Re-analysis and meta-analysis of summary statistics from gene-environment interaction studies.

MOTIVATION: statistics from genome-wide association studies enable many valuable downstream analyses that are more efficient than individual-level data analysis while also reducing privacy concerns. As growing sample sizes enable better-powered analysis of gene-environment interactions, there is a need for gene-environment interaction-specific methods that manipulate and use summary statistics. RESULTS: We introduce two tools to facilitate such analysis, with a focus on statistical models containing multiple gene-exposure and/or gene-covariate interaction terms. REGEM (RE-analysis of GEM summary statistics) uses summary statistics from a single, multi-exposure genome-wide interaction study to derive analogous sets of summary statistics with arbitrary sets of exposures and interaction covariate adjustments. METAGEM (META-analysis of GEM summary statistics) extends current fixed-effects meta-analysis models to incorporate multiple exposures from multiple studies. We demonstrate the value and efficiency of these tools by exploring alternative methods of accounting for ancestry-related population stratification in genome-wide interaction study in the UK Biobank as well as by conducting a multi-exposure genome-wide interaction study meta-analysis in cohorts from the diabetes-focused ProDiGY consortium. These programs help to maximize the value of summary statistics from diverse and complex gene-environment interaction studies. AVAILABILITY AND IMPLEMENTATION: REGEM and METAGEM are open-source projects freely available at https://github.com/large-scale-gxe-methods/REGEM and https://github.com/large-scale-gxe-methods/METAGEM.

Urinary sphingolipids in adolescents and young adults with youth-onset diabetes.

BACKGROUND: This study evaluated urinary sphingolipids as a marker of diabetic kidney disease (DKD) in adolescents and young adults with youth-onset type 1 and type 2 diabetes. METHODS: A comprehensive panel of urinary sphingolipids, including sphingomyelin (SM), glucosylceramide (GC), ceramide (Cer), and lactosylceramide (LC) species, was performed in patients with youth-onset diabetes from the SEARCH for Diabetes in Youth cohort. Sphingolipid levels, normalized to urine creatinine, were compared in 57 adolescents and young adults with type 1 diabetes, 59 with type 2 diabetes, and 44 healthy controls. The association of sphingolipids with albumin-to-creatinine (ACR) ratio and estimated glomerular filtration rate (eGFR) was evaluated. RESULTS: The median age (interquartile range [IQR]) of participants was 23.1 years (20.9, 24.9) and the median duration of diabetes was 9.3 (8.5, 10.2) years. Urinary sphingolipid concentrations in patients with and without DKD (ACR ≥ 30 mg/g) were significantly elevated compared to healthy controls. There were no significant differences in sphingolipid levels between participants with type 1 and type 2 diabetes. In multivariable analysis, many sphingolipid species were positively correlated with ACR. Most significant associations were evident for the following species: C18 SM, C24:1 SM, C24:1 GC, and C24:1 Cer (all p < 0.001). Sphingolipid levels were not associated with eGFR. However, several interaction terms (diabetes type*sphingolipid) were significant, indicating diabetes type may modify the association of sphingolipids with eGFR. CONCLUSION: Urinary sphingolipids are elevated in adolescents and young adults with youth-onset diabetes and correlate with ACR. Urinary sphingolipids may therefore represent an early biomarker of DKD.

Lipoprotein(a): a genetically determined risk factor for Cardiovascular disease.

Lipoprotein(a) is a complex lipoprotein with unique characteristics distinguishing it from all the other apolipoprotein B-containing lipoprotein particles. Its lipid composition and the presence of a single molecule of apolipoprotein B per particle, render lipoprotein(a) similar to low-density lipoproteins. However, the presence of a unique, carbohydrate-rich protein termed apolipoprotein(a), linked by a covalent bond to apolipoprotein B imparts unique characteristics to lipoprotein(a) distinguishing it from all the other lipoproteins. Apolipoprotein(a) is highly polymorphic in size ranging in molecular weight from <300 KDa to >800 kDa. Both the size polymorphism and the concentration of lipoprotein(a) in plasma are genetically determined and unlike other lipoproteins, plasma concentration is minimally impacted by lifestyle modifications or lipid-lowering drugs. Many studies involving hundreds of thousands of individuals have provided strong evidence that elevated lipoprotein(a) is genetically determined and a causal risk factor for atherosclerotic cardiovascular disease. The concentration attained in adulthood is already present in children at around 5 years of age and therefore, those with elevated lipoprotein(a) are prematurely exposed to a high risk of cardiovascular disease. Despite the large number of guidelines and consensus statements on the management of lipoprotein(a) in atherosclerotic cardiovascular disease published in the last decade, lipoprotein(a) is still seldom measured in clinical settings. In this review, we provide an overview of the most important features that characterize lipoprotein(a), its role in cardiovascular disease, and the importance of adding the measurement of lipoprotein(a) for screening adults and youths to identify those at increased risk of atherosclerotic cardiovascular disease due to their elevated plasma concentration of lipoprotein(a).

