Saracino, Amanda; (2020) The molecular aetiopathogenesis of adult morphoea. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background: The precise aetiopathogenesis of morphoea is poorly understood. Importantly, the initiating genetic / molecular mechanisms remain unclear. Linear morphoea (LM) likely follows Blaschko’s-lines and hence may represent epidermal mosaicism. This, along with insights from related fibrotic processes, support the key role of the epidermis in disease initiation and propagation. The goal of this thesis was to investigate the genetic aetiologic and pathogenic basis of morphoea, focusing specifically on epidermal genomic variation and differential epidermal and dermal transcriptomic expression profiles. LM provides a unique template for this work, by enabling intra-individual comparisons of clinically affected and contralateral site-matched unaffected skin, with background genetic homogeneity. Methodology: After clinical and histological validation of the LM cohort (n=133), paired skin biopsies were collected from 27 participants. Epidermis and dermis were isolated using a two-step separation protocol. Whole genome sequencing (WGS; n=4 epidermal; Illumina HiSeqX Ten System, 60-70×) and RNA-seq (n=5 epidermal, n=5 dermal; BGISEQ-500, 20 million reads-per-sample) were performed on isolated tissue pairs. Gene set enrichment, PANTHER enrichment testing and STRING pathway analyses were employed. RT-qPCR and whole-skin immunohistochemistry were used to validate key results. Results: Epidermal WGS confirmed no commonly affected gene or developmental somatic mutation. However potentially disease relevant protein coding pathogenic variants were seen in ADAMTSL1 and ADAMTS16; with known links to fibrosis and LM fibroblasts. Epidermal transcriptomic-profiling demonstrated a wounded, proliferative, inflammatory and profibrotic signature, with over-expression of TNF-α via NFkB, TGF-β, IL-6/JAKSTAT and IFN-signaling. Epidermal IFI27 was upregulated and its induction of IFNγ-related-apoptosis could act as an initiating ‘damage’ signal. IFI27 also negatively regulates NR4A1, which enables persistent TGF-β-signaling; NR4A1 was under-expressed in the dermis. Morphoea dermis demonstrated a strong B-cell and IFN-signature. Upregulation of morphogenic development genes such as WNT was also seen, with concordant dermal upregulation of the frizzled Wnt-receptor SFRP2. Wnt-signaling may facilitate epidermal-dermal communication. Dysregulation of HOX, SOX and PAX genes was present in both skin layers, potentially providing clues to dermal mosaic mechanisms in morphoea. Conclusion: Based on NGS of paired-skin biopsies, this thesis provides a possible genetic and molecular understanding of the underlying morphoea aetiopathogenesis which supports potential complex polygenic epidermal mosaicism and the presence of key potential inciting and disease driving epidermal mechanisms.

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