Allwsh TA, Kucher DS. Role of soluble alpha-klotho as a novel biomarker for characterizing children with autism spectrum disorder in Kurdistan, Iraq. World J Clin Pediatr 2026; 15(2): 112164 [DOI: 10.5409/wjcp.v15.i2.112164]
Corresponding Author of This Article
Thikra A Allwsh, PhD, Professor, Department of Chemistry, University of Mosul, Al-Zahour, Mosul 41003, Nineveh, Iraq. thekraaliallwsh@uomosul.edu.iq
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Biochemistry & Molecular Biology
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Case Control Study
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Jun 9, 2026 (publication date) through May 16, 2026
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World Journal of Clinical Pediatrics
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2219-2808
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Allwsh TA, Kucher DS. Role of soluble alpha-klotho as a novel biomarker for characterizing children with autism spectrum disorder in Kurdistan, Iraq. World J Clin Pediatr 2026; 15(2): 112164 [DOI: 10.5409/wjcp.v15.i2.112164]
Author contributions: Allwsh TA contributed to the study design and data interpretation, and wrote the initial draft of the article; Kucher DS performed the data collection (by conducting a clinical study) and statistical analyses; All authors contributed to revisions to the final manuscript and approved the final version to be published.
Institutional review board statement: This study was approved by the Scientific Research Committee, College of Science, University of Mosul, Iraq (No. 28082024-7-1).
Informed consent statement: All patients gave informed consent.
Conflict-of-interest statement: The authors have no conflicts of interest to declare.
STROBE statement: The authors have read the STROBE Statement-checklist of items, and the manuscript was prepared and revised according to the STROBE Statement-checklist of items.
Corresponding author: Thikra A Allwsh, PhD, Professor, Department of Chemistry, University of Mosul, Al-Zahour, Mosul 41003, Nineveh, Iraq. thekraaliallwsh@uomosul.edu.iq
Received: July 21, 2025 Revised: August 16, 2025 Accepted: November 25, 2025 Published online: June 9, 2026 Processing time: 298 Days and 11 Hours
Abstract
BACKGROUND
The identification of bioindicators for the detection and monitoring of autism spectrum disorder (ASD) still remains a major challenge in clinical medicine. The protein klotho has been linked to various neurological disorders.
AIM
To investigate the evaluation of soluble alpha-klotho (S-KLα) as a new indicator for the diagnosis and monitoring of patients with ASD. This study was conducted in the absence of any prior studies or research on the link between klotho and ASD, nor on how it affects the characteristics of those impacted. To address this gap, we considered this study.
METHODS
The case-control study involved 256 individuals of both sexes, aged between 2 years and 15 years, divided into two groups: 156 children with ASD from autism centers in Dohuk and Zakho cities, Kurdistan Region/Iraq, and 100 healthy individuals serving as the control group. Serum S-KLα level was measured using enzyme-linked immunosorbent assay. Additionally, levels of hemoglobin, iron, glucose, uric acid, creatinine, and vitamin D3 were estimated, with all measurements conducted in duplicate. Afterwards, statistical analyses were performed. An additional component was a questionnaire containing information about the participants.
RESULTS
The results showed a significant decrease in S-KLα levels in the sera of patients with ASD (97.5 ± 19.6) compared to the control group (133.7 ± 32.4). Additionally, the area under the curve (0.91) from the receiver operating characteristic analysis confirms the potential of using S-KLα as a marker for ASD. Furthermore, a notable decline in S-KLα was observed with increasing age, and higher levels of S-KLα were found in girls compared to boys, in both the control and patient groups (P < 0.001). The S-KLα levels in patients with ASD were not influenced by family history or birth weight. However, a significant reduction was seen in patients who experienced a difficult birth (dystocia), as well as in preterm births (< 37 weeks) and post-term births (> 42 weeks). It was observed that there was a significantly negative correlation between S-KLα and glucose level. By contrast, positive correlations were found with hemoglobin, iron, and vitamin D3 (P < 0.01); however, no relationship was detected with creatinine and uric acid levels in patients with ASD.
