Increased Genomic Copy Number of DEFA1/DEFA3 Is Associated with Susceptibility to Severe Sepsis in Chinese Han Population

QiXing Chen, M.D.,* Matthew Hakimi, M.Sc.,† ShuiJing Wu, M.D.,‡ Yue Jin, M.Sc.,§ ʈ ʈ

BaoLi Cheng, M.D., Ph.D., HaiHong Wang, M.D., Ph.D., GuoHao Xie, M.D.,‡ Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/6/1428/251619/0000542-201006000-00022.pdf by guest on 24 September 2021 Tomas Ganz, M.D., Ph.D.,# Rose M. Linzmeier, Ph.D.,** XiangMing Fang, M.D.††

Ϫ ABSTRACT with an increased risk of severe sepsis (P ϭ 2.25 ϫ 10 5, odds Background: Human neutrophil peptides 1–3 are endoge- ratio 2.66, 95% confidence interval 1.69–4.19). This estab- nous cationic antimicrobial peptides implicated in host defense lished association was replicated in a second age- and gender- against microbes. The genes encoding human neutrophil pep- matched case–control cohort (P ϭ 0.02, odds ratio 1.90, 95% tides 1–3 (DEFA1/DEFA3) exhibit copy number variations. confidence interval 1.11–3.27). Furthermore, compared with This study was designed to determine whether DEFA1/DEFA3 those with fewer copies, the patients carrying more than eight copy number variations conferred susceptibility to infection- copies of DEFA1/DEFA3 presented significantly lower plasma induced complications such as severe sepsis. levels of human neutrophil peptides 1–3, tumor necrosis fac- Methods: This case–control study was performed in 179 pa- tor-␣, interleukin-6, and interleukin-10 (P ϭ 0.039, 0.017, tients with severe sepsis and 233 healthy blood donors and was 0.030, and 0.029, respectively). replicated in an independent cohort of 112 cases and 118 Conclusions: DEFA1/DEFA3 is an important genetic com- controls. Plasma levels of human neutrophil peptides 1–3, ponent participating in host immune response to severe sep- tumor necrosis factor-␣, interleukin-6, and interleukin-10 sis. A higher copy number of DEFA1/DEFA3 (Ͼ8 copies) is were detected. significantly associated with the risk of severe sepsis. Results: The genotype of DEFA1/DEFA3 with more than eight copies was more frequent in patients with severe sepsis Ϫ than in controls (55.9% vs. 31.3%; P ϭ 1.13 ϫ 10 6, odds What We Already Know about This Topic ratio 2.77, 95% confidence interval 1.85–4.16). After ad- ❖ Human neutrophil peptides (HNP) 1–3 are antimicrobial pep- justment for age and gender, logistic regression analysis con- tides produced by leukocytes and important to host defense ❖ There is considerable genetic variability in the copy number for firmed the association of the genotype of more than eight copies HNP 1–3 and presumably in their expression and effects during severe infection and sepsis * Senior Research Assistant, ‡ Resident, § Research Assistant, ʈ Research Assistant and Senior Resident, Department of Anesthe- What This Article Tells Us That Is New siology, the First Affiliated Hospital, School of Medicine, Zhejiang ❖ In a case–control series of more than 200 patients, a high University, Hangzhou, China. †† Professor, Department of Anesthe- copy number for the defensin neutrophil peptides 1–3 siology, the First Affiliated Hospital, School of Medicine, and State (DEFA1/DEFA3) was associated with more than two-fold in- Key Laboratory for Diagnosis and Treatment of Infectious Disease, creased risk of severe sepsis Zhejiang University. † Research Assistant, # Professor, ** Senior Research Assistant, Department of Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California. EPSIS is a systemic inflammatory reaction syndrome Received from Department of Anesthesiology, the First Affiliated Sthat occurs during infection and is considered severe Hospital, School of Medicine, Zhejiang University, Hangzhou, when associated with acute organ dysfunction. Sepsis has China. Submitted for publication June 29, 2009. Accepted for pub- 1 lication February 5, 2010. Supported by National Science Fund for become the leading cause of death in critically ill patients. Distinguished Young Scholars (No. 30825037) from National Natural Science Foundation of China, Beijing, China, and sponsored by Zhejiang Provincial Program for the Cultivation of High-level Inno- ᭛ This article is featured in “This Month in Anesthesiology.” vative Health talents, Hangzhou, China. Drs. Linzmeier and Fang are Please see this issue of ANESTHESIOLOGY, page 9A. co-corresponding authors. ᭜ This article is accompanied by an Editorial View. Please see: Address correspondence to Dr. Fang: Department of Anesthesiology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Qing- Russell JA: Are copy number variants of defensins a key Chun Road 79, 310003 Hangzhou, China. [email protected]. This source of variation in the orchestrated response to infection? article may be accessed for personal use at no charge through the ANESTHESIOLOGY 2010; 112:1307–8. Journal Web site, www.anesthesiology.org.

