Patrizia Tarugi

Inherited Apolipoprotein A-V Deficiency in Severe Hypertriglyceridemia

Objective— Mutations in LPL or APOC2 genes are recognized causes of inherited forms of severe hypertriglyceridemia. However, some hypertrigliceridemic patients do not have mutations in either of these genes. Because inactivation or hyperexpression of APOA5 gene, encoding apolipoprotein A-V (apoA-V), causes a marked increase or decrease of plasma triglycerides in mice, and because some common polymorphisms of this gene affect plasma triglycerides in humans, we have hypothesized that loss of function mutations in APOA5 gene might cause hypertriglyceridemia. Methods and Results— We sequenced APOA5 gene in 10 hypertriglyceridemic patients in whom mutations in LPL and APOC2 genes had been excluded. One of them was found to be homozygous for a mutation in APOA5 gene (c.433 C>T, Q145X), predicted to generate a truncated apoA-V devoid of key functional domains. The plasma of this patient was found to activate LPL in vitro less efficiently than control plasma, thus suggesting that apoA-V might be an activator of LPL. Ten carriers of Q145X mutation were found in the patient’s family; 5 of them had mild hypertriglyceridemia. Conclusions— As predicted from animal studies, apoA-V deficiency is associated with severe hypertriglyceridemia in humans. This observation suggests that apoA-V regulates the secretion and/or catabolism of triglyceride-rich lipoproteins.

A Novel Loss of Function Mutation of PCSK9 Gene in White Subjects With Low-Plasma Low-Density Lipoprotein Cholesterol

Objectives—The PCSK9 gene, encoding a pro-protein convertase involved in posttranslational degradation of low-density lipoprotein receptor, has emerged as a key regulator of plasma low-density lipoprotein cholesterol. In African-Americans two nonsense mutations resulting in loss of function of PCSK9 are associated with a 30% to 40% reduction of plasma low-density lipoprotein cholesterol. The aim of this study was to assess whether loss of function mutations of PCSK9 were a cause of familial hypobetalipoproteinemia and a determinant of low-plasma low-density lipoprotein cholesterol in whites. Methods and Results—We sequenced PCSK9 gene in 18 familial hypobetalipoproteinemia subjects and in 102 hypocholesterolemic blood donors who were negative for APOB gene mutations known to cause familial hypobetalipoproteinemia. The PCSK9 gene variants found in these 2 groups were screened in 42 subjects in the lowest (<5th) percentile, 44 in the highest (>95th) percentile, and 100 with the average plasma cholesterol derived from general population. In one familial hypobetalipoproteinemia kindred and in 2 hypocholesterolemic blood donors we found a novel PCSK9 mutation in exon 1 (c.202delG) resulting in a truncated peptide (Ala68fsLeu82X). Two familial hypobetalipoproteinemia subjects and 4 hypocholesterolemic blood donors were carriers of the R46L substitution previously reported to be associated with reduced low-density lipoprotein cholesterol as well as other rare amino acid changes (T77I, V114A, A522T and P616L) not found in the other groups examined. Conclusions—We discovered a novel inactivating mutation as well as some rare nonconservative amino acid substitutions of PCSK9 in white hypocholesterolemic individuals.

PCSK9 Dominant Negative Mutant Results in Increased LDL Catabolic Rate and Familial Hypobetalipoproteinemia

Objective—Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central player in the regulation of cholesterol homeostasis, increasing the low-density lipoprotein (LDL) receptor degradation. Our study aimed at exploring the pathogenic consequences in vivo and in vitro of a PCSK9 prodomain mutation found in a family with hypobetalipoproteinemia (FHBL). Methods and Results—A white 49-year-old diabetic man had profound FBHL (LDLC: 16 mg/dL) whereas his daughter and sister displayed a milder phenotype (LDLC 44 mg/dL and 57 mg/dL, respectively), all otherwise healthy with a normal liver function. A monoallelic PCSK9 double-mutant R104C/V114A cosegregated with FBHL, with no mutation found at other FHBL-causing loci. A dose-effect was also found in FBHL relatives for plasma APOB and PCSK9 (very-low to undetectable in proband, ≈50% decreased in sister and daughter) and LDL catabolic rate (256% and 88% increased in proband and daughter). Transient transfection in hepatocytes showed severely impaired processing and secretion of the double mutant which acted as a dominant negative over secretion of wild-type PCSK9. Conclusion—These results show that heterozygous PCSK9 missense mutations may associate with profound hypobetalipoproteinemia and constitute the first direct evidence in human that decrease of plasma LDLC concentrations associated to PCSK9 LOF mutations are attributable to an increased clearance rate of LDL.

Hypobetalipoproteinemia: genetics, biochemistry, and clinical spectrum.

