Neuropathology and Cellular Pathogenesis of Spinocerebellar Ataxia Type 12.

OBJECTIVE: SCA12 is a progressive autosomal-dominant disorder, caused by a CAG/CTG repeat expansion in PPP2R2B on chromosome 5q32, and characterized by tremor, gait ataxia, hyperreflexia, dysmetria, abnormal eye movements, anxiety, depression, and sometimes cognitive impairment. Neuroimaging has demonstrated cerebellar and cortical atrophy. We now present the neuropathology of the first autopsied SCA12 brain and utilize cell models to characterize potential mechanisms of SCA12 neurodegeneration. METHODS: A fixed SCA12 brain was examined using gross, microscopic, and immunohistochemical methods. The effect of the repeat expansion on PPP2R2B Bß1 expression was examined in multiple cell types by transient transfection of constructs containing the PPP2R2B Bß1 promoter region attached to a luciferase reporter. The neurotoxic effect of PPP2R2B overexpression was examined in transfected rat primary neurons. RESULTS: Neuropathological investigation revealed enlarged ventricles, marked cerebral cortical atrophy and Purkinje cell loss, less-prominent cerebellar and pontine atrophy, and neuronal intranuclear ubiquitin-positive inclusions, consistent with Marinesco bodies, which did not stain for long polyglutamine tracts, alpha-synuclein, tau, or transactive response DNA-binding protein 43. Reporter assays demonstrated that the region of PPP2R2B containing the repeat functions as a promoter, and that promoter activity increases with longer repeat length and is dependent on cell type, repeat sequence, and sequence flanking the repeat. Overexpression of PPP2R2B in primary cortical neurons disrupted normal morphology. CONCLUSIONS: SCA12 involves extensive, but selective, neurodegeneration distinct from Alzheimer's disease, synucleinopathies, tauopathies, and glutamine expansion diseases. SCA12 neuropathology may arise from the neurotoxic effect of repeat-expansion-induced overexpression of PPP2R2B.

Loss of junctophilin-3 contributes to Huntington disease-like 2 pathogenesis.

OBJECTIVE: Huntington disease-like 2 (HDL2) is a progressive, late onset autosomal dominant neurodegenerative disorder, with remarkable similarities to Huntington disease (HD). HDL2 is caused by a CTG/CAG repeat expansion. In the CTG orientation, the repeat is located within the alternatively spliced exon 2A of junctophilin-3 (JPH3), potentially encoding polyleucine and polyalanine, whereas on the strand antisense to JPH3, the repeat is in frame to encode polyglutamine. The JPH3 protein product serves to stabilize junctional membrane complexes and regulate neuronal calcium flux. We have previously demonstrated the potential pathogenic properties of JPH3 transcripts containing expanded CUG repeats. The aim of this study was to test the possibility that loss of JPH3 expression or expanded amino acid tracts also contribute to HDL2 pathogenesis. METHODS: Transcripts from the HDL2 locus, and their protein products, were examined in HDL2, HD, and control frontal cortex. The effect of loss of Jph3 was examined in mice with partial or complete loss of Jph3. RESULTS: Bidirectional transcription occurs at the HDL2 locus, although expression of antisense transcripts with expanded CAG repeats is limited. Protein products with expanded amino acid tracts were not detected in HDL2 brain. However, JPH3 transcripts and full-length JPH3 protein are decreased in HDL2 brain, and Jph3 hemizygous and null mice exhibit abnormal motor function. INTERPRETATION: Our results suggest that the pathogenic mechanism of HDL2 is multifactorial, involving both a toxic gain of function of JPH3 RNA and a toxic loss of JPH3 expression.

Missense mutations in the AFG3L2 proteolytic domain account for ∼1.5% of European autosomal dominant cerebellar ataxias.

Spinocerebellar ataxia type 28 is an autosomal dominant form of cerebellar ataxia (ADCA) caused by mutations in AFG3L2, a gene that encodes a subunit of the mitochondrial m-AAA protease. We screened 366 primarily Caucasian ADCA families, negative for the most common triplet expansions, for point mutations in AFG3L2 using DHPLC. Whole-gene deletions were excluded in 300 of the patients, and duplications were excluded in 129 patients. We found six missense mutations in nine unrelated index cases (9/366, 2.6%): c.1961C>T (p.Thr654Ile) in exon 15, c.1996A>G (p.Met666Val), c.1997T>G (p.Met666Arg), c.1997T>C (p.Met666Thr), c.2011G>A (p.Gly671Arg), and c.2012G>A (p.Gly671Glu) in exon 16. All mutated amino acids were located in the C-terminal proteolytic domain. In available cases, we demonstrated the mutations segregated with the disease. Mutated amino acids are highly conserved, and bioinformatic analysis indicates the substitutions are likely deleterious. This investigation demonstrates that SCA28 accounts for ∼3% of ADCA Caucasian cases negative for triplet expansions and, in extenso, to ∼1.5% of all ADCA. We further confirm both the involvement of AFG3L2 gene in SCA28 and the presence of a mutational hotspot in exons 15-16. Screening for SCA28, is warranted in patients who test negative for more common SCAs and present with a slowly progressive cerebellar ataxia accompanied by oculomotor signs.

