Although the association between type 1 diabetes mellitus (T1DM) and Hashimoto thyroiditis, as well as celiac disease (CD) in children and adolescents, is well documented (1–4), little is known about the association with other autoimmune disorders such as autoimmune gastritis (AG) and pernicious anaemia. AG is characterized by atrophy of the gastric corpus and fundus, autoantibodies to parietal cells, achlorhydria, iron-deficiency anaemia (IDA), hypergastrinaemia, and pernicious anaemia, resulting from vitamin B12 deficiency (5).
Parietal cell antibodies (PCA) are the principal immunological markers of AG and react against the H+/K+ ATPase of gastric parietal cells (6). Chronic autoaggression to the proton pump may result in hypochlorhydria or achlorhydria, hypergastrinemia, and IDA due to decreased gastric secretion and iron absorption (7). PCA may also inhibit intrinsic factor secretion, leading to vitamin B12 deficiency and pernicious anaemia (8).
In adult patients, AG is present in up to 1% to 2% of the general population and is 3- to 5-fold increased in patients with T1DM or autoimmune thyroid disease (5,9). The frequency of PCA in adult patients with T1DM ranges between 9% and 21% (10,11). De Block et al report that PCA-positive patients with T1DM have a high prevalence of gastric autoimmunity and, as a consequence, an increased risk for IDA, pernicious anaemia, and hypochlorhydria (11). They also showed that hypergastrinaemic PCA-positive patients are at an increased risk of developing (pre)malignant gastric lesions (12). Another characteristic marker of AG, low serum level of pepsinogen I, resulting from destruction of chief cells, has also been identified as an early marker of pernicious anaemia in patients with T1DM (13).
Furthermore, some studies demonstrated a strong correlation between gastric and thyroid antibodies (6,14,15). All of these aspects have been examined in adults. Studies about PCA in children are scanty. These studies report prevalence rates of 4% to 15% (5,16,17), but they did not compare the frequency of PCA in children with T1DM to healthy controls, and there is only 1 study that evaluated gastric secretion in PCA-positive patients (17). Therefore, the aims of the present study were to evaluate the frequency of PCA in children and adolescents with T1DM compared to healthy controls and to examine clinical and biochemical markers and biopsy findings in young patients with T1DM positive for PCA.
PATIENTS AND METHODS
From a total of 202 eligible patients attending the paediatric diabetic clinic at the University Hospital Graz, Austria, 170 patients (87 boys, 83 girls; mean age 12.9 years, range 2.7–19.5 years) with a mean duration of diabetes of 4.9 years (range 1.0–16.3 years) were recruited to this cross-sectional study from January 2007 to December 2007 based on the following inclusion criteria: age between 1 and 19 years, duration of T1DM for more than 1 year, or C-peptide negative. Exclusion criteria were evidence of diabetic complications and presence of other chronic illnesses. The patients did not take any medications other than insulin and thyroxin, especially no gastrointestinal-specific medication.
The control group consisted of 101 children (49 boys, 52 girls, mean age 13.0 years, range 3.4–18.9 years) who attended the paediatric unit at the University Hospital Graz for preoperative screening before various elective operations (eg, tonsillectomy and adenoidectomy). All of the control subjects were in good health as assessed by their medical and family history and physical examination and did not take any medications. The study was approved by the local research ethics committee; written informed consent was obtained from all of the parents and assent obtained from all of the children.
In the group of children and adolescents with T1DM, PCA, free T3, free T4, thyroid-stimulating hormone (TSH), thyroid peroxidase antibodies (TPOAb), TgAb, gastrin, pepsiongen I, iron, ferritin, transferrin saturation, vitamin B12, and folate were determined in all of the patients within the annual diabetes review. Because of its invasiveness and the need for sedation in the paediatric age group, gastroscopy with multiple biopsies and the Helicobacter pylori test was carried out only in patients with high (>100 U/mL) PCA levels. In the healthy control group, PCA, free T3, free T4, TSH, TPOAb, and TgAb were measured during the preoperative screening tests; gastroscopy with multiple biopsies was not part of the study protocol.
