South African amaXhosa patients with atopic dermatitis have decreased levels of filaggrin breakdown products but no loss-of-function mutations in filaggrin
Authors: Fatemah Thawer-Esmail, Ivone Jakasa, Gail Todd, Yaran Wen, Sara J. Brown, Karin Kroboth, Linda E. Campbell, Grainne M. O'Regan, W.H. Irwin McLean, Alan D. Irvine, Sanja Kezic, Aileen Sandilands
Journal: Journal of Allergy and Clinical Immunology (2013)

Loss-of-function (LOF) mutations in the filaggrin gene (FLG) are the strongest known genetic risk factors for atopic dermatitis (AD). The genetic architecture of FLG mutations is well established in European, Japanese, and selected Chinese populations, but their contribution to AD in African populations is not well understood. The only data on FLG mutations in Africans come from a recent study conducted in Ethiopia1Winge M.C. Bilcha K.D. Liedén A. Shibeshi D. Sandilands A. Wahlgren C.-F. 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 (80) Google Scholar that studied 103 patients with AD, 7 patients with ichthyosis vulgaris (IV), and 103 healthy controls. This study identified only a single novel mutation (a 2-bp deletion, 632del2), by direct sequencing of FLG in a patient with AD. To investigate the role of filaggrin in the etiology of AD in South Africa, we studied 69 children with AD from the amaXhosa community along with 81 age-, ethnic- and sex-matched controls, with no history of AD. The patients (n = 69) and controls (n = 81) were recruited from tertiary referral AD clinics in Cape Town. Clinical and demographic characteristics of control subjects and patients with AD are outlined in Table I. The study was conducted in accordance with the Helsinki Declaration and was approved by the Human Research Ethics Committee of the Faculty of Health Sciences of the University of Cape Town. Written consent/ascent in the amaXhosa language was obtained from the patients or their parents.Table IClinical and demographic characteristics of control subjects and patients with ADNAge (y), median (range)Male gender, n (%)NESS, median (range)IgE n, median (range)IchthyosisPerifollicular hyperkeratosisPalmar hyper linearityCTRL815 (1-31)43 (53)—————Patients with AD699 (4-29)36 (52)10 (4-15)57, 2114 (12-10500)68 of 6921 of 6960 of 69CTRL, Healthy controls; NESS, Nottingham Eczema Severity Score.*AD was diagnosed by an experienced dermatologist by using the UK diagnostic criteria. Open table in a new tab CTRL, Healthy controls; NESS, Nottingham Eczema Severity Score. *AD was diagnosed by an experienced dermatologist by using the UK diagnostic criteria. The entire coding sequence of the FLG gene was directly sequenced (as described previously2Sandilands A. Terron-Kwiatkowski A. Hull P.R. O'Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (509) Google Scholar) in 31 patients with AD with prominent features of IV, that is, those who should most likely have FLG mutations. Sequencing of PCR products was performed by a core facility (DNA Sequencing and Services, University of Dundee, Dundee, United Kingdom) according to our standard operating procedures. The entire collection was additionally typed for the previously known FLG mutations R501X, 2282del4, R2447X, and S3247X by using custom-made Taqman allelic discrimination assays.2Sandilands A. Terron-Kwiatkowski A. Hull P.R. O'Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (509) Google Scholar Although the primers used for the amplification of the FLG gene were originally optimized for the sequencing of European populations,2Sandilands A. Terron-Kwiatkowski A. Hull P.R. O'Regan G.M. Clayton T.H. Watson R.M. et al.Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema.Nat Genet. 2007; 39: 650-654Crossref PubMed Scopus (509) Google Scholar in the 31 amaXhosa patients with AD who were sequenced, we identified 124 mutations (synonymous and nonsynonymous) throughout exon 3 of the FLG gene by using these methods. Identification of these silent and nonpathogenic missense mutations in the FLG gene indicates minimal allele dropout using these primer sets. None of these identified mutations was predicted to lead to loss of filaggrin at the protein level. Screening of the entire collection of patients for the FLG mutations R501X, 2282del4, R2447X, and S3247X showed that all samples were wild type for these mutations. In addition to gene sequencing, in all patients with AD and controls, we determined the concentrations of filaggrin breakdown products in the stratum corneum (SC). Filaggrin is degraded in the later stages of epidermal differentiation into free amino acids (FAA) and their derivatives; a major proportion of the total SC FAA (70% to 100%) is derived from filaggrin.3Harding C.R. Aho S. Bosko C.A. Filaggrin - revisited.Int J Cosmet Sci. 2013; 35: 412-423Crossref PubMed Scopus (60) Google Scholar The most common amino acid residues in filaggrin repeats are basic amino acids such as histidine (413 of 4061 residues; 10.17%) and arginine (440 of 4061 residues; 10.83%) and the polar residue glutamine (367 of 4061; 9.04%) (see Fig E1 in this article's Online Repository at www.jacionline.org). Histidine is enzymatically deaminated to trans-urocanic acid (trans-UCA).3Harding C.R. Aho S. Bosko C.A. Filaggrin - revisited.Int J Cosmet Sci. 2013; 35: 412-423Crossref PubMed Scopus (60) Google Scholar Trans-UCA, which is converted to cis-UCA on ultraviolet irradiation, functions as a major chromophore and exerts immunomodulatory effects in the skin.3Harding C.R. Aho S. Bosko C.A. Filaggrin - revisited.Int J Cosmet Sci. 2013; 35: 412-423Crossref PubMed Scopus (60) Google Scholar, 4McAleer M.A. Irvine A.D. The multifunctional role of filaggrin in allergic skin disease.J Allergy Clin Immunol. 