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.
Citation: Van Cauwenbergh C, Coppieters F, Roels D, De Jaegere S, Flipts H, De Zaeytijd J, et al. (2017) Mutations in Splicing Factor Genes Are a Major Cause of Autosomal Dominant Retinitis Pigmentosa in Belgian Families. PLoS ONE 12(1): e0170038. https://doi.org/10.1371/journal.pone.0170038
Editor: Alfred S. Lewin, University of Florida, UNITED STATES
Received: October 23, 2016; Accepted: December 27, 2016; Published: January 11, 2017
Copyright: © 2017 Van Cauwenbergh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This study was supported by Belspo (IAP project P7/43 Belgian Medical Genomics Initiative) to EDB, by the Ghent University Special Research Fund (BOF15/GOA/011) to EDB, by the Research Foundation Flanders (FWO) (EYE-splice, G0C6715N) to EDB and FC, by the Research in Ophthalmology (FRO) to CVC. FC is recipient of postdoctoral fellowship of the FWO, EDB and BPL are Senior Clinical Investigators of the FWO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Retinitis pigmentosa (RP) represents the most frequent subtype of inherited dystrophies (iRDs) caused by progressive loss of photoreceptors. The first symptoms in adolescence or early adulthood include night blindness, followed by progressive loss of peripheral visual field in daylight, and eventually culminating in severe visual impairment or blindness after several decades. All modes of Mendelian inheritance can be found in RP, with autosomal dominant (ad) inheritance accounting for 30% to 40% of RP, depending on the population studied . While to date 27 genes and one locus have been identified (RetNet, https://sph.uth.edu/retnet, November 2016), they can only explain 50% to 70% of adRP cases [2,3]. During the last decade, mutation identification studies have shifted from screening of a set of known mutations (e.g. using APEX chip, www.asperbio.com) to targeted next-generation sequencing (NGS) of the coding region of large gene panels [4,5], whole exome sequencing (WES)  and whole genome sequencing .
Here, we report molecular findings in 86 Belgian families with adRP, identifying 48 mutations, including 17 novel mutations in PRPH2, PRPF8, RHO, RP1, SNRNP200, and TOPORS.
Materials and Methods
This study was conducted following the tenets of the Declaration of Helsinki and ethical approval was given by the local ethics committee. All Belgian patients were enrolled in a clinical context. We followed the standard routine practice and obtained verbal consent by the referring physician in agreement with the Belgian legislation. RP was diagnosed based on measurement of best-corrected visual acuity, slit-lamp biomicroscopy, and fundus photography. Additional tests included Goldmann kinetic perimetry, electroretinography, spectral domain optical coherence tomography, and autofluorescence imaging.
Genomic DNA (gDNA) was extracted from leukocytes using the QIAamp DNA mini kit (Qiagen, Antwerp, Belgium), the Gentra Puregene Cell kit (Qiagen, Antwerp, Belgium), or the ReliaPrep Large Volume HT gDNA Isolation System (Promega, Leiden, The Netherlands) according to the manufacturer’s protocols.
Our overall cohort consists of 86 unrelated Belgian index patients, collected over the past ten years, originating from families with at least two affected generations, of which n = 49 with more than two generations, and n = 38 with male-to-male transmission.
APEX chip testing
The commercially available arrayed primer extension microarray chip (APEX chip, Asper Biotech, Tartu, Estonia) was a standard test between November 2007 and December 2012. The initial APEX chip version (v. 2.0) was used from November 2007 to November 2008 and included 353 mutations in 13 adRP genes. This chip was regularly updated with new mutations. The latest version (v. 3.0) included 414 mutations in 16 genes.
Genome-wide linkage analysis and targeted next-generation sequencing (NGS)
Seven families underwent genome-wide linkage analysis. Inclusion criteria were three or more generations of affected members, male-to-male transmission and access to at least six samples from healthy and affected family members. Genome-wide SNP chip genotyping (HumanCytoSNP-12 BeadChip, Illumina) and multipoint linkage analysis (Merlin, dominant model, 95% penetrance, disease allele frequency of 0.0001) was performed on all available family members. A customized microsatellite panel working under uniform PCR conditions was designed for segregation analysis of known adRP genes and the RP63 locus on chromosome 6q23 (S1 Table). Data analysis was performed using the GeneMapper software (Applied Biosystems). Next, adRP genes were selected for downstream NGS analysis based on their presence in loci with the highest LOD-scores.
PCR and Sanger sequencing
All index patients collected between 2006 and 2012 were tested for mutations in the exons and intron-exon boundaries of the four most prevalent adRP genes (RP1, RHO, PRPH2, PRPF31). Mutations found by other techniques were confirmed using PCR and Sanger sequencing (https://pxlence.com; primers available on request).
All index patients were screened for the recurrent NR2E3 mutation c.166G>A p.(Gly56Arg) and the recurrent PROM1 mutation c.1117C>T p.(Arg373Cys).
Starting from 2012, a targeted NGS panel was introduced using a flexible protocol, consisting of singleplex PCR followed by NexteraXT library preparation and sequencing on a MiSeq instrument . The CLC Genomics Workbench v.6 (Qiagen) was employed for read mapping against the hg19 human reference genome and variant calling. To date, our diagnostic panel consists of ten adRP genes (CRX, PRPF6, PRPF8, PRPF31, PRPH2, RDH12, RHO, RPE65, RP1, SNRNP200).
Whole exome sequencing (WES)
Targeted WES was implemented in 2015. Whole exome enrichment was performed using the SureSelectXT human All Exon V5 enrichment kit (Agilent) followed by sequencing on a NextSeq500 (Illumina). The CLC Genomics Workbench (v. 7.5.4, Qiagen) was employed for read mapping against the hg19 human reference genome, and variant calling. Annotation and filtering of variants was done using an in-house developed strategy. Based on variant allele frequency, variants were categorized as heterozygous (20%–70%) or homozygous (>70%). Variant filtering was performed against a list of RetNet genes (gene panel v. 4, 226 genes).
A customized array comparative genomic hybridization platform (arrayCGH), called arrEYE, was used for high-resolution copy number variant analysis of 106 known and 60 candidate genes for iRD and 196 retina-expressed non-coding RNAs (ncRNAs) . The data was processed and analyzed with the ViVar software (http://www.cmgg.be/vivar/).
