Mutational screening of splicing factor genes in cases with autosomal dominant retinitis pigmentosa

Purpose Mutations in genes encoding proteins from the tri-snRNP complex of the spliceosome account for more than 12% of cases of autosomal dominant retinitis pigmentosa (adRP). Although the exact mechanism by which splicing factor defects trigger photoreceptor death is not completely clear, their role in retinitis pigmentosa has been demonstrated by several genetic and functional studies. To test for possible novel associations between splicing factors and adRP, we screened four tri-snRNP splicing factor genes (EFTUD2, PRPF4, NHP2L1, and AAR2) as candidate disease genes. Methods: We screened up to 303 patients with adRP from Europe and North America who did not carry known RP mutations. Exon-PCR and Sanger methods were used to sequence the NHP2L1 and AAR2 genes, while the sequences of EFTUD2 and PRPF4 were obtained by using long-range PCRs spanning coding and non-coding regions followed by next-generation sequencing. Results: We detected novel missense changes in individual patients in the sequence of the genes PRPF4 and EFTUD2, but the role of these changes in relationship to disease could not be verified. In one other patient we identified a novel nucleotide substitution in the 5′ untranslated region (UTR) of NHP2L1, which did not segregate with the disease in the family. Conclusions: The absence of clearly pathogenic mutations in the candidate genes screened in our cohort suggests that EFTUD2, PRPF4, NHP2L1, and AAR2 are either not involved in adRP or are associated with the disease in rare instances, at least as observed in this study in patients of European and North American origin

Two specific mutations are prevalent causes of recessive retinitis pigmentosa in North American patients of Jewish ancestry.

PURPOSE: Retinitis pigmentosa is a Mendelian disease with a very elevated genetic heterogeneity. Most mutations are responsible for less than 1% of cases, making molecular diagnosis a multigene screening procedure. In this study, we assessed whether direct testing of specific alleles could be a valuable screening approach in cases characterized by prevalent founder mutations. METHODS: We screened 275 North American patients with recessive/isolate retinitis pigmentosa for two mutations: an Alu insertion in the MAK gene and the p.Lys42Glu missense in the DHDDS gene. All patients were unrelated; 35 reported Jewish ancestry and the remainder reported mixed ethnicity. RESULTS: We identified the MAK and DHDDS mutations homozygously in only 2.1% and 0.8%, respectively, of patients of mixed ethnicity, but in 25.7% and 8.6%, respectively, of cases reporting Jewish ancestry. Haplotype analyses revealed that inheritance of the MAK mutation was attributable to a founder effect. CONCLUSION: In contrast to most mutations associated with retinitis pigmentosa-which are, in general, extremely rare-the two alleles investigated here cause disease in approximately one-third of North American patients reporting Jewish ancestry. Therefore, their screening constitutes an alternative procedure to large-scale tests for patients belonging to this ethnic group, especially in time-sensitive situations.Genet Med 17 4, 285-290

A missense mutation in PRPF6 causes impairment of pre-mRNA splicing and autosomal-dominant retinitis pigmentosa

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration that leads to progressive visual-field constriction and blindness. Although the disease manifests only in the retina, mutations in ubiquitously expressed genes associated with the tri-snRNP complex of the spliceosome have been identified in patients with dominantly inherited RP. We screened for mutations in PRPF6 (NM_012469.3), a gene on chromosome 20q13.33 encoding an essential protein for tri-snRNP assembly and stability, in 188 unrelated patients with autosomal-dominant RP and identified a missense mutation, c.2185C>T (p.Arg729Trp). This change affected a residue that is conserved from humans to yeast and cosegregated with the disease in the family in which it was identified. Lymphoblasts derived from patients with this mutation showed abnormal localization of endogenous PRPF6 within the nucleus. Specifically, this protein accumulated in the Cajal bodies, indicating a possible impairment in the tri-snRNP assembly or recycling. Expression of GFP-tagged PRPF6 in HeLa cells showed that this phenomenon depended exclusively on the mutated form of the protein. Furthermore, analysis of endogenous transcripts in cells from patients revealed intron retention for pre-mRNA bearing specific splicing signals, according to the same pattern displayed by lymphoblasts with mutations in other PRPF genes. Our results identify PRPF6 as the sixth gene involved in pre-mRNA splicing and dominant RP, corroborating the hypothesis that deficiencies in the spliceosome play an important role in the molecular pathology of this disease

