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

Adam M. Bournazos; Lisa G. Riley; Shobhana Bommireddipalli; Lesley Ades; Lauren S. Akesson; Mohammad Al-Shinnag; Stephen I. Alexander; Alison D. Archibald; Shanti Balasubramaniam; Yemima Berman; Victoria Beshay; Kirsten Boggs; Jasmina Bojadzieva; Natasha J. Brown; Samantha J. Bryen; Michael F. Buckley; Belinda Chong; Mark R. Davis; Ruebena Dawes; Martin Delatycki; Liz Donaldson; Lilian Downie; Caitlin Edwards; Matthew Edwards; Amanda Engel; Lisa J. Ewans; Fathimath Faiz; Andrew Fennell; Michael Field; Mary Louise Freckmann; Lyndon Gallacher; Russell Gear; Himanshu Goel; Shuxiang Goh; Linda Goodwin; Bernadette Hanna; James Harraway; Megan Higgins; Gladys Ho; Bruce K. Hopper; Ari E. Horton; Matthew F. Hunter; Aamira J. Huq; Sarah Josephi-Taylor; Himanshu Joshi; Edwin Kirk; Emma Krzesinski; Kishore R. Kumar; Frances Lemckert; Richard J. Leventer; Suzanna E. Lindsey-Temple; Sebastian Lunke; Alan Ma; Steven Macaskill; Amali Mallawaarachchi; Melanie Marty; Justine E. Marum; Hugh J. McCarthy; Manoj P. Menezes; Alison McLean; Di Milnes; Shekeeb Mohammad; David Mowat; Aram Niaz; Elizabeth E. Palmer; Chirag Patel; Shilpan G. Patel; Dean Phelan; Jason R. Pinner; Sulekha Rajagopalan; Matthew Regan; Jonathan Rodgers; Miriam Rodrigues; Richard H. Roxburgh; Rani Sachdev; Tony Roscioli; Ruvishani Samarasekera; Sarah A. Sandaradura; Elena Savva; Tim Schindler; Margit Shah; Ingrid B. Sinnerbrink; Janine M. Smith; Richard J. Smith; Amanda Springer; Zornitza Stark; Samuel P. Strom; Carolyn M. Sue; Kenneth Tan; Tiong Y. Tan; Esther Tantsis; Michel C. Tchan; Bryony A. Thompson; Alison H. Trainer; Karin van Spaendonck-Zwarts; Rebecca Walsh; Linda Warwick; Stephanie White; Susan M. White; Mark G. Williams; Meredith J. Wilson; Wui Kwan Wong; Dale C. Wright; Patrick Yap; Alison Yeung; Helen Young; Kristi J. Jones; Bruce Bennetts; Sandra T. Cooper; on behalf of the Australasian Consortium for RNA Diagnostics

