RNA Toxicity and Perturbation of rRNA Processing in Spinocerebellar Ataxia Type 2.

BACKGROUND: Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by expansion of a CAG repeat in Ataxin-2 (ATXN2) gene. The mutant ATXN2 protein with a polyglutamine tract is known to be toxic and contributes to the SCA2 pathogenesis. OBJECTIVE: Here, we tested the hypothesis that the mutant ATXN2 transcript with an expanded CAG repeat (expATXN2) is also toxic and contributes to SCA2 pathogenesis. METHODS: The toxic effect of expATXN2 transcripts on SK-N-MC neuroblastoma cells and primary mouse cortical neurons was evaluated by caspase 3/7 activity and nuclear condensation assay, respectively. RNA immunoprecipitation assay was performed to identify RNA binding proteins (RBPs) that bind to expATXN2 RNA. Quantitative PCR was used to examine if ribosomal RNA (rRNA) processing is disrupted in SCA2 and Huntington's disease (HD) human brain tissue. RESULTS: expATXN2 RNA induces neuronal cell death, and aberrantly interacts with RBPs involved in RNA metabolism. One of the RBPs, transducin ß-like protein 3 (TBL3), involved in rRNA processing, binds to both expATXN2 and expanded huntingtin (expHTT) RNA in vitro. rRNA processing is disrupted in both SCA2 and HD human brain tissue. CONCLUSION: These findings provide the first evidence of a contributory role of expATXN2 transcripts in SCA2 pathogenesis, and further support the role of expHTT transcripts in HD pathogenesis. The disruption of rRNA processing, mediated by aberrant interaction of RBPs with expATXN2 and expHTT transcripts, suggest a point of convergence in the pathogeneses of repeat expansion diseases with potential therapeutic implications. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

ATXN2-AS, a gene antisense to ATXN2, is associated with spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis.

OBJECTIVE: Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by a CAG repeat expansion in the gene ataxin-2 (ATXN2). ATXN2 intermediate-length CAG expansions were identified as a risk factor for amyotrophic lateral sclerosis (ALS). The ATXN2 CAG repeat is translated into polyglutamine, and SCA2 pathogenesis has been thought to derive from ATXN2 protein containing an expanded polyglutamine tract. However, recent evidence of bidirectional transcription at multiple CAG/CTG disease loci has led us to test whether additional mechanisms of pathogenesis may contribute to SCA2. METHODS: In this work, using human postmortem tissue, various cell models, and animal models, we provide the first evidence that an antisense transcript at the SCA2 locus contributes to SCA2 pathogenesis. RESULTS: We demonstrate the expression of a transcript, containing the repeat as a CUG tract, derived from a gene (ATXN2-AS) directly antisense to ATXN2. ATXN2-AS transcripts with normal and expanded CUG repeats are expressed in human postmortem SCA2 brains, human SCA2 fibroblasts, induced SCA2 pluripotent stem cells, SCA2 neural stem cells, and lymphoblastoid lines containing an expanded ATXN2 allele associated with ALS. ATXN2-AS transcripts with a CUG repeat expansion are toxic in an SCA2 cell model and form RNA foci in SCA2 cerebellar Purkinje cells. Finally, we detected missplicing of amyloid beta precursor protein and N-methyl-D-aspartate receptor 1 in SCA2 brains, consistent with findings in other diseases characterized by RNA-mediated pathogenesis. INTERPRETATION: These results suggest that ATXN2-AS has a role in SCA2 and possibly ALS pathogenesis, and may therefore provide a novel therapeutic target for these diseases. Ann Neurol 2016;80:600-615.

Quantitative Proteomic Analysis Reveals Similarities between Huntington's Disease (HD) and Huntington's Disease-Like 2 (HDL2) Human Brains.

The pathogenesis of HD and HDL2, similar progressive neurodegenerative disorders caused by expansion mutations, remains incompletely understood. No systematic quantitative proteomics studies, assessing global changes in HD or HDL2 human brain, were reported. To address this deficit, we used a stable isotope labeling-based approach to quantify the changes in protein abundances in the cortex of 12 HD and 12 control cases and, separately, of 6 HDL2 and 6 control cases. The quality of the tissues was assessed to minimize variability due to post mortem autolysis. We applied a robust median sweep algorithm to quantify protein abundance and performed statistical inference using moderated test statistics. 1211 proteins showed statistically significant fold changes between HD and control tissues; the differences in selected proteins were verified by Western blotting. Differentially abundant proteins were enriched in cellular pathways previously implicated in HD, including Rho-mediated, actin cytoskeleton and integrin signaling, mitochondrial dysfunction, endocytosis, axonal guidance, DNA/RNA processing, and protein transport. The abundance of 717 proteins significantly differed between control and HDL2 brain. Comparative analysis of the disease-associated changes in the HD and HDL2 proteomes revealed that similar pathways were altered, suggesting the commonality of pathogenesis between the two disorders.

Phosphorodiamidate morpholino oligomers suppress mutant huntingtin expression and attenuate neurotoxicity.

