Yanning Cai

Clinical profiles of Parkinson's disease associated with common leucine-rich repeat kinase 2 and glucocerebrosidase genetic variants in Chinese individuals

Clinical profiles of Parkinsons disease (PD) related to LRRK2 (LRRK2-PD), and GBA (GBA-PD) genes have not been reported in Chinese individuals. In this study, we have investigated motor and non-motor aspects in 1638 Chinese PD patients who carried LRRK2 G2385R or R1628P (LRRK2-PD, n = 223), GBA L444P variant (GBA-PD, n = 49), or none of the variants (idiopathic PD [IPD], n = 1366). As a result, age at onset and motor and non-motor features of LRRK2-PD patients were similar to IPD patients except for milder non-motor symptoms. In contrast, GBA-PD patients had a significantly younger age at onset and higher Unified Parkinsons Disease Rating Scale scores than LRRK2-PD and IPD patients. In addition, postural instability and gait disorders, motor complications, cognitive decline, hallucination, sexual dysfunction, and constipation were more frequent in GBA-PD than in LRRK2-PD and IPD patients, and GBA-PD patients had a worse performance for social functioning and role-emotional scores. Our study represents the first large-scale clinical study of LRRK2-PD and GBA-PD in ethnic Chinese individuals. The data suggest that both LRRK2-PD and GBA-PD are similar to IPD, except for an earlier age at onset and relatively more common non-motor symptoms in GBA-PD patients. These findings strengthen our understanding of the clinical heterogeneity of PD, and may have implications for molecular classification of the disease.

Promoter methylation analysis of seven clock genes in Parkinson's disease.

The expression of clock genes is altered in leukocytes from patients with Parkinsons disease (PD). However, the underlying mechanisms are unknown. To determine whether abnormal CpG methylation contributes to the dysregulated expression of these genes, the methylation status of the promoters of seven major human clock genes, PER1, PER2, CRY1, CRY2, Clock, NPAS2, and BMAL1, was examined using methylation-specific PCR (MSP) and sequencing in 206 PD patients and 181 healthy controls. This analysis revealed that most clock gene promoters were devoid of methylation. Methylation was only detectable in the CRY1 and NPAS2 promoters. Interestingly, the methylation frequency of the NPAS2 promoter was significantly decreased in PD patients. These results suggest that altered promoter methylation may contribute to the abnormal expression of clock genes in PD.

Pyrosequencing analysis of SNCA methylation levels in leukocytes from Parkinson's disease patients

DNA methylation of a CpG island located in intron 1 of the α-synuclein gene (SNCA) has been reported to play an essential role in the regulation of α-synuclein transcription, and probably has a close association with Parkinsons disease (PD). However, there is no simple and cost effective method to quantify DNA methylation in this region. Additionally, whether this CpG island is hypomethylated in peripheral blood in PD and can be used as PD biomarker is still under debate. In the present study, we developed a set of bisulfite pyrosequencing assays which can be used to examine the DNA methylation level of 13 CpG sites in intron 1 of SNCA. We compared DNA methylation levels at these sites in leukocytes from 50 PD patients and 50 healthy controls. Our results indicated that there were no significant differences in DNA methylation between PD patients and controls.

Decreased expression of Bmal2 in patients with Parkinson's disease.

Bmal1 is one of the central regulators of the clock machinery. Recently, we examined the expression profile of Bmal1 in total leukocytes for a 12h duration during the evening, overnight, and the morning, in subjects with Parkinsons disease (PD) and healthy controls. The results indicate that the expression of Bmal1 is significantly lower in PD patients versus control subjects. However, it is still unclear whether other key regulators of the clock machinery, especially Bmal2, the paralog of Bmal1, are also expressed differently in PD. To address this issue, the expression profiles of Bmal2, Clock, and Dec1 were examined in the same samples using real-time RT-PCR assay. The results show a difference in the expression pattern of Bmal2, but not Clock and Dec1. The expression of Bmal2 is significantly lower in PD at 21:00 h (p=0.005) and 00:00 h (p=0.025). These results together with our previous findings suggest that the molecular clock in total leukocytes is disturbed in PD patients.

Association of ARNTL and PER1 genes with Parkinson's disease: a case-control study of Han Chinese.

Circadian disruptions may result in sleep problems, oxidative stress and an altered inflammatory response. These symptoms may contribute to PD pathogenesis, despite a lack of direct experimental evidence supporting this relationship. Clock genes are essential to drive and maintain circadian rhythm. To elucidate the possible role of circadian disruptions in PD, we investigated 132 tag variants in eight clock genes. We genotyped these tags within 1,394 Chinese cases and 1,342 controls using Illumina GoldenGate chips. We discovered that SNPs in ARNTL (rs900147, Pu2009=u20093.33u2009×u200910−5, ORu2009=u20090.80) and PER1 (rs2253820, Pu2009=u20095.30u2009×u200910−6, ORu2009=u20091.31) genes are significantly associated with PD risk. Moreover, the positive association of the ARNTL rs900147 variant was more robust in tremor dominant (TD) (Pu2009=u20093.44u2009×u200910−4) than postural instability and gait difficulty (PIGD) cases (Pu2009=u20096.06u2009×u200910−2). The association of the PER1 rs2253820 variant was more robust in PIGD (Pu2009=u20095.42u2009×u200910−5) than TD cases (Pu2009=u20094.2u2009×u200910−2). Haplotype analysis also showed that ARNTL and PER1 were associated with PD. Imputation analysis identified more SNPs within ARNTL and PER1 associated with PD, some of which may affect gene expression through altering the transcription factor binding site. In summary, our findings suggest that genetic polymorphisms in ARNTL and PER1 genes, as well as circadian disruptions, may contribute to PD pathogenesis.

