Acute Leukemias

Polymorphisms in the TLR6 gene associated with the inverse association between childhood acute lymphoblastic leukemia and atopic disease

Abstract

Little is known about the etiology of childhood acute lymphoblastic leukemia (ALL). The presence of atopic disease has been shown to protect against developing childhood ALL. The aim of this study was to examine whether single nucleotide polymorphisms (SNPs) in innate immunity genes previously associated with atopic disease, can elucidate the inverse association between childhood ALL and atopic disease. We studied 525 children, including 192 with childhood ALL, 149 with atopic disease and 184 healthy control subjects. We compared genotype distributions of 29 SNPs in genes of TLR2, TLR4, TLR6, TLR9, TLR10 and CD14 between the three groups and corrected for multiple testing. The genotype distributions of two SNPs in the TLR6 gene, rs5743798 and rs6531666, differed significantly between children with ALL, children with atopic disease and control subjects. Particularly in children with atopic eczema, risk alleles for atopic disease were observed more often than in control subjects, and less often in children with ALL than in control subjects. These findings support the immune surveillance hypothesis as an explanation for the protective association of atopic disease on childhood ALL. Further investigation is warranted to examine in more detail the role of innate immunity in the development of childhood ALL.

Introduction

Acute lymphoblastic leukemia (ALL) is the most common form of cancer among children, which accounts for about 30% of all cancer cases in children under the age of 15 years. There is a specific age peak at 2–5 years, mainly due to an excess of pre-B ALL cases in this age range. Little is known about the etiology of childhood ALL. Interestingly, there is growing evidence of an inverse association between childhood ALL and atopic disease.1, 2, 3 Many epidemiological studies have shown a protective association of atopic disease on the risk of childhood ALL. The presence of an atopic condition is thought to increase the vigilance of the immune system in monitoring for, identifying and eliminating malignant cells.4 Therefore, atopy may lead to early elimination of malignant cells and thus prevent the development of malignancies like childhood ALL.

A multifactorial background has been suggested for atopic disease with genetic as well as environmental factors contributing to disease susceptibility. Various studies examined the role of innate immunity genes on the risk of atopic disease. Particularly single nucleotide polymorphisms (SNPs) in toll-like receptor 2 (TLR2), TLR4, TLR6, TLR9, TLR10 and cluster of differentiation 14 (CD14)5, 6, 7, 8, 9 have been associated with atopic disease. TLRs are membrane receptors that act as the gatekeepers of the innate immunity by recognizing microbial components and initiating activation of an adequate immune response. CD14 acts as a co-receptor, along with TLR4, for the detection of bacterial lipopolysaccharide. Variations in genes encoding TLRs and CD14 are suggested to alter the capability to recognize microbes or alter the amount of gene product, leading to inadequate immune responses and increasing the susceptibility for atopic disease.5, 9 In the case of childhood ALL, much less is known about the role of genes involved in innate immunity.10 Examining the genetic basis of the inverse association between childhood ALL and atopic disease might help towards elucidating the etiology of childhood ALL.

The aim of this study was to examine whether genetic variations in the genes of TLR2, TLR4, TLR6, TLR9, TLR10 and CD14 are associated with the inverse association between childhood ALL and atopic disease. We hypothesized that risk alleles for atopic disease are observed more often in atopic children than in healthy controls, and are observed less often in children with ALL than in healthy controls.

Materials and methods

Study population

This study was conducted in three cohorts. The first cohort included 210 children, aged 0–15 years, with ALL who were diagnosed between 1984 and 2001 in Beatrix Children's Hospital of University Medical Center Groningen in Groningen, the Netherlands, or between 1997 and 2001 in Wilhelmina Children's Hospital of University Medical Center Utrecht in Utrecht, The Netherlands. Eighteen subjects were excluded because of their non-Caucasian background.

