Comparison of Serum Usp9x and TGF-ß Levels in Children With Asd Spectrum Disorders With Healthy Controls

doi:10.21203/rs.3.rs-1825047/v1

Introduction:

The etiology of autism spectrum disorder (OSB) is a complicated neurodevelopmental disease that is influenced by a number of unknown factors. Ubiquitin-specific proteins (USP) have been found to alter etiology in recent animal studies. However, there is no human study on the subject that we are aware of. The purpose of this research is to determine the differences and correlations between USP9X and TGFb exposure levels in healthy OSB controls, as well as the violence and correlations.

Methods

The children in the research ranged in age from 3 to 12, with 41 having ASD. The Childhood Autism Rating Scale (CARS), the Gilliam Autism Rating Scale-2, and sociodemographic questionnaires were completed by the participants (GARS TV-2). The parameters USP9X and TGFb were measured in peripheral blood samples using the ELISA technique.

Results

When the levels of USP9X and TGBb in children with ASD and healthy controls were compared, it was shown that the level of USP9X in children with ASD was statistically substantially higher (11.94 vs 8.77, p: 0.001), but the level of TGFb was not significantly different. The difficulty to make eye contact subscale showed a positive link in the correlation study between the CARS and TV-GARS-2 questionnaires and blood measurements.

Discussion

USP9X levels have been found to be high in children with ASD, independent of severity, necessitating additional investigation as a potential etiological target.

Autism

USP9X

TDFb

Etiology

We found the USP9X level to be significantly higher in children with ASD.

A significant positive correlation was found between low eye contact and USP9X and TGF b values in children with ASD.

ASD is a neurodevelopmental disorder marked by a lack of social skills, impairments in verbal and nonverbal communication, and limited and stereotyped interests and activities. Males (m/n = 4:1) are more likely than females to have it (Edition 2013). The prevalence of OSB is increasing as a result of epidemiological study. The frequency of ASD in the general population is estimated to be 1% (Baird et al. 2006). According to data from the United States Center for Disease Control (CDC), the prevalence of ASD was 1/44 in 2021 (Baio 2014). As the OSB shows, etiopathogenesis is a difficult condition to treat. Despite epidemiological studies identifying several risk factors, no single risk factor has been shown to be sufficient for the development of the OSB. Genetic and environmental factors influence the development of OSB (Corrales and Herbert 2011; Rodier 2011)

Although the cause of OSB is uncertain, research has shown that hereditary factors have a role in the disease's progression. The development of DNA decoding techniques has accelerated the discovery of genetic risk factors (Geschwind and State 2015). As these technologies advance, the number of ASD-related genes will increase. The study of OSB genetics holds a lot of potential in terms of improving OSB diagnosis and creating treatment options. Given the current state of knowledge about the OSB, identifying biological markers that can aid in differential diagnosis, prediction, and monitoring of therapy response is crucial. Many studies have been carried out in order to discover chromosomal areas and lobes that may be involved in OSB. No single primary gene that causes OSB has been discovered as a result of these results. This illustrates that OSB is a complex hereditary illness with a wide range of symptoms (Ma et al. 2010).

Post-translational modifications (PTMs) that determine protein function control cell proliferation, differentiation, development, and cell destiny. PTMs serve a role in quantitative modulation of substrate function in response to environmental variables, despite the fact that they are inherently rapid and reversible. Ubiquitylation is a PTM created by the addition of 76 amino acids ubiquitin to the protein structure. It is one of the most well-studied PTMs. In cells, the ubiquitin system is responsible for the degradation of a large number of short-lived proteins. Covalent ligation of proteins to ubiquitin permits them to be at the target spot for destruction. Many activities, including cell cycle, signal transduction, transcriptional regulation, receptor-mediated regulation, signal transduction, synaptic remodeling, and endocytosis, are regulated by the ubiquitin system (Hershko and Ciechanover 1998). Because of the intimate link between the Ubiquitin system and proteins, deterioration of this system can result in neurodevelopmental disorders. Because of covalent interaction between remnants of lyse and the ubiquitin chain, the proteins are identified and destroyed by the ubiquitin-proteasome system (Kerscher et al. 2006).

