Autism spectrum disorder (ASD) is a childhood onset neurodevelopmental disorder characterized by deficits in social communication and interaction, repetitive behaviors, and limited range of interests [1]. Prevalence of ASD has increased in recent years up to 1/68 [2]. Although a range of genetic and environmental factors associated with ASD have been identified in the literature, the pathogenesis and etiology of ASD remained unclear [3]. Latest evidence indicated the role of neuroinflammation and immune system dysfunction in ASD pathophysiology [4-6].

Several post-mortem and imaging studies revealed that patients with ASD had higher levels of microglia in certain brain regions which signal neuroinflammation [5,7,8]. Also, numerous studies have reported increased blood concentrations of inflammatory cytokines in patients with ASD [9-11]. Microglias play a major role in neurodevelopment and synaptic processing [12] and regulate synaptic pruning and synaptogenesis during the early stages of central nervous system (CNS) development [13]. Hence, abnormalities in microglial function could be associated with etiopathogenesis of ASD [14]. Research suggested that chronic microglial activation and abnormal brain inflam-matory response could underlie cognitive dysfunction and neurodegenerative disorders [5,15].

Abnormal cytokine levels in blood and cerebrospinal fluid are observed in children with ASD [16]. These abnormalities were linked to behavioral problems and symp-tom severity [17]. Hence, there has been an increased interest on the biomarkers of ASD which could be helpful for diagnostic process, monitoring prognosis and treatment responses, and early detection of disorder in individuals prone to ASD [18]. Various proteins have been investigated as candidate biomarkers for studying the relationship between inflammation and ASD, although a specific biomarker has not been identified yet [19,20].

Recent research on the role of inflammation in pathogenesis of neurodegenerative and neurodevelopmental disorders primarily focused on galectins. Galectins are a protein family with 15 members present at different cells and tissues [21]. As a family of β-galactoside-binding lectins, galectins play a major role in regulating immune and inflammatory responses [22]. They participate in various biological processes including cell adhesion, migration, proliferation, transformation, apoptosis, angiogenesis, and immune responses [23]. Galectins could regulate or strengthen the inflammatory response in neurological diseases, hence helping damaged CNS tissues regenerate [24]. Galectin-1 and galectin-3 are the most prevalent forms of galectins [21]. Galectin-1 is synthesized by various cell types in immune system such as T and B cells, macrophages, and microglias [25]. Galectin-1 is a significant modulator of CNS homeostasis. Several studies reported that galectin-1 expression was altered in neurological diseases, which was linked to anti-inflammatory processes and neuroregeneration [25-27]. Galectin-1 also facilitates neural protection by inhibiting microglias in CNS [25].

Galectin-3, on the other hand, participate in various cellular processes such as adhesion, activation, growth, differentiation, intercellular interactions, lifecycle, and apoptosis [28,29]. Galectin-3 is also present in various types of immune cells except resting lymphocytes, and it acts as a pro-inflammatory agent [28,29]. There is ample evidence concerning the role of galectin-3 in inflammation and degeneration in CNS [24]. Research indicated that galectin-3 is an initiator of microglia activation and proliferation following CNS damage. It is stated that the interaction of galectin-3 and toll-like receptor 2 on microglia is necessary to initiate neuroinflammation [30,31]. However, excessive galectin-3 levels lead to irregular secretion of proinflammatory cytokines [32]. This could then amplify the glial activation hence resulting in a vicious cycle. This long term inflammatory response may then cause synaptic loss in CNS and neurodegeneration [15].

