IL6 G-174C Associated With Sudden Infant Death Syndrome in a Caucasian Australian Cohort

IL6 G-174C Associated With Sudden Infant Death Syndrome in a Caucasian Australian Cohort Sophia M. Moscovis, Ann E. Gordon, Osama M. Al Madani, Maree Gleeson, Rodney J. Scott, June Roberts-Thomson, Sharron T. Hall, Donald M. Weir, Anthony Busuttil, and C. Caroline Blackwell ABSTRACT: The aims of this study were to analyze IL6 G-174C in relation to high interleukin (IL)-6 concentrations found in some sudden infant death syndrome (SIDS) infants, and to assess the effects of IL6 G-174C, smoking status, and gender on IL-6 responses. SIDS infants, parents of SIDS infants, and populations with high (Aboriginal Australian), medium (Caucasian) or low (Bangladeshi) SIDS incidences were genotyped. Leukocytes were stimulated in vitro with endotoxin and IL-6 responses were assessed in relation to IL6 G-174C genotype, smoking status, and gender. The study findings showed that GG genotype, associated with high IL-6 responses, was predominant among Australian SIDS infants (58%) compared with control subjects (38%, p ⫽ 0.02), as well as Bangladeshis (94%) and Aboriginal Australians (88%) compared with Caucasians (42%, p ⬍ 0.01). GC smokers had higher median IL-6 responses (8.4 ng/ml⫺1) than GG ABBREVIATIONS CSF cerebral spinal fluid E. coli Escherichia coli IL interleukin INTRODUCTION Infection and inflammatory responses to infections have been proposed to trigger the physiologic events leading to sudden infant death syndrome (SIDS) [1–3]. In a From the School of Biomedical Sciences (S.M.M., M.G., R.J.S., J.R.T., S.T.H., C.C.B.), Faculty of Health, University of Newcastle, and Hunter Medical Research Institute, Newcastle, NSW, Australia; Medical Microbiology (A.E.G., O.M.AM., D.M.W.) and Forensic Medicine Unit (A.B.), University of Edinburgh, Edinburgh, United Kingdom; and Immunology (S.T.H.) and Genetics (R.J.S.), Hunter Area Pathology Service, John Hunter Hospital, New Lambton, NSW, Australia. Address reprint requests to: Sophia M. Moscovis, HAPS–Immunology, John Hunter Hospital, Lookout Road, New Lambton Heights, NSW 2305, Australia; Tel: ⫹61 2 49214027; Fax: ⫹61 2 49214196; E-mail: Sophia.Moscovis@newcastle.edu.au. Received May 17, 2006; revised July 10, 2006; accepted July 20, 2006. Human Immunology 67, 819 – 825 (2006) © American Society for Histocompatibility and Immunogenetics, 2006 Published by Elsevier Inc. (3.5 ng/ml⫺1, p ⫽ 0.01) or CC smokers (2.4 ng/ml⫺1, p ⬍ 0.01). GG nonsmokers had higher median IL-6 responses (4.9 ng/ml⫺1) than GG smokers (p ⬍ 0.05). Gender did not affect IL-6 responses. In conclusion, an association between IL6 G-174C and Australian SIDS infants was observed. IL6 G-174C alone cannot explain observed differences in the incidence of SIDS in the Bangladeshi and Aboriginal Australian populations. Further investigations are needed on interactions between smoking and gene polymorphisms in relation to proinflammatory responses implicated in SIDS. Human Immunology 67, 819 – 825 (2006). © American Society for Histocompatibility and Immunogenetics, 2006. Published by Elsevier Inc. KEYWORDS: Sudden infant death syndrome; interleukin-6; ethnicity; cigarette smoke PCR SIDS polymerase chain reaction sudden infant death syndrome Norwegian study [4], half of the SIDS infants had elevated levels of interleukin (IL)-6 in their cerebrospinal fluid (CSF), comparable to levels found in infants dying of infectious diseases such as meningitis and septicemia. SIDS infants with high levels of IL-6 in the CSF also showed signs of immune stimulation in their laryngeal mucosa [5] with increased numbers of IgA immunocytes and increased expression of HLA-DR [6]. Many of these infants also showed signs of infection before death and were found dead in a prone position [7]. IL-6 is an endogenous pyrogen [8] that induces fever, and increased temperatures have been found to influence respiration in infants [9]. The physiologic effect of hyperthermia in relation to SIDS has been reported to be 0198-8859/06/$–see front matter doi:10.1016/j.humimm.2006.07.010 820 particularly significant in relation to infection [10]. Hyperthermia significantly increased production of IL-6 but not IL-1␤ in infant rats. In response to muramyl dipeptide, a surrogate for infection, IL-1␤ was significantly increased, whereas IL-6 