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The pathogenesis of sepsis includes the immune response interleukin (IL)-6 as well as IL-10 in patients with severe and the coagulation–fibrinolysis system as well as elements of sepsis and explored the potential immunomodulating mech- genetic predisposition, which interact mutually in a compli- anisms of DEFA1/DEFA3 CNV in severe sepsis. cated network.2,3 Neutrophils are the first line of host defense against infection. They migrate to the site of infection Materials and Methods early and engulf and kill microbes using oxygen-dependent and -independent mechanisms. Oxygen-independent killing Study Design and Subjects mechanisms depend on neutrophil degranulation, which The study was approved by the Ethics Committee of Zhe- leads to the release of antibiotic proteins such as defensins.4,5 jiang University (Hangzhou, China), and written informed Defensins, classified according to their structure as ␣-de- consent was obtained from the subjects or a close relative of fensins and ␤-defensins, are small cysteine-rich endogenous the patient. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/6/1428/251619/0000542-201006000-00022.pdf by guest on 24 September 2021 antimicrobial peptides produced by certain leukocytes and Discovery Cohort. From December 1, 2003, to November many epithelial cells. The ␣-defensins, human neutrophil 30, 2005, all patients admitted in the postsurgical intensive peptides (HNP) 1–3, are constitutively expressed in neutro- care units (ICUs) at two University Hospitals of Zhejiang phils and comprise 30–50% of the granule proteins. HNP University for more than 24 h were screened for eligibility. 1–3 differ only in a single N-terminal amino acid, and the The diagnosis of sepsis met the criteria recommended by the HNP-2 peptide lacks this residue and is believed to be a American College of Chest Physicians and the Society of 17 proteolytic product of the other two peptides because no Critical Care Medicine Consensus Conference. Severe sep- separate gene has been identified to encode HNP-2.5–7 In sis was defined by sepsis in combination with sepsis-induced addition to their antimicrobial properties, HNP 1–3 may acute organ dysfunction in at least one organ. Acute organ contribute to host innate defenses against invading patho- dysfunction was defined as Sequential Organ Failure Assess- gens by their capacity to enhance phagocytosis by macro- ment score more than 2 for the organ in question. The Se- phages, to promote neutrophil recruitment, and to regulate quential Organ Failure Assessment score was calculated complement activation.8 It has also been proposed that HNP daily. The maximum Sequential Organ Failure Assessment 1–3 participate in the activation of the host-adaptive im- score was defined as the highest Sequential Organ Failure mune system by chemoattracting immature dendritic cells Assessment score reached during the ICU stay and was used and T cells to sites of inflammation and by functioning as to express the worst organ dysfunction status attained during immunoadjuvants.9,10 Because of neutrophil activation dur- the ICU stay. The Acute Physiology and Chronic Health ing sepsis, the levels of HNP 1–3 peptide are greatly in- Evaluation score II was obtained within 24 h of admission. creased in the blood of patients with sepsis and pulmonary The outcome of patients with severe sepsis referred to 28-day infections.11,12 Thus, HNP 1–3 may play an important role survival. All the patients studied were Han Chinese. Notable in infectious and inflammatory diseases. exclusion criteria included the following: age younger than The genes encoding ␣-defensins and eight members of 18 yr, a life expectancy of less than 24 h, human immuno- the ␤-defensin gene family are located on chromosome deficiency virus positive, treatment with long-term cortico- 8p22–23, which is a site of frequent chromosomal rearrange- steroids within 6 months or short-term corticosteroids ments. In this gene cluster, DEFA1 (encoding HNP-1) and within 4 weeks, chemotherapy or radiation therapy