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Background Null mutations within the filaggrin (FLG) gene are established genetic risk factors for atopic dermatitis. Studies of FLG have typically used sequencing or bespoke genotyping. Large-scale population cohorts with genome-wide imputed data offer powerful genetic analysis opportunities, but bespoke FLG genotyping is often not feasible in such studies. Therefore, we aimed to determine the quality of selected FLG null genotype data extracted from genome-wide imputed sources, focussing on UK population data. Methods We compared the allele frequencies of three FLG null mutations that could be detected by imputation (p.Arg501Ter, p.Arg2447Ter and p.Ser3247Ter; commonly referred to as R501X, R2447X and S3247X respectively) in directly genotyped and genome-wide imputed data in the ALSPAC cohort. Logistic regression analysis was used to test the association of atopic dermatitis with imputed and genotyped FLG null mutations in ALSPAC and UK Biobank to investigate the usefulness of imputed FLG data. Results The three FLG null mutations appear to be well imputed in datasets that use the Haplotype Reference Consortium (HRC) for imputation (0.3% discordance compared with directly genotyped data). However, a greater proportion of null alleles failed imputation compared to wild-type alleles. Despite the calling of FLG mutations in imputed data being imperfect, they are still strongly associated with atopic dermatitis (p-values between 7x10-10 and 5x10-75 in UK Biobank). Conclusions HRC imputed data appears to be adequate for UK population-based genetic analysis of selected FLG null mutations (p.Arg501Ter, p.Arg2447Ter and p.Ser3247Ter).

Background: Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cells in vitro provides the opportunity for selected genetic effects to be investigated in detail. Methods: Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown of FLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements. Results: FLG knockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of barrier impairment with FLG knockdown, but the mature organoids showed no difference in transepidermal water loss, water content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins per sample. Gene ontology and pathway analyses identified an increase in transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were represented in both the up-regulated and down-regulated protein groups, as was the term ‘axon guidance’. Conclusions: This work provides further evidence for keratinocyte-specific mechanisms contributing to immune and neurological, as well as structural, aspects of skin barrier dysfunction. Individuals with filaggrin deficiency may derive benefit from future therapies targeting keratinocyte-immune crosstalk and neurogenic pruritus.

Background: Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cells in vitro provides the opportunity for selected genetic effects to be investigated in detail. Methods: Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown of FLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements. Results: FLG knockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed evidence of barrier impairment with FLG knockdown, but the mature organoids showed no difference in transepidermal water loss, water content or dye penetration. There was no difference in epidermal ceramide content. Mass spectrometry proteomic analysis detected >8000 proteins per sample. Gene ontology and pathway analyses identified an increase in transcriptional and translational activity but a reduction in proteins contributing to terminal differentiation, including caspase 14, dermokine, AKT1 and TGF-beta-1. Aspects of innate and adaptive immunity were represented in both the up-regulated and down-regulated protein groups, as was the term ‘axon guidance’. Conclusions: This work provides further evidence for keratinocyte-specific mechanisms contributing to immune and neurological, as well as structural, aspects of skin barrier dysfunction. Individuals with filaggrin deficiency may derive benefit from future therapies targeting keratinocyte-immune crosstalk and neurogenic pruritus.