The second cohort included 149 atopic children, aged 0–8 years, derived from the PIAMA study,11 see Supplementary Table S1. The PIAMA study is a prospective birth cohort study on Prevention and Incidence of Asthma and Mite Allergy. The participating children were born between May 1996 and December 1997 in three regions of the Netherlands. They were selected using a validated screening questionnaire with questions on asthma and respiratory allergies administered to the mother at her first visit to the midwife. The midwives were requested to ask every new pregnant woman to fill in the screening questionnaire. A total of 10 232 pregnant women completed the questionnaire and 2949 (29%) of them reported asthma or respiratory allergy and were defined as ‘allergic’. Based on this screening, 7862 women were invited to participate, and 4146 agreed and gave informed consent. After birth, the baseline study population consisted of 3963 children. All children from allergic mothers (n=1327) and a random sample of children from non-allergic mothers (n=663) were selected for more extensive investigation, including DNA collection at 4 years of age. DNA was obtained from 1046 of these children and genotypes were obtained from 1037 children. Children with a non-Caucasian ethnicity were excluded from the analyses (n=51). Atopic disease was determined by elevated specific IgE levels (0.70 IU/ml) and/or positive allergy skin test, in combination with asthma and/or eczema, as these more stringent definitions of atopy were used in epidemiological studies observing a reverse association between childhood ALL and atopy. Specific IgE to mite (Dermatophagoides pteronyssinus), cat (Fel d1), dog (Can f1), grass (Dactylis glomerata), birch (Betula verrucosa) and mould (Alternaria alternata) was measured by radioallergosorbent test. In PIAMA, allergy skin tests were performed with: Dermatophagoides pteronyssinus, Dermatophagoides farinae, Alternaria alternata, mixed grass pollen, mixed tree pollen, cat and dog. A positive skin test was defined as a mean wheal diameter of 3 mm in response to one or more allergens provided the control was negative (<3 mm) and the positive control was positive (3 mm). Asthma was defined as an episode of wheezing or dyspnea, or use of inhalation steroids in the past 12 months at the age of 8 years. Eczema was defined as having reported an itching rash at least twice in the past 12 months, intermittently present at the usual eczema locations (for example, elbows, back of the knees, front of the ankles, around the eyes or ears, in the neck), for at least 2 years in a row in the age 0–8 years. In all, 82 children (55%) were diagnosed with atopic asthma, and 115 (77.2%) with atopic eczema. Forty-eight of the children (32%) were diagnosed both with asthma and eczema.

The third cohort consisted of 184 healthy control children, aged 0–8 years, also derived from the PIAMA cohort. These children did not have positive skin tests, their specific serum IgE levels were not increased (<0.70 IU/ml), and they were not diagnosed with either asthma or eczema.

The local medical ethics committees of participating institutes approved all studies. Written informed consent was obtained from all participants and/or their parents.

Genotyping

Genomic DNA was extracted from buccal swabs, blood or bone-marrow samples. DNA extraction from blood or bone-marrow samples was performed by using the NucleoSpin- Blood L kit (Macherey-Nagel, Düren, Germany). For frozen samples the NucleoSpin Tissue kit (Macherey-Nagel) was used. Both kits were used according to the manufacturer's protocol. For DNA isolation from buccal swabs, the protocol described in Meulenbelt et al.12 was used. DNA was amplified using REPLI-g UltraFast technology (Qiagen, Venlo, The Netherlands). We selected (potential) functional SNPs complemented with known haplotype-tagging SNPs in the genes for TLR2 (rs3804099, rs3804100, rs4969480), TLR4 (rs4986790, rs6478317, rs10759932, rs11536878, rs11536889, rs1927911, rs10759931, rs2770150), TLR6 (rs1039559, rs5743788, rs5743810, rs6531666, rs5743798), TLR9 (rs352140, rs187084, rs5743836), TLR10 (rs4274855, rs10856839, rs11096956, rs11096957, rs11466652, rs4129009) and CD14 genes (rs2563298, rs2569190, rs256191, rs5744455). Genotyping was performed by Competitive Allele-Specific PCR using KASPar genotyping chemistry, performed under contract by K-Biosciences (Herts, UK). Quality of genotype data was guaranteed by standards of K-Biosciences with extensive quality control as described before.13

