GPIHBP1 C89F Neomutation and Hydrophobic
C-Terminal Domain G175R Mutation in Two
Pedigrees with Severe Hyperchylomicronemia
Sybil Charrie `re, Noe ¨l Peretti, Sophie Bernard, Mathilde Di Filippo, Agne `s Sassolas,
Micheline Merlin, Mireille Delay, Cyrille Debard, Etienne Lefai, Alain Lachaux,
Philippe Moulin, and Christophe Marc ¸ais
Ho ˆpital Louis Pradel (S.C., S.B., P.M.), Fe ´de ´ration d’endocrinologie, maladies me ´taboliques, diabe `te et
nutrition; Centre de Biologie et de Pathologie Est (M.D.F., A.S.), De ´partement de biochimie et biologie
mole ´culaire; and Ho ˆpital Femme-Me `re-Enfant (N.P., A.L.), Service de gastroente ´rologie, He ´patologie et
Nutrition, Hospices Civils de Lyon, Bron F-69677, France; Institut National de la Sante ´ et de la Recherche
Me ´dicale Unite ´ (U) 1060 (S.C., N.P., M.D.F., A.S., C.D., E.L., P.M., C.M.), Institut National des Sciences
Appliquées de Lyon, Institut National de la Recherche Agronomique U1235, Universite ´ de Lyon,
Villeurbanne F-69621 and Oullins F-69600, France; Service d’endocrinologie (S.B.), Ho ˆtel-Dieu du Centre
Hospitalier Universitaire Montre ´al, Montre ´al, Que ´bec, H2W 1R5 Canada; and Centre Hospitalier Lyon-
Sud (M.M., M.D., C.M.), Centre de Biologie Sud, Laboratoire de Biochimie spe ´cialise ´e, Hospices Civils de
Lyon, Pierre-Be ´nite F-69495, France
mutations of the GPIHBP1 gene, C89F and G175R, by systematic sequencing in a cohort of 376
hyperchylomicronemic patients without mutations on the LPL, APOC2, or APOA5 gene.
Design: We performed clinical and genotypic studies of probands and their families. GPIHBP1
functional alterations were studied in CHO pgsA-745 transfected cells.
C89F neomutation and a deletion of the second allele. C89F mutation was associated with a C14F
signal peptide polymorphism on the same haplotype. Both patients had resistant hyperchylomi-
a drastic LPL-binding defect to GPIHBP1. C14F may further potentiate C89F effect.
Conclusions: The emergence of hyperchylomicronemia in the generation after a neomutation
further establishes a critical role for GPIHBP1 in TGRL physiopathology in humans. Our results
highlight the crucial role of C65-C89 disulfide bond in LPL binding by GPIHBP1 Ly6 domain. Fur-
thermore, we first report a mutation of the hydrophobic C-terminal domain that impairs GPIHBP1
membrane targeting. (J Clin Endocrinol Metab 96: E1675–E1679, 2011)
cause hyperchylomicronemia due to LPL activity defect
ipoprotein lipase (LPL) is the key enzyme for intravas-
cular lipolysis of triglycerides-rich lipoproteins
mia in humans remains unexplained.
to heparan sulfate proteoglycans at the luminal surface of
ISSN Print 0021-972X
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/jc.2011-1444 Received May 6, 2011. Accepted July 18, 2011.
First Published Online August 3, 2011
ISSN Online 1945-7197Abbreviations: GPIHBP1, Glycosylphosphatidylinositol-anchored high-density lipoprotein-
binding protein 1; HRP, horseradish peroxidase; LPL, lipoprotein lipase; SNP, single-nucle-
otide polymorphism; TG, triglyceride; TGRL, triglycerides-rich lipoproteins; WT, wild type.
J C E MO N L I N E
B r i e fR e p o r t — E n d o c r i n eR e s e a r c h
J Clin Endocrinol Metab, October 2011, 96(10):E1675–E1679jcem.endojournals.org
endothelial capillaries. In 2007, glycosylphosphatidyli-
nositol-anchored high-density lipoprotein-binding pro-
binding site. Mice lacking gpihbp1 were found to exhibit
severe hyperchylomicronemia with low LPL activity (2).
study of hyperchylomicronemia. A few GPIHBP1 muta-
tions have been recently described in hyperchylomicrone-
mic patients, all but one in the Ly6 domain of the protein,
responsible for the LPL-binding defect (3–6). One muta-
tion (G56R) was described in the acidic N-terminal do-
main, but in vitro studies failed to demonstrate a loss of
function (7, 8).
