Gene Reports 21 (2020) 100843

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Gene Reports
journal homepage: www.elsevier.com/locate/genrep

TGF-β1polymorphism is an inflammatory disease specifier in autism T

spectrum disorders?
Shukur Wasman Smaila, Mahdi Khaled Qadirb, Mustafa Fahmi Rajaba, Iman Idris Ismaila,
Omer Sardar Tahaa, Mudhir Sabir Shekhaa,c, Musarrat Abbas Khand, Muhammad Safdard,
⁎

a
Department of Biology, College of Science, Salahaddin University-Erbil, Iraq
b
Department of Physiotherapy, Erbil Health Technical College, Erbil Polytechnic University, Erbil, Iraq
c
General Directorate of Scientific Research Center, Salahaddin University-Erbil, Iraq
d
Department of Breeding and Genetics, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan

ARTICLE INFO ABSTRACT

Keywords: Background: Autism spectrum disorder (ASD) is a neuropsychological condition that affects brain development
Autism spectrum disorder causing children suffers from social interaction problems, impairment in language and expression, and exhibit
Polymorphism excessive repetitive behaviors. Transforming growth factor beta 1 (TGF-β1) polymorphisms play a significant
Transform growth factor beta 1 part in disease development and progression. The objective of this study is to investigate the involvement of TGF-
PCR-SSP
β1 polymorphism in the development of ASD at two positions; codon 10 and codon 25 in particular.
Method: For this study, 80 samples were collected using sequence-specific primed polymerase chain reaction
technique (PCR-SSP), divided into two groups, 40 autistic children and 40 controls of matched age and gender.
Result: This study showed that there is no association between TGF-β1 codon 10 and ASD, but a significant
association of TGFβ1 in codon 25 and development of ASD.
Conclusion: The polymorphism of TGFβ1 at codon 25 could play an important role in the developing and pro-
gressing of ASD among Kurdish children in Erbil, Iraq.

1. Introduction
Autism spectrum disorder (ASD) is a life-long shift in the neuro-
genesis that interferes with the socialization, comprehension, percep-
tion, and compulsion that would concern 1–2% of children in the world
(Wing and Gould, 1979; Christensen et al., 2016). It is linked to various
factors (Kandeel et al., 2020), but not defined yet. In spite of numerous
advances that have been achieved to determine the exact cause of ASD,
still there is no a clear explanation for its etiology (Gottfried et al.,
2015). For a long time, ASD's feature known as immunological dys-
function had been observed for modifications of the central and per-
ipheral immune system. Therefore, inadequate activation of the im-
mune cells, cytokine/chemokine instability, autoantibodies production,
and higher blood-brain barrier permeability are important examples
that could be involved in these modifications for understanding
(Gladysz et al., 2018).
Transforming growth factor beta (TGF-βs) polymorphisms play a
significant part in driving child to ASD (Baranova et al., 2020; Ashwood
et al., 2008a). TGF-βs are pleotropic cytokines that promote growth in
many organs and tissues, while their expressions are under the control
of tissue-specific patterns (Böttner et al., 2000). Additionally, TGF-βs
can perform various functions such as differentiation, development,
angiogenesis, and hematopoiesis as well in immune system functions
(Böttner et al., 2000; Massague, 1998; Baranova et al., 2020). Also,
TGF-βs are subdivided into three major subfamilies: TGF-β1, TGF-β2,
and TGF-β3 that act via the same receptor signaling systems
(Cordenonsi et al., 2007). The most important TGF-β1 has receptor in
central nervous system promoting brain development (Böttner et al.,
2000). As an evidence to support that idea an experiment was done in
which TGF-β1 was knocked out from the mice, resulted in an extreme
deficit in cortical development with broad raise in nerve cell mortality
and microgliosis (Brionne et al., 2003). In another study, some re-
searchers demonstrated that TGF-β1 level was observed low in the
serum of autistic children (Okada et al., 2007a), and it was proved that
reduction of the cytokine during brain development triggering dis-
rupting immune system and neuropsychological disorders. However,
some authors showed variation in the level of TGF-β1 as feedback
mechanism to prevent inflammation and neuronal degeneration during

