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

2022-01

Bournazos, Adam M

Riley, Lisa G

Bommireddipalli, Shobhana

Ades, Lesley

Akesson, Lauren S

Al-Shinnag, Mohammad

Alexander, Stephen I

Archibald, Alison D

Balasubramaniam, Shanti

Berman, Yemima

Beshay, Victoria

Boggs, Kirsten

Bojadzieva, Jasmina

Brown, Natasha J

Bryen, Samantha J

Buckley, Michael F

Chong, Belinda

Davis, Mark R

Dawes, Ruebena

Delatycki, Martin B

Donaldson, Liz

Downie, Lilian

Edwards, Caitlin

Edwards, Matthew

Engel, Amanda

Ewans, Lisa J

Faiz, Fathimath

Fennell, Andrew

Field, Michael

Freckmann, Mary-Louise

Gallacher, Lyndon

Gear, Russell

Goel, Himanshu

Goh, Shuxiang

Goodwin, Linda

Hanna, Bernadette

Harraway, James

Higgins, Megan

Ho, Gladys

Hopper, Bruce K

Horton, Ari E

Hunter, Matthew F

Huq, Aamira J

Josephi-Taylor, Sarah

Joshi, Himanshu

Kirk, Edwin

Krzesinski, Emma

Kumar, Kishore R

Lemckert, Frances

Leventer, Richard J

Lindsey-Temple, Suzanna E

Lunke, Sebastian

Ma, Alan

Macaskill, Steven

Mallawaarachchi, Amali

Marty, Melanie

Marum, Justine E

McCarthy, Hugh J

Menezes, Manoj P

McLean, Alison

Milnes, Di

Mohammad, Shekeeb

Mowat, David

Niaz, Aram

Palmer, Elizabeth E

Patel, Chirag

Patel, Shilpan G

Phelan, Dean

Pinner, Jason R

Rajagopalan, Sulekha

Regan, Matthew

Rodgers, Jonathan

Rodrigues, Miriam

Roxburgh, Richard H

Sachdev, Rani

Roscioli, Tony

Samarasekera, Ruvishani

Sandaradura, Sarah A

Savva, Elena

Schindler, Tim

Shah, Margit

Sinnerbrink, Ingrid B

Smith, Janine M

Smith, Richard J

Springer, Amanda

Stark, Zornitza

Strom, Samuel P

Sue, Carolyn M

Tan, Kenneth

Tan, Tiong Y

Tantsis, Esther

Tchan, Michel C

Thompson, Bryony A

Trainer, Alison H

van Spaendonck-Zwarts, Karin

Walsh, Rebecca

Warwick, Linda

White, Stephanie

White, Susan M

Williams, Mark G

Wilson, Meredith J

Wong, Wui Kwan

Wright, Dale C

Yap, Patrick

Yeung, Alison

Young, Helen

Jones, Kristi J

Bennetts, Bruce

Cooper, Sandra T

Genetic diagnosis

Noncoding variant

Pre-mRNA splicing

Putative splice variant

Variant classification

Journal Article

0000-0002-8769-2569

0000-0002-1657-6034

10.1016/j.gim.2021.09.001

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). 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. 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. 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.

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Genetics in Medicine : Official journal of the American College of Medical Genetics 2022; 24(1): 130-145

Genetics in Medicine : Official journal of the American College of Medical Genetics