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PITX3 gene polymorphism is associated with Parkinson's disease in Chinese population
BR A I N R ES E A RC H 1 3 9 2 ( 2 01 1 ) 1 1 6 –12 0
available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
PITX3 gene polymorphism is associated with Parkinson's disease in Chinese population Jia Liu a,1 , Qi-ying Sun a,1 , Bei-sha Tang a,b,c , Liang Hu a , Ren-he Yu d , Lei Wang a , Chang-he Shi a , Xin-xiang Yan a,c , Qian Pan b , Kun Xia b , Ji-feng Guo a,c,⁎ a
Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, PR China National Laboratory of Medical Genetics of China, Changsha, Hunan 410008, PR China c Neurodegenerative Disorders Research Center, Central South University, Changsha, Hunan 410008, PR China d School of Public Health, Central South University, Changsha, Hunan 410008, PR China b
A R T I C LE I N FO
AB S T R A C T
Article history:
Genetic variants of PITX3 gene have been reported to be associated with Parkinson's disease
Accepted 27 March 2011
(PD) in several populations. We conducted a case-control study and genotyped the three
Available online 6 April 2011
SNPs of PITX3 gene: rs2281983, rs4919621 and rs3758549 in 512 mainland Chinese PD patients and 506 healthy controls. Our findings show that the PITX3 gene rs3758549
Keywords:
polymorphism is associated with PD (p = 0.02). Moreover, the difference between late onset
Pitx3
PD patients and healthy controls is stronger (p = 0.007). There is no statistical difference in
Single nucleotide polymorphisms
genotype or allele frequencies of rs2281983 or rs4919621 variant in PITX3 gene between
Parkinson's disease
sporadic PD (SPD) group and healthy control group in our study. To assess the possible role
Meta-analysis
of the PITX3 gene rs3758549 polymorphism in PD, we conducted a meta-analysis on the topic. The results of meta-analysis further support that the PITX3 gene rs3758549 polymorphism is associated with PD: Z = 3.09, p = 0.002, OR = 0.89. These findings suggest that the PITX3 gene rs3758549 polymorphism may increase the susceptibility of PD. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Parkinson's disease (PD) is one of the most frequent neurodegenerative disorders caused by loss of dopaminergic neurons in the substantia nigra, which results in decreased dopamine availability in the striatum. Clinical manifestation is characterized by resting tremor, rigidity, bradykinesia and impaired postural reflexes. The majority of the patients are sporadic cases. Approximately 5–10% of patients have genetic factors
(Samii et al., 2004; Lees et al., 2009), yet the etiology of PD remains unclear. To date, 15 loci have been found to be associated with Parkinson's disease, and 11 of them have been cloned (Lees et al., 2009). However, that cannot explain the etiology of the vast majority of patients with an apparently sporadic, late-onset disease. Transcription factor PITX3 is very important for the midbrain dopaminergic (mDA) neurons' terminal differentiation and survival. In mouse embryo, PITX3 is present in the eye lens,
⁎ Corresponding author at: Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, PR China. Fax: +86 731 84327332. E-mail address: [email protected] ( J. Guo). Abbreviations: PD, Parkinson's disease; SPD, Sporadic PD; SNpc, Substantia nigra pars compacta; LB, Lewy body; mDA, Midbrain dopaminergic; TH, Tyrosine hydroxylase; AHD2, Aldehyde dehydrogenase; GDNF, Glial cell line derived neurotrophic factor; BDNF, Brain derived neurotrophic factor; LOPD, Late onset PD; EOPD, Early onset PD; RFLP, Restricted fragment length polymorphism 1 The first two authors contributed equally to the study. 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.03.064
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no statistical difference in genotype or allele frequencies of rs2281983 and rs4919621 variants in PITX3 gene between sporadic PD (SPD) group and healthy control group. In PD group the D′ value between rs4919621 and rs2281983 was 0.984 while in control group it was 0.962. rs4919621 was in strong linkage disequilibrium with rs2281983 (r2 = 0.936). The frequency of rs3758549 variant is significantly higher in SPD group than in healthy control group (p = 0.019), especially in LOPD group (p = 0.007). But there is no statistical difference between EOPD patients and controls (p = 0.274) (summarized in Table 1). The results of meta-analysis further support that the PITX3 rs3758549 polymorphism is associated with PD: Z = 3.09, p = 0.002, OR = 0.89 (Fig. 1). Furthermore, we examined the distribution of sub-haplotypes in PD patients and controls which were compiled as combinations of the three SNPs and found that sub-haplotype T-T-T is associated with Chinese PD patients (p = 0.032) (Table 2), suggesting that it may increase the susceptibility of PD.
