APOE genotype-specific methylation patterns are linked to Alzheimer disease pathology and estrogen response.

The joint effects of APOE genotype and DNA methylation on Alzheimer disease (AD) risk is relatively unknown. We conducted genome-wide methylation analyses using 2,021 samples in blood (91 AD cases, 329 mild cognitive impairment, 1,391 controls) and 697 samples in brain (417 AD cases, 280 controls). We identified differentially methylated levels in AD compared to controls in an APOE genotype-specific manner at 25 cytosine-phosphate-guanine (CpG) sites in brain and 36 CpG sites in blood. Additionally, we identified seven CpG sites in the APOE region containing TOMM40, APOE, and APOC1 genes with P < 5 × 10-8 between APOE ε4 carriers and non-carriers in brain or blood. In brain, the most significant CpG site hypomethylated in ε4 carriers compared to non-carriers was from the TOMM40 in the total sample, while most of the evidence was derived from AD cases. However, the CpG site was not significantly modulating expression of these three genes in brain. Three CpG sites from the APOE were hypermethylated in APOE ε4 carriers in brain or blood compared in ε4 non-carriers and nominally significant with APOE expression in brain. Three CpG sites from the APOC1 were hypermethylated in blood, which one of the 3 CpG sites significantly lowered APOC1 expression in blood using all subjects or ε4 non-carriers. Co-methylation network analysis in blood and brain detected eight methylation networks associated with AD and APOE ε4 status. Five of the eight networks included genes containing network CpGs that were significantly enriched for estradiol perturbation, where four of the five networks were enriched for the estrogen response pathway. Our findings provide further evidence of the role of APOE genotype on methylation levels associated with AD, especially linked to estrogen response pathway.

Genome-wide association and multi-omics studies identify MGMT as a novel risk gene for Alzheimer's disease among women.

INTRODUCTION: Variants in the tau gene (MAPT) region are associated with breast cancer in women and Alzheimer's disease (AD) among persons lacking apolipoprotein E ε4 (ε4-). METHODS: To identify novel genes associated with tau-related pathology, we conducted two genome-wide association studies (GWAS) for AD, one among 10,340 ε4- women in the Alzheimer's Disease Genetics Consortium (ADGC) and another in 31 members (22 women) of a consanguineous Hutterite kindred. RESULTS: We identified novel associations of AD with MGMT variants in the ADGC (rs12775171, odds ratio [OR] = 1.4, P = 4.9 × 10-8 ) and Hutterite (rs12256016 and rs2803456, OR = 2.0, P = 1.9 × 10-14 ) datasets. Multi-omics analyses showed that the most significant and largest number of associations among the single nucleotide polymorphisms (SNPs), DNA-methylated CpGs, MGMT expression, and AD-related neuropathological traits were observed among women. Furthermore, promoter capture Hi-C analyses revealed long-range interactions of the MGMT promoter with MGMT SNPs and CpG sites. DISCUSSION: These findings suggest that epigenetically regulated MGMT expression is involved in AD pathogenesis, especially in women.

Cytokine Levels in Human Vitreous in Proliferative Diabetic Retinopathy.

In this study, we compare the vitreous cytokine profile in patients with proliferative diabetic retinopathy (PDR) to that of patients without PDR. The identification of novel cytokines involved in the pathogenesis of PDR provides candidate therapeutic targets that may stand alone or work synergistically with current therapies in the management of diabetic retinopathy. Undiluted vitreous humor specimens were collected from 74 patients undergoing vitrectomy for various vitreoretinal disorders. Quantitative immunoassay was performed for a panel of 36 neuroinflammatory cytokines in each specimen and assessed to identify differences between PDR (n = 35) and non-PDR (n = 39) patients. Levels of interleukin-8 (IL-8), IL-15, IL-16, vascular endothelial growth factor (VEGF), VEGF-D, c-reactive protein (CRP), serum amyloid-A (SAA), and intracellular adhesion molecule-1 (ICAM1) were significantly increased in the vitreous of PDR patients compared to non-PDR patients (p < 0.05). We report novel increases in IL-15 and IL-16, in addition to the expected VEGF, in the human vitreous humor of patients with PDR. Additionally, we confirm the elevation of ICAM-1, VCAM-1, SAA, IL-8 and CRP in the vitreous of patients with PDR, which has previously been described.

Neurofilament light chain in the vitreous humor of the eye.

