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5Diabetes and Genetic Epidemiology Unit, National Public Health Institute, Helsinki, Finland; 6Clinic of ... genetic effect of IL4RA in T1D, but it is not a major one.
Genes and Immunity (2003) 4, 469–475 & 2003 Nature Publishing Group All rights reserved 1466-4879/03 $25.00 www.nature.com/gene

Testing the possible negative association of type 1 diabetes and atopic disease by analysis of the interleukin 4 receptor gene LM Maier1, RCJ Twells1, JMM Howson1, AC Lam1, DG Clayton1, DJ Smyth1, D Savage2, D Carson3, CC Patterson4, LJ Smink1, NM Walker1, OS Burren1, S Nutland1, H Rance1, E Tuomilehto-Wolf5, J Tuomilehto5, C Guja6, C Ionescu-Tirgoviste6, DE Undlien7, KS Rnningen8, F Cucca9 and JA Todd1 JDRF/WT Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK; Department of Medical Genetics, Queen’s University, Belfast, Northern Ireland, UK; 3Department of Child Health, Queen’s University, Belfast, Northern Ireland, UK; 4Department of Epidemiology and Public Health, Queen’s University, Belfast, Northern Ireland, UK; 5 Diabetes and Genetic Epidemiology Unit, National Public Health Institute, Helsinki, Finland; 6Clinic of Diabetes, Nutrition and Metabolic Diseases ’N Paulescu’, Bucharest, Romania; 7Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway; 8 Laboratory of Molecular Epidemiology, Division of Epidemiology, Norwegian Institute of Public Health, Oslo, Norway; 9Instituto di Clinica e Biologia dell’Eta’ Evolutiva, University of Cagliari, Italy 1 2

Variations in the interleukin 4 receptor A (IL4RA) gene have been reported to be associated with atopy, asthma, and allergy, which may occur less frequently in subjects with type 1 diabetes (T1D). Since atopy shows a humoral immune reactivity pattern, and T1D results from a cellular (T lymphocyte) response, we hypothesised that alleles predisposing to atopy could be protective for T1D and transmitted less often than the expected 50% from heterozygous parents to offspring with T1D. We genotyped seven exonic single nucleotide polymorphisms (SNPs) and the –3223 C4T SNP in the putative promoter region of IL4RA in up to 3475 T1D families, including 1244 Finnish T1D families. Only the 3223 C4T SNP showed evidence of negative association (P ¼ 0.014). There was some evidence for an interaction between 3233 C4T and the T1D locus IDDM2 in the insulin gene region (P ¼ 0.001 in the combined and P ¼ 0.02 in the Finnish data set). We, therefore, cannot rule out a genetic effect of IL4RA in T1D, but it is not a major one. Genes and Immunity (2003) 4, 469–475. doi:10.1038/sj.gene.6364007 Keywords: type 1 diabetes; atopy; promoter; IL4RA

Introduction Several studies have reported on an inverse relation between atopic diseases and type 1 diabetes (T1D),1–4 but the two diseases can also exist together,5 indicating the complexity and impact of common environmental factors involved in the disease process. In the past 50 years, a steady and significant increase in the incidence of T1D and atopy in children has been observed, providing further evidence that common environmental and/or genetic factors and their interaction may influence the incidence of the two diseases.6–8 T1D is caused by autoimmune destruction of pancreatic islet b-cells, a process that is genetically determined and susceptible to major, and as yet unknown, environmental factors.9 The disease is characterised by inflammation of the islets with an immune cell infiltration, in particular CD8+ cytotoxic T lymphocytes.10,11 The HLA class II association in T1D also implicates CD4+ T lymphocytes in disease causality, probably providing Correspondence: Dr JA Todd, Diabetes and Inflammation Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK. E-mail: [email protected]