The functional impact of sequence variants was assessed based on the outcome of in silico predictions performed in Alamut Visual (v. 2.7) or Alamut HT/Alamut Batch (for WES data), including splice prediction tools (SpliceSiteFinder-like, MaxEntScan, NNSPLICE), and missense prediction tools (SIFT, Polyphen-2, Align GVGD and Mutation Taster), assessment of physicochemical distance (Grantham score calculation), evolutionary conservation, location in protein domains, presence in dbSNP build 145 (http://www.ncbi.nlm.nih.gov/SNP/), Exome Variant Server from the NHLBI Exome Sequencing Project (ESP, http://evs.gs.washington.edu/EVS/), ExAC (http://exac.broadinstitute.org) and gnomAD (http://gnomad.broadinstitute.org) . All variants were verified in the public version of the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php) combined with a thorough literature search. The recent ACMG guidelines were applied for classification of the sequence variants . The maximum tolerated reference allele count was calculated for all variants present in public databases (ExAC, gnomAD) using an online calculator (https://jamesware.shinyapps.io/alleleFrequencyApp/) (S2 Table) . HGVS mutation nomenclature was used, with the A of the initiation codon ATG as +1 (http://www.hgvs.org/mutnomen).
Results and Discussion
Mutation detection rate and prevalence of mutations
To date, mutations in 27 adRP genes have been reported in adRP [RetNet, November 2016]. Depending on the technology used, the mutation detection varies from 50 to 70% [2,3]. We applied several screening methods over the past ten years (Table 1). Screening of known mutations (APEX chip) revealed mutations in ten cases. In 2011 a combined approach of genome-wide linkage analysis and targeted NGS on a selected set of seven families identified mutations in known adRP genes in all seven families, with all genes located in regions with the highest LOD score. A retrospective screen of the four most prevalent adRP genes (RHO, RP1, PRPH2, PRPF31), the recurrent NR2E3 p.(Gly56Arg) and PROM1 p.(Arg373Cys) mutations was performed in all 86 index cases initially using Sanger sequencing and subsequently using targeted next-generation sequencing (NGS) on MiSeq. In parallel to targeted NGS of an extended adRP panel, targeted WES (based on integrated variant annotation and filtering of RetNet genes) was introduced. Together, these targeted sequencing approaches revealed mutations in 31 cases (Sanger sequencing n = 16; targeted NGS on MiSeq n = 12; WES n = 3). These molecular screening methods were recently complemented by copy number variant (CNV) analysis using a high-resolution customized array called arrEYE, containing probes for the exonic and entire intronic regions of 106 known iRD genes, including all 27 adRP genes. No copy number variations were identified in the screened adRP cohort so far .
Overall molecular genetic screening revealed mutations in 48 out of 86 cases (56%), 36 of which are distinct mutations. Since only a minority of patients underwent RetNet-based filtering of WES data, this detection rate will probably increase in the coming years. Seventeen mutations are novel and are discussed in this paper (Table 1). Representative fundus pictures of 12 patients with novel mutations in adRP genes are shown in Fig 1.
Overall, the phenotypes shown represent a range of adRP phenotypes varying from milder, classic, to end-stage RP. (A) Age 55 years (FAM_009), RHO mutation, c.1028G>A p.(Ser343Asn) (novel). A classic RP phenotype, including good macular preservation, attenuated retinal vasculature, outer retinal atrophy and predominantly spicular intraretinal pigment migration in the midperiphery. (B) Age 54 years (FAM_010), RHO mutation, c.1028G>A p.(Ser343Asn) (novel). Milder phenotype compared to A. Diffuse outer retinal atrophy in the periphery with good macular preservation. Notice the absence of intraretinal pigment migration. (C) Age 55 years (FAM_002), RHO mutation, c.265G>C p.(Gly89Arg) (novel). End-stage RP with macular atrophy, attenuated retinal vasculature and diffuse intraretinal pigment migration in the midperiphery. (D) Age 53 years (FAM_043), recurrent NR2E3 mutation, c.166G>A p.(Gly56Arg). Outer retinal atrophy with mild intraretinal pigment migration in the periphery and perifoveal outer retinal atrophy. (E) Age 51 years (FAM_017), RP1 mutation, c.2245_2248delinsTGAG p.(Leu749*) (novel). A classic RP phenotype, similar to the description of panel A. (F) Age 72 years (FAM_016), RP1 mutation, c.2200del p.(Ser734Valfs*4) (novel). End-stage RP with complete outer retinal atrophy and intraretinal pigment migration including periphery and macula. (G) Age 72 years (FAM_019), RP1 mutation, c.2597del p.(Leu866*) (novel). Typical yellowish hue due to outer retinal atrophy with intraretinal pigment migration in the periphery and macular preservation. (H) Age 30 years (FAM_022), SNRNP200 mutation, c.1981G>T p.(Val661Leu) (novel). Outer retinal atrophy with spicular intraretinal pigment migration, most pronounced in the retinal midperiphery. (I) Age 38 years (FAM_025), PRPF8 mutation, c.6840C>A p.(Asn2280Lys) (novel). Outer retinal atrophy with intraretinal pigment migration of the spicular type in the midperiphery and a good macular preservation. (J) Age 50 years (FAM_028), PRPF8 mutation, c.6964G>T p.(Glu2322*) (novel). Mild outer retinal atrophy in the periphery with macular preservation, normal retinal vasculature and a normal optic disc. (K) 53 years (FAM_035), PRPF31 mutation, c.541G>T p.(Glu181*). Outer retinal atrophy with macular preservation. (L) 51 years (FAM_048), TOPORS mutation, c.2556_2557del p.(Glu852Aspfs*20) (novel). Pigment epithelium alterations with white dots in the retinal periphery. Notice absence of intraretinal pigment migration and presence of perifoveal atrophy.
The prevalence in our population can only be determined for the four most common disease genes (RHO, RP1, PRPH2, PRPF31) and the recurrent PROM1 and NR2E3 mutations that were screened in the entire cohort. Mutations in the rhodopsin (RHO, NM_000539.3, MIM# 613731) gene are the most common cause of adRP. The prevalence of RHO mutations in the Belgian adRP population is approximately 14%. This is higher than the prevalence in France (10.3%), but lower compared to other populations (European cohorts: 16.5%-30%, American cohorts: approximately 30%) [2,13–19]. Mutations in the retinitis pigmentosa 1 gene (RP1, NM_006269.1, MIM# 603937) account for 10.5% of the Belgian cohort, which is higher than the prevalence in Spain (3.5%), Italy (5%), and France (5.3%), but closer to the prevalence in other cohorts in US (7.7%) and the United Kingdom (8%-10%) [2,17,20–25]. PRPF31 mutations account for 10.5% of adRP in the Belgian cohort. This is higher than the previously reported prevalence in US (5.5%), the United Kingdom (5%) and France (6.7%) [2,26–27]. The prevalence of mutations in genes encoding three splicing factors (PRPF8, PRPF31 and SNRNP200) is high (altogether 19.8%). Together, these mutations represent the most common cause of adRP in the Belgian adRP cohort [2,28]. The prevalence of peripherin 2 (PRPH2) mutations in adRP widely varies depending on the origin, from 0% (Mexican cohort) up to 10.3% (French cohort) [2,29–30]. Here, mutations in PRPH2 account for 4.7%. The prevalence of the recurrent PROM1 mutation p.(Arg373Cys) is higher (3.5%) than anticipated in adRP. Two families have the recurrent NR2E3 mutation p.(Gly56Arg), accounting for 2.3% (Fig 2).
