Arteriosclerosis, Thrombosis, and Vascular Biology

BASIC SCIENCES

Functional Characterization of LIPA (Lysosomal

Acid Lipase) Variants Associated With Coronary
Artery Disease
Trent D. Evans, Xiangyu Zhang, Reece E. Clark, Arturo Alisio, Eric Song, Hanrui Zhang, Muredach P. Reilly,
Nathan O. Stitziel, Babak Razani

OBJECTIVE: LIPA (lysosomal acid lipase) mediates cholesteryl ester hydrolysis, and patients with rare loss-of-function
mutations develop hypercholesterolemia and severe disease. Genome-wide association studies of coronary artery disease
have identified several tightly linked, common intronic risk variants in LIPA which unexpectedly associate with increased
mRNA expression. However, an exonic variant (rs1051338 resulting in T16P) in linkage with intronic variants lies in the
signal peptide region and putatively disrupts trafficking. We sought to functionally investigate the net impact of this locus on
LIPA and whether rs1051338 could disrupt LIPA processing and function to explain coronary artery disease risk.

APPROACH AND RESULTS: In monocytes isolated from a large cohort of healthy individuals, we demonstrate both exonic and
intronic risk variants are associated with increased LIPA enzyme activity coincident with the increased transcript levels. To
functionally isolate the impact of rs1051338, we studied several in vitro overexpression systems and consistently observed
no differences in LIPA expression, processing, activity, or secretion. Further, we characterized a second common exonic
coding variant (rs1051339), which is predicted to alter LIPA signal peptide cleavage similarly to rs1051338, yet is not linked
to intronic variants. rs1051339 also does not impact LIPA function in vitro and confers no coronary artery disease risk.
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CONCLUSIONS: Our findings show that common LIPA exonic variants in the signal peptide are of minimal functional significance
and suggest coronary artery disease risk is instead associated with increased LIPA function linked to intronic variants.
Understanding the mechanisms and cell-specific contexts of LIPA function in the plaque is necessary to understand its
association with cardiovascular risk.

VISUAL OVERVIEW: An online visual overview is available for this article.

Key Words: atherosclerosis ◼ coronary artery disease ◼ human genetics ◼ monocytes ◼ risk

A
therosclerotic plaque progression is the underly- LIPA, encoding for the cholesterol-ester and triglycer-
ing cause of the majority of cardiovascular dis- ide hydrolase lysosomal acid lipase stands out as an
eases including myocardial infarction and stroke, intriguing lead.1–3 In the atherosclerotic macrophage,
leading to tremendous morbidity and mortality world- LIPA (lysosomal acid lipase) contributes substantially to
wide. Understanding the pathophysiology of plaque lipid metabolism to modulate fundamental pathogenic
formation and progression remains an important area hallmarks of atherosclerosis including foam cell forma-
of investigation both scientifically and clinically, serv- tion, inflammatory signaling, and overall plaque pro-
ing as the basis for future therapeutics. One avenue gression.4,5 Indeed, individuals with complete functional
of insight is mechanistic characterization of genetic loss of LIPA develop Wolman disease, characterized by
risk loci identified in GWAS (genome-wide association failure to thrive, hepatosplenomegaly, hyperlipidemia,
studies) of atherosclerosis. Among identified risk loci, and typically infantile death.6 Cholesterol ester storage
 
Correspondence to: Babak Razani, MD, PhD, Washington University, Campus Box 8086, 660 S. Euclid Ave, St. Louis, MO 63110. Email brazani@im.wustl.edu
The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.119.313443.
For Sources of Funding and Disclosures, see page 2490.
© 2019 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb

2480   December 2019 Arterioscler Thromb Vasc Biol. 2019;39:2480–2491. DOI: 10.1161/ATVBAHA.119.313443
 Evans et al
Nonstandard Abbreviations and Acronyms
CAD coronary artery disease
eQTL expression quantitative trait loci
GWAS genome-wide association study
LD linkage disequilibrium
LIPA lysosomal acid lipase
PBMC peripheral blood mononuclear cell
SNP single nucleotide polymorphism
disease is a related disorder with a milder presentation
and is caused by mutations in LIPA leading to subto-
tal loss of function.6,7 These findings, along with similar
observations in animal models with LIPA-deficiency,8
lead to the conclusion that loss of LIPA causes hyper-
cholesterolemia, plaque macrophage dysfunction, and
atherosclerosis.
Therefore, the identification of 2 tightly linked common
variants in LIPA (rs1412444 and rs2246833, ≈30%
allele frequency) strongly associated with an increased
risk for coronary artery disease (CAD) was initially not
surprising. However, these 2 lead variants are intronic and
not associated with lipid levels in the population, suggest-
ing a mechanism independent of hypercholesterolemia.1,2
Additionally, these variants are associated with increased
LIPA mRNA levels in circulating monocytes which
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seemed contrary to the expectation that loss of function
would be atherogenic. Interestingly, in the same linkage
disequilibrium (LD) block, several groups have noted the
existence of a coding polymorphism, rs1051338, result-
ing in T16P with similar prevalence and increased risk
for CAD.9–11 rs1051338 results in a shift from a polar
(Threonine) to nonpolar (Proline) amino acid in the signal
peptide sequence, a region that is characteristically non-
polar and crucial for appropriate LIPA maturation and traf-
ficking.12,13 Therefore, the possibility that this substitution
could adversely impact enzyme trafficking, secretion, and
overall expression presented an attractive explanation for
the association of these LIPA variants with CAD. A recent
study by Morris et al9 proposed that T16P was the caus-
ative variant amounting to a reduction in LIPA expression
and trafficking to lysosomes.
To understand the effects of a LIPA variant on enzyme
function, it is crucial to appropriately understand and
evaluate LIPA trafficking, which is typical of enzymes of
the lysosomal lumen. Following signal peptide cleavage in
the ER, nascent LIPA in the ER/golgi exists as a 56-kDa
propeptide, which can be secreted or routed to the lyso-
some via the mannose-6-phosphate receptor trafficking
system.12 In the lysosome, acidity and proteases result in
propeptide cleavage to yield a ≈41-kDa mature, active
peptide.12–15 Therefore, the relative expression of these
forms carries a great deal of information about subcellular
distribution and processing, and both forms of LIPA can
Arterioscler Thromb Vasc Biol. 2019;39:2480–2491. DOI: 10.1161/ATVBAHA.119.313443
LIPA Variants and CAD
Highlights
Basic Sciences - AL
• LIPA risk SNPs previously identified as conferring
risk for coronary artery disease are associated with
increased LIPA (lysosomal acid lipase) enzyme
expression and activity.
• The sole exonic SNP in this linkage disequilibrum
block (rs1051338 resulting in T16P) does not
impair LIPA expression, activity, lysosomal traffick-
ing, or secretion.
• A functionally similar exonic SNP (rs1051339
resulting in G23R) also does not impact LIPA func-
tion, is not in linkage with risk variants, and does not
associate with coronary artery disease.
• Intronic variants likely drive increased LIPA expres-
sion and highlight the need to explore how increased
LIPA may be pathogenic in this context.
be observed in human monocyte derived macrophages16.
Morris et al9 had evaluated the impact of rs1051338
on LIPA function using a COS7 transient overexpres-
sion model and monocytes/macrophages from human
subjects, concluding that LIPA T16P interferes with
lysosomal trafficking to lead to a net loss of function for
the locus. However, analyses in the Morris study were
restricted to a single LIPA band of unspecified molecular
mass making interpretation of many experiments prob-
lematic regardless of which band was analyzed. Further,
monocyte/macrophage analyses therein were restricted
to n=3 to 4 individuals with the nonrisk versus risk exonic
genotypes, which is highly underpowered to assess typi-
cally smaller effect sizes associated with single nucleo-
tide polymorphisms (SNPs), especially given the already
known effect of this locus on increasing LIPA mRNA.1,2,17
Therefore, in the present study, we sought to evaluate
the functional impact of LIPA CAD risk variants on LIPA
function in monocytes isolated from a sufficiently large
patient cohort (n=114). Further, to isolate the functional
impacts of the exonic variant without the confounding
influence of intronic variants, we studied its impact in
vitro on expression, maturation, and secretion taking into
consideration the discussed relevance of LIPA forms
and subcellular distribution in multiple systems. Last, we
characterize a similar exonic variant (rs1051339) previ-
ously speculated to associate with Wolman’s disease18
that results in the amino acid change G23R with rele-
vance to which SNPs may be causative in the LIPA CAD
risk LD block.
MATERIALS AND METHODS
Data Availability
The authors declare that all supporting data are available within
the article and its online-only Data Supplement.
December 2019   2481
 Evans et al
Subject Recruitment and Human Monocyte
Basic Sciences - AL
Isolation
All protocols were approved by the Washington University in St.
Louis Institutional Review Board. Healthy, young to middle aged
subjects (n=32 male and 82 female) with no known history of
CAD/MI were recruited from the St. Louis Area. Whole blood
was collected from 32 male and 82 female subjects in tubes
containing EDTA (BD Vacutainer). Peripheral blood mononu-
clear cells were isolated via centrifugation with Ficoll-Paque
PLUS (GE Healthcare). A portion of peripheral blood mononu-
clear cells was saved for DNA extraction and SNP genotyping.
CD14+ monocytes were isolated using the EasySep Human
Monocyte Enrichment Kit (19059, StemCell Technologies).
DNA Extraction and SNP Genotyping
DNA was extracted from peripheral blood mononuclear cells
samples using the DNeasy Tissue and Blood DNA Extraction kit
(Qiagen #69506). PCR-based SNP genotyping was performed
using Taqman Human SNP genotyping assays (ThermoFisher
#4351379) for rs1051338 (Assay ID: C___8870360_20),
rs1412444 (Assay ID: C___8870364_10), and rs2246833
(Assay ID: C___2734272_10). For analyses of human monocyte
mRNA and activity segregated by exonic genotype (Figure 1C
and 1D), individuals with dissociated exonic/intronic genotypes
were analyzed separately (Figure IA and IB in the online-only
Data Supplement) because we observed an independent effect
of the intronic variant which obscures the relationship between
the exonic variant and mRNA/activity in these individuals.
RT-qPCR and mRNA Analyses
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Total RNA was extracted using the Ambion Purelink RNA Kit
(Invitrogen) and reverse transcribed using the Superscript Vilo
cDNA synthesis kit (Invitrogen). qPCR was performed using a
ViiA-7 RT-PCR system and SYBR-SelectMaster Mix (Applied
Biosystems). Prevalidated primers were used for amplifica-
tion (Table I in the online-only Data Supplement). Assays were
performed in duplicate and normalized to ribosomal protein
36B4 mRNA levels (in vitro studies) or β-actin (human mono-
cyte studies). LIPA mRNA (Transcripts per Kilobase Million) in
human monocyte-derived macrophages was analyzed using
the publicly available RNA-seq Immunpop Dataset19 available
at http://www.immunpop.com
Lysosomal Acid Lipase Activity Assay
LIPA activity was determined using the fluorogenic substrate
4-methylumbelliferone oleate as previously described.20 Briefly,
10 μL protein lysates (≈1 μg/μL) or conditioned media were
added to 150 μL, 200 μM Sodium Acetate Buffer, pH 5.5, and
preincubated for 10 minutes in the presence or absence (con-
trol vehicle DMSO) of Lalistat-2 (final concentration 10 μM)
a kind gift from Dr Paul Helquist, University of Notre Dame).
4-methylumbelliferone oleate was reconstituted in DMSO
(100 mg/mL) and diluted 1:100 in 4% Triton X-100, and 50
μL added to the assay. Samples were incubated 30 minutes at
37°C, reaction halted with 1 M Tris pH 8 (100 μL), and fluo-
rescence intensity was measured using a fluorometer (excita-
tion 360 nm/emission 460 nm). Values were calculated as the
difference between each sample incubated with and without
2482   December 2019 Arterioscler Thromb Vasc Biol. 2019;39:2480–2491. DOI: 10.1161/ATVBAHA.119.313443
LIPA Variants and CAD
Lalistat-2. Activity values were normalized to exact lysate pro-
tein concentration.
Regulatory Potential Analyses of LIPA SNPs
For regulatory potential analyses of intronic LIPA SNPs, can-
didate-associated SNPs were included according to those in
LD (R2>0.8) with lead SNP rs1412444 in both European and
South Asian populations (separately) according to data from
phase 3 (version 5) of the 1000 Genomes project using the
National Cancer Institute LDlink tool available at https://ldlink.
nci.nih.gov/. Candidate variants were further parsed by exclusion
of variants with low predicted regulatory potential (RegulomeDb
score <4). A description of the meanings of scores (scaled 1–7
where 1a indicates the highest potential for regulatory influ-
ence) can be found at http://www.regulomedb.org/help#score.
SignalP Analyses
All analyses were performed using the SignalP v4.1 tool avail-
able at http://www.cbs.dtu.dk/services/SignalP/. Signal peptide
S-Score values indicate which regions are likely to be included
as part of the signal peptide, and Y-score values indicating the
most probable cleavage sites based on amino acid properties
were obtained through using the long output format with default
D cutoffs and the no transmembrane region settings.
Transient Transfections and Lentiviral
Transductions
Transient transfection vectors containing LIPA variants with a
C-terminus FLAG tag were transfected into HEK-293T cells
with Lipofectamine 3000 (ThermoFisher L3000008). After 12
hours, media was changed to normal media and analyses per-
formed 48-hour post-transfection.
Lentiviral vectors expressing LIPA or empty vector control were
packaged in HEK-293T cells and target 293T, 3T3, and J774 cells
were transduced. Cells were selected in puromycin (4 μg/mL) until
all control nontransduced cells were dead and cells were grown in
normal media for >48 hours before experiments. Two pooled popu-
lations for each variant were matched for mRNA expression and
equivalent comparison. Data for stable transduction experiments
are expressed versus each respective LIPA 16T control. All protein
lysates used for Western Blotting and activity assays, conditioned
media, and mRNA was collected from the same wells.
LIPA Stability and Turnover Assays
Cells were transfected with LIPA T16 or P16 variants. Twenty-
four hour post-transfection, untreated cells were collected. For 6
hours, cells were either left untreated or administered cyclohexi-
mide (100 µg/mL), bortezimib (10 nmol/L), or both as indicated.
Cholesterol Efflux and Ac-LDL Loading
Cholesterol efflux was assessed using the BODIPY (boron-
dipyrromethene)-cholesterol approach as previously described.21–23
Briefly, J774 cells were loaded with BODIPY-Cholesterol labeled
Ac-LDL (acetylated low-density lipoprotein; final concentration 50
µg/mL, Kalen Biomedical 770201-7) in full media for 12 hours
and equilibrated in DMEM+0.2% BSA for 4 hours. Cells were fur-
ther incubated for 6 hours in control (0.2% BSA) or efflux (5%
 Evans et al LIPA Variants and CAD