The genotype data of the healthy control subjects were analyzed for deviations from Hardy–Weinberg equilibrium using 1 degree of freedom chi-square tests. None of the SNPs deviated from HWE (P<0.01). On an average, the call rate was 97%, but one SNP (rs6531666) had a call rate of 86%. We therefore performed an extensive quality control on this SNP: the allele frequencies were similar between the cohorts and compared with near-lying SNPs (P>0.05), and genotypes were in Hardy–Weinberg equilibrium (P0.01). Thus, as the quality of this SNP was good, we included it in the analyses. The 29 SNPs selected for TLR2, TLR4, TLR6, TLR9, TLR10 and CD14, data source and allele frequencies are shown in Table 1. Pair-wise linkage disequilibrium (LD) was assessed between single SNPs by using the r2 statistic as implemented in Haploview version 4.1 (Broad Institute, Cambridge, UK).

Table 1 SNPs genotyped in TLR2, TLR4, TLR6, TLR9, TLR10 and CD14, and their location, minor allele frequency, call rate and deviation from Hardy–Weinberg equilibrium in controls (n=184)

Data analyses

Differences in distribution of genotype and allele frequencies were tested using 4 degree of freedom chi-square test. To account for multiple testing, we calculated the false discovery rate as described by Benjamini and Hochberg,14 using a P-value of 0.05 as a cutoff value. Further, we performed 10 000 permutation tests to correct for false-positives, where we compared the genotype distributions between ALL versus atopic patients for studied SNPs using permute module, implemented in PLINK software (free software, available at http://pngu.mgh.harvard.edu/purcell/plink/). The two subgroups in the atopic cohort, that is, asthmatic children and eczematous children, were analyzed separately in an additive model by using chi-square statistics. Subsequently, we compared children with childhood ALL and children with atopic disease separately with healthy controls by using logistic regression, to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) for the best-fitting model (that is, dominant, recessive or additive). P-values <0.05 were considered statistically significant. The statistical software package used was SPSS 16.0 (SPSS, Chicago, IL, USA).

Results

Study population

Table 2 shows the patient characteristics of the three cohorts. A total of 525 children were included in the study: 192 children with childhood ALL (61% male), 149 children with atopic disease (55% male) and 184 healthy control children (48% male). There was a significant difference in sex distribution among the three cohorts (P=0.041).

Table 2 Patient characteristics of the three cohorts

Association analysis

Table 3 shows the genotype and allele frequencies of the 29 SNPs among the three cohorts. The genotype distribution of two SNPs in the TLR6 gene, rs6531666 (P=0.0025) and rs5743798 (P=0.00047), was significantly different among children with ALL, control subjects and children with atopic disease. Both SNPs sustained correction for multiple testing. When correcting for sex, both rs6531666 and rs5743798 remained significantly different among the three cohorts (P=0.0035 and P=0.0006, respectively). The largest differences in genotype distributions were observed between children with ALL and children with atopic disease. The minor alleles of both rs6531666 and rs5743798 were associated with an increased risk of atopic disease when comparing with the controls, and at the same time associated with a decreased risk of ALL when comparing with the controls. After 10 000 permutation tests, both rs6531666 (P=0.013) and rs5743798 (P=0.0004) sustained their significant differences in their genotype frequencies between ALL and atopic patients (see Supplementary Table S2). SNPs in the other TLR and/or CD14 genes were not associated with childhood ALL or atopy.

Table 3 Comparison of genotype and allele frequencies of SNPs in TLR2, TLR4, TLR6, TLR9, TLR10 and CD14 among children with ALL, healthy control subjects and children with atopic disease

The frequencies of the minor alleles in rs6531666 and rs5743798 were significantly different among the three groups. For rs6531666, the frequency of the minor allele was 23% in children with ALL, 29% in the control subjects and 33% in the atopic children (P=0.028). For rs5743798, the frequency of the minor allele was 19% in children with ALL, 25% in the healthy controls and 34% in the atopic children (P=0.0001, which sustained correction for multiple testing).

