IL12B polymorphisms are linked but not associated with ... - Nature

Genes and Immunity (2008) 9, 405–411 & 2008 Macmillan Publishers Limited All rights reserved 1466-4879/08 $30.00 www.nature.com/gene

ORIGINAL ARTICLE

IL12B polymorphisms are linked but not associated with Plasmodium falciparum parasitemia: a familial study in Burkina Faso M Barbier, A Atkinson, F Fumoux and P Rihet Aix-Marseille universite´, IFR 48, Faculte´ de Pharmacie, Laboratoire de Pharmacoge´ne´tique des Maladies Parasitaires, Marseille, France

Chromosome 5q31–q33 has been linked to Plasmodium falciparum parasitemia in several independent studies. This chromosomal region contains numerous immunoregulatory genes. Among these, IL12B that encodes the p40 subunit of interleukin-12 (IL-12) appeared to be a promising functional candidate gene, and IL12Bpro, a promoter polymorphism, was associated with mortality from severe malaria in children. In this study, we characterized genetic variation in IL12B in 215 individuals belonging to 34 families and evaluated linkage and association of parasitemia with IL12B polymorphisms and haplotypes. We searched for IL12B polymorphisms in the coding regions and the corresponding intron–exon borders. We also examined IL12Bpro and IL12B 30 untranslated region (UTR) polymorphisms, which are thought to influence the production of IL-12. We showed a high level of conservation of IL12B-coding regions and identified five polymorphisms in introns and the two polymorphisms in the promoter and the 30 UTR regions. Although IL12B polymorphisms were linked to parasitemia, there was association of parasitemia with neither polymorphisms nor haplotypes. We cannot exclude that an unknown IL12B cis-regulatory element polymorphism affects both IL-12 production and parasitemia. However, our results suggest that genetic variation in IL12B does not explain differences in parasitemia in individuals living in an endemic area. Genes and Immunity (2008) 9, 405–411; doi:10.1038/gene.2008.31; published online 1 May 2008 Keywords: interleukin-12; Plasmodium falciparum; parasitemia; blood infection levels; family-based association; transmission disequilibrium test

Introduction Malaria remains a major problem of public health in many developing countries, especially in sub-Saharan Africa. Malaria is a complex disease, which likely depends on many host genetic factors.1 Case–control studies have detected associations between severe malaria and several genes, whereas few genes have been associated with either mild malaria or parasitemia.1 This information results from analysis of a small number of genes at the genome scale, and it is likely that some malaria resistance genes remain to be identified. Linkage and association analyses can be viewed as a more general approach to localize and to identify genes involved in malaria resistance, including genes controlling Plasmodium falciparum parasitemia. In humans, linkage analyses mapped a locus-controlling P. falciparum parasitemia to chromosome 5q31–q33.2,3 In mice, a comparative mapping approach demonstrated linkage of P. chabaudi parasitemia to a chromosomal region that shows extensive conservation of synteny with human chromoCorrespondence: Dr P Rihet, Aix-Marseille universite´, IFR 48, Faculte´ de Pharmacie, Laboratoire de Pharmacoge´ne´tique des Maladies Parasitaires-EA 864, 27 Bd Jean Moulin 13385 Marseille Cedex 5, France. E-mail: [email protected] Received 13 February 2008; revised and accepted 25 March 2008; published online 1 May 2008

