APOE Variants Research Articles

Alzheimer’s Disease modeling using hiPSC neurons and microglia

AbstractBackgroundNeuroinflammation is an important topic in neurodegenerative disease research. Scientists have established multiple protocols for differentiating microglia, neurons, and astrocytes from human iPSCs, which enable the study of neuroinflammation in vitro. Here we describe the use of Vala Sciences’ imaging platform, the IC200 Kinetic Image CytometerTM (KIC), to develop screening assays for Alzheimer’s Disease (AD) in cultures with hiPSC‐derived microglia and neurons.MethodWe cultured iCell Glutaneurons with microglia in 384‐well dishes. To detect microglia phagocytosis, we cultured microglia with pHrodo‐Red Zymosan beads or β‐amyloid fragments and then acquired timelapse videos over 5‐24 hours. To measure neuronal activity, we loaded cells with Rhod‐4 calcium dye and imaged at 4fps. We also fixed and immunolabeled cells for neurite and synapse analysis. All assays were scanned using the IC200 KIC. We developed algorithms for our CyteSeer image analysis software to automate neurite tracing, synapse detection, analysis of calcium activity, and microglia phagocytosis and morphology for single cells in a co‐culture system.ResultIn microglia, we demonstrated an increase in uptake of pHrodo‐Red labeled Zymosan beads with increasing concentration of Zymosan particles and in response to IL‐4. We also demonstrated differential engulfment of β‐amyloid 1‐40 and β‐amyloid 1‐42 by microglia. While investigating the effects of microglia on neuronal health, we observed increased neuronal survival, neurite outgrowth, and pre‐synapse count in glutamatergic neurons co‐cultured with microglia when compared to glutamatergic neurons cultured alone. In neuron‐microglia co‐cultures exposed to increasing doses of β‐amyloid 1‐42 fragments, we observed neuronal hyperactivity (increased calcium event frequency), which may reflect disease‐associated stress.ConclusionOur data demonstrate that healthy microglia regulate neuronal heath. Our system can serve as a model for testing inflammatory insults related to AD. To further our research with genetic variants with AD susceptibility, we have created an isogeneic panel of ApoE variants using CRISPR‐Cas9, and plan to characterize them in the culture systems developed above to validate a disease model from these lines.

APOE3 Christchurch mutation carriers from the Colombian kindred with autosomal dominant Alzheimer’s disease due to PSEN1 E280A

AbstractBackgroundOur understanding of the mechanisms of protection mediated by APOE has advanced significantly in the past years. Our group previously showed that APOE3 Christchurch (APOE3ch) homozygosity was associated with the protection against Alzheimer’s disease dementia in an individual who carried the Presenilin‐1 (PSEN1)‐E280A variant for autosomal dominant AD (ADAD) (Arboleda‐Velasquez et al., 2019). Here we present the initial characterization of individuals from the PSEN1‐E280A kindred with the APOE3ch variant.Methods1,000 PSEN1‐E280A variant carriers and 1,000 non‐carriers from the Colombian ADAD kindred were characterized in terms of their common APOE variants and the rare APOEch variant. Participants had previously completed a comprehensive neurological and neuropsychological evaluation. Those who were alive were re‐contacted in 2021 to undergo comprehensive clinical assessments, including the CERAD‐Colombia neuropsychological battery, and MRI and FDG PET imaging as part of the Resist Alzheimer’s Project.ResultsWe discovered 97 ADAD kindred members with the APOE3ch variant (95 heterozygotes), including 84 who are living and 13 who are deceased (7 who were brain donors). 29 of the APOE3ch carriers also had the PSEN1‐E280A variant, including 18 who are living and 11 who are deceased. The age range for all APOE3ch variant carriers was from 17 to 72 years, including 11 APOE3ch + PSEN1‐E280A carriers who were 50‐72 years old (i.e., older than the PSEN1‐E280A variant carriers’ median age at dementia onset). Overall, global cognition and memory performances of the first three APOE3ch + PSEN1‐E280A carriers (older than 50 years of age) assessed fell within the normal range, compared to age‐matched individuals from this kindred.ConclusionsThe prevalence rate of APOE3 Christchurch heterozygosity in the Colombian kindred with ADAD (4.85%) is very high compared to the general population (e.g., gnomAD database reports an allele frequency of 0.00001283; Karczewski et al., 2019). Preliminary clinical and cognitive data from APOE3ch variant carriers are consistent with the hypothesis that heterozygosity may have a mild‐to‐moderate protective effect against cognitive decline. We plan to compare longitudinal clinical, cognitive, biomarker, and post‐mortem neuropathological changes in PSEN1‐E280A carriers with and without the APOE3ch variant to clarify the role of APOE3ch heterozygosity in the protection from ADAD.