To document the potential implication of GPIHBP1 in
unexplained hyperchylomicronemia, we sequenced the
GPIHBP1 gene in a large cohort of 376 hyperchylomi-
cronemic patients without LPL, APOC2, or APOA5 mu-
expression and functional consequences of two new mis-
sense mutations identified in two probands.
Patients and Methods
Patients and population studied
We sequenced GPIHBP1 in a cohort of 376 unrelated pa-
tients with documented episodes of hyperchylomicronemia
without mutation on LPL, APOC2, or APOA5 genes. Hyper-
chylomicronemia was defined by fasting plasma triglycerides
(TG) concentration over 15 mmol/liter with a TG to total cho-
over 10 mmol/liter with a familial history of hypertriglyceride-
mia. Two hundred twenty unrelated normolipidemic controls
were also screened for mutations. A written informed consent
Genomic DNA analyses
Genomic DNA analyses are detailed in Supplemental Mate-
rials and Methods (published on The Endocrine Society’s Jour-
nals Online web site at http://jcem.endojournals.org). Possible
consequences of GPIHBP1 variants on protein function were
tested in silico with different software packages (Supplemental
Materials and Methods and Supplemental Table 1).
LPL and hepatic lipase activities
Postheparin LPL and hepatic lipase activities were deter-
mined as previously described (10). Plasma samples of normo-
lipidemic healthy subjects (n ? 12) were used as controls.
Assessing the amount of GPIHBP1 at the cell
A human Flag-GPIHBP1 pcDNA3 expression vector was
constructed, and C14F, C89F, C14F/C89F, G175R variants
were generated by site-directed mutagenesis. CHO pgsA-745
Culture Collection CRL-2422), were transiently transfected
with the constructs (Supplemental Materials and Methods).
PBS containing 1.0 mmol/liter MgCl2and 1.0 mmol/liter CaCl2
(PBS/Mg/Ca). At the end of the incubation period, cells were
washed six times in ice-cold PBS/Mg/Ca, and cell extracts were
oxidase (HRP)-conjugated goat antimouse IgG (1:1000; Bio-
Rad, Hercules, CA). Total amounts of GPIHBP1 in cells were
also determined by Western blotting (Supplemental Materials
to total GPIHBP1 in cells represents protein transfer efficacy at
the cell surface.
Binding of human LPL to GPIHBP1 variants at the
and a Myc-Flag-tagged human LPL-pCMV6 vector (Origene,
Rockville, MD). Twenty-four hours after transfections, cells were
incubated with a rabbit antibody against c-Myc-tag sequence (1:
500; Sigma), washed, and collected as described above. Western
blots were performed with a HRP-conjugated goat antirabbit
IgG (1:1000; Bio-Rad). Total amounts of GPIHBP1 and LPL in
cells were also determined by Western blotting (Supplemental
Materials and Methods).
in controls and hyperchylomicronemic cohort were compared us-
ing a ?2test (SPSS version 13.0 software). A P value ?0.05 was
considered statistically significant.
Identification of two new GPIHBP1 missense
We screened a cohort of 376 hyperchylomicronemic
patients without LPL, APOC2, and APOA5 mutations
for GPIHBP1 mutations and identified two new muta-
tions (0.53%), C89F and G175R (Supplemental Fig. 1).
These two mutations were not found in normolipemic
controls (n ? 220).
In silico analysis predict all GPIHBP1 C14F, C89F, and
G175R to be potentially damaging (Supplemental Table 2).
Proband AII-1, born in 2003, was diagnosed with hy-
episode of acute pancreatitis (TG, 19.6 mmol/liter). Dur-
ing childhood, TG remained moderately increased (3–7
mmol/liter) under strict diet, with several episodes of
hyperchylomicronemia but without recurrence of acute
Charrie `re et al.
C89F and G175R GPIHBP1 MutationsJ Clin Endocrinol Metab, October 2011, 96(10):E1675–E1679
pancreatitis. His LPL activity was undetectable. Both
parents (AI-1 and AI-2) and his younger brother (AII-2)
had normal lipid profiles (Supplemental Results, Fig. 2,
and Table 3).
Proband BII-2, 35 years old, was referred at the age of
mia (TG, 26 mmol/liter). Then, he remained mostly hy-
perchylomicronemic with recurrent acute pancreatitis.
