Identification of novel mutations in X-linked retinitis pigmentosa families and implications for diagnostic testing

Contains fulltext : 69886.pdf (publisher's version ) (Open Access)PURPOSE: The goal of this study was to identify mutations in X-chromosomal genes associated with retinitis pigmentosa (RP) in patients from Germany, The Netherlands, Denmark, and Switzerland. METHODS: In addition to all coding exons of RP2, exons 1 through 15, 9a, ORF15, 15a and 15b of RPGR were screened for mutations. PCR products were amplified from genomic DNA extracted from blood samples and analyzed by direct sequencing. In one family with apparently dominant inheritance of RP, linkage analysis identified an interval on the X chromosome containing RPGR, and mutation screening revealed a pathogenic variant in this gene. Patients of this family were examined clinically and by X-inactivation studies. RESULTS: This study included 141 RP families with possible X-chromosomal inheritance. In total, we identified 46 families with pathogenic sequence alterations in RPGR and RP2, of which 17 mutations have not been described previously. Two of the novel mutations represent the most 3'-terminal pathogenic sequence variants in RPGR and RP2 reported to date. In exon ORF15 of RPGR, we found eight novel and 14 known mutations. All lead to a disruption of open reading frame. Of the families with suggested X-chromosomal inheritance, 35% showed mutations in ORF15. In addition, we found five novel mutations in other exons of RPGR and four in RP2. Deletions in ORF15 of RPGR were identified in three families in which female carriers showed variable manifestation of the phenotype. Furthermore, an ORF15 mutation was found in an RP patient who additionally carries a 6.4 kbp deletion downstream of the coding region of exon ORF15. We did not identify mutations in 39 sporadic male cases from Switzerland. CONCLUSIONS: RPGR mutations were confirmed to be the most frequent cause of RP in families with an X-chromosomal inheritance pattern. We propose a screening strategy to provide molecular diagnostics in these families

Gene Therapeutic Approach Using Mutation-adapted U1 snRNA to Correct a RPGR Splice Defect in Patient-derived Cells

Retinitis pigmentosa (RP) is a disease that primarily affects the peripheral retina and ultimately causes visual impairment. X-chromosomal forms of RP are frequently caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. We show that the novel splice donor site (SDS) mutation c.1245+3A>T in intron 10 of RPGR cosegregates with RP in a five-generation Caucasian family. The mutation causes in-frame skipping of exon 10 from RPGR transcripts in patient-derived primary fibroblasts. To correct the splice defect, we developed a gene therapeutic approach using mutation-adapted U1 small nuclear RNA (U1). U1 is required for SDS recognition of pre-mRNAs and initiates the splice process. The mutation described herein interferes with the recognition of the SDS by U1. To overcome the deleterious effects of the mutation, we generated four U1 isoforms with increasing complementarity to the SDS. Lentiviral particles were used to transduce patient-derived fibroblasts with these U1 variants. Full complementarity of U1 corrects the splice defect partially and increases recognition of the mutant SDS. The therapeutic effect is U1-concentration dependent as we show for endogenously expressed RPGR transcripts in patient-derived cells. U1-based gene therapeutic approaches constitute promising technologies to treat SDS mutations in inherited diseases including X-linked RP

Localizing the RPGR protein along the cilium: a new method to determine efficacies to treat RPGR mutations

Retinal dystrophies constitute a group of clinically and genetically heterogeneous diseases that cause visual impairment. As treatments are not readily available, readout assays performed in patient-derived cells can aid in the development and comparative analysis of therapeutic approaches. We describe a new method with which the localization of the retinitis pigmentosa GTPase regulator (RPGR) protein along the cilium can be used as a measure for treatment efficacy. In a patient-derived fibroblast cell line, we found that the RPGR protein is mislocalized along the ciliary axoneme. The patient carried a point mutation that leads to skipping of RPGR exon 10. We confirmed that this skipping is causative for the impaired localization of RPGR using a U7 small nuclear RNA (U7snRNA)-based antisense approach in control cells. Treatment of the patient-derived fibroblasts with therapeutic U1snRNA significantly corrected the proteins’ mislocalization. In this proof of principle study, we show that detecting the RPGR protein along the cilium provides a reliable and quantifiable readout assay to evaluate the efficacy of therapies intended to correct or silence RPGR gene mutations. This method opens the possibility to compare different therapeutic agents, and thus facilitate the identification of treatment options for the clinically and molecularly complex RPGR-associated diseases

Identification of Novel and Recurrent Disease-Causing Mutations in Retinal Dystrophies Using Whole Exome Sequencing (WES): Benefits and Limitations

