26 research outputs found
Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4
No full textThe discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1
The ADAMTS18 gene is responsible for autosomal recessive early onset severe retinal dystrophy
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117740.pdf (Publisher’s version ) (Open Access)BACKGROUND: Inherited retinal dystrophies, including Retinitis Pigmentosa and Leber Congenital Amaurosis among others, are a group of genetically heterogeneous disorders that lead to variable degrees of visual deficits. They can be caused by mutations in over 100 genes and there is evidence for the presence of as yet unidentified genes in a significant proportion of patients. We aimed at identifying a novel gene for an autosomal recessive form of early onset severe retinal dystrophy in a patient carrying no previously described mutations in known genes. METHODS: An integrated strategy including homozygosity mapping and whole exome sequencing was used to identify the responsible mutation. Functional tests were performed in the medaka fish (Oryzias latipes) model organism to gain further insight into the pathogenic role of the ADAMTS18 gene in eye and central nervous system (CNS) dysfunction. RESULTS: This study identified, in the analyzed patient, a homozygous missense mutation in the ADAMTS18 gene, which was recently linked to Knobloch syndrome, a rare developmental disorder that affects the eye and the occipital skull. In vivo gene knockdown performed in medaka fish confirmed both that the mutation has a pathogenic role and that the inactivation of this gene has a deleterious effect on photoreceptor cell function. CONCLUSION: This study reveals that mutations in the ADAMTS18 gene can cause a broad phenotypic spectrum of eye disorders and contribute to shed further light on the complexity of retinal diseases
Antisense Oligonucleotide (AON)-based Therapy for Leber Congenital Amaurosis Caused by a Frequent Mutation in CEP290
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109558.pdf (Publisher’s version ) (Open Access)Leber congenital amaurosis (LCA) is the most severe form of inherited retinal degeneration, with an onset in the first year of life. The most frequent mutation that causes LCA, present in at least 10% of individuals with LCA from North-American and Northern-European descent, is an intronic mutation in CEP290 that results in the inclusion of an aberrant exon in the CEP290 mRNA. Here, we describe a genetic therapy approach that is based on antisense oligonucleotides (AONs), small RNA molecules that are able to redirect normal splicing of aberrantly processed pre-mRNA. Immortalized lymphoblastoid cells of individuals with LCA homozygously carrying the intronic CEP290 mutation were transfected with several AONs that target the aberrant exon that is incorporated in the mutant CEP290 mRNA. Subsequent RNA isolation and reverse transcription-PCR analysis revealed that a number of AONs were capable of almost fully redirecting normal CEP290 splicing, in a dose-dependent manner. Other AONs however, displayed no effect on CEP290 splicing at all, indicating that the rescue of aberrant CEP290 splicing shows a high degree of sequence specificity. Together, our data show that AON-based therapy is a promising therapeutic approach for CEP290-associated LCA that warrants future research in animal models to develop a cure for this blinding disease
Homozygosity mapping reveals PDE6C mutations in patients with early-onset cone photoreceptor disorders
No full textCone photoreceptor disorders form a clinical spectrum of diseases that include progressive cone dystrophy (CD) and complete and incomplete achromatopsia (ACHM). The underlying disease mechanisms of autosomal recessive (ar)CD are largely unknown. Our aim was to identify causative genes for these disorders by genome-wide homozygosity mapping. We investigated 75 ACHM, 97 arCD, and 20 early-onset arCD probands and excluded the involvement of known genes for ACHM and arCD. Subsequently, we performed high-resolution SNP analysis and identified large homozygous regions spanning the PDE6C gene in one sibling pair with early-onset arCD and one sibling pair with incomplete ACHM. The PDE6C gene encodes the cone alpha subunit of cyclic guanosine monophosphate (cGMP) phosphodiesterase, which converts cGMP to 5'-GMP, and thereby plays an essential role in cone phototransduction. Sequence analysis of the coding region of PDE6C revealed homozygous missense mutations (p.R29W, p.Y323N) in both sibling pairs. Sequence analysis of 104 probands with arCD and 10 probands with ACHM revealed compound heterozygous PDE6C mutations in three complete ACHM patients from two families. One patient had a frameshift mutation and a splice defect; the other two had a splice defect and a missense variant (p.M455V). Cross-sectional retinal imaging via optical coherence tomography revealed a more pronounced absence of cone photoreceptors in patients with ACHM compared to patients with early-onset arCD. Our findings identify PDE6C as a gene for cone photoreceptor disorders and show that arCD and ACHM constitute genetically and clinically overlapping phenotypes
