Clinical and genetic features of spinocerebellar ataxia type 8

Handbook of Clinical Neurology, Vol. 103 (3rd series) Ataxic Disorders S.H. Subramony and A. Du¨rr, Editors # 2012 Elsevier B.V. All rights reserved

Chapter 31

Clinical and genetic features of spinocerebellar ataxia type 8 1

YOSHIO IKEDA 1,3, LAURA P.W. RANUM 1,3, AND JOHN W. DAY 2,3 * Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA 2

Department of Neurology, University of Minnesota, Minneapolis, MN, USA

3

Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA

INTRODUCTION Spinocerebellar ataxia type 8 (SCA8) is a dominantly inherited, slowly progressive neurodegenerative disorder caused by a CTG●CAG repeat expansion located on chromosome 13q21. By using RAPID cloning, a direct method to identify pathogenic repeat expansions, we were able to identify the SCA8 CTG●CAG expansion from a single ataxia patient without any prior linkage study or extensive family data. This allowed us to find the mutation despite reduced penetrance of the disease. SCA8 is clinically characterized by a predominantly cerebellar ataxia that affects gait, limb and eye coordination in addition to altering speech and voice, but without significant bulbar or limb weakness. These clinical features are consistent with the confined cerebellar atrophy seen by MRI. The SCA8 CTG●CAG expansion has several novel genetic features, including dramatic intergenerational instability. A puzzling and controversial feature of the disease is the high degree of reduced penetrance that is found in all reported SCA8 families, including the large dominant family (MN-A) in which the mutation was first clearly associated with disease (LOD score of 6.8 at y ¼ 0.00). The repeat expansion is bi-directionally expressed as two molecules with expansion motifs previously implicated in disease pathogenesis. A nearly pure polyglutamine expansion protein is expressed in the CAG direction from the ATXN8 gene and a CUG expansion transcript is expressed by the ATXN8OS gene encoded on the opposite strand. The CUG transcript is not translated, raising the possibility that it creates a pathogenic RNA similar to myotonic dystrophy types 1 and 2. The CAG transcript is translated into a nearly pure polyglutamine protein, with neuronal intranuclear inclusions that are ubiquitin positive. The BAC transgenic SCA8 model mice show a progressive

neurological phenotype with impaired cerebellar cortical inhibition. Taken together, these findings demonstrate the direct role of the SCA8 expansion in disease pathogenesis. This recent development makes SCA8 the first disorder that potentially involves both an RNA gain-of-function mechanism similar to DM1 and DM2, caused by CUG expansion transcripts, and pathogenic effects of a polyglutamine expansion protein, ataxin-8 (Moseley et al., 2006).

IDENTIFICATION OF THE SCA8 CTG●CAG REPEAT EXPANSION Because several distinct forms of dominantly inherited spinocerebellar ataxia are caused by polyglutamine repeat expansions, we developed a method to genetically isolate and localize expanded CTG●CAG repeats, called repeat analysis, pooled isolation and detection (RAPID) cloning (Koob et al., 1998). Starting with a repeat expansion detection assay (Koob et al., 1998), RAPID cloning can identify CTG●CAG expansions and flanking sequence from a single individual without needing prior chromosomal localization through linkage analysis (Koob et al., 1998, 1999; Mosemiller et al., 2003). Since linkage studies require identification of large families with highly penetrant disorders, RAPID cloning provided a new strategy for identifying genetic mutations causing less penetrant diseases in small families. We used RAPID cloning to study DNA from a panel of ataxia families, and from a single affected individual we identified an 80 CTG●CAG repeat expansion, preceded by 11 CTA/TAG repeats, on chromosome 13q21. We subsequently identified this SCA8 expansion in a seven-generation ataxia kindred (the MN-A family), and by studying 92 members of

*Correspondence to: John W. Day, MD, PhD, Professor of Neurology, Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA. Tel: (612)-625-6180, Fax: (612) 625-8488. E-mail: [email protected]

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Fig. 31.1. The large SCA8 kindred (MN-A family). Filled symbols indicate individuals with ataxia, symbols with a dot indicate individuals who inherited the CTG expansion but are not clinically affected by ataxia. The CTG repeat lengths of expanded alleles are indicated below the symbols. Haplotype analyses using five short tandem repeat markers confirm that both branches of the family inherited the expanded repeat from a common founder. Family members homozygous for the SCA8 expansion and their affected heterozygous siblings (individuals VI: 24–26) had similar clinical features, with comparable ages of onset and rates of disease progression. (Reproduced from Koob et al., 1999, with permission.)

that family were able to genetically confirm the relationship of the mutation to the clinical disease (Fig. 31.1) (LOD score of 6.8 at y ¼ 0.00) (Koob et al., 1998, 1999; Mosemiller et al., 2003). Our initial analysis of the sequence surrounding the SCA8 CTG●CAG repeat identified no likely open reading frames in the CAG direction, but we showed that the CTG direction was transcribed, that transcripts expressed in the CUG direction are primarily expressed in the central nervous system and cerebellum (Koob et al., 1999), and that the expansion mutation expressed in the CUG direction did not appear to be part of a likely open-reading frame. The SCA8 CTG repeat tract, which is conserved in chimpanzees, gorillas, and orangutans but not mouse (Andres et al., 2003, 2004), is located at the 30 end of a highly alternatively spliced transcript that is expressed at low steady-state levels in the central nervous system (Fig. 31.2). This ATXN8OS transcript is detectable by

RT-PCR but not Northern or in-situ hybridization analysis (Janzen et al., 1999; Koob et al., 1999). The 50 end of the SCA8 ATXN8OS transcript overlaps the 50 end of the Kelch-like 1 (KLHL1) gene, which encodes an actin-binding protein that is transcribed in the opposite direction (Koob et al., 1999; Nemes et al., 2000). A mouse model in which KLHL1 is knocked-out in Purkinje cells showed subtle changes in motor coordination and minor changes in cerebellar molecular layer thickness, indicating that KLHL1 is involved in maintaining normal cerebellar function (He et al., 2006). Understanding the relevance of this model to human SCA8 and determining the effect of the SCA8 CTG expansion on KLHL1 will require additional studies. Although the KLHL1 knock-out model did not examine the pathogenic effect of the CTG expansion, and no functional relationship between the two transcripts has been demonstrated, the genomic organization is formally

Fig. 31.2. Comparison of the genomic organization of the overlapping ATXN8 and ATXN8OS genes in humans and mice. Exons are shown as boxes and various alternative splice forms are indicated by dashed lines. The murine Klhl1as transcript is a partial homolog of the much longer human ATXN8OS transcript. Interestingly, the region of human genomic DNA containing ATXN8OS exons A, B, C1, C2, and C3 is not conserved in the downstream chromosomal region of the murine Klhl1as gene.

