Atopic Dermatitis (AD) is a relapsing chronic pruritic inflammatory disease of skin in which no monogenic cause has been identified so far. Meanwhile Filaggrin (FLG) gene is considered as the most important gene associated with predisposition to the disease.One hundred and six patients with AD and 105 healthy individuals were enrolled in this study. Real time polymerase chain reaction was performed to determine frequencies of alleles and genotype in six variants of FLG gene.Atopic dermatitis (AD) is a relapsing chronic pruritic inflammatory disease of skin for which no monogenic cause has been identified so far. Meanwhile, the filaggrin (FLG) gene is considered as the most important gene associated with predisposition to the disease.One hundred and six patients with AD and 105 healthy individuals were enrolled in this study. Real time polymerase chain reaction was performed to determine frequencies of alleles and genotype in six variants of the FLG gene. The frequencies of allele A in variants of rs3126065, rs2786680, and rs1933063 as well as allele C in variant rs3814300 were 100%. There was no significant difference between allele frequencies in variants rs2485518 and rs3814299. The only genotypes in variants of rs3814299 and rs2485518 were GG and CC, respectively, with no significant difference between the patients and controls. This study demonstrated that there was no significant association between polymorphisms of FLG gene variants and AD.</p

Akbar Fotouhi

Behnaz Mahdaviani

Elham Farhadi

Mehdi Tavakoli

Mohammad Gharagozlou

Mojdeh Khaledi

Nasrin Behniafard

Nima Rezaei

Soheila Sotoudeh

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200 ACTA DERMATOVENEROLOGICA CROATICAFilaggrin Single Nucleotide Polymorphisms in Atopic DermatitisMojdeh Khaledi1,2, Akbar Fotouhi3, Elham Farhadi4,5, Behnaz Mahdaviani1,2, Soheila Sotoudeh6, Mehdi Tavakoli1,2, Nasrin Behniafard5, Mohammad Gharagozlou6, Nima Rezaei2,4 1Growth and Development Research Center, Tehran University of Medical Sciences; 2Research Center for Immunodeficiencies, Children’s Medical Center, Tehran Universi-ty of Medical Sciences; 3Department of Epidemiology and Biostatistics, School of Pub-lic Health, Tehran University of Medical Sciences; 4Molecular Immunology Research Center; and Department of Immunology, School of Medicine, Tehran University of Medical Sciences; 5Deparment of Hematology, School of Allied Medicine, Iran Univer-sity of Medical Sciences; 6Pediatrics Center of Excellence, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, IranActa Dermatovenerol Croat                     2014;22(3):200-204                       CLINICAL ARTICLECorresponding author: Nima Rezaei, MD, PhDChildren’s Medical Center Hospital Dr Quarib StKeshavarz BlvdTeheran 14194, Iranrezaei_nima@tums.ac.irReceived: January 25, 2014Accepted: May 10, 2014SUMMARY Atopic dermatitis (AD) is a relapsing chronic pruritic inflam-matory disease of skin for which no monogenic cause has been identified so far. Meanwhile, the filaggrin (FLG) gene is considered as the most im-portant gene associated with predisposition to the disease.One hundred and six patients with AD and 105 healthy individuals were enrolled in this study. Real time polymerase chain reaction was performed to determine frequencies of alleles and genotype in six variants of the FLG gene. The frequencies of allele A in variants of rs3126065, rs2786680, and rs1933063 as well as allele C in variant rs3814300 were 100%. There was no significant difference between allele frequencies in variants rs2485518 and rs3814299. The only genotypes in variants of rs3814299 and rs2485518 were GG and CC, respectively, with no significant differ-ence between the patients and controls. This study demonstrated that there was no significant association between polymorphisms of FLG gene variants and AD. KeY woRDS: atopic dermatitis, FLG, polymorphismINTRoDUCTIoNAtopic dermatitis (AD) is defined as a relapsing chronic pruritic inflammatory disease of the skin which varies in location and form of the involved skin as well as different ages of infancy, childhood, and adulthood (1). Its prevalence was reported to be 0.73% to 23% in different studies (2). While there is no countrywide study to show the exact prevalence of AD in Iran, some local studies showed a prevalence of about 15% in some areas (3). Although its prevalence varies worldwide (4), AD is a major health problem in both of developing and developed countries. It has a con-siderable impact on quality of life and constitutes a heavy economic burden (5). It is estimated that AD is 201ACTA DERMATOVENEROLOGICA CROATICAKhaledi et al. Acta Dermatovenerol CroatFLG gene polymorphisms in atopic dermatitis  2014;22(3):200-204responsible for $364 million to $3.8 billion in expen-ditures