Lipase maturation factor 1 (LMF1) is a chaperone for lipoprotein lipase (LPL) and critically required for the enzyme to attain lipase activity. LMF1 has been identified as a canonical gene affected in Familial Chylomicronemia Syndrome (FCS) and it has also been extensively analyzed in Multifactorial Chylomicronemia Syndrome (MCS), the polygenic form of severe hypertriglyceridemia (hTG). While recent genetic studies resulted in an explosion in the number of hTG-associated LMF1 variants in different populations, their functional significance remains largely unknown. Here, we present a second-generation LMF1 activity assay allowing the streamlined quantitative functional analysis of LMF1 variants. The assay is based on the reconstitution of lipase maturation in transfected LMF1-deficient cells and fluorescence-based measurement of LPL activity in the culture medium. The use of Gaussia luciferase (GLuc)-LMF1 fusion constructs allows the assessment of LMF1 protein expression and the calculation of LMF1 specific activity. Several variants previously reported as neutral resulted in loss of LMF1 function in the new assay demonstrating increased sensitivity to detect variants of modest effect sizes. Furthermore, the functional analysis of 14 hTG-associated missense mutations identified 12 loss-of-function (LOF) variants. To gain initial insights into the structural determinants of LMF1 function, we analyzed the domain distribution of 73 hTG-associated missense variants and identified Loop C as a critical region in lipase maturation. In conclusion, we developed a simple, non-radioactive and sensitive quantitative assay of LMF1 activity to facilitate the functional analysis of hyperlipidemia-associated genetic variants and structure-function studies.

BACKGROUND:The genetic basis of hypertriglyceridemia (HTG) is complex and includes variants in lipase maturation factor 1 (LMF1), an endoplasmic reticulum (ER)-chaperone involved in the post-translational activation of lipoprotein lipase (LPL). OBJECTIVE:The objective of this study was to identify and functionally characterize biallelic LMF1 variants in patients with HTG. METHODS:Genomic DNA sequencing was used to identify biallelic LMF1 variants in HTG patients without deleterious variants in LPL, apolipoprotein C-II (APOC2), glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) or apolipoprotein A-V (APOA5). LMF1 variants were functionally evaluated by in silico analyses and assessing their impact on LPL activity, LMF1 protein expression, and specific activity in transiently transfected HEK293 cells. RESULTS:We identified four homozygous LMF1 variants in patients with severe HTG: two novel rare variants (p.Asn147Lys and p.Pro246Arg) and two low-frequency variants (p.Arg354Trp and p.Arg364Gln) previously reported at heterozygosity. We demonstrate that all four variants reduce the secretion of enzymatically active LPL by impairing the specific activity of LMF1, whereas p.Asn147Lys also diminishes LMF1 protein expression. CONCLUSION:This study extends the role of LMF1 as a genetic determinant in severe HTG and demonstrates that rare and low-frequency LMF1 variants can underlie this condition through distinct molecular mechanisms. The clinical phenotype of patients affected by partial loss of LMF1 function is consistent with multifactorial chylomicronemia syndrome (MCS) and suggests that secondary factors and additional genetic determinants contribute to HTG in these subjects.

