The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 12 4795-4798
Copyright © 2000 by The Endocrine Society
Type I Hyperlipoproteinemia Due to a Novel Loss of Function Mutation of Lipoprotein Lipase, Cys239
Trp, Associated with Recurrent Severe Pancreatitis
Michael M. Hoffmann,
Stephan Jacob,
Dieter Luft,
Reinhold-M. Schmülling,
Kristian Rett,
Winfried März,
Hans-Ulrich Häring and
Stephan Matthaei
Department of Medicine IV (S.J., D.L., R.-M.S., K.R., H.-U.H.,
S.M.), University of Tübingen, 72076 Tübingen; and
Department of Clinical Chemistry (M.M.H., W.M.), University of
Freiburg, 79106 Freiburg, Germany
Address correspondence and requests for reprints to: Stephan Matthaei, M.D., Department of Medicine IV, University of Tübingen, Otfried-Müller-Str. 10, D-72076 Tübingen, Germany. E-mail: Stephan.Matthaei{at}med.uni-tuebingen.de
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Abstract
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Lipoprotein lipase (LPL) is the major enzyme responsible for the
hydrolysis of triglyceride-rich lipoproteins in plasma. The purpose of
this study was to examine the molecular pathogenesis of type I
hyperlipoproteinemia in a patient suffering from recurrent severe
pancreatitis. Apolipoprotein (apo) CII concentration was normal as well
as apo CII-activated LPL in an in vitro assay. In
postheparin plasma neither LPL mass nor activity was detectable,
whereas hepatic lipase activity was normal. Direct sequencing of all 10
exons of the LPL gene revealed that the patient was homozygous for a
hitherto unknown mutation in exon 6, Cys239
Trp. The
mutation prevents the formation of the second disulfide bridge of LPL,
which is an essential part of the lid covering the catalytic center.
Consequently, misfolded LPL is rapidly degraded within the cells,
causing the absence of LPL immunoreactive protein in the plasma of this
patient. In conclusion, we have identified a novel loss of function
mutation in the LPL gene (Cys239Trp) of a patient with
type I hyperlipoproteinemia suffering from severe recurrent
pancreatitis. After initiation of heparin therapy (10,000 U/day sc),
the patient experienced no more episodes of pancreatitis, although
heparin therapy did not affect serum triglyceride levels.
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Introduction
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THE ETHIOPATHOGENESIS of acute pancreatitis
is multifactorial (1). Ethanol-induced and bile duct
obstruction are the most common causes of acute pancreatitis. Severe
hypertriglyceridemia represents a well known, but more infrequent,
cause of acute pancreatitis. Lipoprotein lipase (LPL; EC 3.1.1.34) is
the rate-limiting enzyme for the hydrolysis of triglycerides in
chylomicrons and very low-density lipoproteins (VLDLs). LPL is active
as a homodimer because full enzymatic activity apolipoprotein (apo) CII
is required as a cofactor. Several mutations occurring predominantly in
exons 4, 5, and 6 of the LPL gene have been shown to cause LPL
deficiency (2, 3, 4, 5, 6). As a result of these defects, the
enzyme is either not produced or becomes catalytically inactive.
Genetic deficiency of LPL causes type I hyperlipoproteinemia, which is
characterized by the presence of chylomicrons in fasting plasma and a
marked increase in plasma triglyceride levels. The patients usually
present with recurrent abdominal pain, pancreatitis, eruptive
xanthomas, lipemia retinalis, and hepatosplenomegaly.
In the LPL molecule functional domains can be distinguished, such as
the catalytic domain and specific binding sites for heparin and lipids
(7). Recently, the role of disulphide bridging within the
LPL molecule was determined in vitro by generating variants
with individually substituted cysteine pairs (8) and by
analysis of a naturally occurring mutation (9). The second
disulphide bridge, linking Cys216 and
Cys239, marks the end of the catalytic groove
covering lid (10, 11) and seems to be of particular
importance. Elimination of this bridge by exchange of both cysteine
residues leads to complete loss of catalytic activity, whereas the
protein is still secreted, albeit at a lower rate (8).
Here, we describe a novel loss of function mutation of LPL,
Cys239Trp, in a patient with type I
hyperlipoproteinemia suffering from recurrent episodes of severe
pancreatitis. After initiation of heparin therapy using 10,000 U/day sc
the patient had no more episodes of pancreatitis, although serum
triglyceride levels were unaffected by heparin therapy.
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Experimental Subject and Methods
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Patient characteristics
The patient is a 47-yr-old Caucasian female who suffered from
recurrent severe pancreatitis. Since the age of 14 she complained of
recurrent abdominal pain; at age 19 she developed her first episode of
pancreatitis. By that time, hyperlipoproteinemia type I (triglycerides,
13.68–45.6 mmol/L) was diagnosed, and a low-fat diet was initiated.
