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1. RNA variant assessment using transactivation and transdifferentiation

Author

Azmanov, Dimitar N., Barnett, Christopher P., Barry, Simon C., Baynam, Gareth, Berkovic, Samuel F., Christodoulou, John, Coman, David J., Cooper, Sandra, Corbett, Mark A., Delatycki, Martin, Dudding, Tracy E., Fletcher, Sue, Gardner, Alison E., Gecz, Jozef, Higgins, Megan J., Hildebrand, Michael S., Jolly, Lachlan A., Lister, Ryan, McGaughran, Julie, Pflueger, Christian, Poulton, Cathryn, Roscioli, Tony, Hamish S. Scott, Ingrid Scheffer, Sinclair, Andrew H., Spurdle, Amanda B., Tan, Tiong Y., van Eyk, Clare L., Voineagu, Irina, Nicolas-Martinez, Emmylou C., Robinson, Olivia, Gardner, Alison, Ritchie, Tarin, Kroes, Thessa, Scheffer, Ingrid E., Barnier, Jean-Vianney, Rousseau, Véronique, Genevieve, David, Haushalter, Virginie, Piton, Amélie, Denommé-Pichon, Anne-Sophie, Bruel, Ange-Line, Nambot, Sophie, Isidor, Bertrand, Grigg, John, Gonzalez, Tina, Ghedia, Sondhya, Marchant, Rhett G., Bournazos, Adam, Wong, Wui-Kwan, Webster, Richard I., Evesson, Frances J., Jones, Kristi J., and Cooper, Sandra T.

Published
2024
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6. Unraveling facets of MECOM-associated syndrome: somatic genetic rescue, clonal hematopoiesis, and phenotype expansion

Author

Venugopal, Parvathy, Arts, Peer, Fox, Lucy C., Simons, Annet, Hiwase, Devendra K., Bardy, Peter G., Swift, Annette, Ross, David M., van Vulpen, Lize F. D., Buijs, Arjan, Bolton, Kelly L., Getta, Bartlomiej, Furlong, Eliska, Carter, Tina, Krapels, Ingrid, Hoeks, Marlijn, Al Kindy, Adila, Al Kindy, Farah, de Munnik, Sonja, Evans, Pamela, Frank, Mahalia S. B., Bournazos, Adam M., Cooper, Sandra T., Ha, Thuong Thi, Jackson, Matilda R., Arriola-Martinez, Luis, Phillips, Kerry, Brennan, Yvonne, Bakshi, Madhura, Ambler, Karen, Gao, Song, Kassahn, Karin S., Kenyon, Rosalie, Hung, Kevin, Babic, Milena, McGovern, Alan, Rawlings, Lesley, Vakulin, Cassandra, Dejong, Lucas, Fathi, Rema, McRae, Simon, Myles, Nicholas, Ladon, Dariusz, Jongmans, Marjolijn, Kuiper, Roland P., Poplawski, Nicola K., Barbaro, Pasquale, Blombery, Piers, Brown, Anna L., Hahn, Christopher N., and Scott, Hamish S.

Published
2024
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7. Severe NAD(P)HX Dehydratase (NAXD) Neurometabolic Syndrome May Present in Adulthood after Mild Head Trauma

Author

Van Bergen, Nicole J., primary, Gunanayagam, Karen, additional, Bournazos, Adam M., additional, Walvekar, Adhish S., additional, Warmoes, Marc O., additional, Semcesen, Liana N., additional, Lunke, Sebastian, additional, Bommireddipalli, Shobhana, additional, Sikora, Tim, additional, Patraskaki, Myrto, additional, Jones, Dean L., additional, Garza, Denisse, additional, Sebire, Dale, additional, Gooley, Samuel, additional, McLean, Catriona A., additional, Naidoo, Parm, additional, Rajasekaran, Mugil, additional, Stroud, David A., additional, Linster, Carole L., additional, Wallis, Mathew, additional, Cooper, Sandra T., additional, and Christodoulou, John, additional

Published
2023
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8. Nemaline myopathy and distal arthrogryposis associated with an autosomal recessive TNNT3 splice variant

Author

Sandaradura, Sarah A., Bournazos, Adam, Mallawaarachchi, Amali, Cummings, Beryl B., Waddell, Leigh B., Jones, Kristi J., Troedson, Christopher, Sudarsanam, Annapurna, Nash, Benjamin M., Peters, Gregory B., Algar, Elizabeth M., MacArthur, Daniel G., North, Kathryn N., Brammah, Susan, Charlton, Amanda, Laing, Nigel G., Wilson, Meredith J., Davis, Mark R., and Cooper, Sandra T.

Published
2018
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9. Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants

Author

Bournazos, Adam M., primary, Riley, Lisa G., additional, Bommireddipalli, Shobhana, additional, Ades, Lesley, additional, Akesson, Lauren S., additional, Al-Shinnag, Mohammad, additional, Alexander, Stephen I., additional, Archibald, Alison D., additional, Balasubramaniam, Shanti, additional, Berman, Yemima, additional, Beshay, Victoria, additional, Boggs, Kirsten, additional, Bojadzieva, Jasmina, additional, Brown, Natasha J., additional, Bryen, Samantha J., additional, Buckley, Michael F., additional, Chong, Belinda, additional, Davis, Mark R., additional, Dawes, Ruebena, additional, Delatycki, Martin, additional, Donaldson, Liz, additional, Downie, Lilian, additional, Edwards, Caitlin, additional, Edwards, Matthew, additional, Engel, Amanda, additional, Ewans, Lisa J., additional, Faiz, Fathimath, additional, Fennell, Andrew, additional, Field, Michael, additional, Freckmann, Mary-Louise, additional, Gallacher, Lyndon, additional, Gear, Russell, additional, Goel, Himanshu, additional, Goh, Shuxiang, additional, Goodwin, Linda, additional, Hanna, Bernadette, additional, Harraway, James, additional, Higgins, Megan, additional, Ho, Gladys, additional, Hopper, Bruce K., additional, Horton, Ari E., additional, Hunter, Matthew F., additional, Huq, Aamira J., additional, Josephi-Taylor, Sarah, additional, Joshi, Himanshu, additional, Kirk, Edwin, additional, Krzesinski, Emma, additional, Kumar, Kishore R., additional, Lemckert, Frances, additional, Leventer, Richard J., additional, Lindsey-Temple, Suzanna E., additional, Lunke, Sebastian, additional, Ma, Alan, additional, Macaskill, Steven, additional, Mallawaarachchi, Amali, additional, Marty, Melanie, additional, Marum, Justine E., additional, McCarthy, Hugh J., additional, Menezes, Manoj P., additional, McLean, Alison, additional, Milnes, Di, additional, Mohammad, Shekeeb, additional, Mowat, David, additional, Niaz, Aram, additional, Palmer, Elizabeth E., additional, Patel, Chirag, additional, Patel, Shilpan G., additional, Phelan, Dean, additional, Pinner, Jason R., additional, Rajagopalan, Sulekha, additional, Regan, Matthew, additional, Rodgers, Jonathan, additional, Rodrigues, Miriam, additional, Roxburgh, Richard H., additional, Sachdev, Rani, additional, Roscioli, Tony, additional, Samarasekera, Ruvishani, additional, Sandaradura, Sarah A., additional, Savva, Elena, additional, Schindler, Tim, additional, Shah, Margit, additional, Sinnerbrink, Ingrid B., additional, Smith, Janine M., additional, Smith, Richard J., additional, Springer, Amanda, additional, Stark, Zornitza, additional, Strom, Samuel P., additional, Sue, Carolyn M., additional, Tan, Kenneth, additional, Tan, Tiong Y., additional, Tantsis, Esther, additional, Tchan, Michel C., additional, Thompson, Bryony A., additional, Trainer, Alison H., additional, van Spaendonck-Zwarts, Karin, additional, Walsh, Rebecca, additional, Warwick, Linda, additional, White, Stephanie, additional, White, Susan M., additional, Williams, Mark G., additional, Wilson, Meredith J., additional, Wong, Wui Kwan, additional, Wright, Dale C., additional, Yap, Patrick, additional, Yeung, Alison, additional, Young, Helen, additional, Jones, Kristi J., additional, Bennetts, Bruce, additional, Cooper, Sandra T., additional, Abdulrasool, Ghusoon, additional, Al Eryani, Ghamdan, additional, Arts, Peer, additional, Bagnall, Richard, additional, Baker, Naomi L., additional, Barnett, Christopher, additional, Beecroft, Sarah, additional, Berbic, Marina, additional, Black, Michael, additional, Blackburn, Jim, additional, Blombery, Piers, additional, Bournazos, Adam M., additional, Branford, Susan, additional, Breen, Jimmy, additional, Burnett, Leslie, additional, Canson, Daffodil, additional, Cheong, Pak, additional, Chew, Edward, additional, Christodoulou, John, additional, Chung, Seo-Kyung, additional, Clark, Mike, additional, Cliffe, Corrina, additional, Cole, Melissa, additional, Collins, Felicity, additional, Compton, Alison, additional, Cooper, Antony, additional, Corbett, Mark, additional, Cowley, Mark, additional, Dudding, Tracy, additional, Eggers, Stefanie, additional, Eyras, Eduardo, additional, Fernandez, Miriam Fanjul, additional, Fellowes, Andrew, additional, Fleischer, Ron, additional, Folland, Chiara, additional, Fox, Lucy, additional, Gaff, Clara, additional, Galea, Melanie, additional, Ghaoui, Roula, additional, Gornanitis, Ilias, additional, Ha, Thuong, additional, Hayashi, Rippei, additional, Hayes, Ian, additional, Henderson, Alex, additional, Hesson, Luke, additional, Heyer, Erin, additional, Hildebrand, Michael, additional, Hipwell, Michael, additional, Hoskins, Cass, additional, Jackson, Matilda, additional, James, Paul, additional, Wong, Justin Jong-Leong, additional, Kassahn, Karin, additional, Kaub, Peter, additional, Kevin, Lucy, additional, Kumble, Smitha, additional, Kummerfeld, Sarah, additional, Laing, Nigel, additional, Lau, Chiyan, additional, Lee, Eric, additional, Leighton, Sarah, additional, Lundie, Ben, additional, Mayoh, Chelsea, additional, McGaughran, Julie, additional, McPhillips, Mary, additional, Meldrum, Cliff, additional, Middleton, Edwina, additional, Mina, Kym, additional, Nisselle, Amy, additional, Oates, Emily, additional, Oshlack, Alicia, additional, Parasivam, Gayathri, additional, Parsons, Michael, additional, Quinn, Michael, additional, Rasko, John, additional, Ravenscroft, Gina, additional, Ravine, Anja, additional, Recsei, Krista, additional, Rehn, Jacqueline, additional, Robertson, Stephen, additional, Ronan, Anne, additional, Ryland, Georgina, additional, Sadedin, Simon, additional, Schreiber, Andreas, additional, Scott, Hamish, additional, Scott, Rodney, additional, Semsarian, Christopher, additional, Simons, Cas, additional, Singer, Emma, additional, Smyth, Renee, additional, Spurdle, Amanda, additional, Sullivan, Patricia, additional, Sundercombe, Samantha, additional, Thorburn, David, additional, Toubia, John, additional, Trent, Ronald, additional, Tudini, Emma, additional, Voneague, Irina, additional, Waddell, Leigh, additional, Walker, Logan, additional, Wallis, Mathew, additional, Warnock, Nick, additional, Weatheritt, Robert, additional, White, Deborah, additional, Winship, Ingrid, additional, Worgan, Lisa, additional, Wu, Kathy, additional, and Ziolowski, Andrew, additional

Published
2022
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10. Clinical impact of whole-genome sequencing in patients with early-onset dementia

Author

Huq, Aamira J, primary, Thompson, Bryony, additional, Bennett, Mark F, additional, Bournazos, Adam, additional, Bommireddipalli, Shobhana, additional, Gorelik, Alexandra, additional, Schultz, Joshua, additional, Sexton, Adrienne, additional, Purvis, Rebecca, additional, West, Kirsty, additional, Cotter, Megan, additional, Valente, Giulia, additional, Hughes, Andrew, additional, Riaz, Moeen, additional, Walsh, Maie, additional, Farrand, Sarah, additional, Loi, Samantha M, additional, Kilpatrick, Trevor, additional, Brodtmann, Amy, additional, Darby, David, additional, Eratne, Dhamidhu, additional, Walterfang, Mark, additional, Delatycki, Martin Bruce, additional, Storey, Elsdon, additional, Fahey, Michael, additional, Cooper, Sandra, additional, Lacaze, Paul, additional, Masters, Colin L, additional, Velakoulis, Dennis, additional, Bahlo, Melanie, additional, James, Paul A, additional, and Winship, Ingrid, additional

Published
2022
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11. Optimising RNA diagnostics for implementation into clinical practice

Author

Bournazos, Adam Michael

Subjects
genetic diagnosis, variant interpretation, variant of uncertain significance, rare disease, RNA sequencing, Splicing variant
Abstract

