Understanding genetic disorders can be challenging, especially when standard DNA tests do not provide clear answers. A team led by researchers from ARUP Laboratories examined how RNA sequencing can fill these gaps and help solve difficult medical cases involving rare diseases.

Traditional genetic testing usually focuses on DNA, but DNA alone cannot always show how a variant affects the way genes actually work. Many important changes happen in regions of the genome that do not code for proteins, such as introns or regulatory sequences. These changes can alter how RNA is made, which in turn affects how proteins are produced.

The research team reviewed 30 complex patient cases from the Utah Penelope Program and the Undiagnosed Diseases Network. In each case, earlier DNA testing could not fully explain the patient’s symptoms or left doctors with uncertain results. To find answers, the researchers analyzed RNA from blood or fibroblast samples to see how gene transcripts were actually behaving inside cells.

RNA sequencing revealed several types of transcript disruptions. These included exon skipping, activation of hidden splice sites, and retention of introns. All of these issues interfere with the normal flow of genetic information. RNA data also helped clarify whether certain genes on the X chromosome were active or silenced, which improved the interpretation of copy number variants.

Overview of cohort characteristics and case outcomes

A Age distribution of pediatric and adult participants who underwent combined DNA and RNA sequencing. Each dot represents an individual and horizontal lines represent median and 95% confidence intervals (CI). Blue circles represent males and red circles represent females. B Comparison of ages between participants with resolved and unresolved cases. Each dot represents an individual and the horizontal lines indicate the median and 95% CI. Participants with resolved cases were significantly younger than those with cases not resolved participants (two-tailed Student’s t-test, P = 0.0054). Blue circles represent males and red circles represent females. C Sample types used for RNA-seq in resolved and not resolved cases, represented as a bar chart. D Parts-of-a-whole chart illustrating clinical categories of participants, distinguishing between those with case resolution (Dx) and those without (No Dx). E Molecular mechanisms for each variant assessed, represented as a bar chart. F Time to case resolution in 10 participants, showing weeks from initial clinical consultation to final resolution and from RNA-seq analysis to resolution. Horizontal bars represent the median and 95% confidence interval. Case 9 was excluded, as RNA-seq was performed concurrently with karyotyping, and the diagnosis was established prior to the return of RNA-seq results

By showing how genetic variants affected RNA, the researchers were able to reclassify multiple variants of uncertain significance and provide clearer diagnoses for some patients. The work highlights how RNA sequencing gives clinicians valuable functional information that DNA sequencing alone cannot provide. When integrated into clinical genetics workflows, this approach can support variant classification, help explain challenging disease cases, and guide patient care and genetic counseling.