Genetic and epigenetic risk factors for asthma in infancy and childhood

Asthma is among the most common non-communicable diseases, with an increasing prevalence worldwide. The pathophysiology behind the disease is heterogeneous, including risk factors like genes, sex, allergies, virus infections, and wheezing episodes. In recent years, epigenetic changes have been found to be associated with the development of asthma, but there is a lack of knowledge about the exact mechanisms of how these genes and epigenetic alterations contribute to disease onset and progression. Furthermore, loss-of-function mutations in the skin barrier gene filaggrin (FLG) are associated with atopic dermatitis (AD) that most often precedes the development of childhood asthma. Airway obstruction is also related to asthma development; nevertheless, less is known about lower lung function and skin barrier impairment in early infancy. Therefore, the overall aim of the thesis was to examine genetic and epigenetic factors for asthma in infancy and childhood.

Study I focused on the genetics of asthma by investigating CST1 and CCL26 to better understand their function in the development of asthma, as these genes were previously identified to have the highest expression in the nasal epithelium of dog dander-sensitized children. CST1 and CCL26 were overexpressed separately in the human alveolar basal epithelial cell line A549. RNA sequencing and protein analyses were performed to investigate the downstream effects. On the RNA level a significant downregulation of type I and III interferons and interferon-stimulated genes was observed in A549 cells overexpressing CST1 or CCL26 compared to the controls. No significant downregulation of the analyzed inflammation proteins was observed due to the overexpression of CST1, however, CXCL11, CCL20, CCL3 and CXCL10 were significantly downregulated due to the overexpression of CCL26. Overall, the overexpression of CCL26 caused a downregulation of interferon related genes and inflammatory proteins.

Study II evaluated the epigenetics of asthma by analyzing 14 hypomethylated CpG sites previously identified in whole blood of asthmatic children. Seven genes that are inferred to be regulated by these CpG sites were selected for deeper analysis. The overall aim of the study was to better understand the downstream effects of the methylation differences on the gene expression and gene function and their contribution to asthma. The seven genes (SLC25A25, MED27, NTNG2, BBLN, PTGES2, NAIF1, LCN2) or their respective control vectors were separately overexpressed via transfection in the airway epithelial cell lines A549 and BEAS-2B. Transcriptomic and proteomic analyses were performed to identify the downstream effects. The overexpression of MED27, NTNG2 and BBLN separately caused mainly an upregulation of type I and III interferon genes as well as interferon-stimulated genes. Due to the location of one asthma associated CpG site within an intronic region of MED27, the overexpression of MED27 was also analyzed at different timepoints and demonstrated to affect the expression of type I and III interferons in a time-dependent manner. On the protein level, the overexpression of MED27 caused an upregulation of CCL3, TGF-alpha and CCL20, whereas CCL3 and CCL20 were downregulated due to the overexpression of NTNG2, BBLN, PTGES2, NAIF1 and LCN2. In general, MED27 might have an important role in the pathophysiology of asthma, due to the location of an asthma associated CpG site within the gene, its time-dependent effect on interferon genes and the increased protein expression which its overexpression causes.

Study III placed the genetics in a clinical context and focused on AD, most often preceding asthma development. It is an epidemiological study of the Scandinavian mother-child cohort PreventADALL including 1836 infants with information on FLG genotyping. The objective was to investigate the role of the most common FLG loss-of-function mutations in the European population on skin barrier function, dry skin, eczema, and AD before one year of age. At 3 months, FLG mutations were associated with eczema and at 6 months, FLG mutation carriers had significantly higher transepidermal water loss (TEWL) than nonmutation carriers. Further, the risk for dry skin on the trunk and extensor limb surfaces was increased at 3 and 6 months for FLG mutation carriers. In conclusion, the study observed associations between FLG mutations and dry skin on the trunk and extensor limb surfaces, eczema, and AD before one year of age.

Study IV was also an epidemiological study of the PreventADALL cohort and combined genetics, asthma, and asthma comorbidities. The study of 1337 children aimed to identify possible associations between early infant lung- and skin barrier function and asthma at age 3 years. Lower lung function and higher TEWL were associated with asthma at age 3 years, while eczema and FLG mutations were not. The strongest association was observed between lower lung function and asthma at age 3 years when the children had a diagnosis of either AD or allergic sensitization by 3 years. Taken together, lung- and skin barrier function at 3 months seemed to have already an effect on asthma at age 3 years.

The four studies of the thesis contribute to the field of genetics and epigenetics in asthma with new findings. Potential functions were identified for genes previously identified in allergic children and for genes regulated by asthma associated CpG sites. The clinical studies of this doctoral thesis discovered that mutations in the skin barrier gene FLG influence the skin already before one year of age and assessed that skin barrier impairment as well as lower lung function in infancy were associated with asthma at age 3 years.

List of scientific papers

I. Hoyer A, Chakraborty S, Lilienthal I, Konradsen JR, Katayama S, Söderhäll C. The functional role of CST1 and CCL26 in asthma development. Immun Inflamm Dis. 2024 Jan;12(1):e1162.
https://doi.org/10.1002/iid3.1162

II. Hoyer A, Chakraborty S, Lilienthal I, Katayama S, Söderhäll C. Overexpression of genes regulated by asthma associated CpG sites increases interferons levels. [Manuscript]

III. Hoyer A, Rehbinder EM, Färdig M, Asad S, Lødrup Carlsen KC, Endre KMA, Granum B, Haugen G, Hedlin G, Monceyron Jonassen C, Katayama S, Konradsen JR, Landrø L, LeBlanc M, Olsson Mägi CA, Rudi K, Skjerven HO, Staff AC, Vettukattil R, Bradley M, Nordlund B, Söderhäll C. Filaggrin mutations in relation to skin barrier and atopic dermatitis in early infancy. Br J Dermatol. 2022 Jul;187(1):132.
https://doi.org/10.1111/bjd.20831

IV. Färdig M, Hoyer A, Almqvist C, Bains KES, Carlsen KCL, Gudmundsdóttir HK, Granum B, Haugen GN, Hedlin G, Jonassen CM, Konradsen JR, Lie A, Rehbinder EM, Skjerven HO, Staff AC, Vettukattil R, Söderhäll C, Nordlund B. Infant lung function and early skin barrier impairment in the development of asthma at age 3 years. Allergy. 2024 Jan 18.
https://doi.org/10.1111/all.16024