help in the form of interferon g (IFNg) and interleukin 2 for pathogenic cytotoxic T lymphocytes (CTLs). In contrast, in atopic illnesses such as asthma, atopic dermatitis, and rhinoconjunctivitis, increased levels of IgE with reduced production of INFg and increased production of interleukin 4 (IL-4), referred to as a humoral immune response, are observed. In the nonobese diabetic (NOD) mouse model of T1D, T1D resistance can be induced by altering the balance between humoral and cellular immune response using IL4.12 In a transgenic model of T1D, disruption of the IL4RA gene protects against disease, suggesting that the inability to respond to IL4 protects against T1D in this model of autoimmunity.13 However, a shift towards a humoral pattern of cytokine expression cannot always be assumed to be the cause of disease resistance but may rather be a consequence.14 The IL4RA gene is a good functional candidate gene for both T1D and atopy, as binding of IL-4 to IL4Ra stimulates development of Th2 lymphocytes, which produce IL-4, IL-5, IL-6, IL-10, and IL-13, while suppressing INFg- and IL-2-producing Th1 cells. Variations in the ligands of IL4Ra, IL-4 and IL-13, have been associated with atopic phenotypes.15,16 Polymorphisms

IL4RA variants in type 1 diabetes LM Maier et al

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in IL4RA have also been reported to be associated with atopic phenotypes: the I50V (+148 A4T), C406R (+1216 T4C), S478P (+1682 T4C, previously S503P), and Q551R (+1902 G4T, previously R576Q) variants have shown association with increased risk for asthma17,18 and atopy.18,19 All polymorphisms except for +148 A4T, which is in exon 5, are located in exon 12. Some of these polymorphisms significantly upregulate receptor response to IL-4, with a resultant increased activation of STAT6, and thus increased cell proliferation and IgE production.17,19 Ober et al18 have also reported an apparent haplotype effect in outbred populations. S411L (+1232 C4T) and S761P (+2281 T4C) are further single nucleotide polymorphisms (SNPs) in exon 12, but have largely been excluded from previous analyses due to low minor allele frequencies and, therefore, low statistical power. A combination of variants, or an additional, as yet unknown variant in the 50 promoter region, the 30 UTR, or the introns, which is in linkage disequilibrium with the exonic polymorphisms, may be associated with atopic phenotypes.18 The C- 3223T (3223 C4T) variant in the 50 promoter region of IL4RA, which disrupts the core sequences of at least two potential transcription factor-binding sites (cAMP response element-binding protein and TCF11/MafG heterodimers), was also investigated. Although it has never been tested for association with an atopic phenotype, it has been associated with reduced levels of a secreted form of IL4Ra, sIL4Ra.20 sIL4Ra lacks both transmembrane and cytoplasmic domains, is expressed in allergic inflammation and is capable of binding and sequestering IL4, countering anti-inflammatory mechanisms. In asthmatic and atopic subjects, the concentration of sIL4Ra is reduced compared to healthy subjects.21 Clinical studies have shown that soluble recombinant IL4Ra is an effective treatment for patients with asthma.22 By reducing sIL4Ra levels and, therefore, enhancing a humoral response, the 3223 T allele may provide some protective effect from T1D. IL4RA is located in the region of chromosome 16p11 that has been linked to T1D in two recent genome-wide linkage scans with LOD scores of 1.74 in 767 affected sibpair families from the UK and USA23 and 2.8 in 408 multiplex families from Scandinavia.24 Association of IL4RA with T1D has previously been investigated in just one study, which examined only one polymorphism in a small data set,25 reporting that the IL4RA +1902 G4T SNP (Q551R) was not linked or associated with T1D. We hypothesised that the atopy-predisposing alleles may be protective for T1D and transmitted less often than the expected 50% from heterozygous parents to offspring with T1D.

