Variation In Apolipoprotein Research Articles

TOMM40-APOE chimera linking Alzheimer's highest risk genes: a new pathway for mitochondria regulation and APOE4 pathogenesis.

The patho-mechanism of apolipoprotein variant, APOE4, the strongest genetic risk for late-onset Alzheimer's disease (AD) and longevity, remains unclear. APOE's neighboring gene, TOMM40 (mitochondria protein transport channel), is associated with brain trauma outcome and aging-related cognitive decline, however its role in AD APOE4-independently is controversial. We report that TOMM40 is prone to transcription readthrough into APOE that can generate spliced TOMM40-APOE mRNA chimera (termed T9A2) detected in human neurons and other cells and tissues. T9A2 translation tethers APOE (normal APOE3 or APOE4) to near-full-length TOM40 that is targeted to mitochondria. Importantly, T9A2-APOE3 boosts mitochondrial bioenergetic capacity and decreases oxidative stress significantly more than T9A2-APOE4 and APOE3, and lacking in APOE4. We describe detailed interactomes of these actors that may inform about the activities and roles in pathogenesis. T9A2 uncovers a new candidate pathway for mitochondria regulation and oxidative stress-protection that are impaired in APOE4 genotypes and could initiate neurodegeneration.

A Review on the Role of SNCA Gene in Neurodegenerative Diseases.

Gene variations significantly impact the development of neurodegenerative disorders, particularly Alzheimer's disease (AD) and Parkinson's disease (PD). In AD, which is marked by amyloid-beta (Aβ) plaques and tau tangles, key genetic contributors such as amyloid beta precursor protein (APP), presenilin(PSEN1), and presenilin 2(PSEN2)play a significant role in early-onset familial AD due to their influence on Aβ accumulation. PD, marked by dopaminergic neuron degeneration and Lewy body formation, is associated with mutations in theSNCAgene encoding alpha-synuclein(α-Syn), as well as other genes such as leucine-rich repeat kinase 2(LRRK2), ParkinRBR E3 ubiquitin-protein ligase(PARK2), PTEN-induced kinase 1(PINK1), and protein deglycase(DJ-1). Genome-wide association studies have identified genetic variants in apolipoprotein(APOE)andSNCAthat increase disease risk.Alpha-synuclein, a protein involved in synaptic function, misfolds and aggregates into toxic forms in neurodegenerative diseases. Aggregates disrupt neuronal functions and propagate in a prion-like manner, withSNCAmutations exacerbatingα-Synaggregation and disease severity.Alpha-synucleinlevels in skin, serum, cerebrospinal fluid, and plasma distinguish PD patients from healthy patients, demonstrating biomarker potential for diagnosis and therapeutic strategies. Furthermore,α-Syn'spresence in neural crest-derived tissues from PD patients and melanoma patients suggests shared pathophysiological features. Ongoing research intoSNCAandα-Synis crucial for advancing diagnostics and therapeutics for neurodegenerative diseases.

Characterization of the interindividual variability of lutein and zeaxanthin concentrations in the adipose tissue of healthy male adults and identification of combinations of genetic variants associated with it.

Lutein (L) and zeaxanthin (Z) are involved in visual function and could prevent age-related macular degeneration and chronic diseases and improve cognitive performances. Adipose tissue is the main storage site for these xanthophylls (Xanth). The factors affecting their concentrations in this tissue remain poorly understood but in animal models, genetic variations in apolipoprotein E and β-carotene oxygenase 2 have been associated with adipose tissue L concentration. Therefore, the aims of this study were to better characterize the interindividual variability of adipose tissue Xanth concentration and to identify single nucleotide polymorphisms (SNPs) associated with it. Periumbilical subcutaneous adipose tissue samples were collected on 6 occasions in 42 healthy adult males and L and Z concentrations were measured by HPLC. Participants had their whole genome genotyped and the associations of 3589 SNPs in 49 candidate genes with the concentrations of L and Z were measured. Mean L and Z concentrations were 281 ± 27 and 150 ± 14 nmol g-1 proteins, respectively. There was no significant correlation between plasma and adipose tissue Xanth concentrations, although the correlation for L approached significance (Pearson's r = 0.276, p = 0.077). Following univariate filtering, 109 and 97 SNPs were then entered into a partial least squares regression analysis to identify the combination of SNPs that explained best adipose tissue concentration of L and Z, respectively. A combination of 7 SNPs in ELOVL5, PPARG, ISX and ABCA1, explained 58% of the variability in adipose tissue L concentration while 11 SNPs located in or near PPARG, ABCA1, ELOVL5, CXCL8, IRS1, ISX, MC4R explained 53% of the variance in adipose tissue Z concentration. This suggests that some genetic variations influence the concentrations of these Xanth in adipose tissue and could therefore indirectly influence the health effects of these compounds. Clinical Trial Registry: https://ClinicalTrials.gov registration number NCT02100774.