Apo(a) and ApoB Interact Noncovalently Within Hepatocytes: Implications for Regulation of Lp(a) Levels by Modulation of ApoB Secretion.

BACKGROUND: Elevated plasma Lp(a) (lipoprotein(a)) levels are associated with increased risk for atherosclerotic cardiovascular disease and aortic valve stenosis. However, the cell biology of Lp(a) biosynthesis remains poorly understood, with the locations of the noncovalent and covalent steps of Lp(a) assembly unclear and the nature of the apoB-containing particle destined for Lp(a) unknown. We, therefore, asked if apo(a) and apoB interact noncovalently within hepatocytes and if this impacts Lp(a) biosynthesis. METHODS: Using human hepatocellular carcinoma cells expressing 17K (17 kringle) apo(a), or a 17KΔLBS7,8 variant with a reduced ability to bind noncovalently to apoB, we performed coimmunoprecipitation, coimmunofluorescence, and proximity ligation assays to document intracellular apo(a):apoB interactions. We used a pulse-chase metabolic labeling approach to measure apo(a) and apoB secretion rates. RESULTS: Noncovalent complexes containing apo(a)/apoB are present in lysates from cells expressing 17K but not 17KΔLBS7,8, whereas covalent apo(a)/apoB complexes are absent from lysates. 17K and apoB colocalized intracellularly, overlapping with staining for markers of endoplasmic reticulum trans-Golgi, and early endosomes, and less so with lysosomes. The 17KΔLBS7,8 had lower colocalization with apoB. Proximity ligation assays directly documented intracellular 17K/apoB interactions, which were dramatically reduced for 17KΔLBS7,8. Treatment of cells with PCSK9 (proprotein convertase subtilisin/kexin type 9) enhanced, and lomitapide reduced, apo(a) secretion in a manner dependent on the noncovalent interaction between apo(a) and apoB. Apo(a) secretion was also reduced by siRNA-mediated knockdown of APOB. CONCLUSIONS: Our findings explain the coupling of apo(a) and Lp(a)-apoB production observed in human metabolic studies using stable isotopes as well as the ability of agents that inhibit apoB biosynthesis to lower Lp(a) levels.

Lipoprotein(a): A Genetically Determined, Causal, and Prevalent Risk Factor for Atherosclerotic Cardiovascular Disease: A Scientific Statement From the American Heart Association.

High levels of lipoprotein(a) [Lp(a)], an apoB100-containing lipoprotein, are an independent and causal risk factor for atherosclerotic cardiovascular diseases through mechanisms associated with increased atherogenesis, inflammation, and thrombosis. Lp(a) is predominantly a monogenic cardiovascular risk determinant, with ≈70% to ≥90% of interindividual heterogeneity in levels being genetically determined. The 2 major protein components of Lp(a) particles are apoB100 and apolipoprotein(a). Lp(a) remains a risk factor for cardiovascular disease development even in the setting of effective reduction of plasma low-density lipoprotein cholesterol and apoB100. Despite its demonstrated contribution to atherosclerotic cardiovascular disease burden, we presently lack standardization and harmonization of assays, universal guidelines for diagnosing and providing risk assessment, and targeted treatments to lower Lp(a). There is a clinical need to understand the genetic and biological basis for variation in Lp(a) levels and its relationship to disease in different ancestry groups. This scientific statement capitalizes on the expertise of a diverse basic science and clinical workgroup to highlight the history, biology, pathophysiology, and emerging clinical evidence in the Lp(a) field. Herein, we address key knowledge gaps and future directions required to mitigate the atherosclerotic cardiovascular disease risk attributable to elevated Lp(a) levels.

