A 60-y-old chylomicronemia patient homozygous for missense mutation
(G188E) in the lipoprotein lipase gene showed no accelerated atherosclerosis
Tetsu Ebaraa,b, Yoriko Endoa, Shouichi Yoshiikeb, Masatomi Tsujib,
Susumu Taguchib, Toshio Murasea, Minoru Okuboa,c,⁎
aOkinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan
bDepartment of Internal Medicine, Showa University Northern Yokohama hospital, 35-1 Chigasaki, Tsuzuki-ku, Yokohama, Kanagawa 224-8503, Japan
cDepartment of Endocrinology and Metabolism, Toranomon Hospital, Tokyo, Japan
Received 10 May 2007; received in revised form 25 August 2007; accepted 27 August 2007
Available online 1 September 2007
Background: Familial lipoprotein lipase (LPL) deficiency is a rare autosomal recessive disorder caused by mutations in the LPL gene. Patients
with LPL deficiency have chylomicronemia; however, whether they develop accelerated atherosclerosis remains unclear.
Methods: We investigated clinical and mutational characteristics of a 60-y-old Japanese patient with chylomicronemia.
Results: The patient's fasting plasma triglyceride levels were N9.0 mmol/l. In postheparin plasma, one fifth of the normal LPL protein mass was
present; however, LPL activity was undetectable. Molecular analysis of the LPL gene showed the patient to be a homozygote of missense
mutation replacing glycine with glutamine at codon 188 (G188E), which had been known to produce mutant LPL protein lacking lipolytic activity.
Ultrasonographic examination of the patient's carotid and femoral arteries showed no accelerated atherosclerosis. Moreover, 64-slice mechanical
multidetector-row computer tomography (MDCT) angiography did not detect any accelerated atherosclerotic lesions in the patient's coronary
arteries. The patient had none of the risk factors such as smoking, hypertension, and diabetes.
Conclusions: Our case suggests that accelerated atherosclerosis may not develop in patients with LPL deficiency, when they have no risk factors.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Lipoprotein lipase deficiency; Chylomicronemia; Point mutation; Atherosclerosis; Mechanical multidetector-row computer tomography
the catabolism of triglyceride-rich lipoprotein particles. The
enzyme hydrolyses triglycerides in chylomicrons and VLDL
using apolipoprotein (apo) CII as a cofactor . LPL is mainly
synthesized by adipose tissue and muscle, transported, and an-
chored to the luminal surface of the capillary endothelium by
heparin sulfate proteoglycans. It is postulated that LPL functions
as a potent bridge between lipoproteins and proteoglycans on
vessel walls, retaining atherogenic lipoproteins on endothelial
cells, exclusive of its enzymatic activity . Some animal model
experiments, using mice and rabbits, showed that macrophage
LPL promotes foam cell formation and atherosclerosis [3,4]. On
the contrary, systemic overexpression of LPL cDNA in mice is
in susceptibility to atherosclerosis, and whether LPL protein is
pro-atherogenic or anti-atherogenic remains unclear in humans.
Defects in LPL-apo CII lipolysis system result in severe
accumulation of chylomicrons in plasma. Familial LPL deficien-
cy is a rare autosomal recessive disorder, estimated to be about
one in one million persons. Patients with LPL deficiency show
severe hypertriglyceridemia, eruptive xanthoma, hepatospleno-
megaly, and recurrent attacks of pancreatitis. Many patients died
from pancreatitis at an early age in the past, but today, LPL
deficiency is diagnosed in childhood and the maintenance of a
low-fat diet enables patients lead a longer life . Whether
patients with LPL deficiency develop premature atherosclerosis
is, consequently, becoming a concern.
Clinica Chimica Acta 386 (2007) 100–104
⁎Corresponding author. Okinaka Memorial Institute for Medical Research, 2-
2-2 Toranomon, Minato-ku, Tokyo 105-8470, Japan. Tel.: +81 3 3588 1111;
fax: +81 3 3582 7068.
E-mail address: QFG00550@nifty.com (M. Okubo).
0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
It has generally been thought that patients with LPL
deficiency do not predispose to develop atherosclerosis, since
chylomicrons were believed to be too large to penetrate vessel
walls. A study using a mouse model for chylomicronemia
supports this view . However, Benlian et al. raised a question
on this assumption, based on an observation that premature
atherosclerosis develops in four patients with LPL deficiency,
atherosclerosis . In contrast, we reported that there was no
evidence of accelerated atherosclerosis in a 66-y-old chylomi-
cronemia patient homozygous for nonsense mutation . It has
thus been suggested a hypothesis that type of LPL mutation may
affect atherosclerosis: LPL mutations leading to null LPL
protein do not accelerate atherosclerosis, while missense
mutations with retained LPL protein promote atherosclerosis
Here we report a 60-y-old patient with LPL deficiency, who
had non-catalytic LPL protein and did not have accelerated
atherosclerosis. Our case provides a unique opportunity to
explore clinical atherosclerosis associated with homozygous
2. Materials and methods
A 60-y-old Japanese female was assessed in this study. She was brought up
in the Kanto district in Japan and received a medical checkup for the first time
when she was 49 y. Her plasma triglyceride concentration was then 17.1 mmol/l.
