Huntingtin HTT1a is generated in a CAG repeat-length-dependent manner in human tissues.

BACKGROUND: The disease-causing mutation in Huntington disease (HD) is a CAG trinucleotide expansion in the huntingtin (HTT) gene. The mutated CAG tract results in the production of a small RNA, HTT1a, coding for only exon 1 of HTT. HTT1a is generated by a block in the splicing reaction of HTT exon 1 to exon 2 followed by cleavage in intron 1 and polyadenylation. Translation of HTT1a leads to the expression of the highly toxic HTT exon 1 protein fragment. We have previously shown that the levels of HTT1a expression in mouse models of HD is dependent on the CAG repeat length. However, these data are lacking for human tissues. METHODS: To answer this question, we developed highly sensitive digital PCR assays to determine HTT1a levels in human samples. These assays allow the absolute quantification of transcript numbers and thus also facilitate the comparison of HTT1a levels between tissues, cell types and across different studies. Furthermore, we measured CAG repeat sizes for every sample used in the study. Finally, we analysed our data with ANOVA and linear modelling to determine the correlation of HTT1a expression levels with CAG repeat sizes. RESULTS: In summary, we show that HTT1a is indeed expressed in a CAG repeat-length-dependent manner in human post mortem brain tissues as well as in several peripheral cell types. In particular, PBMCs show a statistically significant positive correlation of HTT1a expression with CAG repeat length, and elevated HTT1a expression levels even in the adult-onset CAG repeat range. CONCLUSIONS: Our results show that HTT1a expression occurs throughout a wide range of tissues and likely with all CAG lengths. Our data from peripheral sample sources demonstrate that HTT1a is indeed generated throughout the body in a CAG repeat-length-dependent manner. Therefore, the levels of HTT1a might be a sensitive marker of disease state and/or progression and should be monitored over time, especially in clinical trials targeting HTT expression.

Variation of FMRP Expression in Peripheral Blood Mononuclear Cells from Individuals with Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability and autism spectrum disorder. The syndrome is often caused by greatly reduced or absent protein expression from the fragile X messenger ribonucleoprotein 1 (FMR1) gene due to expansion of a 5'-non-coding trinucleotide (CGG) element beyond 200 repeats (full mutation). To better understand the complex relationships among FMR1 allelotype, methylation status, mRNA expression, and FMR1 protein (FMRP) levels, FMRP was quantified in peripheral blood mononuclear cells for a large cohort of FXS (n = 154) and control (n = 139) individuals using time-resolved fluorescence resonance energy transfer. Considerable size and methylation mosaicism were observed among individuals with FXS, with FMRP detected only in the presence of such mosaicism. No sample with a minimum allele size greater than 273 CGG repeats had significant levels of FMRP. Additionally, an association was observed between FMR1 mRNA and FMRP levels in FXS samples, predominantly driven by those with the lowest FMRP values. This study underscores the complexity of FMR1 allelotypes and FMRP expression and prompts a reevaluation of FXS therapies aimed at reactivating large full mutation alleles that are likely not capable of producing sufficient FMRP to improve cognitive function.

Generation of three myotonic dystrophy type 1 patient iPSC lines (CBRCULi018-A, CBRCULi019-A, CBRCULi020-A) derived from lymphoblastoid cell lines for disease modelling and therapeutic research.

Myotonic dystrophy type 1 (DM1) is the most prevalent adult-onset muscular dystrophy affecting 1 in 8,000 individuals. It is characterized by multisystemic symptoms, primarily myopathy. The root cause of DM1 is a heterozygous CTG triplet expansion beyond the normal size threshold in the non-coding region of the DM1 protein kinase gene (DMPK). In our study, we generated and characterized three distinct DM1 induced pluripotent stem cell (iPSC) lines with CTG repeat expansions ranging from 900 to 2000 in the DMPK gene. These iPSC lines maintained normal karyotypes, exhibited distinctive colony morphology, robustly expressed pluripotency markers, differentiated into the three primary germ layers, and lacked residual viral vectors.

Mitochondrial Dysfunction Causes Cell Death in Patients Affected by Fragile-X-Associated Disorders.

