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Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 cause the skipping of exon 39–40 and exon 33–34, respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C, and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4 , providing an important step to advance the development of gene therapy to treat STGD1.

Fuchs endothelial corneal dystrophy (FECD) is caused by a corneal endothelial cell loss, leading to corneal edema and visual impairment. The most significant genetic risk factor for FECD is an expansion of the CTG18.1 locus in transcription factor 4 (TCF4). The current treatment for severe FECD is corneal transplantation, with Descemet stripping automated keratoplasty (DSAEK) as a common surgical method. Although successful in most cases, the risk for transplant failure due to diverse causes must be considered. In this study, we investigated if presence of TCF4 CTG18.1 expansion with more than 31 (n ≥ 31) repeats in donated corneal grafts could be a reason for corneal transplant failure after DSAEK. For this, nine consecutively failed DSAEK corneal grafts were genotyped for CTG18.1 repeat length. One-sided Mann–Whitney U test was performed to evaluate if failed DSAEK corneal grafts had longer CTG18.1 repeats than healthy controls from the same population. All failed corneal grafts had CTG18.1 n ≤ 27 with a median of 18 (IQR 8.0) repeats for the longest allele. There was no statistical difference in CTG18.1 repeat lengths between failed corneal grafts and the geographically matched healthy control group. In conclusion, none of the nine failed corneal grafts in our material had CTG18.1 repeat lengths ≥ 31, a cut-off known to have a biological relevance in FECD. Thus, our results suggest that the assessment of donors and inspection of the corneal tissue before the decision for procurement is sufficient, in terms of recognizing FECD in the donor.

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.

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.

Purpose: Fuchs' endothelial corneal dystrophy (FECD) has been considered a genetically heterogeneous disease but is increasingly associated with the transcription factor 4 (TCF4) gene. This study investigates the prevalence of the cytosine-thymine-guanine (CTG)n repeat expansion in TCF4 among FECD patients in northern Sweden coupled to the phenotype.

Methods: Blood samples were collected from 85 FECD cases at different stages. Short tandem repeat PCR and triplet repeat-primed PCR were applied in order to determine TCF4 (CTG)n genotype.

Results: A (CTG)n repeat expansion (n > 50) in TCF4 was identified in 76 of 85 FECD cases (89.4%) and in four of 102 controls (3.9%). The median (CTG)n repeat length was 81 (IQR 39.3) in mild FECD and 87 (IQR 13.0) in severe FECD (p = 0.01). A higher number of (CTG)n repeats in an expanded TCF4 allele increased the probability of severe FECD. Other ocular surgery was overrepresented in FECD cases without a (CTG)n repeat expansion (44.4%, n = 4) compared with 3.9% (n = 3) in FECD cases with an (CTG)n repeat expansion (p < 0.001).

Conclusion: In northern Sweden, the FECD phenotype is associated with (CTG)n expansion in the TCF4 gene, with nearly 90% of patients being hetero- or homozygous for (CTG)n expansion over 50 repeats. Furthermore, the severity of FECD was associated with the repeat length in the TCF4 gene. Ocular surgery might act as an environmental factor explaining the clinical disease in FECD without a repeat expansion in TCF4.

Background: Sequence variants in the NOS1AP gene have repeatedly been reported to influence QTc, albeit with moderate effect sizes. In the long QT syndrome (LQTS), this may contribute to the substantial QTc variance seen among carriers of identical pathogenic sequence variants. Here we assess three non-coding NOS1APsequence variants, chosen for their previously reported strong association with QTc in normal and LQTS populations, for association with QTc in two Swedish LQT1 founder populations.

Methods: This study included 312 individuals (58% females) from two LQT1 founder populations, whereof 227 genotype positive segregating either Y111C (n = 148) or R518* (n = 79) pathogenic sequence variants in the KCNQ1 gene, and 85 genotype negatives. All were genotyped for NOS1AP sequence variants rs12143842, rs16847548 and rs4657139, and tested for association with QTc length (effect size presented as mean difference between derived and wildtype, in ms), using a pedigree-based measured genotype association analysis. Mean QTc was obtained by repeated manual measurement (preferably in lead II) by one observer using coded 50 mm/s standard 12-lead ECGs.

Results: A substantial variance in mean QTc was seen in genotype positives 476 ± 36 ms (Y111C 483 ± 34 ms; R518* 462 ± 34 ms) and genotype negatives 433 ± 24 ms. Female sex was significantly associated with QTc prolongation in all genotype groups (p < 0.001). In a multivariable analysis including the entire study population and adjusted for KCNQ1 genotype, sex and age, NOS1AP sequence variants rs12143842 and rs16847548 (but not rs4657139) were significantly associated with QT prolongation, +18 ms (p = 0.0007) and +17 ms (p = 0.006), respectively. Significant sex-interactions were detected for both sequent variants (interaction term r = 0.892, p < 0.001 and r = 0.944, p < 0.001, respectively). Notably, across the genotype groups, when stratified by sex neither rs12143842 nor rs16847548 were significantly associated with QTc in females (both p = 0.16) while in males, a prolongation of +19 ms and +8 ms (p = 0.002 and p = 0.02) was seen in multivariable analysis, explaining up to 23% of QTc variance in all males.

