Targeted long-read cDNA sequencing reveals novel splice-altering pathogenic variants causing retinal dystrophies

Area of Science:

• Genetics
• Ophthalmology
• Molecular Biology

Background:

• Splice-altering variants contribute to the heritability of inherited retinal diseases (IRDs).
• Interpreting these variants is challenging due to the need for functional validation of pathogenicity.
• Current diagnostic methods may not fully capture splicing defects.

Purpose of the Study:

• To evaluate the diagnostic utility of targeted long-read cDNA sequencing (lrcDNA-seq) for investigating IRD-associated splicing defects.
• To identify novel splice-altering variants and uncover the genetic basis of unexplained IRD cases.

Main Methods:

• RNA isolation from blood samples of affected individuals.
• Generation of cDNA amplicons spanning open reading frames or multiple exons for candidate genes.
• Application of long-read sequencing to analyze cDNA amplicons for splicing abnormalities.

Main Results:

• Validation of the lrcDNA-seq approach using known pathogenic splice-altering variants in IRD genes.
• Identification of novel splice-altering variants in six previously unexplained IRD cases with NMNAT1 variants.
• Detection of SVA_F retrotransposon partial inclusion in NMNAT1 mRNA in four subjects, leading to premature stop codons.

Conclusions:

• Targeted lrcDNA-seq is effective for characterizing splice defects and identifying novel splice-altering variants in IRDs.
• This technique uncovered the genetic basis for six previously unexplained IRD subjects.
• Implementation of lrcDNA-seq has the potential to increase the diagnostic rate of IRDs.