Background: Coronary heart disease (CHD) remains a leading cause of death and has been associated with alterations in plasma lipoprotein particles and inflammation markers. This study aimed to evaluate and compare standard and advanced lipid parameters and inflammatory biomarkers in CHD cases and matched control subjects. We hypothesized that incorporating advanced lipid and inflammatory biomarkers into risk models would improve CHD risk prediction beyond the standard lipid measures. Methods: CHD cases (n = 227, mean age 61 years, 47% female) and matched controls (n = 526) underwent fasting blood collection while off lipid-lowering medications. Automated chemistry analyses were performed to measure total cholesterol (TC), triglycerides (TGs), low-density lipoprotein-C (LDL-C), small dense LDL-C (sdLDL-C), apolipoproteins (apos) A-I and B, lipoprotein(a) (Lp(a)), high-sensitivity C-reactive protein (hsCRP), serum amyloid-A (SAA), myeloperoxidase (MPO), and apoA-I in HDL particles (via 2-dimensional electrophoresis and immunoblotting). Univariate, multivariate, and machine learning analyses compared the CHD cases with the controls. Results: The most significant percent differences between male and female cases versus controls were for hsCRP (+78%, +200%), MPO (+109%, +106%), SAA (+84%, +33%), sdLDL-C (+48%; +43%), Lp(a) (+43%,+70%), apoA-I in very large α-1 HDL (−34%, −26%), HDL-C (−24%, −27%), and apoA-I in very small preβ-1 HDL (+17%; +16%). Total C, non-HDL-C, and direct and calculated LDL-C levels were only modestly higher in the cases. Multivariate models incorporating advanced parameters were statistically superior to a standard model (C statistic: men: 0.913 vs. 0.856; women: 0.903 versus 0.838). Machine learning identified apoA-I in preβ-1-HDL, α-2-HDL, α-1-HDL, α-3-HDL, MPO, and sdLDL-C as the top predictors of CHD. Conclusions: This study introduces a novel approach to CHD risk assessment by integrating advanced HDL particle analysis and machine learning. By assessing HDL subpopulations (α-1, α-2, preβ-1 HDL), inflammatory biomarkers (MPO, SAA), and small dense LDL, we provide a more refined stratification model. Notably, preβ-1 HDL, an independent risk factor reflecting impaired cholesterol efflux from the artery wall, is highlighted as a critical marker of CHD risk. Our approach allows for earlier identification of high-risk individuals, particularly those with subtle lipid or inflammatory abnormalities, supporting more personalized interventions. These findings demonstrate the potential of advanced lipid profiling and machine learning to enhance CHD risk prediction.

BACKGROUND The apolipoprotein L1 (APOL1) G1 and G2 gene variants, highly prevalent among the African American population (rare in other racial groups), are linked to increased risk of kidney disease, sepsis, and potentially coronary heart disease (CHD). Their role in tobacco-related CHD remains unclear. OBJECTIVE To investigate the effect of APOL1 risk variants on the association between tobacco smoking and prevalent CHD in African American adults. METHODS We conducted a cross-sectional study involving 519 African American adults recruited through the University of California San Francisco Lipid Clinic. Using multivariable logistic regression, we assessed the association between tobacco smoking and CHD, overall and with its most severe subtype, myocardial infarction (MI), among all participants and APOL1 genotype subgroups. RESULTS Among participants, 41% were current (14%) or former (27%) smokers, 54% carried APOL1 risk variants (1 or 2 alleles), and 28% had CHD, including 16% having MI. Current smokers with APOL1 risk variants had 3.3 times higher odds of CHD compared to nonsmokers (95% CI: 1.6, 6.8), with the strongest effect observed in those with 2 risk alleles (odds ratio [OR]: 7.3, CI: 1.1, 48.6) and a substantial effect in carriers of a single risk allele (OR: 3.2, CI: 1.5, 7.2). Among non-carriers, current smoking was not significantly associated with CHD (OR: 1.3). A similar trend was observed for MI. Former smoking was associated with CHD (OR: 2.0), independent of APOL1 genotype. CONCLUSION African American smokers with APOL1 G1 and/or G2 risk variants may be at greater risk of CHD; this relationship appears to follow an additive model.