skeletal muscles and mDA neurons for a short time (Smidt et al., 1997). After birth, PITX3 is only expressed in the mDA neurons (Smidt et al., 1997), which shows the importance of PITX3 on the mDA neurons' development. It has been found that PITX3 interacts with orphan nuclear receptor NURR1 (Martinat et al., 2006; Jacobs et al., 2009a,b), and also regulates the expression of tyrosine hydroxylase (TH) (Cazorla et al., 2000; Lebel et al., 2001), aldehyde dehydrogenase (AHD2) (Jacobs et al., 2007), glial cell line derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF) (Peng et al., 2007), which are all critical for the DA neurons' growth, differentiation and survival. Furthermore, PITX3 deals in a negative feedback circuit that includes an miRNA, miR-133b, which is specifically expressed in mDA neurons and regulates the maturation and function of mDA neurons (Kim et al., 2007). Aphakia mouse was characterized by small eyes and lens defect, which was first described by Varnum and Stevens (1968) (Varnum and Stevens, 1968). The genetic variation of the aphakia mouse was two deletions of PITX3 gene. One is in the upstream enhancer region (Semina et al., 2000); another covers the promoter area, exon 1 and part of intron 1 (Rieger et al., 2001). The mDA neurons of aphakia mouse is less than those of wild blind mouse (Smidt et al., 2004), which is similar with the pattern of PD patients. Moreover, aphakia mouse gets some movement defects which can be reversed by L-DOPA (Hwang et al., 2005). Recently, genetic variants of PITX3 gene have been reported to be associated with PD in Caucasian population (Fuchs et al., 2009; Bergman et al., 2010; Haubenberger et al., 2011; Le et al., 2009), especially the three SNPs: rs2281983, rs4919621, and rs3758549. There are also some reports that failed to confirm the association for some of these SNPs (de Mena et al., 2010). To verify these findings and investigate the possible association between the PITX3 gene and PD in the mainland Chinese population, we detected the above-mentioned three SNPs (rs2281983, rs4919621, and rs3758549) using a case control study design.
2.
3.
Discussion
We examined the possible role of the PITX3 polymorphisms in PD in an ethnically homogeneous Chinese population. We found that the promoter polymorphism rs3758549 was strongly associated with Chinese PD patients, consistent with the previous reports (Fuchs et al., 2009; Haubenberger et al., 2011). In the promoter area of PITX3 gene there are some binding sites in touch with other genes to regulate the expression of signal molecules such as TH gene through a direct high-affinity binding site (Cazorla et al., 2000) then influence the process of mDA neuron development, finally playing a role in the pathogenesis of PD. Moreover, the association was stronger while in the LOPD patients as compared with healthy controls, but no statistical difference was found between EOPD and controls. The interaction of aging and genetic factors may be an explanation. To assess the possible role of the PITX3 gene rs3758549 in PD, we conducted a meta-analysis on the topic. The results of meta-analysis show that the PITX3 rs3758549 polymorphism is associated with PD: Z = 3.09, p = 0.002, OR = 0.89.
Results
All SNPs were in Hardy–Weinberg equilibrium, both in the patient and in the control group. Our data show that there is
Table 1 – Allele frequency of studied PITX3 SNPs in controls, PD patients, patients with early onset PD (≤50 years) and late onset (>50 years), respectively. SNP
Genotype
Case
Controls
Genotype count (frequency) rs2281983
rs4919621
rs3758549
a
CC TC TT AA TA TT CC TC TT
PD group vs. controls.
27 189 296 27 188 297 330 167 15
(5.3) (36.9) (57.8) (5.3) (36.7) (58.0) (64.5) (32.6) (2.9)
30 (5.9) 187 (37.0) 289 (57.1) 34 (6.4) 182 (36.0) 290 (57.3) 366 (72.3) 125 (24.7) 15 (3.0)
SPD a
EOPD a
LOPD a
SPD a
EOPD a
LOPD a
Genotype association
Allele dosage effect model
p-Values
p-Values
0.897
0.458
0.682
0.721
0.322
0.888
0.622
0.420
0.501
0.573
0.186
0.944
0.019
0.274
0.007
0.019
0.100
0.032
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Fig. 1 – Meta-analysis of the PITX3 gene rs3758549: C > T in PD. The T allele has a significant association with PD: Z = 3.09, p = 0.002.
