Congenital heart defects affect 1-2% of newborns and are the leading cause of death in infants under one year of age . While the overwhelming majority of congenital heart malformations do not segregate in Mendelian ratios, they do show familial aggregation, which suggests that genetic factors play a role in their development [2, 3]. Despite this, a limited number of CHD-causing genes have been identified so far .
Isolated D-Transposition of great arteries (D-TGA, OMIM 608808) accounts for 5% of all congenital heart diseases . Its incidence is estimated at 1 in 3,500-5,000 live births . Most D-TGA cases are sporadic, but familial cases have also been reported . A discrete number of causing genes have been identified so far (ZIC3, CFC1, THRAP2, GDF1, NODAL), but their mutation explains only a minority of cases [8–13]. Interestingly, many of these genes participate in embryonic left-right axis patterning . Moreover, D-TGA has been observed to be frequently related to laterality defects (failure to establish a normal left-right asymmetry during embryonic development), in particular, in patients with asplenia/right isomerism. Conversely, one of the most prevalent types of CHD in lateralisation defects is D-TGA .
Transcriptome analysis using DNA microarrays has become a standard approach for investigating the molecular basis of human disease in both clinical and experimental settings, as the pattern of transcriptional deregulation may provide insights into the cause of abnormal phenotypes, including congenital defects [16–20].
In the present study we have analysed the transcriptome of mouse embryos whose development was dramatically altered by temporarily blocking retinoic acid signalling and of embryos in which the abnormal developmental phenotype was rescued by a concomitant supplementation with folic acid [21, 22].
We previously administered to pregnant mice BMS189453, a synthetic retinoic acid (RA) antagonist having good (82-98%) oral bioavailability in rats and monkeys . BMS189453 binds, but does not activate, the α, β, and γ retinoid receptors . Oral administration of BMS189453 to pregnant mice twice, at 7.25/7.75 dpc (days post coitum), induces cardiac defects (81%), thymic abnormalities (98%) and neural tube defects (20%) at birth . Concomitant oral supplementation with FA, during pregnancy, partially rescues this abnormal phenotype . In particular, FA reduces congenital heart diseases from 81.3% to 64.8%, neural tube defects from 20.3% to 3.7% and thymic abnormalities from 98.4% to 27.8%, restoring a normal number of differentiated thymic cells .
To better identify genes/transcripts involved in the pathogenesis of the congenital defects observed in our mouse models, we performed a global microarray analysis on embryos. To identify the best developmental stage for microarray screening, we first analysed the gene expression pattern of Rarα, a retinoic acid responsive gene in mouse embryos, at 8.5, 9.5 and 11.5 dpc. At 8.5 dpc, all embryos analysed showed down-regulation of Rarα mRNA, compared to only 70% of the embryos at 9.5 dpc and 50% of embryos at 11.5 dpc (data not shown). Thus, we thus decided to analyse the gene expression pattern in 8.5 dpc embryos.
The data presented in this paper reveal that changes in the expression level of Hif1α (hypoxia-inducible factor 1 alpha subunit) during mouse embryogenesis are associated with CHD observed in our mouse models.