Deciphering novel TCF4-driven mechanisms underlying a common triplet repeat expansion-mediated disease.

Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.

Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease.

The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human ex vivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability.

Generation of Rhesus Macaque Embryos with Expanded CAG Trinucleotide Repeats in the Huntingtin Gene.

Huntington's disease (HD) arises from expanded CAG repeats in exon 1 of the Huntingtin (HTT) gene. The resultant misfolded HTT protein accumulates within neuronal cells, negatively impacting their function and survival. Ultimately, HTT accumulation results in cell death, causing the development of HD. A nonhuman primate (NHP) HD model would provide important insight into disease development and the generation of novel therapies due to their genetic and physiological similarity to humans. For this purpose, we tested CRISPR/Cas9 and a single-stranded DNA (ssDNA) containing expanded CAG repeats in introducing an expanded CAG repeat into the HTT gene in rhesus macaque embryos. Analyses were conducted on arrested embryos and trophectoderm (TE) cells biopsied from blastocysts to assess the insertion of the ssDNA into the HTT gene. Genotyping results demonstrated that 15% of the embryos carried an expanded CAG repeat. The integration of an expanded CAG repeat region was successfully identified in five blastocysts, which were cryopreserved for NHP HD animal production. Some off-target events were observed in biopsies from the cryopreserved blastocysts. NHP embryos were successfully produced, which will help to establish an NHP HD model and, ultimately, may serve as a vital tool for better understanding HD's pathology and developing novel treatments.

CAG repeat mosaicism is gene specific in spinocerebellar ataxias.

Expanded CAG repeats in coding regions of different genes are the most common cause of dominantly inherited spinocerebellar ataxias (SCAs). These repeats are unstable through the germline, and larger repeats lead to earlier onset. We measured somatic expansion in blood samples collected from 30 SCA1, 50 SCA2, 74 SCA3, and 30 SCA7 individuals over a mean interval of 8.5 years, along with postmortem tissues and fetal tissues from SCA1, SCA3, and SCA7 individuals to examine somatic expansion at different stages of life. We showed that somatic mosaicism in the blood increases over time. Expansion levels are significantly different among SCAs and correlate with CAG repeat lengths. The level of expansion is greater in individuals with SCA7 who manifest disease compared to that of those who do not yet display symptoms. Brain tissues from SCA individuals have larger expansions compared to the blood. The cerebellum has the lowest mosaicism among the studied brain regions, along with a high expression of ATXNs and DNA repair genes. This was the opposite in cortices, with the highest mosaicism and lower expression of ATXNs and DNA repair genes. Fetal cortices did not show repeat instability. This study shows that CAG repeats are increasingly unstable during life in the blood and the brain of SCA individuals, with gene- and tissue-specific patterns.

Precise editing of pathogenic nucleotide repeat expansions in iPSCs using paired prime editor.

Nucleotide repeat expansion disorders, a group of genetic diseases characterized by the expansion of specific DNA sequences, pose significant challenges to treatment and therapy development. Here, we present a precise and programmable method called prime editor-mediated correction of nucleotide repeat expansion (PE-CORE) for correcting pathogenic nucleotide repeat expansion. PE-CORE leverages a prime editor and paired pegRNAs to achieve targeted correction of repeat sequences. We demonstrate the effectiveness of PE-CORE in HEK293T cells and patient-derived induced pluripotent stem cells (iPSCs). Specifically, we focus on spinal and bulbar muscular atrophy and spinocerebellar ataxia type, two diseases associated with nucleotide repeat expansion. Our results demonstrate the successful correction of pathogenic expansions in iPSCs and subsequent differentiation into motor neurons. Specifically, we detect distinct downshifts in the size of both the mRNA and protein, confirming the functional correction of the iPSC-derived motor neurons. These findings highlight PE-CORE as a precision tool for addressing the intricate challenges of nucleotide repeat expansion disorders, paving the way for targeted therapies and potential clinical applications.

AAV-Mediated CAG-Targeting Selectively Reduces Polyglutamine-Expanded Protein and Attenuates Disease Phenotypes in a Spinocerebellar Ataxia Mouse Model.

Polyglutamine (polyQ)-encoding CAG repeat expansions represent a common disease-causing mutation responsible for several dominant spinocerebellar ataxias (SCAs). PolyQ-expanded SCA proteins are toxic for cerebellar neurons, with Purkinje cells (PCs) being the most vulnerable. RNA interference (RNAi) reagents targeting transcripts with expanded CAG reduce the level of various mutant SCA proteins in an allele-selective manner in vitro and represent promising universal tools for treating multiple CAG/polyQ SCAs. However, it remains unclear whether the therapeutic targeting of CAG expansion can be achieved in vivo and if it can ameliorate cerebellar functions. Here, using a mouse model of SCA7 expressing a mutant Atxn7 allele with 140 CAGs, we examined the efficacy of short hairpin RNAs (shRNAs) targeting CAG repeats expressed from PHP.eB adeno-associated virus vectors (AAVs), which were introduced into the brain via intravascular injection. We demonstrated that shRNAs carrying various mismatches with the CAG target sequence reduced the level of polyQ-expanded ATXN7 in the cerebellum, albeit with varying degrees of allele selectivity and safety profile. An shRNA named A4 potently reduced the level of polyQ-expanded ATXN7, with no effect on normal ATXN7 levels and no adverse side effects. Furthermore, A4 shRNA treatment improved a range of motor and behavioral parameters 23 weeks after AAV injection and attenuated the disease burden of PCs by preventing the downregulation of several PC-type-specific genes. Our results show the feasibility of the selective targeting of CAG expansion in the cerebellum using a blood-brain barrier-permeable vector to attenuate the disease phenotype in an SCA mouse model. Our study represents a significant advancement in developing CAG-targeting strategies as a potential therapy for SCA7 and possibly other CAG/polyQ SCAs.

