Differences in the contribution of the CTLA4 gene to susceptibility to fulminant and type 1A diabetes in Japanese patients.

OBJECTIVE: To examine the contribution of the CTLA4 gene in the susceptibility to fulminant type 1 diabetes and compare it with classic type 1A diabetes. RESEARCH DESIGN AND METHODS: We genotyped the +49G>A and CT60G>A variants of the CTLA4 gene in fulminant type 1 diabetic patients (n = 55), classic type 1A diabetic patients (n = 91), and healthy control subjects (n = 369). We also assessed serum levels of the soluble form of CTLA4 (sCTLA4).
RESULTS: The +49GG and CT60GG genotypes were associated with type 1A diabetes (P < 0.001). In contrast, the CT60AA genotype, but not the +49G>A variation, was associated with fulminant type 1 diabetes (P < 0.05), especially in patients carrying HLA-DR4 (P < 0.01). Serum levels of sCTLA4 were significantly decreased in patients with fulminant type 1 diabetes (P < 0.05).
CONCLUSIONS: These results suggest that CTLA4 CT60 affects the genetic susceptibility to fulminant type 1 diabetes. Furthermore, the contribution of CTLA4 to disease susceptibility is distinct between fulminant type 1 diabetes and classic type 1A diabetes.

Fulminant type 1 diabetes is a subtype of type 1 diabetes characterized by extremely rapid onset that can be classified as type 1B diabetes (1). Although frequent flu-like symptoms prior to onset suggest the contribution of virus infection in the etiology of fulminant type 1 diabetes, both environmental and genetic factors are largely unknown. Susceptibility to classic type 1A diabetes is determined by multiple genes within the HLA region and non-HLA genes, including INS-VNTR, CTLA4, and PTPN22 (2). Among them, CTLA4 is associated with autoimmunity, cancer, allergy, and infectious disease. In the CTLA4 region, a number of variants, such as +49G>A and CT60, have shown type 1 diabetes association (3). Although the association between class II HLA and fulminant type 1 diabetes has been reported (4), the contribution of the non-HLA genes to the susceptibility to fulminant type 1 diabetes has not been investigated. In this study, we examined the genetic contribution of the CTLA4 gene to fulminant type 1 diabetes compared with classic type 1A diabetes.

RESEARCH DESIGN AND METHODS

We examined 55 patients with fulminant type 1 diabetes (49% female, median age at onset 35.0 years), 91 patients with classic type 1A diabetes (57% female, median age at onset 17.0 years), and 369 healthy control subjects. Diagnostic criteria for fulminant type 1 diabetes are described elsewhere (1). The criteria for the recruitment of type 1A diabetic patients were presence of diabetic ketosis at onset, duration of hyperglycemic symptoms <3 months prior to initiation of insulin therapy, and positive for at least one of the anti-islet autoantibodies. This study was approved by the appropriate ethics committees, and informed consent was obtained from all subjects.

Genotyping of two single nucleotide polymorphisms in the CTLA4 gene, +49G>A (rs231775) and CT60 (rs3087243), was performed as reported previously (5). Serum concentration of sCTLA4 was measured by enzyme-linked immunosorbent assay using human soluble CTLA4 (sCTLA4) kit (MedSystems Diagnostics, Vienna, Austria), according to the manufacturer's protocol. Sera from type 1 diabetic patients were obtained at disease onset and stored at −20°C until use.

The significance of differences in the distribution of genotypes between case and control subjects was determined by χ2 test or Fisher's exact probability test. Comparisons of the sCTLA4 levels were made by ANOVA with phenotypic group alone and ANOVA with phenotypic group and CTLA4 genotype. P < 0.05 was considered to be statistically significant.

RESULTS

The +49G>A variant was associated with classic type 1A diabetes but not with fulminant type 1 diabetes (Table 1). In contrast, the contribution of CT60 to disease is distinct from that of +49G>A. The frequency of the CT60AA genotype in fulminant type 1 diabetic patients was significantly higher than in control subjects (P = 0.021) and type 1A diabetic patients (P = 0.031). CT60GG was associated with type 1A diabetes (P = 0.008). Because of the strong association of HLA-DR4 in both patient groups (1), the effect of CTLA4 on type 1 diabetes susceptibility relative to HLA-DR4 was also examined. Among DR4-positive individuals, the frequency of the CT60AA genotype was significantly increased in patients with fulminant type 1 diabetes (P = 0.005). However, stratification of patients by the presence or absence of HLA-DR4 did not affect the association between the +49GG genotype and type 1A diabetes (Table 1).

