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Introduction
Diabetes mellitus is the most common endocrine disorder related to metabolic dysfunctions characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. There are several types of diabetes mellitus, but the main types of diabetes mellitus are type 1 (T1DM) and type 2 (T2DM) [1]. Type 1 diabetes mellitus is characterized by a lack of insulin, leading to persistent hyperglycemia and glycosuria as the pancreatic β cells are destroyed by the immune system. Type 1 diabetes mellitus is one of the most prevalent chronic illnesses affecting children [2–5].
Over the past four decades, the global incidence of type 1 diabetes mellitus has nearly doubled. It is currently estimated at approximately 15 per 100,000 individuals per year, with rates varying from 0.1 per 100,000 in Venezuela and China to 40.9 per 100,000 in Finland [6–8]. In Iraq, the prevalence of T1DM increased from 7.8 per 100,000 in 1995 to 14.2 per 100,000 in 2000 and to 24.7 per 100,000 in 2014 in children under 15 years old [8]. Additionally, a study performed in Iraq evaluated 60 T1DM patients, which found that 75% were positive for anti-GAD antibodies [4].
The incidence rates are higher in females aged 5 to 9 and males aged 10 to 14. Overall, the sex ratio is about even, with a slight male bias [6].
Pathophysiology is not entirely understood; however, it is generally believed to be influenced by several factors, such as genetic predisposition and the environment [9, 10]. The autoimmune process has been linked to certain environmental factors, including vitamin D3 insufficiency, cow’s milk proteins, and viral infections, among genetically susceptible patients. Nonetheless, none of these factors has been definitively proven to cause diabetes [11].
Several studies have demonstrated that hereditary factors contribute significantly to the development of T1DM. The HLA complex accounts for 40–50% risk of acquiring T1DM [11]. Autoantibodies against GAD65 are present in 70–80% of individuals with T1DM. These antibodies may manifest years before the disease’s clinical manifestation, serving as a valuable prognostic marker for autoimmune diabetes [12].
The pancreatic enzyme glutamate decarboxylase (GAD) catalyzes the decarboxylation of L-glutamic acid to produce γ-aminobutyric acid (GABA), an inhibitory neurotransmitter. GABA regulates cell function through signaling. It has two different isoforms, GAD65 and GAD67, encoded by genes on chromosomes 10p11 and 2q31, respectively. GAD65 is the primary autoantigen in T1DM [10].
Glycation, the chemical process by which glucose is joined to hemoglobin, gives hemoglobin A1c its name. The relationship between blood glucose and hemoglobin reflects the structure of high blood glucose, which is typically associated with diabetes mellitus. Since red blood cells have a lifespan of approximately four months, the glycated hemoglobin (HbA1c) test is only valid for a 3-month median. Glycated hemoglobin is produced at normal levels when glucose levels are normal and increases predictably as plasma glucose levels rise [13, 14].
This study aimed to evaluate HbA1c levels and to determine the prevalence of glutamic acid decarboxylase 65 autoantibodies (anti-GAD65) in newly diagnosed Kurdish children with T1DM.
Material and methods
Selection of subjects
This cross-sectional study included 148 children, aged 1–18 years, who were recently diagnosed with T1DM (59 females and 89 males). The study was conducted in Duhok, Kurdistan Region, Iraq, with data collected from the Private Diabetology Clinic in Duhok between October 1, 2022, and October 1, 2023, at the College of Medicine, University of Zakho.
Patient information
Before blood sample collection, patient information was obtained individually, with consent obtained from their parents. A questionnaire was established to collect data such as age, sex, ethnicity, religion, residence, kinship, anthropometric measurements (height, weight, body mass index), and family history of diabetes. Laboratory tests included measurements of blood glucose, HbA1c, and anti-GAD65.
Study inclusion and exclusion
Inclusion criteria: Patients with T1DM, aged 1–18 years, who were clinically free of other chronic diseases, were enrolled in this study.
Exclusion criteria: Participants over 18 and those diagnosed with T2DM or other chronic conditions, such as cancer, were excluded from this study.
Definition of variable
Diabetes diagnosis was established, based on the International Society for Pediatric and Adolescent Diabetes (ISPAD) criteria. Diagnostic criteria for all types of diabetes in children and adolescents are based on laboratory measurement of blood glucose levels (BGL) and the presence or absence of symptoms. BGL testing with a glucometer should not be used to diagnose diabetes. A marked elevation of the plasma glucose concentration confirms the diagnosis of diabetes, including a random plasma glucose ≥ 11.1 mmol/l (200 mg/dl) or fasting plasma glucose (≥ 126 mg/dl) in the presence of overt symptoms [14, 15]. Anti-GAD65 levels were considered positive when the level exceeded 1.0 U/ml [16].
Sample collection
Each patient underwent sterile venipuncture to obtain five milliliters of peripheral venous blood, which was then divided evenly between vacuum gel tubes and K2 EDTA tubes (BD Vacutainer, Franklin Lakes, NJ).
The K2 EDTA tubes were mixed by either flipping ten times or by using a rotatory mixer. The vacuum gel tubes were left at room temperature for 30 minutes and then centrifuged at 5,000 rpm for 10 minutes to extract the serum.
