Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
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GENE POLYMORPHISIM OF TRANSFORMING GROWTH
FACTOR Beta 1(TGF-β1) IN AUTISM SPECTRUM DISORDER
ASD IN BASRAH
Khulood, Abdulrazaq Kaleel* Wijdan, Nazar Ibraheim*
* Department of Microbiology, College of Medicine, University of Basrah, Basrah,
Iraq
Corresponding Author: [email protected]
Key words: ASD, TGFB1, gene polymorphism
ABSTRACT
Transforming growth factor-β1 (TGF-β1) is an important immune regulator
critical for immune homeostasis. Accumulating evidence suggests that TGF-β1 has a
crucial regulatory role in CNS development and potential implications for
neurogenesis in a variety of TGF-β1-related CNS diseases: so the aim of the study to
investigate the association of the TGFβ-1 gene polymorphism with its plasma protein
plasma level (TGFB-1)in ASD patients, It's a case – control study atotal of 94 patients
with ASD, their age ranging from 2 to 13 years and 100 apparently healthy children
were used as a control which were matched by age and sex. TGFβ-1 levels was
measured by ELISA and TGF-β1(Codon 10 +869 C/T) and TGF-β1(Codon 25:
+915*G/C)Gene Polymorphism were detected by specific sets of primers.The mean
value of TGF-β1 was significantly low in the autistic group (95.91pg / mm) as
compare with the control one (117.08 pg / mm) TGF-β1(Codon 10: +869*T/C) gene
polymorphism showed heterogeneous results between autistic group and control
group
INTRODUCTION
Autism Spectrum Disorder (ASD) etiology has long been a widely debated topic
and stills weakly understood (1). Double studies supply proof that ASD
susceptibility may have genetic, heritability, in addition to significant environmental
components (2,3). A strong inflammatory state associated with ASD reported
increasingly in the recent years(4). The inflammatory disorder is also associated with
dysfunction of the immune system (5).
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There is abundant knowledge that disturbance normal levels of cytokine plays
a major role as a risk factor for many neurodevelopmental disorders, including autism
and schizophrenia(6). A significant immune regulator critical to immune homeostasis
is the transformation growth factor-β1 (TGF-β1). Transforming growth factor-β1
(TGF-β1) has been found to play a crucial role in early central nervous system
development. Several studies have illustrated decreased TGF-β1 levels in sera and
brains of autistic children. Two point mutations in the TGF-β1 signal peptide at
869T/C and 915G/C have been reported to influence TGF-β1 expression (7). TGF-
β10has therefore been0generally accepted as a cytokine that make a response to injury
of brain. Many researches have documented increases in TGF-β1 levels in brain and
autistic serum (8,9).
Transforming growth factor Beta (TGFB) represents a family of cytokines
with closely related isoforms, encoded by three different genes. TGFB1,
TGFB2 and TGFB3 are expressed in several central nervous system (CNS) cell types,
including neurons, astrocytes, and microglia (10). The TGF-βs are involved in a
variety of biological functions in cellular activities, fibrosis, and immune responses, in
addition to their crucial roles in tissue homeostasis (11). Accumulating evidence
suggests that TGF-β1 has a crucial regulatory role in CNS development and potential
implications for neurogenesis in a variety of TGF-β1-related CNS diseases (12). TGF-
β1 has been extensively investigated in immunity, because TGF-β1 is
expressed predominantly by various immune cells (13) and deficiency of TGFβ-1
results in fatal systemic autoimmune disease(14).
TGF- β controls the magnitude and type of immune responses against
microbes, and has fundamentally important roles in maintaining immune tolerance
and homeostasis against self- and benign antigens at steady state (15) so the aim of
this study to investigate the association of the TGFβ-1 gene polymorphism with its
plasma protein level in ASD patients.
MATERIAL AND METHODS
This is a case – control study in which (94) Autistic children (patients group)
and(100) apparently healthy control individual (control group) were enrolled from
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the same population living in Basra providence in Iraq during the period between
December 2018 and October 2019.they were matched by age and sex.
