Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative...

Area of Science:

• Molecular Biology
• Genetics
• Diagnostic Medicine

Background:

• Aberrant premessenger RNA splicing due to genetic variants is a growing cause of genetic disorders.
• Accurate RNA diagnostics are crucial for identifying these variants and informing patient management.
• Standardized protocols are needed for reliable RNA testing using accessible clinical specimens.

Purpose of the Study:

• To develop and validate standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics.
• To assess the utility of these diagnostics in identifying causal variants for genetic disorders.
• To evaluate the impact of RNA diagnostics on genetic counseling and clinical decision-making.

Main Methods:

• Developed standardized PCR-based RNA diagnostic protocols for blood, fibroblasts, urothelia, and biopsy specimens.
• Triaged 74 families with diverse monogenic conditions into RNA testing, with RNA sequencing for 19 cases.
• Utilized comparative RNA sequencing and PCR amplicons for variant phasing and splicing event interpretation.

Main Results:

• Obtained informative RNA assay data for 96% of cases, enabling variant reclassification for 75%.
• RNA diagnostics provided a genetic diagnosis for 64% of recruited cases.
• Demonstrated high reproducibility of variant-associated mis-splicing and identified PCR amplicons as vital for clinical interpretation.

Conclusions:

• Standardized PCR-based RNA diagnostics are effective in identifying splicing variants and diagnosing genetic disorders.
• This approach offers advantages over RNA sequencing for phasing RNA splicing events and analyzing a broad range of genes.
• The Australasian Consortium for RNA Diagnostics (SpliceACORD) provides clinically-endorsed protocols for RNA assay interpretation.