was not. Muramyl dipeptide in combination with hyperthermia significantly increased mortality of the infant rats [11]. Pyrogenic toxins of Staphylococcus aureus have been identified in more than half of SIDS infants from five countries [1, 12]. These toxins induce powerful inflammatory responses, including production of IL-6 [13–15]. In addition, it has been reported that males produce higher levels of IL-6 than females [16], and a higher proportion of SIDS infants are male [17]. High levels of IL-6 observed in SIDS infants could be caused by differences in individual responses to various stimuli, genetic polymorphisms or a deficient interplay in the cytokine network, for example, reduced production of IL-10 associated with genetic or environmental factors [1, 15]. A single nucleotide polymorphism in the promoter region of the IL6 gene, IL6 G-174C, has been reported to affect the level of gene transcription; the G allele was associated with higher levels of IL-6 [18]. The first objective of this study was to assess the distribution of IL6 G-174C among the following groups: SIDS infants; parents of SIDS infants; and populations with high (Aboriginal Australian) [19], medium (European), and low (Bangladeshi) [20] incidences of SIDS. The second objective was to determine whether cigarette smoke exposure or male gender— both important risk factors for SIDS [21]—affected IL-6 responses of leukocytes of donors with different genotypes of IL6 G-174C after stimulation with endotoxin. MATERIALS AND METHODS Ethics Approval Approval for the study was obtained from the Lothian Health Ethics Committee (UK), Hunter Area Research Ethics Committee and the University of Newcastle Human Research Ethics Committee (Australia). Informed consent was obtained from the parents and control subjects recruited for the study. Subjects and Sample Collections Buccal epithelial cells were collected from Caucasian parents (mother / father or both) of SIDS infants from Britain (n ⫽ 36) and Australia (n ⫽ 60), and their ethnically matched control subjects with no history of SIDS in the family (Britain, n ⫽ 58; Australia, n ⫽ 63). Control subjects from Britain and Australia were combined to form a European Caucasian control group. Fixed samples of tissue were obtained from Caucasian SIDS infants from Australia (n ⫽ 19), Hungary (n ⫽ 20), and S.M. Moscovis et al. Germany (n ⫽ 47). Australian infants had no relation to Australian parents assessed in the study. DNA had been previously extracted from stored frozen blood samples from Aboriginal Australians (n ⫽ 107) and buccal epithelial cells from Bangladeshis (n ⫽ 32). The ethnicity of the Bangladeshi subjects was unknown; however, the majority of Bangladeshis (98%) are of Bengali ethnic origin [22]. Extraction of DNA From Epithelial Cells and Fixed Tissues DNA from buccal swabs was extracted using the QIAamp® DNA Mini Kit (QIAGEN GmbH, Germany) according to the manufacturer’s instructions. DNA from the Hungarian and German SIDS infant tissue samples was extracted by the modified salt precipitation method as previously described [15]. DNA from the Australian SIDS infant tissue samples was extracted on the BioRobot® M48 machine (QIAGEN GmbH); 200 ␮L of lysed tissue solution was processed according to the manufacturer’s instructions. Analysis of IL6 G-174C by Polymerase Chain Reaction An allelic discrimination polymerase chain reaction (PCR) assay was developed to genotype IL6 G-174C (GenBank accession no. AY170325.1). Primers (Invitrogen, Frederick, USA) used to amplify the sequence surrounding the polymorphism were: sense, 5= GCTGCACTTTTCCCCCTAGTT 3=; and antisense, 5= GCTGATTGGAAACCTTATTAAGATTGT 3=. Specific, fluorescent-labeled minor groove binding probes used to identify each allele were: 5= 6-FAM-TGTCTTGCGATGCTA 3= for the G allele; and VIC-TGTCTTGCCATGCTA 3= for the C allele (PE Applied Biosystems, Foster City, USA). Each PCR mixture contained 50 ng of sample DNA, 100 nM of each minor groove binding probe, 400 nM of each primer, and 5 ␮L 2x TaqMan Universal PCR Master Mix (PE Applied Biosystems) made up to a final volume of 10 ␮l with sterilized MilliQ water (Millipore, Sydney, Australia). PCR was performed using the ABI PRISM 7900HT sequence detection system (PE Applied Biosystems) under the following thermal cycling conditions: 40 cycles of 50°C for 2 