Interplay between PTPN22 C1858T polymorphism and cow's milk formula exposure in type 1 diabetes

Journal of Autoimmunity 33 (2009) 155–164

Contents lists available at ScienceDirect

Journal of Autoimmunity journal homepage: www.elsevier.com/locate/jautimm

Interplay between PTPN22 C1858T polymorphism and cow’s milk formula exposure in type 1 diabetes Johanna Lempainen a, b, *, Outi Vaarala c, Miia Ma¨kela¨ a, d, Riitta Veijola e, Olli Simell f, Mikael Knip g, h, Robert Hermann a, i, Jorma Ilonen a, j a

¨katu 6A, 20520 Turku, Finland Immunogenetics Laboratory, University of Turku, MediCity, BioCity 4th Floor, Tykisto Department of Paediatrics, Satakunta Central Hospital, Pori, Finland Laboratory for Immunobiology, National Institute for Health and Welfare, Helsinki, Finland d Department of Virology, University of Turku, Turku, Finland e Department of Paediatrics, University of Oulu, Oulu, Finland f Department of Paediatrics, University of Turku, Turku, Finland g Department of Paediatrics, Tampere University Hospital, Tampere, Finland h Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland i Immunogenomics Laboratory, Semmelweis University, Budapest, Hungary j Department of Clinical Microbiology, University of Kuopio, Kuopio, Finland b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 March 2009 Received in revised form 28 April 2009 Accepted 30 April 2009

Genetic heterogeneity may affect the analysis of risk factors associated with type 1 diabetes (T1D). We studied the effect of the INS 23A/T, PTPN22 1858C/T, and CTLA-4 þ49A/G polymorphisms on the emergence of T1D-associated autoimmunity in children exposed to cow’s milk (CM) based formula during early or late infancy. The study comprised of 156 children from the Finnish DIPP cohort who had developed  2 types of autoantibodies (ICA, IAA, GADA or IA-2A) or clinical T1D and 563 control children. The PTPN22 1858T allele was associated with the appearance of the autoantibodies and clinical T1D among children exposed to CM formula before the age of 6 months (PTPN22: for all P  0.001, Log Rank test), but not among children exposed later on. Cox regression analysis showed an interaction between early CM exposure and 1858T allele and enhanced appearance of ICA, IAA and IA-2A (for all P  0.04). Our results imply that the PTPN22 polymorphism affects the development of T1D-associated autoimmunity only if children are exposed to CM formula during early infancy suggesting an interplay between genetic and environmental factors. This may provide an explanation for the contradictory findings on the significance of CM formula exposure in T1D. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Autoantibody appearance Cow’s milk formula exposure Interaction PTPN22 Type 1 diabetes

1. Introduction The destruction of the insulin-producing pancreatic beta-cells in type 1 diabetes (T1D) is perceived as a T-cell mediated autoimmune process. Both genetic and environmental factors are involved in the disease process. HLA genes, especially the class II region, explain about 50% of the familial clustering of T1D [1]. In addition to the HLA region, several other loci have been associated with the disease

Abbreviations: CM, cow’s milk; EIA, enzyme immunoassay; GADA, antibodies to the 65 kD isoform of glutamic acid decarboxylase; IA-2A, antibodies to the protein tyrosine phosphatase-related IA-2 molecule; IAA, insulin autoantibodies; ICA, islet cell antibodies; T1D, type 1 diabetes. * Corresponding author at: Immunogenetics Laboratory, University of Turku, MediCity, BioCity 4th Floor, Tykisto¨katu 6A, 20520 Turku, Finland. Tel.: þ358 2 333 7010; fax: þ358 2 333 7000. E-mail address: johanna.lempainen@utu.fi (J. Lempainen). 0896-8411/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2009.04.003

process [2,3]. Among these loci with known T1D association the best described so far are the polymorphisms in the insulin (INS), CTLA-4 and PTPN22 genes [2–5]. T1D frequently develops at a young age and serum autoantibodies against beta-cell autoantigens reflecting the initiation of the autoimmune disease process may appear early in life. Autoantibodies present already at the age of 2 years strongly predict the disease [6]. Insulin is the only beta-cell-specific autoantigen and insulin autoantibodies (IAA) often appear as the first autoantibody and associate with T1D diagnosed in young children. There is increasing evidence that an insulin-induced immune response may be crucially involved in the induction of autoimmunity [7]. Immunisation to dietary bovine insulin in cow’s milk (CM) based infant formulas has been implicated to play a role in the early steps of the induction of beta-cell autoimmunity [8–10]. Bovine insulin is immunogenic and cross-reactive with human insulin [10] and could accordingly break tolerance towards human insulin. It

156

J. Lempainen et al. / Journal of Autoimmunity 33 (2009) 155–164

Table 1 The significance of the genetic polymorphisms analysed on the appearance of various autoantibodies and development of clinical T1D (n of subjects positive for autoantibody or progressing to T1D/n total, P value, Kaplan–Meier analysis, Log Rank test). Autoantibody analysed

ICA IAA GADA IA-2A T1D

PTPN22 þ1858C/T

INS 23 Hph A/T

CTLA4 þ49 A/G

TT/CT vs. CC

AA vs. AT/TT

GG vs. AG/AA

105/444, 46/271 0.03 89/444, 35/271 0.01 82/444, 37/271 0.08 76/444, 36/271 0.11 63/444, 20/271 0.005