Laboratory procedures
The Cobas C 311 Roche analyzer was used with the K2-EDTA tubes to estimate the percentage of HbA1c (DCCT/NGSP) and mmol/mol HbA1c (IFCC) in human venous whole blood through quantitative photometric transmission measurement. The analyzer was fully automated and calibrated using manufacturer-supplied reagents.
Vacuum gel tubes were used to estimate glucose levels with a Cobas C 311 Roche analyzer, using fully automated electrochemiluminescence (ECL) technology, which was calibrated and regulated by manufacturer-provided reagents. Anti-GAD65 autoantibodies were measured using commercial ELISA kits (RSR Limited, Cardiff, UK), following the manufacturer’s instructions.
Statistical analysis
Data were analyzed using IBM SPSS version 26 software. For categorical data, the chi-squared test and t-test were utilized. Data were presented as mean ± standard deviation (MSD) values. The chi-squared test was employed to determine associations between variables. A p-value of less than 0.05 was considered statistically significant.
Bioethical standards
The ethics committee at the College of Medicine, University of Zakho, approved the proposed study. Before sample collection, written informed consent was obtained from the parents of all participants to ensure their agreement to take part in this study. This approval is documented by a letter issued on July 18, 2022, with the reference number (JULY 20/E07).
Results
This study included 148 children from Duhok City, Kurdistan Region of Iraq, with males outnumbering females by 89 (60.1%) to 59 (39.9%). The overall mean age with standard deviation was 9.97 ±4.68. Kurds were the predominant ethnic group, comprising 142 (95.9%), while Arabs accounted for only 6 (4.1%). In terms of religion, Muslims were the majority, representing 130 (87.8%) of the sample, followed by Yazidis, 15 (10.1%), and Christians, 3 (2%). Most participants lived in urban areas, 118 (79.7%), while 26 (17.65%) resided in rural regions, and 4 (2.7%) were refugees. Of the 148 participants, 42 (28.4%) had a family history of diabetes, whereas the remaining 106 (71.6%) had no family history, as seen in Table I.
Table I
Sociodemographic information of participant TIDM patients
The overall rate of autoantibody positivity across all age categories was 70 (47.3%), while 78 (52.7%) of the 148 children did not have anti-GAD65 antibodies. The age group of 1–6 years had the highest percentage of positivity, with 32 (45.7%), followed by the 7–12 years group with 29 (41.4%), and the 13–18 years group with 9 (12.9%). The relationship between age groups and anti-GAD65 positivity was statistically significant, with a p-value of 0.011 (Table II).
Table II
The age-group-specific distribution of T1DM patients with positive and negative anti-GAD antibodies
| Age groups | +ve anti-GAD65 No. (%) | –ve anti-GAD65 No. (%) |
|---|---|---|
| 1–6 years | 32 (45.71) | 21 (26.92) |
| 7–12 years | 29 (41.43) | 33 (42.31) |
| 13–18 years | 9 (12.86) | 24 (30.77) |
| Total | 70 (100) | 78 (100) |
| p-value | 0.011 | |
In Table III, of the 70 children positive for anti-GAD65, 37 (52.9%) were males and 33 (47.1%) were females. Conversely, of the 78 children negative for anti-GAD65, 52 (66.7%) were males and 26 (33.3%) were females. The relationship between sex and anti-GAD65 status was not statistically significant, with a p-value of 0.093.
Table III
The sex-specific distribution of T1DM patients with positive and negative anti-GAD65
| Sex | +ve anti-GAD65 No. (%) | –ve anti-GAD65 No. (%) |
|---|---|---|
| Male | 37 (52.9) | 52 (66.7) |
| Female | 33 (47.1) | 26 (33.3) |
| p-value | 0.093 | |
In our study, the overall mean HbA1c level among patients newly diagnosed with T1DM was 11.33 ±2.33, as shown in Table IV. There was no significant difference between males and females, with a p-value of 0.974. Males had a mean of 11.31 ±2.33, while females had a mean of 11.37 ±2.36.
Table IV
The sex-specific distribution of HbA1c levels
| Sex | HbA1c level (mean ±SD) % |
|---|---|
| Male | 11.31 ±2.33 |
| Female | 11.37 ±2.36 |
| Total | 11.33 ±2.33 |
| t-test | 0.974 |
The current study demonstrated modest associations between anti-GAD65 and HbA1c levels, with a p-value of 0.516. These associations were more pronounced in patients with negative results for anti-GAD65, who had a mean of (11.61 ±0.27), compared to a mean of (11.01 ±0.28) in patients positive for anti-GAD65. These results are presented in Table V.
Discussion
The autoimmune process is responsible for the development of T1DM. Anti-GAD antibodies have been extensively studied in population samples over recent years. It has been known that anti-GAD is positive in more than 70% of children with recent onset of T1DM, and its level seems to decrease with the duration of the disease and decreasing number of residual β cells [17].
