GENETIC AND FUNCTIONAL INVESTIGATION OF LPL INDEPENDENT PATHWAYS OF TG-RICH
LIPOPROTEINS CATABOLISM IN SEVERE HYPERTRIGLYCERIDEMIA AND CHYLOMICRONEMIA
Daniel Gaudet, MD, PhD, Diane Brisson, PhD, Karine Tremblay, PhD
Université de Montréal Community Genomic Medicine Centre and ECOGENE-21 Clinical and Translational Research Centre Department of Medicine, Saguenay, QC, Canada
Background
Lipoprotein lipase (LPL) is a key enzyme of triglyceride (TG)-rich lipoproteins
metabolism. LPL deficiency (LPLD) is a rare mendelian cause of severe
hypertriglyceridemia and chylomicronemia (CM) [1-4]. Recent data obtained in
individuals with LPLD suggest APOC3 as a key regulator of LPL-independent
pathways of TG-rich lipoproteins metabolism [5]. The aim of this study was to
investigate gene expression profiles in CM across a spectrum of LPL activity.
Methods
A total of 53 subjects participated in this study and were divided into three groups: 19
LPLD homozygotes for null LPL mutations and less than 5% of LPL activity (HoLPL);
20 CM heterozygotes for null LPL mutations (HeLPL) and 14 normolipemic controls
(wild-type LPL) (Table 1). Whole blood RNA samples were hybridized on Affymetrix®
Human Gene ST 2.0 microarrays (Santa Clara, CA, USA). RMA was applied on raw
intensities [6]. Differential expression moderated T-tests between studied groups were
performed using a linear model of the Bioconductor package Limma. False discovery
rate (FDR) was controlled using the Benjamini-Hochberg procedure [7]. Data were
analyzed using QIAGEN’s Ingenuity® Pathway Analysis (IPA®, QIAGEN Redwood
City, CA, USA).
Results
HoLPL subjects presented significantly lower BMI and HDL-C mean values as well as
higher prevalence of acute pancreatitis than HeLPL subjects (p<0.001, Table 1). Gene
expression analyses revealed that at a p-value <0.01, a FDR of 5% and a ≥2-fold
change expression significance level, 142 detected gene probes were differentially
expressed in HoLPL and 67 in HeLPL compared to wild type LPL (Table 2). Of the
identified probes, 29 are shared by HoLPL and HeLPL and 48 are specific to HoLPL
(Fig. 1). Most of the HoLPL specific annotated biomarkers are involved in
inflammatory, immune, circadian or signalling pathways, docking systems or receptormediated clearance mechanisms (Figures 2 and 3).
Comparisons
Nb of
probes a at
|FC| ≥ 2
HoLPL vs Wild type LPL
5,462
142
HeLPL vs Wild type LPL
5,323
67
0.3
5.3
HeLPLvsNormal
-log(p-value)
HoLPLvsNormal
HeLPL = Heterozygous LPL; HoLPL = Homozygous LPL; LPL = Lipoprotein Lipase; FC = Fold change;
FDR = False discovery rate.
a From Affymetrix® Human Gene 2.0 ST microarrays (on a total of 48,226 detected probes).
GranulocyteAdhesionandDiapedesis
ComplementSystem
AgranulocyteAdhesionandDiapedesis
GlycogenDegrada=onIII
IL-8Signaling
Oncosta=nMSignaling
FcEpsilonRISignaling
Inhibi=onofMatrixMetalloproteases
GαiSignaling
SalvagePathwaysofPyrimidineDeosyribonucleo=des
PPARα/RXRαAc=va=on
NADBiosynthesisIII
α-tocopherolDegrada=on
Roleofmacrophages,FibroblastsandEndothelialCells
AcetateConversiontoAcetyl-CoA
GlycerolDegrada=onI
IL-10Signaling
LPS/IL-1MediatedInhibi=onofRXRFunc=on
RoleofOsteoblasts,OsteoclastsandChondrocytes
cAMP-mediatedSignaling
PPARSignaling
G-ProteinCoupledReceptorSignaling
FaQyAcidAc=va=on
P38MAPKSignaling
Oxida=veEthanolDegrada=onIII
IL-6Signaling
LXR/RXRAc=va=on
MitochondrialL-carni=neShuQlePathway
γ-linolenateBiosynthesisII
EthanolDegrada=onIV
Figure 2: IPA® top canonical pathway analyses. Heatmap presents a
comparison of HoLPL and HeLPL vs normal LPL.
