Epistasis between FLG and IL4R genes on the risk of allergic sensitization: results from two population-based birth cohort studies

Immune-specifc genes as well as genes responsible for the formation and integrity of the epidermal barrier have been implicated in the pathogeneses of allergic sensitization. This study sought to determine whether an epistatic efect (gene-gene interaction) between genetic variants within interleukin 4 receptor (IL4R) and flaggrin (FLG) genes predispose to the development of allergic sensitization. Data from two birth cohort studies were analyzed, namely the Isle of Wight (IOW; n=1,456) and the Manchester Asthma and Allergy Study (MAAS; n=1,058). In the IOW study, one interaction term (IL4R rs3024676×FLG variants) showed statistical signifcance (interaction term: P=0.003). To illustrate the observed epistasis, stratifed analyses were performed, which showed that FLG variants were associated with allergic sensitization only among IL4R rs3024676 homozygotes (OR, 1.97; 95% CI, 1.27–3.05; P=0.003). In contrast, FLG variants efect was masked among IL4R rs3024676 heterozygotes (OR, 0.53; 95% CI, 0.22–1.32; P=0.175). Similar results were demonstrated in the MAAS study. Epistasis between immune (IL4R) and skin (FLG) regulatory genes exist in the pathogenesis of allergic sensitization. Hence, genetic susceptibility towards defective epidermal barrier and deviated immune responses could work together in the development of allergic sensitization

A mechanistic target of rapamycin complex 1/2 (mTORC1)/V-Akt murine thymoma viral oncogene homolog 1 (AKT1)/cathepsin H axis controls filaggrin expression and processing in skin, a novel mechanism for skin barrier disruption in patients with atopic dermatitis

BACKGROUND: Filaggrin, encoded by the FLG gene, is an important component of the skin’s barrier to the external environment and genetic defects in FLG strongly associate with Atopic Dermatitis (AD). However, not all AD patients have FLG mutations. OBJECTIVE: We hypothesised that these patients may possess other defects in filaggrin expression and processing, contributing to barrier disruption and AD, and therefore present novel therapeutic targets for this disease. RESULTS: We describe the relationship between the mTORC1 protein subunit RAPTOR, the serine/threonine kinase AKT1 and the protease cathepsin H, for which we establish a role in filaggrin expression and processing. Increased RAPTOR levels correlated with decreased filaggrin expression in AD. In keratinocyte cell culture, RAPTOR up-regulation or AKT1 shRNA knockdown reduced the expression of the protease cathepsin H. Skin of cathepsin H-deficient mice and CTSH shRNA knockdown keratinocytes showed reduced filaggrin processing and the mouse showed both impaired skin barrier function and a mild proinflammatory phenotype. CONCLUSION: Our findings highlight a novel, potentially treatable, signalling axis controlling filaggrin expression and processing which is defective in AD

The Genetics of Atopic Eczema in the Bangladeshi population of East London

PhDAtopic Eczema (AE) is a common, complex, genetic skin disease. It usually begins in infancy and can affect any part of the body but often occurs in the flexures of the elbows and knees. The cohort used in this study is of Bangladeshi origin and all subjects reside in East London. Using a combination of techniques, such as the Illumina goldengate assay, PCR and sequencing, both novel and previously associated genes have been studied in this thesis. Previously associated genes were used to validate this population and also to investigate the variation of genes associated in different ethnic populations. Six of eleven previously associated genes were replicated in this population. In order to identify novel genes of interest in AE, sixteen novel genes were chosen for investigation. Of these sixteen, eight showed association with AE. A recently identified gene involved in the pathogenesis of AE, Filaggrin (FLG), was also investigated. This was done using a combination of PCR, sequencing and Taqman SNP assays. Only six families out of 80 in this population were found to harbour the two known common FLG mutations. These families were clinically reassessed for Ichthyosis Vulgaris (IV) which is also associated to the same FLG mutations. After this reassessment the FLG mutations were shown to be associated with IV in this population with variable penetrance. No association with AE was found. ABCA12 was also investigated as a candidate gene for AE, again using the Illumina goldengate assay and microsatellite linkage markers. Association was observed with this gene and AE. Harlequin Ichthyosis (HI) mutations were also screened in this gene for twelve additional HI patients. This thesis has provided a greater insight into the variation of gene associations between populations with AE, highlighting novel genes, including KY-NU and JAK3/INSL3, which need to be investigated in other populations

