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

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

Molecular Genetics of FAM161A in North American Patients with Early-Onset Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a hereditary disease that leads to the progressive degeneration of retinal photoreceptor cells and to blindness. It is caused by mutations in several distinct genes, including the ciliary gene FAM161A, which is associated with a recessive form of this disorder. Recent investigations have revealed that defects in FAM161A represent a rather prevalent cause of hereditary blindness in Israel and the Palestinian territories, whereas they seem to be rarely present within patients from Germany. Genetic or clinical data are currently not available for other countries. In this work, we screened a cohort of patients with recessive RP from North America to determine the frequency of FAM161A mutations in this ethnically-mixed population and to assess the phenotype of positive cases. Out of 273 unrelated patients, only 3 subjects had defects in FAM161A. A fourth positive patient, the sister of one of these index cases, was also identified following pedigree analysis. They were all homozygous for the p.T452Sfx3 mutation, which was previously reported as a founder DNA variant in the Israeli and Palestinian populations. Analysis of cultured lymphoblasts from patients revealed that mutant FAM161A transcripts were actively degraded by nonsense-mediated mRNA decay. Electroretinographic testing showed 30 Hz cone flicker responses in the range of 0.10 to 0.60 microvolts in all cases at their first visit (age 12 to 23) (lower norm  =  50 μV) and of 0.06 to 0.32 microvolts at their most recent examination (age 27 to 43), revealing an early-onset of this progressive disease. Our data indicate that mutations in FAM161A are responsible for 1% of recessive RP cases in North America, similar to the prevalence detected in Germany and unlike the data from Israel and the Palestinian territories. We also show that, at the molecular level, the disease is likely caused by FAM161A protein deficiency