within 4 DEFA3 (encoding HNP-3) display copy number variations weeks, or a history of bone marrow or liver transplantation. (CNV) that are independent of a second set of CNV involv- The control group consisted of 233 ethnic-matched healthy ing several ␤-defensin genes (DEFB4, DEFB103, and blood donors. DEFB104 encoding human ␤-defensin 2, 3, and 4, respec- Replication Cohort. The cases in this cohort were selected tively).13–15 The genomic copy number of DEFA1/DEFA3, from those admitted to the postsurgical ICUs for more than which significantly correlates with the protein levels of HNP 24 h at the University Hospitals of Zhejiang University be- 1–3 in neutrophils,15 could contribute to individual varia- tween March 1, 2007, and May 30, 2008. The inclusion and tions in host responses during infection and inflammation. A exclusion criteria as well as the record of the clinical data were previous study suggested that single nucleotide polymor- same as those in the discovery cases described earlier (Discov- phisms in the human ␤-defensin-1 gene were associated with ery Cohort section). The control group comprised 118 age- severe sepsis,16 pointing to the defensin gene cluster as a and gender-matched healthy individuals. potential genetic locus for susceptibility to sepsis. In view of their multifunctional activity, the genotype–phenotype cor- Samples relation, and the genetic location, DEFA1 and DEFA3 are Ten milliliters of peripheral whole blood was drawn from also candidate genes for genetic predisposition to sepsis. each patient within 24 h after diagnosis of severe sepsis. Therefore, we carried out an association study between Plasma was isolated after the blood was centrifuged at 3,000g DEFA1/DEFA3 CNV and severe sepsis in a Chinese Han for 3 min and stored at Ϫ80°C for further analysis. Genomic cohort and replicated the findings in a second independent DNA was extracted using QIAamp DNA Blood Mini Kit cohort. We also measured the plasma levels of the proteins (QIAGEN, Valencia, CA) according to the manufacturer’s HNP 1–3, cytokines tumor necrosis factor (TNF)-␣, and instructions. The DNA concentration was measured with a

Chen et al. Anesthesiology, V 112 • No 6 • June 2010 1429 DEFA1/DEFA3 CNVs and Severe Sepsis spectrophotometer, and the DNA samples were stored at ates. The frequencies of more than eight copies of DEFA1/ Ϫ80°C until the next analysis. DEFA3 between cases and controls in the replication cohort were compared using the Fisher exact test. Determination of DEFA1/DEFA3 Gene Copy Number For comparisons of the plasma levels of HNP 1–3 as The genomic copy number of DEFA1/DEFA3 was deter- well as cytokines TNF-␣, IL-6, and IL-10, 44 patients mined by means of quantitative real-time polymerase chain were randomly selected from the discovery cases using reaction (PCR) as described previously.15 To improve the Microsoft Office Excel 2003 program (Microsoft Corpo- accuracy of the copy number assay, two primer sets were ration, Redmond, WA). The Mann–Whitney test was ap- chosen to amplify the different regions of the target gene. plied to all comparisons. Furthermore, to increase accuracy, two reference genes, All hypothesis tests in this study were two tailed. Statisti-

TATA box-binding protein and myeloperoxidase, which are cal analyses were performed using SPSS16.0 for Windows Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/6/1428/251619/0000542-201006000-00022.pdf by guest on 24 September 2021 single copy and have no known pseudogenes, were used. The (SPSS, Inc., Chicago, IL) and GraphPad Prism 3.00 for sequences and gene locations of the primers for both target Windows (GraphPad Software, Inc., La Jolla, CA). A value and reference genes were described previously.15 of P less than 0.05 was considered statistically significant. Quantitative real-time PCR was performed on an iCycler Thermal Cycler instrument (Bio-Rad Laboratories, Her- Results cules, CA). The PCR contained 1 ϫ iQ SYBR Green Super- mix (Bio-Rad Laboratories) and 250 nM each forward and Discovery Cohort reverse primer in 20-␮l volumes. Five-fold