CONCLUSION
This is the first case-control study to confirm the strong potential of serum S-KLα as a predictive biomarker in autism risk profiling. The proposals outlined by the study suggest new directions and strategies for future research, linking klotho as a potential diagnostic marker for ASD and its complications, as well as its possible role as a therapeutic target.
Core Tip: This is the first study to examine the relationship between soluble alpha-klotho (S-KLα) and autism spectrum disorder (ASD). It involves two groups aged 2-15 years: One healthy group and another comprising patients from autism centers in the Kurdistan Region, Iraq. We compared these groups and analyzed risk factors associated with ASD. The results showed a significant reduction in S-KLα levels in the sera of patients with ASD. S-KLα levels were affected by several risk factors and demonstrated a significant correlation with multiple clinical markers, suggesting a role for S-KLα in ASD and as a predictive biomarker in ASD risk assessment.
Citation: Allwsh TA, Kucher DS. Role of soluble alpha-klotho as a novel biomarker for characterizing children with autism spectrum disorder in Kurdistan, Iraq. World J Clin Pediatr 2026; 15(2): 112164
Autism spectrum disorder (ASD) is a neurodevelopmental condition that influences communication, social interaction, and behavior. It usually starts in childhood and has lifelong effects, with individuals displaying repetitive actions and facing challenges in learning, employment, and social situations. While some people with ASD can live quite independently, others may require ongoing specialized support due to learning difficulties. ASD is often co-occurring with other conditions such as anxiety, depression, and attention deficit hyperactivity disorder[1,2]. However, the causes of ASD are still being studied and may involve complex interactions[3]. Many demographic factors influence ASD, highlighting the complexity of socioeconomic, environmental, and genetic influences in the development of ASD[4].
The social, health, and cultural background of parents affects the development of ASD, especially the mother’s health and diet. Educating parents about risk factors is essential in preventing children from developing the condition[5]. There is limited information about ASD risk factors[6]. In 1997, Japanese scientists discovered a new protein after identifying a gene-disrupted mouse model that showed symptoms such as a short lifespan, infertility, atherosclerosis, skin mottling, osteoporosis, and emphysema. This protein was identified as a senescence suppressor and named klotho. Klotho exists in three subfamilies: Alpha-klotho (KLα), KLb, and KLg, with KLα having two distinct forms (a membrane-bound form, m klotho, and a soluble form, s klotho)[7]. It was later found to be a product of the Klotho gene, a key regulator of various physiological processes, including aging, phosphate metabolism, and vascular health[7,8].
Although the KLα protein plays a crucial role in kidney health, it also influences brain activities and mechanisms. When present in higher amounts in the body, it helps protect the brain in various disease conditions like seizures, strokes, Alzheimer’s, amyotrophic lateral sclerosis, multiple sclerosis, and Parkinson’s disease[9].
No research has been conducted on the association of KLα protein with ASD or its impact on the demographic and clinical characteristics, mental and learning abilities of children with ASD. To address this gap, this study explored a new perspective on KLα by using its level as a marker for diagnosing ASD. This approach could enhance our understanding of the underlying pathophysiology, help identify individuals at higher risk, and examine its relationship with the demographic and clinical features of patients.
MATERIALS AND METHODS
Study design
This study was designed as a case-control study, including children with ASD who visited the autism centers in Kurdistan (Dohuk and Zakho cities)/Iraq, from October 2024 to February 2025. The diagnoses of the cases were confirmed by specialist doctors, and a second group of healthy children without any clinical symptoms was also included. The study protocol was approved by the Dohuk Directorate of Health and the Scientific Committee for Postgraduate Student Research/Biochemistry and Medical Specialities at the University of Mosul (Nineveh, Iraq).