Lipidomic analysis of isogenic ApoE‐variant iPSC‐derived glia uncovers coupling between inflammatory signaling and lipid metabolism.

AbstractBackgroundApoE is the major genetic risk factor for Alzheimer’s disease and the major regulator of brain lipid metabolism. In the brain ApoE is primarily expressed in glia. How different damaging or protective ApoE variants exactly affects glial lipid metabolism, and/or how this correlates with inflammatory signaling is not known.MethodWe generated iPSC‐derived astrocytes and microglia from isogenic induced pluripotent stem cells (iPSC) with ApoE3/3 (reference), ApoE4/4 (increased risk), ApoE2/2 (decreased risk) and ApoEnull/null (control) genotypes. On these cells we performed unbiased lipidomics and transcriptional profiling to measure inflammatory pathways.ResultWe observed strong accumulation of stored lipids in human ApoE4/4, E2/2 and ApoEnull/null glia compared to ApoE3/3 glia. The composition of the exact species of accumulating lipids differed between ApoE4/4, E2/2 and Enull/null glia, and between astrocytes and microglia. At the transcriptional level, lipid accumulation in some genotypes was associated with increased proinflammatory signaling in astrocytes and microglia.ConclusionsApoE genotypes has a major effect on glial lipid metabolism and specific changes in lipid metabolism associate with inflammatory signaling pathways. Using lipid‐targeting interventions we are currently probing a causal relationship between altered lipid metabolism and downstream increases inflammatory signaling

Polygenic score is associated with accelerated tau pathology accumulation in Alzheimer’s disease

AbstractBackgroundProgression of fibrillar tau is a key driver of neurodegeneration and cognitive decline in Alzheimer’s disease (AD), however which factors predict the rate of tau accumulation at the patient‐level is unclear. Here we propose to employ a polygenic score (PGS) for the prediction of tau accumulation and cognitive changes in AD, which could be useful for selection of individuals with faster tau progression in clinical trials.MethodsWe included 231 ADNI participants with longitudinal measurements of tau‐PET, amyloid‐PET, cognitive data and genotype data. We computed a PGS based on 85 independent lead SNPs from two recent large GWAS (DOI:10.1101/2020.10.01.20200659; DOI:10.1038/s41588‐021‐00921‐z), excluding all APOE variants. Using linear mixed effect models, we computed the individual rates of change in each of the biomarkers. In linear regression models we tested whether PGS can predict the rate of tau‐PET changes in Braak‐stage ROIs (Figure 1A) and cognitive changes. In order to assess whether amyloid mediates the effect of PGS on tau accumulation we performed sensitivity analyses in subgroups categorized by amyloid status as well as tested the interaction between the PGS and global amyloid‐PET. We further estimated sample size required for detection of hypothetical treatment effect on the rate tau accumulation using power analysis.ResultsHigher PGS was associated with higher accumulation rates of tau‐PET in cortical regions (Braak 3+4:β=0.306, pFDR<0.001; Braak 5+6:β=0.262, pFDR<0.001; Figure 1B). A higher PGS was further associated with faster decline in episodic memory (β=‐0.223, pFDR<0.001; Figure 1C) and global cognition (β=0.280, pFDR<0.001; Figure 1C). The observed effects of PGS on cognitive changes were mediated by higher tau‐PET accumulation (Figure 2). We found a synergistic effect between PGS and elevated amyloid‐PET levels on the rate of tau‐PET accumulation such that the PGS effects on tau‐PET accumulation were stronger particularly at elevated amyloid levels (Figure 3). Selection of individuals with highest PGS yielded a 32‐33% saving in required sample size to detect a treatment effect on tau accumulation.ConclusionHigher polygenic score is associated with faster tau accumulation and may be a useful tool for risk stratification in disease‐modifying treatments on tau.

