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Fibroblast growth factor 20 (FGF20) polymorphism is a risk factor for Parkinson’s disease in Chinese population
Parkinsonism and Related Disorders 18 (2012) 629e631
Contents lists available at SciVerse ScienceDirect
Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis
Short communication
Fibroblast growth factor 20 (FGF20) polymorphism is a risk factor for Parkinson’s disease in Chinese population Jing Pan a, b,1, Hui Li a,1, Ying Wang a, Jian-Fang Ma a, Jin Zhang a, Gang Wang a, Jun Liu a, Xi-Jin Wang a, Qin Xiao a, *, Sheng-Di Chen a, b, * a
Department of Neurology & Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, PR China Lab of Neurodegenerative Diseases, Institute of Health Science, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) & Shanghai Jiao Tong University School of Medicine, Shanghai 200025, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 May 2011 Received in revised form 21 January 2012 Accepted 25 January 2012
The etiology of Parkinson’s disease (PD) is not well established. Genetic variation in fibroblast growth factor 20 (FGF20) might influence the risk of PD occurrence and development. In this study, Two DNA polymorphisms at genetic variation in FGF20, rs2720208 (C/T) and rs1721100 (C/G), were genotyped by direct sequencing in Han Chinese population, including 394 PD patients and 383 healthy controls. Stastistical analyses revealed that for rs1721100 (C/G) polymorphism, there were significant differences in genotype distribution between PD and healthy-matched controls. For rs12720208 (C/T) polymorphism, there was no significant difference in genotype distribution and gender and age-related differences between PD and control group. Results in this study revealed that the rs1721100(C/G) polymorphism is a risk factor for PD in Han Chinese population, while rs12720208(C/T) polymorphism is not significantly associated with PD. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Fibroblast growth factor 20 Polymorphism Parkinson’s disease
Parkinson’s disease (PD) is a common neurodegenerative disorder that affects 2% of people gt; 65 years [1]. PD is characterized by a loss of dopaminergic neurons (DNs) within the substantia nigra (SN) and both acquired and inherited risk factors have been implicated in DN death [2]. The existence of affected relatives is a risk factor for developing PD, and linkage analysis in families with several affected members has identified at least 16 loci/genes implicated in PD. Most cases of PD are sporadic, and genetic predisposition resides in the variation of several candidate genes. A complete genome screening identified a chromosome in the 8 region positively associated with PD [3]. Further studies reported a significant association with single nucleotide polymorphisms (SNPs) in the Fibroblast Growth Factor 20 (FGF20) gene, with the strongest risk linked to allele T of SNP rs12720208(C/T) in the 30 untranslated region (30 UTR) [4]. FGF20 is a member of the highly conserved growth factor polypeptides that regulate central nervous development and function [5]. Specifically, FGF20 is a neurotrophic factor that is
* Corresponding authors. Department of Neurology & Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, PR China. Tel./fax: þ86 21 6445 7249. E-mail addresses: [email protected] (Q. Xiao), [email protected] (S.-D. Chen). 1 The two authors made equal contributions to this work. 1353-8020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.parkreldis.2012.01.017
preferentially expressed within dopaminergic neurons of the midbrain SN within rat brain and is also expressed in human cerebellum and SN tissues [6]. Evidence suggests that expression of FGF20 significantly enhances the survival of rat midbrain DNs and is a strong biological and positional candidate for investigating its role in the susceptibility to PD. The FGF20 gene is approximately 9.3 kb, and is located approximately 6.2 Mb from a peak marker D8S520 on chromosome 8p22ep21.3 [7]. Two polymorphisms, rs1721100(C/G) and rs12720208(C/T), are known to influence expression from the FGF20 gene [4]. In a family-based genetic study, van der Walt et al. reported association of FGF20 SNP with increased risk for PD [4]. Two SNPs located in the 30 untranslated region (UTR) of the gene, rs12720208(C/T) (T allele positively associated, P ¼ 0.0008) and rs1721100(C/G) (C allele positively associated, P ¼ 0.02) showed association with PD. One of these SNPs, rs12720208, was subsequently shown in tissue culture to affect the translation of FGF20 by disrupting a putative microRNA binding site within the 30 untranslated region. Specifically, the risk associated allele in FGF20 removed this binding domain and resulted in relatively greater levels of FGF20 protein and presumably greater FGF20 effects. Subsequently, Clarimon et al. sought to replicate the association of the FGF20 gene with PD by performing a caseecontrol association study with four SNPs [rs1989756, rs1989754, rs1721100, and rs12720208] using Finnish and Greek samples. They failed to