Inherited retinal dystrophies (IRDs) are Mendelian diseases with tremendous genetic and phenotypic heterogeneity. Identification of the underlying genetic basis of these dystrophies is therefore challenging. In this study we employed whole exome sequencing (WES) in 11 families with IRDs and identified disease-causing variants in 8 of them. Sequence analysis of about 250 IRD-associated genes revealed 3 previously reported disease-associated variants in RHO, BEST1 and RP1. We further identified 5 novel pathogenic variants in RPGRIP1 (p.Ser964Profs*37), PRPF8 (p.Tyr2334Leufs*51), CDHR1 (p.Pro133Arg and c.439-17G>A) and PRPF31 (p.Glu183_Met193dup). In addition to confirming the power of WES in genetic diagnosis of IRDs, we document challenges in data analysis and show cases where the underlying genetic causes of IRDs were missed by WES and required additional techniques. For example, the mutation c.439-17G>A in CDHR1 would be rated unlikely applying the standardWES analysis. Only transcript analysis in patient fibroblasts confirmed the pathogenic nature of this variant that affected splicing of CDHR1 by activating a cryptic splice-acceptor site. In another example, a 33-base pair duplication in PRPF31 missed by WES could be identified only via targeted analysis by Sanger sequencing. We discuss the advantages and challenges of using WES to identify mutations in heterogeneous diseases like IRDs

Structural and functional abnormalities of retinal ribbon synapses due to Cacna2d4 mutation

PURPOSE: In a spontaneous mutant substrain of C57BL/10 mice, severely affected retinal ribbon-type synapses have been described. The retinopathy was accompanied by a substantial loss in the activities of the second-order neurons. Rod photoreceptor responses were maintained with reduced amplitude, whereas cone activities were absent. This study was conducted to identify the genetic defect underlying this hitherto unknown autosomal recessive cone-rod dysfunction. METHODS: Genome-wide linkage analysis and screening of positional candidate genes were used to identify the causative mutation. Tissue-specific transcriptional activity of the defective gene was determined by Northern blot analysis and RT-PCR approaches. The number of cone photoreceptors was estimated by immunohistochemistry. RESULTS: The mutation was localized to a 275-kb region of chromosome 6. Within this candidate interval, a homozygous frameshift mutation (c.2367insC) was identified in the Cacna2d4 gene of affected animals. This gene codes for an L-type calcium channel auxiliary subunit of the alpha2delta type. The mutation introduces a premature stop codon that truncates one third of the predicted Cacna2d4 protein. A severe reduction in Cacna2d4 transcript levels observed in mutant retinas probably results in the lack of Cacna2d4 protein. The mutation leads to significant loss of rods, whereas the number of cone cells remains unaffected until 6 weeks of age. CONCLUSIONS: The Cacna2d4 mutation underlies a novel channelopathy leading to cone-rod dysfunction in the visual system of mice and provides a new candidate gene for human retinal disorders including night blindness, retinitis pigmentosa, and cone-rod dystrophies

Analysis of alternative splicing <i>in vitro</i>.

<p><b>(a)</b> Primer design to capture putative intron retention due to c.439-17G>A variant in <i>CDHR1</i>. <b>(b)</b> Agarose gel electrophoresis of RT-PCR showing a 140bp PCR product only in the patient cell-line (white asterix). No products were seen in control cell line or water control. <b>(c)</b> Bottom: Sequence chromatograph shows a clear retention of 15bp from intron 5 in the RT-PCR product, generated due to the cryptic splice-site activation. Top: Schematic representing the exon-intron boundaries as observed in the RT-PCR product.</p

Clinical phenotypes of patient, variant descriptions and pathogenicity prediction of the variants identified in this study.

<p>Clinical phenotypes of patient, variant descriptions and pathogenicity prediction of the variants identified in this study.</p

<i>CDHR1</i> intronic variant.

<p>Snapshot of Alamut visual showing an intronic variant (c.439-17G>A) in patient 26007 (green rectangle). The bam alignment file clearly shows a heterozygous variant 17bp upstream from exon 6. Inset: Snapshot of splicing prediction algorithms (<i>in silico</i>) shows a strong cryptic splice acceptor gain at the site of the variant (blue rectangle). Values are comparable to that of canonical splice acceptor site. Red rectangle shows a stop codon in-frame to the cryptic splice activator site.</p

Prediction scores of canonical (c.439) and cryptic splice site (c.439-17) by five prediction algorithms due to the variant c.439-17G>A in <i>CDHR1</i> gene.

<p>Prediction scores of canonical (c.439) and cryptic splice site (c.439-17) by five prediction algorithms due to the variant c.439-17G>A in <i>CDHR1</i> gene.</p