(A-D): Schematic representation of the novel mutations identified in this study. (A) RP1 gene. The five mutations are located within the mutational hotspot (nucleotides 1490–3216), indicated with a black horizontal line. E = exon. Grey rectangles are coding regions and orange rectangles are 5’ untranslated region (5’ UTR) and 3’ UTR. (B) RP1 protein. Both truncating mutations identified in this study belong to Class II mutations (amino acids 500–1053), indicated with a black line. The Drosophila melanogaster (BIF) domain (amino acids 486–635) is depicted as a blue rectangle. aa = amino acid. (C) SNRNP200 protein. The two novel mutations identified in this study are both located within the first DExD/H box helicase-like domain (amino acids 477–690). Both the first and the second (amino acids 1324–1528) DExD/H box helicase domains are represented as blue rectangles. Both Sec63-like domains (amino acids 981–1286 and 1812–2124) are indicated as golden rectangles. aa = amino acids. (D) PRPF8 protein. The novel mutation identified here is located within the highly conserved region C-terminal to the Jab1/MPN domain (amino acids 2099–2233), depicted as a blue rectangle. aa = amino acid. (E) Prevalence of causative mutations in adRP genes in a Belgian adRP cohort. The ‘unknown’ part may include new disease genes and mutation mechanisms as well as known disease genes not screened in the course of this study.
Sequence and Copy Number Variations
We identified nine distinct RHO (NM_000539.3, MIM# 613731) mutations, four of which are novel: c.265G>C p.(Gly89Arg), c.532T>G p.(Tyr178Asp), c.911T>A p.(Val304Asp) and c.1028G>A p.(Ser343Asn) (n = 2), (Table 1) [31–35]. All four missense substitutions change a highly conserved amino acid and are predicted to be deleterious. For the missense variant c.532T>G p.(Tyr178Asp), different changes of the same amino acid p.(Tyr178Asn) and p.(Tyr178Cys) have been reported [32,36]. Out of a total of 12 identified mutations, the two novel missense substitutions p.(Val304Asp) and p.(Ser343Asn) were the only variants not present in previously affected amino acids of the RHO protein (http://www.retina-international.org/files/sci-news/rhomut.htm). Interestingly, the p.(Ser343Asn) substitution disrupts the phosphorylation site closest to the C-terminus and is known to play a crucial role in promoting the binding of the rod-specific arrestin to rhodopsin [37,38]. Moreover, this substitution resides within a hot spot region between p.(Thr340) and p.(Pro347), with mutations reported in all consecutive amino acids except for p.(Ser343). The p.(Val304Asp) variant is located in the seventh transmembrane segment within the highly conserved NPxxY-motif (Asn302/Pro303/Val304/Ille305/Tyr306). A key function of this motif is to mediate several inter-helical interactions that might have a potentially stabilizing role to maintain the ground state structure of RHO [39–41]. The pathogenicity of p.(Val304Asp) is uncertain based on its position within the motif. The most prevalent European RHO mutation, p.(Pro347Leu), was not observed in our cohort .
Seven distinct pathogenic variants were found in the RP1 gene (NM_006269.1, MIM# 603937), five of which are novel (Table 1) [20,42]. A heterozygous indel mutation c.2245_2248delinsTGAG, replacing the nucleotides CTCA by their reverse complement TGAG p.(Leu749*), and four heterozygous deletions: c.2305_2317del p.(Lys769Phefs*2), c.2026del p.(Ser676Leufs*6) (n = 2); c.2200del p.(Ser734Valfs*4); c.2597del p.(Leu866*) were found. These five novel mutations create a premature termination codon (PTC) in the last exon that is predicted to escape nonsense-mediated decay (NMD) and to lead to a truncated protein. This is in line with the majority of RP1 alleles that generate PTCs located in a mutational hotspot region (c.1490-c.3216) within the last exon . Indeed, all seven mutations identified here are located within this hotspot region (Fig 2). The numerous deletions and insertions can be explained by the multiple A nucleotides flanking the mutation sites, possibly causing slipped strand mispairing during replication . Chen et al. proposed four classes of RP1 mutations . Truncations located between amino acid 500 and 1053 within the last exon are NMD-insensitive and belong to ‘Class II’ mutations, making up the majority of PTC mutations. A loss of the C-terminal half to one third of the RP1 protein may have a deleterious effect through the exposure of the Drosophila melanogaster bifocal (BIF) domain (amino acids 486–635) (Fig 2). This eventually results in a potential dominant negative effect rather than haploinsufficiency as an underlying mechanism .
Mutations in splicing factor genes SNRNP200, PRPF8, PRPF31
Today, seven ubiquitously expressed adRP genes involved in nuclear pre-messenger RNA (pre-mRNA) splicing have been described (RetNet). Six of them encode components of the U4/U6-U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex of the spliceosome, highlighting its important role in adRP pathogenesis . Overall, mutations in splicing factor genes are the second most common cause of adRP . We identified heterozygous mutations in adRP splicing genes (SNRNP200, PRPF8, PRPF31) in 17 probands (Table 1) [2,26,28,47–49].
A novel heterozygous missense variant was revealed in SNRNP200 (NM_014014.4, MIM# 601664), c.1981G>T p.(Val661Leu). The Val residue is highly conserved up to Baker’s yeast and the mutation is predicted to be deleterious (Table 1). The splicing factor SNRNP200 encodes BRR2, a stable component of the U5 snRNP that is essential for the unwinding of the U4/U6 and U2/U6 snRNAs [50,51]. BRR2 interacts extensively with the U5-specific protein PRPF8 . The SNRNP200 mutations found in Belgian cases are located in the first of two DExD/H box helicase-like domains, in line with most previously described mutations (Fig 2) [53–56].
