To discover susceptibility genes of late-onset Alzheimer's disease (LOAD), we conducted a 3-stage genome-wide association study (GWAS) using three populations: Japanese from the Japanese Genetic Consortium for Alzheimer Disease (JGSCAD), Koreans, and Caucasians from the Alzheimer Disease Genetic Consortium (ADGC). In Stage 1, we evaluated data for 5,877,918 genotyped and imputed SNPs in Japanese cases (n = 1,008) and controls (n = 1,016). Genome-wide significance was observed with 12 SNPs in the APOE region. Seven SNPs from other distinct regions with p-values <2×10−5 were genotyped in a second Japanese sample (885 cases, 985 controls), and evidence of association was confirmed for one SORL1 SNP (rs3781834, P = 7.33×10−7 in the combined sample). Subsequent analysis combining results for several SORL1 SNPs in the Japanese, Korean (339 cases, 1,129 controls) and Caucasians (11,840 AD cases, 10,931 controls) revealed genome wide significance with rs11218343 (P = 1.77×10−9) and rs3781834 (P = 1.04×10−8). SNPs in previously established AD loci in Caucasians showed strong evidence of association in Japanese including rs3851179 near PICALM (P = 1.71×10−5) and rs744373 near BIN1 (P = 1.39×10−4). The associated allele for each of these SNPs was the same as in Caucasians. These data demonstrate for the first time genome-wide significance of LOAD with SORL1 and confirm the role of other known loci for LOAD in Japanese. Our study highlights the importance of examining associations in multiple ethnic populations.
High sensitivity liquid chromatography mass spectrometry (LC-MS/MS) was recently introduced to measure amyloid-β (Aβ) species, allowing for a simultaneous assay that is superior to ELISA, which requires more assay steps with multiple antibodies.We validated the Aβ1-38, Aβ1-40, Aβ1-42, and Aβ1-43 assay by LC-MS/MS and compared it with ELISA using cerebrospinal fluid (CSF) samples to investigate its feasibility for clinical application.CSF samples from 120 subjects [8 Alzheimer's disease (AD) with dementia (ADD), 2 mild cognitive dementia due to Alzheimer's disease (ADMCI), 14 cognitively unimpaired (CU), and 96 neurological disease subjects] were analyzed. Aβ species were separated using the Shimadzu Nexera X2 system and quantitated using a Qtrap 5500 LC-MS/MS system. Aβ1-40 and Aβ1-42 levels were validated using ELISA.CSF levels in CU were 666±249 pmol/L in Aβ1-38, 2199±725 pmol/L in Aβ1-40, 153.7±79.7 pmol/L in Aβ1-42, and 9.78±4.58 pmol/L in Aβ1-43. The ratio of the amounts of Aβ1-38, Aβ1-40, Aβ1-42, and Aβ1-43 was approximately 68:225:16:1. Linear regression analyses showed correlations among the respective Aβ species. Both Aβ1-40 and Aβ1-42 values were strongly correlated with ELISA measurements. No significant differences were observed in Aβ1-38 or Aβ1-40 levels between AD and CU. Aβ1-42 and Aβ1-43 levels were significantly lower, whereas the Aβ1-38/1-42, Aβ1-38/1-43, and Aβ1-40/Aβ1-43 ratios were significantly higher in AD than in CU. The basic assay profiles of the respective Aβ species were adequate for clinical usage.A quantitative LC-MS/MS assay of CSF Aβ species is as reliable as specific ELISA for clinical evaluation of CSF biomarkers for AD.
α-Synucleinopathy is an entity of neurodegenerative diseases such as Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), that involves accumulation of α-synuclein in the brain. PINK1 (PTEN induced kinase 1) is a novel gene recently identified as causative in autosomal recessive early onset parkinsonism (PARK6). In the present study, we examined the localisation of PINK1 in the brains of patients with α-synucleinopathy and found PINK1 in glial cytoplasmic inclusions (GCIs) in MSA, as well as in Lewy bodies (LBs) in PD and DLB. These findings imply that PINK1 may be involved in the formation of LBs and GCIs, suggesting that PINK1 is one of the major pathological proteins in α-synucleinopathy.