BACKGROUND: Neurofilament light chain (NfL) is a promising biomarker of neurodegeneration in the cerebrospinal fluid and blood. This study investigated the presence of NfL in the vitreous humor and its associations with amyloid beta, tau, inflammatory cytokines and vascular proteins, apolipoprotein E (APOE) genotypes, Mini-Mental State Examination (MMSE) scores, systemic disease, and ophthalmic diseases. METHODS: This is a single-site, prospective, cross-sectional cohort study. Undiluted vitreous fluid (0.5-1.0 mL) was aspirated during vitrectomy, and whole blood was drawn for APOE genotyping. NfL, amyloid beta (Aß), total Tau (t-Tau), phosphorylated Tau (p-Tau181), inflammatory cytokines, chemokines, and vascular proteins in the vitreous were quantitatively measured by immunoassay. The main outcome measures were the detection of NfL levels in the vitreous humor and its associations with the aforementioned proteins. Linear regression was used to test the associations of NfL with other proteins, APOE genotypes, MMSE scores, and ophthalmic and systemic diseases after adjustment for age, sex, education level, and other eye diseases. RESULTS: NfL was detected in all 77 vitreous samples. NfL was not found to be associated with ophthalmic conditions, APOE genotypes, MMSE scores, or systemic disease (p > 0.05). NfL levels were positively associated with increased vitreous levels of Aß40 (p = 7.7 × 10-5), Aß42 (p = 2.8 × 10-4), and t-tau (p = 5.5 × 10-7), but not with p-tau181 (p = 0.53). NfL also had significant associations with inflammatory cytokines such as interleukin-15 (IL-15, p = 5.3 × 10-4), IL-16 (p = 2.2 × 10-4), monocyte chemoattractant protein-1 (MCP1, p = 4.1 × 10-4), and vascular proteins such as vascular endothelial growth factor receptor-1 (VEGFR1, p = 2.9 × 10-6), Vegf-C (p = 8.6 × 10-6), vascular cell adhesion molecule-1 (VCAM-1, p = 5.0 × 10-4), Tie-2 (p = 6.3 × 10-4), and intracellular adhesion molecular-1 (ICAM-1, p = 1.6 × 10-4). CONCLUSION: NfL is detectable in the vitreous humor of the eye and significantly associated with amyloid beta, t-tau, and select inflammatory and vascular proteins in the vitreous. Additionally, NfL was not associated with patients' clinical eye condition. Our results serve as a foundation for further investigation of NfL in the ocular fluids to inform us about the potential utility of its presence in the eye.

CpG-related SNPs in the MS4A region have a dose-dependent effect on risk of late-onset Alzheimer disease.

CpG-related single nucleotide polymorphisms (CGS) have the potential to perturb DNA methylation; however, their effects on Alzheimer disease (AD) risk have not been evaluated systematically. We conducted a genome-wide association study using a sliding-window approach to measure the combined effects of CGSes on AD risk in a discovery sample of 24 European ancestry cohorts (12,181 cases, 12,601 controls) from the Alzheimer's Disease Genetics Consortium (ADGC) and replication sample of seven European ancestry cohorts (7,554 cases, 27,382 controls) from the International Genomics of Alzheimer's Project (IGAP). The potential functional relevance of significant associations was evaluated by analysis of methylation and expression levels in brain tissue of the Religious Orders Study and the Rush Memory and Aging Project (ROSMAP), and in whole blood of Framingham Heart Study participants (FHS). Genome-wide significant (p < 5 × 10-8 ) associations were identified with 171 1.0 kb-length windows spanning 932 kb in the APOE region (top p < 2.2 × 10-308 ), five windows at BIN1 (top p = 1.3 × 10-13 ), two windows at MS4A6A (top p = 2.7 × 10-10 ), two windows near MS4A4A (top p = 6.4 × 10-10 ), and one window at PICALM (p = 6.3 × 10-9 ). The total number of CGS-derived CpG dinucleotides in the window near MS4A4A was associated with AD risk (p = 2.67 × 10-10 ), brain DNA methylation (p = 2.15 × 10-10 ), and gene expression in brain (p = 0.03) and blood (p = 2.53 × 10-4 ). Pathway analysis of the genes responsive to changes in the methylation quantitative trait locus signal at MS4A4A (cg14750746) showed an enrichment of methyltransferase functions. We confirm the importance of CGS in AD and the potential for creating a functional CpG dosage-derived genetic score to predict AD risk.

