|Tipo di tesi||Tesi di dottorato di ricerca|
|Autore||MAGNOLO, ANTONIA LUCIA|
|Titolo||Caratterizzazione funzionale di nuove mutazioni nei geni APOB e ANGPTL3 nell’ipobetalipoproteinemia familiare e nella ipolipidemia familiare combinata|
|Titolo in inglese||Functional characterization of novel mutations in APOB and ANGPTL3 genes in familial hypobetalipoproteinemia and familial combined hypolipidemia|
|Settore scientifico disciplinare||MED/04 - PATOLOGIA GENERALE|
|Corso di studi||Scuola di D.R. in MEDICINA MOLECOLARE E RIGENERATIVA|
|Data inizio appello||2013-04-09|
|Disponibilità||Accessibile via web (tutti i file della tesi sono accessibili)|
Introduzione: L’ipobetalipoproteinemia familiare (FHBL) è un disordine dominante caratterizzato da ridotti livelli plasmatici di colesterolo, lipoproteine a bassa densità (LDL) e apolipoproteina B (apoB), valori normali di trigliceridi e lipoproteine ad altà densità (HDL). Nella maggior parte dei casi FHBL è dovuta a mutazioni del gene APOB che portano alla formazione di apoB troncate di lunghezza variabile. Rare mutazioni del gene APOB che comportano sostituzioni amminoacidiche non-conservative sono descritte come causa di FHBL. In vitro, queste mutazioni provocano una ritenzione intracellulare della proteina mutata e riducono la secrezione delle lipoproteine contenenti apoB (LpB). Recentemente è stata identificata una nuova condizione di ipobetalipoproteinemia associata a bassi livelli plasmatici di HDL. Questa condizione denominata Ipolipidemia Familiare Combinata è dovuta a mutazioni con perdita-di-funzione del gene ANGPTL3, codificante la proteina angiopoietina-like 3 (angplt3), una proteina plasmatica secreta dal fegato e nota inibire la lipasi lipoproteica e endoteliale.
Introduction: Familial Hypobetalipoproteinemia (FHBL) is a dominant disorder characterized by low plasma levels of low density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (apoB), normal values of triglycerides (TG) and high density lipoprotein-cholesterol (HDL-C). In most cases FHBL is due to mutations in APOB gene resulting in truncated apoBs of various size. Some APOB gene mutations resulting in non-conservative amino acid substitutions were reported to cause FHBL. In vitro, these mutations induce the intracellular retention of the mutant apoB and impair the secretion of apoB-containing lipoproteins. Recently a new condition of hypobetalipoproteinemia, characterized by low total cholesterol, LDL-C and apoB levels associated with low plasma HDL-C has been identified. This condition, designated Familial Combined Hypolipidemia, is due to loss of function mutations of ANGPTL3 gene, encoding angiopoietin-like 3 protein (angplt3), a plasma protein secreted by the liver and known to inhibit lipoprotein lipase and endothelial lipase. Methods: In two FHBL subjects we identified two novel amino acid variants (Thr26_27delinsAsn and Tyr102Cys) located in the N-terminal 1000 amino acids of apoB, a crucial domain for proper folding of apoB, assembly of lipids with nascent apoB and secretion. To investigate the functional effect of these amino acid changes, the corresponding mutations were introduced in plasmids containing human apoB-48 cDNA by site directed mutagenesis. McA-RH7777 rat hepatoma cells were transiently and stably transfected with wild type or mutant human apoB-48. The secretion efficiency of human apoB-48 was determined by immunoblotting of apoB and the incorporation of apoB into medium lipoproteins. Immunocytochemistry was used to monitor the intracellular localization of the mutant proteins.The post-translational stability of mutant apoB-48 was determined in cells cultured in the presence of a protein synthesis inhibitor. The intracellular degradation pathways (autophagy, proteasomal degradation) of mutant apoB-48 was also evaluated. ANGPLT3 gene was resequenced in three subjects with Familial Combined Hypolipidemia, negative for mutations in APOB and PCSK9 gene. To investigate the effect of a splice site mutation of ANGPTL3 (IVS6+1G>T) two minigenes (mutant and wild type) were constructed by PCR-amplification of the appropriate ANGPLT3 gene regions. Minigenes were transfected into COS-1 cells and their transcripts obtained by RT-PCR. The transcripts were separated by 2% agarose gel electrophoresis, and sequenced. Results and conclusions. The APOB mutation Thr26_27delinsAsn strongly reduced the secretion of apoB-48 from transiently transfected cells; the mutant apoB-48 appears to be retained in endoplasmic reticulum. In stably transfected cells the secretion defect of mutant apoB-48 was confirmed by the absence of apoB-48 containing lipoproteins in the medium. The addiction of a proteasome inhibitor partially blocked the decay of cellular mutant apoB-48 suggesting that a significant proportion of mutant protein was degraded by proteasomes. The mutation Thr26_27delinsAsn alters the structure of the beta-barrel of N-terminal domain of apoB (the first 267 amino) preventing the secretion of apoB-containing lipoproteins. By contrast the mutation Tyr102Cys had no effect on apoB-48 secretion. The sequence of ANGPTL3 in subjects with familial combined hypolipidemia allowed the identification of a mutation involving the donor splice site of intron 6 (c.1198 +1 G>T). In vitro studies demonstrated that the splice site mutation causes a partial retention of intron 6 in mature mRNA leading to a frameshift with the formation of a premature termination codon. The trascription product of the abnormal mRNA is a short protein of 403 amino acids devoid of function.