Health Care Transition to Adult Care in Type 1 Diabetes: Associations With Student and Employment Status-The SEARCH for Diabetes in Youth Study.

Successful transition from a pediatric to adult diabetes care provider is associated with reduced ambulatory diabetes care visits and increased acute complications. This study aimed to determine whether the degree of independence in diabetes care and the rate of acute complications after transition to adult diabetes care were associated with individuals' student or employment status. Nonstudents were found to be less likely than students to be independent with diabetes care, and employed nonstudents were at lower risk of diabetic ketoacidosis than unemployed nonstudents. Additional support may be needed for young adults who are not students or are unemployed to improve independence and reduce the risk for acute complications.

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.

Sortilin enhances secretion of apolipoprotein(a) through effects on apolipoprotein B secretion and promotes uptake of lipoprotein(a).

Elevated plasma lipoprotein(a) (Lp(a)) is an independent, causal risk factor for atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Lp(a) is formed in or on hepatocytes from successive noncovalent and covalent interactions between apo(a) and apoB, although the subcellular location of these interactions and the nature of the apoB-containing particle involved remain unclear. Sortilin, encoded by the SORT1 gene, modulates apoB secretion and LDL clearance. We used a HepG2 cell model to study the secretion kinetics of apo(a) and apoB. Overexpression of sortilin increased apo(a) secretion, while siRNA-mediated knockdown of sortilin expression correspondingly decreased apo(a) secretion. Sortilin binds LDL but not apo(a) or Lp(a), indicating that its effect on apo(a) secretion is likely indirect. Indeed, the effect was dependent on the ability of apo(a) to interact noncovalently with apoB. Overexpression of sortilin enhanced internalization of Lp(a), but not apo(a), by HepG2 cells, although neither sortilin knockdown in these cells or Sort1 deficiency in mice impacted Lp(a) uptake. We found several missense mutations in SORT1 in patients with extremely high Lp(a) levels; sortilin containing some of these mutations was more effective at promoting apo(a) secretion than WT sortilin, though no differences were found with respect to Lp(a) internalization. Our observations suggest that sortilin could play a role in determining plasma Lp(a) levels and corroborate in vivo human kinetic studies which imply that secretion of apo(a) and apoB are coupled, likely within the hepatocyte.

PCSK9 inhibition with alirocumab decreases plasma lipoprotein(a) concentration by a dual mechanism of action in statin-treated patients with very high apolipoprotein(a) concentration.

BACKGROUND: Inhibition of proprotein convertase subtilisin/kexin type 9 with alirocumab decreases plasma lipoprotein(a) [Lp(a)] levels. The kinetic mechanism for lowering Lp(a) by alirocumab may differ according to pre-treatment apolipoprotein(a) [apo(a)] levels. METHODS: The effect of 12-week alirocumab (150 mg subcutaneously fortnightly) on the kinetics of apo(a) was compared in statin-treated patients with high (n = 10) and very high Lp(a) concentrations (n = 11). RESULTS: In patients with high apo(a) concentrations, alirocumab lowered plasma apo(a) pool size (-17%, p < 0.01) chiefly by increasing the fractional catabolic rate (FCR) of apo(a) (+27%, p < 0.001). By contrast in patients with very high apo(a) concentrations, alirocumab significantly lowered plasma apo(a) pool size (-32%, p < 0.001) by both increasing apo(a) FCR (+30%, p < 0.001) and lowering production rate (-11%, p < 0.05). CONCLUSIONS: In statin-treated patients with very high apo(a) concentrations, alirocumab lowers plasma Lp(a) concentration by a dual mode of action that increases the clearance and decreases the production of Lp(a) particles.

Household food insecurity is associated with diabetic ketoacidosis but not severe hypoglycemia or glycemic control in youth and young adults with youth-onset type 2 diabetes.

