SNP 668C (−44) alters a NF-κB1 putative binding site in non-coding strand of human β-defensin 1 (DEFB1) and is associated with lepromatous leprosy

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Abstract

Leprosy is an infectious disease caused by Mycobacterium leprae. The peptide human β-defensin 1 is an antimicrobial effector of innate epithelial immunity. A study was done on the association of three single nucleotide polymorphisms (SNPs) in the β-defensin 1 gene (DEFB1) – 668 C/G (−44 C/G or rs1800972; in 5′ UTR), 692 A/G (−20 A/G or rs11362; in 5′ UTR) and A1836G (rs1800971; in 3′ UTR) – with leprosy susceptibility per se and clinical leprosy variants. The SNPs were genotyped by real-time polymerase chain reaction (rt-PCR) and PCR-restriction fragment length polymorphisms. Subjects were of Mexican mestizo ethnicity from Sinaloa state, México. Analysis was done on borderline leprosy, lepromatous leprosy (L-lep) and indeterminate leprosy subgroups compared with healthy controls. Results: The genotypes associated with L-lep and no other leprosy subgroup after Bonferroni correction were those that contain 668C in a dominant model (OR = 3.06, 95% CI 1.47–6.4, p = 0.024). Estimated haplotype CGA is over-represented in L-lep (p = 0.009; OR = 2.25, 1.23–4.03). Five NF-κB1 putative binding sites (NPBSs) were identified with JASPAR software in non-coding strand spanning the 5′ UTR and intron 1 of DEFB1, including one which is altered when SNP 668C is present. SNP 668C probably abrogates NF-κB-dependent DEFB1 upregulation leading to L-lep variant.

Introduction

Leprosy (or Hansen's disease) is a chronic granulomatous infectious disease caused by the obligate intracellular organism Mycobacterium leprae. The disease affects the skin and peripheral nerves, and complications secondary to the neuropathy can result in deformity and disability (Walker and Lockwood, 2006). Reported worldwide leprosy incidence was as high as 265,661 new cases in 2006 and 254,525 new cases in 2007 (WHO, 2007, WHO, 2008). Incidence in Mexico is low (0.25/100,000), although Sinaloa state is an exception with a rate almost eightfold higher (1.91/100,000) than the national rate (Secretaría de Salubridad y Asistencia, 2005).

Host immune response largely determines the disease's clinical spectrum. Patients with the tuberculoid (T-lep) or paucibacillary form are relatively resistant to the pathogen, exhibiting localized infection and lesions characterized by expression of type 1 (Th1) T-helper lymphocyte-related cytokines (cell-mediated immunity). Patients infected with the lepromatous (L-lep) or multibacillary form are more susceptible to the pathogen and exhibit systemic infection and lesions expressing Th2 cytokines (humoral immunity) (Krutzik et al., 2005). Dimorphic or borderline leprosy (B-lep) is a phenotype intermediate between the T-lep and L-lep forms (Ridley and Jopling, 1966).

Despite the variations in M. leprae genome (Shin et al., 2000, Groathouse et al., 2004, Monot et al., 2005, Matsuoka et al., 2005), it is exceptionally well conserved and highly clonal (Monot et al., 2005); indeed, it exhibits genome downsizing and reductive evolution (Cole et al., 2001). It is improbable, therefore, that observed differences in human leprosy susceptibility or severity result from genetic variability in M. leprae.

Evidence does suggest that leprosy susceptibility per se is influenced by host genetic variants, mainly in locus 20p12 (Tosh et al., 2002) and the genes TNF-α (Shaw et al., 2001), PARK2 and PACRG (Mira et al., 2004, Alcais et al., 2005). Leprosy type is influenced by loci 10p13 (Siddiqui et al., 2001) and 21q22 (Wallace et al., 2004), as well as genes MHC (de Vries et al., 1976); VDR (Roy et al., 1999); SLC11A1, MICA, COL3A1, CTLA4, C4B, HSPA1, TAP2, LTA (Fitness et al., 2002); LAMA2 (Wibawa et al., 2002); MBL2 (de Messias-Reason et al., 2007); IL12B (Ohyama et al., 2005, Morahan et al., 2007); and TLR1 (Misch et al., 2008, Johnson et al., 2007). Fitness et al. (2002) provided an extensive review of genetic susceptibility to leprosy.

Antimicrobial peptides (AMPs) are small (∼12–80 aa), evolutively conserved molecules expressed mainly in epithelia and leukocytes (Ganz, 2003, Selsted and Ouellette, 2005). The AMPs produced in skin are dermcidin, psoriasin, LL-37 and β-defensins (Schroeder and Harder, 2006, Yamasaki and Gallo, 2008). The β-defensin family consists of cationic amphipathic peptides of ∼36–48 residues with a typical α/β cysteine-stabilized scaffold (Torres and Kuchel, 2004). Pazgier et al. (2006) have provided a review of human β-defensins. Of the skin AMPs, expressions of β-defensins are considered an elemental part of initial host immune response since they act as antimicrobials and play a vital role as “microchemokines” by regulating adaptive immunity responses (Yang et al., 1999, Yang et al., 2007) in a Toll-like receptor (TLR)-dependent (Froy, 2005, Kumar et al., 2006) and TLR-independent recognition of pathogen-associated molecular patterns (Froy, 2005, Voss et al., 2006). AMPs are clearly important in skin immunity (Schroeder and Harder, 2006, Schittek et al., 2008) and particularly in the resistance to Mycobacterium tuberculosis (Liu et al., 2007, Méndez Samperio, 2008, Rivas-Santiago et al., 2008), but no research has been done to date on the role of polymorphisms in an AMP gene and leprosy susceptibility.