We identified a novel heterozygous missense mutation in PRPF8 (NM_006445.3, MIM# 600059), c.6840C>A p.(Asn2280Lys). This variant alters a highly conserved amino acid (up to Baker’s yeast) and is predicted to have a possible effect on the protein structure or function. A second, novel PRPF8 nonsense mutation c.6964G>T p.(Glu2322*) was found (Table 1), introducing a PTC in the last exon, predicted to escape NMD and to lead to a truncated protein. The U5 snRNP protein PRPF8 is crucial for the formation of the catalytic center in the spliceosome and interacts via its C-terminus with the DExD/H domain, suggesting that mutations might affect the PRPF8-BBR2 interaction . Indeed, PRPF8 mutations that lead to adRP cluster within the highly conserved region C-terminal to the Jab1/MPN domain. The three mutations found here are located within the same C-terminal domain (Fig 2). In yeast this specific domain forms a complex with BRR2 and stimulates its helicase activity [57–60].
PRPF31 (NM_015629.3, MIM# 600138) encodes an U4/U6-specific protein that interacts with the U4 snRNA and facilitates the formation of tri-snRNP by physically tethering U4/U6 and U5 snRNPs [61–63]. A novel heterozygous PRPF31 nonsense substitution was identified in two unrelated probands, c.34G>T p.(Glu12*), likely subjecting the transcript to NMD. In total, nine patients were found to have mutations in PRPF31, seven nonsense mutations, one out-of-frame deletion, and one splice-site mutation (Table 1). The greater part of PRPF31 mutations described in literature are (large) deletions, insertions, duplications, nonsense and splice-site mutations leading to haploinsufficiency . No large rearrangements of PRPF31 were identified in the studied cohort, however . Variable expression or non-penetrance have been reported in adRP families with mutations in the PRPF31 gene (for review see Rose and Bhattacharya, 2016) . Two out of nine Belgian families exhibited apparent non-penetrance. The c.528-1G>A mutation (FAM_034) segregates with the disease in the family, notably one carrier family member, the sib of the index patient exhibited no clinical signs. In a three generation family (FAM_035, c.541G>T p.[Glu181*]), two obligate carrier females of the second generation have both affected children, but do not show clinical signs.
We identified a novel deletion, c.2556_2557del p.(Glu852Aspfs*20), in the topoisomerase I-binding arginine-serine rich gene (TOPORS, NM_005802.4, MIM# 609923) (Table 1). The majority of the reported TOPORS mutations are located within the same region of the last exon, lead to a PTC and are predicted to escape NMD. The lack of a truncated protein in patients’ lymphoblastoid cells however indicates an unstable mutant protein, suggesting haploinsufficiency, rather than a dominant negative effect as a disease mechanism .
Three out of four variants found in the PRPH2 gene have previously been described in adRP patients (Table 1) [16,67,68]. A novel out-of-frame duplication was found, c.382_382dup p.(Thr129Lysfs*49), likely subjecting the mRNA to NMD. The PRPH2 gene encodes a transmembrane glycoprotein located at the rim regions of photoreceptor outer segment discs . It forms a homo-oligomeric structure that subsequently assembles into homo-tetramers, or forms hetero-tetrameric complexes with its paralogous protein, rod outer segment protein 1 (ROM1) . These protein structures have an important role in photoreceptor disc morphogenesis and stabilization . The majority of PRPH2 mutations are sequence variants. Different mechanisms, including aberrant mRNA splicing, protein mislocalization, and protein degradation may cause a reduced expression of the protein in the rod outer segment .
Recurrent NR2E3 and PROM1 mutations
The recurrent nuclear receptor subfamily 2, group E, member 3 (NR2E3, NM_014249.2, MIM# 611131) mutation p.(Gly56Arg) was found in two index patients (Table 1) . In addition, the recurrent prominin 1 (PROM1, NM_006017.2, MIM# 608051) mutation p.(Arg373Cys) was found in three cases (Table 1) . Patients with the recurrent PROM1 mutation are known to have phenotypes ranging from isolated macular dystrophy, rod dystrophy, rod-cone dystrophy and cone-rod dystrophy. Most reported cases present with a bull’s eye maculopathy [74–75]. The index patients in our cohort were referred with a tentative diagnosis of adRP and adRP with macular involvement. A reclassification may be required based on a detailed clinical examination of family members. Since extraocular phenotypes have been described in some patients with the recurrent PROM1 mutation, this finding may have clinical implications .
Copy number variants
In 2016 we developed and implemented arrEYE, a microarray-based platform for high-resolution copy number analysis in iRD . Using this approach we previously identified a novel heterozygous deletion of exons 7 and 8 of the Heparan-Alpha-Glucosaminide N-Acetyltransferase (HGSNAT, NM_152419.2, MIM# 616544) gene: c.634-408_820+338delinsAGAATATG, p.(Glu212Glyfs*2) in a simplex RP patient. A second variant p.(Arg615Thr) was identified on the other allele . No disease-causing CNVs were found in the adRP cohort studied so far.
Classification of variants
Variants were classified following the ACMG standards and guidelines, categorizing them in one of five classes (pathogenic, likely pathogenic, uncertain significance, likely benign and benign) . For the majority of variants, this categorization is in line with former classifications based on in silico predictions and literature searches. The classification was debatable for several variants that are listed in ExAC and gnomAD (beta, December 2016). Recently, Sharon et al. highlighted the importance of population frequency thresholds for the filtering and classification of variants found with WES. It was estimated that the allele frequency of a true adRP disease-causing variant should be lower than 1 in 100,000 (<0.001%), taking into account the heterogeneous nature of the disease, rare incidences of reduced penetrance and undiagnosed individuals mistaken as controls . A statistical framework for a frequency-based filtering was presented by Whiffin et al. (2016; online calculator; https://jamesware.shinyapps.io/alleleFrequencyApp/) . These calculations estimate the maximum tolerated allelic count for a variant in a reference dataset (e.g. ExAC, gnomAD), i.e. a threshold for assessing whether a variant is to commonly present in a reference dataset to be disease causing. The calculation is based on several parameters, including inheritance, disease prevalence, maximum allelic contribution, penetrance and number of screened reference alleles.
