INTRODUCTION

Early childhood neurodevelopmental disorder known as autism spectrum disorder (ASD) is characterized by a shared spectrum of social interaction, qualitative impairments, and communication deficits in varying degrees. It is also characterized by noticeably restricted interests and repetitive behaviors [1]. The disease starts developing about the time a child turns three and persists throughout life.  There is growing evidence and concern that abnormal immune responses contribute significantly to the pathophysiology and pathogenesis of ASD [2]. The pathogenesis of ASD is still unknown, and there are no definitive biochemical or clinical indicators for identifying ASD, despite significant research efforts over the last several years. Numerous studies have examined proteome patterns in the peripheral blood of ASD patients in search of possible biomarkers [3]. Numerous factors are thought to be involved in the development of ASD, including genetic, infectious, neurological, metabolic, environmental, and immunological factors [4]. The neurological cause of ASD is still unknown, and further research is needed to understand the significance of immunological abnormalities and autoimmune diseases.

The relationships between the immunological and nervous systems are well understood and have been the subject of much investigation. Numerous studies have shown that autism suffers from immune system abnormalities, including irregular synthesis of cytokines, immune cells, and antibodies [5]. Numerous neuroactive substances have also been shown to have potent immunomodulatory effects and to be crucial in the development of illness [6, 7].

By detecting markers suggesting autoimmune, as well as cytokines and other biomarkers that signal pro- and inflammatory states, many studies have connected autism to autoimmunity. Additionally, they found a correlation between illness severity and blood levels of autoantibodies and pro-inflammatory indicators in certain Saudi autistic individuals [8, 9].

AIRE-p, a transcription factor found on chromosome 21, promotes autoimmunity by controlling the expression of promiscuous genes (pGE) [10]. By aiding the negative selection of T cells in the thymus, creating the thymic microarchitecture, and generating a particular fraction of regulatory T cells, AIRE-p plays a crucial part in modeling central immunological tolerance [11]. Additionally, AIRE-p inhibits autoimmunity by encouraging the development of self-antigens in medullary thymic epithelial cells, causing developing T cells that identify these self-antigens to undergo clonal deletion in the thymus [12]. However, since the AIRE-p gene causes severe organ-specific autoimmune diseases [13], it is one of the finest choices to explain the complicated picture of autoimmunity [14].

AIRE-p has a significant impact in the expression of a number of tissue-restricted Ags (TRAs) in medullary thymic epithelial cells (mTEC), according to a research using a mouse model [15]. The negative selection process, in which self-reactive thymocytes with enhanced affinity for TRAs are eliminated before they enter peripheral blood circulation, depends on the expression of TRAs. Therefore, any alteration in AIRE-p levels may have complicated repercussions on thymic selection. It could result in a rise in the prevalence of autoimmune symptoms [16].

Previous research suggested that other elements interact with AIRE-p and support its activity. Abramson et al.'s investigation discovered two AIRE-p complexes that controlled the pre-mRNA processing of tissue-restricted antigens (TRAs) and one complex that was based on DNA-dependent PDNA-dependent protein Kinase (DNAPK), which seemed to increase TRA transcription [17, 18]. Another work revealed a cascade regulatory mechanism for these proteins by demonstrating how AIRE-p functions via the gene node Gucy2d, which is connected to the TRAs in medullary thymic epithelial cells (mTECs) [19]. In contrast, a research found that the transactivation of AIRE-p is inhibited by the interaction between AIRE-p and the DAXX protein [20]. As a result, interactions with other variables may be necessary for AIRE-p to transactivate.

Numerous autoimmune disorders and neurological illnesses have been said to be primarily caused by AIRE gene mutations and protein abnormalities [21]. In order to prevent autoimmunity, testosterone upregulates AIRE-p p-mediated thymic tolerance, according to Zhu et al. [22]. They discovered that men express more thymic AIRE-p than females, which suggests that the administration of androgens and male gender prevent autoimmunity in a multiple sclerosis mouse model in a way that is AIRE-dependent.

