Clinica Chimica Acta 454 (2016) 143–185

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Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/clinchim

Update on the molecular biology of dyslipidemias

I. Ramasamy
Department of Biochemistry, Worcester Royal Hospital, Worcester, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history: Dyslipidemia is a commonly encountered clinical condition and is an important determinant of cardiovascular
Received 2 September 2015 disease. Although secondary factors play a role in clinical expression, dyslipidemias have a strong genetic compo-
Received in revised form 24 October 2015 nent. Familial hypercholesterolemia is usually due to loss-of-function mutations in LDLR, the gene coding for low
Accepted 30 October 2015
density lipoprotein receptor and genes encoding for proteins that interact with the receptor: APOB, PCSK9 and
Available online 4 November 2015
LDLRAP1. Monogenic hypertriglyceridemia is the result of mutations in genes that regulate the metabolism of tri-
Keywords:
glyceride rich lipoproteins (eg LPL, APOC2, APOA5, LMF1, GPIHBP1). Conversely familial hypobetalipoproteinemia
Dyslipidemia is caused by inactivation of the PCSK9 gene which increases the number of LDL receptors and decreases plasma
Familial hypercholesterolemia cholesterol. Mutations in the genes APOB, and ANGPTL3 and ANGPTL4 (that encode angiopoietin-like proteins
Autosomal recessive hypercholesterolemia which inhibit lipoprotein lipase activity) can further cause low levels of apoB containing lipoproteins.
Lipoprotein lipase deficiency Abetalipoproteinemia and chylomicron retention disease are due to mutations in the microsomal transfer protein
Hypertriglyceridemia and Sar1b-GTPase genes, which affect the secretion of apoB containing lipoproteins. Dysbetalipoproteinemia
Dysbetalipoproteinemia stems from dysfunctional apoE and is characterized by the accumulation of remnants of chylomicrons and
Familial hypobetalipoproteinemia
very low density lipoproteins. ApoE deficiency can cause a similar phenotype or rarely mutations in apoE can
Abetalipoproteinemia
be associated with lipoprotein glomerulopathy. Low HDL can result from mutations in a number of genes regu-
Familial combined hypolipidemia
Chylomicron retention disease lating HDL production or catabolism; apoAI, lecithin: cholesterol acyltransferase and the ATP-binding cassette
Tangier disease transporter ABCA1. Patients with cholesteryl ester transfer protein deficiency have markedly increased HDL cho-
lesterol. Both common and rare genetic variants contribute to susceptibility to dyslipidemias. In contrast to rare
familial syndromes, in most patients, dyslipidemias have a complex genetic etiology consisting of multiple genet-
ic variants as established by genome wide association studies. Secondary factors, obesity, metabolic syndrome,
diabetes, renal disease, estrogen and antipsychotics can increase the likelihood of clinical presentation of an in-
dividual with predisposed genetic susceptibility to hyperlipoproteinemia. The genetic profiles studied are far
from complete and there is room for further characterization of genes influencing lipid levels. Genetic assessment
can help identify patients at risk for developing dyslipidemias and for treatment decisions based on ‘risk allele’
profiles. This review will present the current information on the genetics and pathophysiology of disorders
that cause dyslipidemias.
© 2015 Elsevier B.V. All rights reserved.

Contents

1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
1.1. Overview of lipid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
1.2. Regulation of triglyceride production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
1.2.1. Hormonal control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
1.2.2. Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
1.2.3. Glucagon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
1.3. Nuclear hormone receptors and lipid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
2. Inherited dyslipidemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
2.1. Increase in concentrations of lipid or lipoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
2.1.1. Familial hypercholesterolemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
2.2. Primary hypertriglyceridemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
2.2.1. Triglycerides and risk of disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
2.3. Primary monogenic hypertriglyceridemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
2.3.1. LPL deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
2.3.2. ApoCII deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
2.3.3. LMF1 deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

http://dx.doi.org/10.1016/j.cca.2015.10.033
0009-8981/© 2015 Elsevier B.V. All rights reserved.
144 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185

2.3.4. GPIHBP1 deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

2.3.5. ApoAV deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
2.3.6. LPL inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
2.3.7. Polygenic causes of hypertriglyceridemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
2.3.8. Novel rare genetic diseases associated with hypertriglyceridemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
2.4. ApoE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
2.4.1. ApoE and lipoprotein metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
2.4.2. Pathophysiology of dysbetalipoproteinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
2.4.3. Clinical presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
2.4.4. Lipoprotein glomerulopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
2.5. Decrease in concentration of lipid or lipoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
2.5.1. Familial hypobetalipoproteinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
2.5.2. Abetalipoproteinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
2.5.3. Familial combined hypolipidemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
2.5.4. Other candidate genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
2.5.5. Chylomicron retention disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
2.5.6. ApoCIII deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
3. Disorders of HDL metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
3.1. HDL and cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
3.2. Decrease in HDL concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
3.2.1. Monogenic disorders associated with decreased HDL levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
3.3. Increased HDL concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3.3.1. Monogenic disorders associated with increased HDL levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3.4. Polygenic causes of HDL variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
4. Secondary dyslipidemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
4.1. Obesity, metabolic syndrome and diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
4.2. Hypothyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
4.3. Renal disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
4.4. Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
5. Treatment of dyslipidemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
6. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

1. Background
1.1. Overview of lipid metabolism
Understanding the mechanisms of lipoprotein metabolism has
important clinical indications as lipoproteins are risk factors for
atherosclerosis. In both liver and intestine, apolipoprotein B (apoB) is
cotranslationally translocated to the endoplasmic reticulum (ER)
lumen where facilitated by microsomal transfer protein (MTP), lipid is
added to apoB to form primordial apoB containing particles. In humans
apoB48 is secreted exclusively by the intestine in chylomicrons and
apoB100 is secreted exclusively by the liver in very low density lipopro-
teins (VLDL). ApoB100 has 4536 amino acids. ApoB48 is identical to the
amino-terminal 48% of the apoB100 and is produced by a unique mRNA
editing process. Cotranslational targeting of apoB for degradation is reg-
ulated by the availability of lipids. Newly formed but defective apoB100
particles are cleared by mechanisms including ER associated degrada-
tion or autophagosome formation. Both VLDL and chylomicrons (CM)
require specialized vesicles to traffic from ER through to the Golgi
prior to being secreted.
The lipase maturation factor 1 (LMF1), the glycosylphosphati
dylinositol-anchored high density lipoprotein binding protein 1
(GPIHBP1) and an angiopoietin like protein play a role in lipoprotein li-
pase (LPL) mediated hydrolysis of TG so that the right amount of fatty
acids is delivered to the right tissue at the right time. Hydrolysis of CM
and VLDL by LPL results in chylomicron remnants, intermediate density
lipoproteins (IDL) and low density lipoproteins (LDL) particles, which
bind to specific receptors in the liver, the LDL receptor (LDLR), VLDL
receptor and LDL receptor related proteins (LRP) prior to removal
from the plasma (Figs. 1, 2 and 3).
Reverse cholesterol transport occurs when lipid free apoAI and apoE
recruit cholesterol and phospholipid to assemble HDL particles. The
ATP-binding cassette (ABC) transporter protein, ABCA1 is the key cellu-
lar membrane protein for the generation of new HDL particles. Further
cholesterol enrichment of these particles is through ABCG1 and scaven-
ger receptor class B type I (SR-BI). In the case of SR-BI the flux of choles-
terol is bidirectional. In humans, HDL cholesterol can be returned to the
liver either by direct uptake by SR-BI or through cholesteryl ester trans-
fer protein (CETP) exchange of cholesteryl ester for triglycerides (TG) in
apoB lipoproteins, followed by hepatic uptake of apoB containing parti-
cles. In the circulation several proteins and enzymes modulate HDL. Lec-
ithin: cholesterol acyltransferase (LCAT) esterifies free cholesterol into
cholesteryl esters that are hydrophobic and therefore sequestered into
the core of particles. LCAT is activated primarily by apoAI but can also
be activated by apoAIV, apoCI and apoE [1]. It has been postulated that
upon esterification of cholesterol in HDL the gradient of free cholesterol
between the cellular membrane and the surface of the HDL particle is
maintained. This is thought to generate a continuous flow of cholesterol
from the cell to lipoproteins. CETP transfers cholesteryl esters from HDL
to apoB containing proteins. Phospholipid transfer protein (PLTP) trans-
fers phospholipid between HDL and VLDL as well as different HDL par-
ticles (Fig. 4). The reader is referred to several reviews including those
by the author for a more detailed discussion on physiological lipopro-
tein metabolism [2,3].
Experimental data suggest that the exchangeable lipoproteins not
only play a role extracellularly (in the circulation) in modulating
lipoprotein clearance but also have an impact on the rate and effi-
ciency of lipoprotein assembly and secretion. Exchangeable lipopro-
teins are those capable of moving from one lipoprotein particle to
another (apoAI, AII, AIV, AV, CI, CII, CIII) and as opposed to the non-
exchangeable apolipoproteins that remain attached to one lipopro-
tein molecule from synthesis and catabolism (apoB100 and apoB48)
(Table 1). The exchangeability of the apolipoproteins is attributed
to the high content of their amphipathic α-helical structure which
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185 145

