Yih Ru Wu

Analysis of heat-shock protein 70 gene polymorphisms and the risk of Parkinson’s disease

Parkinson’s disease (PD) involves several genetic and environmental components. Heat-shock proteinxa070, a chaperone that is up-regulated in stress responses and that refolds protein, may be involved in the pathogenesis of PD. We have investigated the association of polymorphisms −110 A/C, +190 G/C, +1267 A/G, +2074 G/C, and +2437 G/C in the 5’ and coding regions of the HSP70-1, HSP70-2, and HSP70-hom genes with the risk of PD by screening DNA samples from 274 PD patients and 183 controls in assays based on the polymerase chain reaction. There was no statistically significant difference in genotype distribution between patients and controls for the three coding-region polymorphisms in HSP70-2 and HSP70-hom. However, for HSP70-1, the overall genotype distribution was significantly different at the −110 site (P=0.004) and tended to be different at the +190 site (P=0.012) between patients and controls. The frequencies of the −110 CC and +190 CC genotypes were significantly higher in PD patients than in controls (P=0.001 and 0.006, respectively). Both −110 CC (odds ratio: 2.91; 95% CI: 1.51–5.96; P=0.002) and +190 CC (odds ratio: 3.59; 95% CI: 1.53–9.88; P=0.006) genotypes were significantly associated with PD. Reporter constructs containing the −110 A allele cloned into a luciferase reporter plasmid drove marginally higher transcriptional activity of HSP70-1 compared with the −110 C allele in both control and heat-shocked IMR32 and 293 cells. Therefore, −110 A/C may be a functional polymorphism in the 5’ promoter region of HSP70-1 and may affect susceptibility to PD.

Interleukin-1α and -1β Promoter Polymorphisms in Taiwanese Patients with Dementia

Background: Inflammatory events may contribute to the pathogenesis of dementia and interleukin-1 (IL-1) may exert both neurotoxic and neuroprotective effects. We investigated whether IL-1α –889 C/T and IL-1β –511 C/T promoter polymorphisms are associated with the risk of Alzheimer’s disease (AD) and vascular dementia (VaD). Methods: AD patients (n = 219) and VaD patients (n = 82), who fulfilled the criteria of the NINCDS-ADRDA and NINDS-AIREN, and ethnic-matched and nondemented controls (n = 209) were analyzed by means of genotype association method. Results: No significant difference in the genotype distribution of the analyzed single nucleotide polymorphisms was found between AD or VaD cases and controls. However, the frequency of the IL-1α –889 CT genotype was notably lower in VaD patients aged over 70 years than the age-matched controls (9.1 vs. 22.9%, p = 0.036) andtheIL-1α –889 CT genotype demonstrated a trend toward decrease in risk of developing VaD (odds ratio: 0.34; 95% confidence interval: 0.12–0.83, p = 0.026). Multivariate analysis revealed that the IL-1β –511T-carrying genotype slightly strengthens the negative association of the IL-1α –889 CT genotype with VaD (odds ratio: 0.26; 95% confidence interval: 0.08–0.79, p = 0.024). Conclusion: Our data suggest a protective role of the IL-1α –889 CT genotype in VaD susceptibility among Taiwanese aged over 70 years.

The CAG repeat in SCA12 functions as a cis element to up-regulate PPP2R2B expression

PPP2R2B, a protein widely expressed in neurons, regulates the protein phosphatase 2A (PP2A) activity for dephosphorylation of tau and other substrates. CAG repeat expansion at the 5′-end of the PPP2R2B gene causes autosomal dominant spinocerebellar ataxia type 12. In the present study, we investigated the roles of CAG repeats and flanking cis elements and the associated proteins in controlling PPP2R2B expression. Deletion/site-directed mutagenesis, in silico searches and cDNA overexpression revealed that CREB1 and SP1 bind to the conserved sequence upstream the CAG repeats to up-regulate PPP2R2B expression, whereas TFAP4 binds to the conserved sequence downstream the CAG repeats to down-regulate PPP2R2B expression. The binding of CREB1, SP1, and TFAP4 to the PPP2R2B promoter was further confirmed by DNA pull-down and ChIP-PCR assays. CAG repeats itself also function as a cis element to up-regulate PPP2R2B expression as AT repeat length has no effect on PPP2R2B expression. Together, our data provide evidence that CREB1, SP1, and TFAP4 play roles in modulating PPP2R2B expression, thus offering a mechanism of regulating PP2A activity as the treatment of neurodegenerative diseases associated with abnormal PP2A activity.