Deubiquitile enzymes (DUB) invert the ubiquitin-substrate protein relationship by inhibiting the binding of ubiquitin to the substrate protein. The human genome encodes around 95 DUB. A substrate-specific protease (USP) is the largest class, and it governs cellular activities precisely (Nijman et al. 2005). USP9X (Ubiquitin specific protein 9) genes for a very big deubiuitile enzyme with a 2550 amino acid sequence (Wood et al. 1997). We are constrained in our understanding of protein structure, despite the fact that complex is a structure. At the molecular level, USP9X has two distinct domains: the catalytic region, which is similar to that of other USPs and is 300 to 800 amino acids long, separated by cysteine and histidine catalytic motifs at the beginning and end, respectively, and the ubiquitin-like module in the protein's N-terminal extension. USP9X protein structure is substantially conserved across a wide range of animals, from Drosophila to humans. The degree of conservation of USP9X (90%) in vertebrates is comparable to that of critical developmental promoter genes like pax-6 and b-catenin, and the protection extends to the whole protein region (Murtaza et al. 2015). USP9X was shown to be significantly expressed in neural stem cells/progenitors (NSC/NP) as well as in vivo in mouse embryos as a consequence of studies on the function of USP9X in mice, and mutations in USP9X have affected neuronal cell migration and proliferation (Fischer-Vize et al. 1992; Murtaza et al. 2015). USP9X is a potential gene for human neurodevelopmental disorders include lissencephaly, epilepsy, X-linked intellectual disability, and autism spectrum disorders (ASD) (Homan et al. 2014). The underlying structural and link abnormalities in USP9X knockout mice (USP9x-/y) were analyzed in an up-to-date study with USP9X knockout mice (USP9x-/y). Although it is known that loss of function in the human USP9X can cause neurodevelopmental syndrome, including the OSB, the underlying structural and link abnormalities were analyzed in an up-to-date study with USP9X knockout mice (USP9x- Usp9x-/y mice exhibited aberrant communication and social interaction behavior, and the lack of Usp9x led in brain shrinkage in various areas (Kasherman et al. 2021).

TGF-b (Transforming Growth Factor) is a protein that converts cytokines into growth factors. It's a crucial cytokine, especially for the immune system, but it's also been linked to non-immune functions (Borovcanin et al. 2016). TGF binds to TGF receptors I and II, which are serine/threonine kinases that phosphorylate intracellularly. Ankyrin-G strengthens its interaction with TGF- as a result of TGF-dependent phosphorylation of Usp9X. TGF- controls the subsynaptic localization of Ankyrin-G and Usp9X. S/T kinases and the TGF signaling pathway, as well as downstream s/t kinases such smads, JUN N-terminal kinase, and MAP kinase (mitogen-activated protein kinase). Usp9X has been demonstrated to regulate transcription factor characteristics in the nucleus via deubiquitinating Smad4 proteins, which facilitates TGF signaling. TGF- has thus been demonstrated to have a significant role in neuronal growth and function. The pathogenesis of ASD, as well as cancer, heart disease, obesity, and diabetes, is linked to abnormal TGF-signaling. TGF-; USP9X-mediated deubiquitylation leads to ankyrin-G stability by enhancing its efficacy against its substrates, and TGF- could be used as a therapeutic method to repair synaptic deficits in neurodevelopmental diseases (Yoon et al. 2020). In our assessment of the literature, we discovered that USP9X is a peripheral protein that has yet to be examined in children with ASD. As a result, the levels of USP9X and perhaps associated TGF- in Turkish children with ASD and healthy controls were compared. According to the research we conducted, USP9X has been on the radar but has yet to be tested in children with OSB. As a result, the TGF- levels that may be linked to USP9X have been assessed in Turkish OSB children and healthy controls.