Study population
The study includes 256 children aged between 2 years and 15 years, divided into two groups: Children with ASD (128 males and 28 females). The control group involved 100 healthy children (55 males and 45 females). The clinical diagnosis of ASD is based on behavioral assessments as outlined in the American Psychiatric Association’s Diagnostic and Statistical Manual of Mental Disorders criteria[10]. A verified survey was submitted for the enrolled subjects, which included their ages, sex, family history, medications, and information about birth and parents, for all children and their families.
Inclusion criteria
In this study, children under 2 years of age, children with diabetes, genetic diseases, intellectual disabilities, or any inflammatory condition were considered as exclusion criteria. The study adhered to patient privacy and confidentiality regulations throughout the research process. Strict security protocols were used to safeguard the personal information of research participants.
Collection of blood samples
Five milliliters of venous blood were collected from the participants in the study. The blood was divided into two types of tubes: One for measuring hemoglobin (HGB) and the other for preparing serum, which was then allowed to clot in clot activator gel tubes and separated by centrifuging at 3000 rpm for 10 minutes to collect the serum.
Laboratory parameter assessments
The practical work was conducted in the biochemistry laboratories of the Chemistry Department at the College of Science, University of Mosul, as well as in several private laboratories within Duhok city. The blood biomarker assessments in patients and control groups included: Human soluble KLα (S-KLα) was measured in serum using an enzyme-linked immunosorbent assay kit from Sunlong Company (HangZhou, China). HGB, iron, glucose, uric acid, and creatinine were measured using kits from Biolabo Company (Maizy, France). The level of vitamin D3 was determined using the mini VIDAS device, which operates on the enzyme-linked fluorescent assay principle. This involves using a specific kit from a French company (bioMérieux, Quebec, Canada).
Ethics review
The study complied with all relevant legal and ethical standards and requirements. Approval to include patients was obtained from the Dohuk Directorate of Health, Kurdistan/Iraq (Reference No. 28082024-7-1). The study’s ethics adhered to the Declaration of Helsinki, with informed verbal consent obtained after requesting permission from the children and their guardians to participate. All participants and their legal representatives received written information and signed a consent form to take part.
Statistical analyses
Descriptive and analytic statistics were performed using the SPSS software 2019 (SPSS Inc., Armonk, NY, United States). Descriptive statistics include mean ± SD and percentages for categorical variables. χ2 and t-tests were used to compare the quantitative parameters. Additionally, a two-way analysis of variance (ANOVA) and post-hoc testing (Tukey’s honestly significant difference [HSD]) were conducted to examine the main and subgroups effects of the group. A multiple logistic regression analysis was used to calculate odds ratios (ORs) for S-KLα in ASD, adjusting for various biochemical and demographic covariates. Statistical significance was determined using regression coefficients, standard errors, Z values, and P values, with 95% confidence intervals (95%CIs) indicating estimate precision. The predictive ability of S-KLα indicator was assessed using receiver operating characteristic (ROC) curve analysis and optimal cutoff points were determined by maximizing the Youden index (sensitivity + specificity - 1). A scatter plot and Pearson’s correlation coefficient (r) were calculated between S-KLα and parameters of the ASD group. P ≤ 0.05 was considered statistically significant.
RESULTS
Demographic features of patients with ASD
The patients in this study included 156 children with ASD from centers in Kurdistan/Iraq. They were divided into three different age groups, as shown in Table 1. The results indicate that the highest percentage of patients participating in this study was aged 6-10 years, comprising 54%. The 2-5 ages group made up 22% and the 11-15 ages group accounted for 24%. Additionally, the highest percentage of patients with ASD in this study were boys (82%), while 18% were girls. The percentage of children with a family history of the disorder is approximately 14%. In comparison, the percentage of children without a family history of autism is approximately 86%. The highest incidence of children with autism who experienced a challenging birth (dystocia) was 52%. We found that the incidence percentages based on gestational age (number of weeks) were 38%, 34%, and 28% among children born early (< 37 weeks), late (≥ 42 weeks), and within the normal range (37-42 weeks), respectively. The highest incidence among high-birth-weight children (> 4 kg), followed by low-birth-weight (< 2.5 kg) and normal-birth-weight (2.5-4 kg) were 36%, 33%, and 31%, respectively.