OBJECTIVE: To examine the association between household food insecurity (HFI), glycemic control, severe hypoglycemia and diabetic ketoacidosis (DKA) among youth and young adults (YYA) with youth-onset type 2 diabetes. RESEARCH DESIGN AND METHODS: This cross-sectional study included 395 YYA with type 2 diabetes from the SEARCH for Diabetes in Youth Study (2015-2019). HFI was reported by young adult participants or parents of minor participants via the US Household Food Security Survey Module. Glycemic control was assessed by HbA1c and analyzed as a continuous and categorical variable (optimal: <7.0%, suboptimal: ≥7.0%-9.0%, poor: >9.0%). Acute complications included self-reported severe hypoglycemia or DKA in the last 12 months. Adjusted logistic and linear regression were used for binary and continuous outcomes, respectively. RESULTS: Approximately 31% reported HFI in the past 12 months. Mean HbA1c among those with HFI was 9.2% compared to 9.5% without HFI. Of those with HFI, 56% had an HbA1c >9.0% compared to 55% without HFI. Adjusted models showed no associations between HFI and glycemic control. Of those with HFI, 14.4% reported experiencing DKA and 4.7% reported severe hypoglycemia. YYA with HFI had 3.08 times (95% CI: 1.18-8.06) the odds of experiencing DKA as those without HFI. There was no association between HFI and severe hypoglycemia. CONCLUSIONS: HFI was associated with markedly increased odds of DKA but not with glycemic control or severe hypoglycemia. Future research among YYA with type 2 diabetes should evaluate longitudinally whether alleviating HFI reduces DKA.

Quantification of LDL-Cholesterol Corrected for Molar Concentration of Lipoprotein(a).

PURPOSE: Cholesterol in lipoprotein(a) [Lp(a)-C] is commonly estimated as 30% of the measured Lp(a) mass. However, difficulties in the accurate measurement of Lp(a) mass, along with the inaccuracy of the 30% assumption, produce erroneous values when LDL-C is corrected for Lp(a) [LDL-CLp(a)corr]. Our aim was to develop a new formula for LDL-CLp(a)corr to reduce this error. METHODS: We developed a new formula to calculate Lp(a)-C from the molar measurement of Lp(a), which is Lp(a) nmol/L × 0.077 = Lp(a)-C mg/dL. The calculated Lp(a)-C is subtracted from LDL-C to obtain LDL-CLp(a)corr. The results obtained with our novel formula versus the conventional formula were compared in 440 samples from 239 participants enrolled in the BANTING study. RESULTS: With the conventional formula, approximately 7% of samples with low LDL-C resulted in negative LDL-CLp(a)corr values. With the new formula, no negative LDL-CLp(a)corr values occurred. Among groups with the highest Lp(a)/apoB ratio (p < 0.001) and smaller apolipoprotein(a) isoform size (p < 0.006), LDL-CLp(a)corr was significantly underestimated by the conventional formula, which may result in the undertreatment of some patients. CONCLUSION: The new formula provides more reliable estimates of LDL-CLp(a)corr than the conventional formula. TRIAL REGISTRATION: ClinicalTrials.gov NCT02739984.

Development of an LC-MS/MS Proposed Candidate Reference Method for the Standardization of Analytical Methods to Measure Lipoprotein(a).

BACKGROUND: Use of lipoprotein(a) concentrations for identification of individuals at high risk of cardiovascular diseases is hampered by the size polymorphism of apolipoprotein(a), which strongly impacts immunochemical methods, resulting in discordant values. The availability of a reference method with accurate values expressed in SI units is essential for implementing a strategy for assay standardization. METHOD: A targeted LC-MS/MS method for the quantification of apolipoprotein(a) was developed based on selected proteotypic peptides quantified by isotope dilution. To achieve accurate measurements, a reference material constituted of a human recombinant apolipoprotein(a) was used for calibration. Its concentration was assigned using an amino acid analysis reference method directly traceable to SI units through an unbroken traceability chain. Digestion time-course, repeatability, intermediate precision, parallelism, and comparability to the designated gold standard method for lipoprotein(a) quantification, a monoclonal antibody-based ELISA, were assessed. RESULTS: A digestion protocol providing comparable kinetics of digestion was established, robust quantification peptides were selected, and their stability was ascertained. Method intermediate imprecision was below 10% and linearity was validated in the 20-400 nmol/L range. Parallelism of responses and equivalency between the recombinant and endogenous apo(a) were established. Deming regression analysis comparing the results obtained by the LC-MS/MS method and those obtained by the gold standard ELISA yielded y = 0.98*ELISA +3.18 (n = 64). CONCLUSIONS: Our method for the absolute quantification of lipoprotein(a) in plasma has the required attributes to be proposed as a candidate reference method with the potential to be used for the standardization of lipoprotein(a) assays.