The disease prevalence of RP is one in 5000, with about 30% to 40% having a dominant inheritance [1,77]. We applied a (maximum) disease frequency of 1 in 12,500 for adRP. Since only rather small populations were screened for all known adRP genes, and the contribution of each individual mutation (i.e. allelic heterogeneity) is not well known, we assumed that the maximum contribution of each gene (i.e. the prevalence of the disease gene) is the maximum possible allelic contribution. This is an overestimation since adRP is not only characterized by locus heterogeneity, but also by allelic heterogeneity, whereby most variants within a gene only account for a small percentage of cases. Although most alleles are fully penetrant, non-penetrance has been reported. Calculations were made for a variant penetrance of 1, 0.95 and 0.5. The predicted maximum allele count was calculated for all genes with variants present in ExAC or gnomAD (S2 Table). Only one variant exceeded the predicted maximum allele count in the reference databases. The variant, c.424C>T p.(Arg142Trp) in PRPH2, is predicted to be deleterious by several in silico predictions and has been reported as pathogenic multiple times (also known as R142W), which would qualify it as likely pathogenic . However, the minor allele frequency (MAF) of 0.0021% (gnomAD: present in 6 out of 282,618 alleles) exceeds the expected allele frequency of a dominant mutation in the general population (Table 1) and the allele count is above the threshold (S2 Table). Following this reasoning, this variant should be reclassified as variant of uncertain significance. Moreover, the p.(Arg142Trp) variant was reported in an autosomal recessive RP family with a homozygous pathogenic PDE6B mutation. The PRPH2 variant might explain the more severe phenotype seen in the individual with variants in both disease genes . The p.(Arg142Trp) variant is frequently reported in patients with Central Areolar Choroidal Dystrophy (CACD) (29 out of 60) , with a mean age of onset of 46 years .
The variant allele counts in the reference datasets for the remainder mutations did not exceed the maximum tolerated allele count (S2 Table). It cannot be excluded that any of the individuals in ExAC and gnomAD with mutations in adRP genes are too young to express the gene-associated adRP or display non-penetrance or a minimal expression.
Altogether, this illustrates that weighting the MAF and the maximum tolerated allele count of variants in genomic databases as a parameter for variant classification cannot be done in an absolute way in the context of dominant diseases with a later age of onset, as variant databases of supposedly control individuals do not contain information on the age of individuals or on phenotypes.
To summarize, this is the first comprehensive molecular genetic study on adRP-causing mutations in a Belgian cohort of 86 patients. We obtained a molecular diagnosis of adRP in 48 out of 86 cases (56%), with the highest mutation prevalences in RHO (14%), RP1 (10.5%) and PRPF31 (10.5%). A striking observation is that mutations in splicing factor genes represent the most common cause of adRP in the Belgian cohort (19.8%). Finally, we identified 17 novel mutations in the RP1, RHO, PRPH2, PRPF31, PRPF8, SNRNP200, and TOPORS genes, thereby expanding their molecular spectrum. Classification of variants following ACMG guidelines allows a systematic categorization although variant allele frequencies and allele count in public genomic databases should be assessed with caution.
S1 Table. AdRP microsatellite panel.
Three to four pairs of primers were designed for microsatellites flanking an adRP gene within a distance of one to two megabases (Mbs) up- or downstream (Primer3plus; http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Six primer pairs were designed for the RP63 locus on chromosome 6q23, with adjacent primers less than one Mb apart. Each forward primer is tagged with a M13-tail. Sequence of the M13-tail: 5’-cacgacgttgtaaaacgac-3’.
S2 Table. Calculation of maximum tolerated allele count.
The maximum tolerated allele count was computed using an online calculator (https://jamesware.shinyapps.io/alleleFrequencyApp/). The allele count represents the count of each variant in ExAC or gnomAD for the entire studied population (indicated as ‘all’) and for the individual population groups in which the variant was found. The reference allele number is the total allele count screened in the reference population(s). Inheritance is monoallelic. The prevalence was calculated based on the prevalence of RP (1/5000), with about 30% to 40% having a dominant inheritance. We assumed that the maximum possible allelic contribution (maximum allelic heterogeneity) is the maximum genetic contribution (as described in Whiffin et al. for genes with a less characterized allelic heterogeneity). A range was taken for the maximum allelic heterogeneity; with minimum and maximum value being the in literature reported minimum and maximum contribution of a gene in adRP. Calculations were also made for the prevalence in the Belgian adRP population (B). Since non-penetrance has been described for several genes, we assumed three different penetrance values (1, 0.95 and 0.50). Maximum tolerated ref. AC = Maximum tolerated reference allele count. Blue shaded cells: Values needed for calculation of the maximum tolerated reference allele count. Orange shaded cells: variant counts that exceeded the maximum tolerated reference allele count .
The authors gratefully acknowledge the Belgian families who participated in this study.
- Conceptualization: CVC FC BPL EDB.
- Data curation: CVC.
- Formal analysis: CVC EF.
- Funding acquisition: EDB.
- Investigation: CVC SDJ HF CC EF.
- Methodology: CVC FC EF GVC BPL EDB.
- Project administration: CVC FC EDB.
- Resources: CVC FC DR JDZ SW GVC FD IC TDR BPL EDB.
- Software: CVC FC EF.
- Supervision: EDB.
- Validation: CVC HF SDJ.
- Visualization: CVC DR.
- Writing – original draft: CVC.
- Writing – review & editing: CVC FC BPL EDB.
- 1. Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa. The Lancet. 2006;368: 1795–1809.
- 2. Sullivan LS, Bowne SJ, Birch DG, Hughbanks-Wheaton D, Heckenlively JR, Lewis RA, et al. Prevalence of disease-causing mutations in families with autosomal dominant retinitis pigmentosa: a screen of known genes in 200 families. Invest Ophthalmol Vis Sci. 2006;47: 3052–3064. pmid:16799052
- 3. Hamel CP. Gene discovery and prevalence in inherited retinal dystrophies. C R Biol. 2014;337(3): 160–166. pmid:24702842
- 4. Daiger SP, Bowne SJ, Sullivan LS, Blanton SH, Weinstock GM, Koboldt DC, et al. Application of Next-Generation sequencing to Idenify Genes and Mutations causing Autosomal Dominant Retinitis Pigmentosa (adRP). Adv Exp Med Biol. 2014;801: 123–129. pmid:24664689
- 5. Fernandez-San Jose P, Corton M, Blanco-Kelly F, Avila-Fernandez A, Lopez-Martinez MA, Sanchez-Navarro I, et al. Targeted Next-Generation Sequencing Improves the Diagnosis of Autosomal Dominant Retinitis Pigmentosa in Spanish Patients. Invest Ophthalmol Vis Sci. 2015;56: 2173–2182. pmid:25698705
- 6. Xu Y, Guan L, Shen T, Zhang J, Xiao X, Jiang H, et al. Mutations of 60 known causative genes in 157 families with retinitis pigmentosa based on exome sequencing. Hum Genet. 2014;133: 1255–1271. pmid:24938718
- 7. Nishiguchi KM, Tearle RG, Liu YP, Oh EC, Miyake N, Benaglio P, et al. Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene. Proc Natl Acad Sci USA. 2013;110: 16139–16144. pmid:24043777
- 8. Leeneer K, Hellemans J, Steyaert W, Lefever S, Vereecke I, Debals E, et al. Flexible, scalable, and efficient targeted resequencing on a benchtop sequencer for variant detection in clinical practice. Hum Mutat. 2015;36: 379–387. pmid:25504618
- 9. Van Cauwenbergh C, Van Schil K, Cannoodt R, Bauwens M, Van Laethem T, De Jaegere S, et al. arrEYE: a customized platform for high-resolution copy number analysis of coding and noncoding regions of known and candidate retinal dystrophy genes and retinal noncoding RNAs. Gen Med. 2016. Available from: http://dx.doi.org/10.1038/gim.2016.119.