some 5q31–q33.4 Several groups have investigated some polymorphisms of candidate genes that are located within chromosome 5q31–q33. Polymorphisms of IL4, IL12B and IL13 have been associated with severe malaria,5–7 whereas the IL4-590T allele has been associated with infection prevalence, but not with parasitemia.8 Moreover, IRF-1 polymorphisms that influence the expression of both IL4 and IL12B were associated with carriage of P. falciparum infection and severe malaria.9 Chromosome 5q31–q33 contains numerous immunoregulatory genes, which may be involved in the control of P. falciparum infection.2 One of the most promising candidate genes was IL12B that encodes the 40-kDa subunit of interleukin (IL)-12 and IL-23. IL-12, which was first described as natural killer cell stimulatory factor,10 is a primary inducer of the development of T-helper 1 (Th1) lymphocytes characterized by the production of IFNg, and IL-23 drives the development of Th17 lymphocytes characterized by the production of IL-17.11 There is a growing body of evidence for a key role of IL-12 in the immune response directed against Plasmodium species, whereas IL-23 was only recently proposed to be involved in malaria.12 Neutralization of endogenous IL-12 with anti-IL-12p40 and anti-IL-12p35 antibodies in P. chabaudiinfected mice resulted in an increase of parasitemia,13 whereas administration of recombinant IL-12 in mice and monkeys conferred a protection from Plasmodium species infection.13,14 Studies with IL-12p40-deficient

IL12B in human malaria M Barbier et al

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mice confirmed the effect of IL-12 in the protection against P. chabaudi.15,16 In humans, low levels of IL-12 have been associated with severe malaria17,18 and increased parasitemia.19 In addition, a promoter polymorphism in IL12B (rs17860508), which is thought to influence the production of IL-12,20,21 was associated with increased mortality from cerebral malaria in Tanzanian children.6 Furthermore, a stronger association was found between cerebral malaria and an haplotype based on IL12Bpro and one polymorphism in the 30 untranslated region (UTR) of IL12B (rs3212227).6 Interestingly, the IL12B 30 UTR polymorphism has been associated with several diseases related to immune responses,22,23 and with variation in the production of IL-12.22 To our knowledge, this was the only report of an association between IL12B and human malaria. The hypothesis underlying this study was that a cis-regulatory element polymorphism affects both IL-12 production and susceptibility to malaria, and the authors did not examine genetic variation in IL12B coding regions. Several variants in IL12B-coding regions have been shown to be involved in susceptibility to mycobacterial infections,24 and such variants might also influence susceptibility to malaria. We, therefore, searched for the two functional IL12B polymorphisms in the promoter and 30 UTR regions and for polymorphisms in all the coding regions and the corresponding intron–exon borders. We surveyed 215 subjects belonging to 34 families living in Burkina Faso and we evaluated linkage association between IL12B polymorphisms and P. falciparum parasitemia.

Results Screening of IL12B variants We genotyped polymorphisms in 215 individuals belonging to 34 families. We analyzed genetic variation in the promoter region and all the coding regions by sequencing (Figure 1). In addition, we genotyped the IL12B 30 UTR polymorphism by RFLP and we confirmed the genotypes by sequencing PCR products in nine samples. In all, we searched for 18 known polymorphisms and for unknown polymorphisms within the studied fragments (Table 1). As shown in Figure 1, we identified seven IL12B genetic polymorphic sites that have been described previously. In particular, we found frequent polymorphisms at loci IL12B 30 UTR and IL12Bpro, which are a substitution in the 30 UTR region and a complex insertion/deletion in the promoter.27 (Supplementary Figure S1). We did not identify any polymorphism in coding regions (Table 1). In particular, we did not find the polymorphisms that result in protein sequence change or IL-12-p40 deficiency.24,25 All genotypes passed a Mendelian check with the program FBAT.28 Using multipoint engine for rapid likelihood inference (MERLIN),29 we further searched for improbable recombination events from dense single-nucleotide polymorphisms (SNPs) maps to detect genotyping errors. The detectable genotype errors in the sample were less than 0.1%. Genotype coverage was from 92 to 98%. There was no deviation from Hardy–Weinberg equilibrium (P40.5). Genes and Immunity

Figure 1 Location of the polymorphisms detected within IL12B. The intron/noncoding exon (&)/coding exon ( ) structure of the gene is shown. Exons 2–7 and the corresponding intron–exon borders were sequenced, and two key cis-regulatory element polymorphisms were genotyped (IL12Bpro/rs17860508 and IL12B 30 UTR/rs3212227).