Abbreviations: ASD, autism spectrum disorder; TGF-β1, transforming growth factor beta 1; PCR-SSP, sequence-specific primed polymerase chain reaction
⁎
Corresponding author: Department of Breeding and Genetics, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan
E-mail address: msafdar@cuvas.edu.pk (M. Safdar).

https://doi.org/10.1016/j.genrep.2020.100843
Received 28 July 2020; Received in revised form 10 August 2020; Accepted 19 August 2020
Available online 21 August 2020
2452-0144/ © 2020 Elsevier Inc. All rights reserved.
S.W. Smail, et al. Gene Reports 21 (2020) 100843

brain injuries (Lindholm et al., 1992; Prehn et al., 1993).
The TGF-β1 gene locates on chromosome 19 q13.1-3. Its coding
sequence involves three polymorphisms: 869 T/C, 915 G/C and 1628
C/A while its promoter region compromises two polymorphisms: 800
G/A and 509 C/T (Fujii et al., 1986; Awad et al., 1998). In the present
study, 869 T/C and 915 G/C polymorphisms were selected in relation to
ASD which are located in exon 1 on TGF-β1 gene. The point mutation of
869 T/C causes substitution of arginine (G) with proline (C) at codon 25
while point mutation of 915 G/C at codon 10, results in replacement of
leucine (T) by proline (C). So polymorphism of these two regions affects
the concentration of TGF-β1 in the serum of autistic children (Fujii
et al., 1986; Awad et al., 1998; Hutchinson et al., 1998; Suthanthiran
et al., 2000).
The arginine and leucine in both codon 10 and 25 results in high
production of TGF-β1 while proline in these codons causes low pro-
duction of the cytokine. Some controversies are found regarding codon
10, (Hutchinson et al., 1998) found that leucine is high producer of
TGF-β1, conversely genotype (Suthanthiran et al., 2000; Yamada et al.,
1998) revealed in their study that proline in codon 10 is correlated with
high amounts of TGF-β1. There is no doubt that polymorphism of 869
T/C and 915 G/C alter the concentration of serum TGF-β1 and mod-
ulate the susceptibility of the child to develop autism (Okada et al.,
2007a; Vargas et al., 2005).
In this study we investigated to know whether TGF-β1 poly-
morphisms at codon 10 and codon 25 are risk factor for causing ASD
among Kurdish children in Erbil, Iraq.
2. Material and methods
2.1. Subjects
80 children have been enrolled in this study which has been split
into two groups: 40 autism groups (mean age = 7.088 ± 0.447) and
40 healthy children (mean age = 7.925 ± 0.364), with no statistical
difference in their age (Table 1). The children in the autistic group were
diagnosed with DMS-5 criteria by a pediatric neurologist. Three ml of
blood was taken from them and then moved into EDTA tube and stored
at −80 °C before extraction of DNA. The hospital's Ethics Committee
approved the human subjects protocol and the Research protocol per-
mitted by a University Research Board. All subjects gave agreement to
the study participation.
2.2. DNA extraction
The DNA was isolated from the collected blood samples according
kit manufacturers' protocol (Add prep Genomic DNA extraction Kit;
Add bio, Daejeon, South Korea).
know the point mutation at codon 10 and codon 25 by using the fol-
lowing primers (Khakzad et al., 2015).
5′-GTGCTGACGCCTGGCCG-3′ (G25),
5′-GTGCTGACGCCTGGCCC-3′ (C25),
5′-GGCTCCGGTTCTGCACTC-3′ (common codon 25).and.
5′-CGGGCTGCGGCTGCTGCC-3′ (T10),
5′-CGGGCTGCGGCTGCTGCT-3′ (C10),
5′-TTTCGTTGTGGGTTTCCACCATTAG-3′ (common codon 10)
The thermocycler was optimized using PCR tubes with 20 μl reac-
tion mixtures compromising 2 μl of genomic DNA, 1 μl of each common,
forward, and reverse primers (for each codon), 10 μl of master mix
(Ampliqon, Denmark) and 5 μl of deionized distill water. MiniAmp
thermal cycler (Applied biosystem, USA) have been applied for ampli-
fication of DNA which optimized by the following conditions: initial
denaturation for 10 min at 94 °C followed by 35 cycles of denaturation
(30 s at 94 °C), annealing (20 s at 65 °C), extension (30 s at 63 °C) and a
final extension at 72 °C for 5 min. After that 2% agarose gel was used
for loading PCR products and run at 130 mV for about 30 min and
visualized under gel doc (Bio Rad, USA).
2.4. Statistical analysis
The Graph-Pad Prism 6 (La Jolla, CA, USA) was used for doing
statistical analysis. For determine the association of genotypes, alleles
and haplotypes to ASD in control and autistic children, chi square was
used. Odd ratio (OR) and confidence interval (CI) were calculated in chi
square. For knowing any deviation from Hardy-Weinberg Equilibrium
(Chang, 2005), also chi square was utilized. For socioeconomic para-
meters, data were normally distributed since they fulfilled criteria of
parametric tests (Kolmogorov-Smirnov, D Agostino and Shapiro-Wilk).
Independent t-test was performed to know the difference between
parameters of socioeconomic in controls and autistic group. The P-value
less than 0.5 was considered as statistically significant.
3. Results
3.1. Demographic and anthropometric characteristic of participants
The demographic and anthropometric parameters of both ASDs and
controls were summarized in Table 2. The mean age of ASDs patients
was 7.088 ± 0.447 years, and of the control group was
7.925 ± 0.364 years; yielding no statistically significant difference in
their ages. Regarding to BMI, the BMI of ASDs patients were sig-
nificantly lower than that of control participants.
3.2. Association analysis

