Reduced infection by mycobacteria, including Mycobacterium tuberculosis, may be partly responsible for increased prevalence of allergic and autoimmune diseases in developed countries. In a murine model of innate resistance to mycobacteria, the Nramp1 gene has been shown to affect asthma susceptibility. From this observation, it was proposed that human NRAMP1 may be a modulator of asthma risk in human populations. To experimentally test the candidacy of NRAMP1 in asthma susceptibility, we characterized five genetic variants of NRAMP1 (5′CAn, 274C>T, 469+14G>C, D543N, and 1729+del4) in an asthma family-based cohort from northeastern Quebec. We did not observe any significant association between NRAMP1 variants (either allele or haplotype specific) with asthma, atopy, or serum immunoglobulin E levels. These results demonstrate that, in spite of direct involvement of Nramp1 in a murine asthma model, in human populations NRAMP1 is not likely to be a major contributor to the genetic etiology of asthma and asthma-related phenotypes.
In 1989, David Strachan reported the findings of a prospective study of 17 414 British children from birth to 23 years of age.1 The study found a significant inverse correlation between the number of older siblings in the household and the prevalence of hay fever at age 11 and 23 years. A similar correlation was also observed for eczema in the first year of life. Subsequent epidemiologic studies confirmed the inverse association between family size and atopic markers such as skin prick positivity and specific immunoglobulin (Ig) E titers.2 To explain the inverse correlation between family size and atopy, it was suggested that declining family size, improved household amenities, and higher standards of personal cleanliness, all associated with improved hygiene, had reduced the opportunities of cross-infection, and this may have increased the prevalence of atopic diseases. This explanation is now known as the hygiene hypothesis and it has been extended to include autoimmune diseases in general.
One proposed mechanism underlying the hygiene hypothesis is that microbial exposures to viruses, bacteria, and parasites elicit an immune response towards the maturation of T helper type 1 cells (Th1), and the subsequent production of cytokines such as interleukin (IL)-2 and interferon-γ. This predominating Th1 immune response suppresses the production of Ig E and IgG1, as well as cytokines IL4, IL5, IL9, and IL13, characteristic of type 2 (Th2) responses. The possibility of a Th1/Th2 imbalance and an association between Mycobacterium tuberculosis infection and atopy expression was first shown by a study of Japanese school children.3 The study reported an inverse association between delayed hypersensitivity to tuberculin, a broad measure of exposure to mycobacteria (Th1 response), and serum IgE levels (Th2 response), and concluded that exposure to M. tuberculosis may inhibit the development of atopic diseases. In humans, M. tuberculosis is the cause of tuberculosis (TB). However, not all individuals exposed to M. tuberculosis will become infected, and of those infected only a small proportion will develop clinical disease.4, 5 It is now well established that both genetic and environmental factors determine the progression from exposure to infection and from infection to disease. If M. tuberculosis infection protects against atopy, then genetic risk factors for TB susceptibility may be protective for asthma and vice versa. A well-established TB susceptibility gene is the natural resistance associated macrophage protein 1 gene (NRAMP1). In addition to its known role in TB susceptibility, its associations with numerous immune-mediated disorders such as rheumatoid arthritis, type I diabetes, and multiple sclerosis are making NRAMP1 a prime candidate to test the hypothesis of inverse genetic control of asthma and TB susceptibility.6, 7
Data from the mouse support the candidacy of NRAMP1 as an asthma susceptibility gene. It has been shown that Nramp1-resistant (Nramp1r) mice have a lower IgE and IL-4 response compared to Nramp1-susceptible (NRAMP1s) mice after infection with an attenuated strain of Salmonella typhimurium,8 demonstrating that Nramp1 can modulate Th1/Th2 host responsiveness. Moreover, in M. vaccae and allergen-sensitized mice, subsequent allergen challenge triggered higher levels of Th2 cytokines (IL-4, IL-5, IL-13) and IgE in Nramp1s as compared to Nramp1r mice.9 This finding implies that the ability to develop atopy-associated Th2 responses is dependent on susceptibility to infection, dictated in this model by Nramp1. Finally, M. vaccae is more efficient in lowering allergic and asthmatic symptoms in allergen-challenged Nramp1s than in Nramp1r mice,10 directly demonstrating that Nramp1 can modify immune responses following mycobacterial infection. Taken together, these observations provide a direct experimental link between genetically controlled resistance to infection and altered asthma susceptibility, and constitute the main motivating force for the present study.