630
J. Pan et al. / Parkinsonism and Related Disorders 18 (2012) 629e631
replicate the association of the FGF20 gene with PD [8]. In the Satake et al. study, the three SNPs (rs12718379, rs1989754, and rs1721100) showed some degree of difference between patients and controls [9]. Mena et al. found no association between FGF20 rs12720208(C/T) SNP and PD in Spanish patients [10]. Thus, it is still controversial as to whether or not the FGF20 gene is a susceptibility gene for PD. We conducted a caseecontrol association study using samples from Chinese patients in order to evaluate the association of the FGF20 gene with the risk for PD. Recently, FGF20 genes’ polymorphisms have been reported to be associated with Alzheimer’s disease and schizophrenia, which indicated that FGF20 might contribute to the progress of neurodegenerative disorders. Whether there is an association between the genetic polymorphisms of FGF20 and the development of sporadic PD is unknown. To clarify the role of FGF20 promoter polymorphisms in PD, we analyzed the rs12720208(C/T) and the rs1721100(C/G) polymorphisms of FGF20 from a Han Chinese population in a caseecontrol study. 1. Subjects A total of 777 subjects were recruited from the outpatient Clinic at the Department of Neurology, Ruijin Hospital affiliated with Shanghai Jiao Tong University School of Medicine, China. The PD group (PD group) consisted of 394 patients (age at onset 56.85 10.83) with a mean age of 63.62 11.63 years. PD was diagnosed by two or more experienced neurologists according to the UK brain bank criteria [11]. The healthy control group consisted of 383 subjects from China (mean age 64.48 9.43 years). All the controls were free of neurological or psychiatric disorders by medical history, physical and laboratory examinations. Gender proportion between cases and controls was statistically matched. Each participant was informed of the purpose of the investigation, and informed consent was obtained from each participant. The study was approved by the Research Ethics Committee, Ruijin Hospital affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China. 2. Methods Genomic DNA was extracted from peripheral blood through standardized phenol/chloroform extraction method. PCR primers used for SNP rs1721100(C/G) and rs12720208(C/T) genotyping were as follows: the forward primer 50 CCAGGTCCAAGAGGCATCAG-30 and the reverse primer 50 -AGTAAACATAATCCAGAGAGGTGAGG-30 . PCR was performed in a 50 ul reaction (94 C for 5 min, followed by 36 cycles of 94 C for 30 s, 60 C for 30 s, and 72 C for 40 s, with a final extension at 72 C for 10 min). The PCR products were purified and sequenced on an ABI 3700xl automated sequencer (Applied Biosystems, Foster City, CA, USA).
3. Statistical analysis Statistical analysis was performed with SPSS16.0. Goodness-offit to the HardyeWeinberg equilibrium (HWE) and differences in genotype and allele frequencies between PD and CON groups were examined by Chi-squared analysis. One-way ANOVA was used to detect the difference in onset age between three genotypes group. Odds ratio (OR) together with 95% confidence interval (CI) was estimated. Exact logistic regression was performed in the multivariate analysis to see whether age or gender would influence the distribution of the two SNPs’ polymorphisms. The criterion for significance was set at P value < 0.05 for all the tests. 4. Results Table 1 provides an overview on the socio-demographic variables of the sample. Examination of FGF20 gene polymorphisms
was carried out in 394 patients, who were diagnosed with sporadic PD, and 383 healthy controls. Distributions of the rs1721100(C/G) and rs12720208(C/T) polymorphisms in both the PD and the CON group were in HardyeWeinberg equilibrium. No significant difference was found in distributions of gender and age between two groups (P > 0.05). The allele and genotype frequencies of SNP rs1721100(C/G) in the two groups are given in Table 2. We found the minor alleles frequencies of rs1721100(C/G) within FGF20 were distributed differently in PD group and CON group (OR ¼ 1.270, 95% CI: 1.040e1.550, P ¼ 0.019), and there were significant differences in genotype distribution between two groups (P ¼ 0.046), indicating that the rs1721100(C/G) polymorphism may be a risk factor for PD. Exact logistic regression showed that the results of rs1721100(C/G) (OR ¼ 1.300, 95% CI: 1.054e1.603, P ¼ 0.014) were not influenced by age (P ¼ 