The identification of a RNA splice variant in TULP1 in two siblings with early‐onset photoreceptor dystrophy

Abstract

Background Early‐onset photoreceptor dystrophies are a major cause of irreversible visual impairment in children and young adults. This clinically heterogeneous group of disorders can be caused by mutations in many genes. Nevertheless, to date, 30%–40% of cases remain genetically unexplained. In view of expanding therapeutic options, it is essential to obtain a molecular diagnosis in these patients as well. In this study, we aimed to identify the genetic cause in two siblings with genetically unexplained retinal disease. Methods Whole exome sequencing was performed to identify the causative variants in two siblings in whom a single pathogenic variant in TULP1 was found previously. Patients were clinically evaluated, including assessment of the medical history, slit‐lamp biomicroscopy, and ophthalmoscopy. In addition, a functional analysis of the putative splice variant in TULP1 was performed using a midigene assay. Results Clinical assessment showed a typical early‐onset photoreceptor dystrophy in both the patients. Whole exome sequencing identified two pathogenic variants in TULP1, a c.1445G>A (p.(Arg482Gln)) missense mutation and an intronic c.718+23G>A variant. Segregation analysis confirmed that both siblings were compound heterozygous for the TULP1 c.718+23G>A and c.1445G>A variants, while the unaffected parents were heterozygous. The midigene assay for the c.718+23G>A variant confirmed an elongation of exon 7 leading to a frameshift. Conclusion Here, we report the first near‐exon RNA splice variant that is not present in a consensus splice site sequence in TULP1, which was found in a compound heterozygous manner with a previously described pathogenic TULP1 variant in two patients with an early‐onset photoreceptor dystrophy. We provide proof of pathogenicity for this splice variant by performing an in vitro midigene splice assay, and highlight the importance of analysis of noncoding regions beyond the noncanonical splice sites in patients with inherited retinal diseases.

Findings from a Genotyping Study of over 1000 People with Inherited Retinal Disorders in Ireland

“…This approach is particularly effective for testing exonic and intronic variants that are likely to disrupt correct splicing, as these variants will have a distinct impact on the RNA product when compared to the wild-type equivalent. This method is also particularly useful for the interpretation of variants that are located within introns, yet outside of canonical splice sites, and additionally to examine rare synonymous exonic variants proximal to exon junctions [32,78].…”

Whole genome sequencing and in vitro splice assays reveal genetic causes for inherited retinal diseases

“…CNVs were called using Control-FREEC, which detects copy number changes and allelic imbalances based on the read depth 62 . SVs and CNVs were annotated using an in-house developed pipeline 63 , i.e., annotations for chromosomal location and position, gene(s) and their component (e.g., exonic), type of SV (gain/loss), percentage overlap and frequency of various population frequency databases (e.g., gnomAD-SV 22 , GoNL 53 , Decipher 64 , Wellderly 54 , 1000genomes 55 ), and disease OMIM description. Short tandem repeats (STRs) for 33 known pathogenic sites were detected using Expansion Hunter V.3.1.2.…”

“…After completing the data analysis of 100 cases, 10 deep-intronic and 3 NCSS candidate variants in 14 individuals were identified, which adhered to the stringent criteria defined above for noncoding variants. Subsequently, a midigene or minigene splice assay was employed as described previously to assess potential splicing defects in the presence of variants 63,72 . In short, the regions of interest of a genomic DNA sample were amplified by primers that contain attB1 and attB2 tags at their 5′ end to facilitate the Gateway cloning.…”

Next-Generation Sequencing Applications for Inherited Retinal Diseases

“…WES exclusively captures the protein-coding exons, but only accounts for approximately 1% of the genome. It is important to note that exon-based sequencing is likely to also reliably detect intronic variants located close to the targeted exons, such as non-canonical splice site variants, which are known causes of IRDs [ 28 , 29 , 30 ]. WGS is significantly more comprehensive including introns, promoters, and intergenic regions; in principle sequencing every nucleotide possible in a sample.…”