Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants.
Bournazos AM., Riley LG., Bommireddipalli S., Ades L., Akesson LS., Al-Shinnag M., Alexander SI., Archibald AD., Balasubramaniam S., Berman Y., Beshay V., Boggs K., Bojadzieva J., Brown NJ., Bryen SJ., Buckley MF., Chong B., Davis MR., Dawes R., Delatycki M., Donaldson L., Downie L., Edwards C., Edwards M., Engel A., Ewans LJ., Faiz F., Fennell A., Field M., Freckmann M-L., Gallacher L., Gear R., Goel H., Goh S., Goodwin L., Hanna B., Harraway J., Higgins M., Ho G., Hopper BK., Horton AE., Hunter MF., Huq AJ., Josephi-Taylor S., Joshi H., Kirk E., Krzesinski E., Kumar KR., Lemckert F., Leventer RJ., Lindsey-Temple SE., Lunke S., Ma A., Macaskill S., Mallawaarachchi A., Marty M., Marum JE., McCarthy HJ., Menezes MP., McLean A., Milnes D., Mohammad S., Mowat D., Niaz A., Palmer EE., Patel C., Patel SG., Phelan D., Pinner JR., Rajagopalan S., Regan M., Rodgers J., Rodrigues M., Roxburgh RH., Sachdev R., Roscioli T., Samarasekera R., Sandaradura SA., Savva E., Schindler T., Shah M., Sinnerbrink IB., Smith JM., Smith RJ., Springer A., Stark Z., Strom SP., Sue CM., Tan K., Tan TY., Tantsis E., Tchan MC., Thompson BA., Trainer AH., van Spaendonck-Zwarts K., Walsh R., Warwick L., White S., White SM., Williams MG., Wilson MJ., Wong WK., Wright DC., Yap P., Yeung A., Young H., Jones KJ., Bennetts B., Cooper ST., Australasian Consortium for RNA Diagnostics .
PurposeGenetic variants causing aberrant premessenger RNA splicing are increasingly being recognized as causal variants in genetic disorders. In this study, we devise standardized practices for polymerase chain reaction (PCR)-based RNA diagnostics using clinically accessible specimens (blood, fibroblasts, urothelia, biopsy).MethodsA total of 74 families with diverse monogenic conditions (31% prenatal-congenital onset, 47% early childhood, and 22% teenage-adult onset) were triaged into PCR-based RNA testing, with comparative RNA sequencing for 19 cases.ResultsInformative RNA assay data were obtained for 96% of cases, enabling variant reclassification for 75% variants that can be used for genetic counseling (71%), to inform clinical care (32%) and prenatal counseling (41%). Variant-associated mis-splicing was highly reproducible for 28 cases with samples from ≥2 affected individuals or heterozygotes and 10 cases with ≥2 biospecimens. PCR amplicons encompassing another segregated heterozygous variant was vital for clinical interpretation of 22 of 79 variants to phase RNA splicing events and discern complete from partial mis-splicing.ConclusionRNA diagnostics enabled provision of a genetic diagnosis for 64% of recruited cases. PCR-based RNA diagnostics has capacity to analyze 81.3% of clinically significant genes, with long amplicons providing an advantage over RNA sequencing to phase RNA splicing events. The Australasian Consortium for RNA Diagnostics (SpliceACORD) provide clinically-endorsed, standardized protocols and recommendations for interpreting RNA assay data.