APMIS 123: 169–174
© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12334
No association between rs6897932 in the gene encoding interleukin-7 receptor a and low-grade inflammation or self-reported health – results from the Danish Blood Donor Study HANS J. HARTLING,1,2 CECILIE J. SØRENSEN,3 ANDREAS S. RIGAS,1 KRISTOFFER S. BURGDORF,1 SUSANNE D. NIELSEN,2 OLE B. PEDERSEN,4 MIKKEL S. PETERSEN,3 3 LISE W. THØRNER,1 HENRIK HJALGRIM,5 ERIK SØRENSEN,1 SEBASTIAN KOTZE, CHRISTIAN ERIKSTRUP3 and HENRIK ULLUM1 1 Department of Clinical Immunology, Rigshospitalet, Copenhagen University Hospital, Copenhagen; Viro-Immunology Research Unit, Department of Infectious Diseases, Rigshospitalet, Copenhagen University Hospital, Copenhagen; 3Department of Clinical Immunology, Aarhus University Hospital, Aarhus; 4 Department of Clinical Immunology, Næstved Sygehus, Næstved; and 5Department of Epidemiology Research, Statens Serum Institut, Copenhagen, Denmark
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Hartling HJ, Sørensen CJ, Rigas AS, Burgdorf KS, Nielsen SD, Pedersen OB, Petersen MS , Sørensen E, Kotze S, Thørner LW, Hjalgrim H, Erikstrup C, Ullum H. No association between rs6897932 in the gene encoding interleukin-7 receptor a and low-grade inflammation or self-reported health – results from the Danish Blood Donor Study. APMIS 2015; 123: 169–174. The T-allele in the single nucleotide polymorphism rs6897932 in the gene encoding the IL-7 receptor a (IL7RA) is associated with reduced risk of autoimmune diseases including multiple sclerosis and also affects the course of HIV infection. Low-grade inflammation (LGI) and self-reported, health-related quality of life (HRQL) are often associated with chronic diseases and widely used in assessing and monitoring health status. The aim of the present study was to evaluate whether the T-allele in rs6897932 is associated with reduced risk of LGI (hsCRP 3–10 mg/L), history of infectious mononucleosis (IM), and HRQL in healthy individuals. A total of 17, 293 healthy Danish individuals from the Danish Blood Donor Study were included in the analyses. We tested rs6897932 as a predictor of LGI, self-reported IM, and HRQL in univariable and multivariable models stratified by sex. No associations between rs6897932 and LGI, self-reported IM or HRQL were found in men or women. This suggests that rs6897932 is not associated with general inflammation, and the reported associations between the T-allele in rs6897932 with several autoimmune diseases may be mediated through effects on a restricted part of the immune system. Key words: Infectious mononucleosis; interleukin-7 receptor alpha; lowgrade; inflammation; rs6897932; self-reported; health-related quality of life. Hans Jakob Hartling, Viro-immunology Research Unit, Department of Infectious Diseases, University Hospital of Copenhagen, Blegdamsvej 9b, 2100 Copenhagen, Denmark. e-mail: [email protected]
Interleukin-7 (IL-7) is essential for T-cell development, survival, and proliferation. The responsiveness of IL-7 depends on the IL-7 receptor (IL-7R), which is a heterodimer consisting of an a-subunit (IL-7Ra or CD127) and the common c-chain (CD132) (1). The gene encoding IL-7Ra (IL7RA) is polymorphic, and single nucleotide polymorphisms Received 31 January 2014. Accepted 22 September 2014
(SNPs) in IL7RA have recently been associated with a wide range of diseases. The polymorphism, known as rs6897932, has caught attention, inasmuch as genome-wide association studies and metaanalyses document associations between the T-allele in rs6897932 and reduced risk of multiple sclerosis (MS) and type 1 diabetes (2–4) as well as reduced risk of atopic dermatitis and inhalation allergy in smaller cross-sectional studies (5, 6). Furthermore, 169
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the T-allele in rs6897932 affects the course of both untreated (7) and treated HIV infection (8). C-reactive protein (CRP) is a non-specific marker of systemic inflammation (9) and is routinely used in the clinical setting with a cut-off at 10 mg/L (10). However, the high-sensitivity C-reactive protein (hsCRP) assay has a lower limit of detection of about 0.15 mg/L. A hsCRP level above 3 mg/L and below 10 mg/L is associated with increased risk of atherosclerosis, future risk of cardiovascular disease, cancer, diabetes, and all-cause mortality (11, 12). A hsCRP of 3–10 mg/L is therefore proposed as a definition of low-grade inflammation (LGI) in apparently healthy individuals and is considered a candidate marker for assessing and monitoring health (13). Health-related quality of life (HRQL) is used to monitor physical and mental health (14). It is a self-reported assessment associated with chronic diseases and is a strong predictor of survival and cardiovascular disease (15, 16). Recently, we have shown that LGI is also a predictor of physical HRQL (unpublished data). Because the T-allele in rs6897932 is associated with reduced susceptibility