Clinical characteristics of the study participants have been reported previously.11 Briefly, 1139 individuals between ages 3 and 88 years were recruited. The median age of onset for index cases and their affected siblings is 5 years (2–46 years). Of the 570 subjects with asthma and 569 without asthma, 419 (74%) and 218 (38%) were atopic, respectively. The male to female ratios in probands, affected and unaffected family members are 1 : 1.2, 1 : 1.4, and 1 : 1.2, respectively. Index cases have higher IgE levels and coexistence of atopy compared to other affected family members.
Genetic variants of the NRAMP1 locus
We characterized five polymorphisms of the NRAMP1 gene in the family-based cohort (Figure 1). Variant 5′(CA)n is a promoter dinucleotide repeat polymorphism; variant rs2276631 (reference SNP identifier from the National Center for Biotechnology Information database) is a synonymous C>T polymorphism in exon 3 (common alias 274 C>T); rs3731865 involves a G>C base change in intron 4 (common alias 469+14G>C); variant D543N is a G>A substitution resulting in an aspartic acid to asparagine amino-acid change in exon 15, and variant 1729+55del4 is a TGTG tetranucleotide deletion polymorphism in the 3′ untranslated region (Table 1). All variants have previously been described.32 NRAMP1 variants were selected due to their known associations with susceptibility to infectious and autoimmune diseases. In the French–Canadian families, variants in the 5′ NRAMP1 region (5′(CA)n, 274C>T, and 469+14G>C) are polymorphic, with minor allele frequencies of 0.31, 0.28, and 0.31, respectively, and all variants are in Hardy–Weinberg equilibrium. Variants 1729+del4 and D543N were uninformative (minor allele frequency = 0.012 and 0.017, respectively). Hence, these two 3′ end variants were not further analyzed.
Family-based association analysis of NRAMP1 with asthma, atopy, and IgE levels
The three informative NRAMP1 polymorphisms located in the 5′ region of the gene were tested individually for association with asthma, atopy, and IgE levels (Table 2). IgE serum levels were analyzed as dichotomous trait, independent of atopy status. Subjects were classified either as high or low responders according to their IgE levels. Based on the normal distribution of the log (IgE) values, a cutoff point of 100 mg/l divided the subjects into low (two-thirds of participants) and high (one-third) responders. We tested allelic associations under additive and dominant genetic models. No allele was significantly (P<0.05) transmitted nonrandomly to offspring with asthma or atopy, or to high IgE responders. The 469+14G allele, under an additive model, was nonsignificantly overtransmitted to asthmatic offspring (P=0.08).
Haplotype-specific association analysis
Association among the three variants was assessed by measuring pairwise linkage disequilibrium (LD) using D′. The three variants are strongly associated, as evidenced by a high degree of LD (D′>0.90) among them (data not shown). Alleles of the three variants are likely to be transmitted together as groups, or haplotypes. Within this region of high LD, two haplotypes with frequency >0.1 are observed: haplotype (5′(CA)n) allele 3-(274)C-(469+14)G (frequency=0.683) and haplotype allele 2-T-C (0.258). Other less frequently occurring haplotypes are allele 2-C-G (0.021), allele 2-C-C (0.015), and allele 3-C-C (0.010). All haplotypes were assessed for nonrandom transmissions in the asthma families using family-based association testing software (FBAT), version 1.533, 34 (Table 3). Under additive and dominant genetic models, all haplotypes were randomly transmitted to offspring with either asthma, atopy, or high IgE responsiveness.
