No abstract

We identified a family with a functional mutation in SR-BI. The mutation carriers had increased HDL cholesterol levels and a reduction in cholesterol efflux from macrophages but no significant increase in atherosclerosis. Reduced SR-BI function was associated with altered platelet function and decreased adrenal steroidogenesis. (Funded by the European Community and others.).

This article is available online at http://www.jlr.org the imagination. Because of a general consensus that HDL protects against atherosclerosis, what we shall term the HDL hypothesis, strategies have been developed to raise plasma HDL levels or to improve HDL function. However, it is increasingly questioned whether such interventions will indeed reduce the risk of atherosclerosis. This review summarizes the reported evidence that supports the HDL hypothesis. EPIDEMIOLOGYA low plasma HDL-C concentration is among the strongest, statistically independent risk factors for cardiovascular disease (CVD) (2). In a widely cited meta-analysis of four large studies (total number of individuals studied: 15,252), a 1 mg/dl increase of HDL-C levels was reported to be associated with a 2-3% decreased CVD risk (3). This result provides an epidemiological argument in favor of therapeutically raising HDL-C levels. One may draw parallels with the detrimental consequences of elevated lowdensity lipoprotein cholesterol (LDL-C) levels and blood pressure, which have been successfully controlled through therapy, resulting in signifi cant reductions of cardiovascular mortality and morbidity (4, 5) . It should be noted, however, that the associations between elevated LDL-C and blood pressure and increased CVD risk refl ect causal relationships, whereas such a relation between low HDL-C levels and increased CVD is not undisputed (6, 7). This is related to the fact that HDL-C levels are infl uenced by many different variables that also affect CVD risk: 1 ) Men have on average lower HDL-C levels than women (8). (1) reported that plasma levels of high-density lipoprotein cholesterol (HDL-C) were reduced in patients with coronary artery disease. In 1977, Gordon et al. (2) subsequently showed that low HDL-C is a risk factor for coronary heart disease in the Framingham study. These important fi ndings have given rise to a large number of diverse HDL studies over the last few decades. The numerous different and apparently unrelated beneficial effects that have since been ascribed to HDL appeal to

Background-Prospective epidemiological studies have shown that low plasma levels of HDL cholesterol (HDL-C) are associated with an increased risk for cardiovascular disease (CVD). Despite nearly 40 years of research, however, it is unclear whether this also holds true for individuals with severely reduced levels of HDL-C due to mutations in the lecithin:cholesterol acyltransferase (LCAT) gene. Better insight into CVD risk in these individuals may provide clues toward the potential of LCAT as a pharmaceutical target to raise HDL-C levels. Methods and Results-Lipids, lipoproteins, high-sensitivity C-reactive protein (CRP), and carotid artery intima-media thickness (IMT) were assessed in 47 heterozygotes for LCAT gene mutations and 58 family controls. Compared with controls, heterozygotes presented with a mean 36% decrease in HDL-C levels (PϽ0.0001), a 23% increase in triglyceride levels (PϽ0.0001), and a 2.1-fold increase in CRP levels (PϽ0.0001). Mean carotid IMT was significantly increased in heterozygotes compared with family controls (0.623Ϯ0.13 versus 0.591Ϯ0.08 mm). After adjustment for age, gender, and alcohol use, this difference proved statistically significant (PϽ0.0015). Conclusions-The data show that heterozygosity for LCAT gene defects is associated with low HDL-C levels and elevated concentration of triglycerides and CRP in plasma. This phenotype underlies increased IMT in carriers versus controls, which suggests that LCAT protects against atherosclerosis. This in turn indicates that targeting LCAT to raise HDL-C may reduce CVD risk.

Disorders of Golgi homeostasis form an emerging group of genetic defects. The highly heterogeneous clinical spectrum is not explained by our current understanding of the underlying cell-biological processes in the Golgi. Therefore, uncovering genetic defects and annotating gene function are challenging. Exome sequencing in a family with three siblings affected by abnormal Golgi glycosylation revealed a homozygous missense mutation, c.92T>C (p.Leu31Ser), in coiled-coil domain containing 115 (CCDC115), the function of which is unknown. The same mutation was identified in three unrelated families, and in one family it was compound heterozygous in combination with a heterozygous deletion of CCDC115. An additional homozygous missense mutation, c.31G>T (p.Asp11Tyr), was found in a family with two affected siblings. All individuals displayed a storage-disease-like phenotype involving hepatosplenomegaly, which regressed with age, highly elevated bone-derived alkaline phosphatase, elevated aminotransferases, and elevated cholesterol, in combination with abnormal copper metabolism and neurological symptoms. Two individuals died of liver failure, and one individual was successfully treated by liver transplantation. Abnormal N- and mucin type O-glycosylation was found on serum proteins, and reduced metabolic labeling of sialic acids was found in fibroblasts, which was restored after complementation with wild-type CCDC115. PSI-BLAST homology detection revealed reciprocal homology with Vma22p, the yeast V-ATPase assembly factor located in the endoplasmic reticulum (ER). Human CCDC115 mainly localized to the ERGIC and to COPI vesicles, but not to the ER. These data, in combination with the phenotypic spectrum, which is distinct from that associated with defects in V-ATPase core subunits, suggest a more general role for CCDC115 in Golgi trafficking. Our study reveals CCDC115 deficiency as a disorder of Golgi homeostasis that can be readily identified via screening for abnormal glycosylation in plasma.

Recent evidence suggests that tumor necrosis factor ␣ (TNF␣) signaling in vascular cells can have antiatherogenic consequences, but the mechanisms are poorly understood. TNF␣ is released by free cholesterol-loaded apoptotic macrophages, and the clearance of these cells by phagocytic macrophages may help to limit plaque development. Macrophage cholesterol uptake induces ATP-binding cassette (ABC) transporter ABCA1 promoting cholesterol efflux to apolipoprotein A-I and reducing atherosclerosis. We show that TNF␣ induces ABCA1 mRNA and protein in control and cholesterolloaded macrophages and enhances cholesterol efflux to apolipoprotein A-I. The induction of ABCA1 by TNF␣ is reduced by 65% in I B kinase ␤-deficient macrophages and by 30% in p38␣-deficient macrophages, but not in jun kinase 1 (JNK1)-or JNK2-deficient macrophages. To evaluate the potential pathophysiological significance of these observations, we fed TNF␣-secreting free cholesterol-loaded apoptotic macrophages to a healthy macrophage monolayer (phagocytes). ABCA1 mRNA and protein were markedly induced in the phagocytes, a response that was mediated both by TNF␣ signaling and by liver X receptor activation. Thus, TNF␣ signals primarily through NF-B to induce ABCA1 expression in macrophages. In atherosclerotic plaques, this process may help phagocytic macrophages to efflux excess lipids derived from the ingestion of cholesterol-rich apoptotic corpses.atherosclerosis ͉ cytokine ͉ ATP-binding cassette transporter A BCA1 belongs to the ATP-binding cassette (ABC) transporter superfamily (1, 2) and promotes efflux of cholesterol and phospholipids from cellular membranes to apolipoprotein A-I (apoA-I) (3). In macrophages, oxysterol-activated liver X receptor (LXR) (4) and retinoid X receptor form a heterodimer that binds to a direct repeat 4 sequence (5, 6) located in the proximal promoter of the ABCA1 gene, resulting in increased gene transcription and increased cholesterol and phospholipid efflux to apoA-I (7). Bone marrow transplantation studies have shown that the expression of ABCA1 in macrophage foam cells has antiatherogenic consequences (8, 9).Atherosclerosis represents an inflammatory reaction in the arterial wall, initiated by the retention of lipoprotein lipids (10, 11). One of the most studied inflammatory cytokines is tumor necrosis factor ␣ (TNF␣), which is active in both human and rodent atherosclerotic plaques (10, 12). Many of the inflammatory properties of TNF␣ suggest that TNF␣ signaling is proatherogenic (13). Paradoxically, other studies have shown that signaling by means of the TNF␣ receptor I (p55) may have an overall atheroprotective effect (14) and moreover that TNF␣ signaling through NF-B in macrophages and vascular smooth muscle cells may be antiatherogenic (14-17). However, the mechanisms of atheroprotective effects of TNF␣ signaling in macrophages are not well understood. In this work, we show that TNF␣ induces ABCA1 through NF-B in macrophages and in phagocytes ingesting apoptotic cells, revealing a previously undescribed antiat...

Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously complicated by the large number of proteins involved. As part of a strategy to identify human homologs of yeast proteins that are known to be involved in Golgi homeostasis, we identified uncharacterized transmembrane protein 199 (TMEM199, previously called C17orf32) as a human homolog of yeast V-ATPase assembly factor Vph2p (also known as Vma12p). Subsequently, we analyzed raw exome-sequencing data from families affected by genetically unsolved CDGs and identified four individuals with different mutations in TMEM199. The adolescent individuals presented with a mild phenotype of hepatic steatosis, elevated aminotransferases and alkaline phosphatase, and hypercholesterolemia, as well as low serum ceruloplasmin. Affected individuals showed abnormal N- and mucin-type O-glycosylation, and mass spectrometry indicated reduced incorporation of galactose and sialic acid, as seen in other Golgi homeostasis defects. Metabolic labeling of sialic acids in fibroblasts confirmed deficient Golgi glycosylation, which was restored by lentiviral transduction with wild-type TMEM199. V5-tagged TMEM199 localized with ERGIC and COPI markers in HeLa cells, and electron microscopy of a liver biopsy showed dilated organelles suggestive of the endoplasmic reticulum and Golgi apparatus. In conclusion, we have identified TMEM199 as a protein involved in Golgi homeostasis and show that TMEM199 deficiency results in a hepatic phenotype with abnormal glycosylation.

SUMMARY Genome-wide association studies have identified GALNT2 as a candidate gene in lipid metabolism, but it is not known how the encoded enzyme ppGal-NAc-T2, which contributes to the initiation of mucin-type O-linked glycosylation, mediates this effect. In two probands with elevated plasma high-density lipoprotein cholesterol and reduced triglycerides, we identified a mutation in GALNT2. It is shown that carriers have improved postprandial triglyceride clearance, which is likely attributable to attenuated glycosylation of apolipoprotein (apo) C-III, as observed in their plasma. This protein inhibits lipoprotein lipase (LPL), which hydrolyses plasma triglycerides. We show that an apoC-III-based peptide is a substrate for ppGalNAc-T2 while its glycosylation by the mutant enzyme is impaired. In addition, neuraminidase treatment of apoC-III which removes the sialic acids from its glycan chain decreases its potential to inhibit LPL. Combined, these data suggest that ppGalNAc-T2 can affect lipid metabolism through apoC-III glycosylation, thereby establishing GALNT2 as a lipid-modifying gene.