2013; 131: 280-291Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar UCA maintains an acidic pH in the skin, which is crucial for the optimal function of several enzymes in the SC and antimicrobial defence.3Harding C.R. Aho S. Bosko C.A. Filaggrin - revisited.Int J Cosmet Sci. 2013; 35: 412-423Crossref PubMed Scopus (60) Google Scholar, 4McAleer M.A. Irvine A.D. The multifunctional role of filaggrin in allergic skin disease.J Allergy Clin Immunol. 2013; 131: 280-291Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar Another abundant amino acid glutamine is further converted into pyrrolidone-5-carboxylic acid (PCA). PCA is highly hygroscopic and is one of the major components of the natural moisturizing factor, thus providing a humectant effect by retaining water in the SC.3Harding C.R. Aho S. Bosko C.A. Filaggrin - revisited.Int J Cosmet Sci. 2013; 35: 412-423Crossref PubMed Scopus (60) Google Scholar, 4McAleer M.A. Irvine A.D. The multifunctional role of filaggrin in allergic skin disease.J Allergy Clin Immunol. 2013; 131: 280-291Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar Filaggrin degradation products thus have multiple functions. FLG mutations lead to reduced levels of filaggrin degradation products in the SC in a dose-dependent fashion.5Kezic S. O'Regan G.M. Yau N. Sandilands A. Chen H. Campbell L.E. et al.Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity.Allergy. 2011; 66: 934-940Crossref PubMed Scopus (208) Google Scholar It has been shown that moderate-to-severe AD also has an effect on SC filaggrin expression6Howell M.D. Kim B.E. Gao P. Grant A.V. Boguniewicz M. DeBenedetto A. et al.Cytokine modulation of atopic dermatitis filaggrin skin expression.J Allergy Clin Immunol. 2007; 124: R7-12Abstract Full Text Full Text PDF Scopus (323) Google Scholar as well as on the levels of filaggrin degradation products5Kezic S. O'Regan G.M. Yau N. Sandilands A. Chen H. Campbell L.E. et al.Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity.Allergy. 2011; 66: 934-940Crossref PubMed Scopus (208) Google Scholar possibly due to the systemic TH2 immune response.6Howell M.D. Kim B.E. Gao P. Grant A.V. Boguniewicz M. DeBenedetto A. et al.Cytokine modulation of atopic dermatitis filaggrin skin expression.J Allergy Clin Immunol. 2007; 124: R7-12Abstract Full Text Full Text PDF Scopus (323) Google Scholar In the present study, the levels of both PCA and UCA and their sum were significantly decreased in the SC of patients with AD than in control subjects (Fig 1; see Table E1 in this article's Online Repository at www.jacionline.org). The magnitude of reduction between cases and controls in this study was approximately 20%. Similar results were obtained when comparing total FAA content as well as total FAA including their derivatives, PCA, UCA, citrulline, and ornithine (Fig 1; Table E1). While FLG mutations are the major determinants of the level of filaggrin breakdown products in the SC, it has been previously shown that their levels are significantly reduced in European populations both in nonlesional skin of patients with AD with FLG mutations and in patients without FLG mutations.5Kezic S. O'Regan G.M. Yau N. Sandilands A. Chen H. Campbell L.E. et al.Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity.Allergy. 2011; 66: 934-940Crossref PubMed Scopus (208) Google Scholar In this study, we have replicated these filaggrin breakdown product findings in patients with AD without FLG mutations in an African population. This is consistent with the in vitro findings of Howell et al6Howell M.D. Kim B.E. Gao P. Grant A.V. Boguniewicz M. DeBenedetto A. et al.Cytokine modulation of atopic dermatitis filaggrin skin expression.J Allergy Clin Immunol. 2007; 124: R7-12Abstract Full Text Full Text PDF Scopus (323) Google Scholar and Pellerin et al7Pellerin L. Henry J. Hsu C.-Y. Balica S. Jean-Decoster C. Méchin M.