Huntington's 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.

Pathogenic insights from Huntington's disease-like 2 and other Huntington's disease genocopies.

PURPOSE OF REVIEW: Huntington's disease-like 2 (HDL2) is a rare, progressive, autosomal dominant neurodegenerative disorder that genetically, clinically, and pathologically closely resembles Huntington's disease. We review HDL2 pathogenic mechanisms and examine the implications of these mechanisms for Huntington's disease and related diseases. RECENT FINDINGS: HDL2 is caused by a CTG/CAG repeat expansion in junctophilin-3. Available data from cell and animal models and human brain suggest that HDL2 is a complex disease in which transcripts and proteins expressed bidirectionally from the junctophilin-3 locus contribute to pathogenesis through both gain-and loss-of-function mechanisms. Recent advances indicate that the pathogenesis of Huntington's disease is equally complex, despite the emphasis on toxic gain-of-function properties of the mutant huntingtin protein. SUMMARY: Studies examining in parallel the genetic, clinical, neuropathological, and mechanistic similarities between Huntington's disease and HDL2 have begun to identify points of convergence between the pathogenic pathways of the two diseases. Comparisons to other diseases that are phenotypically or genetically related to Huntington's disease and HDL2 will likely reveal additional common pathways. The ultimate goal is to identify shared therapeutic targets and eventually develop therapies that may, at least in part, be effective across multiple similar rare diseases, an essential approach given the scarcity of resources for basic and translational research.

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.

A natural antisense transcript at the Huntington's disease repeat locus regulates HTT expression.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of huntingtin (HTT). Relatively little attention has been directed to the genomic features of the antisense strand at the HD locus, though the presence of a transcript from this strand has been suggested by a survey of the entire transcriptome and the existence of several EST tags. In this study, we identified huntingtin antisense (HTTAS), a natural antisense transcript at the HD repeat locus that contain the repeat tract. HTTAS is 5' capped, poly (A) tailed and contains three exons, alternatively spliced into HTTAS_v1 (exons 1 and 3) and HTTAS_v2 (exons 2 and 3). Exon 1 includes the repeat. HTTAS_v1 has a weak promoter, and is expressed at low levels in multiple tissue types and throughout the brain. Reporter assays indicate that while efficient promoter activity requires a short repeat, repeat expansion reduces promoter efficiency. Consistent with the reporter assays, levels of HTTAS_v1 are reduced in human HD frontal cortex. In cell systems, overexpression of HTTAS_v1 specifically reduces endogenous HTT transcript levels, while siRNA knockdown of HTTAS_v1 increases HTT transcript levels. Minigene constructs of the HD locus confirm the regulatory effect of HTTAS_v1 on HTT, and demonstrate that the effect is dependent on repeat length and is at least partially Dicer dependent. Together, these findings provide strong evidence for the existence of a gene antisense to HTT, with properties that include regulation of HTT expression.

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.

Brain, blood, and iron: perspectives on the roles of erythrocytes and iron in neurodegeneration.

The terms "neuroacanthocytosis" (NA) and "neurodegeneration with brain iron accumulation" (NBIA) both refer to groups of genetically heterogeneous disorders, classified together due to similarities of their phenotypic or pathological findings. Even collectively, the disorders that comprise these sets are exceedingly rare and challenging to study. The NBIA disorders are defined by their appearance on brain magnetic resonance imaging, with iron deposition in the basal ganglia. Clinical features vary, but most include a movement disorder. New causative genes are being rapidly identified; however, the mechanisms by which mutations cause iron accumulation and neurodegeneration are not well understood. NA syndromes are also characterized by a progressive movement disorder, accompanied by cognitive and psychiatric features, resulting from mutations in a number of genes whose roles are also basically unknown. An overlapping feature of the two groups, NBIA and NA, is the occurrence of acanthocytes, spiky red cells with a poorly-understood membrane dysfunction. In this review we summarise recent developments in this field, specifically insights into cellular mechanisms and from animal models. Cell membrane research may shed light upon the significance of the erythrocyte abnormality, and upon possible connections between the two sets of disorders. Shared pathophysiologic mechanisms may lead to progress in the understanding of other types of neurodegeneration.

A high-throughput screening to identify small molecules that suppress huntingtin promoter activity or activate huntingtin-antisense promoter activity.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of huntingtin (HTT). While there are currently no disease-modifying treatments for HD, recent efforts have focused on the development of nucleotide-based therapeutics to lower HTT expression. As an alternative to siRNA or oligonucleotide methods, we hypothesized that suppression of HTT expression might be accomplished by small molecules that either (1) directly decrease HTT expression by suppressing HTT promoter activity or (2) indirectly decrease HTT expression by increasing the promoter activity of HTT-AS, the gene antisense to HTT that appears to inhibit expression of HTT. We developed and employed a high-throughput screen for modifiers of HTT and HTT-AS promoter activity using luminescent reporter HEK293 cells; of the 52,041 compounds tested, we identified 898 replicable hits. We used a rigorous stepwise approach to assess compound toxicity and the capacity of the compounds to specifically lower huntingtin protein in 5 different cell lines, including HEK293 cells, HD lymphoblastoid cells, mouse primary neurons, HD iPSCs differentiated into cortical-like neurons, and HD hESCs. We found no compounds which were able to lower huntingtin without lowering cell viability in all assays, though the potential efficacy of a few compounds at non-toxic doses could not be excluded. Our results suggest that more specific targets may facilitate a small molecule approach to HTT suppression.