0.374) or gender (P ¼ 0.133). Then, the PD group was subdivided into two groups, patients with early-onset PD (EOPD, diagnosed 45 years of age) and lateonset PD patients (LOPD, diagnosed > 45 years of age). For SNP rs1721100(C/G), Chi-squared analysis did not detect significant difference between EOPD and LOPD groups in terms of both allele and genotype distribution (data not shown). We found no significant difference in mean age of onset between the genotypes in the patients (F ¼ 0.326, P ¼ 0.722). To test the possible influence of FGF20 on the risk of PD, we also sequenced another SNP, rs12720208(C/T). We compared the frequencies of allele and genotype between patients and controls groups, and we found no significant difference between the two groups (Table 2). Nor did we find significant difference between patients with EOPD and LOPD (data not shown). 5. Discussion The etiology of PD is complex and involves both environmental and genetic factors. FGF20 and its family members are expressed in both embryonic and adult tissues, suggesting that they are critical factors in brain development and cell homeostasis [5]. Studies have shown that FGF20 acts as a neurotrophic factor in dopaminergic cells, substantially enhancing their survival in culture [10]. This effect is mediated by the binding of FGF20 to its receptor, FGFR-1c, which activates the mitogen-activated protein kinase pathway, resulting in the phosphorylation of extracellular regulated kinase [3]. Given the role that FGF20 plays in the survival of dopaminergic neurons, FGF20 presents an excellent candidate susceptibility gene for PD risk. Studies have clearly shown that FGF20 is critical to the survival of dopaminergic neurons within the substantia nigra; however, the mechanism of this protective action and its regulation has not been elucidated. A recent study has shown that the protective effect of the related FGF20 polypeptide acts with glialcell-lineederived neurotrophic factor (GDNF) to promote repair of lesioned hippocampal cells [12]. GDNF is also a key neurotrophic factor important for the maintenance and development of dopaminergic neurons. Expression levels of FGF20 may be vital to a signaling cascade involved in the repair of damaged dopaminergic neurons. Table 1 Demographic detail of the sample set.
Total sample Male Female Age at study years UPDRS III score Hoehn and Yahr stage
PD group
Con group
394 220 174 63.62 11.63 15(5e36) 2(1e4)
383 191 192 64.48 9.48 e e
The ages are means and standard deviations.
J. Pan et al. / Parkinsonism and Related Disorders 18 (2012) 629e631
631
Table 2 Genotypes’ distribution and alleles frequencies of SNP rs1721100(C/G) and SNP rs12720208(C/T) in PD and CON groups. SNP rs1721100(C/G)
SNP rs12720208 (C/T)
Genotype (n,%)
PD group CON group
c2 P OR CI(95%) a
Minor allele frequency (n,%)
Genotype (n,%)
Minor allele frequency (n,%)
CC
CG
GG
G
CC
CT
TT
T
85(21.57) 104(27.15) 6.142 0.046a e e
212(53.81) 209(54.57)
97(24.62) 70(18.28)
406(51.52) 349(45.56) 5.526 0.019a 1.270 1.040e1.550
389(98.73) 371(96.87) 3.154 0.089 e e
5(1.27) 12(3.13)
0 0
5(0.63) 12(1.57) 3.154 0.077 0.401 0.141e1.144
P < 0.05.
We have demonstrated that one of SNPs located within the 30 UTR of FGF20 (rs1721100(C/G)) is significantly associated with the risk for PD. Our results are consistent with the reports by van der Walt et al. and Satake et al. [4,9], which showed an association between the FGF20 gene and the risk for PD. The significance of the FGF20 gene for PD susceptibility in our study, however, was not as strong as that shown by van der Walt or Satake et al. [4,9]. This discrepancy may be due to (1) the ethnic difference between the Chinese patients and American ones; or (2) the difference in epidemiological approach taken between our study and theirs. i.e., we performed a caseecontrol association study by the x2 test in unrelated samples, while they analyzed family-based samples using the pedigree disequilibrium test (PDT). However, the finding of Clarimon et al.’s study does not convincingly disprove the association of the FGF20 gene with PD if its influence on the development of PD in the Greek and Finnish populations is similar to our Chinese sample. The sample size of their studies was considerably smaller than ours (Finnish series, 144 patients and 135 controls; Greek series, 151 patients and 186 controls in their study, compared with 394 patients and 383 controls in our study). The possibility of type 2 errors in their study could not be excluded as an explanation for this negative finding. Another explanation for the lack of replication could be genetic heterogeneity; there might be no association between the FGF20 gene and PD in the Greek and Finnish populations, whereas there might be an association