Genetics, epigenetics and functional mechanisms in inherited corneal and retinal dystrophies

Sammanfattning: 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.