We examined the possibility that the failure to detect association of NRAMP1 variants with asthma and related phenotypes was due to insufficient power of the study sample. Power was calculated for 60 sets of parameters defined by susceptibility allele frequency (q) and genetic attributable fraction (GAF) for an additive disease model with disease prevalence set at 0.10 (Figure 2). The result shows that with the present cohort of 1139 individuals there is excellent power (>80%) to detect variants across a wide allele frequency range (0.20–0.50) for a heterozygotes odds ratio (HET OR) >1.6. For example, for q=0.20, power >80% is achieved with GAF ≥0.20, corresponding to a HET OR >1.6 (Figure 2). For the low allele frequencies of 0.05 and 0.10, power >80% is achieved for a HET OR >1.8. For the high allele frequency of 0.70, power >80% is achieved for a HET OR >1.7 (data not shown). By contrast, power to detect variants with a HET OR <1.4 is poor (<60%) for the allele frequency range of 0.10–0.70, although such small-risk modifiers may still account for a substantial proportion of cases, especially if the risk variant is present at high allele frequencies. For low allele frequencies <0.05, power is poor even for a HET OR between 1.6 and 1.8. Overall, the results of the power calculation argue against NRAMP1 alleles being asthma susceptibility factors with relative risk >1.8 in the French Canadian population.
In light of the hygiene hypothesis and the Th1/Th2 paradigm in TB and asthma pathogeneses, the established TB susceptibility gene, NRAMP1, is a strong candidate gene for asthma susceptibility. In mice, the Nramp1 gene encodes a 90–100 kDa transmembrane protein and its mRNA is expressed in primary macrophages and granulocytes. The Nramp1 protein is found at the late endosomal/lysosomal compartment of macrophages.35, 36 Nramp1 is a major determinant of innate host resistance to infection. The gene affects intracellular replication of a wide range of pathogens including S. typhimurium,37 Leishmania donovani,38 M. lepraemurium,39 M. intracellulare,40 M. avium,41 and the TB vaccine strain M. bovis — Bacillus Calmette-Guérin (BCG).42 Specifically, a glycine-to-aspartic-acid change at amino acid 169 (G169D), located in the predicted transmembrane domain number 4 (TM 4), has removed the host's ability to inhibit pathogen growth.43, 44 Comparative sequence analysis of the Nramp gene family suggested that Nramp1 functions as a divalent cation transporter.45 Kinetic studies demonstrated that Nramp1 transports cations out of the phagosomes, and, consequently, mediates depletion of nutrients potentially essential for survival of pathogens in the host cell phagosome.46, 47 In addition, Nramp1 is critical to overcome pathogen-triggered blockages of intracellular vesicle trafficking.48 The exact function of human NRAMP1 is not known. However, due to its high sequence homology with mouse Nramp1 (93% overall sequence similarity and 88% sequence identity), the NRAMP1 protein is likely to be a divalent cation transporter.49, 50, 51, 52, 53 The amino-acid sequences of Nramp1 and NRAMP1 in predicted TM 4 are identical, but the homologous G169D variant in TM 4 has not been found in human NRAMP1.49 However, genetic variants at the 5′ and 3′ ends of the human gene are associated with infectious and autoimmune disease susceptibility.6, 7
Based on the previous findings in a murine model of asthma demonstrating that the propensity to mount an atopic immune response is dependent on Nramp1, we carried out a family-based association study to test if, in humans, NRAMP1 genetic variants are associated with asthma and related phenotypes, such as atopy and IgE levels. We selected five NRAMP1 variants that had previously been shown to be associated with susceptibility to a variety of immune disorders and infectious diseases (Table 1). Specifically, polymorphisms in the 5′ region of NRAMP1 have been found to be risk modifiers for TB in multiple populations. This suggests that a variant located in the 5′ NRAMP1 region impacts on NRAMP1 function. The 5′(CA)n promoter variant has been linked to variable NRAMP1 mRNA expression.54, 55 Promoter allele 3 drives a higher mRNA expression compared to other alleles such as allele 2 (Table 1) in the absence of any stimulant. When stimulated with interferon-γ, alleles 2, 3, and other rare alleles demonstrate enhanced mRNA expression. When co-stimulated with interferon-γ and bacterial antigen lipopolysaccharide, expression by allele 2 is reduced, while that of allele 3 is further enhanced.54 Interestingly, allele 3 has been found to be associated with TB protection, and with risk for type I diabetes, an autoimmune disorder.6
Despite supportive evidence from a mouse model of atopic disease, we failed to detect an impact of human NRAMP1 on asthma and related phenotypes. Our findings suggest that NRAMP1 genetic variants do not play a major role in human atopic disease. Detailed power calculation indicated that our cohort is of sufficient size (power >80%) to detect associations of allelic variants with weak impact on disease risk (HET OR >1.6). However, cohort size is insufficient (power <60%) to detect variants with very low impact on disease risk (HET OR <1.4). The inability to detect genetic risk factors with such low impact on disease risk is not unique to the present study. Due to unfavourable cost–benefit ratios, genetic studies are rarely powered to detect risk variants with OR <2.