Table 1 Demographic features for control and autism spectrum disorder groups.
Assessment of S-KLa levels in control and patients with ASD
Two-way ANOVA results: ANOVA model included group (control and patients with ASD), sex, and their interaction term (group-sex) as independent variables, with S-KLα level as the dependent variable. Type II sums of squares were used. The results are shown in Figure 1 and Table 2.
HSD: Post-hoc tests are necessary to determine specific group differences. HSD test was applied for pairwise comparisons. The results are shown in Table 3. Two-way ANOVA demonstrated significant main effects for both group and sex, indicating that S-KLα levels differ significantly between control and patient groups [F(1, 145) = 449.68; P < 0.001], and between males and females [F(1, 145) = 771.46; P < 0.001]. Crucially, a significant interaction effect between group and sex [F(1, 145) = 35.99; P < 0.001] was also observed, indicating that the effect of group on S-KLα levels is dependent on sex, and vice versa. Post-hoc Tukey HSD tests further elucidated these differences, showing various significant pairwise comparisons across group-sex combinations.
To address the potential confounding effect of sex imbalance, a stratified analysis was performed, separating the data by sex (Table 4). The stratified analysis effectively addresses the concern that the group’s male predominance could solely drive lower S-KLα levels in patients. By analyzing males and females separately, we showed that the significant difference in S-KLα levels between control and patient groups holds true within both sexes. This indicates that the observed lower S-KLα levels in patients are not merely an artifact of sex imbalance but represent a genuine difference between the control and patients, regardless of sex.
Table 4 Sex bias adjustment and stratified analysis.
Table 5 presents the S-KLα serum levels in age groups ranging from 2 years to 15 years. They were divided into age groups of 2-5, 6-10, and 11-15 years. Additionally, it was observed that the concentration of S-KLα in the serum of both healthy and patient groups decreases with age, with a significant difference at P ≤ 0.001, reaching its lowest level in the third group. The average levels of Klotho in the control group were 133.7 ± 32.4. In contrast, its average in the patient group was significantly lower, reaching 97.5 ± 19.6. Statistical analysis showed that this difference was highly statistically significant (P ≤ 0.001). Additionally, when comparing the two groups based on similar age and sex, it is noted that the S-KLα level is lower in patients compared to healthy individuals. Additionally, when comparing the two groups based on similar age and sex, it is observed that the S-KLα level is lower in patients compared to healthy individuals.
Table 5 Levels of soluble alpha-klotho by age in control and autism spectrum disorder patient groups, mean ± SD.
The impact of some demographic characteristics on the level of S-KLα in patients with ASD
There is no significant difference in the level of S-KLα in the serum of children with ASD who have a family history compared to those without, indicating that S-KLα levels are not influenced by family history. However, there is a significant decrease in serum S-KLα levels in children who experienced a challenging birth (dystocia) compared to those with a normal, uncomplicated birth, at a level of P < 0.01, as shown in Table 6. The results also indicate a notable reduction in serum S-KLα levels among children with ASD born prematurely (< 37 weeks) and post-term (> 42 weeks), both at a probability level of P ≤ 0.01. Conversely, there was no significant difference in the level of S-KLα in children with ASD based on their birth weight, whether normal, low, or high. Assessment of clinical indicators in ASD.
Table 6 Effect of some autism spectrum disorder patients’ characteristics on the concentration of (soluble alpha-klotho), mean ± SD.