There has been a rising interest on the relationship between galectins and neurodegenerative diseases. However, very few studies have investigated the relationship between galectins and psychiatric disorders, and even fewer have focused on children with neurodevelopmental disorders. The aim of this study was to compare serum levels of galectin-1 and galectin-3 between patients with ASD, their unaffected siblings, and healthy controls. We hypothesize that serum galectin-1 and galectin-3 levels, which are potential neuroinflammatory biomarkers, will be higher in case group than their unaffected siblings and healthy controls. Similarly, it is also expected that unaffected siblings will have higher serum levels of galectin-1 and galectin-3 compared to healthy controls. Finally, it is hypothesized that serum galectin-1 and galectin-3 levels will be correlated to ASD severity and behavioral problems accompanying ASD. To the best of the researchers’ knowledge, this was the first study to investigate galectin-1 and galectin-3 levels in patients with ASD.

Participants

The present study was conducted in Istanbul University Medical Faculty Child and Adolescent Outpatient Clinic between October 2021 and March 2022. The current study included three groups as a case group, unaffected siblings group, and a healthy control group to examine whether galectins could be endophenotypes of ASD and to compare inflammatory responses in unaffected siblings with the case group and healthy controls. At the beginning of the study 108 ASD patients aged between 2−12 years and their healthy siblings closest in age were invited to the study. ASD diagnosis was made based on the Diagnostic and Statistical Manual of Mental Disorders 5th edition (DSM-5) diagnostic criteria. Diagnosis of ASD was confirmed by experienced faculty members (M.C and Y.T). For the case group, the following exclusion criteria were used: the presence of schizophrenia, bipolar disorders, metabolic, genetic, neurologic and/or gastrointestinal disorders, regular medication use due to chronic illnesses and/or any other psychiatric disorders, intake of supplements such as vitamins and fish oil during the last month, active presence of infection or history of infection in the last month. For both control groups, in addition to the above exclusion criteria, those with attention deficit hyperactivity disorder (ADHD) and intellectual disabilities were also excluded from the study. Overall, 42 ASD patients, and 42 unaffected siblings (control group 1, CG1), and age and gender-matched 42 healthy children (control group 2, CG2) took part in the study. Healthy controls were recruited among 94 children during their visits to Istanbul University Medical Faculty Pediatrics Outpatient Clinics. Figure 1 shows flowchart of the study subjects.

Assessment

Participants were given a survey form consisting of questions on sociodemographic and clinical information. Psychiatric diagnoses for all participants were assessed by Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and Lifetime Version (K-SADS-PL). For case group, Childhood Autism Rating Scale (CARS) was used to evaluate the severity of ASD symptoms. The reliability and validity of the K-SADS- PL/DSM-5 and the CARS have been established for the Turkish population [33,34].

Sociodemographic Data Form

A sociodemographic data form that was prepared by the researchers was used to obtain information about sociodemographic characteristics, developmental and medi-cal history of the participants.

K-SADS-PL/DSM-5

The K-SADS-PL is a semi-structured clinical interview designed to assess current and past episodes of psychopathology in children and adolescents according to DSM-5 criteria [35,36]. It was designed to promote earlier diagnosis of mental disorders in children and adolescents in a way that incorporates reports by both the child and parent and a clinician’s clinical judgment. A reliability and validity study of K-SADS-PL/DSM-5 for the Turkish population was conducted by Ünal et al. [33].

CARS

It is a behavioral rating scale developed by Schopler et al. [37] to distinguish children with autism from children with other developmental disorders, specifically children with intellectual disabilities from children with autism. This scale is filled on the basis of information obtained from the family and observation of the child by the clinician. The scale, which consists of a total of 15 items, is a diagnostic assessment method that rates individuals on a scale ranging from normal to severe, and yields a composite score ranging from non-autistic to mildly autistic, moderately autistic, or severely autistic. CARS was adapted to Turkish by İncekaş Gassaloğlu et al. [34].

The study was approved by Istanbul University Medical Faculty Clinical Research Ethics Committee on December 4, 2020 (project ID: TTU-2021-38129). All participants and their parents were informed about the study and written consent was obtained from the parents.

