Biology subjects

Tuft, S. J.

Publications and source records attributed to Tuft, S. J..

3 recordsLinked to original sources

bioRxiv2024

Tissue-Specific Dynamics of TCF4 Triplet Repeat Instability Revealed by Optical Genome Mapping

Here, we demonstrate the utility of optical genome mapping (OGM) to interrogate the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (termed CTG18.1) and gain novel insights into the tissue-specific nature of the disease. Genomic DNA (gDNA) samples derived from peripheral blood leukocytes and primary corneal endothelial cells (CECs) were analysed by OGM. Concurrently, all samples were genotyped by standard PCR-based methods to classify their expansion status. Individuals with one or more CTG18.1-expanded alleles ([≥]50 CTG repeats) detected in their leukocyte-derived gDNA were classified as expansion-positive. A customised bioinformatics pipeline was developed to perform CTG18.1-targeted OGM analysis. All linearised gDNA molecules containing labels flanking CTG18.1 were extracted, corrected for the repeats on the reference human genome and sized. Analysis of paired bio-samples revealed that expanded CTG18.1 alleles behave dynamically, regardless of cell-type origin, but displayed significantly higher levels of instability within the diseased corneal endothelium. Clusters of CTG18.1 molecules of approximately 1,800-11,900 repeats, beyond the ranges observed in individual-matched leukocyte samples, were detected in all CEC gDNA samples from expansion-positive cases. In conclusion, OGM is a powerful method to analyse the somatically unstable CTG18.1 locus. More generally, this work exemplifies the broader utility of OGM in exploring somatically unstable short tandem repeat loci. Furthermore, this study has highlighted the extreme levels of tissue-specific CTG18.1 somatic instability occurring within the diseased corneal endothelium, which we hypothesise plays a pivotal role in driving downstream pathogenic mechanisms of CTG18.1-mediated FECD.

genomics↗

bioRxiv2023

Deciphering novel TCF4-driven molecular origins and 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. Authors summaryFuchs endothelial corneal dystrophy (FECD) leads to vision loss and is the most common repeat expansion-mediated disease characterised to date; most individuals with FECD harbour a non-coding CTG repeat expansion within the gene TCF4. FECD represents an important paradigm for other rare and devastating neurological repeat-mediated diseases, given its relatively mild and tissue-specific nature. Intriguingly, despite TCF4 being ubiquitously expressed, individuals with FECD only experience corneal disease, and the biological reason for this tissue-specificity remains elusive. Here, we use tissue from 31 individuals with FECD to perform complementary long-read, short-read and spatial transcriptomic analyses to enhance our understanding of mechanisms underpinning this disease. These data highlight that at least two mechanisms, RNA toxicity and TCF4 isoform dysregulation, underlie the disease state in affected corneal cells. Furthermore, TCF4 isoform skewing, with evidence of downregulation, suggests this mechanism in part may explain the unique vulnerability of the cornea. In addition, 7/134 FECD expansion negative cases were identified to harbour rare and potentially deleterious TCF4 variants, further supporting the hypothesis that dysregulation of TCF4 may be key to FECD pathophysiology. Biological insights presented here will guide the development of personalised FECD therapies and may inform the development of repeat-expansion mediated therapies more broadly.

molecular biology↗

bioRxiv2019

Frequency and Distribution of Corneal Astigmatism and Keratometry Features: Methodology and Findings of the UK Biobank Study

PurposeTo describe corneal astigmatism in the UK Biobank population, to look for associations with other biometric variables and socio-demographic factors, and to report the proportion with abnormal keratometry and irregular astigmatism suggestive of pathological corneal ectasias such as keratoconus.\n\nMethodsCross-sectional data were obtained from UK Biobank (www.ukbiobank.ac.uk/). A subsample of 107,452 participants from UK communities had undergone an enhanced ophthalmic examination including autorefractor keratometry (Tomey RC 5000, Tomey Corp., Nagoya, Japan). After quality control and applying relevant exclusions, data on corneal astigmatism on 83,751 participants was available for analysis. Potential associations were tested through univariable regression and significant parameters carried forward for multivariable analysis.\n\nResultsIn a univariable analysis, the characteristics significantly protective against corneal astigmatism were gender (male), older age, darker skin colour and increased alcohol intake (all p<0.001). The parameters significantly associated with increased corneal astigmatism were older age at completion of full time education, use of UV protection and lower corrected visual acuity. After inclusion in the multivariable analysis, age, gender, age at completion of full time education, corrected visual acuity and skin colour remained significant (all p<0.001). Increased corneal astigmatism was also found to be significantly associated with amblyopia or strabismus. No individuals with abnormal keratometry or irregular astigmatism were reported.\n\nConclusionsThis analysis of associations with astigmatism in a large cohort of volunteers confirms previous associations including adverse associations with younger age and female gender, and identified novel associations including darker skin colour and frequency of alcohol intake. The highest risk group for corneal astigmatism were younger females of lighter skin colour, having completed full time education later, with higher logMAR corrected visual acuity. We also confirmed that corneal astigmatism is a high risk factor for amblyopia and strabismus. Finally since no cases of keratoconus were identified, this would suggest that simple keratometry indices may not be sufficient for population screening of keratoconus.

epidemiology↗