Genetic Variants Identified in Retinitis Pigmentosa
Researchers have identified novel genetic variants in RNU4-2 and RNU6 paralogs as underlying causes of autosomal dominant Retinitis Pigmentosa (adRP).
RNU4-2 Variants
Initial analysis of a nonconsanguineous family (M1-A) with adRP, where known retinal disease genes were negative, revealed a single-nucleotide insertion in the RNU4-2 gene (n.18_19insA). Subsequent screening of a cohort of 1,891 individuals with RP or Leber congenital amaurosis identified three additional families with this variant. The n.18_19insA allele was significantly enriched in the RP cohort compared to control databases.
Further screening of RNU4-2 identified another recurrent variant (n.56T>C) in eight individuals from four families, which was also significantly enriched in patients. Additional screening of 2,830 RP cases uncovered another patient with n.18_19insA (de novo) and six families with the n.56T>C variant.
- Total RNU4-2 cases: 41 affected individuals from 15 families.
- Incomplete Penetrance: Observed in nine obligate carriers without visual symptoms.
- RNU4-1 Comparison: The paralog RNU4-1 showed higher tolerance to genetic variation and no significant enrichment of variants in RP cases, suggesting RNU4-2 is more critical.
RNU6 Paralogs Variants
Based on the interaction between U4 and U6 snRNAs in the spliceosome, the analysis was extended to five paralogous U6 snRNA genes (RNU6-1, RNU6-2, RNU6-7, RNU6-8, RNU6-9).
- Key Variants: An insertion (n.55_56insG) was recurrently found in RNU6-2, RNU6-8, and RNU6-9. Another insertion (n.56_57insG) was identified in RNU6-2 and RNU6-9.
- Total RNU6 cases: These two variants were detected in 112 affected individuals from 52 families, involving all RNU6 paralogs except RNU6-7.
- De Novo Events: The n.55_56insG insertion was confirmed as a de novo event in eight individuals and suspected in 14 additional pedigrees.
Overall Disease Impact
Variants in RNU4-2 or RNU6 paralogs were identified in a total of 67 families with de novo or inherited dominant RP. The observed phenotype aligned with classical RP, typically manifesting in adolescence.
- Associated Ocular Features: Cystoid macular edema (55.9%), non-age-related lens opacities (23.6%), and vitreomacular complications (30.6%) were noted.
- Prevalence: These RNU4- and RNU6-associated RP variants are estimated to account for approximately 1.4% of all molecularly undiagnosed RP individuals and 3.0% of undiagnosed adRP families.
Predicted Structural and Functional Effects
All identified RP variants are predicted to cluster within the three-way junction of the U4/U6 duplex, a region distinct from variants associated with neurodevelopmental disorders (NDD).
- RNU4-2 Variants: Predicted to impact the overall stability of the U4/U6 duplex and modify the orientation of the 5′ stem-loop.
- RNU6 Variants: Predicted to extend stem-I, reduce the internal loop, and drastically alter the orientation of the 5′ stem-loop.
- Protein Interactions: Cryo-electron microscopy data confirmed that these variants reside in a region critical for binding splicing factors PRPF31, PRPF3, and PRPF8, all previously linked to adRP. The mutated nucleotides directly participate in hydrogen bonds with these proteins.
Gene Expression and Transcriptional Activity
Analysis of human retinal tissues showed higher expression of RNU4-2 compared to RNU4-1, and overall RNU6 expression was higher than RNU4. The neurosensory retina (NSR) and retinal pigment epithelium (RPE) exhibited elevated RNU4 and RNU6 expression, indicating high demand for snRNAs in these tissues. Chromatin accessibility and histone modification data (ATAC-seq and H3K27ac ChIP–seq) confirmed active transcription marks in all functional RNU4 and RNU6 paralogs in the retina, while most pseudogenes lacked these signatures.
Transcriptome Analysis in Patients
Transcriptome analysis of circulating leukocytes from affected individuals carrying RNU4-2 or RNU6 variants showed no major differences in global gene expression compared to controls. Some differentially expressed splice sites were found, but with minimal effect sizes, and no specific nucleotide frequency biases were identified.
Functional Effects on Spliceosome Assembly
Immunopurification studies revealed that RP pathogenic variants in U4 and U6 snRNAs led to an increased association with SART3 and partially with PRPF31 (markers for the U6 snRNP and U4/U6 di-snRNP), while interaction with SNRNP200 (a tri-snRNP marker) was unchanged or reduced. This suggests that these RP variants dominantly affect snRNP biogenesis, delaying the assembly process at the di-snRNP stage.