serial dilutions From December 1, 2003, to November 30, 2005, a total of were made to generate the three DNA starting quantities of 2,231 critically ill patients who were admitted to postsurgical 50, 10, and 2 ng. The same DNA dilutions were used to ICUs for more than 24 h were screened for severe sepsis. One amplify the reference and target genes. The PCR cycling hundred four patients were excluded because of age younger parameters were set as follows: 1 cycle at 95°C for 3 min, 40 than 18 yr (n ϭ 17), a life expectancy of less than 24 h (n ϭ cycles at 95°C for 30 s and 54°C for 30 s, and 1 cycle at 95°C 27), treatment with long-term corticosteroids within 6 for 1 min, followed by a melting curve initiating at 55°C and months (n ϭ 12) or short-term corticosteroids within 4 increasing in 0.5°C increments for 80 steps. Both reference weeks (n ϭ 11), chemotherapy or radiation therapy genes were amplified simultaneously in separate wells for within 4 weeks (n ϭ 17), a history of bone marrow (n ϭ 5) every DNA sample. PCRs were run in triplicate. Quantifica- or liver transplantation (n ϭ 12), and no informed con- ϭ tion was performed by the comparative threshold cycle (Ct) sent (n 3). The incidence of severe sepsis was 8.02%, method, as described previously.15 The reported gene copy which was consistent with our previous report.18 Patients number is the average of the results obtained for the three with severe sepsis included 106 men and 73 women, with different DNA concentrations used. a median age of 64 (interquartile range, 47.5–75) yr. Con- trols consisted of 103 men and 130 women, with a median Enzyme-linked Immunosorbent Assay for HNP 1–3, age of 48 (interquartile range, 40–57) yr. The difference TNF-␣, IL-6, and IL-10 in gender and age between the two groups is statistically The plasma levels of HNP 1–3 (Hycult Biotechnology, significant (P ϭ 0.002 for gender; P Ͻ 0.001 for age). The Uden, The Netherlands) as well as cytokines TNF-␣, IL-6, detailed characteristics of patients with severe sepsis are and IL-10 (R&D Systems, Inc., Minneapolis, MN) were given in table 1. detected by means of an enzyme-linked immunosorbent The copy number of DEFA1/DEFA3 in the cohort stud- assay. ied was detected using quantitative real-time PCR analysis. In the control group, DEFA1/DEFA3 copy number ranged Statistical Analysis from 2 to 15 per genome, with a median number of seven A comparison of the median copy number of DEFA1/ copies. The median copy number of DEFA1/DEFA3 in the DEFA3 between patients with severe sepsis and controls was patients with severe sepsis was nine copies per genome performed using the Mann–Whitney test. After calculating (range, 4–16 copies), which was significantly higher than the median copy number of DEFA1/DEFA3 in the overall that in controls (P Ͻ 0.001). However, in both the groups, objects, which was eight copies, two categories of genotypes the distribution of the DEFA1/DEFA3 copy number was (Յ8 copies and Ͼ8 copies) were applied in further analyses. independent of gender and age of the related population In the discovery cohort, differences in the distributions of (data not shown). Details of the copy number frequencies in copy number between cases and controls as well as between both the groups are given in table 2. surviving and nonsurviving cases were analyzed by the Fisher Notably, 55.9% of cases with severe sepsis carried the exact test, in which the Bonferroni correction for multiple genotype of DEFA1/DEFA3 with more than eight copies, comparisons was used, and P Ͻ 0.025 was required for sta- compared with a frequency of only 31.3% of this genotype Ϫ tistical significance. Logistic regression was used to model the detected in controls (P ϭ 1.13 ϫ 10 6; odds ratio, 2.77, effect of copy number of DEFA1/DEFA3 on the incidence of 95% confidence interval [CI], 1.85–4.16). Logistic re- severe sepsis when gender and age were included as covari- gression analysis of the genotype adjusted for age and

