SNPs associated with Schizophrenia: Evidence from Iranian patients

Journal Pre-proof SNPs associated with Schizophrenia: Evidence from Iranian patients

Ali Molaei, Zahra Noormohammadi, Iman Salahshourifar, Niloufar Mahdavi Hezaveh PII:

S2214-5400(19)30092-1

DOI:

https://doi.org/10.1016/j.mgene.2019.100633

Reference:

MGENE 100633

To appear in:

Meta Gene

Received date:

9 July 2019

Revised date:

1 September 2019

Accepted date:

7 November 2019

Please cite this article as: A. Molaei, Z. Noormohammadi, I. Salahshourifar, et al., SNPs associated with Schizophrenia: Evidence from Iranian patients, Meta Gene(2018), https://doi.org/10.1016/j.mgene.2019.100633

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Journal Pre-proof SNPs associated with Schizophrenia: Evidence from Iranian patients Ali Molaei1, Zahra Noormohammadi1*, Iman Salahshourifar1, Niloufar Mahdavi Hezaveh2 1 Department of Biology, science and Research Branch, Islamic Azad University, Tehran, iran 2 Department of Psychiatry, Imam Hossein Educational Hospital, School of Medicine, Shahid Beheshti University of medical Sciences, Tehran, Iran. *corresponding author

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Email address: [email protected] [email protected]

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ORCID: 0000-0003-3890-9001 Tel: +98 21 44865939

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Abstract

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Schizophrenia (SCZ) is a complex chronic brain disorder. The brain-derived neurotrophic factor

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(BDNF) is a member of the neurotrophins family, which plays a role in neuron proliferation. In the present work, we aimed to 1- Provide evidence on the association of BDNF variants and

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Schizophrenia, and 2- Investigate the association between MIR-204 with BDNF. We investigated

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the association between single-nucleotide changes in rs112062096 of MIR-204 and rs11030099 in 3 'UTR of the BDNF gene obtained by PCR-RFLP technique on 100 SCZ patients and 100

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healthy individuals. The highest genotype frequency in rs11030099 was C/C genotype in both case and control groups. The Chi-square test showed that rs11030099 in the BDNF gene is associated significantly with the co-dominant and dominant inheritable model. The Logistic regression revealed that the A/C genotype in rs11030099, increased the risk factor by 2.33 times, while the A/A genotype increase by 5.29 times. In dominant inheritance, the A allele increased the risk factor by 2.46 times, while in the recessive model, the risk factor increased by 4.12. The highest genotype frequency in rs112062096 of the MIR-204 gene was A/A genotype (81%) in the case and G/G (69%) genotype in the control group. The results produced a significant

Journal Pre-proof difference between two groups (P <0.0001). However, logistic regression revealed a significant difference between groups studied for the allele frequency. The risk factor was increased in the presence of G/G Genotype by 9.49 times. This is the first report on the association of the studied SNPs and schizophrenia. Keywords: BDNF, MIR-204, polymorphism, schizophrenia 1.Introduction

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Schizophrenia (SCZ) is a complex chronic brain disorder, that may produce delusions,

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hallucinations, trouble with thinking and concentration, and lead to a lack of motivation.

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However, most people with schizophrenia are not dangerous or violent. The frequency of

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Schizophrenia occurrence among men and women is almost equal, it has an earlier onset in males(Giacobbo et al., 2018).

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The Schizophrenia (SCZ) is a complicated, heterogeneous mental disorder that occurs in about

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1% of the world population. Based on family investigations, a relatively high heritability was

2015).

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reported for SCZ (> 60%), while based on twin studies it showed 80% heritability(Chen et al.,

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The findings of the genome-wide association studies (GWASs) and next-generation DNA sequencing technologies, identified a large number of genes with common risk alleles, as well as many rare copy-number variations (CNVs), and de novo mutations (DNMs), that are involved with SCZ disease (Chen et al., 2015). About 30 loci across the whole genome were identified to be related to SCZ. Among them, we can list the genes for transcription factor 4 (TCF4), neurogranin (NRGN), and dyhidropyrimidine dehydrogenase/MicroRNA-317 (DPYD/MIR317), which play an important role in brain development(Chen et al., 2015).