Fig. 1. Pathways of triglyceride rich lipoprotein metabolism. Intestinal enterocytes absorb dietary lipids and package most of it into chylomicrons. Endothelial bound LPL hy-
drolyses triglycerides in CM to generate chylomicron remnants which are rapidly cleared by the liver. Intrahepatic lipids are repackaged and secreted as VLDL which are sub-
strates for LPL. Glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) plays a role in LPL mediated hydrolysis of TG. Remnants formed
by hydrolysis of VLDL are either taken up by the liver or converted to LDL. The hepatic LDL receptor (LDLR) is responsible for clearing LDL from human plasma. The LDL re-
ceptor related lipoprotein (LRP) has a role in triglyceride rich lipoprotein clearance. In addition heparan sulfate proteoglycans (HSPG) act in the clearance of triglyceride-rich
lipoproteins.

displays different affinities towards phospholipid on the surface of
lipoproteins [4]. Accumulating evidence suggests that exchangeable
lipoproteins play a role in lipid mobilization and recruitment during
lipoprotein assembly and trafficking through the ER-Golgi secretory
compartments and that this role is independent of their role in lipo-
protein clearance [5]. Recent suggestions are that variations in sin-
gle nucleotide polymorphisms (SNP) in the ApoAI/CIII/AIV/AV gene
cluster are associated with altered postprandial lipid metabolism
[6].
1.2. Regulation of triglyceride production
1.2.1. Hormonal control
In humans the production of intestinal and hepatic lipoproteins is
subject to both substrate supply and hormonal regulation. Regulation
of TG production by substrate availability and summarized in Figs. 5
and 6 [22,23].
1.2.2. Insulin
Lipoprotein secretion is a complex process in which substrate avail-
ability and insulin play key roles. Studies in cells, small animals and
humans have demonstrated a key role for insulin in hepatic VLDL secre-
tion. Insulin acts as an anabolic hormone stimulating energy storage in
liver and adipose tissue. Insulin signaling acutely suppresses both lipol-
ysis and VLDL secretion. Although insulin acutely promotes hepatic lipo-
genesis it inhibits VLDL secretion. Insulin may indirectly regulate VLDL
production by reducing plasma FA as a consequence of inhibiting
adipose tissue lipolysis. In healthy individuals acute inhibition of VLDL
secretion is only partly due the suppression of FA and further FA inde-
pendent processes may play a part [24]. Decreasing endogeneous
VLDL production during postprandial conditions is important for
regulating whole body TG flux when dietary TG are entering the sys-
temic circulation via chylomicrons.
Acute increases in insulin can decrease the secretion of apoB by
targeting the protein for intracellular degradation. Insulin suppresses he-
patic VLDL secretion through activation of phosphatidylinositol 3 kinase
(PI3K). Binding of insulin to hepatic insulin receptors, leads to activation
of insulin receptor tyrosine kinase and tyrosine phosphorylation of in-
sulin receptor substrates. Tyrosine phosphorylated insulin receptor
substrates complex with and activate PI3K. Activated PI3K interacts
with its substrate phosphotidylinositide [4,5] bisphosphate with the
formation of phosphatidylinositide [3,4,5]triphosphate. Results suggest
that insulin signaling through PI3K inhibited the maturation phase of
VLDL [25]. Insulin may upregulate genes responsible for shifting TG
from VLDL formation to cytosolic lipid formation [26]. Another site for
insulin action is the MTP. Insulin inhibits MTP gene transcription
through the mitogen activated protein kinase (MAPKerk) cascade but
not through the PI3K pathway [27], other studies suggest that insulin
regulates hepatic MTP expression through the forkhead box 01 (Fox01)
transcription factor [28]. Besides regulating MTP gene transcription,
both MAPKerk and insulin upregulate LDL-receptor gene transcription.
Evidence suggests that sterol responsive element binding proteins
(SREBPs) are the downstream effectors of MAPKerk to transactivate
the LDL-receptor gene [29,30].
Insulin regulates metabolic activity and gene transcription by modu-
lating the activity of several intracellular signaling pathways. Evidence
suggests that insulin regulates lipoprotein metabolism through acute
and adaptive methods. Activation of the MAPKerk in the liver has a ben-
eficial effect on plasma lipid profiles as this would induce LDL-receptor
and suppress hepatic MTP expression. In addition there is potential for
cross talk between the signaling pathways which may act as fine tuning
of the lipid profile in response to several other stimuli [31].
146 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185

Fig. 2. Biosynthesis of chylomicrons. In the intestinal cell newly synthesized apoB48 follows one of two itineraries. In the presence of phospholipids, cholesterol and triacylglycerol, apoB48
is chaperoned by microsomal transfer protein (MTP), to form newly synthesized prechylomicron particles. In the absence of lipids it is rapidly degraded. The prechylomicron particle buds
from the endoplasmic reticulum and is transported to the Golgi as the prechylomicron transport vesicle (PCTV). Within the Golgi complex the prechylomicron forms the mature chylo-
micron particles. The mature chylomicron exits the Golgi complex in large transport vesicles to fuse with the basolateral membrane and is secreted. The mature chylomicrons contain
apoAI, apoAIV and apoB48. TG = triglycerides, PL = phospholipid, Chol: cholesterol.

Acute elevations of plasma FA stimulates not only hepatic but also
intestinally derived apoB48 containing lipoprotein particles in fed
humans [32]. Intestinal apoB48 containing lipoprotein production is
acutely suppressed by insulin, and this may involve insulin's direct ef-
fects as well as insulin-mediated suppression of circulating FA [33].
This suggests that factors involved in the regulation of hepatic VLDL
may regulate chylomicron production.
1.2.3. Glucagon
Glucagon is a pancreatic hormone that opposes the action of insulin,
and stimulates gluconeogenesis and glycogenolysis to promote glucose
liberation in hepatocytes. Glucagon actions are transduced through the
glucagon receptor (Gcgr). In mice, it has been suggested that glucagon
action is responsible for multiple pathways regulating lipid homeostasis
[34]. The role of glucagon in regulating hepatic and intestinal
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185 147

Fig. 3. Biosynthesis of VLDL. Biosynthesis of VLDL starts when apoB100 is translocated to the lumen of the ER and interacts with MTP. In the absence of lipids and microsomal transfer
protein (MTP), apoB100 is targeted for proteosomal degradation. MTP driven lipidation gives rise to pre-VLDL particles. Interaction with molecular chaperones enhance/modify
apoB100 degradation. Pre-VLDL is transported to the Golgi apparatus in a COPII dependent fashion. Pre-VLDL is converted to VLDL in the Golgi prior to secretion. Pre-VLDL which is
not converted to mature lipoprotein are sorted out for autophagy through post-ER presecretory proteolysis (PERPP). TG = triglycerides, PL = phospholipid, Chol: cholesterol.

lipoprotein metabolism is not clear. Though recent studies suggest particle production and clearance with no net effect on concentra-
that glucagon acutely regulates hepatic but not intestinal lipoprotein tion. The mechanism of its effect on apoB100 containing particles is
particle metabolism in humans, both by decreasing lipoprotein unknown [35].
148 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185

Fig. 4. Biosynthesis of HDL. Biosynthesis of HDL begins with the interaction of lipid poor apoAI with ABCA1. Lipidated apoAI is the major acceptor for SR-BI and ABCG1 pathways. CETP
promotes the transfer of cholesteryl esters from HDL to VLDL and LDL. PLTP transfers phospholipid between VLDL and HDL and remodels HDL into larger and smaller particles with
the dissociation of lipid poor or lipid free apoAI. Solid arrows indicate cholesterol/cholesteryl ester transfer. chylomicrons (CM); very low density lipoproteins (VLDL); high density lipo-
protein (HDL); apoprotein (apo); lipoprotein lipase(LPL), Hepatic Lipase (HL), ATP-binding cassette (ABC); ABC transporters (ABCA1 and ABCG1), lecithin cholesterol acyl transferase
(LCAT); acyl-CoA:cholesterol acyltransferase (ACAT), cholesteryl ester transfer protein (CETP); Phospholipid transfer protein (PLTP); scavenger receptor BI (SR-BI).