Identification of a novel risk variant in the FUS gene in essential tremor

Objective: A nonsense mutation in the amyotrophic lateral sclerosis gene FUS has been found to segregate in a large family with essential tremor (ET). Coding variants in this gene have not been comprehensively evaluated in ET. We conducted a genetic analysis of FUS for pathogenic and novel coding variants in 2 case-control cohorts among ethnic Chinese. Methods: In a study that involved 7,548 subjects, we first sequenced all the exon and exon-intronic boundaries of FUS in 84 ET samples. Potential causative variants that were identified were then genotyped in 2 separate case-control cohorts involving 263 additional ET samples and 5,919 controls (set 1) and 250 ET cases and 250 controls (set 2), and 782 diseased controls of Chinese ethnicity from 2 different Asian countries. Results: We identified a novel variant, Met392Ile, in exon 12 of the FUS gene. This variant was associated with ET in set 1 (odds ratio = 4.72 [95% confidence interval = 1.90–11.71], p = 0.0037). The association was subsequently validated in set 2 (joint analysis odds ratio = 3.92 [95% confidence interval = 1.57–9.82], p = 8.6 × 10−4). No association was observed in another neurologic cohort of patients with Parkinson disease, and no other potential pathogenic mutations were identified. Conclusion: We identified a novel risk variant, Met392Ile, in the FUS gene that increases susceptibility of ET among ethnic Chinese. Further studies in other ethnic populations are needed to determine whether this is an ethnic-specific risk factor.

PPP2R2B CAG repeat length in the Han Chinese in Taiwan: Association analyses in neurological and psychiatric disorders and potential functional implications.

PPP2R2B, a protein widely expressed in neurons throughout the brain, regulates the protein phosphatase 2A (PP2A) activity for the microtubule‐associated protein tau and other substrates. Altered PP2A activity has been implicated in spinocerebellar ataxia 12, Alzheimers disease (AD), and other tauopathies. Through a case‐control study and a reporter assay, we investigated the association of PPP2R2B CAG repeat polymorphism with Taiwanese AD, essential tremor (ET), Parkinsons disease (PD), and schizophrenia and clarified the functional implication of this polymorphism. The distribution of the alleles was not significantly different between patients and controls, with 68.6–76.1% alleles at lengths of 10, 13, and 16 triplets. No expanded alleles were detected in either group. However, the frequency of the individuals carrying the short 5‐, 6‐, and 7‐triplet alleles was notably higher in patients with AD (5/180 [2.8%], Fishers exact test, Pu2009=u20090.003; including 2 homozygotes) and ET (4/132 [3.0%], Fishers exact test, Pu2009<u20090.001) than in the controls (1/625 [0.2%]). The PPP2R2B transcriptional activity was significantly lower in the luciferase reporter constructs containing the (CAG)5–7 allele than in those containing the common 10‐, 13‐, and 16‐triplet alleles in both neuroblastoma and embryonic kidney cells. Therefore, our preliminary results suggest that the PPP2R2B gene CAG repeat polymorphism may be functional and may, in part, play a role in conferring susceptibility to AD and ET in Taiwan.