Journal Pre-proof The SCZ is also known to be associated with the brain-derived neurotrophic factor (BDNF). This is a member of the neurotrophins family, which plays a role in neuron proliferation and differentiation(Guillin et al., 2007). The BDNF primarily binds to a low-affinity cell surface receptor (LNGFR) but mediates its neurotrophic properties by signaling through a high-affinity cell surface receptor called BDNF/NT-3 growth factors receptor (gp145/TrkB). It is finally expressed as the C-terminal

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portion of an amino acid polypeptide precursor, which also contains a signal sequence of 18

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amino acid residues.

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The BDNF modulates the expression of dopamine D3 receptors and plays a role in the

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maturation of dopaminergic pathways. It also modulates the firing rates of serotonergic neurons

schizophrenia(Kim and Kim, 2018).

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within the raphe nuclei, which in turn through the serotonergic signaling contributes to

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In humans, BDNF shows a change in its level in several psychiatric diseases and therefore, but it is still unclear if these changes are the cause or the result of the normal brain

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malfunction(Giacobbo et al., 2018). For instance, heterozygous BDNF mice had a change in the

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BDNF level as well as an increase in weight gain, aggressiveness, anxiety, and contextual memory impairment (Giacobbo et al., 2018). Although BDNF is considered as a suitable biomarker for diagnostic and prognostic purposes for schizophrenia, its specificity with the disease should be confirmed (Nurjono et al., 2012). The expression of different genes regulates at the post-transcriptional level, by microRNAs (miRNAs). The normal brain development requires fine regulation of miRNA expression, therefore, any disturbance in its expression and function may result in different mental diseases, such as SCZ (Mellios et al., 2009).

Journal Pre-proof Recent investigations revealed that BDNF gene expression, as well as function, may be potentially regulated by several hundreds of miRNAs, primarily via its 3′ UTR region. However, though several miRNAs are suggested to target BDNF, only a few have been investigated in detail(Cattaneo et al., 2016). Imam et al. (2012) reported that the over-expression of the MIR-204 resulted in the reduction of the endogenous BDNF mRNA and protein levels, while its inhibition, increased neurotrophin

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expression. Moreover, the occurrence of mutations in the predicted binding site of miR-204 led

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to the suppression of luciferase expression via the full-length BDNF 3′ UTR region.

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In general association studies carried out to investigate the relationship between the BDNF gene

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and schizophrenia, produced heterogeneous findings. For example, researchers found a relationship between GT polymorphism in the 1 kb upstream from the transcription site of the

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BDNF gene and schizophrenia( Fanous et al., 2004; Krebs et al., 2000; Muglia et al., 2003), but

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some other studies did not show such association(Hawi et al., 1998;Sasaki et al., 1997;Virgos et al., 2001).

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The purpose of the present study was to provide evidence concerning the association of

BDNF.

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BDNFvariants with Schizophrenia and also to investigate the association between MIR-204 with

Therefore, we investigated two novel gene variants 1- rs11030099 of the gene BDNF and 2rs112062096 of the gene MIR-204, within the Iranian population. There has been no report on the association of these gene variants and schizophrenia up to know. 2.Materials and Methods In the present study, 100 healthy individuals with mental health which was approved by the Positive and Negative Syndrome Scale (PANSS) test, were selected as the control group. The

Journal Pre-proof case group included 100 schizophrenic patients who referred to the psychiatric ward of Imam Hossein and Artesh 506 hospitals in Iran. Their disorders were determined via diagnostic tools such as demographic questionnaire (patients completed questionnaires containing questions about age, education, gender, and treatment length, Table 1) and PANSS test, a clinical interview was approved by a psychiatrist based on the Diagnostic and Statistical Manual of Mental Disorders(DSM-IV-TR). Individuals with a history of drug and alcohol abuse were excluded

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from samples.

