Fuchs endothelial corneal dystrophy (FECD) is a common disease for which corneal transplantation is the only treatment option in advanced stages, and alternative treatment strategies are urgently required. Expansion (≥50 copies) of a non-coding trinucleotide repeat in TCF4 confers >76-fold risk for FECD in our large cohort of affected individuals. An FECD subject-derived corneal endothelial cell (CEC) model was developed to probe disease mechanism and investigate therapeutic approaches. The CEC model demonstrated that the repeat expansion leads to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing. Antisense oligonucleotide (ASO) treatment led to a significant reduction in the incidence of nuclear foci, MBNL1 recruitment to the foci, and downstream aberrant splicing events, suggesting functional rescue. This proof-of-concept study highlights the potential of a targeted ASO therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease.

PurposeTo demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1).MethodsWe applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis.ResultsCRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically.ConclusionCRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.

Maintenance of protein homeostasis is vitally important in post-mitotic cells, particularly neurons. Neurodegenerative diseases such as polyglutamine expansion disorders-like Huntington's disease or spinocerebellar ataxia (SCA), Alzheimer's disease, fronto-temporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease-are often characterized by the presence of inclusions of aggregated protein. Neurons contain complex protein networks dedicated to protein quality control and maintaining protein homeostasis, or proteostasis. Molecular chaperones are a class of proteins with prominent roles in maintaining proteostasis, which act to bind and shield hydrophobic regions of nascent or misfolded proteins while allowing correct folding, conformational changes and enabling quality control. There are many different families of molecular chaperones with multiple functions in proteostasis. The DNAJ family of molecular chaperones is the largest chaperone family and is defined by the J-domain, which regulates the function of HSP70 chaperones. DNAJ proteins can also have multiple other protein domains such as ubiquitin-interacting motifs or clathrin-binding domains leading to diverse and specific roles in the cell, including targeting client proteins for degradation via the proteasome, chaperone-mediated autophagy and uncoating clathrin-coated vesicles. DNAJ proteins can also contain ER-signal peptides or mitochondrial leader sequences, targeting them to specific organelles in the cell. In this review, we discuss the multiple roles of DNAJ proteins and in particular focus on the role of DNAJ proteins in protecting against neurodegenerative diseases caused by misfolded proteins. We also discuss the role of DNAJ proteins as direct causes of inherited neurodegeneration via mutations in family genes.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integratedperspective'.

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The predominant cause of Fuchs endothelial corneal dystrophy (FECD) is a CTG repeat expansion (termed CTG18.1) situated within an intron of the transcription factor encoding gene,TCF4. Here we use a primary FECD case-derived corneal endothelial cell (CEC) system to enhance our understanding of multiple pathogenic processes underlying CTG18.1-mediated FECD. We define differential gene expression and alternative splice events using long- and short-read RNA-seq datasets generated from 15 biologically independent primary CEC lines, comprehensively characterizing aberrant splicing-related aspects of the disease. Further targeted analysis of these data, alongside a complementary spatial transcriptomics approach, reveals a unique and distinctive pattern ofTCF4-specific dysregulation that underpins expansion-positive FECD and isolates downstream consequences of this shift as an important pathogenic component of disease. To explore whetherTCF4dysregulation, irrespective of CTG18.1 expansions, could cause FECD we also interrogated exome data generated from a large (n=141) genetically refined cohort of CTG18.1 expansion-negative FECD cases. We identify four rare and predicted deleterious (minor allele frequency <0.005; CADD>15)TCF4variants all encompassed by only a small fraction (10/93) of totalTCF4transcripts suggesting that, in rare instances, disruption of a specific subset of TCF4 isoforms may also confer CEC-specific disease independent of CTG18.1 expansions. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity andTCF4isoform-specific dysregulation, 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.