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Figure 1. LIPA SNP variants associate with higher human monocyte mRNA and activity.
A, Intron-exon map of LIPA highlighting an exonic variant (rs1051338) and the lead intronic variant (rs1412444) associated with coronary
artery disease (CAD). B, SNP alleles, minor allele frequencies, and associations with CAD in the CardioGRAM study. C, LIPA (lysosomal
acid lipase) mRNA expression in human monocytes in individuals with given genotype (n=38, 37, 8). D, LIPA enzymatic activity in human
monocytes in individuals with given genotype (n=44, 46, 9). E, Western Blot of LIPA expression in individuals with given genotypes (n=10, 8).
F, Quantification of LIPA expression normalized to Ponceau loading control. Data are mean±SEM. *Indicates statistical significance using linear
regression (C and D) or Student unpaired t test (F).

FBS+0.2% BSA) media over 6 hours. Cells were rinsed in PBS, Free and esterified cholesterol were measured using a com-
detached, fixed, and analyzed using flow cytometry (BD LSR-II mercially available kit (Thermo Fisher A12216) and standard
and FlowJo) measuring BODIPY-Cholesterol Signal (median fluo- protocol24 with or without acetylated low-density lipoprotein
rescent intensity) in at least 10 000 cells. loading (50 µg/mL, Kalen Biomedical 770201-7) for 12 hours.

Arterioscler Thromb Vasc Biol. 2019;39:2480–2491. DOI: 10.1161/ATVBAHA.119.313443 December 2019   2483
 Evans et al
Western Blotting
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Cells were lysed in RIPA buffer and protein concentration
assessed using BCA Protein Assay Kit (Pierce, Cat# 23225).
Equal amounts of protein were separated by SDS-PAGE
and transferred onto nitrocellulose membranes. LIPA Protein
expression was detected using the appropriate primary anti-
body: Human LIPA (OriGene, Cat# TA309730, 1:1000) and
corresponding secondary antibody (1:4000). Signals were
visualized by SuperSignal West Pico PLUS Chemiluminescent
Substrate (Thermo Fisher Scientific, Cat# 34577) or LiCOR
IRdye secondary fluorescent antibodies to allow detection of
FLAG/LIPA within the same blot, and imaged using either a
Biorad Chemidoc MP, LiCor Odyssey Classic system, or via
film exposure. Band intensities were quantified relative to
Ponceau-S total protein staining used as a loading control.
Immunofluorescence Analyses
Cells were fixed with 4% paraformaldehyde, blocked and per-
meabilized (1% BSA, 0.2% milk powder, 0.3% Triton X-100
in TBS; pH 7.4), and incubated with antibodies sequentially.
Specificity of staining was tested in control experiments by omit-
ting primary antibodies. The following primary antibodies were
used in 1:250 dilutions: LIPA (Origene Cat# TA309730) and
Lamp-1 (Santa Cruz Cat# sc-20011). Species-specific fluores-
cent secondary antibodies were obtained from Invitrogen/Life
Technologies (1:250). A Zeiss LSM-700 confocal microscope
was used for image acquisition and images quantified using
ZEN microscope software (Carl Zeiss AG). At least 50 cells per
group spanning 3 separately stained coverslips were imaged
and analyzed. For each analyzed cell, LIPA foci were analyzed
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by a blinded observer for colocalization to lysosomes (Lamp1+)
and scored as the % of Lamp1+LIPA foci for each cell.
Lysosomal Inhibitor Treatment
Cells were transiently transfected with the LIPA 16T variant as
described. Thirty-six hour post-transfection, cells were treated
with the lysosomal inhibitors Bafilomycin (50 nmol/L) or chlo-
roquine (50 µM) for 12 hours to demonstrate inhibition of LIPA
propeptide cleavage in the lysosome.
Statistics
Results are expressed as mean±SEM. Data were examined
for non-normality using the Shapiro-Wilk test. Comparisons
between 2 groups were performed using 2-tailed Student t
test or Wilcoxon signed rank test for parametric and nonpara-
metrically distributed data, respectively. Comparisons among
3 genotypes were performed with linear regression. P <0.05
were considered to indicate statistically significant differences.
RESULTS
Intronic and Exonic LIPA Risk Alleles Are
Associated With Increased Monocyte LIPA mRNA
Expression and Enzyme Activity
GWASs for coronary artery disease have reproducibly
identified LIPA rs1412444 and rs2246833 as lead
SNPs out of many in moderate to tight LD at the LIPA
risk locus. However, nearly all of these risk variants
2484   December 2019 Arterioscler Thromb Vasc Biol. 2019;39:2480–2491. DOI: 10.1161/ATVBAHA.119.313443
LIPA Variants and CAD
are intronic and perplexingly associated with higher
LIPA mRNA expression, contrary to what would be
expected based on the many known pathologies associ-
ated with loss of LIPA function.6,8 Using data from the
CardioGRAMplusC4D GWAS of CAD,11 we and oth-
ers had identified a sole exonic variant in this LD block,
rs1051338, as a candidate for explaining the pathoge-
nicity of this locus. rs1051338 is in fairly strong LD with
rs1412444 (R2=0.859 in all individuals), and here we
focus on rs1412444 as a representative surrogate for
many tightly linked intronic variants including rs2246833.
Our analyses reveal rs1051338 is associated with CAD
to a degree similar to rs1412444 in pooled data from
the CardioGRAM meta-analysis. Genomic locations, risk
alleles, minor allele frequencies, and association with
CAD are presented in Figure 1A and 1B.
We sought to study whether rs1051338 could nega-
tively impact LIPA function in humans, potentially explain-
ing CAD risk. To this end, we isolated and studied CD14+
classical monocytes as a highly disease-relevant cell
type from a large cohort of healthy young to middle-aged
individuals with no known history of cardiovascular dis-
ease. First, rs1051338 is dose-dependently associated
with higher human monocyte LIPA mRNA expression
(Figure 1C), consistent with the known cis-expression
quantitative trait loci (eQTL) effect at this locus.1,2,17 Con-
trary to any possibility of overt loss of function, we dem-
onstrate for the first time that the risk exonic genotype
also dose-dependently associates with a gain of function
in monocyte LIPA enzyme activity (Figure 1D). Comple-
mentary to this data, individuals with homozygous risk
versus nonrisk alleles have significantly increased LIPA
expression (Figure 1E, quantified in Figure 1F) of simi-
lar magnitude to the increase in activity. This suggests
that any subtle effects of LIPA risk SNPs in this locus
on overall protein translation or degradation are largely
trumped by the eQTL effect, which directly translates to
a straightforward increase in enzyme activity.
Dissecting which variants, in isolation or in combina-
tion, may be responsible for these factors is a challenge
in any functional genetics study. In addition, it has been
proposed that the observed mRNA increase could be
compensating for a loss of enzyme function caused by
rs1051338. However, our data constitute strong evi-
dence that rs1412444 or closely associated intronic
variants are instead responsible for the eQTL effect.
Among all individuals homozygous for the major (non-
risk) allele for rs1051338 (the exonic SNP), those with
a dissociated (heterozygous) genotype for rs1412444
(the intronic SNP) displayed significantly higher LIPA
mRNA and activity versus the major homozygous individ-
uals for rs1412444 (Figure IA and IB in the online-only
Data Supplement). RegulomeDb, a tool that identifies
DNA features and regulatory element alterations by
SNPs, suggests that multiple intronic SNPs in high LD
with the GWAS lead SNP rs1412444 are likely to be
 Evans et al LIPA Variants and CAD