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.

FMR1 Protein Expression Correlates with Intelligence Quotient in Both Peripheral Blood Mononuclear Cells and Fibroblasts from Individuals with an FMR1 Mutation.

Fragile X syndrome (FXS) is the most common heritable form of intellectual disability and is caused by CGG repeat expansions exceeding 200 (full mutation). Such expansions lead to hypermethylation and transcriptional silencing of the fragile X messenger ribonucleoprotein 1 (FMR1) gene. As a consequence, little or no FMR1 protein (FMRP) is produced; absence of the protein, which normally is responsible for neuronal development and maintenance, causes the syndrome. Previous studies have demonstrated the causal relationship between FMRP levels and cognitive abilities in peripheral blood mononuclear cells (PBMCs) and dermal fibroblast cell lines of patients with FXS. However, it is arguable whether PBMCs or fibroblasts would be the preferred surrogate for measuring molecular markers, particularly FMRP, to represent the cognitive impairment, a core symptom of FXS. To address this concern, CGG repeats, methylation status, FMR1 mRNA, and FMRP levels were measured in both PBMCs and fibroblasts derived from 66 individuals. The findings indicated a strong association between FMR1 mRNA expression levels and CGG repeat numbers in PBMCs of premutation males after correcting for methylation status. Moreover, FMRP expression levels from both PBMCs and fibroblasts of male participants with a hypermethylated full mutation and with mosaicism demonstrated significant association between the intelligence quotient levels and FMRP levels, suggesting that PBMCs may be preferable for FXS clinical studies, because of their greater accessibility.

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.

A CAG repeat threshold for therapeutics targeting somatic instability in Huntington's disease.

The Huntington's disease mutation is a CAG repeat expansion in the huntingtin gene that results in an expanded polyglutamine tract in the huntingtin protein. The CAG repeat is unstable and expansions of hundreds of CAGs have been detected in Huntington's disease post-mortem brains. The age of disease onset can be predicted partially from the length of the CAG repeat as measured in blood. Onset age is also determined by genetic modifiers, which in six cases involve variation in DNA mismatch repair pathways genes. Knocking-out specific mismatch repair genes in mouse models of Huntington's disease prevents somatic CAG repeat expansion. Taken together, these results have led to the hypothesis that somatic CAG repeat expansion in Huntington's disease brains is required for pathogenesis. Therefore, the pathogenic repeat threshold in brain is longer than (CAG)40, as measured in blood, and is currently unknown. The mismatch repair gene MSH3 has become a major focus for therapeutic development, as unlike other mismatch repair genes, nullizygosity for MSH3 does not cause malignancies associated with mismatch repair deficiency. Potential treatments targeting MSH3 currently under development include gene therapy, biologics and small molecules, which will be assessed for efficacy in mouse models of Huntington's disease. The zQ175 knock-in model carries a mutation of approximately (CAG)185 and develops early molecular and pathological phenotypes that have been extensively characterized. Therefore, we crossed the mutant huntingtin allele onto heterozygous and homozygous Msh3 knockout backgrounds to determine the maximum benefit of targeting Msh3 in this model. Ablation of Msh3 prevented somatic expansion throughout the brain and periphery, and reduction of Msh3 by 50% decreased the rate of expansion. This had no effect on the deposition of huntingtin aggregation in the nuclei of striatal neurons, nor on the dysregulated striatal transcriptional profile. This contrasts with ablating Msh3 in knock-in models with shorter CAG repeat expansions. Therefore, further expansion of a (CAG)185 repeat in striatal neurons does not accelerate the onset of molecular and neuropathological phenotypes. It is striking that highly expanded CAG repeats of a similar size in humans cause disease onset before 2 years of age, indicating that somatic CAG repeat expansion in the brain is not required for pathogenesis. Given that the trajectory for somatic CAG expansion in the brains of Huntington's disease mutation carriers is unknown, our study underlines the importance of administering treatments targeting somatic instability as early as possible.

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.

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.

Phenotypic variation of FXN compound heterozygotes in a Friedreich ataxia cohort.

OBJECTIVE: Most individuals with Friedreich ataxia (FRDA) have homozygous GAA triplet repeat expansions in the FXN gene, correlating with a typical phenotype of ataxia and cardiomyopathy. A minority are compound heterozygotes carrying a GAA expansion on one allele and a mutation on the other. The study aim was to examine phenotypic variation among compound heterozygotes. METHODS: Data on FXN mutations were obtained from the Friedreich Ataxia Clinical Outcome Measures Study (FA-COMS). We compared clinical features in a single-site FA-COMS cohort of 51 compound heterozygous and 358 homozygous patients, including quantitative measures of cardiac, neurologic, and visual disease progression. RESULTS: Non-GAA repeat mutations were associated with reduced cardiac disease, and patients with minimal/no function mutations otherwise had a typical FRDA phenotype but with significantly more severe progression. The partial function mutation group was characterized by relative sparing of bulbar and upper limb function, as well as particularly low cardiac involvement. Other clinical features in this group, including optic atrophy and diabetes mellitus, varied widely depending on the specific type of partial function mutation. INTERPRETATION: These data support that the typical FRDA phenotype is driven by frataxin deficiency, especially severe in compound heterozygotes with minimal/no function mutations, whereas the heterogeneous presentations of those with partial function mutations may indicate other contributing factors to FRDA pathogenesis.

Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation-suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.

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.