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Autoimmune gastritis (ABG) and pernicious anemia (PA) are prototypical, organ-specific autoimmune diseases whose prevalence in the general population are 2 and 0.15-1%, respectively. The incidence of disease increases with age and with is frequently associated with other autoimmune disorders such as type 1 diabetes (T1D). Early diagnosis of ABG/PA is essential for the prevention and/or treatment before manifestations of chronic disease are irreversible. Parietal cell autoantibody (PCA) detection via ELISA is currently the most widely used biomarker of disease with diagnosis confirmed by subsequent immunohistochemistry via biopsy.
To improve the assay we designed a specific, molecularly defined radioimmunoprecipitation assay (RIA) for early detection of ABG targeting its major antigen ATP4A.
The major antigenic domain in ATP4A was tested against a panel of sera from new onset T1D patients which tested positive for the gold standard T1D autoantibodies (IAA, IA2A, GAD65A, and ZnT8A). Significant immunoreactivity to ATP4A was measured (25%) while, 6% of first-degree relatives of T1D subjects who were sero-negative for T1D autoantigens were positive for ATP4A autoantibodies. ATP4A antibody prevalence increased with age of onset of T1D, which is atypical of other T1D autoantibodies. Immunoreactivity to ATP4A, unlike that of T1D antigens, demonstrates a significant gender bias in newly diagnosed T1D individuals.
Although the utility of the assay as a biomarker for T1D is likely limited, it may serve as an improved indicator of ABG.
Even before an autoimmune basis for T1D was confirmed (1), a significant incidence of PCAs in insulin-dependent diabetic patients was noted (2). The presence of circulating parietal cell autoantibodies (PCAs) is indicative of atrophic body gastritis (ABG), an autoimmune disease which in its chronic form manifests as perniciouos anemia (PA). Although vitamin B12 replacement therapy is an effective cure for PA, the disease is associated with numerous devastating complications including irreversible neuropathy and gastric adenocarcinoma. A major breakthrough in understanding the pathogenesis of ABG/PA was the demonstration that gastric H+/K+-ATP4 (ATP4) is the major antigen recognized by PCAs. It is an α/β-heterodimeric integral membrane protein that is responsible for secretion of hydrogen ions in exchange for K+ ions in parietal cells (3) which serves to acidify gastric juice (4, 5, 6). PCAs have been shown to bind to both the ~100kD α-subunit (SU) and the 60-90kD heavily glycosylated β-SU of ATP4B (7), with the major epitopes in the human β-SU critically dependent on a full complement of N-linked glycans for immunoreactivity (8, 9). The major humoral epitopes for the human α and β subunits have been identified by immunization of mice with overlapping human peptides and selection of those that induce inflammatory infiltration specifically in the gastric mucosa in mice, followed by loss of acid-secreting parietal cells and the appearance of circulating autoantibodies directed to ATP4 (10). We created a customized assay using a molecularly optimized ATP4A probe that exhibits exquisite sensitivity and specificity (Wenzlau and Hutton manuscript in preparation). Given our experience with RIA development for novel antigens in T1D and the clear association of PA with T1D and other autoimmune diseases, we sought to test the prevalence of PCAs in newly diagnosed T1D individuals and their association with the gold standard T1D autoantibodies as well as gender.
Serum samples were acquired after informed consent from patients, relatives and controls attending The Barbara Davis Center in compliance with IRB-approved protocols. Radioimmunoprecipitation assays (RIAs) were performed according to published procedures (11) using ATP4A derivative antigen probes. Assays were conducted with 16 matched control samples and a pool of human sera with high-titer ATP4A antibodies. Cut-off indices were determined by the mean +/− 5 SD of the intra-assay control values. The immunoprecipitation index was calculated by: sample − negative control mean/positive control mean − negative control mean.
We previously cloned the full length ATP4A gene and empirically determined the region of major immunoreactivity to optimize immune complex formation while minimizing background reactivity (Wenzlau and Hutton manuscript in preparation). The major antigenic domain of ATP4A was subcloned into pDNA3.1 (Invitrogen), a vector which we have previously employed for coupled in vitro transcripton/translation reactions to generate 35S-labelled antigen probes for use in radioimmunoprecipitation assays (RIAs) (11).