to chronic autoimmune diseases and affects the course of HIV infection, it is plausible that the T-allele in rs6897932 plays a similar role for LGI and HRQL. Furthermore, infectious mononucleosis (IM) increases the risk of MS (17, 18) and MS is highly associated with rs6879392. To our knowledge, possible associations between rs6897932 and LGI, HRQL, and IM have not been evaluated. We hypothesize that the T-allele in rs6897392 is associated with reduced risk of LGI and selfreported IM as well as a better score in the self-reported HRQL. We therefore determined the rs6897932 allele in a subset of healthy individuals from the Danish Blood Donor Study and evaluated the polymorphism as predictor of LGI, self-reported IM, and HRQL. MATERIALS AND METHODS Study population Study participants were identified in the Danish Blood Donor Study, which is a large, prospective cohort comprising more than 85 000 healthy blood donors as previously described (19). The Danish Blood Donor Study began in March 2010 and is still recruiting participants from the Danish blood banks. At present, DNA has been isolated from more than 20 000 individuals (19), and the rs6897932 alleles have been determined in 18, 148 individuals enrolled between March 1, 2010 and December 31, 2010. All individuals included in the Danish Blood Donor Study period were invited to complete a questionnaire including health-related quality of life as well as a
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question regarding previous diagnosis with IM. Furthermore, hsCRP was measured in 17 235 (95.0%) of the individuals for whom the rs6897932 was known. We excluded individuals with hsCRP above 10 mg/L (N = 162), which suggests an ongoing infection or inflammation (12, 20) or individuals with two or more missing items in HRQL (N = 285), or who reported extreme anthropometric data (height below 100 cm or above 220 cm; or weight below 50 kg or above 200 kg) (N = 408), leaving a total of 17 293 individuals for analyses.
Ethics statement The study was performed in accordance with the ethical guidelines of the Helsinki Declaration and approved by the Scientific Ethical Committee of Central Denmark (M-20090237) and the Danish Data Protection Agency (2007-58-0015).
Genotyping Genotyping was performed by competitive allele-specific PCR using the KASPâ method at LGC Genomics (LGC, Middlesex, UK). The rs6897932 polymorphism was in Hardy–Weinberg equilibrium (HW) (p = 0.071) in the total population (N = 18 148).
hsCRP and low-grade inflammation (LGI) hsCRP was determined in thawed ethylenediaminetetraacetic acid (EDTA) plasma samples on an Ortho Vitrosâ 5600 analyser (Ortho Clinical Diagnostics, Rochester, NY, USA). Low-grade inflammation (LGI) was defined as a hsCRP level of 3–10 mg/L.
Questionnaire including SF-12 and question regarding mononucleosis The questionnaire included questions on baseline characteristics (including anthropometric measure, smoking status, and menstruation cycle), and the 12-item ‘short-form health survey 12’-questionnaire (SF-12), which yields a physical (PCS) and a mental component summary score (MCS). PCS and MCS were determined by weighting and summing the individual items and adding the sum to 57.65693 and 60.58847, respectively. Furthermore, we asked whether the donor had ever been diagnosed with IM by a physician. An answer of ‘yes’ was considered to indicate IM with a degree of confidence similar to that used in most studies examining a potential association between self-reported IM and MS (18).
Statistics Logistic regression was used to evaluate the genotype in rs6897932 as a predictor of LGI and self-reported IM, and general linear models were used for the outcomes of MCS and PCS. The analyses were stratified by sex both in univariable models, and in multivariable models with age, obesity (BMI ≥ 30 kg/m2), current smoking, physical activity at work (high vs low), leisure-time physical © 2014 APMIS. Published by John Wiley & Sons Ltd
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activity (high vs low), and current use of oral contraceptive for women, as covariates (20). Results are presented as odds-ratios with 95%-confidence intervals (95% CI) or means (95% CI). Two-tailed p-values below 0.05 were considered significant. All statistical analyses were performed using SAS (version 9.2 SAS Institute, Copenhagen, Denmark).
RESULTS Study population
Characteristics of the study population are presented in Table 1 (20). The study included data from a total of 17 293 individuals: 46.8% women and 53.2% men. A total of 17.1% of women and 15.8% of men were smokers (Table 1). The allele frequencies of rs6897932 in the total study population were 52.6% CC (N = 9092), 39.4% CT (N = 6820), and 8.0% TT (N = 1381) resulting in HW (p = 0.042). The ideal genotype distribution for the population according to the HW principle (p2 + 2pq + q2 = 1) was 52.3% CC (N = 9039), 40.0% CT (N = 6926), and 7.7% TT (N = 1326).