Background: Genetic variants that elicit aberrant splicing of pre-messenger RNA (pre-mRNA) are recognised as causative variants in ~30-50% of genetic disorders. However, it is still not possible to predict reliably if and how a variant will impact splicing, limiting the application of in silico splice prediction tools in variant interpretation. Most splicing variants fall outside the essential splice site and, in the absence of RNA testing, remain classed variants of uncertain significance (VUS) according to ACMG-AMP (American College of Medical Genetics and Genomics and Association for Molecular Pathology) guidelines. Sequence analysis of spliced messenger RNA (mRNA) is the only definitive means to determine the precise nature of variant associated mis-splicing. Tissues with limited accessibility, for instance vital organs, present a challenge for RNA testing of genes with tissue specific expression. Fortunately, clinically accessible tissues such as blood and fibroblasts can be used to infer variant associated mis-splicing outcomes in the manifesting tissue. A further challenge arises from the lack of guidance on how functional evidence (PS3/BS3 criteria) from RNA studies should be applied to variant interpretation within the current ACMG-AMP framework. There is an urgent need to establish ACMG-AMP aligned quality standards and guidelines for complex RNA assay data for accurate and consistent variant interpretation between clinical laboratories. Methods: Families were recruited from local area health districts across Australia and New Zealand using inclusion criteria to ascertain putative splicing variants with high clinical suspicion of causality. More than 120 families with diverse monogenic conditions were triaged into PCR-based RNA testing, with comparative RNA-sequencing for 38 cases. Consensus ascertainment criteria, standard practices for PCR-based RNA testing, and RNA assay interpretation rubric were devised through consultation with the clinical and molecular genetics community via surveys, live polls and SpliceACORD consortium (Australasian Consortium for RNA Diagnostics) meetings. Results: Informative RNA assay data was obtained for 96% cases, enabling variant re-classification for 75% of variants. RNA testing reports were used to guide clinical care and genetic counselling, and 75% of diagnosis were clinician-reported to have a positive impact for the family. PCR-based RNA diagnostics has the capacity to analyse 81.3% of clinically significant genes and to allow phasing of RNA splicing events. Variant associated mis-splicing was highly reproducible between affected individuals and heterozygotes, and between different biospecimens. Discussion: We provide a standardised protocol for PCR-based RNA testing and ACMG-AMP aligned recommendations for the interpretation of RNA assay data. Our study demonstrates the significant diagnostic and health benefits of RNA analysis as adjunct testing to extend diagnostic yield from genomic testing.

Published
2022

12. Recurrent TTN metatranscript-only c.39974-11T>G splice variant associated with autosomal recessive arthrogryposis multiplex congenita and myopathy

Author

Bryen, Samantha J, Ewans, Lisa J, Pinner, Jason, MacLennan, Suzanna C, Donkervoort, Sandra, Castro, Diana, Töpf, Ana, O'Grady, Gina, Cummings, Beryl, Chao, Katherine R, Weisburd, Ben, Francioli, Laurent, Faiz, Fathimath, Bournazos, Adam M, Hu, Ying, Grosmann, Carla, Malicki, Denise M, Doyle, Helen, Witting, Nanna, Vissing, John, Claeys, Kristl G, Urankar, Kathryn, Beleza-Meireles, Ana, Baptista, Julia, Ellard, Sian, Savarese, Marco, Johari, Mridul, Vihola, Anna, Udd, Bjarne, Majumdar, Anirban, Straub, Volker, Bönnemann, Carsten G, MacArthur, Daniel G, Davis, Mark R, Cooper, Sandra T, Bryen, Samantha J, Ewans, Lisa J, Pinner, Jason, MacLennan, Suzanna C, Donkervoort, Sandra, Castro, Diana, Töpf, Ana, O'Grady, Gina, Cummings, Beryl, Chao, Katherine R, Weisburd, Ben, Francioli, Laurent, Faiz, Fathimath, Bournazos, Adam M, Hu, Ying, Grosmann, Carla, Malicki, Denise M, Doyle, Helen, Witting, Nanna, Vissing, John, Claeys, Kristl G, Urankar, Kathryn, Beleza-Meireles, Ana, Baptista, Julia, Ellard, Sian, Savarese, Marco, Johari, Mridul, Vihola, Anna, Udd, Bjarne, Majumdar, Anirban, Straub, Volker, Bönnemann, Carsten G, MacArthur, Daniel G, Davis, Mark R, and Cooper, Sandra T

Abstract

We present eight families with arthrogryposis multiplex congenita and myopathy bearing a TTN intron 213 extended splice-site variant (NM_001267550.1:c.39974-11T>G), inherited in trans with a second pathogenic TTN variant. Muscle-derived RNA studies of three individuals confirmed mis-splicing induced by the c.39974-11T>G variant; in-frame exon 214 skipping or use of a cryptic 3' splice-site effecting a frameshift. Confounding interpretation of pathogenicity is the absence of exons 213-217 within the described skeletal muscle TTN N2A isoform. However, RNA-sequencing from 365 adult human gastrocnemius samples revealed that 56% specimens predominantly include exons 213-217 in TTN transcripts (inclusion rate ≥66%). Further, RNA-sequencing of five fetal muscle samples confirmed that 4/5 specimens predominantly include exons 213-217 (fifth sample inclusion rate 57%). Contractures improved significantly with age for four individuals, which may be linked to decreased expression of pathogenic fetal transcripts. Our study extends emerging evidence supporting a vital developmental role for TTN isoforms containing metatranscript-only exons.

Published
2020

13. Improving genetic diagnosis in Mendelian disease with transcriptome sequencing

Author

Broad Institute of MIT and Harvard, Lincoln Laboratory, Cummings, Beryl, Marshall, Jamie L., Tukiainen, Taru, Lek, Monkol, Donkervoort, Sandra, Foley, A. Reghan, Bolduc, Veronique, Waddell, Leigh B., Sandaradura, Sarah A., O’Grady, Gina L., Estrella, Elicia, Reddy, Hemakumar M., Zhao, Fengmei, Weisburd, Ben, Karczewski, Konrad, O’Donnell-Luria, Anne H., Birnbaum, Daniel, Sarkozy, Anna, Hu, Ying, Gonorazky, Hernan, Claeys, Kristl, Joshi, Himanshu, Bournazos, Adam, Oates, Emily C., Ghaoui, Roula, Davis, Mark R., Laing, Nigel G., Topf, Ana, Kang, Peter B., Beggs, Alan H., North, Kathryn N., Straub, Volker, Dowling, James J., Muntoni, Francesco, Clarke, Nigel F., Cooper, Sandra T., Bönnemann, Carsten G., MacArthur, Daniel G., Broad Institute of MIT and Harvard, Lincoln Laboratory, Cummings, Beryl, Marshall, Jamie L., Tukiainen, Taru, Lek, Monkol, Donkervoort, Sandra, Foley, A. Reghan, Bolduc, Veronique, Waddell, Leigh B., Sandaradura, Sarah A., O’Grady, Gina L., Estrella, Elicia, Reddy, Hemakumar M., Zhao, Fengmei, Weisburd, Ben, Karczewski, Konrad, O’Donnell-Luria, Anne H., Birnbaum, Daniel, Sarkozy, Anna, Hu, Ying, Gonorazky, Hernan, Claeys, Kristl, Joshi, Himanshu, Bournazos, Adam, Oates, Emily C., Ghaoui, Roula, Davis, Mark R., Laing, Nigel G., Topf, Ana, Kang, Peter B., Beggs, Alan H., North, Kathryn N., Straub, Volker, Dowling, James J., Muntoni, Francesco, Clarke, Nigel F., Cooper, Sandra T., Bönnemann, Carsten G., and MacArthur, Daniel G.