Blood Collection and Quantification

Venous blood samples were collected from the participants after 8−12 hours of fasting. The blood samples were centrifuged at 4,000 rpm for 10 minutes, and the separated sera were aliquoted into Eppendorf tubes and stored at −80°C until the time of analysis. Serum galectin-1 and galectin-3 levels were measured with double-antibody sandwich enzyme-linked immunosorbent assay kits (Invitrogen) according to the manufacturer’s instructions. Values of galectin-1 and galectin-3 are expressed in ng/ml.

Statistical Analysis

All analyses were carried out using SPSS Statistics, version 21 (IBM Co.). Descriptive statistics are reported as mean ± standard deviation. The Kolmogorov-Smirnov test was employed to evaluate whether galectin-1 and galectin-3 levels are normally distributed. Serum galectin-1 levels were non-normally distributed, hence were log- transformed. One-way analysis of variance test was used to evaluate group differences in variables. Serum log-galectin-1 and galectin-3 levels of the case and control groups were compared with the multivariate analysis of covarianc (MANCOVA) test. Age, sex, and body mass index (BMI), which are thought to affect biochemical parameters, were used as covariates. Bonferroni correction was used as a post-hoc test. Statistical significance was set at p < 0.05.

In total, 42 patients with ASD (case group, mean age = 71.59 ± 28.65 months), 42 unaffected siblings (CG1, mean age = 73.92 ± 27.71 months) and 42 healthy controls (CG2, mean age = 86.26 ± 38.66 months) participated in the study. All participants were Turkish origin. The case group and control group 2 consisted of 8 girls and 34 boys, whereas the control group 1 consisted of 20 girls and 22 boys. Majority of the subjects in case group had comorbid diagnosis of intellectual disability (n = 30; 71.4%) and ADHD (n = 22; 54.2%). The demographic and clinical characteristics of the three groups are shown in Table 1.

The mean galectin-1 levels were 876.7 ± 932.8 ng/ml in the case group, 172.9 ± 337.06 ng/ml in CG1 and 159.06 ± 303.1 ng/ml in CG2. The mean galectin-3 levels were 7.375 ± 2.69 ng/ml in case group, 6.677 ± 2.61 ng/ml in control group 1 and 5.536 ± 3.47 ng/ml in control group 2. MANCOVA results revealed significant intergroup differences in serum galectin-1 and galecitn-3 levels controlling for age, sex (V [Pillai’s trace] = 0.313, F = 11.145, p < 0.001, ηp² = 0.157). Separate univariate ANCOVAs disclosed important variations in serum levels of galectin-1 and galectin-3 between the three groups (F [2.120] = 20.799, p < 0.001, ηp² = 0.257; F [2.120] = 4.268, p = 0.016, ηp² = 0.066, respectively) (Table 2). Post-hoc pairwise comparisons with the Bonferroni correction showed that serum galectin-1 levels were significantly higher in the case group compared to the CG1 (p < 0.001), and CG2 (p < 0.001), but there was no significant difference between the CG1 and CG2 (p = ns) (Fig. 2A). Serum galectin-3 levels were significantly higher in the case group compared to CG2 (p = 0.013), but there was no statistically significant difference between the case group and CG1 (p = 0.761). There was no significant difference between CG1 and CG2 (p = 0.309) (Fig. 2B). No significant correlations were found between serum galectin-1 and galectin-3 levels and CARS scores (p = 0.802 and p = 0.107, respectively). Ttest results showed that within the case group, galectin-1 and galectin-3 levels were similar for those with and without ADHD comorbi-dity (p = 0.262; p = 0.860, respectively).

In the current study we investigated serum galectin-1 and galectin-3 levels in children with ASD comparing with their unaffected siblings, and the sex, age, and BMI matched healthy controls. To our knowledge, there has been no study on this subject so far and findings of the current study may have some further research and clinical implications. We found that children with ASD had significantly higher galectin-1 serum levels than their unaffected siblings and healthy controls and, higher galectin-3 serum levels than healthy controls which may indicate possible roles of galectin-1 and galectin-3 in the pathophysiology of ASD.