Atrial fibrillation (AF) is the most common heart rhythm abnormality and is a leading cause of heart failure and stroke. This large-scale meta-analysis of genome-wide association studies increased the power to detect single-nucleotide variant associations and found more than 350 AF-associated genetic loci. We identified candidate genes related to muscle contractility, cardiac muscle development and cell-cell communication at 139 loci. Furthermore, we assayed chromatin accessibility using assay for transposase-accessible chromatin with sequencing and histone H3 lysine 4 trimethylation in stem cell-derived atrial cardiomyocytes. We observed a marked increase in chromatin accessibility for our sentinel variants and prioritized genes in atrial cardiomyocytes. Finally, a polygenic risk score (PRS) based on our updated effect estimates improved AF risk prediction compared to the CHARGE-AF clinical risk score and a previously reported PRS for AF. The doubling of known risk loci will facilitate a greater understanding of the pathways underlying AF.

BACKGROUND:Substantial data support a heritable basis for supraventricular tachycardias, but the genetic determinants and molecular mechanisms of these arrhythmias are poorly understood. We sought to identify genetic loci associated with atrioventricular nodal reentrant tachycardia (AVNRT) and atrioventricular accessory pathways or atrioventricular reciprocating tachycardia (AVAPs/AVRT).METHODS:We performed multiancestry meta-analyses of genome-wide association studies to identify genetic loci for AVNRT (4 studies) and AVAP/AVRT (7 studies). We assessed evidence supporting the potential causal effects of candidate genes by analyzing relations between associated variants and cardiac gene expression, performing transcriptome-wide analyses, and examining prior genome-wide association studies.RESULTS:Analyses comprised 2384 AVNRT cases and 106 489 referents, and 2811 AVAP/AVRT cases and 1,483 093 referents. We identified 2 significant loci for AVNRT, which implicate NKX2-5 and TTN as disease susceptibility genes. A transcriptome-wide association analysis supported an association between reduced predicted cardiac expression of NKX2-5 and AVNRT. We identified 3 significant loci for AVAP/AVRT, which implicate SCN5A, SCN10A, and TTN/CCDC141. Variant associations at several loci have been previously reported for cardiac phenotypes, including atrial fibrillation, stroke, Brugada syndrome, and electrocardiographic intervals.CONCLUSIONS:Our findings highlight gene regions associated with ion channel function (AVAP/AVRT), as well as cardiac development and the sarcomere (AVAP/AVRT and AVNRT) as important potential effectors of supraventricular tachycardia susceptibility.

Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis 1 . These lesions are precursors for blood cancers 2 – 6 , but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2 , ASXL1 , SF3B1 and SRSF2 , but this effect was not seen in clones with driver mutations in DNMT3A . TCL1A was not expressed in normal or DNMT3A -mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimental knockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

Background African American smokers have 2.5 times higher risk for stroke compared with nonsmokers (higher than other races). About 50% of the African American population carry 1 or 2 genetic variants (G1 and G2; rare in other races) of the apolipoprotein L1 gene (APOL1). Studies showed these variants may be associated with stroke. However, the role of the APOL1 risk variants in tobacco-related stroke is unknown.Methods and Results In a cross-sectional study, we examined whether APOL1 risk variants modified the relationship between tobacco smoking and stroke prevalence in 513 African American adults recruited at University of California, San Francisco. Using DNA, plasma, and questionnaires we determined APOL1 variants, smoking status, and stroke prevalence. Using logistic regression models, we examined the association between smoking (ever versus never smokers) and stroke overall, and among carriers of APOL1 risk variants (1 or 2 risk alleles), and noncarriers, separately. Among participants, 41% were ever (current and past) smokers, 54% were carriers of the APOL1 risk variants, and 41 had a history of stroke. The association between smoking and stroke differed by APOL1 genotype (P-interaction term=0.014). Among carriers, ever versus never smokers had odds ratio (OR) 2.46 (95% CI, 1.08-5.59) for stroke (P=0.034); OR 2.00 (95% CI, 0.81-4.96) among carriers of 1 risk allele, and OR 4.72 (95% CI, 0.62-36.02) for 2 risk alleles. Among noncarriers, smoking was not associated with a stroke.Conclusions Current and past smokers who carry APOL1 G1 and/or G2 risk variants may be more susceptible to stroke among the African American population.