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2.1.DNA extraction and PCR-RFLP

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The peripheral blood was collected, and kept in at -20 ° C. Extraction kit (Zhino Gene

The

PCR

amplification

was

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Pazhoohan, Iran) was used for DNA extraction according to the manufacturer’s instructions. performed

using

the

forward

primer

5'

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TGCTTATCCCTCACCCTACT 3'and the reverse primer 5' CCACACTCAAGCCGACTTAT3'

primer

5'

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for rs11030099 in the 3'UTR region of the BDNF gene (NC_000011.10) as well as the forward TTCATGTCATGGTTATCCCAATGC

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TTATACAGTTGATGAAATGGCACCTC

3'

for

3'and

the

rs112062096

reverse in

primer

MIR-204

5' gene

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(NC_000009.12). The PCR reaction was conducted in 25μl volume containing 2x PCR, 0.4 μl of dNTP (10 mM) (CinnaGen Co.), 0.5 μl MgCl2 (1.5 mM, CinnaGen Co.), 0.4 μL of each primer (10pM), 2 units Taq polymerase (CinnaGen Co.), and 2.5 μl DNA template (≤100 ng). The PCR thermocycler was performed as an initial denaturation at 94 ° C for 5 min following 40 cycles of 3 steps, including, 94 ° C for 30 Sec, 56 ° C and 57.3 ° C for the BDNF and MIR-204 SNPs for 40 Sec, the final extension was at 72 C for 40 °sec.

Journal Pre-proof The expected PCR fragment lengths were 657bp and 911 bp for the BDNF the MIR-204 genes respectively. PCR products were run on 1.5% agarose gel and visualized by Sybergreen and UV illuminator (Figure 1). The PCR products of rs11030099 were digested 0.1 μl (10 units/μl) the MseI restriction enzyme at 65 ° C for 16 hours and 0.1 μl (10 units / μl) Tsp45I enzyme at 37 ° C for 16 hours. The MseI and Tsp45I enzymes were deactivated by EDTA at a concentration of 0.5 Mm and at 65 ° C for

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20 min respectively. The digested products were visualized on a 2% agarose gel with Green

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Viewer 0.5 μg / ml (ParsTous, Iran).

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The digested products based on genotypes were randomly selected for sequencing by the ABI

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capillary sequencing machine (Faza Pazhooh Company, Iran). The sequences were visualized by Bio Edit software (http://www.mbio.ncsu.edu/BioEdit).

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2.2. Statistical analysis

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We used χ2 or Fisher's exact test for differences in two SNPs' frequencies between control and case samples. We further used a crude odds ratio (COR) and 95% confidence interval (CI) as a

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calculating the association between genotypes and disease. Hardy–Weinberg equilibrium was

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estimated with the standard procedure by using the chi-square test. Haplotype frequency for three SNPs' genotypes as well as haplotype association between control and case samples were estimated using SNPstats software. 3.Results The enzymatic digestion of rs11030099 by MseI, produced two fragments with 603bp and 54bp size length in the presence of the C allele (ancestral allele), while three fragments with 333bp, 270bp and 54bp size length were obtained in the presence of A allele.

Journal Pre-proof The enzymatic digestion of rs112062096 by Tsp45I, produced two fragments with 770bp and 141bp size length in the presence of A allele (ancestral allele), while three fragments with 770bp, 121bp and 20bp size length were obtained in the presence of G allele (Figure 2). These fragments were also identified by sequencing (Figure3). Details of allele and genotype frequencies, the inheritance model as well as the Hardy Weinberg equilibrium test result for the rs11030099 of BDNFgene, are provided in Table 2. The highest

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genotype frequency in rs11030099 was C/C genotype in both case and control group (78% and

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59%, respectively).

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A significant difference occurred between case and control groups for both co-dominant and

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dominant models (P = 0.011, and 0.0036 respectively).

Logistic regression revealed that the A/C genotype increased the risk factor 2.33 times, while the

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A/A genotype increases it 5.29 times. In dominant inheritance, the A allele increased the risk

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factor 2.46 times, while in the recessive model, the A/A genotype increased the risk factor by 4.12. All the studied samples were in Hardy Weinberg equilibrium.

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Details of allele and genotype frequencies, the inheritance models, as well as Hardy Weinberg

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equilibrium, related to the rs112062096 in the MIR-204 gene, are given in Table 3. The highest genotype frequency in rs112062096 of the MIR-204 gene was A/A genotype (81%) in the case group and G/G (69%) genotype in the control group. Due to the lack of heterozygote genotype (A/G), co-dominant, dominant and recessive inheritance models could not be investigated. However, logistic regression revealed a significant difference between The case and control samples for the allele frequency (P = 0.0001).