Conclusions: Sex was identified as a moderator of the association between NOS1AP sequence variants and QTc in two LQT1 founder populations. This finding may contribute to QTc sex differences and affect the usefulness of NOS1AP as a marker for clinical risk stratification in LQTS.

BACKGROUND: Autopsy of sudden cardiac death (SCD) in the young shows a structurally and histologically normal heart in about one third of cases. Sudden death in these cases is believed to be attributed in a high percentage to inherited arrhythmogenic diseases. The purpose of this study was to investigate the value of performing post-mortem genetic analysis for autopsy-negative sudden unexplained death (SUD) in 1 to 35 year olds.

METHODS AND RESULTS: From January 2009 to December 2011, samples from 15 cases suffering SUD were referred to the Department of Clinical Genetics, Umeå University Hospital, Sweden, for molecular genetic evaluation. PCR and bidirectional Sanger sequencing of genes important for long QT syndrome (LQTS), short QT syndrome (SQTS), Brugada syndrome type 1 (BrS1), and catecholaminergic polymorphic ventricular tachycardia (CPVT) (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, and RYR2) was performed. Multiplex ligation-dependent probe amplification (MLPA) was used to detect large deletions or duplications in the LQTS genes. Six pathogenic sequence variants (four LQTS and two CPVT) were discovered in 15 SUD cases (40%). Ten first-degree family members were found to be mutation carriers (seven LQTS and three CPVT).

CONCLUSION: Cardiac ion channel genetic testing in autopsy-negative sudden death victims has a high diagnostic yield, with identification of the disease in 40 of families. First-degree family members should be offered predictive testing, clinical evaluation, and treatment with the ultimate goal to prevent sudden death.

Background: Long QT syndrome (LQTS) is an inherited arrhythmic disorder characterised by prolongation of the QT interval on ECG, presence of syncope and sudden death. The symptoms in LQTS patients are highly variable, and genotype influences the clinical course. This study aims to report the spectrum of LQTS mutations in a Swedish cohort.

Methods: Between March 2006 and October 2009, two hundred, unrelated index cases were referred to the Department of Clinical Genetics, Umea University Hospital, Sweden, for LQTS genetic testing. We scanned five of the LQTS-susceptibility genes (KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2) for mutations by DHPLC and/or sequencing. We applied MLPA to detect large deletions or duplications in the KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes. Furthermore, the gene RYR2 was screened in 36 selected LQTS genotype-negative patients to detect cases with the clinically overlapping disease catecholaminergic polymorphic ventricular tachycardia (CPVT).

Results: In total, a disease-causing mutation was identified in 103 of the 200 (52%) index cases. Of these, altered exon copy numbers in the KCNH2 gene accounted for 2% of the mutations, whereas a RYR2 mutation accounted for 3% of the mutations. The genotype-positive cases stemmed from 64 distinct mutations, of which 28% were novel to this cohort. The majority of the distinct mutations were found in a single case (80%), whereas 20% of the mutations were observed more than once. Two founder mutations, KCNQ1 p.Y111C and KCNQ1 p.R518*, accounted for 25% of the genotype-positive index cases. Genetic cascade screening of 481 relatives to the 103 index cases with an identified mutation revealed 41% mutation carriers who were at risk of cardiac events such as syncope or sudden unexpected death.

Conclusion: In this cohort of Swedish index cases with suspected LQTS, a disease-causing mutation was identified in 52% of the referred patients. Copy number variations explained 2% of the mutations and 3 of 36 selected cases (8%) harboured a mutation in the RYR2 gene. The mutation panorama is characterised by founder mutations (25%), even so, this cohort increases the amount of known LQTS-associated mutations, as approximately one-third (28%) of the detected mutations were unique.

Introduction: The aim of this study is to use genetic mutation analysis to determine the cause of sudden unexpected death in young (SUDY) persons with normal autopsy findings, and to provide relatives with an identified cardiac mutation with suitable cardiovascular prevention. Methods: We performed mutation analysis on blood samples from first-degree relatives of 25 cases with normal autopsy findings identified in the national Swedish study of sudden cardiac death in 15- to 35-year-olds from 1992 to 1999. Results: We found three families with long QT syndrome through mutation screening, and the mutations were verified in two of the deceased. Eight family members were found to be mutation carriers and have been provided with suitable cardiovascular prevention. Mutation screening also identified a number of common polymorphisms in the individuals screened. Clinical history revealed one family each with short QT syndrome and hypertrophic cardiomyopathy, respectively, but no mutations were found in the family members or in the deceased. Two SCDs each had occurred in two of the affected families. Conclusion: Cardiac/genetic evaluation of relatives long after SUDY can reveal a diagnosis in 5/25 (20%) of cases. Since DNA extraction of formalin fixed paraffin embedded samples is unreliable, it is important that blood or tissue samples be stored at autopsy of such cases. This can facilitate establishing a diagnosis and thereby save lives in the future. (C) 2012 Elsevier Ireland Ltd. All rights reserved.