Gene Reports: A B A A A A C D D
Gene Reports: A B A A A A C D D
Gene Reports: A B A A A A C D D
Gene Reports
journal homepage: www.elsevier.com/locate/genrep
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
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 many organs and tissues, while their expressions are under the control
of tissue-specific patterns (Böttner et al., 2000). Additionally, TGF-βs
Autism spectrum disorder (ASD) is a life-long shift in the neuro- can perform various functions such as differentiation, development,
genesis that interferes with the socialization, comprehension, percep- angiogenesis, and hematopoiesis as well in immune system functions
tion, and compulsion that would concern 1–2% of children in the world (Böttner et al., 2000; Massague, 1998; Baranova et al., 2020). Also,
(Wing and Gould, 1979; Christensen et al., 2016). It is linked to various TGF-βs are subdivided into three major subfamilies: TGF-β1, TGF-β2,
factors (Kandeel et al., 2020), but not defined yet. In spite of numerous and TGF-β3 that act via the same receptor signaling systems
advances that have been achieved to determine the exact cause of ASD, (Cordenonsi et al., 2007). The most important TGF-β1 has receptor in
still there is no a clear explanation for its etiology (Gottfried et al., central nervous system promoting brain development (Böttner et al.,
2015). For a long time, ASD's feature known as immunological dys- 2000). As an evidence to support that idea an experiment was done in
function had been observed for modifications of the central and per- which TGF-β1 was knocked out from the mice, resulted in an extreme
ipheral immune system. Therefore, inadequate activation of the im- deficit in cortical development with broad raise in nerve cell mortality
mune cells, cytokine/chemokine instability, autoantibodies production, and microgliosis (Brionne et al., 2003). In another study, some re-
and higher blood-brain barrier permeability are important examples searchers demonstrated that TGF-β1 level was observed low in the
that could be involved in these modifications for understanding serum of autistic children (Okada et al., 2007a), and it was proved that
(Gladysz et al., 2018). reduction of the cytokine during brain development triggering dis-
Transforming growth factor beta (TGF-βs) polymorphisms play a rupting immune system and neuropsychological disorders. However,
significant part in driving child to ASD (Baranova et al., 2020; Ashwood some authors showed variation in the level of TGF-β1 as feedback
et al., 2008a). TGF-βs are pleotropic cytokines that promote growth in 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: [email protected] (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). know the point mutation at codon 10 and codon 25 by using the fol-
The TGF-β1 gene locates on chromosome 19 q13.1-3. Its coding lowing primers (Khakzad et al., 2015).
sequence involves three polymorphisms: 869 T/C, 915 G/C and 1628
C/A while its promoter region compromises two polymorphisms: 800 5′-GTGCTGACGCCTGGCCG-3′ (G25),
G/A and 509 C/T (Fujii et al., 1986; Awad et al., 1998). In the present 5′-GTGCTGACGCCTGGCCC-3′ (C25),
study, 869 T/C and 915 G/C polymorphisms were selected in relation to 5′-GGCTCCGGTTCTGCACTC-3′ (common codon 25).and.
ASD which are located in exon 1 on TGF-β1 gene. The point mutation of 5′-CGGGCTGCGGCTGCTGCC-3′ (T10),
869 T/C causes substitution of arginine (G) with proline (C) at codon 25 5′-CGGGCTGCGGCTGCTGCT-3′ (C10),
while point mutation of 915 G/C at codon 10, results in replacement of 5′-TTTCGTTGTGGGTTTCCACCATTAG-3′ (common codon 10)
leucine (T) by proline (C). So polymorphism of these two regions affects
the concentration of TGF-β1 in the serum of autistic children (Fujii The thermocycler was optimized using PCR tubes with 20 μl reac-
et al., 1986; Awad et al., 1998; Hutchinson et al., 1998; Suthanthiran tion mixtures compromising 2 μl of genomic DNA, 1 μl of each common,
et al., 2000). forward, and reverse primers (for each codon), 10 μl of master mix
The arginine and leucine in both codon 10 and 25 results in high (Ampliqon, Denmark) and 5 μl of deionized distill water. MiniAmp
production of TGF-β1 while proline in these codons causes low pro- thermal cycler (Applied biosystem, USA) have been applied for ampli-
duction of the cytokine. Some controversies are found regarding codon fication of DNA which optimized by the following conditions: initial
10, (Hutchinson et al., 1998) found that leucine is high producer of denaturation for 10 min at 94 °C followed by 35 cycles of denaturation
TGF-β1, conversely genotype (Suthanthiran et al., 2000; Yamada et al., (30 s at 94 °C), annealing (20 s at 65 °C), extension (30 s at 63 °C) and a
1998) revealed in their study that proline in codon 10 is correlated with final extension at 72 °C for 5 min. After that 2% agarose gel was used
high amounts of TGF-β1. There is no doubt that polymorphism of 869 for loading PCR products and run at 130 mV for about 30 min and
T/C and 915 G/C alter the concentration of serum TGF-β1 and mod- visualized under gel doc (Bio Rad, USA).
ulate the susceptibility of the child to develop autism (Okada et al.,
2007a; Vargas et al., 2005). 2.4. Statistical analysis
In this study we investigated to know whether TGF-β1 poly-
morphisms at codon 10 and codon 25 are risk factor for causing ASD The Graph-Pad Prism 6 (La Jolla, CA, USA) was used for doing
among Kurdish children in Erbil, Iraq. statistical analysis. For determine the association of genotypes, alleles
and haplotypes to ASD in control and autistic children, chi square was
2. Material and methods used. Odd ratio (OR) and confidence interval (CI) were calculated in chi
square. For knowing any deviation from Hardy-Weinberg Equilibrium
2.1. Subjects (Chang, 2005), also chi square was utilized. For socioeconomic para-
meters, data were normally distributed since they fulfilled criteria of
80 children have been enrolled in this study which has been split parametric tests (Kolmogorov-Smirnov, D Agostino and Shapiro-Wilk).
into two groups: 40 autism groups (mean age = 7.088 ± 0.447) and Independent t-test was performed to know the difference between
40 healthy children (mean age = 7.925 ± 0.364), with no statistical parameters of socioeconomic in controls and autistic group. The P-value
difference in their age (Table 1). The children in the autistic group were less than 0.5 was considered as statistically significant.
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 3. Results
at −80 °C before extraction of DNA. The hospital's Ethics Committee
approved the human subjects protocol and the Research protocol per- 3.1. Demographic and anthropometric characteristic of participants
mitted by a University Research Board. All subjects gave agreement to
the study participation. 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
2.2. DNA extraction
7.925 ± 0.364 years; yielding no statistically significant difference in
their ages. Regarding to BMI, the BMI of ASDs patients were sig-
The DNA was isolated from the collected blood samples according
nificantly lower than that of control participants.
kit manufacturers' protocol (Add prep Genomic DNA extraction Kit;
Add bio, Daejeon, South Korea).
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 %
ASDs: autism spectrum disorders OD: odds ratio CI: confidence interval HWE: Hardy-Weinberg-Equilibrium.
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. Gene Reports 21 (2020) 100843
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Informed consent 2 diabetes and diabetic nephropathy. Acta Biochim. Biophys. Sin. 45 (4), 330–338.
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Khakzad, M.R., Salari, F., Javanbakht, M., Hojati, M., Varasteh, A., Sankian, M., et al.,
2015. Transforming growth factor beta 1 869T/C and 915G/C polymorphisms and
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