ISSN 0163-7258/96 $32.00
PI1 SOl63-7258(96)00005-S
Pharmacol. Ther. Vol. 70, No. 2, pp. 101-135, 1996
Copynght 0 1996 Elsev~er Science Inc.
ELSEVIEK
Editor: D. Shugur
Associate
Molecular Pathobiology of
the Human Lipoprotein Lipase Gene
Ven Murthy,*t Pierre Julien,+ and Claude Gag&
*MOLECULAR
BIOLOGY LABORATORY ON HUMAN DISEASES.
DEPARTMENT OF BIOCHEMISTRY,
FACULTY OF MEDICINE, LAVAL UNIVERSITY,
STE-FOY, QUEBEC GIK 7P4, CANADA
+DEPARTMENT OF MEDICINE AND CENTRE DE RECHERCHE
MALADIES
SUR LES
Lll'lDlQUES.LAVALUNIVERSITY MEDICAL CENTRE,STE-FOY,QUEBEC
GlV 4G2,CANADA
is a key enzyme in the metabolism of lipids. Many
ABSTRACT.
Lipoprotein
lipase (LPL; E.C. 3.1.1.34)
diseases, including obesity, coronary
heart disease, chylomicronemia
(pancreatitis),
and atherosclerosis,
appear to be directly or indirectly related to abnormalities
in LPL function. Human LPL is a member
of a superfamily
of lipases that includes hepatic lipase and pancreatic
lipase. These lipases are characterized by extensive homology, both at the level of the gene and the mature protein, suggesting that they
have a common
evolutionary
origin. A large number of natural mutations have been discovered in the
human LPL gene, which are located at different sites in the gene and affect different functions of the
mature protein. There is a high prevalence
of two of these mutations
(207 and 188) in the Province
of Quebec, and one of them (207) is almost exclusive to the French-Canadian
population.
A study of
these and other naturally occurring
mutant LPL molecules,
as well as those created in vitro by sitedirected mutagenesis,
indicate that the sequence of LPL is organized into multiple structural and functional units that act in concert in the normal enzyme. In this review, we discuss the interrelationships
of LPL structure and its function,
the molecular etiology of abnormal LPL in humans, and the clinical
and therapeutic
aspects of LPL deficiency. PHARMACOL.
THER.
70(2): 101-135. 1996.
KEY WORDS.
Familial lipoprotein
lipase deficiency,
micronemia
syndrome,
gene mutations.
hepatic
lipase, pancreatic
lipase,
lipase gene family,
chylo-
CONTENTS
1. INTRODUCTION
. . . . . .. . . . . . . . . .. . .
2. THE LIPASE SUPERFAMILY
. . . . . . . . .. .
2.1. HUMAN
LIPASES AND
THEIRFUNCTION
. . . . . . . .. . . . . .
2.2. GENEORGANIZATIONOFLIPASES
..
2.3. CONSERVATION
ANDEVOLUTION
OF
LIPASES ANDTHEIR
STRUCTURALDOMAINS
.........
3. LIPOPROTEIN LIPASE . . . . . . . . . . . . . . .
3.1. FUNCTIONALANATOMYOFTHE
LIPOPROTEIN LIPASE MOLECULE
...
3.2. MOLECULARPHYLOGENYOF
LIPOPROTEIN LIPASE . . . . . . . . . . .
3.3. MUTATIONSINVOLVINGTHE
NONCODINGSEQUENCESOFTHE
HUMAN
LIPOPROTEIN
LIPASEGENE
. . . . . . . . . . .. . . . . .
3.4. MUTATIONS INVOLVING THE
CODING SEQUENCES OF THE
HUMAN LIPOPROTEIN LIPASE GENE
4. THE CHYLOMICRONEMIA
SYNDROME
..
4.1. DESCRIPTION
. . . . . . .. . . . . . . . .
4.2. DIAGNOSIS . . . . . . . . . . . . . . . . . . .
4.3. CLINICAL MANIFESTATIONS
......
4.3.1. SYMPTOMS
OF
CHYLOMICRONEMIA
. . .. ..
102
102
102
103
105
107
107
109
113
114
118
118
118
119
119
4.3.2.
SIGNSOFCHYLOMICRONEMIA
4.3.3. ANOMALOUS
LABORATORY
FINDINGSIN
CHYLOMICRONEMIA
. . .. . .
4.4. CAUSESOFCHYLOMICRONEMIA
SYNDROME
. . . . . .. . . . . . . . .. . . .
5. PRIMARY
LIPOPROTEIN LIPASE
DEFICIENCY
. .. . . . . . . . . . .. . . . . . . . .
5.1. HOMOZYGOTESTATEOFLIPOPROTEIN
LIPASE DEFICIENCY . . . . . . . . . . . . .
5.2. HETEROZYGOTESTATEOF
LIPOPROTEIN LIPASE DEFICIENCY . .
5.3. ORIGIN ANDDISSEMINATION
OF
LIPOPROTEIN LIPASEGENEDEFECTS
INQUeBEC
. . . . .. . . . . . . .. . . . . .
6. TREATMENT
OF LIPOPROTEIN LIPASE
DEFICIENCY . . . . . . . . . . . . . . . . . . . . . .
6.1. IDENTIFICATION AND CORRECTION
OFSECONDARYFACTORS
. . . . . .. . .
6.2. FAMILY SCREENING AND
COUNSELING
. . . . . . . . .. . . . . . . .
6.3. DIETARYREGIMEN
. . . . . . . . .. . . .
6.4. DRUGTHERAPY
. . . . . . . . . . . .. .
6.5. PROSPECTS•
FGENETHERAPY . . . .
ACKNOWLEDGEMENTS
. . . . . . .. . . . . . . . .
REFERENCES
. . . . . . . . . .. . . . . .. . . . . . . .
120
121
121
122
122
122
124
125
125
125
125
126
127
127
127
ABBREVIATIONS,
apo, apolipoprotein; FCH, familial combined
hyperlipidemia;
HDL, high-density
lipoprotein;
HL, hepatic lipase; IDL, intermediate-density
lipoprotein;
LDL, low-density
lipoprotein;
LPL, lipoprotein lipase; LRP, LDL receptor-related
protein; MCT, medium-chain
TG; PL, pancreatic
lipase; TG, triglyceride;
VLDL,
very low-density
lipoprotein.
~Corresponding
author.
V. Murthy et al
102
1, INTRODUCTION
Lipoprotein lipase (LPL; E.C. 3.1.1.34) is the enzyme responsible for the influx of free fatty acids into peripheral tissues
for storage in the form of triglycerides (TGs) or as a source
of energy. It is known that the action of LPL on TG-rich
lipoproteins promotes the exchanges of lipids between lipoproteins and that LPL is, thus, indirectly involved in the
maturation of the majority of plasma lipoproteins (Brunzell,
1989). There is evidence to suggest that LPL has a contributory role in the manifestation of a number of pathologic
conditions
related to the metabolism ofTG-rich
lipoproteins
(Bergeron et uI., 1991). Dysfunction and deficiency of LPL
activity thus has been associated with the pathogenesis of
various dyslipoproteinemias
and with the production of
atherogenic
particles, such as intermediate-density
lipopro-
teins (IDL) and low-density lipoproteins (LDL) (Brunzell,
1989; Eckel, 1989). Because of its physiological importance
and its possible role in lipid-related pathologies, LPL has
been intensively examined in many in s~ivoand in vitro studies
in both human and animal models.
The functions
of LPL and its regulation have been high-
lighted in a number of excellent and detailed recent articles
(Borensztajn, 1987; Brunzell, 1989; Eckel, 1989; Bensadoun,
1991; Auwerx et al., 1992; Braun and Severson,
1992;
Dolphin, 1992; Lalouel et al., 1992; Santamarina-Fojo,
1992;
Wang et (II., 1992; Olivecrona and Bengtsson-Olivecrona,
1993; Santamarina-Fojo
and Dugi, 1994). The objective of
the present review is to discuss the molecular basis of various LPL deficiencies known to occur in humans and to provide an assessment
of the possible therapeutic
approaches
that are currently available for the treatment of these pathologic conditions.
(PL; EC. 3.1.1.3). All three enzymes hydrolyze lipid emulsions and have similar aqueous-lipid interfacial catalytic activity. The three lipases show several individual characteristics,
as well as differences in their properties and physiological
functions (Table 1). LPL is synthesized in the parenchymal
cells of several tissues, including skeletal muscle, heart, adipose tissue, lung, lactating mammary gland, etc. (Borensztajn,
1987). It is expressed transiently
in the embryonic
liver, but
not in the adult (Vilaro et al., 1988; Gimenez Llort et al.,
1991). It is transported from the site of synthesis to the luminal
surface of vascular endothelia, where it is anchored by ion
interaction
with heparin
sulfate proteoglycans
and/or by
glycosyl phosphatidylinositol
(Braun and Severson, 1992).
It can be released from the bound form into the circulation
by the administration of heparin. This property has been
widely used to release LPL from cell membranes and to purify
LPL by heparin-Sepharose affinity chromatography (Bengtsson and Olivecrona, 1977). In its active form, LPL is a glycosylated noncovalent homodimer (Osborne et al., 1985)
chat reversibly dissociates to form inactive monomers under
physiological
conditions,
as well as under the effects of pH,
ionic strength, and temperature (Olivecrona and BengtssonOlivecrona, 1987). For enzyme activity, it has an obligatory
requirement for apolipoprotein (apo) CII, a small protein
of 79 amino acid residues. Each LPL subunit contains a site
for heparin binding, as well as a site for interaction with
ape CII.
The physical and kinetic properties of LPL enzyme have
been
reviewed
in detail by Olivecrona
and Bengtsson-
Olivecrona (1987). The enzymatic activity is optimal at a
pH between 8.0 and 8.5 and it is stabilized by the presence
of lipids or by binding to lipid-water interfaces and detergents, such as deoxycholate. Although LPL is activated by
its cofactor, apo C-II, it is inhibited by various factors, including fatty acids, apo CIII and, possibly, apo E. It is inhibited
2. THE LIPASE SUPERFAMILY
2.1. Human Lipases and Their Function
Apart from LPL, there are two other lipases with important
also by high salt concentrations (1 M NaCI), an observation
that is used to differentiate between plasma LPL and HL
lipolytic activities: hepatic lipase (HL) and pancreatic
activities. The later remains unaffected
TABLE
lipase
1. Properties of the Enzymes of the Human Lipase Family
HL
LPL
Site of synthesis
Site of action
Subunits
Glycosylation
Binding
to cell surface
Released
into circulation
Activators
Inhibitors
Aqueous-lipid
interfacial
activity
Lipoprotein-ligand
activity
Substrates
for lypolysis
Parenchymal
cells
Capillary
endothelia
of several
Homodimcr
+
Glycosaminoglycan
links
By heparin
Apo CII
Apo CIII and possibly
apo E
f
+
Chylomicrons,
VLDL
Enzyme
TG
Specificity
under these condi-
activity
lipasc,
Sri-1 and
minor
phospholipase
Sri-3 ester
bonds
tissues
Hepatocytes
Liver sinusoids
M onomer
+
Glycosaminoglycan
By heparin
Possible stimulation
PL
links
Exocrine
pancreas
Duodenum
Monomer
+
Free
by apoE
Colipase
+
+
Chylomicron
remnants,
IDL, HDLL
TG lipasc, phospholipase
Sri-1 and
Sri-3 ester
bonds
+
Alimentary
TGs
TG lipase, variable
phospholipase
Sn-1 and Sn-3
ester bonds
Human Lipoprotein Lipase Gene
103
tions. In vitro, LPL can hydrolyze a number
such as long- and short-chain
of substrates,
glycerides, phospholipids,
and
transports PL and procolipase, along with other pancreatic
hydrolases, into the duodenum, where the enzyme completes
various synthetic substrates, but at much lower rates than
TG (see Olivecrona and Bengtsson-Olivecrona,
1987, for
a review). Chylomicrons
and very-low-density lipoproteins
(VLDL) constitute the major substrates for lipolysis by LPL
the hydrolysis
lipase (Verger,
the PL to the
lytic rate (van
in viva (Wang et al., 1992). In the resulting
of apo CII in LPL activity. Like LPL and HL, PL acts preferentially on the sn-1 and sn-3 bonds of TGs (Dolphin, 1992).
reaction,
LPL
shows a high degree of substrate specificity. The sn-1 and
sn-3 bonds of lipoprotein TGs are preferentially hydrolyzed,
generating sn-2 monoglycerides and free fatty acids (Dolphin,
1992). The sn-2 bond is attacked subsequently after nonenzymatic conversion
to the sn-1 isomeric form. In addition
to its lipolytic activity, LPL also fulfills other important functions in lipid metabolism. It recently has emerged that lipoproteins may bind to cell surfaces through lipase-mediated
binding, a ligand function independent of the enzymatic
activities of the lipases (Beisiegel et al., 1994). The heparinbinding capacity of LPL (shared by HL, but not by PL) may
facilitate the uptake and internalization of plasma lipoproteins through specific receptors (Beisiegel, 1995). Both transcriptional
and
posttranscriptional
steps
of LPL
gene
expression appear to be regulated by various environmental,
dietary, and developmental factors and by hormones such
as insulin, thyroid hormone, and glucocorticoids (Pykalisto
et al., 1976; Nilsson-Ehle et al., 1980; Cryer, 1981).
In contrast to LPL, the synthesis of HL is confined mainly
to liver where it is localized on the surface of liver sinusoids
through glycosaminoglycan links (Table 1) (Olivecrona and
Bengtsson-Olivecrona,
1993). Treatment with heparin, therefore, leads to the release of HL, as well as LPL, into circu-
of alimentary TGs begun anteriorly by lingual
1984). The colipase, which helps to anchor
lipid-water interface, does not affect its cataTilbeurgh et al., 1992), in contrast to the role
It shows variable phospholipase activity, depending on the
animal species. The active enzyme is glycosylated like the
other two lipases. LPL and HL share two conserved
glyco-
sylation sites, whereas the glycosylation site of PL is in a
different position (Olivecrona and Bengtsson-Olivecrona,
1990; Ben Zeev et al., 1994). Like HL, PL is active in the
monomeric form, but unlike the other two lipases, it is not
anchored to membrane surfaces, but acts as free molecules.
2.2.
Gene Organization of Lipases
The exon-intron
organization
and nucleotide
sequences of
human LPL and HL genes have been studied by several investigators (Martin et al., 1988; Cai et al., 1989; Deeb and Peng,
1989; Kirchgessner et al., 1989; Ameis et al., 1990). Some
of the salient features of their gene structure are shown in
Table 2. The two lipases are situated on two different chromosomes (8~22 for LPL and 15q21 for HL). The size of the HL
gene is twice that of LPL, due mainly to the longer introns
in HL. The LPL gene contains 10 exons separated by 9
as a
introns, and the HL gene has 9 exons separated by 8 introns.
Intron 9 is present only in LPL and interrupts the termination codon TG/A. The LPL and HL mRNAs code for
monomer. Although glycosylation has been shown to critically affect the secretion and the activity of LPL, it does
signal peptides of 27 and 22 amino acid residues and mature
proteins of 448 and 477 amino acid residues, respectively.
not seem essential for HL catalytic activity (Stahnke et ul.,
1991; Ben Zeev et u1., 1992). HL possesses both TG lipase
and phospholipase activities (Deckelbaum et al., 1992). It
acts on TGs of chylomicron remnants to further reduce their
The greater length of HL is due mainly to the larger number
of amino acid residues coded by its exons 2 and 8. The LPL
lation.
HL is a glycosylated
protein
and is active
size, on the TGs of IDL to produce LDL, and on the TGs
and phospholipids of the high-density lipoproteins (HDLs)
of HDL: to form HDLI. The conversion of HDLL to HDLi
is important in the reverse cholesterol transport process, a
mechanism believed to be necessary for the protection of
extrahepatic tissues from accumulating excess cholesterol.
HL has no requirement for an obligatory activator, unlike
LPL, which needs apo CII for its function, but the activity
of HL may be stimulated
by apo E. In contrast,
as noted
earlier, apo CIII and, possibly, apo E may act as LPL inhibitors. It has been suggested that the opposite effects of apo
E on LPL and HL could serve to direct the action of these
two plasma lipases toward specific lipoproteins (Thuren et
ul., 1992). Thus, the preferred substrates of LPL are the apo
CII-rich lipoproteins, chylomicrons,
and VLDL, whereas
those of HL are the TGs of apo E-containing lipoproteins,
i.e., chylomicron remnants, IDL, and HDL2.
PL is synthesized by the acinar cells of the exocrine pancreas (Table I), which also synthesize the precursor molecule of its protein activator, colipase. The pancreatic duct
mRNA occurs in two isoforms in the human (3.75 kb and
3.35 kb), but only a single isoform of HL mRNA (1.7 kb)
is observed. This is due to the existence of two alternate
polyadenylation sites in LPL mRNA and only one in HL
mRNA. Although the LPL gene is only half the size of the
HL gene, each of the two LPL mRNAs is twice as long as
the HL mRNA. This difference in mRNA size is primarily
due to the very large noncoding exon 10 that is present in
LPL, but absent in HL. Apart from these size differences,
the other 9 exons of LPL and the 9 exons of HL are very
similar in size and are organized in a very similar fashion.
Except for introns 1, 4, and 9, all LPL introns are located
at sites identical to HL (Cai et al., 1989). There are multiple
TATA Box and CAAT Box consensus sequences in the HL
gene, but there is one major transcription
initiation site
for LPL, as well as for HL. Both genes possess potential
5’-elements responsive to glucocorticoids,
cyclic AMP, calcium ions, and adipocyte specific enhancer motifs.
The gene organization of human PL is not known. However, such data are available for canine PL, whose transcriptional unit is 10 times larger than the mature mRNA
and is organized into 13 exons (Mickle et al., 1989). Exon
104
TABLE
2. Organization of Human
LPL and HL Genesa
Chromosome
LPL
HL
8p22
15q21
(kb)
30
60
Number
of exons
10
9
Number
of introns
9
8
9
37.5
Gene
length
Intron/exon
Major
ratio
mRNA
Exon
size (kb)
3.75;
1
3.35
1.7
130 nt
275 nt
peptideh
Uncleaved
43 nt
188 nt
Untranslated
Signal
portion
81 nt: 27 aa
Met-!’