CLINICAL AND GENETIC FEATURES OF SPINOCEREBELLAR ATAXIA TYPE 8 consistent with the possibility that the SCA8 CTG transcript (ATXN8OS) could regulate KLHL1 transcripts through an antisense mechanism (Nemes et al., 2000). A much shorter version of the SCA8 transcript without the repeat region has been found in mice (Fig. 31.2) (Benzow and Koob, 2002). More recent studies have shown that not only transcripts with the CUG repeats (ATXN8OS) but also CAG-repeat-containing transcripts from the reciprocal strand at the SCA8 locus (ATXN8) can be expressed in the central nervous system, so the disease may involve mechanisms of both polyglutamine (ataxin-8) and toxic RNA molecules (Fig. 31.2) (Moseley et al., 2006).

CLINICAL, MRI, AND NEUROPATHOLOGICAL FEATURES OF SCA8 Clinical features of SCA8 SCA8 typically presents as a slowly progressive cerebellar ataxia that largely spares brainstem and cerebral function (Koob et al., 1999; Day et al., 2000; Ikeda et al., 2000a; Juvonen et al., 2000; Cellini et al., 2001; Brusco et al., 2002; Tazon et al., 2002; Topisirovic et al., 2002; Mosemiller et al., 2003; Schols et al., 2003; Zeman et al., 2004; Lilja et al., 2005). In the MN-A family, disease onset ranges from 13 to 65 years, with gait incoordination the most frequently reported initial symptom. Consistent with slow disease progression, the need for mobility aids usually occurs 20 years after the presentation of initial symptoms. Speech is dysarthric with ataxic and spastic components (Day et al., 2000). Oculomotor involvement is commonly found in moderate to severely affected patients (Day et al., 2000; Anderson et al., 2002). Hyperreflexia is also common, with Babinski signs observed in rare severely affected individuals. Occasionally, intermittent low amplitude myoclonic jerks in the fingers and arms and mild athetotic movements of extended fingers are detected. Mild sensory loss was observed in a subset of affected individuals, indicated by decreased vibratory perception (Day et al., 2000). In SCA8 individuals other than the MN-A family members, predominant features remain cerebellar ataxia with occasional hyperreflexia (Ikeda et al., 2004a). Maschke et al. (2005) showed a predominant cerebellar syndrome in 11 affected individuals from four SCA8 families, and suggested that myoclonus, which is also sometimes observed in the MN-A family, could be a typical feature of SCA8. Additional reports of SCA8 support the view that it is primarily a cerebellar syndrome (Ikeda et al., 2000a; Juvonen et al., 2000; Cellini et al., 2001; Brusco et al., 2002; Tazon et al., 2002; Topisirovic et al., 2002; Schols et al., 2003; Zeman et al., 2004; Lilja et al., 2005), with

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extracerebellar symptoms and signs reported in a subset of SCA8 patients. Formal neuropsychological studies demonstrated impaired attention, information processing, and executive functions in SCA8 (Lilja et al., 2005), which support other studies showing that cognitive decline and psychiatric abnormalities can occur in SCA8 (Juvonen et al., 2000; Stone et al., 2001; Zeman et al., 2004). Mental retardation can occur in SCA8 patients with early-onset disease (Silveira et al., 2000; Izumi et al., 2003).

MRI of SCA8 MRI analysis of SCA8 has consistently shown atrophy of the cerebellar vermis and hemispheres, frequently severe, with sparing of the brainstem and cerebrum (Koob et al., 1999; Day et al., 2000; Ikeda et al., 2000a; Juvonen et al., 2000; Cellini et al., 2001; Brusco et al., 2002; Tazon et al., 2002; Topisirovic et al., 2002; Schols et al., 2003; Zeman et al., 2004; Lilja et al., 2005). Infrequently, SCA8 has also been reported with brainstem or cerebral atrophy on MRI (Juvonen et al., 2000; Schols et al., 2003; Lilja et al., 2005). An affected MN-A family member revealed little change in MRI appearance over 9 years, consistent with the slowly progressive disease course (Fig. 31.3) (Day et al., 2000). In contrast, Zeman et al. (2004) reported a patient in whom an initial scan was normal, while a scan 4 years later showed clear

Fig. 31.3. Serial MRI scans of an affected individual. Horizontal (A1, B1) and sagital (A2, B2) MRI scans from an affected individual at ages 26 (A) and 35 (B) years. The earlier image is 9 years after onset (17 years). There is marked cerebellar atrophy, minimal brainstem atrophy, and no evidence of cerebral involvement. There is very little change over the 9-year period between scans, which is consistent with the slow progression of the disease. (Reproduced from Day et al., 2000, with permission.)

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cerebellar atrophy. Importantly, MRI analysis of a clinically unaffected 71-year-old individual with an SCA8 expansion showed mild cerebellar atrophy, indicating that some of the reduced penetrance of the disease can reflect ability to compensate for mild cerebellar loss (Ikeda et al., 2000b).