His LPL activity was undetectable. His mother (BI-2) and
daughter (BIII-1) had normal lipid parameters (Supple-
mental Results, Fig. 2, and Table 3).
GPIHBP1 mutations and haplotype studies in
In family A, proband AII-1 appeared homozygous, but
haplotype studies revealed he was C89F hemizygous.
C89F is a neomutation on the paternal allele associated
with a deleted GPIHBP1 maternal allele (Supplemental
Results). The haplotype with C89F also included C14F
(rs11538389), c.138T3G (rs11538388) and IVS3 ?
27c3t (rs56046179) single-nucleotide polymorphisms
(SNP). In family B, proband BII-2 was homozygous for
C14F association with hyperchylomicronemia
The genotype distribution followed Hardy-Weinberg
equilibrium among controls (n ? 220) and hyperchylo-
micronemic patients (n ? 376). C14F allele was signifi-
cantly more frequent in hyperchylomicronemic patients
quent in hyperchylomicronemic patients than in controls
(21.2 vs. 7.8%, P ? 0.001).
Studies of GPIHBP1 variants in CHO pgsA-745 cells
Expression of GPIHBP1 variants at the cell surface
Total amounts of GPIHBP1 in cells were not different
from wild-type (WT)-GPIHBP1 for G175R variant but
were mildly reduced for C14F, C89F, and C14F/C89F
variants (?24 to ?32%) (Fig. 1, A and B). Amounts of
GPIHBP1 at the cell surface were significantly decreased
for all variants compared with WT-GPIHBP1 (C14F,
?53 ? 4%; C89F, ?35 ? 8%; C14F/C89F, ?45 ? 7%;
G175R, ?48 ? 4%) (Figs. 1A and 2B). A significant re-
duced transfer efficacy at the cell surface was shown only
for C14F (?32 ? 9%) and G175R (?49 ? 9%) variants
LPL binding to GPIHBP1 variants
As expected, LPL was unable to bind cells not express-
modified by C14F variant. LPL binding to GPIHBP1-
C89F was dramatically altered (?78 ? 2%) and even
to GPIHBP1-G175R was reduced by about half but was
conserved after adjustment on cell surface expression of
GPIHBP1-G175R (Fig. 2, A and B). Signal normalization
FIG. 1. Assessing the amount of GPIHBP1 at the cell surface. A,
Western blot analysis. CHO pgsA-745 cells were transiently transfected
with an empty vector or an expression vector encoding for WT human
GPIHBP1 (WT), GPIHBP1-C14F, GPIHBP1-C89F, GPIHBP1-C14F/C89F,
or GPIHBP1-G175R. All constructs contained an amino-terminal Flag
tag. The amount of GPIHBP1 at the surface of cells was determined
after incubation of cells with a mouse monoclonal antibody against
Flag-tag, revealed in Western blot by a HRP-conjugated goat
antimouse IgG. Total amount of GPIHBP1 in cells was determined with
an antibody against GPIHBP1. Actin was used as control. The results
are a representative series of three independent experiments. B–D,
Quantification of Western blots; B, total amounts of GPIHBP1 in cells;
C, amounts of GPIHBP1 at the cell surface; D, transfer efficacy of
GPIHBP1 at the cell surface. Amounts of GPIHBP1 at the cell surface
are reported relative to total amounts GPIHBP1 in cells. All signals were
normalized to actin signals and expressed relative to the ratio for WT
GPIHBP1 (set at 100%). Data are expressed as mean ? SEM of three
independent experiments. *, P ? 0.05 compared with WT.
J Clin Endocrinol Metab, October 2011, 96(10):E1675–E1679 jcem.endojournals.org
on total LPL amount in cells did not significantly modify
the results (Fig. 2C).
two patients with severe defect in LPL activity among
376 hyperchylomicronemic patients. The homozygous
G175R is the first mutation described in the C-terminal
domain of the protein. The second mutation, C89F, is a
sociated with a large deletion of GPIHBP1 on the ma-
ternal allele. Its pathogenic role is strongly suggested by
the occurrence of hyperchylomicronemia in the first gen-
eration after neomutation.
C89 is one of 10 highly conserved cysteines of the cru-
hyperchylomicronemic patients (4–6). The new C89F
jor alteration of LPL binding. Our findings, consistent
with previous reports (4–6, 11), confirm the critical im-
portance of the C65-C89 disulfide bond in GPIHBP1
structure and function.
endothelial binding site but also promotes LPL transport
(12). Specifically, C89A impaired LPL transfer to the en-
dothelial surface in an endothelial cell model (13). There-
fore, as a consequence of Ly6 mutations, dramatic TGRL
to the endothelial surface.