The reason why we could not replicate the mouse findings in a human population is unknown. However, it is becoming increasingly clear that the majority of common human diseases are multifactorial and complex, and that animal systems might not be able to accurately model all aspects of human diseases. In the case of NRAMP1, differences between the mouse model of innate resistance/susceptibility to mycobacteria and human mycobacterial diseases are well known. In mice, Nramp1 controls intracellular replication of several atypical mycobacteria and BCG, but does not seem to affect resistance to M. tuberculosis.56, 57 In humans, NRAMP1 has been shown in multiple studies to be a risk modifier of TB. This species-dependent permissiveness in mycobacterial replication may be due to dosage and route of pathogen administration in mice that do not accurately mimic the natural infection in humans. Likewise, if resistance to M. tuberculosis is under different genetic controls in mice and humans, it is possible that asthma susceptibility may also be under different control in mice and humans. Another major difference between mouse Nramp1 and human NRAMP1 is the tissue-specific gene expression. In humans, NRAMP1 mRNA expression is more pronounced in the lung than in the spleen and liver,49 whereas in mice, Nramp1 mRNA is expressed strongly in the spleen and liver, with almost no detectable expression in the lung.46, 58, 59, 60 Since M. tuberculosis infection in the mouse manifests itself as progressive lung disease, low or absent Nramp1 expression in the lung of mice coincides with their inability to control M. tuberculosis infection. It is possible that similar, but presently unknown differences, in tissue expression impact on asthma susceptibility in mice and humans. Finally, the mouse findings that provided the rationale for our study used M. vaccae as the mycobacterial stimulant. At present, the effect of NRAMP1 on M. vaccae susceptibility in humans is unknown and it is possible that, like M. tuberculosis, M. vaccae is under different genetic controls in mice and humans.
In conclusion, the results of our study have two implications. First, even in well-developed animal models of complex human diseases such as atopy, the genetic control elements may differ between humans and the model system. Second, it seems unlikely that a single inverse relationship exists between variants that predispose to asthma/atopy and those that predispose to TB. Hence, reduced M. tuberculosis infection may not be the driving force behind increased asthma/atopy prevalence in developed countries, and, if it is, the genetic mechanism is not likely to have included NRAMP1.
Patients and methods
Families are from the Saguenay-Lac-St-Jean region of northeastern Quebec, Canada. The recruitment scheme has been described previously.11 Briefly, probands were recruited if they fulfilled at least two of the following three criteria: (1) a minimum of three clinic visits for acute asthma within 1 year; (2) two or more asthma-related hospital admissions within 1 year; or (3) steroid dependency, as defined by either 6 months of oral, or 1 year of inhaled corticosteroid use. Families were included for study if at least one parent was available for phenotypic assessment, at least one parent was unaffected, and all four grandparents were of French Canadian origin. When possible, grandparents and other relatives were also recruited to the study.
The affection status of all study participants was determined by clinical evaluation and the completion of a standard respiratory questionnaire that was modified to include questions about asthma and atopy severity, family history of asthma and/or atopy, age-of-onset and the presence of other respiratory failure diagnoses.61 We defined participants as asthmatics if (1) a reported history of asthma (questionnaire-based) and a history of physician-diagnosed asthma (past/current) were available, or (2) confirmation of diagnosis by a positive methacholine provocation test was obtained (only on subjects older than 12 years of age). Subjects were deemed atopic if they had at least one positive response (wheel diameter ≥3 mm at 10 min) to skin-prick tests. The family participation rate was approximately 60% and all subjects gave informed consent. A total of 223 independent families (1139 individuals) with family size ranging from 3 to 17 and number of affected family members (including probands) ranging from 1 to 10 were analyzed.