- 10. Lek M, Karczewski K, Minikel E, Samocha K, Banks E, Fennell T, et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016;536, 285–291. pmid:27535533
- 11. Richards CS, Bale S, Bellissimo DB, Das S, Grody WW, Hegde MR, et al., Molecular Subcommittee of the ACMG Laboratory Quality Assurance Committee. ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007. Genet Med. 2008;10(4): 294–300. pmid:18414213
- 12. Whiffin N, Minikel E, Walsh R, O'Donnell-Luria A, Karczewski K, Ing AY, et al. Using high-resolution variant frequencies to empower clinical genome interpretation. bioRxiv. 2016. Available from: https://doi.org/10.1101/073114.
- 13. Inglehearn CF, Keen TJ, Bashir R, Jay M, Fitzke F, Bird AC, et al. A completed screen for mutations of the rhodopsin gene in a panel of patients with autosomal dominant retinitis pigmentosa. Hum Mol Genet. 1992;1: 41–45. pmid:1301135
- 14. Shastry BS. Retinitis pigmentosa and related disorders: phenotypes of rhodopsin and peripherin/RDS mutations. Am J Med Genet. 1994;52: 467–474. pmid:7747760
- 15. Bareil C, Hamel C, Pallarès-Ruiz N, Arnaud B, Demaille J, Claustres M. Molecular analysis of the rhodopsin gene in southern France: identification of the first duplication responsible for retinitis pigmentosa, c. 998^999ins4. Ophthalmic Genet. 1999;20: 173–182. pmid:10521250
- 16. Sohocki MM, Daiger SP, Bowne SJ, Rodriquez JA, Northrup H, Heckenlively JR, et al. Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies. Hum Mutat. 2001;17: 42–51. pmid:11139241
- 17. Ziviello C, Simonelli F, Testa F, Anastasi M, Marzoli SB, Falsini B, et al. Molecular genetics of autosomal dominant retinitis pigmentosa (ADRP): a comprehensive study of 43 Italian families. J Med Genet. 2005;42: e47. pmid:15994872
- 18. Audo I, Manes G, Mohand-Saïd S, Friedrich A, Lancelot ME, Antonio A, et al. Spectrum of Rhodopsin Mutations in French Autosomal Dominant Rod–Cone Dystrophy Patients. Invest Ophthalmol Vis Sci. 2010;51: 3687–3700. pmid:20164459
- 19. Fernandez-San Jose P, Blanco-Kelly F, Corton M, Trujillo-Tiebas MJ, Gimenez A, Avila-Fernandez A, et al. Prevalence of Rhodopsin mutations in autosomal dominant Retinitis Pigmentosa in Spain: clinical and analytical review in 200 families. Acta Ophthalmol. 2015;93: e38–44. pmid:25408095
- 20. Bowne SJ, Daiger SP, Hims MM, Sohocki MM, Malone KA, McKie AB, et al. Mutations in the RP1 gene causing autosomal dominant retinitis pigmentosa. Hum Mol Genet. 1999;8: 2121–2128. pmid:10484783
- 21. Guillonneau X, Piriev NI, Danciger M, Kozak CA, Cideciyan AV, Jacobson SG, et al. A nonsense mutation in a novel gene is associated with retinitis pigmentosa in a family linked to the RP1 locus. Hum Mol Genet. 1999;8: 1541–1546. pmid:10401003
- 22. Pierce EA, Quinn T, Meehan T, McGee TL, Berson EL, Dryja TP. Mutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pigmentosa. Nat Genet. 1999;22: 248–254. pmid:10391211
- 23. Berson EL, Grimsby JL, Adams SM, McGee TL, Sweklo E, Pierce EA, et al. Clinical features and mutations in patients with dominant retinitis pigmentosa-1 (RP1). Invest Ophthalmol Vis Sci. 2001;42: 2217–2224. pmid:11527933
- 24. Gamundi MJ, Hernan I, Martínez-Gimeno M, Maseras M, García-Sandoval B, Ayuso C, et al. Three novel and the common Arg677Ter RP1 protein truncating mutations causing autosomal dominant retinitis pigmentosa in a Spanish population. BMC Med Genet. 2006;7: 1–10.
- 25. Audo I, Mohand-Saïd S, Dhaenens CM, Germain A, Orhan E, Antonio A, et al. RP1 and autosomal dominant rod-cone dystrophy: novel mutations, a review of published variants, and genotype-phenotype correlation. Hum Mutat. 2012;33: 73–80. pmid:22052604
- 26. Waseem NH, Vaclavik V, Webster A, Jenkins SA, Bird AC, Bhattacharya SS. Mutations in the gene coding for the pre-mRNA splicing factor, PRPF31, in patients with autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2007 Mar 1;48: 1330–1334. pmid:17325180
- 27. Audo I, Bujakowska K, Mohand-Saïd S, Lancelot ME, Moskova-Doumanova V, Waseem NH, et al. Prevalence and novelty of PRPF31 mutations in French autosomal dominant rod-cone dystrophy patients and a review of published reports. BMC Med Genet. 2010;11: 1–9.