Hypobetalipoproteinemias (HBL) represent a heterogeneous group of disorders characterized by reduced plasma levels of total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (apoB) below the 5th percentile of the distribution in the population. HBL are defined as primary or secondary according to the underlying causes. Primary monogenic HBL are caused by mutations in several known genes (APOB, PCSK9, MTP, SARA2) or mutations in genes not yet identified. Familial hypobetalipoproteinemia (FHBL) is the most frequent monogenic form of HBL with a dominant mode of inheritance. It may be due to loss-of-function mutations in APOB or, less frequently, in PCSK9 genes. The rare recessive forms of primary monogenic HBL are represented by abetalipoproteinemia (ABL) and chylomicron retention disease (CMRD) due to mutations in MTP and SARA2 genes, respectively. The clinical phenotype of heterozygous FHBL is usually mild, being frequently characterized by fatty liver. The clinical phenotype of homozygous FHBL, ABL, and CMRD is usually severe being characterized by intestinal lipid malabsorption and fat-soluble vitamin deficiency. Secondary HBL are due to several nongenetic factors such as diet, drugs, and disease-related conditions. The aim of this review is to discuss the biochemistry, genetics, and clinical spectrum of HBL and to provide a clinical and laboratory diagnostic algorithm.

Characterization of Three Kindreds With Familial Combined Hypolipidemia Caused by Loss-of-Function Mutations of ANGPTL3

Background— Angiopoietin-like protein 3 (ANGPTL3) affects lipid metabolism by inhibiting the activity of lipoprotein and endothelial lipases. Angptl3 knockout mice have marked hypolipidemia, and heterozygous carriers of ANGPLT3, loss-of-function mutations were found among individuals in the lowest quartile of plasma triglycerides in population studies. Recently, 4 related individuals with primary hypolipidemia were found to be compound heterozygotes for ANGPTL3 loss-of-function mutations. Methods and Results— We resequenced ANGPTL3 in 4 members of 3 kindreds originally identified for very low levels of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol (0.97±0.16 and 0.56±0.20 mmol/L, respectively) in whom no mutations of known candidate genes for monogenic hypobetalipoproteinemia and hypoalphalipoproteinemia had been detected. These subjects were found to be homozygous or compound heterozygous for ANGPTL3 loss-of-function mutations (p.G400VfsX5, p.I19LfsX22/p.N147X) associated with the absence of ANGPTL3 in plasma. They had reduced plasma levels of triglyceride-containing lipoproteins and of HDL particles that contained only apolipoprotein A-I and pre-&bgr;–high-density lipoprotein. In addition, their apolipoprotein B–depleted sera had a reduced capacity to promote cell cholesterol efflux through the various pathways (ABCA1-, SR-BI–, and ABCG1-mediated efflux); however, these subjects had no clinical evidence of accelerated atherosclerosis. Heterozygous carriers of the ANGPTL3 mutations had low plasma ANGPTL3 and moderately reduced low-density lipoprotein cholesterol (2.52±0.38 mmol/L) but normal plasma high-density lipoprotein cholesterol. Conclusions— Complete ANGPTL3 deficiency caused by loss-of-function mutations of ANGPTL3 is associated with a recessive hypolipidemia characterized by a reduction of apolipoprotein B and apolipoprotein A-I–containing lipoproteins, changes in subclasses of high-density lipoprotein, and reduced cholesterol efflux potential of serum. Partial ANGPTL3 deficiency is associated only with a moderate reduction of low-density lipoprotein.

Nonsynonymous Mutations within APOB in Human Familial Hypobetalipoproteinemia: EVIDENCE FOR FEEDBACK INHIBITION OF LIPOGENESIS AND POSTENDOPLASMIC RETICULUM DEGRADATION OF APOLIPOPROTEIN B*

Five nontruncating missense APOB mutations, namely A31P, G275S, L324M, G912D, and G945S, were identified in heterozygous carriers of familial hypobetalipoproteinemia (FHBL) in the Italian population. To test that the FHBL phenotype was a result of impaired hepatic secretion of mutant apoB proteins, we performed transfection studies using McA-RH7777 cells stably expressing wild type or mutant forms of human apolipoprotein B-48 (apoB-48). All mutant proteins displayed varied impairment in secretion, with G912D the least affected and A31P barely secreted. Although some A31P was degraded by proteasomes, a significant proportion of it (although inappropriately glycosylated) escaped endoplasmic reticulum (ER) quality control and presented in the Golgi compartment. Degradation of the post-ER A31P was achieved by autophagy. Expression of A31P also decreased secretion of endogenous apoB and triglycerides, yet the impaired lipoprotein secretion did not lead to lipid accumulation in the cells or ER stress. Rather, expression of genes involved in lipogenesis was down-regulated, including liver X receptor α, sterol regulator element-binding protein 1c, fatty acid synthase, acetyl-CoA carboxylase 1, stearoyl-CoA desaturase 1, and lipin-1. These results suggest that feedback inhibition of hepatic lipogenesis in conjunction with post-ER degradation of misfolded apoB proteins can contribute to reduce fat accumulation in the FHBL liver.