During the following 3 yr she experienced a total of five episodes of
severe pancreatitis until a therapy of 10,000 U heparin sc per day was
started. During the following 25 yr after the initiation of heparin
until the date of the examination, during which the present data were
collected (1973–1998), no further episodes of pancreatitis occurred,
although triglyceride levels remained in the range as indicated above.
Between 1970 and 1998 the body weight of the patient was remarkably
stable (50–52 kg). Furthermore, during that time period the patient
did not change the recommended low-fat diet, the degree of exercise was
constantly moderate, and the patient did not receive further
lipidlowering medication. The patient is the only child of her
parents. LPL-sequence analysis was also done in the mother; no material
was available from the patient’s father. Informed consent was obtained
from the patient and her mother.
Lipids, lipoproteins, and apolipoproteins
Blood of fasting individuals was drawn into tubes containing
EDTA-K2 (final concentration, 1.5–2 g/L). Plasma
was recovered by centrifugation. Cholesterol (total and nonesterified),
triglycerides, and phospholipids were measured in duplicates using
enzymatic reagents (Roche, Mannheim, Germany; and WAKO,
Neuss, Germany). VLDL (d < 1.0063 kg/L), intermediate density
lipoprotein (1.0063 kg/L < d < 1.019 kg/L), and LDL (1.019
kg/L < d < 1.065 kg/L) were isolated by preparative
ultracentrifugation. High-density lipoprotein cholesterol was
determined by precipitating apo B containing lipoproteins in the d
> 1.0063 kg/L infranate. Apo AI, apo B, apo CII, apo CIII, and apoE
were measured by automated rate nephelometry (Array Protein System;
Beckman Coulter, Inc. Instruments, Brea, CA).
LPL genotyping and sequencing
DNA was extracted from white blood cells using "blood PCR"
DNA isolation cartridges (Qiagen GmbH, Hilden, Germany)
according to the manufacturer’s instructions. Individual exons and a
part of the promoter of the LPL gene were amplified by PCR. Typical
reaction conditions were as: 20–50 ng DNA, 50 mM KCl, 10
mM Tris-HCl (pH 8.3), 1.5 mM
MgC12, 0.001% (wt/vol) gelatin, 200
mM dNTPs, 1 mM each primer, and 0.5 U
Taq polymerase (Roche) in a 50 mL reaction.
Amplification was achieved using an initial denaturing step of 95 C for
3 min and then 36 cycles of 95 C for 45 sec, 55 C for 1 min, 72 C for 1
min, followed by a final elongation step of 72 C for 10 min. Purified
PCR products were directly sequenced using the dideoxy chain
termination method (BigDye Sequencing Kit; Perkin-Elmer Corp., Norwalk, CT) on a ABI 377 sequencer.
The substitution CG created a new NlaIv restriction site.
To genotype other family members exon 6 was amplified and the PCR
fragments were digested with 1U NlaIv (NEB; Schwalbach,
Germany) for 3 h at 37 C and analyzed electrophoretically on a 3%
agarose gel.
LPL mass and activity
LPL was measured in plasma obtained 10 min and 20 min after iv
administration of heparin (50 U/kg). LPL mass was determined three
times by a sandwich enzyme immunoassay (MARKIT-F LPL; Dainippon
Pharmaceutical, Osaka, Japan). LPL activity was determined using
a radiolabeled 14C-triolein/phosphatidylcholine
emulsion, containing 10% (vol/vol) human heat-inactivated serum as a
source of apo CII. Labeled free fatty acids liberated by the reaction
were measured by scintillation counting. LPL activity was taken as the
fraction of the total lipolytic activity inhibited by 1 M
NaCl.
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Results
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The patient presented with severe type I hyperlipoproteinemia; the
current serum triglycerides were 13.54 mmol/L. During the outpatient
care at our clinic from 1970–1998 serum triglyceride levels were in
the range of 11.4–45.6 mmol/L (see also other lipoprotein data in
Table 1
). The apo CII concentration was
normal (6.9 mg/dL). To exclude a functional defect of apo CII as a
cause of hyperlipidemia we tested the ability of the patient’s apo CII
to activate bovine LPL in an in vitro assay. Serum from the
patient was diluted 1:10 and added to a reaction mixture containing
purified bovine LPL and labeled triolein. LPL was activated 
5-fold
by the patient’s apo CII. In postheparin plasma (10 and 20 min after
iv heparin bolus) neither LPL mass nor activity was detectable, whereas
hepatic lipase activity was normal (Table 2). Direct sequencing of all 10 exons and
of the promoter region of the LPL gene lead to the detection of a
hitherto unknown mutation in exon 6, an exchange of C to G at
nucleotide position 972 (Fig. 1).