In our research, we didn't find the association of rs2281983 or rs4919621 variant with Chinese PD patients, and failed to duplicate the results of Swedish and North American population studies (Le et al., 2009; Bergman et al., 2010). These different findings could be due to the general genetic background or environmental factors of population-specific differences. As summary, the PITX3 gene rs3758549 may be a risk factor in the pathogenesis of PD in China. Further study may provide new methods for the prevention and treatment of PD.
PD patients were divided into early onset PD (an age at onset ≤50 years, n = 172) and late onset PD (an age at onset > 50 years, n = 340). Information on family history, demographic characteristics, clinical data and neurological examination were completed for each patient. All patients were informed regarding the results of the analysis. A control group of 506 healthy mainland Chinese individuals from the same geographic areas was obtained and matched for age and sex (53.18 ± 15.62 years, ranging from 12 to 76 years old) with the PD patients' sample. Each patient and control was asked to provide written informed consent and a blood sample.
4.
Experimental procedures
4.2.
4.1.
Clinical materials
Five hundred and twelve mainland Chinese PD patients were collected from the outpatient neurology clinics of Xiangya Hospital and the National Lab of Medical Genetics of China from October, 2003, to December, 2009. PD was diagnosed by two or more experienced neurologists according to the United Kingdom brain-bank criteria (Hughes et al., 1992). None had a history of neurologic or psychiatric conditions other than PD. Only one patient was selected from each family. These PD patients came from Hunan, Hubei, Jiangxi and Chongqi provinces of China. Three hundred and sixteen (61.7%) of the patients were men, and 196 (38.3%) were women. The mean age at disease onset in the patient group was 54.71 years (SD 11.64 years, ranging from 8 to 79). According to the onset age,
Table 2 – The haplotype frequencies in patients and controls. Haplotypes a
SPD (n, %)
Control (n, %)
χ2
p
CAC TTC TTT
236.58 (23.1) 583.52 (57.0) 194.46 (19.0)
239.88 (23.7) 600.33 (59.3) 154.60 (15.3)
0.176 1.664 4.603
0.675 0.197 0.032
a
The haplotype frequency < 0.03 is not calculated.
Genetic analysis
After extracting genomic DNA from peripheral blood through standard protocols, we screened all PD patients and controls for the three SNPs, rs2281983, rs4919621 and rs3758549, using PCR-restricted fragment length polymorphism (RFLP) method. PCR products were digested with restriction enzymes and the resulting fragments were submitted to electrophoresis using 12% polyacrylamide gel. The PCR primers and restriction enzymes are listed (Supplementary table 1). In order to ensure the accuracy of the results, we sequenced 10% DNA samples directly. Differences of the SNP frequencies between groups were analyzed by means of the χ2 test or Fisher's exact test. p < 0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS 13.0 (SPSS, Inc., Chicago, IL) and Haploview 4.1.
4.3.
Meta-analysis
To assess the possible role of the PITX3 gene rs3758549 polymorphism in PD, we conducted a meta-analysis on the topic. We conducted a literature search in PubMed for all relevant papers published from 1966 to September 2010 using the search terms “PITX3,” “rs3758549,” or “PITX3 polymorphisms” combined with “Parkinson's disease” or “PD.” Furthermore, we reviewed the reference lists from retrieved articles to search for more studies. The following criteria were used to identify relevant studies for the meta-analysis: first, clinical diagnosis of PD was established according to the
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Table 3 – The summary of the PITX3 gene rs3758549 analysis in PD. Study
Population studied
Fuchs et al., 2009 Fuchs et al., 2009 Simon-Sanchez et al., 2009 Haubenberger et al., 2011 Bergman et al., 2010 Jia, current study Total
Germany Germany USA, Germany Austria Sweden China
PD patients C
5.
