ABSTRACT
Variants in ABCA4 are responsible for autosomal‐recessive Stargardt disease and cone‐rod dystrophy. Sequence analysis of ABCA4 exons previously revealed one causative variant in each of 45 ...probands. To identify the “missing” variants in these cases, we performed multiplex ligation‐dependent probe amplification‐based deletion scanning of ABCA4. In addition, we sequenced the promoter region, fragments containing five deep‐intronic splice variants, and 15 deep‐intronic regions containing weak splice sites. Heterozygous deletions spanning ABCA4 exon 5 or exons 20–22 were found in two probands, heterozygous deep‐intronic variants were identified in six probands, and a deep‐intronic variant was found together with an exon 20–22 deletion in one proband. Based on ophthalmologic findings and characteristics of the identified exonic variants present in trans, the deep‐intronic variants V1 and V4 were predicted to be relatively mild and severe, respectively. These findings are important for proper genetic counseling and for the development of variant‐specific therapies.
A person with Stargardt disease (STGD1) carries a deletion of exons 20‐22 in one of two ABCA4 gene copies and a deep‐intronic mutation (c.5196+1137G>A) in the other copy. Both types of mutations cannot be identified using standard Sanger sequencing of the protein coding segments of the gene and require specific analyses. Deletions and deep‐intronic variants in ABCA4 can constitute up to 20% of ABCA4 mutations.
Purpose
To assess the longitudinal vision‐related quality of life among patients with CRB1‐associated inherited retinal dystrophies.
Methods
In this longitudinal questionnaire study, the National Eye ...Institute Visual Function Questionnaire (39 items, NEI VFQ‐39) was applied at baseline, two‐year follow‐up, and 4‐year follow‐up in patients with pathogenic CRB1 variants. Correction added on 20 November 2023, after first online publication: The preceding sentence has been updated in this version. Classical test theory was performed to obtain subdomain scores and in particular ‘near activities’ and ‘total composite’ scores. The Rasch analysis based on previous calibrations of the NEI VFQ‐25 was applied to create visual functioning and socio‐emotional subscales.
Results
In total, 22 patients with a CRB1‐associated retinal dystrophy were included, … with a median age of 25.0 years (interquartile range: 13–31 years) at baseline and mean follow‐up of 4.0 ± 0.3 years. Correction added on 20 November 2023, after first online publication: The preceding sentence has been updated in this version. A significant decline at 4 years was observed for ‘near activities’ (51.0 ± 23.8 vs 35.4 ± 14.7, p = 0.004) and ‘total composite’ (63.0 ± 13.1 vs 52.0 ± 12.1, p = 0.001) subdomain scores. For the Rasch‐scaled scores, the ‘visual functioning’ scale significantly decreased after 2 years (−0.89 logits; p = 0.012), but not at 4‐year follow‐up (+0.01 logits; p = 0.975). Correction added on 20 November 2023, after first online publication: In the preceding sentence, “…after 4 years…” has been corrected to “…after 2 years…” in this version. The ‘socio‐emotional’ scale also showed a significant decline after 2 years (−0.78 logits, p = 0.033) and 4 years (−0.83 logits, p = 0.021).
Conclusion
In the absence of an intervention, a decline in vision‐related quality of life is present in patients with pathogenic CRB1 variants at 4‐year follow‐up. Patient‐reported outcome measures should be included in future clinical trials, as they can be a potential indicator of disease progression and treatment efficacy.
The majority of the genetic causes of autosomal-recessive (ar) cone-rod dystrophy (CRD) are currently unknown. A combined approach of homozygosity mapping and exome sequencing revealed a homozygous ...nonsense mutation (c.565C>T p.Glu189∗) in RAB28 in a German family with three siblings with arCRD. Another homozygous nonsense mutation (c.409C>T p.Arg137∗) was identified in a family of Moroccan Jewish descent with two siblings affected by arCRD. All five affected individuals presented with hyperpigmentation in the macula, progressive loss of the visual acuity, atrophy of the retinal pigment epithelium, and severely reduced cone and rod responses on the electroretinogram. RAB28 encodes a member of the Rab subfamily of the RAS-related small GTPases. Alternative RNA splicing yields three predicted protein isoforms with alternative C-termini, which are all truncated by the nonsense mutations identified in the arCRD families in this report. Opposed to other Rab GTPases that are generally geranylgeranylated, RAB28 is predicted to be farnesylated. Staining of rat retina showed localization of RAB28 to the basal body and the ciliary rootlet of the photoreceptors. Analogous to the function of other RAB family members, RAB28 might be involved in ciliary transport in photoreceptor cells. This study reveals a crucial role for RAB28 in photoreceptor function and suggests that mutations in other Rab proteins may also be associated with retinal dystrophies.