Polymorphism selection and genotyping
Five polymorphisms of the NRAMP1 gene were selected based on their known association with disease. Two of the variants have reference SNP identifiers (rs#) from the NCBI database. In this report, we referred to the common aliases of the variants: 5′(CA)n, 274C>T, 469+14G>C, D543N and 1729+del4.32 Variant 5′(CA)n was genotyped by length polymorphism analysis using ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems, CA, USA). Primers for amplifying polymerase chain reaction (PCR) products were designed using Primer3 software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). PCR products of sizes ranging from 168 basepairs (bp) to 182 bp were amplified using primer pair: IndexTerm5′-AACGAGGGGTCTTGGAACTC-3′ and IndexTerm5′-gcctcccaagttagctctga-3′. PCR reactions were carried out in PTC-100® Peltier thermal cyclers (MJ Research, MA, USA) under the following condition: 10 ng of genomic DNA was added to 20 μl reaction mixture containing 1 × PCR buffer, 2.5 mM of MgCl2, 0.5 U of Platinum Taq polymerase (Qiagen, CA, USA), 0.50 mM of dinucleotides and 0.30 μM of primers. PCR was initiated by denaturing the samples at 96°C for 10 min, followed by 30 cycles of denaturation at 96°C for 25 s, annealing at 67°C for 1 min. Final extension was carried out at 72°C for 5 min. Finally, 1 μl of PCR product was mixed with 0.30 μl of formamide and 10 μl of GeneScan™–500 Liz™ Size Standard (Applied Biosystems, CA, USA) before being denatured at 95°C for 5 min and injected into ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems, CA, USA). Fluorescence signal was read and analyzed using ABI PRISM® GeneMapper™ Software version 3.5 (Applied Biosystems, CA, USA).
Variants 274C>T and 469+14G>C were genotyped using HEFP™ (Molecular Devices),62 a single-base extension (SBE) fluorescence polarization platform, as previously described.11 Briefly, PCR and SBE primers were designed using the Primer3 software. PCR reactions were carried out using primer set 1 (274C>T): IndexTerm5′-GCCAGCCTGAAGATCTGACT-3′, IndexTerm5′-GGACCCCCTCACTCTACTCC-3′ and set 2 (469+14G>C): IndexTerm5′-ATCGTGGAAGCTGAAAATGG-3′, IndexTerm5′-GCGAGGTCTGCCATCTCTAC-3′. A total of 6 ng of genomic DNA was added to 8 μl reaction mixture containing 2.5 mM of MgCl2, 25 mM of dinucleotides, 0.2 U of HotstartTaq DNA polymerase (Qiagen), and 100 nM of primers. PCR was initiated by denaturing the samples at 94°C for 15 min, followed by 45 cycles of denaturation at 94°C for 30 s, annealing at 56°C (primers specific) for 30 s and extension at 72°C for 30 s. Final extension was done at 72°C for 6 min. PCR products were treated with Exonuclease I and Shrimp Alkaline Phosphatase as recommended by the manufacturer (AcycloPrime-FP SNP Detection Kit, Perkin-Elmer, Wellesley, MA, USA). SBE detection primers used were (274C>T): (sense) IndexTerm5′-GGAAAGCAATGCTCATGAG-3′, (antisense) IndexTerm5′-TTCACGGGGCCTGGCTT-3′, (469+14G>C): (sense) IndexTerm5′-TGGTTCTCCCTGTCCAGG-3′ and (antisense) IndexTerm5′-TAAGGTGAGCTTGGGGG-3′. FP-SBE reactions were performed in one or both orientations as suggested by the manufacturer (AcycloPrime-FP SNP Detection Kit, Perkin-Elmer, Wellesley, MA, USA). After the addition of reading buffer, the plates were read using the Analyst HT® reader (Molecular Devices, CA, USA) as described previously.63
Variants D543N and 1729+55del4 were genotyped by TaqMan assays.64 Each variant was analyzed using two sets of oligonucleotides (external primers and internal probes) designed using the Primer3 software. The internal probes were labelled with fluorescent dyes: TAMRA (6-carboxytetramethyl-rhodamine) at 3′ ends, FAM (6-carboxy-fluorescein) and TET (6-carboxy-4,7,2′,7′-tetrachlorofluorescein) (one per oligonucleotide) at 5′ ends. For variant D543N, primers set IndexTerm5′-CCACCACCACTTCCTGTATG-3′ and IndexTerm5′-CACGTCATACATGCCACTCC-3′ and probes set IndexTerm5′-FAM-CCCTTTCTGGTCCTCTTCAAGGA-TAMRA-3′ and IndexTerm5′-TET-CCCTTTCTGGTTCTCTTCAAGGAGC-TAMRA were used. For