Mitochondria are involved in multiple aspects of neurodevelopmental processes and play a major role in the pathogenetic mechanisms leading to neuro-degenerative diseases. Fragile-X-related disorders (FXDs) are genetic conditions that occur due to the dynamic expansion of CGG repeats of the FMR1 gene encoding for the RNA-binding protein FMRP, particularly expressed in the brain. This gene expansion can lead to premutation (PM, 56-200 CGGs), full mutation (FM, >200 CGGs), or unmethylated FM (UFM), resulting in neurodegeneration, neurodevelopmental disorders, or no apparent intellectual disability, respectively. To investigate the mitochondrial mechanisms that are involved in the FXD patients, we analyzed mitochondrial morphology and bioenergetics in fibroblasts derived from patients. Donut-shaped mitochondrial morphology and excessive synthesis of critical mitochondrial proteins were detected in FM, PM, and UFM cells. Analysis of mitochondrial oxidative phosphorylation in situ reveals lower respiration in PM fibroblasts. Importantly, mitochondrial permeability transition-dependent apoptosis is sensitized to reactive oxygen species in FM, PM, and UFM models. This study elucidated the mitochondrial mechanisms that are involved in the FXD phenotypes, and indicated altered mitochondrial function and morphology. Importantly, a sensitization to permeability transition and apoptosis was revealed in FXD cells. Overall, our data suggest that mitochondria are novel drug targets to relieve the FXD symptoms.

The genetic landscape and phenotypic spectrum of GAA-FGF14 ataxia in China: a large cohort study.

BACKGROUND: An intronic GAA repeat expansion in FGF14 was recently identified as a cause of GAA-FGF14 ataxia. We aimed to characterise the frequency and phenotypic profile of GAA-FGF14 ataxia in a large Chinese ataxia cohort. METHODS: A total of 1216 patients that included 399 typical late-onset cerebellar ataxia (LOCA), 290 early-onset cerebellar ataxia (EOCA), and 527 multiple system atrophy with predominant cerebellar ataxia (MSA-c) were enrolled. Long-range and repeat-primed PCR were performed to screen for GAA expansions in FGF14. Targeted long-read and whole-genome sequencing were performed to determine repeat size and sequence configuration. A multi-modal study including clinical assessment, MRI, and neurofilament light chain was conducted for disease assessment. FINDINGS: 17 GAA-FGF14 positive patients with a (GAA)≥250 expansion (12 patients with a GAA-pure expansion, five patients with a (GAA)≥250-[(GAA)n (GCA)m]z expansion) and two possible patients with biallelic (GAA)202/222 alleles were identified. The clinical phenotypes of the 19 positive and possible positive cases covered LOCA phenotype, EOCA phenotype and MSA-c phenotype. Five of six patients with EOCA phenotype were found to have another genetic disorder. The NfL levels of patients with EOCA and MSA-c phenotypes were significantly higher than patients with LOCA phenotype and age-matched controls (p < 0.001). NfL levels of pre-ataxic GAA-FGF14 positive individuals were lower than pre-ataxic SCA3 (p < 0.001) and similar to controls. INTERPRETATION: The frequency of GAA-FGF14 expansion in a large Chinese LOCA cohort was low (1.3%). Biallelic (GAA)202/222 alleles and co-occurrence with other acquired or hereditary diseases may contribute to phenotypic variation and different progression. FUNDING: This study was funded by the National Key R&D Program of China (2021YFA0805200 to H.J.), the National Natural Science Foundation of China (81974176 and 82171254 to H.J.; 82371272 to Z.C.; 82301628 to L.W.; 82301438 to Z.L.; 82201411 to L.H.), the Innovation Research Group Project of Natural Science Foundation of Hunan Province (2020JJ1008 to H.J.), the Key Research and Development Program of Hunan Province (2020SK2064 to H.J.), the Innovative Research and Development Program of Development and Reform Commission of Hunan Province to H.J., the Natural Science Foundation of Hunan Province (2024JJ3050 to H.J.; 2022JJ20094 and 2021JJ40974 to Z.C.; 2022JJ40783 to L.H.; 2022JJ40703 to Z.L.), the Project Program of National Clinical Research Center for Geriatric Disorders (Xiangya Hospital, 2020LNJJ12 to H.J.), the Central South University Research Programme of Advanced Interdisciplinary Study (2023QYJC010 to H.J.) and the Science and Technology Innovation Program of Hunan Province (2022RC1027 to Z.C.). D.P. holds a Fellowship award from the Canadian Institutes of Health Research (CIHR).