BackgroundRetinitis pigmentosa (RP) shows substantial genetic heterogeneity. It has been estimated that in approximately 60%–80% of RP cases, the genetic diagnosis can be found using whole exome ...sequencing (WES). In this study, the purpose was to identify causative variants in individuals with genetically unexplained retinal disease, which included one consanguineous family with two affected siblings and one case with RP.MethodsTo identify the genetic defect, WES was performed in both probands, and clinical analysis was performed. To obtain insight into the function of KIAA1549 in photoreceptors, mRNA expression, knockdown and protein localisation studies were performed.ResultsThrough analysis of WES data, based on population allele frequencies, and in silico prediction tools, we identified a homozygous missense variant and a homozygous frameshift variant in KIAA1549 that segregate in two unrelated families. Kiaa1549 was found to localise at the connecting cilium of the photoreceptor cells and the synapses of the mouse retina. Both variants affect the long transcript of KIAA1549, which encodes a 1950 amino acid protein and shows prominent brain expression. The shorter transcript encodes a 734 amino acid protein with a high retinal expression and is affected by the identified missense variant. Strikingly, knockdown of the long transcript also leads to decreased expression of the short transcript likely explaining the non-syndromic retinal phenotype caused by the two variants targeting different transcripts.ConclusionIn conclusion, our results underscore the causality of segregating variants in KIAA1549 for autosomal recessive RP. Moreover, our data indicate that KIAA1549 plays a role in photoreceptor function.
Exome sequencing revealed a homozygous missense mutation (c.317C>G p.Arg106Pro) in POC1B, encoding POC1 centriolar protein B, in three siblings with autosomal-recessive cone dystrophy or cone-rod ...dystrophy and compound-heterozygous POC1B mutations (c.199_201del p.Gln67del and c.810+1G>T) in an unrelated person with cone-rod dystrophy. Upon overexpression of POC1B in human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type protein localized to the basal body of the primary cilium, whereas this localization was lost for p.Arg106Pro and p.Gln67del variant forms of POC1B. Morpholino-oligonucleotide-induced knockdown of poc1b translation in zebrafish resulted in a dose-dependent small-eye phenotype, impaired optokinetic responses, and decreased length of photoreceptor outer segments. These ocular phenotypes could partially be rescued by wild-type human POC1B mRNA, but not by c.199_201del and c.317C>G mutant human POC1B mRNAs. Yeast two-hybrid screening of a human retinal cDNA library revealed FAM161A as a binary interaction partner of POC1B. This was confirmed in coimmunoprecipitation and colocalization assays, which both showed loss of FAM161A interaction with p.Arg106Pro and p.Gln67del variant forms of POC1B. FAM161A was previously implicated in autosomal-recessive retinitis pigmentosa and shown to be located at the base of the photoreceptor connecting cilium, where it interacts with several other ciliopathy-associated proteins. Altogether, this study demonstrates that POC1B mutations result in a defect of the photoreceptor sensory cilium and thus affect cone and rod photoreceptors.
A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early ...days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.
Leber congenital amaurosis (LCA) is a rare inherited retinal disorder affecting approximately 1:50,000 people worldwide. So far, mutations in 25 genes have been associated with LCA, with
(encoding ...the Centrosomal protein of 290 kDa) being the most frequently mutated gene. The most recurrent LCA-causing
mutation, c.2991+1655A>G, causes the insertion of a pseudoexon into a variable proportion of
transcripts. We previously demonstrated that antisense oligonucleotides (AONs) have a high therapeutic potential for patients homozygously harbouring this mutation, although to date, it is unclear whether rescuing one single allele is enough to restore CEP290 function. Here, we assessed the AON efficacy at RNA, protein and cellular levels in samples that are compound heterozygous for this mutation, together with a protein-truncating mutation in
. We demonstrate that AONs can efficiently restore splicing and increase protein levels. However, due to a high variability in ciliation among the patient-derived cell lines, the efficacy of the AONs was more difficult to assess at the cellular level. This observation points towards the importance of the severity of the second allele and possibly other genetic variants present in each individual. Overall, AONs seem to be a promising tool to treat
-associated LCA, not only in homozygous but also in compound heterozygous carriers of the c.2991+1655A>G variant.