Abstract

Exome and whole-genome sequencing are becoming increasingly routine approaches in Mendelian disease diagnosis. Despite their success, the current diagnostic rate for genomic analyses across a variety of rare diseases is approximately 25 to 50%. We explore the utility of transcriptome sequencing [RNA sequencing (RNA-seq)] as a complementary diagnostic tool in a cohort of 50 patients with genetically undiagnosed rare muscle disorders. We describe an integrated approach to analyze patient muscle RNA-seq, leveraging an analysis framework focused on the detection of transcript-level changes that are unique to the patient compared to more than 180 control skeletal muscle samples. We demonstrate the power of RNA-seq to validate candidate splice-disrupting mutations and to identify splice-altering variants in both exonic and deep intronic regions, yielding an overall diagnosis rate of 35%. We also report the discovery of a highly recurrent de novo intronic mutation in COL6A1 that results in a dominantly acting splice-gain event, disrupting the critical glycine repeat motif of the triple helical domain. We identify this pathogenic variant in a total of 27 genetically unsolved patients in an external collagen VI-like dystrophy cohort, thus explaining approximately 25% of patients clinically suggestive of having collagen VI dystrophy in whom prior genetic analysis is negative. Overall, this study represents a large systematic application of transcriptome sequencing to rare disease diagnosis and highlights its utility for the detection and interpretation of variants missed by current standard diagnostic approaches. 2017, National Institutes of Health (Grant GM096911), National Institute of General Medical Sciences (Grants F32GM115208 and 4T32GM007748)

Published
2020

14. Loss of calpains-1 and -2 prevents repair of plasma membrane scrape injuries, but not small pores, and induces a severe muscular dystrophy

Author

Piper, Ann-Katrin, Sophocleous, Reece, Ross, Samuel, Evesson, Frances, Saleh, Omar, Bournazos, Adam, Yasa, Joe, Reed, Claudia, Woolger, Natalie, Sluyter, Ronald, Greer, Peter, Biro, Mate, Lemckert, Frances, Cooper, Sandra, Piper, Ann-Katrin, Sophocleous, Reece, Ross, Samuel, Evesson, Frances, Saleh, Omar, Bournazos, Adam, Yasa, Joe, Reed, Claudia, Woolger, Natalie, Sluyter, Ronald, Greer, Peter, Biro, Mate, Lemckert, Frances, and Cooper, Sandra

Abstract

Copyright © 2020 the American Physiological Society. The ubiquitous calpains, calpain-1 and -2, play important roles in Ca2+-dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca2+-dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 (CAPNS1∼/∼) virtually ablates Ca2+-dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptoly-sin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 (CAPN1∼/∼) or -2 (CAPN2∼/∼) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 (CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca2+-signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.

Published
2020

15. WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase

Author

Waddell, Leigh B., primary, Bryen, Samantha J., additional, Cummings, Beryl B., additional, Bournazos, Adam, additional, Evesson, Frances J., additional, Joshi, Himanshu, additional, Marshall, Jamie L., additional, Tukiainen, Taru, additional, Valkanas, Elise, additional, Weisburd, Ben, additional, Sadedin, Simon, additional, Davis, Mark R., additional, Faiz, Fathimath, additional, Gooding, Rebecca, additional, Sandaradura, Sarah A., additional, O'Grady, Gina L., additional, Tchan, Michel C., additional, Mowat, David R., additional, Oates, Emily C., additional, Farrar, Michelle A., additional, Sampaio, Hugo, additional, Ma, Alan, additional, Neas, Katherine, additional, Wang, Min-Xia, additional, Charlton, Amanda, additional, Chan, Charles, additional, Kenwright, Diane N., additional, Graf, Nicole, additional, Arbuckle, Susan, additional, Clarke, Nigel F., additional, MacArthur, Daniel G., additional, Jones, Kristi J., additional, Lek, Monkol, additional, and Cooper, Sandra T., additional

Published
2021
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16. Rapid exome sequencing and adjunct RNA studies confirm the pathogenicity of a novel homozygous ASNS splicing variant in a critically ill neonate

Author

Akesson, Lauren S., primary, Bournazos, Adam, additional, Fennell, Andrew, additional, Krzesinski, Emma I., additional, Tan, Kenneth, additional, Springer, Amanda, additional, Rose, Katherine, additional, Goranitis, Ilias, additional, Francis, David, additional, Lee, Crystle, additional, Faiz, Fathimath, additional, Davis, Mark R., additional, Christodoulou, John, additional, Lunke, Sebastian, additional, Stark, Zornitza, additional, Hunter, Matthew F., additional, and Cooper, Sandra T., additional

Published
2020
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17. Loss of calpains-1 and -2 prevents repair of plasma membrane scrape injuries, but not small pores, and induces a severe muscular dystrophy

Author

Piper, Ann-Katrin, primary, Sophocleous, Reece A., additional, Ross, Samuel E., additional, Evesson, Frances J., additional, Saleh, Omar, additional, Bournazos, Adam, additional, Yasa, Joe, additional, Reed, Claudia, additional, Woolger, Natalie, additional, Sluyter, Ronald, additional, Greer, Peter, additional, Biro, Maté, additional, Lemckert, Frances A., additional, and Cooper, Sandra T., additional

Published
2020
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18. Recurrent TTN metatranscript‐only c.39974–11T>G splice variant associated with autosomal recessive arthrogryposis multiplex congenita and myopathy

Author

Bryen, Samantha J., primary, Ewans, Lisa J., additional, Pinner, Jason, additional, MacLennan, Suzanna C., additional, Donkervoort, Sandra, additional, Castro, Diana, additional, Töpf, Ana, additional, O'Grady, Gina, additional, Cummings, Beryl, additional, Chao, Katherine R., additional, Weisburd, Ben, additional, Francioli, Laurent, additional, Faiz, Fathimath, additional, Bournazos, Adam M., additional, Hu, Ying, additional, Grosmann, Carla, additional, Malicki, Denise M., additional, Doyle, Helen, additional, Witting, Nanna, additional, Vissing, John, additional, Claeys, Kristl G., additional, Urankar, Kathryn, additional, Beleza‐Meireles, Ana, additional, Baptista, Julia, additional, Ellard, Sian, additional, Savarese, Marco, additional, Johari, Mridul, additional, Vihola, Anna, additional, Udd, Bjarne, additional, Majumdar, Anirban, additional, Straub, Volker, additional, Bönnemann, Carsten G., additional, MacArthur, Daniel G., additional, Davis, Mark R., additional, and Cooper, Sandra T., additional

Published
2019
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19. Importance of muscle biopsy to establish pathogenicity of DMD missense and splice variants

Author

Jones, Hannah F, primary, Bryen, Samantha J, additional, Waddell, Leigh B, additional, Bournazos, Adam, additional, Davis, Mark, additional, Farrar, Michelle A, additional, McLean, Catriona A, additional, Mowat, David R, additional, Sampaio, Hugo, additional, Woodcock, Ian R, additional, Ryan, Monique M, additional, Jones, Kristi J, additional, and Cooper, Sandra T, additional

Published
2019
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20. Congenital Titinopathy: Comprehensive characterization and pathogenic insights

