ATP-binding cassette subfamily A, member 4 intronic variants c.4773+3A>G and c.5461-10T>C cause Stargardt disease due to defective splicing.

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.

Mutations in collagen, type XVII, alpha 1 (COL17A1) cause epithelial recurrent erosion dystrophy (ERED).

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 heterogeneity and clinical outcome in a Swedish family with retinal degeneration caused by mutations in CRB1 and ABCA4 genes.

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.

Novel mutations in CRB1 and ABCA4 genes cause Leber congenital amaurosis and Stargardt disease in a Swedish family.

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.

Ocular phenotype of CORD5, an autosomal dominant retinal dystrophy associated with PITPNM3 p.Q626H mutation.

PURPOSE: To describe in detail the phenotype of CORD5 in two families segregating a mutation c.1878G>C (p.Q626H) in the PITPNM3 gene. METHODS: The study included 35 individuals from two different families of Swedish origin, all heterozygous for a PITPNM3 p.Q626H mutation. All participants underwent ophthalmological examination including kinetic perimetry, and in selected cases adaptometry, colour vision tests and optical coherence tomography (OCT). Electrophysiological studies were also performed. In some cases, the data were obtained from medical records. RESULTS: The majority of patients showed subnormal visual acuity and light sensitivity from childhood. Early signs of macular degeneration were also observed. There was a progressive decrease in visual acuity leading to legal blindness in early adulthood. Electrophysiological testing showed a progressive loss of photoreceptor function restricted mainly to the cones. OCT revealed decreased macular thickness with flattened and enlarged fovea. CONCLUSION: Our observations of the PITPNM3 p.Q626H mutation carriers confirm that CORD5 is a disease not to mix with other retinal degenerations mapped to 17p13. The results of our clinical evaluation so far indicate that CORD5 is characterized by predominant cone dysfunction without signs of general involvement of the retinal pigment epithelium. The rod system also seems to be unaffected. In this sense, CORD5 is different from other autosomal dominant CORDs where rod involvement is present to some degree in a late phase of the disease. Some intra- and inter-familial differences regarding the severity of the clinical picture were observed.

Transthyretin-related vitreous amyloidosis in different endemic areas.

BACKGROUND: to investigate the vitreous opacity in patients with familial amyloidotic polyneuropathy (FAP) in two major endemic areas, Japan and Sweden. METHODS: we obtained clinical data for 90 patients with vitreous opacity that was associated with FAP amyloidogenic transthyretin (ATTR) Val30Met; 18 Japanese patients and 72 Swedish patients. We reviewed medical records at Kumamoto University Hospital in Japan and Umeå University Hospital in Sweden. We evaluated the characteristics of the patients, systemic and ocular histories, clinical findings and treatment. RESULTS: swedish patients were significantly older at the onset of vitreous opacity (mean age 67.8 years) than were Japanese patients (47.6 years). A similar age difference was found for the onset of polyneuropathy. In addition, Swedish patients without polyneuropathy were significantly older (74.1 years) at the onset of vitreous opacity than those with polyneuropathy (64.6 years). A significant difference in the occurrence of vitreous opacity as the only manifestation of FAP was seen for Swedish patients (35%) compared with Japanese patients (6%). CONCLUSIONS: swedish FAP ATTR Val30Met patients appeared to develop vitreous opacity later and more frequently compared with Japanese patients.

PITPNM3 is an uncommon cause of cone and cone-rod dystrophies.

The first mutation in PITPNM3, a human homologue of the Drosophila retinal degeneration (rdgB not not) gene was reported in two large Swedish families with autosomal dominant cone dystrophy. To establish the global impact that PITPNM3 has on retinal degenerations we screened 163 patients from Denmark, Germany, the UK, and USA. Four sequence variants, two missence mutations and two intronic changes were identified in the screen. Thus, mutations in PITPNM3 do not appear to be a major cause of cone or cone-rod dystrophy.

Mutation spectra in autosomal dominant and recessive retinitis pigmentosa in northern Sweden.

Retinal degenerations represent a heterogeneous group of disorders affecting the function of the retina. The frequency of retinitis pigmentosa (RP) is 1/3500 worldwide, however, in northern Sweden it is 1/2000 due to limited migration and a 'founder' effect. In this study we identified genetic mechanisms underlying autosomal dominant and recessive RP present in northern Sweden. Several novel mutations unique for this region were found. In an autosomal recessive form of RP, Bothnia dystrophy caused by mutations in the RLBP1 gene, bi-allelic mutations R234W, M226K and compound heterozygosity, M226K+R234W was detected.In dominant form of RP mapped to 19q13.42 a 59 kb genomic deletion including the PRPF31 and three other genes was found.These data provide additional information on the molecular mechanisms of RP evolvement and in the future might be useful in development of therapeutic strategies. Identification of the disease-causing mutations allowed introducing molecular genetic testing of the patients and their families into the clinical practice.

Breakpoint characterization of a novel approximately 59 kb genomic deletion on 19q13.42 in autosomal-dominant retinitis pigmentosa with incomplete penetrance.