Genetic Variants of LRRK2 in Taiwanese Parkinson’s Disease

Genetic variants of leucine-rich repeat kinase 2 (LRRK2) were reported to alter the risk for Parkinson’s disease (PD). However, the genetic spectrum of LRRK2 variants has not been clearly disclosed yet in Taiwanese population. Herein, we sequenced LRRK2 coding region in 70 Taiwanese early onset PD patients (age at onset ≤ 50), and found six amino acid-changing single nucleotide polymorphisms (SNPs, N551K, R1398H, R1628P, S1647T, G2385R and M2397T), one reported (R1441H) and 2 novel missense (R767H and S885N) mutations. We examined the frequency of identified LRRK2 variants by genotyping 573 Taiwanese patients with PD and 503 age-matched control subjects. The results showed that PD patients demonstrated a higher frequency of G2385R A allele (4.6%) than control subjects (2.1%; odds ratio = 2.27, 95% confidence interval: 1.38–3.88, P = 0.0017). Fewer PD patients (27.7%) carried the 1647T-2397T haplotype as compared with the control subjects (33.0%; odds ratio = 0.80, 95% confidence interval: 0.65–0.97, P = 0.0215). However, the frequency of 1647T-2385R-2397T haplotype (4.3%) in PD patients was still higher than in control subjects (1.9%, odds ratio: 2.15, 95% confidence interval: 1.27–3.78, P = 0.0058). While no additional subject was found to carry R767H and R1441H, one more patient was observed to carry the S885N variant. Our results indicate a robust risk association regarding G2385R and a new possible protective haplotype (1647T-2397T). Gene-environmental interaction and a larger cohort study are warranted to validate our findings. Additionally, two new missense mutations (R767H and S885N) regarding LRRK2 in PD patients were identified. Functional studies are needed to elucidate the effects of these LRRK2 variants on protein function.

Ubiquitin specific proteases USP24 and USP40 and ubiquitin thiolesterase UCHL1 polymorphisms have synergic effect on the risk of Parkinson's disease among Taiwanese

BACKGROUNDnImpaired ubiquitin-proteasome system function may contribute to the pathogenesis of Parkinsons disease (PD).nnnMETHODSnWe conducted a case-control study in a cohort of 517 PD cases and 518 ethnically matched controls to investigate the association of ubiquitin specific proteases USP24 rs487230 C>T, USP40 rs1048603 C>T, and ubiquitin thiolesterase UCHL1 rs5030732 A>C polymorphisms with the risk of PD.nnnRESULTSnNo significant difference in the genotype or allele distribution was found between PD and controls. After stratification by age, the genotype and allele frequencies of USP24 rs487230 are significantly different between PD and controls >or=60 years of age (P=0.035 and 0.013, respectively). Multivariable logistic regression with adjusting for onset age and sex showed that, in a dominant model, USP24 T-carrying genotype was associated with risk reduction in developing PD in individuals >or=60 years of age (OR=0.61; 95% CI=0.41-0.90, P=0.010). This is also true for T allele (OR=0.64; 95% CI=0.44-0.91, P=0.023). When examining the interaction between genes on PD risk without age stratification, the protective effect of USP24 CT/TT genotype on PD risks was strengthened by the USP40 T-carrying genotype (OR=0.42; 95% CI=0.22-0.81, P=0.009) and UCHL1 C-carrying genotype (OR=0.67; 95% CI=0.47-0.97, P=0.032).nnnCONCLUSIONSnOur results suggest that USP24 alone plays a role in PD susceptibility among Taiwanese people >or=60 years of age, or acting synergistically with USP40 and UCHL1 in the total subjects.

