Long non-coding RNA polymorphisms influence susceptibility to endemic pemphigus foliaceus

1. Long non-coding RNA polymorphisms influence susceptibility to endemic pemphigus foliaceus S.C. Lobo-Alves1, D.G. Augusto1,2, W.C.S. Magalhães3,4,E. Tarazona-Santos4,M.F. Lima-Costa5, M.L. Barreto6, B.L. Horta7, R.C. de Almeida8, M.L. Petzl-Erler1 1Laboratório de Genética Molecular Humana, Departamento de Genética, Universidade Federal do Paraná, Curitiba, Brazil; 2Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz, Ilhéus, Brazil; 3Núcleo de Ensino e Pesquisa - NEP, Instituto Mário Penna, Belo Horizonte, Minas Gerais, Brazil; 4Instituto de Ciências Biológicas, Departamento de Biologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 5Instituto de Pesquisa Rene Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil; 6Instituto de Saúde Coletiva, Universidade Federal da Bahia, Salvador, BA, Brazil; 7Programa de Pós-Graduação em Epidemiologia, Universidade Federal de Pelotas, Pelotas, RS, Brazil; 8Department of Biomedical Data Sciences, Section Molecular Epidemiology, Leiden University Medical Center, Leiden, The Netherlands.] British Journal of Dermatology. DOI: 10.111/bjd.17640

2. Sara Cristina Lobo-Alves FirstAuthor

3. Introduction What’s already known? • The multifactorial autoimmune blistering skin disease pemphigus foliaceus presents a genetic susceptibility component that is not fully understood. • Although pemphigus foliaceus is rare worldwide, it reaches the prevalence of 1.5% to 3% in some regions of Brazil - among the highest ever reported for an autoimmune disease. This endemic form is known as fogoselvagem. • LncRNA polymorphisms have been associated with some complex diseases but were not yet studied in any form of pemphigus.

4. Objective • To investigate whether genetic variants in lncRNA genes are associated with differential susceptibility to endemic PF.

5. Methods • GENOME WIDE DATA • 6,910 subjects • (229 PF patients + 6,681 controls) • Search for SNPs present in lncRNA SNP database • Data integration • lncRNASNP DATABASE • http://bioinfo.life.hust.edu.cn/lncRNASNP • List of SNPs localized in lncRNA genes • IN SILICO ANALYSIS • Secondary structure prediction of PF-associated lncRNAs for each SNP allele • RNAfold Web Server – Vienna Package • Impact prediction of SNPs on • miRNA-lncRNA interaction • lncRNASNP2 database • Functional annotation of associated SNPs • RegulomeDb Database, Haploreg v.4.1 tool • and GTEX portal • ASSOCIATION ANALYSIS • Logistic regression P < 0.001 • Additive model • Adjusted for population structure as covariant

6. Results Association-study results: • SNP rs7144332 (OR = 1.63, P = 2.8 x 10-6), located in the lncRNA gene AL110292.1 is strongly associated with pemphigus foliaceus. • Other five SNPs show suggestive association (P < 0.001).

7. Results In silico analysis results: • Different secondary structures were predicted for each variant of the rs7195536 in AC009121.1, rs6942557 in linc01176 and rs1542604 in AC133785.1. • The rs1542604 T, rs6942557 C, and rs17774133 Tvariants were predicted tocreate a new miRNA binding site in their respectively lncRNAs.

8. Results Functional annotation results: • According RegulomeDB database and the Haploreg v. 4.1 tool: • SNPs rs7144332, rs6095016, rs7195536, rs1542604, and rs6942557 alter motifs for transcription factors. • SNPs rs7195536, rs1542604, rs6942557, and rs17774133 overlap with enhancers that are important in several tissues. • According to the GTEx portal (www.gtexportal.org) • SNP rs7195536 located in lncRNA AC009121.1 is an eQTL for three nearby genes RMI2, RP11485G7.5 and CTD-3088G3.4.

9. Discussion There are several hypothesis for explain genetic association between lncRNA SNPs and diseases: • The SNP changes the lncRNA secondary structure • in silico analysis predicted that the structure of three lncRNAs differed according to the PF associated single nucleotide variants. • The SNP changes motifs of interaction with other molecules, as miRNAs and proteins. • in silico analysis suggested that rs1542604, rs6942557, and rs17774133 can create or disrupt binding sites for miRNAs. • In pancreatic cancer the associated rs11655237 G>A SNP in LINC00673 creates a miR-1231 binding site that affects the lncRNA levels in a variant-specific manner (Zheng et al., 2016). • In celiac disease the associated rs917997 T variant alters lnc13 affinity to hnRNPD (Castellanos-Rubio et al., 2016).

10. Discussion III. The SNP changes regulatory regions, as promoters and enhancers, and influences lncRNA and/or nearby genes expression. Functional annotation analysis revealed that the SNP rs7195536 located in lncRNA AC009121.1 is an eQTL for three nearby genes (RMI2, RP11485G7.5 and CTD-3088G3.4). The cervical cancer- associated variant rs920778 T located in an intronic HOTAIR enhancer regulates its expression(Guo et al, 2016).

11. ConclusionsWhat does this study add? • We showed, for the first time, that variation in lncRNA genes may influence pemphigus foliaceus pathogenesis. • Genetic variation of lncRNA may influence susceptibility to pemphigus foliaceus via its effect on lncRNA structure, on transcription of nearby genes, and on microRNA-lncRNA interactions. • This work adds valuable information to the growing research about susceptibility and pathogenesis of pemphigus and suggests possible targets for functional validation.

13. The research team Danillo G. Augusto Rodrigo Coutinho de Almeida Sara Cristina Lobo-Alves Maria-Luiza Petzl-Erler Wagner. C.S. Magalhães,Eduardo Tarazona-Santos – UFMG, Belo Horizonte Maria Fernanda Lima-Costa – Fundação Oswaldo Cruz, Belo Horizonte Mauricio L. Barreto – UFBA, Salvador Bernardo L. Horta – UFPEL, Pelotas

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