Linkage disequilibrium We generated haplotypes by the method of Abecassis et al.,29 using the genotype data of seven polymorphic sites. As shown in Supplementary Table 1, 12 haplotypes could be inferred. We then calculated pairwise disequilibrium coefficients for the seven genetic markers. As shown in Table 2, we found significant linkage disequilibrium (LD) between most variants. A complete LD was detected between rs3213099 and rs3213105. In addition, strong pairwise LDs were found between IL12Bpro, rs2288831 and IL12B 30 UTR. In contrast, no polymorphism was in LD with rs3181221. Linkage and association analyses of IL12B polymorphisms with parasitemia We evaluated linkage of parasitemia to IL12B polymorphims by using MERLIN. As shown in Table 3, the analysis yielded significant P-values at each locus marker. We further assessed association in the presence of linkage between parasitemia and the polymorphisms. As shown in Table 3, there was no significant association by using FBAT under the additive, dominant and recessive models. We further evaluated association between IL12B polymorphims and parasitemia after age stratification. There was no association when analyzing siblings aged 1–9 years (n ¼ 50), siblings aged 10–14 years (n ¼ 43) and siblings older than 14 years (n ¼ 60). Furthermore, we did not find any association between parasitemia and haplotypes based on the seven SNPs identified under the additive (P ¼ 0.29), dominant (P ¼ 0.11) and recessive (P ¼ 0.74) models. Similarly, no association was found between parasitemia and haplotypes when excluding the less common SNP rs3181221 under the additive (P ¼ 0.54), dominant (P ¼ 0.30) and recessive (P ¼ 0.73) models, and when analyzing only the cis-regulatory polymorphisms (IL12Bpro and IL12B 30 UTR) under the additive (P ¼ 0.83), dominant (P ¼ 0.94) and recessive (P ¼ 0.40) models.

Discussion We have previously obtained strong evidence for linkage of P. falciparum parasitemia to chromosome 5q31–q33.2,3 Although IL12B was not located within the peak of


Table 1

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Polymorphisms analyzed in IL12B

External name

SNP ID

Localization in IL12B

Molecular or functional change

IL12pro20

rs17860508 rs3213096 — rs11317523 rs2288831 —

50 -UTR exon 3 exon 3 exon 3 intron III del [intron III-intron V] intron IV exon 5 exon 5 exon 5 exon 5 intron V intron V intron VI intron VI exon 7 intron VII 30 -UTR

IL12B mRNA level Val4Ile IL-12p40 deficiency IL-12p40 deficiency — IL-12p40 deficiency

0.30 0 0 0 0.29 0

TTAGAG4GC — — — A4G —

— IL-12p40 deficiency Gly4Arg Glu4Asp Ser4Asn — — — — Val4Phe — IL12B mRNA level

0.07 0 0 0 0 0 0.03 0.07 0.28 0 0 0.30

G4C — — — — — T4C G4A C4T — — A4C

297del824 315insA24 g.482+82 856-854del24

rs3213099 — rs10045130 — — rs34446290 rs3181221 rs3213105 rs11574790 rs3213119 rs11574791 rs3212227

526del224 Glu186Asp25 Ser226Asn25

IL12B 30 UTR26

Minor allele frequencya

Allelesb

a

Allele frequency that was calculated in the study population is shown. Major allele4minor allele.

b

Table 2

Pairwise linkage disequilibrium (r2) IL12pro

IL12pro rs2288831 rs3213099 rs3181221 rs3213105 rs11574790

rs2288831

rs3213099

rs3181221

rs3213105

rs11574790

IL12B 30 UTR

0.61***

0.034 0.05*

0.01 0.02 0

0.034 0.05* 1*** 0

0.10*** 0.15*** 0.2*** 0.01 0.2***

0.57*** 0.74*** 0.04* 0.01 0.04* 0.16***

*Po0.05; ***Po0.001.