Low levels of high density lipoprotein-cholesterol (HDL-C) are associated with an elevated risk of arteriosclerotic coronary heart disease. Heritability of HDL-C levels is high. In this research discovery study, we used whole-exome sequencing to identify damaging gene variants that may play significant roles in determining HDL-C levels. We studied 204 individuals with a mean HDL-C level of 27.8 ± 6.4 mg/dl (range: 4–36 mg/dl). Data were analyzed by statistical gene burden testing and by filtering against candidate gene lists. We found 120 occurrences of probably damaging variants (116 heterozygous; four homozygous) among 45 of 104 recognized HDL candidate genes. Those with the highest prevalence of damaging variants were ABCA1 (n = 20), STAB1 (n = 9), OSBPL1A (n = 8), CPS1 (n = 8), CD36 (n = 7), LRP1 (n = 6), ABCA8 (n = 6), GOT2 (n = 5), AMPD3 (n = 5), WWOX (n = 4), and IRS1 (n = 4). Binomial analysis for damaging missense or loss-of-function variants identified the ABCA1 and LDLR genes at genome-wide significance. In conclusion, whole-exome sequencing of individuals with low HDL-C showed the burden of damaging rare variants in the ABCA1 and LDLR genes is particularly high and revealed numerous occurrences in HDL candidate genes, including many genes identified in genome-wide association study reports. Many of these genes are involved in cancer biology, which accords with epidemiologic findings of the association of HDL deficiency with increased risk of cancer, thus presenting a new area of interest in HDL genomics.

Despite the association between cognitive impairment and delirium, little is known about whether genetic differences that confer cognitive resilience also confer resistance to delirium. To investigate whether older adults without postoperative delirium, compared with those with postoperative delirium, are more likely to have specific single nucleotide polymorphisms (SNPs) in the FKBP5, KIBRA , KLOTHO, MTNR1B , and SIRT1 genes known to be associated with cognition or delirium. This prospective nested matched exploratory case–control study included 94 older adults who underwent orthopedic surgery and screened for postoperative delirium. Forty-seven subjects had incident delirium, and 47 age-matched controls were not delirious. The primary study outcome was genotype frequency for the five SNPs. Compared with participants with delirium, those without delirium had higher adjusted odds of KIBRA SNP rs17070145 CT/TT [vs. CC; adjusted odds ratio (aOR) 2.80, 95% confidence interval (CI) 1.03, 7.54; p = 0.04] and MTNR1B SNP rs10830963 CG/GG (vs. CC; aOR 4.14, 95% CI 1.36, 12.59; p = 0.01). FKBP5 SNP rs1360780 CT/TT (vs. CC) demonstrated borderline increased adjusted odds of not developing delirium (aOR 2.51, 95% CI 1.00, 7.34; p = 0.05). Our results highlight the relevance of KIBRA, MTNR1B, and FKBP5 in understanding the complex relationship between delirium, cognition, and sleep, which warrant further study in larger, more diverse populations.

Diabetes mellitus is a complex disease. We are increasingly gaining a better understanding of its mechanisms at the molecular level. From these new insights, better therapeutic approaches should emerge. Diabetes mellitus is a syndrome with many associated subphenotypes. These include mitochondrial disorders, lipodystrophies, and inflammatory disorders involving cytokines. Levels of sphingosine-1-phosphate, which has recently been shown to play a role in glucose homeostasis, are low in diabetics, whereas levels of ceramides are increased. Major phenotypes associated with diabetes mellitus are dyslipidemias, notably hypertriglyceridemia and low high density lipoprotein cholesterol levels. Both diabetes and dyslipidemia are strongly associated with increased risk for atherosclerotic vascular disease.

