TY - JOUR
T1 - Identification and functional analysis of missense mutations in the lecithin cholesterol acyltransferase gene in a Chilean patient with hypoalphalipoproteinemia
AU - Tobar, Hugo E.
AU - Cataldo, Luis R.
AU - González, Trinidad
AU - Rodríguez, Ricardo
AU - Serrano, Valentina
AU - Arteaga, Antonio
AU - Álvarez-Mercado, Ana
AU - Lagos, Carlos F.
AU - Vicuña, Lucas
AU - Miranda, José P.
AU - Pereira, Ana
AU - Bravo, Carolina
AU - Aguilera, Concepción M.
AU - Eyheramendy, Susana
AU - Uauy, Ricardo
AU - Martínez, Álvaro
AU - Gil, Ángel
AU - Francone, Omar
AU - Rigotti, Attilio
AU - Santos, José L.
N1 - Funding Information:
This work was supported by Proyecto FONDECYT 1150416 and Proyecto Interdisciplina VRI-PUC II15024 from the Dirección de Investigación, Pontificia Universidad Católica de Chile. Genotyping of GOCS was performed in the in the Human Genotyping laboratory at the Spanish National Cancer Research Centre, a member of CeGen (PRB2-ISCIII), and was supported by grant PT13/ 0001/0005 of PE I + D + i 2013-2016 funded by ISCIII and ERDF (Fondo Eur-opeo de Desarrollo Regional). This research was partially supported by the supercomputing infrastructure of the NLHPC (ECM-02). L.V. and C.B. were supported by VRI, Pontificia Universidad Católica de Chile (Proyecto Investiga-ción Interdisciplinaria VRI-PUC II15024). TG was supported by “Beca de Magís-ter Nacional” CONICYT. L.V. was additionally supported by FONDECYT postdoctoral grant 3170038. We express our gratitude to the proband and relatives.
Publisher Copyright:
© 2019 The Author(s).
PY - 2019/6/5
Y1 - 2019/6/5
N2 - Background: Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol in high- and low-density lipoproteins (HDL and LDL). Mutations in LCAT gene causes familial LCAT deficiency, which is characterized by very low plasma HDL-cholesterol levels (Hypoalphalipoproteinemia), corneal opacity and anemia, among other lipid-related traits. Our aim is to evaluate clinical/biochemical features of a Chilean family with a proband showing clinical signs of familial LCAT deficiency, as well as to identify and assess the functional effects of LCAT mutations. Methods: An adult female proband with hypoalphalipoproteinemia, corneal opacity and mild anemia, as well as her first-degree relatives, were recruited for clinical, biochemical, genetic, in-silico and in-vitro LCAT analysis. Sequencing of exons and intron-exon boundaries was performed to identify mutations. Site-directed mutagenesis was carried out to generate plasmids containing cDNA with wild type or mutant sequences. Such expression vectors were transfected to HEK-239 T cells to asses the effect of LCAT variants in expression, synthesis, secretion and enzyme activity. In-silico prediction analysis and molecular modeling was also used to evaluate the effect of LCAT variants. Results: LCAT sequencing identified rare p.V333 M and p.M404 V missense mutations in compound heterozygous state in the proband, as well the common synonymous p.L363 L variant. LCAT protein was detected in proband's plasma, but with undetectable enzyme activity compared to control relatives. HEK-293 T transfected cells with vector expression plasmids containing either p.M404 V or p.V333 M cDNA showed detectable LCAT protein expression both in supernatants and lysates from cultured cells, but with much lower enzyme activity compared to cells transfected with the wild-type sequence. Bioinformatic analyses also supported a causal role of such rare variations in LCAT lack of function. Additionally, the proband carried the minor allele of the synonymous p.L363 L variant. However, this variant is unlikely to affect the clinical phenotype of the proband given its relatively high frequency in the Chilean population (4%) and its small putative effect on plasma HDL-cholesterol levels. Conclusion: Genetic, biochemical, in vitro and in silico analyses indicate that the rare mutations p.M404 V and p.V333 M in LCAT gene lead to suppression of LCAT enzyme activity and cause clinical features of familial LCAT deficiency.
AB - Background: Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol in high- and low-density lipoproteins (HDL and LDL). Mutations in LCAT gene causes familial LCAT deficiency, which is characterized by very low plasma HDL-cholesterol levels (Hypoalphalipoproteinemia), corneal opacity and anemia, among other lipid-related traits. Our aim is to evaluate clinical/biochemical features of a Chilean family with a proband showing clinical signs of familial LCAT deficiency, as well as to identify and assess the functional effects of LCAT mutations. Methods: An adult female proband with hypoalphalipoproteinemia, corneal opacity and mild anemia, as well as her first-degree relatives, were recruited for clinical, biochemical, genetic, in-silico and in-vitro LCAT analysis. Sequencing of exons and intron-exon boundaries was performed to identify mutations. Site-directed mutagenesis was carried out to generate plasmids containing cDNA with wild type or mutant sequences. Such expression vectors were transfected to HEK-239 T cells to asses the effect of LCAT variants in expression, synthesis, secretion and enzyme activity. In-silico prediction analysis and molecular modeling was also used to evaluate the effect of LCAT variants. Results: LCAT sequencing identified rare p.V333 M and p.M404 V missense mutations in compound heterozygous state in the proband, as well the common synonymous p.L363 L variant. LCAT protein was detected in proband's plasma, but with undetectable enzyme activity compared to control relatives. HEK-293 T transfected cells with vector expression plasmids containing either p.M404 V or p.V333 M cDNA showed detectable LCAT protein expression both in supernatants and lysates from cultured cells, but with much lower enzyme activity compared to cells transfected with the wild-type sequence. Bioinformatic analyses also supported a causal role of such rare variations in LCAT lack of function. Additionally, the proband carried the minor allele of the synonymous p.L363 L variant. However, this variant is unlikely to affect the clinical phenotype of the proband given its relatively high frequency in the Chilean population (4%) and its small putative effect on plasma HDL-cholesterol levels. Conclusion: Genetic, biochemical, in vitro and in silico analyses indicate that the rare mutations p.M404 V and p.V333 M in LCAT gene lead to suppression of LCAT enzyme activity and cause clinical features of familial LCAT deficiency.
KW - HDL-cholesterol
KW - Hypoalphalipoproteinemia
KW - Lecithin-cholesterol acyltransferase
KW - Variants
UR - http://www.scopus.com/inward/record.url?scp=85066821908&partnerID=8YFLogxK
U2 - 10.1186/s12944-019-1045-0
DO - 10.1186/s12944-019-1045-0
M3 - Article
C2 - 31164121
AN - SCOPUS:85066821908
SN - 1476-511X
VL - 18
JO - Lipids in Health and Disease
JF - Lipids in Health and Disease
IS - 1
M1 - 132
ER -