Nested PCR as an economical and robust approach for detecting aberrant splicing events in clinically accessible tissues

Abstract

Purpose : RNA isolated from clinically accessible tissues, such as blood or fibroblasts, often contains few transcripts from genes expressed in the retina. This limitation hinders the diagnostic utility of RNA-seq for inherited retinal diseases (IRDs). Here, we demonstrate a PCR-based method to assess the effect of splice variants in lowly expressed IRD-associated genes.

Methods : Candidate splice variants discovered by next-generation sequencing in four genes (TULP1, IMPG2, TCOF1, and CEP164) were assessed using blood (n=7) and skin biopsy samples (n=2) from four families. The eyeIntegration database was used to evaluate expression levels in different tissues. Total RNA was isolated from whole blood and fibroblasts, followed by rRNA depletion, poly-A selection, and Illumina sequencing (150 bp paired-end reads) with a projected 50 million clusters per sample. The first strand cDNA was synthesized using gene-specific primers and Luna Reverse Transcriptase, followed by nested PCR and Sanger sequencing.

Results : RNA-seq resulted in 100-150 million unique reads for each sample. No splice alterations were detected in the RNA-seq data for three out of four genes. TULP1 had no reads at the variant site for samples from blood or fibroblasts. IMPG2 had fewer than five reads, TCOF1 had 151 reads, and CEP164 had 136 reads at the variant site. RNA-seq identified an intron retention event caused by an intronic variant in CEP164. Nested PCR followed by Sanger sequencing of cDNA confirmed splicing defects caused by all five variants in these four genes in both blood and fibroblast cells.

Conclusions : Nested PCR is a more cost-effective method than RNA-seq for detecting splice alterations. Limitations of the nested PCR approach include being labor-intensive and low throughput. Nested PCR of cDNA from blood or fibroblast samples is a robust method for detecting aberrant splicing in genes associated with eye diseases.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.