PCA levels (normal range <15 U/mL) were measured using an indirect noncompetitive enzyme immunoassay for the semiquantitative and qualitative determination of parietal cell antibodies in human serum or plasma (Medical Diagnostics Phadia GmbH, Freiburg, Germany). Serum gastrin (normal range 28–185 mU/mL) and serum pepsinogen I levels (normal range >25 μg/L) were measured using enzyme-linked immunosorbent assays (Biohit Oyj, Helsinki, Finland). Thyroid hormones and thyroid antibodies in serum were determined by commercial kits. Free T4 (normal range 9.5–24.0 pmol/L), free T3 (normal range 3.0–6.3 pmol/L), and TSH (normal range 0.1–4.0 μU/mL) were measured using luminescence immunoassays (Bayer, Leverkusen, Germany). TPOAb (upper normal value 50 U/L) and TgAb (upper normal value 50 U/L) were determined by enzyme-linked immunosorbent assays (IASON, Graz, Austria). Vitamin B12 (normal range 180–1000 pg/mL) and folate levels (normal range 2.7–34 ng/mL) were determined using luminescence immunoassay (Siemens, Austria). IDA was defined as microcytic hypochromic anaemia with a transferrin saturation of <20% and decreased iron (normal range 60–180 μg/dL) and ferritin levels (normal range 10–140 ng/mL). Pernicious anaemia was defined as macrocytic anaemia with subnormal vitamin B12 levels and positive PCA.
Gastroscopy and Histopathology
Gastroscopy (GIF Q140, Olympus) was performed in patients with T1DM having PCA higher than 100 U/mL. At least 2 biopsies were taken from the gastric antrum, corpus, and fundus for histopathology. The degree of gastritis was assessed according to the updated Sydney system (18). Atrophy of the gastric body mucosa was defined as focal or complete loss of oxyntic gland and/or replacement by metaplastic pylori or intestinal glands. To each graded variable, the following scores were assigned: 0 = absence, 1 = mild, 2 = moderate, and 3 = severe, as previously reported (19). Antrum, corpus, and fundus biopsies were examined for H pylori colonisation using a modified Giemsa stain and/or immunostaining. In addition, a urease test was performed (Heipha Diagnostics, Heidelberg, Germany).
Calculations and Statistical Analysis
A priori sample size estimations based on the literature and data of adults showed that assuming a prevalence of PCA of 20% in patients with diabetes and of 2% in healthy controls, 47 patients would be needed per study group to demonstrate a significant between-group difference with a power of 80% at an alpha fault of 0.05.
Descriptive statistics including absolute and relative frequencies, means, and ranges were calculated using a commercially available software program (SPSS version 14.0; SPSS Inc, Chicago, IL). The Shapiro-Wilk test was used to test the normal distribution of the parameters. Differences in distribution and the significance of association of categorical data were analysed by the Fisher exact test. The Wilcoxon rank sum test was used to analyse differences in the centres of location for 2 independent groups. The Spearman rank correlation test was used to analyse correlations between the various parameters. The value of P < 0.05 or less was considered significant.
There were no differences in age and sex distribution between diabetic patients and control subjects. Of the 170 T1DM patients, 5 patients had diagnosis of CDs, all of them were consuming a gluten-free diet, and tested negative for tissue transglutaminase antibodies (tTGA). None of the patients positive for CD had positive PCA.
Reviewing our annual review charts, we found that of 170 patients with T1DM, 1 girl from the PCA-positive group and 1 girl from the PCA-negative group complained about gastrointestinal symptoms such as occasional abdominal pain.
PCA Frequency and Levels
The frequency of PCA was higher (5.29%, n = 9) in patients with T1DM than that (1.98%, n = 2) in healthy controls, although this did not reach statistical significance (P = 0.22). Among 170 patients with T1DM, 9 patients (2 boys, 7 girls) had positive PCA levels. Four of them, all girls, had high (>100 U/mL) levels, and the other 5 showed a weak to moderate reaction (Table 1). Only 2 of 101 control subjects (both boys) had positive PCA, of whom 1 showed high (>100 U/mL) levels, whereas the other 1 had a weak reaction.
Risk Factors for PCA
Positive PCAs were seen more often in girls than in boys (7 vs 2 patients, respectively), but without statistical significance (P = 0.09). No correlation was found between PCA and age (r = 0.081), age at diabetes onset (r = 0.017), or diabetes duration (r = 0.021). Details of PCA-positive and PCA-negative patients are listed in Table 2.
