Serum Cingulin Levels are Increased in Children with Autism Spectrum Disorder (ASD)

doi:10.21203/rs.3.rs-2082557/v1

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Serum Cingulin Levels are Increased in Children with Autism Spectrum Disorder (ASD)

https://doi.org/10.21203/rs.3.rs-2082557/v1

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Background:Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders in which the underlying pathogenesis and etiologic factors are not fully understood. The blood brain barrier (BBB) plays a critical role in central nervous system (CNS) defense by limiting access to circulating solutes, macromolecules, and cells that can negatively affect neuronal activity. The loss of BBB integrity is likely to be seen as a common pathologic finding for many psychiatric disorders such as schizophrenia, ASD, and mood disorders. In this study, we aimed to investigate whether serum cingulin levels are associated with ASD.

Subjects and Methods: A total of 40 treatment-naive children with ASD and 40 healthy controls were included in the present study. The Schedule for Affective Disorders and Schizophrenia for School-Aged Children, Present and Lifetime Version-DSM-5 (K-SADS-PL-DSM-5) has been used to screen healthy controls for psychiatric disorders by a psychiatrist after a physical examination by a paediatrician. The clinical severity of the ASD symptoms has been assessed by the Childhood Autism Rating Scale (CARS). Venous blood samples were collected and serum cingulin levels were measured.

Results: When the ASD and control groups were compared, CARS and CINGULIN values of the patient group were statistically higher than the healthy group. There is a statistically positive correlation between CARS and Cingulin values.

Discussion: To the best of our knowledge, this study is a first in the literature conducted about the serum cingulin levels, which is a component of BBB, among patients with ASD. Our findings demonstrate that serum cingulin levels are meaningfully higher in ASD group compared to the healthy control group. It has been also indicated that there has been a meaningful relationship between serum cingulin levels and ASD symptom severity.

ASD

CINGULIN LEVELS

BLOOD BRAIN BARRIER

Autism Spectrum Disorders (ASD) is a group of neurodevelopmental disorder that leads to narrow behavioural patterns and the deterioration of communication and social interactions[1]. According to the analysis of the data obtained in 2018 by Centre for Disease Control and Prevention (CDC), it has been found out that, in the USA, about 1 of 44 (2,3%) children at the age of 8 were diagnosed with autism spectrum disorder. The aetiology of ASD is complicated and mostly unknown; and studies show interactions among various elements including genetic, epigenetic and environmental factors[1].

Studies make researchers think that a combination of risk factors of genetics, autoimmune, environmental, and maybe in utero which lead to neuroinflammation might contribute to the pathogenesis of ASD[2]. Beside ASD, the extreme expression of acute phase protein in serum, brain and cerebrospinal fluid of individuals with schizophrenia (sch) makes us think that inflammation appears in the pathogenesis of these disorders[2]^,[3]. In spite of all the given efforts to research, there has been no certain explanation about how environmental triggers might lead to these neurobehavioral conditions. According to a hypothesis depending upon the connection of gut-brain axis, it has been claimed that an inappropriate antigenic leakage passes through the deteriorated gut barrier and it follows the transition of antigens or activated immunity complexes through a permitted blood-brain barrier (BBB). This might be a part of the chain that causes the inflammation, and consequently the disorder[4]. BBB plays a critical role in the defence of central nervous system (CNS) by restricting the access of cells that might negatively affect the solute in circulation, macromolecules and neuronal activity. The dysfunction of BBB has been associated with numerous neurologic disorders such as stroke, epilepsy, multiple sclerosis, Parkinson and Alzheimer diseases[5]. It has been claimed that the changes in substantia grisea that are observed in ASD reflect the changes in veins and in many components of CNS including dendritic density, glial cell numbers and morphology[6]. The studies on ASD transcriptome have determined a disorder of vascular improvement in autism (beside other processes, including synapsis and the regulation of the inflammation)[7]. It has been demonstrated that the BBB deteriorates in ASD[4]. BBB forms a critical interface between the brain and bloodstream. The loss of BBB integrity is likely to be seen as a common pathologic finding for many psychiatric disorders such as schizophrenia, ASD, and mood disorders[8].

BBB is a highly regulated interface that separates peripheral circulation and CNS. Primarily, it functions as a selective diffusion barrier on cerebral microvascular endothelial level. BBB is generally defined as privatized endothelial cells that structurally covers the intraluminal part of brain capillaries. However, another more dynamic and functional definition accepts the periendothelial accessory structures as inseparable components of BBB. In addition to endothelial cells, BBB consists of astrocytes, pericytes, neurons and extracellular matrix. This established neurovascular unit is needed to preserve the underlying brain cells and the CNS homeostasis that is required for a stable and coordinated neuronal activity[9]. The structure of BBB is complicated and all the components form a functional neurovascular unit together.

