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Objectives: To clarify a possible association between schizophrenia (SZ) and single nucleotide polymorphism (SNP) rs821616 within disrupted in schizophrenia 1 (DISC1) gene in a Kinh population in Vietnam.
Journal of military pharmaco-medicine No7-2017 NO ASSOCIATION BETWEEN RS821616 OF DISC1 GENE AND SUSCEPTIBILITY TO SCHIZOPHRENIA IN A VIETNAMESE POPULATION Dang Tien Truong*; Nguyen Duy Bac*; Pham Minh Dam*; Tran Hai Anh* SUMMARY Objectives: To clarify a possible association between schizophrenia (SZ) and single nucleotide polymorphism (SNP) rs821616 within disrupted in schizophrenia (DISC1) gene in a Kinh population in Vietnam Methods: We genotyped rs821616 of DISC1 gene using direct sequencing method Results: Our case-control analysis in 201 participants (100 schizophrenic patients and 101 healthy controls of Kinh ethnicity) found no significant difference in allele frequencies and genotyping distributions between case and control groups Conclusion: The results providing an initial evidence that SNP rs821616 has no association to susceptibility for SZ in a limited population of Kinh ethnicity in Vietnam * Keywords: Schizophrenia; Single nucleotide polymorphism; rs821616; DISC1 INTRODUCTION Schizophrenia (SZ) is a severe mental disorder influencing multiple brain functions The heritability is accounted about 80% in SC, with around 10-fold risk increase in first degree relatives Genetics in SZ has long been assumed as a major component basing on family and adoption studies Substantial evidence from genetics studies supports an important role of disrupted in schizophrenia (DISC1) in regulation for neural development [6, 8], and of single nucleotide polymorphism (SNP) rs821616 (Ser704Cys) within DISC1 gene in pathophysiology of SZ [1] Ser704Cys associated to poor concentration in SZ patients [3], frontal and temporal gray matter reduction [9], and lower hippocampal volume [2], etc However, results on DISC1 from different populations have been inconsistent, especially between and among European and Asian populations [2, 3, 4] Since there have still no DISC1 related data from Vietnamese people, this early study aimed to: Elucidate a possible association between SZ and rs821616 of DISC1 gene in a Kinh population in Vietnam SUBJECTS AND METHODS Subjects 100 schizophrenic patients and 101 healthy controls of Kinh ethnicity were participated in this case-control study Schizophrenia cases were collected from Department of Psychiatry of 103 Hospital, Vietnam Military Medical University The patients were diagnosed by criteria of Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) * Vietnam Military Medical University Corresponding author: Tran Hai Anh (anhhtr@yahoo.com) Date received: 04/07/2017 Date accepted: 17/08/2017 48 Journal of military pharmaco-medicine no7-2017 Methods Kits used in the study including Qiagen Blood Mini kit, Qiagen master mix kit, QIA quick PCR Purification Kit (Qiagen, Germany), and Bigeye Terminator 3.1 and POP (ABI, USA) * SNP genotyping: Peripheral blood samples were collected from all 201 subjects Genomic DNA was extracted using Qiagen Blood Mini kit, following the manufacturer’s protocol and stored at -20oC The obtained product containing SNP was amplified using polymerase chain reaction (PCR) method The PCR reaction was carried out in a total volume of 50 µL containing 50 - 100 ng genomic DNA as the template, 0.2 µM of each primer (synthesized by IDT, USA), and Master mix Qiagen kit 1X PCR amplification was performed in a Master Cycler® thermal cycler (Eppendorf, Germany) with an initial denaturation step at 98°C for 15 minutes, followed by 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 60 seconds, and a final extension step at 72°C for minutes The primer sequence for the forward and reverse primers were 5’-TGACCAGCTGACTTTTAGCC-3’ and 5’- AAGTTTATATGCTCAATGGGAAGC-3’, respectively The PCR products were separated on 2% agarose gel PCR products were perfricated and genotyped by Bigdye Terminator 3.1 and sequenced on ABI 3130XL * Statistical analysis: Hardy-Weinberg equilibrium (HWE) for the SNP was assessed in both cases and controls Frequencies and alleles of genotypes were calculated and compared using a chi-square test, and Fisher exact test * Ethical approval: This study was approved by the ethics review committee of Vietnam Military Medical University RESULTS AND DISCUSSION Demographics of participants Some characteristics of subjects were showed in table Table 1: Demographics of participants Groups Age Gender (n, %) (mean ± SD) Male female SZ (100) 34.58 ± 11.64 61 (61.00) 39 (39.00) Control (101) 32.91 ± 6.44 63 (62.38) 38 (37.62) p = 0.21 X = 0.04; p = 0.84 The results in table showed that there was no different mean age and gender between SZ and control groups, respectively p = 0.21 and p = 0.84 49 Journal of military pharmaco-medicine No7-2017 Results of genotyping of rs821616 Genomic DNA extracted and purified, then amplified to PCR products for further genotyping analyses is demonstrated in figure A B Figure 1: Artwork of DNA agarose electrophoresis results in SZ group (A) and control group (B) Identical lanes in figure illustrated that PCR products were specific and sufficient for genotyping The results for genotyping of rs821616 (homozygous and heterozygous types) are shown in figure Figure 2: Peaks (arrows) of