Table 1. Characteristic of the cohort stratified by sex Women N = 8092 Men N = 9201 37.6 (27.2–47.9) 40.1 (30.5–49.9) Age, years, median (IQR) Rs6897932, N (%) CC 4245 (52.5%) 4847 (52.7%) CT 3205 (39.6%) 3615 (39.3%) TT 624 (7.9%) 739 (8.0%) Current smoking, 1356 (17.1) 1431 (15.8) yes, N (%) 23.5 (21.6–26.2) 25.1 (23.4–27.5) BMI, kg/m2, median (IQR) 790 (9.8%) 968 (10.5%) Obesity, BMI≥30 kg/m2, N (%) PCS, points, 56.1 (53.8–57.4) 56.2 (53.8–57.3) median (IQR) MCS, points, 54.2 (50.0–56.7) 54.8 (51.2–56.8) median (IQR) 1709 (21.1%) 2338 (25.4%) High physical activity, work, N (%) 5350 (66.1%) 6366 (69.2%) High physical activity, leisure, N (%) hsCRP, mg/L, 0.69 (0.19–1.90) 0.42 (0.11–1.10) median (IQR) 1120 (14.6%) 530 (6.1%) LGI (hsCRP>3 mg/L), N (%) 2311 (28.6%) – Combined oral contraceptive pill (yes), N (%) IQR, Interquartile range; hsCRP, high-sensitive C-reactive protein; LGI, Low-grade inflammation. © 2014 APMIS. Published by John Wiley & Sons Ltd
Thus, heterozygotic individuals were slightly underrepresented in the total study population. Men and women did not differ with respect to rs6897932 allele frequencies (p = 0.904). Low-grade Inflammation (LGI) and infectious mononucleosis (IM)
The median hsCRP was 0.69 mg/L (0.19–1.9) in women and 0.42 mg/L (0.11–1.10) in men, and the frequency of LGI was higher in women than in men (13.9% vs 5.7%) as previously reported (20). No effect of rs6897932 alleles on the presence of LGI was found in women or men (Table 2, p > 0.05). Similarly, the rs6897932 allele did not predict IM (Table 2, p > 0.05). PCS and MCS
HRQL was assessed by the physical (PCS) and mental component summary score (MCS). The PCS was 56.1 (53-8–57.4) in women and 56.2 (53.8– 57.3) in men and the MCS was 54.2 (50.0–56.7) in women and 54.8 (51.2–56.8) in men (Table 1). No effect of rs6897932 genotype on PCS or MCS was found in women or men (Table 2, p > 0.05).
DISCUSSION In a large cohort of 17 293 healthy, Danish blood donors the IL7RA, rs6897932, was tested as a predictor of LGI, history of IM, and HRQL. The study population was stratified by sex. No effect of the genotype in rs6897932 was found in the analyses. The T-allele in rs6897392 reduces susceptibility to MS, diabetes type 1, atopic dermatitis, and inhalation allergy and increases the risk of relapse after hematopoietic cell transplantation. However, the rs6897392 T-allele has also been associated with increased risk of chronic inflammatory arthropathies, although this was not confirmed in a genomewide association study (21–23). Thus, the T-allele seems to be protective for inflammatory disease, but the results are ambiguous. Furthermore, we have presented data suggesting an association between the T-allele in rs6897932 and increased mortality in a Zimbabwean cohort of untreated HIV-infected individuals (7), and a faster rate of CD4 T cell increase after initiation of combined antiretroviral therapy in a Caucasian cohort of HIV-infected individuals (8). Finally, gain-of-func tion mutations in IL7RA adjacent to rs6897392 acting as oncogenes were found in childhood T-cell acute lymphoblastic leukemia (24, 25). The
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Table 2. rs6897932 as predictor of LGI, self-reported infectious mononucleosis, PCS and MCS Women Men OR (95% CI) OR (95% CI) OR (95% CI) 1 Adjusted Un-adjusted Un-adjusted rs6897932 LGI CC, N = 604 (14.5%) 1.0 1.0 N = 276 (6.0%) 1.0 CT, N = 425 (13.9%) 0.92 (0.81–1.05) 0.93 (0.80–1.07) N = 217 (6.3%) 1.05 (0.88–1.27) TT, N = 91 (15.0%) 1.01 (0.79-1.28) 1.04 (0.80–1.35) N = 37 (5.3%) 0.88 (0.62–1.25) Self-reported IM CC, N = 549 (13.0%) 1.0 1.0 N = 519 (10.8%) 1.0 CT, N = 383 (12.0%) 0.91 (0.79–1.05) 0.90 (0.78–1.04) N = 384 (10.7%) 0.99 (0.86–1.14) TT, N = 79 (12.3%) 0.94 (0.73–1.21) 0.94 (0.73–1.22) N = 62 (8.4%) 0.76 (0.58–1.01) Mean (95% CI)
Un-adj. p-value,1 Adj. p-value
Mean (95% CI)
OR (95% CI) 1 Adjusted
1.0 1.07 (0.88–1.29) 0.93 (0.65–1.34) 1.0 0.99 (0.86–1.14) 0.76 (0.57–1.01) Un-adj. p-value,1 Adj. p-value