35/189, 33/189, 30/189, 29/189, 20/189,

57/190, 52/190, 43/190, 42/190, 35/190,

95/529 73/529 77/529 71/529 48/529

< 0.001 < 0.001 0.006 0.002 < 0.001

has also been shown that IAA often preferentially binds to nonhuman insulin and a portion of IAA is of the IgA class suggesting its mucosal origin [11]. This hypothesis may well explain the association of short exclusive breastfeeding and early CM exposure in infancy with the development of diabetes-associated autoimmunity [12–17]. Few studies have so far focused on the combined effect of T1Dassociated gene polymorphisms and CM exposure on the risk of T1D. In three studies, the effect of early CM and solid food exposure on disease risk has been observed to be dependent on the presence of HLA-DQ genotypes conferring susceptibility to T1D [18–20]. In the Diabetes Prediction and Prevention (DIPP) Study the participants were selected on the base of HLA-DQ genotypes conferring the risk for the disease. We could thus not study the HLA gene effect but were able to look for the effect of the disease predisposing polymorphisms in the INS, CTLA-4 and PTPN22 genes, all with a demonstrated effect on disease susceptibility also in the Finnish population [5,21,22]. Effect of various genotypes on the appearance of humoral beta-cell autoimmunity and clinical T1D was thus separately analysed in children exposed and not-exposed to CMbased infant formula and interaction between genetic factors and formula exposure was also studied using Cox regression analysis. In addition, the association of these gene polymorphisms with the formation of bovine insulin-binding antibodies in infancy was analysed. 2. Subjects and methods 2.1. Subjects The study subjects were derived from the Finnish Diabetes Prediction and Prevention (DIPP) study that is an ongoing prospective birth cohort study in Turku, Oulu and Tampere. According to the study protocol, recent parents are invited to have their newborn infant screened for HLA-conferred susceptibility to T1D. Families with infants carrying the HLA-DQB1 genotypes *02/ *0302, *0302/x [xs*02, *0301 or *0602 (*0602/3 in the early phase of the study)] and boys born in Turku with the genotype DQB1*02/y DQA1*05/z (ys0301, *0302, *0602, 0603; zs*0201) were invited to take part in a close follow-up at 3–12 month intervals to monitor the development of T1D-associated autoantibodies [islet cell

116/529 0.38 91/529 0.97 89/529 0.79 83/529 0.96 62/529 0.63

antibodies (ICA), IAA, antibodies to the 65 kD isoform of glutamic acid decarboxylase (GADA) and antibodies to the protein tyrosine phosphatase-related IA-2 molecule (IA-2A)] in their children [23]. If a child tested positive for at least two autoantibodies in two consecutive samples, the family was invited to take part in a randomized double-blinded intervention trial to assess whether daily administration of nasal insulin would decrease the progression rate to clinical T1D [23]. The duration of breastfeeding and the age at introduction of CM-based formula were assessed with sequential questionnaires. The local ethics committees approved the study and informed consent was obtained from the parents of the participants. The study population comprised of 719 subjects (320 girls) born between December 1994 and June 2002 taking part in the DIPP study in two cities in Finland, Turku and Oulu. Two-hundred thirteen of the 719 subjects carried the DQB1 02/0302-DQA1*05 genotype, 491 subjects carried the HLA-DQB1 *0302/x genotype, and 15 children carried the DQB1*02/y-DQA1*05/z genotype. Fifteen of the subjects carried the PTPN22 1858TT genotype, 175 were PTPN22 1858CT heterozygous and 529 of the subjects were homozygous for the PTPN22 1858CC genotype. Four-hundred fortyfour children were homozygous for the INS 23HphI AA genotype, 240 were AT heterozygous and 31 subjects carried the TT genotype. One-hundred eighty-nine subjects were homozygous for GG, 396 heterozygous for GA and 133 homozygous for AA. Four subjects could not be successfully genotyped for the 23HphI polymorphism and one participant for the CTLA4 þ49 polymorphism. One-hundred fifty-six subjects developed positivity for at least two of the T1D-associated autoantibodies. Eighty-three of these subjects developed clinical T1D during the follow-up. In addition, 563 autoantibody-negative subjects with similar distribution of HLA-DQB1 genotypes, gender, date and place of birth were analysed. The median follow-up time for the autoantibody appearance was 9.0 years (range 0.5–14.0 years), for the development of clinical T1D 10.4 years (range 6.2–13.8 years). One-hundred of the 156 children who developed multiple autoantibodies took part in the placebo-controlled intervention trial aimed at assessing the efficacy of nasal insulin in the prevention of T1D. Fifty-one of these subjects received intranasal insulin. No significant differences were observed in the progression rate to diabetes between the two intervention arms [24].

Fig. 1. In this case-control series the PTPN22 Arg620Trp (C1858T) polymorphism was associated with the appearance of autoantibodies only among subjects exposed to CM-based formula before 6 months of age (group 1, figures a, c, e, g and i). In this subgroup 38 of the 107 subjects carrying the 1858T variant (dashed line) developed positivity for ICA and some of the biochemically defined autoantibodies compared to 56 of the 338 subjects with 1858CC genotype (continuous line) (P < 0.001, Log Rank test) (a). In contrast, among subjects exposed to CM-based formula later in infancy (group 2, figures b, d, f, h and j) seven of the 41 subjects with the TT or CT genotype (dashed line) compared to 20 of the 100 subjects with the CC genotype (continuous line) developed autoantibody positivity (P ¼ 0.73) (b). The corresponding numbers for the appearance of IAA were 35 of the 107 subjects with the TT or CT genotype and 45 of the 338 subjects with the CC genotype in group 1 (P < 0.001) (c) and six of 41 subjects with the TT or CT genotype and 16 of the 100 subjects with the CC genotype in group 2 (P ¼ 0.85) (d). For the appearance of GADA the numbers were 29 of the 107 subjects with the TT or CT genotype and 46 of the 338 subjects with the CC genotype in group 1 (P ¼ 0.001) (e) and six of the 41 subjects with the TT or CT genotype and 16 of the 100 subjects with the CC genotype in group 2 (P ¼ 0.84) (f) and for the appearance of IA-2A 28 of the 107 subjects with the TT or CT genotype and 42 of the 338 subjects with the CC genotype in group 1 (P < 0.001) (g) and three of the 41 subjects with the TT or CT genotype and 15 of the 100 subjects with the CC genotype in group 2 (P ¼ 0.26) (h). In group 1, 23 of the 107 subjects with the TT or CT genotype progressed to clinical T1D compared to 27 of the 338 subjects with the CC genotype (P < 0.001) (i) and in group 2 six of the 41 subjects carrying the 1858T variant presented with T1D compared to 9 of the 100 CC homozygous subjects (P ¼ 0.33) (j).