Ethical manifestation:
This study received approval by the Primary Health Care Centers manager and
family of both patients and control children. Its procedures and purpose were
explained to all studied population, and every individual member of the
family has obtained written informed consent.
Collection of Blood Samples:
Three mL of venous blood was drawn from each Participants using a sterile
disposable syringe and divided as following :1 ml of the sample was emptied into an
anticoagulant EDTA tube (Ethyline Diamine Tetra Acetic acid) which used in whole
DNA extraction to detected TGFβ-1 gene polymorphisim, and remaining blood
emptied in other EDTA tube , last tube was centrifuged at 3000 rpm for 10 mints and
plasma transferred to sterile plane tube to use in ELISA technique.
Assessment of plasma TGFβ-1 levels:
Plasma levels of TGFβ-1 were evaluated using an ELISA (Elabscience, USA,
Catalog No: E-EL-0162), according to manufacture protocol.
Extraction of Whole DNA:
From patients and control samples, Whole DNA was extracted by using DNA
Blood Mini Kit (Favorgen -Europe Cat.No: HB10.03.10 UK) depend on kit
manufacture supplied with kit, the DNA extraction stored at -20 until use.
Primers used to detect TGF β-1 Genes :
Eight specific primers was used to detect TGF-β1(Codon 10 +869 *C/T)
and TGF-β1(Codon 25: +915*G/C)Gene Polymorphism according to reference
(Bazzaz et al., 2014) which listed in table below: primers were prepared depending on
manufacturer instruction (alpha DNA Company).
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Table (1): Oligonucleotide Primers Sequences Used for PCR Amplification of TGF
β1 Gene Polymorphism(26).
Amplification of TGF β-1 Gene Polymorphism:
The TGF-β1(Codon 10 +869 *C/T) and TGF-β1(Codon 25: +915*G/C)Gene
Polymorphism were studied by using Eppendrofe thermo cycler with specific primers
(Table 1). The PCR reaction mixture was prepared according to reference (26) as in
table (2) below:
Table (2) The Reaction mixture (25 μl) for TGF β1 gene polymorphism.
Primer name Sequence (5’-3’)
TGF-β1(Codon 10: +869*C/T) (Generic)
5′- TCCGTGGGATACTGAGACAC-3′
TGF-β1(Codon 10: +869*C/T) (C allele) 5′- GCAGCGGTA GCAGCAGCG-3′
TGF-β1(Codon 10: +869*C/T) (T allele) 5′- AGCAGCGGTAGCAGCAGCA-3′
TGF-β1(Codon 25: +915*G/C) (Generic) 5′- GGCTCCGGTTCTGCACTC-3′
TGF-β1(Codon 25: +915*G/C) (Gallele ) 5′- GTGCTGACG CCTGGCCG-3′
TGF-β1(Codon 25: +915*G/C) (C allele ) 5′- GTGCTGACG CCTGGCCC-3′
TGF-β1- Internal control (Forward) 5′-GCCTTCCCAACCATTCCCTTA- 3′
TGF-β1- Internal control (Reverse) 5′-TCACGGATTTC TGTTGTGTTTC- 3′
DNA templates 2 μl
Master mix 12.5 μl
Primers( forward, reverse of gene and forward,
reverse of internal control) (50 picomole)
2μl for each primer
Free nuclease D.W 2.5 μl
Final volume 25 μl
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For each single sample the following steps were processed: For detection of TGF-
β1gene polymorphisim codon 10+869 C/T, and codon 25+915 G/C four tubes were
prepared, for detection of (T) allele / (C ) allele, (C) allele and G one respectively.
Internal control Forward and Reverse primers were used to amplify The
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that was counted as control for
the purpose of comparison. All tubes then shacked well by vortex for 10 seconds. The
PCR tubes were transferred to the thermalcycler to start the amplification reaction
according to specific program table (3).
Table(3) PCR amplification program for TGFβ-1 Gene Polymorphism detection.
PCR product was analyzed by gel electrophoresis in 1.5% agarose containing
ethidium bromide 0.5mg/ml.