Methods

Participants

This study is a sectional study aimed at comparing the levels of serum TGF-β and USP9X of healthy controls with children with 3 to 12 years of OSB Spectrum disorder who apply for Gaziantep University Şahinbey Research and Practice Hospital Child and Adolescent Psychiatry Outpatient Clinic. It is a cross-sectional study aiming at the relationship. Approval for the study was obtained from Gaziantep University Clinical Research Medical Ethics Committee with the decision number 2021/347 on 12.01.2022. All patients who met the inclusion criteria among the patients who applied to the Child and Adolescent Psychiatry Outpatient Clinic between January and March 2022 were included in the study. The inclusion criteria for the study are as follows: 1. Being diagnosed with ASD spectrum disorder according to DSM-5 criteria 2. Being between the ages of 3–12. Inclusion criteria of the volunteers taken as the control group into the study 1- Volunteering to participate in the study 2- Being between the ages of 3–12. Exclusion criteria from the study (valid for the case and control group) 1-Having a history of alcohol or smoking 2-Using any drug except psychotropic drug use in the last 2 weeks 3-Using anti-inflammatory and antioxidant 4-Acute or have any known chronic metabolic disease, genetic disease, neurological disease, cardiovascular disease, respiratory disease, liver or kidney disease, oncological disease, hematological disease, infective disease, autoimmune disease, allergic disease, or other medical disease. In addition to the exclusion criteria, the control group should not have a known mental illness.

Method

The study was informed about working with the parents of the selected OSB-diagnosed children and control group. An Informed Voluntary Consent Form was signed by the parents who were informed about the study and agreed to participate in the study. For the control group, informed consent was obtained from the children over the age of 6 who could read and write. Both the patient and control group children were evaluated by a child psychiatrist, and the sociodemographic data form was filled by the clinician by asking the parents. Socio-demographic Form Using a semi-structured interview schedule, the names, surnames, ages, mode of birth, working status of parents, and presence of psychopathology in parents of mothers of children with ASD and healthy children were questioned. In addition, the Childhood Autism Rating Scale (CARS) was filled in by the clinician for children with ASD based on the information obtained both by asking the parents and by observing the child(Schopler et al. 2010). CARS is a valid and reliable scale widely used in the diagnosis of PDD. CARS is a 15-item scale developed to diagnose ASD, as well as to distinguish between developmentally disabled children without ASD and children with ASD. The items in the scale are under the headings of relationship with people, imitation, emotional reactions, use of the body, reaction to change, visual reactions, resting reactions, use of taste, smell and touch, fear/irritability, verbal communication, non-verbal communication, mental activities and general impressions. are collected and each item is graded with a half-point score between 1 and 4. CARS is a validated and reliable scale in our country (İNcekaŞ GassaloĞLu et al. 2016).

Gilliam Autistic Disorder Rating Scale-2 (Gilliam Autism Rating Scale-2, TV-GARS-2) was filled in by one of the parents of children with ASD (mother or father; the parent with better knowledge about the child was preferred). GARS-2-; It consists of three subscales, which include items that include specific, observable and measurable behaviors, including Stereotype Behaviors, Communication and Social Interaction. There are a total of 42 items in GARS-2, 14 of which are in each subscale. Items are scored with a four-point rating based on opinion. As a result of the evaluation of GARS-2, a standard score called Autistic Disorder Index (Score) is obtained as in individual intelligence tests (Gilliam 2006). The Turkish version of GARS-2 is a valid and reliable scale(Diken et al. 2012).

Collection of Blood Samples and Laboratory Work

Blood was collected from volunteers at the blood collection unit of Gaziantep University Medical Faculty Hospital. The blood samples were placed in a vacuumed plastic gel tube with a yellow tip and centrifuged at 4000 rpm for 10 minutes, after which the serum was separated and stored at -80 degrees Celsius until the analysis period. TGF- and USP9X levels were determined in the Medical Biochemistry Department of Gaziantep University and analyzed using an ELISA approach with a ready-made kit after the samples were completed.