Triglyceride metabolism at the whole body level is summarized in
Fig. 6.
1.3. Nuclear hormone receptors and lipid metabolism
Dietary fats regulate gene expression in a hormone-independent
manner. Since the description of dietary fat as a regulator of gene
expression, many transcription factors were identified as prospective
targets for fatty acid regulation including peroxisome proliferator acti-
vated receptors (PPARα, β, γ1 and γ2), SREBP-1c, hepatic nuclear fac-
tors (HNF-4α and γ), retinoid X receptor (RXRα), LXRα and others
[36]. Fatty acids bind directly to PPARs, LXR and HNF-4α leading to
changes in the trans activating activity of these transcription factors.
PPARs regulate several genes involved in metabolic processes. PPARα
is expressed mainly in the liver, digestive tract and kidney, PPARβ is
ubiquitously expressed, PPARγ is mainly expressed in the adipose tis-
sue. PPARs bind to specific DNA response elements, the PPAR response
elements as heterodimers with RXR. The fact that fatty acids are activa-
tors of PPARs suggests that lipoproteins serve as ligand carriers for
PPARs. Studies suggest that PPARγ regulates adipose tissue triglyceride
lipase (ATGL) in adipocytes [37]. In the liver PPARα directly regulates
genes involved in fatty acid uptake and oxidation [38]. Rakhshanderoo
et al. [39] report that several genes involved in lipid metabolism were
PPARα targets as were several putative novel genes which included
CREBL3 (cAMP responsive element binding protein 3-like 3). CREBL3
encodes the liver expressed mammalian transcription factor CREBH
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185 149

Table 1
Apolipoproteins.

Apolipoprotein Molecular weight Lipoproteins Metabolic functions Synthesis Reference

ApoAI 28,016 HDL, chylomicrons Structural component of HDL, LCAT activator Liver, intestine [3]
ApoAII 17,414 HDL, chylomicrons Liver [7]
ApoAIV 46,465 HDL, chylomicrons Involved in chylomicron assembly and secretion Intestine in humans [8,9]
ApoAV HDL, VLDL, chylomicrons Effects on plasma triglyceride concentrations are Predominantly in the liver [10,11]
complex and variable.
Activator of intravascular hydrolysis by LPL.
Modulates hepatic triglyceride metabolism
ApoB48 264,000 chylomicrons Necessary for assembly and secretion of Intestine [3]
chylomicrons from the small intestine
ApoB100 540,000 VLDL, IDL, LDL Necessary for assembly and secretion of VLDL from Liver [3]
liver. Structural protein of VLDL, IDL and LDL.
Ligand for LDL receptor
ApoCI 6630 Chylomicrons, VLDL, IDL, HDL ApoCI inhibits lipoprotein binding to its receptors. Liver [12,13]
Potent inhibitor of cholesteryl ester transfer
protein.
ApoCII 8900 Chylomicrons, VLDL, IDL, HDL Activator of lipoprotein lipase [19]
ApoCIII 8800 Chylomicrons, VLDL, IDL, HDL Inhibits lipoprotein lipase; increases VLDL Synthesized in the liver and to [12,14,15,16]
secretion. a lesser extent in the intestine
ApoCIII can also stimulate several processes
involved in atherogenesis and vascular
inflammation.
Interferes with remnant lipoprotein clearance.
ApoE 34,145 Chylomicrons, VLDL, IDL, HDL LDL receptor ligand for LDL and chylomicron Predominantly in the liver [17,18]
remnants. Ligand for LRP. Role in reverse
cholesterol transport.
Apo(a) 250,000–800,000 Lp(a) Unknown Liver [20]
ApoM 26,000 Predominantly HDL and also Synthesis of HDL Liver and kidney [21]
found in CM, VLDL, LDL

(cAMP-responsive element binding protein, hepatocyte specific).
Proteolysis results in CREBH activation and nuclear translocation,
where it increases expression of the hepatic gluconeogenetic enzyme,
phosphoenol pyruvate carboxykinase [40].
LXRs are activated in response to cellular cholesterol levels,
allowing them to act as cholesterol sensors. The two isoforms of
LXR, α and β are highly homologous and form obligate heterodimers
with the RXR. Once activated LXRs induce the expression of an array
of genes involved in cholesterol absorption, efflux, transport and ex-
cretion. They also induce the expression of inducible degrader of
LDLR (IDOL), an E3 ubiquitin ligase that catalyzes the ubiquitination
and degradation of the LDLR [41]. Hepatic lipogenic genes known to
be under direct LXR transcriptional control include fatty acid syn-
thase, steoryl CoA desaturase and SREBP1c. LXR activation mediated
changes in insulin receptor substrate phosphorylation as well as in-
creases in protein tyrosine phosphatase (PTP)1B. Tyrosine phospha-
tases including PTP1B can regulate the biological effects of insulin.
LXR activation can dysregulate hepatic insulin signaling and lead to
an increase in VLDL particles. [42,43]. Tumor suppressor phosphatase
with tensin homology (PTEN) may further regulate insulin sensitivity
as in its absence the PI3-kinase pathway can become constitutively
active [44].

Fig. 5. Hepatic fatty acid trafficking. In the postabsorptive state, FAs formed from lipolysis in the adipose tissue enter the liver and mix with fatty acids from the cytosolic TG pool and DNL.
FAs are then diverted to the mitochondrial oxidative pathway to form acetyl CoA. FAs are also esterified and enter the cytosolic TG storage pool. TG are assembled from FAs derived from
plasma NEFAs, chylomicron remnants or DNL and are either secreted as VLDL or stored in the cytosol to be used at a later stage. The postprandial increase in insulin shifts the cellular me-
tabolism of FAs away from oxidative pathways towards esterification, and increased DNL. DNL—denovo lipogenesis; ER: endoplasmic reticulum.
150 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185

Fig. 6. Metabolism of TG. In the postprandial state CM from the enterocyte enters into systemic circulation. CM are hydrolyzed by LPL and the FA is taken up by muscle (for oxidation),
adipose tissue (for storage) and liver. There is insulin dependent suppression of FA release from the adipose tissue, and VLDL release from the liver. In the postprandial state hepatic
DNL is increased. In the postabsorptive phase there is net release of FAs from the adipose tissue, that is taken up by the liver and muscle. In the liver FA is partitioned for oxidation or
re-esterification and storage or VLDL secretion. CM: chylomicron; CMR: chylomicron remnant; DNL: de novo lipogenesis; LPL: lipoprotein lipase. : VLDL; CM.

