Abstract
BackgroundSignaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus.MethodsOne-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay.ResultsWe describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls.ConclusionIn summary, our screen indicates that ALDH1A2 genetic variation is present in TOF patients, suggesting a possible causal role for this gene in rare cases of human CHD, but does not support the hypothesis that variation at the ALDH1A2 locus is a significant modifier of the risk for CHD in humans.
Highlights
Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development
Mutation screening in congenital heart disease (CHDs) To test whether the ALDH1A2 gene is a target for mutations in human CHD we employed DHPLC [31] followed by bi-directional DNA sequencing in a group of patients with multiple CHD etiologies
This first screening in a heterogeneous CHD setting revealed 6 alterations: one c.A453G transition previously characterized as a synonymous, p.Ala151Ala, polymorphism associated with spina bifida [24] and five intronic alterations (Table 1)
Summary
Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. It is likely that the instability caused by insufficient dosage of genes that are critical or permissive throughout cardiac development weakens cardiac morphogenesis, predisposing the embryo to a host of morphogenetic defects [4] In this probabilistic view, defects could be triggered by unfavorable combinations of allelic variants of minor effect of cardiac development genes, by environmental insults such as changes in nutritional status (e.g. vitamin A) [11], or by exposure to toxic chemicals (e.g. alcohol) [12]. Defects could be triggered by unfavorable combinations of allelic variants of minor effect of cardiac development genes, by environmental insults such as changes in nutritional status (e.g. vitamin A) [11], or by exposure to toxic chemicals (e.g. alcohol) [12] In this sense, the morphological consequences of these alterations would be contingent on the particular developmental processes that were at play when regulatory mechanisms were overcome
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