A study of fatty liver disease and plasma lipoproteins in a kindred with familial hypobetalipoproteinemia due to a novel truncated form of apolipoprotein B (APO B-54.5)

BACKGROUND/AIMS Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by reduced plasma levels of low-density lipoproteins. It can be caused by mutations in the gene encoding apolipoprotein B-100 (apo B), leading to the formation of truncated apo Bs which have a reduced capacity to export lipids from the hepatocytes as lipoprotein constituents. Case reports suggest the occurrence of liver disease in FHBL, but there are no studies of liver involvement in FHBL with defined apo B gene mutations. The presence of fatty liver disease was investigated in a large FHBL kindred. METHODS Plasma lipoprotein and apolipoprotein analysis, liver function tests, and apo B gene sequence were performed in 16 members of a FHBL kindred. The presence of fatty liver was assessed by ultrasound and computed tomography scanning. RESULTS The proband, a non-obese heavy drinker male with hypobetalipoproteinemia, had steatohepatitis with fibrosis. He was heterozygous for a novel non-sense mutation of apo B gene producing a truncated apo B of 2745 amino acids (designated apo B-54.5, having half the size of normal apo B-100). Seven other members of his kindred carried apo B-54.5. Although all of them were hypolipidemic, their lipid levels showed a large inter-individual variability not accounted for by polymorphisms of genes involved in apo B metabolism. Four carriers (two heavy drinkers and two teetotallers), irrespective of their plasma lipid levels, had ultrasonographic evidence of fatty liver. In the other four carriers no evidence of fatty liver was found. CONCLUSIONS In this kindred apo B-54.5 predisposes to fatty liver, which however may require some additional factors to become clinically relevant.

Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma

We report a very rapid and simple isocratic reversed-phase HPLC separation of malondialdehyde (MDA) in normal human plasma without previous purification of the MDA-2-thiobarbituric acid (TBA) complex. The separation of MDA-TBA complex was performed using a 250x4.6 mm Nucleosil-5C18 column with a mobile phase composed of 35% methanol and 65% 50 mM sodium phosphate buffer, pH 7.0. Samples of 50 microl (composed of 100 microl plasma mixed with 1.0 ml of 0.2% 2-thiobarbituric acid in 2 M sodium acetate buffer containing 1 mM diethylenetriaminepentaacetic acid, pH 3.5, and 10 microl of 5% 2,6-di-tert.-butyl-4-methylphenol in 96% ethanol, incubated at 95 degrees C for 45 min [K. Fukunaga, K. Takama and T. Suzuki, Anal. Biochem., 230 (1995) 20] were injected into the column. The MDA-TBA complex was eluted at a flow-rate of 1 ml/min and monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Analysis of groups of normal male and female volunteers gave plasma levels of MDA of 1.076 nmol/ml with a coefficient of variation of about 58%. No significant statistical differences were found between male and female groups, and no correlation was discovered on the age.

Mechanisms and genetic determinants regulating sterol absorption, circulating LDL levels, and sterol elimination: implications for classification and disease risk

This review integrates historical biochemical and modern genetic findings that underpin our understanding of the low-density lipoprotein (LDL) dyslipidemias that bear on human disease. These range from life-threatening conditions of infancy through severe coronary heart disease of young adulthood, to indolent disorders of middle- and old-age. We particularly focus on the biological aspects of those gene mutations and variants that impact on sterol absorption and hepatobiliary excretion via specific membrane transporter systems (NPC1L1, ABCG5/8); the incorporation of dietary sterols (MTP) and of de novo synthesized lipids (HMGCR, TRIB1) into apoB-containing lipoproteins (APOB) and their release into the circulation (ANGPTL3, SARA2, SORT1); and receptor-mediated uptake of LDL and of intestinal and hepatic-derived lipoprotein remnants (LDLR, APOB, APOE, LDLRAP1, PCSK9, IDOL). The insights gained from integrating the wealth of genetic data with biological processes have important implications for the classification of clinical and presymptomatic diagnoses of traditional LDL dyslipidemias, sitosterolemia, and newly emerging phenotypes, as well as their management through both nutritional and pharmaceutical means.

Novel LMF1 Nonsense Mutation in a Patient with Severe Hypertriglyceridemia

CONTEXT Lipase maturation factor 1 (LMF1) gene is a novel candidate gene in severe hypertriglyceridemia. Lmf1 is involved in the maturation of lipoprotein lipase (LPL) and hepatic lipase in endoplasmic reticulum. To date only one patient with severe hypertriglyceridemia and related disorders was found to be homozygous for a nonsense mutation in LMF1 gene (Y439X). OBJECTIVE The objective of the study was to investigate LMF1 gene in hypertriglyceridemic patients in whom mutations in LPL, APOC2, and APOA5 genes had been excluded. RESULTS The resequencing of LMF1 gene led to the discovery of a novel homozygous nonsense mutation in one patient with severe hypertriglyceridemia and recurrent episodes of pancreatitis. The mutation causes a G>A substitution in exon 9 (c.1395G>A), leading to a premature stop codon (W464X). LPL activity and mass were reduced by 76 and 50%, respectively, compared with normolipidemic controls. The proband over the years has shown a good response to treatment. The probands son, heterozygous for the W464X, shows normal plasma triglyceride levels. CONCLUSIONS We identified the second novel pathogenic mutation in LMF1 gene in a patient with severe hypertriglyceridemia. LPL deficiency in our patient was milder than in the carrier of the Y439X previously described.