J. Lempainen et al. / Journal of Autoimmunity 33 (2009) 155–164

a

b

c

d

e

f

g

h

i

j

157

158

J. Lempainen et al. / Journal of Autoimmunity 33 (2009) 155–164

a

b

c

d

e

f

g

h

i

j

J. Lempainen et al. / Journal of Autoimmunity 33 (2009) 155–164

Data on the exposure to CM formula in early infancy was available on 586 children. Four-hundred forty-five of these subjects were exposed to CM before the age of 6 months and 141 of the subjects were exposed to CM at the age of 6 months or later. 2.2. The autoantibody assays The antibody assays have been previously described in detail [25]. The detection limit for ICA was 2.5 Juvenile Diabetes Foundation Units (JDFU; sensitivity 100%, specificity 98%). The cut-off limits for IAA, GADA and IA-2A positivity were 1.56 RU, 5.36 RU and 0.43 RU, respectively, representing the 99th percentiles in a series comprising of more than 370 non-diabetic Finnish children and adolescents. The disease sensitivity of the IAA, GADA and IA-2A assays was 58, 82, and 72%, respectively, while the specificity values were 98, 96, and 100%, respectively, based on the 2005 Diabetes Autoantibody Standardization Program Workshop. 2.3. The EIA for bovine insulin-binding antibodies Bovine insulin-binding IgG antibodies were detected using an EIA method [8]. Briefly, microtitre plates (Combiplate Enhanced Binding, Labsystems, Helsinki, Finland) were coated with bovine insulin (Sigma, St. Louis, MO) (1 mg/well in PBS) and incubated at þ4  C overnight. The plates were washed with a buffer containing 0.05% Tween 20 in PBS and residual-coated with 1% human serum albumin. Samples diluted 1:10 in PBS þ0.2% human serum albumin þ0.05% Tween 20 were incubated at RT for 2 h. After washes, alkaline phosphatase-conjugated rabbit anti-human IgG antibody (Vector Laboratories, Burlingame, CA) was added in a 1:100 dilution and the plates were incubated at RT for 90 min P-nitrophenyl phosphatase tablets (Sigma) were used as a substrate and the absorbance was read on a spectrophotometer.

159

Table 2 Cox regression analysis for the combined effect of the PTPN22 þ1858TT/CT or þ1858CC genotype and early or late exposure to cow’s milk based formula during infancy on the appearance of T1D-associated autoimmunity. An interaction between PTPN22 þ1858T allele and early exposure to cow’s milk-based formula on the appearance of humoral signs of beta-cell autoimmunity was observed. Autoantibody analysed

P value

HRa

95% CI

ICA IAA GADA IA-2A T1D

0.03 0.04 0.11 0.02 0.37

2.8 3.0 2.4 4.7 1.7

1.1–7.3 1.1–8.5 0.8–6.7 1.2–17.8 0.5–5.6

a

HR for PTPN22 and CM exposure interaction term.

INS 23A/T (rs689) and CTLA-4 þ49A/G (rs231775) polymorphisms [21,28]. For PTPN22 þ 1858C/T (rs2476601) assay the one-step assay based on asymmetric amplification and subsequent time-resolved fluorescence measurement was used. Upon hybridizing to the PCR product the probes dehybridize from their complementary quenchers and become capable of emitting fluorescence [5,29]. 2.5. Statistical analysis All statistical analysis were performed with SPSS 15.0 (SPSS Inc., Chicago, IL). The Log Rank test was used in the Kaplan–Meier analysis to compare the appearance of autoantibodies or T1D between the groups. The correcting factor for multiple tests within this analysis was not used. Cox regression analysis was employed to analyse the combined effect of genetic factors and cow’s milk formula exposure on the appearance of autoimmunity. ANOVA for repeated measurements was used to compare the antibody levels between the groups over time and the Mann–Whitney U-test to analyse the differences in the antibody levels at specific timepoints.

2.4. Genetic analysis 3. Results Blood spots dried on filter paper and stored at room temperature were used as starting material for all genotypings. For HLA analyses a 3-mm disk was punched directly into 96-well PCR plates where the amplification mix was added. Polymorphic parts of the second exon of HLA-DQB1 and –DQA1 genes were amplified using primer pairs with biotinylated 3’ primer. The biotinylated PCR products were transferred to streptavidin-coated microtitration plates, denatured and hybridized with sequence-specific probes labelled with lanthanide chelates of europium (Eu), terbium (Tb) or samarium (Sm). After incubation, enhancement and washing steps, three-colour time-resolved fluorescence was measured to detect specific hybridization to bound PCR products. Details of the procedure including used probes and primers are described in earlier publications [26,27]. For SNP assays, DNA was extracted by sodium hydroxide from disks punched from blood spots. Similarly to HLA assays, the principle of microtitration plate-bound biotinylated amplification products and lanthanide labelled probes was used in SNP assays for

3.1. The effect of PTPN22, INS and CTLA4 on the appearance of T1D-associated autoantibodies and clinical T1D The effect of the genetic polymorphisms studied on the development of beta-cell autoimmunity was first analysed in the whole series of children with autoantibodies and their matched controls. Survival analyses demonstrated that both the INS gene 23AA genotype and T-positive PTPN22 þ1858 genotypes TT and CT were associated with the development of autoantibodies and clinical disease (Table 1). In the Log Rank test the PTPN22 þ1858CT/TT genotypes were associated with the emergence of all autoantibodies and the development of overt T1D. The INS 23AA risk genotype was associated with the development of all the autoantibodies studied, except GADA, and also with progression to clinical disease. The CTLA-4 þ49 GG genotype was not significantly associated with the development of autoantibodies or clinical T1D in this analysis.

