1 Department of Human Genetics, Roche Molecular Systems, Alameda, California
2 Division of Biostatistics, Department of Health Research and Policy, Stanford University School of Medicine, Stanford, California
3 Childrens Hospital Oakland Research Institute, Oakland, California
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ABSTRACT |
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INTRODUCTION |
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We genotyped 282 type 1 diabetic multiplex families at eight SNPs in the IL4R gene (Table 1) using a multiplex PCR assay coupled with sequence-specific hybridization to an immobilized probe array (Fig. 1). The 282 Human Biological Data Interchange (HBDI) families used in this study are nuclear families of Caucasian ethnicity, with unaffected parents and two ascertained affected siblings (14). The two-generation family structure and the absence of recombination among SNPs enabled us to deduce IL4R haplotypes for all individuals and assess transmission patterns. Herein, we will use the term "haplotype" to represent the ordered set of IL4R SNP alleles present on a single chromosome. The "haplotype" notation is representative of the primary sequence of the protein and is consistent with terminology used by others (8, 1113).
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In our analyses of the IL4R data, we stratified the families into two groups: those in which either or both of the affected sibs are DR3/4 versus those in which neither of the sibs is DR3/4 (see RESEARCH DESIGN AND METHODS). Since the HLA DR3/4 genotype confers such a large genetic risk for type 1 diabetes, our approach was to look in families without the high-risk HLA DR-DQ genotypes so that other less strong linkage and association signals might be detected. Since below we present results together for the full dataset and for the two subgroups, we apply a conservative Bonferroni adjustment to the unadjusted P values to take account of these multiple comparisons.
The transmission disequilibrium test (TDT) (19) was performed on the IL4R genotype data to test for the association of the individual alleles of the eight IL4R SNPs to type 1 diabetes. The results of the SNP locus TDT are shown in Table 2. Nearly statistically significant evidence of excess transmission is only seen at SNP 6 (S478P) and only in "neither sib DR3/4" families (unadjusted P = 0.03, adjusted P = 0.10), although among all families, the data show a strong trend for SNP 6. The transmissions of the " reference" allele (Table 1) of SNPs 3, 4, 5, and 7 are elevated to the same extent (58%) as for SNP 6. We note that the comparisons of the eight SNPs presented are not independent statistical tests, as the SNPs are in very strong linkage disequilibrium (LD) (Table 1).
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Genome-wide linkage scans have identified many potential regions of linkage of type 1 diabetes in addition to the HLA region (IDDM1) and INS (IDDM2). Whereas evidence exists for a linked locus at 16q22-q24 (logarithm of odds [LOD] = 3.93) in U.K. and U.S. families, support for linkage has also been reported at 16p11-q12 (LOD = 1.74), where IL4R resides (21). Our IBD sharing data (online appendix) provide evidence consistent with those reports for linkage of IL4R with type 1 diabetes in the "neither sib DR3/4" group. One explanation for the lower LOD score near IL4R in those other genome-wide scans is that they may not have had sufficient power to detect loci with a s < 1.3.
Association of IL4R haplotypes with type 1 diabetes was assessed using a modified TDT haplotype association analysis (Table 3). We evaluated the nine most frequent eight-locus haplotypes using a two-sided modified TDT for the null hypothesis of no association and no linkage (see RESEARCH DESIGN AND METHODS). The TDT analyses (Table 3) revealed significant evidence of undertransmission of a single eight-locus IL4R haplotype (labeled "21222221" or H-3) in the full cohort (unadjusted P = 0.007, adjusted P = 0.021) and the "neither sib DR3/4" group (unadjusted P = 0.005, adjusted P = 0.015). In the full cohort, the H-3 haplotype has an OR of 0.63 (unadjusted 95% CI = 0.440.89), and in the "neither sib DR3/4" group, the OR = 0.49 (unadjusted 95% CI = 0.280.81) (Table 3). The haplotype H-3 can thus be regarded as conferring protection to type 1 diabetes.
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The use of multiplex families such as the HBDI cohort increases the power to detect a genetic effect precisely because sibships are more likely to share a genetic factor. Because of this ascertainment method, the observed association of IL4R in type 1 diabetes might apply only to a subset of the general population.
A recent report by Reimsnider et al. (7) investigated the association of IL4R and type 1 diabetes using the same HBDI family cohort (among others), but concluded that there was none. There are several possible explanations for the differences between the results of that report and ours. First, our IBD sharing results revealed marginal evidence of linkage only when DR3/4 stratification was applied. Second, in that previous study, only a single IL4R SNP (no. 7, Q551R) was genotyped (7), and it was not the SNP (no. 6, S478P) for which we detected the best evidence for a transmission bias (Table 2). More importantly, the observation of eight-SNP haplotypes increases the informativeness of the locus by converting biallelic SNPs into one multiallelic locus (Table 3).