The findings of this study demonstrated that (47.3%) of individuals with recently diagnosed T1DM were positive for anti-GAD65 antibodies. This result aligns with a prevalence of 48% in Pakistani children, and the Al-Diwaniyah study demonstrated that 42% of T1DM patients were positive for anti-GAD65 [14, 18]. However, higher percentages have been reported in Thi-Qar province in Iraq with (89.04%) positivity rate for anti-GAD antibodies, and other countries: Qatar (59.7%), Sudan (77.5%), USA (73.2%), Bandung of Indonesia (78.7%), and Tunisia (84.6%) [19–24]. On the other hand, lower percentages were reported in India at (25%), and in Bosnia at (35.8%), and a multicenter cross-sectional study performed on 276 patients with T1DM showed anti-GAD65 autoantibody positivity in 37.31% of individuals [25–27].
A global scoping review showed that the weighted mean prevalence of GAD65 antibody in patients with new onset of T1DM was highest in Latin America and lowest in Africa [28]. This variability may be attributed to several factors, such as differences in sample populations, genetic differences among individuals, ethnic diversity, and the influence of regional cultural practices and habits. These elements contribute to the variability observed across different studies.
Several studies on the age distribution of anti-GAD IgG have shown that the prevalence of anti-GAD was 78.3% in patients with a disease duration of less than five years, with a decline after 12 years of illness due to the steady decrease of islet cell autoantibodies over age progression [17].
Our study found a significant correlation between positive GAD65 autoantibody and age, consistent with Amina Elkadhi et al. [24], Zecevic-Pasic [27], Farhan et al. [29], and Almeida et al. [30]. While this study showed a prominent gradual reduction in this antibody with the advancement of age, Farhan et al. [29] reported a stronger association of pathological values with age. However, there was a continuous increase in anti-GAD with age. The correlation between age and anti-GAD sero-positivity and negativity in the control study by Faisal et al. [20], and the uncontrolled study by Khan et al. [31] was not significant. The continued increase in anti-GAD may be attributed to several factors, including the population sample characteristics, immune system maturity, organ development, and improved adaptation to the disease. Additionally, increased awareness and education regarding T1DM, its management, associated risk factors, and potential complications may also contribute to these differences. Enhanced understanding of treatment strategies and preventive measures can play a role in improved disease management outcomes.
Our findings showed no significant variation in the distribution of T1DM and anti-GAD65 positivity between the sexes. This aligns with results from a study that was conducted in Bosnia and Herzegovina, which also reported no significant gender-based variation in anti-GAD65 levels [27]. Another study showed no significant difference in GAD-autoantibody IgG levels between male and female T1DM patients, though there was a slight tendency toward higher values in females [17]. While the control study by Faisal et al. showed a slight tendency of higher anti-GAD positive values in males than females, that was not significant [20]. There was no significant correlation between gender and anti-GAD positivity in the studies by Khan et al. [31], and Almeida et al. [30]. Boys diagnosed before the age of 10 are less likely to have GAD65 antibodies, whereas both boys and girls demonstrate diagnostic sensitivity of 80% in older children, teenagers, and young adults [32]. The difference in prevalence between genders may be related to the lack of significant hormonal changes before puberty, as hormonal fluctuations play a critical role in disease progression. The absence of these fluctuations before puberty may result in similar prevalence rates between males and females.
Alam et al. [26], Zecevic-Pasic et al. [27], and Awchi et al. found no significant correlation between HbA1c levels and gender, consistent with the findings of this study [14]. The measurement of HbA1c levels is an important biochemical test widely used for screening and monitoring diabetes mellitus [14, 33]. All individuals in this study had elevated HbA1c levels above the normal range. However, there was no significant difference between HbA1c levels between patients positive for anti-GAD antibodies and those negative for anti-GAD antibodies, consistent with studies conducted in India, Iraq, and Indonesia [20, 26]. Comparably, studies from Saudi Arabia and Iraq demonstrated a significant correlation between HbA1c and increased levels of glutamic acid decarboxylase antibodies (GADA) [14, 29], with Saudi Arabia reporting higher autoantibody levels in patients with HbA1c above 7.2% [29]. The variability in these results could be attributed to several factors, including technical and laboratory errors, and the limitations of current assays to measure GADA, which may reduce the accuracy of antibody detection.
Limitations of the study
The study focused exclusively on anti-GAD65 antibodies, without assessing other significant islet autoantibodies such as anti-IA-2, anti-insulin, and zinc transporter autoantibodies, which could provide a more comprehensive evaluation of autoimmune markers. Additionally, the limited sample size and data collection from a single center may restrict the generalizability of the findings to other regions or populations with differing genetic and environmental backgrounds. The study also did not collect data on environmental factors such as diet, viral infections, vitamin D levels, or genetic susceptibility (e.g., HLA typing), which are known to influence autoantibody development and disease onset. Moreover, the absence of follow-up data limits the ability to assess the impact of autoantibody status on disease progression, glycemic control, or treatment response over time.
Conclusions
The study found that autoantibody positivity was prevalent across all age groups, with the highest percentage observed in younger children. These results suggest that further study is required, taking into consideration other antibodies that are indicative of T1DM. Sex and changes in HbA1c did not significantly influence the levels of antibodies against GAD65, indicating that these two variables are unlikely to be risk factors.