in the Chinese and the United States-based populations. In this study, we demonstrated an association between FGF20 gene polymorphisms and the risk for developing sporadic PD. After multiple tests, however, a significant association was detected only in FGF20 (rs1721100 (C/G)). For FGF20 (rs12720208(C/T)) polymorphisms, there was no significant difference in genotype distribution between PD and control. We provide additional information of the potential relevance for the understanding of the gender and age-related differences in the susceptibility of PD. Exact logistic regression showed that the results of rs1721100(C/G) were not influenced by age or gender. Based on the data presented above, i.e., the polymorphism at the rs12720208(C/T) positions of FGF20, there was no significant difference in genotype distribution. For rs1721100 polymorphism, there were no significant difference in genotype distribution between LOPD and EOPD groups, Molecular studies are under way to verify the causative role of rs1721100 SNPs and whether these polymorphisms impact the risk for PD independently or collectively. In conclusion, our findings demonstrate that the rs1721100 alleles may increase the risk for PD in the Han Chinese population, while rs12720208 alleles do not increase the risk for developing sporadic PD in the Han Chinese population. Our data might be the first report of the association of FGF20 polymorphism with PD and provide preliminary evidence that reducing FGF20 protein
production or activity could be a useful treatment observed in patients with idiopathic PD in Chinese population. However, further studies with more subjects and/or other ethnicities will be needed before the conclusions of this study can be generalized. Acknowledgments This work was supported by grants from the National Program of Basic Research of China (2010CB945200, 2011CB504104), National Natural Science Fund (34900454, 30872729, 30900454, 30971031), Key Discipline Program of Shanghai Municipality (S30202), Shanghai Key Project of Basic Science Research (10411954500), and Program for Outstanding Medical Academic Leader (LJ 06003). Research Fund for the Doctoral Program of Higher Education of China (20090073120090), Special funding for original sci-tech research supported by Shanghai Municipal Education Commission (09YZ87). References [1] de Rijk MC, Launer LJ, Berger K, Breteler MM, Dartigues JF, Baldereschi M, et al. Prevalence of Parkinson’s disease in Europe: a collaborative study of population-based cohorts. Neurologic Diseases in the Elderly Research Group. Neurology 2000;54:S21e3. [2] Steece-Collier K, Maries E, Kordower JH. Etiology of Parkinson’s disease: genetics and environment revisited. Proc Natl Acad Sci U S A 2002;99: 13972e4. [3] Scott WK, Nance MA, Watts RL, Hubble JP, Koller WC, Lyons K, et al. Complete genomic screen in Parkinson disease: evidence for multiple genes. JAMA 2001;286:2239e44. [4] van der Walt JM, Noureddine MA, Kittappa R, Hauser MA, Scott WK, McKay R, et al. Fibroblast growth factor 20 polymorphisms and haplotypes strongly influence risk of Parkinson disease. Am J Hum Genet 2004 Jun;74: 1121e7. [5] Dono R. Fibroblast growth factors as regulators of central nervous system development and function. Am J Physiol Regul Integr Comp Physiol 2003;284: R867e81. [6] Ohmachi S, Mikami T, Konishi M, Miyake A, Itoh N. Preferential neurotrophic activity of fibroblast growth factor-20 for dopaminergic neurons through fibroblast growth factor receptor-1c. J Neurosci Res 2003;72:436e43. [7] Hauser MA, Li YJ, Takeuchi S, Walters R, Noureddine M, Maready M, et al. Genomic convergence: identifying candidate genes for Parkinson’s disease by combining serial analysis of gene expression and genetic linkage. Hum Mol Genet 2003;12:671e7. [8] Clarimon J, Xiromerisiou G, Eerola J, Gourbali V, Hellström O, Dardiotis E, et al. Lack of evidence for a genetic association between FGF20 and Parkinson’s disease in Finnish and Greek patients. BMC Neurol 2005;5:11. [9] Satake W, Mizuta I, Suzuki S, Nakabayashi Y, Ito C, Watanabe M, et al. Fibroblast growth factor 20 gene and Parkinson’s disease in the Japanese population. Neuroreport 2007;18:937e40. [10] de Mena L, Cardo LF, Coto E, Miar A, Díaz M, Corao AI, et al. FGF20 rs12720208 SNP and microRNA-433 variation: no association with Parkinson’s disease in Spanish patients. Neurosci Lett 2010;479:22e5. [11] Hughes AJ, Daniel SE, Kilford L, Lees AJ. Accuracy of clinical diagnosis of idiopathic Parkinson’s disease: a clinico-pathological study of 100 cases. J Neurol Neurosurg Psychiatry 1992;55:181e4. [12] Lemaitre H, Mattay VS, Sambataro F, Verchinski B, Straub RE, Callicott JH, et al. Genetic variation in FGF20 modulates hippocampal biology. J Neurosci 2010; 30:5992e7.
