Autism Spectrum Diseases (ASD) were neurodevelopmental diseases characterized by abnormalities in communicative and non-communicative abilities, diminished social interaction and restricted interests with stereotypies [1]. Despite an interaction between genetic, neurological and environmental factors is widely accepted as a disease-causing factor, the pathogenesis and etiology remain unknown [2]. Three to eight times more males than females are diagnosed with ASD [3,4]. Young brains are significantly more actively metabolizing than adult brains and account for roughly 60 percent of the body's total energy consumption [5]. Neurological t is dependent on early healthful nutrition [6,7]. Deficiencies in proteins, fatty acids, different types of minerals and vitamins (which include vitamins A, D, B6, B9 and B12) may impair brain function during critical periods due to the role these nutrients serve in signaling cascades that alter neuronal abilities to function [8]. The relationship between vitamin D and autism arose as evidence of a higher prevalence of autism in children residing in areas with less Ultraviolet-B radiation than in sunny areas grew [9]. Over the past 20 years, there has been a steady rise in the prevalence of ASD. ASD was projected to affect 1 in 150 children in 2000 by the Autism and Developmental Disabilities Monitoring Network of the Centers for Disease Control and Prevention. The consistent increase in the prevalence of ASD may also be attributed to higher diagnostic standards, more precise behavioral and neuropsychological scales to evaluate the behavior and symptoms of ASD and more thorough ASD screening in both children and adults [10]. Genetic susceptibility in the form of multiple genes leads to many biological disorders (nature) in favor of the negative neurological influence of many environmental hazards [11]. Dietary vitamin D is a hormone that produces steroids. Vitamin D is synthesized by the epidermis in response to exposure to sunlight and is hence the principal source of vitamin D in the human body. Vitamin D is necessary for normal brain and nervous system development and for antioxidant responses to take place. Cerebro-spinal fluids contain vitamin D and enzymes necessary for vitamin D production in the brain [12]. In addition, exposure to sunlight, skin pigmentations, time of year of birth and site latitude are all possible causes of low vitamin D levels, which have been linked to mental problems [13]. Understanding the role of genes in determining autism risk is an intriguing area of inquiry. An increased risk for autism has been linked to variations in genes involved in vitamin D's metabolism, catabolism, transport and coupling [14]. Individuals acquire vitamin D3 primarily via endogenous synthesis in the epidermis, which begins with exposure to ultraviolet B (UVB) irradiance (280-315 nanometers) and constitutes up to 80-90 percent of vitamin D3 in European [15]. Nutrition and supplements provide lesser quantities of vitamin D3 in comparison. Two hydroxylation pathways in the liver and kidneys metabolize vitamin D to 1,25(OH)D, which is the active form [16]. Multiple aspects of brain growth, including the early development of cognition, are governed by the neuroactive hormone calcitriol [17]. Vitamin D2 (ergocalciferol) originates from invertebrates including plants, mushrooms and yeast, whereas vitamin D3 (cholecalciferol) is produced from animal sources including meat, milk, fish and eggs [18]. Vitamin D2 differs structurally from vitamin D3 due to the presence of an additional double bond and methyl group [19]. Ultraviolet radiation converts 7-dehydro-cholesterols (7-DHC) to the pre-vitamins D3, which undergo thermal isomerization to vitamin D3 within two to three days of initial solar exposure [20]. Moreover, both vitamin D2 and D3 have a short half-life of 10-20 hours after ingestion because of how they are absorbed in the small intestine and transported to the liver. The predominant circulating metabolite, having a half-life of 1 to 2 weeks, is vitamin D binding protein (DBP). 1,25 (OH) 2D coupled to DBP is rapidly transported to the target tissues which may have effects both of transcriptional as well as non-transcriptional. Vitamin D's influence on gene transcription [21]. In this study, it was aimed to determine the association of polymorphism in the Vitamin D3 and receptor gene TaqI (rs731236) in Iraqi children with autism spectrum disorder and Study the effect of polymorphism for Vitamin D3 on their serum levels in Baghdad, Iraq.

Study Design

This research was a CASE-CONTROL study during the period 1st of November 2022 and 31st of April 2023 from the laboratories in welfare children teaching hospital/medical city/Baghdad. School-aged (4-12 years old) children diagnosed with autism spectrum disorders were the target of this study. Because of the ethical issues related to, control and organize the contact with the kid’s population which forbid blood collection from children directly for research purposes. Samples were grouped as targets and controls according to a collection of criteria.