The sample group consists of a total of 82 individuals, including 41 children with ASD between the ages of 3–12 and 41 healthy children from the same age group. The mean age of the control group was 7.83 (± 2.37), and the mean age of the case group was 7.20 (± 2.66), and no statistically significant difference was found (p > 0.05). When the gender distribution is examined, 65 (79.2%) of 82 children are boys and 17 (19.1%) are girls. The male/female ratio is 4/1. There was no statistically significant difference between the case and control groups when the children were evaluated in terms of gender (p > 0.05). When the case and control groups were evaluated in terms of delivery type, no statistically significant difference was found (p > 0.05). Considering the developmental stages reported by the parents, 3 (7.4%) children in the control group experienced delays in their developmental stages, while 38 (92.6%) children completed their developmental stages on time; In the case group, it was learned that the developmental stages of 37 (90.2%) children were delayed, and only 4 (9.8%) children completed their developmental stages on time. When the walking, speaking and toilet training developmental stages were examined on time or delayed between the case and control groups, a statistically significant difference was found in terms of all 3 developmental stages (p < 0.05), and all 3 developmental stages were delayed more in the case group compared to the control group. A statistically significant difference was found between the case and control groups in terms of working status of the mother and father (p < 0.05). When the educational status of the parents of the children in the case and control groups was evaluated, no statistically significant difference was found (p > 0.05). No statistically significant difference was found when the two groups were compared according to the presence or absence of psychiatric disease in the mothers and fathers of the participants (p > 0.05). When the case and control groups were evaluated in terms of hand use, a statistically significant difference was found (p < 0.05). While 40 (97.5%) of the participants in the control group were right-handed, 1 (2.5%) was left-handed. On the other hand, 27 (65.8%) of the participants in the case group were right-handed, 9 (21.9%) were left-handed, and 5 (12.1%) were using both hands. When the first degree relatives of the case and control groups were evaluated in terms of psychiatric disease states, a statistically significant difference was found (p < 0.05). While 14 (34.1%) of the participants in the control group had a family history of psychiatric disease, 27 (65.9%) had no family history of psychiatric disease. While 25 (60.9%) of the participants in the case group had a family history of psychiatric disease, 16 (39.1%) had no family history of psychiatric disease. When the case and control groups were evaluated in terms of the presence of ASD in the family, a statistically significant difference was found (p < 0.05). While 1 (2.5%) of the participants in the control group had a family history of ASD, 40 (97.5%) had no family history of ASD. While 10 (24.3%) of the participants in the case group had a family history of ASD, 31 (75.7%) had no family history of ASD. When the case and control groups were evaluated in terms of the presence of ADHD in the family, no statistically significant difference was found (p > 0.05). The continuation of the data is given in Table 1 in detail.

When the GARS-2 scale scores filled in by the parents of the patients are calculated and the scores corresponding to the Autistic Disorder Index are examined, 1 (2.5%) patient is unlikely to have ASD, 3 (7.3%) patients are likely to have ASD, and 9 (21.9%) patients have a high probability of ASD. high (mild level), 17 (41.4%) patients had a very high probability of ASD (moderate), and 11 (26.8%) patients had a very high probability of ASD (severe). According to the CARS scores filled by the clinician, it was seen that 18 (43.9%) patients met mild-moderate ASD scores and 23 (56.1%) patients met severe ASD scores. (Table 2)

As a result of the comparison of USP9X and TGBb levels of children with ASD and healthy controls, it was found that the level of USP9X was statistically significantly higher in children with ASD (11.94 vs 8.77, p: 0.001), while the level of TGFb did not differ significantly between the two groups. (Table 3) In the correlation analysis between the CARS and GOBDÖ scores of the cases and the USP9X and TGFb values, no statistically significant difference was found except for the 7th question of CARS, the Visual response subscale. USP9X and TGFb correlations were r:0.337, p:0.031, respectively; There was a moderate positive correlation with r: 0.390, p: 0.012. According to this result, it was observed that the USP9X and TGFb values increased as the eye contacts of the cases became abnormal.

The levels of USP9X and TGFb in the sera of children with ASD and matched healthy controls were investigated in this study, as well as the association between these markers and the severity of ASD. In our research, we discovered a statistically significant difference in USP9X levels between children with ASD and healthy controls, as well as a link between decreased eye contact and an increase in USP9x and TGFb levels.

