Association Studies of Common Variants of TCF4 Gene Conferring Risk of Schizophrenia in Pakistani Patients

is a psychiatric disorder and it is strongly inherited disease with a heritability of 80% or more. Rare genetic mutations are more frequent in schizophrenia patients. These genetic variations interfere with brain development and include hundreds of distinct genes. Transcription factor 4 (TCF4) has been emphasized as major players for disruption of brain development as well as function and consequently, the onset of schizophrenia. The dysregulation of TCF4 gene expression in brain affects the process of pre pulse inhibition (PPI) and consequently profound reduction in sensor motor gating that may results in to the onset of schizophrenia. Objective: To �nd out the genetic association of common variants of TCF4 gene conferring risk of schizophrenia. Methods: It was a case control study in which statistically signi�cant number of blood samples of con�rmed diagnosed schizophrenic patients as well as age matched healthy control subjects were analyzed to screen out selected Single Nucleotide Polymorphisms (rs9960767, rs4309482, rs12966547, and rs2958182) of TCF4 gene for their association with schizophrenia. Results: Out of these four SNPs rs9960767 and rs4309482 were signi�cantly associated with schizophrenia. p-values for SNPs rs12966547 and rs2958182 were greater than 0.05 in both healthy controls and in patients. Conclusions: The results of this study offer compelling evidence for the link between particular TCF4 gene polymorphisms and schizophrenia. Two SNPs, rs9960767 and rs4309482, were found to have a strong correlation with schizophrenia in the research population, according to the analysis.


I N T R O D U C T I O N
Schizophrenia is a psychiatric disorder and it is strongly inherited disease with a heritability of 80% or more. Rare genetic mutations are more frequent in schizophrenia patients. These genetic variations interfere with brain development and include hundreds of distinct genes. Transcription factor 4 (TCF4) has been emphasized as major players for disruption of brain development as well as function and consequently, the onset of schizophrenia. The dysregulation of TCF4 gene expression in brain affects the process of pre pulse inhibition (PPI) and consequently profound reduction in sensor motor gating that may results in to the onset of schizophrenia. Objective: To nd out the genetic association of common variants of TCF4 gene conferring risk of schizophrenia. Methods: It was a case control study in which statistically signi cant number of blood samples of con rmed diagnosed schizophrenic patients as well as age matched healthy control subjects were analyzed to screen out selected Single Nucleotide Polymorphisms (rs9960767, rs4309482, rs12966547, and rs2958182) of TCF4 gene for their association with schizophrenia. Results: Out of these four SNPs rs9960767 and rs4309482 were signi cantly associated with schizophrenia. p-values for SNPs rs12966547 and rs2958182 were greater than 0.05 in both healthy controls and in patients. Conclusions: The results of this study offer compelling evidence for the link between particular TCF4 gene polymorphisms and schizophrenia. Two SNPs, rs9960767 and rs4309482, were found to have a strong correlation with schizophrenia in the research population, according to the analysis. association studies motivated for testing of selected Single Nucleotide Polymorphisms rs9960767, rs4309482, rs12966547and rs2958182) of TCF4 gene for association with schizophrenia in Pakistani population. In this study, the genetic associations of common variants of TCF4 gene (rs9960767, rs4309482, rs12966547, and rs2958182) conferring risk of schizophrenia were carried out in casecontrol study pattern (case n=60, control n=60) in Pakistani population. The identi cation of disease susceptibility loci may lead to a better understanding of the biological mechanism of schizophrenia that will pave a way for the better diagnostic, prevention and treatment of schizophrenia.
hallucinations and orid thought disorders [1]. Schizophrenia may cause by duplications or deletions of DNA sequences in genes that play role in brain development or neuronal signalization. Recent genomewide association studies highlight schizophrenia as highly polygenic disorder and abnormal connection between different genes network can also leads to schizophrenia [3]. One of the most well-known schizophrenia risk genes is transcription factor 4 (TCF4). The TCF4 gene (Entrez Gene ID 6925; ensemble ENSG00000196628) is a basic helixturn-helix (bHLH) Ephrussi-box (also known as "E-box" or "E-") protein transcription factor that is found on chromosome 18q21.2. TCF4 also goes by the names E2-2, ITF2, and bHLHb19. It is 437 kb long, has 41 exons, and has a human protein length of between 511 and 773 amino acids [4]. TCF4 has been actively expressed in brain, especially in t h a l a m i c n e u r o n s a n d p l a ys i m p o r t a n t r o l e i n differentiation of glial cells, especially the maturation of oligodendrocyte progenitors [5]. The microRNAmir137, a regulator of neuronal development targets TCF4 and disrupts the tissue speci c tuning of its expression that may leads to Schizophrenia [4]. Furthermore, SNPs in miR-137 and its target genes have shown association with risk of s c h i z o p h r e n i a , a s r e s u l t o f d y s r e g u l a t e d neurodevelopment [6]. The cognitive impairments and de cits in pre-pulse inhibition has been reported in mice with over expressing TCF4 in the forebrain [7]. The signi cant mRNA over expression of TCF4has been reported in psychosis patients in comparison to controls as well as positive correlation with positive-and negativesymptom levels. Therefore, involvement of TCF4 variants in p s y c h o s i s p a t h o l o g y e s p e c i a l l y a b n o r m a l neurodevelopment has been suggested in previous studies [8]. Furthermore, the increase in obstetric complications in children who later develop schizophrenia, cortical thickness reduction, enlargement of ventricles in the early phases of the disease, cognitive dysfunction, positive and negative symptoms support the neuro-developmental hypothesis for the onset of schizophrenia [8]. Most recent studies have found out the importance of common variants of TCF4 gene and their association with schizophrenia [9][10][11]. The signi cant association of SNP (rs9960767) of TCF4 gene has been observed with risk of schizophrenia in American and German populations [36]. Further evidence for the involvement of the same variant (rs9960767) for the risk of schizophrenia has been provided in British population [11]. Another SNP (rs2958182) of gene TCF4 appeared to be signi cantly associated with schizophrenia among Han Chinese population in recent studies [10]. The replication study of SNPs (rs4309482, rs12966547) showed their signi cant association with schizophrenia in Nor wegian population [9]. The above-mentioned