Author

Oates, Emily, Jones, Kristi, Donkervoort, Sandra, Charlton, Amanda, Brammah, Susan, Smith, John, Ware, James, Yau, Kyle, Swanson, Lindsay, Whiffin, Nicola, Peduto, Anthony, Bournazos, Adam, Waddell, Leigh, Farrar, Michelle, Sampaio, Hugo, Teoh, Hooi Ling, Lamont, Phillipa, Mowat, David, Fitzsimons, Robin, Corbett, Alastair, Ryan, Monique, O'Grady, Gina, Sandaradura, Sarah, Ghaoui, Roula, Joshi, Himanshu, Marshall, Jamie, Nolan, Melinda, Kaur, Simranpreet, Punetha, Jaya, Töpf, Ana, Harris, Elizabeth, Bakshi, Madhura, Genetti, Casie, Marttila, Minttu, Werlauff, Ulla, Streichenberger, Nathalie, Pestronk, Alan, Mazanti, Ingrid, Pinner, Jason, Vuillerot, Carole, Grosmann, Carla, Camacho, Ana, Mohassel, Payam, Leach, Meganne, Foley, A Reghan, Bharucha-Goebel, Diana, Collins, James, Connolly, Anne, Gilbreath, Heather, Iannaccone, Susan, Castro, Diana, Cummings, Beryl, WEBSTER, Richard, Lazaro, Leila, Vissing, John, Coppens, Sandra, Deconinck, Nicolas, Luk, Ho-Ming, Thomas, Neil, Foulds, Nicola, Illingworth, Marjorie, Ellard, Sian, Mclean, Catriona, Phadke, Rahul, Ravenscroft, Gianina, Witting, Nanna, Hackman, Peter, Richard, Isabelle, Cooper, Sandra, Kamsteeg, Erik-Jan, Hoffman, Eric, Bushby, Kate, Straub, Volker, Udd, Bjarne, Ferreiro, Ana, North, Kathryn, Clarke, Nigel, Lek, Monkol, Beggs, Alan, Bönnemann, Carsten, MacArthur, Daniel, Granzier, Henk, Davis, Mark, Laing, Nigel, Foley, A. Reghan, Bharucha‐Goebel, Diana, Luk, Ho‐Ming, Kamsteeg, Erik‐Jan, The University of Sydney, Institute for Neuroscience and Muscle Research, Westmead Hospital [Sydney], Prologue, Hospices Civils de Lyon (HCL), Washington University in St Louis, Hôpital Femme Mère Enfant [CHU - HCL] (HFME), Department of Neurology, Rady Children's Hospital San Diego, National Institute of Neurological Disorders and Stroke [Bethesda] (NINDS), National Institutes of Health [Bethesda] (NIH), German Research Centre for Geosciences - Helmholtz-Centre Potsdam (GFZ), Duke University [Durham], Rothamsted Research, Département de médecine de l'enfant et de l'adolescent, University of Copenhagen = Københavns Universitet (KU), Université Libre de Bruxelles [Bruxelles] (ULB), University of Birmingham [Birmingham], Our Lady's hospital for Sick Children, Our Lady's Hospital for Sick Children, Royal Devon and Exeter Foundation Trust, State Neuropathology Service, Department of Pathology, University of Melbourne, The University of Western Australia (UWA), Haartman Institute [Helsinki], Faculty of Medecine [Helsinki], University of Helsinki-University of Helsinki, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Neurology Department, Physiopathologie et thérapie du muscle strié, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR14-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Neuroscience, Uppsala University, Boston Children's Hospital, Washington State University (WSU), University of East London & Glasgow Caledonian University, Centre for Medical Research, Richard, Isabelle, Institut NeuroMyoGène (INMG), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Washington University in Saint Louis (WUSTL), Université de Lyon, Le CHCB, Centre Hospitalier de la Côte Basque, École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Unité de Biologie Fonctionnelle et Adaptative (BFA (UMR_8251 / U1133)), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Université libre de Bruxelles (ULB), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Centre Hospitalier de la Côte Basque (CHCB), and École Pratique des Hautes Études (EPHE)

Subjects
Cardiomyopathy, Dilated, Male, MESH: Connectin, MESH: Mutation, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, education, Muscle Proteins, [SDV.GEN.GH] Life Sciences [q-bio]/Genetics/Human genetics, MESH: Protein Isoforms, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Phenotype, Article, MESH: Muscle Proteins, Humans, Protein Isoforms, Connectin, Muscle, Skeletal, MESH: Cardiomyopathy, Dilated, health care economics and organizations, MESH: Muscle, Skeletal, MESH: Humans, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Male, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, Phenotype, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Mutation, Female, MESH: Female
Abstract

International audience; Objective: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder.Methods: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients.Results: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder.Interpretation: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.

Published
2018

23. Congenital Titinopathy:Comprehensive characterization and pathogenic insights

Author

Oates, Emily C., Jones, Kristi J., Donkervoort, Sandra, Charlton, Amanda, Brammah, Susan, Smith, John E., Ware, James S., Yau, Kyle S., Swanson, Lindsay C., Whiffin, Nicola, Peduto, Anthony J., Bournazos, Adam, Waddell, Leigh B., Farrar, Michelle A., Sampaio, Hugo A., Teoh, Hooi Ling, Lamont, Phillipa J., Mowat, David, Fitzsimons, Robin B., Corbett, Alastair J., Ryan, Monique M., O'Grady, Gina L., Sandaradura, Sarah A., Ghaoui, Roula, Joshi, Himanshu, Marshall, Jamie L., Nolan, Melinda A., Kaur, Simranpreet, Punetha, Jaya, Töpf, Ana, Harris, Elizabeth, Bakshi, Madhura, Genetti, Casie A., Marttila, Minttu, Werlauff, Ulla, Streichenberger, Nathalie, Pestronk, Alan, Mazanti, Ingrid, Pinner, Jason R., Vuillerot, Carole, Grosmann, Carla, Camacho, Ana, Mohassel, Payam, Leach, Meganne E., Foley, A. Reghan, Bharucha-Goebel, Diana, Collins, James, Connolly, Anne M., Gilbreath, Heather R., Iannaccone, Susan T., Castro, Diana, Cummings, Beryl B., Webster, Richard I., Lazaro, Leïla, Vissing, John, Coppens, Sandra, Deconinck, Nicolas, Luk, Ho Ming, Thomas, Neil H., Foulds, Nicola C., Illingworth, Marjorie A., Ellard, Sian, McLean, Catriona A., Phadke, Rahul, Ravenscroft, Gianina, Witting, Nanna, Hackman, Peter, Richard, Isabelle, Cooper, Sandra T., Kamsteeg, Erik Jan, Hoffman, Eric P., Bushby, Kate, Straub, Volker, Udd, Bjarne, Ferreiro, Ana, North, Kathryn N., Clarke, Nigel F., Lek, Monkol, Beggs, Alan H., Bönnemann, Carsten G., MacArthur, Daniel G., Granzier, Henk, Davis, Mark R., Laing, Nigel G., Oates, Emily C., Jones, Kristi J., Donkervoort, Sandra, Charlton, Amanda, Brammah, Susan, Smith, John E., Ware, James S., Yau, Kyle S., Swanson, Lindsay C., Whiffin, Nicola, Peduto, Anthony J., Bournazos, Adam, Waddell, Leigh B., Farrar, Michelle A., Sampaio, Hugo A., Teoh, Hooi Ling, Lamont, Phillipa J., Mowat, David, Fitzsimons, Robin B., Corbett, Alastair J., Ryan, Monique M., O'Grady, Gina L., Sandaradura, Sarah A., Ghaoui, Roula, Joshi, Himanshu, Marshall, Jamie L., Nolan, Melinda A., Kaur, Simranpreet, Punetha, Jaya, Töpf, Ana, Harris, Elizabeth, Bakshi, Madhura, Genetti, Casie A., Marttila, Minttu, Werlauff, Ulla, Streichenberger, Nathalie, Pestronk, Alan, Mazanti, Ingrid, Pinner, Jason R., Vuillerot, Carole, Grosmann, Carla, Camacho, Ana, Mohassel, Payam, Leach, Meganne E., Foley, A. Reghan, Bharucha-Goebel, Diana, Collins, James, Connolly, Anne M., Gilbreath, Heather R., Iannaccone, Susan T., Castro, Diana, Cummings, Beryl B., Webster, Richard I., Lazaro, Leïla, Vissing, John, Coppens, Sandra, Deconinck, Nicolas, Luk, Ho Ming, Thomas, Neil H., Foulds, Nicola C., Illingworth, Marjorie A., Ellard, Sian, McLean, Catriona A., Phadke, Rahul, Ravenscroft, Gianina, Witting, Nanna, Hackman, Peter, Richard, Isabelle, Cooper, Sandra T., Kamsteeg, Erik Jan, Hoffman, Eric P., Bushby, Kate, Straub, Volker, Udd, Bjarne, Ferreiro, Ana, North, Kathryn N., Clarke, Nigel F., Lek, Monkol, Beggs, Alan H., Bönnemann, Carsten G., MacArthur, Daniel G., Granzier, Henk, Davis, Mark R., and Laing, Nigel G.