Impact of mutational profiles on response of primary oestrogen receptor-positive breast cancers to oestrogen deprivation

Pre-surgical studies allow study of the relationship between mutations and response of oestrogen receptor-positive (ER+) breast cancer to aromatase inhibitors (AIs) but have been limited to small biopsies. Here in phase I of this study, we perform exome sequencing on baseline, surgical core-cuts and blood from 60 patients (40 AI treated, 20 controls). In poor responders (based on Ki67 change), we find significantly more somatic mutations than good responders. Subclones exclusive to baseline or surgical cores occur in ∼30% of tumours. In phase II, we combine targeted sequencing on another 28 treated patients with phase I. We find six genes frequently mutated: PIK3CA, TP53, CDH1, MLL3, ABCA13 and FLG with 71% concordance between paired cores. TP53 mutations are associated with poor response. We conclude that multiple biopsies are essential for confident mutational profiling of ER+ breast cancer and TP53 mutations are associated with resistance to oestrogen deprivation therapy

ROLE OF ALLERGIC SENSITIZATION, FILAGGRIN VARIANTS, AND DNA

Background: Allergic disorders, including eczema, asthma, and rhinitis, have emerged as a global public health concern due to their elevated prevalence and the associated clinical morbidity. Environmental, immunologic, and genetic factors have been implicated in the pathogenesis of allergic disorders. Allergic sensitization (representing deviated immune responses) and filaggrin gene (FLG) variants (leading to dysfunctional epidermal barrier) have shown to be common predisposing factors in the development of allergic disorders. However, there is a lack of knowledge on their joint effects on the development of single and multiple (coexistence) allergic disorders. More recently, epigenetic mechanisms, such as DNA methylation, have emerged as potentially important factors in the development of such complex diseases; however, the extent to which DNA methylation associates with allergic disorders is unclear. Objectives: This dissertation sought to (i) determine whether eczema and/or allergic sensitization is an effect modifier of the association between ‘FLG variants and asthma’ and ‘FLG variants and rhinitis’, (ii) test whether FLG variants and allergic sensitization jointly predispose to the comorbidity of eczema, asthma and rhinitis, and (iii) examine associations between DNA methylation across the epidermal differentiation complex (EDC) genomic region with eczema status. Methods: The Isle of Wight (IOW) birth cohort, a population-based sample of 1,456 infants born between January 1989 and February 1990, was prospectively assessed at ages 1, 2, 4, 10, and 18 years. Repeated measurements of eczema, asthma, rhinitis, and allergic sensitization (documented by skin prick tests) were available for all follow-ups. FLG variants R501X, 2282del4, and S3247X were genotyped in 1,150 participants. Logbinomial regression models were applied to test for associations and statistical interactions on multiplicative scale. On the other hand, DNA methylation was measured in a subsample (n = 367) of the IOW participants at age 18 years (discovery cohort) and in two semi-independent samples (replication cohorts I and II). Associations between eczema status and DNA methylation were assessed using linear regression. Results: FLG variants were associated with increased risk of asthma and rhinitis. Both eczema status (RRinteraction = 1.96, Pinteraction = 0.006) and allergic sensitization (RRinteraction= 1.58, Pinteraction = 0.013) modified the association between FLG variants and asthma, but not the association with rhinitis. The combined effect of both risk factors increased the risk of coexisting “eczema and asthma” (RR = 13.67, 95% CI: 7.35 – 25.42), “asthma andrhinitis” (RR = 7.46, 95% CI: 5.07 – 10.98), and “eczema, asthma, and rhinitis” (RR =23.44, 95% CI: 12.27 – 44.78). On the other hand, Differential DNA methylation of CpG site cg12048339 (located within promoter of S100A6 gene) was associated with eczema specifically among female participants of all study cohorts; whereas, aberrant DNA methylation of cg10959711 (located within promoter of S100A11 gene) associated with eczema among male participants in all study samples. Conclusions: Allergic sensitization and eczema modulated the association between FLG variants and asthma, but not rhinitis; implying that the mechanisms and pathways through which FLG variants predispose to increased risk of asthma and rhinitis may be different. Moreover, the coexistence of allergic disorders is frequent and allergic sensitization and FLG variants jointly increased risk of allergic comorbidities, which may represent more severe and complex clinical phenotypes. Results of an exploratory investigation demonstrated that DNA methylation of the EDC locus could be an important factor in the development of eczema in a sex-specific manner. Future studies corroborating our findings are needed

