Connection of TF and TCF4 Gene Polymorphisms with ASD

Though the prevalence of autism spectrum disorder (ASD) is increasing day by day, there is still a lack of a proper way to diagnose or prevent ASD. There is no study carried out in the Bangladeshi children with ASD to evaluate the association of Transferrin (TF) and Transcription Factor 4 (TCF4) genetic polymorphisms. This genetic association study was designed to explore the association of rs1867503 polymorphism of TF and rs9951150 polymorphism of TCF4 genes with ASD. We collected blood from 96 children with ASD and 118 healthy children of very similar age differences. Genotyping of these SNPs was performed by the PCR-RFLP method. SPSS (version 16) was used to estimate the odds ratio (OR) and their 95% condence intervals (CI). The frequency of mutant allele G for rs1867503 and rs9951150 polymorphisms was found 48% and 44%, respectively. In our analysis, both TF and TCF4 polymorphisms showed an increased risk for the development of ASD. AG heterozygote, GG mutant homozygote, AG+GG combined genotype, and G mutant allele of TF rs1867503 showed a signicantly elevated risk of ASD development (OR=3.18, p=0.0003; OR=2.62, p=0.0128; OR=2.98, p=0.0002; and OR=1.94, p=0.001, respectively). Likewise, AG heterozygote, GG mutant homozygote, AG+GG combined genotype, and G minor allele of TCF4 rs9951150 also showed a signicantly elevated risk of ASD development (OR=2.92, p=0.0007; OR=2.36, p=0.0273; OR=2.72, p=0.0005; and OR=1.92; p=0.0014, respectively). Our results indicate that TF rs1867503 and TCF4 rs9951150 polymorphisms are strongly associated with the development of ASD in Bangladeshi children.


Introduction
Autism spectrum disorders (ASD) are neurodegenerative disorders that are mainly diagnosed based on the behaviors of children, whose symptoms include de cit to develop normal social interaction with other people, impaired development of communicative ability, lack of imaginative ability, and repetitive, stereotyped movements (Casanova et al. 2002). Some changes occur in the anatomy and physiology of brain, such as overgrowth of the frontal cortex during the prenatal period in ASD (Casanova et al. 2002;Talkowski et al. 2012). On the other side, underdeveloped parts in cognitive areas affect decision making, communication and language (Talkowski et al. 2012). Abnormal growth of the hippocampus can affect the development of language syntax, semantics, and the capacity of creativity in language generation and a better understanding of words of a child. One in forty-two boys and one in 189 girls children have ASD worldwide (Autism Speaks, 2018) and the prevalence has increased 10 folds in the last 40 years (Hansen et al. 2015). A new statistic of 2017 shows that the prevalence of ASD among children in the selected countries was found 168, 161, 152, 100, 100, 69, 67, 49, 27, 9.2 per 10000 for USA, Japan, Canada, UK, Ireland, Denmark, Australia, China, Brazil, Portugal, respectively (Hansen et al. 2015). In 2013, a pilot study in Bangladesh found a prevalence of ASD was 0.15% (3% in Dhaka city and 0.07% in the rural area), and the ratio of boys and girls was 4:1 (Global autism movement and Bangladesh, 2014). It is still a case today that diagnosis of ASD lacks unifying theory (Mullegama et al. 2015). Early theories mainly focused on substandard parenting (Mullegama et al., 2015). Newschaffer et al. (2007) suggested that causes of ASD mainly fall into three categories, genetic, environmental and neurobiological. Some other factors like toxicity, teratogenic effect, trauma, infections can also cause ASD (Newschaffer et al. 2007).
Transferrin (TF) (chromosomal location: 3q22.1) is one of the genes which has the most substantial evidence of ASD susceptibility with several independent studies (Davis et al. 2003;Konstantynowicz et al. 2012). TF is an iron transporting plasma glycoprotein that controls the iron level in the biological uid (Davis et al. 2003). It has two iron binding sites, and these irons accumulate rapidly at the onset of myelination. A very recent study suggested that an elevated extent of oxalate in plasma might play a role in ASD by binding to the bilobal iron transport protein transferrin (hTF) and thereby interfering with iron metabolism by inhibiting iron delivery to cells (Konstantynowicz et al. 2012) So, genetic modi cation in the transferrin gene may manifest during the generation of ASD (Luck et al. 2013). An investigation was carried out on rs1867503 of transferrin gene and reported that genetic polymorphism of transferrin gene plays a signi cant role in generating cognitive disorders like ASD (Chaste et al. 2015).
These studies proved the association of these genes with ASD in some ethnic groups.
However, there is no study carried out in the Bangladeshi children with ASD to validate the association of rs9951150 variant of the TCF4 gene and rs1867503 of the TF gene. Considering the current situation of ASD in Bangladeshi children, this study was performed with a polymerase chain reaction (PCR) based ampli cation followed by restriction fragment length polymorphism (RFLP) method to detect TF (rs1867503) and TCF4 (rs9951150) association with ASD, and we hope it will help to understand ASD and to improve their diagnosis and treatment procedure.