Contents lists available at SciVerse ScienceDirect
Parkinsonism and Related Disorders journal homepage: www.elsevier.com/locate/parkreldis
Short communication
Fibroblast growth factor 20 (FGF20) polymorphism is a risk factor for Parkinson’s disease in Chinese population Jing Pan a, b,1, Hui Li a,1, Ying Wang a, Jian-Fang Ma a, Jin Zhang a, Gang Wang a, Jun Liu a, Xi-Jin Wang a, Qin Xiao a, *, Sheng-Di Chen a, b, * a
Department of Neurology & Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, PR China Lab of Neurodegenerative Diseases, Institute of Health Science, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) & Shanghai Jiao Tong University School of Medicine, Shanghai 200025, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 May 2011 Received in revised form 21 January 2012 Accepted 25 January 2012
The etiology of Parkinson’s disease (PD) is not well established. Genetic variation in fibroblast growth factor 20 (FGF20) might influence the risk of PD occurrence and development. In this study, Two DNA polymorphisms at genetic variation in FGF20, rs2720208 (C/T) and rs1721100 (C/G), were genotyped by direct sequencing in Han Chinese population, including 394 PD patients and 383 healthy controls. Stastistical analyses revealed that for rs1721100 (C/G) polymorphism, there were significant differences in genotype distribution between PD and healthy-matched controls. For rs12720208 (C/T) polymorphism, there was no significant difference in genotype distribution and gender and age-related differences between PD and control group. Results in this study revealed that the rs1721100(C/G) polymorphism is a risk factor for PD in Han Chinese population, while rs12720208(C/T) polymorphism is not significantly associated with PD. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Fibroblast growth factor 20 Polymorphism Parkinson’s disease
Parkinson’s disease (PD) is a common neurodegenerative disorder that affects 2% of people gt; 65 years [1]. PD is characterized by a loss of dopaminergic neurons (DNs) within the substantia nigra (SN) and both acquired and inherited risk factors have been implicated in DN death [2]. The existence of affected relatives is a risk factor for developing PD, and linkage analysis in families with several affected members has identified at least 16 loci/genes implicated in PD. Most cases of PD are sporadic, and genetic predisposition resides in the variation of several candidate genes. A complete genome screening identified a chromosome in the 8 region positively associated with PD [3]. Further studies reported a significant association with single nucleotide polymorphisms (SNPs) in the Fibroblast Growth Factor 20 (FGF20) gene, with the strongest risk linked to allele T of SNP rs12720208(C/T) in the 30 untranslated region (30 UTR) [4]. FGF20 is a member of the highly conserved growth factor polypeptides that regulate central nervous development and function [5]. Specifically, FGF20 is a neurotrophic factor that is
* Corresponding authors. Department of Neurology & Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, PR China. Tel./fax: þ86 21 6445 7249. E-mail addresses: [email protected] (Q. Xiao), [email protected] (S.-D. Chen). 1 The two authors made equal contributions to this work. 1353-8020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.parkreldis.2012.01.017
preferentially expressed within dopaminergic neurons of the midbrain SN within rat brain and is also expressed in human cerebellum and SN tissues [6]. Evidence suggests that expression of FGF20 significantly enhances the survival of rat midbrain DNs and is a strong biological and positional candidate for investigating its role in the susceptibility to PD. The FGF20 gene is approximately 9.3 kb, and is located approximately 6.2 Mb from a peak marker D8S520 on chromosome 8p22ep21.3 [7]. Two polymorphisms, rs1721100(C/G) and rs12720208(C/T), are known to influence expression from the FGF20 gene [4]. In a family-based genetic study, van der Walt et al. reported association of FGF20 SNP with increased risk for PD [4]. Two SNPs located in the 30 untranslated region (UTR) of the gene, rs12720208(C/T) (T allele positively associated, P ¼ 0.0008) and rs1721100(C/G) (C allele positively associated, P ¼ 0.02) showed association with PD. One of these SNPs, rs12720208, was subsequently shown in tissue culture to affect the translation of FGF20 by disrupting a putative microRNA binding site within the 30 untranslated region. Specifically, the risk associated allele in FGF20 removed this binding domain and resulted in relatively greater levels of FGF20 protein and presumably greater FGF20 effects. Subsequently, Clarimon et al. sought to replicate the association of the FGF20 gene with PD by performing a caseecontrol association study with four SNPs [rs1989756, rs1989754, rs1721100, and rs12720208] using Finnish and Greek samples. They failed to