BBS1 Mutations in a Wide Spectrum of Phenotypes Ranging From Nonsyndromic Retinitis Pigmentosa to Bardet-Biedl Syndrome
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Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4
No full textThe high allelic heterogeneity in Stargardt disease (STGD1) complicates the design of intervention strategies. A significant proportion of pathogenic intronic ABCA4 variants alters the pre-mRNA splicing process. Antisense oligonucleotides (AONs) are an attractive yet mutation-specific therapeutic strategy to restore these splicing defects. In this study, we experimentally assessed the potential of a splicing modulation therapy to target multiple intronic ABCA4 variants. AONs were inserted into U7snRNA gene cassettes and tested in midigene-based splice assays. Five potent antisense sequences were selected to generate a multiple U7snRNA cassette construct, and this combination vector showed substantial rescue of all of the splicing defects. Therefore, the combination cassette was used for viral synthesis and assessment in patient-derived photoreceptor precursor cells (PPCs). Simultaneous delivery of several modified U7snRNAs through a single AAV, however, did not show substantial splicing correction, probably due to suboptimal transduction efficiency in PPCs and/or a heterogeneous viral population containing incomplete AAV genomes. Overall, these data demonstrate the potential of the U7snRNA system to rescue multiple splicing defects, but also suggest that AAV-associated challenges are still a limiting step, underscoring the need for further optimization before implementing this strategy as a potential treatment for STGD1
Novel insights into the molecular pathogenesis of CYP4V2-associated Bietti's retinal dystrophy
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153526.pdf (Publisher’s version ) (Open Access)Bietti's crystalline dystrophy (BCD) is a rare, autosomal recessive retinal degenerative disease associated with mutations in CYP4V2. In this study, we describe the genetic and clinical findings in 19 unrelated BCD patients recruited from five international retinal dystrophy clinics. Patients underwent ophthalmic examinations and were screened for CYP4V2 mutations by Sanger sequencing and quantitative polymerase chain reaction (qPCR) copy number variation screening. Eight CYP4V2 mutations were found in 10/19 patients, including three patients in whom only monoallelic mutations were detected. Four novel mutations were identified: c.604G>A; p.(Glu202Lys), c.242C>G; p.(Thr81Arg), c.604+4A>G; p.(?), and c.1249dup; p.(Thr417Asnfs*2). In addition, we identified a heterozygous paternally inherited genomic deletion of at least 3.8 Mb, encompassing the complete CYP4V2 gene and several other genes, which is novel. Clinically, patients demonstrated phenotypic variability, predominantly showing choroidal sclerosis, attenuated vessels, and crystalline deposits of varying degrees of severity. To our knowledge, our study reports the first heterozygous CYP4V2 deletion and hence a novel mutational mechanism underlying BCD. Our results emphasize the importance of copy number screening in BCD. Finally, the identification of CYP4V2-negative patients with indistinguishable phenotypes from CYP4V2-positive patients might suggest the presence of mutations outside the coding regions of CYP4V2, or locus heterogeneity, which is unreported so far
Maternal Uniparental Isodisomy of Chromosome 6 Reveals a TULP1 Mutation as a Novel Cause of Cone Dysfunction