The association of haplotype H-3 with type 1 diabetes may reflect the combined effect of the eight SNPs that compose the haplotype. Alternatively, the eight IL4R SNPs studied here may not capture all the variation defining the type 1 diabetes-protective haplotype, and other SNPs on or in LD with haplotype H-3 may be involved. We have found that haplotype H-3 is associated with protection from type 1 diabetes, with a similar OR, in a Filipino population (T. Bugawan, D.B.M., A.M.V., A. Panelo, C. Solfelix, P. Pozzilli, R. Buzetti, H.A.B., manuscript in preparation) and favor the hypothesis that this specific variant of the IL4R protein confers protection to type 1 diabetes. An alternative hypothesis, in which H-3 acts as a marker in LD with other causal variation(s), seems less likely because the LD patterns and haplotype distributions among Filipinos are different from those of the Caucasian HBDI population.
Our results suggest that a specific combination of the eight SNPs, rather than any individual SNP, is responsible for the observed protection from type 1 diabetes (Table 3). The evidence for the negative association of the H-3 haplotype with type 1 diabetes is stronger than that seen with any of the individual SNPs (33 vs. 42% transmission in the "neither DR3/4" group; unadjusted P = 0.005 vs. 0.03, respectively). The effect of individual SNPs in a haplotype seems to depend on the nature of the other SNPs, which is biologically plausible, as these SNPs encode allelic variants of the same IL4R protein. For example, haplotypes H-1 and -2 differ only at SNP 1 and have the same OR (Table 3); H-3 and -5 also differ only at SNP1, although H-5 does not appear to influence susceptibility to type 1 diabetes, whereas H-3 does. The role of SNP 1 in susceptibility to type 1 diabetes is therefore context dependent, highlighting the value of using haplotype data.
How might haplotype H-3 confer protection to type 1 diabetes given the known role of IL4R variation in Th2-related disease? Although numerous studies report associations of individual IL4R SNP alleles and genotypes with asthma and atopy, the results are not consistent. In some studies, the variant allele is associated with a disease-related phenotype, while in others it is the reference allele. If, in fact, the variant alleles (or haplotypes bearing them, such as H-3) are associated with a Th2 phenotype, then the protection to type 1 diabetes reported here may reflect a shift of the balance between Th1 and Th2 responses toward Th1. If, on the other hand, the reference alleles (or haplotypes bearing them) are associated with asthma and atopy, then this simple model of Th1 versus Th2 balance does not fit the data. Perhaps, therefore, variations in IL4R influence the regulation of both Th1 and Th2 responses, as elaborated further below.
The SNPs we have used to define IL4R haplotypes do not fully coincide with those used by others in assessing haplotypic association to asthma and atopy phenotypes. Nonetheless, what Ober et al. (11) called haplotype "222122" and Hackstein et al. (8) called IL4RA*03 both correspond to our haplotype H-3. IL4RA*03 was reported to be associated with lower total IgE levels in nonatopics than were more common haplotypes (equivalent to our haplotypes H-1, -2, and -4) (8). Also, haplotype "222122" was observed by TDT to be significantly undertransmitted to asthmatics in an isolated Hutterite population, although in that study the TDT was used as a test of linkage (11). These observations suggest that H-3 might be associated with a dampened T-helper cell response in either the Th1 or Th2 direction. In vitro and in vivo studies comparing the functions of the receptor molecules expressed from fully defined IL4R haplotypes will be instructive. Whatever the mechanism, allelic variants of the IL4R protein, one of which is encoded by haplotype H-3, are likely to be modifiers of immune disease susceptibility.
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RESEARCH DESIGN AND METHODS |
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Definition of DR3/4, stratification.
Families were stratified into two groups based on the HLA genotype of the two ascertained affected children. A child possesses the high-risk HLA-DR3/DR4 genotype if he/she carries a DRB1* 0301-DQB1*02 haplotype and a DR4 allele that is not DRB1*0403 (a protective allele) in a haplotype with DQB1*02 or DQB1*0302 (but not *0301 or *0304). In "either/both sib DR3/4" families (n = 135), one or two children possess the DR3/DR4 genotype. In "neither sib DR3/4" families (n = 147), neither child possesses the DR3/DR4 genotype.
IL4R genotyping
List of SNPs detected.
The eight SNPs in the human IL4R gene are listed in Table 1. The SNPs will subsequently be referred to by the SNP number listed in Table 1. The "reference" allele for each SNP is that present in GenBank accession X52425.1 and is denoted by a "1" in subsequent tables; the variant allele for each SNP is denoted by a "2."
PCR primers and amplification.