T
574 (0.844) 1139 (0.851) 2850 (0.832) 568 (0.782) 572 (0.829) 827 (0.808) 6530 (0.828)
United Kingdom brain-bank criteria (Hughes et al., 1992). Second, they had a case-control study design. Third, the PITX3 gene rs3758549 was detected among PD patients and controls. We identified 9 relevant studies concerning the PITX3 polymorphisms and Parkinson's disease (Fuchs et al., 2009; Bergman et al., 2010; Haubenberger et al., 2011; Le et al., 2009; Fung et al., 2006; Mizuta et al., 2006; Pankratz et al., 2009; Simon-Sanchez et al., 2009; de Mena et al., 2010). Five studies (Le et al., 2009; Fung et al., 2006; Mizuta et al., 2006; Pankratz et al., 2009; de Mena et al., 2010) were excluded because they did not detect rs3758549. Thus, the meta-analysis on the PITX3 gene rs3758549 and PD included four papers in total. We extracted the following data from each publication: the first author's name, the year of publication, population studied, the frequencies of the PITX3 gene rs3758549 among PD patients and the control group (summarized in Table 3). Meta-analyses were performed using RevMan4.2. p < 0.05 was considered statistically significant.
Conflict of interest statement
The authors declare that there are no competing interests.
Acknowledgments This work was supported by grant 2011CB510000 from the Major State Basic Research Development Program of China (973 Program) (to Dr. Bei-sha Tang), grant 2006AA02A408 from the National “863” High-Tech Research and Development Program of China (to Dr. Bei-sha Tang), grants 30570638, 30770735 and 30971035 from the National Natural Science Foundation of China (to Dr. Bei-sha Tang), and grant 30900469 from the National Natural Science Foundation of China (to Dr. Ji-feng Guo). The authors thank all the PD patients and healthy controls for participating in the study. Author contributions: study conception, design and organization (Liu, Sun, and Guo); acquisition of data (Liu, Sun, Tang, Hu, Wang, Shi, Pan, Xia, and Yan); analysis and interpretation of data (Liu, Sun, Tang, Hu, Wang, Shi, Pan, Xia, Yan, and Guo); drafting of the manuscript (Liu, Sun, Tang, and Guo); critical revision of the manuscript for important intellectual content (Liu, Sun, Tang, and Guo); statistical analysis (Liu, Sun, Tang, Hu, Wang, Shi, Pan, Xia, Yan, and
Controls
106 199 576 158 118 197 1354
(0.156) (0.149) (0.168) (0.218) (0.171) (0.192) (0.172)
C 1098 1088 6392 653 461 857 10,549
(0.807) (0.813) (0.803) (0.804) (0.829) (0.847) (0.809)
T 262 (0.193) 250 (0.187) 1564 (0.197) 159(0.196) 95 (0.171) 155 (0.153) 2485 (0.191)
Guo); obtained funding (Tang and Guo); administrative, technical, and material support (Liu, Sun, Tang, Hu, Wang, Shi, Pan, Xia, Yan, and Guo); and study supervision (Liu, Sun, Tang, and Guo).
Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.brainres.2011.03.064.
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3,4-Dihydroxyphenylalanine reverses the motor deficits in Pitx3-deficient aphakia mice: behavioral characterization of a novel genetic model of Parkinson's disease. J. Neurosci. 25, 2132–2137. Jacobs, F.M., Smits, S.M., Noorlander, C.W., von Oerthel, L., van der Linden, A.J., Burbach, J.P., Smidt, M.P., 2007. Retinoic acid counteracts developmental defects in the substantia nigra caused by Pitx3 deficiency. Development 134, 2673–2684. Jacobs, F.M., van der Linden, A.J., Wang, Y., von Oerthel, L., Sul, H. S., Burbach, J.P., Smidt, M.P., 2009a. Identification of Dlk1, Ptpru and Klhl1 as novel Nurr1 target genes in meso-diencephalic dopamine neurons. Development 136, 2363–2373. Jacobs, F.M., van Erp, S., van der Linden, A.J., von Oerthel, L., Burbach, J.P., Smidt, M.P., 2009b. Pitx3 potentiates Nurr1 in dopamine neuron terminal differentiation through release of SMRT-mediated repression. Development 136, 531–540. Kim, J., Inoue, K., Ishii, J., Vanti, W.B., Voronov, S.V., Murchison, E., Hannon, G., Abeliovich, A., 2007. A MicroRNA feedback circuit in midbrain dopamine neurons. Science 317, 1220–1224. Le, W., Nguyen, D., Lin, X.W., Rawal, P., Huang, M., Ding, Y., Xie, W., Deng, H., Jankovic, J., 2009. Transcription factor PITX3 gene in Parkinson's disease. Neurobiol. Aging. Lees, A.J., Hardy, J., Revesz, T., 2009. Parkinson's disease. Lancet 373, 2055–2066. Lebel, M., Gauthier, Y., Moreau, A., Drouin, J., 2001. Pitx3 activates mouse tyrosine hydroxylase promoter via a high-affinity binding site. J. Neurochem. 