- 28. Martínez-Gimeno M, Gamundi MJ, Hernan I, Maseras M, Millá E, Ayuso C, et al. Mutations in the pre-mRNA splicing-factor genes PRPF3, PRPF8, and PRPF31 in Spanish families with autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2003;44: 2171–2177. pmid:12714658
- 29. Matias-Florentino M, Ayala-Ramirez R, Graue-Wiechers F, Zenteno JC. Molecular screening of rhodopsin and peripherin/RDS genes in Mexican families with autosomal dominant retinitis pigmentosa. Curr EYE Res. 2009;34: 1050–1056. pmid:19958124
- 30. Manes G, Guillaumie T, Vos WL, Devos A, Audo I, Zeitz C, et al. High prevalence of PRPH2 in autosomal dominant retinitis pigmentosa in France and characterization of biochemical and clinical features. Am J Ophthalmol. 2015;159: 302–314. pmid:25447119
- 31. Kranich H, Bartkowski S, Denton MJ, Krey S, Dickinson P, Duvigneau C, et al. Autosomal dominant ‘sector’retinitis pigmentosa due to a point mutation predicting an Asn-15-Ser substitution of rhodopsin. Hum Mol Genet. 1993;2: 813–814. pmid:8353500
- 32. Sung CH, Davenport CM, Hennessey JC, Maumenee IH, Jacobson SG, Heckenlively JR, et al. Rhodopsin mutations in autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci USA. 1991;88: 6481–6485. pmid:1862076
- 33. Macke JP, Davenport CM, Jacobson SG, Hennessey JC, Gonzalez-Fernandez F, et al. Identification of novel rhodopsin mutations responsible for retinitis pigmentosa: implications for the structure and function of rhodopsin. Am J Med Genet. 1993;53: 80–89.
- 34. Inglehearn CF, Bashir R, Lester DH, Jay M, Bird AC, Bhattacharya SS. A 3-bp deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa. Am J Med Genet. 1991;48: 26–30.
- 35. Dryja TP, Hahn LB, Cowley GS, McGee TL, Berson EL. Mutation spectrum of the rhodopsin gene among patients with autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci USA. 1991;88: 9370–9374. pmid:1833777
- 36. Souied E, Gerber S, Rozet JM, Bonneau D, Dufier JL, Ghazi I, et al. Five novel missense mutations of the rhodopsin gene in autosomal dominant retinitis pigmentosa. Hum Mol Genet. 1994;3: 1433–1434. pmid:7987331
- 37. Kennedy MJ, Lee KA, Niemi GA, Craven KB, Garwin GG, Saari JC, et al. Multiple phosphorylation of rhodopsin and the in vivo chemistry underlying rod photoreceptor dark adaptation. Neuron. 2001;31: 87–101. pmid:11498053
- 38. Zhang L, Sports CD, Osawa S, Weiss ER. Rhodopsin phosphorylation sites and their role in arrestin binding. J Biol Chem. 1997;272: 14762–14768. pmid:9169442
- 39. Sakmar TP, Menon ST, Marin EP, Awad ES. Rhodopsin: insights from recent structural studies. Annu Rev Biophys Biomol Struct. 2002;31: 443–484. pmid:11988478
- 40. Fritze O, Filipek S, Kuksa V, Palczewski K, Hofmann KP, Ernst OP. Role of the conserved NPxxY (x) 5, 6F motif in the rhodopsin ground state and during activation. Proc Natl Acad Sci USA. 2003;100: 2290–2295. pmid:12601165
- 41. Standfuss J, Edwards PC, D’Antona A, Fransen M, Xie G, Oprian DD, et al. The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature. 2011;471: 656–660. pmid:21389983
- 42. Jacobson SG, Cideciyan AV, Iannaccone A, Weleber RG, Fishman GA, Maguire AM, et al. Disease expression of RP1 mutations causing autosomal dominant retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2000;41: 1898–1908. pmid:10845615
- 43. Payne A, Vithana E, Khaliq S, Hameed A, Deller J, Abu-Safieh L, et al. RP1 protein truncating mutations predominate at the RP1 adRP locus. Invest Ophthalmol Vis Sci. 2000;41: 4069–4073. pmid:11095597
- 44. Chen LJ, Lai TY, Tam PO, Chiang SW, Zhang X, Lam S, et al. Compound heterozygosity of two novel truncation mutations in RP1 causing autosomal recessive retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2010;51: 2236–2242. pmid:19933189
- 45. Liu Q, Zuo J, Pierce EA. The retinitis pigmentosa 1 protein is a photoreceptor microtubule-associated protein. J Neurosci. 2004;24: 6427–6436. pmid:15269252
- 46. Daiger SP, Sullivan LS, Bowne SJ. Genes and mutations causing retinitis pigmentosa. Clin Genet. 2013;84: 132–141. pmid:23701314
- 47. Benaglio P, McGee TL, Capelli LP, Harper S, Berson EL, Rivolta C. Next generation sequencing of pooled samples reveals new SNRNP200 mutations associated with retinitis pigmentosa. Hum Mutat. 2011;32: 2246–2258.
- 48. McKie AB, McHale JC, Keen TJ, Tarttelin EE, Goliath R, van Lith-Verhoeven JJ, et al. Mutations in the pre-mRNA splicing factor gene PRPC8 in autosomal dominant retinitis pigmentosa (RP13). Hum Mol Genet. 2001;10: 1555–1562. pmid:11468273
- 49. Pomares E, Riera M, Permanyer J, Méndez P, Castro-Navarro J, Andrés-Gutiérrez Á, et al. Comprehensive SNP-chip for retinitis pigmentosa-Leber congenital amaurosis diagnosis: new mutations and detection of mutational founder effects. Eur J Hum Genet. 2010;18: 118–124. pmid:19584904
- 50. Zhao C, Bellur DL, Lu S, Zhao F, Grassi MA, Bowne SJ, et al. Autosomal-dominant retinitis pigmentosa caused by a mutation in SNRNP200, a gene required for unwinding of U4/U6 snRNAs. Am J Med Genet. 2009;85: 617–627.