Huntington's disease like-2: review and update.

Huntington's Disease-like 2 (HDL2), like Huntington's disease (HD), is an adult onset, progressive, neurodegenerative autosomal dominant disorder clinically characterized by abnormal movements, dementia, and psychiatric syndromes. Like HD, the neuropathology of HDL2 features prominent cortical and striatal atrophy and intranuclear inclusions. HDL2 is generally rare, accounting for only a few percent of HD-like cases in which the HD mutation has already been excluded. However, the rate is considerably higher among individuals of African ancestry, and is almost as common as HD in Black South Africans. The disorder is caused by a CTG/CAG expansion mutation on chromosome 16q24.3, with normal and expanded repeat ranges similar to HD, and a correlation between repeat length and onset age very similar to HD. Surprisingly, the available evidence suggests that HDL2 is not a polyglutamine disease. Rather, the repeat expansion is located within Junctophilin-3 in the CTG orientation. The phenotypic similarities between HD and HDL2 suggest that understanding the pathobiology of HDL2 may shed new light on the pathogenesis of HD and other disorders of striatal neurodegeneration.

Detection of large pathogenic expansions in FRDA1, SCA10, and SCA12 genes using a simple fluorescent repeat-primed PCR assay.

At least 18 human genetic diseases are caused by expansion of short tandem repeats. Here we describe a successful application of a fluorescent PCR method for the detection of expanded repeats in FRDA1, SCA10, and SCA12 genes. Although this test cannot give a precise estimate of the size of the expansion, it is robust, reliable, and inexpensive, and can be used to screen large series of patients. It proved useful for confirming the presence of large expansions in the Friedreich ataxia gene following an ambiguous result of long-range PCR, as well as rapid pre-screening for large repeat expansions associated with Friedreich ataxia and SCA10 and the shorter repeat expansions associated with SCA12.

Spinocerebellar ataxia type 12.

SCA12 is a late-onset, autosomal dominant, slowly progressive disorder. Action tremor is the usual presenting sign. Subsequent development of ataxia and hyperreflexia suggests spinocerebellar ataxia. In the index SCA12 kindred, which resides in North America and is of German ancestry, parkinsonism, anxiety, depression, and cognitive dysfunction are not uncommon. SCA12 is linked to a CAG repeat expansion mutation in exon 7 of PPP2R2B, a gene that encodes Bß, a regulatory subunit of protein phosphatase 2A (PP2A). CAG repeats number 7-28 in normal individuals and 55-78 in SCA12 patients. The mechanism by which this mutation leads to SCA12 has not been determined. The CAG expansion in PPP2R2B has promoter function in vitro. CAG length correlates with increased Bß expression. There is no evidence that this CAG expansion results in polyglutamine production. In addition to the North. American SCA12 kindred, multiple SCA12 families have been found in Northern India that are not related to the index SCA12 kindred. SCA12 has been reported, rarely, in Singapore and China. Action tremor, anxiety, and depression in SCA12 have responded to usual treatments for these disorders. SCA12 may be considered in patients who present with action tremor and later develop signs of cerebellar and cortical dysfunction.

Long tandem repeats as a form of genomic copy number variation: structure and length polymorphism of a chromosome 5p repeat in control and schizophrenia populations.

OBJECTIVES: Genomic copy number variations (CNVs) are a major form of variation in the human genome and play an etiologic role in several neuropsychiatric diseases. Tandem repeats, particularly with long (>50 bp) repeat units, are a relatively common yet underexplored type of CNV that may significantly contribute to human genomic variation and disease risk. We therefore carried out a pilot experiment to explore the potential role of long tandem repeats as risk factors in psychiatric disorders. METHODS: A bacterial artificial chromosome-based array comparative genomic hybridization (aCGH) platform was used to examine CNVs in genomic DNA from 34 probands with schizophrenia or schizoaffective disorder. RESULTS: The aCGH screen detected an apparent deletion on 5p15.1 in two probands, caused by the presence in each proband of two low copy number (short) alleles of a tandem repeat that ranges in length from fewer than 10 to greater than 50 3.4 kb units in the population examined. Short alleles partially segregate with schizophrenia in a small number of families, though linkage was not significant. An association study showed no significant difference in repeat length between 406 schizophrenia cases and 392 controls. CONCLUSION: Although we did not demonstrate a relationship between the 5p15.1 repeat and schizophrenia, our results illustrate that long tandem repeats represent an intriguing type of genetic variation that have not been studied in earlier connection with psychiatric illness. aCGH can detect a small subset of these repeats, but systematic investigation will require the development of specific arrays and improved analytical methods.