Abstract

Objective: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. Methods: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. Results: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. Interpretation: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105–1124.

Published
2018

25. Recurrent TTN metatranscript‐only c.39974–11T>G splice variant associated with autosomal recessive arthrogryposis multiplex congenita and myopathy.

Author

Bryen, Samantha J., Ewans, Lisa J., Pinner, Jason, MacLennan, Suzanna C., Donkervoort, Sandra, Castro, Diana, Töpf, Ana, O'Grady, Gina, Cummings, Beryl, Chao, Katherine R., Weisburd, Ben, Francioli, Laurent, Faiz, Fathimath, Bournazos, Adam M., Hu, Ying, Grosmann, Carla, Malicki, Denise M., Doyle, Helen, Witting, Nanna, and Vissing, John

Abstract

We present eight families with arthrogryposis multiplex congenita and myopathy bearing a TTN intron 213 extended splice‐site variant (NM_001267550.1:c.39974‐11T>G), inherited in trans with a second pathogenic TTN variant. Muscle‐derived RNA studies of three individuals confirmed mis‐splicing induced by the c.39974‐11T>G variant; in‐frame exon 214 skipping or use of a cryptic 3′ splice‐site effecting a frameshift. Confounding interpretation of pathogenicity is the absence of exons 213‐217 within the described skeletal muscle TTN N2A isoform. However, RNA‐sequencing from 365 adult human gastrocnemius samples revealed that 56% specimens predominantly include exons 213‐217 in TTN transcripts (inclusion rate ≥66%). Further, RNA‐sequencing of five fetal muscle samples confirmed that 4/5 specimens predominantly include exons 213‐217 (fifth sample inclusion rate 57%). Contractures improved significantly with age for four individuals, which may be linked to decreased expression of pathogenic fetal transcripts. Our study extends emerging evidence supporting a vital developmental role for TTN isoforms containing metatranscript‐only exons. [ABSTRACT FROM AUTHOR]

Published
2020
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30. Lack of MG53 in human heart precludes utility as a biomarker of myocardial injury or endogenous cardioprotective factor

Author

Lemckert, Frances A., primary, Bournazos, Adam, additional, Eckert, Daniel M., additional, Kenzler, Manuel, additional, Hawkes, Joanne M., additional, Butler, Tanya L., additional, Ceely, Bradley, additional, North, Kathryn N., additional, Winlaw, David S., additional, Egan, Jonathan R., additional, and Cooper, Sandra T., additional

Published
2016
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31. Nemaline myopathy and distal arthrogryposis associated with an autosomal recessive <italic>TNNT3</italic> splice variant.

Author

Sandaradura, Sarah A., Bournazos, Adam, Mallawaarachchi, Amali, Cummings, Beryl B., Waddell, Leigh B., Jones, Kristi J., Troedson, Christopher, Sudarsanam, Annapurna, Nash, Benjamin M., Peters, Gregory B., Algar, Elizabeth M., MacArthur, Daniel G., North, Kathryn N., Brammah, Susan, Charlton, Amanda, Laing, Nigel G., Wilson, Meredith J., Davis, Mark R., and Cooper, Sandra T.

Abstract

Abstract: A male neonate presented with severe weakness, hypotonia, contractures and congenital scoliosis. Skeletal muscle specimens showed marked atrophy and degeneration of fast fibers with striking nemaline rods and hypertrophy of slow fibers that were ultrastructurally normal. A neuromuscular gene panel identified a homozygous essential splice variant in TNNT3 (chr11:1956150G > A, NM_006757.3:c.681+1G > A). TNNT3 encodes skeletal troponin‐Tfast and is associated with autosomal dominant distal arthrogryposis. TNNT3 has not previously been associated with nemaline myopathy (NM), a rare congenital myopathy linked to defects in proteins associated with thin filament structure and regulation. cDNA studies confirmed pathogenic consequences of the splice variant, eliciting exon‐skipping and intron retention events leading to a frameshift. Western blot showed deficiency of troponin‐Tfast protein with secondary loss of troponin‐Ifast. We establish a homozygous splice variant in TNNT3 as the likely cause of severe congenital NM with distal arthrogryposis, characterized by specific involvement of Type‐2 fibers and deficiency of troponin‐Tfast. [ABSTRACT FROM AUTHOR]

Published
2018
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33. Improving genetic diagnosis in Mendelian disease with transcriptome sequencing

Author

Cummings, Beryl B., Marshall, Jamie L., Tukiainen, Taru, Lek, Monkol, Donkervoort, Sandra, Foley, A. Reghan, Bolduc, Veronique, Waddell, Leigh B., Sandaradura, Sarah A., O’Grady, Gina L., Estrella, Elicia, Reddy, Hemakumar M., Zhao, Fengmei, Weisburd, Ben, Karczewski, Konrad J., O’Donnell-Luria, Anne H., Birnbaum, Daniel, Sarkozy, Anna, Hu, Ying, Gonorazky, Hernan, Claeys, Kristl, Joshi, Himanshu, Bournazos, Adam, Oates, Emily C., Ghaoui, Roula, Davis, Mark R., Laing, Nigel G., Topf, Ana, Kang, Peter B., Beggs, Alan H., North, Kathryn N., Straub, Volker, Dowling, James J., Muntoni, Francesco, Clarke, Nigel F., Cooper, Sandra T., Bönnemann, Carsten G., and MacArthur, Daniel G.