Hypertriglyceridemia was,in retrospect, evident in her medical recordas shown in
Table 1, but she did not pay attention to it. Her body mass index was 22.4 kg/m2
and her blood pressure was within normal range. The patient's fasting blood
glucose and hemoglobin A1c levels were also normal. She has never experienced
anginapectoris. At the ageof60 y, shesuffered from acutepancreatitis. Beforethe
admission, she attended several parties and took large amounts of meals
containingfatand carbohydrates, whichsheused to refrain from. On dietary habit,
she has taken a traditional Japanese diet, which consisted of 17% protein, 23% fat,
and 60% carbohydrate, that is, equivalent of diet recommended by the national
cholesterol education program. After discharge, sheis keepingonlow-fat diet
b10% of total calories. She has no habit of smoking and drinking alcohol. Her
coronary heart disease or pancreatitis, to our knowledge.
2.2. Lipids and apoproteins
A blood sample was obtained from the patient after an overnight fast.
Plasma total cholesterol and triglyceride concentrations were measured by
enzymatic methods. High-density lipoprotein (HDL) cholesterol was
quantified in the plasma after the polyanion precipitation of apo B-con-
taining lipoproteins. Low-density lipoprotein (LDL) cholesterol was mea-
sured by a homogeneous method using a direct LDL-cholesterol assay kit
(Daiichi Pure Chemicals Co., Tokyo, Japan) . Plasma apo AI, AII,
B, CII, CIII, and E were determined by an immunoturbidometric assay
(Daiichi Pure Chemicals Co., Tokyo, Japan), according to the manufacturer's
2.3. LPL mass and activity
Postheparin plasma from the patient was collected 10 min after a bolus
injection of heparin (30 units/kg body weight). The LPL mass was determined
to the manufacturer's instructions. LPL activity and hepatic triglyceride lipase
(HL) activity were assayed as described previously .
2.4. Nucleotide sequence analysis of the LPL gene
Genomic DNA was extracted from the peripheral blood sample after
informed consent was obtained from the patient. The PCR fragments con-
taining each exon and exon–intron boundary of the LPL gene were am-
plified as described previously . Direct sequencing of the PCR
products was performed using a Big Dye Terminator cycle sequencing kit
on a genetic analyzer PRISM 310 (Applied Biosystems, CA, USA). The
nucleotides of LPL cDNAwere numbered from the AUG codon according to
GenBank accession no. NM_000237. The study was approved by our ethics
2.5. Detection of the mutation by restriction fragment length
To verify the G-to-A transition at nucleotide 818 in exon 5, a pair of a sense
primer (5′-atc tgt gtt cct gct ttt ttc c-3′) in intron 4 and an antisense primer (5′-
aag agt cac att taa ttc gct tc-3′) in intron 5 were used. PCR was carried out by
30 cycles of denaturation, annealing, and extension at 94 °C for 30 s, 54 °C for
1 min, and 72 °C for 2 min, respectively. The fragments then were digested
with restriction endonuclease Ava II and analyzed on a 5% polyacrylamide
gel. In the presence of the mutation, the 256-bp PCR product was uncleaved by
Ava II, whereas it was cleaved into 168- and 88-bp fragments with the normal
Biochemical data for a lipoprotein lipase deficient patient
VariableAdmissionDay 1018 days after discharge10 months after dischargeNormal range
Total cholesterol (mmol/l)
LDL cholesterol (mmol/l)
HDL cholesterol (mmol/l)
Apo AI (mg/dl)
Apo B (mg/dl)
Apo CII (mg/dl)
Apo CIII (mg/dl)
Apo E (mg/dl)
Apo E genotype
LPL mass (ng/ml)
ND, not determined.
aNormal values are expressed as mean±S.D.
101T. Ebara et al. / Clinica Chimica Acta 386 (2007) 100–104
2.6. Apo E genotype
Apo E genotype was determined by digestion of PCR-amplified fragments
with restriction enzyme Hha I as described .
2.7. Clinical examination of the patient
The exercise-tolerance electrocardiogram was judged according to the
Minnesota standards. Intima–media thickness (IMT) was measured at the
patient's bilateral common and internal carotid arteries, and femoral
arteries by standard B-mode ultrasonography as described previously .
In order to screen coronary artery lesions, 64-slice mechanical multi-
detector-row computer tomography (MDCT) angiography (Toshiba, Tokyo,
Japan) was performed for the patient. A volume data set was acquired
covering the region from the pulmonary hilum to the diaphragmatic surface
of the heart. Computed tomography gantry rotation time was 330 ms. Tube
voltage and effective tube current–time product were set to 120 kV and
880 mA s.