Generation of two human induced pluripotent stem cell lines, IGIBi012-A and IGIBi013-A from Friedreich's ataxia (FRDA) patients with homozygous GAA repeat expansion in FXN gene.

Friedreich's ataxia is a neurodegenerative disorder caused by the hyper expansion of (GAA-TTC)n triplet repeats in the first intron of the FXN gene. Here, we generated iPSC lines from two individuals with FRDA, both of whom have homozygous GAA repeat expansion in the first intron of FXN gene. Both iPSC lines demonstrated characteristics of pluripotency, including expression of pluripotency markers, stable karyotypes and ability to develop into all three germ layers, and presence of GAA repeat expansion with reduced FXN mRNA expression. These iPSC lines will serve as invaluable tools for investigating the pathophysiology and phenotypes of FRDA.

BREACHing new grounds in fragile X syndrome: Trinucleotide expansion linked to genome-wide heterochromatin domains and genome misfolding.

In a recent study in Cell, Malachowski et al.1 show that the trinucleotide expansion in the FMR1 gene underlying fragile X syndrome triggers formation of large heterochromatin domains across the genome, resulting in the repression of synaptic genes housed within these domains.

Attenuated huntingtin gene CAG nucleotide repeat size in individuals with Lynch syndrome.

DNA mismatch repair (MMR) is thought to contribute to the onset and progression of Huntington disease (HD) by promoting somatic expansion of the pathogenic CAG nucleotide repeat in the huntingtin gene (HTT). Here we have studied constitutional HTT CAG repeat size in two cohorts of individuals with Lynch syndrome (LS) carrying heterozygous loss-of-function variants in the MMR genes MLH1 (n = 12/60; Lund cohort/Bochum cohort, respectively), MSH2 (n = 15/88), MSH6 (n = 21/23), and controls (n = 19/559). The sum of CAG repeats for both HTT alleles in each individual was calculated due to unknown segregation with the LS allele. In the larger Bochum cohort, the sum of CAG repeats was lower in the MLH1 subgroup compared to controls (MLH1 35.40 CAG repeats ± 3.6 vs. controls 36.89 CAG repeats ± 4.5; p = 0.014). All LS genetic subgroups in the Bochum cohort displayed lower frequencies of unstable HTT intermediate alleles and lower HTT somatic CAG repeat expansion index values compared to controls. Collectively, our results indicate that MMR gene haploinsufficiency could have a restraining impact on constitutional HTT CAG repeat size and support the notion that the MMR pathway is a driver of nucleotide repeat expansion diseases.

High-Resolution NMR Structures of Intrastrand Hairpins Formed by CTG Trinucleotide Repeats.

The CAG and CTG trinucleotide repeat expansions cause more than 10 human neurodegenerative diseases. Intrastrand hairpins formed by trinucleotide repeats contribute to repeat expansions, establishing them as potential drug targets. High-resolution structural determination of CAG and CTG hairpins poses as a long-standing goal to aid drug development, yet it has not been realized due to the intrinsic conformational flexibility of repetitive sequences. We herein investigate the solution structures of CTG hairpins using nuclear magnetic resonance (NMR) spectroscopy and found that four CTG repeats with a clamping G-C base pair was able to form a stable hairpin structure. We determine the first solution NMR structure of dG(CTG)4C hairpin and decipher a type I folding geometry of the TGCT tetraloop, wherein the two thymine residues form a T·T loop-closing base pair and the first three loop residues continuously stack. We further reveal that the CTG hairpin can be bound and stabilized by a small-molecule ligand, and the binding interferes with replication of a DNA template containing CTG repeats. Our determined high-resolution structures lay an important foundation for studying molecular interactions between native CTG hairpins and ligands, and benefit drug development for trinucleotide repeat expansion diseases.