When looking through the literature, it is clear that demographic factors like age and gender are frequently matched in order to control the effect of age and gender on study results when selecting the sample. In our study, groups were matched by age and gender, and case and control groups were chosen, with no statistical differences found between them. When comparing the case and control groups in terms of total development steps, we discovered a statistically significant difference. When the case and control groups were compared in terms of mother and father working situations, a statistically significant difference was found. According to a study conducted in the United States, the prevalence of ASD is higher in states with a high socioeconomic position (Thomas et al. 2012). In our investigation, a statistically significant difference in hand dominance was discovered between the case and control groups. Left-handedness, or the lack of hand dominance, was shown to be more common in the case group than the control group. This finding backs up prior research into how children with autism use their hands. When we look at the research on this topic, we can find that left hand dominance and two-handedness rates have risen in specific disease groups, compared to the general population. When the first degree relative was assessed for psychiatric disease in our investigation, a statistically significant difference was discovered between the case and control groups. In a large case-control study conducted in Finland, psychiatric disorders were found to be more common in first-degree relatives of children with ASD (Jokiranta et al. 2013). In our study, a statistically significant difference was found between the case and control groups in terms of the presence of ASD in the family. Studies show that close relatives of a child with ASD have a significantly increased risk of ASD.

When the USP9X and TGFb levels were compared between the case and control groups in our investigation, the USP9X level was found to be significantly higher, but there was no significant change in TGFb levels. The process of neural stem cells dividing, migrating, and differentiating is known as neurogenesis. Although the great majority of neural stem cells are depleted shortly after birth, a population of these cells exists in two primary places inside the cerebral cortex that supports lifelong neurogenesis. The lateral ventricles and the hippocampus dentate gyrus are covered by the subventricular region. Learning and memory are aided by neurogenesis in the adult dentate gyrus, and impairments in neurogenesis have been linked to cognitive decline. USP9X, a deubiquitinating enzyme, has recently been discovered to play a role in neural stem cell identity. Furthermore, Usp9x-deficient animals have a significantly reduced total size of the adult dentate gyrus(Oishi et al. 2016). TGFb signaling is lost in USP9X missense variants. In neurodevelopmental signaling pathways, this results in a reduction in substrate levels. While the USP9X missense mutation inhibits TGFb-mediated axonogenesis, it causes mTOR-mediated decreased neural stem cell proliferation and notch signaling disruption with wnt-mediated neural proliferation and differentiation. Axonogenesis is inhibited by TGFB, resulting in corpus callosum agenesis, enlarged ventricles (ventriculomegaly), and a variety of brain abnormalities. This causes hypotonia, motor defects, and visual defects, as well as decreased grip strength, body tone, gait, and visual location (Johnson et al. 2020). Mutations in USP9X are thought to cause neurodevelopmental problems in mammals, including humans. Intellectual disability, autism, epilepsy, and lissencephaly have all been linked to the loss of USP9X function (Oishi et al. 2016).

In a 2007 study by Kyoko et al., it was said that there may be a link between low tgf b levels and autism (Okada et al. 2007). Despite the fact that there was no significant difference in tgf b values between the case and control groups in our study, the case group's tgf b level was lower. According to findings from an Egyptian study, there may be a link between the severity of autistic symptoms and tgf b levels. As the TGFb level dropped further, behavioral symptoms such as adaptive behavior, stereotypy, irritability, and hyperactivity could worsen (Hashim et al. 2013).

While prior research suggests that low TGFB levels in the blood of children with autism are linked to the severity of the disease, TGFb levels could be employed as an early screening tool for ASD. Irritability, hyperactivity, stereotyped behavior, social disengagement, and incorrect speech have all been found to improve when risperidone is used with pentoxifylline, a proinflammatory cytokine inhibitor and immunomodulatory medication (Akhondzadeh et al. 2010). Another adjunctive trial in children with ASD combining risperidone and pioglitazone found that the adjuvant therapy group had less irritability, social withdrawal, and hyperactivity than the risperidone-only group, implying pioglitazone's antineuroinflammatory effects (Ghaleiha et al. 2015). Furthermore, when combined with the nonsteroidal anti-inflammatory medicine celecoxib, risperidone has been shown to generate significant regressions in the irritability, social disengagement, and stereotypy subscales of autism (Asadabadi et al. 2013).

The levels of USP9x and TGFb were shown to be linked to the limiting of eye contact. Eye contact limitation is one of the red flag signs of ASD, and it is well recognized that eye contact can be compromised even in moderate cases. Although no link was discovered between ASD severity and USP9X, the positive correlation with eye contact led us to believe that USP9X could be linked to ASD diagnosis regardless of severity. However, more research is required on this topic.

