Exome Sequencing of Filaggrin and Related Genes in African-American Children with Atopic Dermatitis

Abstract

atopic dermatitis Filaggrin minor allelic frequency S100-fused-like protein TO THE EDITOR Atopic dermatitis (AD) is a common chronic relapsing disease. There is a considerable body of evidence supporting a genetic basis for AD (Bussman et al., 2011Bussman C. Weidinger S. Novak N. Genetics of atopic dermatitis.J German Soc Dermatol. 2011; 9: 670-679Google Scholar; Ellinghaus et al., 2013Ellinghaus D. Baurecht H. Esparza-Gordillo J. et al.High-density genotyping study indentifies four new susceptibility loci for atopic dermatitis.Nat Genet. 2013; 45: 808-812Crossref PubMed Scopus (134) Google Scholar). Mutations in the Filaggrin (FLG) gene have been consistently found to be associated with AD in people of European and Asian ancestry (Brown and McLean, 2012Brown S.J. McLean W.H. One remarkable molecule: filaggrin.J Invest Dermatol. 2012; 132: 751-762Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). More than 40 FLG loss-of-function mutations have been described in Europeans and Asians (Brown and McLean, 2012Brown S.J. McLean W.H. One remarkable molecule: filaggrin.J Invest Dermatol. 2012; 132: 751-762Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). However, FLG loss-of-function mutations have not commonly been found in Africans or African Americans (Winge et al., 2011Winge M.C. Bilcha K.D. Lieden A. et al.Novel filaggrin mutation but no other loss-of-function variants found in Ethiopian patients with atopic dermatitis.Br J Dermatol. 2011; 165: 1074-1080Crossref PubMed Scopus (69) Google Scholar; Brown and McLean, 2012Brown S.J. McLean W.H. One remarkable molecule: filaggrin.J Invest Dermatol. 2012; 132: 751-762Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar; Margolis et al., 2012Margolis D.J. Apter A.J. Gupta J. et al.The persistence of atopic dermatitis and Filaggrin mutations in a US longitudinal cohort.J Allergy Clin Immunol. 2012; 130: 912-917Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Loss-of-function mutations in exon 3 of FLG result in diminished or absent filaggrin protein, most often due to a premature stop codon or a frameshift mutation resulting in a stop codon further downstream. Interestingly, the absence of profilaggrin protein (precursor of filaggrin) has also been noted in keratohyalin granules in the majority of individuals with ichthyosis vulgaris of European and Asian ancestry (Fleckman and Brumbaugh, 2002Fleckman P. Brumbaugh S. Absence of granular layer and keratohylin define a morphologically distinct subset of individuals with ichthyosis vulgaris.Exper Dermatol. 2002; 11: 327-336Crossref PubMed Scopus (44) Google Scholar; Perusquia-Ortiz et al., 2013Perusquia-Ortiz A.M. Oji V. Sauerland M.C. et al.Complete filaggrin deficiency in ichthyosis vulgaris is associated with only moderate changes in epidermal permeability barrier function profile.J Eur Acad Dermatol Venereol. 2013; 27: 1552-1558Crossref PubMed Scopus (28) Google Scholar; Thyssen et al., 2013Thyssen J.P. Godoy-Gijon E. Elias P.M. Ichthyosis vulgaris: the filaggrin mutation disease.Br J Dermatol. 2013; 168: 155-158Crossref Scopus (90) Google Scholar). FLG is located on chromosome 1q21 in a region called the epidermal differentiation complex. It is part of a family of genes that code for S100-fused-like proteins (SFTP). The SFTPs include the proteins profilaggrin (coded by FLG), hornerin (HRNR), filaggrin-2 (FLG2), repetin (RPTN), cornulin (CRNN), trichohyalin (TCHH), and trichohyalin-like 1(TCHHL1; Henry et al., 2012Henry J. Toulza E. Hsu C.Y. et al.Update on the epidermal differentiation complex.Front Biosci. 2012; 17: 1517-1532Crossref PubMed Scopus (100) Google Scholar). These genes are very similar to one another with respect to structure and function, and lie in close proximity to each other in the epidermal differentiation complex (Marenholz et al., 2011Marenholz I. Rivera V.A. Esparza-Gordillo J. et al.Association screening in the Epidermal Differentiation Complex (EDC) identifies an SPRR3 repeat number variant as a risk factor for eczema.J Investig Dermatol. 2011; 131: 1644-1649Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar; Henry et al., 2012Henry J. Toulza E. Hsu C.Y. et al.Update on the epidermal differentiation complex.Front Biosci. 