Investigation of the independent role of a rare APOE variant (L28P; APOE*4Pittsburgh) in late-onset Alzheimer disease

A rare missense APOE variant (L28P; APOE*4Pittsburgh), which is present only in populations with European ancestry, has been reported to be a risk factor for late-onset Alzheimer's disease (LOAD). However, due to the complete linkage disequilibrium of L28P with APOE*4 (C112R), its independent genetic association is uncertain. The original association study implicating L28P with LOAD risk was carried out in a relatively small sample size. In the current study, we have re-evaluated this association in a large case-control sample of 15,762 White U.S. subjects and investigated its independent effect in APOE 3/4 subjects, as L28P has been observed only in the heterozygous state of APOE*4 carriers and 3/4 is the most common genotype containing the APOE*4 allele. The heterozygous carrier frequency of L28P, all with APOE*4, was about 3-fold higher in AD cases than in cognitively intact controls (0.845% vs. 0.277%). The age- and sex-adjusted meta-analysis odds ratio (OR) was 2.87 (95% CI: 1.34 – 6.13; = 0.0066). Among APOE 3/4 subjects, age- and sex-adjusted meta-analysis OR was 1.53 (95% CI: 0.70 – 3.36; p = 0.28), indicating its effect was independent of APOE*4. The lack of statistical significance appears mainly due to the low power of 4138 subjects with the 3/4 genotype (12% power at α= 0.05) compared to the required sample of 139,088 subjects with the 3/4 genotype to detect an OR of 1.5 at α= 0.05 and 80% power. Our data suggesting that L28P has an independent genetic effect on AD risk is reinforced by earlier experimental findings showing that this mutation leads to significant structural and conformational changes in the ApoE4 molecule and can induce functional defects associated with neuronal Aβ42 accumulation and oxidative stress. Additional functional studies in cell-based systems and animal models will help to delineate its functional significance in the etiology of AD.

TREM2-independent microgliosis promotes tau-mediated neurodegeneration in the presence of ApoE4

In addition to tau and Aβ pathologies, inflammation plays an important role in Alzheimer's disease (AD). Variants in APOE and TREM2 increase AD risk. ApoE4 exacerbates tau-linked neurodegeneration and inflammation in P301S tau mice and removal of microglia blocks tau-dependent neurodegeneration. Microglia adopt a heterogeneous population of transcriptomic states in response to pathology, at least some of which are dependent on TREM2. Previously, we reported that knockout (KO) of TREM2 attenuated neurodegeneration in P301S mice that express mouse Apoe. Because of the possible common pathway of ApoE and TREM2 in AD, we tested whether TREM2 KO (T2KO) would block neurodegeneration in P301S Tau mice expressing ApoE4 (TE4), similar to that observed with microglial depletion. Surprisingly, we observed exacerbated neurodegeneration and tau pathology in TE4-T2KO versus TE4 mice, despite decreased TREM2-dependent microgliosis. Our results suggest that tau pathology-dependent microgliosis, that is, TREM2-independent microgliosis, facilitates tau-mediated neurodegeneration in the presence of ApoE4.

Apolipoprotein E imbalance in the cerebrospinal fluid of Alzheimer’s disease patients