Results The present study reports on the analysis of one SNP in the 50 promoter region of IL4RA, one SNP in exon 5, and six SNPs in exon 12 in up to 3475 T1D families, which comprise families from the UK, Finland, the USA, Norway, Romania, and Sardinia. With this large data set, we had greater than 99.99% power to detect an effect at P ¼ 0.05 for a disease allele with 5% frequency and a relative risk of 1.5. The +2281 T4C (S761P) variant was Genes and Immunity

rare (allele frequency 1%) and, therefore, provided 80% power. All loci were in Hardy–Weinberg equilibrium in parents from all populations. In an attempt to detect new variants and confirm previously detected polymorphisms, we resequenced all 12 exons of the IL4RA gene including 50 bp up- and downstream of each exon in both the forward and reverse directions. Sequences were obtained from 64 T1D subjects originating from the UK that show linkage to chromosome 16 and 32 control subjects. We confirmed all previously known polymorphisms and discovered novel SNPs (ie not available in dbSNP) (Table 1). Significant heterogeneity for allele frequencies in the six populations was obtained for all SNPs, except for +1124 A4C (E375A) and +1216 T4C (C406R) (Table 2). For the exonic SNPs, there was no evidence to suggest association with T1D (Table 3). Note that, as families with one affected sibling and families with more than one affected sibling were analysed together, we employed robust variance estimates, which allow for nonindependence of sibs. We also tested for the association of genotypes with TID, but obtained no results with Po0.05. The 3223 T allele in the 5’ promoter region of IL4RA was transmitted 47.5% of the time to diabetic offspring from heterozygous parents (PTDT ¼ 0.014, PGTRR ¼ 0.024), with an estimated relative risk of 0.90 (95% CI 0.84–0.98), and most of this undertransmission was largely restricted to maternal meioses (data not shown). This latter observation, although not significant on its own, is consistent with previously published evidence of excess sharing of maternal alleles at asthma susceptibility loci,26–28 including markers flanking IL4RA.29 There was no evidence that the potential disease association of 3223 C4T differed between the six populations (not shown). As shown by a linkage disequilibrium map in Figure 1, the disease association of the 3223 C4T SNP does not appear to be due to disease association of the +148 A4G (I50V) variant as previously reported,20 since the 3223 C4T SNP is not in linkage disequilibrium with +148 A4G (I50V). Having obtained Po0.05 for the 3223 C4T SNP in the total data set, we investigated the interaction between the 3223 T allele and the known T1D susceptibility loci IDDM1/HLA and IDDM2/INS VNTR. Using multinomial logistic regression analysis, we analysed six HLA-DR subgroups (DR3/DR3, DR3/DR4, DR4/DR4, DR3/nonDR4, DR4/non-DR3, non-DR3/non-DR4), and the two INS VNTR subgroups, the T1D susceptible class I homozygous genotype and the combined class I/III + III/III subgroup. 3223 C4T genotype results were also

-3,223 C>T +148 A>G +1,124 A>C +1,216 T>C +1,232 C>T +1,682 T>C +1,902 G>A +2,281 T>C

-3,223 C>T 0.003 0.224 0.144 0.777 0.309 0.268 0.984

+148 A>G 0.057 0.001 0.003 0.030 0.004 0.000 0.078

+1,124 A>C 0.473 0.033 0.913 1.000 0.906 0.914 0.926

+1,216 T>C 0.379 0.058 0.955 1.000 0.740 0.793 0.002

+1,232 +1,682 C>T T>C 0.881 0.556 0.174 0.062 1.000 0.952 1.000 0.860 0.943 0.889 0.957 0.804 1.000 1.000

+1,902 G>A 0.517 0.017 0.956 0.890 0.978 0.897 1.000

+2,281 T>C 0.992 0.279 0.962 0.040 1.000 1.000 1.000 -

Figure 1 Pairwise linkage disequilibrium map. D0 values are shown in the upper-right triangle, r2 values in the lower-left triangle. D0 values above 0.8 and r2 values above 0.5 are shown in bold.