To date, serum galectin-1 and galectin-3 levels have been examined in few psychiatric disorders including schizophrenia, depression and ADHD. Several studies reported different findings on galectins levels in schizophrenia patients; such as higher galectin-3 levels in chronic schizophrenia patients [38], lower galectin-3 levels in first-episode schizophrenia patients and relapse cases but higher in cases with remission [39], and higher levels of galectin-1 in the unaffected siblings compared to both the patient group and the healthy control group while higher galectin-3 levels in the sibling group relative to the patient group [40]. In another study using a large community sample, King et al. [41] found that galectin- levels were positively linked to scores on depressive symptoms scale. Previous literature also focused on the link between galectin-3 levels and ADHD [42-44]. For example, Wu et al. [42] found lower levels of galectin-3 levels in rodent models of ADHD. Similarly, another study by the same research group revealed that children with ADHD had lower levels of galectin-3 compared to healthy controls [42,43]. Conversely, Isık et al. [44] found that children with ADHD had higher levels of galectin-3 compared to healthy controls. These mixed results could be due to methodological differences in study designs. Con-sidering the positive link between inflammation and galectin levels [40], the findings of the present study are generally compatible with the recent literature [38,41,44] suggesting the role of galectin-1 and galectin-3 in the pathophysiology of ASD. Because this is the first study investigating galectin-1 and galectin-3 levels in ASD, it may not be possible to directly compare our findings with the literature.

There has been a rising interest on immune system disorders and neuroinflammation due to complex etiology of ASD [4,5]. A number of studies confirmed the positive association between ASD and chronic and disrupted neuroinflammatory response [5,45]. In addition, post-mortem studies in ASD patients using positron emission tomo-graphy scans showed higher intensity of microglial cells in certain brain regions [5,6]. Microglias are key agents in synaptic development and functioning [12,19], synaptogenesis and neuronal pruning during early stages of CNS development [13]. Hence, disrupted microglial functioning could indicate ASD and other neurodevelopmental disorders [14,46]. Considering galectin-1 and galectin-3 expression in microglias for chronic illnesses affecting CNS [25] and the role of inflammation in ASD pathogenesis [14] our results are supporting the neuroinflam-mation hypothesis of ASD. Increased galectin-1 concentrations in the case group may indicate that galectin-1 may have a role in the etiopathogenesis of ASD. Galectin- 1 up-regulation in inflammatory cells inhibit microglia infiltration and migration, resulting in anti-inflammatory effect [25]. Therefore, galectin-1 production could be an attempt to reestablish the homeostatis in response to inflammatory processes [47]. In line with this idea, higher galectin-1 levels in case group in the present study could be due to chronic inflammation in ASD patients. Several studies support the anti-inflammatory role of galectin-1, whereas a limited number of studies found that inflam-mation and stress in CNS led to increase in galectin-1 levels [48,49]. In the present study, although a certain mechanism was not identified for galectin-1 increase, higher galectin levels could be interpreted as an attempt to control inflammatory response.

Research suggests that similar to galectin-1, galectin-3 may also play a part in the etiopathogenesis of ASD. Pa-tients with ASD had higher levels of proinflammatory cytokines compared to controls [9-11]. Galectin-3 is suggested to cause increased inflammatory response by inhibiting anti-inflammatory cytokine interleukin (IL)-10 production [50]. An in vitro study reported that galectin-3 induced IL-6 expression and galectin-3 inhibition reduced IL-1b expression. These findings suggest that galectin-3 regulates the expression levels of cytokines related to ASD [51]. Since these cytokines play a role in facilitating inflammation, increased galectin-3 may signal increased neuroinflammation in ASD. Hence, galectin-3 appears to be a significant potential biomarker of neurological diseases both in terms of diagnosis and progression [21]. In line with the available research, significantly higher galectin-3 levels in case group compared to healthy controls but not to unaffected siblings may indicate a role of galectin-3 in the etiology of ASD.