doi:10.1016/j.gim.2021.09.001

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

Adam M. Bournazos, Lisa G. Riley, Shobhana Bommireddipalli, Lesley Ades, Lauren S. Akesson, Mohammad Al-Shinnag, Stephen I. Alexander, Alison D. Archibald, Shanti Balasubramaniam, Yemima Berman, Victoria Beshay, Kirsten Boggs, Jasmina Bojadzieva, Natasha J. Brown, Samantha J. Bryen, Michael F. Buckley, Belinda Chong, Mark R. Davis, Ruebena Dawes, Martin DelatyckiLiz Donaldson, Lilian Downie, Caitlin Edwards, Matthew Edwards, Amanda Engel, Lisa J. Ewans, Fathimath Faiz, Andrew Fennell, Michael Field, Mary Louise Freckmann, Lyndon Gallacher, Russell Gear, Himanshu Goel, Shuxiang Goh, Linda Goodwin, Bernadette Hanna, James Harraway, Megan Higgins, Gladys Ho, Bruce K. Hopper, Ari E. Horton, Matthew F. Hunter, Aamira J. Huq, Sarah Josephi-Taylor, Himanshu Joshi, Edwin Kirk, Emma Krzesinski, Kishore R. Kumar, Frances Lemckert, Richard J. Leventer, Suzanna E. Lindsey-Temple, Sebastian Lunke, Alan Ma, Steven Macaskill, Amali Mallawaarachchi, Melanie Marty, Justine E. Marum, Hugh J. McCarthy, Manoj P. Menezes, Alison McLean, Di Milnes, Shekeeb Mohammad, David Mowat, Aram Niaz, Elizabeth E. Palmer, Chirag Patel, Shilpan G. Patel, Dean Phelan, Jason R. Pinner, Sulekha Rajagopalan, Matthew Regan, Jonathan Rodgers, Miriam Rodrigues, Richard H. Roxburgh, Rani Sachdev, Tony Roscioli, Ruvishani Samarasekera, Sarah A. Sandaradura, Elena Savva, Tim Schindler, Margit Shah, Ingrid B. Sinnerbrink, Janine M. Smith, Richard J. Smith, Amanda Springer, Zornitza Stark, Samuel P. Strom, Carolyn M. Sue, Kenneth Tan, Tiong Y. Tan, Esther Tantsis, Michel C. Tchan, Bryony A. Thompson, Alison H. Trainer, Karin van Spaendonck-Zwarts, Rebecca Walsh, Linda Warwick, Stephanie White, Susan M. White, Mark G. Williams, Meredith J. Wilson, Wui Kwan Wong, Dale C. Wright, Patrick Yap, Alison Yeung, Helen Young, Kristi J. Jones, Bruce Bennetts, Sandra T. Cooper, on behalf of the Australasian Consortium for RNA Diagnostics

Paediatrics Monash Health

Research output: Contribution to journal › Article › Research › peer-review

27 Citations (Scopus)

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.

Original language	English
Pages (from-to)	130-145
Number of pages	16
Journal	Genetics in Medicine
Volume	24
Issue number	1
DOIs	https://doi.org/10.1016/j.gim.2021.09.001
Publication status	Published - Jan 2022

Keywords

Genetic diagnosis
Noncoding variant
Pre-mRNA splicing
Putative splice variant
Variant classification

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10.1016/j.gim.2021.09.001

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Bournazos, A. M., Riley, L. G., Bommireddipalli, S., Ades, L., Akesson, L. S., Al-Shinnag, M., Alexander, S. I., Archibald, A. D., Balasubramaniam, S., Berman, Y., Beshay, V., Boggs, K., Bojadzieva, J., Brown, N. J., Bryen, S. J., Buckley, M. F., Chong, B., Davis, M. R., Dawes, R., ... on behalf of the Australasian Consortium for RNA Diagnostics (2022). Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants. Genetics in Medicine, 24(1), 130-145. https://doi.org/10.1016/j.gim.2021.09.001

@article{aa5d50059eea414e8f0ea293312d5ebc,

title = "Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants",

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 = "Genetic diagnosis, Noncoding variant, Pre-mRNA splicing, Putative splice variant, Variant classification",