The relationship between traffic-related air pollution exposures and allostatic load score among youth with type 1 diabetes in the SEARCH cohort.

OBJECTIVE: We investigated the effects of chronic exposures to particulate and traffic-related air pollution on allostatic load (AL) score, a marker of cumulative biological risk, among youth with type 1 diabetes. RESEARCH DESIGN AND METHODS: Participants were drawn from five clinical sites of the SEARCH for Diabetes in Youth (SEARCH) study (n = 2338). Baseline questionnaires, anthropometric measures, and a fasting blood test were taken at a clinic visit between 2001 and 2005. AL was operationalized using 10 biomarkers reflecting cardiovascular, metabolic, and inflammatory risk. Annual residential exposures to PM2.5 and proximity to heavily-trafficked major roadways were estimated for each participant. Poisson regression models adjusted for sociodemographic and lifestyle factors were conducted for each exposure. RESULTS: No significant associations were observed between exposures to PM2.5 or proximity to traffic and AL score, however analyses were suggestive of effect modification by race for residential distance to heavily-trafficked major roadways (p = 0.02). In stratified analyses, residing <100, 100-<200 and 200-<400 m compared to 400 m or more from heavily-trafficked major roadways was associated with 11%, 26% and 14% increases in AL score, respectively (95% CIs: -4, 29; 9, 45; -1, 30) for non-white participants compared to 6%, -2%, and -2% changes (95% CIs: -2, 15; -10, 7; -8, 6) for white participants. CONCLUSIONS: Among this population of youth with type 1 diabetes, we did not observe consistent relationships between chronic exposures to particulate and traffic-related air pollution and changes in AL score, however associations for traffic-related pollution exposures may differ by race/ethnicity and warrant further examination.

Trends in Prevalence of Type 1 and Type 2 Diabetes in Children and Adolescents in the US, 2001-2017.

Importance: Changes in the prevalence of youth-onset diabetes have previously been observed. Objective: To estimate changes in prevalence of type 1 and type 2 diabetes in youths in the US from 2001 to 2017. Design, Setting, and Participants: In this cross-sectional observational study, individuals younger than 20 years with physician-diagnosed diabetes were enumerated from 6 areas in the US (4 geographic areas, 1 health plan, and select American Indian reservations) for 2001, 2009, and 2017. Exposures: Calendar year. Main Outcomes and Measures: Estimated prevalence of physician-diagnosed type 1 and type 2 diabetes overall and by race and ethnicity, age, and sex. Results: Among youths 19 years or younger, 4958 of 3.35 million had type 1 diabetes in 2001, 6672 of 3.46 million had type 1 diabetes in 2009, and 7759 of 3.61 million had type 1 diabetes in 2017; among those aged 10 to 19 years, 588 of 1.73 million had type 2 diabetes in 2001, 814 of 1.85 million had type 2 diabetes in 2009, and 1230 of 1.85 million had type 2 diabetes in 2017. The estimated type 1 diabetes prevalence per 1000 youths for those 19 years or younger increased significantly from 1.48 (95% CI, 1.44-1.52) in 2001 to 1.93 (95% CI, 1.88-1.98) in 2009 to 2.15 (95% CI, 2.10-2.20) in 2017, an absolute increase of 0.67 per 1000 youths (95%, CI, 0.64-0.70) and a 45.1% (95% CI, 40.0%-50.4%) relative increase over 16 years. The greatest absolute increases were observed among non-Hispanic White (0.93 per 1000 youths [95% CI, 0.88-0.98]) and non-Hispanic Black (0.89 per 1000 youths [95% CI, 0.88-0.98]) youths. The estimated type 2 diabetes prevalence per 1000 youths aged 10 to 19 years increased significantly from 0.34 (95% CI, 0.31-0.37) in 2001 to 0.46 (95% CI, 0.43-0.49) in 2009 to 0.67 (95% CI, 0.63-0.70) in 2017, an absolute increase of 0.32 per 1000 youths (95% CI, 0.30-0.35) and a 95.3% (95% CI, 77.0%-115.4%) relative increase over 16 years. The greatest absolute increases were observed among non-Hispanic Black (0.85 per 1000 youths [95% CI, 0.74-0.97]) and Hispanic (0.57 per 1000 youths [95% CI, 0.51-0.64]) youths. Conclusions and Relevance: In 6 areas of the US from 2001 to 2017, the estimated prevalence of diabetes among children and adolescents increased for both type 1 and type 2 diabetes.