A mechanistic target of rapamycin complex 1/2 (mTORC1)/V-Akt murine thymoma viral oncogene homolog 1 (AKT1)/cathepsin H axis controls filaggrin expression and processing in skin, a novel mechanism for skin barrier disruption in patients with atopic dermatitis

Background Filaggrin, which is encoded by the filaggrin gene (FLG), is an important component of the skin's barrier to the external environment, and genetic defects in FLG strongly associate with atopic dermatitis (AD). However, not all patients with AD have FLG mutations. Objective We hypothesized that these patients might possess other defects in filaggrin expression and processing contributing to barrier disruption and AD, and therefore we present novel therapeutic targets for this disease. Results We describe the relationship between the mechanistic target of rapamycin complex 1/2 protein subunit regulatory associated protein of the MTOR complex 1 (RAPTOR), the serine/threonine kinase V-Akt murine thymoma viral oncogene homolog 1 (AKT1), and the protease cathepsin H (CTSH), for which we establish a role in filaggrin expression and processing. Increased RAPTOR levels correlated with decreased filaggrin expression in patients with AD. In keratinocyte cell cultures RAPTOR upregulation or AKT1 short hairpin RNA knockdown reduced expression of the protease CTSH. Skin of CTSH-deficient mice and CTSH short hairpin RNA knockdown keratinocytes showed reduced filaggrin processing, and the mouse had both impaired skin barrier function and a mild proinflammatory phenotype. Conclusion Our findings highlight a novel and potentially treatable signaling axis controlling filaggrin expression and processing that is defective in patients with AD

Meta-analysis identifies seven susceptibility loci involved in the atopic March

Eczema often precedes the development of asthma in a disease course called the a 'atopic march'. To unravel the genes underlying this characteristic pattern of allergic disease, we conduct a multi-stage genome-wide association study on infantile eczema followed by childhood asthma in 12 populations including 2,428 cases and 17,034 controls. Here we report two novel loci specific for the combined eczema plus asthma phenotype, which are associated with allergic disease for the first time; rs9357733 located in EFHC1 on chromosome 6p12.3 (OR 1.27; P=2.1 × 10 a'8) and rs993226 between TMTC2 and SLC6A15 on chromosome 12q21.3 (OR 1.58; P=5.3 × 10 a'9). Additional susceptibility loci identified

The Role of Epidermal Enhancer 923 in the Chromatin Architecture and Transcriptional Regulation of the Epidermal Differentiation Complex