In mammals the cholesterol content of the cell membranes is 2. Inherited dyslipidemias

controlled by a family of membrane bound transcription factors des-
ignated as SREBPs. Fatty acids do not bind to SREBP proteins but in-
duce changes in the nuclear abundance of the transcription factor
[45,46].
In vivo studies suggest that multiple PPARα and SREBP1c target
genes are regulated by fatty acids. These mechanisms control hepatic
lipid composition and the lipids available for VLDL synthesis [47].
The synthesis of cholesterol and fatty acids begins with their precur-
sor acetyl-CoA. Acetyl-CoA is converted to cholesterol in a pathway
involving at least 23 enzymes. Acetyl-CoA forms fatty acids in a path-
way involving up to 12 enzymatic steps Many of these pathways are
regulated by the SREBP family of transcription factors [48]. The three
SREBP isoforms are designated SREBP1a, SREBP1c and SREBP2. The
relative activities of three isoforms differ. SREBP1a is a potent activa-
tor of all SREBP-responsive genes, owing to its long transactivation
domain encoded by the first domain. In SREBP1c this exon encodes
a shorter transactivation domain that is less potent than SREBP1a.
Although they differ in potency, SREBP1a and 1c both activate
genes involved in the synthesis of fatty acids and their incorporation
into triglycerides and phospholipids. SREBP2 encoded by a different
gene preferentially activates genes for cholesterol synthesis and
LDLR [49]. The challenge in the future will be to integrate these
different pathways for a rational model for further understanding of
lipid metabolism.
2.1. Increase in concentrations of lipid or lipoprotein
2.1.1. Familial hypercholesterolemia
2.1.1.1. Clinical presentation. Elevated plasma levels of LDL cholesterol
have been shown to be a risk factor for the development of atheroscle-
rosis and associated ischemic heart disease (IHD). In men, a rise in total
cholesterol from 5.2 to 6.2 mmol/L is associated with a threefold in-
creased risk of death from IHD [50]. Raised serum cholesterol and LDL
cholesterol are characteristic of familial hypercholesterolemia (FH,
OMIM 143890). In the pre-genomic era the commonly used system
for classification of dyslipidemias was based on the Frederickson or
World Health Organization International Classification of Diseases of li-
poprotein phenotypes [51]. FH presents as hyperlipoproteinemia (HLP)
type 2A, elevated LDL cholesterol and normal TG levels. Most HLP type
2A patients have no defined monogenic defect, only about ~ 10% are
monogenic. Causal alleles for monogenic disorders are highly penetrant
and often lead to severe phenotypes. Alleles that cause complex poly-
genic traits have a more subtle effect on disease. It is likely that the poly-
genic form involves a multiplicity of loci with a range of effects. The
monogenic syndromes are generally distinguished from the polygenic
forms by the severity of LDL cholesterol elevation, by the inheritance
pattern and the severity of IHD risk [52].
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
Raised cholesterol levels initiate changes that lead to the develop-
ment of atherosclerotic lesions. FH leads often lead to the accumulation
of cholesterol in the skin, xanthomas in the tendons, xanthelasmata
around the eyes and presenile corneal arcus around the cornea [53].A
number of molecular defects have been shown to underlie extreme
levels of LDL cholesterol. Defects in the LDLR gene is the most frequent
cause [54,55]. Defects in other genes, the gene for apoB, proprotein
convertase subtilisin kexin type 9 (PCSK9), and a protein that causes in-
ternalization of the LDLR protein can cause a similar phenotype. Not all
clinically diagnosed patients have a known defect, suggesting that other
genes remain to be discovered [56]. In a majority of cases monogenic FH
is inherited as an autosomal dominant disorder. The gene causing the
rare autosomal recessive hypercholesterolemia (ARH), LDLRAP1 which
encodes an adapter protein named LDLRAP1 that is required for the in-
ternalization of the LDLR has recently been characterized [57]. The clin-
ical phenotype from these mutations is variable, the apoB mutation is
less severe [57,58]. The homozygous forms are more severely affected
than the heterozygous forms [59]. Carriers of mutations assumed to re-
sult in complete obliteration of LDLR activity had a more severe pheno-
type with higher LDL cholesterol and higher prevalence of tendon
xanthomatosis and coronary heart disease than those with residual
LDLR activity [60]. Gradient of severity of phenotype presentation has
been stated as mutations in LDLR = PCSK9 N APOB [61]. The clinical phe-
notype of ARH is milder than receptor negative LDLR and resembles that
observed in the receptor defective LDLR patients [62], and is more re-
sponsive to lipid lowering treatment [63].
There is no single internationally accepted set of criteria for the clin-
ical diagnosis of FH. The most commonly used are the US (Make Early
Diagnosis to Prevent Early Death) MEDPED, the UK (The Simon Broome
register) and the Dutch Lipid Clinic criteria. Criteria developed by the
groups in the UK and The Netherlands include family and personal his-
tory, physical signs and genetic studies in addition to cholesterol levels,
and are classified as definite, probable and possible diagnosis of FH,
while the US criteria only uses lipid levels [64,65]. The estimated preva-
lence worldwide is 1/500 for the heterozygous form and 1 in 1 million
for the homozygous form [66,67]. Recently, the prevalence of the molec-
ular defined homozygous variant was estimated at 1: 300,000 in The
Netherlands with a more variable clinical phenotype [68]. The frequen-
cy of the APOB mutation varies from 2–10% of patients with clinical
criteria for monogenic hypercholesterolemia compared to 52–76% of
LDLR mutations and 2% PCSK9 and 2% LDLRAP1 mutations [69].
2.1.1.2. Low density lipoprotein receptor. The classical cause of FH is mu-
tations in the LDLR (OMIM 606945) gene. Pathogenic changes in LDLR
result in impaired uptake and processing of LDL particles which leads
to elevated serum cholesterol levels. The LDLR gene which is located at
19p13.2 is composed of 18 exons spanning 45 kb, the transcript is
5.3 kb long and encodes a peptide of 860 residues. Functional domains
of the peptide correspond with the exons as follows: signal sequence-
exon 1, ligand binding domain-exons 2–6, epidermal growth factor
(EGF) precursor like domain-exons 7–14, O-linked carbohydrate
domain-exon 15, transmembrane domain-exon 16 and 41 bp of exon
17 and cytoplasmic domain-remainder of exon 17 and exon 18. LDLR
is expressed ubiquitously under the control of sterol regulated negative
feedback, mediated by three 16 bp imperfect repeats (sterol regulatory
element) and a TATA like sequence in the promoter. [66]. As of 2012, the
LDLR variant database contained over 1288 LDLR gene events. The
variants include 55% exonic substitutions, 22% exonic small rearrange-
ments (b100 bp), 11% large rearrangements (N100 bp), 2% promoter
variants, 10% intronic variants and 1 variant in the 3ˈ untranslated se-
quence. The LDLR variant database is recorded in the University College
London database and has been transferred to the Leiden Open Source
Variation Database (LOVD) platform. (https://grenada.lumc.nl/LOVD2/
UCL-Heart/home.php?select_db=LDLR) and a further database is main-
tained by INSERM (http://www.umd.be/LDLR/). FH associated variants
continue to be reported along the length of the LDLR gene by groups
151
around the world, the proportion of variants distributed equally
among the exons. Pathogenic predictions and experimental/family
studies were used to study the consequences of these changes, some
of which are dominant and fully penetrant causes of hypercholesterol-
emia while others seem to have little or no effect on the protein [70].
Ideally, the putative mutant LDLR should be expressed in heterologous
cell in vitro and its activity compared with normal activity. Though the
absence of the variant from a large number of normolipidemic individ-
uals, its cosegregation with raised cholesterol in a patient's family and
the nature of the amino acid variation may provide sufficient evidence
for its pathogenicity [71]. Mutations can either affect LDLR activity,
LDLR transcription [66] or prevent the internalization of the receptor
through the clathrin coated pit machinery [72].
2.1.1.3. Polygenic hypercholesterolemia. The Global Lipid Genetic Consor-
tium meta-analysis of genome wide association studies (GWAS, up-
dated in the website www.ebi.ac.uk/gwas) identified several loci