Statistical analysis:
The Statistical Package of Social Science (SPSS, version 23) were utilized to
examine and processed data. The relation between polymorphism of TGFβ-1with its
protein level in the plasms was analyzed by utilizing χ2 test , and TGFβ-
1concentration was tested for autistic and control group utilizing the T-test.
RESULTS
Stage
Steps Temperature( C˚) Time( sec )
First Denature template 95 1
Second
I Initial denaturation 95
10 cycles
15
II Annealing 65 15
III Extension 72 40
I Initial denaturation 95
20 cycles
20
II Annealing 56 20
III Extension 72 50
Third Final Extension 72 7 min
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Plasma Concentration TGF-β- 1 in Study Groups:
The mean value of TGF-β1 was significantly low in the autistic group (95.91pg
/ mm) as compare with the control one (117.08 pg / mm) as illustrate in Table (4). The
P value was < 0.05 as estimated by Independent T test.
Table (4) Plasma Concentration of TGFβ-1 in the study Population
Autistic group Control group
Test No.=94 No. =100 P value
Mean ± SD Mean ± SD
TGFβ-1
95.91 ± 6.37
117.08 ± 7.58
0.035
Figure (1) Plasma TGFβ-1 Concentration in the Study Population Genetic
polymorphism of TGF-β1 A-(Codon 10: +869*T/C).
The results of the electrophoresis of TGF-β1(Codon 10: +869*T/C) gene
polymorphism amplification were shown two alleles T/ C and 3 genotyping (TT,
TC, CC) in patients and control samples.
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Figure (2) 2.5% Gel Electrophoresis of TGF-β1(Codon 10 T/C): lane 1
Ladder(1.5kb), lane 2&3 patient1, lane 4&5 patient 2and lane 6&7 patient 3
Results of iterative distribution of TGF-β1(Codon 10: +869*T/C) gene
polymorphism showed heterogeneous results between autistic group and control
group, as the T allele was scored in autistic group 85.1% in compared with C allele
which scored 76.6%, while T allele was scored 34% in compared with C allele which
scored 67% in control group figure 3 .The difference in repetitive distribution of T
allele between patients and control group was statistically significant (p value =
0.000). While no significant differences (p value = 0.000) in the iterative distribution
of C when compared with control group , also table 4 showed significant differences
in repetitive distribution of T allele between patients and control group by using
Fisher’s test, and the OR of T allele was 0.249 ( less than one) . While OR of C allele
was 1.2. (more than one) could be considered as preventive fraction (not associated
with the disease).
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Figure (3) Frequency of TGFβ-1 Codon 10 T/C alleles in Study Population.
Results of genetic analysis of TGF-β1(Codon 10 T/C) for SNP-PCR were showed 3
genotypes (TT,TC and CC) in patients and control samples. The results showed
contrast in genotypes repetition between autistic group and control group. As
heterogeneous genotype TC showed higher frequency among autistic patients when
compared to control subjects,(60.6% vs 1% respectively),while homogenous genotype
CC showed lower frequency among autistic patients when compared to control
subjects,(14.9% vs 66% respectively),and TT genotypes scored 24.4% in autistic
patients and 33% was scored in control group figure 4.
Figure 4 Frequency of TGFβ-1 Codon 10 Genotyping in Study Population .
On the other hand the results of the electrophoresis of TGF-β1(Codon 25 G/C) gene
polymorphism amplification were shown two alleles G/ C and 3 genotyping
(GG,GC,CC) in patients and control samples,
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference.
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Figure (5) 2.5% Gel Electrophoresis of TGF-β1(Codon 25 G/C): lane 1
Ladder(1.5kb), lane 2&3 patient1, lane 4&5 patient 2, lane 6&7 patient 3 and
lane 8&9 patient 4.
Results of iterative distribution of TGF-β1(Codon 25 G/C) gene polymorphism
showed heterogeneous results between autistic group and control group, as the G
allele was scored in autistic group 41.5% in compared with C allele which scored
61.7%, while G allele was scored 97% in compared with C allele which scored 34%
in control group figure 5 .The difference in repetitive distribution of G allele and C
allele between patients and control group was statistically significant (p value =
0.000) by chi square test which scored (41.5%, 97% and 61.7%, 34%) respectively .