Genetic variants of the PITX3 gene are not associated with late-onset sporadic Parkinson's disease in a Chinese population

PITX3 is a transcription factor which determines the survival of dopaminergic neurons in the substantia nigra, and is considered a candidate gene for Parkinsons disease (PD). Recent association studies indicated that three PITX3 single nucleotide polymorphisms (SNPs), including rs2281983, rs4919621, and rs3758549 are likely to be associated with PD. However, no similar associations in Chinese populations have been reported. To examine the nature of the association in Chinese individuals we analyzed DNA samples for these three PITX3 SNPs from 509 late-onset sporadic PD patients, and 494 healthy controls, using a ligase detection reaction (LDR). Results indicated that allele and genotype frequencies did not differ between patients and controls for all three SNPs, suggesting that these SNP sites do not contribute to the risk of developing PD in late-onset sporadic PD in this Chinese population.

PITX3 polymorphism is not associated with Parkinson's disease in a Chinese population

Several studies have indicated that three PITX3 single nucleotide polymorphisms (SNPs), rs2281983, rs4919621 and rs3758549, are likely to be associated with Parkinsons disease (PD) in Caucasians. Some studies also suggested an age-of-onset effect. We recently reported that allele and genotype frequencies did not differ between late-onset PD (LOPD) patients and controls for all three SNPs. To extend the analysis to early-onset PD (EOPD) patients, and to test whether an age-of-onset effect exists in Chinese, we genotyped these SNPs in 290 Chinese EOPD patients using a ligase detection reaction (LDR). For all three SNPs, allele and genotype frequencies did not differ between total PD patients and controls, between LOPD patients and controls, between EOPD patients and controls, or between LOPD and EOPD patients. Our results suggest that these PITX3 SNPs do not contribute to the risk of developing PD in EOPD or LOPD in Chinese.

Oscillation development for neurotransmitter-related genes in the mouse striatum.

The aims of this study were to determine (i) whether striatal neuropeptides (dynorphin, enkephalin 1, substance P, cholecystokinin) and dopamine receptors 1 and 2 (D1r and D2r) are regulated by the molecular clock; and (ii) when their oscillations start after birth. Twenty-four-hour mRNA oscillations of these genes were evaluated in the mouse striatum at early postnatal stage (postnatal day 3), preweaning stage (postnatal day 14), and adult (postnatal day 60). At P3, no daily oscillations were observed. A significant time effect was present for D2r, dynorphin, and enkephalin 1 at P14, and for all genes except D1r, at P60. In conclusion, circadian expression of these neurotransmitter-related genes develops in the mouse striatum after birth gradually.

Epigenetic analysis of mPer1 promoter in peripheral tissues

E-boxes targeted by BMAL1-CLOCK dimers are crucial for the rhythmic expression of mPer1. We investigated whether DNA methylation, especially E-box CpG methylation, plays a role in the regulation of mPer1 oscillations in mice. E-box-containing amplicons were examined in liver, thymus and testis around-the-clock using bisulfite sequencing, combined bisulfite restriction analysis (COBRA) and bisulfite pyrosequencing. Results indicated that mPer1 E-boxes were only hypomethylated in all tissues at all circadian stages, suggesting that E-boxes are open to BMAL1-CLOCK dimers at all times, and thus E-box methylation is most likely not involved in global regulation of the mPer1 rhythm. In addition, low but noticeable methylation of E-box 3 and/or 4 and a sub-group of CpGs nearby were identified in all three tissues, indicating this region as area of preferred methylation, and may have a role in silencing mPer1 transcription.

Circadian E-boxes and surrounding CpG islands are free from methylation throughout the day

Circadian E-boxes in the promoters of mPer1, mPer2, mCry1 and mDBP play a key role in regulating rhythmic gene expression by recruiting the BMAL1/CLOCK heterodimer. However, each of these circadian E-boxes seems different from one another. In the mouse liver, BMAL1/CLOCK binds to E-boxes in the mPer1 and mPer2 promoters constantly but binds with dynamic daily changes to mCry1 and mDBP promoters. It is unclear whether DNA methylation of these circadian E-boxes and the surrounding CpG islands would affect protein/DNA interactions and lead to distinct binding features. In the present study, bisulfite sequencing analysis indicated that all E-boxes and their surrounding CpG islands were free from methylation, suggesting that DNA methylation does not determine distinct binding features. The methylation status of the E-box in the MAP kinase phosphatase 1 (MKP1) promoter was also examined. Our results indicated that DNA methylation does not regulate the tissue-specific clock control of MKP1.