Anatomically, as they have unique and distinguishing specialities, endothelial cells of BBB can be separated from endothelial cells of peripheral tissue. Inter-endothelial gaps of cerebral microvasculature are characterized with the existence of a junction complex that is formed by tight junctions (TJs), which limits the paracellular permeability through the endothelium and adherens junctions (AJs)[9].

Astrocytes, in shape of a star, are cellular elements of neuroglia (literally a nerve glue or a nerve cement) which is a non-neuronal supportive tissue of CNS. Glial cells own end feet that form a thin lamellar cage which separates capillary vessels from neurons because they are settled near the exterior surface of endothelium. These cells act as a skeleton, and they lead the neurons to the appropriate places during the function of BBB and they also lead the vessels. Astrocytic glia plays an important role in the stimulation and continuation of BBB phenotype. Astrocyte-endothelial interaction and intercellular signals are required for optimal BBB function and there has been increasing evidence that endothelial cells have a mutual inductive effect on astrocytes[10].

TJs have a critical importance to create and maintain the barriers between different body parts in vertebrata epithelium and endothelium. In molecular level, TJs include transmembrane proteins from different categories[11]. These are four-pass membrane proteins (occludin, claudins, tri-cellulin and marvelD3), junctional adhesion molecules like lg and others (BVES and angulins)[11]; cytoplasmic scaffold proteins including zonula occludens (ZO) proteins (ZO-1, ZO-2 and ZO-3), cingulin (CGN) and paracingulin[12]; and signal proteins including kinases and phosphatases[11].

Cingulin (~ 140 kDa) is a non-PDZ cytoplasmic scaffold TJ protein and it exists as a parallel homodimer of two sub-units, each of which consists of a N-terminal globular head domain, a long coiled-coil rod domain and a small globular globe[13]. While the head domain, ZOs, of cingulin seems to control the fertile intake of cingulin into the cell-cell interactions through the interaction of JAM-A and actin, the rod domain is essential in the dimerization of cingulin[13]. Cingulin is a protein localized in the cytoplasmic region of TJ of BBB. The function of cingulin in BBB is to interact with ZO-1 and JAM and to provide TJ proteins to adhere actin-based cellular structures. As a result, its function is to strengthen BBB and regulate BBB permeability[13]. Cingulin has showed that there has been a deterioration of BBB among knockout mice[14].

There have been a few studies related to claudin levels, which is another TJ protein that regulates BBB, although there have been no studies related to the relationship between psychiatric and neurologic diseases and cingulin levels, which is also a TJ protein that regulates BBB.

To the best of our knowledge, in the literature, there hasn’t been a study related to cingulin levels in ASD. It is known that BBB deteriorates in ASD. In this study, it is hypothesised that as BBB deteriorates in ASD, TJ protein cingulin levels, which is also a component of BBB, would be higher in blood levels among children with ASD when compared to healthy controls. It has been planned that the serum cingulin levels of children with ASD and healthy controls will be examined under the control of parameters such as age, gender and body mass index (BMI) percentiles because it is known that these parameters might affect cingulin levels.

Study Sample

Samples for the study are obtained from Uşak University, Training and Research Hospital, Child and Adolescent Psychiatry Outpatient Clinic. 40 children, between 36-59 months-old, who have been diagnosed with ASD, have medical afflicted reports and get special education, have been examined as the ASD group. Having a crucial medical condition (such as diabetes mellitus, obesity, diagnosed GI disease), and having allergic or neurologic (like epilepsy) diseases that are diagnosed as additional genetic syndromes related to ASD are determined as comorbidity and included in the exclusion criteria for ASD group. Children who are mentally disabled (controlled Z clinically), have BMI over 95%, have taken, in any time, corticosteroid or a medication that affects immunologic system and have an infection history within the last month are also excluded from the study. Besides, cases who have gone under a psychotropic medical treatment, especially within the last 6 months, are also excluded from the study. The comorbidity of ADHD has been evaluated through clinical examination and family interviews. Cases who have clinical findings or suspicious history related to ADHD are excluded from the study. Control group is formed by the cases from the paediatric outpatient department of the same hospital. After the paediatric examination, a psychiatrist evaluated the cases in the control group clinically according to DSM-5. For the control group the same exclusion criteria which is determined for the ASD group is used. Moreover, the existence of a chronic disease and any psychiatric disorders such as schizophrenia, bipolar syndrome, major depression, obsessive compulsive disorder and anxiety are also added to the exclusion criteria for the control group. As a result, 40 children, between 36-59 months-old, who matches the exclusion criteria, are formed as the control group.