- Ala-’
66 nt: 22 aa
Met-22 - Ala-I
6 nt: 2 aa
Ala’ - Asp?
21 nt: 7 aa
Leu’ - Glu’
Exon
2’
162 nt: 54 aa
Gln’(c/!A) - Thri”
186 nt: 62 aa
G~u”(G/AG)- Se@
Exon
3
180 nt: 60 aa
Va15’ - GIu”~
183 nt: 61 aa
Val’(’ - Glu”@
Exon
4
111 nt: 37 aa
117 nt: 39 aa
Glu”’ - Thr’h”
Exon
5
Glulli
-
Thrli'
234 nt: 78 aa
Asn?”
234 nt:78 aa
Glyli’ _ Gly”l
G~Y”“(G/GG) -
Exon
6
243 nt: 81 aa
As~?‘?(c/AT) - Lvs”:
243 nt: 81 aa
A~~?‘~(G/cc) - Lys jjh
Exon
7
120 nt: 40 aa
V~~“)(G/TC) - Thr’sl
Vd'*"(G/TT) -
Exon
8
Exon
9
-
Lcu'~"(cT/G) -
LyS’”
I05 nt: 35 aa
L~s~‘~(AA/G)- Gly++”
Termination
codon
Noncoding
sequence
Exon
219 nt: 73 aa
Gln’-‘l’
183 nt: 61 aa
LeUii3(CT/G)
3 nt (TGA)
3 nt (TG/A)
sequence
Noncoding
Number
sequence
None
49 nt
None
None
1948 nt
None
4’75
499
of aa coded by mRNA
Total
Signal peptide
Mature
protein
5’-flanking
Start
27
23
448
476
elementsc
-43
-188 nt
of transcription
TATA
Box
215 to -210
nt
CAAT
Box
253 to -249
nt
Nuclear
factor-A
Glucocorticoid
Cal+
111 nt: 37 aa
Arg4+‘(AG/A) - Arg”;’
IO”
Coding
Cyclic
117 nt: 39 aa
Thr”‘;
AMP
-424 to -419 nt
-512 to -508 nt
- 1332 to ~ 1327 nt
-234
-768
to -227 nt
to -761
nt
responsive
-832
to -827
nt
~ 1022 to 1008 nt
-560
to -554
nt
-577
responsive
-242 to -235 nt
_ 550 to 543 nt
Adipocyte
specific
i’-flanking
elements?
t 2952 to + 2957 nt
signals
Polyadenylation
nt
to -66 nt
to -111 nt
binding
responsive
Poly-A
-70
-116
sites
+3348
to +3353
nt
t2981
nt; +3376
nt
to -571
113 to -91
+1525
nt
nt
to t1531
+1545
a Compiled from Deeh and Peng (1989) and Kirchgessner et ui. (1989) for LPL, and Marnn et ui. (I%?),
Cai et al. (1989) and Am& et ul. (1990) for HL.
’ The ammo acids are numbered starting with the first residue (+ 1) nf the mature protein.
’ Codons that are split between two succcss~vc exons and the correspondmg armn~ acids arc shown
in parentheses.
d The number of nucleotides in exon 10 of LPL ~ncludcs the third nuclrotidc of the i’-terminal sr<q~
codon (TG/A) and up to nr 3376 (the second polyadenylatlon
site).
’ The position of the 5’- and 3’-flank’mg eIements
areidentltied m the s<ienrlfic ltteraturcusinga wmetv
of startmg sites. ~g., the first nucieotide of a given cDNA clone (Wion et ul., 1987) or the transcnpnon
initiation site (Deb and Peng, 1989; A meis et al., 1990). Because these reference points are not unique
and may be subject to change, we have numbered the nuclcotidea in this xwew using the first nucleotlde
of the Initiator codon (ATG) as + I, becaux there occurs only one such codon 111both LPL and HL.
nt, nucleotides; aa, amino acids.
Human Lipoprotein Lipase Gene
105
1 codes for all of the untranslated mRNA sequence, and
Exon 2 starts with the initiator codon ATG and encodes
1990; Winkler et al., 1990). The sequences of these three
lipases are aligned in Fig. 1 so as to obtain maximum homol-
the major part of the signal peptide. The stop codon,
ogy with the minimum number of gaps. The putative struc-
polyadenylation
signal, and the 3’ untranslated
the
sequence
are contained in exon 13. The presence of an intron immediately upstream of the initiator codon may afford a mechanism for the use of alternate promoters and differential
splicing to achieve tissue-specific expression of PL.
From a comparison of the intron-exon organization
of
PL, HL, and LPL, it has been proposed that these three major
lipolytic enzymes may belong to a superfamily of lipase genes
and may have descended from a single ancestral gene (Kirchgessner et al., 1989). On the basis of amino acid sequence
homology, the membership of this superfamily is occasionally extended to include certain conserved regions of Drosophila yolk proteins YPl, YP2, and YP3 (Kirchgessner et al.,
tural and functional
also indicated.
The sequence
domains common
similarity between
to these lipases are
LPL and HL (53%) is
much greater than that between either LPL and PL (35%)
or HL and PL (36%) (Table 3). It is reasonable to assume
that amino acid sequences that are associated with critical
functions
in an enzyme protein may be much better con-
served than others. This, indeed, appears to be the case for
certain functional and structural domains of the lipase
family (Table 4). For example, the catalytic triad consisting
of serine, aspartate, and histidine is identical and even the
regions surrounding the triad residues are well conserved
between
the members of the lipase family (Fig. I) (Kirch-
1987; Komaromy and Schotz, 1987; Datta et al., 1988; Kirchgessner et al., 1989). The Drosophila yolk proteins, however,
show no lipolytic or other activities in common with the
gessner et al., 1989). A second series of conserved regions
are those identified as the potential lipid binding sites. Two
putative N-linked glycosylation sites are present in HL and
three lipases in the group. Two other lipase-like enzymes,
lecithin-cholesterol
acyltransferase and lingual lipase are
found to have no significant homology with LPL, HL, and
PL and, presumably, do not belong to the same family
(Komaromy and Schotz, 1987).
LPL, which are conserved
2.3.
Conservation
of the lid and its structural relation to other parts
of the enzyme may be stabilized by the disulfide bridge that
links the two conserved cysteines flanking the lid. Other
Lipases and their Structural Domains
Of the three human lipases, only the three-dimensional
(A2
and A4), and only one such consensus sequence is identified
in PL (A3) (Hide et al., 1992). The sequence of the lid covering the active site is not well conserved, although the conformation
and Evolution of
and in the same positions
and A5 in Fig. I). However, HL appears to have two additional potential sites where N-glycosylation can occur (Al
struc-
ture of PL has been established by X-ray crystallography so
far (Winkler et al., 1990). The results show the presence of
two distinct domains in the PL molecule. The N-terminal
domain (residues l-336) consists of a central P-sheet surrounded by ol-helices (the cr/P-hydrolase fold) and encloses
the three amino acid residues of the catalytic triad: Ser153,
Asp”‘,
HisZh4 (in the porcine enzyme, the catalytic triad is
numbered Ser152, Asp *7h, HisZh4, because the amino acid
residue Serj’ of human PL is deleted in this species). The
C-terminal domain (residues 337-449) has a &sandwich fold
and contains the main colipase binding site. The active site
in the N-terminal domain is shielded by a flap or a lid consisting of an amphipathic helix. On binding to the substrate,
the helical lid is supposed to roll back upon the body of
the molecule, thus enhancing the hydrophobicity
around
the active site. This movement of the lid is related to interfacial activation, a phenomenon by which the lipase activity is increased in the presence of lipid-water interface (Ollis
et al., 1992; van Tilbeurgh et al., 1992, 1993). Colipase, the
protein activator of PL, helps to bind the enzyme to the
interface in the presence of bile salts in the intestine that
might otherwise interfere with this binding. Similar lids are
also presumably present in all three enzymes of the lipase
family.
The amino acid sequences of human LPL, HL, and PL
have all been established either by direct sequencing of
selected polypeptide regions or by deciphering from the corresponding cDNA sequences (Wion et al., 1987; Ameis et ui.,
putative functional structures that do not show marked conservation are the p-5 loop (or hydrophobic flanking wings)
and the oxyanion hole, presumed to be involved in controlling access of the substrate to the active site.
Proline is an amino acid that can have profound effects
on protein structure.
The &-tram
isomerism of this amino
acid generally influences the conformation of proteins. LPL
and HL have a striking resemblance in regard to the frequency and distribution of proline residues, but are different from PL in this regard (Table 5). The four proline residues conserved in all three lipases (Pro’60, I’ro1T3, ProZ07, and
Pro214 of LPL) are all clustered within a region that contains
the lipid-binding domains B3 and B4 and the lid (Fig. 1).
There are also other proline residues that are not conserved,
but that are present within various lipid-binding domains
of LPL: e.g., Pro95, Pro157, and Prolgo.
Cysteine residues contribute to protein conformation and
stability by their capacity to form covalent disulfide linkages
that bring distant regions of the protein molecule into closer
proximity. Not all cysteines, however, have such a role (Fig. 1,
Table 5). The eight cysteines conserved in all the three lipases
participate in the formation of disulfide bridges. One of these,
involving CY~*‘~ and Cyszj9 of LPL, encloses the cr-helical
flap or lid region of the lipases (Fig. 1). The two additional
cysteines that LPL shares with HL, but not with PL (Cys*7
and Cys40 of LPL), are not linked by an S-S bond, but are
highly conserved in LPL and HL molecules of different
species. Of the six cysteines unique to PL, four are linked
by S-S bridges not found in LPL or HL. Of the other two,
106
++
hLPL:----ADQRRD
hHL: -------LGQ
hPL: KEVCYERLGC
Al
+
+
++
---------RTPEDT---AQAVETmL
HEMK----TR
TE--RPLHIL
PWSPKDVNTR
++
FIDIESKFAL
SLKPEPFGRR
FSDDSPWSGI
+++
++
___------
+
A
FLLGETNQ-FLLY-TNENP
EDTCHLIPGV
--GCQIRINH
NNFQEVAA-D
33
45
56
O------o
A2
+ ++ ++**
+
+ *+
hLPL:AESVATCHFNSKTFMVI
H
SSLPLVMII Jd
hHL: PDTLQECGFN
TNRKTRFIIH
hPL: SSSISGSNFK
D
*+ + *
*+
GWTVTGMYES
GWSVDGVLEN
GFIDKGE-EN
A
Bl
+*+ +
+++ +** +**
hLPL:mVSAGYTK
LVGQDVARFI
hHL: =IAVRNTR
LVGKEVAALL
hPL: UQASQNIR
IVGAEVAYFV
B2
A3
++**++*+* ****++* ** t
++++ + ++
+
+
NWMEEEFNYP
LD -GYS
LGAHAAGIAG
S----LTNKK
148
RWLEESVQLS
RS SAHVSGFAG
SSIGG--THK
163
EFLQSAFGYS
PS HVHVIGHS
J&AHAAGEAG
RRTNG----T
169
AA
B3
++******+*
+* *++
hLPL:VNRITGLDJ?A
GPNFEYAEAP
hHL: IGRITGLDAALFEGSAPS
hPL: IGRITGLDPA
EPCFQGTPEL
A
**+*+** * **++**+++
SRLSPDDADF
VDVLHTFTBS;
NRLSPDDANF
VDAIHTFTPF,
VRLDPSDAKF
VDVIHTDGAP
C
***
++***
hLPL:NGGTFQPGm
hHL: NGGSFQPGhPL: NGGVEMPGLDF
0
+
*
++*+
++
*
++
RrJaGDVD-OL
HGFMIT-OT
+ ++
ICEAIRVIAF.
+*
+++*+
*
+++*
FIVPKLVAALY
WIWQMVAALK
WLANVCKNLF
0
+
*+
+ +
*++
KREPDS-NV1
SQPAQPVNVG
KVE--SVNCI
B4
+++ *+
SP--Q
HM-GJSVGLK
IVPNJaaGMS
B5
+*+*+** + +++**+++
VKCSHERSIH
LFIDSUNEE
ESHERSVH
LF-HAG
SNHLRSYKNPD
.A
+++*
*
++
++t
+++**+*++*
KPVGHVDIYP
QPIGHYDFYP
QWGHLDFFP
+ +++*
207
222
229
+
NPSKAYRCSS
TQSMAYPCGD
GF-AGFPCAS
+*++++
++
A5
** *++
* *++
+++* +*+++
+** +*+*
+++++ +
+
+
IGELLMLKLK
AFEISLYGTV
AESENIPFTL
P--EVSTm
YSFLIYTEVD
IGELIMIKFK
TFTMSLLGTK
EKMQKIPITL
GKGIA-Sm
YSFLITLDVD
VGDLQMVKFI
---VSLFGNK
GNSKQYEIFK
GTLKPDS--T
HSNEFDSDVD
+
+++*
hLPL:APAVFVKCHD
hHL: QEKIFVKCEI
hPL: VLLTLTPC-0
----DSYFSW
WDTVQTIIPW
--INPTL---
++
+ +
+
KSLNKKSG-KSKTSKRKIR
-----_____
++
SDWWSSPGFA
STGPRHSGLV
------PRVG
448
476
449
+*+*++++
IQKIRVKAGE
LKTIRVKAGE
ASKIIVET-N
+++
+**
TQKKVIFCSR
TQQRMTPCSE
VGKQFNFCSP
266
281
28 8
l-
A4
+
++++
hLPL:GTESETHTNQ
hHL: ImETPIQT
hPL: GKKVTGHIL-
+
KTRSQMPYKV
VTRAQSPFKV
DTGDASNFAR
92
105
113
SCRKNRCNNL
SCKKGRCNTL
PCPSGGCPQM
o--a
*
R---SSKMYL
K---SKRLFL
TNDVGQKFYL
***
VVDWLSRim
LVDWITLAm
CVDWKGGSm
0
hLPL:KEAFEKGLCL
hHL: MNSFSQGLCL
hPL: YNVFTANKCF
d
hLPL:WKS------hHL: WENSAVWANV
hPL: WYNNV-----
GYEINKVRAK
GYHVRQEPRS
GHYADRYPGK
+
+
FHYQVKIHFS
YHYQLKIQFWRYKVSVTLS
+++
323
337
348
381
396
402
+ +
EKVSHLQKGK
NTDDLLLRPT
ETV----REE
430
456
441
Human Lipoprotein Lipase Gene
107
TABLE 3. Amino Acid Sequence Homology
in Human Lipases
Homology
Lipases
(“4
LPL and HL
LPL and PL
HL and PL
,’ From Hdc
53
35
36
and Divergence
Dayhoff
distancea
(W
72
132
130
er ui. (1992).
tions of the lipases. On the basis of these data, a number
of structural domains with possible specific functions have
been identified in the LPL molecule (Table 4). Thus, by analogy with PL, LPL is considered to be organized into two
large and distinct amino terminal (N: residues 1-312) and
carboxy terminal (C: residues 313-448) structural domains.
The N-domain is the seat of many important activities of
LPL, including catalysis, and the C-domain appears to be
implicated in such functions as the initial interaction with
lipoproteins
one (Cys’02 of LPL) is situated within the sequence bordered
by one of the unique S-S bridges of PL, and the other
(Cys’HJ of LPL) is part of one of the presumed lipid binding
domains (B3, Fig. 1).
LPL and HL are much closer to each other than they
and the LPL-mediated
uptake of lipoproteins
by cell surface receptors.
The active site of LPL can be considered to be made up
of (a) the catalytic triad, (b) the oxyanion hole, (c) the lipid
binding site, (d) the lid, and (e) the p-5 loop. Except for the
catalytic triad, most residues in the catalytic sites of LPL
are to PL in both their amino acid sequence homology and
their evolutionary divergence (Table 3). In the course of evo-
and PL are hydrophobic and their main chains are not readily accessible for hydrogen bonding. This has the effect of
diminishing the affinity of the catalytic site for the phos-
lution of the lipase gene
gene branched off earlier
PL gene has the highest
imately the same number
phoryl groups of the phospholipids (van Tilbeurgh et al.,
1994), thus explaining the lower phospholipase activities of
these enzymes, as compared with their TG lipase activities.
The catalytic triad of LPL is made up of the three amino
family, it is probable that the PL
than the other two. Although the
number of introns, it has approxof codons as the other two lipases
(e.g., 448, 477, and 449 amino acid residues in mature pro-
acid residues Ser’jz, Asp’j6, and Hi@‘,
teins of hLPL, hHL, and hPL). As seen in Fig. 1, the functional domains of the lipases often coincide with specific
in all the three members of the human lipase family (LPL,
HL, and PL) (Fig. 1) and in all other species so far studied
(Hide et al., 1992). The oxyanion hole in LPL is probably
axons. Based on these observations, it has been suggested
that the molecular evolution of the lipases could have occurred through gene duplication events, selective loss of
introns, and fusion of exons (Kirchgessner et al., 1989; Hide
that has also been applied to
explain the evolution of other multidomain proteins, such
as the LDL receptor (Sudof et al., 1985) and serine proteases
et ul., 1992), a mechanism
(Rogers,
3.
3.1.
LIPOPROTEIN
may also show hydrophobic
interactions
with the lipid
a mobile surface loop in the putative
three-dimensional
structure of LPL that covers the catalytic
site and is rearranged to permit the substrate to have access
LII’ASE
Functional Anatomy
the Lipoprotein
formed by the main chain nitrogens of Trpis and Leu”‘,
which are next to Ser”: of the catalytic triad (van Tilbeurgh
et al., 1994). The lipid-binding site constitutes a very hydrophobic groove that probably binds the aliphatic chain of
the acyl-enzyme intermediate. Tyry4, ProiS’, AlalSH, and Ile’“’
substrates.