Abstract

Transcriptome sequencing improves the diagnostic rate for Mendelian disease in patients for whom genetic analysis has not returned a diagnosis.

Published
2017
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34. Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants

Author

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, Ghusoon Abdulrasool, Ghamdan Al Eryani, Peer Arts, Richard Bagnall, Naomi L. Baker, Christopher Barnett, Sarah Beecroft, Marina Berbic, Michael Black, Jim Blackburn, Piers Blombery, Susan Branford, Jimmy Breen, Leslie Burnett, Daffodil Canson, Pak Cheong, Edward Chew, John Christodoulou, Seo-Kyung Chung, Mike Clark, Corrina Cliffe, Melissa Cole, Felicity Collins, Alison Compton, Antony Cooper, Mark Corbett, Mark Cowley, Tracy Dudding, Stefanie Eggers, Eduardo Eyras, Miriam Fanjul Fernandez, Andrew Fellowes, Ron Fleischer, Chiara Folland, Lucy Fox, Clara Gaff, Melanie Galea, Roula Ghaoui, Ilias Gornanitis, Thuong Ha, Rippei Hayashi, Ian Hayes, Alex Henderson, Luke Hesson, Erin Heyer, Michael Hildebrand, Michael Hipwell, Cass Hoskins, Matilda Jackson, Paul James, Justin Jong-Leong Wong, Karin Kassahn, Peter Kaub, Lucy Kevin, Smitha Kumble, Sarah Kummerfeld, Nigel Laing, Chiyan Lau, Eric Lee, Sarah Leighton, Ben Lundie, Chelsea Mayoh, Julie McGaughran, Mary McPhillips, Cliff Meldrum, Edwina Middleton, Kym Mina, Amy Nisselle, Emily Oates, Alicia Oshlack, Gayathri Parasivam, Michael Parsons, Michael Quinn, John Rasko, Gina Ravenscroft, Anja Ravine, Krista Recsei, Jacqueline Rehn, Stephen Robertson, Anne Ronan, Georgina Ryland, Simon Sadedin, Andreas Schreiber, Hamish Scott, Rodney Scott, Christopher Semsarian, Cas Simons, Emma Singer, Renee Smyth, Amanda Spurdle, Patricia Sullivan, Samantha Sundercombe, David Thorburn, John Toubia, Ronald Trent, Emma Tudini, Irina Voneague, Leigh Waddell, Logan Walker, Mathew Wallis, Nick Warnock, Robert Weatheritt, Deborah White, Ingrid Winship, Lisa Worgan, Kathy Wu, Andrew Ziolowski, Bournazos, Adam M, Riley, Lisa G, Bommireddipalli, Shobhana, Ades, Lesley, Cooper, Sandra T, Toubia, John, and Australasian Consortium for RNA Diagnostics

Subjects
Adult, Adolescent, RNA Splicing, Genetic counseling, putative splice variant, Biology, law.invention, genetic diagnosis, law, Exome Sequencing, Biopsy, medicine, Humans, variant classification, Gene, Genetics (clinical), Polymerase chain reaction, Genetics, medicine.diagnostic_test, Sequence Analysis, RNA, noncoding variant, RNA, Heterozygote advantage, Amplicon, Child, Preschool, Mutation, RNA splicing, pre-mRNA splicing
Abstract

usc Refereed/Peer-reviewed 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.

Published
2022

35. Deep Sequencing and Phenotyping in an Australian Tuberous Sclerosis Complex "No Mutations Identified" Cohort.

Author

Chung CWT, Bournazos AM, Chan LCD, Sarkozy V, Lawson J, Kennedy SE, Cooper ST, Kirk EP, and Mowat D

Subjects
Humans, Female, Male, Australia, Child, Adolescent, Adult, Child, Preschool, High-Throughput Nucleotide Sequencing methods, Infant, Mutation, Mosaicism, Tuberous Sclerosis genetics, Tuberous Sclerosis pathology, Tuberous Sclerosis diagnosis, Phenotype, Tuberous Sclerosis Complex 2 Protein genetics, Tuberous Sclerosis Complex 1 Protein genetics
Abstract

Tuberous sclerosis complex (TSC) is a variable multisystem disorder. The "no mutations identified" (NMI) group are reportedly phenotypically milder than those with an identified molecular cause, and often have mosaic or intronic variants not detected by standard sequencing methods., Methods: We describe the phenotypes in an Australian TSC NMI group (n = 18) and a molecular testing strategy implementable in a diagnostic laboratory. Massively parallel sequencing (MPS) of the whole genomic regions of TSC1 and TSC2 was performed using DNA extracted from multiple tissue samples per participant., Results: Our study showed that the phenotype in TSC NMI individuals can be similar to those with heterozygous, particularly TSC1, variants. Although neurodevelopmental outcomes can be less severe, the number of organ systems involved was similar to the non-mosaic groups. A diagnostic yield of 72% (13/18) was achieved, with the majority (10/13) being mosaic variants and the remainder heterozygous variants missed on previous testing., Conclusion: Testing DNA from multiple tissue samples allowed for validation of otherwise discarded low-level mosaic variants and detection of mosaic variants by MPS without excessive cost or the need for specialised techniques. Implementing this approach in a diagnostic setting is viable and allows optimal clinical care of patients with NMI TSC., (© 2024 The Author(s). Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Published
2024
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36. RNA variant assessment using transactivation and transdifferentiation.

Author

Nicolas-Martinez EC, Robinson O, Pflueger C, Gardner A, Corbett MA, Ritchie T, Kroes T, van Eyk CL, Scheffer IE, Hildebrand MS, Barnier JV, Rousseau V, Genevieve D, Haushalter V, Piton A, Denommé-Pichon AS, Bruel AL, Nambot S, Isidor B, Grigg J, Gonzalez T, Ghedia S, Marchant RG, Bournazos A, Wong WK, Webster RI, Evesson FJ, Jones KJ, Cooper ST, Lister R, Gecz J, and Jolly LA

Subjects
Humans, RNA genetics, RNA metabolism, CRISPR-Cas Systems, Cell Transdifferentiation genetics, Fibroblasts metabolism, Fibroblasts cytology, Transcriptional Activation, Neurons metabolism, Neurons cytology
Abstract