Inclusion Criteria

Children with ages of 4 to 12 years, confirmed diagnosed with ASD by a psychiatrist, having a record in the autism center, not diagnosed with genetic or metabolic disorders.

Exclusion Criteria

Individuals with genetic, metabolic, or cancer diagnoses, in addition to individuals with no autism history or symptoms.

Blood samples were collected from (50) autistic patients in the autism center in the mentioned hospital and (50) age-matched non-autistic normal developed children outpatient visitors in the same hospital.

The history of each patient was obtained from the records in the autism center. Blood and serum samples had been stored at -20°C after being divided and labeled.

Methods

To achieve the study objectives, three main techniques had been used which are:

• Cobas E411 was used to determine vitamin D3 target concentrations in the serum of both groups of samples using a single kit

• Real-time PCR, by utilizing particular primers and probes targeting SNPS in the TaqI gene to estimate the frequency of the gene polymorphism among both sample and control groups

• Statistical analysis, all findings with a significant level (p≤0.05) were analyzed using Chi-Square

Patient Age Groups and Gender

The current study was conducted on 100 blood samples collected from autism spectrum disorder (n = 50) and healthy control group (n = 50). 

The sex distribution among both study groups was around 70% for boys in contrast to about 30 % for girls between the age of 4-12 years old. As it is shown in Table 1.

Estimation of Serum Vitamin D3

Table 2 shows a slightly highly statistically significant elevation of serum vitamin D3 among control group samples (mean = 21.64 ng/ml) ranging from 6.98 ng\ml to 79.4 ng\ml in contrast to ASD patient’s serum samples (mean = 16.73 ng/ml) ranging from 5.1 ng\ml to 46.01 ng\ml. The results show statistically significant elevated serum vitamin D3 in the control group in contrast to the ASD group at p<0.04.

The etiology of ASD may be significantly influenced by vitamin D. The growth and operation of the brain are thought to be impacted by vitamin D, a neuroactive steroid. It plays a crucial part in myelination, which is crucial for brain connectivity. Reduced vitamin D levels in patients, reduced vitamin D levels in pregnant women and reduced sun exposure have all been linked to an increased risk of ASD, according to studies.

Additionally, supplementing with vitamin D may help with symptoms of autism and overall functioning. When compared to children without ASD, children with ASD were found to have vitamin inadequacies [22]. ASD symptoms may significantly improve with mineral and vitamin supplementation according to some researchers [23].

According to research, the participants' CARS scores. After 15 weeks, the vitamin D-treated kids' CARS total scores dropped substantially more than those in the placebo group [24]. Furthermore, supplementing with vitamin D dramatically reduced the signs of almost all neurodevelopmental abnormalities that children displayed. According to these studies, vitamin D supplementation may help with ASD symptoms. However, serum IL-6 or serotonin levels in kids with ASD were not statistically significantly affected by vitamin D. These results agreed with research achieved by the research were showed that Serum levels of vitamin D3 has been lower in ASD children compared to the healthy control group [25]. Additionally, these results disagreed with a study achieved by research in Turkey, which showed that the concentration of vitamin D3 in ASD patients’ (79.3±25.8 ng/ml) was significantly higher than in the Control healthy group (65.2±23.8 ng/ml) [26].

Table 1: Study Population Characteristics

Table 2: Statistical Evaluation of Serum Vitamin D3 in ASD and Control Groups

*: statistical significance (p≤0.05)

TaqI Gene Polymorphism (rs731236) Frequencies in ASD Patients and Control Groups

Table 3 showed the distribution of each examined allele and genotype in both ASD patient and control groups.