Analytical Performance Specifications for Lipoprotein(a), Apolipoprotein B-100, and Apolipoprotein A-I Using the Biological Variation Model in the EuBIVAS Population.

BACKGROUND: With increased interest in lipoprotein(a) (Lp[a]) concentration as a target for risk reduction and growing clinical evidence of its impact on cardiovascular disease (CVD) risk, rigorous analytical performance specifications (APS) and accuracy targets for Lp(a) are required. We investigated the biological variation (BV) of Lp(a), and 2 other major biomarkers of CVD, apolipoprotein A-I (apoA-I) and apolipoprotein B-100 (apoB), in the European Biological Variation Study population. METHOD: Serum samples were drawn from 91 healthy individuals for 10 consecutive weeks at 6 European laboratories and analyzed in duplicate on a Roche Cobas 8000 c702. Outlier, homogeneity, and trend analysis were performed, followed by CV-ANOVA to determine BV estimates and their 95% CIs. These estimates were used to calculate APS and reference change values. For Lp(a), BV estimates were determined on normalized concentration quintiles. RESULTS: Within-subject BV estimates were significantly different between sexes for Lp(a) and between women aged <50 and >50 years for apoA-I and apoB. Lp(a) APS was constant across concentration quintiles and, overall, lower than APS based on currently published data, whereas results were similar for apoA-I and apoB. CONCLUSION: Using a fully Biological Variation Data Critical Appraisal Checklist (BIVAC)-compliant protocol, our study data confirm BV estimates of Lp(a) listed in the European Federation of Clinical Chemistry and Laboratory Medicine database and reinforce concerns expressed in recent articles regarding the suitability of older APS recommendations for Lp(a) measurements. Given the heterogeneity of Lp(a), more BIVAC-compliant studies on large numbers of individuals of different ethnic groups would be desirable.

PCSK9 loss-of-function variants and Lp(a) phenotypes among black US adults.

The pharmacologic inhibition of proprotein convertase subtilisin-kexin type 9 (PCSK9) lowers lipoprotein (a) [Lp(a)] concentrations. However, the impact of genetic PCSK9 loss-of-function variants (LOFVs) on Lp(a) is uncertain. We determined the association of PCSK9 LOFVs with Lp(a) measures among black adults. Genotyping for PCSK9 LOFVs was conducted in 10,196 black Reasons for Geographic and Racial Differences in Stroke study participants. Among 241 participants with and 723 randomly selected participants without PCSK9 LOFVs, Lp(a) concentations, apo(a) kringle IV (KIV) repeats (a proxy for isoform size), and oxidized phospholipid (OxPL) apoB levels were measured using validated methods. Median Lp(a) concentrations among participants with and without PCSK9 LOFVs were 63.2 and 80.4 nmol/l, respectively (P = 0.016). After adjusting for age, sex, estimated glomerular filtration rate, LDL cholesterol, and statin use, participants with versus without a PCSK9 LOFV had a lower median Lp(a) concentration [Δ = -18.8 nmol/l (95% CI: -34.2, -3.3)]. Median apo(a) isoform sizes were 24 and 23 KIV repeats (P = 0.12) among participants with and without PCSK9 LOFVs, respectively [Δ = 1.1 (95% CI: 0.2, 2.0) after adjustment]. Median OxPL-apoB levels among participants with and without PCSK9 LOFVs were 3.4 and 4.1 nM (P = 0.20), respectively [Δ = -1.2 nM (95% CI -2.4, -0.04) after adjustment]. Among black adults, PCSK9 LOFVs were associated with lower Lp(a) concentration and OxPL-apoB levels.

Non-traditional biomarkers and incident diabetes in the Diabetes Prevention Program: comparative effects of lifestyle and metformin interventions.