ObjectiveThe purpose of this study was to examine the levels of cerebrospinal fluid (CSF) apolipoprotein E (apoE) species in Alzheimer’s disease (AD) patients.MethodsWe analyzed two CSF cohorts of AD and control individuals expressing different APOE genotypes. Moreover, CSF samples from the TgF344-AD rat model were included. Samples were run in native- and SDS-PAGE under reducing or non-reducing conditions (with or without β-mercaptoethanol). Immunoprecipitation combined with mass spectrometry or western blotting analyses served to assess the identity of apoE complexes.ResultsIn TgF344-AD rats expressing a unique apoE variant resembling human apoE4, a ~35-kDa apoE monomer was identified, increasing at 16.5 months compared with wild-types. In humans, apoE isoforms form disulfide-linked dimers in CSF, except apoE4, which lacks a cysteine residue. Thus, controls showed a decrease in the apoE dimer/monomer quotient in the APOE ε3/ε4 group compared with ε3/ε3 by native electrophoresis. A major contribution of dimers was found in APOE ε3/ε4 AD cases, and, unexpectedly, dimers were also found in ε4/ε4 AD cases. Under reducing conditions, two apoE monomeric glycoforms at 36 kDa and at 34 kDa were found in all human samples. In AD patients, the amount of the 34-kDa species increased, while the 36-kDa/34-kDa quotient was lower compared with controls. Interestingly, under reducing conditions, a ~100-kDa apoE complex, the identity of which was confirmed by mass spectrometry, also appeared in human AD individuals across all APOE genotypes, suggesting the occurrence of aberrantly resistant apoE aggregates. A second independent cohort of CSF samples validated these results.ConclusionThese results indicate that despite the increase in total apoE content the apoE protein is altered in AD CSF, suggesting that function may be compromised.

Metabolomic and lipidomic signatures in autosomal dominant and late-onset Alzheimer's disease brains.

The identification of multiple genetic risk factors for Alzheimer's disease (AD) suggests that many pathways contribute to AD onset and progression. However, the metabolomic and lipidomic profiles in carriers of distinct genetic risk factors are not fully understood. The metabolome can provide a direct image of dysregulated pathways in the brain. We interrogated metabolomic signatures in the AD brain, including carriers of pathogenic variants in APP, PSEN1, and PSEN2 (autosomal dominant AD; ADAD), APOE ɛ4, and TREM2 risk variant carriers, and sporadic AD (sAD). We identified 133 unique and shared metabolites associated with ADAD, TREM2, and sAD. We identified a signature of 16 metabolites significantly altered between groups and associated with AD duration. AD genetic variants show distinct metabolic perturbations. Investigation of these metabolites may provide greater insight into the etiology of AD and its impact on clinical presentation. APP/PSEN1/PSEN2 and TREM2 variant carriers show distinct metabolic changes. A total of 133 metabolites were differentially abundant in AD genetic groups. β-citrylglutamate is differentially abundant in autosomal dominant, TREM2, and sporadic AD. A 16-metabolite profile shows differences between Alzheimer's disease (AD) genetic groups. The identified metabolic profile is associated with duration of disease.

Association of Alzheimer’s disease polygenic risk scores with amyloid accumulation in cognitively intact older adults

BackgroundEarly detection of individuals at risk for Alzheimer’s disease (AD) is highly important. Amyloid accumulation is an early pathological AD event, but the genetic association with known AD risk variants beyond the APOE4 effect is largely unknown. We investigated the association between different AD polygenic risk scores (PRS) and amyloid accumulation in the Flemish Prevent AD Cohort KU Leuven (F-PACK).MethodsWe calculated PRS with and without the APOE region in 90 cognitively healthy F-PACK participants (baseline age 67.8 (52–80) years, 41 APOE4 carriers), with baseline and follow-up amyloid-PET (time interval 6.1 (3.4–10.9) years). Individuals were genotyped using Illumina GSA and imputed. PRS were calculated using three p-value thresholds (pT) for variant inclusion: 5 × 10−8, 1 × 10−5, and 0.1, based on the stage 1 summary statistics from Kunkle et al. (Nat Genet 51:414–30, 2019). Linear regression models determined if these PRS predicted amyloid accumulation.ResultsA score based on PRS excluding the APOE region at pT = 5 × 10−8 plus the weighted sum of the two major APOE variants (rs429358 and rs7412) was significantly associated with amyloid accumulation (p = 0.0126). The two major APOE variants were also significantly associated with amyloid accumulation (p = 0.0496). The other PRS were not significant.ConclusionsSpecific PRS are associated with amyloid accumulation in the asymptomatic phase of AD.