630
J. Pan et al. / Parkinsonism and Related Disorders 18 (2012) 629e631
replicate the association of the FGF20 gene with PD [8]. In the Satake et al. study, the three SNPs (rs12718379, rs1989754, and rs1721100) showed some degree of difference between patients and controls [9]. Mena et al. found no association between FGF20 rs12720208(C/T) SNP and PD in Spanish patients [10]. Thus, it is still controversial as to whether or not the FGF20 gene is a susceptibility gene for PD. We conducted a caseecontrol association study using samples from Chinese patients in order to evaluate the association of the FGF20 gene with the risk for PD. Recently, FGF20 genes’ polymorphisms have been reported to be associated with Alzheimer’s disease and schizophrenia, which indicated that FGF20 might contribute to the progress of neurodegenerative disorders. Whether there is an association between the genetic polymorphisms of FGF20 and the development of sporadic PD is unknown. To clarify the role of FGF20 promoter polymorphisms in PD, we analyzed the rs12720208(C/T) and the rs1721100(C/G) polymorphisms of FGF20 from a Han Chinese population in a caseecontrol study. 1. Subjects A total of 777 subjects were recruited from the outpatient Clinic at the Department of Neurology, Ruijin Hospital affiliated with Shanghai Jiao Tong University School of Medicine, China. The PD group (PD group) consisted of 394 patients (age at onset 56.85 10.83) with a mean age of 63.62 11.63 years. PD was diagnosed by two or more experienced neurologists according to the UK brain bank criteria [11]. The healthy control group consisted of 383 subjects from China (mean age 64.48 9.43 years). All the controls were free of neurological or psychiatric disorders by medical history, physical and laboratory examinations. Gender proportion between cases and controls was statistically matched. Each participant was informed of the purpose of the investigation, and informed consent was obtained from each participant. The study was approved by the Research Ethics Committee, Ruijin Hospital affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China. 2. Methods Genomic DNA was extracted from peripheral blood through standardized phenol/chloroform extraction method. PCR primers used for SNP rs1721100(C/G) and rs12720208(C/T) genotyping were as follows: the forward primer 50 CCAGGTCCAAGAGGCATCAG-30 and the reverse primer 50 -AGTAAACATAATCCAGAGAGGTGAGG-30 . PCR was performed in a 50 ul reaction (94 C for 5 min, followed by 36 cycles of 94 C for 30 s, 60 C for 30 s, and 72 C for 40 s, with a final extension at 72 C for 10 min). The PCR products were purified and sequenced on an ABI 3700xl automated sequencer (Applied Biosystems, Foster City, CA, USA).
3. Statistical analysis Statistical analysis was performed with SPSS16.0. Goodness-offit to the HardyeWeinberg equilibrium (HWE) and differences in genotype and allele frequencies between PD and CON groups were examined by Chi-squared analysis. One-way ANOVA was used to detect the difference in onset age between three genotypes group. Odds ratio (OR) together with 95% confidence interval (CI) was estimated. Exact logistic regression was performed in the multivariate analysis to see whether age or gender would influence the distribution of the two SNPs’ polymorphisms. The criterion for significance was set at P value < 0.05 for all the tests. 4. Results Table 1 provides an overview on the socio-demographic variables of the sample. Examination of FGF20 gene polymorphisms
was carried out in 394 patients, who were diagnosed with sporadic PD, and 383 healthy controls. Distributions of the rs1721100(C/G) and rs12720208(C/T) polymorphisms in both the PD and the CON group were in HardyeWeinberg equilibrium. No significant difference was found in distributions of gender and age between two groups (P > 0.05). The allele and genotype frequencies of SNP rs1721100(C/G) in the two groups are given in Table 2. We found the minor alleles frequencies of rs1721100(C/G) within FGF20 were distributed differently in PD group and CON group (OR ¼ 1.270, 95% CI: 1.040e1.550, P ¼ 0.019), and there were significant differences in genotype distribution between two groups (P ¼ 0.046), indicating that the rs1721100(C/G) polymorphism may be a risk factor for PD. Exact logistic regression showed that the results of rs1721100(C/G) (OR ¼ 1.300, 95% CI: 1.054e1.603, P ¼ 0.014) were not influenced by age (P ¼ 0.374) or gender (P ¼ 0.133). Then, the PD group was subdivided into two groups, patients with