Somatic CAG Repeat Stability in a Transgenic Sheep Model of Huntington's Disease.

Somatic instability of the huntingtin (HTT) CAG repeat mutation modifies age-at-onset of Huntington's disease (HD). Understanding the mechanism and pathogenic consequences of instability may reveal therapeutic targets. Using small-pool PCR we analyzed CAG instability in the OVT73 sheep model which expresses a full-length human cDNA HTT transgene. Analyses of five- and ten-year old sheep revealed the transgene (CAG)69 repeat was remarkably stable in liver, striatum, and other brain tissues. As OVT73 sheep at ten years old have minimal cell death and behavioral changes, our findings support instability of the HTT expanded-CAG repeat as being required for the progression of HD.

Combinatorial effects of ion channel mis-splicing as a cause of myopathy in myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder caused by an unstable expanded CTG repeat located in the 3'-UTR of the DM1 protein kinase (DMPK) gene. The pathogenic mechanism results in misregulated alternative splicing of hundreds of genes, creating the dilemma of establishing which genes contribute to the mechanism of DM1 skeletal muscle pathology. In this issue of the JCI, Cisco and colleagues systematically tested the combinatorial effects of DM1-relevant mis-splicing patterns in vivo and identified the synergistic effects of mis-spliced calcium and chloride channels as a major contributor to DM1 skeletal muscle impairment. The authors further demonstrated the therapeutic potential for calcium channel modulation to block the synergistic effects and rescue myopathy.

Fragile X syndrome in the largest world clustering. I. Genetic epidemiology and founder effect outline.

The FMR1 5' regulation gene region harbors a CGG trinucleotide repeat expansion (CGG-TRE) that causes Fragile X syndrome (FXS) when it expands to more than 200 repetitions. Ricaurte is a small village in southwestern Colombia, with an FXS prevalence of 1 in 38 men and 1 in 100 women (~100 times higher than the worldwide reported prevalence), defining Ricaurte as the largest FXS cluster in the world. In the present study, using next-generation sequencing of whole exome capture, we genotype 55 individuals from Ricaurte (49 with either full mutation or with premutation), four individuals from neighboring villages (with either the full mutation or with the premutation), and one unaffected woman, native of Ricaurte, who did not belong to any of the affected families. With advanced clustering and haplotype reconstruction, we modeled a common haplotype of 33 SNPs spanning 83,567,899 bp and harboring the FMR1 gene. This reconstructed haplotype was found in all the men from Ricaurte who carried the expansion, demonstrating that the genetic conglomerate of FXS in this population is due to a founder effect. The definition of this founder effect and its population outlining will allow a better prediction, follow-up, precise and personalized characterization of epidemiological parameters, better knowledge of the disease's natural history, and confident improvement of the clinical attention, life quality, and health interventions for this community.

The FGF14 GAA repeat expansion in Greek patients with late-onset cerebellar ataxia and an overview of the SCA27B phenotype across populations.

A pathogenic GAA repeat expansion in the first intron of the fibroblast growth factor 14 gene (FGF14) has been recently identified as the cause of spinocerebellar ataxia 27B (SCA27B). We herein screened 160 Greek index cases with late-onset cerebellar ataxia (LOCA) for FGF14 repeat expansions using a combination of long-range PCR and bidirectional repeat-primed PCRs. We identified 19 index cases (12%) carrying a pathogenic FGF14 GAA expansion, a diagnostic yield higher than that of previously screened repeat-expansion ataxias in Greek LOCA patients. The age at onset of SCA27B patients was 60.5 ± 12.3 years (range, 34-80). Episodic onset (37%), downbeat nystagmus (32%) and vertigo (26%) were significantly more frequent in FGF14 expansion-positive cases compared to expansion-negative cases. Beyond typical cerebellar signs, SCA27B patients often displayed hyperreflexia (47%) and reduced vibration sense in the lower extremities (42%). The frequency and phenotypic profile of SCA27B in Greek patients was similar to most other previously studied populations. We conclude that FGF14 GAA repeat expansions are the commonest known genetic cause of LOCA in the Greek population and recommend prioritizing testing for FGF14 expansions in the diagnostic algorithm of patients with LOCA.

