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Genomics 88 (2006) 122–126 www.elsevier.com/locate/ygeno

Short Communication Copy number polymorphisms are not a common feature of innate immune ⁎ Rose M. Linzmeier , Tomas Ganz

Departments of Medicine and Pathology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095-1690, USA Received 22 November 2005; accepted 6 March 2006 Available online 17 April 2006

Abstract

Extensive copy number polymorphism was recently reported for innate immunity-related α- genes DEFA1 and DEFA3 and β-defensin genes DEFB4, DEFB103, and DEFB104. To establish whether such polymorphisms are a common feature of innate immune genes we used quantitative real-time PCR to determine the copy numbers of seven genes whose products have important innate immune functions. The genes encoding lysozyme, lactoferrin, cathelicidin antimicrobial peptide (hCAP18/LL-37), cathepsin G, bactericidal/permeability-increasing , azurocidin (CAP37/heparin-binding protein), and neutrophil elastase were each found to be single copy per haploid genome. These findings, along with the recent observation that defensin genes DEFA4, DEFA5, DEFA6, and DEFB1 are single copy, suggest that copy number polymorphisms are not a common feature of the innate immune genome but are restricted to a small subset of innate immunity-related genes. © 2006 Elsevier Inc. All rights reserved.

Keywords: Real-time PCR; Copy number polymorphisms; Large-scale copy number variation; Host defense

Approximately 5–10% of the consists of be CNP [9]. These genes were carried on the same low-copy repeats (LCRs) [1]. Recombination between LCRs in approximately 240-kb repeating unit and so vary in concert regions containing duplicated DNA sequences can lead to the with copy number ranging from 2 to 12 per diploid genome expansion and deletion of surrounding chromatin [2]. System- (PDG). Comparing the number of DEFB4 copies with atic scans of the entire human genome for areas that are copy the amount of DEFB4 mRNA for seven lymphoblastoid cell number polymorphic (CNP) have been made using microarray lines, a direct correlation was found between DEFB4 gene technology-based comparative genomic hybridization (CGH) copies and DEFB4 transcription levels. Also using MAPH, [3–5]. Among the large-scale copy number variations identified the number of DEFA1 and DEFA3 gene copies was found to in these studies, multiple sites associated with genetic diseases range from 4 to 11 in a sample of 111 subjects [10]. The and cancers were found. The methodology of CNP discovery DEFA3 allele was absent completely in 10% of these and the role CNPs play in creating phenotypic differences are subjects. In a previous study, we used real-time PCR to emerging areas of study [6,7]. quantify the number of DEFA1 and DEFA3 genes for 27 Among genes involved in innate immunity, the copy subjects and found that the combined number ranged from 5 number variability of several defensin genes has been well to 14 copies PDG [11]. The DEFA3 allele was absent in 26% established. The DEFA1 and DEFA3 CNP and variable of these subjects. The amount of defensin peptides HNP 1–3, presence of the DEFA3 gene was demonstrated using the products of the DEFA1 and DEFA3 genes, isolated from quantitative Southern hybridization [8]. Using the multiplex circulating neutrophils was directly proportional to the amplifiable probe hybridization (MAPH) technique the combined copy number of the DEFA1 and DEFA3 genes. DEFB4, DEB103, and DEFB104 genes were also found to In the same study the number of DEFB4 and DEFB103 genes ranged from 2 to 8 DPG. Although this has not yet been ⁎ Corresponding author. Fax: +1 310 206 8766. specifically demonstrated for DEFB4 and DEFB103, it is E-mail address: [email protected] (R.M. Linzmeier). reasonable to expect that as observed for DEFA1 and DEFA3,