The aim of this study was to identify and characterize the underlying molecular mechanisms in autosomal-dominant retinitis pigmentosa (adRP) with incomplete penetrance in two Swedish families. An extended genealogical study and haplotype analysis indicated a common origin. Mutation identification was carried out by multiplex ligation-dependent probe amplification (MLPA) and sequencing. Clinical examinations of adRP families including electroretinography revealed obligate gene carriers without abnormalities, which indicated incomplete penetrance. Linkage analysis resulted in mapping of the disease locus to 19q13.42 (RP11). Sequence analyses did not reveal any mutations segregating with the disease in eight genes including PRPF31. Subsequent MLPA detected a large genomic deletion of 11 exons in the PRPF31 gene and, additionally, three genes upstream of the PRPF31. Breakpoints occurred in intron 11 of PRPF31 and in LOC441864, 'similar to osteoclast-associated receptor isoform 5.' An almost 59 kb deletion segregated with the disease in all affected individuals and was present in several asymptomatic family members but not in 20 simplex RP cases or 94 healthy controls tested by allele-specific PCR. A large genomic deletion resulting in almost entire loss of PRPF31 and three additional genes identified as the cause of adRP in two Swedish families provide an additional evidence that mechanism of the disease evolvement is haploinsufficiency. Identification of the deletion breakpoints allowed development of a simple tool for molecular testing of this genetic subtype of adRP.

Ocular manifestations in liver transplant recipients with familial amyloid polyneuropathy.

PURPOSE: To evaluate postoperative ocular involvement in Swedish liver transplant (LT) recipients with familial amyloid polyneuropathy (FAP). METHODS: Routine ophthalmological examinations were performed in 48 LT recipients, with particular attention given to amyloid deposition in the anterior segment and the vitreous body. Medical records were scrutinized for information regarding neurological impairment at the time of the LT. The diagnosis was secured in all cases by examining for amyloid deposits in biopsy specimens and positive genetic testing for amyloidogenic transthyretin (ATTR) Val30Met mutation. RESULTS: Six patients (12.5%) developed vitreous opacities within the post-LT observation period. The first opacities were seen 40 months after transplantation, 8 years after the onset of systemic disease. Four patients (8%) developed secondary glaucoma, the first of which was observed 18 months after the procedure and 6.5 years after the onset of disease. Sixteen patients (33%) developed deposits on the anterior surface of the lens. Scalloped pupillary margins were noted in 10 patients (21%). CONCLUSION: The prevalence of eye complications increases with time after LT and regular follow-up is necessary, especially to disclose the development of glaucoma--a complication with insidious symptoms of which patients are normally unaware.

Carrier of R14W in carbonic anhydrase IV presents Bothnia dystrophy phenotype caused by two allelic mutations in RLBP1.

PURPOSE: Bothnia dystrophy (BD) is an autosomal recessive retinitis pigmentosa (arRP) associated with the c.700C>T mutation in the RLBP1 gene. Testing of patients with BD has revealed the c.700C>T mutation on one or both alleles. The purpose of this study was to elucidate the underlying genetic mechanisms along with a clinical evaluation of the heterozygous patients with BD. METHODS: Patients with BD heterozygous for the RLBP1 c.700C>T were tested for 848 mutations by arrayed primer-extension technology. Further mutation detection was performed by PCR-restriction fragment length polymorphism (RFLP), sequencing, denaturing (d)HLPC and allelic discrimination. The ophthalmic examinations were performed in all c.700C>T heterozygotes. RESULTS: The clinical findings in 10 BD heterozygotes were similar to those in the homozygotes. The presence of a second mutation, c.677T>A, corresponding to p.M226K was detected in all 10 cases. Segregation analysis showed that the mutations were allelic, and the patients were compound heterozygotes [c.677T>A]+[c.700C>T]. One of those patients was also a carrier of the c.40C>T corresponding to the p.R14W change in carbonic anhydrase IV (CAIV) associated with autosomal dominant RP, RP17. His mother, a carrier of the identical change was declared healthy after ophthalmic examination. This sequence variant was found in 6 of 143 tested blood donors. CONCLUSIONS: The high frequency of arRP in northern Sweden is due to two mutations in the RLBP1 gene: c.677T>A and c.700C>T. BD is caused by the loss of CRALBP function due to changed physical features and impaired activity of retinoid binding. The CAIV p.R14W sequence variant found in one of the patients with a BD phenotype is a benign polymorphism in a population of northern Sweden.

Effects of prolonged dark adaptation in patients with retinitis pigmentosa of Bothnia type: an electrophysiological study.

Bothnia dystrophy (BD) is a variant of recessive retinitis punctata albescens (RPA), caused by the missense mutation R233W in cellular retinaldehyde-binding protein (CRALBP), which is localized in the retinal pigment epithelium (RPE) and Müller cells of the retina. The purpose of this study was, by examining the electrophysiological responses of the retina, to evaluate the capacity of recovery of the whole retinal area and different cell types induced by extremely prolonged dark adaptation (DA) in BD disease and to gain further understanding of the pathogenesis of BD. Six young patients underwent bilateral full-field ERGs after 24 h of DA in one eye and standard DA in the fellow eye. The results were also compared with the effect of prolonged DA (10 h), previously studied in the same patients. After extremely prolonged DA (24 h) the rod b-wave and the mixed rod-cone a-wave responses reached normal though delayed amplitudes. An increase, up to normal level, in the oscillatory response was found. There was no obvious recovery of the cone response. We conclude that in young BD patients during extremely prolonged DA there is a significant additional capacity of recovery of rod function and also significant gain of activity in the inner retinal layer. A continuous but slow regeneration of rod photopigment seems to occur at least up to 24 h. The visual process in the RPE is retarded and CRALBP acts in this process; also, the Müller cells of the retina seem to be involved. The findings also support an extremely slow synthesis of photopigments and irreversibly disturbed cone function early in BD.

Mutation in the PYK2-binding domain of PITPNM3 causes autosomal dominant cone dystrophy (CORD5) in two Swedish families.

Autosomal dominant cone dystrophy (CORD5) (MIM 600977) is a rare disease predominantly affecting cone photoreceptors. Here we refine the CORD5 locus previously mapped to 17p13 from 27 to 14.3 cM and identified a missense mutation, Q626H in the phosphatidylinositol transfer (PIT) membrane-associated protein (PITPNM3) (MIM 608921) in two Swedish families. PITPNM3, known as a human homologue of the Drosophila retinal degeneration B (rdgB), lacks the N-terminal PIT domain needed for transport of phospholipids, renewal of photoreceptors membrane and providing the electroretinogram (ERG) response to light. In our study, the mutation causing CORD5 is located in the C-terminal region interacting with a member of nonreceptor protein tyrosine kinases, PYK2. Our finding on the first mutation in the human homologue of Drosophila rdgB indicates novel pathways and a potential important role of the PITPNM3 in mammalian phototransduction.

Tinted contact lenses in Bothnia dystrophy.

PURPOSE: To determine whether tinted contact lenses can improve visual function in patients with Bothnia dystrophy (BD), a genetically defined retinal dystrophy with prolonged dark adaptation. METHODS: Twelve patients with BD were fitted with the same type of soft contact lenses tinted dark brown. Visual acuity (VA), contrast vision, near vision and visual fields were tested before and 1 month after contact lens fitting. The patients completed a visual function questionnaire. The physical properties of the contact lenses were tested using spectrophotometry. RESULTS: The patients with the lowest VA described the most obvious improvement in visual function. This group of patients preferred darker contact lenses and continued wearing their contact lenses after the study ended. The patients with the best VA preferred lighter contact lenses and a few patients in this group discontinued contact lens wear upon completion of the study. CONCLUSIONS: Visual function in BD patients was improved by dark tinted contact lenses. The optimal colour for lenses varies, depending on the season and the individual. Other patient groups with retinal dystrophies associated with prolonged dark adaptation or dysfunction of the cone system, such as cone dystrophies or achromatopsia, may also benefit from this type of contact lens.

Associations between specific measures of vision and vision-related quality of life in patients with bothnia dystrophy, a defined type of retinitis pigmentosa.

PURPOSE: To assess the relationship between objective tests of visual function and vision-related quality of life in patients with Bothnia dystrophy (BD), a retinal dystrophy of retinitis pigmentosa type with progressive maculopathy. METHODS: Forty-nine patients were tested. Weighted distance logMAR visual acuity (WVA), weighted logMAR low contrast VA (WCS), and binocular visual field (VF) areas were calculated. Vision-related quality of life (VRQL) was assessed using the 25-item National Eye Institute-Visual Function Questionnaire (NEI-VFQ-25). Correlation statistics were used and adjusted analyses of the relationship between the composite score and the objective visual function tests were performed with multiple linear regression. RESULTS: VRQL was significantly correlated with age, WVA, WCS, and binocular VF areas (P < 0.001). Calculation of partial correlation coefficients showed age to be significantly correlated only with VF (V-4-e) area (P < 0.0001). Multiple linear regression analyses revealed age and WVA to be significantly associated with the NEI-VFQ-25 composite score (P < 0.02 and P = 0.001, respectively). WVA alone was the strongest predictor of self-reported experience of total visual function in BD patients (r 2= 0.69). CONCLUSIONS: A strong relationship between objective tests of visual function and patient perceived VRQL as assessed by a questionnaire was found. WVA was the strongest predictor and together with age explained almost 70% of the variability of the composite score of the questionnaire.

Retinal function in Bothnia dystrophy. An electrophysiological study.

Using prolonged dark adaptometry, standard dark adaptation (DA) and prolonged DA full-field electroretinograms (ERGs), we analysed the retinal function in patients with Bothnia dystrophy (BD), a variant of recessive retinitis punctata albescens (RPA). A compromised rod and cone function, a likely dysfunction of the Müller cells, and indications of disturbed neuronal function of the inner retina, were found. With prolonged DA, a gradual increase in retinal sensitivity to light and an improvement of the ERG components occurred. The findings indicate a prolonged synthesis of photopigments, retardation of the visual process in the retinal pigment epithelium (RPE), and a loss of retinal cells, probably starting at a relatively early age in BD.

Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions.

Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cis-retinol acceptor and as a substrate carrier.