Non-coding Cause of Congenital Heart Defects: Abnormal RNA Splicing with Multiple Isoforms As a Mechanism for Heterotaxy

Overview

Journal HGG Adv

Specialty Genetics

Date 2024 Sep 14

PMID 39275801

Authors

John R Wells

Maria B Padua

Allison M Haaning

Amanda M Smith

Shaine A Morris

Muhammad Tariq

Stephanie M Ware

Affiliations

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Abstract

Heterotaxy is a disorder characterized by severe congenital heart defects (CHDs) and abnormal left-right patterning in other thoracic or abdominal organs. Clinical and research-based genetic testing has previously focused on evaluation of coding variants to identify causes of CHDs, leaving non-coding causes of CHDs largely unknown. Variants in the transcription factor zinc finger of the cerebellum 3 (ZIC3) cause X-linked heterotaxy. We identified an X-linked heterotaxy pedigree without a coding variant in ZIC3. Whole-genome sequencing revealed a deep intronic variant (ZIC3 c.1224+3286A>G) predicted to alter RNA splicing. An in vitro minigene splicing assay confirmed the variant acts as a cryptic splice acceptor. CRISPR-Cas9 served to introduce the ZIC3 c.1224+3286A>G variant into human embryonic stem cells demonstrating pseudoexon inclusion caused by the variant. Surprisingly, Sanger sequencing of the resulting ZIC3 c.1224+3286A>G amplicons revealed several isoforms, many of which bypass the normal coding sequence of the third exon of ZIC3, causing a disruption of a DNA-binding domain and a nuclear localization signal. Short- and long-read mRNA sequencing confirmed these initial results and identified additional splicing patterns. Assessment of four isoforms determined abnormal functions in vitro and in vivo while treatment with a splice-blocking morpholino partially rescued ZIC3. These results demonstrate that pseudoexon inclusion in ZIC3 can cause heterotaxy and provide functional validation of non-coding disease causation. Our results suggest the importance of non-coding variants in heterotaxy and the need for improved methods to identify and classify non-coding variation that may contribute to CHDs.

References

Bedard J, Purnell J, Ware S . Nuclear import and export signals are essential for proper cellular trafficking and function of ZIC3. Hum Mol Genet. 2006; 16(2):187-98. DOI: 10.1093/hmg/ddl461. View

Li H, Durbin R . Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14):1754-60. PMC: 2705234. DOI: 10.1093/bioinformatics/btp324. View

Mukhopadhyay S, Wen X, Ratti N, Loktev A, Rangell L, Scales S . The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic hedgehog pathway via cAMP signaling. Cell. 2013; 152(1-2):210-23. DOI: 10.1016/j.cell.2012.12.026. View

Li A, Hanchard N, Azamian M, DAlessandro L, Coban-Akdemir Z, Lopez K . Genetic architecture of laterality defects revealed by whole exome sequencing. Eur J Hum Genet. 2019; 27(4):563-573. PMC: 6460585. DOI: 10.1038/s41431-018-0307-z. View

Lin A, Krikov S, Riehle-Colarusso T, Frias J, Belmont J, Anderka M . Laterality defects in the national birth defects prevention study (1998-2007): birth prevalence and descriptive epidemiology. Am J Med Genet A. 2014; 164A(10):2581-91. PMC: 4462240. DOI: 10.1002/ajmg.a.36695. View

Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J . 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(5):405-24. PMC: 4544753. DOI: 10.1038/gim.2015.30. View

Robinson M, McCarthy D, Smyth G . edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2009; 26(1):139-40. PMC: 2796818. DOI: 10.1093/bioinformatics/btp616. View

Bedard J, Haaning A, Ware S . Identification of a novel ZIC3 isoform and mutation screening in patients with heterotaxy and congenital heart disease. PLoS One. 2011; 6(8):e23755. PMC: 3157443. DOI: 10.1371/journal.pone.0023755. View

Morcos P, Li Y, Jiang S . Vivo-Morpholinos: a non-peptide transporter delivers Morpholinos into a wide array of mouse tissues. Biotechniques. 2009; 45(6):613-4, 616, 618 passim. DOI: 10.2144/000113005. View

10.

Robinson J, Thorvaldsdottir H, Winckler W, Guttman M, Lander E, Getz G . Integrative genomics viewer. Nat Biotechnol. 2011; 29(1):24-6. PMC: 3346182. DOI: 10.1038/nbt.1754. View

11.

Merveille A, Davis E, Becker-Heck A, Legendre M, Amirav I, Bataille G . CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs. Nat Genet. 2010; 43(1):72-8. PMC: 3509786. DOI: 10.1038/ng.726. View

12.

Mizugishi K, Aruga J, Nakata K, Mikoshiba K . Molecular properties of Zic proteins as transcriptional regulators and their relationship to GLI proteins. J Biol Chem. 2000; 276(3):2180-8. DOI: 10.1074/jbc.M004430200. View

13.

Cowan J, Tariq M, Ware S . Genetic and functional analyses of ZIC3 variants in congenital heart disease. Hum Mutat. 2013; 35(1):66-75. PMC: 3946352. DOI: 10.1002/humu.22457. View

14.

Trimouille A, Tingaud-Sequeira A, Lacombe D, Duelund Hjortshoj T, Kreiborg S, Hove H . Description of a family with X-linked oculo-auriculo-vertebral spectrum associated with polyalanine tract expansion in ZIC3. Clin Genet. 2020; 98(4):384-389. DOI: 10.1111/cge.13811. View

15.

Sakai-Kato K, Ishiguro A, Mikoshiba K, Aruga J, Utsunomiya-Tate N . CD spectra show the relational style between Zic-, Gli-, Glis-zinc finger protein and DNA. Biochim Biophys Acta. 2008; 1784(7-8):1011-9. DOI: 10.1016/j.bbapap.2008.01.013. View

16.

Saba T, Geddes G, Ware S, Schidlow D, Del Nido P, Rubalcava N . A multi-disciplinary, comprehensive approach to management of children with heterotaxy. Orphanet J Rare Dis. 2022; 17(1):351. PMC: 9463860. DOI: 10.1186/s13023-022-02515-2. View

17.

Wang Y, Dai X, Liu H, Peng J, Chen J . A novel ZIC3 mutation in a Chinese family with heterotaxy and multiple types of congenital heart defect. Prenat Diagn. 2022; 43(3):275-279. DOI: 10.1002/pd.6294. View

18.

Ware S, Peng J, Zhu L, Fernbach S, Colicos S, Casey B . Identification and functional analysis of ZIC3 mutations in heterotaxy and related congenital heart defects. Am J Hum Genet. 2003; 74(1):93-105. PMC: 1181916. DOI: 10.1086/380998. View

19.

Hilger A, Halbritter J, Pennimpede T, van der Ven A, Sarma G, Braun D . Targeted Resequencing of 29 Candidate Genes and Mouse Expression Studies Implicate ZIC3 and FOXF1 in Human VATER/VACTERL Association. Hum Mutat. 2015; 36(12):1150-4. PMC: 4643331. DOI: 10.1002/humu.22859. View

20.

Destici E, Zhu F, Tran S, Preissl S, Farah E, Zhang Y . Human-gained heart enhancers are associated with species-specific cardiac attributes. Nat Cardiovasc Res. 2023; 1(9):830-843. PMC: 9937543. DOI: 10.1038/s44161-022-00124-7. View