77, 558–567. Martinat, C., Bacci, J.J., Leete, T., Kim, J., Vanti, W.B., Newman, A.H., Cha, J.H., Gether, U., Wang, H., Abeliovich, A., 2006. Cooperative transcription activation by Nurr1 and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype. Proc. Natl. Acad. Sci. U.S.A. 103, 2874–2879. Mizuta, I., Satake, W., Nakabayashi, Y., Ito, C., Suzuki, S., Momose, Y., Nagai, Y., Oka, A., Inoko, H., Fukae, J., Saito, Y., Sawabe, M., Murayama, S., Yamamoto, M., Hattori, N., Murata, M., Toda, T., 2006. Multiple candidate gene analysis identifies alpha-synuclein as a susceptibility gene for sporadic Parkinson's disease. Hum. Mol. Genet. 15, 1151–1158. Pankratz, N., Wilk, J.B., Latourelle, J.C., DeStefano, A.L., Halter, C., Pugh, E.W., Doheny, K.F., Gusella, J.F., Nichols, W.C., Foroud, T., Myers, R.H., 2009. Genomewide association study for
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available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
PITX3 gene polymorphism is associated with Parkinson's disease in Chinese population Jia Liu a,1 , Qi-ying Sun a,1 , Bei-sha Tang a,b,c , Liang Hu a , Ren-he Yu d , Lei Wang a , Chang-he Shi a , Xin-xiang Yan a,c , Qian Pan b , Kun Xia b , Ji-feng Guo a,c,⁎ a
Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, PR China National Laboratory of Medical Genetics of China, Changsha, Hunan 410008, PR China c Neurodegenerative Disorders Research Center, Central South University, Changsha, Hunan 410008, PR China d School of Public Health, Central South University, Changsha, Hunan 410008, PR China b
A R T I C LE I N FO
AB S T R A C T
Article history:
Genetic variants of PITX3 gene have been reported to be associated with Parkinson's disease
Accepted 27 March 2011
(PD) in several populations. We conducted a case-control study and genotyped the three
Available online 6 April 2011
SNPs of PITX3 gene: rs2281983, rs4919621 and rs3758549 in 512 mainland Chinese PD patients and 506 healthy controls. Our findings show that the PITX3 gene rs3758549
Keywords:
polymorphism is associated with PD (p = 0.02). Moreover, the difference between late onset
Pitx3
PD patients and healthy controls is stronger (p = 0.007). There is no statistical difference in
Single nucleotide polymorphisms
genotype or allele frequencies of rs2281983 or rs4919621 variant in PITX3 gene between
Parkinson's disease
sporadic PD (SPD) group and healthy control group in our study. To assess the possible role
Meta-analysis
of the PITX3 gene rs3758549 polymorphism in PD, we conducted a meta-analysis on the topic. The results of meta-analysis further support that the PITX3 gene rs3758549 polymorphism is associated with PD: Z = 3.09, p = 0.002, OR = 0.89. These findings suggest that the PITX3 gene rs3758549 polymorphism may increase the susceptibility of PD. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Parkinson's disease (PD) is one of the most frequent neurodegenerative disorders caused by loss of dopaminergic neurons in the substantia nigra, which results in decreased dopamine availability in the striatum. Clinical manifestation is characterized by resting tremor, rigidity, bradykinesia and impaired postural reflexes. The majority of the patients are sporadic cases. Approximately 5–10% of patients have genetic factors
(Samii et al., 2004; Lees et al., 2009), yet the etiology of PD remains unclear. To date, 15 loci have been found to be associated with Parkinson's disease, and 11 of them have been cloned (Lees et al., 2009). However, that cannot explain the etiology of the vast majority of patients with an apparently sporadic, late-onset disease. Transcription factor PITX3 is very important for the midbrain dopaminergic (mDA) neurons' terminal differentiation and survival. In mouse embryo, PITX3 is present in the eye lens,
⁎ Corresponding author at: Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, PR China. Fax: +86 731 84327332. E-mail address: [email protected] ( J. Guo). Abbreviations: PD, Parkinson's disease; SPD, Sporadic PD; SNpc, Substantia nigra pars compacta; LB, Lewy body; mDA, Midbrain dopaminergic; TH, Tyrosine hydroxylase; AHD2, Aldehyde dehydrogenase; GDNF, Glial cell line derived neurotrophic factor; BDNF, Brain derived neurotrophic factor; LOPD, Late onset PD; EOPD, Early onset PD; RFLP, Restricted fragment length polymorphism 1 The first two authors contributed equally to the study. 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.03.064
