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, ML; 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

Author(s): 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, ML; 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;
Details: Publication Year 2022-01,Volume 24,Issue #1,Page 130-145
Journal Title: Genetics in Medicine
Publication Type: Research article
Abstract: PURPOSE: 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.
Keywords: Adolescent; Adult; Child, Preschool; Humans; Mutation; *RNA/genetics; *RNA Splicing/genetics; Sequence Analysis, RNA; Exome Sequencing; Genetic diagnosis; Noncoding variant; Pre-mRNA splicing; Putative splice variant; Variant classification
Department(s): Pathology
PubMed ID: 34906502
Publisher's Version: https://doi.org/10.1016/j.gim.2021.09.001
Terms of Use/Rights Notice: Refer to copyright notice on published article.

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