Study Procedures

Sociodemographic and clinical information have been written down on a form by the authors. BMI values are calculated manually for each case. A psychiatrist of child and adolescent has evaluated the patients and the controls in terms of any psychiatric condition. The clinical severity of ASD symptoms has been evaluated with Childhood Autism Rating Scale (CARS). CARS is found to be valid and reliable for Turkish society²¹. It consists of 14 domains that evaluate autism related behaviours and 15 scales interpret autism general review. Each scale is graded from 1 to 4. Higher numbers are associated with more severe deterioration. Total points range from 15 to 60 and points under 30 demonstrate that the individual is not in the autistic spectrum. While points between 30 and 36,5 demonstrate mild and moderate autism, points between 37 and 60 show severe autism. The psychometrics of CARS is well-documented[15].

The determination of serum CINGULIN levels

Venous blood samples were taken from left radial artery between 8 and 9 a.m. after avoiding eating and drinking during the previous night. In order to remove the plasma, blood samples were centrifuged for ten minutes at 4 C°, 3.000 rpm. Until the analysis, the samples were preserved at -80 C °. In order to measure serum CINGULIN concentrations, Elabscience Instant ELISA kit (E-EL-H3651) was used according to the producer’s instructions. Serum CINGULIN levels were recorded as pg/mL.

STATISTICAL METHOD

The obtained data have been evaluated with IBM SPSS Statistics Standard Concurrent User V 26 (IBM Corp., Armonk, New York, the USA), a statistic packaged software. Descriptive statistics are given as number of units (n), percentage (%), mean ± standard deviation, median (M), minimum (min) and maximum (max). Normal distribution of the data that belong to numeric variables has been evaluated with Shapiro Wilk normality test. The comparison between groups for age and CARS has been done with independent (two sample) t test, and the comparison between groups for Cingulin has been done with Mann- Whitney U test. The comparison between groups for gender has been done with Pearson’s chi-square test, exact method. The relationship between age, CARS and Cingulin has been evaluated with Pearson and Spearman correlation analysis. p<0,05 value has been accepted as statistically significant.

According to Table 1, gender and age distribution of the groups are statistically similar. CARS and Cingulin levels of the ASD group are statistically higher than the control group.

Table 1: The comparisons of groups in terms of variables in the study

	Groups		Test Statistics
	ASD	Control	Test Value	p value
Gender, n (%) Male Female	34 (85,0) 6 (15,0)	31 (77,5) 9 (22,5)	χ²=0,738	0,568
Age, (month) ±ss M (min-max)	46,0±6,5 45,0 (36,0-59,0)	45,2±6,7 44,0 (36,0-59,0)	t=0,505	0,615
CARS ±ss M (min-max)	41,9±7,0 41,0 (32,0-62,0)	23,6±5,5 25,0 (15,0-32,0)	t=12,894	<0,001
Cingulin ±ss M (min-max)	1723,5±1018,6 1699,0 (186,5-1725,0)	964,1±697,5 899,0 (150,0-2561,5)	z=3,341	0,001

: Average, ss: Standard deviation, M: Median, χ²: Pearson’s chi-square test statistics, t: independent (two sample) t test, z: Mann-Whitney U test

According to Table 2, there is a statistically positive correlation between CARS and Cingulin levels in the whole group. Other correlation coefficients in the table are statistically insignificant.

Table 2: The relationship between the age, CARS and Cingulin values of volunteers

	Whole Group N=80		ASD n=40		Control n=40
	CARS	Cingulin	CARS	Cingulin	CARS	Cingulin
Age	r=0,012; p=0,919	rho=0,186; p=0,099	r=0,071; p=0,663	rho=0,431; p=0,006	r=-0,227; p=0,159	rho= -0,141; p=0,385
Cingulin	rho=0,379; p=0,001	-	rho=0,061; p=0,708	-	rho=0,180; p=0,265	-

r: Pearson’s coefficient of correlation, rho: Spearman’s coefficient of correlation

To the best of our knowledge, this study is a first in the literature conducted about the serum cingulin levels, which is a component of BBB, among patients with ASD. Our findings demonstrate that serum cingulin levels are meaningfully higher in ASD group compared to the healthy control group. It has been also indicated that there has been a meaningful relationship between serum cingulin levels and ASD symptom severity.