RNA structure promotes liquid-to-solid phase transition of short RNAs in neuronal dysfunction.

In nucleotide expansion disorders, RNA foci are reportedly associated with neurodegenerative disease pathogeneses. Characteristically, these RNAs exhibit long poly-RNA repeats, such as 47 × CAG, 47 × CUG, or 29 × GGGGCC, usually becoming abnormal pathological aggregations above a critical number of nucleotide repeats. However, it remains unclear whether short, predominantly cellular RNA molecules can cause phase transitions to induce RNA foci. Herein, we demonstrated that short RNAs even with only two repeats can aggregate into a solid-like state via special RNA G-quadruplex structures. In human cells, these solid RNA foci could not dissolve even when using agents that disrupt RNA gelation. The aggregation of shorter RNAs can be clearly observed in vivo. Furthermore, we found that RNA foci induce colocalization of the RNA-binding protein Sam68, a protein commonly found in patients with fragile X-associated tremor/ataxia syndrome, suppressing cell clonogenicity and eventually causing cell death. Our results suggest that short RNA gelation promoted by specific RNA structures contribute to the neurological diseases, which disturb functional cellular processes.

CAG repeat expansions create splicing acceptor sites and produce aberrant repeat-containing RNAs.

Expansions of CAG trinucleotide repeats cause several rare neurodegenerative diseases. The disease-causing repeats are translated in multiple reading frames and without an identifiable initiation codon. The molecular mechanism of this repeat-associated non-AUG (RAN) translation is not known. We find that expanded CAG repeats create new splice acceptor sites. Splicing of proximal donors to the repeats produces unexpected repeat-containing transcripts. Upon splicing, depending on the sequences surrounding the donor, CAG repeats may become embedded in AUG-initiated open reading frames. Canonical AUG-initiated translation of these aberrant RNAs may account for proteins that have been attributed to RAN translation. Disruption of the relevant splice donors or the in-frame AUG initiation codons is sufficient to abrogate RAN translation. Our findings provide a molecular explanation for the abnormal translation products observed in CAG trinucleotide repeat expansion disorders and add to the repertoire of mechanisms by which repeat expansion mutations disrupt cellular functions.

Viral-based animal models in polyglutamine disorders.

Polyglutamine disorders are a complex group of incurable neurodegenerative disorders caused by an abnormal expansion in the trinucleotide cytosine-adenine-guanine tract of the affected gene. To better understand these disorders, our dependence on animal models persists, primarily relying on transgenic models. In an effort to complement and deepen our knowledge, researchers have also developed animal models of polyglutamine disorders employing viral vectors. Viral vectors have been extensively used to deliver genes to the brain, not only for therapeutic purposes but also for the development of animal models, given their remarkable flexibility. In a time- and cost-effective manner, it is possible to use different transgenes, at varying doses, in diverse targeted tissues, at different ages, and in different species, to recreate polyglutamine pathology. This paper aims to showcase the utility of viral vectors in disease modelling, share essential considerations for developing animal models with viral vectors, and provide a comprehensive review of existing viral-based animal models for polyglutamine disorders.

Phase transition and lysosomal degradation of expanded CAG repeat RNA suppress global protein synthesis.

Phase transitions (PT) of biomolecules are heavily involved in neurodegenerative disorders. Almost all previous studies were focusing on the PT of misfolded proteins whereas RNA molecules containing expanded repeats such as the CAG repeats are also able to undergo PT in vitro, a process called RNA gelation. Meanwhile, the expanded CAG repeat (eCAGr) RNA forms condensates that are largely observed only in the nuclei and exhibit liquid-like properties without obvious gelation. Thus, whether eCAGr RNA gelation occurs in cells and what function it is involved in remained elusive. We recently discovered that eCAGr RNA forms solid-like RNA gels in the cytoplasm, but they are rapidly cleared by the lysosomes via an autophagy-independent but LAMP2C-depdent pathway, making their presence in the cytoplasm difficult to be observed. We further revealed that these RNA gels sequester EEF2 in the cells and thus suppress global protein synthesis. In vivo expression of eCAGr RNA alone without detectable protein expression in the mouse model led to neurodegeneration-relevant electrophysiological and behavioral phenotypes, demonstrating its possible pathogenic roles.

