Guidelines by professional organizations for assessing variant pathogenicity include the recommendation to utilize biologically relevant transcripts; however, there is variability in transcript selection by laboratories.
We describe three patients whose genomic results were incorrect, because alternative transcripts and tissue expression patterns were not considered by the commercial laboratories.
In individual 1, a pathogenic coding variant in a brain-expressed isoform of CKDL5 was missed twice on sequencing, because the variant was intronic in the transcripts considered in analysis. In individual 2, a microdeletion affecting KMT2C was not reported on microarray, since deletions of proximal exons in this gene are seen in healthy individuals; however, this individual had a more distal deletion involving the brain-expressed KMT2C isoform, giving her a diagnosis of Kleefstra syndrome. Individual 3 was reported to have a pathogenic variant in exon 10 of OFD1 on exome, but had no typical features of the OFD1-related disorders. Since exon 10 is spliced from the more biologically relevant transcripts of OFD1, it was determined that he did not have an OFD1 disorder.
These examples illustrate the importance of considering alternative transcripts as a potential confounder when genetic results are negative or discordant with the phenotype.
Subscribe to Journal
Get full journal access for 1 year
only $41.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–424.
Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 2008;40:1413–1415.
Baralle FE, Giudice J. Alternative splicing as a regulator of development and tissue identity. Nat Rev Mol Cell Biol. 2017;18:437–451.
Zimmermann MT. The importance of biologic knowledge and gene expression context for genomic data interpretation. Front Genet. 2018;9:670.
Bodian DL, Kothiyal P, Hauser NS. Pitfalls of clinical exome and gene panel testing: alternative transcripts. Genet Med. 2019;21:1240–1245.
McCarthy DJ, Humburg P, Kanapin A, et al. Choice of transcripts and software has a large effect on variant annotation. Genome Med. 2014;6:26.
Shashi V, Schoch K, Spillmann R, et al. A comprehensive iterative approach is highly effective in diagnosing individuals who are exome negative. Genet Med. 2019;21:161–172.
DePristo MA, Banks E, Poplin R, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011;43:491–498.
Hector RD, Dando O, Landsberger N, et al. Characterisation of CDKL5 Transcript Isoforms in human and mouse. PLoS One. 2016;11:e0157758.
Archer HL, Evans J, Edwards S, et al. CDKL5 mutations cause infantile spasms, early onset seizures, and severe mental retardation in female patients. J Med Genet. 2006;43:729–734.
Bodian DL, Schreiber Schreiber, Vilboux T, Khromykh A, Hauser NS. Mutation in an alternative transcript of CDKL5 in a boy with early-onset seizures. Cold Spring Harb Mol Case Stud. 2018;4:a002360.
Diebold B, Delepine C, Gataullina S, Delahaye A, Nectoux J, Bienvenu T. Mutations in the C-terminus of CDKL5: proceed with caution. Eur J Hum Genet. 2014;22:270–272.
Koemans TS, Kleefstra T, Chubak MC, et al. Functional convergence of histone methyltransferases EHMT1 and KMT2C involved in intellectual disability and autism spectrum disorder. PLoS Genet. 2017;13:e1006864.
Coene KL, Roepman R, Doherty D, et al. OFD1 is mutated in X-linked Joubert syndrome and interacts with LCA5-encoded lebercilin. Am J Hum Genet. 2009;85:465–481.
Epilepsy Genetics Initiative. De novo variants in the alternative exon 5 of SCN8A cause epileptic encephalopathy. Genet Med. 2018;20:275–281.
DiStefano MT, Hemphill SE, Cushman BJ, et al. Curating clinically relevant transcripts for the interpretation of sequence variants. J Mol Diagn. 2018;20:789–801.
Shashi V, McConkie-Rosell A, Schoch K, et al. Practical considerations in the clinical application of whole-exome sequencing. Clin Genet. 2016;89:173–181.
van der Donk R, Jansen S, Schuurs-Hoeijmakers JHM, et al. Next-generation phenotyping using computer vision algorithms in rare genomic neurodevelopmental disorders. Genet Med. 2019;21:1719–1725.
