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.

Epstein-Barr virus (EBV) reactivation in allogeneic stem-cell transplantation: relationship between viral load, EBV-specific T-cell reconstitution and rituximab therapy.

BACKGROUND: Monitoring of Epstein-Barr virus (EBV) reactivation after allogeneic hematopoietic stem-cell transplantation markedly improved with quantitative real-time polymerase chain reaction amplification of EBV DNA and visualization of EBV-specific CD8+ T cells with peptide-human leukocyte antigen (HLA) class I tetramers. We decided to combine these methods to evaluate posttransplant EBV reactivation and rituximab therapy. METHODS: We followed 56 patients treated with an HLA-genoidentical sibling (n=32), an HLA-matched unrelated donor (MUD, n=19), or an unrelated cord-blood transplant (n=5). EBV DNA was quantified in plasma and in peripheral blood mononuclear cells (PBMC). Patient CD8+ T cells were stained with a panel of eight tetramers. RESULTS: EBV DNA was detected in half of the patients, mainly in the MUD group (17/19). In 19 patients, viral DNA was detected only in the cellular compartment. All patients who controlled reactivation without rituximab and despite a viral load of greater than 500 genome equivalents (gEq)/150,000 PBMC mounted an EBV-specific CD8+ T-cell response in greater than 1.4% of CD3+CD8+ T cells. Plasmatic EBV genome was found in nine patients preceded by a high cellular viral load. Three of these patients controlled the reactivation before or without the introduction of rituximab, and they all developed a significant and increasing EBV-specific T-cell response. Patients with EBV-specific T cells at the onset of reactivation controlled viral reactivation without rituximab. CONCLUSION: This study emphasizes the benefit of an early and close monitoring of EBV reactivation and CD8+-specific immune responses to initiate rituximab only when necessary and before the immune response becomes overwhelmed by the viral burden.

NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.

The discovery of autosomal dominant hypercholesterolemic patients with mutations in the PCSK9 gene, encoding the proprotein convertase NARC-1, resulting in the missense mutations suggested a role in low density lipoprotein (LDL) metabolism. We show that the endoplasmic reticulum-localized proNARC-1 to NARC-1 zymogen conversion is Ca2+-independent and that within the zymogen autocatalytic processing site SSVFAQ [downward arrow]SIP Val at P4 and Pro at P3' are critical. The S127R and D374Y mutations result in approximately 50-60% and > or =98% decrease in zymogen processing, respectively. In contrast, the double [D374Y + N157K], F216L, and R218S natural mutants resulted in normal zymogen processing. The cell surface LDL receptor (LDLR) levels are reduced by 35% in lymphoblasts of S127R patients. The LDLR levels are also reduced in stable HepG2 cells overexpressing NARC-1 or its natural mutant S127R, and this reduction is abrogated in the presence of 5 mm ammonium chloride, suggesting that overexpression of NARC-1 increases the turnover rate of the LDLR. Adenoviral expression of wild type human NARC-1 in mice resulted in a maximal approximately 9-fold increase in circulating LDL cholesterol, while in LDLR-/- mice a delayed approximately 2-fold increase in LDL cholesterol was observed. In conclusion, NARC-1 seems to affect both the level of LDLR and that of circulating apoB-containing lipoproteins in an LDLR-dependent and -independent fashion.