Neuropathology of SCA8 Autopsy of an MN-A family member with 110 combined CTA/CTG repeats, and of a second unrelated ataxia patient with 140 combined repeats, showed moderate to severe Purkinje cell loss, and loss of granule cells and olivary nuclei without neurodegeneration in other parts of the brain or spinal cord (Ikeda et al., 2004b; Moseley et al., 2006). Interestingly, Purkinje cells, medullary and cerebellar dentate neurons had intranuclear inclusions and pan-nuclear staining with antibodies against ubiquitin and with the 1C2 antibody that labels polyglutamine expansions (Fig. 31.4). Control tissue had no similar staining, and the inclusions were not labeled with antibody against TATA-binding protein that contains a polyglutamine stretch used to generate the 1C2 antibody, which along with additional expression studies demonstrated that the inclusions resulted from expression of ataxin8 (ATXN8), the protein encoded on the CAG strand of the SCA8 expansion (Moseley et al., 2006). Purkinje cells

REDUCED DISEASE PENETRANCE IS A GENETIC FEATURE OF SCA8 Disease penetrance in MN-A family is affected by SCA8 repeat length Within the large MN-A family there are 22 individuals with an expanded repeat who were not clinically affected at the time of evaluation. The age at examination of these asymptomatic carriers ranged from 14 to 74 years, with a mean (43  17 years) that was comparable to the mean age at examination of the affected family members. The repeat lengths among these asymptomatic carriers are significantly (p<10-8) shorter than those in affected individuals (means ¼ 90 and 116 repeats, respectively), indicating that disease penetrance in the MN-A family is influenced by SCA8 repeat length. All but one of the individuals with a combined repeat tract > 110 repeats is clinically affected. This 42-year-old individual with 143 combined repeats has exhibited no signs of ataxia during repeated neurological examinations, which may reflect the fact that SCA8 is an adult-onset disorder with a documented age of onset as old as 65 years of age. These data demonstrate that disease penetrance is affected by SCA8 repeat length in the MN-A family (Koob et al., 1999). Medullary neuron

Dentate neuron

Human SCA8

Human Ctl

Purkinje cells

Purkinje cells

Pontine neurons

BAC-Exp Tg

BAC-Ctl Tg

Fig. 31.4. 1C2-positive intranuclear inclusions and pan-nuclear staining are found in Purkinje, medullary, and dentate neurons in SCA8 autopsy brain, while no visible staining is found in control brain. 1C2-positive intranuclear inclusions are evident in the Purkinje cells and pontine neurons of BAC-Exp mice but not in those of BAC-Ctl mice. Scale bars each ¼ 10 mm. (Reproduced with permission from Moseley et al., 2006.)

CLINICAL AND GENETIC FEATURES OF SPINOCEREBELLAR ATAXIA TYPE 8

Reduced penetrance in other ataxia families In the MN-A family, SCA8 is transmitted in an autosomal dominant pattern with alleles that have fewer than 110 combined repeats showing reduced penetrance. Other SCA8 families, however, show a complex inheritance pattern in which only a subset of individual carrying long

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expansions are affected (Koob et al., 1999; Day et al., 2000; Ikeda et al., 2000b; Cellini et al., 2001; Topisirovic et al., 2002). Representative SCA8 pedigrees that have presented with apparently distinct inheritance patterns are shown in Fig. 31.5 (Ikeda et al., 2004a). Families A and B appear to have transmitted ataxia in a dominant pattern, with affected individuals in multiple generations.

Fig. 31.5. SCA8 pedigrees with varying degrees of disease penetrance. Symbols for individuals affected by ataxia are blackened, and unaffected expansion carriers are indicated by symbols with a dot inside them. A diagonal line through a symbol denotes an individual who is deceased. The size of the expanded and unexpanded SCA8 alleles is shown below the individuals. The numbers designating each family correspond to those that appear in Fig. 31.9A. Individuals indicated with an asterisk are negative for CTG expansion by Southern analysis. (Reproduced from Ikeda et al., 2004a, with permission.)

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In contrast, families C and D appear recessive, with multiple affected individuals in a single generation; the affected individuals in families E and F appear as sporadic cases with no other family members clinically affected. In contrast to the relatively large number of affected patients in the MN-A family (n ¼ 13), the 36 smaller SCA8 ataxia families have significantly fewer affected individuals, with only two families having three affected individuals, nine families having two affected individuals, and 25 families with only a single affected individual. Although only a subset of the expansion carriers in the MN-A family develop ataxia (13/35), disease penetrance is significantly higher in the MN-A pedigree than in the 36 smaller ataxia families we have studied, as well as in families reported by other groups worldwide (Koob et al., 1999; Day et al., 2000; Ikeda et al., 2000b, 2004a; Juvonen et al., 2000; Silveira et al., 2000; Stevanin et al., 2000; Worth et al., 2000; Cellini et al., 2001; Brusco et al., 2002; Tazon et al., 2002; Topisirovic et al., 2002; Schols et al., 2003; Zeman et al., 2004; Corral et al., 2005). Within the reported SCA8 ataxia families, repeat sizes of affected and unaffected expansion carriers overlap and often exceed the pathogenic threshold found in the MN-A family (Ikeda et al., 2004a). The tight correlation between repeat size and pathogenesis in the MN-A family is not present in these other ataxia families – SCA8 expansions vary dramatically in size and the presence of an SCA8 expansion cannot be used to predict whether or not an asymptomatic individual will develop ataxia. (Ranum et al., 1999; Moseley et al., 2000; Worth et al., 2000; Juvonen et al., 2002; Ikeda et al., 2004a).

SCA8 expansions on control chromosomes SCA8 expansions have also been found in control samples that we and others have screened (Koob et al., 1999; Day et al., 2000; Ikeda et al., 2000a, 2004a; Juvonen et al., 2000, 2005; Silveira et al., 2000; Stevanin et al., 2000; Vincent et al., 2000a, b; Worth et al., 2000; Cellini et al., 2001; Sobrido et al., 2001; Tazon et al., 2002; Topisirovic et al., 2002; Izumi et al., 2003; Schols et al., 2003; Brusco et al., 2004; Zeman et al., 2004; Corral et al., 2005). In a screen of 2626 unrelated control chromosomes, we identified ten SCA8 alleles (0.4%) that had expansions larger than 74 combined repeats, which is the smallest expansion found in an ataxia patient (Ikeda et al., 2004a). One of these control expansions was from a CEPH grandmother (family 1416) (Fig. 31.5G). Medical histories indicate that neither this woman nor her son (54 years, 800 repeats) was affected by ataxia. All six of the SCA8 expansion carriers in this family were

