The impact of nationwide folic acid fortification on genetic variants associated with conotruncal heart defects

Technical Summary: The Impact of Nationwide Folic Acid Fortification on Genetic Variants Associated with Conotruncal Heart Defects

Problem Statement
Congenital heart diseases (CHDs) are the leading cause of birth defect-associated morbidity and mortality, yet the etiology for most cases remains unclear, likely involving complex interactions between polygenic risk and environmental factors. While periconceptional folic acid fortification is established to reduce the risk of neural tube defects and conotruncal heart defects (CTHD), the specific mechanisms remain partially understood. Previous genetic epidemiology studies investigating folate-related gene variants in CTHD have yielded inconclusive results, often due to difficulties in accounting for maternal folate deficiency and small sample sizes. Crucially, no study had directly compared genetic variant distributions among children with CTHD born before and after the 1998 United States mandate for folic acid fortification of grains and cereals. This study addresses the gap in understanding how a population-level nutritional intervention (folic acid fortification) may have altered the penetrance or expressivity of genetic risk variants for CTHD.

Methodology
The study utilized genomic sequencing data from the Pediatric Cardiac Genomics Consortium (PCGC), accessed via the dbGaP repository.

• Cohort Definition: The analysis included 1,285 individuals of European-inferred ancestry. The cohort was stratified into three groups:
  1. Full analytic group (all participants, n=1,285).
  2. CTHD subset (n=534), including tetralogy of Fallot, truncus arteriosus, D-transposition of the great arteries, double outlet right ventricle, and interrupted aortic arch type B.
  3. Non-CTHD subset (n=751), including septal defects, left-sided obstruction lesions, and single ventricle defects.
• Exposure Classification: Participants were categorized based on birth year relative to the folic acid fortification policy. Those born in 1997 or earlier were classified as "pre-fortification," and those born in 2000 or later as "post-fortification." Birth years 1998 and 1999 were excluded to account for the policy transition period.
• Data Processing: Sequencing data were aligned to GRCh38/hg38 and processed jointly to minimize batch effects. Quality control (QC) filters included genotype quality >20, allele read depth >10, minor allele frequency >0.01, and Hardy-Weinberg equilibrium p > 1x10⁻⁶.
• Statistical Analysis: Genome-wide association studies (GWAS) were performed using logistic-Firth hybrid regression under an additive genetic model (PLINK v2.0). The top ten principal components were included as covariates to control for population stratification. Suggestive significance was defined as p < 1 x 10⁻⁵.
• Functional Enrichment: Gene set enrichment analysis was conducted using DAVID (v6.8) on the top 100 ranked genes from each model. Significance was assessed using modified Fisher's exact tests with Benjamini-Hochberg false discovery rate (FDR) adjustment.

Key Results

• GWAS Findings:
  • Full Group (Model 1): 94 variants were nominally enriched (p < 10⁻⁵). Most variants were enriched in the pre-fortification era (OR < 1), suggesting a population-wide shift where certain genetic variants became less frequent in CHD cases after fortification.
  • CTHD Subset (Model 2): 26 variants were identified. Notably, two variants in the DHRS3 gene (rs7551703, OR 2.10; rs6541043, OR 2.21) and four variants in the PPARGC1β locus (specifically within the intronic miRNA MIR378A) were nominally enriched in the post-fortification group (OR 4.47–4.51).
  • Non-CTHD Subset (Model 3): 59 variants were identified. Variants in MIR548A1HG and NKX1-1 were enriched in the pre-fortification group, similar to Model 1, suggesting these may represent shared risk factors for broader cardiac phenotypes rather than CTHD-specific risks.
  • Folate Pathway Genes: No variants classically associated with folate transport or one-carbon metabolism (e.g., MTHFR, MTRR, MTR) were detected in any model.
• Functional Enrichment:
  • In the CTHD group, enriched biological pathways included Rho protein signaling, cytoskeletal dynamics, cell-cell junctions, and transcriptional regulation, consistent with cardiac developmental processes.
  • In the non-CTHD group, enriched terms included chromatin binding and oxidative phosphorylation.
  • The authors note that these enrichment results are hypothesis-generating as p-values were not corrected for multiple testing in the final interpretation.

Significance and Claims
The paper claims to be the first to directly compare genetic variants in CTHD cases born before and after the 1998 US folic acid fortification mandate. The study posits that folic acid fortification acts as a "natural experiment" that may have modified the genomic landscape of folate-responsive loci.

• Gene-Nutrient Interaction: The identification of DHRS3 and PPARGC1β variants enriched specifically in the post-fortification CTHD group suggests a potential gene-nutrient interaction. DHRS3 is involved in retinoid homeostasis, and the authors note that folate deficiency and retinoic acid imbalance are both linked to CTHD, suggesting folate may modulate retinoid pathways. PPARGC1β is linked to cardiac metabolic maturation; its association in the post-fortification era may reflect survival bias due to improved clinical outcomes over time or specific interactions with the altered nutritional environment.
• Population Shift: The finding that many variants were less frequent in the post-fortification group across the broader cohort suggests that fortification may have altered the penetrance of genetic risk factors at a population level.
• Modest Interpretation: The authors explicitly state that the results are "hypothesis-generating" rather than confirmatory due to the use of suggestive significance thresholds (p < 10⁻⁵) and the lack of individual-level maternal folate data. They acknowledge that the observed shifts could be influenced by secular trends, such as improved survival rates for complex lesions or changes in maternal characteristics (e.g., obesity, smoking) over the study period.

In conclusion, the study provides evidence that folic acid fortification may have influenced the distribution of genetic risk variants for CTHD, highlighting a complex interplay between maternal folate status, embryonic retinoid levels, and cardiac developmental pathways.