Journal Pre-proof The risk factor was increased in the presence of G/G Genotype by 9.49 times, however, the studied samples were not in the Hardy Weinberg equilibrium. This may be due to the absence of the heterozygote genotype (A/G), in this study. Chi-square test, performed between the case and control groups with regard to the gender, did not produce a significant difference in the genotype frequency in both rs11030099 and rs112062096(P = 0.209, and P = 0.319, respectively) (Table 4).

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We found no significant difference between the genotypes regarding the manner of response to

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treatment (P = 0.229) in rs11030099, but a significant difference occurred in rs112062096 (P =

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0.016) (Table 5).

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The haplotype analysis for two SNPs studied showed that the highest frequency of haplotypes belonged to C-A (0.485, rs11030099 and rs112062096 respectively) as ancestral genotypes with

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OR= 1.00. The estimated risk in 95% CI for C-G and A-G haplotypes showed significant

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association in SCZ patients [OR (95% CI) =2.75 (1.87 - 4.03), P<0.0001 and OR (95% CI) =8.27

groups (Table 6).

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(2.84 - 24.05) respectively] while A-A haplotypes showed no significant association between

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Linkage disequilibrium (LD) based on D and D´ value, r and associated p- value was analyzed between SNP pairs. These two SNPs of the BDNF gene and MIR204 showed linked together (D value = 0.020, D´ value=0.212, r = 0.108, P = 0.030). 4.Discussion Many association studies concerned with psychiatric disorders such as schizophrenia and BDNF SNP genotypes; have been carried out, which produced different and controversial results. For example, an association study performed on Scottish patients diagnosed with schizophrenia, and the Allele G of 196G/A revealed strong association(Neves-Pereira et al., 2005). But a similar

Journal Pre-proof investigation in the Asian population, produced negative results(Naoe et al., 2007). Similarly, Kim and Kim (2018), investigated the association between two SNPs, 196G/A and 11757G/C with schizophrenia patients and found no significant association. The present study revealed a significant association between rs11030099 of the gene BDNF and SCZ. Moreover, Logistic regression revealed that the genotype AC in the co-dominant inheritance model increases the risk factor up to 33.2 %, much higher than the genotype AA with

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a 3.5 % risk factor. In the case of rs112062096 of the gene MIR-204 also we found a significant

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association. The genotype GG had a 50% higher risk factor compared to that of AA genotype.

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These results and those obtained in dominance, as well as recessive genetic models, indicate that

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genetic polymorphism within the BDNF gene and the SNP genotypes are associated with SCZ disorder in the Iranian population. We consider the present findings as a preliminary report as we

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need to perform this study in a much larger sample size.

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It is suggested that population structure and stratification may affect on the false positive or negative outcomes in case-control studies(Tang et al., 2005). The frequency of the risk allele

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may differ in different ethnic groups, while the specific allele may be a risk factor in one ethnic

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population but be normal in the other groups. Though the present study was on Iranian patients, we do have different ethnic populations within the country, therefore, our next investigation may be conducted on an ethnic group-base investigation. The linkage disequilibrium (LD) situation of the studied SNPs may also affect the results(Kim and Kim, 2018). In the present study, we found that rs11030099 genotypes were in LD in both schizophrenia and control persons, while the studied samples were not in LD for rs112062096. These may also be under the influence of the small sample size investigated. However, we found

Journal Pre-proof low haplotype diversity in the two studied genes and that these genotypes did not differ significantly among the sexes. Kim and Kim (2018), investigated 196G/A of the Brain-Derived Neurotrophic Factor Gene Polymorphisms and suggested that BDNF gene allele and genotype can serve as a predictor for suicide attempts in schizophrenia patients. The complex nature of genetics in SCZ also exists in BDNF expression. For example, BDNF

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expression was increased in SCZ patient’s brains, while its expression in the other investigations

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showed that the expression of BDNF, and of its receptor TrkB, is significantly down-regulated in

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schizophrenic patients (Durany et al., 2001;Hashimoto et al., 2005;Weickert et al., 2003). The

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same heterogeneity was reported for alterations in BDNF gene expression and protein levels in the blood of patients affected by SCZ (Durany and Thome, 2004;Pillai et al., 2008). This

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heterogeneity is suggested to be due to the chronicity of the illness and antipsychotic therapies

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(Cattaneo et al., 2016). But, a meta-analysis revealed an association between reduced BDNF gene expression and protein levels and SCZ, whereas the treatment with antipsychotics does not

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exert any significant effects on BDNF levels (Green et al., 2011;Fernandes et al., 2015).