AIMS/HYPOTHESIS: We compared the associations of circulating biomarkers of inflammation, endothelial and adipocyte dysfunction and coagulation with incident diabetes in the placebo, lifestyle and metformin intervention arms of the Diabetes Prevention Program, a randomised clinical trial, to determine whether reported associations in general populations are reproduced in individuals with impaired glucose tolerance, and whether these associations are independent of traditional diabetes risk factors. We further investigated whether biomarker-incident diabetes associations are influenced by interventions that alter pathophysiology, biomarker concentrations and rates of incident diabetes. METHODS: The Diabetes Prevention Program randomised 3234 individuals with impaired glucose tolerance into placebo, metformin (850 mg twice daily) and intensive lifestyle groups and showed that metformin and lifestyle reduced incident diabetes by 31% and 58%, respectively compared with placebo over an average follow-up period of 3.2 years. For this study, we measured adiponectin, leptin, tissue plasminogen activator (as a surrogate for plasminogen activator inhibitor 1), high-sensitivity C-reactive protein, IL-6, monocyte chemotactic protein 1, fibrinogen, E-selectin and intercellular adhesion molecule 1 at baseline and at 1 year by specific immunoassays. Traditional diabetes risk factors were defined as family history, HDL-cholesterol, triacylglycerol, BMI, fasting and 2 h glucose, HbA1c, systolic blood pressure, inverse of fasting insulin and insulinogenic index. Cox proportional hazard models were used to assess the effects of each biomarker on the development of diabetes assessed semi-annually and the effects of covariates on these. RESULTS: E-selectin, (HR 1.19 [95% CI 1.06, 1.34]), adiponectin (0.84 [0.71, 0.99]) and tissue plasminogen activator (1.13 [1.03, 1.24]) were associated with incident diabetes in the placebo group, independent of diabetes risk factors. Only the association between adiponectin and diabetes was maintained in the lifestyle (0.69 [0.52, 0.92]) and metformin groups (0.79 [0.66, 0.94]). E-selectin was not related to diabetes development in either lifestyle or metformin groups. A novel association appeared for change in IL-6 in the metformin group (1.09 [1.021, 1.173]) and for baseline leptin in the lifestyle groups (1.31 [1.06, 1.63]). CONCLUSIONS/INTERPRETATION: These findings clarify associations between an extensive group of biomarkers and incident diabetes in a multi-ethnic cohort with impaired glucose tolerance, the effects of diabetes risk factors on these, and demonstrate differential modification of associations by interventions. They strengthen evidence linking adiponectin to diabetes development, and argue against a central role for endothelial dysfunction. The findings have implications for the pathophysiology of diabetes development and its prevention.

Lp(a) (Lipoprotein(a)) Levels Predict Progression of Carotid Atherosclerosis in Subjects With Atherosclerotic Cardiovascular Disease on Intensive Lipid Therapy: An Analysis of the AIM-HIGH (Atherothrombosis Intervention in Metabolic Syndrome With Low HDL/High Triglycerides: Impact on Global Health Outcomes) Carotid Magnetic Resonance Imaging Substudy-Brief Report.

OBJECTIVE: To assess whether Lp(a) (lipoprotein(a)) levels and other lipid levels were predictive of progression of atherosclerosis burden as assessed by carotid magnetic resonance imaging in subjects who have been treated with LDL-C (low-density lipoprotein cholesterol)-lowering therapy and participated in the AIM-HIGH trial (Atherothrombosis Intervention in Metabolic Syndrome With Low HDL/High Triglycerides: Impact on Global Health Outcomes). APPROACH AND RESULTS: AIM-HIGH was a randomized, double-blind study of subjects with established vascular disease, elevated triglycerides, and low HDL-C (high-density lipoprotein cholesterol). One hundred fifty-two AIM-HIGH subjects underwent both baseline and 2-year follow-up carotid artery magnetic resonance imaging. Plaque burden was measured by the percent wall volume (%WV) of the carotid artery. Associations between annualized change in %WV with baseline and on-study (1 year) lipid variables were evaluated using multivariate linear regression and the Bonferroni correction to account for multiple comparisons. Average %WV at baseline was 41.6±6.8% and annualized change in %WV over 2 years ranged from -3.2% to 3.7% per year (mean: 0.2±1.1% per year; P=0.032). Increases in %WV were significantly associated with higher baseline Lp(a) (ß=0.34 per 1-SD increase of Lp(a); 95% confidence interval, 0.15-0.52; P<0.001) after adjusting for clinical risk factors and other lipid levels. On-study Lp(a) had a similar positive association with %WV progression (ß=0.33; 95% confidence interval, 0.15-0.52; P<0.001). CONCLUSIONS: Despite intensive lipid therapy, aimed at aggressively lowering LDL-C to <70 mg/dL, carotid atherosclerosis continued to progress as assessed by carotid magnetic resonance imaging and that elevated Lp(a) levels were independent predictors of increases in atherosclerosis burden.