regulatory (Figure IC in the online-only Data Supple- Second, T16P results in a shift from a polar amino acid
ment), providing premise for future functional genomic (Thr) to a nonpolar one (Pro) in a region which is char-

interrogation of the causal SNPs underlying the GWAS
and eQTL association.25 Last, LIPA expression is known
to be induced on monocyte-to-macrophage differen-
tiation,16 potentially lessening the relative impact of this
locus on increasing expression in macrophages. How-
ever, using publicly available RNA-seq data, we observe
the intronic variant rs1412444 is associated with simi-
larly increased LIPA mRNA levels in human monocyte-
derived macrophages.19
Exonic LIPA Risk SNP rs1051338 Does Not Alter
LIPA Enzyme Trafficking or Function in Isolation
Out of many SNPs in the LIPA risk locus for CAD,
rs1051338 stood out as a strong candidate for explain-
ing pathogenicity. rs1051338 causes a coding amino
acid change from threonine to proline at amino-acid
16 (T16P). Several aspects of this change pointed to
a possibility for disruption of enzyme function or traf-
ficking, which may not be reflected in our whole mono-
cyte enzyme activity analyses. First, T16P lies in the
signal peptide domain of LIPA. This region is crucial for
proper sorting and trafficking to LIPA’s main subcellular
destinations, the secretory pathway and the lysosome.
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acteristically nonpolar.
To experimentally assess the isolated impact of
rs1051338/T16P on LIPA maturation and trafficking, we
studied a transfection model with overexpression of either
LIPA variant with a C-Terminus FLAG tag. We observed
no absolute or relative differences in proLIPA (ER/Golgi,
≈56 kDa), mature LIPA (Lysosomal, ≈41 kDA), or FLAG
expression (Figure 2A, quantified in Figure 2B), in cells
well matched for degree of mRNA expression (Figure
IIA in the online-only Data Supplement). Whole cell LIPA
activity (Figure 2C) and LIPA secretion as assessed by
LIPA activity in supernatant media (Figure 2D) were also
unaffected. We further confirmed the dependence of
LIPA propeptide cleavage on lysosomal hydrolytic activity
by treating cells with the lysosomal inhibitors bafilomy-
cin and chloroquine, demonstrating impaired LIPA pro-
peptide cleavage with either treatment (Figure IIB in the
online-only Data Supplement).
If the risk variant were to have reduced stability or
mistrafficking, pulse-chase assays could reveal such a
defect. We assessed the stability, turnover, and degrada-
tion of LIPA variants using a cycloheximide pulse-chase
assay (Figure 2E). We also applied a proteasome inhibitor,

Figure 2. rs1051338 does not impact LIPA (lysosomal acid lipase) in transfected cells.
A, Western Blot of HEK-293T cells transduced with either FLAG-tagged LIPA variant. Each lane represents a transfection replicate with a
single representative experiment. B, Quantified densitometry of LIPA expression normalized to loading control. C, LIPA activity in whole-cell
lysates. D, LIPA secretion measured in conditioned media. E, Pulse-chase analyses of LIPA stability in exonic variants using cycloheximide
(CHX) and/or bortezimib (Bort.) where indicated. All parameters (expression, activity, secretion, mRNA) are measured within the same well and
are representative of 3 or more independent experiments performed on separate occasions. Data are mean±SEM.

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 Evans et al
bortezimib, to assess whether any mistrafficked LIPA
would be subject to proteasomal degradation. At 6 hours,
Basic Sciences - AL
cycloheximide treatment nearly completely abolished pro-
peptide expression similarly in either variant as expected
given this is the nascent form that is rapidly trafficked
to the lysosome. Mature LIPA expression was modestly
reduced with cycloheximide but clearly had a longer half-
life than the propeptide and was preserved likely due to
existing propeptide maturation that balanced turnover.
Overall, T16P did not result in lower LIPA stability. Addi-
tionally, the addition of bortezimib did not preserve LIPA
stability in the absence or presence of cycloheximide in
either variant, indicating LIPA is not subject to cytosolic
mistrafficking or proteasomal degradation (Figure 2E).
A potential limitation of transfection models is the
high degree of overexpression, which could saturate
pathways involved in LIPA trafficking and mask modest
impairments caused by variants like T16P. Typical trans-
fection experiments showed a somewhat favored relative
accumulation of pro-LIPA versus empty vector trans-
fected control (Figure IIC in the online-only Data Supple-
ment). Despite obvious increases in both pro-LIPA and
LIPA in overexpression models, the C terminus FLAG tag
only marked pro-LIPA (Figure IA and IIC in the online-
only Data Supplement), likely due to instability of FLAG
tags in the lysosome.
To study T16P in a system using a more modest
degree of overexpression and confirm findings suggest-
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ing minimal functional impacts, we generated HEK-293T
and NIH-3T3 cell lines stably overexpressing either vari-
ant in 2 cell types. In this system, fractionally more over-
expressed LIPA was trafficked to the lysosome but still no
differences with T16P were observed in either pro-LIPA
or LIPA expression in either cell type (Figure 3A, quanti-
fied in Figure 3B). No difference in cellular LIPA activity
(Figure 3C) or secretion (Figure 3D was observed, and
cell lines were well matched for degree of overexpres-
sion (mRNA, Figure IIIA in the online-only Data Supple-
ment). Last, immunofluorescent colocalization analyses
revealed the vast majority of LIPA foci to be colocalized
to lysosomes (Lamp1+) with either variant (Figure IIIB
and IIIC in the online-only Data Supplement)
Exonic SNP rs1051339 Disrupts the LIPA Signal
Peptide but Is Not Functionally Significant
A key challenge in identifying causative variants within
genomic risk loci is the frequently strong linkage dis-
equilibrium among variants. As an independent test of
whether a signal peptide variant like T16P could disrupt
LIPA function or associate with CAD independent of link-
age with intronic variants, we identified the nearby SNP
rs1051339 as a functionally similar variant meeting
these criteria. This SNP is exonic and also causes a func-
tional shift from glycine (nonpolar) to arginine (charged,
polar) at amino acid 23 (G23R), and several aspects of
this SNP are informative in relation to the LIPA risk locus
2486   December 2019 Arterioscler Thromb Vasc Biol. 2019;39:2480–2491. DOI: 10.1161/ATVBAHA.119.313443
LIPA Variants and CAD
for CAD and evaluating causality of rs1051338/T16P.
Further, rs1051339 has been found in compound homo-
zygosity with other detrimental mutations in patients with
Wolman’s disease, and it has been suggested that G23R
(annotated therein as G5R) results in major defects in
LIPA secretion.18
We first used SignalP, an in silico computational tool
to contrast the functional impacts of T16P and G23R.
The shift in S-score, an index of likelihood of an amino
acid residue’s inclusion in the signal peptide, was nearly
identical between these 2 variants (Figure 4A). Similarly,
the Y-score, an index taking into account local amino
acid properties to predict cleavage site, suggested T16P
and G23R both shift the cleavage site identically (Fig-
ure 4A). We also sought to test the functional impacts of
G23R experimentally using a similar LIPA-FLAG over-
expression system. Like T16P, G23R had no impact on
LIPA expression or trafficking as assessed by Western
Blot expression of proLIPA (ER/golgi localized), FLAG,
and mature (lysosomal) LIPA (Figure 4B, quantified in
Figure 4C). G23R also did not impact enzyme activity
(Figure 4D) or secretion (Figure 4E), in cells that were
well matched for degree of expression (Figure IVA in
the online-only Data Supplement). We also analyzed the
CardioGRAM GWAS study for CAD risk and found that
rs1051339 is common (maf=0.116), yet not in linkage
disequilibrium with rs1051338 or other intronic SNPs
(rs1412444) at the LIPA risk locus (LD, R2<0.09). Most
importantly, despite these similarities with T16P, G23R
carries no association with CAD (Figure 4F), strongly
implying the mild signal peptide disruption associated
with either variant is of minimal functional significance,
and that CAD risk associated with T16P is merely as a
result of its association with the intronic LIPA variants.
With the conclusion that the LIPA CAD risk locus is
associated with increased expression, we sought to con-
duct a preliminary evaluation of whether an increase in
LIPA expression could modulate cholesterol metabolism.
An important downstream phenotype of LIPA loss of
function is reduced cholesterol efflux, which has been
demonstrated in both fibroblasts from cholesterol ester
storage disease fibroblasts, mouse peritoneal macro-
phages, and human-induced pluripotent stem-cell derived
macrophages.16,26,27 One way through which this occurs is
reduced expression of the cholesterol efflux transporter
ABCA1, which could be rescued to baseline levels with
recombinant enzyme addition.27 No differences were
observed in monocyte ABCA1 mRNA expression across
individuals segregated by LIPA rs1412444 genotype
(Figure VA in the online-only Data Supplement). To eval-
uate whether modest increases in LIPA expression (as is
seen in the CAD risk haplotype) could modulate choles-
terol metabolism, we overexpressed LIPA in J774 cells
(Figure VB in the online-only Data Supplement). Small
increases in LIPA expression did not increase cholesterol
efflux (Figure VC in the online-only Data Supplement),
 Evans et al LIPA Variants and CAD