IL4RA variants in type 1 diabetes LM Maier et al

Table 1 List of variants known to date in the regions sequenced in this study and novel variants discovered by resequencing of IL4RA in 96

471

individuals Variant DIL1071 DIL1077 DIL1078 DIL1079 DIL1080 DIL1081 DIL1099 DIL1102 DIL1103 DIL1073 DIL1074 DIL1075 DIL1076 DIL1105 DIL1106 DIL1107 DIL1111 DIL1110 DIL1112 DIL1113 DIL1114 DIL1115 DIL1140 DIL1141 DIL1142 DIL1143 DIL1144 DIL1484 DIL1087 DIL1088 DIL1089 DIL1486 DIL1090 DIL1091 DIL1092 DIL1093 DIL1094 DIL1096 DIL2398 DIL1097 DIL1086 DIL1082 DIL1083 DIL1084

Flanking sequence or dbSNP ID

Location

Minor allele frequency

GCCTTTGGACCTGCTCCCAGGACTG[C/T] CGGAGCAGTCACAGATAAAGTCTGG CAAACGCAACTGCCCCGGCGCAAAA [C/T] GACTACATGAGCATCTCTACTTGCG GATTCCAGTCGCCCAGCCCTCCCCC[A/C] ACAAACGCAACTGCCCCGGCGCAAAA GATTCCAGTCGCCCAGCCCTCCCCCA[A/C] CAAACGCAACTGCCCCGGCGCAAAA TCTCCGCTGGGCGTGACCTCGGGCT[A/G] CAGCGTGGGAGGAAGCGCGCGGCAAG CTCCGCTGGGCGTGACCTCGGGCTGC[A/G] GCGTGGGAGGAAGCGCGCGGCAAGA GATTCCAGTCGCCCAGCCCTCCCCCA[A/C] CAAACGCAACTGCCCCGGCGCAAAA rs2107356 GATGAAACCCCATCTCTACTAAAAA[C/T] ACAAAAATTAGTTGGGCATGGTGGC rs2283563 rs3024543 rs3024544 CCCTCACGCATTGAGTTCCTGGGCC[G/T] CTCAGGCTGCTCCTGTGTCTCCCCA rs3024558 rs2074572 rs2072130 CAGGAGCCTGGGAGGCAAGCCCTGG[A/G] GCTGGATAGCAAATCCCAGGAGCTA rs3024571 rs2301807 rs3024575 rs3024576 rs3024577 rs3024634 rs3024633 rs3024632 rs3024630 rs3024629 rs3024636 rs3024612 rs3024611 rs3024610 TGCAGTCCTCTCAGTCAATAATACG[T/C] ATTTACTGAGCAGCTACTACACACC AGTGTCGAAACTGAACCCTGACCAA[A/C] CTTTGCTTTTGCAGACACTGGAAGA rs2234897 rs1805011 rs2234898 rs1805012 rs1805013 rs1801275 rs1805015 CTTGCTTTTGCAGACACTGGAAGA[A/G]TG AGCAAGAAATATATTCC rs2234924 rs1049631 rs2057768

50 region

34.7%

50 region

2.6%

0

5 region

54.7%

50 region

54.7%

0

5 region

37.2%

50 region

6.7%

0

5 region

45.4%

50 region Intron 2

38.5% 30.5%

Intron Intron Intron Intron

2 3 3 3

41.0% 16.1% 15.7% 1.1%

Exon 5 Intron 5 Intron 5 Intron 5

46.2% 36.0% 37.6% 1.6%

Exon 6 Intron 6 Intron 6 Intron 6 Intron 6 Intron 7 Intron 7 Intron 7 Intron 7 Intron 7 Intron 7 Intron 7 Intron 7 Intron 7 Intron 9

9.7% 6.0% 38.2% 8.5% 44.8% 14.1% 8.9% 9.0% 8.2% 8.2% 8.4% 6.8% 15.8% 46.3% 15.1%

Intron 11

18.9%

Exon 12, synonymouschange [Phe > Phe] Exon 12, non-synonymous change [Glu > Ala] Exon 12, synonymous change [Leu >Leu] Exon 12, non-synonymous change [Cys >Arg] Exon 12, non-synonymous change [Ser >Leu] Exon 12, non-synonymous change [Gln >Arg] Exon 12, non-synonymous change [Ser >Pro]