Table 1

CTLA4 polymorphisms in fulminant type 1 diabetic patients, type 1A diabetic patients, and healthy control subjects

	Fulminant	Type 1A	Control	P value			OR (95% CI)
				Fulminant vs. control	Type 1A vs. control	Fulminant vs. type 1A	Fulminant vs. control	Type 1A vs. control	Fulminant vs. type 1A
+49 G>A
n	55	91	369
AA	13 (23.6)	7 (7.7)	61 (16.5)
AG	23 (41.9)	36 (39.6)	186 (50.4)
GG	19 (34.5)	48 (53.7)	122 (33.1)	NS	0.0013	0.012
GG	19 (34.5)	48 (53.7)	122 (33.1)	NS	0.0005	0.033		2.26 (1.41–3.60)	0.47 (0.24–0.94)
AA + AG	36 (65.5)	43 (46.3)	247 (66.9)
CT60 G>A
n	55	91	369
AA	8 (14.5)	4 (4.4)	22 (6.0)
AG	26 (47.3)	28 (30.8)	165 (44.7)
GG	21 (38.2)	59 (64.8)	182 (49.3)	0.043	0.029	0.0036
GG	21 (38.2)	59 (64.8)	182 (49.3)	NS	0.008	0.0017		1.89 (1.18–3.05)	0.33 (0.17–0.67)
AA + AG	34 (61.8)	32 (35.2)	187 (50.7)
AA	8 (14.5)	4 (4.4)	22 (6.0)	0.021	NS	0.031	2.68 (1.13–6.37)		3.70 (1.06–12.9)
AG + GG	47 (85.5)	87 (95.6)	347 (94.0)
DR4 (+)
n	42	52	121
+49GG	14 (33.3)	27 (51.9)	38 (31.4)	NS	0.011	NS		2.35 (1.21–4.59)
CT60AA	8 (19.0)	2 (3.8)	6 (5.0)	0.005	NS	0.018	4.51 (1.46–13.9)		5.88 (1.18–29.4)
DR4 (−)
n	13	36	183
+49GG	5 (38.5)	19 (52.8)	61 (33.3)	NS	0.026	NS		2.24 (1.08–4.61)
CT60AA	0 (0.0)	3 (8.3)	16 (8.7)	NS	NS	NS

Data are n (%). The interaction between CTLA4 polymorphisms and HLA-DR4 was assessed by a χ2 test with a 2 × 2 contingency table (+49 GG vs. AG + AA or CT60 AA vs. AG + GG) in DR4-positive or -negative patients and the corresponding control subjects.

It has been reported that the CT60G allele might be associated with lower production of sCTLA4 mRNA (3). We therefore determined serum sCTLA4 levels. The mean sCTLA4 levels in fulminant type 1 diabetic patients (0.56 ± 0.24 ng/ml [mean ± SD], n = 36) was significantly lower than those in type 1A diabetic patients (0.94 ± 0.87 ng/ml, n = 45) and control subjects (0.89 ± 0.76 ng/ml, n = 23) (P = 0.043). A mixed-model ANOVA using phenotypic group (fulminant type 1 diabetes, type 1A diabetes, and control) and CT60 genotype (GG and GA+AA) as factorial fixed effects revealed no differences in sCTLA4 levels between CT60 genotypes (P = 0.76) or phenotype/genotype interactions (P = 0.40).

CONCLUSIONS

CTLA4, which delivers inhibitory signals to T-cell activation, is expressed on the surface of activated T-cells and regulatory T-cells, and the lack of CTLA4 results in uncontrolled T-cell–mediated lymphoproliferative disease (6). Furthermore, CTLA4 also has a significant biological role in attenuating T-cell responses in the context of an inflammatory environment, such as infection with a pathogen (7). We showed that CTLA4 is associated with an increased risk of fulminant type 1 diabetes and that its contribution is distinct from classic type 1A diabetes. As reported previously (5,8), a significant association between classic type 1A diabetes and +49GG and CT60GG genotype was also found in the present study. However, the CT60AA genotype contributes to the susceptibility to fulminant type 1 diabetes. Moreover, it is implicated that HLA-DR4 influences the association of fulminant type 1 diabetes with the CT60AA genotype.

In this study, we also revealed that serum sCTLA4 level in fulminant type 1 diabetic patients were significantly lower than those in type 1A diabetic patients and control subjects. Although it remains unknown how sCTLA4 regulates T-cell activation, recombinant sCTLA4 inhibits T-cell proliferation in vitro. Furthermore, sCTLA4 is constitutively expressed in nonstimulated T-cells, and its expression is downregulated after T-cell activation (9). Therefore, the decreased levels of sCTLA4 might indicate a lower potential of T-cell inhibition in fulminant type 1 diabetes, which might be caused by functional defects leading to reduced production of sCTLA4 or ongoing activation of the immune system eventually leading to decreased levels of sCTLA4.

In conclusion, the present study implicates that CTLA4 confers susceptibility to fulminant type 1 diabetes. Furthermore, the different contributions of CTLA4 to susceptibility to fulminant and classic type 1A diabetes indicate that the underlying immune process–primed β-cell injury might be distinct between these subtypes of type 1 diabetes.