Table 7 shows that the serum glucose level was significantly higher in ASD at a P value of ≤ 0.01 compared to healthy individuals. It turns out serum glucose levels (127.63 ± 17.47 mg/dL) are close to the range associated with the pre-diabetic stage. A significant decrease in HGB levels was observed in people with ASD compared to healthy individuals at the probability level P ≤ 0.01. They also found a significant reduction in iron levels in patients with ASD compared to healthy people, with a P value of ≤ 0.01. Notably, the iron level in patients remained within the normal blood range. Additionally, uric acid levels were significantly decreased in patients with ASD at a P value of ≤ 0.01. However, no significant difference was observed in serum creatinine levels between patients with ASD and healthy individuals. The serum vitamin D3 level was significantly lower in ASD at a P value of ≤ 0.01 compared to healthy individuals. It appears that vitamin D3 levels (20.69 ± 13.87 ng/mL) were close to the range of moderate deficiency.
Table 7 Clinical parameters for the control and autism spectrum disorder groups, mean ± SD.
ROC analysis: ROC analysis was performed to evaluate the diagnostic accuracy of S-KLα in predicting ASD (Figure 2 and Table 8). The area under the curve (AUC), standard error, 95%CI, P value, optimal cutoff, sensitivity, specificity, and Youden index were calculated for each parameter. Based on the AUC (0.91, P < 0.0001 for S-KLα), S-KLα is considered an excellent marker for diagnosing ASD (AUC > 0.91) (Figure 2). Patients at risk for ASD could be identified with a sensitivity of 0.822 and a specificity of 0.943 when their serum S-KLα level was below 120 pg/mL, which is the recommended cutoff value, as shown in Table 8. These results support the strong potential of serum S-KLα as a predictive biomarker in ASD risk assessment. Other parameters such as vitamin D3, creatinine, glucose, uric acid, iron, HGB, and age were also analyzed, with glucose demonstrating the highest AUC (0.97). Age, despite being included, showed lower discriminative ability (AUC = 0.58).
Multiple regression analysis: Multiple linear regression analysis was performed to calculate crude and adjusted OR for the effect of S-KLα on ASD (Figure 3 and Table 9). The crude analysis for the protein S-KLα showed a strong protective effect against ASD (coefficient = -2.5825, OR = 0.076, 95%CI: 0.066-0.087, Z = -36.61, P < 0.001), indicating that higher S-KLα levels are associated with a significantly lower risk of ASD before adjusting for other variables. After adjustment for potential confounders including glucose, HGB, iron, uric acid, creatinine, vitamin D3, age, time of birth, and birth weight, S-KLα remained significantly protective (coefficient = -1.1061, OR = 0.331, 95%CI: 0.288-0.380, Z = -15.68, P < 0.001), suggesting that each unit increase in S-KLα reduces the risk of ASD by approximately 67%. Among the other adjusted variables, higher glucose was associated with increased risk (OR = 8.55), while HGB, iron, uric acid, and vitamin D3 were protective (OR < 1). Creatinine showed an increased risk (OR > 1). Age, time of birth, and birth weight were not statistically significant (P = 0.127, 0.012, 0.919, respectively).
Table 10 displays the correlation coefficient of S-KLα with clinical parameters in the ASD patient group, calculated using the Pearson’s coefficient. The results revealed a significantly negative correlation between S-KLα and glucose levels. Conversely, a significant positive correlation was observed between S-KLα and HGB, iron, and vitamin D3 (P < 0.01). No association was found between S-KLα and serum creatinine and uric acid levels in patients with ASD, also no significant association between S-KLα and age, time of birth and birth weight.
Table 10 Correlation between soluble alpha-klotho and parameters in autism spectrum disorder groups by using Pearson’s coefficient.
The results showed that the highest percentage of patients with ASD in this study were aged 6-10 years, accounting for 54%. This aligns with a report by the Centers for Disease Control and Prevention, which stated that most ASD diagnoses occur between the ages of 4 years and 8 years[11]. A second study by Chen et al[12] indicated that the peak age at diagnosis is between 5 years and 9 years, attributed to increased awareness and early detection programs. The findings also revealed a decline in the percentage of patients with ASD in the 2-year-old to 5-year-old age group, reaching 22%. This may be due to the challenges in diagnosing young children, often because there is no specific medical test. Instead, diagnosis depends on careful observation of the child’s behavior, social interactions, and skills. Consequently, early signs are detected in this age group, which can lead to underdiagnosis. Additionally, the results showed that the 11-15 ages group had a 24% representation; this lower incidence might be mainly because many children have already been diagnosed at younger ages[13].