Huntington's disease--like 2 is associated with CUG repeat-containing RNA foci.

OBJECTIVE: Huntington's disease-like 2 (HDL2) is caused by a CAG/CTG expansion mutation on chromosome 16q24.3. The repeat falls, in the CTG orientation, within a variably spliced exon of junctophilin-3 (JPH3). The existence of a JPH3 splice variant with the CTG repeat in 3' untranslated region suggested that transcripts containing an expanded CUG repeat could play a role in the pathogenesis of HDL2, similar to the proposed pathogenic role of expanded CUG repeats in myotonic dystrophy type 1 (DM1). The goal of this study, therefore, was to test the plausibility of an RNA gain-of-function component in the pathogenesis of HDL2. METHODS: The presence and composition of RNA foci in frontal cortex from HDL2, Huntington's disease, DM1, and control brains were investigated by in situ hybridization and immunohistochemistry. An untranslatable JPH3 transcript containing either a normal or an expanded CUG repeat was engineered and expressed in human embryonic kidney 293 and HT22 cells to further test the toxic RNA hypothesis. The formation of RNA foci and the extent of cell death were quantified. RESULTS: RNA foci resembling DM1 foci were detected in neurons in HDL2 cortex and other brain regions. Similar to DM1, the foci colocalize with muscleblind-like protein 1, and nuclear muscleblind-like protein 1 in HDL2 cortical neurons is decreased relative to controls. In cell experiments, expression of a JPH3 transcript with an expanded CUG repeat resulted in the formation of RNA foci that colocalized with muscleblind-like protein 1 and in cell toxicity. INTERPRETATION: These results imply that RNA toxicity may contribute to the pathogenesis of HDL2.

Clinical signs and symptoms in a large hereditary spastic paraparesis pedigree with a novel spastin mutation.

The most common form of autosomal dominant hereditary spastic paraparesis (HSP), SPG4, is caused by mutations in the spastin gene on chromosome 2p. This disease is characterized by intra- and interfamilial phenotypic variation. To determine the predictive values of clinical signs and symptoms in SPG4, we examined 43 members of a large pedigree with autosomal dominant HSP. We then identified the genetic etiology of the disorder in this family, a novel nonsense mutation in exon 1 of spastin, carried by 24 of the examined family members. The best clinical predictors of positive gene status were the presence of hyperreflexia in the lower extremities, >2 beats of ankle clonus, pes cavus, bladder symptoms and increased tone in the legs. The mean age of onset was 32.2 +/- 7.4 years, but the age of onset was earlier in children from 10 of 12 child-parent gene-positive pairs, with a mean difference of 10.8 +/- 3.3 years. The finding of leg weakness was especially common in older-onset affected family member with leg hyperreflexia. These results suggest that specific clinical signs and symptoms may be of value in differentiating individuals affected with SPG4 from family members with nonspecific neurological findings.

Huntington's Disease-like 2 (HDL2) in North America and Japan.

Huntington's Disease-like 2 (HDL2) is a progressive, autosomal dominant, neurodegenerative disorder with marked clinical and pathological similarities to Huntington's disease (HD). The causal mutation is a CTG/CAG expansion mutation on chromosome 16q24.3, in a variably spliced exon of junctophilin-3. The frequency of HDL2 was determined in nine independent series of patients referred for HD testing or selected for the presence of an HD-like phenotype in North America or Japan. The repeat length, ancestry, and age of onset of all North American HDL2 cases were determined. The results show that HDL2 is very rare, with a frequency of 0 to 15% among patients in the nine case series with an HD-like presentation who do not have the HD mutation. HDL2 is predominantly, and perhaps exclusively, found in individuals of African ancestry. Repeat expansions ranged from 44 to 57 triplets, with length instability in maternal transmission detected in a repeat of r2=0.29, p=0.0098). The results further support the evidence that the repeat expansion at the chromosome 16q24.3 locus is the direct cause of HDL2 and provide preliminary guidelines for the genetic testing of patients with an HD-like phenotype.