Controlled study of the effect of proprotein convertase subtilisin-kexin type 9 inhibition with evolocumab on lipoprotein(a) particle kinetics.

Aims: Lipoprotein(a) [Lp(a)], a low-density lipoprotein (LDL) particle covalently bound to apolipoprotein(a) [apo(a)], is a potentially potent heritable risk factor for cardiovascular disease. We investigated the mechanism whereby evolocumab, a monoclonal antibody against proprotein convertase subtilisin-kexin type 9 (PCSK9), lowers Lp(a). Methods and results: We studied the kinetics of Lp(a) particles in 63 healthy men, with plasma apo(a) concentration >5 nmol/L, participating in an 8-week factorial trial of the effects of evolocumab (420 mg every 2 weeks) and atorvastatin (80 mg daily) on lipoprotein metabolism. Lipoprotein(a)-apo(a) kinetics were studied using intravenous D3-leucine administration, mass spectrometry, and compartmental modelling; Lp(a)-apoB kinetics were also determined in 16 subjects randomly selected from the treatment groups. Evolocumab, but not atorvastatin, significantly decreased the plasma pool size of Lp(a)-apo(a) (-36%, P < 0.001 for main effect). As monotherapy, evolocumab significantly decreased the production of Lp(a)-apo(a) (-36%, P < 0.001). In contrast, in combination with atorvastatin, evolocumab significantly increased the fractional catabolism of Lp(a)-apo(a) (+59%, P < 0.001), but had no effect on the production of Lp(a)-apo(a). There was a highly significant association between the changes in the fractional catabolism of Lp(a)-apo(a) and Lp(a)-apoB in the substudy of 16 subjects (r = 0.966, P < 0.001). Conclusions: Evolocumab monotherapy lowered the plasma Lp(a) pool size by decreasing the production of Lp(a) particles. In combination with atorvastatin, evolocumab lowered the plasma Lp(a) pool size by accelerating the catabolism of Lp(a) particles. This dual mechanism may relate to an effect of PCSK9 inhibition on Lp(a)-apo(a) production and to marked up-regulation of LDL receptor activity on Lp(a) holoparticle clearance. Clinical Trial Registration Information: NCT02189837.

Effects of mipomersen, an apolipoprotein B100 antisense, on lipoprotein (a) metabolism in healthy subjects.

Elevated lipoprotein (a) [Lp(a)] levels increase the risk for CVD. Novel treatments that decrease LDL cholesterol (LDL-C) have also shown promise for reducing Lp(a) levels. Mipomersen, an antisense oligonucleotide that inhibits apoB synthesis, is approved for the treatment of homozygous familial hypercholesterolemia. It decreases plasma levels of LDL-C by 25% to 39% and lowers levels of Lp(a) by 21% to 39%. We examined the mechanisms for Lp(a) lowering during mipomersen treatment. We enrolled 14 healthy volunteers who received weekly placebo injections for 3 weeks followed by weekly injections of mipomersen for 7 weeks. Stable isotope kinetic studies were performed using deuterated leucine at the end of the placebo and mipomersen treatment periods. The fractional catabolic rate (FCR) of Lp(a) was determined from the enrichment of a leucine-containing peptide specific to apo(a) by LC/MS. The production rate (PR) of Lp(a) was calculated from the product of Lp(a) FCR and Lp(a) concentration (converted to pool size). In a diverse population, mipomersen reduced plasma Lp(a) levels by 21%. In the overall study group, mipomersen treatment resulted in a 27% increase in the FCR of Lp(a) with no significant change in PR. However, there was heterogeneity in the response to mipomersen therapy, and changes in both FCRs and PRs affected the degree of change in Lp(a) concentrations. Mipomersen treatment decreases Lp(a) plasma levels mainly by increasing the FCR of Lp(a), although changes in Lp(a) PR were significant predictors of reductions in Lp(a) levels in some subjects.