Unlike AMPs such as hBD-2 and LL-37, which are only overexpressed in inflammatory processes (Ong et al., 2002), human β-defensin 1 (hBD-1) is constitutively expressed in skin keratinocytes (Harder et al., 2004) and is considered the major AMP in human epithelia (Zhu et al., 2003), one of the main infection sites of M. leprae (Walker and Lockwood, 2006). It is also active against and upregulated by mycobacteria (Fattorini et al., 2004, Zhu et al., 2003). We hypothesize that SNPs with functional consequences in the DEFB1 gene could lead to leprosy susceptibility or a better/worse immunological response to M. leprae and therefore may affect clinical outcome.

The DEFB1 gene is located at the 8p23.2–p23.1 locus (MIM *602056) and spans ∼7.3 kb (SNP Browser 2.0, Applied Biosystems, Foster City, CA, USA). We decided to study three of the SNPs reported in the DEFB1 gene (ENSG00000164825, www.ensembl.org) because they are in UTRs, and variants in these regions can determine gene expression by influencing mRNA stability and translational efficiency (Hughes, 2006) and they may have functional consequences in response to M. leprae: 668 C/G (rs1800972, also known as −44 C/G according to the initiation of codon-based nomenclature) and 692 A/G (rs11362 or −20 A/G) lies in exon 1 and within the 5′ UTR (Selsted and Ouellette, 2005, Sun et al., 2006) and 1836 A/G (rs1800971) in exon 2 within the 3′ UTR (Jurevic et al., 2002).

The present study aim was to analyze the association of the above mentioned DEFB1 polymorphisms with leprosy per se and/or with leprosy clinical variants in a population from Sinaloa state, Mexico. The genotypes that contain 668C allele and haplotype CGA were found to be associated with L-lep, and in the 5′ UTR of DEFB1 and unique intron, five NF-κB1 (p50/p105) putative binding sites were identified in non-coding DEFB1 strand, including one which is altered by polymorphism 668C.

Section snippets

Subjects and DNA extraction

Peripheral blood samples were obtained from 75 patients diagnosed according to Ridley and Jopling's criteria (Ridley and Jopling, 1966) and who were admitted to the Genomic Medicine Center, Culiacán General Hospital, México. Patients were recruited on a consecutive basis and met five criteria: (i) patient, parents and grandparents born in Sinaloa; (ii) unrelated; (iii) regular clinical follow-up; (iv) under treatment; (v) Mexican mestizo ethnicity. Control samples were obtained from

Results

A total of 75 leprosy patients (I-lep, n = 11; B-lep, n = 18; L-lep, n = 46) were recruited, and 151 unrelated otherwise healthy individuals were recruited as controls. Tuberculoid patients were excluded from analysis because they were only few (n = 4). Leprosy patient age range was 24–88 (mean 51.42 ± 16.48) years with a male–female ratio of 1:0.68, whereas for controls the age range was 18–70 (mean 26.78 ± 9.41) years and the sex ratio was 1:0.96. Analysis was done on B-lep, I-lep, L-lep, B-lep + I-lep and

Discussion

Human β-defensin 1 is constitutively expressed in keratinocytes, and therefore may function to limit M. leprae infection in these primary host defense cells since these mycobacteria exhibit cutaneous tropism. The in vitro antimicrobial activity of hBD-1 has been proven against Gram-positive and Gram-negative bacteria (Schroeder and Harder, 2006), fungi (Pazgier et al., 2006), viruses (Sun et al., 2006) and M. tuberculosis (Fattorini et al., 2004), but antimicrobial activity against M. leprae

Acknowledgements

This study was partially supported by a Consejo Nacional de Ciencia y Tecnología-Gobierno del Estado de Sinaloa grant to J.S.V.F. (SIN-2005-CO1-02, FOMIX-CONACYT). E.P.M. was a Research Fellow at the Instituto Mexicano del Seguro Social (IMSS, 99145989), received a National Council of Science and Technology Ph.D. scholarship (CONACYT, México, 176803) and was a recipient of a Wellcome Trust Sanger Institute Advanced Course complete support. The authors thank Dr. Iribe Martínez and Dr. Castro

References (90)

  • M. Matsuoka et al.

    Polymorphism in the rpoT gene in Mycobacterium leprae isolates obtained from Latin American countries and its possible correlation with the spread of leprosy

    FEBS Microbiol. Lett.

    (2005)
  • P. Méndez Samperio

    Role of antimicrobial peptides in host defense against mycobacterial infections

    Peptides

    (2008)
  • H. Sherman et al.

    Expression of human β-defensin 1 is regulated via c-Myc and the biological clock

    Mol. Immunol.