Various studies showed that galectin-1 and galectin-3 levels increase in chronic diseases such as neoplastic disease, heart failure, kidney failure, hepatitis, and diabetes mellitus [52,53]. In our study, participants went under a thorough medical examination, and none was diagnosed with a medical illness. Also, those with a history of serious medical illness was excluded from the study. Hence, it is unlikely that higher galectin levels in case group is due to the presence of a medical condition. Similarly, participants using medication and/or supplements were not included in the study. All three groups had similar BMIs. Hence, high serum galectin-1 and galectin-3 levels in ASD cases are not attributable to the differences in physical illness, medication use, or BMI.

The inclusion of unaffected siblings into the current study allowed us to examine whether galectins could be endophenotypes of ASD and to assess inflammatory responses in unaffected siblings. A recent study showed that children with ASD and their unaffected siblings had higher levels of proinflammatory and anti-inflammatory cytokines than healthy controls, suggesting immune deficiencies are a potential indicator of autism endophenotype [54]. Although the findings of the present study showed higher galectin levels in unaffected siblings compared to healthy controls, the difference was not statistically sig-nificant. Hence, it may not be possible to consider galectins as potential endophenotypic markers of ASD based on the findings of the current study.

Our results showed that galectin-1 and galectin-3 levels was not associated with the severity of ASD symptoms mea-sured by CARS. Similarly, there was no relationship between galectin levels and several clinical characteristics such as self-mutilation and history of regression. However, given the study limitations including relatively small sample size, it may not be possible to document, if any, associations between galectin levels and the severity or associated factors of ASD. Further research is needed on this area.

The present study may have some strengths and limita-tions. First of all, to our knowledge, no previous study was conducted on galectin-1 and galectin-3 levels in ASD. Also, the current study included unaffected siblings of the children with ASD in addition to healthy controls, which may allow to evaluate whether galectins could be potential endophenotypes in individuals genetically inclined to ASD. In addition, a number of factors that may influence galectin levels such as chronic medical, neurological or gastrointestinal disorders, and use of supplements or medications were used as exclusion criteria. Regarding the study limitations, it may be important to note that our sample consisted of Turkish participants which limits the generalizability of the findings to other ethnic or racial groups. The relatively small sample size may have limited the statistical power of the study. Also, infection presence, an exclusion criteria, was clinically but not biochemically tested, which could be an important limitation. The cross- sectional, single-centered, and non-randomized nature of the present study are among the other limitations. The presence of intellectual developmental disorder and ADHD comorbidities in a significant portion of the children with ASD can be stated as another limitation of the study, since it may affect galectin levels. In addition, there has been no previous study on the levels of galectin-1 and galectin-3 in children with autism, making it difficult to compare the findings of this study with previous research. The absence of a clear mechanism for a role of increased galectin-1 and galectin-3 levels in the pathophysiology of autism and inconsistent findings in previous studies examining the relationship between galectin-3 levels and other psychiatric disorders can also be cited as other limitations.

Higher levels of galectin-1 in case group compared to both unaffected sibling and healthy control, and higher levels of galectin-3 in case group compared to healthy control may show potential roles of galectins in the pathophysiology of ASD. Further studies with larger, and more diverse samples are needed to determine the role of galectin-1 and galectin-3 in the pathophysiology of ASD, and whether they can be biomarkers or targeted interventions for ASD.

This study was supported by a grant from Istanbul University, Unit of Scientific Research (TTU-2021-38129).

No potential conflict of interest relevant to this article was reported.

Conceptualization: Murat Coskun, Yasar Tanir. Data acquisition: Zeynep Nur Karadogan. Formal analysis: Zeynep Nur Karadogan, Canan Kucukgergin, Ali Karayagmurlu. Supervision: Murat Coskun, Yasar Tanir. Writing—original draft: Zeynep Nur Karadogan, Yasar Tanir. Writing—reciew & editing: Murat Coskun.