A targeted long-read RNA sequencing approach may help improve the interpretation of genetic variants and support diagnosis in patients with rare diseases, according to a study published in Science Advances.
Current genetic testing methods, including exome and genome sequencing, identify the cause of disease in around 20 to 50 percent of cases. This leaves many patients without a confirmed molecular diagnosis. RNA sequencing can provide additional information by showing how genetic variants affect gene expression and splicing.
In this study, researchers developed a workflow called STRIPE (Sequencing Targeted RNAs Identifies Pathogenic Events). The method focuses sequencing on selected disease-related genes and uses long-read sequencing to analyze full-length RNA transcripts. This allows variants and their effects to be assessed together and linked to specific parental alleles.
The researchers applied the method to skin fibroblast samples from 88 individuals, including patients with congenital disorders of glycosylation and primary mitochondrial diseases. In patients with known genetic diagnoses, the approach identified all previously reported pathogenic variants and provided additional information about how these variants affected RNA processing.
Compared with short-read sequencing, long-read sequencing enabled analysis across larger regions of DNA, helping to determine whether variants occur on the same or different alleles. This can be difficult with standard methods, particularly when variants are far apart.
The study also showed that variants affecting splice sites can lead to a wider range of RNA changes than expected. In more than half of the splice site variants examined, the effects were more complex than simple exon skipping. Some variants were associated with early termination of RNA transcripts due to activation of alternative polyadenylation sites.
Importantly, the method helped identify disease-causing variants in several patients who had not previously received a genetic diagnosis, including cases where standard RNA sequencing approaches were inconclusive.
Because the approach targets specific genes, it can achieve greater sequencing depth and improve detection of low-level transcripts in clinically accessible tissues such as fibroblasts. However, it is limited to the genes included in the panel and may not detect variants in genes outside the selected set.
The authors note that further work will be needed before the method can be implemented in clinical laboratories, including standardizing workflows and integrating the approach into existing diagnostic pathways.
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