Association of TNF-α gene with spontaneous deep intracerebral hemorrhage in the Taiwan population: A case control study

BackgroundGenetic factors may play a role in susceptibility to spontaneous deep intracerebral hemorrhage (SDICH). Previous studies have shown that TNF-α gene variation was associated with risks of subarachnoid hemorrhage in multiple ethnicities. The present case-control study tested the hypothesis that genetic variations of the TNF-α gene may affect the risk of Taiwanese SDICH. We examined the association of SDICH risks with four single nucleotide polymorphisms (SNPs) within the TNF-α gene promoter, namely T-1031C, C-863A, C-857T, and G-308A.MethodsGenotyping was determined by PCR-based restriction and electrophoresis assay for 260 SDICH patients and 368 controls. Associations were tested by logistic regression or general linear models with adjusting for multiple covariables in each gender group, and then in combined. Multiplicative terms of gender and each of the four SNPs were applied to detect the interaction effects on SDICH risks. To account for the multiple testing, permutation testing of 1,000 replicates was performed for empirical estimates.ResultsIn an additive model, SDICH risks were positively associated with the minor alleles -1031C and -308A in men (OR = 1.9, 95% CI 1.1 to 3.4, p = 0.03 and OR = 2.6, 95% CI 1.3 to 5.3, p = 0.005, respectively) but inversely associated with -863A in females (OR = 0.5, 95% CI 0.2 to 0.9, p = 0.03). There were significant interaction effects between gender and SNP on SDICH risks regarding SNPs T-1031C, C-863A, and G-308A (p = 0.005, 0.005, and 0.007, respectively). Hemorrhage size was inversely associated with -857T in males (p = 0.04).ConclusionsIn the Taiwan population, the associations of genetic variations in the TNF-α gene promoter with SDICH risks are gender-dependent.

SCA17 repeat expansion: mildly expanded CAG/CAA repeat alleles in neurological disorders and the functional implications.

BACKGROUNDnSpinocerebellar ataxia type 17 (SCA17) involves the expression of a CAG/CAA expansion mutation in the gene encoding TATA-box binding protein (TBP), a general transcription initiation factor. The spectrum of SCA17 clinical presentation is broad.nnnMETHODSnWe screened for triplet expansion in the TBP gene in Taiwanese Parkinsons disease (PD), Alzheimers disease (AD) and atypical parkinsonism and investigated the functional implication of expanded alleles using lymphoblastoid cells as a model.nnnRESULTSnA total of 6 mildly expanded alleles (44-46) were identified in patients group. The frequency of the individuals carrying expanded alleles in PD (3/602 [0.5%]), AD (2/245 [0.8%]) and atypical parkinsonism (1/44 [2.3%]) is not significant as compared to that in the control subjects (0/644 [0.0%]). In lymphoblastoid cells, HSPA5, HSPA8 and HSPB1 expression levels in cells with expanded TBP were significantly lower than that of the control cells. Although not significantly, the levels of PARK7 protein isoforms 6.1 and 6.4 are notably increased in SCA17 lymphoblastoid cells. Treatment of TBH (tert-butyl hydroperoxide) significantly increases cell death in the cells with mildly expanded TBP.nnnCONCLUSIONSnOur findings expand the spectrum of SCA17 phenotype and may contribute to our understanding of the disease.

Genetic analysis of NFE2L2 promoter variation in Taiwanese Parkinson's disease

Oxidative stress has been reported as one of the pathogeneses of Parkinsons disease (PD). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates the expression of the target genes involved in antioxidant pathway. The promoter polymorphisms of the Nrf2-encoding gene, NFE2L2, have been shown to affect the promoter activity. Inconsistent findings of the associations between NFE2L2 promoter polymorphisms and PD have been reported in a multiple candidate gene study, a whole-genome association study, and a case-control study in multiple ethnicity groups. This study enrolled a total of 1006 individuals composed of 480 PD patients and 526 controls to evaluate if there was an association of the NFE2L2 promoter polymorphisms with PD susceptibility in the Taiwan population. Three promoter single nucleotide polymorphisms (SNPs) rs35652124, rs6706649, and rs6721961 were examined using polymerase chain reaction and restriction analysis. The associations of each of the SNPs and the haplotypes constructed by the variations with PD susceptibility were examined. In spite of adequate statistic power, we observe no association of the three SNPs and their haplotypes with PD in a Taiwanese population. One of the reasons for the association disparity may include the genomic differences in ethnicities and environmental factors in different geographical regions.