Sample and Data Collection
Two groups of children were selected. One group consisted of 96 ASD children (aged 3-15 years) recruited as cases from the different schools for ASD children in Chittagong and Dhaka. Total 118 healthy children (aged 3-15 years) were recruited as controls from the different areas of Dhaka and Chittagong, Bangladesh. All of them were selected to investigate the risk of ASD due to polymorphisms of TF (rs1867503) and TCF4 (rs9951150). The genotyping analysis was performed in the Laboratory of Pharmacogenomics and Molecular Biology, Department of Pharmacy, Faculty of Science, Noakhali Science and Technology University, Noakhali, Bangladesh. The study was directed as per the International Conference of Harmonization (ICH) for Good Clinical Practice (GCP) and in compliance with the Declaration of Helsinki and its further amendments (World Medical Association Declaration of Helsinki, 2013).

DNA extraction and genotyping
About 3 ml of blood was drawn into a tube containing ethylenediaminetetraacetic acid disodium from all the patients and controls and stored at −80°C until the isolation of genomic DNA (Daly et al. 1998;Islam et al. 2013). Genomic DNA was isolated from 96 children with ASD and 118 controls by a kit method using a Favorprep DNA isolation kit. Genotyping of the selected SNPs was performed by a PCR-RFLP method. The PCR condition for rs1867503 consisted of an initial denaturation at 95 0 C for 3 m, 35 cycles of 95 0 C for 20 s, 55 0 C for 30 s and 72 0 C for 30 s and a single step nal extension at 72 0 C for 5 m. The PCR condition for the ampli cation of rs9951150 was the same, except the annealing temperature was 57 0 C instead of 55 0 C. After completion of PCR ampli cation, two PCR products of 299 and 446 bp were obtained for rs1867503 and rs9951150, respectively, and these products were visualized in 1% (w/v) agarose gel. Targeted polymorphisms were identi ed by the digestion with the respective restriction enzymes and conditions mentioned in Table 1.    Table 4.  Figure 1).  Figure 2).

Discussion
Though the prevalence of ASD is increasing day by day, there is still a lack of a proper way to diagnose or prevent ASD. The heritability of ASD is 90%. However, it is challenging to identify relevant genes, which are liable for the development of ASD (Bailey et al. 1995). Multiple studies are going on to identify the responsible genes and already hundreds of genes are found positively accountable for the development of ASD, and these genes are following various biochemical pathways to show their functions (Davis et al. 2003;Konstantynowicz et al. 2012;Luck et al. 2013). It was the rst-ever attempt in Bangladesh, and here, we reported our initial ndings on the association of TF and TCF4 genes polymorphisms with ASD from the perspective of Bangladesh.
Several studies considered TF (rs1867503) and TCF4 (rs9951150) as role players in a variety of psychiatric symptoms and diseases, including phobic anxiety, obsessive-compulsive disorder, schizophrenia, and attention-de cit hyperactivity disorder. Polymorphism of TF causes an increase or decrease of oxygen free radicals, which are responsible for oxidative stress associated with the neurodegenerative disorder by causing damage of neurons with excess production of lipid peroxidation (Onyango et al. 2010). This polymorphism also causes more formation of ferrous, which stimulates hydroxyl formation and leads to brain cell damage (Bjørklund et al. 2020 an elevated level of lipid peroxidation in autistic children compared to their non-autistic siblings. They also found increased oxidative stress, which is caused by reduced transferrin. This reduced transferrin is also responsible for language di culties in ASD children. Luck et al. (2013) conducted a study in which they described oxalate in plasma could play a role in ASD to interfere with iron transport by binding with transferrin (hTF), and this high oxalate can cause iron de ciency anemia (IDA) in children with ASD. Our SNP nding study has also suggested such kind of relation to ASD.
Polymorphism of TCF4 disrupts the columnar and laminar structure of the cortex, which is activitydependent. It also hampers calcium activity, which is responsible for neuronal excitability. These incidents result in different autistic syndrome in children (Page et al. 2018 From this result, we can say that the rs1867503 and rs9951150 SNPs are strongly associated with the development of ASD. As we have identi ed the genetic basis of the Bangladeshi children with ASD, we hope it will be helpful to understand the etiology of ASD. However, some limitations of this study should be noted. Only two known SNPs were selected from a public database without novel SNP. Another limitation is that the study population we present here is not large enough to represent the actual scenario of the country. Though we have found a strong association, a large-scale study may provide stronger evidence.

Conclusion
This case-control study reveals that TF rs1867503 and TCF4 rs9951150 polymorphisms are signi cantly associated with ASD in Bangladeshi children. However, it is the rst study for these SNPs in Bangladesh with a limited number of cases and controls, and the results are signi cant. This study will be bene cial for further studies with a large-scale population. Figure 1 Forest plot of rs1867503 allele of TF gene in the study population