early-onset PD (EOPD, diagnosed 45 years of age) and lateonset PD patients (LOPD, diagnosed > 45 years of age). For SNP rs1721100(C/G), Chi-squared analysis did not detect significant difference between EOPD and LOPD groups in terms of both allele and genotype distribution (data not shown). We found no significant difference in mean age of onset between the genotypes in the patients (F ¼ 0.326, P ¼ 0.722). To test the possible influence of FGF20 on the risk of PD, we also sequenced another SNP, rs12720208(C/T). We compared the frequencies of allele and genotype between patients and controls groups, and we found no significant difference between the two groups (Table 2). Nor did we find significant difference between patients with EOPD and LOPD (data not shown). 5. Discussion The etiology of PD is complex and involves both environmental and genetic factors. FGF20 and its family members are expressed in both embryonic and adult tissues, suggesting that they are critical factors in brain development and cell homeostasis [5]. Studies have shown that FGF20 acts as a neurotrophic factor in dopaminergic cells, substantially enhancing their survival in culture [10]. This effect is mediated by the binding of FGF20 to its receptor, FGFR-1c, which activates the mitogen-activated protein kinase pathway, resulting in the phosphorylation of extracellular regulated kinase [3]. Given the role that FGF20 plays in the survival of dopaminergic neurons, FGF20 presents an excellent candidate susceptibility gene for PD risk. Studies have clearly shown that FGF20 is critical to the survival of dopaminergic neurons within the substantia nigra; however, the mechanism of this protective action and its regulation has not been elucidated. A recent study has shown that the protective effect of the related FGF20 polypeptide acts with glialcell-lineederived neurotrophic factor (GDNF) to promote repair of lesioned hippocampal cells [12]. GDNF is also a key neurotrophic factor important for the maintenance and development of dopaminergic neurons. Expression levels of FGF20 may be vital to a signaling cascade involved in the repair of damaged dopaminergic neurons. Table 1 Demographic detail of the sample set.
Total sample Male Female Age at study years UPDRS III score Hoehn and Yahr stage
PD group
Con group
394 220 174 63.62 11.63 15(5e36) 2(1e4)
383 191 192 64.48 9.48 e e
The ages are means and standard deviations.
J. Pan et al. / Parkinsonism and Related Disorders 18 (2012) 629e631
631
Table 2 Genotypes’ distribution and alleles frequencies of SNP rs1721100(C/G) and SNP rs12720208(C/T) in PD and CON groups. SNP rs1721100(C/G)
SNP rs12720208 (C/T)
Genotype (n,%)
PD group CON group
c2 P OR CI(95%) a
Minor allele frequency (n,%)
Genotype (n,%)
Minor allele frequency (n,%)
CC
CG
GG
G
CC
CT
TT
T
85(21.57) 104(27.15) 6.142 0.046a e e
212(53.81) 209(54.57)
97(24.62) 70(18.28)
406(51.52) 349(45.56) 5.526 0.019a 1.270 1.040e1.550
389(98.73) 371(96.87) 3.154 0.089 e e
5(1.27) 12(3.13)
0 0
5(0.63) 12(1.57) 3.154 0.077 0.401 0.141e1.144
P < 0.05.
We have demonstrated that one of SNPs located within the 30 UTR of FGF20 (rs1721100(C/G)) is significantly associated with the risk for PD. Our results are consistent with the reports by van der Walt et al. and Satake et al. [4,9], which showed an association between the FGF20 gene and the risk for PD. The significance of the FGF20 gene for PD susceptibility in our study, however, was not as strong as that shown by van der Walt or Satake et al. [4,9]. This discrepancy may be due to (1) the ethnic difference between the Chinese patients and American ones; or (2) the difference in epidemiological approach taken between our study and theirs. i.e., we performed a caseecontrol association study by the x2 test in unrelated samples, while they analyzed family-based samples using the pedigree disequilibrium test (PDT). However, the finding of Clarimon et al.’s study does not convincingly disprove the association of the FGF20 gene with PD if its influence on the development of PD in the Greek and Finnish populations is similar to our Chinese sample. The sample size of their studies was considerably smaller than ours (Finnish series, 144 patients and 135 controls; Greek series, 151 patients and 186 controls in their study, compared with 394 patients and 383 controls in our study). The possibility of type 2 errors in their study could not be excluded as an explanation for this negative finding. Another explanation for the lack of replication could be genetic heterogeneity; there might be no association between the FGF20 gene and PD in the Greek and Finnish populations, whereas there might be an association in the Chinese and the United States-based