GGC expansions in NOTCH2NLC contribute to Parkinson disease and dopaminergic neuron degeneration.

BACKGROUND AND PURPOSE: The role of GGC repeat expansions within NOTCH2NLC in Parkinson's disease (PD) and the substantia nigra (SN) dopaminergic neuron remains unclear. Here, we profile the NOTCH2NLC GGC repeat expansions in a large cohort of patients with PD. We also investigate the role of GGC repeat expansions within NOTCH2NLC in the dopaminergic neurodegeneration of SN. METHODS: A total of 2,522 patients diagnosed with PD and 1,085 health controls were analyzed for the repeat expansions of NOTCH2NLC by repeat-primed PCR and GC-rich PCR assay. Furthermore, the effects of GGC repeat expansions in NOTCH2NLC on dopaminergic neurons were investigated by using recombinant adeno-associated virus (AAV)-mediated overexpression of NOTCH2NLC with 98 GGC repeats in the SN of mice by stereotactic injection. RESULTS: Four PD pedigrees (4/333, 1.2%) and three sporadic PD patients (3/2189, 0.14%) were identified with pathogenic GGC repeat expansions (larger than 60 GGC repeats) in the NOTCH2NLC gene, while eight PD patients and one healthy control were identified with intermediate GGC repeat expansions ranging from 41 to 60 repeats. No significant difference was observed in the distribution of intermediate NOTCH2NLC GGC repeat expansions between PD cases and controls (Fisher's exact test p-value = 0.29). Skin biopsy showed P62-positive intranuclear NOTCH2NLC-polyGlycine (polyG) inclusions in the skin nerve fibers of patient. Expanded GGC repeats in NOTCH2NLC produced widespread intranuclear and perinuclear polyG inclusions, which led to a severe loss of dopaminergic neurons in the SN. Consistently, polyG inclusions were presented in the SN of EIIa-NOTCH2NLC-(GGC)98 transgenic mice and also led to dopaminergic neuron loss in the SN. CONCLUSIONS: Overall, our findings provide strong evidence that GGC repeat expansions within NOTCH2NLC contribute to the pathogenesis of PD and cause degeneration of nigral dopaminergic neurons.

Exonic trinucleotide repeat expansions in ZFHX3 cause spinocerebellar ataxia type 4: A poly-glycine disease.

Autosomal-dominant ataxia with sensory and autonomic neuropathy is a highly specific combined phenotype that we described in two Swedish kindreds in 2014; its genetic cause had remained unknown. Here, we report the discovery of exonic GGC trinucleotide repeat expansions, encoding poly-glycine, in zinc finger homeobox 3 (ZFHX3) in these families. The expansions were identified in whole-genome datasets within genomic segments that all affected family members shared. Non-expanded alleles carried one or more interruptions within the repeat. We also found ZFHX3 repeat expansions in three additional families, all from the region of Skåne in southern Sweden. Individuals with expanded repeats developed balance and gait disturbances at 15 to 60 years of age and had sensory neuropathy and slow saccades. Anticipation was observed in all families and correlated with different repeat lengths determined through long-read sequencing in two family members. The most severely affected individuals had marked autonomic dysfunction, with severe orthostatism as the most disabling clinical feature. Neuropathology revealed p62-positive intracytoplasmic and intranuclear inclusions in neurons of the central and enteric nervous system, as well as alpha-synuclein positivity. ZFHX3 is located within the 16q22 locus, to which spinocerebellar ataxia type 4 (SCA4) repeatedly had been mapped; the clinical phenotype in our families corresponded well with the unique phenotype described in SCA4, and the original SCA4 kindred originated from Sweden. ZFHX3 has known functions in neuronal development and differentiation n both the central and peripheral nervous system. Our findings demonstrate that SCA4 is caused by repeat expansions in ZFHX3.