asymptomatic at the time of clinical evaluation; however, given that the individuals in generation III were children when they were last clinically evaluated, it is not known if they have or will develop ataxia (Ikeda et al., 2004a). Expansions containing > 74 combined repeats occurred on 12/292 (4.1%) independent ataxia chromosomes in our collection of probands from genetically undefined ataxia families (Ikeda et al., 2004a). Although this frequency of expansions in ataxia probands is significantly higher than occurs in the general population (4.1% compared to 0.4%, p ¼ 410-25), the relative frequency of alleles with > 74 combined repeats in the general population ( 0.4%) is higher than that of all forms of ataxia ( 1/10 000), indicating that not all expansions of this size cause ataxia. Zeman et al. (2004) screened the SCA8 expansions in normal, disease control, and ataxic Scottish populations and similarly showed expansions (> 91 combined repeats) in 0.3% of control subjects as compared with 3.5% of unrelated ataxia subjects. Because SCA8 expansions occur in the general population without causing ataxia, they can also occur in apparently non-hereditary neurodegenerative disorders, in which case the role of SCA8 expansion in disease pathogenesis is unclear.

SCA8 REPEAT INSTABILITY MAY UNDERLIE THE REDUCED PENETRANCE Repeat instability during transmission Intergenerational changes in SCA8 expansion size are large compared to other dominantly inherited SCAs (Tsilfidis et al., 1992; Chung et al., 1993; Maciel et al., 1995; Maruyama et al., 1995; Cancel et al., 1997; David et al., 1997; Jodice et al., 1997; Zhuchenko et al., 1997; Koob et al., 1999; Moseley et al., 2000; Mosemiller et al., 2003). Gender differences of intergenerational changes are significant in SCA8, with paternal transmissions commonly resulting in contraction ( 86 to þ 7 CTGs), and maternal transmissions resulting in expansion ( 11 to þ 900). The largest maternally transmitted alleles include increases of þ 250, þ 375, þ 600, and þ 900 CTG repeats (Koob et al., 1999; Corral et al., 2005). The maternal bias for SCA8 repeat expansions has not been observed in other SCAs, but is similar to the maternal expansion biases for two other non-coding expansion disorders – fragile X syndrome and DM1 (Groenen and Wieringa, 1998; Koob et al., 1999; Jin and Warren, 2000; Mosemiller et al., 2003). Examples of intergenerational changes for maternal and paternal transmissions are shown in Fig. 31.6. In the MN-A family, the maternal expansion and paternal deletion biases clearly affect disease penetrance, with 90% of maternal transmissions resulting

CLINICAL AND GENETIC FEATURES OF SPINOCEREBELLAR ATAXIA TYPE 8

Fig. 31.6. Intergenerational variation in repeat number for maternal is shown as a decrease or an increase of CTG repeat units. Maternal gray and black bars, respectively. (Reproduced with permission et al., 1999. An untranslated CTG expansion causes a novel form 21:379–84.)

in clinically evident ataxia (Fig. 31.1) (Koob et al., 1999; Mosemiller et al., 2003). In contrast, 16 of the 22 asymptomatic individuals who carried repeat expansions received the SCA8 expansion from their father. The maternal penetrance bias in the MN-A family is likely caused by the transmission of expansions above the pathogenic threshold of  110 combined repeats, while paternal transmissions tend to result in contractions below the pathogenic threshold for that family (Koob et al., 1999; Day et al., 2000). Although the maternal penetrance bias is striking for the MN-A family, this bias is not found in most of other SCA8 families that have been reported (Ikeda et al., 2000a; Juvonen et al., 2000; Silveira et al., 2000; Cellini et al., 2001; Tazon et al., 2002; Topisirovic et al., 2002; Mosemiller et al., 2003; Brusco et al., 2004; Ikeda et al., 2004a; Corral et al., 2005)

En masse SCA8 repeat contractions in sperm Southern analysis of sperm DNA has shown massive contraction of SCA8 repeat length to a size less often associated with ataxia (e.g., from 500 to  80 and from 800 to  100) (Fig. 31.7A), consistent with the paternal contractions seen with intergenerational studies (Moseley et al., 2000; Mosemiller et al., 2003). Similar trends in men with smaller somatic expansions led to contractions in sperm to below  100 repeats (Fig. 31.7B). The equal intensities of the normal and

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and paternal transmissions. Repeat variation and paternal transmissions are represented by from Koob MD, Moseley ML, Schut LJ, of spinocerebellar ataxia (SCA8). Nat Genet

expanded allele bands in the Southern analyses suggest that nearly all of the expanded alleles contract in sperm, which is likely to contribute to the reduced penetrance in some SCA8 families (Moseley et al., 2000; Silveira et al., 2000).

Interruptions within the SCA8 repeat expansion Distinct from other pathogenic repeats, expanded SCA8 alleles commonly are not purely CTG●CAG, but often contain one or more CCG, CTA, CTC, CCA, or CTT interruptions within the CTG tract (Moseley et al., 2000; Tazon et al., 2002; Mosemiller et al., 2003; Martins et al., 2005). Surprisingly, these interruptions, which are often located near the 50 end of the CTG tract, often duplicate or change during transmission, resulting in offspring with alleles that vary from their affected parent in both repeat tract length and sequence configuration (Fig. 31.8). In contrast to SCA1 and FMR1, most normal SCA8 repeat tracts with < 50 repeats do not have sequence interruptions (Moseley et al., 2000), although Sobrido et al. (2001) have reported a single normal allele with 23 combined repeats, in which the CTG tract has a CAG interruption. While both interrupted and pure CTG repeat tracts are found in SCA8 ataxia families, it is possible that these sequence interruptions play some role in disease penetrance (Moseley et al., 2000; Mosemiller et al., 2003).

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Repeat number

A

polymorphic short tandem repeat (STR) markers flanking the SCA8 repeat over  1 Mb of DNA, we identified two ancestrally related haplotypes (A and A0 ) in the Caucasian population, which included 31 SCA8 families as well as controls and psychiatric patients, indicating a common ancestral origin for pathogenic and non-pathogenic expansions among Caucasians (Fig. 31.9A) (Ikeda et al., 2004a). Additionally, Japanese and Mexican ataxia families showed two other distinct haplotypes (B and C, respectively) (Fig. 31.9A,B). The identification of independently arising SCA8 expansions among ataxia families with various ethnic backgrounds further supports the direct role of the CTG expansion in disease pathogenesis.