rs6897932 PCS CC 55.0 (54.8–55.1) 0.206, 10.210 54.9 (54.8–55.0) 0.570, 10.468 CT 54.8 (54.7–55.0) 54.8 (54.7–55.0) TT 55.1 (54.7–55.4) 54.9 (54.6–55.2) MCS CC 52.2 (51.9–52.4) 0.790, 10.793 53.4 (53.2–53.5) 0.354, 10.452 CT 52.2 (51.9–52.4) 53.3 (53.1–53.5) TT 52.4 (51.8–52.9) 53.7 (53.2–54.2) LGI, Low-grade inflammation (hsCRP>3 mg/L); IM, Infectious mononucleosis; PCS, Physical component summary score; MCS, Mental component summary score; OR, Odds-ratio; CI, Confidence Interval. 1 Adjusted analysis included age, obesity (BMI≥30 kg/m2), current smoking, physical activity at work, physical activity in leisure, and use of oral contraceptive (only women) as covariates.
rs6897392 thus seems to play a role in T-cell homeostasis and T-cell mediated diseases, and may also affect the general health status. LGI and HRQL are associated with increased risk of chronic diseases for which rs6897392 is predictive (11, 12, 26), and we hypothesized an association between the rs6897932 T-allele and LGI and HRQL. However, we found no evidence to support our hypothesis. Although, IL-7R is essential for thymopoeisis (27), and severe combined immunodeficiency (SCID) patients with a specific mutation in IL7RA was characterized by T-cell lymphopenia (T-B+NK+ phenotype) (28), the full biological function of the rs6897932 polymorphism is not clear. T cells play a major role in several of the diseases (MS, diabetes type 1, HIV infection, T-cell acute lymphoblastic leukemia) associated with rs6897392. However, CRP is a general inflammation marker and primarily a part of the innate immune system and correlates with HRQL as an assessment of a general health status. Thus, the lack of associations between rs6897392 and LGI and HRQL may reflect the non-specificity of LGI and HRQL which are affected by a wide range of diseases and circumstances in which T cells are of minor importance. Furthermore, the results may be affected by the characteristic of the study population, inasmuch as blood donors represent a selected population with respect to chronic diseases, medications, and vacci172
nations. In addition, individuals volunteering as blood donors may share many characteristics. Thus, the hypotheses of the present study are based on immunological studies of diseased individuals, whereas the present study population most likely represents a population of homogeneous, healthy individuals. Confirmation of the presented results is warranted in independent cohorts. We also tested rs6897932 as a predictor of selfreported IM. Primary Epstein-Barr virus infection is typically asymptomatic or subclinical. However, the infection is sometimes accompanied by IM with clinical manifestations such as pharyngitis, cervical lymphadenopathy, fever, hepatomegaly, and splenomegaly (29). A total of 11.5% of the individuals in the present study reported having been diagnosed with mononucleosis. IM is an independent risk factor of MS, and CD8+ T cells play a significant role in the pathogenesis (29). However, we could not document an association between rs6897932 and a history of IM. The biological impact of rs6897932 on MS is not understood, but the present data suggest that the underlying mechanisms are not to be found in IM. Although the study benefits from the large number of participants, a limitation of the study is the aspect of self-reported IM diagnosed by a physician, and the lack of antibody titers to confirm IM. The association between MS and IM is found in data based on self-reported © 2014 APMIS. Published by John Wiley & Sons Ltd
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IM similar to findings based on IM verified by blood samples (18). Irrespective, the present data can be biased by poor recollection of past disease episodes, and EBV-related diseases (such as infection with Cytomegalovirus) can be over-reported. Our findings must therefore be interpreted with caution. In conclusion, the IL7RA rs6897932 was not associated with LGI or self-reported IM, nor with HRQL (PCS or MCS) in healthy individuals. These results suggest that the previously observed association between rs6897932 and several diseases is mediated by the T-cell compartment.
8.
9. 10.
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FUNDING
12.
The Danish Blood Donor Study has been supported by The Danish Council for Independent Research (09-069412) and by Danish Regions (02/2611).
13. 14.
DISCLOSURES The authors have no conflicts to disclose.
15.
We thank the Danish blood donors for the participation in the study.
