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| Status |
Public on Sep 04, 2024 |
| Title |
Non-coding cause of congenital heart defects: Abnormal RNA splicing with multiple isoforms as a mechanism for heterotaxy |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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 by-pass 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.
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| Overall design |
CRISPR/Cas9 was used in H1-OCT4-eGFP cells (a male human embryonic stem cell line) to produce two cell clones containing the ZIC3 intronic variant (NM_003413.4:c.1224+3286A>G) termed ZIC3 AtoG_C1 and ZIC3 AtoG_C2. Two ZIC3 knockout cell clones were also produced (ZIC3 KO_C1: NM_003413.4:c.190_200delinsG; ZIC3 KO_C2: NM_003413.4:c.200_201del). Unedited H1-OCT4-eGFP cells were termed ZIC3 WT. Short-read RNA sequencing was completed for ZIC3 WT ( n=1), ZIC3 AtoG_C1 (n=1), ZIC3 AtoG_C2 (n=1), ZIC3 KO_C1 (n=1), and ZIC3 KO_C2 (n=1) H1-OCT4-EGFP cells using the Illumina sequencing platform. Long-read RNA sequencing was completed in ZIC3 WT (n=3), ZIC3 AtoG_C1 (n=3), and ZIC3 KO_C1 (n=3) H1-OCT4-EGFP cells using the Oxford Nanopore sequencing platform.
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| Contributor(s) |
Wells JR, Padua MB, Haaning AM, Smith AM, Morris SA, Tariq M, Ware SM |
| Citation(s) |
39275801 |
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| Submission date |
Apr 08, 2024 |
| Last update date |
Sep 14, 2024 |
| Contact name |
John Robert Wells |
| E-mail(s) |
[email protected]
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| Organization name |
Indiana University School of Medicine
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| Department |
Department of Medical & Molecular Genetics
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| Lab |
Stephanie Ware Lab
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| Street address |
1044 W Walnut St
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| City |
Indianapolis |
| State/province |
IN |
| ZIP/Postal code |
46202 |
| Country |
USA |
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| Platforms (2) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
| GPL26167 |
PromethION (Homo sapiens) |
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| Samples (17)
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| GSM8191367 |
ZIC3 WT, short-read, biol rep 1 of 1 |
| GSM8191368 |
ZIC3 WT, short-read, CHX treated, biol rep 1 of 1 |
| GSM8191369 |
ZIC3 AtoG_C1, short-read, biol rep 1 of 1 |
| GSM8191370 |
ZIC3 AtoG_C1, short-read, CHX treated, biol rep 1 of 1 |
| GSM8191371 |
ZIC3 AtoG_C2, short-read, biol rep 1 of 1 |
| GSM8191372 |
ZIC3 AtoG_C2, short-read, CHX treated, biol rep 1 of 1 |
| GSM8191373 |
ZIC3 KO_C1, short-read, biol rep 1 of 1 |
| GSM8191374 |
ZIC3 KO_C2, short-read RNA-seq, biol rep 1 of 1 |
| GSM8191375 |
ZIC3 WT, long-read, biol rep 1 of 3 |
| GSM8191376 |
ZIC3 WT, long-read, biol rep 2 of 3 |
| GSM8191377 |
ZIC3 WT, long-read, biol rep 3 of 3 |
| GSM8191378 |
ZIC3 AtoG_C1, long-read, biol rep 1 of 3 |
| GSM8191379 |
ZIC3 AtoG_C1, long-read, biol rep 2 of 3 |
| GSM8191380 |
ZIC3 AtoG_C1, long-read, biol rep 3 of 3 |
| GSM8191381 |
ZIC3 KO_C1, long-read, biol rep 1 of 3 |
| GSM8191382 |
ZIC3 KO_C1, long-read, biol rep 2 of 3 |
| GSM8191383 |
ZIC3 KO_C1, long-read, biol rep 3 of 3 |
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| Relations |
| BioProject |
PRJNA1097502 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE263414_Long_Read_Log_CPM_EdgeR.txt.gz |
1.8 Mb |
(ftp)(http) |
TXT |
| GSE263414_Short_Read_Raw_Counts_All_Samples.txt.gz |
653.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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