3.7% 13.7% 13.7% 13.5% 4.2% 28.5 19.1% 45.8%

30 UTR 30 UTR 30 UTR

1.6% 34.1% 47.8

evaluated on the basis of age at onset of disease and sex of affected siblings (Table 4). The only interaction showing Po0.05 was with the INS VNTR (P ¼ 0.001), whereby the T allele was undertransmitted to T1D cases who were VNTR class I homozygous (42.9% transmission; P ¼ 0.0004; OR ¼ 0.83, 95% CI 0.73–0.93). P values o0.05 for the interaction between the 3223 C4T variant were also obtained when a second statistical method,

the pseudocase–control approach, was employed.30 For completeness, we compared the association in DR3/4 cases vs the rest and found no difference (not shown). Finland is a founder population in which alleles that are rare in other countries can be significantly more common in the Finnish population. For the eight IL4RA SNPs analysed here, none showed a significantly increased frequency in Finland. Nevertheless, because Genes and Immunity

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Table 2

Population allele frequencies of minor alleles in parents of T1D offspring and P values obtained from heterogeneity tests for allele frequencies 3223 C>T

+148 A>G (I50 V)

+1124 A>C (E375A)

+1216 T>C (C406R)

UK USA Sardinia Finland Norway Romania

31.2 (1623) 27.2 (1101) 26.5 (825) 26.6 (1662) 29.7 (550) 26.1 (566)

52.2 (1456) 44.2 (1040) 46.9 (739) 44.3 (1387) 52.0 (487) 53.7 (538)

11.0 (979) 11.5 (1094) 11.0 (858) 10.2 (1767) 12.2 (679) 8.7 (548)

11.3 (1354) 10.8 (1066) 11.5 (802) 9.7 (1799) 12.9 (287) 10.5 (370)

P value

2  104

3.3  1011

0.06

0.14

+1232 C>T (S411L)

+1682 T>C (S478P)

+1902 G>A (Q551R)

+2281 T>C (S761P)

UK USA Sardinia Finland Norway Romania

5.4 (1186) 4.3 (1100) 2.8 (916) 2.6 (1891) 4.0 (700) 3.8 (553)

17.0 (1182) 16.6 (714) 15.3 (625) 12.8 (1662) 15.3 (472) 12.7 (508)

21.5 (1189) 20.8 (1341) 13.7 (697) 19.0 (1926) 21.7 (655) 19.7 (563)

0.3 (918) 1.3 (1267) 1.2 (904) 0.2 (1520) 0.5 (645) 0.8 (179)

P value

2  106

1  104

2  104

3  105

The number of independent individuals used to calculate allele frequencies are given in parentheses.

Table 3 Association analysis of SNPs previously associated with atopic phenotype in T1D families with at least one affected offspring Marker

Position

Allele

Na

p

T

NT

%T

P

3223 C>T +148 A>G (I50 V) +1124 A>C (E375A) +1216 T>C (C406R) +1232 C>T (S411L) +1682 T>C (S478P) +1902 G>A (Q551R) +2281 T>C (S761P)

5 region Exon 5

T Gb

2505 (3228) 2161 (3198)

27.9 48.9

1173 1189

1297 1262

47.5 48.5

0.014 0.141

Exon 12

Cb

2250 (2693)

10.8

506

527

49.0

0.525

Exon 12

Cb

2294 (3067)

11.2

492

524

48.4

0.322

Exon 12

T

2508 (2789)

3.8

243

240

50.3

0.895

Exon 12

Cb

2026 (2520)

17.5

655

653

50.1

0.956

Exon 12

Ab

2433 (2964)

19.4

934

900

50.9

0.425

Exon 12

C

1980 (2287)