The result of the current study showed a significant difference in the frequency of rs731236 SNP toward mutant type in ASD individuals in comparison to a control group. The homozygous wild genotype (TT) showed more prevalence among the control healthy group (53.0%) in compare to (32.0%) among ASD patients. The genotype of rs731236 showed a high prevalence of heterozygous mutant type TC/CT (53.0%) in the ASD group than individuals in the control group (37.0%). Moreover, the genotype of rs731236 showed a significantly higher prevalence of homozygous mutant type CC (15.0%) in ASD children than the control group (9.0%). The present study revealed a correlation between the presence of the T allele at location rs731236 of the TaqI gene (p≤0.05) and a lower incidence of ASD. Also C alleles at location rs731236 in the TaqI gene along with an increased incidence of autism spectrum disorder. The result in the current study agreed with the study achieved by researchers, who demonstrated the correlation between TaqI, FokI and BsmI polymorphisms and ASD [27,28]. Other studies showed that there was a significant association of rs731236 (TaqI) polymorphism with ASD which showed that the C allele of the rs731236 gene might be a risk factor for autism [29]. These results agreed with a study achieved by research, which showed that the genotype of rs731236 of the TaqI TT, TC and CC in ASD patients was (44.7%, 37.5% and 17.7%) respectively [26]. Whereas in the control group was (46.5%, 44. % and 9,0%) respectively. These results disagreed with the result in the current study only in TC (‘Tt’) which showed that higher than TT (‘TT’). Another study in Iran, reported that the alleles and genotypes distribution of TT (p≤0. 045) and tt (p≤0. 013) genotypes between ASD and healthy control groups showed a significant difference. The results provide early evidence that genetic alterations in the VDR (TaqI) gene may alter children's susceptibility to ASD [30].

The Relation of Serum Serotonin and the HTR2A rs 6313 Prevalence in ASD Patients

As it is shown in Table 4 there was a significant association between lower vitamin D3 levels and ASD patients with mutant allele (C) of TaqI rs731236 in both homozygous and heterozygous mutant genotypes.

The result in the present study showed that lower serum levels of vitamin D3 in CC (‘tt’) and TC (‘Tt’) genotypes compared with the TT (‘TT’) in both ASD and control groups. 

Additionally, the result in the present study showed that the prevalence of the TC (‘Tt’) genotype was significantly higher than TT (‘TT’) and CC (‘tt’) genotype in ASD patients compared with the control group. The result in the current study agreed with the study achieved by research in Iraq, the incidence of the CC genotype and the C allele at VDR TaqI rs731236 was significantly higher in patients than in controls (15.6% vs. 1.8%, p≤0.001) and (34.6% vs. 23.2%, p≤0.001), respectively [25]. In addition, vitamin D and VDR levels were significantly lower in ASD patients than in controls. These results agreed with a study achieved by research, who showed that the genotype of rs731236 of the percent TaqI TT, TC and CC in ASD patients was (44.7%, 37.5% and 17.7%) respectively and serum levels of vitamin d3 was (75.59±26.85, 21.56±3.94, 26.65±8.43) [26]. Whereas in the control group was (46.5%, 44. % and 9,0%) respectively. These results disagreed with the result in the current study only in TC (‘Tt’) which showed that the percentage of the TC genotype (53.0%) higher than TT genotype (32%). Moreover, the result in the current study showed that the presence of at least a T (‘T’) allele was associated with lower serum levels of vitamin D3 because TaqI is proximal to the 3'UTR region of exon-9. The UTR region regulates VDR mRNA stability and posttranscriptional processes, which supports the role of TaqI polymorphism in modifying protein structure and vitamin D binding specificity [31]. Further research is required to clarify the precise function of vitamin D metabolism in autism spectrum disorder (ASD) due to inconsistencies in study results that may have been caused by population heterogeneity, geographic and ethnic diversity [32].

Table 3: TaqI rs731236 SNP Distribution Among ASD and Control Groups

*: Statistical Significance (p≤0.05), **: High Statistical Significance (p≤0.001)

Table 4: The Association of the Prevalence of TaqI rs731236 SNPs with the Serum Level of Vitamin D3

*: Statistical Significance (p≤0.05)

• Slightly highly statistically significant elevation of serum vitamin D3 in the control group in comparison to ASD patients

• The genotype of rs731236 showed a high prevalence of heterozygous mutant type TC/CT (53.0%) in the ASD group than individuals in the control group (37.0%). Moreover, the genotype of rs731236 showed a significantly higher prevalence of homozygous mutant type CC (15.0%) in ASD children than the control group (9.0%)

• There was a significant association between lower vitamin D3 levels and ASD patients with mutant allele (C) of TaqI rs731236 in both homozygous and heterozygous mutant genotypes

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