Genetic and secondary causes of severe HDL deficiency and cardiovascular disease.

We assessed secondary and genetic causes of severe HDL deficiency in 258,252 subjects, of whom 370 men (0.33%) and 144 women (0.099%) had HDL cholesterol levels <20 mg/dl. We excluded 206 subjects (40.1%) with significant elevations of triglycerides, C-reactive protein, glycosylated hemoglobin, myeloperoxidase, or liver enzymes and men receiving testosterone. We sequenced 23 lipid-related genes in 201 (65.3%) of 308 eligible subjects. Mutations (23 novel) and selected variants were found at the following gene loci: 1) ABCA1 (26.9%): 2 homozygotes, 7 compound or double heterozygotes, 30 heterozygotes, and 2 homozygotes and 13 heterozygotes with variants rs9282541/p.R230C or rs111292742/c.-279C>G; 2) LCAT (12.4%): 1 homozygote, 3 compound heterozygotes, 13 heterozygotes, and 8 heterozygotes with variant rs4986970/p.S232T; 3) APOA1 (5.0%): 1 homozygote and 9 heterozygotes; and 4) LPL (4.5%): 1 heterozygote and 8 heterozygotes with variant rs268/p.N318S. In addition, 4.5% had other mutations, and 46.8% had no mutations. Atherosclerotic cardiovascular disease (ASCVD) prevalence rates in the ABCA1, LCAT, APOA1, LPL, and mutation-negative groups were 37.0%, 4.0%, 40.0%, 11.1%, and 6.4%, respectively. Severe HDL deficiency is uncommon, with 40.1% having secondary causes and 48.8% of the subjects sequenced having ABCA1, LCAT, APOA1, or LPL mutations or variants, with the highest ASCVD prevalence rates being observed in the ABCA1 and APOA1 groups.

A longitudinal circulating tumor DNA-based model associated with survival in metastatic non-small-cell lung cancer.

One of the great challenges in therapeutic oncology is determining who might achieve survival benefits from a particular therapy. Studies on longitudinal circulating tumor DNA (ctDNA) dynamics for the prediction of survival have generally been small or nonrandomized. We assessed ctDNA across 5 time points in 466 non-small-cell lung cancer (NSCLC) patients from the randomized phase 3 IMpower150 study comparing chemotherapy-immune checkpoint inhibitor (chemo-ICI) combinations and used machine learning to jointly model multiple ctDNA metrics to predict overall survival (OS). ctDNA assessments through cycle 3 day 1 of treatment enabled risk stratification of patients with stable disease (hazard ratio (HR) = 3.2 (2.0-5.3), P < 0.001; median 7.1 versus 22.3 months for high- versus low-intermediate risk) and with partial response (HR = 3.3 (1.7-6.4), P < 0.001; median 8.8 versus 28.6 months). The model also identified high-risk patients in an external validation cohort from the randomized phase 3 OAK study of ICI versus chemo in NSCLC (OS HR = 3.73 (1.83-7.60), P = 0.00012). Simulations of clinical trial scenarios employing our ctDNA model suggested that early ctDNA testing outperforms early radiographic imaging for predicting trial outcomes. Overall, measuring ctDNA dynamics during treatment can improve patient risk stratification and may allow early differentiation between competing therapies during clinical trials.

Genetic variation at the NPC1L1 gene locus, plasma lipoproteins, and heart disease risk in the elderly.

Niemann-Pick C1-like 1 protein (NPC1L1) plays a critical role in intestinal cholesterol absorption. Our objective was to examine whether five variants (-133A>G, -18A>C, L272L, V1296V, and U3_28650A>G) at the NPC1L1 gene have effects on lipid levels, prevalence, and incidence of coronary heart disease (CHD) and lipid-lowering response to pravastatin. We studied 5,804 elderly participants from the PROSPER study, who were randomized to prava-statin 40 mg/day or placebo and were followed on average for 3.2 years. In the adjusted gender-pooled analyses, homozygous carriers of the minor alleles at four NPC1L1 sites (-18A>C, L272L, V1296V, and U3_28650A>G, minor allele frequencies 0.15-0.33) had 2-8% higher LDL-cholesterol (LDL-C) levels at baseline than homozygous carriers of the common alleles (P < 0.05). Homozygotes for the rare alleles also had a significant increase in the risk of CHD events on trial (range of hazard ratios 1.50-1.67; P < 0.02), regardless of the treatment regimen. The -133 A>G polymorphism and not other variants was associated with 6 month LDL-C lowering (P = 0.02). Our data indicate that variation in the NPC1L1 gene is associated with plasma total and LDL-C levels and CHD risk.

