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Louise McClelland 13th October 2010 Filaggrin Polyprotein profilaggrin Epidermal differentiation cDNA library Somatic hybrids M13 sequencing Long range PCR Ichthyosis vulgaris Atopic dermatitis (eczema) Asthma Ancient mutations Filaggrin protein Processing and function Gene ID and characterisation Mutation analysis Filaggrin is synthesised as a giant precursor protein, profilaggrin Filament-aggregating protein Interact with keratin intermediate filaments in terminally differentiating mammalian epidermis Very important in facilitating epidermal differentiation and maintaining skin barrier function Implicated in a no. of keratinizing disorders 1981 - Term Filaggrin (FLG) coined Described a class of structural proteins that have been isolated from the stratum corneum 1984-1985 – Filaggrin and profilaggrin protein characterisation 1989 – FLG gene cloned and mapped 1992-1993 Gene characterisation 2006 – LOF mutations ID’d in ichthyosis vulgaris and atopic dermatitis 2007 – Further mutation analysis 1980 1990 2000 2010 >400 kDa N-terminal domain 2 subdomains ?role in regulation of profilaggrin by Ca2+ Repeat region 10-12 tandemly arranged filaggrin repeats Linker regions between repeats C-terminal domain 1984-1985 1980 1990 2000 2010 During terminal differentiation of granular cells profilaggrin is proteolytically cleaved into; Multiple 37 kDa (342 AA) filaggrin peptides N and C domains Linker regions between repeats Phosphorylation of profilaggrin believed to prevent premature cleavage Aggregates to keratin intermediate filaments in cytoskeleton Creates a dense insoluble protein-lipid keratin matrix cross linked by transgluaminases Causes collapse of granular cells into flattened anuclear squames Scaffold for the attachment of cornified cell envelope (CCE) proteins and lipids CCE Outermost barrier layer of skin Prevents water loss Impedes allergens, toxic chemicals and infectious agents Post translational conversion of arginine to citrulline (Deimination/citrulination) Promotes unfolding and degradation into hygroscopic AA and derivatives Contributes to Natural moisturising factor (NMF) NMF responsible for maintaining hydration in low environment humidity Differences between mouse and human FLG complicated ID Anti-mouse FLG antibodies do not recognise human FLG cDNA clones encoding mouse FLG do not recognise human FLG cDNA library of human foreskin epidermal tissue created in a λ phage Screened library using human anti-filaggrin monoclonal antibody Antibody positive clones purified from plaques (12 clones) 1989 1980 1990 2000 2010 Griffiths, 1999 cDNA inserted into M13 vectors Strachan and Read, 2010 Longest clone contained ORF of 416 AA Beginning and end share homology Showed encodes a polyprotein precursor of filaggrin Identified linker sequences are removed to create monomers Somatic cell hybrids Recorded what human chromosomes retained in each hybrid Examined hybrids DNA by Southern blot Filaggrin sequence probe Only hybrids retaining chromosome 1 Griffiths, 1999 RNA probes for in situ hybridisation Synthesised from earlier plaques (in pGEM-3B vector) Refined chromosomal position to 1q Cloned profilaggrin cDNA was compared to genomic profilaggrin to identify the exon/intron boundaries Contains 3 exons 1992-1993 1980 1990 2000 2010 Dry, flaky skin First investigated in ichthyosis vulgaris (IV) Most common and mildest ichthyosis Poorly formed stratum corneum prone to water loss Predisposition to eczema and associated asthma Keratosis pilaris Smith et al., 2006 1980 Palmar hyperlinearity of palms and soles 1990 2006 2000 2010 Absence of filaggrin in keratinocytes of IV patients by immunoblotting Decreased FLG mRNA Recessive mouse model flaky tail (ft) Histologically similar to IV Strong genetic linkage to mouse FLG locus IV linkage to 1q21 in an American family 15 kindred examined Irish, Scottish and Americans of European decent Long range PCR of FLG R501X 2284del4 Allele F = ~4% in European ancestry Both cause PTC in 1st filaggrin repeat Semi dominant inheritance Heterozygotes = mild phenotype Homozygotes = severe phenotype Noted several patients had atopic dermatitis and a few had asthma Atopic dermatitis (eczema) Allergies Asthma FLG mutations suspected in atopic disease because of link between IV and eczema Identified R501X and 2284del4 mutations in atopic disease Significant association between these mutations and; Atopic dermatitis Asthma in the context of atopic dermatitis Carried by 9% of people of European origin Palmer et al., 2006 1980 2006 1990 2000 2010 Several mutations now identified Other populations with FLG mutations identified Japanese, Mexican, Basque, Indian Distinct haplotypes associated with some mutations All common mutations null mutants R501X, 2284del4, R2447X, S3247X R501X and 2284del4 absent from non-Europeans Ancient mutations Similar pathogenicity of 3’ mutations as 5’ Immunoblotting showed still greatly reduced filaggrin 2007 Sandilands et al., 2007 1980 1990 2000 2010 Major predisposing gene for atopic disease Complex disease model emerging Refocused attention on skin barrier rather than immunity in eczema research FLG mutations may be modifying factors in other conditions E.g. X-linked ichthyosis Reduced no. of FLG repeats may predispose to dry skin Hypothesis of why mutations so common in Europe (particularly Ireland) Mutation advantage at some point in history Allows a degree of natural vaccination against antigens e.g. plague Identifying when the mutations arose may help explain why so prevalent Brown, Genome, BIOS, 1st edition, 1999 Griffiths et al., Modern Genetic Analysis, Freeman and Co. NY, 1st edition, 1999 McKinley-Grant et al, PNAS 86: 4848-4852; 1989 Palmer et al., Nat Genet 38: 441-446; 2006 Presland et al., J Biol Chem 267: 23772-23781; 1992 Sandilands et al., Nat Genet 39: 650-654; 2007 Sandilands et al., J Cell Sci 122: 1285-1294; 2009 Good review Smith et al., Nat Genet 38: 337; 2006 Steinert et al., PNAS 78: 4097-4101; 1981 Strachan and Read, Human Molecular Genetics, Garland Science, 4th edition, 2010