minutes, 95°C for 10 minutes, 92°C for 15 seconds, and 60°C for 1 minute. In Vitro Assessment of IL-6 Responses to Endotoxin Blood samples (10 –20 ml) were collected from Caucasian British donors. Samples were collected in the morning to limit the effect of circadian variation on cytokine production. The blood was transported to the laboratory at room temperature. All samples were coded and tested without knowing gender or smoking status of the do- 821 Interleukin-6 and Sudden Infant Death Syndrome TABLE 1 Distribution of IL6 G-174C Genotypes in Each Study Group Distribution of IL6 G-174C (%) Ethnicity Group British British Australian Australian Australian Hungarian German Combined German Bangladeshi Aboriginal Australian European Caucasian SIDS Control SIDS Control SIDS SIDS SIDS SIDS Control Control Control Control Parents Parents Parents Parents Infants Infants Infants Infants GG GC CC Sample Size (n) 31 47 35 38 58 25 19 29 34 94 88 42 56 38 45 44 21 50 64 51 45 6 11 41 14 16 20 17 21 25 17 20 20 0 1 17 36 58 60 63 19 20 47 86 207 32 107 121 pⴱ 0.23 0.92 0.02a 0.57b 0.10c ⬍0.01d 0.97e 0.06f ⬍0.01g ⬍0.01h ⴱ Chi-square or Fisher’s exact test, where appropriate, was used to assess the distribution of IL6 G-174C in sudden infant death syndrome (SIDS) infants, parents of SIDS infants, and between ethnic groups. a Australian SIDS infants vs. Australian control parents; b Hungarian SIDS infants vs. German SIDS infants; c Hungarian SIDS infants vs. Australian SIDS infants; d German SIDS infants vs. Australian SIDS infants; e Combined SIDS infants vs. European Caucasian control; f German SIDS infants vs. German control; g Bangladeshi control vs. European Caucasian control; h Aboriginal Australian control vs. European Caucasian control. nors. Leukocytes collected from blood samples were stimulated in vitro with 0.01 ␮g ml⫺1 or 1 ␮g ml⫺1 Escherichia coli (E. coli) endotoxin (Sigma, Poole, Dorset, UK) for 24 hours. Cell culture conditions are described in Moscovis et al. [15]. IL-6 production was assessed by enzyme-linked immunosorbent assays (ELISA) as previously described [13]. The results were expressed as ng/ ml⫺1 derived from the standard curves obtained using a recombinant human IL-6 standard. Statistical Methods Data were analyzed using the statistical software package Statistics/Data Analysis (STATA) Version 8.0 (Stata Corporation, College Station, TX). The Chi-square test or Fisher Exact test, where appropriate, was used to assess the distribution of IL6 G-174C in SIDS infants, parents of SIDS infants, and between ethnic groups. Student’s t-test was used on log-transformed data to assess differences in IL-6 responses of smokers (n ⫽ 45) and nonsmokers (n ⫽ 74) and of males (n ⫽ 46) and females (n ⫽ 73) in relation to genotype. The significance level for all tests was set at p ⬍ 0.05. RESULTS The distribution of IL6 G-174C for each study group is summarized in Table 1. Distribution of IL6 G-174C Among SIDS Infants The distribution of IL6 G-174C varied among SIDS infants from different countries. The predominant genotype among Australian SIDS infants was GG (11/19, 58%); however, the majority of Hungarian (10/20, 50%) and German (30/47, 64%) infants possessed the GC genotype. There was no significant difference in the distribution of IL6 G-174C for Hungarian and German SIDS infants (p ⫽ 0.57). The distribution differed significantly between the German and the Australian SIDS infants (p ⬍ 0.01) but not between the Hungarian and Australian infants (p ⫽ 0.10). The distribution of IL6 G-174C for the Australian control population differed significantly from that observed for Australian SIDS infants (p ⫽ 0.02). Only 24/63 (38%) of Australian control subjects had the GG genotype compared with 58% of SIDS infants. No significant differences were detected between the distributions of IL6 G-174C for the combined SIDS infant group compared with the European Caucasian control subjects (p ⫽ 0.97). The distribution of IL6 G-174C among 207 healthy German control subjects (Table 1) was obtained from studies previously published [23]. The distribution of IL6 G-174C for the 47 German SIDS infants was not significantly different from the German control popula- 822 S.M. Moscovis et al. tion; however, there was an increased proportion of infants with the GC genotype (64%), compared with the control