where common variants affect LDL cholesterol concentration and the
results of another study showed that a proportion of individuals carry-
ing several LDL cholesterol raising single nucleotide polymorphisms
(SNPs) have LDL cholesterol concentrations that exceed the diagnostic
LDL cholesterol threshold for the diagnosis of FH. It has been suggested
that in a number of patients with FH without a known mutation, raised
LDL cholesterol may have a polygenic cause [73]. Studies have identified
loci with evidence for association between common variants (those of
ApoB, ApoE, ApoCI, ApoCIV, ApoCII, PCSK9, ABCA1, ApoAI-ApoCIII-ApoAIV-
ApoAV, CETP, LIPC (hepatic lipase), LPL and ANGTPL4) and lipid or lipo-
protein concentrations [74,75]. In a further study a combination of
rare and common variants in the SORT1 (sortilin), APOB, LDLR, APOE
and PCSK9 genes were found to contribute to variation in LDL cholester-
ol levels [76]. In patients with FH with a known causative mutation, an
additional polygenic contribution can explain a highly increased LDL
cholesterol concentration. A polygenic component can explain the
large overlap in LDL cholesterol concentration between mutation carrier
and non-carrier relatives [77].
2.1.1.4. Apolipoprotein B. Defects in other genes can cause a similar phe-
notype to the LDLR defect. Familial defective apoB100 (FDB, OMIM
144010), characterized by a mutation in the APOB gene, the gene coding
for apoB the ligand for the receptor on the surface of LDL is probably the
second most frequent cause of the FH phenotype. The 43 kb APOB gene
is located on the short arm of chromosome 2. It consists of 29 exons of
which exon 26, with 7572 bp, is the largest and encodes more than
one half of the full length protein. ApoB100 appears to be arranged as
a series of amphipathic α-helices and β-sheet domains (NH2-βα1-β1-
α2-β2-α3-COOH). ApoB48 comprises the first two domains. It is thought
that the βα1 domain binds MTP and that the carboxy terminal binds
LDLR. The mature protein contains 25 cysteine residues of which 16
are involved in intramolecular linkage [78,79]. Studies of the three
dimensional structure of apoB100 by immunoelectron microscopy sug-
gests that apoB100 enwraps LDL like a belt, completing the encircle-
ment by about amino acid 4050 and that the carboxy terminal forms a
bow that crosses backwards over the chain between residues 3000–
3500, the suggested binding site for the LDLR [80]. ApoB is subject to
both transcriptional and posttranscriptional control. APOB transcription
is regulated by a promoter in combination with either the liver-specific
regulatory region or the intestinal enhancer. Thus two separate regula-
tory elements operate to control the expression of apoB in liver and in-
testine [81]. ApoB mRNA levels remain relatively stable despite the
presence of a number of factors known to influence apoB secretion,
suggesting post-translational regulation of apoB. [82]. The ability of
apoB100 to interact with the LDLR depends on both sequence and con-
formation [79]. In patients with FDB, LDL had a lower LDLR receptor
binding capacity than normal LDL [83].
In contrast to the large heterogeneity of the LDLR locus only a few
mutations near the vicinity of codon 3527 of APOB were known to
152 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
prevent LDL from binding to LDLR. A predominant mutation CCG to CAG
mutation in the amino acid 3500 (R3500Q or now termed R3527Q) is on
a chromosome with a rare haplotype at the apoB locus suggesting that
most probands descended from a common ancestor who lived in
Europe about 7000 years ago [84]. However, different haplotypes have
been reported suggesting an independent R3500Q mutation [85]. The
frequency of the R3500Q mutation has been estimated from a four cen-
ter study to be 1 in 543 [86]. Three further mutations R3500W, R3531C,
R3480W located within a stretch of 51 amino acids and result in the loss
of an arginine have been linked to defective LDLR binding. One hypoth-
esis is that arginine 3500 interacts with tryptophan 4369 and this inter-
action is essential for the correct conformation of the carboxy terminal
tail of apoB100. Disruption of this interaction results in a conformational
change that disrupts receptor binding. One model suggests that trypto-
phan 4369 interacts with arginines in addition to 3500 in apoB100 dur-
ing the conversion of VLDL to LDL and that this functions as a modulator
element that inhibits VLDL from interacting with the LDLR [79]. Muta-
tions with less of a severe impairment of binding can cause less of an
increase in serum cholesterol [86]. A new mutation H3543Y with a
prevalence of heterozygotes as 0.47% compared to 0.12% for the
R3500Q mutation has been reported in German patients with coronary
artery disease [87]. The advances in DNA enrichment and next genera-
tion sequencing technology have made it possible to sequence the
whole ‘exome’ ie the protein coding portion of the genome. Whole se-
quencing of APOB in FDB families identified R1164T, Q4494 del,
R3059C, K3394N mutations that can attenuate cellular LDL uptake.
These results emphasize the need to study the whole APOB to identify
new patients with FDB [88,89]. In some cases mutations are susceptibil-
ity mutations and enhance the effect of other mutations (eg an LDLR de-
fect) and there is an absence of cosegregation of the APOB mutation with
hypercholesterolemia [90].
The rare mutations in APOB confer a risk of IHD in the mutation car-
riers, their effect at the population level is minimal. Conversely, single
nucleotide polymorphisms (SNPs) may have a population effect that is
far from negligible despite a weak effect at the individual level. ApoB
is a highly polymorphic gene and in individuals without rare mutations,
polymorphisms may have a role in determining apoB and cholesterol
level. Large epidemiological studies have indicated that the increased
apoB serum levels are strong predictors for IHD [91]. Using isotope la-
beling techniques both mutations and polymorphisms have been
shown to affect LDL metabolism in vivo. However, APOB polymorphisms
studied so far have shown no association with IHD, suggesting other
functionally important SNPs have not been identified [92]. Metabolism
of apoB containing particles are dependent on gender, APOE genotype,
LDLR mutations, estrogen levels and LPL activity which may contribute
to inter individual variation in apoB levels.
2.1.1.5. Gain of function mutations in PCSK9. PCSK9 is mainly of hepatic
origin but is also well expressed in the intestine and kidneys. PCSK9
contains a signal sequence (1–30 amino acids), a prodomain (amino
acids 31–152), a catalytic domain (amino acids 153–425) and a
C-terminal domain (amino acids 426–694) [93]. In the ER, proPCSK9 is
autocatalytically cleaved at Q152 resulting in a secreted enzymatically
inactive complex of PCSK9 with its inhibitory prosegment [94]. PCSK9
gene transcription has been shown to be under the control of SREBPs,
HNF-1 [95] and PPARγ [96].
LDL bound to LDLR is internalized in clathrin coated pits and subse-
quently undergoes lysosomal degradation. Following dissociation of
LDL, the LDLR is recycled back to the plasma membrane where it can
bind more LDL. Internalization and shuttling of the receptor towards
the plasma membrane is a continuous process. PCSK9 plays a role in
this process because it promotes the degradation of the LDLR [97].
PCSK9 binds to the extracellular EGF-A domain of LDLR in a post ER
compartment and prevents the receptor recycling to the surface [98].
Models suggest that circulating PCSK9 in sera bind to LDLR. Acidification
of the pH in the endosomes increases PCSK9 affinity to the LDLR and
directs the trafficking of LDLR to lysosomes for degradation [99]. It has
been suggested that amyloid precursor protein-like protein 2 is in-
volved in mediating postendocytic delivery of PCSK9 to lysosomes
[100].
In 2003 gain of function mutations in PCSK9 (OMIM 603776) were
shown to cosegregate in two FH families in which mutations to LDLR
and APOB had been excluded. The mutation was mapped to the 1p34-
32 region [101]. An online database of PCSK9 mutation is now available
PCSK9 @ www.ucl.ac.uk/ldlr/LOVDv.1.1.0/. The clinical features of FH
linked to PCSK9 mutations are similar to LDLR mutations, though excep-
tions exist. PCSK9 patients with the D374Y variant had a more severe
phenotype than the FH patients with known mutations in LDLR, even
those selected as having null mutations. The D374Y mutations showed
higher baseline and post statin treatment cholesterol levels [102]. Bio-
chemical and cellular analysis revealed that the gain of function muta-
