Dobrila D. Rudnicki

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

Huntingtons 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.

An Antisense CAG Repeat Transcript at JPH3 Locus Mediates Expanded Polyglutamine Protein Toxicity in Huntington's Disease-like 2 Mice

Huntingtons disease-like-2 (HDL2) is a phenocopy of Huntingtons disease caused by CTG/CAG repeat expansion at the Junctophilin-3 (JPH3) locus. The mechanisms underlying HDL2 pathogenesis remain unclear. Here we developed a BAC transgenic mouse model of HDL2 (BAC-HDL2) that exhibits progressive motor deficits, selective neurodegenerative pathology, and ubiquitin-positive nuclear inclusions (NIs). Molecular analyses reveal a promoter at the transgene locus driving the expression of a CAG repeat transcript (HDL2-CAG) from the strand antisense to JPH3, which encodes an expanded polyglutamine (polyQ) protein. Importantly, BAC-HDL2 mice, but not control BAC mice, accumulate polyQ-containing NIs in a pattern strikingly similar to those in the patients. Furthermore, BAC mice with genetic silencing of the expanded CUG transcript still express HDL2-CAG transcript and manifest polyQ pathogenesis. Finally, studies of HDL2 mice and patients revealed CBP sequestration into NIs and evidence for interference of CBP-mediated transcriptional activation. These results suggest overlapping polyQ-mediated pathogenic mechanisms in HD and HDL2.

A comparison of huntington disease and huntington disease-like 2 neuropathology.

Abstract Huntington disease-like 2 (HDL2) is an autosomal dominant disorder characterized by adult-onset, progressive motor abnormalities, psychiatric disturbances, and dementia ending in premature death. Clinically, it most closely resembles Huntington disease (HD), although a subset of affected individuals have parkinsonian features. Here, we systematically compare 5 HDL2 and 5 HD brains with the hypothesis that, reflecting the clinical presentation, the neuropathology of the 2 diseases would be similar. Gross and microscopic examination revealed prominent striatal neuron loss and astrocytic gliosis in a dorsal to ventral gradient in each disorder and cortical atrophy. Nuclear protein aggregates were as common in HDL2 as in HD, and the ultrastructural features of HDL2 and HD aggregates were similar. Electron microscopy also revealed degenerating neurons, some with evidence of autophagy, in both HDL2 and HD. Small ribonuclear foci, previously associated with potentially neurotoxic RNA transcripts in HDL2, rarely colocalized with protein aggregates in HDL2 brain, although the protein aggregates were stained by anti-TATA-box binding protein antibodies. Overall, the neuropathologic features of HDL2 and HD are very similar but not identical, suggesting that the pathogenetic mechanisms of the 2 diseases may partially overlap.

Huntington’s disease-like 2 can present as chorea-acanthocytosis

Three patients from a previously described family with autosomal dominant chorea-acanthocytosis were found to have the CTG trinucleotide repeat expansion mutation of the junctophilin-3 gene associated with Huntington’s disease–like 2 (HDL2). One of six previously identified patients with HDL2 had acanthocytosis on peripheral blood smear, suggesting that HDL2 should be considered in the differential of chorea-acanthocytosis.

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

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.

Nuclear retention of full-length HTT RNA is mediated by splicing factors MBNL1 and U2AF65

Huntington’s disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. Recent evidence suggests that HD is a consequence of multimodal, non-mutually exclusive mechanisms of pathogenesis that involve both HTT protein- and HTT RNA-triggered mechanisms. Here we provide further evidence for the role of expanded HTT (expHTT) RNA in HD by demonstrating that a fragment of expHTT is cytotoxic in the absence of any translation and that the extent of cytotoxicity is similar to the cytotoxicity of an expHTT protein fragment encoded by a transcript of similar length and with a similar repeat size. In addition, full-length (FL) expHTT is retained in the nucleus. Overexpression of the splicing factor muscleblind-like 1 (MBNL1) increases nuclear retention of expHTT and decreases the expression of expHTT protein in the cytosol. The splicing and nuclear export factor U2AF65 has the opposite effect, decreasing expHTT nuclear retention and increasing expression of expHTT protein. This suggests that MBNL1 and U2AF65 play a role in nuclear export of expHTT RNA.

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.

Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity

Huntingtons disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene. Disease pathogenesis derives, at least in part, from the long polyglutamine tract encoded by mutant HTT. Therefore, considerable effort has been dedicated to the development of therapeutic strategies that significantly reduce the expression of the mutant HTT protein. Antisense oligonucleotides (ASOs) targeted to the CAG repeat region of HTT transcripts have been of particular interest due to their potential capacity to discriminate between normal and mutant HTT transcripts. Here, we focus on phosphorodiamidate morpholino oligomers (PMOs), ASOs that are especially stable, highly soluble and non-toxic. We designed three PMOs to selectively target expanded CAG repeat tracts (CTG22, CTG25 and CTG28), and two PMOs to selectively target sequences flanking the HTT CAG repeat (HTTex1a and HTTex1b). In HD patient-derived fibroblasts with expanded alleles containing 44, 77 or 109 CAG repeats, HTTex1a and HTTex1b were effective in suppressing the expression of mutant and non-mutant transcripts. CTGn PMOs also suppressed HTT expression, with the extent of suppression and the specificity for mutant transcripts dependent on the length of the targeted CAG repeat and on the CTG repeat length and concentration of the PMO. PMO CTG25 reduced HTT-induced cytotoxicity in vitro and suppressed mutant HTT expression in vivo in the N171-82Q transgenic mouse model. Finally, CTG28 reduced mutant HTT expression and improved the phenotype of Hdh(Q7/Q150) knock-in HD mice. These data demonstrate the potential of PMOs as an approach to suppressing the expression of mutant HTT.

Junctophilin 3 (JPH3) expansion mutations causing Huntington disease like 2 (HDL2) are common in South African patients with African ancestry and a Huntington disease phenotype.

Huntington disease (HD) is a progressive autosomal dominant neurodegenerative disorder, characterized by abnormal movements, cognitive decline, and psychiatric symptoms, caused by a CAG repeat expansion in the huntingtin (HTT) gene on chromosome 4p. A CAG/CTG repeat expansion in the junctophilin‐3 (JPH3) gene on chromosome 16q24.2 causes a Huntington disease‐like phenotype (HDL2). All patients to date with HDL2 have some African ancestry. The present study aimed to characterize the genetic basis of the Huntington disease phenotype in South Africans and to investigate the possible origin of the JPH3 mutation. In a sample of unrelated South African individuals referred for diagnostic HD testing, 62% (106/171) of white patients compared to only 36% (47/130) of black patients had an expansion in HTT. However, 15% (20/130) of black South African patients and no white patients (0/171) had an expansion in JPH3, confirming the diagnosis of Huntington disease like 2 (HDL2). Individuals with HDL2 share many clinical features with individuals with HD and are clinically indistinguishable in many cases, although the average age of onset and diagnosis in HDL2 is 5 years later than HD and individual clinical features may be more prominent. HDL2 mutations contribute significantly to the HD phenotype in South Africans with African ancestry. JPH3 haplotype studies in 31 families, mainly from South Africa and North America, provide evidence for a founder mutation and support a common African origin for all HDL2 patients. Molecular testing in individuals with an HD phenotype and African ancestry should include testing routinely for JPH3 mutations.

Repeat expansion and autosomal dominant neurodegenerative disorders: consensus and controversy.

Repeat-expansion mutations cause 13 autosomal dominant neurodegenerative disorders falling into three groups. Huntingtons disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA), and spinocerebellar ataxias (SCAs) types 1, 2, 3, 7 and 17 are each caused by a CAG repeat expansion that encodes polyglutamine. Convergent lines of evidence demonstrate that neurodegeneration in these diseases is a consequence of the neurotoxic effects of abnormally long stretches of glutamines. How polyglutamine induces neurodegeneration, and why neurodegeneration occurs in only select neuronal populations, remains a matter of intense investigation. SCA6 is caused by a CAG repeat expansion in CACNA1A, a gene that encodes a subunit of the P/Q-type calcium channel. The threshold length at which the repeat causes disease is much shorter than in the other polyglutamine diseases, and neurodegeneration may arise from expansion-induced change of function in the calcium channel. Huntingtons disease-like 2 (HDL2) and SCAs 8, 10 and 12 are rare disorders in which the repeats (CAG, CTG or ATTCT) are not in protein-coding regions. Investigation into these diseases is still at an early stage, but it is now reasonable to hypothesise that the net effect of each expansion is to alter gene expression. The different pathogenic mechanisms in these three groups of diseases have important implications for the development of rational therapeutics.