Fig. 2. When the association of the INS 23HphI A/T polymorphism with the appearance of autoantibodies and clinical diabetes was analysed separately among subjects exposed to CM-based formula before 6 months of age (group 1, figures a, c, e, g and i) or later in infancy (group 2, figures b, d, f, h and j) the association of the INS 23Hph polymorphism with the emergence of autoantibodies was observed only in group 1. In this group, 67 of the 274 subjects with the 23HphI AA genotype (dashed line) developed ICA and some of the biochemically defined autoantibodies during the follow-up compared to 26 of the 168 subjects with AT or TT genotype (continuous line) (P ¼ 0.02, Log Rank test) (a). Fifty-eight of the 274 subjects with the AA genotype developed IAA compared to 21 of the 168 subjects with the AT or TT genotype (P ¼ 0.02) (c), 55 (274) with the AA genotype compared to 19 (168) with the AT or TT genotype developed GADA (P ¼ 0.01) (e) and 50 (274) with the AA genotype compared to 19 (168) with the AT or TT genotype developed IA-2A (P ¼ 0.04) (g). Clinical diabetes emerged in 40 (274) subjects with the AA genotype compared to 10 (168) subjects with the AT or TT genotype (P ¼ 0.006) (i). In contrast, in group 2 18 (83) subjects with the AA genotype (dashed line) developed ICA with any of the other autoantibodies vs. nine (58) subjects with the AT or TT genotype (continuous line) (P ¼ 0.38) (b) and the corresponding numbers for IAA were 15 (83) vs. seven (58), respectively (P ¼ 0.32) (d), for GADA 13 (83) vs. nine (58), respectively (P ¼ 0.91) (f), for IA-2A 11 (83) vs. seven (58), respectively (P ¼ 0.83) (h) and for T1D 11 (83) vs. four (58), respectively (P ¼ 0.22) (j).

160

J. Lempainen et al. / Journal of Autoimmunity 33 (2009) 155–164

a

b

c

d

e

f

g

h

i

j

J. Lempainen et al. / Journal of Autoimmunity 33 (2009) 155–164

3.2. The effect of the polymorphisms on the appearance of T1D autoimmunity in groups categorised according to the cow’s milk formula exposure in infancy The effect of early nutrition was thereafter analysed by categorising the children according to their feeding pattern, i.e. exposure to CM-based formula nutrition at 3 and 6 months of age. When children who had received formula feeding before 3 or 6 months of age were compared to those exposed to CM formula later on, no significant differences were observed in the appearance of autoantibodies or clinical diabetes (data not shown). However, when the effect of the genetic markers studied was considered, the Kaplan–Meier analysis demonstrated significant associations between the PTPN22 and INS genotypes and the appearance of autoantibodies and T1D in children who had been exposed to formula before the age of 6 months, but not if CM formula was introduced to diet later in infancy (Figs. 1 and 2). The appearance of ICA, IAA, GADA, IA-2A and progression to T1D differed significantly between individuals with different PTPN22 genotypes among the children exposed to formula before 6 months of age (P<0.001, <0.001, 0.001, <0.001 and <0.001, respectively), but not among subjects exposed to CM-based formula at the age of 6 months or later (P ¼ 0.73, 0.85, 0.84, 0.26 and 0.33, respectively). Similarly, the appearance of ICA, IAA, GADA, IA-2A and clinical diabetes differed significantly between the subjects with different INS genotypes among the children exposed to formula before 6 months of age (P ¼ 0.02, 0.02, 0.01, 0.04 and 0.006, respectively) whereas no significant difference was observed among the subjects with late introduction of CM-based formula (P ¼ 0.38, 0.32, 0.91, 0.83 and 0.22, respectively). No significant effect on the appearance of betacell autoimmunity or overt T1D was seen for CTLA-4 gene polymorphism in any of the groups studied (data not shown). Cox regression analysis was also used to look for the combined risk effect of the different gene polymorphisms and CM exposure during the first 6 months of life. An interaction between the PTPN22 T allele and CM exposure was observed when the appearance of ICA together with any of the biochemically defined autoantibodies or the appearance of IAA or IA-2A was analysed (for ICA together with any of the biochemically defined autoantibodies: P ¼ 0.03, HR 2.8, 95% CI 1.1–7.3, for IAA: P ¼ 0.04, HR 3.0, 95% CI 1.1–8.5 and for IA2A: P ¼ 0.02, HR 4.7, 95% CI 1.2–17.8, Cox regression analysis) (Table 2). No significant interaction between the INS gene or CTLA-4 gene polymorphisms with CM exposure on the appearance of T1Dassociated autoimmunity was detected (data not shown). The effect of CM exposure on the appearance of T1D-associated autoimmunity was then analysed among carriers of the PTPN22 þ1858T allele and subjects with CC genotype separately. Here, among subjects with 1858TT/CT genotype early exposure to CM was associated with enhanced emergence of ICA with any of the biochemically defined autoantibodies, IAA and IA-2A (P ¼ 0.03, 0.03 and 0.02, respectively, Log Rank test) but no significant difference on the appearance of GADA or clinical T1D was observed (Fig. 3). No effect of CM formula exposure on the appearance of humoral signs of autoimmunity could be observed among subjects with þ1858CC genotype.

161

3.3. The association of bovine insulin antibodies with the appearance of autoantibodies and T1D Bovine insulin-binding antibody levels were analysed at the age of 3, 6, 12, 18 and 24 months. Children who received CM-based formula during the first 3 months of life had significantly higher levels of bovine insulin-binding antibodies than children exclusively breast-fed at the age of 3 months (P<0.001, MWU-test), and the levels were higher also when looked over the total observation period (P ¼ 0.007, ANOVA for repeated measurements). The bovine insulin antibody levels were significantly higher over time among subjects developing multiple autoantibodies or clinical diabetes compared to subjects remaining negative for each autoantibody and non-diabetic during the follow-up (P<0.001, ANOVA for repeated measurements). 3.4. The association of bovine insulin antibodies with the PTPN22 C1858T, INS 23 Hphl A/T, and CTLA-4 þ49 A/G polymorphisms The bovine insulin-binding antibody levels did not differ between subjects carrying the INS 23HphI AA genotype compared to the carriers of the AT or TT genotype (P ¼ 0.49, ANOVA for repeated measurements). When the subjects exposed to CM-based formula before the age of 6 months and those exposed to formula later were analysed separately no association of bovine insulinbinding antibodies and the INS genotype was observed (P ¼ 0.285 and 0.11, respectively). In contrast elevated levels of bovine insulinbinding antibodies were seen in carriers of the PTPN22 T allele when compared to subjects with the 1858CC wild-type genotype in the whole series (P ¼ 0.02, ANOVA for repeated measurements) (Fig. 4a). When the effect of the PTPN22 polymorphism on the bovine insulin-binding antibody levels was analysed taking the age at formula exposure into consideration, the association of the 1858T allele with increased bovine insulin-binding antibody levels was observed only among subjects exposed to CM-based formula before the age of 6 months (P ¼ 0.001, ANOVA for repeated measurements) (Fig. 4b), but not among subjects exposed to formula later in infancy (P ¼ 0.77) (Fig. 4c). No effect of the CTLA4 þ49 A/G polymorphism was observed on bovine insulin-binding antibody levels. 4. Discussion Despite the definite genetic background there is strong evidence supporting the critical role of environmental factors in the pathogenesis of T1D. The geographic variation in diabetes incidence is wide even among Caucasian populations varying from the low incidence of 3.2 cases/100 000 children under 15 years of age in Macedonia [30] to the high incidence of more than 60 cases/ 100 000 children during recent years in Finland [31]. A conspicuous increase in disease incidence that cannot be explained by genetic factors has been observed in most countries with available data [32]. Moreover, the proportion of newly diagnosed patients carrying diabetes-associated high-risk HLA genotypes has decreased among affected patients over the last decades suggesting

