Identification and characterization of new gain-of-function mutations in the PCSK9 gene responsible for autosomal dominant hypercholesterolemia.
BACKGROUND
The identification of mutations in PCSK9 (proprotein convertase subtilisin kexin9) in autosomal dominant hypercholesterolemia (ADH), has revealed the existence of a new player in cholesterol homeostasis. PCSK9 has been shown to enhance the degradation of the LDL receptor (LDLR) at the cell surface. Gain-of-function mutations of PCSK9 induce ADH and are very rare, but their identification is crucial in studying PCSK9's role in hypercholesterolemia, its detailed trafficking pathway and its impact on the LDLR.


METHODS
In order to identify new mutations and understand the exact mechanisms of action of mutated PCSK9, PCSK9 was sequenced in 75 ADH patients with no mutations in the LDLR or APOB genes. Functional analyses in cell culture were conducted and the impact of novel PCSK9 mutations on the quantitative and qualitative features of lipoprotein particles and on the HDL-mediated cellular cholesterol efflux was studied.


RESULTS
Among these 75 ADH probands with no mutations in the LDLR or APOB genes, four gain-of-function mutations of PCSK9 were identified, of which two were novel: the p.Leu108Arg and the p.Asp35Tyr substitutions. In vitro studies of their consequences on the activity of PCSK9 on cell surface levels of LDLR showed that the p.Leu108Arg mutation clearly results in a gain-of-function, while the p.Asp35Tyr mutation created a novel Tyr-sulfation site, which may enhance the intracellular activity of PCSK9.


CONCLUSIONS
These data further contribute to the characterization of PCSK9 mutations and to better understanding of the impact on cholesterol metabolism of this new therapeutic target.
Institut National de la Santé et de la Recherche Médicale, Inserm UMR698, Hemostasis, Bio-Engineering and Cardiovascular Remodelling, Hôpital Bichat-Claude Bernard, 46 Rue Henri Huchard, 75877 Paris Cedex 18, France. marianne.abi-fadel@inserm.fr
Marianne
M
Abifadel
Maryse
M
Guerin
Suzanne
S
Benjannet
Jean-Pierre
JP
Rabès
Wilfried
W
Le Goff
Zélie
Z
Julia
Josée
J
Hamelin
Valérie
V
Carreau
Mathilde
M
Varret
Eric
E
Bruckert
Laurent
L
Tosolini
Olivier
O
Meilhac
Philippe
P
Couvert
Dominique
D
Bonnefont-Rousselot
John
J
Chapman
Alain
A
Carrié
Jean-Baptiste
JB
Michel
Annik
A
Prat
Nabil G
NG
Seidah
Catherine
C
Boileau
D001055:Apolipoproteins B / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000097:blood; D008076:Cholesterol, HDL / Q000097:blood; D004252:DNA Mutational Analysis; D005260:Female; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D008297:Male; D009154:Mutation; D010297:Paris; D010375:Pedigree; D010641:Phenotype; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000235:genetics* / Q000378:metabolism; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D001055:Apolipoproteins B; D015415:Biomarkers; D008076:Cholesterol, HDL; C573412:LDLR protein, human; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases