Ian Tietjen

Novel mutations in scavenger receptor BI associated with high HDL cholesterol in humans

Brunham LR, Tietjen I, Bochem AE, Singaraja RR, Franchini PL, Radomski C, Mattice M, Legendre A, Hovingh GK, Kastelein JJP, Hayden MR. Novel mutations in scavenger receptor BI associated with high HDL cholesterol in humans.

Increased risk of coronary artery disease in Caucasians with extremely low HDL cholesterol due to mutations in ABCA1, APOA1, and LCAT.

Mutations in ABCA1, APOA1, and LCAT reduce HDL cholesterol (HDLc) in humans. However, the prevalence of these mutations and their relative effects on HDLc reduction and risk of coronary artery disease (CAD) are less clear. Here we searched for ABCA1, APOA1, and LCAT mutations in 178 unrelated probands with HDLc <10th percentile but no other major lipid abnormalities, including 89 with ≥1 first-degree relative with low HDLc (familial probands) and 89 where familial status of low HDLc is uncertain (unknown probands). Mutations were most frequent in LCAT (15.7%), followed by ABCA1 (9.0%) and APOA1 (4.5%), and were found in 42.7% of familial but only 14.6% of unknown probands (p=2.44∗10(-5)). Interestingly, only 16 of 24 (66.7%) mutations assessed in families conferred an average HDLc <10th percentile. Furthermore, only mutation carriers with HDLc <5th percentile had elevated risk of CAD (odds ratio (OR)=2.26 for 34 ABCA1 mutation carriers vs. 149 total first-degree relative controls, p=0.05; OR=2.50 for 26 APOA1 mutation carriers, p=0.04; OR=3.44 for 38 LCAT mutation carriers, p=1.1∗10(-3)). These observations show that mutations in ABCA1, APOA1, and LCAT are sufficient to explain >40% of familial hypoalphalipoproteinemia in this cohort. Moreover, individuals with mutations and large reductions in HDLc have increased risk of CAD. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Aminoadamantanes with persistent in vitro efficacy against H1N1 (2009) influenza A.

A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy against strains of influenza A including those having an S31N mutation in M2 proton channel that confer resistance to amantadine and rimantadine. The addition of as little as one CH2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1). Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding. But electrophysiology using the full length S31N M2 protein in HEK cells showed no blockade. A wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to representative drugs within one passage with mutations in M2 TMD, but A/Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and the resistant virus had no mutations in M2 TMD. The results indicate that 2-alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.

The impact of partial and complete loss-of-function mutations in endothelial lipase on high-density lipoprotein levels and functionality in humans

Background—Endothelial lipase is a phospholipase with activity against high-density lipoprotein. Although a small number of mutations in LIPG have been described, the role of LIPG in protection against atherosclerosis is unclear. Methods and Results—We identified 8 loss-of-function (LOF) mutations in LIPG in individuals with high-density lipoprotein cholesterol. Functional analysis confirmed that most rare mutations abolish lipase activity in vitro, indicating complete LOF, whereas 2 more common mutations N396S and R476W reduce activity by ≈50%, indicating partial LOF and implying ≈50% and ≈75% remaining endothelial lipase function in heterozygous complete LOF and partial LOF mutation carriers, respectively. complete LOF mutation carriers had significantly higher plasma high-density lipoprotein cholesterol levels compared with partial LOF mutation carriers. Apolipoprotein B-depleted serum from complete LOF carriers showed significantly enhanced cholesterol efflux acceptor capacity, whereas only trends were observed in partial LOF carriers. Carriers of LIPG mutations exhibited trends toward reduced coronary artery disease in 4 independent cohorts (meta-analysis odds ratio, 0.7; P=0.04). Conclusions—Our data suggest that the impact of LIPG mutations is directly related to their effect on endothelial lipase function and support that antagonism of endothelial lipase function improves cardioprotection.

Segregation of LIPG, CETP, and GALNT2 Mutations in Caucasian Families with Extremely High HDL Cholesterol

To date, few mutations are described to underlie highly-elevated HDLc levels in families. Here we sequenced the coding regions and adjacent sequence of the LIPG, CETP, and GALNT2 genes in 171 unrelated Dutch Caucasian probands with HDLc≥90th percentile and analyzed segregation of mutations with lipid phenotypes in family members. In these probands, mutations were most frequent in LIPG (12.9%) followed by GALNT2 (2.3%) and CETP (0.6%). A total of 6 of 10 mutations in these three genes were novel (60.0%), and mutations segregated with elevated HDLc in families. Interestingly, the LIPG mutations N396S and R476W, which usually result in elevated HDLc, were unexpectedly found in 6 probands with low HDLc (i.e., ≤10th percentile). However, 5 of these probands also carried mutations in ABCA1, LCAT, or LPL. Finally, no CETP and GALNT2 mutations were found in 136 unrelated probands with low HDLc. Taken together, we show that rare coding and splicing mutations in LIPG, CETP, and GALNT2 are enriched in persons with hyperalphalipoproteinemia and segregate with elevated HDLc in families. Moreover, LIPG mutations do not overcome low HDLc in individuals with ABCA1 and possibly LCAT and LPL mutations, indicating that LIPG affects HDLc levels downstream of these proteins.

Screening of the pan-African Natural Product Library identifies ixoratannin A-2 and boldine as novel HIV-1 inhibitors

The continued burden of HIV in resource-limited regions such as parts of sub-Saharan Africa, combined with adverse effects and potential risks of resistance to existing antiretroviral therapies, emphasize the need to identify new HIV inhibitors. Here we performed a virtual screen of molecules from the pan-African Natural Product Library, the largest collection of medicinal plant-derived pure compounds on the African continent. We identified eight molecules with structural similarity to reported interactors of Vpu, an HIV-1 accessory protein with reported ion channel activity. Using in vitro HIV-1 replication assays with a CD4+ T cell line and peripheral blood mononuclear cells, we confirmed antiviral activity and minimal cytotoxicity for two compounds, ixoratannin A-2 and boldine. Notably, ixoratannin A-2 retained inhibitory activity against recombinant HIV-1 strains encoding patient-derived mutations that confer resistance to protease, non-nucleoside reverse transcriptase, or integrase inhibitors. Moreover, ixoratannin A-2 was less effective at inhibiting replication of HIV-1 lacking Vpu, supporting this protein as a possible direct or indirect target. In contrast, boldine was less effective against a protease inhibitor-resistant HIV-1 strain. Both ixoratannin A-2 and boldine also inhibited in vitro replication of hepatitis C virus (HCV). However, BIT-225, a previously-reported Vpu inhibitor, demonstrated antiviral activity but also cytotoxicity in HIV-1 and HCV replication assays. Our work identifies pure compounds derived from African plants with potential novel activities against viruses that disproportionately afflict resource-limited regions of the world.

Identification of four novel genes contributing to familial elevated plasma HDL cholesterol in humans

While genetic determinants strongly influence HDL cholesterol (HDLc) levels, most genetic causes underlying variation in HDLc remain unknown. We aimed to identify novel rare mutations with large effects in candidate genes contributing to extreme HDLc in humans, utilizing family-based Mendelian genetics. We performed next-generation sequencing of 456 candidate HDLc-regulating genes in 200 unrelated probands with extremely low (≤10th percentile) or high (≥90th percentile) HDLc. Probands were excluded if known mutations existed in the established HDLc-regulating genes ABCA1, APOA1, LCAT, cholesteryl ester transfer protein (CETP), endothelial lipase (LIPG), and UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2). We identified 93 novel coding or splice-site variants in 72 candidate genes. Each variant was genotyped in the proband’s family. Family-based association analyses were performed for variants with sufficient power to detect significance at P < 0.05 with a total of 627 family members being assessed. Mutations in the genes glucokinase regulatory protein (GCKR), RNase L (RNASEL), leukocyte immunoglobulin-like receptor 3 (LILRA3), and dynein axonemal heavy chain 10 (DNAH10) segregated with elevated HDLc levels in families, while no mutations associated with low HDLc. Taken together, we have identified mutations in four novel genes that may play a role in regulating HDLc levels in humans.

Two novel mutations in apolipoprotein C3 underlie atheroprotective lipid profiles in families.

Apolipoprotein C3 (APOC3) mutations carriers typically display high plasma high‐density lipoprotein cholesterol (HDL‐C) and low triglycerides (TGs). We set out to investigate the prevalence and clinical consequences of APOC3 mutations in individuals with hyperalphalipoproteinemia. Two novel mutations (c.‐13‐2A>G and c.55+1G>A) and one known mutation (c.127G>A;p.Ala43Thr) were found. Lipid profiles and apoCIII isoform distributions were measured. c.55+1G>A mutation carriers displayed higher HDL‐C percentiles (35.6 ± 35.8 vs 99.0 ± 0, p = 0.002) and lower TGs (0.51 (0.37–0.61) vs 1.42 (1.12–1.81) mmol/l, p = 0.007) and apoCIII levels (4.24 ± 1.57 vs 7.33 ± 3.61 mg/dl, p = 0.18). c.‐13‐2A>G mutation carriers did not display significantly different HDL‐C levels (84.0 ± 30.0 vs 63.7 ± 45.7, p = 0.50), a trend towards lower TGs [0.71 (0.54 to 0.78) vs 0.85 (0.85 to –) mmol/l, p = 0.06] and significantly lower apoCIII levels (3.09 ± 1.08 vs 11.45 ± 1.06 mg/dl, p = 0.003). p.Ala43Thr mutation carriers displayed a trend towards higher HDL‐C percentiles (91.2 ± 31.8 vs 41.0 ± 29.7 mmol/l, p = 0.06) and significantly lower TGs [0.58 (0.36–0.63) vs 0.95 (0.71–1.20) mmol/l, p = 0.02] and apoCIII levels (4.92 ± 2.33 vs 6.60 ± 1.60, p = 0.25). Heterozygosity for APOC3 mutations results in high HDL‐C and low TGs and apoCIII levels. This favourable lipid profile in patients with genetically low apoCIII levels holds promise for current studies investigating the potential of apoCIII inhibition as a novel therapeutic in cardiovascular disease prevention.

Unciaphenol, an Oxygenated Analogue of the Bergman Cyclization Product of Uncialamycin Exhibits Anti-HIV Activity.

Unciaphenol (2), an oxygenated analogue of the Bergman cyclization product of the enediyne uncialamycin (1), has been isolated along with 1 from cultures of the actinomycete Streptomyces uncialis. It is proposed that the C-22 OH substituent in 2 might arise from the attack of a nucleophilic oxygen species on the p-benzyne diradical intermediate IA in the Bergman cyclization of 1. 2 shows in vitro anti-HIV activity against viral strains that are resistant to clinically utilized anti-retroviral therapies.

ABCA8 Regulates Cholesterol Efflux and High-Density Lipoprotein Cholesterol Levels

Objective— High-density lipoproteins (HDL) are considered to protect against atherosclerosis in part by facilitating the removal of cholesterol from peripheral tissues. However, factors regulating lipid efflux are incompletely understood. We previously identified a variant in adenosine triphosphate–binding cassette transporter A8 (ABCA8) in an individual with low HDL cholesterol (HDLc). Here, we investigate the role of ABCA8 in cholesterol efflux and in regulating HDLc levels. Approach and Results— We sequenced ABCA8 in individuals with low and high HDLc and identified, exclusively in low HDLc probands, 3 predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G and p.Thr741Stop [T741X]). HDLc levels were lower in heterozygous mutation carriers compared with first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P=0.005). HDLc levels were significantly decreased by 29% (P=0.01) in Abca8b−/− mice on a high-cholesterol diet compared with wild-type mice, whereas hepatic overexpression of human ABCA8 in mice resulted in significant increases in plasma HDLc and the first steps of macrophage-to-feces reverse cholesterol transport. Overexpression of wild-type but not mutant ABCA8 resulted in a significant increase (1.8-fold; P=0.01) of cholesterol efflux to apolipoprotein AI in vitro. ABCA8 colocalizes and interacts with adenosine triphosphate–binding cassette transporter A1 and further potentiates adenosine triphosphate–binding cassette transporter A1–mediated cholesterol efflux. Conclusions— ABCA8 facilitates cholesterol efflux and modulates HDLc levels in humans and mice.