2.3. Polymorphism analysis In this case-control study, 40 ASDs patients and 40 controls com-
pared according to two SNPs on the TGF-β gene one of them on codon
Single Specific Primer-PCR (SSP-PCR) technique was applied to 10 and the other on codon 25 by SS-PCR method. The genotype, hap-
lotypes and allele frequencies of both SNPs on codon 10 and codon 25
Table 1 of TGF-β gene were given in Tables 2, and 3. The genotype distribution
Demographic characteristics of children with ASD and controls. of both SNPs codon 10 and codon 25 of the TGF-β gene were in
(N = 40) P value agreement with Hardy-Weinberg Equilibrium with a p-value of 0.59
and 0.80 respectively.
Control Autism In case of polymorphism in codon 10 of TGF-β gene, there is not a
Age 7.925 ± 0.364 7.088 ± 0.447 0.150
significant association between ASD and control groups. From the 40
Gender ASD patients, 11 had the TT genotype (27.50%), the 25 TC genotype
Male 19 27 – (62.50%) and 4 the CC genotype (10%). Of the 50 healthy control
Female 21 13 – subjects, 13 had the TT genotype (32.50%), 21 had the TC genotype
Height 1.243 ± 0.021 1.213 ± 0.033 0.422
(52.50%) and 6 had CC genotype (15%), the homozygous (CC) was not
Weight 25.41 ± 1.519 27.57 ± 2.544 0.444
BMI 15.86 ± 0.472 17.84 ± 0.783 0.025 associated significantly with ASDs patients (OR = 2.423,
95%CI = 0.57 to 10.25, p = 0.286), whereas the heterozygous (TC)
BMI: body mass index, ASD: autism spectrum disorders. was also not associated significantly with ASDs patients (OR = 0.710,

2
S.W. Smail, et al. Gene Reports 21 (2020) 100843

Table 2
Genotypes and allele frequencies distribution of TGF-β1 gene in codon 10 869T/C polymorphism in ASD patients and controls.
Polymorphism ASDs (N = 40) Control (N = 40) OR 95% CI P value HWE

No % No %

TT 11 27.50 13 32.50 1 – – 0.59

TC 25 62.50 21 52.50 0.710 0.26 to 1.914 0.498
CC 4 10.00 6 15.00 2.423 0.57 to 10.25 0.286
TC + CC 29 72.50 27 67.5 0.787 0.30 to 2.056 0.625
TT + TC 26 90 34 85 0.620 0.15 to 2.44 0.492
T 47 67.14 47 58.75 1.435 0.73 to 2.80 0.289
C 23 32.86 33 41.25

ASDs: autism spectrum disorders OD: odds ratio CI: confidence interval HWE: Hardy-Weinberg-Equilibrium.

