Characterisation of filaggrin gene and results of mutation screening

1. Characterisation of filaggrin gene and results of mutation screening Louise McClelland 13th October 2010

2. Key words • Filaggrin • Polyprotein profilaggrin • Epidermal differentiation • cDNAlibrary • Somatic hybrids • M13 sequencing • Long range PCR • Ichthyosis vulgaris • Atopic dermatitis (eczema) • Asthma • Ancient mutations

3. Filaggrin Overview • Filaggrin protein • Processing and function • Gene ID and characterisation • Mutation analysis

4. Filaggrin protein • 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

5. Epidermal differentiation

6. Time line 1980 1990 2000 2010 • 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

7. Profilaggrin polyprotein 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

8. Profilaggrin processing • 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

9. Filaggrin • 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

10. Filaggrin processing in squames • 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

12. ID of filaggrin gene 1980 1990 2000 2010 • 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

13. DNA libraries Griffiths, 1999

14. M13 mediated sequencing • cDNA inserted into M13 vectors Strachan and Read, 2010

15. 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

16. Mapping 1 • 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

17. Mapping 2 • RNA probes for in situ hybridisation • Synthesised from earlier plaques (in pGEM-3B vector) • Refined chromosomal position to 1q

18. characterisation 1980 1990 2000 2010 • Cloned profilaggrin cDNA was compared to genomic profilaggrin to identify the exon/intron boundaries • Contains 3 exons 1992-1993

19. Dry, flaky skin Mutation screening 1980 1990 2000 2010 • 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 Palmar hyperlinearity of palms and soles Keratosis pilaris Smith et al., 2006 2006

20. FLG mutations suspected in IV because; • 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

21. FLG Mutation screening:Ichthyosis vulgaris • 15 kindred examined • Irish, Scottish and Americans of European decent • Long range PCR of FLG • R501X • 2284del4 • 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 Allele F = ~4% in European ancestry

22. Atopic disease (AD) • Atopic dermatitis (eczema) • Allergies • Asthma • FLG mutations suspected in atopic disease because of link between IV and eczema

23. FLG Mutation screening:Atopic Disease 1980 1990 2000 2010 • 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 2006 Palmer et al., 2006

24. Further analysis 1980 1990 2000 2010 • Several mutations now identified • All common mutations null mutants • Other populations with FLG mutations identified • Japanese, Mexican, Basque, Indian • Distinct haplotypes associated with some mutations • 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

25. conclusions • 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

26. References • Brown, Genome, BIOS, 1st edition, 1999 • Griffiths et al., Modern Genetic Analysis, Freeman and Co. NY, 1st edition, 1999 • McKinley-Grant et al, PNAS86: 4848-4852; 1989 • Palmer et al., Nat Genet38: 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 Sci122: 1285-1294; 2009 • Good review • Smith et al., Nat Genet38: 337; 2006 • Steinert et al., PNAS78: 4097-4101; 1981 • Strachan and Read, Human Molecular Genetics, Garland Science, 4th edition, 2010