PCA and Thyroid Antibodies
TPOAb and/or TgAb were found significantly more often in children and adolescents with T1DM (14.70%, n = 25) and compared to children of the healthy control group (1.98%, n = 2; P < 0.01). Overall, thyroid autoimmunity (positive for TPOAb and/or TgAb) in young patients with T1DM was significantly higher in girls (18 patients, 72%) than in boys (7 patients, 28%) (P < 0.01). In the subgroup of patients with high PCA levels, 3 of 4 patients had positive thyroid antibodies (Table 1). In the control group, none of the patients with positive PCA had positive thyroid antibodies. In the group of children and adolescents with T1DM, a significant association was observed between PCA positivity and TPOAb (P = 0.01), but not TgAb (P = 0.24).
Gastrin and Pepsinogen I Levels in T1DM Patients
Gastrin levels were available in 158 patients with T1DM and in all of the patients who were positive for PCA. Hypergastrinemia was seen in 2 patients (22.22%) of the PCA-positive group compared to none (0%) of the PCA-negative group (Table 2). These 2 patients had high (>100 U/mL) PCA levels (Table 1). We found a positive association between PCA and gastrin (P = 0.003). Pepsinogen I levels, available in a total of 139 patients and in all of the patients with positive PCA, were within normal ranges, although 2 patients presented with rather low levels (28.7 and 27.8 μg/L, respectively). These 2 patients were from the PCA-negative group. We found no association between pepsinogen I levels and PCA.
Iron-deficiency Anemia, Vitamin B12, and Folate Levels in Patients with T1DM
IDA was present in 4 of 9 patients from the PCA-positive group compared to 5 patients of 161 from the PCA-negative group, yet this did not reach statistical significance. We found a relation between PCA and ferritin (P = 0.029), showing that PCA-positive patients have lower ferritin levels (27.4 ± 18.1) compared to PCA-negative patients (51.3 ± 37.7). All 4 patients with high (>100 U/mL) PCA levels experienced IDA compared to none of the 5 patients with low to moderate PCA levels. Of the 170 T1DM patients, only 1 girl, who was PCA negative, had decreased vitamin B12 levels. None of the children with T1DM and adolescents had decreased folate levels or signs of pernicious anaemia.
Histopathological Findings and Helicobacter Test
Gastroscopy with multiple biopsies was carried out in all 4 patients with T1DM and high (>100 U/mL) PCA levels. Mild atrophic gastritis (grade 1) was diagnosed in 1 girl. She tested H pylori positive but had normal gastrin and pepsinogen I levels (Table 1). The other 3 patients showed mild signs of chronic superficial gastritis, with positive H pylori tests in 2 of them. None of the patients had (pre)malignant gastric lesions.
Associated autoimmune diseases in patients with T1DM are common, but not all of the patients develop them and different pathogenic mechanisms may exist between patients with isolated diabetes and those with a more general autoimmune disease. Several risk factors have been identified for a number of autoimmune disorders, including sex, age, age at diabetes onset, and duration of diabetes, which may influence the development of autoantibodies (5,15).
In our study, we observed a higher frequency of PCA in young patients with T1DM, compared to healthy controls, although this did not reach statistical significance. Although the frequency of PCA in controls was 1.98%, which is in line with the data of the general population (5), the frequency of PCA in patients with T1DM was 5.29%. This is in line with a paediatric study reporting a prevalence of 4.0% (16), but it is much lower compared to the data from adult patients, showing a prevalence of up to 20% (5,11).
In our study, children and adolescents with T1DM did not show any correlation between PCA and sex, age at diabetes onset, or diabetes duration. We also found no correlation with age in the group of children, which probably reflects the fact that PCA develop later in life, and this has also been suggested by studies in adults (5,11). These studies also did not note any correlation between PCA and age at diabetes onset nor diabetes duration (5,11). The reports on a sex-specific association of PCA in adults are controversial, and a female preponderance for PCA positivity (5,10) has not been consistently observed (5,11,13). In our paediatric study, although not statistically significant, girls tend to be more often positive for PCA. Compared to other associated autoimmune diseases in children and adolescents with T1DM, PCA have a similar frequency as CD, and the average prevalence of CD in 26 reports was 4.5% (0.97%–16.4%) (20). In an Austrian study, the frequency of CD was 2.9% (21). Compared to associated thyroid diseases in children and adolescents with T1DM, with reported prevalence rates from 7% to 40% (2,22), the frequency of PCA is much lower and it is also much lower compared to prevalence rates of PCA in children and adolescents with autoimmune thyroid disorders, which was 14% to 30% (7).