We found that boys were more likely than girls to be diagnosed with ASD among the participants in this study, with rates reaching 82% and 18%, respectively, in autism centers in the cities of Duhok and Zakho in the Kurdistan Region of Iraq. Genovese and Butler[14] determined that for every four boys diagnosed with ASD, there was one girl. Mahboub et al[15] confirmed that the incidence of ASD is higher in boys than in girls. This is due to genetic factors, as autism is linked to several genes located on the X chromosome. Since girls have two X chromosomes, while boys have one X chromosome, boys are more susceptible to infection when damage occurs in the X chromosome, as they lack another X chromosome to compensate for the defect[16]. This is consistent with Gong et al[17], who reported that several mutations occurring on the X chromosome, including the RPL10 gene in the Xq28 region and the SHANK3 gene on chromosome 22 (q13.3), have been identified as contributing to autism.
The results showed that about 14% of children have a family history of ASD. Conversely, about 86% do not. However, not having a family history of ASD does not necessarily mean there is no risk, as ASD can occur due to spontaneous genetic mutations (de novo mutations)[18] or environmental factors such as exposure to pollutants, heavy metals, and chemicals during pregnancy, or complications during birth, which can contribute to the development of autism[19].
We observed a higher rate of ASD (53%) in children who experienced a challenging birth process (dystocia), making up more than half of the cases. This is due to various reasons, including that dystocia can cause a lack of oxygen (neonatal asphyxia), leading to brain damage and neurological issues that threaten neurons and increase the risk of ASD[20]. Birth stress (stress during birth) also triggers inflammatory and physiological changes that impact the child’s brain development[21]. Although dystocia (difficult birth) is not the sole cause of autism, it is linked to an increased risk, according to a study by Kaiser Permanente. Specific birth complications such as birth asphyxia and preeclampsia are strongly associated with ASD, while other factors like breech or transverse fetal positions have also been linked to fetal dystocia. Therefore, ensuring proper healthcare for both mother and fetus during pregnancy and delivery is essential to prevent these complications[22].
In addition, the timing of birth was observed to influence the incidence rate, with the proportions among children in the early birth (< 37 weeks), late birth (≥ 42 weeks), and normal birth (37-42 weeks) categories being 38%, 34%, and 28%, respectively. This suggests that both preterm and late births may be contributing factors. This aligns with Crump et al[23], who reported an association between preterm birth and an increased risk of ASD, noting that the earlier a child is born, the higher the risk. This is likely due to impaired brain development in preterm infants during the last 3 months of pregnancy. Jenabi et al[24] confirmed the association of late birth with a higher risk of ASD. This finding is consistent with a systematic review by Guo et al[25], which indicated that ASD occurred in 0.6% of children born after 42 weeks of pregnancy. Another study by Martini et al[26] also suggested that birth after 42 weeks of pregnancy is a risk factor for ASD. Delayed delivery causes perinatal problems and placental insufficiency, which predisposes to a lack of oxygen and nutrients, leading to abnormal fetal growth and neurological dysfunction associated with autism[24].
The highest incidence percentage was among high-birth-weight children (> 4 kg, 36%), followed by low-birth-weight (< 2.5 kg, 33%), and then normal-birth-weight (2.5-4 kg, 31%). This indicates that a child’s weight influences the occurrence of the disease, and children with either high or low birth weight are more prone to developing ASD. This aligns with Brumbaugh et al[27], where both very low and very high birth weights are linked to increased rates of ASD. The elevated risk is also believed to be connected to whether the child was born prematurely. A uterus that is too small or too large may be associated with fetal growth issues, which could lead to brain cell alterations or neurodevelopmental challenges. It may also be related to environmental factors, such as exposure to pollutants, toxins, and dietary influences, as well as maternal diabetes and obesity, all of which can contribute to fetal birth weight disorders; these factors may raise the risk of ASD[28]. Because of the lack of research connecting S-KLα levels to ASD, we sought to address this gap. This was our study, and our conclusion was based on research similar to it.