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Table 1. Characteristics of Patients with Severe Sepsis Table 2. Distributions of Genomic Copy Number of DEFA1/DEFA3 and Its Effect on the Odds Ratio of Discovery Cases Validation Cases Severe Sepsis Characteristics (n ϭ 179) (n ϭ 112) Discovery Cohort Validation Cohort Age, yr 64 (47.5–75.0) 63 (45.0–73.0) Male 106 (59.2%) 75 (67.0%) Copy Cases Controls Cases Controls 28-Day survival 88 (49.2%) 62 (55.4%) Number (n ϭ 179) (n ϭ 233) (n ϭ 112) (n ϭ 118) APACHE II 20 (15–25) 19 (12–25) SOFAmax 8 (6–10) 8 (5–10) 20511 Initial diagnosis 301812 Severe acute 30 (16.8%) 20 (17.8%) 421527 5 6 20 6 21

pancreatitis Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/6/1428/251619/0000542-201006000-00022.pdf by guest on 24 September 2021 Gastrointestinal 44 (24.6%) 23 (20.5%) 6 14332017 perforation or 7 28361614 intestinal fistula 8 29331520 9 30211814 after abdominal 10 18 21 7 4 operation 11 18 9 8 9 Pneumonia 35 (19.6%) 11 (9.8%) 12 15 13 7 4 Trauma 29 (16.2%) 36 (32.1%) 132431 Biliary duct or liver 18 (10.1%) 13 (11.6%) 149230 infection 154311 Others 23 (12.8%) 9 (8.0%) 164001 Source of infection 180011 Lung 126 (70.4%) 74 (66.1%) 200001 Abdomen 90 (50.3%) 62 (55.4%) 220030 Blood 69 (38.5%) 33 (29.5%) Յ8 79 160 61 82 Wound 29 (16.2%) 27 (24.1%) Ͼ8 100 73 51 36 Invasive vessel 28 (15.6%) 14 (12.5%) P value 1.13 ϫ 10Ϫ6 0.02 Urinary tract 14 (7.8%) 7 (6.3%) OR (95%CI) 2.77 (1.85–4.16) 1.90 (1.11–3.27) Other locus 10 (5.6%) 1 (0.9%) Infectious organisms CI ϭ confidence interval; OR ϭ odds ratio. Gram-positive 107 (59.8%) 59 (52.7%) bacteria Gram-negative 118 (65.9%) 72 (64.3%) in a second independent cohort containing 112 patients who bacteria had severe sepsis and 118 controls. The age and gender of the Fungi 69 (38.5%) 32 (28.6%) individuals in the two groups were comparable (cases: age 63 Nondocumented 29 (16.2%) 20 (17.8%) [interquartile range, 45–73] yr and 75 men; controls: age 62 infection [interquartile range, 43–80] yr and 78 men). The median copy number of DEFA1/DEFA3 was nine in patients with The values are given as mean (interquartile range) or n (%). Ͻ APACHE II ϭ Acute Physiology and Chronic Health Evaluation sepsis and seven in controls (P 0.001). Individuals carrying score II; SOFAmax ϭ maximum Sequential Organ Failure As- more than eight copies of DEFA1/DEFA3 showed more pre- sessment score. disposition to severe sepsis (P ϭ 0.02; odds ratio, 1.90; 95% CI, 1.11–3.27). gender confirmed the association of the genotype of more than eight copies with an increased risk of severe sepsis in Ϫ this discovery cohort (P ϭ 2.25 ϫ 10 5; odds ratio, 2.66; Biologic Plausibility 95% CI, 1.69–4.19). To test for evidence of biologic plausibility of association To analyze whether there was any association between the between high copy number of DEFA1/DEFA3 and incidence copy number of DEFA1/DEFA3 and the outcome of severe of severe sepsis, the plasma levels of HNP 1–3 as well as sepsis, septic patients were divided into survivors and non- cytokines TNF-␣, IL-6, and IL-10 were measured in a sub- survivors. The median copy number in both the groups was group of 44 patients with severe sepsis randomly selected nine copies per genome (range, 4–16 copies). The frequen- from the discovery cases. When the copy number of DEFA1/ cies of genotype with more than eight copies of DEFA1/ DEFA3 increased, the plasma levels of HNP 1–3 was not DEFA3 were 60.2% in survivors and 51.6% in nonsurvivors. elevated linearly (fig. 1A). Meanwhile, the HNP 1–3 levels No significant difference in distribution of the copy number were gender and age independent (P Ͼ 0.05, fig. 1, B and C). of DEFA1/DEFA3 was observed between the two groups We further divided the patients into two groups according to (P ϭ 0.29). the defined genotypes (Յ8 copies and Ͼ8 copies). As shown in figure 1D, the HNP 1–3 levels in patients carrying more Replication Cohort than eight copies (n ϭ 17) of DEFA1/DEFA3 were signifi- To evaluate the established association in the discovery co- cantly lower than those in the groups with Յ8 copies (n ϭ hort, we then examined the copy number of DEFA1/DEFA3 27), when analyzed by the Mann–Whitney test (P ϭ 0.039).