The cDNA of PINK1, corresponding to 112–520 amino acids of the protein, was subcloned in a vector pET30(a) with a His tag. Anti-PINK1 antibody was generated against recombinant His tagged PINK1 by immunising a rabbit. The obtained antibody was affinity purified. A postmortem brain sample from a normal patient was homogenised, subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a membrane. After blocking in Tris buffered saline with 5% dry milk, the membrane was incubated with anti-PINK1 antibody (1:1000). The membrane was then incubated with a secondary antibody (1:2500; Amersham, Buckinghamshire, UK), and visualised with an enhanced chemiluminescent substrate (Pierce, Rockford, Illinois, USA). Immunohistochemical analysis was carried out with paraffin embedded midbrain sections from patients with sporadic PD, …
In previous studies, patients with severe peri-ovarian adhesions have been found to show low pregnancy rates and a poor response to gonadotrophin stimulation during in-vitro fertilization (IVF) treatment. The purpose of this retrospective pharmacokinetic study was to assess the diffusion of exogenous human chorionic gonadotrophin (HCG) in patients with peri-ovarian adhesions by examining the concentration of exogenous HCG in the follicular fluid in patients undergoing down-regulation and IVF due to infertility. The patients underwent laparoscopic examination for the scoring of peri-ovarian adhesions (using the classification of adnexal adhesions adopted by the American Fertility Society, a score of 0 means no adhesions, and a score of 32 represents bilateral expanded dense adhesions). Oocytes were recovered after human menopausal gonadotrophin-human chorionic gonadotrophin (HMG-HCG) stimulation with gonadotrophin-releasing hormone agonist. Serum and follicular fluid were collected at the time of oocyte recovery for measuring the HCG ratio (the follicular HCG concentration to the serum HCG concentration; a reflection of the diffusion of exogenous gonadotrophin) by time-resolved fluoroimmunoassay. A negative correlation was found between the number of oocytes recovered and the peri-ovarian adhesion score (r = -0.62, P < 0.01). In a given patient, the follicular HCG concentration was always lower than the serum HCG at the time of oocyte recovery. The HCG ratio in all samples was 0.9 or less (0.51 +/- 0.20; range, 0.09-0.90). Significant negative correlations were found between the peri-ovarian adhesion score and both the follicular HCG concentration (r = -0.80, P < 0.01) and the HCG ratio (r = -0.75, P < 0.01). In conclusion, severe peri-ovarian adhesions interfered with the diffusion of exogenous gonadotrophin into the follicular fluid during IVF treatment. Thus, the diffusion of exogenous gonadotrophin into the follicular fluid may represent a new parameter in the assessment of ovarian blood flow and IVF outcome.
APOE4 is the strongest risk factor for Alzheimer's disease (AD). However, limited information is currently available on APOE4 and the pathological role of plasma apolipoprotein E (ApoE) 4 remains unclear.The aims of the present study were to measure plasma levels of total ApoE (tE), ApoE2, ApoE3, and ApoE4 using mass spectrometry and elucidate the relationships between plasma ApoE and blood test items.We herein examined plasma levels of tE, ApoE2, ApoE3, and ApoE4 in 498 subjects using liquid chromatograph-mass spectrometry (LC-MS/MS).Among 498 subjects, mean age was 60 years and 309 were female. tE levels were distributed as ApoE2/E3 = ApoE2/E4 >ApoE3/E3 = ApoE3/E4 >ApoE4/E4. In the heterozygous group, ApoE isoform levels were distributed as ApoE2 >ApoE3 >ApoE4. ApoE levels were not associated with aging, the plasma amyloid-β (Aβ) 40/42 ratio, or the clinical diagnosis of AD. Total cholesterol levels correlated with the level of each ApoE isoform. ApoE2 levels were associated with renal function, ApoE3 levels with low-density lipoprotein cholesterol and liver function, and ApoE4 levels with triglycerides, high-density lipoprotein cholesterol, body weight, erythropoiesis, and insulin metabolism.The present results suggest the potential of LC-MS/MS for the phenotyping and quantitation of plasma ApoE. Plasma ApoE levels are regulated in the order of ApoE2 >ApoE3 >ApoE4 and are associated with lipids and multiple metabolic pathways, but not directly with aging or AD biomarkers. The present results provide insights into the multiple pathways by which peripheral ApoE4 influences the progression of AD and atherosclerosis.
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