We measured ATP4A antibodies in a panel of 116 sera from subjects with T1D of less than 6 months duration (new onset) who were followed prospectively and had been stratified on the basis of immunoreactivity to the T1D autoantibodies for insulin (INS, MIAA), the 65-kD form of glutamatic acid decarboxylase (GAD65), zinc transporter 8 (ZnT8) and insulinoma autoantigen-2 autoantibodies (IA2) (Figure 1). Individual autoantibodies demonstrated their characteristic age of onset prevalence profile. As expected, the prevalence of GAD65 autoantibodies remained consistent independent of age of T1D onset while insulin autoantibodies (typically) declined. Prevalence of IA2 (ICA512) autoantibodies likewise diminish with advanced age of onset frequently in parallel (albeit not significant) with ZnT8 autoantibodies. In striking contrast, ATP4A autoantibodies do not mimic the established profiles for islet cell autoantibodies or those associated with other related autoimmune diseases such as Addison’s and celiac disease (data not shown) but rather demonstrate a consistent increase in prevalence with age of onset of T1D.
We monitored a second collection of sera derived from another cohort of newly diagnosed T1D individuals (<6 months, n=463) for the prevalence of autoantibodies against ATP4A, indicative of autoimmune gastritis, and INS, IA2, GAD65, and ZnT8 associated with T1D. Twenty-five percent of sera from these patients demonstrated significant immunoreactivity to the ATP4A antigen (not shown). In contrast, RIAs conducted with sera obtained from first degree relatives of T1D individuals negative for the classic T1D autoantibodies, demonstrated 5% positivity for ATP4A autoantibodies (not shown). The non-T1D controls group (n=180) had a mean age of 12.5 yrs. The cut off was chosen determined at the 95th percentile. It is possible that the presence of the ATP4A autoantibodies are due to the loss of immune tolerance that impacts more than one tissue (gastric mucosa and pancreatic islets). As ATP4A autoantibodies are found in a number of autoimmune disorders, the prevalence among first degree relatives may be a consequence of genetic predisposition linked to HLA and/or other interactions between specific at-risk genetic alleles or epigenetic factors linked to environmental agents.
When the indices for autoantibody positive samples were stratified according to gender, there was a significant gender bias in titers where the mean index for females and males is 0.5130 and 0.2820, respectively (p=0.0136; n=34 females, n=37 males) for individuals positive for ATP4A autoantibodies (Figure 2). The female gender bias extended to the negative control group for ATP4A autoantibodies (n=77 females) (n=103 males) where p=.0211. There was no appreciable gender bias for any of the gold standard T1D autoantibodies: INS (p=0.8258) showing a mean index of 0.1940 for females (n=126) and 0.2164 for males (n=196), GAD (p=0.2865) with a mean index of 0.3238 for females (n=141) and 0.2508 for males (n=139), IA2A (p=0.2261) having a mean index of 0.6961 for females (n=161) and 0.7507 for males (n=182), and ZnT8 (p=0.8488) displaying a mean index of 0.5920 for females (n=60) and 0.5815 for males. The significance of the female gender bias in the binding index for ATP4A autoantibodies is presently currently underway and is presently being validated with a larger collection of sera to increase the power of the study.
We have employed a customized ATPase autoantibody assay, molecularly optimized to target the major antigen ATP4A of ABG. A significant proportion of new onset T1D individuals harbor autoantibodies to ATP4A which may serve as a very early biomarker for impending PA. The antibody prevelance profile of ATP4A autoantibodies is distinctive relative to those for the gold standard T1D autoantibodies in that the prevalence increases with increasing age of diagnosis. Moreover, a significant gender bias (females/males) is apparent for the mean index for ATP4A autoantibodies whereas no gender bias exists for autoantibodies associated with T1D. Implementation in clinical settings of the RIA will facilitate early ABG diagnosis and the identification of individuals, a subset of which are also at risk of T1D, who may benefit from simple, efficient, and cost-effective preventative therapies such as prophylactic vitamin B12.
This work was supported by a JDRF autoimmunity prevention consortium center grant (4-2007-1056), NIH R01 DK052068 (to JCH), NIH R56 DK052068 (to JCH and HWD), the UCHSC Diabetes and Endocrinology Research Center (P30 DK57516), and the Children's Diabetes Foundation of Denver.