The epidermis covers the surface of the skin and provides a functional barrier across the entire body. Epidermal cells or keratinocytes proliferate in the innermost basal layer and migrate upwards into the suprabasal spinous and granular layers as they differentiate, and finally into the terminally differentiated outermost stratum corneum. Keratinocytes undergoing terminal differentiation are marked by tissue-specific concomitant expression of genes encoded in the Epidermal Differentiation Complex (EDC) locus. The EDC genes are organized into four gene families - S100, Sprr, Lce, and Flg-like, which are coordinately expressed upon activation of the terminal differentiation program in keratinocytes. The molecular mechanisms that govern the activation of the EDC during epidermal differentiation are poorly understood. The synteny and colinearity of the locus across multiple mammalian species and the coordinate expression of EDC genes upon keratinocyte differentiation suggest molecular mechanisms operating at the chromatin level. I hypothesize coordinate activation of the EDC by an enhancer regulatory element. Enhancers are non-coding regulatory DNA sequences that upon binding specific transcription factors, are able to increase expression of a proximal or distal target gene. Previous work in our lab identified an epidermal-specific enhancer, CNE 923, that was active in in cell-based luciferase assays and transgenic mice. Here, I examine the function of the 923 enhancer for epidermal differentiation. Using an independent transgenic mouse line, I identified spatiotemporal sensitivity of the 923 enhancer that correlated with the patterning of epidermal barrier formation during mouse embryonic development. To determine if 923 formed chromatin interactions with the EDC gene promoters, I performed chromosome conformation capture (3C) assays in proliferating and differentiated primary mouse keratinocytes. The 3C studies identified physiologically sensitive chromatin interactions between 923 and EDC gene promoters. The data supports a dynamic EDC chromatin topology during keratinocyte differentiation. A requirement for c-Jun/AP-1 in relation to 923-mediated EDC chromatin remodeling for normal EDC gene expression during keratinocyte differentiation was further determined by chromatin immunoprecipitation, 3C, and RNA-seq upon pharmacological inhibition of AP-1 binding. To further determine the function of 923 in vivo, I generated a series of mutation alleles using CRISPR/Cas9 genome editing in mice. Cas9 nuclease activity targeted to the flanking ends of the 923 enhancer in mouse zygotes by a pair of guide RNAs, coupled with homologous recombination-mediated loxP insertions, generated 1 floxed (923flox), 2 independent deletions (923delA, 923delB), and 1 partial deletion (923pdel) alleles for the 923 enhancer. My results from the 923 knockout mice identified decreased expression of nearby Smcp, Lce6a, and involucrin gene expression, decreased distal Crnn and Lce gene family members, and a correlative increase in expression of Sprr gene family members. To identify the chromatin interactions for the 923 enhancer on a genome-wide scale, I performed high-throughput circular chromosome conformation capture (4C-seq) assays with respect to the 923 enhancer and an additional Flg promoter viewpoint in proliferating and differentiated keratinocytes and P5424 T-cells. My results revealed 923 enhancer-mediated chromatin interactions indicative of a topologically associated domain encompassing the EDC. However, an enrichment of 923 mediated chromatin interactions within the EDC, were identified in keratinocytes relative to the T-cells, specifically between the 923 enhancer and the Sprr and Lce gene families, and with non-coding regions in the gene desert between the S100 and Sprr gene families. Of note was a 923 interaction with another putative enhancer near Crct1, enriched specifically in proliferating keratinocytes, and suggesting cross-talk between enhancers. Keratinocyte-specific trans-interactions identified by MACS and GREAT algorithms included genes important for epidermal function including Trp63, an important regulator of keratinocyte differentiation. Together, my 4C-seq identifies unique chromatin architectures of the EDC in keratinocytes and T cells, including keratinocyte-specific enhancer-enhancer crosstalk in cis and interactions between transcriptionally active loci in trans. My studies identify, for the first time, a link between the 923 enhancer and proximal (Ivl, Smcp, Lce6a) and distal genes (Crnn, distal Lce family), the loss of which coincides with upregulation of other epidermal differentiation genes (Sprr family) to maintain skin barrier function. Together, my work has identified 923 as an epidermal-specific enhancer that participates in a chromatin looping network to co-regulate expression of genes important for epidermal development, as a mechanism for maintaining skin barrier integrity