APOE Alleles With Tau and Aβ Pathology in Patients With Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex in the Kii Peninsula.

To examine the association of the APOE ε4 and ε2 alleles with the pathologic features of patients with amyotrophic lateral sclerosis and parkinsonism-dementia complex cases in the Kii peninsula of Japan (Kii ALS/PDC). We analyzed APOE variants in 18 autopsy patients with ALS/PDC, consisting of 9, 8, and 1 patient with PDC, ALS, and PDC followed by ALS, respectively. Moreover, we revealed the relationship between APOE variants and Aβ and tau pathologies. The frequency of the ε4 allele was not different between patients with Kii ALS/PDC and control participants. APOE ε4 was associated with increased Aβ pathology (p = 0.005 by the χ 2 test), but not with increased tau pathology (p = 0.984). The frequency of the ε2 allele was apparently higher than that of control participants (p = 0.254). The APOE ε2 allele was associated with increased tau pathology (p = 0.009) and not with reduced Aβ pathology (p = 0.383) in patients with Kii ALS/PDC. Although there was no overrepresentation of the frequency of the ε4 or ε2 allele, our findings suggest that the ε2 allele is associated with increased tau pathology and not with reduced Aβ pathology in patients with Kii ALS/PDC.

Common human genetic variants of APOE impact murine COVID-19 mortality.

Clinical outcomes of severe acute respiratory syndrome 2 (SARS-CoV-2) infection are highly heterogeneous, ranging from asymptomatic infection to lethal coronavirus disease 2019 (COVID-19). The factors underlying this heterogeneity remain insufficiently understood. Genetic association studies have suggested that genetic variants contribute to the heterogeneity of COVID-19 outcomes, but the underlying potential causal mechanisms are insufficiently understood. Here we show that common variants of the apolipoprotein E (APOE) gene, homozygous in approximately 3% of the world's population1 and associated with Alzheimer's disease, atherosclerosis and anti-tumour immunity2-5, affect COVID-19 outcome in a mouse model that recapitulates increased susceptibility conferred by male sex and advanced age. Mice bearing the APOE2 or APOE4 variant exhibited rapid disease progression and poor survival outcomes relative to mice bearing the most prevalent APOE3 allele. APOE2 and APOE4 mice exhibited increased viral loads as well as suppressed adaptive immune responses early after infection. In vitro assays demonstrated increased infection in the presence of APOE2 and APOE4 relative to APOE3, indicating that differential outcomes are mediated by differential effects of APOE variants on both viral infection and antiviral immunity. Consistent with these in vivo findings in mice, our results also show that APOE genotype is associated with survival in patients infected with SARS-CoV-2 in the UK Biobank (candidate variant analysis, P = 2.6 × 10-7). Our findings suggest APOE genotype to partially explain the heterogeneity of COVID-19 outcomes and warrant prospective studies to assess APOE genotyping as a means of identifying patients at high risk for adverse outcomes.

Polygenic Scores of Alzheimer's Disease Risk Genes Add Only Modestly to APOE in Explaining Variation in Amyloid PET Burden.

Brain accumulation of amyloid-β is a hallmark event in Alzheimer's disease (AD) whose underlying mechanisms are incompletely understood. Case-control genome-wide association studies have implicated numerous genetic variants in risk of clinically diagnosed AD dementia. To test for associations between case-control AD risk variants and amyloid PET burden in older adults, and to assess whether a polygenic measure encompassing these factors would account for a large proportion of the unexplained variance in amyloid PET levels in the wider population. We analyzed data from the Mayo Clinic Study of Aging (MCSA) and the Alzheimer's Disease Neuroimaging Initiative (ADNI). Global cortical amyloid PET burden was the primary outcome. The 38 gene variants from Wightman et al. (2021) were analyzed as predictors, with PRSice-2 used to assess the collective phenotypic variance explained. Known AD risk variants in APOE, PICALM, CR1, and CLU were associated with amyloid PET levels. In aggregate, the AD risk variants were strongly associated with amyloid PET levels in the MCSA (p = 1.51×10-50) and ADNI (p = 3.21×10-64). However, in both cohorts the non-APOE variants uniquely contributed only modestly (MCSA = 2.1%, ADNI = 4.4%) to explaining variation in amyloid PET levels. Additional case-control AD risk variants added only modestly to APOE in accounting for individual variation in amyloid PET burden, results which were consistent across independent cohorts with distinct recruitment strategies and subject characteristics. Our findings suggest that advancing precision medicine for dementia may require integration of strategies complementing case-control approaches, including biomarker-specific genetic associations, gene-by-environment interactions, and markers of disease progression and heterogeneity.