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118781.pdf (Publisher’s version ) (Open Access)PURPOSE: The majority of the genetic causes of autosomal recessive (ar) cone dystrophy (CD) and cone-rod dystrophy (CRD) are currently unknown. We used a high-resolution homozygosity mapping approach in a cohort of patients with CD or CRD to identify new genes for ar cone disorders. DESIGN: Case series. PARTICIPANTS: A cohort of 159 patients with ar CD and 91 patients with CRD. METHODS: The genomes of 83 patients with ar CD and 73 patients with CRD were analyzed for homozygous regions using single nucleotide polymorphism (SNP) microarrays. One patient showed homozygosity of SNPs across chromosome 6, and segregation analysis was performed using microsatellite markers. Direct sequencing of all retinal disease genes on chromosome 6 revealed a novel pathogenic TULP1 mutation in this patient. A cohort of 159 individuals with CD and 91 individuals with CRD was screened for this particular mutation using the restriction enzyme HhaI. The medical history of patients carrying the TULP1 mutation was reviewed and additional ophthalmic examinations were performed, including electroretinography (ERG), perimetry, optical coherence tomography (OCT), fundus autofluorescence (FAF), and fundus photography. MAIN OUTCOME MEASURES: TULP1 mutations, age at diagnosis, visual acuity, fundus appearance, color vision defects, visual field, ERG, FAF, and OCT findings. RESULTS: In 1 patient, homozygosity mapping and subsequent segregation analysis revealed maternal uniparental disomy (UPD) of chromosome 6. A novel homozygous missense mutation (p.Arg420Ser) was identified in TULP1, whereas no mutations were detected in other retinal disease genes on chromosome 6. The mutation affects a highly conserved amino acid residue in the Tubby domain and is predicted to be pathogenic. The same homozygous mutation was also identified in an additional, unrelated patient with CRD. Both patients carrying the p.Arg420Ser mutation presented with a bull's eye maculopathy. The first patient had progressive loss of visual acuity with a relatively preserved ERG, whereas the second patient developed loss of visual acuity, peripheral degeneration, and severely reduced ERG responses in a cone-rod pattern. CONCLUSIONS: Maternal UPD of chromosome 6 unmasked a mutation in the TULP1 gene as a novel cause of cone dysfunction. This expands the disease spectrum of TULP1 mutations from Leber congenital amaurosis and early-onset retinitis pigmentosa to cone-dominated disease. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article
A missense mutation in the splicing factor gene DHX38 is associated with early-onset retinitis pigmentosa with macular coloboma
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137807.pdf (Publisher’s version ) (Open Access)BACKGROUND: Retinitis pigmentosa (RP) is the most frequent inherited retinal disease, which shows a relatively high incidence of the autosomal-recessive form in Pakistan. METHODS: Genome-wide high-density single-nucleotide polymorphism (SNP) microarrays were used to identify homozygous regions shared by affected individuals of one consanguineous family. DNA of three affected and two healthy siblings was used for SNP genotyping. Genotyping data were then analysed by Homozygosity Mapper. DNA of the proband was further analysed employing exome sequencing. RESULTS: Homozygosity mapping revealed a single homozygous region on chromosome 16, shared by three affected individuals. Subsequent exome sequencing identified a novel missense mutation, c.995G>A; p.(Gly332Asp), in DHX38. This mutation was found to be present in a homozygous state in four affected individuals while two healthy siblings and the parents of the affected persons were heterozygous for this mutation. This variant thereby yields a logarithm of the odds (LOD) score of 3.25, which is highly suggestive for linkage. This variant was neither detected in 180 ethnically matched control individuals, nor in 7540 Africans or Caucasians and an in-house database that contained the exome data of 400 individuals. CONCLUSIONS: By combining genome-wide homozygosity mapping and exome sequencing, a novel missense mutation was identified in the DHX38 gene that encodes the pre-mRNA splicing factor PRP16, in a Pakistani family with early-onset autosomal-recessive RP. The phenotype is different from those associated with other retinal pre-mRNA splicing factors and DHX38 is the first pre-mRNA splicing gene that is putatively associated with autosomal-recessive inherited RP
Antisense Oligonucleotide-based Splice Correction for USH2A-associated Retinal Degeneration Caused by a Frequent Deep-intronic Mutation
No full textUsher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G) was reported in 2012, leading to the insertion of a pseudoexon (PE40) into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs) to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation
