Achromatopsia is a rare autosomal recessive cone disorder characterized by color vision defects, photophobia, nystagmus, and severely reduced visual acuity. The disease is caused by mutations in ...genes encoding crucial components of the cone phototransduction cascade (CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H) or in ATF6, involved in the unfolded protein response. CNGB3 encoding the beta subunit of the cyclic nucleotide‐gated ion channel in cone photoreceptors is the major achromatopsia gene. Here, we present a comprehensive spectrum of CNGB3 mutations and their prevalence in a cohort of 1074 independent families clinically diagnosed with achromatopsia. Of these, 485 (45.2%) carried mutations in CNGB3. We identified a total of 98 different potentially disease‐causing CNGB3 variants, 58 of which are novel. About 10% of patients with CNGB3 mutations only harbored a single heterozygous variant. Therefore, we performed quantitative real‐time PCR in 43 of such single heterozygotes in search of the missing allele, followed by microarray‐based comparative genomic hybridization and breakpoint mapping. We discovered nine different heterozygous copy number variations encompassing one to 10 consecutive exons in 16 unrelated patients. Moreover, one additional patient with a homozygous CNGB3 deletion encompassing exons 4−18 was identified, highlighting the importance of CNV analysis for this gene.
CNGB3 mutation spectrum including CNVs.
Sorsby fundus dystrophy (SFD) is a macular degeneration caused by mutations in TIMP3, the majority of which introduce a novel cysteine. However, the exact molecular mechanisms underlying SFD remain ...unknown. We aimed to provide novel insights into the functional consequences of a distinct N‐terminal mutation. Haplotype reconstruction in three SFD families revealed that the identified c.113C>G, p.(Ser38Cys) mutation is a founder in Belgian and northern French families with a late‐onset SFD phenotype. Functional consequences of the p.(Ser38Cys) mutation were investigated by high‐resolution Western blot analysis of wild type and mutant TIMP3 using patient fibroblasts and in vitro generated proteins, and by molecular modeling of TIMP3 and its interaction partners. We could not confirm a previous hypothesis on dimerization of mutant TIMP3 proteins. However, we identified aberrant intramolecular disulfide bonding. Our data provide evidence for disruption of the established Cys36‐Cys143 disulfide bond and formation of a novel Cys36‐Cys38 bond, possibly associated with increased glycosylation of the protein. In conclusion, we propose a novel pathogenetic mechanism underlying the p.(Ser38Cys) TIMP3 founder mutation involving intramolecular disulfide bonding. These results provide new insights into the pathogenesis of SFD and other retinopathies linked to mutations in TIMP3, such as age‐related macular degeneration.
Sorsby fundus dystrophy (SFD) is a macular degeneration caused by mutations in TIMP3, the majority of which introduce a novel cysteine. Most studies hypothesize that mutant TIMP3 proteins with unpaired cysteines form abnormal disulfide‐bonded dimers. As opposed to this, we propose a novel pathogenetic mechanism for the N‐terminal p.(Ser38Cys) mutation, identified in three families with late‐onset SFD, comprising the formation of a novel intramolecular disulfide bond with a potential effect on TIMP3 glycosylation.
CEP290-associated Leber congenital amaurosis type 10 (LCA10) is a retinal disease resulting in childhood blindness. Sepofarsen is an RNA antisense oligonucleotide targeting the c.2991+1655A>G variant ...in the CEP290 gene to treat LCA10. In this open-label, phase 1b/2 ( NCT03140969 ), 12-month, multicenter, multiple-dose, dose-escalation trial, six adult patients and five pediatric patients received ≤4 doses of intravitreal sepofarsen into the worse-seeing eye. The primary objective was to evaluate sepofarsen safety and tolerability via the frequency and severity of ocular adverse events (AEs); secondary objectives were to evaluate pharmacokinetics and efficacy via changes in functional outcomes. Six patients received sepofarsen 160 µg/80 µg, and five patients received sepofarsen 320 µg/160 µg. Ten of 11 (90.9%) patients developed ocular AEs in the treated eye (5/6 with 160 µg/80 µg; 5/5 with 320 µg/160 µg) versus one of 11 (9.1%) in the untreated eye; most were mild in severity and dose dependent. Eight patients developed cataracts, of which six (75.0%) were categorized as serious (2/3 with 160 µg/80 µg; 4/5 with 320 µg/160 µg), as lens replacement was required. As the 160-µg/80-µg group showed a better benefit-risk profile, higher doses were discontinued or not initiated. Statistically significant improvements in visual acuity and retinal sensitivity were reported (post hoc analysis). The manageable safety profile and improvements reported in this trial support the continuation of sepofarsen development.
Abstract
Exome-based testing for genetic diseases can reveal unsolicited findings (UFs), i.e. predispositions for diseases that exceed the diagnostic question. Knowledge of patients’ interpretation ...of possible UFs and of motives for (not) wanting to know UFs is still limited. This lacking knowledge may impede effective counselling that meets patients’ needs. Therefore, this article examines the meaning of UFs from a patient perspective. A qualitative study was conducted and an interpretative phenomenological analysis was made of 14 interviews with patients with an inherited retinal disease. Patients assign a complex meaning to UFs, including three main components. The first component focuses on result-specific qualities, i.e. the characteristics of an UF (inclusive of actionability, penetrance, severity and age of onset) and the consequences of disclosure; the second component applies to a patient’s lived illness experiences and to the way these contrast with reflections on presymptomatic UFs; the third component addresses a patient’s family embedding and its effect on concerns about disease prognosis and genetic information’s family relevance. The complex meaning structure of UFs suggests the need for counselling procedures that transcend a strictly clinical approach. Counselling should be personalised and consider patients’ lived illness experiences and family context.
Autosomal dominant retinitis pigmentosa (adRP) is characterized by an extensive genetic heterogeneity, implicating 27 genes, which account for 50 to 70% of cases. Here 86 Belgian probands with ...possible adRP underwent genetic testing to unravel the molecular basis and to assess the contribution of the genes underlying their condition.
Mutation detection methods evolved over the past ten years, including mutation specific methods (APEX chip analysis), linkage analysis, gene panel analysis (Sanger sequencing, targeted next-generation sequencing or whole exome sequencing), high-resolution copy number screening (customized microarray-based comparative genomic hybridization). Identified variants were classified following American College of Medical Genetics and Genomics (ACMG) recommendations.
Molecular genetic screening revealed mutations in 48/86 cases (56%). In total, 17 novel pathogenic mutations were identified: four missense mutations in RHO, five frameshift mutations in RP1, six mutations in genes encoding spliceosome components (SNRNP200, PRPF8, and PRPF31), one frameshift mutation in PRPH2, and one frameshift mutation in TOPORS. The proportion of RHO mutations in our cohort (14%) is higher than reported in a French adRP population (10.3%), but lower than reported elsewhere (16.5-30%). The prevalence of RP1 mutations (10.5%) is comparable to other populations (3.5%-10%). The mutation frequency in genes encoding splicing factors is unexpectedly high (altogether 19.8%), with PRPF31 the second most prevalent mutated gene (10.5%). PRPH2 mutations were found in 4.7% of the Belgian cohort. Two families (2.3%) have the recurrent NR2E3 mutation p.(Gly56Arg). The prevalence of the recurrent PROM1 mutation p.(Arg373Cys) was higher than anticipated (3.5%).
Overall, we identified mutations in 48 of 86 Belgian adRP cases (56%), with the highest prevalence in RHO (14%), RP1 (10.5%) and PRPF31 (10.5%). Finally, we expanded the molecular spectrum of PRPH2, PRPF8, RHO, RP1, SNRNP200, and TOPORS-associated adRP by the identification of 17 novel mutations.
We describe both phenotype and pathogenesis in two male siblings with typical retinitis pigmentosa (RP) and the potentially X-linked RP (XLRP) carrier phenotype in their mother. Two affected sons, ...two unaffected daughters, and their mother underwent detailed ophthalmological assessments including Goldmann perimetry, color vision testing, multimodal imaging and ISCEV-standard electroretinography. Genetic testing consisted of targeted next-generation sequencing (NGS) of known XLRP genes and whole exome sequencing (WES) of known inherited retinal disease genes (RetNet-WES). Variant validation and segregation analysis were performed by Sanger sequencing. The mutational load of the RHO variant in the mother was assessed in DNA from leucocytes, buccal cells and hair follicles using targeted NGS. Both affected sons showed signs of classical RP, while the mother displayed patches of hyperautofluorescence on blue light autofluorescence imaging and regional, intraretinal, spicular pigmentation, reminiscent of a carrier phenotype of XLRP. XLRP testing was negative. RetNet-WES testing revealed RHO variant c.404G > C p.(Arg135Pro) in a mosaic state (21% of the reads) in the mother and in a heterozygous state in both sons. Targeted NGQSS of the RHO variant in different maternal tissues showed a mutation load between 25.06% and 41.72%. We report for the first time that somatic mosaicism of RHO variant c.404G > C p.(Arg135Pro) mimics the phenotype of a female carrier of XLRP, in combination with heterozygosity for the variant in the two affected sons.