95%, CI = 0.2639 to 1.914, p = 0.498) (Table 3). Table 4

Regarding the polymorphism of codon 25 in TGF-β genotypes,
haplotypes and alleles were associated with an increased risk of ASD as
compared to the control. Regarding haplotypes, we found non-sig-
nificant association in dominant model comparison (GG/GC vs. CC:
OR = 0. 3.353, 95%CI: 0.633 to 17.74; P = 0.1360), but a significant
association was found in the recessive model (GG vs. GC/CC:
OR = 6.00, 95% CI: 2.206 to 16.32; P = 0.0003). Regarding genotypes,
there was significant association in heterozygote model and homo-
zygote variants (GC vs. TT: OR = 6.000, 95%CI: 1.993 to 18.06;
P = 0.0008) and (CC vs. GG: OR = 6.000, 95%CI: 1.086 to 33.16;
P = 0.0255) respectively, if compared to wild homozygote. Moreover,
the carrier frequency of the mutant G allele was significantly higher in
the patient group in comparison to the control group. Finally, there was
statistical difference in the frequencies of the two alleles in codon 25 of
TGF-β (G vs. C: OR = 4.20, 95%CI: 1.882 to 9.372; P = 0.0003)
(Table 4).
4. Discussion
Autism spectrum disorder (ASD) is disorder of nervous system
which affects the psychology and behavior of the children who exhibit
abnormal repetitive movements and skills (Carter and Scherer, 2013). It
affects more male than female (Loomes et al., 2017). The etiologies of
ASD are unknown (Ruthsatz et al., 2015), but it may be affected by
environmental and genetics factors (Chaste and Leboyer, 2012). The
genetics basis of this disease was investigated by cytogenetics, linkage
disequilibrium and polymorphism (Vorstman et al., 2006). There are
many polymorphisms that were studied and confirmed by Genome-
wide association studies (GWAS) (Glessner et al., 2014; Peyre et al.,
2020; Anney et al., 2010; Wang et al., 2009; Weiss and Arking, 2009).
TGF-β is considered the most important candidate that is im-
munosuppressive and anti-inflammatory cytokine (Abbas et al., 2019).
Beside anti-inflammatory, it has critical role in brain development,
neuronal migration, synaptogenesis, microglial control and astrocyte
specializations (El Gohary et al., 2015). Some researchers demonstrated
that TGF-β1 (from other TGF-β) was decreased in ASD children, so the
Demographic and anthropometric parameters of ASDs patients and controls.
Demographic and anthropometric (N = 40) P value
parameters
Control ASDs Patients
Age 7.925 ± 0.364 7.088 ± 0.447 0.150
BMI 15.86 ± 0.472 17.84 ± 0.783 0.025
BMI: body mass index, ASD: autism spectrum disorders.
brain development was impaired and the inflammation was flared-up
(Okada et al., 2007b; Hashim et al., 2013; Moaaz et al., 2019). A study
showed interesting findings such as polymorphisms in TGF-β1 i.e.
-509C/T (rs1800469), −800 TGF-β1 located in promoter region while
+869 T/C (rs1800470), and +915G/C (rs1800471) located in signal
sequences of the cytokine, finally +72 located in un-translated region
(Chen et al., 2016). In our case, polymorphism of +869T/C and
+915G/C (at codon 10 and codon 25 respectively), were selected in
relation to ASD because the SNP of these genes was responsible for
modification the expression of TGF-β1 in the serum (Khakzad et al.,
2015), thereby they affected T lymphocyte activation, brain develop-
ment, and neuronal differentiation (Ashwood et al., 2008). In addition,
the SNP of TGF-β1 in the codon 25 and codon 10 was associated with
many diseases and disorders such as: asthma, diabetes mellitus, cancer
and ASD (Shah et al., 2006; ten Dijke and Hill, 2004). But importantly,
there was no association between codon 10 of TGF-β1 and ASD as
showed in another study in Iran (Okada et al., 2007b). While there was
a strong association between codon 25 polymorphism of TGF-β1 and
ASD, this result was not similar to Khakzad, Salari (Khakzad et al.,
2015) in Iran (Okada et al., 2007b). So we believed that the poly-
morphism of +915G/C of TGF-β1 could be considered as risk factor for
developing ASD in Erbil, Iraq. In another study showed that the
+915G/C polymorphism of TGF-β1 lead to decrease of TGF-β in vitro
and in vivo (Awad et al., 1998; El-Sherbini et al., 2013).
When we looked at the +915G/C polymorphism of TGF-β1 that
lead to substitution of G (arginine) with C (proline) at codon 25 so it
may cause decreasing the circulatory TGF-β in the serum of autistic