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The present study revealed a significant association between rs11030099 of the BDNF gene and MIR-204. Though there is no other report on the association between these two genes, we can expect to have inconsistent results in other population-based investigations, as is the case in the other association studies(Lin et al., 2013). It has been suggested that the interaction between multi-genes is probably playing the main role in the pathogenesis of schizophrenia, but not a single gene(Lin et al., 2013). 5.Declarations

Journal Pre-proof The informed consent was obtained from members of the case and control groups or their supervisors. This study was approved by the Ethical committee of Islamic Azad University, Science and Research Branch, Tehran (approval No. IR.IAU.SRB.REC.1398.004). Informed consent was obtained from patients before commencement of the research. 6.Competing interests "The authors declare that they have no competing interests"

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7.Authors' contributions

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"AM collected the samples and performed the PCR tests, ZN conceptualization of the project and

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data analyzed and interpreted. IS data analyzed and interpreted. NMH was co-advisor and

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psychiatrist regards to diagnose patients. All authors were contributors in writing the manuscript and read and approved the final manuscript."

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8.Acknowledgements

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We would like to thank Ms. Mahtash Malekian and Ava Lajevardi for collecting samples. We

9. No fund is available

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10. References

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also appreciate all patients to contribute in this research.

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(DAT1), and BDNF: a family based association study. American Journal of Medical Genetics

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of antipsychotics: meta-analysis and implications. Molecular psychiatry 20(9), 1108.

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Green, M., Matheson, S., Shepherd, A., Weickert, C., Carr, V., 2011. Brain-derived neurotrophic factor levels in schizophrenia: a systematic review with meta-analysis. Molecular psychiatry 16(9), 960. Guillin, O., Demily, C., Thibaut, F., 2007. Brain‐ derived neurotrophic factor in schizophrenia and its relation with dopamine. International review of neurobiology 78, 377-395. Hashimoto, T., Bergen, S.E., Nguyen, Q.L., Xu, B., Monteggia, L.M., Pierri, J.N., Sun, Z., Sampson, A.R., Lewis, D.A., 2005. Relationship of brain-derived neurotrophic factor and its

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Journal Pre-proof race/ethnicity, and confounding in case-control association studies. The American Journal of Human Genetics 76(2), 268-275. Virgos, C., Martorell, L., Valero, J.n., Civeira, F., Joven, J., Labad, A., Vilella, E., Figuera, L.d., 2001. Association study of schizophrenia with polymorphisms at six candidate genes. Schizophrenia research 49(1-2), 65-71. Weickert, C., Hyde, T., Lipska, B., Herman, M., Weinberger, D., Kleinman, J., 2003. Reduced

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5.1. Tables

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5. Tables and Figures

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Neuropsychiatric Genetics 141(4), 387-393.

Table 1. Demographic characteristics of case and control group Variables

Case

Control

40.80 ± 11.298

32.92 ± 7.391

35

37

Continuous Variable Age Discontinuous Variables Sex Female

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65

63

Treatment Response Complete Response

8

-

Partial Response

89

-

No Response

3

-

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equilibrium of rs11030099 in thecase and control groups

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Table 2. Frequency of the genotypes, inheritable models and exact test for Hardy-Weinberg

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rs11030099

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Frequency of the genotype in the control group and case group All

Case

Genotype

subjects(n=200)

Count Proportion

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Allele

Count Proportion

177 0.88

155 0.78

23 0.12

45 0.22

78 0.78

59 0.59

58 0.29

21 0.21

37 0.37

5 0.02

1 0.01

4 0.04

332 0.83

A

68 0.17

C/C

137 0.68

A/C A/A

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C

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Count Proportion

Control

Inheritable Models Model

Genotype

status=ca

status=co

OR (95% CI)

P-value

Codominant

C/C

78 (78%)

59 (59%)

1.00

0.011

A/C

21 (21%)

37 (37%)