Fig. 3. Exposure to cow’s milk-based formula nutrition by 6 months of age was found to be associated with humoral signs of T1D autoimmunity among carriers of the PTPN22 1858T allele predisposing to the development of T1D (figures a, c, e, g and i) but not among subjects with the 1858CC genotype (figures b, d, f, h and j). Here, among the group with the 1858TT or CT genotype 38 of the 107 subjects exposed to CM formula in early infancy (dashed line) developed ICA autoantibodies together with any of the biochemically defined autoantibodies IAA, GADA or IA-2A, compared to seven of the 41 subjects in the later CM exposure group (continuous line) (P ¼ 0.03, Log Rank test) (a). Among subjects with 1858CC genotype, no difference in the appearance of humoral autoimmunity was observed between the two dietary groups [56/338 subjects in group 1 (dashed line), 20/100 in group 2 (continuous line), P ¼ 0.53] (b). Similarly, among subjects with the disease predisposing allele, early formula nutrition was associated with the enhanced appearance of IAA (35/107 vs. 6/41 subjects in group 1, P ¼ 0.03) (c) but no effect was observed among subjects with the CC genotype (45/338 vs. 16/100 in group 2, P ¼ 0.58) (d). The result remained similar when analysing the appearance of GADA [29/107 vs. 6/41 in group 1, P ¼ 0.12 (e), 46/338 vs. 16/100 in group 2, P ¼ 0.66 (f)] and IA-2A [28/107 vs. 3/41 in group 1, P ¼ 0.02 (g), 42/338 vs. 15/100 in group 2, P ¼ 0.64 (h)]. However, no significant effect of formula exposure on the progression to clinical diabetes was observed in any of the groups [23/107 vs. 6/41 in group 1, P ¼ 0.35 (i), 27/338 vs. 9/100 in group 2, P ¼ 0.79 (j)].

162

J. Lempainen et al. / Journal of Autoimmunity 33 (2009) 155–164

a

b

c

Fig. 4. The bovine insulin-binding IgG class antibodies were higher among subjects carrying the PTPN22 þ1858T variant (white boxes) compared to subjects homozygous for the CC genotype (grey boxes) (P ¼ 0.02, ANOVA for repeated measurements) (a). When time-points were analysed separately, the antibody levels were higher among the T variant carriers at the age of 12 and 18 months (P ¼ 0.04 and 0.002, respectively) and tended to be higher at 24 months (P ¼ 0.06, MWU-test). When the subjects exposed to CM-based formula before the age of 6 months (group 1) and subjects exposed to CM formula later in infancy (group 2) were analysed separately, the association of the PTPN22 þ1858T variant with increased bovine insulin-binding antibody levels was observed only in group 1 (P ¼ 0.001; ANOVA for repeated measurements) (b). When specific time-points were analysed separately among group 1, the þ1858T variant was associated with elevated antibody levels at the age of 12, 18 and 24 months (P ¼ 0.001, 0.003 and 0.005, respectively). No association with the PTPN22 T variant and the antibody levels was observed in group 2 (P ¼ 0.77; ANOVA for repeated measurements) (c). Median value in each box is shown with a horizontal line in the box. The boxes delineate values between the 25th and 75th percentiles and the whiskers values between the 10th and the 90th.