The lid represents
1985).
which are identical
of
Lipase Molecule
to the catalytic domain. The lid sequence is contained between two conserved cysteines (Cys*‘h to Cysz”), which
The structural and functional domains of LPL and HL have
mostly been extrapolated from the available physical information on PL. Some of these inferences have been further
corroborated by (a) nucleotide and amino acid homologies
form one of the four disulfide bridges in the LPL molecule
and could have the effect of stabilizing the lid within the
protein during its action. Dugi et al. (1992) have identified
two highly amphiphilic a-helical structures (residues Asn?ii
to Arg2ls and Asp?‘? to Lys?‘” connected by a p-turn) in the
existing among the three lipases, (b) analysis of degrees of
conservation in different regions of the enzyme molecules,
(c) identification of naturally occurring human mutations
and characterization
of the biochemical lesions involved,
and (d) production of in vitro site-directed mutations and
lid with opposite polar and hydrophobic faces. In addition
to its role in the hydrolysis of TGs and phospholipids, the
lid may be essential for specifying the lipase substrate. For
example, there are found to be differences in the open (active)
conformation of LPL and PL, which may account for their
examination
different substrate specificities (van Tilbeurgh
of their effect on the catalytic or other func-
et al., 1994).
FIGURE 1. Amino acid sequences of human LI’L (hLPL), human HL (hHL), and human PL (hPL). The amino acid sequences
are shown in single letter notation and are aligned according to Hide et al. (1992). Amino acid identity between all three sequences is indicated by an asterisk (*) and identity between any two of the three sequences is indicated by a plus sign (+). The
following putative functional sites are underlined: (Al-AS)
N-linked glycosylation, (Bl-BS) lipid-binding domain, (C) o-helical
lid, and (d) p-5 loop. The three amino acids representing the catalytic triad (serine, aspartate, and histidine) are indicated by
solid triangles (A). Amino acids of the oxyanion hole are indicated by open triangles (A). The four cysteine pairs involved in
disulfide linkages in all three lipases (0) and the two pairs unique to PL (0) are each connected by continuous lines.
108
V. Murthy
TABLE
4. Functional and Structural Anatomy
Functional
Catalytic
domain
Amino
triad
Oxyanion
Trp5j,
site
Lid
p-loop
site,’
Heparin-binding
clusters
Cys?‘h
up to cysl’q
interaction
Dimerization
van Tilbeurgh
YS
Lys’WArg?W,
148
2110, A$82
C-terminal
residues
56 amino
et al., 1994
Wong et ul., 1991; Dichek et al.,
Bengtsson
Olivecrona,
1993
acid
van
uptake of
by cell surface
et al., 1992;
et ul., 1993
Semenkovich
et al., 1990; Ben Zecv et al., 1994;
Busca et ul., 1995
site
LPL-mediated
lipoproteins
receptors
al., 1992;
Lys4”‘, Lys”l)
Asn-“-His44-Ser-‘5
Asn~jY_Lys’h@_Thr’hl
with lipoproteins
1994
1990; Hide et
et al., 1994
Davis et al., 1992; Berryman and Bensadoun,
1993;
Dichek et al., 1993; Hata et al., 1993; Ma et al.,
1994a; van Tilbeurgh
et al., 1994
LYs’O”
Arg151
Lys’“‘, Arg4”;,
Lys+“-Lys4’+
sitesh
et al.,
et al., 1991a,b;
et al., 1992
Yang et al., 1989; Davis et al., 1992; Dichek
Arg""_L
"i-Lys
Tilbeurgh
Dugi et al., 1992; Tashiro
Henderson et al., 1993
up to Trp”4
&I"',
et nl., 1989; Faustinella
et al., 1992; Faustinella
Winkler
et al.,
van Tilbeurgh
14'_Lys14"
LYS
LYS
Kirchgessner
Emmerich
van
up to ProYi
up to Prolh”
up to Ile””
up to Gly”q
up to Leulj’
Arg:“‘,
Initial
Reference
Leu”’
Glu9’
Pro’57
ArglR;
Vallzb
Ser*“”
Glyj’
Apo CII binding
Glycosylation
LPL Gene
acid residues implicated
Ser’32, AsP’~~, His*4’
hole
Lipid-binding
of the Human
et al.
Tilbeurgh
et ul.,
1993; Lookene
and
1994
Intact carboxy terminal
folding domain
Williams et al., 1992; Al-Haideri
et nl., 1993; Nykjaer
et ul., 1993; Zhang et al., 1994a
Prolines
Residues: 19, 31,
122, 157, 160,
190, 199, 207,
310, 350, 354,
Bruin
Cysteines
Residues:
27, 40, 216, 239,
264, 275, 278, 283, 418,
438
66,
168,
214,
397,
77, 95,
173,
258,
432
et al.,
1994a
:’ LyslqY of LPL in the human is changed to Thr m the gutnea pig.
h Ser” 1s changed to Thr m chicken, and Lys “” is changed to Asn m the guinea pig. Asn”‘-Pro”‘~Srr”‘,
(Won et al., 1987), may not be eflic~ent because of the presence of proline (Marshall, 1974).
Using
deletion
Henderson
of charge
ments
and
tion.
Ile225Thr
tion
that
The
periodicity
in the
ogy with
the &loop
rendering
occupies
Tilbeurgh
hole
its protein
upon
into
segthe
to this func-
human
LPL
muta-
region.
in the LPL protein
Hisi
to Trph”. In anal-
on opening
of the LPL lid,
the core of the protein,
accessible
a catalytically
necessary
for
believed
activator,
The
thus
and bringing
competent
position
sequence
functional
catalysis.
One
to be responsible
apo CII.
The
domains
of them
the
N-terminal
sites
other
The
in the endothelial
These
heparin-binding
presence
consensus
proteins,
of two S-S bridges
Cys?“‘)
in LPL
may confer
heparin
binding.
Additional
heparin-binding
(in the N-terminal
region)
Lysj’j,
His””
is the
site,
tetrapeptide
of
region).
Lys”‘q,
A noncharged
the consensus
sulfated
and
sequence
proteoglycans,
including
and X is a small
are found
on these
and CysL;“regions
charged
are: Lys’q’,
and Lys”“,
clusters
Lys”“,
with
Arg’j’
C-terminal
Trp’“P up to Trp’“j
Trp-Set--Asp-Trp,
is also
upon
LYS~“‘, Arg”Oi, LyP,
up to GIYJ”~ (in the
tetrapeptide,
in
apo E and apo B.
(Cy~?~‘-Cys?‘~
positively
two
heparin-
and X-B-B-B-X-X-
residue
sequences
activity
in addition
and for the
wall. The
hypothetical
stability
in medi-
for the interaction
X-B-B-X-B-X
charged
site
LPL.
vessel
to the
sequences,
B is a positively
residue.
and
sites are essential
correspond
consensus
neutral
implicated
apo CII
cell wall glycosaminoglycans
of LPL
where
as a pClsn1ble glycosylatlorl
has been
between
of activities
for the binding
C-terminal
with
localization
B-X,
Glu-Glu)
interaction
heparin-binding
of LPL
possible
in a complex
multiple
the
binding
loop
site even more
is a participant
LYS ‘t7-Lys’qH,
minimally
region
that
distal
and that
of apo CII (Lys-Gly
ating
et al., 1994).
and has, therefore,
to those
the
may fold back
the active
this
and
catalysis
occurring
of the lid,
the maintenance
proximal
mobile
PL, it is supposed
oxyanion
LPL
within
is another
parts
that
for normal
is a naturally
is located
that
different
shown
of the loop contribute
p-5 loop
structure
(van
affecting
have
of the lid is crucial
apical residues
the
mutants
et al. (1993)
proposed
present
capable
in the
with
of binding
C-terminal
Human
region.
Lipoprotein
Lipase Gene
The relative importance
109
of these various sites in hep-
arin binding is not clear, although it may be presumed that
they may have differential activities, depending upon different reaction conditions. As compared with LPL, PL, which
has no significant interaction with heparin, shows a more
symmetrical charge distribution (van Tilbeurgh et al., 1994).
Two putative glycosylation sites have been identified in
human LPL. In the absence of glycosylation at the N-terminal
domain (Asn” up to Serq5), LPL is completely inactive and
the enzyme is not secreted (Ben Zeev et al., 1992). In contrast, glycosylation at the C-terminal domain (Asn3jy up to
Thr36’) does not appear to affect either enzyme activity or
secretion (Semenkovich
Busca et al., 1995).
The initial interaction
et al., 1990; Ben Zeev et al., 1994;
of lipoprotein substrates with LPL,
which is a necessary prerequisite for their subsequent
change in the enzyme mole-
cule, leading to the opening of the lid that normally masks
the catalytic triad. This opening exposes the hydrophobic
residues of the lid in a process called interfacial activation
(Tashiro et aI., 1992) and forms a cleft lined by the hydrophobic amino acid side chains of the LPL backbone or the
amphiphilic helices of the lid (Dugi et al., 1992). The fatty
acid chains of TGs are thought to bind to this hydrophobic
cleft, with the glycerol moiety occupying the oxyanion hole.
The hydrolysis of the TG is then brought about with the
participation of the catalytic triad.
The active form of LPL is a homodimer
(Osborne
5. Frequency
and Distribution
line Residues
in Human
Lipasesa
et al.,
1985). Both head-to-head and head-to-tail dimeric forms have
been considered. Based on structural analysis of LPL and
pancreatic lipase, van Tilbeurgh et al. (1994) have proposed a
head-to-tail dimeric form in which the N-terminal domain of
each monomer is in contact with the C-terminal domain
of the other in such a manner that the heparin binding sites
are available for reaction and both lids are free to open upon
interfacial activation. The specific amino acid residues in
the two monomers that may participate in dimer formation
have not been identified.
In addition to its action on TGs, LPL may also play a
role as an intermediary in the uptake and degradation of
lipoproteins by cells. Although LPL, by itself, may mediate
the binding of lipoproteins to cell surfaces by the interaction
of the free heparin binding sites of the LPL-lipoprotein complexes with the membrane proteoglycans, it appears that
cell surface receptors, such as the LDL receptor or the LDL
receptor-related protein (LRP), may also have a role in this
phenomenon
(Williams et al., 1992; Nykjaer et al., 1993).
Using various deletions, spanning positively charged amino
acids, in the C-terminal region of LPL, Zhang et al. (1994a)
found that the binding of lipoproteins to LRP was independent of catalytic function and heparin binding properties
of LPL, but was dependent on the region Ile404 up to Lys4’4,
which includes the very highly conserved residue Glyq”“.
of Cysteine
Cysteines
Total number
LPL
HL
PL
Unique
LPL
HL
PL
Common to:
LPL and HL
LPL and PL
HL and PL
LPL, HL and PL
;’ Derlvcd
3.2.
and ProProlines
10 (4 SS)
10 (4 SS)
20
21
14 (6 SS)
26
0
0
6 (2 SS)
11
10
16
to:
hydro-
lysis, is believed to be mediated by the carboxy terminal
domain of LPL, particularly the region containing the last
56 amino acids (Lookene and Bengtsson Olivecrona, 1993).
The interaction of LPL with the lipoproteins is supposed
to result in a conformational
TABLE
10
8
8
8
(4
(4
(4
(4
SS)
SS)
SS)
SS)
7
6
8
4
from Fig. 1. SS, disulfide linkages.
Molecular Phylogeny
of Lipoprotein
Lipase
The amino acid sequences of chicken, guinea-pig, mouse,
rat, bovine, and human LPL are shown in Fig. 2. There are
two extra amino acid residues at the N-terminus of the mature
LPL of bovine, sheep, and chicken. In chicken, there are
also 15 additional amino acids following the stop codon of
the human LPL. In rat and mouse, there is deletion of the
residue corresponding to Asn444 in the human. The sheep,
rat, mouse, guinea-pig, and chicken LPL mRNAs code for
28, 27, 27, 17, and 23 amino acids of the signal peptide as
compared with 27 in the human LPL. The bovine signal
peptide has not yet been determined. The bovine and sheep
LPL, both of which have 450 amino acid residues in the
mature protein, differ from each other by only one residue
in exon 1 (Glys in the ox replacing Arg5 in the sheep). The
rodents, rat and mouse, differ from each other in 11 of their
447 residues. In spite of these differences, there exists a very
high proportion of identical amino acids (92-94%) and high
degree of sequence similarity (96-97%) in the LPLs of the
rodents and the ruminants (sheep and ox) as compared with
the human (Table 6). The identity and similarity are less
in the guinea-pig (87% and 92%) and even less in the chicken
(74% and 82%). These differences between the species are
consistent with their evolutionary divergence, as determined
by the Dayhoff distance (Hide et al., 1992).
The proportions of amino acid residues in the various
LPL species, identical or similar to the human enzyme, are
the highest in the middle exons and they tend to be lower
towards the N-terminal or C-terminal exons. The similarity
is even greater if only those regions of the LPL molecule
are considered that are presumed to subserve specific functions of the enzyme.
There are also specific amino acids, such as proline and
cysteine, that may have a disproportionate influence on protein conformation and remain unchanged even within certain regions of the LPL molecule that are not generally well
conserved. For example, Proi60, Proiij, ProZoi, and Pro214 of
LPL are not only conserved in all species of LPL, but also
in HL and PL (Figs. 1 and 2). Prolines in positions 95, 157,
Exon 1
hLPL:
- ------ N-
_--__-_fJ--
"--__--___
_-sy_-----
TlhLPL:
ADQRRDFIDI
I
bLPL:DRITGGK--R-sLPL:DRITRGK--R---GG---S-rLPL:
--AG-m-S-mLPL:
gLPL:
-NCQK-YT-cLPL:SDPEAEMN-EG-
ESKFALRTPE
DTAEDTCHLI
----------------se-
-------------------
____----___--------
PGVAESVATC
HFNXSKTFM
_--T----N---T-_--N-
__-----___-----_---
--L-j-j--SN-
--L-j-,--SN-
-----_---v
-----
------------------
V
-----R-m-----S----A
N-"------EPD--V-Y-V
--QMD-L-Q-
N---T----V
TKLVGQDVAR
_--T----N-
+---
-----i---____-,--------A----
hLPL:
hLPL:
YEVPKLVA
ALYKREPDSN
VIVVDWLSRA
QEHYPVSAGY
bLPL:
sLPL:
rLPL:
mLPL:
gLPL:
cLPL:
_--------________-_
_____-----------------------
--------__
_____----__------__
--------__
_-------__
-------_-------------____y-_------y__
-Q--------
----m----K
-M---AD_--
-Q----___-
---------K
+,--_f(D---
-Q____----
-----N__-_
-a----____
----L_,____
-----N_---
----_+__K
-------R__
-H---E--D_
-----E___-
_--------D
-----___--
__A_-e-V--
-Q------A-
-----K---M
Exon
hLPL:
hLPL:
bLPL:
sLPL:
rLPL:
mLPL:
gLPL:
cLPL:
----------
-----R--S-
82
YPLDNVHLLG
YSLGAHAAGI
A
A
--- G -------------_--- G ---------------
4
-E-------_
AGSLTNKKVN
RI
-------------------
--D-------
-C---T---G
NFEYAEAPSR
LSPDDADFVD
-------------------
----_____----___---
-------------------
--------._v
----------
----------
----------
---------v
----------
----------
----------
-sv-----_-
-------m-V
---R--T--S
-------T-s
------Q---
---N-_-__-
----------
-----K-_--
T----D--I-
----______
__f_______
Exon
hLPL:
R
_-Q-_--E_-
__-TE---_-
-a-ES-L-@-
-----S----
0---T----C
EM
AIRVIAERGL
5
- -_--__ aI
GDVDQLVKCS
hLPL:
VLHTFTRGSP
GRSIGIQKPV
GHVDIYPNGG
TFQPGCNIGE
bLPL:
sLPL:
rLPL:
mLPL:
gLPL:
cLPL:
---------_
-_--__---_
---------_
-------------------
____-----____---------------------------------
___----____-----_-----------------------------
-------------------------------------
----------------
S-------QD
-L---SQK-F
_
M-------
----y____-
D---------
--I-----__
G_____-L__
-L-L---K-F
S
_-------
-L-__-----
-+--------
-I--------
-L___-----
K --
-t-------- --------
f
i3
hLPL:
hLPL:
bLPL:
sLPL:
rLPL:
mLPL:
gLPL:
cLPL:
--HT-----N
B5
HERSIHLFID
A
P ----
CA------R_
TH---P----
______----
-----
SLLNEENPSK
AYRCSSKEAF
0
EKGLCLSCRK
0.4
NRCNNLGYEI
-_--V----_
----------
----N---_-
------____
-----
-_--V----_
_-------__
----N-m---
----__----
-----M----
----------
----------
----N--___
------____
-----
----------------------------
--------__
------------Y--K--M
----N-m-----_N-------_NT_---
-_____--------_--------_---_
M ----
V -----___---------V__---------KV
S__------NKVRAKRSSK
---------___----------------
-__----_----_------R--T--NT-
Human
Lipoprotein
Exon
hLPL:
111
Lipase Gene
6
--------em
--
7
Exon
I
_____--
I
----__----
---T-____-
____------
1
_I@------
I
hLPL
:
MYLKTRSQMP
YKVFHYQVKI
HFSGTESETH
TNQAFEISLY
GTVAESENIP
FTLPEVSTNK
bLPL
:
---______-
--
-------
_______N-y
-____-----
----_-----
--
SLPL
:
----------
--
----_--
-------N-y
_____----_
____------
--
-------
rLPL
:
-________-
--
-------
------NDKQ
N------m-m
____------
--
-------
mLPL
gLPL
:
:
CLPL
:
i
A2
-----__
-----____-
--
-------
------NGKQ
H------s-m
____------
--
-------
----------
--
----e-s
y----_TT-y
----------
----------
--
----A-N
--____A__-
--
----_--
--F-KTNV-K
VD-p-L----
--LD------
--+___s--
------__--
l-3
-fi-----___
---------_
Exon
---------
DIGELLMLKL
KWKSDSYFSW
SDWWSSPGFA
IQKIRVKAGE
TQKKVIFCSR
_N--_----D
_G__------
---
-N-------D
-G--
t
a ---
hLPL:
hLPL
:
bLPL
:
V-----
TYSFLIYTEV
sLPL:
___--L---_
----------
--I---__--
-----L---_
----------
--
----------
-------
M --
----------
-----_-s-v
_E__------
M --
I
-------
:
mLPL
:
----------
-------
--M_------
p----_-s-v
_ER_------
gLPL
:
----------
----------
--ITE-____
-S--GR-T-T
-E-_------
CLPL
:
_F-------_
---
Q-EK-TF---
-N--TPFA-T
hLPL
:
----------
----------
hLPL
:
EKVSHLQKGK
bLPL
:
--M-Y-----
SLPL
:
:
:
_--------_
DR--------
---X-__
_____----_
DS--------
---X---
----K--__-
EAP--___--
:
DGS-R-G--E
EA-I----
rLPL
mLPL
-1 ------
-----------
--RV---S--
9
Exon
------
R
--M-Y--__-
gLPL:
CLPL
---
______
rLPL
D ------
8
V
FIGURE
2. Amino acid replacements
in LPL sequences
of various species and in human mutant LPL. Note that hLPL, bLPL,
sLPL, rLPL, mLPL, gLPL, and cLPL refer to mature LPL sequences
from human (Wion et al., 1987), bovine (Senda et al., 1987),
sheep (Edwards et al., 1993), rat (Bra&
et al., 1992), mouse (Kirchgessner
et al., 1987), guinea-pig
(Enerback
et al., 1987), and
chicken (Cooper etal., 1989), respectively.