2.33 (1.24-4.39)

A/A

1 (1%)

4 (4%)

5.29 (0.58-48.56)

Journal Pre-proof Dominant

C/C

78 (78%)

59 (59%)

1.00

A/C-A/A

22 (22%)

41 (41%)

2.46 (1.33-4.57)

C/C-A/C

99 (99%)

96 (96%)

1.00

A/A

1 (1%)

4 (4%)

4.12 (0.45-37.53)

N11

N12

N22

N1

N2

P-value

All subjects

137

58

5

332

68

1

status=ca

78

21

1

177

23

1

status=co

59

37

4

155

45

0.77

Recessive

0.0036

0.16

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Hardy-Weinberg equilibrium

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OR; Odd ratio, CI; Confidence interval

Table 3. Frequency of the genotypes, Inheritable Models and exact test for Hardy-Weinberg

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equilibrium of rs112062096 in the case and control groups

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rs112062096

Frequency of the genotype in the control group and case group All

Genotype

subjects(n=200)

Case

Control

Count Proportion

Count Proportion

Jo

ur

Allele

Count Proportion A

224 0.56

162 0.81

62 0.31

G

176 0.44

38 0.19

138 0.69

A/A

112 0.56

81 0.81

31 0.31

G/G

88 0.44

19 0.19

69 0.69

Inheritable Models

Journal Pre-proof Model

Genotype

status=ca

status=co

OR (95% CI)

P-value

---

A/A

81 (81%)

31 (31%)

1.00

<0.0001

G/G

19 (19%)

69 (69%)

9.49(4.93-18.27)

N11

N12

N22

N1

N2

P-value

All subjects

112

0

88

224

176

<0.0001

status=ca

81

0

19

162

38

<0.0001

status=co

31

0

69

62

138

<0.0001

ro

of

Hardy-Weinberg equilibrium

-p

OR; Odd ratio, CI; Confidence interval

re

Table 4. The association between rs11030099 and rs112062096 genotypes with the sex of patients and healthy individuals.

26 8 1

Male

52 13 0

Female

23 12 2

ur

Jo

Control

Male

2

0.360

0.319

2

0.697

0.699

na

Female Case

lP

rs11030099 CC AC AA df Pearson Chi-Square Likelihood Ratio

36 25 2

rs112062096 AA GG df Pearson Chi-Square Likelihood Ratio Female

26 9

Case

Control

Male

55 10

Female

11 26

Male

20 43

1

0.209

0.216

1

0.833

0.833

Journal Pre-proof

df; Degrees of freedom Table 5. The association between rs11030099 and rs112062096 genotypes with the treatment response of Patients rs11030099 CC AC AA df Pearson Chi-Square Likelihood Ratio Complete 611

ro

Relative

of

Response

70 19 0

-p

Case

210

0.017

0.229

na

Response

4

lP

No

re

Response

Complete

ur

rs112062096 AA GG df Pearson Chi-Square Likelihood Ratio

44

Jo

Response

Relative

76 13

Case Response

No Response df; Degrees of freedom

12

2

0.005

0.016

Journal Pre-proof Table 6. Haplotype association of two SNPs in two groups studied (n=200)

1 2 3 4

rs11030099 rs112062096 Freq OR (95% CI) P-value C A 0.485 1.00 --C G 0.345 2.75 (1.87 - 4.03) <0.0001 A G 0.095 8.27 (2.84 - 24.05) <0.00001 A A 0.075 1.63 (0.72 - 3.69) 0.24

5.2. Figures

ur

na

lP

re

-p

ro

of

Separately in “illustrations” file

Jo

Fig. 1. PCR products of two studied SNPs A) 657 bp band for rs11030099 PCR product, B) 911 bp band for rs112062096 PCR product, M, 100bp ladder, N, no DNA

ro

of

Journal Pre-proof

Fig. 2.A) Digestion of rs11030099 PCR products by MseI, Ladder 100bp and No DNA. B)

Jo

ur

na

lP

re

-p

Digestion of rs112062096 PCR products by Tsp45I, No DNA and Ladder 100bp.

Fig.3.Sequencing examination of rs11030099 and rs112062096 Conflict of interest: The authors have no conflict of interest

Figure 1

Figure 2

Figure 3