Understanding the impact of splicing and nonsense variants on RNA is crucial for the resolution of variant classification as well as their suitability for precision medicine interventions. This is primarily enabled through RNA studies involving transcriptomics followed by targeted assays using RNA isolated from clinically accessible tissues (CATs) such as blood or skin of affected individuals. Insufficient disease gene expression in CATs does however pose a major barrier to RNA based investigations, which we show is relevant to 1,436 Mendelian disease genes. We term these "silent" Mendelian genes (SMGs), the largest portion (36%) of which are associated with neurological disorders. We developed two approaches to induce SMG expression in human dermal fibroblasts (HDFs) to overcome this limitation, including CRISPR-activation-based gene transactivation and fibroblast-to-neuron transdifferentiation. Initial transactivation screens involving 40 SMGs stimulated our development of a highly multiplexed transactivation system culminating in the 6- to 90,000-fold induction of expression of 20/20 (100%) SMGs tested in HDFs. Transdifferentiation of HDFs directly to neurons led to expression of 193/516 (37.4%) of SMGs implicated in neurological disease. The magnitude and isoform diversity of SMG expression following either transactivation or transdifferentiation was comparable to clinically relevant tissues. We apply transdifferentiation and/or gene transactivation combined with short- and long-read RNA sequencing to investigate the impact that variants in USH2A, SCN1A, DMD, and PAK3 have on RNA using HDFs derived from affected individuals. Transactivation and transdifferentiation represent rapid, scalable functional genomic solutions to investigate variants impacting SMGs in the patient cell and genomic context., Competing Interests: Declaration of interests S.T.C. has no paid advisory roles to declare. S.T.C. is a volunteer member of ClinGen Expert Panels: Muscular Dystrophies and Myopathies GCEP and Limb Girdle Muscular Dystrophy VCEP. S.T.C. is named inventor of intellectual property (IP) relating to novel methods and biomarkers to identify DNA variants that alter pre-messenger RNA splicing: (1) PCT no. 2018904348 and (2) Australian Patent no. 2019379868. PCT no. 2019900836. This IP is unrelated to the data and outcomes described within this manuscript., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Published
2024
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37. Loss of calpains-1 and -2 prevents repair of plasma membrane scrape injuries, but not small pores, and induces a severe muscular dystrophy.

Author

Piper AK, Sophocleous RA, Ross SE, Evesson FJ, Saleh O, Bournazos A, Yasa J, Reed C, Woolger N, Sluyter R, Greer P, Biro M, Lemckert FA, and Cooper ST

Subjects
Animals, Bacterial Proteins pharmacology, Calcium Signaling, Calpain genetics, Cell Membrane drug effects, Cell Membrane pathology, Disease Models, Animal, Dysferlin deficiency, Dysferlin genetics, Female, HEK293 Cells, Humans, Male, Mice, Knockout, Muscle, Skeletal pathology, Muscular Dystrophies, Limb-Girdle genetics, Muscular Dystrophies, Limb-Girdle pathology, Muscular Dystrophy, Animal genetics, Muscular Dystrophy, Animal pathology, Saponins pharmacology, Severity of Illness Index, Streptolysins pharmacology, Calpain deficiency, Cell Membrane enzymology, Muscle, Skeletal enzymology, Muscular Dystrophies, Limb-Girdle enzymology, Muscular Dystrophy, Animal enzymology
Abstract

The ubiquitous calpains, calpain-1 and -2, play important roles in Ca 2+ -dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca 2+ -dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 ( CAPNS1 -/- ) virtually ablates Ca 2+ -dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 ( CAPN1 -/- ) or -2 ( CAPN2 -/- ) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 ( CAPN3 ). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca 2+ -signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.

Published
2020
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38. Cullin-3 dependent deregulation of ACTN1 represents a new pathogenic mechanism in nemaline myopathy.

Author

Blondelle J, Tallapaka K, Seto JT, Ghassemian M, Clark M, Laitila JM, Bournazos A, Singer JD, and Lange S

Subjects
Animals, Cullin Proteins genetics, Disease Models, Animal, Gene Expression Regulation, Developmental, Genetic Predisposition to Disease genetics, Humans, Membrane Proteins metabolism, Mice, Mice, Knockout embryology, Muscle Proteins genetics, Muscle Weakness embryology, Muscle Weakness genetics, Muscle Weakness metabolism, Muscle, Skeletal embryology, Muscular Diseases metabolism, Muscular Diseases pathology, Mutation, Myopathies, Nemaline embryology, Myopathies, Nemaline genetics, Myopathies, Nemaline pathology, Neuromuscular Junction growth & development, Neuromuscular Junction metabolism, Neuromuscular Junction pathology, Ubiquitin-Protein Ligases metabolism, Actinin metabolism, Cullin Proteins metabolism, Muscle Proteins metabolism, Muscle, Skeletal metabolism, Myopathies, Nemaline metabolism
Abstract

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy and presence of nemaline bodies within myofibers. However, the understanding of underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40 and KLHL41, three substrate adaptors for the E3-ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for the disease development. Using Cullin-3 knockout mice, we identified accumulation of non-muscle alpha-Actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in KBTBD13 patients. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis and acetylcholine receptor clustering; features that we characterized in Cullin-3 deficient mice. Taken together, our data highlight the importance for Cullin-3 mediated degradation of ACTN1 for muscle development, and indicate a new pathomechanism for the etiology of myopathies seen in Cullin-3 knockout mice and nemaline myopathy patients.

Published
2019
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39. Congenital Titinopathy: Comprehensive characterization and pathogenic insights.

Author

Oates EC, Jones KJ, Donkervoort S, Charlton A, Brammah S, Smith JE 3rd, Ware JS, Yau KS, Swanson LC, Whiffin N, Peduto AJ, Bournazos A, Waddell LB, Farrar MA, Sampaio HA, Teoh HL, Lamont PJ, Mowat D, Fitzsimons RB, Corbett AJ, Ryan MM, O'Grady GL, Sandaradura SA, Ghaoui R, Joshi H, Marshall JL, Nolan MA, Kaur S, Punetha J, Töpf A, Harris E, Bakshi M, Genetti CA, Marttila M, Werlauff U, Streichenberger N, Pestronk A, Mazanti I, Pinner JR, Vuillerot C, Grosmann C, Camacho A, Mohassel P, Leach ME, Foley AR, Bharucha-Goebel D, Collins J, Connolly AM, Gilbreath HR, Iannaccone ST, Castro D, Cummings BB, Webster RI, Lazaro L, Vissing J, Coppens S, Deconinck N, Luk HM, Thomas NH, Foulds NC, Illingworth MA, Ellard S, McLean CA, Phadke R, Ravenscroft G, Witting N, Hackman P, Richard I, Cooper ST, Kamsteeg EJ, Hoffman EP, Bushby K, Straub V, Udd B, Ferreiro A, North KN, Clarke NF, Lek M, Beggs AH, Bönnemann CG, MacArthur DG, Granzier H, Davis MR, and Laing NG

Subjects
Female, Humans, Male, Mutation genetics, Phenotype, Protein Isoforms genetics, Cardiomyopathy, Dilated congenital, Connectin genetics, Muscle Proteins genetics, Muscle, Skeletal pathology
Abstract

Objective: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder., Methods: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients., Results: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder., Interpretation: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124., (© 2018 American Neurological Association.)

Published
2018
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