increasing environmental pressure over genetic determinants [33,34]. The importance of environmental factors is also supported by data of monozygotic twins where concordance of about 50% is found. The concordance is also higher when the index case has been diagnosed at early age [35]. A multitude of environmental factors have been implicated in contributing to the increasing risk of T1D. These include microbial infections or in contrast, lack of the immune stimulation provided by such infections as well as dietary factors. The association of short duration of breastfeeding or early exposure to CM-based formula with the emergence of beta-cell autoimmunity has been reported in several studies but other investigations have not been able to confirm such findings, not even when analysed in the same populations [12,14,17,36–38]. The only intervention study with outcome data thus far, i.e. the second pilot of the TRIGR (Trial to Reduce Diabetes in Genetically at Risk) study, observed a reduced emergence of beta-cell autoantibodies when the infants with HLAconferred disease susceptibility and a positive family history for T1D were weaned to a casein hydrolysate formula instead of a conventional CM-based formula [12]. In the setting of the Finnish DIPP study recruiting a follow-up cohort based on HLA-conferred susceptibility to T1D, it is not possible to assess the effect of the HLA-DQ genotype on diabetes risk in children with a diverse history of infant feeding. However, we were able to analyse the impact of polymorphisms in other known susceptibility loci. We observed a definite effect of both the PTPN22 þ1858C/T and the INS 23 A/T polymorphism on the appearance of autoantibodies and progression to clinical T1D, whereas the CTLA-4 þ49 G/A polymorphism did not have any demonstrable effect in the cohort studied. This is not unexpected, since the CTLA-4 effect predisposing to T1D has been weak in several populations of Northern European origin, although in other populations it seems to provide a rather strong contribution to T1D risk [39]. In our own unpublished case-control series the odds ratio associated with the CTLA-4 þ49 GG genotype was 1.4 compared to an odds ratio of 2.6 for INS 23 AA genotype [21] and 3.1 for the PTPN22 þ1858TT genotype and 2.9 for the CT genotype [5]. The effect of the PTPN22 gene polymorphisms on the risk of beta-cell autoimmunity and clinical T1D differed between infants who had received formula feeding before the age of 6 months compared to those exposed to CM-based formula later in infancy. The predisposing genotypes enhanced the risk of autoantibody formation and progression to overt disease only in children who had received CM-based infant formula before the age of 6 months. Similar phenomenon was observed also when analysing the effect of the INS gene polymorphism, but the result of regression analysis remained non-significant. The CTLA-4 polymorphism did not confer increased risk of beta-cell autoimmunity or clinical T1D irrespective of the infant feeding pattern. These results are in accordance with our earlier results indicating that both PTPN22 and INS gene polymorphisms especially modulate the production of IAA and autoimmunity initiated by insulin [5,39]. Interestingly, dietary insulin has also been shown to be crucial for the formation of tolerance towards insulin [8,10,40]. The PTPN22 þ1858C/T variant is leading to the replacement of arginine by trypthophan in a position 620 of the lymphoid tyrosine phosphatase. This molecule modulates the activation of protein kinases involved in early T-cell-receptor mediated signalling events. Accordingly, the T1D assocaited Arg620Trp variant has been shown to be related to decreased responsiveness of T and B-cell populations including decreased interleukin-2 secretion and decreased intracellular calcium mobilisation in T cells [41–43]. The poor activation of T cells and impaired IL-2 secretion may contribute to decreased activation of regulatory T cells. The PTPN22 þ1858C/T variant has been associated with several other

J. Lempainen et al. / Journal of Autoimmunity 33 (2009) 155–164

autoimmune disorders including rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus and autoimmune thyreoiditis and recently also with celiac disease [44]. Although T cells are perceived as mediators of target tissue destruction in T1D many of the above listed diseases are predominantly associated with humoral immunity indicating that the basic functions of immune regulation are affected. We observed an association between the variant predisposing to disease and the development of beta-cell autoimmunity only among subjects exposed to CM-based formula during their first 6 months of life suggesting that the mechanisms of tolerance in the developing gut are especially vulnerable to the functional derangements associated with the PTPN22 þ1858C/T variant. Moreover, CM exposure in early infancy was associated with the enhanced appearance of T1Dassociated autoantibodies among the carriers of the PTPN22 þ1858T variant whereas no effect of early CM exposure on the autoantibody appearance was observed among subjects with 1858CC genotype. Also the levels of antibodies binding to dietary insulin were modulated by PTPN22 polymorphism when dietary insulin was introduced during early life. In the individuals exposed to CM formula after the age of 6 months the PTPN22 gene polymorphisms did not modulate the levels of serum insulin-binding antibodies, the risk of beta-cell autoimmunity or the risk of progression to clinical disease which suggests that CM induced effects on insulin immunity and beta-cell autoimmunity are regulated by different mechanisms depending on the age of exposure and gut maturation accordingly. The INS gene polymorphisms have been shown to be associated with insulin transcription levels in the thymus resulting in decreased insulin expression [45–52]. Negative thymic selection deletes autoreactive T cells, and it has been hypothesised that the insulin gene polymorphism leading to low expression of insulin in the thymus would result in the escape of insulin-reactive T cells that eventually may react against pancreatic beta-cells [52]. Alternatively, the expression level may interfere with the development of natural regulatory T cells in the thymus. Our findings of increased levels of beta-cell autoantibodies in individuals carrying the risk associated INS gene polymorphism and exposed early in life to bovine insulin could be related to impaired down-regulation of the insulin-specific immunity, but further investigation is needed to confirm the finding. Our results suggest an interplay between genetic determinants and dietary factors in the development of insulin-specific immunity and in the autoimmune process leading to loss of beta-cell tolerance and finally to T1D. The findings emphasise the fact that the effect of environmental factors may be variable and the identified environmental risk factors might not be involved in the disease process in all cases. These findings may also explain the diverse and contradictory findings concerning the role of early CM exposure or long breastfeeding in the development of beta-cell autoimmunity. The effect of early exposure to CM-based formula seems to be more crucial among subjects carrying the PTPN22 þ1858T variant and to some extent also among subjects carrying the insulin gene 23 AA genotype. It is interesting to note that the frequency of INS and PTPN22 risk genotypes are particularly high in Finland compared to other populations of European origin [5,21]. Acknowledgements This study was supported by the Juvenile Diabetes Research Foundation, the Sigrid Juselius Foundation, the Turku Graduate School of Biomedical Sciences, the Finnish Cultural Foundation, the Novo Nordisk Fund, the Research and Science Foundation of Farmos and the Satakunta Central Hospital District. We thank Mia Karlsson, Eija Nirhamo, Pia Nurmi, Terhi Laakso, Ritva Suominen, Anneli

163

Suomela, Sirpa Anttila, Tanja Haapala, Riitta Pa¨kkila¨, and Pa¨ivi Salmija¨rvi for their skilful technical assistance. We wish to thank Tero Vahlberg MSc for valuable help with the statistics. We acknowledge the entire DIPP study personnel and all DIPP study children and their families for their irreplaceable contribution.