in the United States annually (6).Atopy and atopic march are strongly associated with AD (5). Atopic march refers to food allergies in in-fants followed by AD in first years of life and asthma, rhinitis, and other allergic disorders in later years (7).Although worldwide variation in AD could be sug-gestive of the influence of environmental factors (7), considerable genetic factors were introduced as an-other etiology (8,9). Indeed, AD is known as a multi-factorial disease (10).Recently, Genome Wide Association Studies (GWAS) were used in order to investigate genes sus-pected of causing AD (5) and the whole genome was scanned in order to determine genetic variants asso-ciated with AD (11). Genes with a role in skin barrier function were primary candidates in this regard (12).Epidermal Differentiation Complex (EDC), located on chromosome 1q21, is a group of genes involved in creation of stratum corneum as the most important component of the skin barrier. The filaggrin protein, a critical protein in this regard, is encoded by the FLG gene and located in EDC (2,7,9,10).Filaggrin has an important role in moisturizing the skin and protecting from penetrating allergens (2,4,7). Loss of intact skin barrier, which can be due to FLG gene problems, leads to allergen penetra-tion through the epidermal layer, which causes aller-gic response and immune sensitization to allergens (1,4,5,13).Recent studies have pointed to an association be-tween FLG mutations and AD (8), and it is now con-sidered the most important predisposing gene for AD (14,15).Several studies were performed to evaluate the role of Single Nucleotide Polymorphisms (SNPs), lo-cated on the FLG gene, in susceptibility to AD in dif-ferent parts of the world (13,16-22). Our study was designed to investigate, for the first time, the associa-tion between 6 functional SNPs in the FLG gene and AD among the Iranian population.  MeThoDSSubjectsWe conducted a case control study among 106 AD patients and 105 healthy subjects as controls. All of the patients were selected from patients with Cau-casian ethnic group older than 6 months who were referred to the Immunology Clinic of the Children’s Medical Center Hospital, the Pediatrics Center of Ex-cellence, Iran, and diagnosed with AD according to Hanifin and Rajka’s standard criteria in the Dermatol-ogy Clinic. Patients with mild AD were excluded from the study. Severity of their disease was evaluated by the Scoring of Atopic Dermatitis (SCORAD) index. The Position Alleles GenotypePatients (n=106) Controls (n=105)number numberrs2485518C 212 210G - -CC 106 105rs3126065A 212 210G - -AA 106 105rs2786680A 212 210T - -AA 106 105rs3814300C 212 210T - -CC 106 105rs3814299G 212 210A - -GG 106 105rs1933061A 212 210T - -AA 106 105Table 1. Frequencies of alleles and genotypes in different FLG positions in patients with atopic dermatitis and controls202 ACTA DERMATOVENEROLOGICA CROATICAeczematous skin with pruritis and discharge in the most affected areas of the body, depending on the patient’s age, was the phenotype of all patients with severe AD.The control group was selected from healthy in-dividuals without history of atopy who volunteered for this study. We attempted to select control subjects who were free from any kind of clinical atopy, such as rhinitis, asthma, and food allergy. Subjects with per-sonal or familial history of atopy were excluded from the study as well.This study was approved by the Ethics Commit-tee of Tehran University of Medical Sciences. Written informed consent was signed by the parents of all en-rolled patients.Genetic studyPeripheral venous blood sampling was done for all the enrolled subjects. Real time polymerase chain reaction (PCR) was performed to determine frequen-cies of alleles and genotypes in six variants of the FLG gene, namely rs2485518, rs1933061, rs3126065, rs2786680, rs3814300, and rs3814299. These variants were selected based on the aforementioned poly-morphisms in the FLG gene identified by research done in other countries. The aim in this study was to determine whether the same results would be found in Iranian patients.Statistical AnalysisAllele frequencies of each variant in both patient and control groups were determined by direct count-ing. The odds ratio (OR) and 95% confidence interval (CI) were calculated for each allele and genotype. Chi square test was used to compare differences between the two groups. A P value less than 5% was consid-ered significant. ReSUlTSAs shown in table 1, the frequency of allele A in variant rs3126065 was 100% in both of patients and controls; none of the individuals had a G allele in this variant. Allele A in variants rs2786680 and rs1933063 and allele C in variant rs3814300 had frequencies of 100% as well. The only genotype in variant rs3814299 was GG