M E T H O D S
In k3 EDTA vials, blood samples from 60 Institute of Mental Health, Lahore con rmed diagnosed schizophrenia patients and 60 age-matched healthy control persons were taken. The preparation of these samples for molecular analysis. Following all ethical guidelines, blood samples were taken from patients, and clinical data were gathered from patient les. Blood samples were drawn by the knowledgeable paramedical staff. Using a DNA extraction by Thermo sher for puri cation of genomic DNA or by using the organic method of DNA isolation methodology outlined by Maniatis et al., (1982), the DNA was isolated from the fresh blood samples [12]. Agarose gel electrophoresis was used to assess the DNA's quantity and quality. The quantity and purity of the DNA were evaluated using agarose gel electrophoresis. Using the Primer 3 program from the internet (https://primer3.ut.ee/), allele speci c primers for allelic variations of TCF4 were created. For each marker, two universal primers and one allele-speci c primer were created. Method of Hirotsu et al., (2010) was followed for creating allele-speci c primers, and the primer sequences and annealing temperatures are listed in Table 1 [13].   This study comprised of Pakistani Schizophrenia patients (n=60) and age-matched normal healthy control subjects (n=60). DNA isolation was performed from fresh blood samples, and four variants of TCF4 gene containing (rs9960767, rs2958182, rs12966547&rs4309482) were genotyped. The allele speci c extension method was exploited to amplify the variant regions of TCF4 gene and ampli ed products were analyzed by agarose gel electrophoresis. The ampli ed PCR product for all four SNPs of TCF4 (two possible variants of each genetic marker) are shown in gel image ( Figure 1).

