A Leaky Deep Intronic Splice Variant in CLRN1 Is Associated with Non-Syndromic Retinitis Pigmentosa

Abstract

Background: Inherited retinal diseases (IRDs) are clinically complex and genetically heterogeneous visual impairment disorders with varying penetrance and severity. Disease-causing variants in at least 289 nuclear and mitochondrial genes have been implicated in their pathogenesis.

Methods: Whole exome sequencing results were analyzed using established pipelines and the results were further confirmed by Sanger sequencing and minigene splicing assay.

Results: Exome sequencing in a 51-year-old Ashkenazi Jewish patient with non-syndromic retinitis pigmentosa (RP) identified compound heterozygous variants in the CLRN1 gene: a known pathogenic missense [p.(N48K)] and a novel deep intronic variant c.254-643G>T. A minigene splicing assay that was performed aiming to study the effect of the c.254-643G>T variant on CLRN1 pre-mRNA splicing revealed the inclusion of a pseudo-exon that was also reported to be included in the transcript due to an adjacent variant, c.254-649T>G. However, unlike the reported c.254-649T>G variant, c.254-643G>T showed aberrant splicing in a leaky manner, implying that the identified variant is not totally penetrant.

Conclusion: We report on a novel deep intronic variant in CLRN1 causing non-syndromic RP. The non-syndromic phenotype observed in this index case may be attributed to the leaky nature of this variant, which is causing some normal transcripts to be produced.

Keywords: CLRN1; deep intronic; inherited retinal diseases; pseudo-exon; retinitis pigmentosa; splicing.

Figure 1

Variant detection and familial segregation analysis. (A) Two-generation family pedigree; (B) BAM files showing the two CLRN1 variants: c.144T>G and c.254-643G>T; (C,D) Familial segregation analysis in proband (C) and one of his unaffected siblings (D). Dotted red lines shows the identified variants.

Figure 2

Retinal imaging of MOL377-1 at the age of 40 (A–F) and 50 (G–L). A-B and G-H represent ultra-wide-field pseudocolor and autofluorescence (FAF) fundus photos, respectively, taken using the Optos Panoramic 200 Optomap Fundus Camera. Characteristic peripheral dense BSPs mixed with retinal atrophy encroaching the temporal vascular arcades can be seen. (C,D) and (I,J) show heterogeneous autofluorescence compatible with the atrophic retina along with the hyperfluorescent ring surrounding the fovea. (E,F,K,L) are horizontal optical coherence tomography (OCT) sections showing preserved foveal islands surrounded by retinal thinning and loss of the outer retinal layers in the macular area. Cystoid macular edema (CME) was observed in the LE and RE in the first and the last follow-up visits, respectively.

Figure 3

Analyzing the effect of two CLRN1 variants (c.254-643G>T and c.254-649T>G) on its pre-mRNA splicing. (A) Graphical representation of pET01 minigene plasmid with a 792 bp CLRN1 intron 1 insert. (B) A representative agarose gel image of cDNA analysis from HeLa cells transfected with different plasmid constructs. A 1.5% agarose gel was used to separate the PCR products; the experiment was performed in biological triplicates. Bands labeled as TS1-3 represent the different transcripts shown in panel (C). (C) Graphical representation of the different splicing patterns in the pET01-CLRN1 minigene due to the two CLRN1 variants c.254-643G>T and c.254-649T>G. (D) A graphical representation of the human CLRN1 gene (top panel) and the insertion of a pseudo-exon due to the c.254-643G>T and c.254-649T>G variants (bottom panel) is labelled in orange color, with the sequences flanking the activated cryptic splice site. The wild-type flanking sequence is labelled in black; the previously reported c.254-649T>G variant is labelled in blue; the variant reported in the current study, c.254-643G>T, is labelled in red. A naturally occurring 83 bp CLRN1 pseudo-exon is labelled in green. Int—intron; TS—transcript; Ex—exon; WT—wild-type.

Figure 4

Sanger sequencing of the wild-type and mutant CLRN1 transcripts. (A) Sanger sequencing of transcript TS3 containing pseudo-exon 1; (B) Sanger sequencing of transcript TS2 containing pseudo-exon 2; (C) Sanger sequencing of transcript TS1 (wild-type transcript). Light blue shadows indicate the exon-exon junctions.

Figure 5

Detailed analysis of CLRN1 transcripts using TapeStation. (A) Automated gel electrophoresis of the PCR products using Agilent Technologies 4200 TapeStation. The left lane is a DNA marker. “Wild type” shows transcripts produced by the normal CLRN1 minigene, a high-intensity wild-type TS1 transcript and a very low-intensity TS2 transcript. “c.254-643G>T” shows transcripts produced by the CLRN1 minigene carrying the 254-643G>T variant, a high-intensity TS3 transcript and a low-intensity wild-type TS1 transcript. c.254-649T>G shows transcripts produced by the CLRN1 minigene carrying the c.254-649T>G variant, a high-intensity TS3 transcript alone. “Empty plasmid” shows transcripts produced by the pET01 empty plasmid, a high-intensity TS1 transcript alone. (B) Bar graph showing the intensity of each band in WT compared to the two studied variants.