References [1] Vyse TJ, Todd JA. Genetic analysis of autoimmune disease. Cell 1996;85:311–8. [2] Jahromi MM, Eisenbarth GS. Genetic determinants of type 1 diabetes across populations. Ann N Y Acad Sci 2006;1079:289–99. [3] Maier LM, Wicker LS. Genetic susceptibility to type 1 diabetes. Curr Opin Immunol 2005;17:601–8. [4] Bottini N, Musumeci L, Alonso A, Rahmouni S, Nika K, Rostamkhani M, et al. A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet 2004;36:337–8. [5] Hermann R, Lipponen K, Kiviniemi M, Kakko T, Veijola R, Simell O, et al. Lymphoid tyrosine phosphatase (LYP/PTPN22) Arg620Trp variant regulates insulin autoimmunity and progression to type 1 diabetes. Diabetologia 2006;49:1198–208. [6] Hummel M, Bonifacio E, Schmid S, Walter M, Knopff A, Ziegler AG. Brief communication: early appearance of islet autoantibodies predicts childhood type 1 diabetes in offspring of diabetic parents. Ann Intern Med 2004;140:882–6. [7] Zhang L, Nakayama M, Eisenbarth GS. Insulin as an autoantigen in NOD/ human diabetes. Curr Opin Immunol 2008;20:111–8. [8] Paronen J, Knip M, Savilahti E, Virtanen SM, Ilonen J, Åkerblom HK, et al. Effect of cow’s milk exposure and maternal type 1 diabetes on cellular and humoral immunization to dietary insulin in infants at genetic risk for type 1 diabetes. Finnish Trial to Reduce IDDM in the Genetically at Risk Study Group. Diabetes 2000;49:1657–65. [9] Tiittanen M, Paronen J, Savilahti E, Virtanen SM, Ilonen J, Knip M, et al. Dietary insulin as an immunogen and tolerogen. Pediatr Allergy Immunol 2006;17:538–43. [10] Vaarala O, Knip M, Paronen J, Ha¨ma¨la¨inen AM, Muona P, Va¨a¨ta¨inen M, et al. Cow’s milk formula feeding induces primary immunization to insulin in infants at genetic risk for type 1 diabetes. Diabetes 1999;48:1389–94. [11] Koczwara K, Muller D, Achenbach P, Ziegler AG, Bonifacio E. Identification of insulin autoantibodies of IgA isotype that preferentially target non-human insulin. Clin Immunol 2007;124:77–82. [12] Åkerblom HK, Virtanen SM, Ilonen J, Savilahti E, Vaarala O, Reunanen A, et al. Dietary manipulation of beta cell autoimmunity in infants at increased risk of type 1 diabetes: a pilot study. Diabetologia 2005;48:829–37. [13] Holmberg H, Wahlberg J, Vaarala O, Ludvigsson J. Short duration of breastfeeding as a risk-factor for beta-cell autoantibodies in 5-year-old children from the general population. Br J Nutr 2007;97:111–6. [14] Kimpima¨ki T, Erkkola M, Korhonen S, Kupila A, Virtanen SM, Ilonen J, et al. Short-term exclusive breastfeeding predisposes young children with increased genetic risk of Type I diabetes to progressive beta-cell autoimmunity. Diabetologia 2001;44:63–9. [15] Rosenbauer J, Herzig P, Giani G. Early infant feeding and risk of type 1 diabetes mellitus-a nationwide population-based case-control study in pre-school children. Diabetes Metab Res Rev 2008;24:211–22. [16] Virtanen SM, Kenward MG, Erkkola M, Kautiainen S, Kro¨nberg-Kippila¨ C, Hakulinen T, et al. Age at introduction of new foods and advanced beta cell autoimmunity in young children with HLA-conferred susceptibility to type 1 diabetes. Diabetologia 2006;49:1512–21. [17] Ziegler AG, Schmid S, Huber D, Hummel M, Bonifacio E. Early infant feeding and risk of developing type 1 diabetes-associated autoantibodies. Jama 2003;290:1721–8. [18] Pe´rez-Bravo F, Carrasco E, Gutierrez-Lopez MD, Martinez MT, Lopez G, GarciadelosRios M. Genetic predisposition and environmental factors leading to the development of insulin-dependent diabetes mellitus in Chilean children. J Mol Med 1996;74:105–9. [19] Kostraba JN, Cruickshanks KJ, Lawlerheavner J, Jobim LF, Rewers MJ, Gay EC, et al. Early exposure to cow’s milk and solid foods in infancy, genetic predisposition, and risk of IDDM. Diabetes 1993;42:288–95. [20] Virtanen SM, La¨a¨ra¨ E, Hyppo¨nen E, Reijonen H, Ra¨sa¨nen L, Aro A, et al. Cow’s milk consumption, HLA-DQB1 genotype, and type 1 diabetes: a nested casecontrol study of siblings of children with diabetes. Childhood diabetes in Finland study group. Diabetes 2000;49:912–7. [21] Laine AP, Holmberg H, Nilsson A, Ortqvist E, Kiviniemi M, Vaarala O, et al. Two insulin gene single nucleotide polymorphisms associated with type 1 diabetes risk in the Finnish and Swedish populations. Dis Markers 2007;23:139–45. [22] Doroudis, K., Laine, A.P., Heinonen, M., Hermann, R., Lipponen, K., Veijola, R., Simell, O., Knip, M., Uibo, R., Ilonen, J., Kisand, K. Association of CTLA4 but not ICOS polymorphisms with type 1 diabetes in two populations with different disease Rates. Hum Immunol, in press. [23] Kupila A, Muona P, Simell T, Arvilommi P, Savolainen H, Ha¨ma¨la¨inen AM, et al. Feasibility of genetic and immunological prediction of type I diabetes in a population-based birth cohort. Diabetologia 2001;44:290–7.