Apolipoprotein E Receptor 2 is associated with Alzheimer’s disease pathogenesis

AbstractBackgroundGenetic variations to apolipoprotein E (apoE) underlie the strongest risk for Alzheimer´s disease (AD) after aging but it is not yet known how apoE confers risk for AD. While most research is focusing on dysregulations associated with the presence of the risk allele of apoE, several aspects of apoE biology remain poorly understood. In particular, the physiological relevance of apoE interaction with members of the low‐density lipoprotein (LDL) receptor gene family is not well understood.MethodFerroptosis was induced in the N27 neuronal cell model of ferroptosis using multiple agents (erastin, RSL3, iron, cysteine depletion). Genetic variants of apolipoprotein E receptor 2 (apoER2) were generated via site‐directed mutagenesis and expressed in N27 neurons. The severity of ferroptosis was measured by several toxicity assays (MTT, LDH) and lipid peroxide probe (C11BODIPY). The association between apoER2 polymorphism and cognition were investigated in two longitudinal studies: the Alzheimer’s Disease Neuroimaging Initiative (ADNI) and the Australian Imaging Biomarkers and Lifestyle Study of Ageing (AIBL).ResultWe recently reported that apoE is a potent inhibitor of ferroptosis in neuronal cells in a mechanism that involves a receptor‐mediated activation of the PI3K/AKT signaling pathway. In this study, we identified genetic variants of apoER2 (also known as LRP8, a member of the LDL receptor family) that were associated with slower cognitive decline in clinical cohorts of AD. In preliminary work, we show that one apoER2 variant was associated with an increased protection from ferroptosis in an apoE‐dependent process.ConclusionWe present evidence for a biological role of apoER2 and its genetic variation on pathologies associated with Alzheimer’s disease, which may help develop new diagnostic and therapeutic approaches towards the disease.

A Novel ELISA for the Quantification of Serum Levels of 2 Glycosylated Variants of Apolipoprotein J: Biomarkers for Myocardial Ischemia.

Previous studies have pointed out a potential role of ApoJ-Glyc as a biomarker of cardiac ischemia. The aim of this study was to validate the analytical performance of 2 novel ELISAs against 2 different glycosylated ApoJ variants, ApoJ-GlycA2 and ApoJ-GlycA6. The analytical measuring range, limit of blank (LoB), lower limit of quantification (LoQ), precision, accuracy, recovery, cross-reactivity, and stability were evaluated in serum samples. The analytical measuring range was 500-16 000 ng/mL for ApoJ-GlycA2 and 125-4000 ng/mL for ApoJ-GlycA6, with a LoB of 455 ng/mL and 121 ng/mL for ApoJ-GlycA2 and ApoJ-GlycA6, respectively. The LoQ was 500 ng/mL for ApoJ-GlycA2 and 125 ng/mL for ApoJ-GlycA6. The assay performance fulfills the acceptance criteria established in the European Medicines Agency Guideline on bioanalytical method validation. Specifically, the calibration range variability is <15% for ApoJ-GlycA2 and ApoJ-GlycA6; the accuracy is <15% for ApoJ-GlycA2 and ApoJ-GlycA6; the between- and within-run precision is <15% for ApoJ-GlycA6 and ≤20% for ApoJ-GlycA2; and the total allowable error is <30% for ApoJ-GlycA2 and ApoJ-GlycA6. Cross-reactivity studies revealed the absence of cross-reactivity with endogenous components of the matrix (using ApoJ-depleted serum), with nonglycosylated ApoJ and with transferrin (as a high abundant N-glycosylated serum protein). Both ApoJ-GlycA2 and ApoJ-GlycA6 measurements were stable after storage of serum samples at -80°C for 24 months. The newly developed ELISAs to quantify ApoJ-GlycA2 and ApoJ-GlycA6 serum levels showed an acceptable analytical performance according to European Medicines Agency guidelines on bioanalytical method validation in terms of precision, accuracy, recovery, cross-reactivity, and stability.