HeLPL
(n = 20)
56.6 (1.8)
13:7
30.0 (0.8) 1
7 (30.0)
7 (38.9)
8.7 (2.9) 1
7.2 (1.2) 1
1.4 (0.2) 1
0.8 (0.0) 1
6.1 (0.2)
4 (20.0)
14 (70.0) 1
8 (40.0) 1
9 (45.0)
4 (20.0)
HoLPL
(n = 19)
46.1 (3.2)
7:12
22.2 (1.0) 2
6 (31.6)
14 (73.7)
21.6 (2.6) 1, 2
7.5 (0.9) 1
1.3 (0.1) 1
0.4 (0.0) 1, 2
5.3 (0.2) 2
19 (100.0) 1, 2
2 (10.5) 2
4 (21.0)
6 (31.6)
4 (21.1)
p-value c
0.063
NS
<0.001
0.061
0.088
<0.001
0.006
0.001
<0.001
0.017
<0.001
<0.001
0.023
NS
NS
Data are mean (SE), unless otherwise stated. LPL = Lipoprotein Lipase; HDL = High-Density Lipoprotein; HeLPL (Heterozygous) HoLPL (Homozygous) loss-offunction LPL gene mutations carrier; LDL = Low-Density Lipoprotein.
a Geometric means and p-values obtained on log -transformed data. b Post hoc analyses performed using a C Dunnett significance level due to the inegal variance
10
c
of the distribution. For continuous parameters: One-way ANOVA followed by Bonferroni adjusted post hoc tests. All significant p-values (p < 0.05) remain
significant when using non-parametric Kruskal-Wallis analyses followed by Mann-Whitney T-tests. For dichotomous parameters: Pearson Chi-square followed by
Bonferroni-adjusted Z-tests or Exact Fischer Chi-Square. Significantly difference from 1 Normal LPL activity or 2 Defective LPL activity.
NS: p ≥ 0.1
HoLPL
Up !
Down "
44 !
98 "
30 !
37 "
Wild type LPL
(n = 14)
52.8 (3.2)
7:7
24.9 (0.9)
0 (0.0)
9 (64.3)
1.1 (0.1)
4.5 (0.2)
2.6 (0.2)
1.3 (0.1)
4.9 (0.3)
0 (0.0)
2 (14.3)
0 (0.0)
2 (14.3)
0 (0.0)
Age, years
Men:Women
Body Mass Index (BMI), Kg/m2
Alcohol abstinence, n (%)
Ex or current smoker, n (%)
Total triglycerides, mmol/L a
Total cholesterol, mmol/L a, b
LDL-cholesterol, mmol/L
HDL-cholesterol, mmol/L
Glucose, mmol/L a
Pancreatitis, n (%)
Obesity, n (%)
Diabetes, n (%)
Arterial hypertension, n (%)
Cardiovascular disease, n (%)
Table 2: Differential expression analysis results
Nb of probes a at
p < 0.01 and 5%
FDR
Results – Cont’d
Table 1: Characteristics of study participants
Common
HeLPL
ACSL1
ADGRG3
ALPL
ANXA3
BACH1
BASP1
CASP5
CEACAM8
CPD
CSF2RB
CXCR1
CXCR2
CYP4F3
DDX11L2
DOCK4
DOCK5
FAM129A
FCER1A
FFAR2
FPR2
GK
GZMK
HCAR2
HIST1H3H
IGKC
IL1R2
IL1RAP
KCNJ15
LINC00189
LOC729040
LOC100507639
LOC101927851
LOC102724190
LUCAT1
MME
NAMPT
NOP56
REPS2
RPL27
SLED1
C5AR1
CHI3L1
CPA3
DEFA1
GAS5
LINC00694
LRG1
MANSC1
MGAM
MGAM2
MIR4802
MMP25
MS4A2
MS4A3
PI3
RBMX
RNASE3
RPL23A
SERPING1
SNHG1
SNHG16
SNHG6
SNORD116-1
SNORD116-15
CDA
CLEC4E
HIST1H4L
LOC100507006
LOC102723340
METTL12
SIGLEC5
SNORD41
SNORD54
SNORD60
SNORD82
SNORD119
TECPR2
TRPM6
WDFY3
SNORD116-24
SNORD44
SNORD59A
SNORD71
SNRPN
Figure 1: Differentially expressed biomarkers among HoLPL and HeLPL (|FC| ≥ 2). Adaptation
of Venn diagram obtained from IPA® comparisons analyses: 29 biomarkers are shared by HoLPL
and HeLPL, 48 are specific to HoLPL and 7 are specific to HeLPL.