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Figure 3. rs1051338 does not impact LIPA (lysosomal acid lipase) in stable overexpression systems.
A, Western Blot analyses of National Institutes of Health (NIH) 3T3 and HEK-293T cells transduced with either LIPA variant. - indicates empty
vector transduction control and T1 P1 T2 P2 represent paired sets of cell lines transduced with either variant and matched for mRNA expression.
All data are expressed relative to respective T16 variant control. B, Quantified densitometry of LIPA expression. C, LIPA activity in transduced
cell lines. D, LIPA secretion measured in conditioned media from transduced cell lines. All parameters (expression, activity, secretion, mRNA)
within each replicate are measured within the same well and were assessed on 2 separate occasions. Data are mean±SEM.

or affect levels of free or esterified cholesterol with or
without acetylated-LDL loading state (Figure VD in the
online-only Data Supplement).
DISCUSSION
The finding that LIPA was a risk locus for Coronary Artery
Disease highlighted the need to better understand the
functional impacts of risk variants on LIPA expression
and function, and in turn, better understand the function
of LIPA in CAD. In the present study, we demonstrate the
LIPA risk haplotype associates with both increased LIPA
mRNA and enzyme activity in primary human CD14+
monocytes. We found rs1051338, an exonic variant in
tight linkage disequilibrium with lead intronic SNPs, to be
of minimal functional significance. Similarly, rs1051339,
a nearby exonic SNP also affecting the signal peptide
region, did not affect LIPA function. Overall, our results
provide strong support for a model wherein the LIPA risk
haplotype is associated with a gain of function in LIPA
likely driven by one or more intronic SNPs.

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 Evans et al LIPA Variants and CAD
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Figure 4. LIPA (lysosomal acid lipase) SNP rs1051339 does not affect expression, trafficking, activity, or secretion and is not
associated with coronary artery disease (CAD).
A, SignalP v. 4.1 in silico analyses of LIPA signal peptide property alterations with G23R and T16P. S (Signal Peptide) Score indicates the
probability of a region’s inclusion in the signal peptide. Y score takes into account local amino acid properties to predict the signal peptide
cleavage site (C.S.) and the predicted C.S. shift with G23R and T16P is shown. Note that the 23R/16P haplotype does not occur in humans.
B, Western Blot of HEK-293T cells transfected with either FLAG-tagged LIPA variant. Each lane represents an independent transfection
replicate. C, Quantified densitometry of LIPA expression. D, LIPA activity in whole-cell lysates. E, LIPA secretion measured in conditioned
media. All parameters (expression, activity, secretion, mRNA) are measured within the same well and represent at least 2 independent
experiments. Data are mean±SEM. F, rs1051338 and rs1051339 coding amino acid changes, predicted signal peptide cleavage sites, minor
allele frequencies, and association with intronic SNPs and CAD.

In our large human cohort, we replicated key find-
ings demonstrating the LIPA risk haplotype associated
with increased mRNA and crucially extend these find-
ings to demonstrate for the first time that this trans-
lates to increased LIPA enzyme activity. Such functional
cis-eQTL effects are common in many studies associat-
ing GWAS loci and gene expression with disease. How-
ever, we discovered that an exonic variant, rs1051338,
was in tight linkage disequilibrium with the intronic vari-
ants and provided a plausible candidate mechanism for