Mele M, Ferreira PG, Reverter F, et al. Human genomics. The human transcriptome across tissues and individuals. Science. 2015;348:660–665.
GTEx Consortium. Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans. Science. 2015;348:648–660.
DiStefano MT, Hemphill SE, Oza AM, et al. ClinGen expert clinical validity curation of 164 hearing loss gene-disease pairs. Genet Med. 2019;21:2239–2247.
We thank the families who participated in this study. Research reported in this paper was supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director under award number U01HG007672. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The Undiagnosed Diseases Network members are Maria T. Acosta, Margaret Adam, David R. Adams, Pankaj B. Agrawal, Mercedes E. Alejandro, Justin Alvey, Laura Amendola, Ashley Andrews, Euan A. Ashley, Mahshid S. Azamian, Carlos A. Bacino, Guney Bademci, Eva Baker, Ashok Balasubramanyam, Dustin Baldridge, Jim Bale, Michael Bamshad, Deborah Barbouth, Gabriel F. Batzli, Pinar Bayrak-Toydemir, Anita Beck, Alan H. Beggs, Edward Behrens, Gill Bejerano, Jimmy Bennet, Beverly Berg-Rood, Raphael Bernier, Jonathan A. Bernstein, Gerard T. Berry, Anna Bican, Stephanie Bivona, Elizabeth Blue, John Bohnsack, Carsten Bonnenmann, Devon Bonner, Lorenzo Botto, Brenna Boyd, Lauren C. Briere, Elly Brokamp, Gabrielle Brown, Elizabeth A. Burke, Lindsay C. Burrage, Manish J. Butte, Peter Byers, William E. Byrd, John Carey, Olveen Carrasquillo, Ta Chen Peter Chang, Sirisak Chanprasert, Hsiao-Tuan Chao, Gary D. Clark, Terra R. Coakley, Laurel A. Cobban, Joy D. Cogan, F. Sessions Cole, Heather A. Colley, Cynthia M. Cooper, Heidi Cope, William J. Craigen, Andrew B. Crouse, Michael Cunningham, Precilla D'Souza, Hongzheng Dai, Surendra Dasari, Mariska Davids, Jyoti G. Dayal, Matthew Deardorff, Esteban C. Dell'Angelica, Shweta U. Dhar, Katrina Dipple, Daniel Doherty, Naghmeh Dorrani, Emilie D. Douine, David D. Draper, Laura Duncan, Dawn Earl, David J. Eckstein, Lisa T. Emrick, Christine M. Eng, Cecilia Esteves, Tyra Estwick, Marni Falk, Liliana Fernandez, Carlos Ferreira, Elizabeth L. Fieg, Paul G. Fisher, Brent L.Fogel, Irman Forghani, Laure Fresard, William A. Gahl, Ian Glass, Rena A. Godfrey, Katie Golden-Grant, Alica M. Goldman, David B. Goldstein, Alana Grajewski, Catherine A. Groden, Andrea L. Gropman, Irma Gutierrez, Sihoun Hahn, Rizwan Hamid, Neil A. Hanchard, Kelly Hassey, Nichole Hayes, Frances High, Anne Hing, Fuki M. Hisama, Ingrid A. Holm, Jason Hom, Martha Horike-Pyne, Alden Huang, Yong Huang, Rosario Isasi, Fariha Jamal, Gail P. Jarvik, Jeffrey Jarvik, Suman Jayadev, Jean M. Johnston, Lefkothea Karaviti, Emily G. Kelley, Jennifer Kennedy, Dana Kiley, Isaac S. Kohane, Jennefer N. Kohler, Deborah Krakow, Donna M. Krasnewich, Elijah Kravets, Susan Korrick, Mary Koziura, Joel B. Krier, Seema R. Lalani, Byron Lam, Christina Lam, Brendan C. Lanpher, Ian R. Lanza, C. Christopher Lau, Kimberly LeBlanc, Brendan H. Lee, Hane Lee, Roy Levitt, Richard A. Lewis, Sharyn A. Lincoln, Pengfei Liu, Xue