    (2008)
  • A.M. Torres et al.

    The beta-defensin-fold family of polypeptides

    Toxicon

    (2004)
  • E. Voss et al.

    NOD2/CARD15 mediates induction of the antimicrobial peptide human beta-defensin-2

    J. Biol. Chem.

    (2006)
  • H. Abdi

    The Bonferroni and Sidák corrections for multiple comparisons

  • D. Altschuler et al.

    Genetic mapping in human disease

    Science

    (2008)
  • P.Y. Bochud et al.

    Cutting edge: a Toll-like receptor 2 polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling

    J Immunol.

    (2003)
  • L. Braida et al.

    A single-nucleotide polymorphism in the human beta-defensin 1 gene is associated with HIV-1 infection in Italian children

    AIDS

    (2004)
  • J.C. Bryne et al.

    JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update

    Nucleic Acids Res.

    (2008)
  • R. Cagliani et al.

    The signature of long-standing balancing selection at the human defensin beta-1 promoter

    Genome Biol.

    (2008)
  • R.M. Cerda-Flores et al.

    Genetic admixture in three Mexican Mestizo populations based on D1S80 and HLA-DQA1 loci

    Am. J. Hum. Biol.

    (2002)
  • Q.X. Chen et al.

    Genomic variations within DEFB1 are associated with the susceptibility to and the fatal outcome of severe sepsis in Chinese Han population

    Genes Immun.

    (2007)
  • W.O. Chung et al.

    Innate immune response of oral and foreskin keratinocytes: utilization of different signaling pathways by various bacterial species

    Infect. Immun.

    (2004)
  • W.O. Chung et al.

    Differential utilization of nuclear factor-kappaB signaling pathways for gingival epithelial cell responses to oral commensal and pathogenic bacteria

    Oral Microbiol. Immunol.

    (2008)
  • S.T. Cole et al.

    Massive gene decay in the leprosy bacillus

    Nature

    (2001)
  • I.J. de Messias-Reason et al.

    The association between mannan-binding lectin gene polymorphism and clinical leprosy: new insight into an old paradigm

    J. Infect. Dis.

    (2007)
  • M. Del Pero et al.

    Beta-defensin 1 gene variability among non-human primates

    Immunogenetics

    (2002)
  • L. Excoffier et al.

    Arlequin (version 3.0): an integrated software package for population genetics data analysis

    Evol. Bioinf.

    (2005)
  • L. Excoffier et al.

    Maximum-likelihood estimation of molecular haplotype frequencies in a diploid population

    Mol. Biol. Evol.

    (1995)
  • X.M. Fang et al.

    Differential expression of alpha- and beta-defensins in human peripheral blood

    Eur. J. Clin. Invest.

    (2003)
  • J. Fitness et al.

    Genetics of susceptibility to leprosy

    Genes Immun.

    (2002)
  • O. Froy

    Regulation of mammalian defensin expression by Toll-like receptor-dependent and independent signalling pathways

    Cell. Microbiol.

    (2005)
  • T. Ganz

    Defensins: antimicrobial peptides of innate immunity

    Nat. Rev. Immunol.

    (2003)
  • N.A. Groathouse et al.

    Multiple polymorphic loci for molecular typing of strains of Mycobacterium leprae

    J. Clin. Microbiol.

    (2004)
  • S.W. Guo et al.

    Performing the exact test of Hardy–Weinberg proportion for multiple alleles

    Biometrics

    (1992)
  • S. Gustincich et al.

    A fast method for high-quality genomic DNA extraction from whole human blood

    Biotechniques

    (1991)
  • Instituto Nacional de Estadística, Geografía e Informática (INEGI), censo 2000....
  • C.M. Johnson et al.

    Cutting edge: a common polymorphism impairs cell surface trafficking and functional responses of TLR1 but protects against leprosy

    J. Immunol.

    (2007)
  • R.J. Jurevic et al.

    Single-nucleotide polymorphisms and haplotype analysis in beta-defensin genes in different ethnic populations

    Genet. Test.

    (2002)
  • R.J. Jurevic et al.

    Single-nucleotide polymorphisms (SNPs) in human beta-defensin 1: high-throughput SNP assays and association with Candida carriage in type I diabetics and nondiabetic controls

    J. Clin. Microbiol.

    (2003)
  • S.R. Krutzik et al.

    TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells

    Nat. Med.

    (2005)
  • T.F. Leung et al.

    Asthma and atopy are associated with DEFB1 polymorphisms in Chinese children

    Genes Immun.

    (2006)
  • C.M. Lewis

    Genetic association studies: design, analysis and interpretation

    Brief. Bioinform.

    (2002)
  • P.T. Liu et al.

    Cutting edge: vitamin D-mediated human antimicrobial activity against Mycobacterium tuberculosis is dependent on the induction of cathelicidin

    J. Immunol.

    (2007)
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    Current address: Molecular Biology Laboratory, Southeast Unit, Research Center in Technology and Design Assistance of Jalisco State (CIATEJ, A.C.), National Council of Science and Technology (CONACYT), Mérida, Yucatán, Mexico.

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