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A Novel Genotyping Method for Detection of the Muscarinic Receptor M1 Gene rs2067477 Polymorphism and Its Genotype/Allele Frequencies in a Turkish Population.

Objectives
Gene variation in the cholinergic muscarinic receptor 1 (CHRM1) has potential to become a candidate biomarker in the development of several disorders as well as drug response. In this study, a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed to determine the C to A single nucleotide polymorphism at position 267 in the CHRM1 gene.


Materials and Methods
A new reverse primer and a mismatched forward primer were designed to obtain 125 bp PCR products. The PCR products were then digested with the Hae III restriction enzyme to detect the rs2067477 polymorphism that comprises a C to A base change. The novel assay developed was tested in 51 Turkish schizophrenia patients.


Results
The genotyping assay was successfully performed in patients with schizophrenia in order to confirm the accuracy and validity of this method. The frequency of CC, CA, and AA genotypes was 72.5%, 25.5%, and 2%, respectively. On the basis of these findings, the allele frequency of C was 0.85 and the allele frequency of A was 0.15.


Conclusion
This genotyping assay is practical for screening the CHRM1 C267A polymorphism in pharmacogenetic studies. The present polymorphism may be used as a candidate biomarker to determine genetic susceptibility to related diseases and may contribute to the implementation of individualized drug therapy for M1-related diseases.


INTRODUCTION
Prenatal and perinatal risks, negative early life events, and genetic predisposition may cause neurodevelopmental alterations and sensitize the dopamine system in the brain, and the presence of these factors may contribute to the development of schizophrenia. 1,2 The prevalence of schizophrenia varies from 3 to 7 per 1000 worldwide and the average lifetime prevalence is 4/1000 while the lifetime risk is 7.2 per 1000. 3,4 However, studies about the prevalence of schizophrenia have shown that the disorder differs in all societies and can vary according to the characteristics of the society. 5,6 A systematic review based on a limited number of general population surveys conducted in Turkey showed that the prevalence of schizophrenia was 8.9 in 1000. 7 The risk of schizophrenia is 10% for first-degree relatives and 40% for children if both parents have schizophrenia. 8 In addition to heredity in the development of this disease, the gene differences involved in the pharmacokinetics and pharmacodynamics of the drugs used in the treatment of schizophrenia also play a major role in treatment, response, and adverse drug reactions.
Antipsychotic drugs used in the treatment of schizophrenia such as clozapine (CLZ) and olanzapine have been found to be antagonistic to muscarinic receptors. 9 CLZ is prescribed especially in treatment-resistant schizophrenia patients and it is a weak muscarinic receptor 1 (M1) agonist, while its active metabolite, N-desmethylclozapine (NCLZ), is a potent M1 agonist receptor. 9 In addition, M1 receptor agonist DCLZ plays an important role in determining the clinical effects and pharmacotherapy in the treatment of psychotic disorders. Studies have also pointed out that a decreased density of M1 receptors particularly in the neocortical regions was associated with schizophrenia. 10 Similarly, some studies showed reduced M1 receptor mRNA levels in brain samples from schizophrenia patients. 11 Considering all of these, M1 receptor is an important target in the development and also treatment of schizophrenia.
There are five types of cholinergic muscarinic receptors, designated as M1 to M5. Among these, M1 is mostly located in the nervous system. M1 is typically found in the parasympathetic ganglia, cortical and hippocampal regions of the brain, and less in airway epithelial cells and is involved in cognitive functions such as learning and memory, as well as regulation of cardiac contractions. 12,13 M1 is encoded by the CHRM1 gene located on chromosome 11q12.3. There are 15 single nucleotide polymorphisms (SNPs) in the CHRM1 gene region; one of them is the C267A (rs2067477) base change. This polymorphism is a silent mutation that is a transversion of cytosine (C) to adenine (A) at position 267 in the CHRM1 gene region. It is in the wobble site of the codon (GGC→GGA), so the protein sequence is preserved. 13,14 In short, the determination of the SNPs in the gene regions that are potentially involved in schizophrenia are important because they could affect disease susceptibility, cognitive performance, drug response, or adverse drug reactions. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay is one of the most common, simple, effective, fast, and inexpensive methods used to determine SNPs. Thus, our aim was to develop a novel PCR-RFLP method for genotyping the CHRM1 C267A polymorphism. Subsequently, the PCR-RFLP assay developed was performed for validation of the method and determination of genotype and allele frequencies in Turkish patients with schizophrenia.

Study subjects and DNA isolation
Whole blood samples were obtained from 51 consecutive Turkish schizophrenia outpatients admitted to Ankara University Medical Faculty Psychiatry Department and diagnosed using the Diagnostic and Statistical Manual of Mental Disorders fourth edition 15 between October 2016 and April 2018. The inclusion criteria were being between 18 and 65 years of age and having signed the written informed consent. Patients with any additional psychiatric diagnosis or general medical comorbidity were excluded. Informed consent was obtained from all subjects and the protocol was approved by the Research Ethics Committee of the Medical Faculty, Ankara University. Genomic DNA was extracted with the high salt method from the peripheral blood of the 51 subjects. 16 The absorbance level of DNA samples for 260 and 280 nm was detected with spectrophotometric analysis and the purity of the samples was between 1.7 and 2.0.