The human mutant LPL molecule is indicated by hLPL (in italics). The LPL sequences
are aligned according
to Hide et al. (1992). The amino acid sequence of the hLPL is shown in full; for others, only those amino
acids that differ from the human sequence
are indicated
at the appropriate
sites. The nine coding exons of human LPL are
identified
by the corresponding
numbers
at their C-termini
and a vertical line following
each number.
When a vertical line
passes through the letter symbol of an amino acid, it indicates that the codon for that amino acid is split between two successive
exons by an intronic
sequence
within the gene. Mutations
in the human LPL gene involving
amino acid substitutions
are indicated by amino acid letter symbols, stop codons by p, frame shift mutations
by 0, and silent mutations
by a. Specific amino
acid or polypeptide
regions of the human LPL protein suspected
of being involved in the spatial structure
and functions
of
the active enzyme (Table 10) are overlined with the following identifications:
(4) N-linked glycosylation;
(g) lipid-binding
domain;
(c)
lid; (B) the p-5 loop. The th ree amino acids of the catalytic
triad (Ser13Z, As~‘~~, and HisZG1) are denoted by solid triangles
(A). Amino acids of the oxyanion
hole are indicated
by open triangles (A). The four cysteine pairs involved in disulfide linkages
are denoted by solid circles (o), and the corresponding
pairs are connected
by continuous
lines.
and
190 of LPL are conserved
necessarily
lines
in other
in positions
LPL by alanine
plete
160,
to have
168,
effect
by another
tion) or replacement
of another
similar
397,
of pro-
in almost
com-
substitutions
and
432
et al., 1994a).
amino
acid
(Type
of
were found
(Bruin
amino
but not
and 310 of human
to result
but
199, 350,
of LPL,
substitution
214, 258,
reported
activity,
173,
no significant
of proline
in all species
Site-directed
157, 190, 207,
has been
loss of catalytic
prolines
ment
lipases.
Replace-
A substitu-
acid by proline
(Type B
substitution)
may both
LPL
Two naturally
activity.
mutations
are Prol57Arg
activate
enzyme
present
in human
replaced
in the
animals
with
B mutations
are Ser266Pro
activity
have
undesirable
occurring
no evident
in humans
on
LPL
and Pro207Leu,
(Table
LPL (Pro’??,
natural
consequences
Type A human
LPL
Pro’@,
that
Proj9i
molecules
adverse
and Leu286Pro.
both
7). Certain
effect.
of which
other
and Pro432) are
of other
Examples
lead to the inactivation
This
in-
prolines
loss of enzyme
species
of
of Type
of LPL
activity
V. Murthv et al.
112
TABLE
6. Amino
Acid Replacements
in the LPL of Different Species as Compared
with the Human
Enzymea
Exon 1
LPL
(species)
Exon
Signal
Uncleaved
2
3
4
5
6
7
8
9
Mature
protein
27
2
54
60
37
78
81
40
61
35
448
UK?
4-o-4
0
0
54-2-5
87
91
60-3-l
93
98
37-1-1
95
81-3-O
96
100
40-O-2
95
95
61-2-3
92
95
35-2-4
83
89
450-14-20
92
96
115-3-O
97
78-1-O
99
100
Human
Number of
amino acids
Bovine
T-C-NJ
% identity
% homologyi
Functional
regionsb
96
100
Sheep
T-C-N
% identity
% homology
28-l-5
79
82
4-o-4
0
0
54-2-5
87
91
60-3-l
93
98
37-1-1
95
Y7
78-1-O
99
100
81-3-O
96
100
40-O-2
95
95
61-2-3
92
95
35-2-4
83
89
450-14-20
92
96
115-3-O
Rat
T-C-N
% identity
% homology
27-3-2
81
93
2-o-o
100
100
54-3-5
85
91
60-3- 1
93
98
37-1-o
97
100
78-1-O
99
100
81-2-O
98
100
40-3-2
88
95
61-3-l
93
98
34-o-4
88
88
447-16-13
94
97
115-2-l
97
99
Mouse
T-C-N
% identity
% homology
27-3-2
81
93
2-o-o
100
100
54-3-5
85
91
60-2-I
95
98
37-1-l
95
97
78-0-O 81-I-O
40-I-4
88
90
61-4-3
89
95
34-o-4
88
88
447-12-18
93
96
115-2-O
Guinea-pig
T-C-N
% identity
B homology
17-l-16
0
6
2-1-o
50
100
54-4-6
81
89
60-l-4
92
93
37-4-4
78
89
78-4-6
87
92
81-2-I
96
09
40-O-4
90
90
61-2-Y
82
85
34-3-3
82
91
448-21-37
Chicken
T-C-N
% identity
o/ohomology
23-2-21
0
9
4-I-3
0
25
54-6- 16
59
60- 3-4
88
78-8-5
83
70
93
37-1-2
92
95
8 l-4-8
85
YO
40-4-8
70
80
61-7-10
72
84
50-7-26
34
48
465-4 l-82
74
82
Y9
100
100
100
94
97
100
98
100
115-4-10
88
Yl
87
92
I 15-5-h
YO
95
,’ Derived from Fig. 2.
” Onlv those regions of LPL presumed
to participate in specific funcm)ns (If rhc CIIZWWare taken into ionslderation (Table 4. Fig. 2).
LWhen an amino aud m human LPL 1s coded bv a codon split between two successive axons, due to an ~nrervcn~ng sequrnce (intron), it IS nrbitrarlly
assigned to the following
cxon in calculating
the number of amino acids III each coon. The same rule is applied to other LPL sequences,
in the absence
of specific mformation
on their gene organization.
J T 3C I and N refer to the total number of eron ammo acids and the number of ronscrvat1ve or nonconscr~x~v~
replacements
in the var~~b LPL m&c&s.
cThe bovine signal peptide has nor been idenuficd (Senda er ui., 1957).
f Homology
is calculated
by adding the number of ammo acids that are &ntlcnl
and those that represent conservative
suhstlturlons.
The amino xld
changes
are classified as conscrx~ative or nonconservative
from the Venn diagram of ammo acid subsets &rived
by Taylor (lY86), hxxd on Dayhoff’s
amino acid changes are arhltrarily
defined as those involving
not more than a single property.
mutation
odds matrix (Davhoff et al., 1978). C onservative
Substitutions
of one amino acld by another differing m more than one propertv
are rlarsed as nonconscrwtlw.
Extra amino RCI~\ or deletions,
compared
with the normal human
LPL, are also considered
nonconservative
seems
line
to be due to the appearance
and
not
because
due
substitutions
amino
acids
species
other
of LPL
On the other
line
with
at certain
present
in
Ala”’
sites
species
the effect
by proline
in the chicken
to inactivation
LPL
of the
(Table
7).
acid by proappears
to
Ser’““,
Alaq”,
and
of substitution
may not always
also on the nature
For example,
effect
its replacement
human
In view of the importance
replacement
because
of
it occurs
by threonine
leads
and stability
proteins,
residues
in the for-
of the three-dimensional
it is to be expected
by amino
acid
Replacement
occurring
(Table
inactivation
SerliZCys,
highly
S-S
in
conserved
affect
Pro’“.
(Table
8), results
interference
with
cases of human
the
introduction
of
other
amino
Arg”’
or other
to loss of cata-
from
that
Ser’;’
is in close
encloses
to His?“’ of the catalytic
by cysteine
of a naturally
leading
and SerZ5ICys.
amino
triad.
acids,
in enzyme
of new
acids,
proximity
the
e.g.,
is next to the
to an
lid sequence,
Substitution
such
and/or
of
as histidine
inactivation,
the S-S bridge
as
such as LPL.
is the cause
LPL,
of
of cystcines
the structure,
enzyme
are also known
place
(Cy~~‘~-Cys!~‘)
and is close
ing that
8). There
Arg243Cys,
bridge
A$”
in human
resulting
residues
cysteine
would
by serine
of Cys””
structures
loss or gain
of a multidomain
mutation
lytic activity
that
substitutions
well as the function,
or leucine
LPL.
of cysteine
mation
LPL
are naturally
but may depend
involved.
but
by
in other
molecule
(Ser’“‘,
proacids,
Leu?“”
activity
substitutions
has no apparent
LPL,
and
of an amino
in the
such
amino
naturally
no loss of catalytic
LPL
residue
S@h
occur
substitution
due to proline,
of the other
original
same
proline
because
However,
be entirely
of the
other
some
of a new additional
loss of the
than
hand,
be well tolerated
Se+?).
to the
indicatthe His”’
113
Human
Lipoprotein
Lipase Gene
TABLE
Various
7. Amino Acid Substitutions
Involving
Proline
LPLs as Compared
with the Human Species
Site
Ser
Ax
Thr
LW
Phe
Arg
Ser
Ala
LPL
species
guinea-pig
human mutant
guinea-pig
human mutant
chicken
rat
mouse
guinea-pig,
chicken
Site
Ala’
Ala’
Thr12
Se+
Se+”
LeuLsh
LeuL8”
Led”6
Ala”+
Aias3’
Serj”’
Ser”h
Ser396
Ala”’
Ala”’
Serqq2
Replacing
amino
acid
k-0
Ile
Pro
Pro
Thr
Pro
Met
Val
Pro
Thr
Pro
Pro
A rg
Pro
Val
Pro
LPL
species
chicken
bovine, sheep
chicken
human mutant
chicken
human mutant
bovine, sheep
rat, guinea-pig
chicken
human mutant
mouse
chicken
guinea-pig
guinea-pig
bovine,
sheep
chicken
All amino acid residues are numbered as in hLPL. Conserved proline
sites in LPL are 19, 31, 66, 77, 95, 160, 173, 190, 199, 214, 258, 310,
350, and 354.
of the catalytic triad may be involved in this action. SerZ5’
forms a part of one of the lipid-binding domains (B5 in
Fig. 2). Among the LPL species so far investigated, the guineapig LPL is the only natural molecule that contains 11 cysteines instead of 10. This extra cysteine, which replaces Glu’
of human LPL, occurs very near the extreme N-terminus
of exon 1 and, therefore, may be expected to have little effect
on enzyme activity.
3.3. Mutations Involving the Noncoding
Sequences of the Human Lipoprotein Lipase Gene
Although intron nucleotides do not code for amino acids,
at least some parts of their sequence, particularly those near
the intron-exon junctions, play a critical role in the processing of the mRNA precursor and in the correct splicing of
the coding exons. Mutations in the introns, therefore, may
affect the maturation and turnover of the mRNA, as well
as its size, its translatability, and the nature and number
of the protein products
TABLE
Various
formed. The 5’ and 3’ noncoding
sequences also contain various regulatory elements, including the transcription initiation site, polyadenylation site, etc.
(see Table 2 for a description of the elements present in the
human LPL gene), whose activity may be influenced positively or negatively by a given polymorphism. Several mutations have been discovered in the human LPL gene involving
transitions or transversions in the nucleotide sequence of
introns or in the flanking regions (Table 9). Some of these
occur in, or near, the splice acceptor or splice donor sites
and are found to interfere with gene expression by creating
8. Amino Acid Substitutions
Involving Cysteine
LPLs as Comoared
with the Human Soecies
Amino acid to cysteine
other amino acids
Cysteine to other
amino acids
Amino acid to proline
or other amino acids
Proline to other
amino acids
Replacing
amino
acid
in
Site
Cys216
in
or
Replacing
amino
acid
LPL
soecies
Site
Replacing
amino
acid
Ser
human
mutant
Glu3
Ser’7L
Cys
guinea-pig
CYS
human
human
human
human
mutant
mutant
mutant
mutant
human
mutant
ArgZ4j Cys
Arg2q3 His
Arg2q7 Leu
Serz5’
CYS
LPL
soecies
All armn~ acid residues are numbered as in human LPL. Conserved cyst&e
sites in LPL are 27, 40, 239, 264, 275, 278, 283, 418, and 438.
new splice sites and leading to the formation of aberrantly
spliced mRNAs. In a recent report, Yang et al. (1995) have
described a naturally occurring mutation in the LPL gene,
which occurs in the binding site of the transcription
Ott-I and results in a highly reduced promoter
Although mutations of the coding sequences
factor
activity.
are often
revealed by their effects on enzyme mass and/or function,
mutations of the noncoding sequences may have more subtle
effects and may require indirect approaches for detection
and investigation. The occurrence of alternate types of
nucleotides in the same position in the nucleic acid sequence,
with no concomitant
apparent phenotypic differences, is
generally referred to as polymorphism.
Such polymorphisms
in nucleic acids can be detected easily if they lead to alterations in restriction sites. In fact, many of the polymorphisms
in the noncoding sequences of the human LPL gene, which
have now been characterized in terms of the actual base
change, were originally detected by restriction fragment analysis and, even now, continue
to be identified
in the same
manner. A number of studies have been published supporting or refuting claims that certain restriction polymorphisms
of the LPL gene may be associated with various lipid-related
pathologies
in humans.
The most extensively
investigated
of these are the PvuII and Hind111 sites. Variable results have
been reported on the possible association between the PvuII
polymorphism and plasma TG levels (Chamberlain
et al.,
1989; Ahn et al., 1993a), which may be due to ethnic differences in test subjects. The Hind111 site is reported to be associated with hypertriglyceridemia
(Chamberlain et al., 1989;
Ahn et aI., 1993b), levels of total and HDL cholesterol (Heizmann et at., 1991; Mitchell et al., 1994), coronary heart disease (Chamberlain
et al., 1989; Thorn et al., 1990; Mattu
et al., 1994), and insulin resistance (Ahn et al., 1993a; Cole
et al., 1993). Even though the association between a particular LPL polymorphic site and a pathology is confirmed, it
remains to be clarified whether this association is due to
the LPL gene itself or due to its metabolic or genetic linkage
to a separate unidentified gene that may be primarily responsible for the observed abnormality.
114
V. Murthy
TABLE
9.
Mutations
in the Noncoding
Location
of the Human
Alternate
Type
1 (SD site)
Intron
Sequences
1 bp SB
ACCgta
-
LPL
Gene
Restriction
site
forms
Reference
Chimienti et al., 1992; Pepe
and Chimienti,
1993
ACCcta
-
Gotoda
2 (SD site)”
1 bp SB
GACGgt
Intron
2-Exon
3 (SA site)h
1 bp SB
agGTAAC
-
aaGTAAC
Hata et al., 1990a
Intron
3 (20 bp from SD site)
GAGACT
-
GAGCCT
Gotoda
3 (SA site)
3 (6 bp from SA site)
Intron
4 (SD site)
1 bp SB
G -
Intron
6 (SA site)’
1 bp SB
C-A
Intron
6 (1.57 kb from SA site)”
cttcagGT
Intron
8 (495 bp from SD site)
Intron
9’
-
tttcagGT
(TTTA)n,
AAGCTT
-
TAGCTG
Fisher rt al., 1987; Li er al.,
1988a; Gotoda et ul., 1989;
Oka et al., 1989; Gotoda
er al., 199213
PVUII
Zuliani and Hobbs,
Ahn et al., 1992
n = 9- 13
-
AAGCGT
101:
element”
et al., 1992
Holzl et ul., 1994
1990;
Hind111
Heinzmann et al., 1987;
Oka et al., 1990, 1991;
Gotoda et al., 1992b
XbaI
Heizmann
BstNI
Funke
BamHI
et al.,
1991
et cd., 1988
Repeats
(CA)n
Fisher et al., 1987;
Chamberlain
et u1., 1989
Li et al., 1988b
Hegele et al., 1989a
Hegele et al., 198913
Hegele et al., 198913
Narcisi er al., 1993
1 bp SB
T-C
Yang et ul., 1995
BstI
BgIII
PstI
TaqI
Promoter
et ul., 1994
Wiebusch
Inn-on 9
3’ flank of exon
et al., 1992
Nakamura
Mb011
A
CAGCTG
Repeats
rt ul., 1992b
Wiebusch
C -T
1 bp SB
Intron
Intron
Intron 6, at the 3’-end of an
alu sequence’
GACGat
et cd., 1990, 1991a,b
2-Intron
Exon
et al.
L’This mutatwn,
involwng
the first nucleotide
of intron 2 at the junction
of exon 2 and intron 2, creates multiple
cryptic splice sites, givq
rise to
several aberrantly
sphced mRNAs.
h This represents
a transition mutation of the last nucieotlde
of mtron 2 in rhe splice acceptor we at the lunction
of intron 2 and exon 3.
LThis mutation causes aberrant splicing, resulting in the deletion of c‘xons 6 through 9 in the mRNA.
d The region containmg
the PvuII site resembles the splicing sire in its homology
towards the consensus
sequence required for 3’-splicing and formation
of lariat structure,
suggesting
that the C - T change may interfere with the correct splicing of mRNA.
v A total of five different
alleles were found containing
this tetranucleotide
repeat.