Updated APOE and TOMM40 rs2075650 genotype effects on cognition: a UK Biobank study

AbstractBackground58‐79% of late‐onset Alzheimer’s disease (AD) is due to genetic heritability. The epsilon 4 (ε4) variant of Apolipoprotein E (APOE) is the strongest genetic risk factor for AD, yet only accounts for 20% of AD heritability. In addition, the APOE ε2 or ε3 variants appear to have either a protective or neutral effect on AD risk respectively. Translocase of Outer Mitochondrial Membrane 40 (TOMM40) is next to APOE and may modulate different APOE variant effects, such as with the Single Nucleotide Polymorphism (SNP) at rs2075650. Thus, we explored main and interaction effects of TOMM40 and APOE on longitudinal measures of executive function and memory.Method10,347 adults (40‐70y) in the UK Biobank were genotyped for APOE and TOMM40 ‘650. Executive function and memory were tested at baseline and twice over a period of 6‐10 years. Main effects within APOE haplotypes and TOMM40 SNP genotypes (A,G) were analyzed, as well as interactions between these genetic markers.ResultHaving the TOMM40 ‘650 G risk variant was related to better baseline cognitive scores regardless of APOE haplotype (ps &lt;.05), but a greater decline over time in APOE4 carriers (ps &lt;.05) The effects of the APOE haplotype were strongly modulated by sex. Men vs. women had less protective or more detrimental effects based on APOE2 or APOE4 haplotype respectively.ConclusionThe data suggests that TOMM40 modulates APOE, regardless of its haplotype. Future studies should stratify APOE by haplotype to re‐test our findings. In addition, more research needs to be done on the effect of having what we term a “mixed” APOE haplotype (carrying a copy of both the protective and risk gene variants).

Reduced binding of apoE4 to complement factor H promotes amyloid‐β oligomerization and neuroinflammation

The APOE4 variant of apolipoprotein E (apoE) is the most prevalent genetic risk allele associated with late‐onset Alzheimer's disease (AD). ApoE interacts with complement regulator factor H (FH), but the role of this interaction in AD pathogenesis is unknown. Here we elucidate the mechanism by which isoform‐specific binding of apoE to FH alters Aβ1‐42‐mediated neurotoxicity and clearance. Flow cytometry and transcriptomic analysis reveal that apoE and FH reduce binding of Aβ1‐42 to complement receptor 3 (CR3) and subsequent phagocytosis by microglia which alters expression of genes involved in AD. Moreover, FH forms complement‐resistant oligomers with apoE/Aβ1‐42 complexes and the formation of these complexes is isoform specific with apoE2 and apoE3 showing higher affinity to FH than apoE4. These FH/apoE complexes reduce Aβ1‐42 oligomerization and toxicity, and colocalize with complement activator C1q deposited on Aβ plaques in the brain. These findings provide an important mechanistic insight into AD pathogenesis and explain how the strongest genetic risk factor for AD predisposes for neuroinflammation in the early stages of the disease pathology.

APOBEC3A/B Polymorphism Is Not Associated with Human Papillomavirus Infection and Cervical Carcinogenesis.

The persistence of a high-risk Human papillomavirus (HPV-HR) infection of the cervix results in different manifestations of lesions depending on the immunologic capacity of the host. Variations in apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC)-like genes, such as the APOBEC3A/B deletion hybrid polymorphism (A3A/B), may contribute to cervical malignancy in the presence of HPV. The aim of this study was to investigate the association between the A3A/B polymorphism and HPV infection and the development of cervical intraepithelial lesions and cervical cancer in Brazilian women. The study enrolled 369 women, who were categorized according to the presence of infection and subdivided according to the degree of intraepithelial lesion and cervical cancer. APOBEC3A/B was genotyped by allele-specific polymerase chain reaction (PCR). As for the A3A/B polymorphism, the distribution of genotypes was similar between groups and among the analyzed subgroups. There were no significant differences in the presence of infection or development of lesions, even after exclusion of confounding factors. This is the first study to show that the A3A/B polymorphism is not associated with HPV infection and the development of intraepithelial lesions and cervical cancer in Brazilian women.