Aldose Reductase Polymorphisms, Fasting Blood Glucose, and Age-Related Cortical Cataract.

Purpose: To determine whether there is an association between polymorphisms of the AKR1B1 gene and cortical cataract in the presence of hyperglycemia. Methods: In the second cross section of the Blue Mountains Eye Study (BMES), 3508 participants (2334 at 5-year follow-up and 1174 newly recruited participants) were examined during 1997 to 2000. Cataract was graded from lens photographs using the Wisconsin Cataract Grading System. Fasting blood glucose (FBG) was measured. Continuous imputed dosages of minor alleles of 17 AKR1B1 single nucleotide polymorphisms (SNPs) were assessed for associations with prevalent cortical cataract. Gene-environment interactions between SNPs and FBG were examined. Odds ratios (OR) and 95% confidence intervals (CI) for prevalent cortical cataract were estimated using logistic regression adjusting for age, sex, smoking, hypertension, education, and myopia. A P value of 0.005 was considered statistically significant after correction for 10 independent tests. Replication of significant associations found in the BMES sample was conducted in the Singapore Epidemiology of Eye Diseases (SEED) study (n = 10,033). Results: No polymorphism was associated with prevalent cortical cataract. A significant interaction was observed between rs9640883 and FBG (Pinteraction = 0.004), with increased cortical cataract prevalence associated with rs9640883 minor allele dosage in those with FBG >6.0 mM (strata-specific OR 1.72, 95% CI 1.09-2.72). No similar association was found in participants with normal FBG (OR 0.85, 95% CI 0.69-1.04). This interaction was not evident in the SEED study. Conclusions: The identified interaction between rs9640883 and FBG in relation to cortical cataract was not replicated but may warrant further investigation.

Genome-wide pleiotropy analysis of neuropathological traits related to Alzheimer's disease.

BACKGROUND: Simultaneous consideration of two neuropathological traits related to Alzheimer's disease (AD) has not been attempted in a genome-wide association study. METHODS: We conducted genome-wide pleiotropy analyses using association summary statistics from the Beecham et al. study (PLoS Genet 10:e1004606, 2014) for AD-related neuropathological traits, including neuritic plaque (NP), neurofibrillary tangle (NFT), and cerebral amyloid angiopathy (CAA). Significant findings were further examined by expression quantitative trait locus and differentially expressed gene analyses in AD vs. control brains using gene expression data. RESULTS: Genome-wide significant pleiotropic associations were observed for the joint model of NP and NFT (NP + NFT) with the single-nucleotide polymorphism (SNP) rs34487851 upstream of C2orf40 (alias ECRG4, P = 2.4 × 10-8) and for the joint model of NFT and CAA (NFT + CAA) with the HDAC9 SNP rs79524815 (P = 1.1 × 10-8). Gene-based testing revealed study-wide significant associations (P ≤ 2.0 × 10-6) for the NFT + CAA outcome with adjacent genes TRAPPC12, TRAPPC12-AS1, and ADI1. Risk alleles of proxy SNPs for rs79524815 were associated with significantly lower expression of HDAC9 in the brain (P = 3.0 × 10-3), and HDAC9 was significantly downregulated in subjects with AD compared with control subjects in the prefrontal (P = 7.9 × 10-3) and visual (P = 5.6 × 10-4) cortices. CONCLUSIONS: Our findings suggest that pleiotropy analysis is a useful approach to identifying novel genetic associations with complex diseases and their endophenotypes. Functional studies are needed to determine whether ECRG4 or HDAC9 is plausible as a therapeutic target.

Two novel loci, COBL and SLC10A2, for Alzheimer's disease in African Americans.

INTRODUCTION: African Americans' (AAs) late-onset Alzheimer's disease (LOAD) genetic risk profile is incompletely understood. Including clinical covariates in genetic analyses using informed conditioning might improve study power. METHODS: We conducted a genome-wide association study (GWAS) in AAs employing informed conditioning in 1825 LOAD cases and 3784 cognitively normal controls. We derived a posterior liability conditioned on age, sex, diabetes status, current smoking status, educational attainment, and affection status, with parameters informed by external prevalence information. We assessed association between the posterior liability and a genome-wide set of single-nucleotide polymorphisms (SNPs), controlling for APOE and ABCA7, identified previously in a LOAD GWAS of AAs. RESULTS: Two SNPs at novel loci, rs112404845 (P = 3.8 × 10-8), upstream of COBL, and rs16961023 (P = 4.6 × 10-8), downstream of SLC10A2, obtained genome-wide significant evidence of association with the posterior liability. DISCUSSION: An informed conditioning approach can detect LOAD genetic associations in AAs not identified by traditional GWAS.

