Bournazos, Adam M.Riley, Lisa G.Bommireddipalli, ShobhanaAdes, LesleyAkesson, Lauren S.Al-Shinnag, MohammadAlexander, Stephen I.Archibald, Alison D.Balasubramaniam, ShantiBerman, YemimaBeshay, VictoriaBoggs, KirstenBojadzieva, JasminaBrown, Natasha J.Bryen, Samantha J.Buckley, Michael F.Chong, BelindaDavis, Mark R.Dawes, RuebenaDelatycki, MartinDonaldson, LizDownie, LilianEdwards, CaitlinEdwards, MatthewEngel, AmandaEwans, Lisa J.Faiz, FathimathFennell, AndrewField, MichaelFreckmann, Mary LouiseGallacher, LyndonGear, RussellGoel, HimanshuGoh, ShuxiangGoodwin, LindaHanna, BernadetteHarraway, JamesHiggins, MeganHo, GladysHopper, Bruce K.Horton, Ari E.Hunter, Matthew F.Huq, Aamira J.Josephi-Taylor, SarahJoshi, HimanshuKirk, EdwinKrzesinski, EmmaBreen, JimmyEyras, EduardoHayashi, Rippei2025-05-252025-05-251098-3600PubMed:34906502ORCID:/0000-0003-0793-6218/work/164351544ORCID:/0000-0002-5848-9019/work/168142702http://www.scopus.com/inward/record.url?scp=85122411169&partnerID=8YFLogxKhttps://hdl.handle.net/1885/733753310Purpose: Genetic 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).  Methods: A 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.  Results: Informative 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.  Conclusion: RNA 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.We thank the families for their participation and invaluable contributions to this research. We also thank the clinicians and health care workers involved in their assessment and management. Special thanks to Naomi L. Baker, Bethany Buckley, Tenielle Clinch, Fiona Cunningham, Ryan L. Davis, Elizabeth Farnsworth, Tegan French, Janette Hayward, Katherine Holman, Cass Hoskins, Anna Jarmolowicz, McKenna Kyriss, Crystle Lee, Sarah Pantaleo, and Emma Wright who were either involved in patient care, counseling, ascertainment, diagnostic laboratory analysis, variant curation, and/or completed surveys. The data sets used for the analyses described in this manuscript were obtained from dbGaP at http://www.ncbi.nlm.nih.gov/gap through dbGaP accession number phs000424.v7.p2. We downloaded the call sets from ENCODE ( https://www.encodeproject.org/) with the following identifiers: (Cerebellum) ENCFF602BYA, ENCFF113PDT and (Camera type eye) ENCFF980GGP, ENCFF883SDA. Sandra T. Cooper is supported by a National Health and Medical Research Council of Australia Senior Research Fellowship under grant APP1136197. This project received funding through the Medical Research Future Fund Rapid Applied Research Translation Program grant awarded to Sydney Health Partners. Adam M. Bournazos is supported by a University of Sydney Research Training Scholarship. Part of this work was supported by Luminesce Alliance––Innovation for Children's Health, a not for profit cooperative joint venture between the Sydney Children's Hospitals Network, the Children's Medical Research Institute, and the Children's Cancer Institute. It has been established with the support of the New South Wales Government to coordinate and integrate pediatric research. Luminesce Alliance is also affiliated with the University of Sydney and the University of New South Wales, Sydney. Carolyn M. Sue is a National Health and Medical Research Council Practitioner Fellow under APP1136800. Conceptualization: A.M.B. L.G.R. S.T.C.; Data Curation and Formal Analysis: A.M.B. R.D. H.J.; Investigation: all authors; Resources: all authors; Validation: all authors; Visualization: A.M.B. L.G.R. S.T.C.; Funding Acquisition: S.T.C.; Methodology: A.M.B. L.G.R. S.B. A.N.; Project Administration: S.T.C.; Writing-original draft: A.M.B. L.G.R. S.T.C.; Writing-review and editing: all authors; Supervision: K.J.J. B.B. S.T.C. Consent for diagnostic genomic testing was supported by governance infrastructure of the relevant local ethics committees of the participating Australian Public Health Local Area Health Districts. Kids Neuroscience Centre's biobanking and functional genomics human ethics protocol was approved by the Sydney Children's Hospitals Network Human Research Ethics Committee (protocol 10/CHW/45 renewed with protocol 2019/ETH11736 [July 2019-2024]) with informed, written consent for all participants. Sandra T. Cooper is supported by a National Health and Medical Research Council of Australia Senior Research Fellowship under grant APP1136197. This project received funding through the Medical Research Future Fund Rapid Applied Research Translation Program grant awarded to Sydney Health Partners. Adam M. Bournazos is supported by a University of Sydney Research Training Scholarship. Part of this work was supported by Luminesce Alliance––Innovation for Children’s Health, a not for profit cooperative joint venture between the Sydney Children’s Hospitals Network, the Children’s Medical Research Institute, and the Children’s Cancer Institute. It has been established with the support of the New South Wales Government to coordinate and integrate pediatric research. Luminesce Alliance is also affiliated with the University of Sydney and the University of New South Wales, Sydney. Carolyn M. Sue is a National Health and Medical Research Council Practitioner Fellow under APP1136800.16enPublisher Copyright: © 2021Genetic diagnosisNoncoding variantPre-mRNA splicingPutative splice variantVariant classification2021-11-3010.1016/j.gim.2021.09.00185122411169