Hypoxia hits APOL1 in the kidney

Individuals of African ancestry carrying two pathogenic variants of apolipoprotein 1 (APOL1) have a substantially increased risk for developing chronic kidney disease. The course of APOL1 nephropathy is extremely heterogeneous and shaped by systemic factors such as a response to interferon. However, additional environmental factors operating in this second-hit model have been less well defined. Here, we reveal that stabilization of hypoxia-inducible transcription factors (HIF) by hypoxia or HIF prolyl hydroxylase inhibitors activates transcription of APOL1 in podocytes and tubular cells. An active regulatory DNA-element upstream of APOL1 that interacted with HIF was identified. This enhancer was accessible preferentially in kidney cells. Importantly, upregulation of APOL1 by HIF was additive to the effects of interferon. Furthermore, HIF stimulated expression of APOL1 in tubular cells derived from the urine of an individual carrying a risk variant for kidney disease. Thus, hypoxic insults may serve as important modulators of APOL1 nephropathy.

Association of candidate genetic variants and circulating levels of ApoE/ApoJ with common neuroimaging features of cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of amyloid-β (Aβ) in brain vessels and is a main cause of lobar intracerebral hemorrhage (ICH) in the elderly. CAA is associated with magnetic resonance imaging (MRI) markers of small vessel disease (SVD). Since Aβ is also accumulated in Alzheimer's disease (AD) in the brain parenchyma, we aimed to study if several single nucleotide polymorphisms (SNPs) previously associated with AD were also associated with CAA pathology. Furthermore, we also studied the influence of APOE and CLU genetic variants in apolipoprotein E (ApoE) and clusterin/apolipoprotein J (ApoJ) circulating levels and their distribution among lipoproteins. The study was carried out in a multicentric cohort of 126 patients with lobar ICH and clinical suspicion of CAA. We observed several SNPs associated with CAA neuroimaging MRI markers [cortical superficial siderosis (cSS), enlarged perivascular spaces in the centrum semiovale (CSO-EPVS), lobar cerebral microbleeds (CMB), white matter hyperintensities (WMH), corticosubcortical atrophy and CAA-SVD burden score]. Concretely, ABCA7 (rs3764650), CLU (rs9331896 and rs933188), EPHA1 (rs11767557), and TREML2 (rs3747742) were significantly associated with a CAA-SVD burden score. Regarding circulating levels of apolipoproteins, protective AD SNPs of CLU [rs11136000 (T) and rs9331896 (C)] were significantly associated with higher HDL ApoJ content in the lobar ICH cohort. APOEε2 carriers presented higher plasma and LDL-associated ApoE levels whereas APOEε4 carriers presented lower plasma ApoE levels. Additionally, we observed that lower circulating ApoJ and ApoE levels were significantly associated with CAA-related MRI markers. More specifically, lower LDL-associated ApoJ and plasma and HDL-associated ApoE levels were significantly associated with CSO-EPVS, lower ApoJ content in HDL with brain atrophy and lower ApoE content in LDL with the extent of cSS. This study reinforces the relevance of lipid metabolism in CAA and cerebrovascular functionality. We propose that ApoJ and ApoE distribution among lipoproteins may be associated with pathological features related to CAA with higher ApoE and ApoJ levels in HDL possibly enhancing atheroprotective, antioxidative, and anti-inflammatory responses in cerebral β-amyloidosis.

Current allele distribution of the human longevity gene APOE in Europe can mainly be explained by ancient admixture.

