Umeå University's logo

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations. The ortholog of Drosophila eyes shut/spacemaker, EYS on chromosome 6q12 is a major genetic cause of recessive RP worldwide, with prevalence of 5 to 30%. In this study, by using targeted NGS, MLPA and Sanger sequencing we uncovered the EYS gene as one of the most common genetic cause of autosomal recessive RP in northern Sweden accounting for at least 16%. The most frequent pathogenic variant was c.8648_8655del that in some patients was identified in cis with c.1155T>A, indicating Finnish ancestry. We also showed that two novel EYS variants, c.2992_2992+6delinsTG and c.3877+1G>A caused exon skipping in human embryonic kidney cells, HEK293T and in retinal pigment epithelium cells, ARPE-19 demonstrating that in vitro minigene assay is a straightforward tool for the analysis of intronic variants. We conclude, that whenever it is possible, functional testing is of great value for classification of intronic EYS variants and the following molecular testing of family members, their genetic counselling, and inclusion of RP patients to future treatment studies.

Purpose

Inherited retinal dystrophies (IRDs) represent a group of progressive conditions affecting the retina. There is a great genetic heterogeneity causing IRDs, and to date, more than 260 genes are associated with IRDs. Stargardt disease, type 1 (STGD1) or macular degeneration with flecks, STGD1 represents a disease with early onset, central visual impairment, frequent appearance of yellowish flecks and mutations in the ATP‐binding cassette subfamily A, member 4 (ABCA4) gene. A large number of intronic sequence variants in ABCA4 have been considered pathogenic although their functional effect was seldom demonstrated. In this study, we aimed to reveal how intronic variants present in patients with Stargardt from the same Swedish family affect splicing.

Methods

The splicing of the ABCA4 gene was studied in human embryonic kidney cells, HEK293T, and in human retinal pigment epithelium cells, ARPE‐19, using a minigene system containing variants c.4773+3A>G and c.5461‐10T>C.

Results

We showed that both ABCA4 variants, c.4773+3A>G and c.5461‐10T>C, cause aberrant splicing of the ABCA4 minigene resulting in exon skipping. We also demonstrated that splicing of ABCA4 has different outcomes depending on transfected cell type.

Conclusion

Two intronic variants c.4773+3A>G and c.5461‐10T>C, both predicted to affect splicing, are indeed disease‐causing mutations due to skipping of exons 33, 34, 39 and 40 of ABCA4 gene. The experimental proof that ABCA4 mutations in STGD patients affect protein function is crucial for their inclusion to future clinical trials; therefore, functional testing of all ABCA4 intronic variants associated with Stargardt disease by minigene technology is desirable.

Purpose: Retinitis pigmentosa (RP) represents a large group of inherited retinal diseases characterized by clinical and genetic heterogeneity. Among patients with RP in northern Sweden, we identified two severely affected siblings and aimed to reveal a genetic cause underlying their disease.

Methods: Whole exome sequencing (WES) was performed on both affected individuals. Sequence variants were filtered using a custom pipeline to find a rare or novel variant predicted to affect protein function. Genome-wide genotyping was used to identify copy number variants (CNVs) and homozygous regions with potential disease causative genes.

Results: WES uncovered a novel heterozygous variant in the MER proto-oncogene, tyrosine kinase (MERTK) gene, c.2309A>G, p.Glu770Gly located in the tyrosine kinase domain and predicted to be likely pathogenic. The second variant, a large heterozygous deletion encompassing exons 1 to 7 of the MERTK gene, was revealed with genome-wide genotyping. The CNV analysis suggested breakpoints of the deletion, in the 5′-untranslated region and in intron 7. We identified genomic sequences at the site of the deletion as part of L1ME4b (LINE/L1) and AluSx3 that indicated a non-homologous recombination as a mechanism of the deletion evolvement.

Conclusions: Patients with RP in this study were carriers of two novel allelic mutations in the MERTK gene, a missense variant in exon 17 and an approximate 91 kb genomic deletion. Mapping of the deletion breakpoints allowed molecular testing of a cohort of patients with RP with allele-specific PCR. These findings provide additional information about mutations in MERTK for molecular testing of unsolved recessive RP cases and highlight the necessity for analysis of large genomic deletions.

Purpose : To describe clinical phenotype in patients of northern Sweden affected by recessive retinitis pigmentosa (ARRP) caused by mutations in Eyes Shut Homolog (Drosophila) (EYS) gene.