The fact that blood parameters are reliant on peripheral blood is the most significant limitation of our research. The study's sectional structure limits our ability to identify how the USP9X level changes over time, and we are unable to do so. Operating constraints can also be attributed to the low number of cases.

As a result, this is the first study to look at USP9X in children with ASD. It has been discovered that the existence of ASD, regardless of severity, affects USP9X levels, which are greater in children with ASD. Today, as DUBs are being evaluated as possible therapy targets in ASD, USP9X should be addressed as well, and additional study should be conducted.

Funding

This article has not funding.

Conflict of interest

The authors have no conflict of interest

Author Contributions

M.K. made substantial contributions to the conception, M.K., F.S.T and S.T. design of the work; Z.C. and Z.H. the acquisition, analysis, M.K. interpretation of data; F.S.T., Z.C. and S.T. the creation of new software used in the work; M.K. and Z.H. have drafted the work or substantively revised it

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TABLE 1 Comparison of participants' sociodemographic information

Patient

Control

p

Age

7.20 (±2.66)

7.83 (±2.37)

0.259

Sex

Female

Male

6 (14.6%)

35 (85.4%)

11 (26.8%)

30 (73.2%)

0.173

Type of delivery

Normal delivery

C-section

14 (34.1%)

27 (65.9%)

21 (51.2%)

20 (48.8%)

0.118

General Developmental Steps (Speaking, Walking, Toilet Training)

On time

delayed

4 (9.8%)

37 (90.2%)

38 (92.7%)

3 (7.3%)

<0.001

Mother working status

Not working

Working

40 (97.6%)

1 (2.4%)

34 (82.9%)

7 (17.1%)

0.026

Father working status

Not working

Working

7 (17.1%)

34 (82.9%)

0 (0%)

41 (100%)

0.012

Mother’s education

High education level

Low wducation level

10 (24.4%)

31 (75.6%)

17 (41.5%)

24 (58.5%)

0.158

Father’s education

High education level

Low education level

19 (46.3%)

22 (53.7%)

14 (34.2%)

27 (65.8%)

0.075

Maternal psychiatric illness

No

Yes

33 (80.5%)

8 (19.5%)

32 (78.0%)

9 (22.0%)

0.785

Paternal psychiatric illness

No

Yes

32 (78.0%)

9 (22.0%)

37 (90.2%)

4 (9.8%)

0.131

Hand dominance

Right

Left

No dominance

27 (65.9%)

9 (22%)

5 (12.2%)

40 (97.6%)

1 (2.4%)

0 (0%)

0.001

Psychiatric illness in first degree relative

No

Yes

16 (39%)

25 (61%)

27 (65.9%)

14 (34.1%)

0.015

A family history of ASD*

No

Yes

31 (75.6%)

10 (24.4%)

40 (97.6%)

1 (2.4%)

0.004

A family history of ADHD**

No

Yes

27 (65.9%9

14 (34.1%)

33 (80.5%)

8 (19.5%)

0.135

Note. Autism Spektrum Disorder*; Attention Deficit Hyperactiviy Disorder**

TABLE 2 ASD Severity of Cases According to CARS and GARS.

		n	%
Decision guide	ASD* is unlikely to be seen	1	2.4
	ADS is possible	3	7.3
	ASD Possibility Quite High-Light	9	21.9
	ASD Probability Fairly High-Medium	17	41.5
	ASD Possibility Quite High-Heavy	11	26.9
GARS-TV2** total point	32.15 (±6.7)
CARS results***	Low-medium	18	43.9
CARS results***	Extremely Autistic	23	56.1
CARS total point	37.6 (±5.9)

Note. Autism Spektrum Disorder*; Gilliam Autism Rating Scale-2**; Childhood Autism Rating Scale***

TABLE 3 Comparison of USP9X and TGFb values between patient and control groups

	Patient	Control	p
USP9X	11.94 (10.41-13.48)	8.77 (7.27-10.26)	0.001
TGFb	466.28 (420.18-512.37)	503.93 (461.48-546.38)	0.209

No competing interests reported.

Comparison of Serum Usp9x and TGF-ß Levels in Children With Asd Spectrum Disorders With Healthy Controls

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Abstract

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Methods

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Discussion

Lay Summary

Introduction

Material And Methods

Results

Discussion

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Tables

Additional Declarations

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