C 800 – 500 –

260 –

100 –

Repeat number

2-1 Sperm

2-1 Blood

1-1 Blood

1-1 Sperm

170 – 160 –

SCA8 expansions co-segregate with ataxia in small families 6-1 Blood 6-1 Sperm 6-2 Blood 6-2 Sperm

5-1 Sperm

5-1 Blood

4-1 Sperm

4-1 Blood

3-1 Sperm

115 – 80 – 3-1 Blood

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B

Fig. 31.7. En masse contraction of SCA8 alleles in sperm. (A) Dramatic repeat length changes in patients 1 and 2 detected by Southern blotting. The repeat lengths of patients 1 and 2 contract from 500 and 800 repeats in blood to 80 and 100 repeats in sperm, respectively. The probe used did not contain the CTG repeat. (B) Southern blots of blood and sperm DNA from patients with smaller expansions in their blood reveal the same trend of contractions of the expanded allele in sperm to much smaller allele sizes, usually below  100 repeats. Again, the equal intensities of the bands representing the normal and expanded alleles indicate that repeat contractions occurred in all or nearly all of the sperm with expanded alleles. (C) PCR analysis of SCA8 contractions in two patients from a family with paternal disease transmission. Although contraction of repeats in sperm is again observed, the resulting alleles remain within a more penetrant size range (>100 CTGs). Approximate repeat numbers are shown at the left of each figure. (Reproduced from Moseley et al., 2000, with permission.)

EVIDENCE OF SCA8 EXPANSION PATHOGENICITY Haplotype analysis of SCA8 expansion chromosomes To better understand the origin of the SCA8 expansion and the reduced penetrance of the disease, we undertook haplotype analysis of a panel of 37 SCA8 ataxia families from the United States, Canada, Japan, and Mexico, 13 SCA8 expansion positive samples sent to Athena Diagnostics for ataxia testing, 7 control samples with expansions, and 14 expansion carriers with psychiatric diseases (Ikeda et al., 2004a). Using seventeen

Genetic studies demonstrate that co-segregation of the SCA8 expansion and ataxia is highly significant in the MN-A family (LOD score ¼ 6.8, y ¼ 0.00) (Koob et al., 1999). We were also able to study co-segregation of ataxia and the SCA8 expansion in 11 families with more than one affected individual (Ikeda et al., 2004a). Twelve of the 13 affected first-degree relatives of the probands in those families also inherited the SCA8 expansion, indicating that the expansion co-segregates with ataxia and is the likely cause of ataxia in these additional families (p ¼ 0.0038). The single exception was a family in which the two affected sisters had rapid progression of a disease characterized by choreiform movements, a severe sensory neuronopathy, and neuromyotonic discharges on electromyography, all of which were distinctly different clinical features than seen in the other SCA8 families. Eliminating this family with a different disease, and performing linkage analysis on the remaining 10 small families with multiple affected individuals, revealed LOD scores that were consistently positive, and when combined, exceeded 2.0 (LOD ¼ 2.02), which is significant for testing linkage to a single specific locus (Ott, 1991; Ikeda et al., 2004a). These results establishing co-segregation of the SCA8 expansion with ataxia, even among families with severely reduced penetrance, further indicate that the SCA8 expansion directly predisposes individuals to developing ataxia (Ikeda et al., 2004a).

SCA8 model mice showed progressive neurological phenotype To further elucidate the molecular mechanisms underlying SCA8 pathogenesis, we generated a transgenic mouse model using a human bacterial artificial chromosome (BAC) that contained the SCA8 gene with either an expanded or a normal CTG repeat length

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Fig. 31.8. An abbreviated pedigree for the large SCA8 kindred (MN-A) showing the different allele configurations found within this family. The two cases in which allele configurations changed over a single generation are noted as case 1 and case 2. The CTA, CTG, and CCG triplets are indicated by black, open, and gray circles, respectively. The number of CTG repeats at the 30 end of each allele is indicated by a subscript number. Filled pedigree symbols indicate individuals with ataxia. Haplotype analysis confirms that both branches of the family inherited the expanded repeat from a common, closely related founder. (Reproduced from Moseley et al., 2000, with permission.)

(Moseley et al., 2006). The SCA8 BAC-expansion mice have the (CTA)3(CTG)118 repeat tract that was cloned from the MN-A family proband, and control mice have a (CTA)8(CTG)11 repeat tract. The BAC construct used to generate the mice includes 220 kb of sequence surrounding the SCA8 gene, including expression regulatory elements, and thus is spatially and temporally expressed in the normal human pattern. All seven SCA8 BAC-expansion lines develop a progressive neurological phenotype that is fatal in high-copy number lines, and results in loss of cerebellar cortical inhibition; the three control lines have no pathological changes. The SCA8 BAC-expansion mice exhibit 1C2-positive, ubiquitin-positive intranuclear polyglutamine inclusions in cerebellar Purkinje cells and brainstem neurons, comparable to those seen in human material (Fig. 31.4). Demonstration of similarly stained intranuclear inclusions in cerebellar Purkinje and brainstem neurons of BAC expansion lines and in human SCA8 autopsy tissue (Fig. 31.4) provide a histopathological link between our mouse model and the human disease, indicating a common molecular mechanism underlying human SCA8 and the transgenic mouse model. The fact that the BAC construct containing an SCA8 expansion, but not with normal-size repeats, causes the cerebellar disease in mice directly demonstrates

the pathogenicity of the expansion (Moseley et al., 2006). Direct measurement showed no change of KLHL1 in BAC-expansion mice, indicating that the SCA8 expansion itself is pathogenic without any secondary effects on KLHL1 levels. Either the CUG transcript could be pathogenic due to toxic effects of the RNA expansion, or the CAG transcript and polyglutamine expression could underlie the disease in humans and the mouse model. Although additional experiments will be necessary to determine the relative significance of each of these mechanisms, the SCA8 BAC transgenic mice establish several aspects of SCA8 pathogenesis: 1) the SCA8 CTG●CAG expansion is pathogenic in mice when expressed in the normal genetic context and under the normal human promoter and regulatory elements; 2) SCA8 in mice and in humans is not associated with any change in cerebellar KLHL1 levels; 3) both strands of the SCA8 CTG●CAG expansion are expressed in cerebellar tissue of humans and BAC transgenic mice; 4) in cerebellar Purkinje cells, the SCA8 CAG transcripts are translated into ataxin-8 (ATXN8), a polyglutamine tract that accumulates as ubiquitinpositive intranuclear inclusions in both mice and human; 5) expression of the SCA8 mutation in mice results in abnormal cerebellar molecular layer inhibition.