0.7

24

33

42.1

0.241

Complete data of two parents and at least one affected offspring (number of families attempted to be genotyped is given in parentheses). Denotes association with increased susceptibility to atopic phenotypes. N, number of families; p; % frequency of minor allele in parents of T1D offspring; T, number of transmissions; NT, number of nontransmission; %T, percentage transmission from heterozygous parents to T1D offspring (obtained by transmission/disequilibrium test (TDT), using robust variance estimates); P, probability value (two-sided). a

b

the Finnish data were the largest data set used in this study (contributing 41% of individuals to the total set), we analysed the Finnish data separately for each SNP (Table 5). The variants +148 A4G (I50V) in exon 5 and +1216 T4C (C406R) in exon 12 showed some evidence of association (both P ¼ 0.03). The only evidence of interaction of these two SNPs was with INS VNTR (P ¼ 0.04 and P ¼ 0.02 for SNPs +148 A4G (I50V) and +1216 T4C (C406R), respectively, when all affected parents and offspring were used for analysis). Ober et al18 showed that certain haplotypes of exonic SNPs in IL4RA were associated with asthma in the Hutterites, most significantly the two-locus haplotype Genes and Immunity

consisting of the +1124 A4C (E375A) and +1682 T4C (S478P) variants, and the six-locus haplotype consisting of +148 G and the wild-type alleles at all other sites. Some of the IL4RA haplotypes have been shown to increase phosphorylation of binding substrates, which might result from a change in the receptor’s charge and conformation, leading to tighter substrate binding.19 However, in our study none of these combinations and indeed, no other haplotype, deviated from the expected Mendelian transmissions (see Table 6 for the five most common haplotypes). After this work was completed, part of the USA data set of this study was analysed by Mirel et al,31

IL4RA variants in type 1 diabetes LM Maier et al

Table 4 P values obtained by multiple logistic regression analysis

Table 6 IL4RA six-locus haplotypes observed in the full data set

of interaction effects between –3223 C>T and HLA status, INS VNTR status, sex of affected offspring, and age at onset of disease (cases only were used for analysis)

Haplotype ID

HLAa INS VNTR Sex Age at onset

Affected parents and offspring

All affected siblings

First affected sibling only

0.25 (0.63) 0.002 0.69 0.95

0.26 (0.47) 0.001 0.69 0.78

0.33 (0.42) 0.002 0.96 0.51

HLA DR genotypes of Romanian subjects were only available in four classes (DR3/DR4, DR3/non-DR4, DR4/non-DR3, and nonDR3/non-DR4). Results obtained from all families including Romanian subjects are given in parentheses.

a

Table 5

H1 H2 H3 H4 H5

Haplotype

Frequency (%)

Relative risk

Relative risk 95% CI

111111 211111 122122 222122 111112

42.7 40.0 3.5 3.2 2.2

1.0 1.06 1.25 0.91 0.82

F 0.91–1.24 0.87–1.81 0.62–1.36 0.51–1.31

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Note that for better comparison with previous data sets, the SNPs shown include exonic SNPs only (ie +148 A>G, +1124 A>C, +1216 T>C, +1232 C>T, +1682 T>C, +1902 G>A). SNP +2281 T>C was excluded from analysis due to low minor allele frequency. ‘1’ denotes the presence of the reference allele and ‘2’ indicates the presence of the variant allele for each SNP. The five most common haplotypes are shown. The most common haplotype was used as the reference haplotype.

Disease association analysis of SNPs in the Finnish data

set only Na

T

NT

%T

P

786 (1044) 594 (1172)

258 265

297 317

46.5 45.5

0.09 0.03

772 (1021)

145

130

55.1

0.39

885 (1244)

135

175

43.6

0.03

850 (1021)

49

43

53.3

0.52

714 (9 3 2)

157

162

49.2

0.79

830 (1112)

242

258

48.4

0.47

686 (8 1 1)

1

4

20.0

0.18

Table 7 IL4RA six-locus haplotypes observed in the USA data set Haplotype ID

3223 C>T +148 A>G (I50 V) +1124 A>C (E375A) +1216 T>C (C406R) +1232 C>T (S411L) +1682 T>C (S478P) +1902 G>A (Q551R) +2281 T>C (S761P)

Complete data of two parents and at least one affected offspring (number of families attempted to be genotyped is given in parentheses). N, number of families; T, number of transmission; NT, number of nontransmissions; %T, percentage transmission from heterozygous parents to T1D offspring (obtained by transmission/disequilibrium test (TDT), using robust variance estimates); P, probability value (two-sided).