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no statistical difference in genotype or allele frequencies of rs2281983 and rs4919621 variants in PITX3 gene between sporadic PD (SPD) group and healthy control group. In PD group the D′ value between rs4919621 and rs2281983 was 0.984 while in control group it was 0.962. rs4919621 was in strong linkage disequilibrium with rs2281983 (r2 = 0.936). The frequency of rs3758549 variant is significantly higher in SPD group than in healthy control group (p = 0.019), especially in LOPD group (p = 0.007). But there is no statistical difference between EOPD patients and controls (p = 0.274) (summarized in Table 1). The results of meta-analysis further support that the PITX3 rs3758549 polymorphism is associated with PD: Z = 3.09, p = 0.002, OR = 0.89 (Fig. 1). Furthermore, we examined the distribution of sub-haplotypes in PD patients and controls which were compiled as combinations of the three SNPs and found that sub-haplotype T-T-T is associated with Chinese PD patients (p = 0.032) (Table 2), suggesting that it may increase the susceptibility of PD.
skeletal muscles and mDA neurons for a short time (Smidt et al., 1997). After birth, PITX3 is only expressed in the mDA neurons (Smidt et al., 1997), which shows the importance of PITX3 on the mDA neurons' development. It has been found that PITX3 interacts with orphan nuclear receptor NURR1 (Martinat et al., 2006; Jacobs et al., 2009a,b), and also regulates the expression of tyrosine hydroxylase (TH) (Cazorla et al., 2000; Lebel et al., 2001), aldehyde dehydrogenase (AHD2) (Jacobs et al., 2007), glial cell line derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF) (Peng et al., 2007), which are all critical for the DA neurons' growth, differentiation and survival. Furthermore, PITX3 deals in a negative feedback circuit that includes an miRNA, miR-133b, which is specifically expressed in mDA neurons and regulates the maturation and function of mDA neurons (Kim et al., 2007). Aphakia mouse was characterized by small eyes and lens defect, which was first described by Varnum and Stevens (1968) (Varnum and Stevens, 1968). The genetic variation of the aphakia mouse was two deletions of PITX3 gene. One is in the upstream enhancer region (Semina et al., 2000); another covers the promoter area, exon 1 and part of intron 1 (Rieger et al., 2001). The mDA neurons of aphakia mouse is less than those of wild blind mouse (Smidt et al., 2004), which is similar with the pattern of PD patients. Moreover, aphakia mouse gets some movement defects which can be reversed by L-DOPA (Hwang et al., 2005). Recently, genetic variants of PITX3 gene have been reported to be associated with PD in Caucasian population (Fuchs et al., 2009; Bergman et al., 2010; Haubenberger et al., 2011; Le et al., 2009), especially the three SNPs: rs2281983, rs4919621, and rs3758549. There are also some reports that failed to confirm the association for some of these SNPs (de Mena et al., 2010). To verify these findings and investigate the possible association between the PITX3 gene and PD in the mainland Chinese population, we detected the above-mentioned three SNPs (rs2281983, rs4919621, and rs3758549) using a case control study design.
2.
3.
Discussion
We examined the possible role of the PITX3 polymorphisms in PD in an ethnically homogeneous Chinese population. We found that the promoter polymorphism rs3758549 was strongly associated with Chinese PD patients, consistent with the previous reports (Fuchs et al., 2009; Haubenberger et al., 2011). In the promoter area of PITX3 gene there are some binding sites in touch with other genes to regulate the expression of signal molecules such as TH gene through a direct high-affinity binding site (Cazorla et al., 2000) then influence the process of mDA neuron development, finally playing a role in the pathogenesis of PD. Moreover, the association was stronger while in the LOPD patients as compared with healthy controls, but no statistical difference was found between EOPD and controls. The interaction of aging and genetic factors may be an explanation. To assess the possible role of the PITX3 gene rs3758549 in PD, we conducted a meta-analysis on the topic. The results of meta-analysis show that the PITX3 rs3758549 polymorphism is associated with PD: Z = 3.09, p = 0.002, OR = 0.89.