AimTo investigate the natural history in a Belgian cohort of CRB1-associated retinal dystrophies.MethodsAn in-depth retrospective study focusing on visual function and retinal structure.ResultsForty ...patients from 35 families were included (ages: 2.5–80.1 years). In patients with a follow-up of >1 year (63%), the mean follow-up time was 12.0 years (range: 2.3–29.2 years). Based on the patient history, symptoms and/or electroretinography, 22 patients (55%) were diagnosed with retinitis pigmentosa (RP), 15 (38%) with Leber congenital amaurosis (LCA) and 3 (8%) with macular dystrophy (MD), the latter being associated with the p.(Ile167_Gly169del) mutation (in compound heterozygosity). MD later developed into a rod-cone dystrophy in one patient. Blindness at initial presentation was seen in the first decade of life in LCA, and in the fifth decade of life in RP. Eventually, 28 patients (70%) reached visual acuity-based blindness (<0.05). Visual field-based blindness (<10°) was documented in 17/25 patients (68%). Five patients (13%) developed Coats-like exudative vasculopathy. Intermediate/posterior uveitis was found in three patients (8%). Cystoid maculopathy was common in RP (9/21; 43%) and MD (3/3; 100%). Macular involvement, varying from retinal pigment epithelium alterations to complete outer retinal atrophy, was observed in all patients.ConclusionBi-allelic CRB1 mutations result in a range of progressive retinal disorders, most of which are generalised, with characteristically early macular involvement. Visual function and retinal structure analysis indicates a window for potential intervention with gene therapy before the fourth decade of life in RP and the first decade in LCA.
ABSTRACT
Leber congenital amaurosis (LCA) is a severe autosomal‐recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. ...Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9‐associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5′UTR variant, c.‐70A>T. Moreover, an adjacent 5′UTR variant, c.‐69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5′UTR variants resulted in decreased NMNAT1 mRNA abundance in patients’ lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE‐1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu‐rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1‐associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat‐mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness.
Leber Congenital Amaurosis (LCA) is a severe autosomal recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1 (LCA9). Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden non‐coding defects or structural variations (SVs). Our study uncovered, for the first time, hidden genetic variation in NMNAT1‐associated LCA and emphasized a shift from coding to non‐coding (e.g. 5'UTR) mutations and repeat‐mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness.
To describe the genetic and phenotypic characteristics of a cohort of patients with PROM1 variants.
Case-case study.
We screened a cohort of 2216 families with inherited retinal dystrophies using ...classical molecular techniques and next-generation sequencing approaches. The clinical histories of 25 patients were reviewed to determine age of onset of symptoms and the results of ophthalmoscopy, best-corrected visual acuity, full-field electroretinography, and visual field studies. Fundus autofluorescence and spectral-domain optical coherence tomography were further assessed in 7 patients.
PROM1 variants were identified in 32 families. Disease-causing variants were found in 18 autosomal recessive and 4 autosomal dominant families. Monoallelic pathogenic variants or variants of unknown significance were identified in the remaining 10 families. Comprehensive phenotyping of 25 patients from 22 families carrying likely disease-causing variants revealed clinical heterogeneity associated with the PROM1 gene. Most of these patients presented cone-rod dystrophy and some exhibited macular dystrophy or retinitis pigmentosa, while all presented with macular damage. Phenotypic association of a dominant splicing variant with late-onset mild maculopathy was established. This variant is one of the 3 likely founder variants identified in our Spanish cohort.
We report the largest cohort of patients with PROM1 variants, describing in detail the phenotype in 25 of them. Interestingly, within the variability of phenotypes related to this gene, macular involvement is a common feature in all patients.
Leber congenital amaurosis (LCA) is a severe autosomal-recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most ...mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9-associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5'UTR variant, c.-70A>T. Moreover, an adjacent 5'UTR variant, c.-69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5'UTR variants resulted in decreased NMNAT1 mRNA abundance in patients' lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE-1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu-rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1-associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat-mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness.
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