A Group I: Haplotype analysis of 37 SCA8 families Marker Repeat Motif No of Alleles Kb from (CTG)n C1 (MN-A) C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13A C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26A C27 C28 C29 C26B C30 C31 C32 J1 J2 J3 J4 C13B M1

D13S275 (CA)n 5 974kb 188 184 184 184 184/182 184 184 184/182 184 182 184 184 182 184 184/170 184/188 184 184/182 184 184 188 184/182 184 182 184/182 184 184 184/188 182 182 182/170 182 182 184/182 184 184/182 182 184 182

YI18 (GATA)n 12 284kb 214 210 210 210 210/242 210/242 210/214 210 210 214 210/206 214 210 210 210/242 210/214 218 210/218 218 218 218 218 218 218 218/234 218 214 214/206 222/206 206 218/210 214 210 214 214 214/210 218 218 214

YI17 (AAAAT)n 6 277kb 243 248 248 n.d. 248/243 248 248/253 248 248 248 248 248/243 248 248 248/243 248 248 248/253 248 248 248/243 248 248 248 248/243 248 248 248 248/258 258 248/253 248 238 248 248 248/238 248 243 248

YI15 (AAT)n 7 156kb 236 233 233 n.d. 233/230 233/230 233/230 233 233 233 230 230 230 236 236/230 236/224 233 233/230 233 233 236 236 236 236 236 236 236 236/230 236 236 236 230 230 230 230 230/233 230 227 233

D13S318 (TATC)n 8 137kb 280 284 284 284 284/272 284/288 284 284/276 284 284 284 284 284/276 284 284/272 284 288 280/272 284 284 284 284 284 284 284 284 284 284/268 284/280 284 288/276 288 288 284 284 284/276 284 284/276 284

YI14 (GT)n 6 112kb 164 164 164 164 164/162 164 164 164 164 164 164 164 164 164 164 164/168 164 164/162 164 164 164 164 164/168 164 164 164 164 164/162 164 164 164/170 168 162 164 164 164/162 162 162 168

CL2 (CAA)n 4 72kb 197 197 197 197 197/200 197 197 197 197 197 197 197 197 197 197 197/200 197 197/200 197 197 197/200 197 197 197 197 197 197 197/200 197 197 197/200 200 200 200 200 200/197 200 200 203

D13S1296 CL4 (CA)n (GAAA)n 13 9 57kb 53kb 175 165 175 165 175 165 175 165 175/185 165 175/183 165/167 175/183 165/167 175 165 175 165 175 165 175 165 175/181 165 175 165 175 165 175/181 165/167 175/179 165/163 175 165 173/181 165/157 177 165 177 165 177 165 177 165 177 165 177 165 177/185 165/169 177 165 177 165 177/181 165/157 177/181 165/167 177 165 177/173 165/169 181 165 185 168 179/183 168 179 168 179/183 167/157 185 168 181 168 185 163

CL6 (GT)n 5 10kb 147 147 147 147/145 147/145 147/145 147/145 147 147 147 147 147/149 147 147/145 147/145 147 147 147/149 147 147 147 147 147 147 147/149 147 147 147 147/145 147 147/149 147 145 145 145 145 145 145 149

(CTG)n 0 73~143 80~88 80~115 110 98 90 134 71~88 208~750 130~735 101~118 90~143 97~120 93~126 91 169 102 212 150 84~588 134~445 845~945 130~1110 177~263 1380 101~950 140 146~1130 75 101~950 150 138 80~84 89~105 95~99 155 95~136 97~120 100

D13S135 (CA)n 7 97kb 186 186 186 186 186/180 186/182 186/180 186 186 186 186 186 186 186 186/180 186/182 186 186/182 180 180 180 180 180 180 180/186 180 180/182 180/186 182/186 180 182 188 190 192/182 192 190/178 190 182 190

Marker Repeat Motif No of Alleles Kb from (CTG)n C1 (MN-A) C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13A C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26A C27 C28 C29 C26B C30 C31 C32 J1 J2 J3 J4 C13B M1

D13S135 (CA)n 7 97kb 186/182 186/190 186/196 186/180 186/182 186/182 186/178 186/190 186/180 182 180/182 180/182 190/182

Marker Repeat Motif No of Alleles Kb from (CTG)n AD1 AD2 AD3 AD4 AD5 AD6 AD7 AD8 AD9 AD10 AD11 AD12 AD13

CL8 CL1 JJ9 JJ10 JJ12 JJ11 D13S135 (CA)n (GT)n (GT)n (CT)n (GT)n (GT)n (CA)n 6 7 6 4 8 7 7 1.1 kb 13.6 kb 17kb 20kb 52kb 80kb 97kb 246/247 156 199/196 251/247 174/162 206/204 186/182 246 156 199 251 174 206 186 246/245 156/164 199/200 251 174 206 186 246/244 156/164 199/200 251/249 174/168 204/210 186/178 246/244 156/164 196/200 247/251 162/168 206/204 180/178 n.d. 156 196 247 162 206/204 180/182 246 156 196 247 162 206 180

Marker Repeat Motif No of Alleles Kb from (CTG)n N1 CEPH1416 N2 N3 N4 N5 CEPH1334

CL8 (CA)n 6 1.1 kb 246 246 246 246 246/245 246/244 246/244 246 245 246 246 246/243 246 246 246/244 246 246 245 246/245 246 246 246 246 246 246/245 246 246 245 246/244 246 246 246 245 245 245 245/244 245 245 244

