeppen AH, Barron KD (1984) The neuropathology of olivopontocerebellar atrophy, in The Olivopontocerebellal Atrophies (Duvoisin RC Plaitakis A, eds), pp 13-38. Raven Press, New York 3. Jackson JF, Currier RD, Terasaki PI, Morton NE (1977) Spinocerebellar ataxia and HLA linkage: risk prediction by HLA typing. N Engl J Med 296:1138-l 141 4. Schut JW (1950) hereditary ataxia: clinical study through six generations. arch Neural Psychiatr G3:535-568 5. Zoghbi HY, Pollack MS, Lyons LA, Ferell RE, Daiger SP, Beaudet AL (1988) Spinocerebellar ataxia: variable age of onset and linkage to human leukocyte antigen in a large kindred. Ann Neurol 23:580584 6. Gispert S, Twells R, Orozco G, Brice A, Weber J, Heredero L, Scheufler K, Riley B, Allotey R, Notbers C, Hillerman R, Lunkes, A, Kbati C, Stevanin G, Hernandfz A, Magarino C,

Presymptomatic diagnosis can be provided to individuals from families with documented expansion of the SCAl CAG repeat. In the event of presymptomatic testing, proper genetic counselling and psychological support are necessary given the progressive and fatal nature of this disease. The biological implications of identifying the SCAl gene are twofold, one involving the understanding of the mechanisms involved in trinucleotide repeat instability and expansions, and the other involving the understanding of the mechanism by which polyglutamine expansions lead to specific neuronal degeneration.

Klockgether T, Durr A, Chneiweiss H, Enczmann J, Farrall M, Beckmann J, Mullan M, Wernet P, Agid Y, Freund H-J, Williamson R, Auburger G, Chamberlain S (1993) Chromosoma1 assignment of the second locus for autosomal dominant cerebellar ataxia (%X2) to chromosome 12q23-24.1. Nature Genet 4:295-299 7. Stevanin G, Le Guern E, Ravise N, Chneiweiss H, Durr A, Cancel G, Vignal A, Both A-L, Ruberg M, Penet C, Pothin Y,

Acknowledgements The authors thank Catherine Tasnier and Dr. Belinda Rossiter for preparation of the illustrations in this manuscript. This work was supported by grants from the National Institutes of Health and the Muscular Dystrophy Association.

Lagroua I, Haguenau M, Rancurel G, Weissenbach J, Agid Y, Brice A (1994) A third locus for autosomal dominant cerebellar ataxia type 1 maps to chromosome 14q24.3qter: evidence for the existence of a fourth locus. Am J Hum Genet 54:11-20 8. Stevanin G, Chneiweiss H, Le Guern E, Ravise N, Durr A, Penet C, Agid Y, Brice A (1993) Genetic heterogeneity of autosomal dominant cerebella ataxis type I; evidence for the existence of a third locus. Hum Mol Genet 2:1483-1483 9. Yakura H, Wakisaka A, Fujimoto S, Itakura K(1974) Hereditary ataxia and HLA genotypes. N Engl J Med 291:154155 10. Zoghbi Hy, Jodice C, Sandkuijl LA, Kwiatkowsi Jr TJ, McCall AE, Huntoon SA, Lulli P, Spadaro M, Litt M, Cann HM, Frontali M, Terrenato L (1991) The gene for autosomal dominant spinocerebellar ataxia (%X1) maps telomeric to HLA complex

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17.

and is closely linked to the D6S89 locus in three large kindreds. Am J Hu Genct 492330 Ranunt LPW, Duvick LA, Rich SS, Schut LJ, Litt M, 0t-r HT (1991) Localization of the autosomal dominant, HLA-linked spinocerebellar atasia (SCAI) locus in two kindreds within an 8cm subregion of chromosome 6p. Am J Hum Genet 49:31-41 Kwiatkowski .Jr TJ, Orr HT. Banfi S, McCall AE, Jodice C, Persichetti F, Novelletto A, LeBorgne-Demarquoy F, Duvick LA, Frontali M, Subramony SM. Beaudet AL, Terrenato L, Zoghbi HY, Ranum LPW (1993) The gene for autosomal dominant spinocerebellar ataxia (SCAI) maps centromeric to D6S89 and shows no recombination, in nine large kindreds, with a dinucleotide repeat at the AM10 locus. Am J Hum Genet 53:391-40 Banfi S, Chung M-y, Kwiatkowski Jr TJ, Ranum LPW, McCall AE, Chinault AC, Orr HT. Zoghbi HY (1993) Mapping and cloning of the critical region for the spinocerebellar ataxia type 1 gene in a yeast artificial chromosome contig spanning I.2 Mb. Genomics 18627-635 Orr H, Chung M-y, Ban6 S, Kwiatkowsi Jr TJ, Servadio A, Beaudet AL, McCall AE, Duvick IA, Rat-mm LPW, Zoghbi HY (1993) Expansion of an unstable trinucleotide (CAG) repeat in spinocerebellar ataxia type 1. Nature Genet 4221-226 Matilla T, Volpini V, Genis, D. Rosell J, Corral J, Davalos A, Molins A, Estivill X (1993) Presymptomatic analysis of spinocerebellar ataxis type 1 (SCAl) via the expansion of the SC41 CAGrepeat in a large pedigree displaying anticipation and parental male bias. Hum Mol Genet 2:21232128 Jodice C, Malaspina P, Persichetti F, Novelletto A, Spadaro M, Giunti P, Morocutti C, Terrenato L, Harding AE, Frontali M (1994) Effect of trinucleotide repeat length and parental sex on phenotypic variation in spinocerebellar ataxia 1. Am J Hum Genet 54959-965 Ranum LPW, Chung M-y, Banfi S, Bryer A, Schut LJ, Ramesar R, Duvick LA, McCall AE, Subramony SH, Goldfarb L, Gomez C, Sandkuijl LA, Orr HT, Zoghbi HY (1994) Molecular and clinical correlations in spinocerebellar ataxia type 1 (SC41): evidence for familial effects on the age of onset. Am J Hum Genet 55:244-252

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