Description of a large family with autosomal dominant hypercholesterolemia associated with the APOE p.Leu167del mutation.

Apolipoprotein (apo) E mutants are associated with type III hyperlipoproteinemia characterized by high cholesterol and triglycerides levels. Autosomal dominant hypercholesterolemia (ADH), due to the mutations in the LDLR, APOB, or PCSK9 genes, is characterized by an isolated elevation of cholesterol due to the high levels of low-density lipoproteins (LDLs). We now report an exceptionally large family including 14 members with ADH. Through genome-wide mapping, analysis of regional/functional candidate genes, and whole exome sequencing, we identified a mutation in the APOE gene, c.500_502delTCC/p.Leu167del, previously reported associated with sea-blue histiocytosis and familial combined hyperlipidemia. We confirmed the involvement of the APOE p.Leu167del in ADH, with (1) a predicted destabilization of an alpha-helix in the binding domain, (2) a decreased apo E level in LDLs, and (3) a decreased catabolism of LDLs. Our results show that mutations in the APOE gene can be associated with bona fide ADH.

Molecular spectrum of autosomal dominant hypercholesterolemia in France.

Autosomal Dominant Hypercholesterolemia (ADH), characterized by isolated elevation of plasmatic LDL cholesterol and premature cardiovascular complications, is associated with mutations in 3 major genes: LDLR (LDL receptor), APOB (apolipoprotein B) and PCSK9(proprotein convertase subtilisin-kexin type 9). Through the French ADH Research Network, we collected molecular data from 1358 French probands from eleven different regions in France.Mutations in the LDLR gene were identified in 1003 subjects representing 391 unique events with 46.0% missense, 14.6% frameshift, 13.6% splice, and 11.3% nonsense mutations, 9.7% major rearrangements, 3.8% small in frame deletions/insertions, and 1.0% UTR mutations. Interestingly,175 are novel mutational events and represent 45% of the unique events we identified, highlighting a specificity of the LDLR mutation spectrum in France. Furthermore, mutations in the APOB gene were identified in 89 probands and in the PCSK9 gene in 10 probands. Comparison of available clinical and biochemical data showed a gradient of severity for ADH-causing mutations:FH=PCSK9>FDB>«Others¼ genes. The respective contribution of each known gene to ADH inthis French cohort is: LDLR 73.9%, APOB 6.6%, PCSK9 0.7%. Finally, in 19.0% of the probands,no mutation was found, thus underscoring the existence of ADH mutations located in still unknown genes.

Epigenetic silencing of lysyl oxidase-like-1 through DNA hypermethylation in an autosomal recessive cutis laxa case.

We have recently reported a case of cutis laxa caused by a fibulin-5 missense mutation (p.C217R). Skin fibroblasts from this individual showed an abnormal pattern of expression of several genes coding for elastic fiber-related proteins, including lysyl oxidase-like-1 (LOXL1). In this study we intended to elucidate the mechanism responsible for LOXL1 downregulation in these fibulin-5-mutant cells. We identified a proximal region (-442/-342) of the human LOXL1 promoter in which two binding sites for the transcription factor specific protein 1 (Sp-1) are required for gene activity in normal fibroblasts. Binding of Sp-1 to these sequences was dramatically reduced within cutis laxa cells, although Sp-1 expression was normal. Further analysis of the promoter sequence found increased methylation levels in cutis laxa cells compared with cells from unaffected individuals. When DNA methyltransferase activity was transiently inhibited in cutis laxa cells using the 5-aza-2'-deoxycytidine, we found a significant increase in LOXL1 expression. In conclusion, besides changes caused by the fibulin-5 mutation, LOXL1 gene regulation is affected by an epigenetic mechanism that can be reversed by an inhibitor of DNA methyltransferase activity. It is not yet known whether LOXL1 gene expression is affected in all cases of cutis laxa arising from fibulin-5 mutation.

A fourth locus for autosomal dominant hypercholesterolemia maps at 16q22.1.

Autosomal dominant hypercholesterolemia (ADH) is characterized by isolated increase in plasmatic low-density lipoprotein (LDL) cholesterol levels associated with high risk of premature cardiovascular disease. Mutations in LDLR, APOB, and PCSK9 genes have been shown to cause ADH. We now report further genetic heterogeneity of ADH through the study of a large French family in which the involvement of these three genes was excluded. A genome-wide scan mapped the disease-causing gene, named HCHOLA4, at 16q22.1 in a 7.89-Mb interval containing 154 genes with a maximum LOD score of 3.9. To reduce the linked region, we genotyped 18 smaller non-LDLR/non-APOB/non-PCSK9-ADH families at the HCHOLA4 locus. Six families did not exclude linkage to the locus, but none allowed reduction of the disease interval. The 154 regional genes were sorted according to the function of the encoded protein and tissue expression profiles, and 57 genes were analyzed through sequencing of their coding region and close flanking intronic parts. No disease-causing mutation was identified in these families, particularly in the LCAT gene. Finally, our results also show the existence of other ADH genes as nine families were neither linked to LDLR, APOB, and PCSK9 genes nor to the new HCHOLA4 locus.