Additionally, it was thought that AIRE-p dysregulation, which reduces promiscuous gene expression (pGE), promotes autoimmunity in Down syndrome (DS) [23]. In thymus samples from DS patients and controls, the expression of AIRE-p and numerous peripheral tissue-specific Ag genes was evaluated. When DS individuals were compared to controls, it was shown that AIRE-p expression was drastically decreased by two times.

This study's goal was to investigate AIRE-p's potential function in ASD patients. Never before has the function of AIRE-p in the pathophysiology of autism been discussed. There hasn't yet been a single research that evaluated the AIRE-p levels in autistic people. We hypothesized that AIRE-p could play a pathogenic role in inflammation and autoimmune disorders given that autoimmunity has been shown to be one of the pathophysiological causes of autism and given its role in the transcription of a wide variety of self-antigens to start the negative selection process in the thymus. These details have motivated us to look for AIRE-p as a possible biomarker that might help identify ASD sooner.

MATERIALS AND METHODS

Participants

In the current study, 37 ASD patients with ages ranging from 2 to 12 (mean SD = 5.47 2.52 years) from the King Saud University in Riyadh, Saudi Arabia, as well as 37 age- and sex-matched healthy children from the pediatric clinic at the King Saud Medical City in Riyadh, Saudi Arabia, made up the control group. The Diagnostic and Statistical Manual of Mental Disorders (DSM-5) was used to make the diagnosis of ASD [24]. The research excluded participants who had epileptic seizures, obsessive-compulsive disorder, affective disorders, fragile X syndrome, or any other mental or neurological conditions. The current research was authorized by the IBR Committee of the King Khalid Hospital at King Saud University in Riyadh, Saudi Arabia. All registered individuals had their parents or legal guardians sign informed written permission on their behalf.

CARS, or the Childhood Autism Rating Scale

Autism severity was evaluated using CARS [25]. It assigns scores for 15 different symptoms or dimensions (such as activity level, object use, body use, imitation, verbal and nonverbal communication, emotional response, relation to others, response to listening, nervousness or fear, reliability and intellectual response level, adaptation to changes, visual responses, responses to touch, smell, and taste, and general impressions) to children ranging in age from one to four. Scores below 30 on this measure strongly suggest the presence of autism. Children with scores between 30 and 36.5 had mild to moderate autism, whereas those with values between 37 and 60 had severe autism [26].

SRS, or the Social Responsiveness Scale 

The SRS is a validated assessment of communication, interpersonal behavior, and stereotyped characteristics in autism. In order to separate clinically relevant ASD from other mental diseases with varied degrees of social impairment, it is employed as a diagnostic tool. There are five subscales that make up this scale: social awareness, social cognition, social communication, social motivation, and autistic mannerisms. Total SRS raw values vary from 0 to 195, with a score of 195 representing a profound social impairment as seen in ASD patients. A clinical diagnosis of autism is highly correlated with a score of 76 or above, which is considered severe. The mild-to-moderate range of social impairment is defined as a score between 60 and 75 [27].

Automated Battery for the Cambridge Neuropsychological Test (CANTAB)

The Cambridge Neuropsychological Test Automated Battery (CANTAB) is a touch-screen computer-based cognitive assessment system that includes a battery of neuropsychological tests that are given to individuals. Among the 25 tests in CANTAB, general memory and learning, working memory and executive function, visual memory, attention and reaction time (RT), semantic/verbal memory, decision-making, and response control are some of the cognitive functions that are tested.

Five subtest models—the Big/Little Circle (BLC), Intra/Extra Dimensional Set Shift (IED), Simple Reaction Time (SRT), Spatial Recognition Memory (SRM), and Motor Screening (MOT)—were used to evaluate the participants' performance on attention and memory tasks. Numerous investigations in the area of autism have employed CANTAB [28].

Collection of Blood Samples

After an overnight fast, blood was taken from 37 children with ASD and 37 similarly aged healthy children. For blood collection, blood was drawn into 3 mL EDTA-containing tubes. Following blood collection, the samples were centrifuged for 20 min. at 4 °C and 3000 g. The plasma was held at 80 °C prior to analysis. A commercial sandwich ELISA kit (Cusabio Biotech Co. Ltd., Wuhan, China) was used to assess the levels of AIRE-p in the plasma of autistic participants in accordance with the manufacturer's recommendations. The mean results of each biochemical analysis were reported after being carried out in triplicate. There was no discernible interference or cross-reactivity.

Analytical Statistics

Software from SPSS Inc., Chicago, Illinois, USA, called Statistical Package for Social Sciences, version 21, was used to analyze the data. Normality The Kolmogorov-Smirnov test was used to determine if the findings were normal. The Mann-Whitney test was employed to compare the non-parametric data. The median and interquartile range (IQR; 25th to 75th percentile) of the results were shown. The independent- samples t-test was used to the normally distributed data to compare the means between the control group and the autistic group. The result was described using descriptive statistics (mean and standard deviation). The Pearson correlation coefficient (r) was used to ascertain the relationship between the various variables (CARS, SRS, and CATAB). In order to explain any relationships between the biomarkers and clinical events by computing odds ratios (OR), multivariate logistic regression analysis of the biomarkers is utilized. A p-value of 0.05 or below was used to determine statistical significance for all tests.

Additionally, a Receiver Operating Characteristics (ROC) curve analysis for the assessment of biomarkers was performed. The area under the curve (AUC), cutoff values, and degree of specificity and sensitivity were all calculated. Predictiveness curves were also used to explain how the scores were distributed.

RESULTS AND DISCUSSION

Table 1 summarizes the overall characteristics of the research subjects, plasma levels of AIRE-p in autistic (n=37) and normal children (n=37), as well as CARS and SARS scores.  The results are shown as the median and IQR. AIRE-p levels were examined between autistic kids with various degrees of autism (mild, moderate, or severe), as well as controls. The recorded CARS scores of the ASD participants, which varied from 30 to 65, were used to categorize them. Children with autism's social traits were also noted by their SRS score.

The plasma level of AIRE-p in autistic children (n = 37) was considerably lower (p= 0.003) than that of normal children (1.144 (0.938) pg/ml).

AIRE-p and Childhood Autism Rating Scale (CARS)

The results of the childhood autism rating scale are shown in Table 1. Thirty-one individuals (83.80%) had mild to moderate autism according to the CARS scoring system, while six people (16.20%) had severe autism according to the mean (40.42 3.65) scores. According to the CARS score, there was no discernible difference between the plasma AIRE-p levels of severe autism (0.365 (1.114) pg/ml) and mild to moderate autism (0.661 (0.666) pg/ml).

The AIRE-p and the Social Responsiveness Scale (SRS)

Table 1 shows the findings of the social responsiveness scale. Using the SRS scoring system, it was determined that 10 individuals (41.70%) had mild to moderate impairment in social skills (mean: 71.30 2.11), and 14 subjects (58.30%) had severe impairment (mean: 78.50 3.11). Due to an age issue, 13 individuals with autism did not match the requirements for using the SRS. Based on the SRS score, there was no discernible difference in plasma AIRE p concentration between severe autism (0.639 (0.690) pg/ml) and mild to moderate autism (0.585 (0.995) pg/ml).

AIRE-p and the Automated Cambridge Neuropsychological Test Battery (CANTAB)

In this research, memory, attention, speed of reaction, and visual discrimination patterns were the five cognitive tasks examined. Median (IQR) values were used to display the results. AIRE-p levels and CANTAB cognitive tests did not significantly correlate in the ASD group compared to the control group. AIRE-p levels and The IED te 1 of CANTAB cognitive tests were found in the group of people with mild to moderate ASD, and they showed a significant positive association according to the CARS scoring system (CC=0.401, P = 0.028) (Table 2).

“AIRE-p levels are not shown in the table but were taken into consideration.

*     Correlation is significant at the 0.05 level.

Additionally, in the mild to moderate subgroup, a significant negative connection between AIRE-p levels and the SRT (mean) (CC=-0.730, P= 0.017) and the SRT (max) (CC=-0.644, P= 0.044) of CANTAB cognitive tests was discovered. This was by the ASD social character measured by the SRS social scale.

Additionally, a connection between the MOT test and ASD severe subgroups was positive (CC= 0.631, P= 0.015) (Table 3).

“AIRE-p levels are not shown in the table but were taken into consideration.

^*Correlation is significant at the 0.05 level.