Mercury Ion Binding to Apolipoprotein E Variants ApoE2, ApoE3, and ApoE4: Similar Binding Affinities but Different Structure Induction Effects.

Mercury intoxication typically produces more severe outcomes in people with the APOE-ε4 gene, which codes for the ApoE4 variant of apolipoprotein E, compared to individuals with the APOE-ε2 and APOE-ε3 genes. Why the APOE-ε4 allele is a risk factor in mercury exposure remains unknown. One proposed possibility is that the ApoE protein could be involved in clearing of heavy metals, where the ApoE4 protein might perform this task worse than the ApoE2 and ApoE3 variants. Here, we used fluorescence and circular dichroism spectroscopies to characterize the in vitro interactions of the three different ApoE variants with Hg(I) and Hg(II) ions. Hg(I) ions displayed weak binding to all ApoE variants and induced virtually no structural changes. Thus, Hg(I) ions appear to have no biologically relevant interactions with the ApoE protein. Hg(II) ions displayed stronger and very similar binding affinities for all three ApoE isoforms, with KD values of 4.6 μM for ApoE2, 4.9 μM for ApoE3, and 4.3 μM for ApoE4. Binding of Hg(II) ions also induced changes in ApoE superhelicity, that is, altered coil–coil interactions, which might modify the protein function. As these structural changes were most pronounced in the ApoE4 protein, they could be related to the APOE-ε4 gene being a risk factor in mercury toxicity.

PILRA polymorphism modifies the effect of APOE4 and GM17 on Alzheimer’s disease risk

PILRA (rs1859788 A > G) has been suggested to be a protective variant for Alzheimer’s disease (AD) and is an entry co-receptor for herpes simplex virus-1. We conducted a nested case–control study of 360 1:1-matched AD subjects. Interactions between the PILRA-A allele, APOE risk variants (ε3/ε4 or ε4/ε4) and GM17 for AD risk were modelled. The associations were cross-validated using two independent whole-genome sequencing datasets. We found negative interactions between PILRA-A and GM17 (OR 0.72, 95% CI 0.52–1.00) and between PILRA-A and APOE risk variants (OR 0.56, 95% CI 0.32–0.98) in the discovery dataset. In the replication cohort, a joint effect of PILRA and PILRA × GM 17/17 was observed for the risk of developing AD (p .02). Here, we report a negative effect modification by PILRA on APOE and GM17 high-risk variants for future AD risk in two independent datasets. This highlights the complex genetics of AD.

Ketogenic diets exacerbating hyperlipidemia in APOE variants

Background and Aims : APOE4 variants are one of the known causes of hypercholesterolemia. We present two patients with markedly elevated LDL suspicious for familial hypercholesterolemia who were subsequently diagnosed with APOE3/4 variants while on ketogenic diets.

Mutations in SORL1 and MTHFDL1 possibly contribute to the development of Alzheimer's disease in a multigenerational Colombian Family.

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, affecting over 50 million people worldwide in 2020 and this number will triple to 152 million by 2050. Much of the increase will be in developing countries like Colombia. In familial forms, highly penetrant mutations have been identified in three genes, APP, PSEN1, and PSEN2, supporting a role for amyloid-β peptide. In sporadic forms, more than 30 risk genes involved in the lipid metabolism, the immune system, and synaptic functioning mechanisms. We used whole-exome sequencing (WES) to evaluate a family of 97 members, spanning three generations, with a familiar AD, and without mutations in APP, PSEN1, or PSEN2. We sequenced two affected and one unaffected member with the aim of identifying genetic variants that could explain the presence of the disease in the family and the candidate variants were validated in eleven members. We also built a structural model to try to determine the effect on protein function. WES analysis identified two rare variants in SORL1 and MTHFD1L genes segregating in the family with other potential risk variants in APOE, ABCA7, and CHAT, suggesting an oligogenic inheritance. Additionally, the structural 3D models of SORL1 and MTHFD1L variants shows that these variants produce polarity changes that favor hydrophobic interactions, resulting in local structural changes that could affect the protein function and may contribute to the development of the disease in this family.