In our study, C89F mutation is associated with C14F, a
poorly documented signal peptide GPIHBP1 SNP. A func-
tional impact of this variant is suggested by the higher prev-
alence of C14F allele carriers in our hyperchylomicronemic
cohort compared with controls and reduced GPIHBP1 cell
may significantly reduce protein translocation efficiency in
APOA5 polymorphism (14). Paradoxically, this reduced
GPIHBP1-C14F expression does not lead to the expected
reduction of LPL binding in the absence of additional GPI-
variant on TGRL lipolysis remains uncertain. Nevertheless,
severe phenotype of our patient.
The G175R mutation is the first mutation described in
ing crucial to cell membrane transfer (15). Likewise, we
showed that the G175R mutation alters the transfer of
may affect GPI anchoring efficacy.
of C-terminal domain hydrophobic region with GPI-
transamidase. Consequently the C-terminal domain is pre-
G175R substitutes within the hydrophobic region a moder-
ately hydrophobic glycine for a highly hydrophilic arginine.
FIG. 2. Binding of human LPL to GPIHBP1 variants at the cell surface. A,
Western blot analysis. CHO pgsA-745 cells were transiently cotransfected
with a human Myc-Tagged LPL vector and an empty vector, an expression
vector for WT human GPIHBP1 (WT), GPIHBP1-C14F, GPIHBP1-C89F,
GPIHBP1-C14F/C89F, or GPIHBP1-G175R. The amount of LPL bound to
the cells was determined after incubation of cells with a rabbit antibody
against Myc tag, revealed in Western blot by a HRP-conjugated goat
antirabbit IgG. The levels of total GPIHBP1 and LPL expression were
assessed with an antibody against the Flag-tag. Actin was used as control.
The results are a representative series of three independent experiments. B
and C, Quantification of Western blots; B, amounts of LPL bound to
GPIHBP1 at the cell surface; C, amounts of LPL bound to GPIHBP1
normalized on total LPL in cells. All signals was normalized to actin signals
and expressed relative to the ratio for WT GPIHBP1 (set at 100%). Data
are expressed as mean ? SEM of three independent experiments. *, P ?
0.05 compared with WT.
Charrie `re et al.
C89F and G175R GPIHBP1 Mutations J Clin Endocrinol Metab, October 2011, 96(10):E1675–E1679
SuchmutationsdiminishingC-terminaldomainhydrophobic- Download full-text
was reduced by about half, and we cannot exclude that endo-
In our large cohort of 376 hyperchylomicronemic pa-
tients, the contribution of GPIHBP1 mutations is marginal
(0.53%), as reported in smaller cohorts (3, 6). Expression
of these mutations is recessive because heterozygous rel-
atives were normolipemic, in agreement with previous
studies (4, 5).
are all characterized by early pediatric hyperchylomi-
mutations (3–6). By contrast, hyperchylomicronemia oc-
curred in the third decade for patients with mutations lo-
cated outside the Ly6 domain, for our G175R patient and
the G56R homozygotes (7). Including our two patients, a
large majority of hyperchylomicronemic children or
adults with GPIHBP1 mutations exhibited acute pancre-
atitis (eight of 10) and were refractory to low lipid diets
(eight of 10) (3–7). Therefore, GPIHBP1 mutations are
characterized by a particular severity of the phenotypes.
of the Ly6 domain cysteines, we identified the C-terminal
domain as a new target for GPIHBP1 alteration in hyper-
chylomicronemic patients, causing defective cell surface
expression. Additionally, phenotypic expression of GPI-
polymorphism associated with hyperchylomicronemia. Ad-
ditional works are needed to explore the functionality of
C14F polymorphism and its association with hypertriglyc-
eridemia. Our results provide additional evidence that GPI-
hyperchylomicronemia in humans.
We thank the Institut National de la Sante ´ et de la Recherche
Me ´dicale and the French Society of Nutrition for their support.
Address all correspondence and requests for reprints to:
Dr. Sybil Charrie `re, Fe ´de ´ration d’endocrinologie, maladies
me ´taboliques, diabe `te et nutrition, Ho ˆpital Louis Pradel, 28
avenue Doyen Le ´pine, 69677 Bron Cedex, France. E-mail:
Disclosure Summary: The authors have nothing to disclose.
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