tion D374Y resulted in a 10–25 fold higher affinity for LDLR [103]. The
reported co-crystal structure of PCSK9 with the EGF-A domain of LDLR
revealed that at acidic pH Asp374 in PCSK9 forms a hydrogen bond
with His306 within the EGF-A domain. The mutation D374Y would po-
sition a hydroxyl group of tyrosine to form a more favorable hydrogen
bond [104]. A natural gain of function mutation L108R enhances the
natural interaction PCSK9 for LDLR [105]. It has been suggested
(i) that LDL from PCSK9 mutant patients have an impaired capacity to
compete for binding to normal LDLRs [102] and that (ii) the secretion
of apoB100 containing particles is increased from cells expressing the
pathogenic PCSK9 variant [106]. It has been suggested that PCSK9
plays a role in TG rich lipoprotein metabolism at least in certain patho-
physiological conditions [107]. It is likely that in patients with mutations
in the PCSK9 gene, as is the case with patients with FH attributable to
mutations in the LDLR, the nature of the molecular defect has an impact
on the severity of hypercholesterolemia. Two further mutations S127R
and D129G segregated with the FH phenotype. Both mutations have re-
duced autocatalytic activity and were not secreted from cells [108]. The
S127R mutant has only a modest increase in affinity for LDLR. It has been
suggested that the S127R mutant may interfere with intracellular traf-
ficking of LDLR to the cell surface [109].
The PCSK9 mutations causing FH are rare, however more frequent
polymorphism variants have been associated with cholesterol levels
and IHD in population studies [110]. In patients the use of steroids or
mutations in the LDLR gene were additive with the PCSK9 mutation.
Mutations are associated with variable phenotypes in families where
lipid disorders show variable degrees of severity suggesting that the
phenotype of hypercholesterolemia may result from a combination of
environmental and genetic factors [111,112].
2.1.1.6. Autosomal recessive hypercholesterolemia. Over the years a num-
ber of rare cases of autosomal recessive rather than autosomal domi-
nant form of FH have been described [113]. Interestingly no defect in
LDLR function could be detected, though subsequently a recessive null
mutation in a novel gene called the LDL receptor adaptor protein 1
(LDLRAP1 also known as ARH) was observed to cosegregate with hyper-
cholesterolemia in members of these rare families [114]. This was con-
firmed by restoring LDLR function in ARH patient lymphocytes with
the expression of normal ARH protein [115]. To date, autosomal reces-
sive hypercholesterolemia (ARH, OMIM 603813) is caused by mutation
in the LDLRAP1 gene, though as other recessive forms of hypercholester-
olemia emerge this term may need to change. The LDLRAP1 protein con-
tains a phosphotyrosine-binding domain that interacts with LDLR via
the internalization sequence (NPVY) in the cytoplasmic tail of LDLR en-
abling the receptor to engage with the clathrin coated pit machinery for
endocytosis [116]. The conserved motifs of the C-terminal portion of
LDLRAP1 bind to clathrins and to the β2-adaptin subunit of AP-2
[117]. Most mutations of LDLRAP1 are null mutations though a mutation
lacking the phosphotyrosine binding domain has been reported
[116]. Whether the role of LDLRAP1 extends beyond sorting of the
LDLR into clathrin vesicles and its subsequent internalization remains
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
Fig. 7. Defects in LDLR cholesterol processing which cause FH. Dominant mutations in LDLR can affect ligand binding and receptor transcription and recycling [1]. Dominant mutations in
the receptor binding domain of apoB reduce its affinity for LDLR [2]. Dominant mutations in PCSK9 reduce the amount of LDLR at the cell surface [3]. Recessive mutations in LDLRAP1 pre-
vent internalization of the receptor [4].
to be solved. It is however involved in escorting LDLR on its complex
recycling route. Additional mutations in LDLRAP1 may account for
more severe phenotype in families with LDLR mutation [118,119]
(Fig. 7).
2.1.1.7. Other candidate genes. A recessive mutation in the CYP7A1 gene
which encodes the enzyme cholesterol 7α-hydroxylase which catalyzes
the initial step in cholesterol metabolism and bile acid synthesis has
been identified in a single family. High levels of cholesterol were seen
in three homozygous subjects [120]. The enzyme 3hydroxy-3-methyl-
glutaryl-coenzyme A reductase (HMGCR) is a rate limiting enzyme in
cholesterol biosynthesis. Burkhardt et al. [121] identified variants in
HMGCR that contribute to LDL cholesterol levels, across populations.
Both SREBP and the SREBP cleavage-activating protein (SCAP) which
are involved in cholesterol and LDLR biosynthesis are candidate genes
for the development of hypercholesterolemia. Though variants in both
genes have been associated with higher LDL cholesterol concentrations
the evidence that they cause the phenotype is not strong [122].
Sterols in the intestinal lumen enter enterocytes through the
Niemann–Pick C1 like-1 (NPC1L1) transporter and are secreted by
the heterodimeric ATP-binding cassette transporter ABCG5/ABCG8.
Humans express ABCG5/ABCG8 on the apical border of intestinal
enterocytes (though ABCG5 has an additional cytoplasmic component)
and in hepatocytes framing the biliary canaliculi cells [123]. Recent pub-
lications suggest that human NPC1L1 variants may modulate serum LDL
cholesterol levels. Rare NPC1L1 alleles with significantly higher LDL
cholesterol levels were associated with increased IHD risk [124]. Con-
versely, variants classified as dysfunctional were identified from low
cholesterol absorbers. Dysfunction of the variants associated with
poor cholesterol uptake was due to impaired recycling, subcellular lo-
calization, glycosylation or stabilization of NPC1L1 [125].
The rare, recessive disorder, sitosterolemia, (OMIM 210250) is
caused by mutations in either of ABCG5 or ABCG8, that heterodimerize
to form a duplex that promotes sterol excretion. Mutations in either
the ABCG5 or ABCG8 result in increased fractional absorption and de-
creased biliary excretion of neutral sterols. Plant sterols (eg sitosterols,
153
campesterol, and stigmasterol) in addition to cholesterol accumulate in
the blood and tissues of patients with this disorder. Cholesterol levels
are more variable (normal or mild or moderately elevated plasma choles-
terol levels) in sitosterolemia than in other genetic hyperlipidemias, but
can be extremely elevated in some patients. Clinical features may vary
from the presence of tendon xanthomas, atherosclerosis and its compli-
cations to a milder phenotype with very few specific symptoms or
signs. Disease is suspected in patients with elevated levels of plant sterols.
Plasma sterols levels in normal or heterozygotes should not be N1 mg/dL
as opposed to N20–30 mg/dL in patients with sitosterolemia, though
higher levels are found in some heterozygotes [126,127]. Several muta-
tions have been described. A few of the mutations are over represented
in certain ethnic groups eg ABCG5 R389H in Japanese and ABCG8
Y361X in Caucasians [128].
2.2. Primary hypertriglyceridemia
2.2.1. Triglycerides and risk of disease
The scientific statement from the American Heart Association states
that TG represent an important biomarker of IHD risk as it is associated
with atherogenic remnant lipoprotein particles [129]. Because many
human cells can degrade TG, but none cholesterol, it seems more plau-
sible that it is the cholesterol content of remnants that is causal for ath-
erosclerosis development. The specific role for TG as an independent
risk factor for IHD has not been straightforward to make. Elevated TG
typically exists with a range of comorbidities such as obesity, metabolic
syndrome and type 2 diabetes, which obscures a direct relationship be-
tween IHD and TG concentration. Further, there is an inverse relation-
ship between HDL and TG concentration [130]. Remnant cholesterol is
the cholesterol content of TG rich lipoproteins composed of VLDL
(following partial lipolysis by LPL) and IDL in the fasting state and addi-
tionally, with chylomicron remnants in the non-fasting state. Several
meta-analyses indicate a link between fasting/non-fasting TG values
and IHD risk [131]. Both case control and prospective studies have
found that elevated remnant cholesterol is associated with increased
risk of IHD. Elevated nonfasting plasma triglyceride is a marker of
154 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
elevated nonfasting remnant cholesterol and is associated with in-
creased risk for IHD. It has been suggested that a 1 mmol/L increase in