Also table 5 showed significant differences in repetitive distribution of G allele
between patients and control group by using Fisher’s test, and the OR of G allele was
0.45 and its preventive fraction and not associated with the disease. While OR of C
allele was 0.349 so this allele could be considered as preventive fraction.
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Figure(6) Frequency of TGFβ-1 Codon 25 G/C alleles in Study Population
Results of genetic analysis of TGF-β1(Codon 25 G/C) for SNP-PCR were
showed 3 genotypes (GG,GC and CC) in patients and control samples. The results
showed contrast in genotypes repetition between autistic group and control group. As
homogeneous genotype CC showed higher frequency among autistic patients when
compared to control subjects,(58.5% vs 3% respectively),while heterogeneous
genotype GC showed lower frequency among autistic patients when compared to
control subjects,(3.1% vs 31% respectively),and GG genotypes scored 38.3% in
autistic patients and 66% was scored in control group. figure (7).
Figure (7) Frequency of TGFβ-1 codon 25 G/C Genotyping in Study Population
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Table (5) Distribution of TGF-β1(Codon 10 and codon 25) alleles in study groups.
Table (6) Correlation between TGF-β1 Plasma Level and TGF-β1(Codon 10 and
codon 25 ) Genotyping among the Study Groups
Genotyping Autistic group Control group
Codon 10 T/C No. TGFB-1 P value
Median
No. TGFB-1
Median
TT 23 75.0 33 88.0 0.000
TC 57 79.0 1 55.0 0.000
CC 14 101.0 66 97.0 0.000
Codon 25 G/C
GG 36 74.5 66 86.5 0.000
GC 3 63.0 31 184.0 0.000
CC 55 84.0 3 66.0 0.000
Allele Autistic group Control group X² df OR P
value
Codon 10 T/C
T allele 80 (85.1 %) 34 (34 %) 52.2 1 0.249 0.000
C allele 71 (75.5%) 67 (67%) 2.195 1 1.2 >0.05
Codon25 G/C
G allele 39 (41.5%) 97 (97%) 71.23 1 0.45 0.000
C allele 58 (61.7%) 34 (34%) 12.8 1 0.349 0.000
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The association of the plasma levels of (TGFβ-1) with its genotypes of the
study population summarized in table 6 which demonstrated that there were
significance differences in the quantity of TGF-β-1 genotypes and its patients plasma
concentration in in the study groups.
DISCUSSION
Over the past decade, numerous reports have noted abnormalities or
alterations of immune system activity in autistics; these include increased serum
levels of inflammatory cytokines and factors such as tumor necrosis factor-α (TNF-α),
interferon-γ (INF-γ) and high sensitivity C-reactive protein .TGF-β1 is considered to
be one of the critical immunosuppressive cytokines in immune homeostasis and T cell
activated unresponsiveness (16).
In this study the mean value of TGFβ-1 significantly low in ASD than in control
group which was (95.91 pg/mm, 117.08 pg/mm) respectively. The result was in
agreement with (8, 17), that suggesting that immune responses in ASD may be
inappropriately regulated due to reductions in TGFβ-1 .Low TGFβ-1 levels may lead
to an inappropriate control of the immune response in these children (8).
Such immune dysregulation may predispose to the development of possible
autoimmune responses and/ or adverse neuroimmune interactions during critical
windows in development. Overall, the data suggested that immune dysfunction and
excessive inflammation play important pathophysiologic roles in autism disorders.
Transforming growth factor-β1 (TGF-β1) has been found to play a crucial role in
early central nervous system development. Furthermore, during the brain
development, glial and neuronal cells produce TGF-β1, which plays a crucial role in
the regulation of early CNS development such as astrocyte differentiation
(18). Several studies have illustrated decreased TGF-β1 levels in sera and brains of
autistic children (7,19). (17) found that serum of ASD patients had decreased level
of TGF- β1as well as increased other inflammatory markers. This suggests wide
range of immune dysregulation in ASD, with an improper balance between regulation
and activation, which could have wide reaching consequences for many systems in
the body.