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© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12334
No association between rs6897932 in the gene encoding interleukin-7 receptor a and low-grade inflammation or self-reported health – results from the Danish Blood Donor Study HANS J. HARTLING,1,2 CECILIE J. SØRENSEN,3 ANDREAS S. RIGAS,1 KRISTOFFER S. BURGDORF,1 SUSANNE D. NIELSEN,2 OLE B. PEDERSEN,4 MIKKEL S. PETERSEN,3 3 LISE W. THØRNER,1 HENRIK HJALGRIM,5 ERIK SØRENSEN,1 SEBASTIAN KOTZE, CHRISTIAN ERIKSTRUP3 and HENRIK ULLUM1 1 Department of Clinical Immunology, Rigshospitalet, Copenhagen University Hospital, Copenhagen; Viro-Immunology Research Unit, Department of Infectious Diseases, Rigshospitalet, Copenhagen University Hospital, Copenhagen; 3Department of Clinical Immunology, Aarhus University Hospital, Aarhus; 4 Department of Clinical Immunology, Næstved Sygehus, Næstved; and 5Department of Epidemiology Research, Statens Serum Institut, Copenhagen, Denmark
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Hartling HJ, Sørensen CJ, Rigas AS, Burgdorf KS, Nielsen SD, Pedersen OB, Petersen MS , Sørensen E, Kotze S, Thørner LW, Hjalgrim H, Erikstrup C, Ullum H. No association between rs6897932 in the gene encoding interleukin-7 receptor a and low-grade inflammation or self-reported health – results from the Danish Blood Donor Study. APMIS 2015; 123: 169–174. The T-allele in the single nucleotide polymorphism rs6897932 in the gene encoding the IL-7 receptor a (IL7RA) is associated with reduced risk of autoimmune diseases including multiple sclerosis and also affects the course of HIV infection. Low-grade inflammation (LGI) and self-reported, health-related quality of life (HRQL) are often associated with chronic diseases and widely used in assessing and monitoring health status. The aim of the present study was to evaluate whether the T-allele in rs6897932 is associated with reduced risk of LGI (hsCRP 3–10 mg/L), history of infectious mononucleosis (IM), and HRQL in healthy individuals. A total of 17, 293 healthy Danish individuals from the Danish Blood Donor Study were included in the analyses. We tested rs6897932 as a predictor of LGI, self-reported IM, and HRQL in univariable and multivariable models stratified by sex. No associations between rs6897932 and LGI, self-reported IM or HRQL were found in men or women. This suggests that rs6897932 is not associated with general inflammation, and the reported associations between the T-allele in rs6897932 with several autoimmune diseases may be mediated through effects on a restricted part of the immune system. Key words: Infectious mononucleosis; interleukin-7 receptor alpha; lowgrade; inflammation; rs6897932; self-reported; health-related quality of life. Hans Jakob Hartling, Viro-immunology Research Unit, Department of Infectious Diseases, University Hospital of Copenhagen, Blegdamsvej 9b, 2100 Copenhagen, Denmark. e-mail: [email protected]
Interleukin-7 (IL-7) is essential for T-cell development, survival, and proliferation. The responsiveness of IL-7 depends on the IL-7 receptor (IL-7R), which is a heterodimer consisting of an a-subunit (IL-7Ra or CD127) and the common c-chain (CD132) (1). The gene encoding IL-7Ra (IL7RA) is polymorphic, and single nucleotide polymorphisms Received 31 January 2014. Accepted 22 September 2014
(SNPs) in IL7RA have recently been associated with a wide range of diseases. The polymorphism, known as rs6897932, has caught attention, inasmuch as genome-wide association studies and metaanalyses document associations between the T-allele in rs6897932 and reduced risk of multiple sclerosis (MS) and type 1 diabetes (2–4) as well as reduced risk of atopic dermatitis and inhalation allergy in smaller cross-sectional studies (5, 6). Furthermore, 169
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the T-allele in rs6897932 affects the course of both untreated (7) and treated HIV infection (8). C-reactive protein (CRP) is a non-specific marker of systemic inflammation (9) and is routinely used in the clinical setting with a cut-off at 10 mg/L (10). However, the high-sensitivity C-reactive protein (hsCRP) assay has a lower limit of detection of about 0.15 mg/L. A hsCRP level above 3 mg/L and below 10 mg/L is associated with increased risk of atherosclerosis, future risk of cardiovascular disease, cancer, diabetes, and all-cause mortality (11, 12). A hsCRP of 3–10 mg/L is therefore proposed as a definition of low-grade inflammation (LGI) in apparently healthy individuals and is considered a candidate marker for assessing and monitoring health (13). Health-related quality of life (HRQL) is used to monitor physical and mental health (14). It is a self-reported assessment associated with chronic diseases and is a strong predictor of survival and cardiovascular disease (15, 16). Recently, we have shown that LGI is also a predictor of physical HRQL (unpublished data). Because the T-allele in rs6897932 is associated with