The study by Prud'homme et al[29] showed that serum KLα levels decrease with age in both children and adults, with this decline observed in healthy individuals as well as those with age-related changes and disorders. Additionally, low KLα levels are linked to age-related changes, including cognitive decline[30]. For the first time, Espuch-Oliver et al[31] provided reference values for KLα in healthy individuals aged 18 years to 85 years, discovering that age was negatively correlated with KLα levels.
Investigating the causes and implications of age-related declines in S-KLα is essential for developing strategies to improve health and physiological function, especially as people age[30]. KLα has been identified in mammals as a component involved in regulating phosphate levels and vitamin D metabolism[32]. Hyperphosphatemia is a key feature of autism and is linked to decreasing klotho levels with age. This recent evidence confirms that KLα functions as a sensitive biomarker of physiological and pathological changes in childhood, and that its decline indicates pathological agitation, ageing, and metabolic stress. These findings closely align with those of this study, which highlight the strong potential of serum klotho as a predictive biomarker in Autism risk profiling.
In children, S-KLα levels do not show significant differences between the sexes[33]. While studying Wang et al[34] observed higher levels in females; this may be attributed to the relationship between S-KLα and factors such as age, sex hormones, puberty, and physiological and health conditions, which may influence these differences. They also found that triglyceride levels and white cell count directly affect S-KLα levels in females compared to males[34]. Tan et al[35] reported that estrogen increases klotho secretion, which may lead to higher S-KLα levels in females. Another study found an inverse correlation between muscle mass and KLα levels in females[36]. Additionally, a survey by Koike et al[37] demonstrated higher KLα levels in females, suggesting that sex-linked S-KLα may be used as a marker for children with growth hormone deficiency. These findings indicate that S-KLα may play a role in various conditions in children, and its relationship to other factors may differ between the sexes, highlighting the importance of considering sex when studying KLα.
Evidence indicates that low klotho levels are associated with cognitive impairment, dementia, and other neurodegenerative conditions. This is because of klotho’s neuroprotective properties, as its deficiency promotes the development of neurodegenerative diseases[38]. Low klotho levels can lead to hyperphosphatemia[39]. Evidence also shows that elevated phosphate levels are observed in individuals with ASD. Disorders of phosphate metabolism further contribute to the development of related diseases and symptoms, due to phosphate’s role in inflammation and neurodevelopment[40], as well as its effects on neuronal growth and function. In the brains of affected patients, it results in increased glial cell development, causing changes in neural circuits, inflammation, and altered immune responses. Hyperphosphatemia is linked with autism-related conditions such as mitochondrial and bone mineral disorders, epilepsy, and certain neurological conditions[41].
Glucose is the brain’s main energy source. Therefore, any changes or disruptions in glucose metabolism affect brain functions, including cognition, emotional behavior, and social communication in people with autism[42]. Guevara-Ramírez et al[43] showed that adolescents with autism have a higher risk of developing type 2 diabetes as they age, as they exhibited phenotypic patterns and signs similar to prediabetes early in life, providing evidence of impaired glucose metabolism in autistic individuals. This may be due to potential links between gut function and glucose metabolism regulation with the behavioral manifestations of autism. Connections have also emerged between ASD symptoms and food preferences through a complex interaction between disease severity and selective eating habits in children with autism[44]. While evidence suggests a specific association, further investigation and research are necessary to understand the physiological and pathological nature of these relationships[45].
Low HGB levels may result from iron deficiency, which is linked to a higher risk of autism, as iron is essential for brain development, neurological function, and neurotransmitter formation[46]. It has also been observed that iron deficiency associated with anemia occurs more frequently in individuals with severe autism and intellectual disabilities[47]. Iron may influence the development of ASD. Research indicates that children with ASD often have iron deficiency. This aligns with our findings, as statistical differences were found between children with autism and the control group during data analysis. This is because iron plays a role in the myelination of white matter and supports the functions of various neurotransmitter systems, such as dopamine, norepinephrine, and serotonin. Therefore, it is believed that iron levels may be connected to the development of various neurological and psychiatric disorders, including autism[48].