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Fig. 1. (A) Plasma levels of human neutrophil peptide (HNP) 1–3 in 44 randomly selected patients who had severe sepsis versus copy number of DEFA1/DEFA3.(B) Plasma levels of HNP 1–3 in men (filled squares,nϭ 29) and women (filled triangles, n ϭ 15) of the 44 patients with severe sepsis, P ϭ 0.83. (C) Plasma levels of HNP 1–3 in the 44 patients with severe sepsis aged less than 55 yr (filled squares,nϭ 15) and those aged 55 yr or older (filled triangles,nϭ 29), P ϭ 0.44. (D) Plasma levels of HNP 1–3 in patients with severe sepsis carrying 8 or fewer copies of DEFA1/DEFA3 (filled square,nϭ 27) and those with more than 8 copies of DEFA1/DEFA3 (filled triangles,nϭ 17), P ϭ 0.039. The line represents the median plasma level of HNP 1–3.

Moreover, compared with those with more than eight copies Discussion Յ of DEFA1/DEFA3, patients carrying 8 copies of DEFA1/ Recently, large-scale CNVs (Ͼ1 kb) were identified ␣ DEFA3 had higher plasma levels of TNF- , IL-6, and IL-10 throughout the entire human genome. The currently known ϭ (P 0.017, 0.030, and 0.029, respectively; fig. 2). CNVs are selectively enriched in genes that are relevant to host-environmental interactions and influence host response to certain environmental stimuli.19–22 CNVs affect gene expression and phenotypic variation by altering gene dosage and cause diseases or confer risk to complex diseases such as human immunodeficiency virus infection and glomerulonephritis.23,24 Because CNVs may not be detectable through single nucleotide polymorphism-based association study or linkage study,20 the effect of CNVs in DEFA1/DEFA3 on disease or disease susceptibility remains unclear. Using a candidate-gene association study in two independent cohorts, the current investigation found that a high copy number of DEFA1/DEFA3 (Ͼ8 copies) was significantly associated with the incidence of severe sepsis. Furthermore, patients with more than eight copies of DEFA1/DEFA3 had lower plasma levels of HNP 1–3 and cytokines compared with those with fewer copies of DEFA1/DEFA3. Fig. 2. Plasma levels of cytokines in patients with severe The ␣-defensins are stored primarily in the azurophil sepsis carrying 8 or fewer copies of DEFA1/DEFA3 (filled granules of neutrophils and released into circulation during squares,nϭ 27) and those with more than 8 copies of DEFA1/DEFA3 (filled triangles,nϭ 17). The line represents the activation of neutrophils in response to phagocytic stim- 5,6 the median plasma level of related cytokines. (A) Tumor ne- uli such as infection. The intracellular levels of HNP 1–3 crosis factor (TNF)-␣, P ϭ 0.017; (B) interleukin (IL)-6, P ϭ in neutrophils are proportional to the copy numbers of 0.030; (C) interleukin (IL)-10, P ϭ 0.029. DEFA1/DEFA3,15 that is, higher copy numbers of DEFA1/

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DEFA3 equate to higher levels of HNP 1–3. The circulating regulating host immune responses due to the production of levels of HNP 1–3 in patients with severe sepsis observed in higher levels of HNP 1–3. this study were comparable with previous reports.11,12 How- Quantitative real-time PCR is a well-established reliable ever, a linear correlation of circulating HNP 1–3 levels with method for detecting CNVs. Using this method, the copy DEFA1/DEFA3 copy number was absent. Compared with number of DEFA1/DEFA3 in healthy individuals was found those with fewer gene copies, the plasma levels of HNP 1–3 to range between 2 and 15 copies, with 5–10 copies being in patients carrying more than eight copies of DEFA1/ most common, which is consistent with the findings of Al- 14 15 DEFA3 were significantly lower. These findings are consis- dred et al., but was different from our previous study. tent with another study that also observed that combined This discrepancy may result from ethnic differences, because expression levels of DEFA1 and DEFA3 were not correlated the copy number of DEFA1/DEFA3 was screened in five with genomic copy number.14 The discrepancy between races in that study and genomic copy number of certain genes Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/112/6/1428/251619/0000542-201006000-00022.pdf by guest on 24 September 2021 31 gene copy number and protein levels may be due to different shows an interpopulation difference. transcriptional mechanisms modulating these two genes or Several limitations of this study must be mentioned. First, an increased distance between the regulators