Autoantibodies against ATP4A are a feature of the abundant autoimmunity that develops in first‐degree relatives of patients with type 1 diabetes

Type 1 diabetes is associated with autoantibodies to different organs that include the gut. The objective of the study was to determine the risk of developing gastric parietal cell autoimmunity in relation to other autoimmunity in individuals with a family history of type 1 diabetes.


| INTRODUCTION
Type 1 diabetes is characterized by the presence of circulating autoantibodies to islet antigens and an increased prevalence of autoantibodies targeting other organs such as the thyroid, gut and adrenal gland. [1][2][3][4] Many of these autoantibodies also appear in first-degree relatives of patients with or without concomitant islet autoantibodies. 5 The increased prevalence of autoimmunity other than islet autoantibodies in patients with type 1 diabetes and their relatives is likely to be in part due to shared genetic susceptibility at HLA and other gene regions. 6 Despite the shared susceptibility, there are differences in the age at which thyroid, transglutaminase, and islet autoantibodies develop, suggesting different etiological factors. 5 Gastric parietal cell antibodies are also more prevalent in patients with type 1 diabetes. 3 Here, we have measured autoantibodies against the parietal cell autoantigen H + /K + ATPase subunit A (ATP4A) in a cohort of over 2000 first-degree relatives of patients with type 1 diabetes prospectively followed from birth to identify genetic and temporal similarities and differences between the manifestation of autoantibodies to different organs and antigens.

| Study population
Children with a first-degree family history of type 1 diabetes recruited to the BABYDIAB or BABYDIET prospective studies between 1989 and 2006 7,8 were included. Children were followed for the development of autoimmunity associated with type 1 diabetes, celiac disease, and thyroid autoimmune disease. 5 Blood samples were obtained at age 9 months, 2 years, 5 years and every 3 years thereafter or, for 150 children in the BABYDIET study, every 3 months until age 3 years and subsequently at age 5 years and every 3 years thereafter. Follow-up was increased to six-monthly intervals in children who developed islet autoantibodies. The median follow-up period from birth was 14.5 years (interquartile range [IQR], 7.9-19.9 years). Children were HLA typed at the HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci as previously described. 9,10 The studies were approved by the Bavarian ethics committees (Bayerische Landesärztekammer no. 95357 and Ludwig-Maximilians University no. 329/00, respectively) and were performed in accordance with the principles of the Declaration of Helsinki, including the provision of written informed consent from the participants or their parents.

| ATP4A autoantibody measurements
The coding sequence was obtained as a synthetic gene (BaseClear B.V., The Netherlands) and was cloned into the pCMV6-AC-IRES-

| Statistical analysis
Participants were considered positive for an autoantibody if the autoantibody was above the positive threshold in two or more consecutive samples or in the last available sample. 1,17 The Kaplan-Meier method was used to determine the cumulative risks of developing autoantibodies and the log rank test was used to compare categories. Follow-up was calculated from birth to the age when autoimmunity first developed or to the last sample in participants who were negative. Univariable and multivariable hazard ratios (HRs) and their 95% confidence intervals (CI) for autoantibody development were determined using Cox proportional hazard models. The incidence of antibodies was expressed as per 1000 person-years and was calculated for the age intervals 0-18 months,

| RESULTS
3.1 | ATP4A autoantibody prevalence and age of appearance ATP4A autoantibodies were measured in 2218 first-degree relatives of patients with type 1 diabetes. ATP4A autoantibodies were observed in 128 participants ranging in first appearance from age 9 months to 23 years. The cumulative prevalence of ATP4A autoantibodies was 4.7% (95% CI, 3.7-5.7) by age 10 years and 8.1% (95% CI, 6.6-9.6) by age 20 years ( Figure 1A). ATP4A autoantibodies incidence rose to a peak of 7.9/100,000 per year at around 2 years of age and remained above 5/100,000 per year until 8 years of age ( Figure 1B). In comparison, the peak incidence for islet autoantibodies was observed at age 9 months to 2 years, TG autoantibodies at age 2 years, and TPO autoantibodies at 14 years of age.

| Factors associated with ATP4A autoantibodies
Sex and HLA class II genotypes were associated with ATP4A autoantibody risk (    (Figure 2A). Any of these autoantibodies was observed in 489 participants ( Figure 2B). autoantibodies (6 prior, 2 at the same age, and 5 after islet autoantibodies). ATP4A autoantibodies appeared after TG autoantibodies in all 6 with both autoantibodies, and most often before (n = 18) or at the same age (n = 9) as TPO autoantibodies in the 31 children with both autoantibodies ( Figure 2C). Islet autoantibodies were more frequently observed before TG autoantibodies (5 prior, 0 at the same age, and 2 after TG autoantibodies), and before TPO autoantibodies (16 prior, 5 at the same age, and 4 after TPO autoantibodies; Figure 2D).  Figure 3C).

| DISCUSSION
Autoantibodies to the gastric parietal cell autoantigen ATP4A were found in 8.1% of relatives of patients with type 1 diabetes by age 20 years.
ATP4A autoantibodies developed throughout childhood and adolescence, as early as age 9 months, and with a peak incidence around age 2 years.
The findings are from a large cohort of first-degree relatives of patients with type 1 diabetes followed from birth for up to 32 years.
The cohort participants are, therefore, genetically at risk for autoimmunity that is associated with type 1 diabetes, which includes ATP4A autoantibodies. 3 A potential limitation of the study is that screening for ATP4A autoantibodies was performed on the last sample available from participants. It is possible, therefore, that participants with transient ATP4A autoantibodies were missed and that the risk for the autoantibodies is slightly underestimated. ATP4A autoantibodies are often detected in the absence of or only mild clinical complications 18,19 Since our study did not have clinical or biochemical data related to pernicious anemia, we cannot determine the clinical significance of ATP4A autoantibodies in the relatives.
The association of ATP4A autoantibodies with female sex, thyroid autoimmunity and HLA DR4 are consistent with previous reports in patients with type 1 diabetes 20 The marked risk in the relatives with the HLA DR4-DQ8/DR4-DQ8 genotype is novel. This genotype is strongly associated with the risk for developing type 1 diabetes and autoantibodies against IA-2 and insulin. [21][22][23] Of interest, there was relatively little overlap between islet and ATP4A autoantibodies in relatives with this genotype, suggesting that the two autoimmune entities are likely to have distinct etiologies. There was also no association between ATP4A and TG autoantibodies, although both target gut autoantigens and both had a peak age of incidence around age 2 years. Unlike ATP4A and islet autoantibodies, the ATP4A and TG autoantibodies had distinct HLA genotype associations suggesting that HLA may determine the likely specificity of gut autoimmunity. A similar scenario is observed for pancreatic islet autoimmunity where HLA DR4-DQ8 is linked to the development of autoantibodies to insulin and HLA DR3 is linked to the development of autoantibodies to GAD. 24,25 A remarkable observation is that around 50% of the relatives of patients with type 1 diabetes and with two HLA DR3 or DR4-DQ8 haplotypes or heterozygous for these haplotypes developed islet, gut or thyroid autoimmunity by age 20 years. Although the three possible genotypes had different autoantibody associations, any autoantibody risk was around 50% for each of the genotypes. Familial autoimmunity is, therefore, strongly determined by genetics and its target specificity determined by HLA, age, and sex. The contribution of common and/or specific environmental factors to the development of each autoimmunity remains to be determined. Bonifacio is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.