Table 3

Linkage and family-based association analyses in the presence of linkage

Polymorphism

Association in the presence of linkageb Linkagea

IL12pro rs2288831 rs3213099 rs3181221 rs3213105 rs11574790 IL12B 30 UTR

Additive model

Dominant model

Recessive model

LOD score

P

w2(df)

P

w2(df)

P

w2(df)

P

1.25 1.38 1.35 1.34 1.31 1.35 1.12

0.008 0.006 0.006 0.007 0.007 0.006 0.012

0.25(1) 1.00(1) 0.02(1) ND 0.97(1) 0.12(1) 0.35(1)

0.61 0.31 0.89 ND 0.32 0.73 0.55

0.53(2) 1.57(2) 1.00(2) ND 1.28(2) 0.12(2) 2.76(2)

0.77 0.45 0.60 ND 0.53 0.94 0.25

0.48(2) 1.98(2) 0.37(2) ND 2.04(2) 0.10(2) 2.23(2)

0.78 0.37 0.83 ND 0.36 0.95 0.33

a

Linkage analysis was performed with MERLIN. Association analysis was performed with FBAT.

b

linkage, it seemed to be a promising candidate gene. It should be emphasized that the predisposing locus may not be located within the peak of linkage and that there is accumulating evidence for a role of IL12 in malaria.13–19 We, therefore, searched for IL12B polymorphisms in the whole coding sequence, including the intron–exon borders, and genotyped cis-regulatory polymorphisms

within the promoter and the 30 UTR region in individuals living in an endemic area. We did not find any polymorphism within the coding sequence, indicating a high level of conservation of IL12B in African populations, as previously suggested in Caucasian populations.26,27 It is very likely that coding genetic variations resulting in IL-12p40 deficiency and Genes and Immunity


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susceptibility to mycobacterial infections are very rare.24 Nevertheless, we genotyped seven polymorphisms in the regulatory regions and the intron–exon borders. The frequencies of the detected polymorphisms in our population were similar to those found in another West African population.30 Using a family-based approach, we detected linkage of parasitemia to IL12B polymorphisms. We further evaluated association of parasitemia with those polymorphisms and haplotypes in the presence of linkage. We found no evidence to support a significant association between IL12B polymorphisms and parasitemia when taking into account linkage. Our results show that common genetic variation within the IL12B-coding sequence did not influence parasitemia in the study population. Our results also indicate that two known cisregulatory element polymorphisms, IL12Bpro and IL12B 30 UTR, did not strongly influence parasitemia, despite the associations between IL-12 levels and P. falciparum parasitemia,19 between IL12Bpro and cerebral malaria6 and the association of several diseases related to immune responses with IL12Bpro and IL12B 30 UTR.20,22,23,31 Since IL12B polymorphisms may interact, such that different haplotypes exhibit different IL-12 levels,23 we also evaluated the association of parasitemia with haplotypes, including the IL12Bpro—IL12B 30 UTR haplotypes; we detected the association of parasitemia with neither haplotypes based on the seven polymorphisms identified nor with IL12Bpro—IL12B 30 UTR haplotypes. This suggests that parasitemia is not influenced by genetic variation in IL12B, including IL12Bpro and IL12B 30 UTR polymorphisms. Although IL12Bpro and IL12B 30 UTR polymorphisms have been associated with IL12 production,22,27,32,33 it remains possible that an unidentified polymorphism that is in LD with either IL12Bpro or IL12B 30 UTR affects the production of IL-12. Therefore, we cannot exclude that unknown cis-regulatory element polymorphisms, including long-range ones, affect both IL12B expression and parasitemia. Under this hypothesis, the power of our study that depends on the LD between the polymorphisms identified and the causal polymorphism might be insufficient to detect the disease allele. Alternatively, the effect of IL12B polymorphisms may depend on other factors, such as sibling age and genetic interactions. In this way, the association between cerebral malaria and IL12Bpro was found in very young children,6 suggesting an age-dependent effect of IL12B polymorphisms. To test this hypothesis, we evaluated the association of IL12B polymorphisms after age stratification; no association was detected. However, we cannot exclude an effect of IL12B polymorphisms in very young children because of the small number of siblings aged 1–5 years. Besides, IL12B polymorphisms may act in interaction with other loci and their effect might be not detected without taking into account the genetic interaction. In this way, clusters of cytokines were found to determine malaria severity in P. falciparum-infected patients from endemic areas in India, whereas the levels of cytokines, including IL-12, were poorly associated with severe malaria when analyzed separately.34 Recently, acquisition of hemozoin by macrophages has been shown to downregulate both IL-12p40 transcripts and circulating IL-12 through an IL-10-dependent mechanisms.35 Furthermore, it has been shown in vitro that the effect of IL12B

