Objective:

To improve the interpretation of genetic variants and support diagnosis in patients with rare diseases using a targeted long-read RNA sequencing approach.

Key Findings:

• The approach identified all previously reported pathogenic variants in patients with known genetic diagnoses and provided additional insights into RNA processing.
• Long-read sequencing enabled analysis across larger DNA regions, facilitating the determination of whether variants occur on the same or different alleles.
• Variants affecting splice sites led to a wider range of RNA changes than expected, with complex effects observed in over half of the splice site variants examined.
• The method identified disease-causing variants in patients who had not previously received a genetic diagnosis.

Interpretation:

The targeted long-read RNA sequencing approach enhances the understanding of genetic variants' effects on gene expression and splicing, potentially leading to better diagnostic outcomes for patients with rare diseases.

Limitations:

• The method is limited to the genes included in the panel and may not detect variants in genes outside the selected set.
• Further work is needed to standardize workflows and integrate the approach into existing diagnostic pathways before clinical implementation.

Conclusion:

The STRIPE method shows promise in improving genetic diagnosis for rare diseases, but further validation and integration into clinical practice are necessary.