‘Occurs
only among Blacks, not in Caucasians,
in the populations
surveyed.
c Two IOCI of CA repeats were found with a total of 10 d&rent
alleles.
‘>This mutation
occurs at the blnding
site of rhe transcriptlon
factor Ocr-I (nt-39 starting from the major transcriptional
start site or nt-227 from the
ATG initiator
codon). It is reported
to inhibit
promotor
activity by 85%.
SA, splice acceptor;
SB, substitution;
SD, sphce donor.
3.4.
Mutations Involving the Coding
the deletions
Sequences of the Human Lipoprotein
Mutations
merous
Table
in the
and thev
coding
continue
10 lists 71 different
that affect LPL protein
a majority
amino
acid
of 46,
to be detected
mutations
structure
gene
at a rapid
in the human
and function.
pace.
These
9 missense
mutations
involving
of various
of various
mutations
in which
the
amino
codon
acid codon
sizes (from
codons
acid.
stops
the
are altered
Although
polypeptide
the
with
creation
and
growth
4
5 silent
no change
in
of a termiimmediately,
of interesting
presented
deductions
in Table
LPL mutations
is higher
in exon
number
many
of
can
be made
from
The
exons
and C-terminal
5 (Fig.
3). The
of mutations
the
distributed.
in the middle
with the N-terminal
it is highest
contain
and even-
10.
are not uniformly
of mutations
the highest
1 bp to 6 kb),
1 bp to 2 kb) and
data
lead to frame-shifts
of translation.
compared
by a termination
sizes (from
A number
the
density
include
substitutions,
and insertions
termination
The
iPL^gene
involving
of an amino
tual
are nu-
mutations
insertions
nation
LPL
are missense
7 deletions
coded
of the
which
the substitution
codon,
regions
Lipase Gene
functional
as
exons,
exons
with
are also those
that
sites
of
the
LPL
molecule.
Some
regions,
to be more
example,
and even some
susceptible
3 of the
specific
to mutations
4 amino
acid
codons,
than
residues
appear
others;
for
situated
be-
Human
Lipoprotein
TABLE
10.
115
Lipase Gene
Mutations
in Coding Sequences of the Human
Tvue
Mutation
Location
LPL Gene
Base or
codon change
Amino acid
change
-
Reference
Rouis et al., 1996; Elbein et ul.,
1994; Mailly et al., 1995
Reina et al., 1992
Gag& et al., 1994
Kobayashi et al., 1994
Gag& et al., 1994
Foubert et al., 1994
Gotoda et al., 1991a
Sprecher et al., 1992
Bruin et al., 199413
Wilson et al., 1993
Wilson et al., 1993
Ishimura Oka et al., 1992a
Henderson et al., 1990
Emi et al., 1990a; Ishimura
Oka et al., 1992a
Gagne et al., 1994; Nevin et al.,
1994
Foubert et al., 1994
Gagne et al., 1994
Emi et al., 1990a
Deeb et al., 1991
Bijvoet et al., 1994
Ameis et al., 1991
Elbein et al., 1994
Bruin et al., 1993
Ma et al., 1992a
Faustinella et al., 1991a
Foubert et al., 1994
Bruin et al., 1992
Gehrisch et al., 1994
Hayden and Ma, 1992; Ma
et al., 1993
Beg et al., 1990
Haubenwallner
et al., 1993
Tenkanen
et al., 1994
Emi et al., 1990b; Monsalve et
al., 1990; Bergeron et al., 1992;
Henderson et al., 1992
Foubert et al., 1994
Dichek er al., 1991; Henderson
et al., 1991; Ma et al., 199413
Hata et nl., 1992
Gehrisch et al., 1994
D9N’
MS
Exon
2
GAC
-
AAC
Asp -
T18”
D2lV’
N43S”
H44Yi
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
Exon
2
2
2
2
2
3
3
3
3
3
3
3
3
Multiple
GAC AAT CAC _
GTC
ACT
TAC
Multiple
Asp - Val
Asn - Ser
His - Tyr
_
Y61Ter
W64Ter
V69Lh
Y73Ter
R75S
W86R
K102’
Q 106Te@
11 bp DL-FS-Ter
MS
MS
MS
1 bp IN
Ter
Ter
MS
Ter
MS
MS
5 bp IN-FS-Ter
Ter
TAT - TAA
TGG - TGA
GTG - CTG
TAC - TAG
AGA - ACT
TCG - CGG
Multiple
CAG - TAG
Vlcw
Silent
Exon
3
GTG
E118E
N120-Y1219
H136R
G139S’@
G142E”
v149v
G154P
D156N”
D156G”
D156H”
PI 57R”
E163D
s172clj
2 bp IN
Silent
4 bp DL-FS-Ter
MS
MS
MS
Silent
MS
MS
MS
MS
MS
MS
MS
Exon 3
Exon 4
Exon 4
Exon 4
Exon 4
Exon 4
Exon 4
Exon 5
Exon 5
Exon 5
Exon 5
Exon 5
Exon 5
Exon 5
GAG AACTAC
CAT GGC CGA GTG GGC CAT CAT CAT CCA GAG TCT -
A176T’”
D180G”
H183QlS
G188E’”
MS
MS
MS
MS
Exon
Exon
Exon
Exon
5
5
5
5
GCA
GAC
CAC
GCG
-
ACA
GAG
CAG
GAG
Ala
Asp
His
Gly
G188R’4
MS
MS
Exon
Exon
5
5
GGG
ATT
-
(A/C)GG
ACT
Gly - Arg
Ile - Thr
MS
Silent
MS
MS
Exon
5
5
5
5
5
CGA
CAT
-
GAA
CAC
Gly His -
GAC
ATT
-
GAG
ACT
Asp - Glu
Ile - Ser
Gotoda et al., 1991a
Reina et al., 1992
MS
Exon
Exon
Exon
Exon
CCC
-
CTG
Pro -
G2092”
C216P
1 bp DL-FS-Ter
MS
Exon
Exon
5
5
CGA
TGT
-
GG
ACT
Multiple
Cys - Ser
A221L”
1 bp DL-FS-Ter
Exon
5
GCT
-
CT
Multiple
1225T2’
MS
Exon
5
ATT
-
ACT
Ile -
6 kb DL
C239Ter2”
R243HL4
Major
Ter
MS
R243P
MS
Ma et al., 1991; Normand et al.,
1992; Levesque et al., 1994
Wiebusch et al., 1992
Hayden and Ma, 1992; Ma
et al., 1992
Deeb ec al., 1991; Goroda et al.,
1992a; Takagi et al., 1992
Henderson et al., 1993; Ma
et al., 1993
Langlois et al., 1989
Takagi et al., 1994
Dichek et al., 1991; Gotoda
et al., 1991a; Ma et al., 1994b
Ma et al., 1994b
1194T’@
G195E
H202H
D204E2’
1205Sz2
P207L:’
DL
-
GTA
Asn
Tyr Trp Val Tyr Arg Trp Multiple
Gln -
Ter
Ter
Leu
Ter
Ser
Arg
Val
Val
-
Ter
_
GAA
- AC
CGT
AGC
GAA
GTC
AGC
AAT
GGT
CAT
CGA
GA(T/C)
TGT
Glu - Glu
Multiple
His - Arg
Gly - Ser
Gly - Glu
Val - Val
Gly - Ser
Asp - Asn
Asp - Gly
Asp - His
Pro - Arg
Glu - Asp
Ser - Cys
- Thr
- Glu
- Gln
- Glu
Glu
His
Leu
Thr
Exons 3-5
Exon 6
Exon 6
Multiple
TGC CCC -
TGA
CAC
Multiple
Multiple
Arg - His
Exon 6
CCC
TGC
Arg -
-
Cys
V. Murthy
116
TABLE
10.
(continued)
Mutation
Type
Location
Base or
codon change
Amino acid
change
Exon
D250N”
MS
MS
MS
6
Exon 6
Exon 6
CCC - CTC
TCC - ACC
GAC - AAC
Arg - Leu
Ser - Thr
Asp - Asn
S251C
L252A’:
S259R
MS
MS
MS
Exon 6
Exon 6
Exon 6
Ser - Cys
Leu - Arg
Ser - Arg
A26lT’:
Y262H”
Y262Ter’ ’
S266P’”
L286P
N291S”
MS
MS
-I-U
MS
MS
MS
Exon
Exon
Exon
Exon
Exon
Exon
TCT
CTG
ACT
or
CCC
TAC
TAC
TCC
CTG
AAT
2 kb IN’j
Duplication
Multiple
Multlple
A3 34Tlh
T3521
L353’7
T36lT
MS
MS
2 bp DL-FS-ter
Silent
Exon 6Inn-on 6
Exon 7
Exon 7
Exon 7
Exon 8
GCC - ACC
ACT - ATT
CTG - G
ACC - ACA
Ala - Thr
Thr - Ile
MultIpIe
Thr - Thr
L365V’”
MS
Exon 8
CTA
-
GTA
Leu -
Val
W382Trr’”
W382Ter’z,“’
Ter
Ter
Exon 8
Exon 8
TGG
TGG
-
TAG
TGA
Trp Trp -
Ter
Ter
E410Vf”
MS
Exon 8
GAG
-
GTG
Glu -
Val
S447Ter-”
Ter
Exon 9
TCA
-
TGA
Ser -
3 kb DL’?
Majpr DL
Exon 9
Multiple
R243LL”
S244T’O
et al.
6
6
6
6
6
6
- TGT
- CGG
- CGT
AG(A/G)
- ACC
- CAC
- TA(A/G)
- CCC
- CCG
- ACT
Ala
Tyr
Tyr
Ser
Leu
Asn
-
Multiple
Thr
His
Ter
Pro
Pro
Ser
Ter
Reference
Appelman et al., 1994
Hata et al., 1990a
Ishimura Oka et cd., lYY2b; Ma
et ul., 199213
Wiebusch et cd., 1992
Ma et al., 1994~
Wiebusch et al., 1992; Foubert
et al., 1994
Ma et al., 1994~
Rouis et ul., 1996
Funke et al., 1990
Wiebusch et ul., 1992
Foubert et ul., 1994
Wiebusch et al., 1992; Ma et al.,
1994~; Reymer et al., 1995
Langlois et ul., 1989; Devlin
er al., 1990
Kobayashi et a!., 1993
Wiebusch et al., 1992
Wiebusch et ul., 1992
Reina et al., 1992; Gagne et al.,
1994; Gehrisch et al., 1994
Wlebusch et al., 1992; Pepe
et UI., 1994
Ma rr al., 1994~
Gotoda et cd., 1991a; Kozaki
er al., 1993
Wiebusch ec al., 1992; Previato
et ul., 1994
Hara et ul., 1990b; Kobayashi et
al., 1992; Kozaki er ucI., 1993
Be&an et al., 1995
The amino acid residue numbers
refer to the mature LPL protein as in Wion et al. (1987). &dons
are numbered
starting
from the imtiator
codon
(ATG), when mentioned in the following text. DL, deletion;
FS, frameshlft;
IN, insertion;
MS, missense; Ter, translation
terminarion.
’ Alters TaqI sate. Appears to contribute to hypertriglyceridemia
in some families. In e‘~tr” expression
shows the presence of both LPL mass and activity.
: Leads to the formation of a truncated protein of only 19 residues.
’ Asp” is highly conserved in all species examined, except guinea-pig and chlcken, where it is replaced by Asn or Glu, both conservative subsritutlons.
The rrpiacemenr
of Asp hv Val residue leads tu a change in charge and generates a new HaeIII site m the exon. in wtro studies show no effect on catalytic activity.
i Asn” is thought
to be an N-hnked
glycosylation
site of the mature LPL proteln.
Expression
studies show that this mutation
affects both enzyme
actlvxy
and secretion.
i HIS+~ is a highly conserved residue. His4iTyr
represents a nonconser\‘anve
wbstltutlon.
Found in two Individuals
wth famihal combined
hypcrlipidemia,
but m rlitro studies show no change m catalytic
activity.
’ Located in a conserved
hydrophobic
region of LPL. Expresa~on of the mutant cDNA prepared by site-directed
mutagenesis
m COS cells shows 80%
reducrion
in catalytic
activity.
(ACC
; Found in an LPL-deficient patient of Malaysian descent, it consists of a 6 hp msertior (TGGGCT)
at th e site of n single base d&non
AC) at the residue Thr”“ in exon 3, causing a frameshlft.
This results in a markedly truncated
LPL protein rhat rrrminates
prematurely
in exon 4 with
a random
sequence of 44 ammo acid residues in the carhoxy terminal
portlo”.
of the first base of
’ Found in suhlects of Polish, German,
and English descent, this mutation
leads to a truncated
LPL molecule due to substitution
with resultmg
suhstirution
of
the Gln codon (CAG) by T. Th c s a me base is replaced by G and by A m normal guinea-pig
and chicken LPL mRNA,
Gln by Glu and Lys. In rat and mouse, both rhe first and the third bases of this codon are replaced to give Asn in place of Gin.
’ This deletion causes a frameshIft mutation by removing the last two nucleorides of Asn”” (AAC - A) and the hrst Two nucleotides of Tyr”’ (TAC C), producing
a truncated
proteln of 142 residues, h>Ith substitution
of 23 C-terminal
ammo acids. This mutation
alters the MSEI restriction
site.
“I Found in an LPL-d&cent
indrvidual
of Spanish
descent. In tiitro mutagenesis
shows that this mutation
completely
abolishes
LPL functmn.
normal amounrs
” The region surrounding
Gly ‘I’ is highly conserved among lipases from dlffcrent specws. Expression m COS-i cells shows that, although
of the proteln
are produced,
It is deticienr m both catalytic
acriviry and secretion.
” This mutation
affects the last base of exon 4 and is sxuated
within the 5’-consensus
sequence for splicing.
It cgeates a new BfaI restrlction
site. It
inactive enzyme when expressed
occurs withln a conserved P-sheet region close to Asp”‘, which forms a part of the catalytic site and produces catalytlcally
in z’ltr”
changes of the first base and the third of
” Three d&rent
mutations
affect Asp”+ of the catalytic triad, SIX”‘, Asp”“, HIS!“, two of them involving
the second base of wdon
181 and resultmg in substxution
of aspartare by asparagine,
histldme,
or glyc~ne. All three mutations
alter the TaQl restriction
site and give rise ta inactlve
LPL when expressed
m vitro.
proline.
It alters a PvuII site.
” This muratmn
1s situated
next to Asp”” and results in the loss of a conserved
Ii Leads ro only partial loss of LPL catalytic
activity,
compared
with several other m~ssensc mutations.
” Also termed LPL-Bethesda,
this mutant
LPL shows loss of catalytic
activity
and alrered affinity for heparln.
Also alters a SfaNl site.
(contmued)
Human
Lipoprotein
117
Lipase Gene
TABLE 10. (continued)
‘7 Although
this amino
acid substitution
is highly conservative,
is no effect on secretion or heparm binding.
Ii: His’“’ hes close to a putanve lipld-blnding
domain
and outside the proposed
of the LPL molecule
active site of LPL, the catalytic
(B4, Fig. 2), and KS replacement
by glutamine
activity
is abolished;
there
leads to charge alteration
and
production of lnacuve LPL. The mutant gene is thought to be of Russian or Swiss origm.
” Glyl88Glu,
highly prevalent in the Province of Quebec, is believed to be a ubiquitous and probably ancient mutation. The frequency of occurrence
and distribuuon
of Glyl8HArg is not known. Both mutations alter Sau961 and AvaII sites.
x Ilc”’ is located wthm a highly conserved, putative lipid-binding domain of LPL (84, Fig. 2). By analogy with pancreatic lipase, it is in the proximity
of both the catalync triad and the lid that covers the hydrolytic pocket of the enzyme. Introduction of the hydroxyl group of threonine provides a potential
site for phosphorylation
or glycosylation that could induce conformational
changes. The mutation results in loss of enzyme activity, but retains normal
heparin affinity. The occurrence of this mutanon in two different DNA haplotypes is suggested to indicate a multicentric
origin.
” Alters a Hincl site in cxon 5.
1LIk?
1s a highly conserved ammo acid and is situated in strand 10 of the proposed three-dimensional
structure of LPL (as do Pro’@’ and Asp”+).
Substitutions
of these residues with other amino acids could disrupt hydrophobic interactions between strands 10 and 11, thus changing the conformation
of the central catalytic domain.
li This mutation, which appears to have originated in the Northwest region of France, is now found almost exclusively in the French-Canadian
population of Quebec. It alters the BslI restriction site.
after 223 residues.
” This involves deletion of the thnd nucleotide of the codon for Gly”“, resulting in termination
:’ This mutatlon destroys a conserved dlsulfide bridge that may be crmcal for LPL structure and function.
“~The frameshift caused by the single bp d&non
leads to termination
of translation
three residues downstream. The deletion of G (GCT - CT)
abolishes an AluI we (AGCT).
I; Ile’!i is located in the proximal xctwn of the amphipathic surface loop shielding the active site of LPL. L oss of LPL activity by this mutation suggests
the rnportance
of maintaining
charge and periodicity in this region of the molecule.
!’ This mutation, which results in total loss of LPL activity or mass, abolishes an HgiAl site and creates an Mbol site.
:” Three mutations have been reported that change Arg”’ (which is close to the catalytic triad: Set”‘, Asp’jh , Hi@‘) to histidine, cyst&e,
ot leucine.
One of them affects the first base of codon 270 and the other two the second base of the same codon. Al1 three mutations alter HhaI and Eco47111 restriction
sites. Arg243His was found m Caucasian, Chinese, and Japanese subjects, and Arg243Cy s was found in those of French and German descent. Haplotype
analysis indicates two separate origins for the ArgZ43Cys mutation. This, together with ArgZ43His, suggests high mutability of this CpG dinucleotide
in the LPL gene.
‘j’ Also occurs near H&l’ of the catalytic wad.
” The location of Asp”” in the proposed three-dimenslonal
structure of LPL suggests that it may be involved in a charge interaction wth an cY-helix
III the armno terminal region of LPL. The mutation alters a TaqI site.