author = "Bournazos, {Adam M.} and Riley, {Lisa G.} and Shobhana Bommireddipalli and Lesley Ades and Akesson, {Lauren S.} and Mohammad Al-Shinnag and Alexander, {Stephen I.} and Archibald, {Alison D.} and Shanti Balasubramaniam and Yemima Berman and Victoria Beshay and Kirsten Boggs and Jasmina Bojadzieva and Brown, {Natasha J.} and Bryen, {Samantha J.} and Buckley, {Michael F.} and Belinda Chong and Davis, {Mark R.} and Ruebena Dawes and Martin Delatycki and Liz Donaldson and Lilian Downie and Caitlin Edwards and Matthew Edwards and Amanda Engel and Ewans, {Lisa J.} and Fathimath Faiz and Andrew Fennell and Michael Field and Freckmann, {Mary Louise} and Lyndon Gallacher and Russell Gear and Himanshu Goel and Shuxiang Goh and Linda Goodwin and Bernadette Hanna and James Harraway and Megan Higgins and Gladys Ho and Hopper, {Bruce K.} and Horton, {Ari E.} and Hunter, {Matthew F.} and Huq, {Aamira J.} and Sarah Josephi-Taylor and Himanshu Joshi and Edwin Kirk and Emma Krzesinski and Kumar, {Kishore R.} and Frances Lemckert and Leventer, {Richard J.} and Lindsey-Temple, {Suzanna E.} and Sebastian Lunke and Alan Ma and Steven Macaskill and Amali Mallawaarachchi and Melanie Marty and Marum, {Justine E.} and McCarthy, {Hugh J.} and Menezes, {Manoj P.} and Alison McLean and Di Milnes and Shekeeb Mohammad and David Mowat and Aram Niaz and Palmer, {Elizabeth E.} and Chirag Patel and Patel, {Shilpan G.} and Dean Phelan and Pinner, {Jason R.} and Sulekha Rajagopalan and Matthew Regan and Jonathan Rodgers and Miriam Rodrigues and Roxburgh, {Richard H.} and Rani Sachdev and Tony Roscioli and Ruvishani Samarasekera and Sandaradura, {Sarah A.} and Elena Savva and Tim Schindler and Margit Shah and Sinnerbrink, {Ingrid B.} and Smith, {Janine M.} and Smith, {Richard J.} and Amanda Springer and Zornitza Stark and Strom, {Samuel P.} and Sue, {Carolyn M.} and Kenneth Tan and Tan, {Tiong Y.} and Esther Tantsis and Tchan, {Michel C.} and Thompson, {Bryony A.} and Trainer, {Alison H.} and {van Spaendonck-Zwarts}, Karin and Rebecca Walsh and Linda Warwick and Stephanie White and White, {Susan M.} and Williams, {Mark G.} and Wilson, {Meredith J.} and Wong, {Wui Kwan} and Wright, {Dale C.} and Patrick Yap and Alison Yeung and Helen Young and Jones, {Kristi J.} and Bruce Bennetts and Cooper, {Sandra T.} and {on behalf of the Australasian Consortium for RNA Diagnostics}",

note = "Funding Information: 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. Funding Information: 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{\textquoteright}s Health, a not for profit cooperative joint venture between the Sydney Children{\textquoteright}s Hospitals Network, the Children{\textquoteright}s Medical Research Institute, and the Children{\textquoteright}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. Publisher Copyright: {\textcopyright} 2021",

year = "2022",

month = jan,

doi = "10.1016/j.gim.2021.09.001",

language = "English",

volume = "24",

pages = "130--145",

journal = "Genetics in Medicine",

issn = "1098-3600",

publisher = "Nature Publishing Group",

number = "1",

}

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 & on behalf of the Australasian Consortium for RNA Diagnostics 2022, 'Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants', Genetics in Medicine, vol. 24, no. 1, pp. 130-145. https://doi.org/10.1016/j.gim.2021.09.001

TY - JOUR

T1 - Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants

AU - Bournazos, Adam M.

AU - Riley, Lisa G.

AU - Bommireddipalli, Shobhana

AU - Ades, Lesley

AU - Akesson, Lauren S.

AU - Al-Shinnag, Mohammad

AU - Alexander, Stephen I.

AU - Archibald, Alison D.

AU - Balasubramaniam, Shanti

AU - Berman, Yemima

AU - Beshay, Victoria

AU - Boggs, Kirsten

AU - Bojadzieva, Jasmina

AU - Brown, Natasha J.

AU - Bryen, Samantha J.

AU - Buckley, Michael F.

AU - Chong, Belinda

AU - Davis, Mark R.