BACKGROUND:Susceptibility to severe hypertriglyceridemia (HTG), defined as plasma triglyceride (TG) levels ≥10 mmol/L (880 mg/dL), is conferred by both heterozygous rare variants in five genes involved in TG metabolism and numerous common single-nucleotide polymorphisms (SNPs) associated with TG levels. OBJECTIVE:To date, these genetic susceptibility factors have been comprehensively assessed primarily in severe HTG patients of European ancestry. Here, we expand our analysis to HTG patients of East Asian and Hispanic ancestry. METHODS:The genomic DNA of 336, 63 and 199 severe HTG patients of European, East Asian and Hispanic ancestry, respectively, was evaluated using a targeted next-generation sequencing panel to screen for: 1) rare variants in LPL, APOA5, APOC2, GPIHBP1 and LMF1; 2) common, small-to-moderate effect SNPs, quantified using a polygenic score; and 3) common, large-effect polymorphisms, APOA5 p.G185C and p.S19W. RESULTS:While the proportion of individuals with high polygenic scores was similar, frequency of rare variant carriers varied across ancestries. Compared with ancestry-matched controls, Hispanic patients were the most likely to have a rare variant (OR = 5.02; 95% CI 3.07-8.21; p < 0.001), while European patients were the least likely (OR = 2.56; 95% CI 1.58-4.13; p < 0.001). The APOA5 p.G185C polymorphism, exclusive to East Asians, was significantly enriched in patients compared with controls (OR = 10.1; 95% CI 5.6-18.3; p < 0.001), showing the highest enrichment among the measured genetic factors. CONCLUSION:While TG-associated rare variants and common SNPs are both found in statistical excess in severe HTG patients of different ancestral backgrounds, the overall genetic profiles of each ancestry group were distinct.

Objective: To determine whether selected genes and plasma markers involved in energy homeostasis are associated with sleep disruption or duration in adults with HIV/AIDS. Methods: A sample of 289 adults with HIV/AIDS wore a wrist actigraph for 72 h to estimate total sleep time (TST) and wake after sleep onset (WASO). Twenty-three single nucleotide polymorphisms (SNP) spanning 5 energy homeostasis genes (adiponectin [ADIPOQ], ghrelin [GHRL], leptin [LEP], peroxisome proliferator-activated receptor-alpha [PPARA], and -gamma [PPARG]) were genotyped using a custom array. Plasma markers of energy homeostasis (adiponectin, ghrelin, leptin) were measured by commercial multiplex assay. Results: After adjusting for demographic and clinical characteristics (race/ethnicity, gender, CD4 cell count, waist circumference, medications), both WASO and TST were associated with SNPs in ADIPOQ (rs182052), LEP (rs10244329, rs3828942), PPARA (rs135551, rs4253655), and PPARG (rs709151). Additional SNPs in ADIPOQ were associated with WASO (rs1501299, rs3821799, rs6773957) and TST (rs2241766). TST was also associated with SNPs in GHRL (rs26802), LEP (rs11760956), PPARA (rs135547, rs8138102, rs4253776), and PPARG (rs12490265, rs796313). Many covariate-adjusted associations involved a significant interaction with markers of HIV (viral load, years since diagnosis). Among plasma markers, higher adiponectin was associated with less WASO, higher ghrelin and glucose levels with shorter TST, and higher leptin with longer TST. Conclusions: Replication of SNPs in all five genes and three plasma markers of energy homeostasis were associated with objective sleep measures. HIV disease influenced many of the associations. Findings strengthen evidence for associations between energy homeostasis genetics and poor sleep, and provide direction for pharmacological intervention research. (C) 2020 Published by Elsevier B.V.