CL1 (GT)n 7 13.6 kb 156 156 156 156 156 156 156 156 156 156 156/158 156 156 156 156 156/158 156 156 156 156 156 156 156 156 156/158 156 156 156/164 158/164 156 156/158 156 160 160 160 162/168 160 160 148

JJ9 (GT)n 6 17kb 199 199 199/196 199 199/196 199/196 199/196 199 199 199 199/196 199/200 199 199 199/196 199/196 199 199/196 196 196/200 196 196 196/200 196/200 196 196 196 196/200 196/200 196 196/198 196 198 198/196 198 198/200 198 199 198

JJ10 (CT)n 4 20kb 251 251 251/247 251 251/247 251/247 251/247 251 251 251 251 251 251 251 251/247 251/247 251 251/247 247 247/251 247 247 247 247 247 247 247 247/251 247 247 247 247 249 249 249 249/251 249 249 249

JJ12 (GT)n 8 52kb 174 174 174 174 174/162 174/162 174/162 174 174 172 174 174 174 174 174/162 176/162 174 174/162 162 162/172 162 162 162 162 162/172 162 162 162/172 162/172 162 162 166 168 168/162 168 168/172 166 168 168/164

JJ11 (GT)n 7 80kb 206 206 206 206 206/204 206/202 206/204 212 206 206 206 206 206 206 206/204 206/204 206 206/204 206 206 206 206 206 206 206 206 206 206 206/202 206 206 210 212 210/204 210 214/204 210/208 204 216

C: Caucasian SCA8 families C13A/C13B and C26A/C26B represent haplotypes of the two different expansion chromosomes in these homozygous individuals. J: Japanese SCA8 families M: Mexican SCA8 family

Group II: Haplotype analysis of 13 diagnostic ataxia samples Marker Repeat Motif No of Alleles Kb from (CTG)n AD1 AD2 AD3 AD4 AD5 AD6 AD7 AD8 AD9 AD10 AD11 AD12 AD13

D13S275 (CA)n 5 974kb 184 184/182 184 184/182 184/182 184 184/182 184/182 182/188 182 184/182 184/182 184/182

YI18 (GATA)n 12 284kb 210/242 210/206 210/206 214/194 210/206 210/202 210/214 214/206 210/190 210/254 218/214 218/210 210/206

YI17 (AAAAT)n 6 277kb 248 248/263 248/243 248 248/238 248/263 248/243 248/258 243/238 248/223 248 248 238/263

YI15 (AAT)n 7 156kb 233/230 233/224 233/236 233/230 230 233/224 233/224 236/224 230/227 233/236 236/230 239/227 230

D13S318 (TATC)n 8 137kb 284/288 284 284/288 284/276 284/280 288/280 288/272 284/280 272/268 284/280 284/280 284/280 288/276

YI14 (GT)n 6 112kb 164 164/168 164 164/162 164/168 164/168 164/168 164/168 164/162 164/162 164 164/168 162

CL2 (CAA)n 4 72kb 197 197/200 197 197/200 197/203 197/200 197/200 197 197/200 197/200 197 197/203 200

D13S1296 CL4 (CA)n (GAAA)n 13 9 57kb 53kb 175/183 165/167 175/179 165/163 175/183 165/167 175/181 165/157 175/183 165/163 175/179 165/163 175/181 165/157 175/179 165/167 175/181 165/157 181/185 163/157 177/193 165/157 177/185 165/163 183/185 168

CL6 (GT)n 5 10kb 147/145 147 147/145 147 147/149 147 147 147/145 147/145 147/151 147/145 147/149 145

(CTG)n 0 88 86 56 103 89 105 137 69 76 62 849 95 96

CL8 (CA)n 6 1.1 kb 246/244 246 246/244 246/245 246/243 246 246/245 246/244 246/245 246/245 246/244 246/243 245

CL1 (GT)n 7 13.6 kb 156 156/158 158/154 156 156/158 156 156/154 156/158 156 152/164 156/148 156/158 160

JJ9 (GT)n 6 17kb 199/196 199/196 199 199/196 199/196 199/196 199/200 199/196 199/196 199/200 196/200 196 198/200

JJ10 (CT)n 4 20kb 251/247 251/247 251 251/247 251/247 251/247 251 251/247 251/247 251 247/249 247 249

JJ12 (GT)n 8 52kb 174/162 174/162 174/170 174/162 174/162 174/162 174/168 174/164 174/162 168/166 162 162 168

JJ11 (GT)n 7 80kb 206/202 206/204 206/208 206/204 206/204 206/204 206/204 206/204 206/204 204 206/204 206/204 216/204

Group III: Haplotype analysis of 5 normal controls and 2 CEPH families Marker Repeat Motif No of Alleles Kb from (CTG)n N1 CEPH1416 N2 N3 N4 N5 CEPH1334

D13S275 (CA)n 5 974kb 184/182 184 184 184/182 184/182 184 182

YI18 (GATA)n 12 284kb 210/206 210 214/190 210/234 218/238 218/210 222

YI17 (AAAAT)n 6 277kb 248 253 248/243 248/243 248/243 248/243 248

YI15 (AAT)n 7 156kb 233/224 233 233/230 233/232 233/232 236/233 236

D13S318 (TATC)n 8 137kb 284/280 284 284/268 280/276 284/276 280 284

YI14 CL2 D13S1296 CL4 CL6 (CTG)n (GT)n (CAA)n (CA)n (GAAA)n (GT)n 6 4 13 9 5 112kb 72kb 57kb 53kb 10kb 0 164/168 197/200 175/183 165/169 147/149 117 164 197 175 165 147 85~800 164/162 197/200 171/181 165/157 147 103 164/168 197 175/191 165/167 147/145 970 164/168 197/200 177/195 165/167 147/149 230 164 197 177/191 165/167 147/149 550 164 197 177 165 147 160~900

Group IV: Haplotype analysis of 14 major psychosis patients Marker Repeat Motif No of Alleles Kb from (CTG)n P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14

D13S275 (CA)n 5 974kb 184 184/182 184/182 184/188 184/182 184/182 184/182 184/182 184/172 184 184/182 184 184/188 184/182