Zhong Liu, Nicola Longo, Sandra K. Loo, Joseph Loscalzo, Richard L. Maas, Ellen F. Macnamara, Calum A. MacRae, Valerie V. Maduro, Marta M. Majcherska, May Christine V. Malicdan, Laura A. Mamounas, Teri A. Manolio, Rong Mao, Kenneth Maravilla, Thomas C. Markello, Ronit Marom, Gabor Marth, Beth A. Martin, Martin G. Martin, Julian A. Martínez-Agosto, Shruti Marwaha, Jacob McCauley, Allyn McConkie-Rosell, Colleen E. McCormack, Alexa T. McCray, Elisabeth McGee, Heather Mefford, J. Lawrence Merritt, Matthew Might, Ghayda Mirzaa, Eva Morava-Kozicz, Paolo M. Moretti, Marie Morimoto, John J. Mulvihill, David R. Murdock, Mariko Nakano-Okuno, Avi Nath, Stan F. Nelson, John H. Newman, Sarah K. Nicholas, Deborah Nickerson, Donna Novacic, Devin Oglesbee, James P. Orengo, Laura Pace, Stephen Pak, J. Carl Pallais, Christina G.S. Palmer, Jeanette C. Papp, Neil H. Parker, John A. Phillips III, Jennifer E. Posey, Lorraine Potocki, Barbara N. Pusey, Aaron Quinlan, Wendy Raskind, Archana N. Raja, Genecee Renteria, Chloe M. Reuter, Lynette Rives, Amy K. Robertson, Lance H. Rodan, Jill A. Rosenfeld, Natalie Rosenwasser, Robb K. Rowley, Maura Ruzhnikov, Ralph Sacco, Jacinda B. Sampson, Susan L. Samson, Mario Saporta, C. Ron Scott, Judy Schaechter, Timothy Schedl, Kelly Schoch, Daryl A. Scott, Prashant Sharma, Vandana Shashi, Jimann Shin, Rebecca Signer, Catherine H. Sillari, Edwin K. Silverman, Janet S. Sinsheimer, Kathy Sisco, Edward C. Smith, Kevin S. Smith, Emily Solem, Lilianna Solnica-Krezel, Rebecca C. Spillmann, Joan M. Stoler, Nicholas Stong, Jennifer A. Sullivan, Kathleen Sullivan, Angela Sun, Shirley Sutton, David A. Sweetser, Virginia Sybert, Holly K. Tabor, Cecelia P. Tamburro, Queenie K.-G. Tan, Mustafa Tekin, Fred Telischi, Willa Thorson, Cynthia J. Tifft, Camilo Toro, Alyssa A. Tran, Brianna M. Tucker, Tiina K. Urv, Adeline Vanderver, Matt Velinder, Dave Viskochil, Tiphanie P. Vogel, Colleen E. Wahl, Stephanie Wallace, Nicole M. Walley, Chris A. Walsh, Melissa Walker, Jennifer Wambach, Jijun Wan, Lee-kai Wang, Michael F. Wangler, Patricia A. Ward, Daniel Wegner, Mark Wener, Tara Wenger, Katherine Wesseling Perry, Monte Westerfield, Matthew T. Wheeler, Jordan Whitlock, Lynne A. Wolfe, Jeremy D. Woods, Shinya Yamamoto, John Yang, Guoyun Yu, Diane B. Zastrow, Chunli Zhao, Stephan Zuchner.
The authors declare no conflicts of interest.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Schoch, K., Tan, Q.KG., Stong, N. et al. Alternative transcripts in variant interpretation: the potential for missed diagnoses and misdiagnoses. Genet Med 22, 1269–1275 (2020). https://doi.org/10.1038/s41436-020-0781-x
- alternative splicing
- exome/genome sequencing
- chromosomal microarray
- transcript expression
Clinical sites of the Undiagnosed Diseases Network: unique contributions to genomic medicine and science
Genetics in Medicine (2020)
Missense variants in the N-terminal domain of the A isoform of FHF2/FGF13 cause an X-linked developmental and epileptic encephalopathy
The American Journal of Human Genetics (2020)