Results
All SNPs were in Hardy–Weinberg equilibrium, both in the patient and in the control group. Our data show that there is
Table 1 – Allele frequency of studied PITX3 SNPs in controls, PD patients, patients with early onset PD (≤50 years) and late onset (>50 years), respectively. SNP
Genotype
Case
Controls
Genotype count (frequency) rs2281983
rs4919621
rs3758549
a
CC TC TT AA TA TT CC TC TT
PD group vs. controls.
27 189 296 27 188 297 330 167 15
(5.3) (36.9) (57.8) (5.3) (36.7) (58.0) (64.5) (32.6) (2.9)
30 (5.9) 187 (37.0) 289 (57.1) 34 (6.4) 182 (36.0) 290 (57.3) 366 (72.3) 125 (24.7) 15 (3.0)
SPD a
EOPD a
LOPD a
SPD a
EOPD a
LOPD a
Genotype association
Allele dosage effect model
p-Values
p-Values
0.897
0.458
0.682
0.721
0.322
0.888
0.622
0.420
0.501
0.573
0.186
0.944
0.019
0.274
0.007
0.019
0.100
0.032
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Fig. 1 – Meta-analysis of the PITX3 gene rs3758549: C > T in PD. The T allele has a significant association with PD: Z = 3.09, p = 0.002.
In our research, we didn't find the association of rs2281983 or rs4919621 variant with Chinese PD patients, and failed to duplicate the results of Swedish and North American population studies (Le et al., 2009; Bergman et al., 2010). These different findings could be due to the general genetic background or environmental factors of population-specific differences. As summary, the PITX3 gene rs3758549 may be a risk factor in the pathogenesis of PD in China. Further study may provide new methods for the prevention and treatment of PD.
PD patients were divided into early onset PD (an age at onset ≤50 years, n = 172) and late onset PD (an age at onset > 50 years, n = 340). Information on family history, demographic characteristics, clinical data and neurological examination were completed for each patient. All patients were informed regarding the results of the analysis. A control group of 506 healthy mainland Chinese individuals from the same geographic areas was obtained and matched for age and sex (53.18 ± 15.62 years, ranging from 12 to 76 years old) with the PD patients' sample. Each patient and control was asked to provide written informed consent and a blood sample.
4.
Experimental procedures
4.2.
4.1.
Clinical materials
Five hundred and twelve mainland Chinese PD patients were collected from the outpatient neurology clinics of Xiangya Hospital and the National Lab of Medical Genetics of China from October, 2003, to December, 2009. PD was diagnosed by two or more experienced neurologists according to the United Kingdom brain-bank criteria (Hughes et al., 1992). None had a history of neurologic or psychiatric conditions other than PD. Only one patient was selected from each family. These PD patients came from Hunan, Hubei, Jiangxi and Chongqi provinces of China. Three hundred and sixteen (61.7%) of the patients were men, and 196 (38.3%) were women. The mean age at disease onset in the patient group was 54.71 years (SD 11.64 years, ranging from 8 to 79). According to the onset age,
Table 2 – The haplotype frequencies in patients and controls. Haplotypes a
SPD (n, %)
Control (n, %)
χ2
p
CAC TTC TTT
236.58 (23.1) 583.52 (57.0) 194.46 (19.0)
239.88 (23.7) 600.33 (59.3) 154.60 (15.3)
0.176 1.664 4.603
0.675 0.197 0.032
a
The haplotype frequency < 0.03 is not calculated.
Genetic analysis
After extracting genomic DNA from peripheral blood through standard protocols, we screened all PD patients and controls for the three SNPs, rs2281983, rs4919621 and rs3758549, using PCR-restricted fragment length polymorphism (RFLP) method. PCR products were digested with restriction enzymes and the resulting fragments were submitted to electrophoresis using 12% polyacrylamide gel. The PCR primers and restriction enzymes are listed (Supplementary table 1). In order to ensure the accuracy of the results, we sequenced 10% DNA samples directly. Differences of the SNP frequencies between groups were analyzed by means of the χ2 test or Fisher's exact test. p < 0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS 13.0 (SPSS, Inc., Chicago, IL) and Haploview 4.1.