” L~u’~‘, like the neighboring AspziO and SC?‘, is a highly conserved amino acid and is replacedonly by residues wth similar size and hydrophobicity
XI other wld-type lipases. Substwutmn with the positively charged arginine may lead to disruption of spatial structure and results in the observed partial
loss of LPL actwty. Pregnancy-induced
chylomicronemia
is reported to be associated with this mutation or one of the other three mutatmns (Ala261Thr,
AsnZUlSer, Trp382Ter)
that result in parnal LPL deficiency.
” Two dlffcrent types of substitutions are reported, involving the third base of the codon of the amino acid Ty?,
one leading to replacement of tyrosine by hlsndine and the other to termination
of translation.
The Tyr262His mutant LPL has reduced heparin-binding
activity and a destabihzmg
effect on dimcr formation.
It can be detected by alteranon in an Awl site in exon 6.
” Ser266Pr0,
a mutation that inactivates human LPL, is caused by T - C change m the serine codon. T - A change in the same codon results in
the suhstltutlon serine - threonine in the normal chicken LPL.
” This mutatwn 1s produced by the juxtaposition
of intron 6 to a partially duplicated exon 6 and involves exon-Alu interchange.
It is found in subjects
of different ancestries and possibly represents an ancient mutation.
“’ In contrast to Ala334Thr,
Ala334Pro apparently has no effect on LPL activny, because this substitution is found in normal chicken LPL.
‘- The codon involved m this mutation is split between exons 7 and 8, but both the deleted bases of the codon are in exon 7. The frameshift leads
to termination
after 355 residues.
ii It is suggested that this mutation interferes with the correct folding and assembly of active LPL homodimers.
“I Two different mutatwns of this codon have been reported, involvmg the second or the thrd base. Both lead to immediate translation termination.
” Glu”“ 1s conserved in LPL and in analogous regions of pancreatic hpase in different species. Replacement
of this residue by mutatcon may affect
formanon of homodlmers. The A to T base change in the codon generates a Mae111 restnction site.
‘! This represents the longest prematurely terminated natural LPL molecule, with only two residues less than the normal protein at the C-terminus.
Less, equal, or higher catalytic activities than normal have been reported for the truncated protein. The mutation generates a MnII restriction site.
” This deletion (2.116 kb) includes the end of intron 8, the whole of exon 9, and about two-thirds of mtron 9. Of the three AIu sequences present
in the normal lntron 9, the mutant LPL contains only the third Alu sequence plus the right arm of the second, suggesting that a stem-loop structure
hetwccn the end of lntron 8 and an Alu sequence m intron 9 may have been involved in this mutation.
tween Gly’j’ and Pro ‘57, 5 of the 8 situated between
HiP2 and Gly?@“, and all the 3 successive residues in
Asp”5”-Serzi’-Leu?iZ
undergo mutations.
As~‘~~, a
member of the catalytic triad, and Arg2”, which is
a close neighbor of another member of the triad,
His!“, each suffer three missense mutations resulting
in three different amino acid substitutions. Two missense mutations affect each of the following amino
acids: Gly’ss, Tyr262, and Trp3sz. One of these,
Gly188Glu, is very prevalent in the Qukbec population (Bergeron et al., 1992).
3. The presence of an amino acid residue with unique
properties at a given position in the protein sequence
may be critical for enzyme activity, and replacement
by any other residue may not be tolerated. Such appears
to be the case for most of the human LPL mutations.
However, in some cases, LPL from other species are
found to contain normal variants of the amino acid
residue whose replacement leads to mutation in the
human LPL. Thus, for example, although Asp9Asn,
AspZlVal, Leu286Pro, and Ala334Thr are mutational
substitutions in the human LPL, Asp9Gly, AspZlAsn
(Giu), Leu286Met(Val),
and Ala334Pro substitutions
are normal variants in other LPL species (Fig. 2).
118
mucosa
and
response
to the consumption
cleared
appear
from
(Cohen,
the
types
lipoproteins,
and
I is defined
the
in which
lipoprotein
the presence
from
FIGURE
3. Distribution
of mutations
in different exons of
the human LPL gene. The mutation density is defined as the
number of mutations
in a specific exon divided by the total
number of amino acid residues coded by that exon.
4.
All LPL
mutations
the same manner.
arin binding
0: these
tural domains
are
also
degrees
found
dimensional
A search
involving
the amino
acid
alternate
acid
and,
therefore,
ceivable
the
depending
the new codon
exons
and disorders
the nucleotide
atic studies
effects
shows
three-
at least
are silent
protein
specify
degree
(Table
the
does
not
same
result
the
consequence
the turnover
have been conducted
of
the rate
rate of synthesis
of such mutations
However,
to examine
polymorphisms
func-
preference
rate of mRNA
action.
in the
but it is con-
may influence
possible
10).
of acceptability
change
hence,
to
amino
The protein
of codon
and,
five
in regard
to the rules
of endonuclease
of codon
acid resi-
overall
to be affected,
on the
of mRNA
ing the sites
that
by alter-
no system-
the quantitative
on LPL mRNA
CHYLOMICRONEMIA
4.1.
Description
The
chylomicronemia
or protein.
syndrome
Brunzell,
presence
(>15
is characterized
hypertriglyceridemia
Chylomicrons
are formed
are the predomiis marked
as various
but
secondary
the
causes
has been
in the plasma
more
defects.
by
resulting
classification
of chylomicrons
from
Type
activity
recently,
it is
The chylomicro-
clinical
consequences
and Bierman,
1982;
of
Chait
and
1992).
4.2.
Diagnosis
Diagnosis
of chylomicronemia
ing the appearance
(lactescent,
white).
in the fasting
state
in the intestinal
After
for 24 hr, chylomicrons
plasma
(Type
in large amounts,
V).
With
tion
and
in specialized
laboratory
4.4,
of heparin
into
the
plasma
assay
U/kg
body
circulation
i.v. holus
which
the
LPL
in S.C. biopsies
mutations
1991,
1992b).
of adipose
syndrome
in
are
by apo
of apo E
and Vernier,
(Table
of LPL
tissue
useful
activation
In the newborn
assay
enzyme
LPL activ-
techniques
of chylomicronemia
of apo CII,
LPL
et al., 1987; Huff et ul., 1990), analysis
(Hixson
gene
LPL
et ul., 1992).
laboratory
diagnosis
releases
mass by immuno-
defective
(Brunzell
Other
using
injection
et cd., 1989). Postheparin
ity can also be determined
1992).
cause
In this procedure,
(Peeva
et al.,
such
by measurement
TG substrate
weight),
if a catalytically
into the plasma
of
evalua-
are available
is one
after
(Brun
causes
As will be discussed
activity.
is also used to measure
to determine
is released
usually
deficiency
10 min
(Type
to formulate
exact
by clinical
with a synthetic
obtained
(50-100
blood
The
that
lipolytic
is measured
samples
LPL
plasma
accuracy
laboratories.
layer
may frequently
and can be confirmed
postheparin
LPL activity
levels
can be pinpointed
techniques
in Section
of plasma
sufficient
surface
are also present
lactescent
TG
approach.
lipid
of chylomicronemia
plasma
with
then,
only
in detail
plasma
therapeutic
chylomicronemia,
VLDL
or plasma
left in the re-
form a creamy
or frankly
practice,
visually,
an immediate
has been
I) or, when
a turbid
some
by observ-
(“cream of tomato”)
the plasma
on clear
CII (Connelly
by the
is easily performed
of the blood
frigerator
genotypes
of marked
mmol/L).
in LPL
and VLDL,
past,
on
of plasma
manifestations.
Type V pattern
of molecular
(Brunzell
into
based
profiles
resulting
metabolism,
arises
chylomicronemia
measurements
SYNDROME
syndrome
In the
based on the detection
the differential
THE
as well
1973).
I-V),
clinical
chylomicrons
in lipid
be estimated
mutation
in the human,
in changing
the
acid by another.
according
Another
to variable
the site of a given
of the mature
of translation
may consist
struc-
mutations
of the amino
literature
is not expected
that
of protein.
functions
and
of the codon
of one amino
Different
and
of LPL.
LPL
therefore,
may be
of LPL
to different
upon
local
published
sequence
forms
replacement
the
structure
mutations
tion,
to
of the
The
these
probably,
and the contribution
affected
nature
the molecule.
to affect
depending,
mutation
due
within
10). This
functions
in
and hep-
causes,
et ul.,
comFredrick-
disorders
(Types
chylomicrons
The
hr
from
and in certain
to Table
of the multifunctional
assignment
function
secretion,
differentially
(see footnotes
a reflection
the
activity,
are affected
combinations
LPL
do not influence
Catalytic
fasting
species.
8-10
results
in Types I and V disorders.
made based on the appearance
nemia
lipoprotein
disorder,
of elevated
genetic
(Brunzell
within
in the plasma.
associated
as a genetic
deficiency
EXON
classified
in
are, then,
large macromolecular
presence
is present
stream
syndrome
and the electrophoretic
Chylomicronemia
nant
of these
have
blood
fat. They
compartment
of dyslipoproteinemias
the concentrations
0
the
chylomicronemia
continued
et al. (1967)
several
in
of dietary
vascular
in the clearance
plexes and their
son
transiently
1989). The
defects
4.
ct ul.
V. Murthv
1990), and detection
10) (Monsalve
with primary
et al.,
of LPL
1990; Ma et ul.,
LPL deficiency,
chylomicro-
Human
Lipoprotein
119
Lipase Gene
TABLE
11.
Symptoms,
Signs, and Laboratory
is present
infants
very
often
after
early
the first
after
milk,
plications
of the disease
children,
diffuse
reported
(Black
patients
have
and
expected
medical
It is presumed
during
the
of LPL
and/or
have
a less severe
clinic,
first
diagnosis
year
of life and
“Cream
trauma,
high
(Fig.
been
make
The
nal pain,
vious
are more
TGs
common
complication
with or without
levels
eating
be elicited.
patients
do not
with comparatively
show
when
clinical
plasma
signs,
TGs
and
are very
4).
major
plasma
the
1995). However,
of plasma
of TGs
habits,
of chylomicronemia
pancreatitis,
(Gagne
et al.,
unusually
The precise
rich
reasons
is abdomi-
and is related
1989).
History
in fatty foods,
for repetitive
to the
of pre-
can often
colicky
abdomi-
tooth
abscess,
the
Two grandmothers
diagnosis
hyperbilirubinemia
conditions
leading
icterus,
for other
and
in our
was unusually
g-o-
newborn
330-
had
led to suspicions
because
than
-
and often
:
n 2O4
L;: lo-
of the aspect
0 -
et al., 1989).
are diverse
purposes
in
investiga-
(Gagne
suspected
*
that have
deficiency
which
to diagnosis
to LPL deficiency,
drawn
asthma,
the clinical
of LPL deficiency
60-
of LPL
blood
conditions
grandchild
in
E
5
Physical
of the
a chylomicronemic
without
and, later, confirmation
pains.
LPL
ages
and,
led to diagnosis
initiated
that their
case,
the
1
ao-
2
was made
screening
mononucleosis,
of primary
they reported
In another
between
appearance
100
disease.
of patients
the diagnosis
have rarely
1983).
to avoid
of the
in 25%
in 50%
adults
et al.,
learned
form
are some of the coincidental
of the blood
to com-
childhood
and
of abdominal
of tomato”
allowed
unrelated
signs
in young
or family
evaluation
patients.
Thus,
early
have
70%,
findings
signs of chylomicronemia
tion when
such
experience,
(Hoeg
was made
In almost
following
“pale?
our
deficiency
fortuitously
individuals
coincidental
pregnancy
In
levels
from
have
1994a)
such
through
epistaxis,
lower
1993). In young
since
et al.,
that
20 years.
deficiency.
related
hemorrhages
1993).
These
findings
TG > 15 mmol/L
Fasting chylomicronemia
Pseudohyponatremia
Pseudohypocalcemia
Pseudo-increased
hemoglobinemia
Normal amylasemia with pancreatitis
Pseudohyperbilirubinemia
1989; Brunzell,
may
1977).
may refrain
problems
observation
is made
of 1 and
others,
pains,
and Sprecher,
(I’erron
diagnosis
In our referral
et al.,
Syndrome
progression.
patients
foods
growth
Sprecher,
growth
Sometimes,
fatty
colicky
(Black
and diagnosis
(Sadan
gastrointestinal
school
and older
have
and
under
normal
feeding,
birth
have repetitive
drinking
Laboratory
Lipemia retinalis
Eruptive xanthomas
Hepatomegaly
Splenomegaly
Abdominal
pains
with or without pancreatitis
Dyspnea
Paresthesias
Flushing with alcohol
Recent memory loss
Peripheral neuropathy
nemia
of Chylomicronemia
Signs
Symptoms
be made
Findings
measurement
5.
I
I
I
I
of lipids.
4.3.
Clinical
Manifestations
4.3.1.
Symptoms
plasma
triglyceridemia
the
chylomicronemia
presence
of one or more
in Table
11. However,
tions
is unpredictable
equal
lence
1995).
of each
Thus,
of these
to or greater
syndrome
the occurrence
for each
than
patient,
and/or
of clinical
and
by
is no consensus
clinical
manifestations
the
signs listed
manifesta-
some
may have no symptoms
there
with
15 mmol/L,
is characterized
of the symptoms
with severe chylomicronemia
(Brunzell,
In patients
of chylomicronemia.
patients
or signs
on the preva(Gagne
et al.,
FIGURE
4. Plasma TG concentrations
and frequency of physical signs of chylomicronemia
syndrome in patients with primary LPL deficiency. The frequency was evaluated on the basis
of the first clinical evaluation of 56 patients, carried out at
the Quebec Lipid Research Clinic (Gagne et al., 1989). The
asterisk (*) indicates a significant difference (I’< 0.05) for the
plasma TG concentration
compared with that of patients with
no clinical signs. (Mean * SD).
V. Murthy
FIGURE
5. Retinal photographs
of normolipidemic
subject (A) and chylomicronemic
ciency (B) showing lipaemia
retinalis.
Eruptive
xanrhomas
in female (C) and male
deficiency.
nal pains
and pancreatitis
presumably
et al.,
related
1967). Pregnancy
ciency
(Ma et al.,
the
ceptives,
production
1992). Rarely,
leads to chronic
and diabetes
of
mellitus
pancreatitis
(Searles
(Howard
with fasting,
plasma
1992).
The
1977).
by which
is still unknown.
to determine
of irritation
microns
in the blood
by the
of pancreatitis
done
after
the acute
rapidly
is generally
(Chait
event;
acids
(Chait
from
but the
leads to pancreatitis
could
and lysolecithins
PL from
capillaries
and Brun-
resulting
of the pancreas
the
circulating
and Brunzell,
This
possibility
elevated
patient homozygote
for familial LPL defi(D) homozygote
patients with familial LPL
is supported
concentrations
in chylomicronemic
Other
mono-
(Chait
and
Brunzell,
Patients
of flushing
symptoms
and
1992).
only
loss
subjects,
often
However,
on treatment
with chylomicronemia
are
unex-
of hypertriglycoccasionally
consumption
of alcohol,
present,
as
com-
mechanisms
manifestations
are rare and, when
elicited
are
memory
of dyslipidemia,
of the extremities.
upon
syndrome
Chylomicronemic
forms
as yet and disappear
eridemia.
1995).
et al.,
and recent
in psychoneuropathic
plained
plain
(Cantin
of chylomicronemia
with other
of numbness
involved
of abnormally
lysophosphatidylcholine
or polyparesthesias,
well as patients
plain
patients
likely symptoms
dyspnea,
by the presence
of plasma
com-
but
are most often
such
subtle
by a questionnaire.
evaluaof pan-
understood,
chylomicronemia
by fatty
quantities
if hyper-
because
of pancreatitis
Inflammation
in large
relation
overlooked
is part of the treatment
pathogenesis
mechanism
The
1973),
TG levels decrease
as cholelithiasis
even sub-
et al.,
is often
such causes
result
et al., 1986; Chait
is often
It is difficult
which
contra-
and alcohol
with fat malabsorption
and Levy,
1964; Cameron
trimesters,
pancreatitis,
is the cause or the consequence
er ul.,
creatitis,
(Kraus
LPL defi-
Oral
types,
(Stuyt
repetitive
are
at increased
is increased.
estrogenic
pancreatitis
1992).
incipient
and third
chylomicronemia
of hyperlipidemia
indeed,
zell,
to
and Ooi,
triglyceridemia
tion
of VLDL
the more
they
(Fredrickson
patients
the second
the risk of pancreatitis
and Brunzell,
clinical,
may also unmask
during
especially
may increase
although
levels
1993) and put these
risk of pancreatitis
when
are unknown,
to TG-chylomicron
et al.
be the
released
4.3.2.
Signs
of chylomicronemia.
tive xanthomas,
nemia
that are observed
Lipemia
retinalis
nal vessels,
due
as revealed
are the
which
result
chylo-
macrophages
1992).
They
have
intermittently
(Table
chylomicrons
of the accumulation
a yellowish
of the reti-
examination,
(Fig.
in groups
in the skin (Parker
erup-
11 and Fig. 4).
appearance
by funduscopic
may cluster
retinalis,
are signs of chylomicro-
refers to the whitish
to circulating
thomas,
Lipcmia
and hepatomegaly
5B).
Eruptive
and become
appearance
1970; Brunzell,
with
xan-
confluent,
of chylomicrons
et al.,
and is
a reddish
by the
1995).
ring
at
Human
Lipoprotein
Lipase
121
Gene
the base, and may become pruritic (Fig. 5C and D). Eruptive xanthomas are found mainly on the extensor surfaces
of the arms, the back, the buttocks,
and the thighs.
Lipemia retinalis, eruptive xanthomas, and hepatomegaly
should be considered signs of very elevated plasma TG levels,
as well as high risk factors for pancreatitis. These signs may
also be used to evaluate the efficacy of therapeutic treatment. In contrast
to these, splenomegaly
often persists in
LPL-deficient patients, despite adequate control of plasma
TG levels (Gagne et al., 1989; Bertrand et al., 1990*). It is
of interest to note that 28% of the patients with splenomegaly had no other clinical sign and their plasma TGs were
only
16.7
f
3.7 mmol/L,
whereas
other
patients
with
TABLE
12.