reduced susceptibility to chronic autoimmune diseases and affects the course of HIV infection, it is plausible that the T-allele in rs6897932 plays a similar role for LGI and HRQL. Furthermore, infectious mononucleosis (IM) increases the risk of MS (17, 18) and MS is highly associated with rs6879392. To our knowledge, possible associations between rs6897932 and LGI, HRQL, and IM have not been evaluated. We hypothesize that the T-allele in rs6897392 is associated with reduced risk of LGI and selfreported IM as well as a better score in the self-reported HRQL. We therefore determined the rs6897932 allele in a subset of healthy individuals from the Danish Blood Donor Study and evaluated the polymorphism as predictor of LGI, self-reported IM, and HRQL. MATERIALS AND METHODS Study population Study participants were identified in the Danish Blood Donor Study, which is a large, prospective cohort comprising more than 85 000 healthy blood donors as previously described (19). The Danish Blood Donor Study began in March 2010 and is still recruiting participants from the Danish blood banks. At present, DNA has been isolated from more than 20 000 individuals (19), and the rs6897932 alleles have been determined in 18, 148 individuals enrolled between March 1, 2010 and December 31, 2010. All individuals included in the Danish Blood Donor Study period were invited to complete a questionnaire including health-related quality of life as well as a
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question regarding previous diagnosis with IM. Furthermore, hsCRP was measured in 17 235 (95.0%) of the individuals for whom the rs6897932 was known. We excluded individuals with hsCRP above 10 mg/L (N = 162), which suggests an ongoing infection or inflammation (12, 20) or individuals with two or more missing items in HRQL (N = 285), or who reported extreme anthropometric data (height below 100 cm or above 220 cm; or weight below 50 kg or above 200 kg) (N = 408), leaving a total of 17 293 individuals for analyses.
Ethics statement The study was performed in accordance with the ethical guidelines of the Helsinki Declaration and approved by the Scientific Ethical Committee of Central Denmark (M-20090237) and the Danish Data Protection Agency (2007-58-0015).
Genotyping Genotyping was performed by competitive allele-specific PCR using the KASPâ method at LGC Genomics (LGC, Middlesex, UK). The rs6897932 polymorphism was in Hardy–Weinberg equilibrium (HW) (p = 0.071) in the total population (N = 18 148).
hsCRP and low-grade inflammation (LGI) hsCRP was determined in thawed ethylenediaminetetraacetic acid (EDTA) plasma samples on an Ortho Vitrosâ 5600 analyser (Ortho Clinical Diagnostics, Rochester, NY, USA). Low-grade inflammation (LGI) was defined as a hsCRP level of 3–10 mg/L.
Questionnaire including SF-12 and question regarding mononucleosis The questionnaire included questions on baseline characteristics (including anthropometric measure, smoking status, and menstruation cycle), and the 12-item ‘short-form health survey 12’-questionnaire (SF-12), which yields a physical (PCS) and a mental component summary score (MCS). PCS and MCS were determined by weighting and summing the individual items and adding the sum to 57.65693 and 60.58847, respectively. Furthermore, we asked whether the donor had ever been diagnosed with IM by a physician. An answer of ‘yes’ was considered to indicate IM with a degree of confidence similar to that used in most studies examining a potential association between self-reported IM and MS (18).
Statistics Logistic regression was used to evaluate the genotype in rs6897932 as a predictor of LGI and self-reported IM, and general linear models were used for the outcomes of MCS and PCS. The analyses were stratified by sex both in univariable models, and in multivariable models with age, obesity (BMI ≥ 30 kg/m2), current smoking, physical activity at work (high vs low), leisure-time physical © 2014 APMIS. Published by John Wiley & Sons Ltd
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activity (high vs low), and current use of oral contraceptive for women, as covariates (20). Results are presented as odds-ratios with 95%-confidence intervals (95% CI) or means (95% CI). Two-tailed p-values below 0.05 were considered significant. All statistical analyses were performed using SAS (version 9.2 SAS Institute, Copenhagen, Denmark).
RESULTS Study population
Characteristics of the study population are presented in Table 1 (20). The study included data from a total of 17 293 individuals: 46.8% women and 53.2% men. A total of 17.1% of women and 15.8% of men were smokers (Table 1). The allele frequencies of rs6897932 in the total study population were 52.6% CC (N = 9092), 39.4% CT (N = 6820), and 8.0% TT (N = 1381) resulting in HW (p = 0.042). The ideal genotype distribution for the population according to the HW principle (p2 + 2pq + q2 = 1) was 52.3% CC (N = 9039), 40.0% CT (N = 6926), and 7.7% TT (N = 1326).
Table 1. Characteristic of the cohort stratified by sex Women N = 8092 Men N = 9201 37.6 (27.2–47.9) 40.1 (30.5–49.9) Age, years, median (IQR) Rs6897932, N (%) CC 4245 (52.5%) 4847 (52.7%) CT 3205 (39.6%) 3615 (39.3%) TT 624 (7.9%) 739 (8.0%) Current smoking, 1356 (17.1) 1431 (15.8) yes, N (%) 23.5 (21.6–26.2) 25.1 (23.4–27.5) BMI, kg/m2, median (IQR) 790 (9.8%) 968 (10.5%) Obesity, BMI≥30 kg/m2, N (%) PCS, points, 56.1 (53.8–57.4) 56.2 (53.8–57.3) median (IQR) MCS, points, 54.2 (50.0–56.7) 54.8 (51.2–56.8) median (IQR) 1709 (21.1%) 2338 (25.4%) High physical activity, work, N (%) 5350 (66.1%) 6366 (69.2%) High physical activity, leisure, N (%) hsCRP, mg/L, 0.69 (0.19–1.90) 0.42 (0.11–1.10) median (IQR) 1120 (14.6%) 530 (6.1%) LGI (hsCRP>3 mg/L), N (%) 2311 (28.6%) – Combined oral contraceptive pill (yes), N (%) IQR, Interquartile range; hsCRP, high-sensitive C-reactive protein; LGI, Low-grade inflammation. © 2014 APMIS. Published by John Wiley & Sons Ltd
Thus, heterozygotic individuals were slightly underrepresented in the total study population. Men and women did not differ with respect to rs6897932 allele frequencies (p = 0.904). Low-grade Inflammation (LGI) and infectious mononucleosis (IM)
The median hsCRP was 0.69 mg/L (0.19–1.9) in women and 0.42 mg/L (0.11–1.10) in men, and the frequency of LGI was higher in women than in men (13.9% vs 5.7%) as previously reported (20). No effect of rs6897932 alleles on the presence of LGI was found in women or men (Table 2, p > 0.05). Similarly, the rs6897932 allele did not predict IM (Table 2, p > 0.05). PCS and MCS
HRQL was assessed by the physical (PCS) and mental component summary score (MCS). The PCS was 56.1 (53-8–57.4) in women and 56.2 (53.8– 57.3) in men and the MCS was 54.2 (50.0–56.7) in women and 54.8 (51.2–56.8) in men (Table 1). No effect of rs6897932 genotype on PCS or MCS was found in women or men (Table 2, p > 0.05).
DISCUSSION In a large cohort of 17 293 healthy, Danish blood donors the IL7RA, rs6897932, was tested as a predictor of LGI, history of IM, and HRQL. The study population was stratified by sex. No effect of the genotype in rs6897932 was found in the analyses. The T-allele in rs6897392 reduces susceptibility to MS, diabetes type 1, atopic dermatitis, and inhalation allergy and increases the risk of relapse after hematopoietic cell transplantation. However, the rs6897392 T-allele has also been associated with increased risk of chronic inflammatory arthropathies, although this was not confirmed in a genomewide association study (21–23). Thus, the T-allele seems to be protective for inflammatory disease, but the results are ambiguous. Furthermore, we have presented data suggesting an association between the T-allele in rs6897932 and increased mortality in a Zimbabwean cohort of untreated HIV-infected individuals (7), and a faster rate of CD4 T cell increase after initiation of combined antiretroviral therapy in a Caucasian cohort of HIV-infected individuals (8). Finally, gain-of-func tion mutations in IL7RA adjacent to rs6897392 acting as oncogenes were found in childhood T-cell acute lymphoblastic leukemia (24, 25). The
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Table 2. rs6897932 as predictor of LGI, self-reported infectious mononucleosis, PCS and MCS Women Men OR (95% CI) OR (95% CI) OR (95% CI) 1 Adjusted Un-adjusted Un-adjusted rs6897932 LGI CC, N = 604 (14.5%) 1.0 1.0 N = 276 (6.0%) 1.0 CT, N = 425 (13.9%) 0.92 (0.81–1.05) 0.93 (0.80–1.07) N = 217 (6.3%) 1.05 (0.88–1.27) TT, N = 91 (15.0%) 1.01 (0.79-1.28) 1.04 (0.80–1.35) N = 37 (5.3%) 0.88 (0.62–1.25) Self-reported IM CC, N = 549 (13.0%) 1.0 1.0 N = 519 (10.8%) 1.0 CT, N = 383 (12.0%) 0.91 (0.79–1.05) 0.90 (0.78–1.04) N = 384 (10.7%) 0.99 (0.86–1.14) TT, N = 79 (12.3%) 0.94 (0.73–1.21) 0.94 (0.73–1.22) N = 62 (8.4%) 0.76 (0.58–1.01) Mean (95% CI)
Un-adj. p-value,1 Adj. p-value
Mean (95% CI)
OR (95% CI) 1 Adjusted
1.0 1.07 (0.88–1.29) 0.93 (0.65–1.34) 1.0 0.99 (0.86–1.14) 0.76 (0.57–1.01) Un-adj. p-value,1 Adj. p-value