2488   December 2019 Arterioscler Thromb Vasc Biol. 2019;39:2480–2491. DOI: 10.1161/ATVBAHA.119.313443
 Evans et al
a loss of function that may not be reflected in whole cell
enzyme activity.
rs1051338 causes the amino acid substitution T16P
in the signal peptide region of the LIPA protein. T16P is
predicted to be disruptive due to the polar to nonpolar
amino acid shift in a region that is characteristically non-
polar. This could affect several aspects of signal peptide
function such as the cleavage site with implications for
downstream trafficking to LIPA’s 2 main subcellular des-
tinations: the secretory pathway and lysosome. Follow-
ing signal peptide cleavage, proLIPA is trafficked to the
lysosome, where the propeptide is cleaved to produce
a mature, active enzyme.12,28 This type of trafficking is
nearly universal to enzymes of the lysosomal lumen,29
and specific evidence for these forms of LIPA includes
35S-Met pulse chase labeling and propeptide cleavage
site mutagenesis studies.12,14,15 Similarly, we provide sup-
port for this concept by demonstrating inhibition of LIPA
propeptide cleavage with treatment of the lysosomal
acidity inhibitors bafilomycin and chloroquine. We also
noted that C-terminal FLAG tags selectively marked
pro-LIPA, likely due to cleavage of these exposed resi-
dues on the mature peptide in the acidic environment of
the lysosome.
To evaluate the functional impacts of rs1051338/
T16P on LIPA enzyme trafficking in the absence of
potentially confounding intronic variants, we studied
allele-specific overexpression in vitro and examined
the overall impacts on LIPA activity and trafficking and
Downloaded from http://ahajournals.org by on June 13, 2022
found that T16P had no detrimental impact on overall
LIPA expression or activity. As evidenced by the identi-
cal expression of the LIPA 41-kDa band in isolation or
considered relative to the proprotein, T16P does not
adversely impact enzyme maturation or trafficking to
the lysosome, nor did it impact secretion. While it is dif-
ficult to entirely exclude a subtle impact of T16P, any
effect clearly has minimal relevance in relation to the
overall increased LIPA activity we observed in humans
with the risk haplotype.
As an independent line of evidence for whether T16P
could be the causative SNP in the LIPA risk locus, we
characterized the functional impacts of rs1051339, a
common SNP also lying in the signal peptide that results
in the amino acid substitution G23R. This SNP is pre-
dicted to disrupt signal peptide function similarly to T16P,
yet is not in linkage disequilibrium with intronic variants,
allowing evaluation of whether this type of disruption
can impact enzyme function or cause CAD in isolation.
Additionally, a previous study of patients with Wolman’s
disease (LIPA loss of function) observed G23R (anno-
tated therein as G5R) in compound homozygosity with
another mutation and demonstrated that G23R alone
profoundly disrupted LIPA secretion when expressed in
insect cells.18 Overall, we observed no differences in LIPA
expression, activity, trafficking, or secretion with G23R
studied in vitro, suggesting this SNP is also of minimal
Arterioscler Thromb Vasc Biol. 2019;39:2480–2491. DOI: 10.1161/ATVBAHA.119.313443
LIPA Variants and CAD
functional relevance. This discrepancy from findings in
insect cells may be due to inappropriate glycosylation
Basic Sciences - AL
and oligosaccharide processing pathways in insect cells30
and emphasizes the importance of studying human, or at
least mammalian cells in this regard. The other identified
homozygous Wolman’s variant (G87V, annotated therein
as G60V) in the Zschenker study in compound homo-
zygosity in conjunction with G23R has been observed
to be sufficient to cause Wolman’s elsewhere.31 Most
importantly, rs1051339 carries no association with CAD,
implying in turn that rs1051338 association with CAD is
merely a result of linkage with intronic variants.
Our conclusions contrast with another recent report
evaluating the functional impacts of rs1051338 on
LIPA function, which suggested that T16P is a signifi-
cant loss of function mutation and acts by mistrafficking
LIPA away from the lysosome.9 In the present study, we
highlight the need to consider expression of pro-LIPA
and mature (lysosomal) LIPA as crucial indices of LIPA
subcellular distribution and trafficking. The identity of
the single Western Blot bands analyzed throughout the
study of Morris et al is unspecified and problematic for
their interpretations drawn. If mature/lysosomal 41 kDa
was the focus of analysis in their study, any differences
with T16P observed in their enriched lysosomal frac-
tion should be of similar magnitude in whole cell lysates.
However, this was not apparent and may therefore be
an artifact associated with their crude lysosomal enrich-
ment protocol. Alternatively, if the 56-kDa proprotein
band was analyzed (likely the dominant form in a trans-
fection-overexpression system), the conclusions about
lysosomal trafficking are hard to justify. Here, we dem-
onstrate C-terminal FLAG-tags nearly exclusively mark
pro-LIPA in overexpression systems. As LIPA and other
enzymes of the lysosomal lumen are exquisitely folded
and glycosylated to withstand lysosomal acidity and
proteases, we interpreted this as meaning that FLAG
tags were unstable in the lysosome. Therefore, the many
FLAG blots used in the Morris study are also likely to
be inappropriate for drawing conclusions regarding LIPA
lysosomal trafficking. Finally, the analyses of human
monocytes/macrophages (n=3–4) in the Morris study
were underpowered to observe the significant increase
in LIPA mRNA and activity we observed in a much larger
cohort.
A key conclusion of our study is the exclusion of
the possibility that rs1051338 causes a loss of func-
tion relevant to overall LIPA expression or activity, espe-
cially in relation to the obvious increase in mRNA and
activity driven by tightly associated intronic variants. One
hypothesis about increased mRNA expression at this
locus was that loss of function caused by rs1051338
caused compensatory LIPA mRNA upregulation. Our
data in individuals with dissociated intronic and exonic
genotypes suggests this is not the case and rs1412444
or other tightly associated intronic variants are sufficient
December 2019   2489
 Evans et al
to explain increase mRNA and activity. The association of
intronic variants with eQTL effects amounting to disease
Basic Sciences - AL
risk is not surprising and is quite common across many
discovered loci for complex diseases. Finally, evidence
about the relationship between loss of LIPA function and
CAD risk has been gleaned from studies of the Wolman’s
disease LIPA Exon 8 Splice Junction Mutation. Whereas
homozygosity leads to a near total loss of function and