Whitebloodcellsgeneexpression
Legend
?
Pancreas
Down-regulatedinHoLPL
IL-8
IL-1
?
1
2
Down-regulatedinHoLPL
Deficiency
IL1RAP/IL1R2
?
CXCR1/CR2
SuspectedrelaPonship
Complementary functional analyses were performed in
key pathways involving differentially expressed genes.
Functional analyses revealed significant differences in
chylomicron, and HDL particles characteristics (e.g.:
oxydation markers or ApoC-III levels) in HoLPL vs
HeLPL with CM. The glycerol kinase (GK) pathway
was down-regulated in HoLPL compared to HeLPL
and controls, and plasma concentration of free glycerol
and markers of lipolysis tended to be lower in HoLPL.
Plasma concentration of pro-inflammatory cytokines
IL-1β and IL-8 were significantly higher in HoLPL
compared to HeLPL (data not shown), which is
consistent with the observed down-regulation of IL-8
(CXCR1/CR2) and IL-1 (IL1R2/IL1RAP) receptor
genes.
Conclusion
These results highlight that gene expression profiling,
combined with functional analyses may contribute to
identify key mechanisms involved in LPL independent
pathways of TG-rich lipoproteins management.
Exploring genetic and functional differences between
HoLPL and HeLPL with CM may also contribute to
reveal or further understand key mechanisms
differentiating the clinical risk trajectory of LPLD vs
other CM sub-phenotypes (e.g.: pancreatitis,
cardiovascular or cardiometabolic risk). This requires a
systems approach. Genetic, epigenetic and functional
investigations are ongoing.
References
[1] Monsalve et al. 1990. J Clin Invest, 86:728-734;
[2] Ma et al. 1991. NEJM, 324:1671-1766;
[3] Mattei et al. 1993. Cytogenet Cell Genet, 63:45-46;
[4] Brunzell and Deeb. 2001. The Metabolic and
Molecular Bases of Inherited Disease, 8Edn,
McGraw-Hill: 2789-2816;
[5] Gaudet et al. 2014. NEJM, 371:2200-2206;
[6] Irizarry et al. 2003. Biostatistics, 4(2); 249-264;
[7] Benjamini and Hochberg. 1995. J Roy Statist Soc
Ser B, 57:289-300.
Acknowledgements
We are thankful to all participants and ECOGENE-21
staff. Microarrays were performed at the Genome
Quebec and McGill University Innovation Center
(Montréal, QC, Canada). Authors have no conflict of
interest to disclose. This study was supported by
ECOGENE-21, a non-for-profit research organization.
3
JNK
Identified pathways
NFkB
?
Contact information:
Daniel Gaudet, MD, PhD
daniel.gaudet@umontreal.ca
1 Inflammation
2
3
4
Liver
Immune
response
RXRα-LXR
3
RXRα-FXR
Cell
signaling
Oxydative
stress
Whitebloodcells
geneexpression
APOC3
Chylomicron
(CM)
VLDL
APOC3
GK ACSL1
4
6
5 Circadian clock
(NAMPT
pathway)
LPL
metabolism
and clearance
7
LPL
FFA
6 Energy
7 LPL metabolism
6
?
?
adipocytes
Muscle cells
Figure 3: Reductionist representation of some IPA® canonical pathways possibly differentiating
severe hyper TG and CM in total absence of LPL activity (HoLPL), when LPL activity is
available but limited (HeLPL) and in normolipidemic controls. Blood GK and interleukins
(IL)-1β and IL-8 receptors genes were down-regulated in HoLPL compared to HeLPL and
controls. Consistently, this was associated with lower free glycerol and higher IL-1β and IL-8
plasma concentration, respectively (data not shown).