Genes and pathways implicated in tetralogy of Fallot revealed by ultra-rare variant burden analysis in 231 genome sequences
Bibliographic record
Abstract
Abstract Recent genome-wide studies of rare genetic variants have begun to implicate novel mechanisms for tetralogy of Fallot (TOF), a severe congenital heart defect (CHD). To provide statistical support for case-only data without parental genomes, we re-analyzed genome sequences of 231 individuals with TOF or related CHD. We adapted a burden test originally developed for de novo variants to assess singleton variant burden in individual genes, and in gene-sets corresponding to functional pathways and mouse phenotypes, accounting for highly correlated gene-sets, and for multiple testing. The gene burden test identified a significant burden of deleterious missense variants in NOTCH1 (Bonferroni-corrected p-value <0.01). These NOTCH1 variants showed significant enrichment for those affecting the extracellular domain, and especially for disruption of cysteine residues forming disulfide bonds (OR 39.8 vs gnomAD). Individuals with NOTCH1 variants, all with TOF, were enriched for positive family history of CHD. Other genes not previously implicated in TOF had more modest statistical support and singleton missense variant results were non-significant for gene-set burden. For singleton truncating variants, the gene burden test confirmed significant burden in FLT4. Gene-set burden tests identified a cluster of pathways corresponding to VEGF signaling ( FDR =0%), and of mouse phenotypes corresponding to abnormal vasculature ( FDR =0.8%), that suggested additional candidate genes not previously identified (e.g., WNT5A and ZFAND5 ). Analyses using unrelated sequencing datasets supported specificity of the findings for CHD. The findings support the importance of ultra-rare variants disrupting genes involved in VEGF and NOTCH signaling in the genetic architecture of TOF. These proof-of-principle data indicate that this statistical methodology could assist in analyzing case-only sequencing data in which ultra-rare variants, whether de novo or inherited, contribute to the genetic etiopathogenesis of a complex disorder. Author summary We analyzed the ultra-rare nonsynonymous variant burden for genome sequencing data from 231 individuals with congenital heart defects, most with tetralogy of Fallot. We adapted a burden test originally developed for de novo variants. In line with other studies, we identified a significant truncating variant burden for FLT4 and deleterious missense burden for NOTCH1 , both passing a stringent Bonferroni multiple-test correction. For NOTCH1 , we observed frequent disruption of cysteine residues establishing disulfide bonds in the extracellular domain. We also identified genes with BH-FDR <10% that were not previously implicated. To overcome limited power for individual genes, we tested gene-sets corresponding to functional pathways and mouse phenotypes. Gene-set burden of truncating variants was significant for vascular endothelial growth factor signaling and abnormal vasculature phenotypes. These results confirmed previous findings and suggested additional candidate genes for experimental validation in future studies. This methodology can be extended to other case-only sequencing data in which ultra-rare variants make a substantial contribution to genetic etiology.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.002 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.002 | 0.002 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".