Variation in apolipoprotein E (APOE) has been shown to have the strongest genetic effect on human longevity. The aim of this study was to unravel the evolutionary history of the three major APOE alleles in Europe by analysing ancient samples up to 12,000 years old. We detected significant allele frequency shifts between populations and over time. Our analyses indicated that selection led to large frequency differences between the earliest European populations (i.e., hunter-gatherers vs. first farmers), possibly due to changes in diet/lifestyle. In contrast, the allele distributions in populations from ~4000 BCE onward can mainly be explained by admixture, suggesting that it also played an important role in shaping current APOE variation. In any case, the resulting allele frequencies strongly influence the predisposition for longevity today, likely as a consequence of past adaptations and demographic processes.

Association of APOE (rs429358 and rs7412) and PON1 (Q192R and L55M) Variants with Myocardial Infarction in the Pashtun Ethnic Population of Khyber Pakhtunkhwa, Pakistan.

Coronary Artery Diseases (CAD) remains the top among Non-communicable Diseases (NCDs). Variations in Apolipoprotein E (APOE) and Paroxonase 1 (PON1) have been associated with Myocardial Infarction (MI) in several populations. However, despite the high prevalence of CAD, no such study has been reported in the Pashtun ethnic population of Pakistan. We have conducted a two-stage (i.e., screening and validation) case-control study in which 200 cases and 100 control subjects have been recruited. In the first stage, Whole Exome Sequencing (WES) was used to screen for pathogenic variants of Myocardial Infarction (MI). In the second stage, selected variants of both APOE and PON1 genes (rs7412, rs429358, rs854560, and rs662) were analyzed through MassARRAY genotyping. Risk Allele Frequencies (RAFs) distribution and association of the selected SNPs with MI were determined using the Chi-square test and logistic regression analysis. WES identified a total of 12 sequence variants in APOE and 16 in PON1. Genotyping results revealed that APOE variant rs429358 (ɛ4 allele and ɛ3/ɛ4 genotype) showed significant association in MI patients (OR = 2.11, p value = 0.03; 95% CI = 1.25-2.43); whereas no significant difference (p˃ 0.05) was observed for rs7412. Similarly, the R allele of PON1 Q192R (rs662) was significantly associated with cases (OR = 1.353, p value = 0.048; 95% CI = 0.959-1.91), with particular mention of RR genotype (OR = 1.523, p value = 0.006; 95% CI = 1.087-2.132). Multiple logistic regression analysis showed that rs429358 (C allele) and rs662 (R allele) have a significantly higher risk of MI after adjustment for the conventional risk factors. Our study findings suggested that the rs429358 variant of APOE and PON1 Q192R are associated with MI susceptibility in the Pashtun ethnic population of Pakistan.

Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms

The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer's disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms largely because the data are potentially confounded by the presence of heterogeneous oligomers. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogeneous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Overall, these data provide a foundation for understanding how ApoE4 differs from nonpathogenic and protective variants of the protein.

Single-molecule spectroscopy of Apolipoprotein E reveals a complex conformational ensemble.

The ε4-allele variant of Apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer's disease, though it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of its pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms, largely due to the potential contribution of data representing heterogenous oligomers in solution. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically-detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. We further investigate the impact of key mutations by inserting serine residues that mimic the ApoE3 and ApoE2 isoform sequence differences, C112S and C112S C158S, respectively. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogenous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Inspection of local conformational changes across different domains, in the context of the full-length protein, reveals that the major difference across isoforms occurs in the conformations adopted by the C-terminal tail. Overall, these data provide a new foundation for understanding how ApoE differs from non-pathogenic and protective variants of the protein.

Predictive link between systemic metabolism and cytokine signatures in the brain of apolipoprotein E ε4 mice

The ε4 variant of apolipoprotein E (APOE) is the strongest and most common genetic risk factor for Alzheimer's disease (AD). While the mechanism of conveyed risk is incompletely understood, promotion of inflammation, dysregulated metabolism, and protein misfolding and aggregation are contributors to accelerating disease. Here we determined the concurrent effects of systemic metabolic changes and brain inflammation in young (3-month-old) and aged (18-month-old) male and female mice carrying the APOE4 gene. Using functional metabolic assays alongside multivariate modeling of hippocampal cytokine levels, we found that brain cytokine signatures are predictive of systemic metabolic outcomes, independent of AD proteinopathies. Male and female mice each produce different cytokine signatures as they age and as their systemic metabolic phenotype declines, and these signatures are APOE genotype dependent. Ours is the first study to identify a quantitative and predictive link between systemic metabolism and specific pathological cytokine signatures in the brain. Our results highlight the effects of APOE4 beyond the brain and suggest the potential for bi-directional influence of risk factors in the brain and periphery.