0888-7543/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2006.03.005 R.M. Linzmeier, T. Ganz / Genomics 88 (2006) 122–126 123 individuals with more gene copies produce higher amounts of number quantification by real-time PCR. We employ the same the encoded peptides. population sample previously used to show copy number Interestingly, we observed that the highly CNP DEFA1 and polymorphism in defensin genes [11]. Each of the selected DEFA3 genes are adjacent to defensin genes consistently found innate immunity genes is a member of a gene family, a group of in two copies PDG: the α-defensin genes DEFA4, DEFA5, and genes with common structural and functional features thought DEFA6 and the β-defensin DEFB1 [11]. We also reported that to have diverged from an ancestral gene. The products of the in the case of the α- DEFA1 and DEFA3 the mutation selected innate immunity genes have been well characterized causing the single-nucleotide polymorphism that distinguishes and shown to have important roles in host defense. The genes these genes likely preceded their independent copy number and some of their characteristics are listed in Table 1.Someof polymorphisms, as the presence or absence of the DEFA3 allele these genes vary in the number of orthologs carried by different is not related to the total DEFA1/3 copy number. In one subject species, as summarized below. the usually minor allele DEFA3 accounted for the majority of Lysozyme was the first recognized antimicrobial protein, the DEFA1/3 gene copies (subject 3, 9 copies of DEFA3 of 13 discovered by Alexander Fleming in 1922. Lysozyme is combined copy number for DEFA1/3 [11]). The size of the abundantly expressed in numerous human tissues and secretions DNA unit duplicated in the DEFA1/3 CNP is approximately [14]. The number of genes encoding lysozyme varies greatly 20 kb (NCBI human genome database), much smaller than the among mammals, with humans and pigs carrying 1 gene, mice approximately 240-kb repeating unit of the closely linked and camels 2, and cows and sheep 10 [15] Recently 3 human β-defensin locus. Unequal crossing over between LCRs in lysozyme-related genes have been reported to be expressed at duplicated regions giving rise to gene families can also account high levels in the epididymis [16]. Lactoferrin is a member of for CNPs [12,13]. While individual variability in resistance to the iron-binding transferrin family, which in humans includes infectious diseases is readily observed, the causes of this serum transferrin and melanotransferrin [17].Thegenes diversity in immune function are poorly understood. One encoding these are closely linked, mapping to 3q21– possible source for this variability is CNP. To investigate q23, 3q21, and 3q28–q29, respectively. Cathelicidin peptides directly the prevalence of copy number polymorphisms in genes form a family recognized by their conserved N-terminal of the innate immune system, we selected seven genes for copy prosequence. The adjacent C-terminal antimicrobial peptide

Table 1 Gene expression and gene product function of the innate immunity target genes and the MPO reference gene Gene symbol Gene name Regulation of expression Site(s) of expression Known functions of protein LYZ Lysozyme Constitutive, upper GI PMN primary and secondary A muramidase, antimicrobial in vitro tract and Paneth cells; granules, monocytes, and in mouse models; enhances may be inducible in macrophages and phagocytosis and binds LPS [27] mouse macrophages [12] epithelia [26] and in skin [24,25] LTF Lactoferrin Constitutive [28] PMN secondary granules, An iron-binding protein antimicrobial glandular epithelial cells [29] against , fungi, and directly and by iron chelation; binds LPS [30] CAMP Cathelicidin Constitutive, PMN and PMN secondary granules, Antimicrobial against gram– and antimicrobial colon [31]; IL-6, ASK1, skin, respiratory epithelia, and gram+bacteria, binds LPS, peptide and vitamin D upregulated, gastric epithelia [35] chemotactic for PMN, skin [32–34] monocytes, and mast cells [36] CTSG Cathepsin G Constitutive PMN primary granules A serine protease, antimicrobial against bacteria [37] and fungi [38] and remodels tissue at wound sites [39] BPI Bactericidal/ Constitutive [40]; PMN primary granules, An LPS-binding protein antimicrobial permeability- lipid mediator-induced mucosal epithelia, and tears against gram– bacteria [44] increasing in human mucosal [42,43] protein epithelia [41] AZU1 Azurocidin Constitutive PMN primary granules An enzymatically inactive protease homolog antimicrobial against gram– bacteria, increases vascular permeability, binds endotoxin, and chemotactic for monocytes [45] ELA2 Neutrophil Constitutive PMN primary granules, A serine protease antimicrobial elastase GI tract [46] against gram– bacteria; also regulates leukocyte trafficking and remodels connective tissue [46,47] MPO Myeloperoxidase Constitutive PMN primary granules Antimicrobial to bacteria and fungi and inactivates neutrophil elastase [48,49] 124 R.M. Linzmeier, T. Ganz / Genomics 88 (2006) 122–126

