John Neidhardt

Hereditary retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different forms of RD can be caused by mutations in >100 genes, including >1600 exons. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. So far, NGS is not routinely used in gene diagnostics. We developed a diagnostic NGS pipeline to identify mutations in 170 genetically and clinically unselected RD patients. NGS was applied to 105 RD-associated genes. Underrepresented regions were examined by Sanger sequencing. The NGS approach was successfully established using cases with known sequence alterations. Depending on the initial clinical diagnosis, we identified likely causative mutations in 55% of retinitis pigmentosa and 80% of Bardet–Biedl or Usher syndrome cases. Seventy-one novel mutations in 40 genes were newly associated with RD. The genes USH2A, EYS, ABCA4, and RHO were more frequently affected than others. Occasionally, cases carried mutations in more than one RD-associated gene. In addition, we found possible dominant de-novo mutations in cases with sporadic RD, which implies consequences for counseling of patients and families. NGS-based mutation analyses are reliable and cost-efficient approaches in gene diagnostics of genetically heterogeneous diseases like RD.

Splicing is an essential cellular process to generate mature transcripts from pre-mRNA. It requires the splice factor U1 small nuclear ribonucleoprotein (U1), which promotes exon recognition by base-pairing interaction with the splice donor site (SD). After U1 dissociation, exon recognition is maintained by U6 small nuclear ribonucleoproteins (U6). It has been shown that SD mutations lower the binding affinity of U1 and cause splice defects in about 10% of patients with monogenetic diseases. U1 isoforms specifically designed to bind the mutated SD with increased affinity can correct these splice defects. We investigated the applicability of this gene therapeutic approach for different mutated SD positions. A minigene-based splicing assay was established to study a typical SD derived from the gene BBS1. We found that mutations at seven SD positions caused splice defects. In four cases, mutation-adapted U1 isoforms completely corrected these splice defects. Partial correction was found for splice defects induced by the mutation at SD position +5. The limited therapeutic efficacy at this position was alleviated by applying a combined treatment with mutation-adapted U1 and U6. The sequence complementarity between U6 and three SD positions (+4, +5,and +6) was relevant for the outcome of the therapy. Between 30 and 100% of the normal transcripts can be restored. The treatment significantly decreased both exon skipping and intron retention. Massive missplicing of off-target transcripts was not detected. Our study helps to assess the therapeutic efficacy of mutation-adapted U snRNAs in gene therapy and illustrates their strong potential to correct splice defects, which cause many different inherited conditions.

Bardet‐Biedl syndrome (BBS) is a multisystem disorder caused by ciliary defects. To date, mutations in 15 genes have been associated with the disease and BBS1 is most frequently affected in patients with BBS. The use of homozygosity mapping in a large consanguineous family allowed us to identify the splice donor site (SD) mutation c.479G>A in exon 5 of BBS1. Clinically affected family members show symptoms of retinitis pigmentosa (RP) but lack other primary features that would clearly support the diagnosis of BBS. In agreement with this exceptionally mild BBS1‐associated phenotype, we did not detect obvious ciliary defects in patient‐derived cells. SDs are bound by the U1 small nuclear RNA (U1), a process that initiates exon recognition during splicing. The mutation described herein interferes with U1 binding and induces aberrant splicing of BBS1. For a gene therapeutic approach, we have adapted the sequence of U1 to increase its complementarity to the mutated SD. Lentiviral treatment of patient‐derived fibroblasts with the adapted U1 partially corrected aberrant splicing of endogenously expressed BBS1 transcripts. This therapeutic effect was dose‐dependent. Our results show that the adaptation of U1 can correct pathogenic effects of splice donor site mutations and suggest a high potential for gene therapy.Hum Mutat 32:815–824, 2011.

Purpose.: To identify the causative mutation leading to autosomal dominant macular dystrophy, cone dystrophy, and cone–rod dystrophy in a five-generation family and to explain the high intrafamilial phenotypic variation by identifying possible modifier genes. Methods.: Fifteen family members were investigated by detailed ophthalmic and electrophysiologic phenotyping. Mutation screening was initially performed with microarrays that detect known mutations in genes associated with retinal degeneration. Furthermore, the patients' genomic DNA was analyzed by sequencing analysis of PRPH2, ABCA4, and ROM1. Results.: Heterozygous mutations were identified in three genes and showed five different combinations within the studied family. All clearly affected family members carried the heterozygous PRPH2 mutation p.R172W. Patients with heterozygous sequence alterations only in ROM1 (p.R229H) or ABCA4 (p.V2050L) showed a mild ocular phenotype and were otherwise asymptomatic. The phenotypic severity of patients carrying the PRPH2 mutation increased with an additional mutation in ROM1. Patients carrying all three mutations were the most severely affected. Conclusions.: Features of a PRPH2-associated phenotype might be modulated by additional mutations in other genes (in this family ABCA4 and/or ROM1) accounting for intrafamilial variability and resulting in a cumulative effect worsening the phenotype. Families showing a variable macular dystrophy phenotype caused by mutations in PRPH2 should be tested for additional mutations in ABCA4 and ROM1, as they may alter the progression of the PRPH2 phenotype. This testing will influence genetic counseling, as patients with additional mutations may be confronted with a faster progression of visual loss.