Figure 1 presents the genotype effects (ORs and 95% CIs) of the heterozygous and homozygous minor allele genotype of TLR6 SNPs rs6531666 and rs5743798 compared with the homozygous major allele genotype on the risk of ALL and atopic disease, using the healthy control subjects as reference group. Subjects homozygous for the minor allele (C) of rs6531666 showed a borderline significant decreased risk of childhood ALL (OR 0.39, 95% CI, 0.12–1.20, P=0.099), whereas they had significantly increased risk of atopic disease (OR 2.61, 95% CI, 1.07–6.39, P=0.035). The same trend was observed for subjects homozygous for the minor allele (T) of rs5743798, with a borderline significant decreased risk of childhood ALL (OR 0.42, 95% CI, 0.14–1.28, P=0.13) and a significantly increased risk of atopic disease (OR 2.88, 95% CI, 1.26–6.58, P=0.012).

Figure 1
figure1

Genotype effects (OR and 95% CI) of the heterozygous and homozygous minor allele genotype of TLR6 SNPs rs6531666 and rs5743798 compared with the homozygous major allele genotype on the risk of ALL and atopic disease, using the healthy control subjects as a reference group.

As the atopic children and the control subjects were examined annually until the age of 8, we also performed the analysis including only the children with ALL who were 0–8 years (n=157). Although numbers were lower, this analysis still showed that the genotype distributions of the same two TLR6 SNPs, that is, rs6531666 (P=0.008) and rs5743798 (P=0.0009), differed significantly among the three groups. Supplementary Table S3 shows the genotype distributions of the children with ALL who were 0–8 years, compared with the controls and the atopic children.

It has been suggested that different subtypes of ALL, in particular, B-lineage ALL, may have different etiologies. Therefore, we performed separate analyses for the groups of children with B-lineage ALL and the children with T-lineage ALL. The results for the group with B-lineage ALL (n=163) were similar to the entire ALL group: the TLR6 SNPs rs6531666 and rs5743798 were significantly different among the three groups (P=0.005 and P=0.001, respectively). Only SNP rs5743798 sustained correction for multiple testing. ORs did not reach significant levels. When comparing the group with T-lineage ALL (n=20) to the controls and the atopic children, none of the SNPs sustained correction for multiple testing, which may be due to the small number of T-ALL patients.

Among the 149 children with atopic disease, 82 (55%) were diagnosed with asthma. When comparing the genotype frequencies of asthmatic children and children with ALL with the controls, the genotype distribution of both TLR6 SNP rs6531666 and rs5743798 were significantly different among the three cohorts (P=0.01 and P=0.02, respectively). Both SNPs did not sustain correction for multiple testing. Of the 149 atopic children, 115 (77%) were diagnosed with eczema. When comparing the children with eczema and the children with ALL with the control subjects, again both TLR6 SNP rs6531666 and rs5743798 were significantly different among the three groups (P=0.001 and P<0.001, respectively). Both SNPs sustained correction for multiple testing.

Finally, we constructed an LD plot of the TLR6 gene, including the SNPs that were associated with the inverse association between childhood ALL and atopic disease. As can be seen in the LD plot of TLR6, rs5743798 and rs6531666 show moderately high LD with an r2 of 0.77 (Figure 2). Minor alleles of these SNPs were both associated with an increased risk of atopy and a decreased risk of childhood ALL, as would be expected with this LD.

Figure 2
figure2

Genetic layout of TLR6 with LD plot (r2).

Discussion

In the present study, we show that two polymorphisms in the TLR6 gene are associated with the inverse association between childhood ALL and atopic disease. At the same time, compared with healthy controls, the minor alleles of these two TLR6 SNPs are associated with an increased risk of atopic disease, as they are also associated with a decreased risk of childhood ALL. Ours was the first study to provide a possible genetic explanation for the protective association of atopic disease on the occurrence of childhood ALL.

