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The Arg499His gain-of-function mutation in the C-terminal domain of PCSK9

  • Author Footnotes
    1 These authors contributed equally to this work.
    Rosa M. Sánchez-Hernández
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Sección de Endocrinología y Nutrición, Complejo Hospitalario Universitario Insular Materno Infantil de Gran Canaria, Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS) de la Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Maria Donata Di Taranto
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli and CEINGE S.C.a r.l, Biotecnologie Avanzate, Napoli, Italy
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Asier Benito-Vicente
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Instituto Biofisika (UPV/EHU, CSIC) and Departamento de Bioquímica, Universidad del País Vasco, Apdo. 644, 48080, Bilbao, Spain
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  • Kepa B. Uribe
    Affiliations
    Instituto Biofisika (UPV/EHU, CSIC) and Departamento de Bioquímica, Universidad del País Vasco, Apdo. 644, 48080, Bilbao, Spain
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  • Itziar Lamiquiz-Moneo
    Affiliations
    Hospital Universitario Miguel Servet. IIS Aragon. CIBERCV. Universidad de Zaragoza, Zaragoza, Spain
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  • Asier Larrea-Sebal
    Affiliations
    Instituto Biofisika (UPV/EHU, CSIC) and Departamento de Bioquímica, Universidad del País Vasco, Apdo. 644, 48080, Bilbao, Spain
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  • Shifa Jebari
    Affiliations
    Instituto Biofisika (UPV/EHU, CSIC) and Departamento de Bioquímica, Universidad del País Vasco, Apdo. 644, 48080, Bilbao, Spain
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  • Unai Galicia-Garcia
    Affiliations
    Instituto Biofisika (UPV/EHU, CSIC), Barrio Sarriena s/n, 48940, Leioa, Bizkaia, Spain
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  • F. Javier Nóvoa
    Affiliations
    Sección de Endocrinología y Nutrición, Complejo Hospitalario Universitario Insular Materno Infantil de Gran Canaria, Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS) de la Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain
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  • Mauro Boronat
    Affiliations
    Sección de Endocrinología y Nutrición, Complejo Hospitalario Universitario Insular Materno Infantil de Gran Canaria, Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS) de la Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain
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  • Ana M. Wägner
    Affiliations
    Sección de Endocrinología y Nutrición, Complejo Hospitalario Universitario Insular Materno Infantil de Gran Canaria, Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS) de la Universidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain
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  • Fernando Civeira
    Affiliations
    Hospital Universitario Miguel Servet. IIS Aragon. CIBERCV. Universidad de Zaragoza, Zaragoza, Spain
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  • César Martín
    Correspondence
    Corresponding author. Present address: Instituto Biofisika (UPV/EHU, CSIC) and Departamento de Bioquímica, Universidad del País Vasco, Apdo. 644, 48080, Bilbao, Spain.
    Affiliations
    Instituto Biofisika (UPV/EHU, CSIC) and Departamento de Bioquímica, Universidad del País Vasco, Apdo. 644, 48080, Bilbao, Spain
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  • Giuliana Fortunato
    Correspondence
    Corresponding author.
    Affiliations
    Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli and CEINGE S.C.a r.l, Biotecnologie Avanzate, Napoli, Italy
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  • Author Footnotes
    1 These authors contributed equally to this work.

      Highlights

      • We identified a novel PCSK9 GOF variant, p.(Arg499His), in two unrelated FH patients from Spain and Italy.
      • p.(Arg499His) PCSK9 variant carriers show high LDL-C concentrations and FH phenotype.
      • In vitro assays revealed reduced LDL receptor expression at membrane surface with p.(Arg499His).
      • p.(Arg499His) is a GOF mutation that causes FH through an intracellular effect reducing LDLr availability.

      Abstract

      Background and aims

      Familial hypercholesterolemia (FH) is a monogenic disease characterized by high levels of low-density lipoprotein cholesterol and premature atherosclerotic cardiovascular disease. FH is caused by loss of function mutations in genes encoding LDL receptor (LDLR), and Apolipoprotein B (APOB) or gain of function (GOF) mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9). In this study, we identified a novel variant in PCSK9, p.(Arg499His), located in the C-terminal domain, in two unrelated FH patients from Spain and Italy.

      Methods

      We studied familial segregation and determined variant activity in vitro.

      Results

      We determined PCSK9 expression, secretion and activity of the variant in transfected HEK293 cells; extracellular activity of the recombinant p.(Arg499His) PCSK9 variant in HEK 293 and HepG2 cells; PCSK9 affinity to the LDL receptor at neutral and acidic pH; the mechanism of action of the p.(Arg499His) PCSK9 variant by co-transfection with a soluble construct of the LDL receptor and by determining total PCSK9 intracellular accumulation when endosomal acidification is impaired and when an excess of soluble LDLr is present in the culture medium. Our results show high LDL-C concentrations and FH phenotype in p.(Arg499His) carriers. In vitro functional characterization shows that p.(Arg499His) PCSK9 variant causes a reduction in LDLr expression and LDL uptake. An intracellular activity for this variant is also shown when blocking the activity of secreted PCSK9 and by inhibiting endosomal acidification.

      Conclusions

      We demonstrated that p.(Arg499His) PCSK9 variant causes a direct intracellular degradation of LDLr therefore causing FH by reducing LDLr availability.

      Graphical abstract

      Keywords

      Abbreviations:

      GOF (gain of function), MLPA (Multiplex Ligation-dependent Probe Amplification), LOF (loss of function), HGVS (Human Genome Variation Society), LOVD (Leiden Open Variation Database), ExAC (Exome Aggregation Consortium), gnomAD (Genome Aggregation Database), EVS (Exome Variant Server)
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