We observed lower uric acid levels in patients with ASD. This aligns with Dai et al[49], who reported that reduced uric acid levels are linked to alterations in purine metabolism and increased oxidative stress, indicating a connection with certain types of ASD. Similarly, Esnafoglu and Yurdakul[50] found lower uric acid levels in individuals with attention deficit hyperactivity disorder and ASD, suggesting that uric acid has distinct roles in the pathophysiology of these conditions, which makes it a potential target for treatment.
There was no significant difference in serum creatinine levels between patients with ASD and healthy controls. This aligns with the study by Harel et al[51], which found no link between serum creatinine levels and ASD. Children with autism have a vitamin D deficiency, due to its vital role in regulating genes linked to brain development, reducing inflammation and oxidative stress, and supporting neuroimmunity[52]. This deficiency may also be associated with other factors, including dietary intake, reduced sun exposure, and problems with vitamin D metabolism[53].
Our results showed that serum klotho level was linked to several clinical variables in the ASD group, including a negative association with glucose. This link can be attributed to KLα protein, which stimulates insulin secretion and supports the health of beta cells in the pancreas, thereby enhancing glucose tolerance in the body[54]. It also promotes glucose uptake and glycogen synthesis in the liver, as well as improving lipid oxidation in adipose tissue and the liver. This aligns with what Gong and Ge[55] reported, that decreased levels of KLα worsen oxidative stress and neuroinflammation.
We observed a positive correlation between S-KLα and HGB in the ASD group, which may be due to klotho’s role in red blood cell production and regulation, potentially serving as a useful marker for detecting anemia[56]. A positive association between klotho and HGB has also been noted in both healthy people and those with chronic kidney disease[57].
Additionally, we observed a positive correlation between S-KLα and iron in the ASD group, which could be linked to a potential connection between iron metabolism and klotho expression, as suggested by Saito et al[58]. The relationship between iron and klotho may involve iron deficiency causing disruptions in immunity and neurological functions. They also work together to reduce free radicals in the body, and their deficiency in children with ASD leads to increased oxidative stress[59], a fact confirmed by Bjørklund et al[60], who demonstrated that redox imbalance and oxidative stress are associated with the pathophysiology of ASD. It has been noted that klotho may influence iron regulation, or the relationship could be the opposite, especially in cases of chronic kidney disease and anemia[61].
The results demonstrated a positive correlation between S-KLα level and vitamin D levels in the ASD group. This may be due to their reciprocal relationship, as Jebreal Azimzadeh et al[62] suggest that vitamin D stimulates klotho production, and klotho can also influence vitamin D efficacy. This is evident through a feedback pathway. This complex bidirectional interaction plays a crucial role in regulating mineral and bone balance[63]. Vitamin D also controls calcium and phosphate absorption, while FGF23 and klotho help prevent hyperphosphatemia[64]. Since autism is linked to hyperphosphatemia, this may account for the positive relationship between klotho and vitamin D in individuals with ASD.
CONCLUSION
This is the first case-control study indicating an important role for S-KLα in identifying children with ASD. It suggests that low serum S-KLα levels are a risk factor for ASD. Notable relationships were found linking various aspects to the level of klotho in patients, paving the way for future research and proposing mechanisms that may have a significant biological impact, especially in the connection between klotho and glucose, HGB, iron, and vitamin D.
ACKNOWLEDGEMENTS
The researchers would like to thank the families of the participants for their valuable contribution to the completion of the research, as well as the staff of the autism centers in the Kurdistan Region for their assistance in collecting data, in addition to the Department of Chemistry, College of Science, University of Mosul, for facilitating the research and supporting the practical aspect.
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