Methods : Whole exome sequencing (WES) and multiple ligation dependent prode amplification (MLPA) were used for identification of EYS sequence variants in a cohort of ARRP patients (n=148) from northern Sweden. The patients with EYS mutations were ophthalmologically examined over time using visual acuity (ETDRS), visual fields, slit lamp and fundus examination and ocular coherence tomography (OCT). Dark adaptometry and full-field electroretionograms (ERG) was performed.

Results : Phenotype characterization was done in 13 ARRP cases with EYS mutations representing five bi-allelic sequence variants, three of which were novel. Only one variant was detected in two cases. The phenotypic outcome was predominately presented as classical RP aggravating in young adulthood. However, among these patients we observed a variation of phenotypic expression with initial paracentral to central macular affection of the retina and areolar retinal degeneration with electrophysiological outcome of only slightly subnormal responses of both rods and cones in late adulthood (60 y/o), clinically defined as areolar atrophy.

Conclusions : The EYS mutations account for 10% of ARRP in northern Sweden. The phenotype presents both typical classical RP and chorioretinal degenerative retinal disease, areolar dystrophy. This suggests that molecular genetic testing of the EYS is crucial when both RP and pattern macular diseases are clinically diagnosed.

Inherited retinal and corneal dystrophies represent a group of disorders with great genetic heterogeneity. Over 250 genes are associated with retinal diseases and 16 genes are causative of corneal dystrophies. This thesis is focused on finding the genetic causes of corneal dystrophy, Leber congenital amaurosis (LCA), Stargardt disease and retinitis pigmentosa in families from northern Sweden.  By whole exome sequencing a novel mutation, c.2816C>T, p.Thr939Ile, in Collagen Type XVII, Alpha 1 chain, COL17A1, gene was identified in several families with epithelial recurrent erosion dystrophy (ERED). We showed that the COL17A1 protein is expressed in the basement membrane of the cornea, explaining the mutation involvement in the corneal symptoms. We could link all the families in this study to a couple born in the late 1700s confirming a founder mutation in northern Sweden. Our finding highlights role of COL17A1 in ERED and suggests screening of this gene in patients with similar phenotype worldwide. Furthermore the genetic causes in several retinal degenerations were identified. In one family with two recessive disorders, LCA and Stargardt disease, a novel stop mutation, c.2557C>T, p.Gln853Stop, was detected in all LCA patients. In the Stargardt patients two intronic variants, the novel c.4773+3A>G and c.5461-10T>C, were detected in the ABCA4 gene. One individual was homozygous for the known variant c.5461-10T>C and the other one was compound heterozygote with both variants present. Both variants, c.4773+3A>G and c.5461-10T>C caused exon skipping in HEK293T cells demonstrated by in vitro splice assay, proving their pathogenicity in Stargardt disease. Finally, in recessive retinitis pigmentosa, Bothnia Dystrophy (BD), we identified a second mutation in the RLBP1 gene, c.677T>A, p.Met226Lys. Thus, BD is caused not only by common c.700C>T variant but also by homozygosity of c.677T>A or compound heterozygosity. Notably, known variant, c.40C>T, p.R14W in the CAIV gene associated with a dominant retinal dystrophy RP17 was detected in one of the compound BD heterozygote and his unaffected mother. This variant appears to be a benign variant in the population of northern Sweden.

In conclusion, novel genetic causes of retinal dystrophies in northern Sweden were found demonstrating the heterogeneity and complexity of retinal diseases. Identification of the genetic defect in COL17A1 in the corneal dystrophy contributes to understanding ERED pathogenesis and encourages refinement of IC3D classification. Our results provide valuable information for future molecular testing and genetic counselling of the families.

Corneal dystrophies are a clinically and genetically heterogeneous group of inherited disorders that bilaterally affect corneal transparency. They are defined according to the corneal layer affected and by their genetic cause. In this study, we identified a dominantly inherited epithelial recurrent erosion dystrophy (ERED)-like disease that is common in northern Sweden. Whole-exome sequencing resulted in the identification of a novel mutation, c.2816C>T, p.T939I, in the COL17A1 gene, which encodes collagen type XVII alpha 1. The variant segregated with disease in a genealogically expanded pedigree dating back 200 years. We also investigated a unique COL17A1 synonymous variant, c.3156C>T, identified in a previously reported unrelated dominant ERED-like family linked to a locus on chromosome 10q23-q24 encompassing COL17A1. We show that this variant introduces a cryptic donor site resulting in aberrant pre-mRNA splicing and is highly likely to be pathogenic. Bi-allelic COL17A1 mutations have previously been associated with a recessive skin disorder, junctional epidermolysis bullosa, with recurrent corneal erosions being reported in some cases. Our findings implicate presumed gain-of-function COL17A1 mutations causing dominantly inherited ERED and improve understanding of the underlying pathology.