First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography-Urgent need for harmonisation of analytical methods.

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

The association between hypercholesterolemia and sitosterolemia, and report of a sitosterolemia kindred.

BACKGROUND: Sitosterolemia is associated with increases in intestinal sterol absorption, low-density lipoprotein cholesterol (LDL-C), and cardiovascular disease risk. OBJECTIVE: We examined the relationship between hypercholesterolemia and sitosterolemia in a large population and report a new sitosterolemia case. METHODS: Plasma sterol concentrations were measured by gas chromatography/mass spectrometry, and LDL-C by direct assay. RESULTS: Of 207,926 subjects tested, 4.3% had LDL-C ≥190 mg/dL. Plasma ß-sitosterol concentrations ≥8.0 mg/L (99th percentile) were found in 4.3% of these subjects vs 0.72% with LDL-C <130 mg/dL. Among all subjects, 0.050% had ß-sitosterol levels ≥15.0 mg/L, consistent with sitosterolemia, while among those with LDL-C ≥190 mg/dL, 0.334% had this rare disorder. A 13-year-old boy with the highest LDL-C (679 mg/dL) of all subjects had planar xanthomas and a ß-sitosterol level of 53.5 mg/L (normal <3.3 mg/L). He was a compound heterozygote for 2 ABCG8 mutations (p.N409D and an intron 11+2T>A splice site mutation). On a low-cholesterol and plant-sterol diet, his LDL-C decreased to 485 mg/dL (-29%) and ß-sitosterol to 44.6 mg/L (-27%). On atorvastatin 20 mg/d, his LDL-C decreased to 299 mg/dL (-38%). With added ezetimibe 10 mg/d, his LDL-C normalized to 60 mg/dL (-80% further decrease); and his ß-sitosterol decreased to 14.1 mg/L (-68% further decrease). CONCLUSIONS: Our data indicate that about 4% of subjects with LDL-C concentrations ≥190 mg/dL have plasma ß-sitosterol concentrations above the 99th percentile and about 0.3% have concentrations consistent with sitosterolemia. Therefore, this diagnosis should be considered in such patients.

Unusual genetic variants associated with hypercholesterolemia in Argentina.

BACKGROUND AND AIMS: Marked hypercholesterolemia, defined as low density lipoprotein cholesterol (LDL-C) levels ≥ 190 mg/dL, may be due to LDLR, APOB, and PCSK9 variants. In a recent analysis, only 1.7% of cases had such variants. Our goal was to identify other potential genetic causes of hypercholesterolemia. METHODS: In a total of 51,253 subjects with lipid testing, 3.8% had elevated total cholesterol >300 mg/dL and/or LDL-C≥190 mg/dL. Of these, 246 were further studied, and 69 without kidney, liver, or thyroid disease and who met Dutch Lipid Clinic Network criteria of ≥6 points had DNA sequencing done at the LDLR, APOB, PCSK9, APOE, LDLRAP1, STAP1, ABCG5, ABCG8, CYP27A1, LIPA, LIPC, LIPG, LPL, and SCARB1 gene loci and also had 10 SNP analysis for a weighted high LDL-C genetic risk score. RESULTS: In the 69 subjects with genetic analyses, the following variants were observed in 37 subjects (53.6%): LDLR (n = 20, 2 novel), ABCG5/8 (n = 7, 2 novel), APOB (n = 3, 1 novel), CYP27A1 (n = 3, 1 novel), LIPA (n = 2, 1 novel), APOE (n = 2), LIPC (n = 1, novel), LIPG (n = 1, novel), and SCARB1 (n = 1); 14 subjects (20.3%) had a high polygenic score, with 4 (5.8%) having no variants. CONCLUSIONS: Our data indicate that in addition to variants in LDLR, APOB, PCSK9, APOE, LDLRAP1, and STAP1, variants in ABCG5/8, CYP27A1, LIPA, LIPC, and LIPG may be associated with hypercholesterolemia and such information should be used to optimize therapy.

Diagnosis and treatment of high density lipoprotein deficiency.

Low serum high density lipoprotein cholesterol level (HDL-C) <40 mg/dL in men and <50 mg/dL in women is a significant independent risk factor for cardiovascular disease (CVD), and is often observed in patients with hypertriglyceridemia, obesity, insulin resistance, and diabetes. Patients with marked deficiency of HDL-C (<20 mg/dL) in the absence of secondary causes are much less common (<1% of the population). These patients may have homozygous, compound heterozygous, or heterozygous defects involving the apolipoprotein (APO)AI, ABCA1, or lecithin:cholesterol acyl transferase genes, associated with apo A-I deficiency, apoA-I variants, Tangier disease , familial lecithin:cholesteryl ester acyltransferase deficiency, and fish eye disease. There is marked variability in laboratory and clinical presentation, and DNA analysis is necessary for diagnosis. These patients can develop premature CVD, neuropathy, kidney failure, neuropathy, hepatosplenomegaly and anemia. Treatment should be directed at optimizing all non-HDL risk factors.