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The role of TGFβ-1 with respect to the immune response is complex. Early during
an immune response, TGFβ-1 has the ability to enhance inflammation. Conversely,
TGFβ-1 has a number of profound down-regulatory effects on T and B cell
development and function, as well as the ability to modulate the differentiation and
activation status of NK cells, dendritic cells, monocytes/macrophages, granulocytes
and mast cells (20) .All these facts can give a hints of the effect of low level of TGFβ-
1 on the immunity and the general health of ASD children.
Given the key role of TGF-β1 in brain development and inflammation, we
investigated the association between TGF-β1 gene polymorphisms and autism.
Consequently, estimated alleles and genotypes frequencies were compared between
autistic patients and normal controls in Basrah providence population. We found
association between the TGF-β1 gene polymorphisms and autism.To date, several
functional single-nucleotide polymorphisms (SNPs) of TGF-β1 have been reported.
Particularly, the −509C/T (rs1800469), codon 25G/C (rs1800471), and codon 10 T/C
(rs1982073).
SNPs are the most widely evaluated polymorphisms (21). It has been
demonstrated that these functional SNPs are associated with the inter-individual
differences of TGF-β1 expression (22, 23). The above facts suggest that −509C/T,
codon 25G/C, and codon 10 T/C SNPs may contribute to TGF-β1-mediated immune
response.
In our study there is a moral repetition result of TC genotype in TGF-β1 gene
polymorphisms codon 10 +860 T/C in autistic patients compared with control
samples( the differences highly significance) : this may give a hint that there is an
association between this genotype and other predisposing factors in causing the
disease ,TC genotype can considered as genetic marker associated with autism
disease, in addition to that interestingly, the TC, allele have been associated with
higher TGF-β1 expression so we found a significant correlation between the plasma
level of (TGF-β) and the TC genotype of Codon 10 in autistic group
In compared to the control group , so the result in harmony with (23) who
published that Transforming growth factor β (TGF-β) signaling pathways are
ubiquitous and essential regulators of cellular processes including proliferation,
differentiation, migration, and survival, as well as physiological processes, including
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embryonic development, angiogenesis, and wound healing. However, alterations in
these pathways, including either germ-line or somatic mutations or alterations in the
expression of members of these signaling pathways often result in human disease.
While (7) reported that the polymorphisms in TGF-β1 gene may not play an
important role in the development of autism. The understanding of mechanisms
behind various signaling pathways in the etiology of ASD may help to facilitate the
identification of potential therapeutic targets and design of new treatment methods
(25).
Another crucial SNP locus is codon 25G/C which may impact the production of
TGF-β1. we found that the CC genotype significantly higher in autistic patients than
the control group and the frequency of allele C at TGF-B1 codon 25 was significantly
higher in patients with ASD than in healthy controls.
Also we found that The plasma level of TGF-β1 in autistic patient with the G allele
were significantly lower than those with the C allele this finding followed the role
of (25) which believed that, the transition of G to C may be correlated with the
reduced level of TGF-β1 in vitro (26).
Results of TGF-β1 gene polymorphism in both codon showed that C allele
strongly associated with risk of autistic disease and interestingly, the C, allele have
been associated with higher TGFβ-1 expression which may link with the
immunological dysregulation . It has also been demonstrated that the TC of codon 10
and CC of codon 25 haplotypes are associated with ASD.
On the other hand, the G allele of TGFβ-1 codon 25 polymorphism may be
interpreted as a protective allele against ASD, Additionally, this polymorphism may
be in linkage disequilibrium with other SNPs within or outside the TGFβ-1 gene, only
acting as a marker of an extended haplotype.
Theoretically, it is possible that subjects with the high TGF-β1 producer phenotype
which is associated with the codon 25 G allele present over suppression in the human
immune response. This mechanism may result in the correlation of the polymorphism
of codon 25G/C and disease (27)
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