in both patients and controls. Also, in variant of rs2485518, the CC genotype was the dominant geno-type among both groups.There was no significant difference in genotype frequency in variants rs3814299 and rs2485518. DISCUSSIoNGenetic factors for allergic diseases in the lit-erature could be categorized into 4 groups. Group 1 consists of parts of the innate immune system rolled in response to environmental exposures; group 2 in-cludes genes related to skin barrier function; group 3 consists of factors affecting the adaptive immune system; and finally group 4 is comprised of  factors modifying the tissue response to inflammation (8).Although most prior studies focused on the in-nate and adaptive immune systems, recent studies were directed at genes related to epidermal barrier dysfunction in order to evaluate the pathophysiology of the disease (23).Skin barrier dysfunction is considered one of the most important etiologies of allergic diseases, includ-ing AD. Stratum corneum and its tight junctions in the epidermal layer of the skin have an important role in acting as skin barrier. Loricrin, involucrin, S100 gene family, small pro-line-rich proteins, peptidoglycan proteins, and filag-grin are some of the proteins coded in the EDC locus on chromosome 1q216, and their problems are as-sociated with skin barrier dysfunction. FLG has been widely studied in literature in this regard (12).Palmer et al. (16) reported for the first time that impaired epidermal barrier due to mutation on the FLG gene could predispose patients to AD. The study done by Sugiura et al., (24) who evaluat-ed some gene expressions in skin biopsy specimens of patients with, revealed down regulation of FLG mRNA in patients with AD compared to the controls. Many of these studies are restricted to studying those two null mutations of R501X (rs61816761) and 2282del4 investigated by Palmer et al., but full se-quencing of FLG has demonstrated polymorphisms with different frequencies in different races (12).These mutations have been shown to predispose the Caucasian population to AD; however, these mu-tations were not found in non-European populations such as Asian or African ethnic groups (2). This is in agreement with the findings of the study by Nomura et al. (25) who found two novel mutations in Japanese patients, 3321delA and S2554X, rather than R501X and 2282del4. Winge et al. (26) conducted a study among Ethiopian patients with AD, in which the FLG mutation of R501X, 2282del4, S3247X, R2447X was not seen. CoNClUSIoNOur study showed that all of the patients enrolled in this study had the same allele in variants rs1933061, Khaledi et al. Acta Dermatovenerol CroatFLG gene polymorphisms in atopic dermatitis  2014;22(3):200-204203ACTA DERMATOVENEROLOGICA CROATICArs3126065, rs2786680, rs3814300, rs2485518, and rs3814299. Our findings regarding the variants listed suggest that these variants should not be considered SNPs among the Iranian population. However, more studies with a larger sample size could increase the power of this result. ACKNowleDGeMeNTThis study was a part of medical student thesis, which was supported by a grant from Tehran Univer-sity of Medical Sciences and Health Services (89-04-80-12136). References1.  Buggiani G, Ricceri F, Lotti T. Atopic dermatitis. Dermatol Ther 2008;21:96-100.2.  Enomoto H, Hirata K, Otsuka K, Kawai T, Takahashi T, Hirota T, et al. Filaggrin null mutations are asso-ciated with atopic dermatitis and elevated levels of IgE in the Japanese population: a family and case-control study. J Hum Gen 2008;53:615-21.3.  Ghaderi R, Tabiee S, Peyrovi S, Jafari Pour M. Pre-valence of atopic dermatitis and its risk factors in 2-5 years old children at kindergartens of Birjand city (2008). Journal of Birjand University of Medi-cal Sciences 2012;19:286-934.  Deckers IA, McLean S, Linssen S, Mommers M, van Schayck CP, Sheikh A. Investigating international time trends in the incidence and prevalence of atopic eczema 1990-2010: a systematic review of epidemiological studies. PloS one 2012;7:e39803.5.  Boguniewicz M, Leung DY. Atopic dermatitis: a di-sease of altered skin barrier and immune dysregu-lation. Immunol Rev 2011;242:233-46.6.  Irvine AD, McLean WH, Leung DY. Filaggrin muta-tions associated with skin and allergic diseases. N Engl J Med 2011;365:1315-27.7.  van den Oord RA, Sheikh A. Filaggrin gene defects and risk of developing allergic sensitisation and allergic disorders: systematic review and meta-analysis. BMJ 2009;339:b2433.8.  Holloway JW, Yang IA, Holgate ST. Genetics of al-lergic disease. 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Filaggrin Single Nucleotide Polymorphisms in Atopic Dermatitis

Filaggrin Single Nucleotide Polymorphisms in Atopic Dermatitis

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