Objective: Genetic determinants of severe hypertriglyceridemia include both common variants with small effects (assessed using polygenic risk scores) plus heterozygous and homozygous rare variants in canonical genes directly affecting triglyceride metabolism. Here, we broadened our scope to detect associations with rare loss-of-function variants in genes affecting noncanonical pathways, including those known to affect triglyceride metabolism indirectly. Approach and Results: From targeted next-generation sequencing of 69 metabolism-related genes in 265 patients of European descent with severe hypertriglyceridemia (≥10 mmol/L or ≥885 mg/dL) and 477 normolipidemic controls, we focused on the association of rare heterozygous loss-of-function variants in individual genes. We observed that compared with controls, severe hypertriglyceridemia patients were 20.2× (95% CI, 1.11–366.1; P =0.03) more likely than controls to carry a rare loss-of-function variant in CREB3L3 , which encodes a transcription factor that regulates several target genes with roles in triglyceride metabolism. Conclusions: Our findings indicate that rare variants in a noncanonical gene for triglyceride metabolism, namely CREB3L3 , contribute significantly to severe hypertriglyceridemia. Secondary genes and pathways should be considered when evaluating the genetic architecture of this complex trait.

Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis. However, guidelines for treatment are defined solely by fasting lipid levels, even though postprandial lipids may be more informative. In the postprandial state, circulating lipids consist of dietary fat transported from the intestine in chylomicrons (CMs; containing ApoB48) and fat transported from the liver in VLDL (containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because of the difficulty in separating these particles by ultracentrifugation. CM fractions have considerable contamination from VLDL (purity, 10%). To separate CMs from VLDL, we produced polyclonal antibodies against ApoB100 and generated immunoaffinity columns. TRLs isolated by ultracentrifugation of plasma were applied to these columns, and highly purified CMs were collected (purity, 90–94%). Overall eight healthy unmedicated adult volunteers (BMI, 27.2 ± 1.4 kg/m2; fasting triacylglycerol, 102.6 ± 19.5 mg/dl) participated in a feeding study, which contained an oral stable-isotope tracer (1-13C acetate). We then used this technique on plasma samples freshly collected during an 8 h human feeding study from a subset of four subjects. We analyzed fractionated lipoproteins by Western blot, isolated and derivatized triacylglycerols, and calculated fractional de novo lipogenesis. The results demonstrated effective separation of postprandial lipoproteins and substantially improved purity compared with ultracentrifugation protocols, using the immunoaffinity method. This method can be used to better delineate the role of dietary sugar and fat on postprandial lipids in cardiovascular risk and explore the potential role of CM remnants in atherosclerosis.

Severe hypertriglyceridemia (HTG) is a relatively common form of dyslipidemia with a complex pathophysiology and serious health complications. HTG can develop in the presence of rare genetic factors disrupting genes involved in the triglyceride (TG) metabolic pathway, including large-scale copy-number variants (CNVs). Improvements in next-generation sequencing technologies and bioinformatic analyses have better allowed assessment of CNVs as possible causes of or contributors to severe HTG. We screened targeted sequencing data of 632 patients with severe HTG and identified partial deletions of the LPL gene, encoding the central enzyme involved in the metabolism of TG-rich lipoproteins, in four individuals (0.63%). We confirmed the genomic breakpoints in each patient with Sanger sequencing. Three patients carried an identical heterozygous deletion spanning the 5 ' untranslated region (UTR) to LPL exon 2, and one patient carried a heterozygous deletion spanning the 5 ' UTR to LPL exon 1. All four heterozygous CNV carriers were determined to have multifactorial severe HTG. The predicted null nature of our identified LPL deletions may contribute to relatively higher TG levels and a more severe clinical phenotype than other forms of genetic variation associated with the disease, particularly in the polygenic state. The identification of novel CNVs in patients with severe HTG suggests that methods for CNV detection should be included in the diagnostic workup and molecular genetic evaluation of patients with high TG levels.