subjects (45%) (p ⫽ 0.06). Assessment of IL6 G-174C Among Parents of SIDS Infants Differences in the distribution of IL6 G-174C among parents of SIDS infants were not statistically significant compared with their respective ethnic control populations. Parents of SIDS infants recruited from Britain showed an increased proportion of individuals with the GC genotype compared with the control subjects, but this was not statistically significant (p ⫽ 0.23). Parents of SIDS infants recruited from Australia had a similar distribution to their control population (p ⫽ 0.92), with the majority of individuals possessing the GC genotype. Distribution of IL6 G-174C in Different Ethnic Groups The distribution of IL6 G-174C varied significantly among individuals from different ethnic groups. The majority of both British and Australian control populations had either the GG or GC genotype, and fewer than 20% of individuals had the CC genotype. There were no differences in the distribution of IL6 G-174C between British and Australian control populations; therefore, these data were combined (European Caucasians) for further comparison with the Bangladeshi and Aboriginal Australian populations. The distribution of IL6 G-174C differed significantly between the European Caucasian group and both Bangladeshis (p ⬍ 0.01) and Aboriginal Australians (p ⬍ 0.01). Approximately 90% of the Bangladeshi and Aboriginal Australian populations were of the GG genotype. Effect of Smoking, Gender, and IL6 G-174C on IL-6 Median Responses The IL-6 responses to 0.01 ␮g ml⫺1or 1.0 ␮g ml⫺1 endotoxin were assessed with leukocytes from Caucasian British donors. There was a median twofold increase in IL-6 responses when endotoxin was increased from 0.01 ␮g ml⫺1 to 1.0 ␮g ml⫺1. In Vitro Stimulation with 0.01 ␮g mlⴚ1 Endotoxin For nonsmokers, the median IL-6 responses observed by genotype were: GG, 4.9 ng/ml⫺1; GC, 6.3 ng/ml⫺1; CC, 8.9 ng/ml⫺1. For smokers, the median IL-6 responses observed by genotype were: GG, 3.5 ng/ml⫺1; GC, 8.4 ng/ml⫺1; CC, 2.4 ng/ml⫺1. The highest median IL-6 response to stimulation with 0.01 ␮g ml⫺1 of endotoxin was observed with leukocytes from nonsmokers with the CC genotype (Figure 1). The response of smokers with the CC genotype was markedly lower; however, the difference was not significant (p ⫽ 0.08). For donors with FIGURE 1 The effect of smoking on median interleukin 6 (IL-6) responses elicited by endotoxin (0.01 ␮g ml⫺1) stimulation of cells obtained from donors in relation to IL6 G-174C genotype (outside values omitted). the GG genotype, nonsmokers exhibited a significantly higher median IL-6 response compared with smokers (p ⬍ 0.05). For smokers, individuals with the GC genotype produced a higher median IL-6 response than those with the GG genotype (p ⫽ 0.01) or CC genotype (p ⬍ 0.01). Although the median IL-6 responses for males (n ⫽ 46, 7.5 ng/ml⫺1) were higher than that for females (n ⫽ 73, 4.9 ng/ml⫺1), the difference was not significant (p ⫽ 0.30). No significant differences between males and females were observed when data were assessed for smoking status and/or gender. In Vitro Stimulation With 1.0 ␮g mlⴚ1 Endotoxin For nonsmokers, the median IL-6 responses observed by genotype were as follows: GG, 11.5 ng/ml⫺1; GC, 15.6 ng/ml⫺1; CC, 15.8 ng/ml⫺1. For smokers, the median IL-6 responses observed by genotype were: GG, 21.3 ng/ml⫺1; GC, 16.8 ng/ml⫺1; CC, 19.0 ng/ml⫺1. Although the median IL-6 responses to 1.0 ␮g ml⫺1 of endotoxin were higher for smokers of all three genotypes, the differences were not significant (Figure 2). The highest median IL-6 responses were observed for smokers with the GG genotype, approximately twofold higher than those of nonsmokers with the GG genotype. The responses of nonsmokers were highly variable and the difference was not significant (p ⫽ 0.71). Although the median IL-6 responses for males (n ⫽ 46, 14.2 ng/ml⫺1) were lower than that for females (n ⫽ 73, 14.7 ng/ml⫺1), the difference was not significant (p ⫽ 0.68). No significant differences between males and females were observed when data were controlled for smoking status and/or gender. Interleukin-6 and Sudden Infant Death Syndrome 823 their immediate families. If IL6 G-174C was associated with SIDS, we would expect to see an association, or trend, within parents of SIDS infants. It is unclear whether