Genetic mechanisms underlying severe retinal dystrophy in a large Swedish family presenting two distinct phenotypes, Leber congenital amaurosis and Stargardt disease were investigated. In the family, four patients with Leber congenital amaurosis were homozygous for a novel c.2557C>T (p.Q853X) mutation in the CRB1 gene, while of two cases with Stargardt disease, one was homozygous for c.5461-10T>C in the ABCA4 gene and another was a compound heterozygous for c.5461-10T>C and a novel ABCA4 mutation c.4773+3 A>G. Sequence analysis of the entire ABCA4 gene in patients with Stargardt disease revealed complex alleles with additional sequence variants.Our results provide evidence of genetic complexity causative of different clinical features present in the same family, which is an obvious challenge for ophthalmologists, molecular geneticists and genetic counsellors.

Purpose: To evaluate phenotypes caused by different RLBP1 mutations in autosomal recessive retinitis pigmentosa of Bothnia type. Methods: Compound heterozygotes for mutations in the RLBP1 gene [c.677T>A]+[c.700C>T] (p.M226K+p.R234W), n=10, aged 7-84years, and homozygotes c.677T>A (p.M226K), n=2, aged 63 and 73years, were studied using visual acuity (VA), low-contrast VA, visual fields (VFs) and optical coherence tomography (OCT). Retrospective VA and VFs, standardized dark adaptation and full-field electroretinograms (ERGs) were analysed and prolonged dark adaptometry and ERG (at 24hr) were performed. Results: Progressive decline of VA and VF areas was age-dependent. Retinal degenerative maculopathy, peripheral degenerative changes and retinitis punctata albescens (RPA) were present. Early retinal thinning in the central foveal, foveal (O 1mm), and inner ring (O 3mm) in the macular region, with homogenous, high-reflectance RPA changes, was visualized in and adjacent to the retinal pigment epithelium/choriocapillaris using OCT. Reduced dark adaptation and affected ERGs were present in all ages. Prolonged dark adaptation and ERG (at 24hr), an increase in final threshold, and ERG rod and mixed rod/cone responses were found. Conclusions: The two RLBP1 genotypes presented a phenotypical and electrophysiological expression of progressive retinal disease similar to that previously described in homozygotes for the c.700C>T (p.R234W) RLBP1 mutation. The uniform phenotypical expression of RLBP1 mutations is relevant information for the disease and of importance in planning future treatment strategies.

This study aimed to identify genetic mechanisms underlying severe retinal degeneration in one large family from northern Sweden, members of which presented with early-onset autosomal recessive retinitis pigmentosa and juvenile macular dystrophy. The clinical records of affected family members were analysed retrospectively and ophthalmological and electrophysiological examinations were performed in selected cases. Mutation screening was initially performed with microarrays, interrogating known mutations in the genes associated with recessive retinitis pigmentosa, Leber congenital amaurosis and Stargardt disease. Searching for homozygous regions with putative causative disease genes was done by high-density SNP-array genotyping, followed by segregation analysis of the family members. Two distinct phenotypes of retinal dystrophy, Leber congenital amaurosis and Stargardt disease were present in the family. In the family, four patients with Leber congenital amaurosis were homozygous for a novel c.2557C>T (p.Q853X) mutation in the CRB1 gene, while of two cases with Stargardt disease, one was homozygous for c.5461-10T>C in the ABCA4 gene and another was carrier of the same mutation and a novel ABCA4 mutation c.4773+3A>G. Sequence analysis of the entire ABCA4 gene in patients with Stargardt disease revealed complex alleles with additional sequence variants, which were evaluated by bioinformatics tools. In conclusion, presence of different genetic mechanisms resulting in variable phenotype within the family is not rare and can challenge molecular geneticists, ophthalmologists and genetic counsellors.