2012; 17: 1517-1532Crossref PubMed Scopus (100) Google Scholar; Pellerin et al., 2013Pellerin L. Henry J. Hsu C.Y. et al.Defects in filaggrin-like proteins in both lesional and nonlesional atopic skin.J Allergy Clin Immunol. 2013; 131: 1094-1102Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). On the basis of previous experiences with FLG, it has been hypothesized that a stop-gain (null) mutation in exon 3 of any of the SFTP genes will result in decreased or absent protein production (Marenholz et al., 2011Marenholz I. Rivera V.A. Esparza-Gordillo J. et al.Association screening in the Epidermal Differentiation Complex (EDC) identifies an SPRR3 repeat number variant as a risk factor for eczema.J Investig Dermatol. 2011; 131: 1644-1649Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar; Henry et al., 2012Henry J. Toulza E. Hsu C.Y. et al.Update on the epidermal differentiation complex.Front Biosci. 2012; 17: 1517-1532Crossref PubMed Scopus (100) Google Scholar; Margolis et al., 2014Margolis D.J. Gupta J. Apter A.J. et al.Filaggrin-2 variation is associated wtih more persistent atopic dermatitis in African American subjects.J Allergy Clin Immunol. 2014; 133: 784-789Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The goal of this study was to identify stop-gain variants in FLG and closely related genes in African Americans with AD from the Pediatric Eczema Elective Registry (PEER; Margolis et al., 2012Margolis D.J. Apter A.J. Gupta J. et al.The persistence of atopic dermatitis and Filaggrin mutations in a US longitudinal cohort.J Allergy Clin Immunol. 2012; 130: 912-917Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). From this cohort we randomly selected 60 subjects for whole-exome sequencing to ensure sufficient power to detect variants with a minor allelic frequency (MAF) of greater than 3%. Sequencing was performed by Ambry Genetics (Aliso Viejo, CA) using whole exome–targeted enrichment by Agilent SureSelectXT Human All Exon 50Mb kit. Quality assessment revealed that most samples were above 50% on target and mean coverage per gene was excellent. The libraries were indexed using 100 base paired ends and processed using Illumina HiSeq2000 at × 100 coverage per exon. Data were assessed using a pipeline generated at the University of Pennsylvania based on the best practices protocol from the Broad Institute (Cambridge, MA). This report focused on stop-gain mutations of exon 3 (i.e., loss-of-function mutations) in the SFTP genes because of their likely functional relevance (Marenholz et al., 2011Marenholz I. Rivera V.A. Esparza-Gordillo J. et al.Association screening in the Epidermal Differentiation Complex (EDC) identifies an SPRR3 repeat number variant as a risk factor for eczema.J Investig Dermatol. 2011; 131: 1644-1649Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar; Brown and McLean, 2012Brown S.J. McLean W.H. One remarkable molecule: filaggrin.J Invest Dermatol. 2012; 132: 751-762Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar; Henry et al., 2012Henry J. Toulza E. Hsu C.Y. et al.Update on the epidermal differentiation complex.Front Biosci. 2012; 17: 1517-1532Crossref PubMed Scopus (100) Google Scholar). Taqman allelic discrimination assays were created for any newly identified FLG loss-of-function mutations, which were then used to genotype an additional random sample of 100 African-American PEER children. Sequencing of the SFTP genes in 60 self-reported (ancestry previously confirmed with ancestral informative markers (Margolis et al., 2012Margolis D.J. Apter A.J. Gupta J. et al.The persistence of atopic dermatitis and Filaggrin mutations in a US longitudinal cohort.J Allergy Clin Immunol. 