Associations of Alzheimer Disease–Protective APOE Variants With Age-Related Macular Degeneration

The association of major lipid genes with and their potential as drug targets for age-related macular degeneration (AMD) is unknown. These associations are important to study because AMD is the leading cause of irreversible late-onset blindness in high-income countries. To determine whether the full range of structural genetic variation in apolipoprotein E (APOE), a master gene in peripheral and cerebral lipid metabolism, is associated with risk of AMD. This cohort study used data from the Copenhagen City Heart Study (CCHS) and the Copenhagen General Population Study (CGPS) cohorts. Participants were followed from study inclusion at the time of blood sampling to occurrence of event, death, emigration, or December 7, 2018, whichever came first. For participants in CCHS, the APOE gene was sequenced, and 9 variants with a heterozygote frequency of at least 0.0002 were genotyped in the CGPS. Observers were masked to patient groupings. Data were analyzed from March to September 2021. The exposure was APOE status, and the direct gene product in plasma, apoE levels, was measured in all participants. Cox regression was applied to estimate risk of AMD associated with APOE genotype. A total of 105 546 participants (mean [SD] age, 57.7 [13.4] years; 58 140 [55%] female participants) were included. Compared with participants with the common ɛ33 genotype, risk of AMD was lower in participants with ε44 (multifactorially adjusted hazard ratio [aHR], 0.66; 95% CI, 0.45-0.96) and ε43 (aHR, 0.80; 95% CI, 0.71-0.90) genotypes and higher in the ε32 (aHR, 1.15; 95% CI, 1.00-1.31) genotype. Compared with noncarriers, risk of AMD was higher for participants with Gly145Asp (aHR, 3.53; 95% CI, 1.14-10.96) and Arg154Cys (aHR, 4.52; 95% CI, 1-13-18.13) heterozygotes. Results were similar after further adjustment for lipid traits and after adjustment for the APOE ε2/ε3/ε4 variant. Combining all common and rare structural variants in a weighted allele score, risk of AMD per 1-mg/dL genetically higher plasma apoE was increased in the adjusted model (aHR, 1.12; 95% CI, 1.05-1.19), the adjusted model plus APOE ɛ2/ɛ3/ɛ4 status (aHR, 1.82; 95% CI, 1.20-2.76), and the adjusted model in individuals with the ε33 genotype only (aHR, 1.77; 95% CI, 1.14-2.75). These findings highlight that structural variation in APOE beyond the ε2/ε3/ε4 variants may be important for risk of AMD in a population of European ancestry. Rare functional ɛ2-like variants in APOE have previously been reported to have protective associations for Alzheimer disease but the present findings suggest a simultaneous high risk of AMD. This would limit the drug target potential of mechanisms resembling these variants.

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.

Development and validation of an isoform-independent monoclonal antibody–based ELISA for measurement of lipoprotein(a)

The study aims were to develop a new isoform-independent enzyme-linked immunoassay (ELISA) for the measurement of lipoprotein(a) [Lp(a)], validate its performance characteristics, and demonstrate its accuracy by comparison with the gold-standard ELISA method and an LC-MS/MS candidate reference method, both developed at the University of Washington. The principle of the new assay is the capture of Lp(a) with monoclonal antibody LPA4 primarily directed to an epitope in apolipoprotein(a) KIV2 and its detection with monoclonal antibody LPA-KIV9 directed to a single antigenic site present on KIV9. Validation studies were performed following the guidelines of the Clinical Laboratory Improvement Amendments and the College of American Pathologists. The analytical measuring range of the LPA4/LPA-KIV9 ELISA is 0.27–1,402 nmol/L, and the method meets stringent criteria for precision, linearity, spike and recovery, dilutability, comparison of plasma versus serum, and accuracy. Method comparison with both the gold-standard ELISA and the LC-MS/MS method performed in 64 samples with known apolipoprotein(a) isoforms resulted in excellent correlation with both methods (r=0.987 and r=0.976, respectively). Additionally, the variation in apolipoprotein(a) size accounted for only 0.2% and 2.2% of the bias variation, respectively, indicating that the LPA4/LPA-KIV9 ELISA is not affected by apolipoprotein(a) size polymorphism. Peptide mapping and competition experiments demonstrated that the measuring monoclonal antibodies used in the gold-standard ELISA (a-40) and in the newly developed ELISA (LPA-KIV9) are directed to the same epitope, 4076LETPTVV4082, on KIV9. In conclusion, no statistically or clinically significant bias was observed between Lp(a) measurements obtained by the LPA4/LPA-KIV9 ELISA and those obtained by the gold-standard ELISA or LC-MS/MS, and therefore, the methods are considered equivalent.