the SIDS infants of these parents had an association with IL6 G-174C which was unable to be detected, or if there is no association within this cohort of infants. FIGURE 2 The effect of smoking on median interleukin 6 (IL-6) responses elicited by endotoxin (1 ␮g ml⫺1) stimulation of cells obtained from donors in relation to IL6 G-174C genotype (outside values omitted). DISCUSSION Four observations prompted this study. The first was the finding of high levels of IL-6 in CSF of SIDS infants compared with infants who died of accidental or nonaccidental causes [4]. The second was the report that cells from individuals with the GG genotype of IL6 G-174C produced higher levels of IL-6 in vitro[18]. In addition, two risk factors for SIDS, namely, overheating and male gender, were also associated with increased levels of IL-6 [11, 16]. In this study, the distribution of IL6 G-174C was assessed for associations with SIDS. The effect of IL6 G-174C on IL-6 responses to endotoxin was also assessed in relation to gender and exposure to cigarette smoke. Distribution of IL6 G-174C in SIDS Infants and Parents Among SIDS infants, the distribution of IL6 G-174C differed for the populations examined and these results emphasise the need for appropriate local ethnically matched control subjects for genetic studies. The Australian population was the only SIDS group for whom appropriate control data were available. The distribution of IL6 G-174C for the Australian control population differed significantly from that observed for Australian SIDS infants. Only 38% of control subjects had the GG genotype compared with 58% SIDS infants. The distribution of the combined SIDS group did not differ significantly from the combined European Caucasian population; however, there were differences in the distribution among the SIDS infants from various European regions. There were no significant differences between the distribution of IL6 G-174C between SIDS parents and parents who had not had a SIDS death in Distribution of IL6 G-174C Among Different Ethnic Groups Major differences in the distribution of IL6 G-174C were observed among ethnic groups assessed. The distribution of the Bangladeshi and the Aboriginal Australian groups did not differ significantly. There was a significant increase in the proportion of individuals with the GG genotype, thought to be associated with increased IL-6 production, when compared with the combined European Caucasian group. Bangladeshis who live in Britain have a significantly lower incidence of SIDS, compared with British Caucasians [20]. If the GG genotype of IL6 G-174C is strongly associated with SIDS, an increase in incidence of SIDS in Bangladeshis would be observed. It is probable that other contributing risk factors, genetic or environmental, also interact with IL6 G-174C to lead to the cause of SIDS. Gene– environment interactions are likely to be implicated in complex diseases including SIDS. The effect of cigarette smoke on IL-6 responses might be relevant to infants heavily exposed to smoky environments. One study found the body fluid of some infants had levels of cotinine (a metabolite of nicotine) equivalent to those of smokers [24]. Previous studies indicate that cigarette smoke significantly reduced anti-inflammatory IL-10 responses, particularly for leukocytes of individuals with the genotype associated with low IL-10 production [15]. The genotype associated with low IL-10 responses was predominant among both Bangladeshi and Australian Aboriginal populations [15]. It was proposed that the high proportion of smokers among Aboriginal women [25] and the low proportion of smokers among Bangladeshi women [26] might help explain the differences in the incidence of SIDS observed for these two groups. Effects of Gender, Cigarette Smoke, and IL6 G-174C on IL-6 Responses Three previous studies using a variety of experimental protocols reported conflicting results for IL-6 responses associated with genotypes of IL6 G-174C [16, 18, 27]. The results observed in our in vitro studies also conflict with previous findings. Fishman et al. conducted transient transfection experiments in HeLa cells with constructs of the IL6 G–174C polymorphism. The cells were stimulated with 10 ␮g ml⫺1 endotoxin [18]. Our model used lower concentrations of endotoxin, and donor cells were stimulated