overt pathology, heterozygosity (resulting in ≈50% loss
of function32–34) carries no association with serum lip-
ids, CAD, or myocardial infarction risk in large GWAS
cohorts.35 This suggests that a small magnitude loss of
function in LIPA hypothetically caused by T16P, which
we did not observe regardless, would still be unlikely to
confer disease risk.
Attention should now be focused on evaluating the
other possibilities through which the LIPA locus could
be causative in mediating CAD risk, including increased
expression. While LIPA deficiency markedly disrupts
cholesterol ester hydrolysis and cholesterol efflux,16,27 we
observed no clear changes with mild overexpression in
J774 cells. Thorough investigation using in vivo models
and taking into account the unique context of the athero-
sclerotic plaque will be required to fully evaluate whether
increased LIPA expression observed in the CAD risk hap-
lotype could be causal. One possibility is that secreted
LIPA (along with other lysosomal enzymes) can induce
LDL modifications such as aggregation and oxidation.5
Downloaded from http://ahajournals.org by on June 13, 2022
Increases in extracellular LIPA are observed in athero-
sclerotic lesions, and some have proposed that a very
significant portion of aggregated LDL hydrolysis occurs
extracellularly in atherosclerotic macrophages.36,37 A
second possibility is that increased LIPA function gener-
ates free cholesterol exceeding the capacity of systems
for lysosomal efflux and reverse cholesterol trans-
port. Excess free cholesterol in the lysosome is known
to increase lysosomal pH and lead to a more general
lysosomal dysfunction5,20 among many other cytotoxic
effects.38 Overall, further work is required to character-
ize whether the increased expression or other yet to be
appreciated possibilities could causally explain the CAD
risk associated with this locus.
ARTICLE INFORMATION
Received December 19, 2018; accepted September 26, 2019.
Affiliations
From the Cardiovascular Division, Department of Medicine (T.D.E., X.Z., R.E.C.,
A.A., E.S., N.O.S., B.R.) and Department of Pathology and Immunology (B.R.),
Washington University in St. Louis School of Medicine, MO; John Cochran VA
Medical Center, St. Louis, MO (B.R.); Department of Medicine, Cardiology Division,
Columbia University Medical Center, New York (H.Z., M.P.R.); and Irving Institute
for Clinical and Translational Research, Columbia University, New York (M.P.R.).
Acknowledgments
T.D. Evans wrote the manuscript and performed experiments. R.E. Clark, X.
Zhang, E. Song, and A. Alisio performed experiments. N.O. Stitziel contributed
and analyzed human GWAS (genome-wide association study) data. T.D. Evans,
2490   December 2019 Arterioscler Thromb Vasc Biol. 2019;39:2480–2491. DOI: 10.1161/ATVBAHA.119.313443
LIPA Variants and CAD
N.O. Stitziel, H. Zhang, M.P. Reilly, and B. Razani contributed to study conception,
discussion, revision, and editing of the manuscript. B. Razani is the guarantor
of this work, and, as such, had full access to all the data in the study and takes
responsibility for the integrity of the data and the accuracy of the data analysis.
Sources of Funding
This work was supported by National Institutes of Health (NIH) CTSA UL1
TR000448 (B. Razani), NIH R01 HL125838 (B. Razani), NIH F31 HL132434
(T.D. Evans), the Washington University Diabetic Cardiovascular Disease Center
(P30 DK020579), VA MERIT I01 BX003415 (B. Razani), and American Diabe-
tes Association ADA #1-18-IBS-029 (B. Razani) grants. H. Zhang is supported
by R00HL130574 and pilot grant through UL1TR001873. M.P. Reilly is support-
ed by R01-HL-132561, R01-HL-113147, and K24-HL-107643. We addition-
ally thank David and Deborah Winston, and the Adipocyte Biology and Molecular
Nutrition Core (P30 DK056341) for support.
Disclosures
B. Razani has previously conducted consulting for Alexion Pharmaceuticals. The
other authors report no conflicts.
REFERENCES
1. Wild PS, Zeller T, Schillert A, Szymczak S, Sinning CR, Deiseroth A,
Schnabel RB, Lubos E, Keller T, Eleftheriadis MS, et al. A genome-
wide association study identifies LIPA as a susceptibility gene for
coronary artery disease. Circ Cardiovasc Genet. 2011;4:403–412. doi:
10.1161/CIRCGENETICS.110.958728
2. Peden JF, Hopewell JC, Saleheen D, Chambers JC, Hager J, Soranzo N,
Collins R, Danesh J, Elliott P, Farrall M, et al. A genome-wide association
study in Europeans and South Asians identifies five new loci for coronary
artery disease. Nat Genet. 2011;43:339–346.
3. Vargas-Alarcón G, Posadas-Romero C, Villarreal-Molina T, Alvarez-León E,
Angeles J, Vallejo M, Posadas-Sánchez R, Cardoso G, Medina-Urrutia A,
Kimura-Hayama E. Single nucleotide polymorphisms within LIPA (Lysosomal
Acid Lipase A) gene are associated with susceptibility to premature coronary
artery disease. a replication in the genetic of atherosclerotic disease (GEA) Mex-
ican study. PLoS One. 2013;8:e74703. doi: 10.1371/journal.pone.0074703
4. Sergin I, Evans TD, Razani B. Degradation and beyond: the macrophage lyso-
some as a nexus for nutrient sensing and processing in atherosclerosis. Curr
Opin Lipidol. 2015;26:394–404. doi: 10.1097/MOL.0000000000000213
5. Dubland JA, Francis GA. Lysosomal acid lipase: at the crossroads of normal
and atherogenic cholesterol metabolism. Front Cell Dev Biol. 2015;3:3. doi:
10.3389/fcell.2015.00003
6. Hoeg JM, Demosky SJ Jr, Pescovitz OH, Brewer HB Jr. Cholesteryl ester stor-
age disease and Wolman disease: phenotypic variants of lysosomal acid cho-
lesteryl ester hydrolase deficiency. Am J Hum Genet. 1984;36:1190–1203.
7. Bernstein DL, Hülkova H, Bialer MG, Desnick RJ. Cholesteryl ester stor-
age disease: review of the findings in 135 reported patients with an under-
diagnosed disease. J Hepatol. 2013;58:1230–1243. doi: 10.1016/j.jhep.
2013.02.014
8. Du H, Heur M, Duanmu M, Grabowski GA, Hui DY, Witte DP, Mishra J. Lyso-
somal acid lipase-deficient mice: depletion of white and brown fat, severe
hepatosplenomegaly, and shortened life span. J Lipid Res. 2001;42:489–500.
9. Morris GE, Braund PS, Moore JS, Samani NJ, Codd V, Webb TR. Coronary
artery disease-associated LIPA coding variant rs1051338 reduces lyso-
somal acid lipase levels and activity in lysosomes. Arterioscler Thromb Vasc
Biol. 2017;37:1050–1057. doi: 10.1161/ATVBAHA.116.308734
10. Brænne I, Civelek M, Vilne B, Di Narzo A, Johnson AD, Zhao Y, Reiz B,
Codoni V, Webb TR, Foroughi Asl H, et al; Leducq Consortium CAD Genom-
ics‡. Prediction of causal candidate genes in coronary artery dis-
ease loci. Arterioscler Thromb Vasc Biol. 2015;35:2207–2217. doi:
10.1161/ATVBAHA.115.306108
11. Nikpay M, Goel A, Won HH, Hall LM, Willenborg C, Kanoni S, Saleheen D,
Kyriakou T, Nelson CP, Hopewell JC, et al. A comprehensive 1,000 genomes-
based genome-wide association meta-analysis of coronary artery disease.