- 51. Hahn D, Beggs JD. Brr2p RNA helicase with a split personality: insights into structure and function. Biochem Soc Trans. 2010;38: 1105–1109. pmid:20659012
- 52. Nguyen TH, Li J, Galej WP, Oshikane H, Newman AJ, Nagai K. Structural basis of Brr2-Prp8 interactions and implications for U5 snRNP biogenesis and the spliceosome active site. Structure. 2013;21: 910–919. pmid:23727230
- 53. Bowne SJ, Sullivan LS, Avery CE, Sasser EM, Roorda A, Duncan JL, et al. Mutations in the small nuclear riboprotein 200 kDa gene (SNRNP200) cause 1.6% of autosomal dominant retinitis pigmentosa. Mol Vis. 2013;19: 2407–2417. pmid:24319334
- 54. Li N, Mei H, MacDonald IM, Jiao X, Hejtmancik JF. Mutations in ASCC3L1 on 2q11. 2 are associated with autosomal dominant retinitis pigmentosa in a Chinese family. Invest Ophthalmol Vis Sci. 2010;51: 1036–1043. pmid:19710410
- 55. Liu T, Jin X, Zhang X, Yuan H, Cheng J, Lee J, et al. A novel missense SNRNP200 mutation associated with autosomal dominant retinitis pigmentosa in a Chinese family. PloS one. 2012;7(9): e45464. pmid:23029027
- 56. Pan X, Chen X, Liu X, Gao X, Kang X, Xu Q, et al. Mutation analysis of pre-mRNA splicing genes in Chinese families with retinitis pigmentosa. Mol Vis. 2014;20: 770–779. pmid:24940031
- 57. Pena V, Jovin SM, Fabrizio P, Orlowski J, Bujnicki JM, Lührmann R, et al. Common design principles in the spliceosomal RNA helicase Brr2 and in the Hel308 DNA helicase. Mol Cell. 2009;35: 454–466. pmid:19716790
- 58. Zhang L, Xu T, Maeder C, Bud LO, Shanks J, Nix J, et al. Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2. Nat Struct Mol Biol. 2009;16: 731–739. pmid:19525970
- 59. Maeder C, Kutach AK, Guthrie C. ATP-dependent unwinding of U4/U6 snRNAs by the Brr2 helicase requires the C terminus of Prp8. Nat Struct Mol Biol. 2009;16: 42–48. pmid:19098916
- 60. Mozaffari-Jovin S, Wandersleben T, Santos KF, Will CL, Lührmann R, Wahl MC. Inhibition of RNA helicase Brr2 by the C-terminal tail of the spliceosomal protein Prp8. Science. 2013;341: 80–84. pmid:23704370
- 61. Makarova OV, Makarov EM, Liu S, Vornlocher HP, Lührmann R. Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6· U5 tri-snRNP formation and pre-mRNA splicing. EMBO J. 2002;21: 1148–1157. pmid:11867543
- 62. Nottrott S, Urlaub H, Lührmann R. Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins. EMBO J. 2002;21: 5527–5538. pmid:12374753
- 63. Liu S, Rauhut R, Vornlocher HP, Lührmann R. The network of protein–protein interactions within the human U4/U6. U5 tri-snRNP. RNA. 2006;12: 1418–1430. pmid:16723661
- 64. Wilkie SE, Vaclavik V, Wu H, Bujakowska K, Chakarova CF, Bhattacharya SS, et al. Disease mechanism for retinitis pigmentosa (RP11) caused by missense mutations in the splicing factor gene PRPF31. Mol Vis. 2008;14: 683–690. pmid:18431455
- 65. Rose AM, Bhattacharya SS. Variant haploinsufficiency and phenotypic non-penetrance in PRPF31-associated retinitis pigmentosa. Clin Genet. 2016;90: 118–126. pmid:26853529
- 66. Chakarova CF, Papaioannou MG, Khanna H, Lopez I, Waseem N, Shah A, et al. Mutations in TOPORS cause autosomal dominant retinitis pigmentosa with perivascular retinal pigment epithelium atrophy. Am J Med Genet. 2007;81: 1098–1103.
- 67. Trujillo MJ, Martinez-Gimeno M, Giménez A, Lorda I, Bueno J, García-Sandoval B, et al. Two novel mutations (Y141H; C214Y) and previously published mutation (R142W) in the RDS. Hum Mutat. 2001;12: 70.
- 68. Bareil C, Delague V, Arnaud B, Demaille J, Hamel C, Claustres M. W179R: A novel missense mutation in the peripherin/RDS gene in a family with autosomal. Genomics. 2000;10: 733–739.
- 69. Arikawa K, Molday LL, Molday RS, Williams DS. Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration. J Cell Biol. 1992;116: 659–667. pmid:1730772
- 70. Loewen CJ, Moritz OL, Molday RS. Molecular characterization of peripherin-2 and rom-1 mutants responsible for digenic retinitis pigmentosa. J Biol Chem. 2001;276: 22388–22396. pmid:11297544
- 71. Goldberg AF. Role of peripherin/rds in vertebrate photoreceptor architecture and inherited retinal degenerations. Int Rev Cytol. 2006;253: 131–175. pmid:17098056
- 72. Becirovic E, Böhm S, Nguyen ON, Riedmayr LM, Koch MA, Schulze E, et al. In Vivo analysis of disease-associated point mutations unveils profound differences in mRNA splicing of peripherin-2 in rod and cone photoreceptors. PLoS Genet. 2016;12: 1–22.
- 73. Coppieters F, Leroy BP, Beysen D, Hellemans J, De Bosscher K, Haegeman G, et al. Recurrent mutation in the first zinc finger of the orphan nuclear receptor NR2E3 causes autosomal dominant retinitis pigmentosa. Am J Med Genet. 2007;81: 147–157.
- 74. Michaelides M, Gaillard MC, Escher P, Tiab L, Bedell M, Borruat FX, et al. The PROM1 Mutation p. R373C Causes an Autosomal Dominant Bull's Eye Maculopathy Associated with Rod, Rod–Cone, and Macular Dystrophy. Invest Ophthalmol Vis Sci. 2010;51: 4771–4780. pmid:20393116
- 75. Oishi M, Oishi A, Gotoh N, Ogino K, Higasa K, Iida K, et al. Next-generation sequencing-based comprehensive molecular analysis of 43 Japanese patients with cone and cone-rod dystrophies. Mol Vis. 2016;22: 150–160. pmid:26957898
- 76. Arrigoni FI, Matarin M, Thompson PJ, Michaelides M, McClements ME, Redmond E, et al. Extended extraocular phenotype of PROM1 mutation in kindreds with known autosomal dominant macular dystrophy. Eur J Hum Genet. 2011;19: 131–137. pmid:20859302
- 77. Sharon D, Kimchi A, Rivolta C. OR2W3 sequence variants are unlikely to cause inherited retinal diseases. Ophthalmic genet. 2016;20: 1–3.
- 78. Smailhodzic D, Fleckenstein M, Theelen T, Boon CJ, van Huet RA, van de Ven JP, et al. Central areolar choroidal dystrophy (CACD) and age-related macular degeneration (AMD): differentiating characteristics in multimodal imaging. Invest Ophthalmol Vis Sci. 2011;52: 8908–8918. pmid:22003107
- 79. Neveling K, Collin RW, Gilissen C, van Huet RA, Visser L, Kwint MP, et al. Next-generation genetic testing for retinitis pigmentosa. Hum Mutat. 2012;33: 963–972. pmid:22334370
- 80. Boon CJ, van Schooneveld MJ, den Hollander AI, van Lith-Verhoeven JJ, Zonneveld-Vrieling MN, Theelen T, et al. Mutations in the peripherin/RDS gene are an important cause of multifocal pattern dystrophy simulating STGD1/fundus flavimaculatus. Brit J Ophthal. 2007;91: 1504–1511. pmid:17504850