Associations Between Methylenetetrahydrofolate Reductase Polymorphisms, Serum Homocysteine Levels, and Incident Cortical Cataract.

IMPORTANCE: Methylenetetrahydrofolate reductase (MTHFR) polymorphisms have been shown to influence homocysteine levels; homocysteine has been implicated as a cataractogenic stressor. OBJECTIVE: To investigate the associations of MTHFR polymorphisms and serum homocysteine levels with incident cortical cataract in an older population. DESIGN, SETTING, AND PARTICIPANTS: From 1992 to 1994, a population-based cohort study, the Blue Mountains Eye Study, was conducted with 3654 residents (82.4% of eligible participants) of the Blue Mountains region aged 49 years and older. At the second (1997-1999, 5-year follow-up) and third (2002-2004, 10-year follow-up) surveys, 2334 (75.8% of survivors) and 1952 (76.7% of survivors) were examined, respectively. For this report, the second survey serves as baseline when homocysteine levels were assessed, and 5-year incidence of cataract refers to incidence estimated from the second to the third survey. After excluding participants with no follow-up data or DNA or who had previous cortical cataract or cataract surgery, 757 participants were included in gene and environment analyses. This current project on associations with cataract was designed initially March 19, 2013, and completed April 14, 2014. Cataract was assessed using the Wisconsin Cataract Grading system. Two MTHFR polymorphisms, C677T (rs1801133) and A1298C (rs1801131), were included. Serum homocysteine levels were assessed following standard methods. MAIN OUTCOMES AND MEASURES: Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals for incident cortical cataract, after adjusting for age, sex, smoking status, hypertension, diabetes, education, and myopia. Path analysis was performed to explore a possible pathway of MTHFR polymorphisms via homocysteine levels to cortical cataract. RESULTS: The mean (SD) age of the 1726 participants in the Blue Mountains Eye Study 2 cohort with normal homocysteine levels was 68.3 (8.1) years and 73.2 (8.5) years for those with elevated homocysteine levels. Both the C677T polymorphism (CT/TT vs CC: OR = 1.50; 95% CI = 1.01-2.23) and elevated homocysteine levels (>15 µmol/L: OR = 2.24; 95% CI = 1.38-3.63) were independently associated with increased risk of cortical cataract. Path analysis showed that the genetic effect on cortical cataract was partially mediated via homocysteine levels. Combined CT/TT genotypes and elevated homocysteine levels were associated with a 3-fold risk of cortical cataract (OR = 3.74; 95% CI = 1.79-7.80). The synergy index of both exposures was 1.34 (95% CI = 0.44-4.01). CONCLUSIONS AND RELEVANCE: MTHFR polymorphism and elevated homocysteine levels contributed separately and jointly to increased risk of cortical cataract. If these findings are confirmed, homocysteine levels may be a therapeutic target to reduce risk of cortical cataract in persons carrying genetic risk.

Meta-analysis of genome-wide association studies in multiethnic Asians identifies two loci for age-related nuclear cataract.

Age-related cataract is a leading cause of blindness worldwide, especially in developing countries where access to cataract surgery remains limited. Previous linkage and candidate gene studies suggested genetic influences on age-related nuclear cataract but few genetic markers have been identified thus far. We conducted genome-wide association studies on 4569 Asians (including 2369 Malays and 2200 Indians), and replicated our analysis in 2481 Chinese from two independent cohorts (1768 Chinese in Singapore and 803 Chinese in Beijing). We confirmed two genome-wide significant loci for nuclear cataract in the combined meta-analysis of four cohorts (n = 7140). The first locus was at chromosome 3q25.31 in KCNAB1 (rs7615568, fixed-effect Pmeta = 2.30 × 10(-8); random-effect Pmeta = 1.08 × 10(-8)). The second locus was at chromosome 21 in the proximity of CRYAA (rs11911275, fixed-effect Pmeta = 2.77 × 10(-8); random-effect Pmeta = 1.98 × 10(-9)), a major protein component of eye lens. The findings were further supported by up-regulation and down-regulation of KCNAB1 and CRYAA in human lens capsule, respectively, as the severity of nuclear cataract increases. The results offer additional insights into the pathogenesis of nuclear cataract in Asians.