Exploring Novel Biologically-Relevant Chemical Space Through Artificial Intelligence: The NCATS ASPIRE Program.

In recent years, artificial intelligence (AI)/machine learning (ML; a subset of AI) have become increasingly important to the biomedical research community. These technologies, coupled to big data and cheminformatics, have tremendous potential to improve the design of novel therapeutics and to provide safe and effective drugs to patients. A National Center for Advancing Translational Sciences (NCATS) program called A Specialized Platform for Innovative Research Exploration (ASPIRE) leverages advances in AI/ML, automated synthetic chemistry, and high-throughput biology, and seeks to enable translation and drug development by catalyzing exploration of biologically active chemical space. Here we discuss the opportunities and challenges surrounding the application of AI/ML to the exploration of novel biologically relevant chemical space as part of ASPIRE.

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

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.

Absence of Acanthocytosis in Huntington's Disease-like 2: A Prospective Comparison with Huntington's Disease.

Background: Huntington's Disease-like 2 (HDL2) is classified as a neuroacanthocytosis; however, this remains unverified. We aim to determine if acanthocytes are present in HDL2 and whether acanthocytes can differentiate HDL2 from Huntington's disease (HD). Methods: We prospectively compared 13 HD and 12 HDL2 cases against 21 unaffected controls in Johannesburg. Blood smears were prepared using international standards and reviewed by at least two blinded reviewers. An acanthocytosis rate of greater than 1.2% in the dry smear or greater than 3.7% in the wet smear was designated a priori as the threshold for clinical significance based on previously established standards. Flow cytometry was performed on all but four of the cases. Red cell membrane protein analysis was performed on all participants. Results: There were 12 HDL2, 13 HD, and 21 controls enrolled. None of the HD or HDL2 participants had defined acanthocytosis or other morphological abnormalities. None of the HD or HDL2 cases had evidence of an abnormal band 3. Discussion: Acanthocytosis was not identified in either HDL2 or HD in our patient population. Our results, based on the first prospective study of acanthocytes in HDL2 or HD, suggest that screening for acanthocytes will not help establish the diagnosis of HD or HDL2, nor differentiate between the two disorders and raises the question if HDL2 should be placed within the neuroacanthocytosis syndromes.

Eighth International Chorea-Acanthocytosis Symposium: Summary of Workshop Discussion and Action Points.

Chorea-Acanthocytosis (ChAc) is a rare hereditary neurological disorder characterized by abnormal movements, red blood cell pathology, and progressive neurodegeneration. Little is understood of the pathogenesis of ChAc and related disorders (collectively Neuroacanthocytosis). The Eighth International Chorea-Acanthocytosis Symposium was held in May 2016 in Ann Arbor, MI, USA, and focused on molecular mechanisms driving ChAc pathophysiology. Accompanying the meeting, members of the neuroacanthocytosis research community and other invited scientists met in a workshop to discuss the current understanding and next steps needed to better understand ChAc pathogenesis. These discussions identified several broad and critical needs for advancing ChAc research and patient care, and led to the definition of 18 specific action points related to functional and molecular studies, animal models, and clinical research. These action points, described below, represent tractable research goals to pursue for the next several years.

Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila.

Polyglutamine (polyQ) diseases represent a group of progressive neurodegenerative disorders that are caused by abnormal expansion of CAG triplet nucleotides in disease genes. Recent evidence indicates that not only mutant polyQ proteins, but also their corresponding mutant RNAs, contribute to the pathogenesis of polyQ diseases. Here, we describe the identification of a 13-amino-acid peptide, P3, which binds directly and preferentially to long-CAG RNA within the pathogenic range. When administered to cell and Drosophila disease models, as well as to patient-derived fibroblasts, P3 inhibited expanded-CAG-RNA-induced nucleolar stress and suppressed neurotoxicity. We further examined the combined therapeutic effect of P3 and polyQ-binding peptide 1 (QBP1), a well-characterized polyQ protein toxicity inhibitor, on neurodegeneration. When P3 and QBP1 were co-administered to disease models, both RNA and protein toxicities were effectively mitigated, resulting in a notable improvement of neurotoxicity suppression compared with the P3 and QBP1 single-treatment controls. Our findings indicate that targeting toxic RNAs and/or simultaneous targeting of toxic RNAs and their corresponding proteins could open up a new therapeutic strategy for treating polyQ degeneration.

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.

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.

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

Repeat-expansion mutations cause 13 autosomal dominant neurodegenerative disorders falling into three groups. Huntington's 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. Huntington's 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.