We speculate that the reason for the lower frequency of PCA among young patients with T1DM in our study, on the one hand, may result from an age-related increase in the prevalence of PCA, which has been shown both in the general population and in adult patients with T1DM. On the other hand, it may be influenced by a strong thyroid-gastric autoimmune association (5,11,15). Regarding risk factors for the development of PCA, studies in adults could identify thyroid peroxidase autoantibodies as an additional immunological risk factor, and studies in adults with T1DM and 1 study in children with thyroid disease suggest a strong association between thyroid and gastric autoimmunity (5,7). In these studies, PCA were found in 22% and 40% of patients with Graves disease and autoimmune thyroiditis, respectively (6). In our study, we were also able to detect a strong association between PCA and TPOAb, but there was no association with TgAb. In contrast to children with T1DM, we found no correlation between PCA and thyroid antibodies in the healthy control group.
H pylori infection may also play an initiating role in the pathogenesis of autoimmune gastritis and pernicious anaemia by inducing autoreactive T cells through gastric H+/K+ ATPase (23,24). As a consequence, a current recommendation suggests that H pylori infection should be tested and treated in adult patients with gastric atrophy (5). So far, a correlation between H pylori and PCA has not been reported by all of the studies (12,25,26), and whether H pylori may definitely trigger autoimmune gastritis remains controversial. In our paediatric study, 3 of 4 patients who underwent gastroscopy tested H pylori positive.
Looking at clinical findings, 4 of 9 patients from the PCA-positive group presented with IDA compared to 5 patients of 161 from the PCA-negative group, but the result did not reach statistical significance. Studies in adults showed that 20% to 30% of patients with IDA with no evidence of gastrointestinal blood loss may have autoimmune gastritis (5). The mechanism leading to IDA in patients with atrophic gastritis is that decreased gastric acidity, caused by chronic autoaggression to parietal H+/K+ ATPase, may reduce the availability of iron for absorption.
Furthermore, of 4 patients with high PCA levels who underwent gastroscopy, 3 had mild signs of superficial gastritis and 1 girl had mild (grade 1) atrophic gastritis. None of the patients had any symptoms of gastritis. Why we found only early signs of AG, as iron deficiency and hypergastrinemia, and why none of the patients showed signs of vitamin B12 or folate deficiency, pernicious anaemia, or low pepsinogen levels seems to be because the progression of AG to pernicious anaemia is likely to span 20 to 30 years (27), as it has been shown in adult studies.
Taking into account that biopsy findings demonstrate only superficial gastritis, in girls IDA may also have resulted at least in part from menstrual losses. Early detection and treatment of iron, vitamin B12, or folate deficiency and the underlying conditions are, however, important and may significantly reduce morbidity. In contrast to CD, which has been reported to influence anthropometric parameters in children and adolescents with T1DM (3,28,29), comorbidities due to PCA seem to develop later in life.
In conclusion, the frequency of PCA in children and adolescents with T1DM is much lower as in adults, but compared to healthy controls, children and adolescents with T1DM, and in particular those with positive TPOAb seem to be at increased risk for developing PCA. Only a few children with T1DM and positive PCA have overt clinical disease because PCA seem to develop later in life (second or third decade), leading to atrophic gastritis later on. Therefore, further longitudinal studies with large numbers of patients are needed to evaluate whether PCA screening should be part of a routine workup in adolescents with T1DM, as it has been recommended for thyroid autoimmunity and celiac disease so far. Nevertheless, physicians should be aware of the possibility of PCA in children and adolescents with T1DM in case of unclear anaemia (microcytic and macrocytic) or gastrointestinal symptoms.
We thank DI Gordana Djuras for assistance in analysing the data. In addition, we thank the children and their families for participating in the present study.
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