Search for age-related macular degeneration risk variants in Alzheimer disease genes and pathways.

Several lines of inquiry point to overlapping molecular mechanisms between late-onset Alzheimer disease (AD) and age-related macular degeneration (AMD). We evaluated summarized results from large genome-wide association studies for AD and AMD to test the hypothesis that AD susceptibility loci are also associated with AMD. We observed association of both disorders with genes in a region of chromosome 7, including PILRA and ZCWPW1 (peak AMD SNP rs7792525, minor allele frequency [MAF] = 19%, odds ratio [OR] = 1.14, p = 2.34 × 10(-6)), and with ABCA7 (peak AMD SNP rs3752228, MAF = 0.054, OR = 1.22, p = 0.00012). Next, we evaluated association of AMD with genes in AD-related pathways identified by canonical pathway analysis of AD-associated genes. Significant associations were observed with multiple previously identified AMD risk loci and 2 novel genes: HGS (peak SNP rs8070488, MAF = 0.23, OR = 0.91, p = 7.52 × 10(-5)), which plays a role in the clathrin-mediated endocytosis signaling pathway, and TNF (peak SNP rs2071590, MAF = 0.34, OR = 0.89, p = 1.17 × 10(-5)), which is a member of the atherosclerosis signaling and the LXR/RXR activation pathways. Our results suggest that AMD and AD share genetic mechanisms.

Systems biology-based analysis implicates a novel role for vitamin D metabolism in the pathogenesis of age-related macular degeneration.

Vitamin D has been shown to have anti-angiogenic properties and to play a protective role in several types of cancer, including breast, prostate and cutaneous melanoma. Similarly, vitamin D levels have been shown to be protective for risk of a number of conditions, including cardiovascular disease and chronic kidney disease, as well as numerous autoimmune disorders such as multiple sclerosis, inflammatory bowel diseases and type 1 diabetes mellitus. A study performed by Parekh et al. was the first to suggest a role for vitamin D in age-related macular degeneration (AMD) and showed a correlation between reduced serum vitamin D levels and risk for early AMD. Based on this study and the protective role of vitamin D in diseases with similar pathophysiology to AMD, we examined the role of vitamin D in a family-based cohort of 481 sibling pairs. Using extremely phenotypically discordant sibling pairs, initially we evaluated the association of neovascular AMD and vitamin D/sunlight-related epidemiological factors. After controlling for established AMD risk factors, including polymorphisms of the genes encoding complement factor H (CFH) and age-related maculopathy susceptibility 2/HtrA serine peptidase (ARMS2/HTRA1), and smoking history, we found that ultraviolet irradiance was protective for the development of neovascular AMD (p = 0.001). Although evaluation of serum vitamin D levels (25-hydroxyvitamin D [25(OH)D]) was higher in unaffected individuals than in their affected siblings, this finding did not reach statistical significance. Based on the relationship between ultraviolet irradiance and vitamin D production, we employed a candidate gene approach for evaluating common variation in key vitamin D pathway genes (the genes encoding the vitamin D receptor [VDR]; cytochrome P450, family 27, subfamily B, polypeptide 1 [CYP27B1]; cytochrome P450, family 24, subfamily A, polypeptide 1 [CYP24A1]; and CYP27A1) in this same family-based cohort. Initial findings were then validated and replicated in the extended family cohort, an unrelated case-control cohort from central Greece and a prospective nested case-control population from the Nurse's Health Study and Health Professionals Follow-Up Studies, which included patients with all subtypes of AMD for a total of 2,528 individuals. Single point variants in CYP24A1 (the gene encoding the catabolising enzyme of the vitamin D pathway) were demonstrated to influence AMD risk after controlling for smoking history, sex and age in all populations, both separately and, more importantly, in a meta-analysis. This is the first report demonstrating a genetic association between vitamin D metabolism and AMD risk. These findings were also supplemented with expression data from human donor eyes and human retinal cell lines. These data not only extend previous biological studies in the AMD field, but further emphasise common antecedents between several disorders with an inflammatory/immunogenic component such as cardiovascular disease, cancer and AMD.