Purpose
Intravitreal anti‐vascular endothelial growth factor (VEGF) injections are an effective treatment for neovascular age‐related macular degeneration (nARMD). Bevacizumab appears to be a ...cost‐effective off‐label anti‐VEGF alternative to ranibizumab, but an optimal injection schedule has not yet been determined. In this study, we investigate whether on‐demand bevacizumab treatment every 8 weeks is non‐inferior to on‐demand bevacizumab every 4 weeks in treating nARMD.
Methods
A total of 120 nARMD patients were randomly assigned to an on‐demand regimen of intravitreal bevacizumab (IVB) every 4 (n = 60) or 8 weeks (n = 60). The primary outcome was visual acuity (VA) change after 1 year of treatment.
Results
Visual acuity (VA) improved between baseline and 1 year in both treatment groups. The mean change in the VA score at 1 year was not significantly different between bevacizumab administration on‐demand every 4 weeks 5.6 ± 10.2 Early Treatment Diabetic Retinopathy Study (ETDRS) letter or 8 weeks (4.6 ± 12.0 ETDRS letters). A reduction in the central retinal thickness was observed in both groups. At 1 year, the mean decrease in central foveal thickness ranged from 61 ± 90 μm in the 4‐week group to 91 ± 83 μm in the 8‐week group (p = 0.07). The mean number of IVB treatments during the study period was 8.7 ± 2.3 in the 4‐week group and 5.9 ± 1.0 in the 8‐week group.
Conclusion
At 1 year, bevacizumab administration on‐demand every 8 weeks was non‐inferior to administration every 4 weeks. The results strongly suggest that bevacizumab acts longer than 4 weeks in ARMD, reducing the burden of injections for patients.
Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify ...the missing heritability.
Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects.
In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects.
Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.
USH2A mutations are an important cause of retinitis pigmentosa (RP) with or without congenital sensorineural hearing impairment. We studied genotype-phenotype correlations and compared visual ...prognosis in Usher syndrome type IIa and nonsyndromic RP.
Clinic-based, longitudinal, multicenter study.
Consecutive patients with Usher syndrome type IIa (n = 152) and nonsyndromic RP (n = 73) resulting from USH2A mutations from ophthalmogenetic clinics in the Netherlands and Belgium.
Data on clinical characteristics, visual acuity, visual field measurements, retinal imaging, and electrophysiologic features were extracted from medical charts over a mean follow-up of 9 years. Cumulative lifetime risks of low vision and blindness were estimated using Kaplan-Meier survival analysis.
Low vision and blindness.
Participant groups had similar distributions of gender (48% vs. 45% males in Usher syndrome type IIa vs. nonsydromic RP; P = 0.8), ethnicity (97% vs. 99% European; P = 0.3), and median follow-up time (6.5 years vs. 3 years; P = 0.3). Usher syndrome type IIa patients demonstrated symptoms at a younger age (median age, 15 years vs. 25 years; P < 0.001), were diagnosed earlier (median age, 26 years vs. 36.5 years; P < 0.001), and became visually impaired 13 years earlier (median age, 41 years vs. 54 years; P < 0.001) based on VF and 18 years earlier based on VA (median age, 54 years vs. 72 years; P < 0.001) than nonsyndromic RP patients. The presence of 2 truncating mutations in USH2A was associated mostly with the syndromic phenotype, whereas other combinations were present in both groups. We found novel variants in Usher syndrome type IIa (25%) and nonsyndromic RP (19%): 29 missense mutations, 10 indels, 14 nonsense mutations, 9 frameshift mutations, and 5 splice-site mutations.
Most patients with USH2A-associated RP have severe visual impairment by age 50. However, those with Usher syndrome type IIa have an earlier decline of visual function and a higher cumulative risk of visual impairment than those without nonsyndromic RP. Complete loss of function of the USH2A protein predisposes to Usher syndrome type IIa, but remnant protein function can lead to RP with or without hearing loss.
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