Nat Genet. 2015;47:1121–1130. doi: 10.1038/ng.3396
12. Zschenker O, Oezden D, Ameis D. Lysosomal acid lipase as a preproprotein.
J Biochem. 2004;136:65–72. doi: 10.1093/jb/mvh093
13. Braulke T, Bonifacino JS. Sorting of lysosomal proteins. Biochim Biophys
Acta. 2009;1793:605–614. doi: 10.1016/j.bbamcr.2008.10.016
14. Sando GN, Rosenbaum LM. Human lysosomal acid lipase/cholesteryl ester
hydrolase. Purification and properties of the form secreted by fibroblasts in
microcarrier culture. J Biol Chem. 1985;260:15186–15193.
 Evans et al
15. Ameis D, Merkel M, Eckerskorn C, Greten H. Purification, characterization
and molecular cloning of human hepatic lysosomal acid lipase. Eur J Bio-
chem. 1994;219:905–914. doi: 10.1111/j.1432-1033.1994.tb18572.x
16. Zhang H, Shi J, Hachet MA, Xue C, Bauer RC, Jiang H, Li W, Tohyama J,
Millar J, Billheimer J, et al. CRISPR/Cas9-mediated gene editing in human
iPSC-derived macrophage reveals lysosomal acid lipase function in human
macrophages-brief report. Arterioscler Thromb Vasc Biol. 2017;37:2156–
2160. doi: 10.1161/ATVBAHA.117.310023
17. Deloukas P, Kanoni S, Willenborg C, Farrall M, Assimes TL, Thompson JR,
Ingelsson E, Saleheen D, Erdmann J, Goldstein BA, et al. Large-scale asso-
ciation analysis identifies new risk loci for coronary artery disease. Nat
Genet. 2013;45:25–33.
18. Zschenker O, Jung N, Rethmeier J, Trautwein S, Hertel S, Zeigler M,
Ameis D. Characterization of lysosomal acid lipase mutations in the signal
peptide and mature polypeptide region causing Wolman disease. J Lipid
Res. 2001;42:1033–1040.
19. Nédélec Y, Sanz J, Baharian G, Szpiech ZA, Pacis A, Dumaine A, Grenier JC,
Freiman A, Sams AJ, Hebert S, et al. Genetic ancestry and natural selec-
tion drive population differences in immune responses to pathogens. Cell.
2016;167:657–669.e21. doi: 10.1016/j.cell.2016.09.025
20. Emanuel R, Sergin I, Bhattacharya S, Turner J, Epelman S, Settembre C,
Diwan A, Ballabio A, Razani B. Induction of lysosomal biogenesis in athero-
sclerotic macrophages can rescue lipid-induced lysosomal dysfunction and
downstream sequelae. Arterioscler Thromb Vasc Biol. 2014;34:1942–1952.
doi: 10.1161/ATVBAHA.114.303342
21. Martel C, Li W, Fulp B, Platt AM, Gautier EL, Westerterp M, Bittman R, Tall AR,
Chen SH, Thomas MJ, et al. Lymphatic vasculature mediates macrophage
reverse cholesterol transport in mice. J Clin Invest. 2013;123:1571–1579.
doi: 10.1172/JCI63685
22. Huang LH, Zinselmeyer BH, Chang CH, Saunders BT, Elvington A, Baba O,
Broekelmann TJ, Qi L, Rueve JS, Swartz MA, et al. Interleukin-17 drives
interstitial entrapment of tissue lipoproteins in experimental psoriasis. Cell
Metab. 2019;29:475–487.e7. doi: 10.1016/j.cmet.2018.10.006
23. Sankaranarayanan S, Kellner-Weibel G, de la Llera-Moya M, Phillips MC,
Asztalos BF, Bittman R, Rothblat GH. A sensitive assay for ABCA1-mediated
cholesterol efflux using BODIPY-cholesterol. J Lipid Res. 2011;52:2332–
2340. doi: 10.1194/jlr.D018051
24. Robinet P, Wang Z, Hazen SL, Smith JD. A simple and sensitive enzymatic
Downloaded from http://ahajournals.org by on June 13, 2022
method for cholesterol quantification in macrophages and foam cells. J Lipid
Res. 2010;51:3364–3369. doi: 10.1194/jlr.D007336
25. Zhang H, Reilly MP. LIPA variants in Genome-Wide Association Studies
of coronary artery diseases: loss-of-function or gain-of-function? Arterio-
scler Thromb Vasc Biol. 2017;37:1015–1017. doi: 10.1161/ATVBAHA.
117.309344
26. Bowden KL, Bilbey NJ, Bilawchuk LM, Boadu E, Sidhu R, Ory DS,
Du H, Chan T, Francis GA. Lysosomal acid lipase deficiency impairs reg-
ulation of ABCA1 gene and formation of high density lipoproteins in
Arterioscler Thromb Vasc Biol. 2019;39:2480–2491. DOI: 10.1161/ATVBAHA.119.313443
LIPA Variants and CAD
cholesteryl ester storage disease. J Biol Chem. 2011;286:30624–30635.
doi: 10.1074/jbc.M111.274381
Basic Sciences - AL
27. Bowden KL, Dubland JA, Chan T, Xu YH, Grabowski GA, Du H, Francis GA.
LAL (Lysosomal Acid Lipase) promotes reverse cholesterol transport In
Vitro and In Vivo. Arterioscler Thromb Vasc Biol. 2018;38:1191–1201. doi:
10.1161/ATVBAHA.117.310507
28. Du H. Characterization of lysosomal acid lipase by site-directed mutagen-
esis and heterologous expression. J Biol Chem. 1995;270:27766–27772.
29. Kornfeld S. Trafficking of lysosomal enzymes in normal and disease states.
J Clin Invest. 1986;77:1–6. doi: 10.1172/JCI112262
30. Jarvis DL. Developing baculovirus-insect cell expression systems for
humanized recombinant glycoprotein production. Virology. 2003;310:1–7.
doi: 10.1016/s0042-6822(03)00120-x
31. Valles-Ayoub Y, Esfandiarifard S, No D, Sinai P, Khokher Z, Kohan M, Kahen T,
Darvish D. Wolman disease (LIPA p.G87V) genotype frequency in people of
Iranian-Jewish ancestry. Genet Test Mol Biomarkers. 2011;15:395–398.
doi: 10.1089/gtmb.2010.0203
32. Wiebusch H, Muntoni S, Funke H, Lu F, Seedorf U, Oberle S, Schwarzer U,
Assmann G. A novel missense mutation (Gly2Arg) in the human lysosomal
acid lipase gene is found in individuals with and without cholesterol ester
storage disease (CESD). Clin Genet. 1996;50:106–107.
33. Muntoni S, Wiebusch H, Funke H, Ros E, Seedorf U, Assmann G. Homozy-
gosity for a splice junction mutation in exon 8 of the gene encoding lyso-
somal acid lipase in a Spanish kindred with cholesterol ester storage disease
(CESD). Hum Genet. 1995;95:491–494. doi: 10.1007/bf00223858
34. Hamilton J, Jones I, Srivastava R, Galloway P. A new method for the mea-
surement of lysosomal acid lipase in dried blood spots using the inhibi-
tor Lalistat 2. Clin Chim Acta. 2012;413:1207–1210. doi: 10.1016/j.cca.
2012.03.019
35. Stitziel NO, Fouchier SW, Sjouke B, Peloso GM, Moscoso AM, Auer PL,
Goel A, Gigante B, Barnes TA, Melander O, et al; National Heart, Lung, and
Blood Institute GO Exome Sequencing Project. Exome sequencing and
directed clinical phenotyping diagnose cholesterol ester storage disease pre-
senting as autosomal recessive hypercholesterolemia. Arterioscler Thromb
Vasc Biol. 2013;33:2909–2914. doi: 10.1161/ATVBAHA.113.302426
36. Singh RK, Barbosa-Lorenzi VC, Lund FW, Grosheva I, Maxfield FR, Haka AS.
Degradation of aggregated LDL occurs in complex extracellular sub-com-
partments of the lysosomal synapse. J Cell Sci. 2016;129:1072–1082. doi:
10.1242/jcs.181743
37. Hakala JK, Oksjoki R, Laine P, Du H, Grabowski GA, Kovanen PT,
Pentikäinen MO. Lysosomal enzymes are released from cultured human
macrophages, hydrolyze LDL in vitro, and are present extracellularly in human
atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2003;23:1430–1436.
doi: 10.1161/01.ATV.0000077207.49221.06
38. Tabas I. Consequences of cellular cholesterol accumulation: basic con-
cepts and physiological implications. J Clin Invest. 2002;110:905–911. doi:
10.1172/JCI16452
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