Genetic Variants at the APOE Locus Predict Cardiometabolic Traits and Metabolic Syndrome: A Taiwan Biobank Study.

Several apolipoprotein genes are located at the APOE locus on chromosome 19q13.32. This study explored the genetic determinants of cardiometabolic traits and metabolic syndrome at the APOE locus in a Taiwanese population. A total of 81,387 Taiwan Biobank (TWB) participants were enrolled to undergo genotype–phenotype analysis using data from the Axiom Genome-Wide CHB arrays. Regional association analysis with conditional analysis revealed lead single-nucleotide variations (SNVs) at the APOE locus: APOE rs7412 and rs429358 for total, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol levels; CLPTM1 rs3786505 and rs11672748 for LDL and HDL cholesterol levels; and APOC1 rs438811 and APOE-APOC1 rs439401 for serum triglyceride levels. Genotype–phenotype association analysis revealed a significant association of rs429358 and rs438811 with metabolic syndrome and of rs7412, rs438811, and rs439401 with serum albumin levels (p < 0.0015). Stepwise regression analysis indicated that CLPTM1 variants were independently associated with LDL and HDL cholesterol levels (p = 3.10 × 10−15 for rs3786505 and p = 1.48 × 10−15 for rs11672748, respectively). APOE rs429358 and APOC1 rs438811 were also independently associated with metabolic syndrome (p = 2.29 × 10−14) and serum albumin levels (p = 3.80 × 10−6), respectively. In conclusion, in addition to APOE variants, CLPTM1 is a novel candidate locus for LDL and HDL cholesterol levels at the APOE gene region in Taiwan. Our data also indicated that APOE and APOC1 variants were independently associated with metabolic syndrome and serum albumin levels, respectively. These results revealed the crucial role of genetic variants at the APOE locus in predicting cardiometabolic traits and metabolic syndrome.

*Ketogenic Diets Exacerbating Hypercholesterolemia in APOE Variants and APOB Mutations-Potential Role for Measuring Intestinal Absorption of Lipids

Lead Author's Financial Disclosures Nothing to disclose. Study Funding None. Background/Synopsis Familial Hypercholesterolemia (FH) is caused primarily by mutations in either LDL receptor, APOB, or PCSK9 genes. There are other genetic causes of LDL elevation including APOE4 variants. Objective/Purpose We present two patients with markedly elevated LDL suspicious for FH who were subsequently diagnosed with APOE3/4 variants while on ketogenic diets. We also present a patient with a known APOB mutation who had a marked elevation of LDL on a ketogenic diet. Methods Case 1: 58-year-old male with no prior medical history was found to have elevated cholesterol of 600mg/dL and LDL>455mg/dL while on a ketogenic diet. Prior to initiating the diet, his cholesterol was 206mg/dL and LDL was 132mg/dL. Genetic testing revealed a ApoE3/4 variant. His most recent cholesterol increased to 706mg/dL and LDL to >500mg/dL. Case 2: 53-year-old male with hypertension cholesterol of 282mg/dL and LDL of 212mg/dL went on a ketogenic diet and lost 100lbs. Follow up lipid panel demonstrated a cholesterol of 400mg/dL and LDL of 317mg/dL. Genetic testing revealed a APOE3/4 variant. Case 3: 45-year-old female with FH and known APOB missense mutation went on a ketogenic diet for weight loss. Prior to initiating the diet, her total cholesterol was 311 mg/dL and LDL was 211 mg/dL. While on the ketogenic diet, her total cholesterol was 550 mg/dL and LDL was 454 mg/dL. Her LDL decreased to 276mg/dl on a less strict ketogenic diet. Results All three patients had a normal exercise stress test, normal carotid duplex ultrasound, and coronary calcium score of zero. They refused the initiation of a statin. All 3 patients had the Boston Heart Cholesterol Balance Test performed that directly measures the absorption markers (beta-sitosterol, campesterol and cholestanol) for circulating plasma cholesterol. Only the patient with the APOB mutation had increased intestinal absorption of cholesterol, suggesting a response to ezetimibe. She agreed to initiate ezetimibe, her LDL subsequently decreased by 52% to 132mg/dl. Conclusions We hypothesize patients with APOE variants and APOB mutations could have an exaggerated elevation of their LDL levels while on ketogenic diets. Although these 3 patients did not demonstrate progressive atherosclerosis, the long-term effect of this diet is unknown. We recommend that all patients on a ketogenic diet have their LDL monitored and if there is a significant increase, undergo genetic testing for FH, APOE and other genetic variants. Since this patient group is often reluctant to start statins, the Cholesterol Balance Test may be useful to identify patients who may respond well to ezetimibe. Nothing to disclose.