and the genes. sepsis is a heterogeneous syndrome with multifactorial cau- 2,3 Alternatively, more severe sepsis-induced neutrophil dys- ses. The incidence of sepsis is much more frequent in function in patients with more copies of DEFA1/DEFA3 elderly (especially those older than 55 yr) and in males, and may dysregulate the release of intracellular HNP 1–3 into the thus, it is difficult to enroll a completely age- and gender- circulation. On the other hand, a similar phenomenon that matched cohort as controls. Currently, healthy blood donors ␤ were deemed acceptable and were adopted by many case– the message RNA levels of a -defensin gene, DEFB4,donot 32 increase linearly with the copy number of DEFB4 was re- control studies investigating genetic associations in sepsis. ported in patients with Crohn disease.21 Furthermore, using Second, the control population was not matched with the an Escherichia coli expression system, our previous study and severe sepsis population in terms of gender or age. However, other studies found that an increase in the repeat copy num- the distributions of DEFA1/DEFA3 copy numbers, as well as the plasma levels of HNP 1–3, were independent of gender ber of the defensin gene or other genes did not lead to a and age. After analysis by multivariant logistic regression, further increase in the related recombinant protein lev- which included age and gender as covariants, the association els.25–27 These findings might be a common phenomenon in in this study is still statistically significant (P ϭ 2.25 ϫ nature because recent studies have also demonstrated that Ϫ 10 5). This established association was further validated in 2–15% of genes were expressed in the opposite direction as an independent age- and gender-matched replication cohort. the copy number changed.28,29 The definite mechanisms for In addition, the cohort studied was entirely of the Chinese this phenomenon both in prokaryotes and in eukaryotes re- Han population. Because the frequency of CNVs within the main unknown and deserve to be further investigated. human genome may differ between populations, this factor Accumulated evidence suggests that HNP 1–3 act as a may limit the generalization of the current findings. Never- multifunctional mediator both in host innate immunity and theless, this is the first study to discover that genetic copy adaptive immunity.5,8 Studies have shown that the biologic 9,10,30 number variants contribute to susceptibility to severe sepsis. activity of HNP 1–3 is dose-dependent. Consequently, Furthermore, based on the biologic functional data, the as- the higher levels of HNP 1–3 in severe septic patients with sociation of DEFA1/DEFA3 CNVs with severe sepsis dem- fewer copies of DEFA1/DEFA3 may result in more efficient onstrated here strongly supports DEFA1/DEFA3 CNVs as a antimicrobial activity and other functions in innate immu- potential risk factor for susceptibility to severe sepsis. nity against infection and inflammation. On the other hand, In summary, an increase in copy number of DEFA1/ in vivo and in vitro studies have demonstrated that HNP 1–3 DEFA3 was associated with predisposition to severe sepsis, released by neutrophils during severe sepsis could also recruit ϩ suggesting that differences in the gene dosage of DEFA1/ immature dendritic cells and T cells, activate CD4 T cells, DEFA3 constitute a genetic basis for interindividual re- 5,9,10 and induce secretion of Th1/Th2-type cytokines. A sig- sponses to severe sepsis. nificant increase in both Th1 (such as TNF-␣) and Th2 cytokines (such as IL-6 and IL-10) in patients with Յ8 copies of DEFA1/DEFA3 was observed in this study. The elevated References level of balanced Th1/Th2 response further enhances cellular 1. Hotchkiss RS, Karl IE: The pathophysiology and treatment immunity and mediates humoral immunity, thus controlling of sepsis. N Engl J Med 2003; 348:138–50 2. Cohen J: The immunopathogenesis of sepsis. Nature 2002; the response to infection and inflammation. It is possible that 420:885–91 the lower concentration of HNP 1–3 in patients with high 3. Annane D, Bellissant E, Cavaillon JM: Septic shock. Lancet copy numbers could not induce a high enough Th1/Th2 2005; 365:63–78 response, leading to amplification of infection and inflam- 4. Brown KA, Brain SD, Pearson JD, Edgeworth JD, Lewis SM, Treacher DF: Neutrophils in development of multiple or- mation and ultimately the development of sepsis. 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