Genes and Immunity

30 UTR and IL12Bpro polymorphisms on IL-12 production depends on IL10 polymorphisms.36,37 The effect of IL12B polymorphisms on IL12 production might also be influenced by polymorphisms in other genes, the expression of which characterizes Th2 responses. Therefore, the association of IL12B polymorphisms with malaria might be affected by genetic variation in other genes, such as IL4 or IL10. Finally, IL-12 may not be critical for the control of parasitemia in individuals living in an endemic area, and genetic variation in a neighbouring gene could explain the linkage of IL12B to parasitemia. This hypothesis is supported by mouse studies showing that IL-12p40deficient mice on a C57BL/6 background mount a protective immunity after repeated P. berghei-irradiated sporozoite immunization, whereas the same mice are not protected against P. berghei infection after a single dose of irradiated sporozoites.38 These results clearly show that IL-12p40 plays an important role in mediating early protection by irradiated sporozoite immunization, but that compensatory immune responses develop in IL12p40-deficient mice after repeated immunization. Therefore, IL-12p40 might be dispensable for the build up of human protective immunity, following repeated infections in endemic areas. It should be stressed, however, that IL-12p40-, IL-12p35- and IFNg-deficient mice on a BALB/c background are unable to mount a protective response against P. yoelii after multiple irradiated sporozoite immunizations, suggesting that the influence of IL-12 depends either on the parasite or host genetic background.39,40 Thus, the role of human IL12 in the control of parasitemia in endemic areas cannot be ruled out. In addition, this hypothesis is supported by the negative correlation between IL-12 levels and asymptomatic parasitemia in Papua New Guineans.19 Further studies in other populations are required to confirm whether low IL-12 levels are associated with increased parasitemia. In conclusion, we have screened IL12B polymorphisms in the whole coding sequence and in the intron–exon borders, genotyped two known cis-regulatory polymorphisms and characterized haplotype structure. Although we provide evidence for linkage of parasitemia to IL12B polymorphisms, we did not find any evidence for association between IL12B polymorphisms and P. falciparum parasitemia in the presence of linkage. This indicates that IL12B polymorphisms did not explain the linkage of chromosome 5q31–q33 to P. falciparum parasitemia. Further studies of genetic variation within this chromosomal region are required to identify the polymorphisms involved. It is not excluded, however, that IL12B and other genes interact and further analysis of polymorphisms within IL12B and other genes involved in the same pathway would be helpful in better defining the effect of IL12B polymorphisms on the control of malarial infection and disease.

Materials and methods Families The study subjects live in an urban district of BoboDioulasso, the second largest town of Burkina Faso, in an endemic area for malaria; P. falciparum transmission occurred only during the rainy season (August to