RNA Hypomethylation and Unchanged DNA Methylation Levels in the Cortex of ApoE4 Carriers and Alzheimer's Disease Subjects.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, and ApoE4 variants are significant risk factors for AD. Epigenetic modifications are involved in AD pathology. However, it is unclear whether DNA/RNA methylation plays a role in AD pathology, and dysregulation of DNA/RNA methylation occurs in ApoE4 carriers. The present study aimed to determine whether dysregulation of DNA/RNA methylation is present in the brains of ApoE4 carriers and AD patients. In this study, postmortem brain tissues from carriers of ApoE4 and ApoE3, from AD and non- AD controls, were used in the analysis of DNA/RNA methylation, methyltransferases, and their demethylases. Immunofluorescence staining indicates that RNA methylation is suppressed in ApoE4 carriers. Further analysis shows that the expression of RNA methyltransferases and an RNA methylation reader is suppressed in ApoE4 carriers, whereas RNA demethylase expression is increased. RNA hypomethylation occurs in NeuN+ neurons in ApoE4 carriers and AD patients. Furthermore, in ApoE4 carriers, both DNA methyltransferases and demethylases are downregulated, and overall DNA methylation levels are unchanged. Our finding indicates that RNA methylation decreased in ApoE4 carriers before AD pathology and AD individuals. The expression of RNA methyltransferases and RNA methylation reader is inhibited, and RNA demethylase is upregulated in ApoE4 carriers, which leads to suppression of RNA methylation, and the suppression precedes the AD pathogenesis and persists through AD pathology.

APOE Molecular Spectrum in a French Cohort with Primary Dyslipidemia

Primary hypercholesterolemia is characterized by elevated LDL-cholesterol (LDL-C) levels isolated in autosomal dominant hypercholesterolemia (ADH) or associated with elevated triglyceride levels in familial combined hyperlipidemia (FCHL). Rare APOE variants are known in ADH and FCHL. We explored the APOE molecular spectrum in a French ADH/FCHL cohort of 5743 unrelated probands. The sequencing of LDLR, PCSK9, APOB, and APOE revealed 76 carriers of a rare APOE variant, with no mutation in LDLR, PCSK9, or APOB. Among the 31 APOE variants identified here, 15 are described in ADH, 10 in FCHL, and 6 in both probands. Five were previously reported with dyslipidemia and 26 are novel, including 12 missense, 5 synonymous, 2 intronic, and 7 variants in regulatory regions. Sixteen variants were predicted as pathogenic or likely pathogenic, and their carriers had significantly lower polygenic risk scores (wPRS) than carriers of predicted benign variants. We observed no correlation between LDL-C levels and wPRS, suggesting a major effect of APOE variants. Carriers of p.Leu167del were associated with a severe phenotype. The analysis of 11 probands suggests that carriers of an APOE variant respond better to statins than carriers of a LDLR mutation. Altogether, we show that the APOE variants account for a significant contribution to ADH and FCHL.