rs6897932 PCS CC 55.0 (54.8–55.1) 0.206, 10.210 54.9 (54.8–55.0) 0.570, 10.468 CT 54.8 (54.7–55.0) 54.8 (54.7–55.0) TT 55.1 (54.7–55.4) 54.9 (54.6–55.2) MCS CC 52.2 (51.9–52.4) 0.790, 10.793 53.4 (53.2–53.5) 0.354, 10.452 CT 52.2 (51.9–52.4) 53.3 (53.1–53.5) TT 52.4 (51.8–52.9) 53.7 (53.2–54.2) LGI, Low-grade inflammation (hsCRP>3 mg/L); IM, Infectious mononucleosis; PCS, Physical component summary score; MCS, Mental component summary score; OR, Odds-ratio; CI, Confidence Interval. 1 Adjusted analysis included age, obesity (BMI≥30 kg/m2), current smoking, physical activity at work, physical activity in leisure, and use of oral contraceptive (only women) as covariates.
rs6897392 thus seems to play a role in T-cell homeostasis and T-cell mediated diseases, and may also affect the general health status. LGI and HRQL are associated with increased risk of chronic diseases for which rs6897392 is predictive (11, 12, 26), and we hypothesized an association between the rs6897932 T-allele and LGI and HRQL. However, we found no evidence to support our hypothesis. Although, IL-7R is essential for thymopoeisis (27), and severe combined immunodeficiency (SCID) patients with a specific mutation in IL7RA was characterized by T-cell lymphopenia (T-B+NK+ phenotype) (28), the full biological function of the rs6897932 polymorphism is not clear. T cells play a major role in several of the diseases (MS, diabetes type 1, HIV infection, T-cell acute lymphoblastic leukemia) associated with rs6897392. However, CRP is a general inflammation marker and primarily a part of the innate immune system and correlates with HRQL as an assessment of a general health status. Thus, the lack of associations between rs6897392 and LGI and HRQL may reflect the non-specificity of LGI and HRQL which are affected by a wide range of diseases and circumstances in which T cells are of minor importance. Furthermore, the results may be affected by the characteristic of the study population, inasmuch as blood donors represent a selected population with respect to chronic diseases, medications, and vacci172
nations. In addition, individuals volunteering as blood donors may share many characteristics. Thus, the hypotheses of the present study are based on immunological studies of diseased individuals, whereas the present study population most likely represents a population of homogeneous, healthy individuals. Confirmation of the presented results is warranted in independent cohorts. We also tested rs6897932 as a predictor of selfreported IM. Primary Epstein-Barr virus infection is typically asymptomatic or subclinical. However, the infection is sometimes accompanied by IM with clinical manifestations such as pharyngitis, cervical lymphadenopathy, fever, hepatomegaly, and splenomegaly (29). A total of 11.5% of the individuals in the present study reported having been diagnosed with mononucleosis. IM is an independent risk factor of MS, and CD8+ T cells play a significant role in the pathogenesis (29). However, we could not document an association between rs6897932 and a history of IM. The biological impact of rs6897932 on MS is not understood, but the present data suggest that the underlying mechanisms are not to be found in IM. Although the study benefits from the large number of participants, a limitation of the study is the aspect of self-reported IM diagnosed by a physician, and the lack of antibody titers to confirm IM. The association between MS and IM is found in data based on self-reported © 2014 APMIS. Published by John Wiley & Sons Ltd
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IM similar to findings based on IM verified by blood samples (18). Irrespective, the present data can be biased by poor recollection of past disease episodes, and EBV-related diseases (such as infection with Cytomegalovirus) can be over-reported. Our findings must therefore be interpreted with caution. In conclusion, the IL7RA rs6897932 was not associated with LGI or self-reported IM, nor with HRQL (PCS or MCS) in healthy individuals. These results suggest that the previously observed association between rs6897932 and several diseases is mediated by the T-cell compartment.
8.
9. 10.
11.
FUNDING
12.
The Danish Blood Donor Study has been supported by The Danish Council for Independent Research (09-069412) and by Danish Regions (02/2611).
13. 14.
DISCLOSURES The authors have no conflicts to disclose.
15.
We thank the Danish blood donors for the participation in the study.
16.
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