as opposed to transfected cell lines. The 824 use of transfected cells with different genotypes of the IL6 polymorphism for analysis would eliminate individual variation resulting from other genetic factors, cigarette smoke exposure, time of collection, or an asymptomatic infection. In 102 fasting healthy volunteers, there was an association found between the genotypes and IL-6 plasma concentrations similar to the results observed for transfected cell lines; the highest levels were found for the GG genotype, intermediate levels for the GC and lowest levels for CC [18]. Endler et al. challenged 76 healthy adult males intravenously with 2 ng kg⫺1 E. coli endotoxin, which elicited increases in plasma IL-6; however, there was no association between IL-6 levels and genotype [27]. Heesen et al. [16] found that the highest of IL-6 responses elicited by E. coli endotoxin from leukocytes of 86 individuals in an in vitro assay were associated with the CC genotype. Intermediate responses were associated with the GG genotype and the lowest levels with the heterozygote. The leukocytes in the study by Heesen et al. were stimulated with 0.1 ␮g ml⫺1 endotoxin and were assessed for IL-6 after an exposure period of 4 hours. In contrast, samples in our study were stimulated with 0.01 ␮g ml⫺1 or 1.0 ␮g ml⫺1 endotoxin and IL-6 levels determined after 24 hours. The finding by Heesen et al. [16] that males had higher IL-6 responses than females was not confirmed by the results of this study. Although we found median IL-6 responses to 0.01 ␮g ml⫺1 endotoxin were higher for males (n ⫽ 46, 7.5 ng/ml⫺1) compared with females (n ⫽ 73, 4.9 ng/ml⫺1) the differences were not significant, and there were no differences noted for responses elicited by 1 ␮g ml⫺1 cells. The conflicting results observed between studies highlight the need for a uniform model to assess cytokine responses to surrogate infections. Concentration, type of stimulant and length of stimulation also need to be comparable. In contrast to previous studies by Heesen et al. and Endler et al., information on smoking status was available in our studies, but not the number of cigarettes smoked per day. In retrospect, the effect of dose needed to be considered to assess the effect of cigarette smoke on inflammatory responses. A quantitative assessment of exposure to cigarette smoke (e.g., measurement of serum cotinine), for both smokers and nonsmokers would have helped to dissect the effects of cigarette smoke exposure. When samples were stimulated with 1.0 ␮g ml⫺1 endotoxin, no significant differences in IL-6 responses were observed between genotypes, or for smoking status. When samples were stimulated with 0.01 ␮g ml⫺1 endotoxin, significant differences in IL-6 responses were observed between genotypes for nonsmokers, and also for smoking status for the GG genotype. Our study found an opposite trend to that reported by Heesen et al. [16]. We observed that cells from nonsmokers with the CC geno- S.M. Moscovis et al. type had the highest IL-6 response to endotoxin (p ⬍ 0.08). The small number of donors with this genotype needs to be considered in interpreting the data. Although no significant differences were observed between genotypes of nonsmokers, significant differences were observed for smokers. The highest response was observed for smokers with the GC genotype. Our data confirms our previous findings that exposure to cigarette smoke alters cytokine responses more significantly for some genotypes than others [15]. IL-6 has significant effects on the physiology of humans and high levels are associated with risk of fatal infection [21]. The high IL-6 responses observed in SIDS infants [4] could be an important clue to the pathology of SIDS. In this study we observed that Aboriginal Australians, who have one of the highest incidences of SIDS, and Australian SIDS infants, are associated with the genotype that reportedly results in increased IL-6 production in transfected cells. The association of this genotype in Bangladeshis, who have one of the lowest incidences of SIDS, indicates that IL6 G-174C is not the only factor leading to SIDS risk. The interaction of gene polymorphisms, including IL6 G-174C, with cigarette smoke exposure and/or other risk factors for SIDS, requires further analysis. 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