Table 1 shows the biochemical data for the patient. The
HDL-cholesterol level was below normal range. Chylomicrons
were observed in the patient's plasma after the overnight storage
apo E genotype was E3/E3. The patient's HbA1c level was
normal. In postheparin plasma, the patient's LPL mass was
45 ng/ml, i.e., one fifth of the mean normal value. However, her
LPL activity was undetectable (normal: 6.4±2.1 μmol FFA/ml/
h), whereas her HL activity was within normal range (6.6 μmol
FFA/ml/h; normal: 8.8±2.9).
The direct sequencing analysis showed a G-to-A substitution
at nucleotide 818, the second base of codon 188, in exon 5 of
the patient's LPL gene. This point mutation replaced glycine
(GGG) with glutamic acid (GAG) (G188E). In Ava II RFLP
analysis, the patient had a 256-bp fragment alone, demonstrat-
ing that the patient was homozygous for the G188E mutation.
Homozygosity was compatible with the fact that her parents
were first cousins. These findings allowed us to diagnose the
patient as having with LPL deficiency.
The ultrasonogram of the carotid arteries indicated that this
nor calcification was detected. Ultrasonographic examination of
femoral arteries did not detect accelerated atherosclerosis either.
MDCT angiography for this patient showed a thin atheromatous
plaque in the right coronary artery. However, this lesion was
consistent with her age and no significant stenosis was detected in
any other segments of coronary arteries. These findings indicated
that thepatienthad noclinical signs ofacceleratedatherosclerosis.
We showed the patient to be homozygous for missense
mutation (G188E) in the LPL gene, and diagnosed her with LPL
deficiency. Previous reports on patients with G188E have de-
monstrated that the mutation causes LPL deficiency. In
Caucasian, G188E has been shown as one of the prevalent LPL
deficient patients of non-Caucasian descent. Our patient is the
third case with G188E and the eldest in Japan, since 2 patients
under 1 y with the same mutation have been reported [17,18].
Patients homozygous for G188E manifested reduced amount of
LPL protein without LPL activity . Moreover, in vitro
expression studies established that G188E produced mutant LPL
protein completely lacking lipolytic activity [20–22]. Our patient
In complete LPL deficient patients, reports focusing on
whether they have accelerated atherosclerosis have been limited
to be associated with atherosclerosis, this hypothesis has been
called into question by Benlian et al. They observed premature
atherosclerosis in 4 patients (patients 2–5 in Table 2) and
Reports focusing on atherosclerosis of LPL deficient patients
Patient no. Age Sex LPL mutation LPL massaCervical atherosclerosis Coronary atherosclerosis Smoking Hypertension Glucose intolerance Reporter
4 67M 33+n.d.+++Benlian
n.d., not described.
aPercentage of the mean normal value.
102T. Ebara et al. / Clinica Chimica Acta 386 (2007) 100–104
proposed that defective lipolysis may increase susceptibility to
atherosclerosis in humans . Saika et al. observed that a patient
with missense mutation L303F suffered from coronary artery
disease and severe systemic atherosclerosis (patient 7) . On
the other hand, we described a patient homozygous for nonsense
mutation who had no evidence of accelerated atherosclerosis
(patient 6) . In addition, Kawashiri et al. showed that a patient
with another nonsense mutation had no clinically significant
atherosclerotic lesions (patient 8) . Accordingly, it has been
argued that atherosclerosis among those patients is attributable to
the type of LPL mutations [7–9,24,25]. Missense mutation resul-
activity) develops accelerated atherosclerosis, while nonsense
mutations do not accelerate atherosclerosis because of the lack of
had no evidence of accelerated atherosclerosis, and those with
LPL mass (patients 2–5, 7) had accelerated atherosclerosis.
In contrast to the hypothesis that catalytically inactive LPL
protein is pro-atherogenic, our patient homozygous for missense
ultrasonography and 64-slice MDCT angiography, and no
evidence of accelerated atherosclerosis was found. This case
suggests that the type of LPL mutation may not be a major factor
determining progression of atherosclerosis in LPL deficient
patients. Presumably, classic risk factors, such as smoking, hyper-
tension, and glucose intolerance, play a larger role on athe-
rosclerosis than LPL. As far as risk factors are concerned, patients
2–4 with missense mutations had some of risk factors. Patient 3
who had angina pectoris. However, patient 8 did not show
accelerated atherosclerosis, despite risk factors. Further study is
necessary to clarify the impact of LPL mutation types on
atherosclerosis in LPL deficient patients.
Decreased LDL-cholesterol levels may prevent premature
atherosclerosis in LPL deficient patient. LPL plays a key role in
production of LDL and the defect of LPL function leads to the
impairment of the conversion from VLDL to LDL. Zambon et al.
reported a patient who is both heterozygous for FH and homo-
level was significantly lower than the levels of noromolipidemic
relatives . The absence of LPL activity lowers plasma LDL-
cholesterol levels and the susceptibility to atherosclerosis.
In summary, the molecular basis of a 60-y-old patient with
chylomicronemia has been characterized, and our case suggests
that accelerated atherosclerosis may not develop in patients with
LPL deficiency, when they have no risk factors.
This study was supported in part by a Research Grant from
the Takeda Science Foundation.
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