164

J. Lempainen et al. / Journal of Autoimmunity 33 (2009) 155–164

[24] Na¨nto¨-Salonen K, Kupila A, Simell S, Siljander H, Salonsaari T, Hekkala A, et al. Nasal insulin to prevent type 1 diabetes in children with HLA genotypes and autoantibodies conferring increased risk of disease: a double-blind, randomised controlled trial. Lancet 2008;372:1746–55. [25] Kimpima¨ki T, Kulmala P, Savola K, Kupila A, Korhonen S, Simell T, et al. Natural history of beta-cell autoimmunity in young children with increased genetic susceptibility to type 1 diabetes recruited from the general population. J Clin Endocrinol Metab 2002;87:4572–9. [26] Sjo¨roos M, Iitia A, Ilonen J, Reijonen H, Lo¨vgren T. Triple-label hybridization assay for type-1 diabetes-related HLA alleles. Biotechniques 1995;18:870–7. [27] Laaksonen M, Pastinen T, Sjo¨roos M, Kuokkanen S, Ruutiainen J, Sumelahti ML, et al. HLA class II associated risk and protection against multiple sclerosisda Finnish family study. J Neuroimmunol 2002;122:140–5. [28] Haller K, Kisand K, Nemvalts V, Laine AP, Ilonen J, Uibo R. Type 1 diabetes is insulin 2221 MspI and CTLA-4 þ49 A/G polymorphism dependent. Eur J Clin Invest 2004;34:543–8. [29] Kiviniemi M, Nurmi J, Turpeinen H, Lo¨vgren T, Ilonen J. A homogeneous highthroughput genotyping method based on competitive hybridization. Clin Biochem 2003;36:633–40. [30] Eurodiab Ace Study Group. Variation and trends in incidence of childhood diabetes in Europe. Lancet 2000;355:873–6. [31] Knip M, Siljander H. Autoimmune mechanisms in type 1 diabetes. Autoimmun Rev 2008;7:550–7. [32] Onkamo P, Va¨a¨na¨nen S, Karvonen M, Tuomilehto J. Worldwide increase in incidence of Type I diabetes–the analysis of the data on published incidence trends. Diabetologia 1999;42:1395–403. [33] Gillespie KM, Bain SC, Barnett AH, Bingley PJ, Christie MR, Gill GV, et al. The rising incidence of childhood type 1 diabetes and reduced contribution of high-risk HLA haplotypes. Lancet 2004;364:1699–700. [34] Hermann R, Knip M, Veijola R, Simell O, Laine AP, Åkerblom HK, et al. Temporal changes in the frequencies of HLA genotypes in patients with Type 1 diabetes–indication of an increased environmental pressure? Diabetologia 2003;46:420–5. [35] Redondo MJ, Yu L, Hawa M, Mackenzie T, Pyke DA, Eisenbarth GS, et al. Heterogeneity of type I diabetes: analysis of monozygotic twins in Great Britain and the United States. Diabetologia 2001;44:354–62. [36] Knip M. Environmental triggers and determinants of beta-cell autoimmunity and type 1 diabetes. Rev Endocr Metab Disord 2003;4:213–23. [37] Norris JM, Beaty B, Klingensmith G, Yu L, Hoffman M, Chase HP, et al. Lack of association between early exposure to cow’s milk protein and beta-cell autoimmunity. Diabetes Autoimmunity Study in the Young (DAISY). Jama 1996;276:609–14. [38] Virtanen SM, Knip M. Nutritional risk predictors of beta cell autoimmunity and type 1 diabetes at a young age. Am J Clin Nutr 2003;78:1053–67.

[39] Hermann R, Laine AP, Veijola R, Vahlberg T, Simell S, La¨hde J, et al. The effect of HLA class II, insulin and CTLA4 gene regions on the development of humoral beta cell autoimmunity. Diabetologia 2005;48:1766–75. [40] Vaarala O, Paronen J, Otonkoski T, Åkerblom HK. Cow milk feeding induces antibodies to insulin in childrenda link between cow milk and insulindependent diabetes mellitus? Scand J Immunol 1998;47:131–5. ¨ ling V, Simell O, et al. Reduced [41] Aarnisalo J, Treszl A, Svec P, Marttila J, O CD4(þ)T cell activation in children with type 1 diabetes carrying the PTPN22/ Lyp 620Trp variant. J Autoimmun 2008. [42] Rieck M, Arechiga A, Onengut-Gumuscu S, Greenbaum C, Concannon P, Buckner JH. Genetic variation in PTPN22 corresponds to altered function of T and B lymphocytes. J Immunol 2007;179:4704–10. [43] Vang T, Congia M, Macis MD, Musumeci L, Orru V, Zavattari P, et al. Autoimmune-associated lymphoid tyrosine phosphatase is a gain-of-function variant. Nat Genet 2005;37:1317–9. [44] Vang T, Miletic AV, Arimura Y, Tautz L, Rickert RC, Mustelin T. Protein tyrosine phosphatases in autoimmunity. Annu Rev Immunol 2007. [45] Kennedy GC, German MS, Rutter WJ. The minisatellite in the diabetes susceptibility locus IDDM2 regulates insulin transcription. Nat Genet 1995;9:293–8. [46] Pugliese A, Zeller M, Fernandez Jr A, Zalcberg LJ, Bartlett RJ, Ricordi C, et al. The insulin gene is transcribed in the human thymus and transcription levels correlated with allelic variation at the INS VNTR-IDDM2 susceptibility locus for type 1 diabetes. Nat Genet 1997;15:293–7. [47] Vafiadis P, Ounissi-Benkalha H, Palumbo M, Grabs R, Rousseau M, Goodyer CG, et al. Class III alleles of the variable number of tandem repeat insulin polymorphism associated with silencing of thymic insulin predispose to type 1 diabetes. J Clin Endocrinol Metab 2001;86:3705–10. [48] Bennett ST, Lucassen AM, Gough SC, Powell EE, Undlien DE, Pritchard LE, et al. Susceptibility to human type 1 diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus. Nat Genet 1995;9:284– 92. [49] Bennett ST, Wilson AJ, Cucca F, Nerup J, Pociot F, McKinney PA, et al. IDDM2VNTR-encoded susceptibility to type 1 diabetes: dominant protection and parental transmission of alleles of the insulin gene-linked minisatellite locus. J Autoimmun 1996;9:415–21. [50] Vafiadis P, Bennett ST, Colle E, Grabs R, Goodyer CG, Polychronakos C. Imprinted and genotype-specific expression of genes at the IDDM2 locus in pancreas and leucocytes. J Autoimmun 1996;9:397–403. [51] Chentoufi AA, Polychronakos C. Insulin expression levels in the thymus modulate insulin-specific autoreactive T-cell tolerance: the mechanism by which the IDDM2 locus may predispose to diabetes. Diabetes 2002;51:1383–90. [52] Vafiadis P, Bennett ST, Todd JA, Nadeau J, Grabs R, Goodyer CG, et al. Insulin expression in human thymus is modulated by INS VNTR alleles at the IDDM2 locus. Nat Genet 1997;15:289–92.