H1 H2 H3 H4 H5

Haplotype

Frequency (%)

Relative risk

Relative risk 95% CI

111111 211111 222122 122122 211222

49.6 34.7 4.8 3.9 3.8

1.0 0.89 0.59 0.75 0.86

F 0.61–1.28 0.28–1.25 0.30–1.86 0.40–1.83

Note that for better comparison with previous data sets, the SNPs shown include exonic SNPs only (ie +148 A>G, +1124 A>C, +1216 T>C, +1232 C>T, +1682 T>C, +1902 G>A). SNP +2281 T>C was excluded from analysis due to low minor allele frequency. ‘1’ denotes the presence of the reference allele and ‘2’ indicates the presence of the variant allele for each SNP. The most common haplotype was used as the reference haplotype.

a

who suggested a specific haplotype to be protective, particularly among individuals not carrying the HLA DR3/DR4 genotype. The study of Mirel et al involved the genotyping of eight IL4RA SNPs in 282 multiplex families from the USA, of which six SNPs coincide with those genotyped by us in up to 3475 T1D families. We have thus aimed to replicate these findings and analysed the USA data separately. Inconsistent with the data reported by Mirel et al., who did not observe any significant results for single-locus analyses, we have obtained P values less than 0.05 for SNPs +1124 A4C (E375A, P ¼ 0.007), +1216 T4C (C406R, P ¼ 0.02), and +1682 T4C (S478P, P ¼ 0.008) in the USA data set. These increased transmissions, however, are not seen in any other population we have studied except for P ¼ 0.03 for SNP +1216 T4C (C406R) in the Finnish data set. This emphasises the importance of including all publicly

available data sets, such as the UK Warren 1 repository, in a study design and highlights that the analysis of a single small data set and its interpretation in terms of possible involvement of a candidate gene in a complex disease may not be sufficient. Some of our findings are consistent with those of Mirel et al 31 in that none of the common haplotypes deviated from the expected 50% transmission. However, haplotype H-3, observed by Mirel et al to be undertansmitted to T1D offspring and which corresponds to our haplotype H-3, does not show disease association in our USA data or any other data set, nor does any other haplotype in our analysis (Table 7). It is further noted here that even though Mirel et al. observed P values less than 0.05 in the ’all families’ and the ’neither sib DR3/4’ groups, no statistical test was used to evaluate any difference among the groups. Indeed, haplotype H-3 in the data obtained by Mirel et al. is also undertransmitted in the group ’either/both sib DR3/4’, which undermines the observation that the protective effect of this haplotype is strongest among individuals not carrying the HLA DR3/4 genotype. In order to exclude the possibility that genotyping errors in our study may have contributed to the inconsistencies observed between Mirel et al’s data and Genes and Immunity

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our data with regard to haplotype frequencies in the USA data set, five exonic SNPs were genotyped a second time in the USA samples. Secondly, five exonic SNPs (as well as the weakly associated 3223 C4T variant in the putative promoter region) were genotyped a third time using a different genotyping technology. Those SNPs previously genotyped by the Invader assay were genotyped using TaqMan probes and those SNPs previously genotyped using TaqMan probes were genotyped using the Invader assay. No inconsistencies were found between the first and second stages of genotyping, and no inconsistencies were found between the first and third stages of genotyping.