populations. In this study, we demonstrated an association between FGF20 gene polymorphisms and the risk for developing sporadic PD. After multiple tests, however, a significant association was detected only in FGF20 (rs1721100 (C/G)). For FGF20 (rs12720208(C/T)) polymorphisms, there was no significant difference in genotype distribution between PD and control. We provide additional information of the potential relevance for the understanding of the gender and age-related differences in the susceptibility of PD. Exact logistic regression showed that the results of rs1721100(C/G) were not influenced by age or gender. Based on the data presented above, i.e., the polymorphism at the rs12720208(C/T) positions of FGF20, there was no significant difference in genotype distribution. For rs1721100 polymorphism, there were no significant difference in genotype distribution between LOPD and EOPD groups, Molecular studies are under way to verify the causative role of rs1721100 SNPs and whether these polymorphisms impact the risk for PD independently or collectively. In conclusion, our findings demonstrate that the rs1721100 alleles may increase the risk for PD in the Han Chinese population, while rs12720208 alleles do not increase the risk for developing sporadic PD in the Han Chinese population. Our data might be the first report of the association of FGF20 polymorphism with PD and provide preliminary evidence that reducing FGF20 protein
production or activity could be a useful treatment observed in patients with idiopathic PD in Chinese population. However, further studies with more subjects and/or other ethnicities will be needed before the conclusions of this study can be generalized. Acknowledgments This work was supported by grants from the National Program of Basic Research of China (2010CB945200, 2011CB504104), National Natural Science Fund (34900454, 30872729, 30900454, 30971031), Key Discipline Program of Shanghai Municipality (S30202), Shanghai Key Project of Basic Science Research (10411954500), and Program for Outstanding Medical Academic Leader (LJ 06003). Research Fund for the Doctoral Program of Higher Education of China (20090073120090), Special funding for original sci-tech research supported by Shanghai Municipal Education Commission (09YZ87). References [1] de Rijk MC, Launer LJ, Berger K, Breteler MM, Dartigues JF, Baldereschi M, et al. Prevalence of Parkinson’s disease in Europe: a collaborative study of population-based cohorts. Neurologic Diseases in the Elderly Research Group. Neurology 2000;54:S21e3. [2] Steece-Collier K, Maries E, Kordower JH. Etiology of Parkinson’s disease: genetics and environment revisited. Proc Natl Acad Sci U S A 2002;99: 13972e4. [3] Scott WK, Nance MA, Watts RL, Hubble JP, Koller WC, Lyons K, et al. Complete genomic screen in Parkinson disease: evidence for multiple genes. JAMA 2001;286:2239e44. [4] van der Walt JM, Noureddine MA, Kittappa R, Hauser MA, Scott WK, McKay R, et al. Fibroblast growth factor 20 polymorphisms and haplotypes strongly influence risk of Parkinson disease. Am J Hum Genet 2004 Jun;74: 1121e7. [5] Dono R. Fibroblast growth factors as regulators of central nervous system development and function. Am J Physiol Regul Integr Comp Physiol 2003;284: R867e81. [6] Ohmachi S, Mikami T, Konishi M, Miyake A, Itoh N. Preferential neurotrophic activity of fibroblast growth factor-20 for dopaminergic neurons through fibroblast growth factor receptor-1c. J Neurosci Res 2003;72:436e43. [7] Hauser MA, Li YJ, Takeuchi S, Walters R, Noureddine M, Maready M, et al. Genomic convergence: identifying candidate genes for Parkinson’s disease by combining serial analysis of gene expression and genetic linkage. Hum Mol Genet 2003;12:671e7. [8] Clarimon J, Xiromerisiou G, Eerola J, Gourbali V, Hellström O, Dardiotis E, et al. Lack of evidence for a genetic association between FGF20 and Parkinson’s disease in Finnish and Greek patients. BMC Neurol 2005;5:11. [9] Satake W, Mizuta I, Suzuki S, Nakabayashi Y, Ito C, Watanabe M, et al. Fibroblast growth factor 20 gene and Parkinson’s disease in the Japanese population. Neuroreport 2007;18:937e40. [10] de Mena L, Cardo LF, Coto E, Miar A, Díaz M, Corao AI, et al. FGF20 rs12720208 SNP and microRNA-433 variation: no association with Parkinson’s disease in Spanish patients. Neurosci Lett 2010;479:22e5. [11] Hughes AJ, Daniel SE, Kilford L, Lees AJ. Accuracy of clinical diagnosis of idiopathic Parkinson’s disease: a clinico-pathological study of 100 cases. J Neurol Neurosurg Psychiatry 1992;55:181e4. [12] Lemaitre H, Mattay VS, Sambataro F, Verchinski B, Straub RE, Callicott JH, et al. Genetic variation in FGF20 modulates hippocampal biology. J Neurosci 2010; 30:5992e7.