In a recent study, Işık and et al. (2020) have found that serum clausin-5 levels are higher in patients with ASD compared to healthy controls[16]. In another study, Fiorentino et al. (2016) have examined the post-mortem cortex and cerebellum of patients with ASD and healthy controls and have presented that claudin-5 protein and claudin-5 gene expression levels are meaningfully higher in the brain parenchyma and cerebellum of patients with ASD compared to healthy controls[4].

An outstanding finding in a recent study is that serum claudin-5 levels in patients with ADHD are meaningfully higher compared to the control group. In that study among the patients with ADHD, it has been found that there has been a positively meaningful relationship between serum claudin-5 levels and attention deficit and depression points[17].

In the literature, it has been informed that the studies on claudin-5 are related to epithelium/ endothelium dysfunction and inflammation in various autoimmune, neurologic and psychiatric disorders[17]^,[18]. As a result of the increasing permeability in BBB, depression-like behaviours are observed among rats. It has been stated that by using antidepressants, claudin-5 loss has been prevented and the endurance in BBB has been increased[19]. Moreover, it has been claimed that the decrease in the expression of endothelium claudin-5 might lead to the deterioration in BBB and depressive symptoms[20]. Previous studies have indicated that there has been an abnormal and deteriorated claudin-5 expression in BBB in schizophrenia, and antipsychotic medication increases according to in vitro and in vivo claudin-5 expression dose[21]. Fiorentino et al. have studied the post-mortem cortex and cerebellum of patients with ASD and healthy controls. In the tissues, cerebral damage/neuroinflammation markers, matrix metalloproteaz-9 (MMP9), translocator protein18 (TLOP-19) gene expression, claudin-5 protein and claudin-5 gene expression levels have been found meaningfully higher among patients with ASD compared to healthy controls. They claimed that the increase in serum claudin-5 levels might be a mechanism that emerges to compensate a probable deterioration in BBB[4].

Yücel and et al. have compared the serum claudin-5 levels in patients with migraine during their attack period to the patients in remission period and healthy controls. Serum claudin-5 levels are found to be meaningfully high during the attack period. It has been informed that the increase in claudin-5 levels might be an indication of a neuroinflammation that triggers the headache depending upon the endothelial dysfunction in BBB[22].

Despite the efforts in researches, there has been no determined explanation about how environmental triggers lead to the neurobehavioral conditions. A hypothesis depending upon the bonding of gut-brain axis each other claims that an inappropriate antigen leakage passes through the deteriorated gut barrier, and after that, from a permitted BBB, it follows the transition of antigens or activated immunity complexes. This might be a part of the chain of events that lead to the neuroinflammation, and thus the disease[4]. The dysfunction of BBB has been associated with numerous neurologic disorders such as stroke, epilepsy, multiple sclerosis, Parkinson and Alzheimer diseases[5].

In our study, it has been indicated that serum cingulin levels among children with ASD are meaningfully higher compared to healthy controls. Serum cingulin levels have a meaningfully positive correlation with the severity of the disease. The loss of BBB integrity is likely to be seen as a common pathologic finding for many psychiatric disorders including schizophrenia, ASD, and mood disorders[8].

It is determined that as BBB has a dysfunction in patients with ASD, cingulin protein, which is a component of BBB, releases to the peripheral circulation. Therefore, serum cingulin levels are seen to be meaningfully higher in children with ASD. We think that serum cingulin levels might have a potential to become a biomarker for ASD. More studies with more participants are required to confirm this result.

This study has a relatively moderate sample size with 40 patients and 40 healthy controls and has been unable to determine the prognostic importance of serum cingulin levels. Examining blood parameters in only serum samples, negative early living conditions, the socioeconomic conditions that might influence the blood parameters and the cross-sectional design of the study have been the main restrictions.

Acknowledgements

None.

Disclosure statement

No potential conflict of interest was reported by the authors.

FUNDİNG

Funding not received for the study

Ethical Approval:

The research has been approved by Süleyman Demrel University Non-Interventional Clinical Research on 28.08.2022. Written consent has been obtained from the participants and their families.

Authors' Contributions:

Abdulbaki Artık data collection and wrote the main manuscript text

Bahar Öztelcan Gündüz methodology and wrote the main manuscript text

Soycan Mızrak performed biochemical analysis

Umit Isik performed statistical analysis

Burak Acıkel wrote the main manuscript text

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No competing interests reported.

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Serum Cingulin Levels are Increased in Children with Autism Spectrum Disorder (ASD)

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