Discussion Biologically, the reduction of soluble IL4Ra associated with the T allele of the 3223 C4T variant could divert the immune response away from islet antigens towards IgE production. Moreover, the INS VNTR has been associated with islet autoantibody development, possibly indicating its involvement in immune tolerance induction,32 at the level of the thymus.33 It is also possible that the –3223 C4T variant is in linkage disequilibrium with the true causative variant, in close proximity of this SNP. Even with a plausible biological rationale for the candidacy of a gene such as IL4RA, P values in the order of 105 are desirable to ensure a relatively low falsepositive rate for disease association studies and Po107– 108 for genome-wide association studies.34–36 Obviously, a second independent even larger data set will need to be genotyped for this SNP to test if the T allele of the –3223 C4T SNP does indeed protect from T1D. New collections of families (http://www.t1dgc.org) and case–controls are under way (http://www.childhooddiabetes.org.uk). Nevertheless, despite the large sample size and the very high statistical power for common variants such as I50 V(499.9% for a relative risk as small as 1.2), which are supposedly associated with IgE production and atopic illness, our failure to detect an association between the exonic variants of IL4RA and TID suggests that if there is the gene polymorphism explanation for the reported negative association of TID and atopic disease then structural polymorphisms of IL4Ra probably do not account for this.

Methods Subjects All families were comprised of white subjects of European descent with two parents and at least one affected child. The families comprised 472 UK families with two affected siblings and 527 UK families with one affected sibling; 317 US families with two affected siblings; 240 Sardinian families with one affected sibling; 359 Norwegian families with one affected sibling; 316 Romanian families with one affected sibling; 1231 Finnish families with one affected sibling; and 13 Finnish families with two affected siblings. All DNA samples were collected with informed consent. Sequencing Exons and 50 bp up- and downstream of each exon were sequenced in 64 T1D subjects that showed linkage to Genes and Immunity

chromosome 16 and 32 control subjects from the UK. Cycle sequencing in both forward and reverse direction was performed using the ABI 3700 capillary sequencer (Perkin Elmer Applied Biosystems, Foster City, CA, USA) and the ABI PRISM BigDye Terminator sequencing kit with AmpliTaq DNA polymerase (Perkin Elmer). Genotyping SNPs were genotyped by the Invader assay (Third Wave Technologies, Inc., Madison, WI, USA) and TaqMan (ABI). Invader probe sets for each locus were designed and synthesised by Third Wave Technologies; TaqMan probes were designed in-house and synthesised by ABI. Full details of primers and probes used for genotyping are available at http://www-gene.cimr.cam.ac.uk/todd/ human_data.shtml. All genotyping data were doublescored. Statistical analysis Allelic association was tested using the transmission/ disequilibrium test (TDT); robust variance estimates were used to allow for nonindependence of sibs. Statistical analysis, including genotype relative risk analyses, was performed using the GenAssoc package within STATA (http://www.stata.com), available from David Clayton’s website (http://www.gene.cimr.cam. ac.uk/clayton/software/). The effects of sex of affected offspring, HLA status, INS VNTR status, age at onset of T1D (analysed as a linear variable) as well as haplotypic effects were evaluated using multinomial logistic regression methods. Method 1 uses cases (affected parents and affected offspring only), and Method 2 compares cases to up to three ‘pseudocontrols’, having the other nontransmitted genotypes.30 Population effects were evaluated using a conditional logistic regression approach with cases and pseudocontrols.30 The analysis of a parentof-origin effect was based on the method proposed by Weinberg.37 P values were not corrected for multiple testing.

Acknowledgements This work was funded by the Wellcome Trust and the Juvenile Diabetes Research Foundation International. LMM is the recipient of a Wellcome Trust Prize Studentship. JT and ET-W were funded by the NIH, the Novo Nordisk Foundation, the Academy of Finland, and the Juvenile Diabetes Research Foundation. DEU was supported by the Novo Nordisk Foundation. We thank Gillian Coleman, Sarah Field, Tasneem Hassanali, Jayne Hutchings, Ruth Lewis, John Lumby, Meera Sebastian, Vin Everett and Geoff Dolman for advice and help.

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