Pharmacogenomics of statin responsiveness.

Statins are widely prescribed and are established as first-line therapy for the primary and secondary prevention of coronary artery disease. However, the benefit of treatment varies between patients. Genetic variation can contribute to interindividual variations in clinical efficacy of drug therapy, and significant progress has been made in identifying common genetic polymorphisms that influence responsiveness to statin therapy. To date, >30 candidate genes related to pharmacokinetics and pharmacodynamics of statins have been investigated as potential determinants of drug responsiveness in terms of low-density lipoprotein cholesterol lowering. Genetic variation at gene loci that affect intestinal cholesterol absorption include apolipoprotein (apo) E; adenosine triphosphate-binding cassette transporter G5 and G8; cholesterol production, such as 3-hydroxy-3-methylglutaryl coenzyme A reductase; and lipoprotein catabolism, such as apoB and the low-density lipoprotein receptor, all may play a role in modulating responsivesness as well genes involved in metabolism of statins such as cytochrome P450. However, there is considerable variation in results reported, and the data suggest that combined analysis of multiple genetic variants in several genes, all of which have possible functional significance, is more likely to give significant results than single gene studies in small sample populations. In the future, pharmacogenomic studies with a greater number of participants (>2,000 participants) should provide a better picture as to who is most likely and who is least likely to benefit from statin therapy.

Case report: A novel apolipoprotein A-I missense mutation apoA-I (Arg149Ser)Boston associated with decreased lecithin-cholesterol acyltransferase activation and cellular cholesterol efflux.

We report a novel heterozygous apolipoprotein A-I (apoA-I) missense mutation (c.517C>A, p.Arg149Ser, designated as apoA-IBoston) in a 67-year-old woman and her 2 sons, who had mean serum high-density lipoprotein (HDL) cholesterol, apoA-I, and apoA-I in very large α-1 HDL that were 10%, 35%, and 16% of normal, respectively (all P < .05). The percentage of HDL cholesterol in the esterified form was also significantly (P < .05) reduced to 52% of control values. Cholesteryl ester tranfer protein (CETP) activity was normal. The mean global, adenosine triphosphate (ATP)-binding cassette transporter A1 and scavenger receptor B type I-mediated cellular cholesterol efflux capacity in apoB-depleted serum from affected family members were 41%, 37%, 47%, 54%, and 48% of control values, respectively (all P < .05). lecithin-cholesterol acyltransferase (LCAT) activity in plasma was 71% of controls, whereas in the cell-based assay, it was 73% of control values (P < .05). The data indicate that this novel apoA-I missense is associated with markedly decreased levels of HDL cholesterol and very large α-1 HDL, as well as decreased serum cellular cholesterol efflux and LCAT activity, but not with premature coronary heart disease, similar to other apoA-I mutations that have been associated with decreased LCAT activity.

Common mitochondrial DNA deletion associated with sudden natural death in adults.

One of the most frequent causes of death in developed countries is sudden natural death (SND), which is the most common indication for medico-legal autopsies. Cardiac diseases are frequently detected among SND. Mitochondrial DNA (mtDNA) is easily damaged by reactive oxygen species, and it may cause dysfunction in tissues, leading to early events in cardiovascular disease. A specific mtDNA deletion of 4977 bp is associated to aging, myocardial dysfunction, and bioenergetic deficit. The potential link between mtDNA damage and SND has not been investigated before. Our aim was to evaluate the accumulation of the common mtDNA4977-deletion in cardiac muscle samples from autopsies of SND in adults (n = 14) in comparison to control samples from unnatural deaths (n = 12). Serial dilution-polymerase chain reaction method was performed to estimate the proportion of the total mtDNA harboring the mtDNA4977-deletion. Coefficient variation intra-assay was 8%, and inter-assay was 12%. MtDNA4977-deletion percentage was higher in samples obtained from victims of SND than in those from subjects who died of unnatural causes (p < 0.05). No differences in mtDNA4977-deletion were found between SND victims 39-51 years old, and no correlation was found between these samples and age, r = 0.30, p = 0.29 while it was significant among control samples, r = 0.68, p < 0.05. The association between mtDNA4977 deletion with SND victims might offer a tool to provide additional information to clarify complex SND investigations.