-C. et al.Defects of 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 (173) Google Scholar and highlights the fact that there is an interplay between the skin barrier and a systemic immunologic process, with systemic TH2 inflammation causing a decrease in SC filaggrin expression. The SC profiles of filaggrin breakdown products are highly informative in this African population because they provide a second look, in addition to direct sequencing, for FLG mutations. In our study, we demonstrate that the diminution of filaggrin breakdown products is consistent with what we have seen in European patients with AD who are wild type for FLG mutations. The magnitude of this reduction is much less than that seen in patients with AD with FLG mutations, even in African patients with clinical findings consistent with IV who could be expected to have FLG mutations. In Europeans with a single FLG LOF mutation, expression of these products is reduced by approximately 50%.5Kezic S. O'Regan G.M. Yau N. Sandilands A. Chen H. Campbell L.E. et al.Levels of filaggrin degradation products are influenced by both filaggrin genotype and atopic dermatitis severity.Allergy. 2011; 66: 934-940Crossref PubMed Scopus (208) Google Scholar We here provide strong and complementary evidence for an absence of FLG mutations in this African population through 2 analytical methodologies. Given that FLG mutations are well established as the strongest and most important risk factor for AD in European, Japanese, and Chinese populations, this work clearly implies significant genetic heterogeneity between AD in the African population and AD in these populations. The genetics of AD is not well studied in African populations; in addition to the Ethiopian study referenced earlier, 2 studies in African American populations have found low frequencies of European-derived FLG mutations,8Gao P.-S. Rafaels N.M. Hand T. Murray T. Boguniewicz M. Hata T. et al.Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum.J Allergy Clin Immunol. 2009; 124: 507-513Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 9Margolis D.J. Apter A.J. Gupta J. Hoffstad O. Papadopoulos M. Campbell L.E. et al.The persistence of atopic dermatitis and filaggrin (FLG) mutations in a US longitudinal cohort.J Allergy Clin Immunol. 2012; 130: 912-917Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar presumably due to European population admixture.10Kidd J.M. Gravel S. Byrnes J. Moreno-Estrada A. Musharoff S. Bryc K. et al.Population genetic inference from personal genome data: impact of ancestry and admixture on human genomic variation.Am J Hum Genet. 2012; 91: 660-671Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar While it is tempting to speculate, on the basis of this work, that there could be a major (non-FLG) African-specific gene for IV and/or AD, African populations will require specific studies and dedicated collections to disclose African population-specific major genetic risks for AD. In conclusion, FLG LOF mutations are not a significant contributor to AD in the amaXhosa population. When combined with previous findings in the Ethiopian population, the contribution of these mutations to AD in Africans seems to be at great variance with their major role in European and Asian populations. Further work is required in African populations to better understand the genetic basis of AD in these populations. We are very grateful to the patients and their families for participating in this study. We appreciate support of the COST Action BM0903 (Skin Barrier in Atopic Diseases, SKINBAD). We also appreciate the efforts of Sister Julie Esterhuizen for overall patient recruitment, organization, and drawing bloods and Sister Anthea Ndyenga and Mr Jackson Tyodi for helping with translating consent forms and interviewing patients, respectively. We are grateful to Maia Lesosky for statistical input when planning this work. Table E1The levels of filaggrin degradation products and differences between healthy controls and patients with AD with corresponding 95% CIsFilaggrin degradation product∗PCA and UCA have been determined by HPLC from the SC samples collected by adhesive tape strips as described elsewhere (Dapic I, Jakasa I, Yau N, Kezic S, Kammeyer A. Evaluation of an HPLC method for the determination of natural moisturizing factors in the human stratum corneum. Anal Lett 2013;46:2134-44.) FAA and their derivatives from the SC tapes were analyzed by using ultra performance liquid chromatography-tandem mass spectrometry. The concentrations are expressed as mmol of each component/g of protein to compensate for variable harvest of the SC. (mmol/g protein)Median (range)Difference (95% CI)P valueCTRLADPCA0.60 (0.11-1.10)0.49 (0.10-1.18)−0.11 (−0.181 to −0.068)<.0001UCA0.19 (0.03-0.33)0.15 (0.03-0.55)−0.043 (−0.063 to −0.017).0006FAA2.32 (0.51-3.70)1.82 (0.62-3.67)−0.51 (−0.60 to −0.18).0005FAA + derivatives3.44 (0.72-5.40)2.82 (0.98-5.34)−0.62 (−0.91 to −0.29).0002∗ PCA and UCA have been determined by HPLC from the SC samples collected by adhesive tape strips as described elsewhere (Dapic I, Jakasa I, Yau N, Kezic S, Kammeyer A. Evaluation of an HPLC method for the determination of natural moisturizing factors in the human stratum corneum. Anal Lett 2013;46:2134-44.) FAA and their derivatives from the SC tapes were analyzed by using ultra performance liquid chromatography-tandem mass spectrometry. The concentrations are expressed as mmol of each component/g of protein to compensate for variable harvest of the SC. Open table in a new tab