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DNA methylation changes and increased mRNA expression of coagulation proteins, factor V and thrombomodulin in Fuchs endothelial corneal dystrophy
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.ORCID iD: 0000-0001-8454-802X
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
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2023 (English)In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 80, no 3, article id 62Article in journal (Refereed) Published
Abstract [en]

Late-onset Fuchs endothelial corneal dystrophy (FECD) is a disease affecting the corneal endothelium (CE), associated with a cytosine-thymine-guanine repeat expansion at the CTG18.1 locus in the transcription factor 4 (TCF4) gene. It is unknown whether CTG18.1 expansions affect global methylation including TCF4 gene in CE or whether global CE methylation changes at advanced age. Using genome-wide DNA methylation array, we investigated methylation in CE from FECD patients with CTG18.1 expansions and studied the methylation in healthy CE at different ages. The most revealing DNA methylation findings were analyzed by gene expression and protein analysis. 3488 CpGs had significantly altered methylation pattern in FECD though no substantial changes were found in TCF4. The most hypermethylated site was in a predicted promoter of aquaporin 1 (AQP1) gene, and the most hypomethylated site was in a predicted promoter of coagulation factor V (F5 for gene, FV for protein). In FECD, AQP1 mRNA expression was variable, while F5 gene expression showed a ~ 23-fold increase. FV protein was present in both healthy and affected CE. Further gene expression analysis of coagulation factors interacting with FV revealed a ~ 34-fold increase of thrombomodulin (THBD). THBD protein was detected only in CE from FECD patients. Additionally, we observed an age-dependent hypomethylation in elderly healthy CE.Thus, tissue-specific genome-wide and gene-specific methylation changes associated with altered gene expression were discovered in FECD. TCF4 pathological methylation in FECD because of CTG18.1 expansion was ruled out.

Place, publisher, year, edition, pages
Springer, 2023. Vol. 80, no 3, article id 62
Keywords [en]
Coagulation factors; DNA methylation; Factor V; Fuchs dystrophy; Thrombomodulin; Transcription factor 4 (TCF4); Trinucleotide repeat disorder
National Category
Medical Genetics
Research subject
Medical Genetics; Medical Genetics
Identifiers
URN: urn:nbn:se:umu:diva-200178DOI: 10.21203/rs.3.rs-1758860/v1ISI: 000929515100001PubMedID: 36773096Scopus ID: 2-s2.0-85147894855OAI: oai:DiVA.org:umu-200178DiVA, id: diva2:1702947
Funder
Region VästerbottenUmeå UniversityStiftelsen Kronprinsessan Margaretas arbetsnämnd för synskadadeThe Kempe Foundations
Note

Originally included in thesis in manuscript form. 

Available from: 2022-10-12 Created: 2022-10-12 Last updated: 2023-09-05Bibliographically approved
In thesis
1. Genetics, epigenetics and functional mechanisms in inherited corneal and retinal dystrophies
Open this publication in new window or tab >>Genetics, epigenetics and functional mechanisms in inherited corneal and retinal dystrophies
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Genetik, epigenetik och funktionella mekanismer i ärftliga corneala och retinala dystrofier
Abstract [en]

Inherited eye disorders (IED) are groups of genetically and clinically heterogenous conditions affecting different tissues in the eye. IED are most often progressive with reduced vision or legal blindness as outcome. This thesis is focused on investigating the underlying mechanisms in Fuchs’ endothelial corneal dystrophy (FECD) and two retinal dystrophies, Stargardt disease (STGD1) and autosomal recessive Retinitis pigmentosa (arRP, RP25).

In FECD, we studied the association between FECD and the (CTG)n repeat expansion at the CTG18.1 locus in the TCF4 gene, in patients from northern Sweden. By using STR-PCR and TP-PCR, we found that 90% of FECD patients carry an expanded CTG18.1 allele, establishing the highest prevalence among FECD patients world-wide. With droplet digital PCR, we showed that transcripts spanning over the CTG18.1 have lower fractions in human corneal endothelium (CE) compared to skin, brain, muscle, and white blood cells. With Illumina Methylation arrays (850K), we detected a decreased global methylation in the CE at advanced age, that could possibly contribute to the late onset of FECD. We also found distinct differences in methylation between FECD patients and controls, that led us to two coagulation factors, found to be over-expressed in the CE from FECD patients.

For the two retinal dystrophies, STGD1 and RP25, we investigated the functional effect of four genetic variants residing adjacent to or in splice consensus sequence of the ABCA4 gene (STDG1) and the EYS gene (RP25). With an in vitro mini-gene splicing assay we showed that all four genetic variants caused exon skipping in Retinal Pigment Epithelial cell line (ARPE-19) and Human Embryonic kidney cell line (HEK293T). Our results functionally proved these variants to be pathogenic and causative of STGD1 and RP25.

In RP25, we also investigated the prevalence of pathogenic EYS variants in a cohort of patients from northern Sweden. DNA from 81 patients with a clinical diagnosis of RP were interrogated with a "cascade-targeted mutation analysis" approach, where NGS, MLPA and Sanger sequencing was used to find common EYS variants in this acknowledged genetically homogenous population. EYS mutations were present in at least 16% of all arRP patients and the most recurrent mutation in the study was an 8-bp deletion, previously found in the Finnish population.

In conclusion, this thesis provides knowledge on disease causative mechanisms in IED and contributes with valuable information for future genetic counselling and genetic testing for affected families.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2022. p. 77
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2187
Keywords
Genetics, Epigenetics, Splicing, Methylation, Ophtalmology, FECD, Stargardt, Retinitis pigmentosa, ABCA4, EYS, TCF4, F5, THBD, Coagulation factor V, Thrombomodulin
National Category
Medical Genetics Ophthalmology
Research subject
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-200205 (URN)978-91-7855-810-0 (ISBN)978-91-7855-811-7 (ISBN)
Public defence
2022-11-18, Sal 933, 9 tr., byggnad 3A, Norrlands universitetssjukhus, Umeå, 13:00 (English)
Opponent
Supervisors
Note

I tryckt spikblad kan stå Filosofie doktorsexamen. I digital version står korrekt: Medicine doktorsexamen. 

Available from: 2022-10-28 Created: 2022-10-12 Last updated: 2022-10-13Bibliographically approved

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Westin, Ida MariaLandfors, MattiasGiannopoulos, AntoniosViberg, AndreasOsterman, PiaByström, BeritDegerman, SofieGolovleva, Irina

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Westin, Ida MariaLandfors, MattiasGiannopoulos, AntoniosViberg, AndreasOsterman, PiaByström, BeritDegerman, SofieGolovleva, Irina
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Medical and Clinical GeneticsPathologyDepartment of Integrative Medical Biology (IMB)OphthalmologyDepartment of Clinical Microbiology
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