Different hypotheses have been proposed to explain the inverse relationship between childhood ALL and atopic disease. The principal factor linking childhood ALL and atopic disease seems to be the rate at which the immune system matures. Hypotheses proposed by Greaves and Kinlen suggest an etiological role for the immune system in the development of childhood leukemia via delayed exposure and abnormal response to childhood infections.15, 16 A hypothesis that has been proposed concerning the inverse association between atopic disease and childhood ALL is the so-called immune surveillance hypothesis. This hypothesis implies that the innate immune system recognizes antigens of malignant cells as foreign and mounts a response to them by triggering adaptive immune responses, which prevent a majority of potential cancers from developing.17 As the vigilance of the immune system may be increased in the presence of atopic disease,4 this could eliminate malignant cells, and prevent the development of malignancies, such as childhood ALL.

Atopic disease is associated with a predominance of T-helper 2 (Th2) cells, essential for the production of IgE, versus T helper 1 (Th1) cells. All infants are born with a Th2-dominated immune profile, characterized by interleukin 4 (IL-4), IL-5, IL-9, IL-10 and IL-13 production. By the age of 2 years, nonatopic infants have gradually migrated to a Th1-dominant profile, which is characterized by: IL-12, IL-18, interferon-gamma and tumour necrosis factor α (TNF-α), whereas infants who develop atopy fail to make this Th2-to-Th1 transition. It has been suggested that one of the driving forces for this immune shift is microbial exposure, which induces innate immunity cells, such as dendritic cells, to produce cytokines important for the development of Th1 responses.18 Dendritic cells express TLRs and are susceptible to TLR ligands, including microbial stimuli, as well as endogenous ligands. Genetic variation in TLRs may influence the activation of T-regulatory cells (responsible for suppressing Th2 responses), and/or skewing of the Th1–Th2 balance. We propose that the two SNPs in the TLR6 gene as found in our study are related to an altered shift in the Th1–Th2 balance, causing an increased risk of developing atopic disease, which by means of increased vigilance protects against childhood ALL.

Ligands for TLR6 include diacyl lipopeptides from Mycoplasma, yeast zymosan from Saccharomyces cerevisiae, and lipoteichoic acid from group B Streptococci and Staphylococci, which are often found in the upper respiratory tract.19 Earlier studies found that the production of tumour necrosis factor α elicited by zymosan and gram-positive bacteria20 is inhibited in TLR6 knockout mice, as is its production in response to mycoplasmal macrophage-activating lipopeptide-2 from Mycoplasma fermentans.21 These data suggest that the TLR6 gene controls Th1 differentiation, whereas the absence of TLR6-mediated signals generates Th2 responses. Moreover, Kormann et al.5 showed another SNP located in the TLR6 gene, that is, rs5743789, to be associated with increased mRNA expression. Carriers of the minor allele of this SNP showed increased Th1 cytokine expression, and reduced Th2 cytokine production after stimulation with its ligand. Unfortunately, at the beginning of our study, there was no information available on the possible importance of TLR6 SNP rs5743789, and it was therefore not included in our study. Alternatively, the TLR6 SNPs may be associated with a decreased risk of childhood ALL more directly via expression of TLR6 on leukemic cells, as has previously been shown for B-chronic lymphoblastic leukemia cells.22 Future research is warranted to define the association between TLR6 and childhood ALL cells.

Recently, various genome-wide association studies have been performed in order to find risk genes for atopic disease.23 Although several candidate gene studies did find TLR6 polymorphisms to be associated with atopy,5, 8, 24 this has to date not yet been confirmed in genome-wide association studies. However, a genome-wide association study of atopic disease in combination with positive skin tests has not been performed to date. Based on previous epidemiological studies, we specifically defined atopic disease relatively strictly by elevated IgE levels and/or positive skin tests in combination with asthma and/or eczema.5, 6, 24, 25, 26 This strengthens our results. Unfortunately, for the children with ALL, no information was available on atopy, as IgE levels and skin tests are not performed regularly in children with leukemia. However, since measuring IgE levels may be influenced by immunosuppressive treatment regimens, measuring IgE levels for the purpose of this study did not seem useful.