Mutations and polymorphisms in the proprotein convertase subtilisin kexin 9 (PCSK9) gene in cholesterol metabolism and disease.

Hypercholesterolemia is one of the major causes of coronary heart disease (CHD). The genes encoding the low-density lipoprotein receptor and its ligand apolipoprotein B, have been the two genes classically implicated in autosomal dominant hypercholesterolemia (ADH). Our discovery in 2003 of the first mutations of the proprotein convertase subtilisin kexin 9 gene (PCSK9) causing ADH shed light on an unknown actor in cholesterol metabolism that since then has been extensively investigated. Several PCSK9 variants have been identified, some of them are gain-of-function mutations causing hypercholesterolemia by a reduction of low-density lipoprotein (LDL) receptor levels; while others are loss-of-function variants associated with a reduction of LDL-cholesterol (LDL-C) levels and a decreased risk of CHD. In this review, we focus on reported variants, and their biological, clinical, and functional relevance. We also highlight the spectrum of hypercholesterolemia or hypobetalipoproteinemia phenotypes that are already associated with mutations in PCSK9. Finally, we present future prospects concerning this therapeutic target that might constitute a new approach to reduce cholesterol levels and CHD, and enhance the effectiveness of other lipid-lowering drugs.

A p.C217R mutation in fibulin-5 from cutis laxa patients is associated with incomplete extracellular matrix formation in a skin equivalent model.

Cutis laxa (CL) is a rare genodermatosis, which is clinically and genetically heterogeneous. It is characterized by redundant, loose, sagging, and inelastic skin. In a consanguineous family from Lebanon with autosomal-recessive transmission, we identified a homozygous missense mutation (c.649T --> C; p.C217R) in the fibulin-5 gene (FBLN5), which was, to our knowledge, previously unreported. Small skin biopsies were performed, which permitted isolation of skin fibroblasts harboring this FBLN5 mutation; they exhibited a deficit in cell growth. A CL skin equivalent (CL-SE) model compared with control SE was successfully developed to define the behavior of CL fibroblasts in a three-dimensional model. There was increased cell death and a global extracellular matrix deficiency in the dermis of this CL-SE model, and a low level of the main elastic fiber expression. There was no basement membrane evident at the ultrastructural level, and type-VII collagen could not be detected at the histological level. This model reproduced some defects of the extracellular matrix and highlighted other defects, which occurred at the time of the basement membrane formation, which were not evident in skin from patients. This CL-SE model could be adapted to screen for therapeutically active molecules.

Novel mutations of the PCSK9 gene cause variable phenotype of autosomal dominant hypercholesterolemia.

Autosomal dominant hypercholesterolemia (ADH) is a frequent (1/500) monogenic inherited disorder characterized by isolated elevation of LDL leading to premature cardiovascular disease. ADH is known to result from mutations at two main loci: LDLR (encoding the low density lipoprotein receptor), and APOB (encoding apolipoprotein B100), its natural ligand. We previously demonstrated that ADH is also caused by mutations of the PCSK9 (proprotein convertase subtilisin/kexin type 9) gene that encodes Narc-1 (neural apoptosis-regulated convertase 1). However, the role of this novel disease locus as a cause of hypercholesterolemia remains unclear. In the present study, we analysed the PCSK9 coding region and intronic junctions in 130 adult or pediatric patients with ADH, previously found as being non LDLR/non APOB mutation carriers. Four novel heterozygous missense variations were found: c.654A>T (p.R218S), c.1070G>A (p.R357H), c.1405C>T (p.R469W), and c.1327G>A (p.A443T). All mutations were absent in 340 normolipidemic controls. Except for the A443T, all mutations are nonconservative and modify a highly conserved residue. Segregation with hypercholesterolemia is incomplete in one pedigree. Type and severity of hyperlipidemia and of cardiovascular disease could vary among subjects from the same family. Finally, the proband carrying the R357H mutation exhibited very high plasma cholesterol during pregnancy, whereas the proband carrying the p.R469W mutation exhibited a severe phenotype of hypercholesterolemia in combination with a LDLR mutation resulting from a frameshift at residue F382 (1209delC). These observations suggest that variations in PCSK9 are a rare cause of non LDLR/non APOB ADH (approximately 2.3%) and that additional environmental or genetic factors may contribute to the phenotype caused by PCSK9 missense mutations in humans.

Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.

Autosomal dominant hypercholesterolemia (ADH; OMIM144400), a risk factor for coronary heart disease, is characterized by an increase in low-density lipoprotein cholesterol levels that is associated with mutations in the genes LDLR (encoding low-density lipoprotein receptor) or APOB (encoding apolipoprotein B). We mapped a third locus associated with ADH, HCHOLA3 at 1p32, and now report two mutations in the gene PCSK9 (encoding proprotein convertase subtilisin/kexin type 9) that cause ADH. PCSK9 encodes NARC-1 (neural apoptosis regulated convertase), a newly identified human subtilase that is highly expressed in the liver and contributes to cholesterol homeostasis.