4.3.
Meta-analysis
To assess the possible role of the PITX3 gene rs3758549 polymorphism in PD, we conducted a meta-analysis on the topic. We conducted a literature search in PubMed for all relevant papers published from 1966 to September 2010 using the search terms “PITX3,” “rs3758549,” or “PITX3 polymorphisms” combined with “Parkinson's disease” or “PD.” Furthermore, we reviewed the reference lists from retrieved articles to search for more studies. The following criteria were used to identify relevant studies for the meta-analysis: first, clinical diagnosis of PD was established according to the
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Table 3 – The summary of the PITX3 gene rs3758549 analysis in PD. Study
Population studied
Fuchs et al., 2009 Fuchs et al., 2009 Simon-Sanchez et al., 2009 Haubenberger et al., 2011 Bergman et al., 2010 Jia, current study Total
Germany Germany USA, Germany Austria Sweden China
PD patients C
5.
T
574 (0.844) 1139 (0.851) 2850 (0.832) 568 (0.782) 572 (0.829) 827 (0.808) 6530 (0.828)
United Kingdom brain-bank criteria (Hughes et al., 1992). Second, they had a case-control study design. Third, the PITX3 gene rs3758549 was detected among PD patients and controls. We identified 9 relevant studies concerning the PITX3 polymorphisms and Parkinson's disease (Fuchs et al., 2009; Bergman et al., 2010; Haubenberger et al., 2011; Le et al., 2009; Fung et al., 2006; Mizuta et al., 2006; Pankratz et al., 2009; Simon-Sanchez et al., 2009; de Mena et al., 2010). Five studies (Le et al., 2009; Fung et al., 2006; Mizuta et al., 2006; Pankratz et al., 2009; de Mena et al., 2010) were excluded because they did not detect rs3758549. Thus, the meta-analysis on the PITX3 gene rs3758549 and PD included four papers in total. We extracted the following data from each publication: the first author's name, the year of publication, population studied, the frequencies of the PITX3 gene rs3758549 among PD patients and the control group (summarized in Table 3). Meta-analyses were performed using RevMan4.2. p < 0.05 was considered statistically significant.
Conflict of interest statement
The authors declare that there are no competing interests.
Acknowledgments This work was supported by grant 2011CB510000 from the Major State Basic Research Development Program of China (973 Program) (to Dr. Bei-sha Tang), grant 2006AA02A408 from the National “863” High-Tech Research and Development Program of China (to Dr. Bei-sha Tang), grants 30570638, 30770735 and 30971035 from the National Natural Science Foundation of China (to Dr. Bei-sha Tang), and grant 30900469 from the National Natural Science Foundation of China (to Dr. Ji-feng Guo). The authors thank all the PD patients and healthy controls for participating in the study. Author contributions: study conception, design and organization (Liu, Sun, and Guo); acquisition of data (Liu, Sun, Tang, Hu, Wang, Shi, Pan, Xia, and Yan); analysis and interpretation of data (Liu, Sun, Tang, Hu, Wang, Shi, Pan, Xia, Yan, and Guo); drafting of the manuscript (Liu, Sun, Tang, and Guo); critical revision of the manuscript for important intellectual content (Liu, Sun, Tang, and Guo); statistical analysis (Liu, Sun, Tang, Hu, Wang, Shi, Pan, Xia, Yan, and
Controls
106 199 576 158 118 197 1354
(0.156) (0.149) (0.168) (0.218) (0.171) (0.192) (0.172)
C 1098 1088 6392 653 461 857 10,549
(0.807) (0.813) (0.803) (0.804) (0.829) (0.847) (0.809)
T 262 (0.193) 250 (0.187) 1564 (0.197) 159(0.196) 95 (0.171) 155 (0.153) 2485 (0.191)
Guo); obtained funding (Tang and Guo); administrative, technical, and material support (Liu, Sun, Tang, Hu, Wang, Shi, Pan, Xia, Yan, and Guo); and study supervision (Liu, Sun, Tang, and Guo).
Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.brainres.2011.03.064.
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