This mutation is on the third position of codon 239 and changes the
amino acid from cysteine (TGC) to tryptophan
(TGG). The patient was homozygous for this mutation. We did
not find any other mutation within the coding sequence. It is unusual
that a newly discovered mutation appears in a homozygous state, in
particular, because there appeared no consanguinity. Therefore, we
performed Southern blot analysis to exclude hemizygosity due to a major
deletion in the LPL gene. We could not detect any difference between
the patient’s DNA and a control (data not shown). As shown by
NlaIV restriction fragment length analysis, the mother of
the patient is heterozygous for this mutation (Fig. 2).
Unfortunately, material from other family members was not
available.
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Figure 2. Restriction isotyping of LPL
Cys239Trp; agarose gel electrophoresis of
NlaIv digested LPL exon 6 fragment. Wild-type allele,
361 bp; mutated allele, 280 bp + 81 bp; lane 1, 100 bp marker; lane 2,
control; lane 3, mother; lane 4, patient.
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Discussion
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We report on a homozygous carrier of a novel
loss-of-function mutation of LPL, LPL
Cys239Trp, leading to severe type I
hyperlipoproteinemia. As shown in Table 2
, no LPL mass or LPL activity
could be detected in the patients’ plasma. Takagi et al.
(12) described another mutation at this position, a
nucleotide C972 to A transversion, resulting in
substitution of a premature termination codon (TGA) for
Cys239 (TGC). These authors were
unable to determine whether a truncated LPL protein was produced or
not.
The mutation identified in our patient prevents the formation of the
second disulphide bridge of LPL, which flanks the end of the lid
covering the catalytic center (10, 11). Recently, the role
of disulphide bridging in the stability of LPL was determined in
vitro by generating variants with individually substituted
cysteine pairs (8). In contrast to our observation the
authors found that LPL, in which the formation of the
Cys216-Cys239 disulphide
bridge was disturbed, was secreted (22% of wild-type mass), whereby
the mutant was completely inactive. Several experiments have shown that
the length, position and amino acid composition of the lid is essential
for LPL activity and substrate specificity (10, 11, 13, 14). It is, therefore, not surprising that the LPL
Cys239Trp variant with the missing second
disulphide bridge completely lost activity. Contrary to the in
vitroexpressed LPL mutation in which cysteine residues were
changed (8), the Cys239Trp mutation lead to
complete absence of protein in plasma. This discrepancy may be
explained by wrong cysteine bridging within the LPL, which is very
probable if only one cysteine is missing, especially if the missing
cysteine is in front of other cysteines (positions 264, 275, 278, and
283). Consistently, Busca et al. (15) showed
that misfolded LPL is rapidly degraded within the cells.
The importance of the correct formation of disulphide bridges within
the LPL is demonstrated by another missense mutation,
Cys418Tyr (9), leading to the
secretion of inactive LPL into the plasma. In this case, the last
disulphide bridge is missing and the folding of the C-terminal domain
of LPL might be impaired.
The mechanism by which heparin prevented further episodes of
pancreatitis without affecting plasma triglyceride levels in our
patient remains unresolved. However, during the last decade evidence
has accumulated suggesting that heparin activity facilitates removal of
triglycerides from the blood by inducing the expression of hepatic
triglyceride lipase (16), which, in concert with apo E, is
involved in hepatic clearance of chylomicrons and VLDL remnant
particles from plasma (17). Furthermore, heparinase
treatment of liver cells almost totally abolishes hepatic clearance of
remnant particles (18). Thus, it is tempting to speculate
that heparin therapy caused the favorable course in our patient by
stimulating the expression and/or activity of different lipases,
possibly of hepatic origin.
As an alternative therapeutic approach, Heaney et al.
recently presented evidence to prevent recurrent pancreatitis in
familial LPL deficiency using high-dose antioxidant therapy
(19). These authors treated three patients with LPL
deficiency using 
-tocopherol (270 IU/day), ß-carotene (9,000
IU/day), vitamin C (540 mg/day), organic selenium (600 µg/day), and
methionine (0.5 g/day). Following initiation of this high-dose
antioxidant therapy no more episodes of pancreatitis occurred in two of
the three patients and were markedly reduced in the third.
In conclusion, we have identified a novel loss-of-function mutation of
LPL in a patient with type I hyperlipoproteinemia and recurrent
episodes of severe pancreatitis. After initiation of therapy with
heparin (10,000 U/day sc) the patient did not have any more episodes of
pancreatitis.
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Figure 3. Schematic map of the translated
LPL-complementary DNA including the positions of disulphide bridges
(blue) and the mutation (red). The
catalytic triad residues are depicted as green,
short bars.
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Received March 28, 2000.
Revised July 18, 2000.
Revised August 29, 2000.
Accepted September 1, 2000.
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Abstract
Introduction
