Little is known about the role of most asthma susceptibility genes during human lung development. Here we present a thorough evaluation of gene expression patterns of current published asthma genes in the developing human and murine lung. While there was no general over-representation of asthma genes among differentially expressed genes, some asthma genes were consistently differentially expressed in multiple developing lung transcriptomes, e.g. NOD1, EDN1, CCL5, RORA and HLA-G suggesting key functional roles in lung development.
Determinants for a normal lung development are critical not only early in life, but also for later lung function. Longitudinal studies have shown that infants with reduced lung function have an increased risk of developing asthma and respiratory illness later in life [97
]. Shared genetic factors for reduced lung function in children with asthma and adults who smoke (e.g. MMP12
variants) emphasize the role of genetics on long term lung function [99
signaling genes (e.g. Wif1, Wisp1
) were not identified as asthma genes in our literature search, and were thus not included in our analyses. In our previous article by Sharma et al, Wif1
were differentially expressed during fetal lung development and polymorphisms in these genes also showed association with lung function measured as FEV1 and FVC, but association to asthma per se was not tested [14
The transcriptional control of lung morphogenesis is key for normal development from primordium to a fully differentiated, functioning organ [100
]. Human lung growth has historically been categorised into five stages based on histological and anatomical characteristics: embryonic (26 days to 5 weeks), pseudoglandular (5-16 weeks), canalicular (16-26 weeks), saccular (26 weeks to birth), and alveolar (birth to 6 months) [100
]. Additional "molecular" phases within the pseudoglandular stage have been observed, which extends our knowledge of lung development beyond traditional embryology [15
GWAS have contributed to important knowledge about underlying functional genetics in many complex diseases [102
]. The majority of trait associated SNPs show weak to moderate effect sizes, which supports earlier evidence that complex diseases result from several genetic and, often, environmental factors. Evidence of a functional role is also lacking for most identified genes. In order to increase our understanding of the mechanism and potential function of asthma susceptibility genes identified in published GWAS and "classic" asthma candidate genes, we evaluated their gene expression patterns in the developing human lung. Comparative analyses also showed that many of the differentially expressed genes in the human data set were also differentially expressed during murine lung development. Among the GWAS asthma genes, ROBO1, RORA, HLA-DQB1, IL2RB
were differentially expressed in the human data. These genes represent a wide range of structural and ontological families with different assumed functions, but their potential involvement in lung development has previously not been thoroughly evaluated. Regulation of cytokine production and cell activation were the most significant bio-ontologic attributes to genes differentialy expressed during lung development.
Using the murine data sets for comparative analyses, RORA
, which encodes for a nuclear hormone receptor, showed the most consistent expression pattern (expression positively correlated with gestational age in all data sets). ROBO1
expression was on the other hand negatively correlated with gestational age in all tested data sets (albeit significant in only 2/3 sets), which indicates an important effect early in the developing lung and then a diminishing effect over time. The ROBO1 protein is involved in axon guidance and neuronal precursor cell migration. PTGDR, WDR36, PRNP, DENND1B, PDE4D, TLE4
also showed weak evidence of differential expression in the human data using adjusted p < 0.05 as cut off (Additional file 1
, Table E2), but none showed consistent gene expression patterns in the murine data sets.
showed the strongest evidence for differential expression in the human data and this pattern was consistent in the C57BL6 strain. However, Nod1
was not represented on the platforms used for analyses on the A/J and SW strains and could thus not be evaluated in these data sets (also true for another asthma gene with consistent expression patterns, PCDH1
encodes for a cytosolic protein which contains an N-terminal caspase recruitment domain (CARD) and plays an important role for recognition of bacterial compounds and initiation of the innate immune response [103
]. Little is known about the role of NOD1
during lung development and our findings indicate that NOD1
could have important contribution.
was the second most differentially expressed asthma gene in the human data set and very consistent expression patterns were found in all murine data sets. Also for the embryonic stage analyses (pseudoglandular vs canalicular), EDN1
was among the most highly differentially expressed genes. In general, embryonic stage results were very similar to the results using time as a continuous variable. EDN1 belong to a family of secreted peptides produced by vascular endothelial cells with multiple effects on cardiovascular, neural, pulmonary and renal physiology [104
]. EDN1 shows involvement in pulmonary hypertension, fibrosis, obstructive diseases and acute lung injury, and is also required for the normal development of several tissues. Mice lacking the Edn1
gene die of respiratory failure at birth and show severe craniofacial abnormalities, as well as cardiovascular defects [106
]. Transgenic mice with lung-specific over-expression of the human EDN1
gene develop, on the other hand, chronic lung inflammation and fibrosis [108
heterozygous knockout mice also show increased bronchial responsiveness and these result link EDN1
functionally to asthma and obstructive diseases [72
]. To date, three studies report significant association between EDN1
and asthma [41
]. Our data, as well as previous studies, point to an important role for EDN1
in normal lung development, which warrants further studies.
Our study has several limitations. Our 38 human lung tissue samples were restricted to the pseudoglandular and canalicular stages. Information about key exposures that could influence gene expression patterns, such as maternal smoking, residential area, and parental allergy is not available. Thirty-eight samples are a relatively small sample size for expression analyses due to human biological variation and fetal lung tissue during the later stages of gestation was not available. It is possible that some asthma genes are important for human lung development during the later stages of gestation, but we were not able to evaluate this with our current data set. To complement the human data, we analysed expression patterns from early gestational to postnatal stages of lung development in three different murine strains. We used this murine data to replicate, in silico, the human results in the early stages and to infer human gene expression pattern in the later stages of the developing lung. Also, the microarray platforms used in the included data sets do not entirely cover the human (and murine) transcriptome and important genes may have been missed (e.g. GPRA/NPSR1
] is not represented on the U133 Plus 2.0 microarray chip and could not be evaluated). Protein analyses could provide a better view to understand specific gene functions and the post-transcriptional regulation level, but such data was not available in our study. Our asthma gene list represents genes that met our predefined criteria for asthma association, and some genes genes may have been missed (e.g. those only captured by the search terms "family based study" AND "asthma"). Given the rapid rate at which novel asthma susceptibility loci are being discovered, some of the most recent asthma genes may have been missed. These may introduce a potential null bias in the analysis.