Sequences of primers
Genomic DNA (10 ng), oligonucleotide primers (0.4 ρM each), dNTPs (200 μM), 1X PCR Buffer, Taq Polymerase (1U) and MgCl (2 mM) were all used in the PCR. The following PCR cycling circumstances were used: With varying annealing temperatures listed in table 1 for 30 seconds each, one cycle was performed at 95°C for 5 minutes, followed by 32 cycles at 95°C for 30 seconds, and one cycle was performed at 72°C for 5 minutes. Products from ampli ed SNPs were separated by gel electrophoresis and staining was performed with Ethidium Bromide (EtBr) and then seen under an Ultra Violet light source. The genotyping of the allelic variations of TCF4 was done using a gel-based approach. Sanger sequencing of puri ed PCR products from chosen samples was carried out to con rm the various allelic variants of TCF4 in order to validate the gelbased approach of SNP identi cation. BigDye Sequencing Kit was used to sequence the puri ed products using universal primers in accordance with the manufacturer's instructions (Applied Biosystems). Sanger sequencing was done at the business facilities. Chi-square, odds ratios (ORs), and 95% con dence intervals (CIs) were performed to estimate the association of genetic variations of TCF4 with schizophrenia. A 0.05 p-value was regarded as statistically signi cant. The haplotypes association with the risk of schizophrenia was assessed using Fisher's exact test. SHEsis, an online statistical analysis tool, was used for all statistical analysis. Four SNPs of TCF4 were genotyped in 60 Pakistani schizophrenic patients and in normal healthy individuals. Chi square analysis was performed for all four SNPs genotyped in schizophrenia patients and in controls. Table  2 shows p-values for each SNP and its association with schizophrenia risk in both case and control individuals. By considering p-values, two SNPs i.e. rs9960767 and rs4309482 of TCF4 showed signi cant association (<0.05) with risk of schizophrenia in Pakistani population p=7.47e-007 and p=0.000537 respectively). However, other two SNPs rs2958182 and rs12966547 were not signi cantly associated with risk of schizophrenia in this population. The strongest association with the schizophrenia was observed for rs9960767(p=7.47e-007) in Pakistani schizophrenia patients.

D I S C U S S I O N
TCF4 is considered as a most susceptible gene that cause schizophrenia. This study comprised of Pakistani Schizophrenia patients (n=60) and age-matched normal healthy control subjects (n=60). Four variants of TCF4 gene containing (rs9960767, rs2958182, rs12966547, rs4309482) were genotyped. In this study of patients of schizophrenia, signi cant association was seen between two SNPs of TCF4 [rs9960767, rs4309482 with p=7.47e-007 and p=0.000537 respectively] and Schizophrenia. These results are similar with the study of Stefansson et al., which showed that rs9960767, an SNP found in an intron of TCF4 (P.4.1_10_9), was one of seven single nucleotide polymorphisms (SNPs) that were reported as being related with schizophrenia at a genomewide level, which included data from different GWAS [14]. This association was replicated in a study of Han Chinese patients using a different SNP (rs2958182, in high linkage disequilibrium (LD) with rs9960767). Another signi cant GWAS found rs4309482, which is intergenically located upstream of CCDC68 and downstream of TCF4 [15]. Finally, two novel TCF4 SNPs (rs17512836, which is located in intron 3 of TCF4, and rs12966547, which is in high LD with  Table 3 shows haplotypes of four SNPs of TCF4. In the four TCF4 SNPs, the haplotype analysis found 15 common haplotypes with frequencies greater than 0.03; 11 of these haplotypes were signi cantly associated with the risk of schizophrenia, demonstrating that patients carrying more risk alleles have a higher risk of developing schizophrenia.   (1) (1) In conclusion, the goal of this study was to determine how Pakistani patients' vulnerability to schizophrenia may be in uenced by the TCF4 gene. The results of this study offer compelling evidence for the link between particular TCF4 gene polymorphisms and schizophrenia. Two SNPs, rs9960767 and rs4309482, were found to have a strong correlation with schizophrenia in the research population, according to the analysis. Overall, this research adds to the growing body of data that TCF4 is a schizophrenia susceptibility gene.