AU - Dawes, Ruebena

AU - Delatycki, Martin

AU - Donaldson, Liz

AU - Downie, Lilian

AU - Edwards, Caitlin

AU - Edwards, Matthew

AU - Engel, Amanda

AU - Ewans, Lisa J.

AU - Faiz, Fathimath

AU - Fennell, Andrew

AU - Field, Michael

AU - Freckmann, Mary Louise

AU - Gallacher, Lyndon

AU - Gear, Russell

AU - Goel, Himanshu

AU - Goh, Shuxiang

AU - Goodwin, Linda

AU - Hanna, Bernadette

AU - Harraway, James

AU - Higgins, Megan

AU - Ho, Gladys

AU - Hopper, Bruce K.

AU - Horton, Ari E.

AU - Hunter, Matthew F.

AU - Huq, Aamira J.

AU - Josephi-Taylor, Sarah

AU - Joshi, Himanshu

AU - Kirk, Edwin

AU - Krzesinski, Emma

AU - Kumar, Kishore R.

AU - Lemckert, Frances

AU - Leventer, Richard J.

AU - Lindsey-Temple, Suzanna E.

AU - Lunke, Sebastian

AU - Ma, Alan

AU - Macaskill, Steven

AU - Mallawaarachchi, Amali

AU - Marty, Melanie

AU - Marum, Justine E.

AU - McCarthy, Hugh J.

AU - Menezes, Manoj P.

AU - McLean, Alison

AU - Milnes, Di

AU - Mohammad, Shekeeb

AU - Mowat, David

AU - Niaz, Aram

AU - Palmer, Elizabeth E.

AU - Patel, Chirag

AU - Patel, Shilpan G.

AU - Phelan, Dean

AU - Pinner, Jason R.

AU - Rajagopalan, Sulekha

AU - Regan, Matthew

AU - Rodgers, Jonathan

AU - Rodrigues, Miriam

AU - Roxburgh, Richard H.

AU - Sachdev, Rani

AU - Roscioli, Tony

AU - Samarasekera, Ruvishani

AU - Sandaradura, Sarah A.

AU - Savva, Elena

AU - Schindler, Tim

AU - Shah, Margit

AU - Sinnerbrink, Ingrid B.

AU - Smith, Janine M.

AU - Smith, Richard J.

AU - Springer, Amanda

AU - Stark, Zornitza

AU - Strom, Samuel P.

AU - Sue, Carolyn M.

AU - Tan, Kenneth

AU - Tan, Tiong Y.

AU - Tantsis, Esther

AU - Tchan, Michel C.

AU - Thompson, Bryony A.

AU - Trainer, Alison H.

AU - van Spaendonck-Zwarts, Karin

AU - Walsh, Rebecca

AU - Warwick, Linda

AU - White, Stephanie

AU - White, Susan M.

AU - Williams, Mark G.

AU - Wilson, Meredith J.

AU - Wong, Wui Kwan

AU - Wright, Dale C.

AU - Yap, Patrick

AU - Yeung, Alison

AU - Young, Helen

AU - Jones, Kristi J.

AU - Bennetts, Bruce

AU - Cooper, Sandra T.

AU - on behalf of the Australasian Consortium for RNA Diagnostics

N1 - Funding Information: 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. Funding Information: 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. Publisher Copyright: © 2021

PY - 2022/1

Y1 - 2022/1

N2 - 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.

AB - 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.

KW - Genetic diagnosis

KW - Noncoding variant

KW - Pre-mRNA splicing

KW - Putative splice variant

KW - Variant classification

UR - http://www.scopus.com/inward/record.url?scp=85122411169&partnerID=8YFLogxK

U2 - 10.1016/j.gim.2021.09.001

DO - 10.1016/j.gim.2021.09.001

M3 - Article

C2 - 34906502

AN - SCOPUS:85122411169

SN - 1098-3600

VL - 24

SP - 130

EP - 145

JO - Genetics in Medicine

JF - Genetics in Medicine

IS - 1

ER -

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

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Keywords

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