As a wide range of atopic conditions exist, it is conceivable that the analysis of allergic subtypes is more accurate than the overall allergy estimate in describing the association, even though numbers are smaller. When comparing the genotype frequencies of the subgroups of children with asthma and eczema to childhood ALL, the strongest inverse association was found between eczematous children and children with childhood ALL. This finding is supported by the literature.1, 2, 3, 27

This study has some limitations. Although the minor allele frequencies of the two TLR6 SNPs (0.25 and 0.28) are considered common, observations were made in a limited number of subjects and therefore need to be interpreted with caution. Even though we showed a robust association between two SNPs in the TLR6 gene and the inverse association between childhood ALL and atopic disease, one could argue that these results were chance findings. This is, however, not very likely as we corrected for multiple testing and performed permutation analysis. Nevertheless, the common problem in candidate gene studies of false positive findings could not be ruled out altogether. Therefore, our study needs to be replicated in study populations of the same or, preferably, larger sample sizes. Furthermore, the control population was derived from the study on Prevention and Incidence of Asthma and MIte Allergy (PIAMA). However, the control population was selected after careful screening for absence of atopic disease, by specific IgE levels, skin-prick tests, asthma and eczema, to make sure our control cohort was not biased.

Moreover, the question arises whether the SNPs in the TLR6 gene that we found are relevant with respect to their function. This clearly requires further study. They are both haplotype-tagging SNPs, implying the effect can be direct or indirect. A direct effect may come from a direct biological influence of (one of) the SNPs, even though they are located in introns. It is now well established that introns may be related to the regulation of gene expression. An indirect effect can by achieved by tagging of a region of the gene that is functional.

Conclusion

We examined the etiology of childhood ALL from a new genetic perspective, and found two TLR6 polymorphisms to be associated with the inverse association between childhood ALL and atopic disease. Our findings support the immune surveillance hypothesis as an explanation for the protective effect of atopic disease on childhood ALL and provide new insight into the mechanism of the inverse association between childhood ALL and atopic disease. Further investigation is warranted to replicate our findings and to examine in more detail the role of innate immunity in the development of childhood ALL.

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Acknowledgements

We acknowledge the help of Dr Titia Brantsma—van Wulfften Palthe from Utrecht for correcting the English. Moreover, we thank Dr Arjan Lankester for revising the manuscript. The Dutch ODAS foundation supported the work of KGE Miedema. EM te Poele was supported by a research grant from the Foundation for Pediatric Oncology Groningen (SKOG 03-001). The Prevention and Incidence of Asthma and Mite Allergy (PIAMA) study is funded by the Netherlands Organization for Health Research and Development, the Netherlands Asthma Foundation, the Netherlands Ministry of Planning, Housing and the Environment, the Netherlands Ministry of Health, Welfare and Sport and the National Institute for Public Health and the Environment.

Author contributions

Karin GE Miedema designed and performed the research, analyzed and interpreted data, performed statistical analysis and wrote the manuscript. Wim JE Tissing designed research, performed research, analyzed and interpreted data and wrote the manuscript. Esther M te Poele designed and performed the research, collected data and revised the manuscript. Willem A Kamps interpreted data and revised the manuscript. Behrooz Z Alizadeh and Marjan Kerkhof analyzed and interpreted data, performed statistical analysis and revised the manuscript. Johan HC de Jongste, Henriëtte A Smit, Anne P de Pagter and Marc Bierings performed research, collected data and revised the manuscript. H Marike Boezen designed research and revised the manuscript. Dirkje S Postma and Eveline SJM de Bont designed the research, interpreted data and revised the manuscript. Gerard H Koppelman designed the research, interpreted data and wrote the manuscript.

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Correspondence to W J E Tissing.

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The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on the Leukemia website

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Miedema, K., Tissing, W., te Poele, E. et al. Polymorphisms in the TLR6 gene associated with the inverse association between childhood acute lymphoblastic leukemia and atopic disease. Leukemia 26, 1203–1210 (2012). https://doi.org/10.1038/leu.2011.341

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Keywords

  • childhood acute lymphoblastic leukemia
  • atopic disease
  • TLR6
  • polymorphisms
  • innate immunity

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