December). The family structures and the area of parasite exposure have been previously extensively described.2,41 Thirty informative pedigrees corresponding to 34 nuclear families and containing at least three available siblings were selected for genotyping. 215 subjects (62 parents and 153 full siblings) were available for genotyping and retained for association analysis. The families had the following distribution of sibship sizes: 8, 7, 15 and 2, and 2 sibships contained 3, 4, 5, 6 and 7 sibs, respectively. The mean age of the siblings was 12.1±6.2 (1–34 years). Determination of parasitemia The determination of parasitemia was described in our previous study.2 Briefly, each family was visited 20 times during the 21 months of the study (from April 1994 to December 1995) and the mean number of parasitemia measurements per subject was 12.8±5.1. Blood samples were taken from all family members present and thick and thin blood films were stained with Giemsa. The parasite determination and numeration were established blindly from two independent readings. More than 95% of parasites identified on thin blood smears were P. falciparum; the others were P. malaria. Only P. falciparum asexual forms were retained to determine parasitemia. The analysis was conducted on a logarithmic transformation of parasite density adjusted for seasonal transmission and for covariates that showed a significant effect on parasitemia. As previously reported,42 age was strongly correlated with parasitemia (Po104) and was retained for data adjustment. Genotyping DNA was extracted from mononuclear cells separated by Ficoll–Hypaque density gradient as described.2 Samples were first subjected to prior whole-genome amplification by primer extension preamplification.43 The DNA fragments were amplified individually. To identify IL12B mutations in the promoter region and all the coding regions (for example, exons 2–7), we performed sequencing analysis of defined PCR products. The primer pairs were designed with the PRIMER 3 program, as shown in Supplementary Table 2. Before starting the sequencing reaction, the PCR products were purified with the Qiagen QIAquick PCR purification kit (Qiagen, Hilden, Germany) and quantified by 2% agarose gel electrophoresis. Sequencing reaction was performed with the CEQ DTCS kit (Beckman Coulter, Fullerton, CA, USA) and a CEQ 8000 automated fluorescent sequencer (Beckman Coulter). In addition, IL12B 30 UTR polymorphism was determined by using an allele-specific restriction enzyme digestion method;44 only the PCR product of the C allele contains a TaqI site. Digestion products were analyzed by electrophoresis on a 2% agarose gel stained by ethidium bromide. Allele frequencies, haplotype reconstruction and linkage disequilibrium analysis Incompatibilities with Mendelian inheritance of marker alleles was checked with the FBAT and MERLIN programs.28,29 All genotypes passed a Mendelian check with the program FBAT. We further searched for improbable recombination events from SNP maps to detect genotyping errors. The detectable genotype errors in the sample were less than 0.1%.

Allele frequencies were calculated by gene counting and deviation from Hardy–Weinberg equilibrium was tested using a w2-test with 1 df. We generated haplotypes based on family genotypic data with MERLIN. Pairwise LD was calculated with graphical overview of linkage disequilibrium (GOLD).45 We tested LD between pairs of biallelic markers by the r2 statistic:

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r2 ¼ ðf11:f22 f12:f21Þ2 =p1:p2:q1:q2 where f11, f22, f12 and f21 were two-locus haplotype frequencies, and p1, p2, q1 and q2 were allele frequencies. r2 is the standard w2 statistic divided by the number of chromosomes in the sample. It ranges from 0 to 1. Linkage and association analyses Linkage analysis was performed by using the MERLIN package.29 We used the nonparametric quantitative trait linkage statistic and calculated a Kong and Cox LOD score and a Kong and Cox P-value at each locus marker. We carried out combined association and linkage analyses of quantitative traits using the FBAT program.28 The FBAT statistics, which uses data from sibships in nuclear families takes into account sibling correlations. The null hypothesis tested was not associated in the presence of linkage by using an empirical variance– covariance estimator that adjusts for the correlation among sibling marker genotypes and for different nuclear families within a single pedigree. We used this empirical variance estimator because we tested for association in an area of known linkage. The statistics under this hypothesis, calculated under the distribution of offspring genotype, is conditional on parental genotype and on trait values for offspring and parents. FBAT calculates a Z score and a two-side P-value based on a normal approximation when the biallelic mode was used and a w2 and a two-side P-value when using the multiallelic mode. FBAT can also perform permutations to calculate empirical P-values, when analyzing haplotypes. Three genetic models (that is, additive, dominant and recessive) were tested for individual and multilocus SNPs.

Acknowledgements We thank all volunteer families of Bobo-Dioulasso (Burkina Faso, West Africa). This work was supported by the French Ministry of Research and Technology (Agence Nationale de la Recherche, Microbiology– Immunology Program). MB and AA were supported by a studentship from the French Ministry of Research and Technology.

Conflict of interest The authors state no conflict of interest.

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