Table 2 Real-time PCR primer sets Primer name Primer sequence Sequence Accession No. Primer position in sequence Location in gene LYZ 1F ATAGCCGCTACTGGTGTAAT X14008 2342–2361 Exon 2 LYZ 1R CTTTCAAGGCTTTTTCATTT X14008 2489–2470 Exon 2 LYZ 2F AAATATTTTGTTTCCCCAAG X14008 5189–5208 Exon 4 LYZ 2R CCACAACCTTGAACATACTG X14008 5340–5321 Exon 4 LTF 1F AGCTCAAAGGTCAGAGTCAT U95626 142814–142833 Exon 6 LTF 1R TGATGGTTTCTCTTTTCACA U95626 142925–142944 Exon 6 LTF 2F AAAGGAAACACCTTCACCTA U95626 129984–130004 Exon 14 LTF 2R TATGAAAGCCCCACTAGTTT U95626 130133–130114 Exon 14 CAMP 1F CCCAGGATAACAAGAGATTT X96735 2758–2777 Exon 4 CAMP 1R GTAGGGCACACACTAGGACT X96735 2906–2887 Exon 4 CAMP 2F TTCAGTCTCACTTGTTTTGC X96735 2963–2982 Downstream CAMP 2R CTACTGCCTTACCCTGTGTC X96735 3112–3091 Downstream CTSG 1F ATAATTTGGCAGCATGTGT J04990 653–671 Intron 1 CTSG 1R CTTTTATCCTCATGGAGCTT J04990 802–783 Intron 1 CTSG 2F CAGACCTACCTCTTGCTTTT J04990 1739–1758 Exon 3 CTSG 2R ATGATGTCATTCTGGATGG J04990 1888–1870 Exon 3 BPI 1F TTCAATGCCAACAAATAAAG AL359555 36852–36871 Intron 1 BPI 1R GGCCAAATAGTAAACTGACC AL359555 37001–36982 Intron 1 BPI 2F GACCTTGCTTTCCTTTAGTG AL499625 6779–6798 Intron 9 BPI 2R CTGTTCTGTGGATGAAGCTA AL499625 6928–6909 Intron 9 AZU1 1F CTAGTCCCAGCTACTCAGGA M96326 2577–2597 Intron 3 AZU1 1R TGCTGTATTGTTTTTGGAAG M96326 2727–2708 Intron 3 AZU1 2F AGTTTGGTCTCGAACTCCT M96326 2929–2947 Intron 3 AZU1 2R GAAGTGTCAGCGACATTAAC M96326 3077–3058 Intron 3 ELA2 1F AACTGAGTCACGGAGAGTTT AC112706 3130–3149 Upstream ELA2 1R AGCTGCATTTATTGAGCAC AC112706 3281–3261 Upstream ELA2 2F CGTGAATTTATTTCATCCTCT AC112706 3060–3080 Upstream ELA2 2R GTTCATATCCTGGTTCTGGT AC112706 3209–3188 Upstream MPO F CCAGCCCAGAATATCCTTGG M17176 13–32 Exon 12 MPO R GGTGATGCCTGTGTTGTCG M17176 162–144 Exon 12 TBP F TGAGAAGATGGATGTTGAGTTG AL031259 76585–76564 Exon 8 TBP R AGATAGCAGCACGGTATGAG AL031259 76438–76457 Exon 8 portions vary greatly in size and structure. While humans and homologous genes have acquired new functions, giving rise to a mice have a single cathelicidin gene, multiple different multigene family [12]. We considered the possibility that the cathelicidin antimicrobial peptides have been found in some LCR DNA sequences that provide the means for the duplication mammalian species [18]. Pigs carry 12, cattle 11, and sheep of an ancestral gene could also promote CNPs of the descendant 8 different cathelicidin genes [18,19]. The genes encoding genes. bactericidal/permeability-increasing protein (BPI) and its func- tional opposite lipopolysaccharide binding protein (LBP) are adjacent to each other, at 20q11.23 [20]. Along with phospholipid transfer protein, also encoded on 20, and cholesteryl ester transfer protein, the gene for which is located on chromosome 16, BPI and LBP are part of a larger family of lipid-binding proteins [21]. The innate immunity genes clustered at 19p13.3, azurocidin, neutrophil elastase, and proteinase 3, have diverged from an ancestral proteinase gene after duplication and mutation [22]. Another member of the granule protease family, cathepsin G, is encoded in a separate serine protease cluster. The 4 protease genes of this cluster, located at 14q11.2, encode neutrophil cathepsin G, lymphocyte CGL-1 and 2 (also known as granzyme B and H), and mast cell chymase [23]. The myeloperoxidase gene, used as a reference Fig. 1. Copy numbers of a panel of innate immunity genes. Gene copy number gene in this study, is adjacent to the eosinophil peroxidase and PDG is shown on the y axis for 30 donors, the target genes are identified on the x lactoperoxidase genes at 17q23.1. These genes share primary axis. The median copy number for each gene is shown by a line within the shaded boxes that demarcate the 25th and 75th percentiles. The 10th and 90th and similar gene organization and their percentiles are marked by error bars. Individual data points outside the 10th and products have similar functional properties [24]. Through 90th percentile are represented by filled circles. The horizontal background line duplication of an ancestral gene, followed by mutation, the indicates the average gene copy number of 2 PDG. R.M. Linzmeier, T. Ganz / Genomics 88 (2006) 122–126 125

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