Analysis of plasma proteins using 2D gels and novel fluorescent probes: in search of blood based biomarkers for Alzheimer\u2019s disease

BackgroundThe Australian Imaging and Biomarker Lifestyle (AIBL) study of aging is designed to aid the discovery of biomarkers. The current study aimed to discover differentially expressed plasma proteins that could yield a blood-based screening tool for Alzheimer’s disease.MethodsThe concentration of proteins in plasma covers a vast range of 12 orders of magnitude. Therefore, to search for medium to low abundant biomarkers and elucidate mechanisms of AD, we immuno-depleted the most abundant plasma proteins and pre-fractionated the remaining proteins by HPLC, prior to two-dimensional gel electrophoresis. The relative levels of approximately 3400 protein species resolved on the 2D gels were compared using in-gel differential analysis with spectrally resolved fluorescent protein detection dyes (Zdyes™). Here we report on analysis of pooled plasma samples from an initial screen of a sex-matched cohort of 72 probable AD patients and 72 healthy controls from the baseline time point of AIBL.ResultsWe report significant changes in variants of apolipoprotein E, haptoglobin, α1 anti-trypsin, inter-α trypsin inhibitor, histidine-rich glycoprotein, and a protein of unknown identity. α1 anti-trypsin and α1 anti-chymotrypsin demonstrated plasma concentrations that were dependent on APOE ε4 allele dose. Our analysis also identified an association with the level of Vitamin D binding protein fragments and complement factor I with sex. We then conducted a preliminary validation study, on unique individual samples compared to the discovery cohort, using a targeted LC-MS/MS assay on a subset of discovered biomarkers. We found that targets that displayed a high degree of isoform specific changes in the 2D gels were not changed in the targeted MS assay which reports on the total level of the biomarker.ConclusionsThis demonstrates that further development of mass spectrometry assays is needed to capture the isoform complexity that exists in theses biological samples. However, this study indicates that a peripheral protein signature has potential to aid in the characterization of AD.

Apolipoprotein E Polymorphism, Cardiac Remodeling, and Heart Failure in the ARIC Study

Backgroundβ-Amyloid has recently been discovered in the myocardium of patients with Alzheimer's disease (AD). Whether genetic variation in apolipoprotein E (APOE) ɛ4, a common variant associated with Alzheimer's disease, is associated with incident heart failure (HF), N-terminal pro-B-type natriuretic peptide (NT-proBNP), and cardiac structure and function is unknown. Methods and ResultsWe studied 15,064 White and Black participants in the Atherosclerosis Risk in Communities, relating genotype status at visit 1 (1987–1989) to incident HF hospitalization using Cox regression. At visits 2, 4, and 5, we assessed NT-proBNP levels by genotype. At visits 3 and 5, we related Aβ peptides to incident HF. At visit 5 (2011–2013, n = 6251), we assessed the relationship of genotype with prevalent HF and echocardiographic parameters. The mean participant age was 54.7 ± 5.8 years, 45% were men, and 73% were White. At visit 5, there was no difference in prevalent HF by genotype. The APOE ε4 carriers did not have increased risk for HF hospitalization. The APOE ε4 genotype was not associated with cardiac structure and function or NT-proBNP levels. The Aβ peptides were not associated with incident HF after multivariable adjustment. ConclusionsA genetic predisposition to Alzheimer's disease through APOE ε4 is not associated with an increased prevalence of HF, HF hospitalization, myocardial remodeling, or biochemical evidence of HF.