Causes
Primary
apo CII deficiency
Familial
LPL inhibitor
Autoimmune
chylomicronemia
Combination
of primary hypertriglyceridemia,
and/or heterozygous state for LPL deficiency
Primq
h~pertriglyceridemia
Familial hypertriglyceridemia
Dysbetalipoproteinemia
Familial combined
hyperlipidemia
4.3.3.
Anomalous
laboratory
findings in chylomicronemia. In chylomicronemic patients, laboratory anomalies
are encountered that are caused either directly by high levels
of blood chylomicrons
or indirectly
due to chylomicron
interference in the analytical
procedures
(Table 11). In vitro hemolysis is often observed when blood is collected from chylomicronemic patients for analysis (Cantin et al., 1995). This
is possibly due to an increased fragility of the erythrocyte
clinical consequences, but can become misleading in terms
of diagnosis. There are other instances where chylomicronemia gives rise to false blood analysis results. Pseudohyponatremia is a well-known anomaly in chylomicronemia. Thus,
a plasma level of 15 mmol/L of TGs may decrease natremia
by about 1 mmol/L in the presence of normal plasma osmality (Steffes and Frier, 1976). Similarly, a concentration
of
12 mmol/L of chylomicron TGs increases hemoglobin measurement by 10 g/L, as determined by the Coulter counter
method (Gagne et al., 1977a). This false increase in hemoglobin concentration
may be corrected by replacing the
plasma with an isotonic solution. Ordinarily, pancreatitis
is associated with increases in plasma amylase concentrations.
Syndrome
Primary LPL deficiency
splenomegaly combined with one or more extra clinical signs
had plasma TG levels of 50.7 f 16.4 mmol/L (mean ? SEM).
membrane resulting from exchange of lipoprotein material
between the membrane and the ambient plasma (Cantin
et ul., 1992). This hemolysis does not appear to have any
of the Chylomicronemia
4.4.
factors
Common secondan factors
Secondary hypertriglyceridemia
Diabetes mellitus
Hvuothvroidism
Nephrotic syndrome
Chronic renal failure
Alcohol
Obesity
Drugs raising the level of
plasma TGs
Estrogens
Oral contraceptives
Corticosteroids
Tamoxifen
Isotretinoids
Diuretics
P-Blockers
Pregnancy
I.
Causes of the Chylomicronemia
The most common
secondary
Syndrome
genetic and nongenetic
causes leading
to chylomicronemia
are listed in Table 12. The catabolism
of chylomicrons and VLDL is dependent on the catalytic
activity of LPL and the availability of its protein activator,
the apo CII. Defects in either LPL or apo CII may lead to
chylomicronemia.
Familial LPL deficiency and apo CII deficiency are autosomal recessive disorders, each of which can
cause massive chylomicronemia
due to complete or partial
loss of LPL activity. Primary LPL deficiency is discussed in
Section 5. Readers are referred to detailed recent reviews
and articles on familial apo CII deficiency
(Breckenridge
et
al., 1978; Breckenridge, 1987; Connelly et al., 1987; Dolphin,
1992; Tuzgol et al., 1994; Brunzell, 1995). An inherited LPL
However, amylasemia has often been found to be normal
in pancreatitis due to chylomicronemia.
This paradoxical
finding has been attributed to an interference factor (Fallat
inhibitor leading to chylomicronemia
and very low postheparin LPL activity has also been reported in a family
et ul., 1973) or to the presence of an enzyme inhibitor (Lesser
and Warshaw, 1975; Warshaw et al., 1975). Real values are
obtained if amylase activity is determined using diluted
disorders (autoantibodies
against LPL) has been reported
in a patient with idiopathic thrombocytopenic
purpura and
Grave’s disease (Kihara et al., 1989), and heparin resistance
plasma (Fallat er al., 1973). Pseudohyperbilurinemia
has also
been observed in chylomicronemia.
These uncertainties in
laboratory analyses may make the diagnosis and the follow-
was noted in a case of disseminated
up of chylomicronemic
patients somewhat difficult. All laboratory results, therefore, should be interpreted with caution in the presence of a lactescent plasma.
*Bertrand. M., Gagne, C.. Bun, L. D., J&en, P., Pineault, S., White, J.,
hlurthy, M. R. V. and Lupien, P. J. (1990) Familial hyperchylomicronemia
and splenomegaly. In: International Symposium on Triglycerides: The Role
in Diabetes and Atherosclerosis, May 23-26, 1990, p. 130, Vienna, Austna.
(Brunzell et al., 1983). Chylomicronemia
due to autoimmune
lupus erythematosus
(Glueck et al., 1969a,b).
Abnormalities
in the LPL and apo CII genes do not
represent the most frequent causes of the chylomicronemia
syndrome in the general population. Familial forms of hypertriglyceridemia, such as familial hypertriglyceridemia,
dysbetalipoproteinemia,
and familial combined hyperlipidemia
(FCH), are often associated with one or more other genetic
and/or nongenetic factors that tend to increase the plasma
TG levels (Table 12). The genetic predisposition to hypertriglyceridemia is exacerbated by secondary factor(s), lead-
V. Murthy
122
ing to the chylomicronemia
syndrome
1992). The same individual
may possess
ent genetic
hypertriglyceridcmic
triglyceridemia,
type,
heterozygocity
Gaudet
rt al.,
Uncontrolled
nemia
1983; Wilson
fasting
(Bierman
1973;
et ul.,
the
Wilson
catalytic
activity
and Brunzell,
(especially
Gleeson
and
1987;
Iverius
(Molitch
(Chait
mild
to moderate
uals,
may
familial
forms
secondary
(Stuyt
et u1.,
1988),
1992),
which
oral
Plasma
TGs
Cholesterol
1.31 t 0.55
4.92 + 0.92
22.35 t
5.44 t
VLDL
TG::
Cholesterol
Cholesterol/npo
concentrations
mmol/L,
36 mmol/L
of these
eliminate
the
When
LPL
activity
LPL
gene,
VLDL
is impaired
(Brunzell,
of these
particles.
of chylomicrons
is unclear,
in the
but it could
of HL or the direct
endothelial
The
and
do
plasma,
that
not
but
there
complete
absence
be the result
continue
reach
exists
mechanism
occurs
a process
of
amount
of ingested
siderable
variations
The
with
between
different
viduals,
over
long-chain
patients,
long
periods
of LPL
(Table
no postheparin
TGs.
as well as within
10) may
Patients
plasma
acids.
The
due
LPL
produce
heterogeneity
have been
activity,
and/or
reported
but
show
profiles
significant
of cholesterol
and HDL
The
(Table
vascular
apos
ably
found
in
B and
apo AI),
that
as
in both
these
particles
reduced
level,
which
relative
atherosclerotic
in the homozygotes
attacks
and the presence
the
HDL
but also drastically
are rarely
cholesterol
explain
been
were reduced
indicating
in recurrent
complications
ratio,
(apo
of chylomicronemia
mainly
low LDL
for
with females,
13).
morbidity
is expressed
have
ratios,
were not only poor in cholesterol,
in number
Except
13.
occa-
the sexes (GagnC et al., 1989).
and
fractions,
and
in Table
level are observed
differences
between
well as the cholesterol-to-apo
the LDL
for females
are shown
chylomicronemia.
profiles
TG
6 and 234
1989).
is lower in males as compared
such as chylomicrons,
of pancreatitis;
observed
cardio-
(Nikkila,
1983).
the low LDL-to-HDL
cho-
of large plasma
lipoproteins,
are nonatherogenic,
freedom
could prob-
of homozygote
complications
(Gagne
patients
et ul.,
1989).
to the
con-
observed
indi-
et ul., 1989). Variations
the secretion
levels
to vary between
et ul.,
(Brun-
the plasma
of 26 mmol/L
(Gagne
to severe
which
activity
the same
value
in another
patients,
total cholesterol
lesterol
However,
activity
found
lipoprotein
in plasma
activity
is related
and normal
a mean
of LPL
TG levels have been
(Gagne
that may influence
levels of plasma
fatty
0.0042
0.0001
0.0001
for males
plasma
Increases
from
in plasma
0.36 * 0.10
0.33 * 0.08
0.45 + 0.06
The
by the reticulo-
of hypcrchylomicronemia
AI
0.27 t 0.04
1.25 ? 0.33
0.78 * 0.10
for the turnover
of the catalytic
of chylomicrons
to
an even-
system.
severity
in factors
also
The
uptake
lesions
chylomicron
hypertriglyceridt-mia
in the
indicating
of genetic
of both
Chylomicrons
indefinitely
equilibrium,
turnover
port
DEFICIENCY
as a result
and massive
1995).
accumulate
tual
is defective
the catabolism
0.021
0.0001
0.0001
have been
the lipoprotein
Homozygote State of Lipoprotein Lipase Deficiency
in the
0.36 k 0.25
0.49 k 0.26
1.19 + 0.35
in one tissue
individ-
Correction
LII’ASE
B
O.lY k 0.11
3.14 t 0.78
3.M k 0.71
TGs :lllii cholesterol values xc expressed as mmol/L. Mean * SD (n = 16).
I,‘, mu slgnificnnt. Datn from CantIn et 01. ClYO?).
sionally
LIPOPROTEIN
B
ns
ns
ns
zell, 1989). In the French-Canadian
condition.
PRIMARY
12.84
3.91
4.33 2 7.41
1.64 rt 3.00
17.80 t 25.12
HDL
T(;s
Cholcstcrol
Cholcs~crol/apo
no statistically
5.1.
0.0001
ns
0.72 + 0.48
0.38 + 0.19
6.79 -’ 8.33
LDL
I-G
Cholesterol
Cholestert-rl/apo
cholesterol,
5.
P
27.39 k 18.23
7.91 k 6.42
Chylomicrons
TGS
Cholesterol
carrying
in normal
to rapidly
Homozygotes
Controls
enzyme
induce
in patients
found
13. Plasma Lipoprotein
Concentrations
in Control
and Homozygotes
for Primary
LPL Deficiency
(Bag-
rt cd, 1986), isotretinoids
et al., 1987), diuretics,
of hypertriglyceridemia.
chylomicronemic
(Ma
et ul., 199513).
Flynn
Brunzell,
has been
et
condition
corticosteroids
chylomicronemia
factors
with
(Pykalisto
(julien
hypertriglyceridemia
produce
failure,
combined
and Brunzell,
(Brun
and
as hypo-
the third trimester)
1974),
1980;
TV (IL.,
renal
as estrogens
et ul.,
and Connolly,
P-blockers
such
physiological
during
such
particles
1992). Chylomicronemia
by obesity
dade rt u1., 1970), tamoxifen
(Dicken
diseases,
when
drugs,
et al.,
produc-
(Brunzcll
and chronic
by a transient
lipid-raising
1986;
of LPL
1983,
et al., 1993), and exacerbated
contraceptives
may induce
to hypertriglyceridemia
may also be induced
Several
and Brunzell,
the hepatic
chylomicronemia
such as pregnancy
use are the
with chylomicron
syndrome,
predisposition
d., 1976; Chait
et ul.,
in the chylomicro-
and alcohol
et al., 1994). V arious
induce
alcohol
by increasing
nephrotic
also
E: geno-
(Julien
1966; Chait
TGs that compete
thyroidism,
familial
and
involved
et al., 1993). Diabetes
saturate
may
mellitus
factors
chylomicronemia
tion of VLDL
and
due to apo
TABLE
Subjects
hyper-
1995).
secondary
syndrome
of differ-
such as familial
for LPL deficiency
diabetes
most frequent
and Brunzell,
an amalgam
traits,
hypertriglyceridemia
and/or
1994;
(Chait
et al.
the transin the
5.2.
Heterozygote State of
Lipoprotein Lipase Deficiency
The
heterozygote
ically
and
zygotes
are
considered
who have
chylomicronemia
abnormal
ease,
and
state
for LPL
biochemically
they
is still
not
characterized.
The
to be
asymptomatic
in regard
and other
escape
deficiency
well
usual manifestations
unambiguous
clinical
clin-
heteroto
of the dis-
identification.
Human
Lipoprotein
TABLE
14.
Lipase Gene
Phenotypic
123
Expression
Subjects
LPL gene mutation
Number of subjects
Male/Female
Reduction
in LPL activity
Total TG
Total cholesterol
VLDL cholesterol
LDL cholesterol
HDL cholesterol
Apo R
Ape AI
Denser LDL
of the Heterozygote
State for LPL
Deficiency
Minnicha
Maillyb
Babirakc
Wilsond
Emi’
Het
291
19
nd
slight
- or
nd
nd
Het
(Obligate Het
9
25
25/o
2Om30%
nd
14
8/6
52%
Het
188
29
II/18
50%
Het
188
11
5/6
- 50%
t
1
- or I
t
nd
t
I
nd
nd
nd
nd
or
t
nd
nd
nd
Julieng
Het
188
8
4/4
62%
Het
207
48
23/25
40-50%
t
- or 1
t
-
Miesenback’
nd
nd
nd
I
nd
nd
1
1 HDLL
1 HDLL
presence
presence
1
Het, heterozygote; nd, not determined;
-, unchanged;
1, increased; I, decreased.
‘Minmch et al. (1995): hMa~llv et al. (1995): <Babirak et al. (1989): dWilson et ai. (19901; ‘E mi et ui. (1990b); ‘Mxsenbock
(1994). (1995~4 (unpubil;hed
results) and Sniderman et al. (1995).
et ui. (1993); EJulien et al.
Babirak et al. (1989) have reported that carriers of LPL defi-
dense in subjects carrying LPL gene mutations
ciency could be identified on the basis of reduced postheparin
(Miesenbbck et al., 1993; Julien et al., 1994, 1995a).
Heterozygotes for LPL gene deficiency show impaired TG
plasma LPL activity and mass. However, in our own study
(Gagne et al., 1977b), even though adipose tissue LPL activity in heterozygotes was half the normal level, we could not
clearly distinguish individual carriers from individual noncarriers based on postheparin plasma LPL activity because
of significant overlapping in LPL activity between these two
groups of subjects. Wilson et al. (1990) also reported that
adipose tissue LPL activity was reduced by 50% in carriers,
but this reduction did not allow reliable identification of
the carriers from the noncarriers.
tainties in biochemical
In view of these uncer-
and clinical data, the only reliable
way to identify the carriers of LPL deficiency
of the gene defects.
is by analysis
tolerance and postprandial lipemia (Miesenbbck et al., 1993).
Postprandial hyperlipidemia is known to be a predisposing
condition
for atherosclerosis
French-Canadian
heterozygotes
is consistent with impaired clearance of plasma TGs, due
to a catalytically defective LPL enzyme. This probably leads
hyperapo
profiles in most
by higher
than normal levels of plasma TG. Hypertriglyceridemia
is
found not only in individuals with a marked decrease in
LPL activity (40-60% of normal), but also in those with only
moderate reduction
in LPL activity (<30%).
is significant heterogeneity
However, there
in hypertriglyceridemia
(normal
do not exhibit increases in
either total apo B or LDL apo B (Sniderman et al., 1995).
The phenotypic expression of the heterozygote state, thus,
LPL activity (Table 14). Plasma lipoprotein
characterized
1979). However,
deficiency could represent a subgroup of FCH, a dyslipidemia
characterized by an overproduction
of apo B. However,
to mild hypcrtriglyceridemia,
are abnormal,
(Zilversmith,
precocious atherogenesis has not been investigated in these
carriers. Babirak et al. (1989, 1992) have suggested that LPL
Phenotypic expression of LPL mutations in heterozygotes
is found to result in a 20-62% decrease in postheparin plasma
of these individuals
188 and 207
the presence
hypoalphalipoproteinemia,
of cholesterol-poor
LDL
particles
and
without
B.
The heterogeneity of triglyceridemia in the heterozygote
carriers of LPL gene mutations indicates that factors other
than LPL deficiency may also play a role in the phenotypic
expression of this familial dyslipoproteinemia. This possibility
is supported by the observation that as high as 13-20% of
French-Canadian
patients with Type IV and V hyperlipo-
to severe), even in subjects carrying the same LPL gene mutation. The major feature of this hypertriglyceridemia
is the
presence of larger than normal VLDL particles (TG- and
proteinemia are carriers of an LPL gene defect (Julien et al.,
1994; Minnich et al., 1995). Age, obesity, hyperinsulinemia,
and lipid-raising drugs have been shown to contribute to
cholesterol-rich
particles) (Julien et al., 1995a). As shown
in Table 14, an investigation of a group of Quebec heterozygotes, the largest of any group studied so far, shows that
the expression of hypertriglyceridemia
in heterozygotes
(Wilson et al., 1990, 1993; Julien et al., 1995b). Thus, severe
hypertriglyceridemia
observed in some heterozygotes could
these heterozygote subjects had reduced HDL cholesterol,
as well as reduced apo AI, indicating that these HDL particles are less numerous and poor in cholesterol. But this
reduction, was not as pronounced as in the homozygotcs.