heterozygous (AT) and homozygous genotypes (TT, AA) of the SNP in SZ samples Test for Hardy Weinberg equilibrium showed that genotypes in the patient (X2 = 1.29) and control (X2 = 1.22) groups were consistent with HWE (p > 0.05) Genotyped allele frequencies of rs821616 in DISC1 gene is shown in table 50 Journal of military pharmaco-medicine no7-2017 Table 2: Allele frequencies of rs821616 in DISC1 gene Groups n SC 200 Control 202 Allele frequency (n) A T 0.94 (188) 0.06 (12) 0.90 (182) 0.10 (20) X = 2.09; p = 0.15; OR = 0.58; 95%CI 0.25 - 1.29 The results in table showed no significant difference in allele frequencies between the patient and control groups (X = 2.09; p = 0.15) The results of genotype prevalence of rs821616 in DISC1 is shown in table Table 3: Genotypic distribution of the rs821616 of the DISC1 gene n (%) AA AT TT SC 100 (49.75) 89 (52.35) 10 (33.33) (100.00) Control 101 (50.25) 81 (47.65) 20 (66.67) (0.00) Total 201 (100.00) 170 (100.00) 30 (100.00) (100.00) Groups Genotype frequency n (%) p = 0.074 Genotyping of rs821616 in table showed no significant difference in genotypic distribution between the total patients and controls (p = 0.074) Our results were in accordance with the results of previous studies on rs821616 and SZ in Korean and Malaysian populations [3, 4] On the other hand, there were some results showed an association between rs821616 and SZ in Chinese Han [7] and in Caucasian populations [2] Nevertheless, a study on a Korean population also indicated that DISC1 variation is associated with the poor concentration phenotype of SZ [3] While rs821616 was reported as having an association with SZ in the Chinese Han population [7], but found no such association in a study on a Malaysian population [4] Several studies reported a relation of symptoms, reduction of gray matter and this rs821616 [2, 3, 9] Those lines of evidence supports the role of rs821616 within DISC1 gene in pathology of schizophrenia [5, 8] To our knowledge, the present work is the first study in our country investigating SNP rs821616 and SC, and found no marked association between them This finding is limited due to the relative small sample size and genetic stratification of an initial investigation Therefore, further studies with a larger size would be needed to firmly elucidate effects of SNP generally, and rs821616 (Ser704Cys), specifically in SZ 51 Journal of military pharmaco-medicine No7-2017 CONCLUSION Single nucleotide polymorphism rs821616 tentatively seems no association to susceptibility for SZ in a limited population of Kinh ethnicity in Vietnam REFERENCES Balu D.T, Coyle J.T Neuroplasticity signaling pathways linked to the pathophysiology of SZ Neurosci Biobehav Rev 2011, 35 (3), pp.848-870 M.A, Penninx B.W, Veltman D.J, Aleman A DISC1 gene and affective psychopathology: a combined structural and functional MRI study J Psychiatr Res 2015, 61, pp.150-157 Palo O.M, Antila M, Silander K, Hennah W, Kilpinen H, Soronen P, Tuulio-Henriksson A, Kieseppä T, Partonen T, Lönnqvist J, Peltonen L, Paunio T Association of distinct allelic haplotypes of DISC1 with psychotic and bipolar spectrum disorders and with underlying cognitive impairments Hum Mol Genet 2007, 16 (20), pp.2517-2528 Callicott J.H, Straub R.E, Pezawas L, Egan M.F, Mattay V.S, Hariri A.R, Verchinski B.A, Meyer-Lindenberg A, Balkissoon R, Kolachana B, Goldberg T.E, Weinberger D.R Variation in DISC1 affects hippocampal structure and function and increases risk for SZ Proc Natl Acad Sci USA 2005, 102 (24), pp.8627-8632 Qu M, Tang F, Yue W, Ruan Y, Lu T, Liu Z, Zhang H, Han Y, Zhang D, Wang F, Zhang D Positive association of the disrupted-in-SZ-1 gene (DISC1) with SZ in the Chinese Han population Am J Med Genet B Neuropsychiatr Genet 2007, 144B (3), pp.266-270 Kim H.J, Park H.J, Jung K.H, Ban J.Y, Ra J, Kim J.W, Park J.K, Choe B.K, Yim S.V, Kwon Y.K, Chung J.H Association study of polymorphisms between DISC1 and SZ in a Korean population Neurosci Lett 2008, 430 (1), pp.60-63 Shao L, Lu B, Wen Z, Teng S, Wang L, Zhao Y, Wang L, Ishizuka K, Xu X, Sawa A, Song H, Ming G, Zhong Y Disrupted-in-SZ-1 (DISC1) protein disturbs neural function in multiple disease-risk pathways Hum Mol Genet 2017, doi: 10.1093/hmg/ddx147 Norlelawati A.T, Kartini A, Norsidah K, Ramli M, Tariq A.R, Wan Rohani W.T Disrupted-in-SZ-1 SNPs and susceptibility to SZ: Evidence from Malaysia Psychiatry Investig 2015, 12 (1), pp.103-111 Trost S, Platz B, Usher J, Scherk H, Wobrock T, Ekawardhani S, Meyer J, Reith W, Falkai P, Gruber O DISC1 (disrupted-inSZ 1) is associated with cortical grey matter volumes in the human brain: a voxel-based morphometry (VBM) study J Psychiatr Res 2013, 47 (2), pp.188-196 Opmeer E.M, van Tol M.J, Kortekaas R, van der Wee N.J, Woudstra S, van Buchem 52 ... rs821616 and SZ in Chinese Han [7] and in Caucasian populations [2] Nevertheless, a study on a Korean population also indicated that DISC1 variation is associated with the poor concentration phenotype... protocol and stored at -20oC The obtained product containing SNP was amplified using polymerase chain reaction (PCR) method The PCR reaction was carried out in a total volume of 50 µL containing... phenotype of SZ [3] While rs821616 was reported as having an association with SZ in the Chinese Han population [7], but found no such association in a study on a Malaysian population [4] Several studies