PCR primers and conditions
The sequence data of the C267A (rs2067477) polymorphism in the human CHRM1 gene region were obtained from the NCBI website (http://www.ncbi.nlm.nih.gov) and the new primers were designed as follows based on the published sequence: forward primer: 5'-TACTTCCTGCTGAGCCTAGCC-3'; reverse primer: 5'-GCCAGCCAGAGGTCACAAGCC-3'. The PCR reaction was carried out in a volume of 25 µL, which contained 10X PCR buffer (Amplicon, Denmark; containing 10X ammonium and 15 mM Mg), 1.1 mM MgCl 2 , 0.1 mM dNTP, 10 pmol from each primer, 1.5 µL of DMSO, 0.45 U of Taq DNA polymerase (Amplicon, Denmark), approximately 100 ng of genomic DNA, and distilled water to complete the final volume to 25 µL. Moreover, 125 bp PCR product was obtained using the following PCR cycling conditions: initial denaturation at 94°C for 3 min, followed by 30 3-graded cycles, which were denaturation at 94°C for 30 s, annealing for 30 s at 59°C, and elongation at 72°C for 45 s. At the end, a final extension for 5 min at 72°C was carried out. The PCR products (125 bp) were visualized under an ultraviolet illuminator on 1% agarose gel stained with ethidium bromide.

Restriction fragment length polymorphism conditions
The RFLP was carried out in a 20-µL volume mixture consisting of 2 µL of 10X buffer, 10 U of Hae III enzyme (New England Biolabs, USA), 10 µL of PCR product, and 7 µL of distilled H 2 O. The reactions were incubated at 37°C overnight and the digested products were visualized under an ultraviolet transilluminator after they had been electrophoresed on 3% agarose gel containing ethidium bromide for 1 h. The digested RFLP products were obtained for a wild-type genotype, while there were undigested RFLP products for a mutant genotype on the agarose gel.
To further assess the reliability of the presented assay, the PCR product of each different genotype was verified by direct sequencing using the same set of primers.

Statistical analysis
Allele and genotype frequencies were calculated by genotype counting method. The observed genotype frequencies of CHRM1 C267A were compared with the expected frequencies according to the Hardy-Weinberg equilibrium. The data obtained were compared with previously reported representative data in other ethnic groups. Differences in allele frequencies between schizophrenic groups were tested by Pearson's chi-square test and a p value <0.05 was considered statistically significant.

RESULTS
A novel PCR-RFLP assay was designed to detect C267A SNP in the CHRM1 gene region in schizophrenic patients. We also evaluated the accuracy and validity of this novel method. New primers were designed and the PCR products were digested with Hae III restriction enzyme for determination of the variant genotypes. A schematic illustration of the assay is given in Figure 1.
The previous genotyping method for rs2067477 by Liao et al. 17 could not be perfectly applied to analyze this SNP due to the difficulties in finding primer sites. This method also did not include any information about PCR product fragments, PCR conditions, or base pairs of the restriction fragments for genotyping. In the present study, a novel genotyping assay was developed and successfully performed by utilizing a reverse primer and mismatch forward primer, which are explained above. As shown in Figure 2, the underlined A (adenine base) is the mismatched base in the forward primer, which was replaced with the ancestral base G (guanine base) to eliminate the recognition site of the Hae III restriction enzyme (GG▼CC) in the primer binding site. This was also confirmed by sequencing (data not shown).
The individuals with the CC genotype (wild type) yielded two bands of 83 bp and 42 bp, while those with the AA genotype (mutant type) gave an undigested band (125 bp) on 3% agarose gel. The agarose gel electrophoresis results of the RFLP products on 3% agarose gel are given in Figure 3.
One sample of each different genotype PCR product was sequenced to confirm the expected sequence of each genotype and the data obtained were consistent with our findings. The sequencing results of the three genotypes are given below in Figure 4. The PCR products of each different genotype sequencing result precisely demonstrated the reliability of our novel assay.
The allele and genotype frequencies in the 51 Caucasian Turkish schizophrenic patients are shown in Table 1 for the C267A polymorphism in the CHRM1 gene. This is the first study to document the frequencies and genotypes of CHRM1 C267A alleles in Turkish patients with schizophrenia. The molecular analyses revealed that, among the 51 patients tested for the C267A genotype, 37 (72.5%) were CC, 13 (25.5%) were CA, and 1 (2%) was AA. On the basis of these data, the allele frequency of C was 0.85 and the frequency of A was 0.15. The distribution of CHRM1 genotypes in our samples is presented in Table 1. The