Abstract Background Homozygous Familial Hypercholesterolemia (HoFH) is an inherited recessive condition associated with extremely high levels of low‐density lipoprotein (LDL) cholesterol in affected individuals. It is usually caused by homozygous or compound heterozygous functional mutations in the LDL receptor (LDLR). A number of mutations causing FH have been reported in literature and such genetic heterogeneity presents great challenges for disease diagnosis. Objective We aim to determine the likely genetic defects responsible for three cases of pediatric HoFH in two kindreds. Methods We applied whole exome sequencing (WES) on the two probands to determine the likely functional variants among candidate FH genes. We additionally applied 10x Genomics (10xG) Linked‐Reads whole genome sequencing (WGS) on one of the kindreds to identify potentially deleterious structural variants (SVs) underlying HoFH. A PCR‐based screening assay was also established to detect the LDLR structural variant in a cohort of 641 patients with elevated LDL. Results In the Caucasian kindred, the FH homozygosity can be attributed to two compound heterozygous LDLR damaging variants, an exon 12 p.G592E missense mutation and a novel 3kb exon 1 deletion. By analyzing the 10xG phased data, we ascertained that this deletion allele was most likely to have originated from a Russian ancestor. In the Mexican kindred, the strikingly elevated LDL cholesterol level can be attributed to a homozygous frameshift LDLR variant p.E113fs. Conclusions While the application of WES can provide a cost‐effective way of identifying the genetic causes of FH, it often lacks sensitivity for detecting structural variants. Our finding of the LDLR exon 1 deletion highlights the broader utility of Linked‐Read WGS in detecting SVs in the clinical setting, especially when HoFH patients remain undiagnosed after WES.

Introduction: Elevated fasting and postprandial triglyceride-rich lipoproteins (TRL) are associated with increased risk for atherosclerosis. In the postprandial state circulating lipids consist of dietary fat transported from the intestine, by chylomicrons (containing ApoB48) and fat transported from the liver, in very low-density lipoproteins (VLDL, containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because current methods are inadequate to fully separate these particles. Specifically, chylomicron fractions have considerable contamination from VLDL. Hypothesis: We hypothesized that the generation of resin crosslinked to specific ApoB100 antibodies unique to VLDL would allow purification of chylomicron particles. Proof-of-principle experiments will determine: 1) individual contributions of newly synthesized fatty acids (de novo lipogenesis (DNL)), to triglycerides found in VLDL and chylomicrons; and 2) the proportion of labeled acetyl-CoA (precursor-pool value (PPV)) used to synthesize new palmitate. A difference in DNL and PPV for the purified VLDL and chylomicron fractions will confirm the successful separation of these particles. Methods: Polyclonal antibodies were produced against the C-terminus of the ApoB100 protein and crosslinked to resin generating ApoB100-specific affinity columns. To yield purified chylomicron and VLDL particles we used this technique in conjunction with stable-isotope tracer methodology in a human feeding study. Two healthy volunteers consumed a standardized diet accompanied by oral dosing of 1- 13 C-acetate every 30 minutes for 8 hours. Plasma collected hourly was used for the isolation of TRL, which was applied to the affinity resin and purified chylomicrons and VLDL collected. Triglycerides were isolated and derivatized. The enrichment of methyl-palmitate was measured by GC/MS and Mass Isotopomer Distribution Analysis was used to calculate DNL and PPV. Results: Western blots confirmed the separation of chylomicrons and VLDL by the described method. From the purified TRL fractions, the DNL-AUC (%DNL x 5 hours) values for subject 1 were chylomicron 4% and VLDL 34%, an 88% difference, and for subject 2 were chylomicron 8% and VLDL 83%, a 90% difference. The PPV calculated for each fraction in subject 1 were chylomicron 10% and VLDL 13% and in subject 2 were chylomicron 10%, VLDL 12%. On average, the PPV in the chylomicron fraction were 18% less than the VLDL fraction. Conclusions: In conclusion, results demonstrate that the immunoaffinity method effectively separated postprandial lipoproteins showing fractionated particles carry unique information regarding tissue of origin . Future applications include investigating the respective contribution of fat and sugar to atherosclerosis risk.