2012; 130: 912-917Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar)) African-American children with AD revealed a total of 289 variants in FLG, 107 variants in FLG2, 339 variants in HRNR, 4 variants in RPTN, 37 variants in CRNN, 88 variants in TCHH, and 14 variants in TCHHL1. However, very few variants resulted in a premature stop codon in exon 3 (Table 1). Each of the three newly identified FLG stop-gain mutations, Q570X, R3409X and S3707X, were observed only once. S2392X and S2377X in FLG2 were noted 1 and 16 times, respectively. In TCHHL1, the variant Q294X was noted twice. All subjects were heterozygous for the mutations. The MAF for variants noted once, twice and 16 times were 0.008, 0.017, and 0.133, respectively. Next, we used Taqman-based allelic discrimination assays to evaluate the three FLG mutations, Q570X, R3409X, and S3707X, in an additional 100 African-American PEER children. However, none of these variants could be detected in other members of our cohort.Table 1Null mutations identified in genes belonging to S100-fused-like protein familyGeneVariantReference alleleAlternate alleleLocation of amino acid changeMAF current studyMAF African ancestry (source: 1000 Genomes)dbSNP designationFLGStop-gainGAFLG:NM_002016:exon3:c.1708C>T:p.Q570X0.008——FLGStop-gainGAFLG:NM_002016:exon3:c.10225C>T:p.R3409X0.008——FLGStop-gainGTFLG:NM_002016:exon3:11120C>A:p.S3707X0.008——FLG2Stop-gainGTFLG2:NM_001014342:exon3:c.7130C>A:p.S2377X0.1330.29rs12568784FLG2Stop-lossCGFLG2:NM_001014342:exon3:c.7175G>C:p.S2392X0.0080.01rs150529054TCHHL1Stop-gainGATCHHL1:NM_001008536:exon3:c.880C>T:p.Q294X0.0170.01rs61749316Abbreviations: dbSNP, Single-Nucleotide Polymorphism Database; MAF, minor allele frequency. Open table in a new tab Abbreviations: dbSNP, Single-Nucleotide Polymorphism Database; MAF, minor allele frequency. This report is from the largest whole-exome sequencing study of African Americans with AD performed to date. Here we have reported results specific to the SFTP genes. We identified a few new null mutations, albeit the ones in FLG had low MAFs. The MAFs noted for S2377X (FLG2) and Q294X (TCHHL1) vary from the healthy subjects in the 1000 Genomes database (The 1000 Genomes Project Consortium, 2012The 1000 Genomes Project ConsortiumAn integrated map of genetic variation from 1,092 human genomes.Nature. 2012; 491: 56-65Crossref PubMed Scopus (5676) Google Scholar; Table 1). S2377X was seen about half as frequently in our cohort compared with that in the healthy 1000 Genomes African population; whereas Q294X, although more common in our cohort, still had a low MAF (i.e., MAF=0.017), suggesting that these variants may not be clinically important with respect to incident AD. Our findings are in agreement with those of Winge et al., 2011Winge M.C. Bilcha K.D. Lieden A. et al.Novel filaggrin mutation but no other loss-of-function variants found in Ethiopian patients with atopic dermatitis.Br J Dermatol. 2011; 165: 1074-1080Crossref PubMed Scopus (69) Google Scholar, who also failed to detect common FLG loss-of-function mutations in people of African ancestry with AD. Our study does have limitations in that we focused only on exon 3 stop-gain mutations in genes. We did not assess copy number variations. We also did not assay protein function. Another point to be noted is that as most African Americans have their origins in West Africa; our findings may not generalize to everyone with African ancestry. However, based on the experiences of others as well as those from our study, which is the largest whole-exome study of African Americans with AD, it seems unlikely that FLG stop-gain mutations have a prominent role with respect to incident AD in African Americans children (Winge et al., 2011Winge M.C. Bilcha K.D. Lieden A. et al.Novel filaggrin mutation but no other loss-of-function variants found in Ethiopian patients with atopic dermatitis.Br J Dermatol. 2011; 165: 1074-1080Crossref PubMed Scopus (81) Google Scholar; Thaswer-Esmail et al., 2014Thaswer-Esmail F. Jakasa I. Todd G. et al.South African amaXhosa patient with atopic dermatitis have decreased levels of filaggrin breakdown products but no loss-of-function mutations in filaggrin.J Allergy Clin Immunol. 2014; 133: 280-282Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The study was approved by the University of Pennsylvania Institutional Review Board. The study was conducted according to Declaration of Helsinki Principles. All participants provided written informed consent. This study was funded by R01-AR0056755 from the National Institute of Arthritis Musculoskeletal and Skin Diseases, a grant from Valeant Pharmaceuticals for the PEER study, and a grant from the Breast Cancer Research Foundation (Nathanson) for informatics analysis.