DISCUSSION
Due to several gene variations that are potentially involved in the physiopathology of mental disorders, the CHRM1 C267A polymorphism has the probability to become a genetic biomarker. In addition, this variation might play a role in psychopharmacotherapy since the muscarinic M1 receptor is a prominent target for a considerable number of medications. There were three primary objectives in the present study. The    Forward and reverse primers are highlighted in gray. The mismatch base (A), which is used to eliminate the recognition site of Hae III in the forward primer, is underlined. The Hae III recognition site is depicted by underlining in the middle of the CHRM1 sequence. This recognition site also includes rs2067477 SNP, which is depicted with capital and bold letters in the recognition site (C). In the case of the ancestral C allele at position 267 of the CHRM1 gene 83 bp and 42 bp DNA fragments are obtained, after Hae III digestion. Conversely, no digestion site for Hae III endonuclease is found, when the C allele is replaced by an A allele at position 267, giving one fragment of 125 bp main purpose was to develop a novel genotyping assay for the CHRM1 C267 polymorphism and to test the accuracy and validity of the developed method. The other two aims were to draw attention to the importance of the CHRM1 gene in the pathology of schizophrenia and to determine the genotype and allele frequencies of the CHRM1 C267 polymorphism in Turkish patients with schizophrenia.
M1 receptors could be important for neuronal disorders and cognitive function in the pathophysiology of schizophrenia due to the location in the medial prefrontal cortex and hippocampus. 18,19 Lower levels of muscarinic receptors in the central nervous system of people with schizophrenia have been found in some studies. 18,20 Scarr et al. 21 showed that decreased M1 levels in the cortical region of the brain could contribute to the pathophysiology of schizophrenia. Thus, a brain imaging test before treatment could be useful in identifying patients with low M1 levels who could be treatment resistant. Another neuroimaging study also showed that muscarinic receptors were extensively decreased in schizophrenia patients under treatment during neuroimaging. 20 At the molecular level, Mancama et al. 22 demonstrated that the levels of CHRM1 cDNA in schizophrenia patients were 28% lower than those in their control group. Moreover, research suggested that there could be a relationship between rs2067477 SNP and a reduction in gray matter volume in patients with schizophrenia. 23 Other studies have shown that rs2067477 might be associated with cognitive performance. In these studies the Wisconsin Card Sorting Test performance, which is a measure of prefrontal and executive functions, was better in heterozygous individuals than in homozygous wildtype carriers. 17,24 In one of these studies, 243 schizophrenic patients were assessed according to the rs2067477 genotype and the genotypes differed in responses in the Wisconsin Card Sorting Test but not in other parameters including age of onset, chlorpromazine equivalents, and Brief Psychiatric Rating Scale. 17 Contrary to these, Cropley et al. 23 indicated that the homozygous CC genotype did not have an impact on attention, visuospatial construction, verbal fluency, or working memory but they did not assess the patients using the Wisconsin Card Sorting Test. All of these studies showed the importance of the determination of CHRM1 C267A alleles in schizophrenic patients. To the best of our knowledge, ours is the first study to document the frequencies of CHRM1 C267A alleles and its genotype distribution in Turkish schizophrenia patients.
In the present study, the genotype distribution and allele frequencies of the CHRM1 C267A polymorphism were obtained from 51 Turkish schizophrenia patients. The data obtained were compared with previously reported representative data in other schizophrenia patients as shown in Table 2. The present results showed that the C and A allele frequencies in Turkish patients with schizophrenia were 0.85 and 0.15, respectively. The C267A variant frequency ranged between 0.07 and 0.11 in Australian patients with schizophrenia or schizoaffective disorder, while it was 0.09 in Chinese schizophrenia patients. 17,[23][24][25][26] The difference in frequency of C267A SNP between Turkish schizophrenia patients and other populations patients was not statistically significant (p>0.05).

CONCLUSION
In summary, a novel, practical, low-cost, and reproducible PCR-RFLP method was developed for genotyping the CHRM1 C267A polymorphism. The method is based on elimination of the recognition site of Hae III in the forward primer binding site by utilizing a mismatch base in the forward primer. As a result of this study, the validity and accuracy of the present novel method have been proven. Thus, the genotype and allele frequencies of the CHRM1 C267 polymorphism in Turkish patients with schizophrenia have been determined for the first time. The number of samples should be increased in further studies for more certain and reliable results. Additionally, the effect of the CHRM1 gene in the pathology and treatment of schizophrenia is explained with the data in the literature. The developed genotyping assay and results could be useful and provide a perspective for future studies.
Financial and Conflict of interest: This work was supported in part by grant from the Research Fund of Ankara University under Project 18L0217001. The author declares no conflict of interest, financial or otherwise. Ethical conduct of research: All authors state that the appropriate institutional review board approval had obtained and the informed consent has been obtained from the participants involved study. The authors state that all experiments had followed the principles outlined in the Declaration of Helsinki.
Conflicts of interest: No conflict of interest was declared by the authors. The authors alone are responsible for the content and writing of the paper.