variant 1729+55del4, primers set IndexTerm5′-GGGAGTGGCATGTATGACG-3′ and IndexTerm5′-TCTATCCTGCTGCCTGCAC-3′, and probes set IndexTerm5′-FAM-TGGCCTGCTGGATGTGGAGTAMRA and IndexTerm5′-TET-TGACTGGCCTGCTGGAGAGGTAMRA were used. For both variants, 10 ng of genomic DNA were added to a 20 μl reaction mixture containing 1 × PCR buffer, 5.0 mM of MgCl2, 0.2 mM of each nucleotide, 0.5 U of HotstartTaq DNA polymerase (Qiagen), 0.03 μM of probes, and 0.30 mM of primers. PCR was initiated by denaturing the samples at 96°C for 10 min followed by 40 cycles of denaturation at 96°C for 30 s, annealing and extending at 60°C for 1 min. Final extension was carried out at 72°C for 5 min. PCR endpoint fluorescence reading was carried out using ABI PRISM® 7700 Sequence Detector System (Applied Biosystems, CA, USA). The fluorescence intensity was adjusted and recorded using Sequence Detector Software version 1.7 (Applied Systems, CA, USA).
Hardy–Weinberg equilibrium was tested in a subset of independent DNA samples (parents of probands) using HAPLOVIEW.65 Allele distribution patterns were assessed by FBAT, (version 1.5).33, 34 This software uses an empirical variance–covariance estimator to account for the possibility of nonindependent allelic transmission to affected sibs.66 Asthma, atopy and IgE level phenotypes were tested separately under additive and dominant genetic models.
Associations between variants were assessed by calculating D′, a measurement of the LD strength67 using HAPLOVIEW.65 Based on the LD strength of variants, haplotypes were inferred and assessed for nonrandom transmission using the ‘hbat’ command of FBAT version 1.5.33, 34 An empirical variance estimator was used.66 Asthma, atopy, and IgE level phenotypes were tested separately under additive and dominant genetic models.
To assess whether the cohort size has sufficient power to detect association between variants and phenotypes, we used Power calculation of the FBAT (PBAT).68, 69 The family design was based on the observed count of nuclear families, according to the number of affected sibs, unaffected sibs, and missing parents. The genetic models considered assumed a constant population prevalence of 0.10, varying susceptibility allele frequencies (q) and varying GAF of the allele GAF, under an additive model for risk. GAF corresponds to a reduction of incidence in the study families when the risk variant is removed, that is, a GAF of 5% indicates that removal of the risk variant would reduce incidence by 5% in the study families. We used values for q ranging from 0.05 to 0.70 and values for GAF ranging from 0.05 to 0.50. For an additive disease model with prevalence at 0.10, the odds ratio for the heterozygotes was calculated for each q and GAF parameter set. The level of significance was set at 0.05.
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We thank all families for their enthusiastic participation in this study. We would like to thank D Gagné and P Bégin for their invaluable participation in the ascertainment of the subjects. We thank Y Renaud, M Girard, and A Verville for technical assistance. We thank A Alcaïs (INSERM U550, Paris) for his comments on statistical analysis. This study was supported by the Canadian Genetic Diseases Network—Network Centres of Excellence (CGDN – NCE). C Laprise is supported by the Fonds pour la recherche en santé du Québec (FRSQ). TJ Hudson is recipient of an Investigator Award from CIHR and a Clinician-Scientist Award in Translational Research from the Burroughs Wellcome Fund. E Schurr is an Investigator of the Canadian Institutes of Health Research.
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Poon, A., Laprise, C., Lemire, M. et al. NRAMP1 is not associated with asthma, atopy, and serum immunoglobulin E levels in the French Canadian population. Genes Immun 6, 519–527 (2005). https://doi.org/10.1038/sj.gene.6364238
- genetic predisposition
- natural resistance-associated macrophages protein 1
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