nonfasting remnant cholesterol is associated with 2.8 fold causal risk
for IHD [132,133]. In a recent study, with a Mendelian randomization
design, genetically reduced concentrations of nonfasting plasma TGs
were associated with reduced all cause mortality [134].
TG rich lipoprotein remnants can drive atherogenesis either by
direct mechanisms contributing to plaque formation or by indirect
mechanisms associated with impaired vasodilation and enhanced in-
flammatory reaction [135]. Mechanistically the explanation for a causal
effect of elevated levels of nonfasting remnants on IHD is that remnant
cholesterol, like LDL, can enter and get trapped in the intima of the arte-
rial wall. In line with this, patients with mutations in the LPL gene caus-
ing chylomicronemia with large CMs and VLDLs have an increased risk
of pancreatitis but not an increased risk of IHD. However, some patients
with chylomicronemia due to LPL deficiency have developed significant
atherosclerosis [136]. It has been suggested that elevated LDL levels
cause atherosclerosis without a major inflammatory component,
whereas the inflammatory component of atherosclerosis is driven by
nonfasting remnant cholesterol levels [137]. The increasing evidence
to support non-fasting TG levels to predict IHD risk identifies a need
for accurate and standardized methodology to measure lipoprotein
remnants. However, as lipoprotein remnants are different in composi-
tion of lipids and apolipoproteins as a result of different stages of TG li-
polysis and exchange of lipoproteins, assays are difficult to standardize.
An important question is whether all forms of hypertriglyceridemia are
equally atherogenic. In addition there is a lack of specific target goals for
each component that is applicable to large scale clinical trials. Data from
randomized clinical trials on the clinical benefits of lowering TG are
much less robust than the benefits of lowering LDL-cholesterol with
statins, thus hampering recommendations for the management of in-
creased TG levels [138].
Plasma TG concentration is a complex trait which follows a right
skewed distribution in a population [139]. The classification of elevated
serum TG is generally based on fasting values and varies in different
guidelines. Fasting TG cut-off values for the diagnosis of hypertriglyc-
eridemia have varied form 1.7–2.3 mmol/L (to convert to mg/dL
multiply by 88.6) and vary in different guidelines [140]. Together with
environmental influences, common and rare variants in multiple
genes may determine plasma TG concentration. A mild to severe eleva-
tion of TG (2–5 mmol/L) is a common feature in subjects with obesity,
the metabolic syndrome or type 2 diabetes. More severe elevations in
TG (N5 and 10 mmol/L) is observed in individuals diagnosed with famil-
ial combined hyperlipidemia (FCH, HLP type 2B, elevated VLDL and LDL,
OMIM 144250) and familial hypertriglyceridemia (FHTG, HLP type 4, el-
evated VLDL, OMIM 144600). Severe hypertriglyceridemia (TG N10
mmol/L) is a hallmark of rare genetic disorders due to mutations in
LPL, APOCII, APOAV, GPIHBP1 or LMF1 [141]. Clinical features of increased
TG concentrations are eruptive, tuberous, or palmar crease xanthomas,
hepatosplenomegaly, and when TG concentrations exceed 10 mmol/L,
lipemia retinalis and pancreatitis [142].
Rarely, severe hypertriglyceridemia is a simple monogenic disorder
with a Mendelian (typically recessive) inheritance, often presenting in
childhood and a result of homozygosity or compound heterozygosity
for large effect genetic mutations. More commonly hypertriglyc-
eridemia develops later in life on a background of genetic variants com-
bined with environmental (poor quality diet) and metabolic stressors
(diabetes, metabolic syndrome, certain medications). Recent advances
in knowledge on the common hypertriglyceridemia have changed the
classification of hypertriglyceridemia from the Frederickson classifica-
tion to one with a molecular genetic foundation [51].
2.3. Primary monogenic hypertriglyceridemia
A few patients with plasma TG more than the 95th percentile
have rare monogenic disorders resulting from loss of function
mutations in LPL, APOCII, APOAV, LMF1, and GHIHBP1 genes. Familial
chylomicronemia is a rare autosomal disorder, diagnosed predomi-
nantly in childhood. The frequency of individuals with these elevat-
ed TG syndromes is less than 1:1,000,000 and they fit the definition
of HLP type 1 (OMIM 238600) with elevated chylomicronemia, skin
and eye abnormalities and pancreatitis. HLP type 1 as a monogenic
disorder is frequently caused by mutations in LPL. Mutations in
GPIHBP1, APOCII and APOAV are rare. In one study among patients
with HLP type 1, 51% had mutations in LPL, 16% had a loss of function
mutation in APOCII, GPIHBP1 or APOAV, 21% had common single nu-
cleotide polymorphisms (SNPs) in LPL or APOAV, and 12% of patients
had no genetic mutation [143]. This would suggest that the molecular
diagnosis of familial chylomicronemia requires analysis of LPL gene as
the first choice.
2.3.1. LPL deficiency
LPL deficiency is associated with an absence or decreased LPL activ-
ity in post heparin plasma. Nearly a hundred clinically significant LPL
mutations have been described. Most LPL deficiency mutations are
missense mutations, although nonsense, deletions, insertion, splicing,
and some mutations localized to the peptide signal sequence have
been reported [144]. Some abolish the production of LPL transcripts or
lead to unstable transcripts; certain missense mutations prevent secre-
tion of LPL from the cells, others interfere with the stability of LPL
homodimers; and others yield LPL homodimers that are stable yet
catalytically inactive [145]. The majority of LPL mutations are in the
amino terminal catalytic domain and have been isolated to exon 5 and
6 (Human Gene Mutation Database; HGMD® home page). Both homo-
zygotes and compound heterozygotes have been demonstrated by
classical cloning techniques. Two LPL missense mutations C418Y and
E421K abolish LPLs ability to bind to GPIHBP1 without interfering with
LPL catalytic activity or binding to heparin [146], suggesting that the
binding of LPL to GPIHBP1 requires the C-terminal portion of LPL. Two
further LPL mutations, G409R and E410V render LPL susceptible to
cleavage at residue 297 (a known furin cleavage site) [147].
Patients with mutations in both alleles of the LPL gene may more fre-
quently be associated with partial LPL activity and for these patients the
lipoprotein abnormalities may be more varied. Normal pregnancy is as-
sociated with alterations in plasma lipoprotein and lipid levels including
a two to threefold increase in triglyceride level which reaches its highest
level during the third trimester. This physiological hypertriglyceridemia
is believed to be induced by an increased level of estrogen during preg-
nancy. A correlation between estrogen levels and hypertriglyceridemia
has also been observed in premenopausal women using estrogen con-
taining contraceptives. In patient homozygous for the S172C mutation
with residual LPL activity pregnancy markedly elevated triglyceride
levels [148]. The natural history of biochemical consequences of hetero-
zygosity for mutations in the LPL varies. Plasma TG in carriers of one
defective LPL allele can be normal or elevated; age, obesity and lipid
raising drugs can be contributory to changes in TG levels. Reduced
HDL cholesterol concentrations have been observed in heterozygotes.
The pronounced hypertriglyceridemia observed by decreased plasma
LPL activity may regulate HDL cholesterol levels by (i) increasing the en-
hanced exchange cholesteryl esters to VLDL (ii) altering the remodeling
and maturation of HDL [149,150,151]. Four novel variants H273R,
R333H (most likely to result in a defect in LPL activity rather than syn-
thesis and secretion), p.A125Gfs*22 and p.T85Kfs*13 (predicted to
form a truncated protein) were described by Martin-Campos et al.
[152]. The authors suggest that heterozygosity for LPL variants may cor-
respond to increased susceptibility to hypertriglyceridemia.
Common mutations in the LPL gene: D9N, N291S, W86R, G188E,
P207L, D250N are associated with increased TG levels and may contrib-
ute to susceptibility factors involved in age and sex dependent pheno-
typic expression of increased TG [153]. In certain regions of Quebec
the prevalence of LPL homozygosity is 200 per million and has been
postulated as due to a founder effect in French Canadians [154]. The
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
G188E, P207L and D250N mutations account for most cases of LPL
deficiency in French Canadians [155].