Purpose.: The majority of patients with X chromosome-linked retinitis pigmentosa (XlRP) carry mutations in the RPGR gene. The authors studied whether patients with RPGR mutations show additional splice defects that may interfere with RPGR properties. Methods.: Patient-derived cell lines with RPGR mutations were raised in suspension. To verify mutations, direct sequencing of PCR products was performed. Patient-specific alterations in RPGR splicing were analyzed by RT-PCR and confirmed by sequencing. Tissue-specific expression levels of RPGR splice variants were quantified by real-time PCR using pools of different human donor tissues. Results.: The authors analyzed the splicing of RPGR in seven RP patient-derived lymphoblastoid cell lines carrying hemizygous RPGR mutations. In three patient cell lines, they identified and characterized splice defects that were present in addition to a mutation. These splice defects were likely to interfere with normal RPGR properties. Furthermore, they identified four novel RPGR transcripts, either containing a new exon termed 11a or skipping the constitutive exons 12, 14, and 15. Novel and known RPGR isoforms were found to be differentially regulated in several human tissues. In human retina, approximately 10% of RPGR transcripts are alternatively spliced between exons 9 and 15. Conclusions.: These findings show that splicing of RPGR is precisely regulated in a tissue-dependent fashion and suggest that mutations in RPGR frequently interfere with the expression of alternative transcript isoforms. These results implicate the importance of RPGR transcript analysis in patients with RP. The authors further discuss RPGR splicing as a modifier of different disease phenotypes described in patients with XlRP.

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Retinitis pigmentosa (RP) is a degenerative retinopathy leading to visual impairment in more than 1.5 million patients worldwide. Splice site (SS) mutations cause various diseases including RP. Most exonic donor splice‐site (DS) mutations are reported at the last nucleotide of an exon and over 95% of them are predicted to result in missplicing. A novel human mutation at the last nucleotide of exon 4 in rhodopsin (RHO, c.936G>A) is shown to generate two misspliced transcripts in COS 7 cells and retinal explants. One of these transcripts skips exon 4 whereas the other activates a cryptic DS. Both are predicted to result in truncated RHO, explaining the pathogenic mechanism underlying the patient's RP phenotype. U1 snRNA‐mediated DS recognition is a key step in the splicing process. As a therapeutic strategy, U1 snRNAs were adapted to the novel RHO mutation and tested for its potential to reverse missplicing. The rescue efficiency for misspliced transcripts of RHO was examined by quantitative RT‐PCR. Using mutation‐adapted U1 snRNA, we observed significantly reduced exon skipping that reached wild‐type levels. Nevertheless, activation of the cryptic splice site (CS) was still detected. To test the feasibility of the strategy for mutations that only cause exon skipping, we inactivated the CS. Indeed, adapted U1 snRNA was able to rescue almost 90% of misspliced transcripts. This study shows that modified U1 snRNAs constitute a promising therapeutic strategy to treat DS mutations. Our findings have implications for various diseases caused by similar mutations. Hum Mutat 0, 1–10, 2008,

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. 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. 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. 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.

Retinitis pigmentosa (RP) constitutes a major cause of blindness and the Retinitis Pigmentosa GTPase Regulator (RPGR) gene accounts for up to 80% of all X-linked RP cases. A novel isoform of RPGR, expressed in the human retina, was identified and characterized. It truncates the Regulator of Chromosome Condensation 1 (RCC1) homologous protein domain (RCC1h) of RPGR and mediates the formation of isoform-specific complexes with the RPGR-interacting protein 1 (RPGRIP1). Immunohistochemistry localized the novel RPGR isoform predominantly to inner segments of cone photoreceptors, where it colocalizes with RPGRIP1 in the human retina. In a patient with a mild RP phenotype, we identified a nucleotide substitution in a splicing regulator, which leads to 3.5 times higher levels of the transcripts coding for the novel RPGR isoform. The nucleotide substitution affects regulated alternative splicing of the novel RPGR isoform and suggests a tight adjustment of splicing as a prerequisite for proper function of photoreceptors. Hum Mutat 28(8), 797–807, 2007.