In two other studies, only the particles in the HDLI subfraction were found to be altered. Generally, no significant
changes were found in total plasma apo B and LDL cholesterol. However, LDL particles have been reported to be more
be the result of a second independently inherited defect in
lipoprotein metabolism (Chait and Brunzell, 1983; Wilson
et al., 1983). Similarly, it has been shown that homozygosity
for LPL deficiency protects familial hypercholesterolemic
patients against increased LDL cholesterol. This indicates
the importance of gene-gene interactions in the phenotypic
expression of dyslipoproteinemia (Zambon et u1., 1993). Furthermore, a variable lipid phenotype has also been dem-
V. Murthy et ul.
124
TABLE
Activity
15. Frequency
of LPL Gene Mutations
Among 82 Homozygous French-Canadian
Number
of alleles
Exon
Mutation
5
pro’“;
j
6
6
9
Gly’s” - Glu
A,$”
- Asn
ASTP - Ser
Se+
- Ter
-
W)
113
37
4
1
Le”
(1992) and unpublished
(69)
(22)
(2)
(1)
0
0
0
0
0
0
data.
onstrated in a family affected by combined
and LPL activities
Plasma
postheparin
LPL activity
1 (1)
8 (5)
Unidentified
(’Data from J&n
and LPL
Probands=
deficiency of HL
(Auwerx et al., 1990). The interaction
of genetic, dietary, and other factors in the phenotypic expression of dyslipidemia (in LPL heterozygote
subjects) has not
tury from the Northwestern
are homozygous for a specific LPL gene haplotype named
H2 (Hind111 and PvuII: + / +), indicating a common origin
and a founder effect for this mutation (Ma et al., 1991). After
arrival in Charlevoix, some of the settlers migrated towards
the Northeast along the St. Lawrence River and then along
the Saguenay River towards Lac St-Jean (Fig. 7B) (Dionne
et al.,
1992). These are the regions now populated by the
descendants of the original immigrants
tion 207 in their LPL gene (Fig. 6).
has led to identification of 4 founders originating from different regions of France, one of them possibly of Scottish oriMutation
occurrence
As mentioned in Sections 3.3 and 3.4, gene analysis of individuals with documented enzyme deficiency has led to the
of a large number of LPL gene variants. These
variants have been found in subjects from different ethnic
origins, including Caucasians, Japanese, Malaysian, and
Black Americans (Lalouel et al., 1992). Detailed studies have
been carried out in two large populations affected by LPL
deficiency,
one North
European
and the other
French-
Canadian (Wilson et al., 1990; Julien et al., 1994). Genealogical reconstructions
of affected families have been attempted in the French-Canadian
The incidence of homozygosity
Northeastern
population of Quebec.
for LPL deficiency in the
region of the Province
of Quebec
et al., 1992; Dionne
et al., 1993).
188 has been shown to be specifically associated
with haplotype Hl (Hind111 and PvuII: +/ -), suggesting
a common origin for this mutation (Ma et al., 1991). The
5.3. Origin and Dissemination of
Lipoprotein Lipase Gene Defects in Qukbec
identification
who carried muta-
The second most common mutation, 188, is present mainly
in Western Quebec (Fig. 6) (Bergeron et al., 1992). Genealogical reconstruction of 14 families carrying mutation 188
gin (Fig. 7A) (Bergeron
yet been investigated.
part of France, especially from
Perche (Fig. 7A) (Dionne et al., 1993). DNA haplotype analysis has revealed that LPL alleles containing mutation 207
has been
calculated to be at least one in 10,000, and the incidence
in the general population is as low as 1 in 1 million (Julien
rt ul., 1994). Five LPL gene defects have been identified to
date in the Province of Quebec, and they all lead to complete loss of plasma postheparin LPL activity (Table 15). Of
the 82 French-Canadian
patients identified, a majority are
of this mutation
in different parts of the world
and in various ethnic groups suggests that it may be an
ancient mutation predating the spread of European and East
Indian populations. The European immigrants to Canada
carrying mutation
188 arrived at the site presently occupied
by the city of Quebec. Their descendants migrated towards
the Mauricie region, where they settled. Some of them, however, also moved along the fertile St. Lawrence Valley towards
Montrkal (Fig. 7B). We find most of the current carriers of
mutation 188 in these areas (Fig. 6). The relative isolation
of different regions of the Province of Quebec and the limited
demographic movements during the past centuries have
served to confine the carriers of mutations 207 and 188, over
a long period of history, to the regions where these immigrants originally
settled (Dionne
et al., 1993).
Haplotype analysis of other French-Canadian
patients
clinically homozygous for LPL deficiency, and carrying still
unidentified mutation(s) of the LPL gene, suggests that there
may exist at least 3 additional mutations, besides 188, 207,
and 250, underlying LPL deficiency in the French-Canadian
homozygotes or compound heterozygotes for mutations 207
and 188.
Most of the French-Canadians
living in the Province of
population (Julien et al., 1995a). Recently, 2 new mutations
at positions 291 and 447 of the LPL protein were identified
Quebec are descendants
nich et ul., 1995).
The total heterozygote carrier rate for all of these LPL
gene mutations has been estimated, based on the HardyWeinberg equilibrium (Table 16) (Julien et al., 1994). The
Saguenay-Lac-St-Jean
(Northeastern Qukbec) and Mauricie
(Western Quebec) regions are found to have the highest carrier rate, with frequencies of l/48 and l/107, respectively.
of approximately
8500 settlers who
migrated from France between 1608 and 1759 (Charbonneau and Robert, 1987). We previously have hypothesized
that the high frequency of familial LPL deficiency in this
population was the result of a founder effect (De Braekeleer
et al., 1991). Genealogical reconstructions of affected families
show two different sets of founders for mutation 188 and
207. Analyses of geographic distributions indicate that mutation 207, which is almost exclusively French-Canadian,
is
more prevalent in the Northeastern region of the Province
(Fig. 6) (Normand et al., 1992). Genealogical reconstruction
of families carrying mutation 207 has enabled us to identify
16 founders who migrated to Quebec in the early 17th cen-
in 2 French-Canadian
homozygote patients (Table 15) (Min-
However, within the Charlevoix area, a subdivision of the
Quebec region, the incidence is estimated to be l/33. On
the basis of the number of homozygote patients referred to
the Lipid Clinics, the total number of carriers for these two
major mutations is believed to be at least 45,000 in the Province of Quebec (J&en et ul., 1994).
125
Human Lipoprotein Lipase Gene
08
Lac St-Jean
Administrative
Regions
01: Bas-St-Laurent
02: Saguenay - Lac St-Jean
03: Quebec
04: Mauricie - Trois-Rivikres
05: Estrie
06: Montreal
07: Outaouais
08: Abitibi - Tkmiscaminque
09: C&e-Nord
10: Nouveau-QuGbec
-
207-m
188-m
250-m
Unknown - 0
Compound
Heterozygote
-
H
FIGURE
6. Geographic
distribution
of LPL-deficient
patients homozygous
for different LPL gene mutations
in the Province
of Quebec. The distribution
is based on the place of birth of each patient. The size of each box is proportional
to the number
of patients identified in each administrative
region. Reprinted from Julien et al. (1994), with permission of the copyright holder,
Canadian
Cardiology
Publications,
Inc., Mississauga, Ontario, Canada.
6.
TREATMENT
LIPOPROTEIN
6.2.
OF
LIPASE
DEFICIENCY
Family Screening and Counseling
Identification and Correction of Secondary Factors
Statistically, the siblings of a patient with primary LPL deficiency have a relative risk of 25% of having the disease and
As mentioned in Section 4.4, primary LPL deficiency is not
the most common cause of chylomicronemia.
Understanding the multiple etiology underlying chronic and transient
50% of being heterozygote. Diagnosis of chylomicronemia
in one individual, therefore, implies family screening. In families where primary LPL deficiency is detected, the only way
chylomicronemia
is necessary for the proper management
of this syndrome, as well as to avoid recurrent clinical complications. The long-term goal of the treatment must be
directed towards the prevention of recurrences. This can
be achieved by maintaining
plasma TG levels below 10
to predict the risk of having a child with the disease is to
identify the mutation at the molecular level in both prospective parents. As discussed in Section 5.2, the measurement
6.1.
mmol/L. Factors that may aggravate hypertriglyceridemia,
such as alcohol consumption,
use of hypertriglyceridemic
drugs, oral contraceptive agents, and estrogens, should be
avoided. Careful attention should be given to detection and
treatment of secondary factors, such as diabetes mellitus and
hypothyroidism. If a woman desires pregnancy, the followup should be planned in advance, as pregnancy may be complicated and has the potential for high risk (Watts et al.,
1992; Ma et al., 1993).
of postheparin LPL activity is not useful for identification
of heterozygotes, because enzyme levels change over time
and overlap with those of normal individuals, although the
mean is lower in heterozygotes
(Gag& et al., 197713).
6.3.
(about 50% of normal levels)
Dietary Regimen
Fat restriction applies to patients with familial LPL deficiency, as well as to patients with secondary hypertriglyceridemia, when secondary causes cannot be treated or treat-
V. Murthy
126
familial
LPL deficiency.
limiting
the intake
it by complex
should
caloric
and minerals.
cess depends,
The
low-fat
increased
by using
micronemia,
administration
a milk
of MCT
formula
amounts
PROVINCE
Formulation
in the
nutrients,
daily
diet.
in women
be limited
in iron.
1993),
The
milk
reduction
reduce
signs
of triglyccridemia.
triglyceridemia
of
chylomicronemia
pancreatitis
TGs
can be achieved
at lo-20
Fat restriction
The
to a level
are
aim of the
where
the
eliminated.
by maintaining
therapy
is to
symptoms
and
Prevention
fasting
plasma
Perron
et
zell,
1992).
emulsions
TG
is progressively
are identified
by other
nutrition
1992;
introduced,
and Brun-
be given,
but lipidic
After
while
and possible
also be desirable
to consider
drugs,
necessary.
in the same
(Chait
lipids are metabolized
be monitored
and corrected.
et al.,
as their
et u1., 1985).
as chylomicrons.
should
Ma
not nurse,
causes
as these
should
and Sprecher,
is treated
should
be avoided,
if judged
et al.,
may
adapted
attention
should
pancreatitis
to iron,
the diet
(Black
in fats (Steiner
mechanisms
levels
be given
Special
(Watts
of
are present
for whom
children
by a
quantities
conditions.
produced
should
must
children
women
Parenteral
be made
be periodically
poor
as that
should
adequate
the acute
alimentation
secondary
For long-term
factors
therapy,
it may
the use of hypotriglyceridemic
mmol/L.
is the only
dietary
treatment
available
TABLE
16. Estimation
of the Heterozygote
Carrier
LPL Gene Mutations
in the Province
of Quebeca
Administrative
of
available
and minerals
with LPL deficiency
is extremely
phase,
for adequate
the
adequate
1990;
diet should
Chylomicron-induced
manner
diet
attention
growing
and pregnant
by the same
is not sufficient
and
er al.,
that
vitamins,
and
to infants,
1993). Women
causes
(Schluter
Special
especially
be given
secondary
MCT
with
For infants,
is commercially
to ensure
tc age and physiological
of these
vein. Chylo-
relieved
et u1., 1992).
mostly
of an adequate
dietitian
all necessary
ment
and
MCT
199413).
professional
FIGURE
7. A: Spread of mutations
188 (solid line) and 207
(dashed line) from France to Quebec in the 17th century.
B: Founding regions for mutations
188 and 207 in the Province of Quebec and movement of settlers along the St. Lawrence
River and the Saguenay.
via the portal
acids
Mead-Johnson)
may be
oils for cook-
into chylomicrons
oil (Shirai
fatty
with the
of the diet
significantly
containing
of essential
(Portagen@,
ill.,
be
to
Suc-
for their catabolism.
transported
can
compliance
TG (MCT)
on LPL activity
thus,
of fat is limited
palatability
fats are not incorporated
fatty acids are directly
the diet
with the usual 40%.
medium-chain
are not dependent
However,
consumption
The
by
and replacing
as well as essential
on the patient’s
diet.
accomplished
acids
intake,
compared
however,
extremely
fatty
and proteins.
adequate
10-l 5% of total calories,
ing. MCT
is efficiently
carbohydrates
provide
vitamins
This
of long-chain
et al.
region
01 Rx St Laurent
02 Saquenay Lac-St-Jean”
03 Quebec (Charlcvolx)”
04 Maurlcie’
06 Montr~ul
00 core Nod
Eastern Quebec”
Western Quebec’
Whole Quebec
Estimated
Rate for
carrier
l/245
l/48
l/l16
l/107
l/24’)
l/166
l/M
l/220
l/l43
for
6.4.
Drug Therapy
Drug
therapy
is used to lower plasma
triglyceridemia,
such as familial
and dysbetalipoproteinemia,
rate
LPL
deficiency
glyceridemia.
effective
action
Fibric
lipolysis
develop
moderate
acid derivatives
is on
plasma
plasma
in adipocytes,
and activation
LDL
in HDL
of fibrates
and high-carbohydrate
when
heterozygotes.
are known
VLDL
However,
hyperFCH,
inhibition
principal
through
of VLDL
cholesterol
produc-
Lowering
with a reduction
(or nicotinic
with
are elevated,
the use of fibrates
of
in denser
(Shepherd,
acid)
diet is useful in lowering
levels
as the most
The
1994).
for
hypertri-
metabolism
of LPL (Davignon,
and an increase
especially
to severe
TG levels.
TG
plasma TG levels is often associated
A combination
in primary
as well as in heterozygotes
drugs for lowering
of fibrates
reduced
tion,
who
TGs
hypertriglyceridemia,
1993).
a low-fat
plasma
TGs,
as in the case of
to treat
homo-
Human
Lipoprotein
zygote
patients
is not
indicated
127
Lipase Gene
for primary
because
LPL-
they
and
apo
respond
Two approaches
(X-deficiencies
only
to low-fat
oped.
diet.
from the patient,
Prospects of Gene Therapy
6.5.
As described
in Sections
of LPL deficiency
restrictions
or
lipidemia.
consists
administration
Both
methods
the part of medical
of the patient.
and,
6.3 and 6.4, the current
in humans
professionals
Chronic
and compliance
use of drugs
in the techniques
genes
normal
cells
of direct
could
means
a cure
of continuous
Several
that
are directly
mouse
(Field,
1993;
Liu et al.,
et al.,
1994),
its activator
and HL (Busch
et al., 1994).
of an optimum
The
model
chylomicrons,
thomas,
and
deficiency
(Jones
inherited
in the
LPL
et al.,
LPL
gene,
zinger
er al.,
bined
lipase-deficient
recessive
1994).
The
glycosylated
that
but
remains
within
cytes
above
pathway,
two models
directly
by defects
l-3
1990).
The
appears
because
could
help
by lesions
in other
genes.
and
for
an
secretory
if
the target
can
sophisticated
to migrate
and
techniques
more work is needed
can
technical
the gene
expressed.
to develop
loss,
to
in a form
Although
is receiving
in clinical
direct
to the target
or qualitative
and to deliver
be tested
of the
a highly
where
(Wilson
method,
cells in viuo. This,
the vector
of attention,
they
disease
consists
to the target
integrated
use of such
in the treat-
An alternative
quantitative
cells,
be properly
the possible
a great deal
them
to a point
situations.
Ackno~,lrdgements-The
authors are deeply indebted co Professor David
Shugar for his interest and careful correction of the drafts of this article.
We wish to express our thanks to Professor Alan Sniderman for reading
the final manuscript and for useful suggestions. We thank Gervais Lapointe,
Merck Frosst Canada, and Car& Bra&, CHUL Research Centre, for assistance in complling the references. Research from the authors’ laboratories
discussed in this review was supported by grants from the Heart and Stroke
Foundation of Canada, Parke-Davis Canada, the Canadian Diabetes Association, the Natural Scirnces and Engineering Research Council of Canada
and the Medical Research Council of Canada. Dr. I? Julien was a Career
Scwnt~st of the Fends de la Recherche en Sante: du Quebec.
et
for the
to the
lipase
glyco-
in cld adipo-
characteristics
the
LPL deficiency
of the
efficacy
of
is caused
itself or indirectly
Ahn, Y. I., Kamboh,
M. I. and Ferrell, R. E. (1992) Tm,o new alleles
in the tetranucleotide
repeat polymorphism
lipase (LPL) locus. Hum. Genet.
Ahn, Y. I., Ferrell, R. E., Hamman,
of lipoprotein
iological components
at the lipoprotein
90: 184.
R. E and Kamboh,
lipase gene variation
of the insulin-resistance
of the San Luis Valley, Colorado.
M. I. (1993a)
with the physsyndrome
Diabetes
in the
Care 16:
1502-1506.
Ahn, Y. I., Kamboh,
M. I., Hamman,
R. F., Cole, S. A. and Ferrell,
R. E. (1993b) Two DNA polymorphisms
gene and their associations
in the lipoprotein
lipase
with factors related to cardiovascu-
lar disease. J. Lipid Res. 34: 421-428.
Al-Haideri,
M., Granot,
E., Schwiegelshoh,
I. J. and Deckelbaum,
B., Vogel, T., Gorecki,
R. J. (1993) Apoprotein
E simulates non receptor triglyceride-rich
Circulation
terization
particle cellular uptake.
88: I-321.
M., Schotz,
protein
(Davis
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abnormally
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normally
no significant
recognize
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LPL gene
gene responsible
in the LPL gene
related
of
appears
inactive
in evaluating
when
and
reticulum
are processed
in situations
LPL
The
to be specific
other
a single,
chylomicronemia
it produces
permit
tech-
hypercholesterol-
lipid-related
1994).
less invasive,
require
site with
population
com-
homozygote
days.
would
that would
Association
for
T/t complex
catalytically
endoplasmic
is
(Gin-
is the
has
in utero
I erent
1990). Th e d’ff
et al.,
LPL gene therapy
either
et al.,
such as adipsin
(Davis
but
the
of Arg
of both
massive
mannose),
glycosylation
processing
proteins
exhibits
normally,
(il., 1990; Masuno
defective
normally
and dies within
(high
LPL
base change
model
et ul., 1983). The
develops
to suckle,
animal
a deficiency
cld mutation
is transcribed
second
causes
the
xan-
deficiency
substitution
the
(Paterniti
show
412 of the LPL protein
within
17 that
at birth,
LPL
mutation
activities
that
to human
by a single
in the
of
LPL transgene
subcutaneous
feline
be much
success
familial
gene
This
for the devel-
of cats
which
chromosome
allowed
models
mouse,
HL
normal
attempted
two animal
similar
The
(cld/cld)
autosomal
of complica-
is eventually
for human
to be caused
acid residue
are useful
of the LPL transgene
activity
resulting
Gly at the amino
studies
the nature
retinalis,
1983).
and is found
and
LPL itself
et al.,
Animal
prevalent
functioning
patient.
partial
from
et al.,
the
devel-
are harvested
that
199413;
is a colony
lipemia
reduced
Zhang
serve as test systems
protocol
first
genes
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