Primers were purchased from Operon (Alameda, CA) and were modified at the 5' phosphate by conjugation with biotin. Pairs of primers were designed and used to amplify the regions encompassing the eight SNPs. Other SNPs not relevant to this report were genotyped concurrently by coamplification within the same multiplex PCR and hybridization to an immobilized probe array (data not shown).
PCR amplification was performed in a total reaction volume of 50100 µl containing the following reagents: 70 mmol/l KCl, 12 mmol/l Tris-HCl (pH 8.3 at 25°C), 3 mmol/l MgCl2, 0.2 ng/µl purified human genomic DNA, 0.2 µmol/l each primer, 200 µmol/l each of dATP, dTTP, dCTP, and dGTP, and 0.25 units/µl AmpliTaq Gold DNA polymerase. Amplification was carried out in a GeneAmp PCR System 9600 thermal cycler (PE Biosystems, Foster City, CA) using the following specific temperature cycling profile: an initial hold at 94°C for 12.5 min; 33 cycles of 95°C for 45 s, 61°C for 30 s, and 72°C for 45 s; and a final extension step of 72°C for 7 min.
Allele-specific detection by probe hybridization.
The detection of allelic variants at each SNP is performed by stringent hybridization of the biotinylated PCR products to the immobilized sequence-specific probes, followed by visualization using an enzyme-catalyzed color development step. The procedure is essentially as described in Cheng et al. (22), except that hybridization and washing were performed at 55°C. An example of linear array results is shown in Fig. 1. Haplotypes were determined as described below.
Inference of haplotypes.
We inferred IL4R haplotypes by a combination of two methods: 1) using the GeneHunter program (Falling Rain Genomics, Palo Alto, CA), which calculates haplotypes from genotype data from pedigrees; and 2) inspecting each HBDI family pedigree individually using the Cyrillic program (Cherwell Scientific Publishing, Palo Alto, CA) to resolve any ambiguous or unsupported haplotype assignments. Haplotypes could be confidently and unambiguously assigned in all but 6 of the 282 families; in the remainder, the phase of SNP 1 could not be determined.
Statistical analysis, methods, and algorithms.
Evidence for linkage of IL4R to type 1 diabetes was assessed by the haplotype sharing method. IBD values of parental IL4R haplotypes in the affected sibs could be determined unambiguously in 256 families. The locus-specific sibling relative risk was calculated using the formula s = 0.25/(proportion of families IBD = 0) (20). CIs were calculated using the standard error of the IBD proportions. The TDT was performed for individual SNPs by the method of Spielman (19). Association of IL4R haplotypes with type 1 diabetes was assessed using the modified TDT haplotype association analysis (described below).
LD among the eight IL4R SNPs was assessed using the pool of AFBAC haplotypes deduced for the entire cohort. The AFBAC pool represents an unbiased estimate of the population haplotype frequencies (23). We calculated the values of two distinct but related metrics for LD, namely D and , by standard methodology.
Modified TDT haplotype association analysis.
We evaluated the nine most frequent eight-locus haplotypes using a two-sided modified TDT for the null hypothesis of no association and no linkage. As obtaining haplotypes for parents based on data from the children can introduce bias (24), we used a statistic that does not rely on this information. The statistic is the proportion of times a haplotype was transmitted among informative transmissions where it was available. When both parents and a child are heterozygous at n 1 loci, the statistic gives weight = (0.5)n to each haplotype arrangement consistent with the observed diplotypes. We used 2,000 randomly permuted datasets to assess significance (25). A childs complementary diplotype is defined as the entire set of alleles remaining when the childs observed diplotype is subtracted from the combined diplotypes of the two parents. The permutation procedure randomly and independently switches the roles of each childs observed and complementary diplotypes to create 2,000 permuted datasets. This procedure is valid when phase is unknown, even if recombination is present. Empirical CIs were based on 2,000 bootstrap samples, created by resampling the observed families with replacement. Intervals for the proportion transmitted were converted to equivalent intervals for the reported ratio, which can be interpreted as an OR for observed segregation relative to Mendelian segregation. The haplotype analysis was performed using the S-PLUS software package (Insightful, Seattle, WA), and S-PLUS bootstrap functions were used for both resampling methods.
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ACKNOWLEDGMENTS |
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We thank Amy White for technical assistance and our colleagues at Roche Molecular Systems for comments and discussion.
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FOOTNOTES |
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Received for publication 8 February 2002 and accepted in revised form 26 July 2002.
HBDI, Human Biological Data Interchange; IBD, identity by descent; IL4, interleukin-4; LD, linkage disequilibrium; LOD, logarithm of odds; OR, odds ratio; SNP, single-nucleotide polymorphism; TDT, transmission disequilibrium test.
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REFERENCES |
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