YI18 (GATA)n 12 284kb 210/214 210/214 214/202 206/250 210/246 210/190 210/202 218/198 214/238 218 218/202 218/222 218 206/246

YI17 (AAAAT)n 6 277kb 248 248 248/263 248/243 253/228 253/243 248/258 248/258 248/243 248 248/263 248 248 248

YI15 (AAT)n 7 156kb 233/230 233/236 233/224 233/232 233/238 233/224 232 230/239 233/230 233 233/227 236/230 236/230 236/230

D13S318 (TATC)n 8 137kb 284 284/268 284 284/276 284/272 288/272 272/268 284/288 288/276 284 284/276 284 284/280 284

YI14 (GT)n 6 112kb 164 164/162 164/168 164/170 164/170 164/170 164/168 164/162 164/162 164 164/162 164 164/168 164

CL2 (CAA)n 4 72kb 197 197/200 197/200 197 197/200 197 197/200 197/203 197/200 197 197/200 197 197/203 197

D13S1296 CL4 (CA)n (GAAA)n 13 9 57kb 53kb 175/181 165/157 175/181 165/157 175 165/163 175/197 165/167 175/177 165/155 175 165 175/181 165/157 175/183 165/163 175/183 165/157 177 165 177/183 165/168 177 165 177/185 165/163 177/183 165/167

CL6 (GT)n 5 10kb 147 147 147 147/145 147 147 147/149 147 147/145 147 147/145 147 147/149 147/145

(CTG)n 0 100 103 180 257 1300 130 600 1140 550 116 130 600 107 106

CL8 (CA)n 6 1.1 kb 246/245 246/245 246 246/244 246/244 246/245 246/245 246/243 246/245 246 246/245 246 246/243 246/244

CL1 (GT)n 7 13.6 kb 156/164 156 156 158/164 156/164 156 156/166 156 156 156 156/160 156 156/158 156

JJ9 (GT)n 6 17kb 199/200 199/196 199 199/196 199/200 199/196 199 199/196 196 196 196/199 196 196 196

conserved + 1 repeat unit – 1 repeat unit > 1 repeat unit difference Recombinant regions that are not conserved among families are uncolored. (CTG)n: Size range of the combined CTA/CTG repeat expansions in the family n.d.: not determined

Fig. 31.9.—Cont’d

JJ10 (CT)n 4 20kb 251/249 251/247 251/247 251/247 251/249 251 251/249 251/247 247/245 247 247/249 247/245 247 247

JJ12 (GT)n 8 52kb 174 174/162 174 168/162 174/164 174 174/168 174/162 162/164 162 162/168 162 162 162

JJ11 (GT)n 7 80kb 206/204 206/202 206/204 204/202 206/200 206/204 206/204 206 204/210 206 206/204 206 206/204 206/202

D13S135 (CA)n 7 97kb 186/188 186/182 186/182 182/178 186/174 186/178 186/178 186/188 182/190 180 180/182 180 180/182 180/182

Marker Repeat Motif No of Alleles Kb from (CTG)n P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14

CLINICAL AND GENETIC FEATURES OF SPINOCEREBELLAR ATAXIA TYPE 8

503

Fig 31.9—Cont’d (A) Haplotype analysis of several SCA8 expansion positive families with and without ataxia. The markers in this figure are ordered according to their physical distance from the CTG repeat. Group I: Haplotypes of 37 SCA8 positive families in which at least one member has been diagnosed with ataxia. Two predominant and probably related SCA8 expansion haplotypes (A and A0 ) were found in 18 and 13 families, respectively. Six families, including four from Japan, have a clearly distinct second haplotype (B), and a Mexican family shows evidence for a third independent haplotype (C). The consensus haplotype A is indicated in yellow. Minor deviations in repeat size of þ/– 1 repeat unit flanked by markers with conserved allele sizes are indicated by alternative colors with a color key located below the figure. Recombinant regions that are not conserved among families are uncolored. Two families (C13 and C26) presented with homozygous expansion positive patients and the separate SCA8 haplotypes for these families are indicated. The microsatellite marker name, its repeat motif and distance from the SCA8 CTG repeat expansion are shown at the top of the figure. The size range of the combined CTA/CTG repeat expansions in the family (or in some cases a single expansion carrier) are shown. Group II: SCA8 expansion haplotypes of 13 samples sent to Athena Diagnostics for testing have either haplotype A or A´ except for a single subject (AD13) with haplotype B. Group III: Haplotypes of seven normal control families including two CEPH families with SCA8 expansions. Four of these families had haplotype A, three had haplotype A´. Group IV: Haplotypes of 14 major psychosis patients with CTG expansions had either haplotype A (n ¼ 8) or haplotype A´ (n ¼ 6). (B) Proposed summary of the ancestral origins based on the analysis of 37 SCA8 ataxia families. The current haplotypes are likely to have arisen from a small number of ancestral recombination and microsatellite instability events. “R” indicates a recombination event and the asterisk symbolizes an area with microsatellite repeat instability. (Reproduced from Ikeda et al., 2004a, with permission.)

CONCLUSIONS Clinically, pathologically, and on imaging studies, SCA8 is a relatively pure cerebellar disorder that typically has an adult onset and very slow disease progression. Although the markedly reduced penetrance of the disease is unexplained, it may relate to several novel genetic features: 1) the CTG●CAG expansion is transcribed in both CTG and CAG directions; 2) the CUG expansion RNA transcripts may involve a pathogenic mechanism that is similar to the myotonic dystrophies; 3) the CAG transcript is translated into a nearly pure polyglutamine protein, with neuronal intranuclear inclusions that are ubiquitin positive; 4) there is dramatic intergenerational instability, with large expansions and contractions; 5) there are interruptions within the SCA8 repeat expansion of pathogenic alleles, and the interruptions can change in motif and number from generation to generation; 6) BAC transgenic mice show that the SCA8 repeat expansion is pathogenic and impairs cerebellar

cortical inhibition. Given the unusual genetic features of this disease, clarifying its molecular pathophysiology will shed light on novel mechanisms of neurodegeneration caused by the CTG●CAG expansion.

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