Table 3
Genotypes and allele frequencies distribution of TGF-β1 gene in codon 25 915G/C polymorphism in ASD patients and controls.
Polymorphism ASDs (N = 40) Control OR 95% CI P value HWE

No % No %

GG 32 80 16 40 1 – – 0.8
GC 6 15 18 45 0.16 0.05–0.50 0.0008
CC 2 5 6 15 0.16 0.11–1.26 0.02
GC + CC 8 20 24 60 6.000 2.20 to 16.32 0.0003
GG + GC 38 95 34 85 3.353 0.63 to 17.74 0.1360
G 70 87.5 50 62.5 0.23 0.24–0.74 0.0003
C 10 12.5 30 37.5

ASD: autism spectrum disorders OD: odds ratio CI: confidence interval HWE: Hardy-Weinberg-Equilibrium.

3
S.W. Smail, et al.
children (El-Sherbini et al., 2013), results in dys-regulation of immune
balance and increasing neuro-inflammation disorders such as ASD
(Ashwood et al., 2008). Also, the decreasing level of TGF-β leads to
improper brain development and neuron differentiation (Ashwood
et al., 2008). Another study proved that increasing the serum level of
TGF-β1 lead to improve the behaviors of ASD children by Auditory
Integrative Training (Hirose et al., 2002). It could be a good prognostic
factor in autistic children.
In conclusion, this study focused on the significance of TGF-β1 gene
polymorphism in the development of ASD and its role in disease pro-
gression. It showed that TGF-β1 polymorphism in codon 10 was not
significantly associated with ASD, but that TGF-β1 polymorphism in
codon 25 was significantly associated with genotypes containing ASD,
G allele and G allele, and that the C allele/CC genotype could be major
risk factor for ASD and its complications. Nevertheless, one of the
limitations of this research was its small size of samples; in addition to
the children of dissimilar ethnic backgrounds, the findings should be
checked in a population with larger number of sample size. Based on
these findings, further study of other immune response gene(s) poly-
morphisms is therefore required to understand the significance of dif-
ferent genes in ASD and its complications.
Ethical approval
We have followed all ethical approvals for this study.
Informed consent
All authors have read and approved the contents and manuscript.
CRediT authorship contribution statement
Shukur Wasman Smail: Conceptualization, Methodology,
Supervision, Validation, Visualization, Writing - original draft, Writing -
review & editing. Mahdi Khaled Qadir: Conceptualization,
Methodology, Visualization, Writing - review & editing. Mustafa
Fahmi Rajab: Conceptualization, Methodology, Visualization, Writing
- review & editing. Iman Idris Ismail: Methodology. Omer Sardar
Taha: Methodology. Mudhir Sabir Shekha: Methodology. Musarrat
Abbas Khan: Validation. Muhammad Safdar: Conceptualization,
Supervision, Validation, Visualization, Writing - review & editing.
Declaration of competing interest
The authors have declared that no competing interests exist.
Acknowledgment
We thank Hana autism center in Erbil-Iraq for their support for
taking permission from children's parent to give blood samples and
CUVAS, Bahawalpur for logistic support for conducting the research.
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