The Genetic Basis of Pericentral Retinitis Pigmentosa—A Form of Mild Retinitis Pigmentosa

Pericentral retinitis pigmentosa (RP) is an atypical form of RP that affects the near-peripheral retina first and tends to spare the far periphery. This study was performed to further define the genetic basis of this phenotype. We identified a cohort of 43 probands with pericentral RP based on a comprehensive analysis of their retinal phenotype. Genetic analyses of DNA samples from these patients were performed using panel-based next-generation sequencing, copy number variations, and whole exome sequencing (WES). Mutations provisionally responsible for disease were found in 19 of the 43 families (44%) analyzed. These include mutations in RHO (five patients), USH2A (four patients), and PDE6B (two patients). Of 28 putatively pathogenic alleles, 15 (54%) have been previously identified in patients with more common forms of typical RP, while the remaining 13 mutations (46%) were novel. Burden testing of WES data successfully identified HGSNAT as a cause of pericentral RP in at least two patients, suggesting it is also a relatively common cause of pericentral RP. While additional sequencing might uncover new genes specifically associated with pericentral RP, the current results suggest that genetically pericentral RP is not a separate clinical entity, but rather is part of the spectrum of mild RP phenotypes

Clinical summary of patients with <i>FAM161A</i> mutations associated with retinitis pigmentosa.

a<p>Visual Acuity: best corrected Snellen visual acuity.</p>b<p>Electroretinograms: full field cone ERG amplitude in microvolts to 30HZ white light (lower norm  = 50 microvolts).</p>c<p>Visual Field: Goldmann total field area to V-4e white test light (lower norm  =  11,399 degrees squared).</p>d<p>Dark adaptation: final threshold in log units above normal after 45 minutes of dark adaptation.</p>e<p>Lens: −, clear lens; +, central posterior subcapsular cataract.</p>f<p>Macula: −, within normal limits; +, granular.</p>g<p>Periphery: bone spicule or clumped pigment in one or more quadrants: +, present; −, absent.</p><p>Abbreviations: F, female; M, male; EO, ethnic origin; OD, right eye; OS, left eye; HM, hand motions; PP, pseudophakia; AS, atrophic scar; NA, not available.</p

Fundus photographs from patient 121–385 (male, 43 years old).

<p>The patient shows the representative phenotype of this mutation with waxy pallor of the optic disc, retinal arteriolar attenuation, granularity of the macula and intra retinal pigment around the midperiphery in a bone spicule or clumped configuration.</p

Schematic representation of alternative splicing events of <i>FAM161A</i> transcripts.

<p>Boxes represent exons, while lines represent splicing events. Coding regions are in black, noncoding regions are white. The canonical forms of <i>FAM161A</i> mRNA are presented in the top panel, as reference. The bottom two panels show newly-discovered splicing isoforms with an alternative 5′UTR in intron 1 (1a).</p

Mutation c.1355_6delCA (p.T452SfsX3) produces transcripts with a premature stop codon that are targets for NMD degradation.

<p>The image shows an agarose gel on which six RT-PCR products were run: two from patients 003-161 and 102-001, respectively, one from a control cell line (CTR), and one from the same control cell line, following a cDNA synthesis reaction performed without the addition of the reverse transcriptase enzyme (RT-). Presence of cycloheximide in cell cultures is indicated with "+", its absence with "−". M, DNA molecular size marker; 18S, RT-PCR loading control (18S rRNA); 1000 bp and 250 bp, bands of the marker corresponding to these specific sizes, respectively.</p

Variants identified in this study.

<p>+, wild-type allele.</p>a<p>http://genetics.bwh.harvard.edu/pph2/.</p>b<p>http://sift.jcvi.org/.</p>c<p>http://mutpred.mutdb.org/</p>d<p>http://mmb2.pcb.ub.es:8080/PMut/.</p