In contrast to other variants the S447X mutant that lacks the
C-terminal serine and glycine is a gain of function mutation. Published
data indicates enhanced post heparin lipolytic activity in the S447X
variant compared to controls though a large number of individuals is
required to unmask this effect [156].
2.3.2. ApoCII deficiency
The importance of apoCII as an LPL activator is established by the ab-
sence of LPL activity in patients with apoCII deficiency (OMIM 207750).
The diagnosis of apoCII deficiency is established by finding a virtual
absence of apoCII in plasma associated with reduced post heparin lipo-
protein lipase activity that is corrected by the addition of apoCII contain-
ing plasma. Since the initial report by Breckenridge et al. [157] many
kindreds with apoCII have been described and the molecular defect
characterized. Patients present with fasting chylomicronemia, hypertri-
glyceridemia, eruptive xanthomas and chronic pancreatitis [158,159].
ApoCII is a constituent of CM, VLDL, LDL and HDL (Table 1). The
human apoCII gene is located on chromosome 19q13.2; it is ~3.4 kb in
length and consists of three introns and four exons [160]. It is a 79
amino acid apolipoprotein. The lipid binding domain of apoCII is located
at the N-terminal and the C-terminal helix is responsible for interaction
with LPL. A variety of nonsense, frameshift and missense mutations
have been uncovered. They are located in the promoter region, the cod-
ing exons or splice junctions. Streicher et al. [161] describe a single nu-
cleotide substitution which diminishes the binding of a transcription
factor to a positive cis-acting element in the promoter region resulting
in the depletion of apoCII in the plasma. Other mutations produce
amino acid exchanges, abnormal RNA splicing, frameshifts or premature
termination codons [162,163,164].
ApoCII deficiency is inherited as an autosomal recessive trait. The
heterozygous state (eg the apoCIIPadova kindred) is not always associat-
ed with abnormalities in lipoprotein pattern, and cannot always be dis-
tinguished by measuring fasting TG or plasma apoCII levels. This
conclusion cannot be extended to all families with apoCII deficiency,
since mutations are heterogeneous, the heterozygote state may have
different levels of apoCII deficiency [165].
2.3.3. LMF1 deficiency
The recent study of a hypertriglyceridemic mouse strain carrying a
combined deficiency of LPL and hepatic lipase (HL) led to the identifica-
tion of a new gene whose protein product is involved in the maturation
of LPL and HL in the ER [26]. The human homolog of this gene, LMF1,
maps on chromosome 16p13.3 and encodes a 567 amino acid protein.
It is a multi-pass transmembrane protein with three soluble domains
protruding into the ER domain. Co-immunoprecipitation studies re-
vealed that LMF1 physically interacts with LPL, HL and endothelial li-
pase (EL) but not with pancreatic lipase. The molecular mechanisms
underlying the lipase chaperone function of LMF1 is unknown. It has
been suggested that LMF1 is involved in the assembly of inactive lipase
monomers to active dimers, either by being responsible for the as-
sembly of dimers or by stabilization of already formed dimers. This
modulation of lipase assembly may be a method of controlling lipase
expression. Further studies are required to explore factors regulating
LMF1 expression and function [166].
A human subject homozygous for a nonsense mutation LMF1
(Y439X) was identified in a patient with markedly increased TG, tuber-
ous xanthomas and repeated episodes of pancreatitis. The severe
hypertriglyceridemia was due to LPL deficiency, as determined by a
93% decrease in post heparin LPL activity in the plasma of the affected
individual, as well as a 50% decrease in HL activity [167]. A second ho-
mozygous nonsense mutation W464X was identified in another patient
with severe hypertriglyceridemia with a decrease in post heparin LPL
activity by 76% [168]. Lipodystrophy (loss of adiposity) was associated
with W464X suggesting diversity in phenotypes. It is conceivable that
155
LMF1 may affect the metabolism of a broader range of proteins other
than lipases but this awaits the identification of other LMF1 client pro-
teins [166]. LMF1 deficiency is inherited as an autosomal recessive syn-
drome, and reports of clinically significant mutations are rare.
2.3.4. GPIHBP1 deficiency
GPIHBP1 is an endothelial cell protein that is hypothesized to serve
as an endothelial cell platform for LPL mediated lipolysis [169]. Mature
GPIHBP1 is a relatively short protein (184 amino acids), with four prin-
cipal domains: a signal peptide, an amino terminal acidic domain, a cys-
teine rich Ly6 motif (a three fingered domain specified by 10 cysteines)
and a hydrophobic carboxy terminal motif that is replaced within the ER
by a GPI anchor, following interaction of the C-terminal domain with
GPI transamidase. GPIHBP1 is cleaved at residue 153 and replaced by a
GPI anchor [170,171]. The negatively charged acidic domain with a
large number of aspartates and glutamates binds LPL that contains pos-
itively charged heparin binding domains. The structural Ly6 domain is
additionally required for LPL binding [172].
During recent years homozygous GPIHBP1 mutations have been un-
covered in patients with severe chylomicronemia. The first GPIHBP1
mutation was identified in a patient with lifelong chylomicronemia.
The patient was homozygous for a Q115P mutation in the Ly6 domain.
Cell culture studies revealed that the mutant GPIHBP1 reached the cell
surface normally but could not bind LPL or chylomicrons [172]. In
some case reports mutations involve the conserved cysteine residue in
the Ly6 domain, C65Y, [173], C65S, C68G [174], C89F [175], C68Y
[176], C14F and C68R [177]. Although the C14F/C68R double homozy-
gous mutations exhibited normal LPL binding activity, the levels of mu-
tant protein were extremely reduced compared to those of wild type
proteins in vitro, possibly due to the instability of mutant GPIHBP1 pro-
teins [177]. Charriere et al. [175] identified a G175R mutant in the C-
terminal domain that targets the GPI anchorage crucial to membrane
transfer. Cell surface expression was significantly decreased for the
G175R variant. Rios et al. [178] identified a 17.5 kb deletion that encodes
the whole of the GPIHBP1 gene and Berge at al. [179] identified dele-
tions of exons 3 and 4 of the GPIHBP1 gene in an extended family ped-
igree with chylomicronemia.
GPIHBP1 mutants frequently present with childhood onset
chylomicronemia and pancreatitis, though probands can present as
adults [178]. Rios et al. [178] report lipemia retinalis and several
eruptive xanthomas on the elbows at presentation, though reports
of eruptive xanthomas are variable. LPL activity in adipose tissue is
normal and breast milk from affected subjects contained normal
LPL activity levels. Enzymatically active LPL entered the plasma, al-
beit with delayed kinetics, after an injection of heparin, although
the increase in LPL activity is lower in affected patients [174]. Het-
erozygotes of affected kindred had normal lipid levels, suggesting
that half normal amounts of a functional GPIHBP1 are sufficient for
normal lipolytic processing of TG rich lipoproteins and that inheri-
tance is autosomal recessive [180].
2.3.5. ApoAV deficiency
In humans ApoAV gene locus forms part of the gene cluster of APOAI/
CIII/AIV/AV gene cluster on chromosome 11q23. The sole site of apoAV
synthesis is the liver where it is translated as a 366 amino acid
preprotein. Following intracellular cleavage of a 23 amino acid signal
peptide, mature apoAV appears in the plasma associated with VLDL,
HDL and to a lesser extent chylomicrons (Table 1). The role of apoAV
in TG metabolism is evolving and the specific mechanism by which
ApoAV reduces plasma TG is still unclear. ApoAV plasma levels are
quite low (~150 μg/L or 4 nM) but a number of observations suggest it
is a strong modulator of plasma triglycerides. It has been suggested
that (i) apoAV binds to GPIHBP1 in vitro and this interaction facilitates
LPL mediated hydrolysis of CM (ii) apoAV may act as a ligand for hepa-
tocellular receptors (iii) apoAV plays a role in intracellular TG storage
and/or mobilization [181,182].
156 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
In GWAS APOAV gene was found to be one of the genes influencing
TG levels [183]. APOAV variants have been linked to polygenic hyperlip-
idemia types 2B, 4 and 5 [184]. Only a few alterations in the APOAV gene
have been found to cause the accumulation of CM, HLP type 1, or accu-
mulation of CM and VLDL, HLP type 5 (OMIM 144650). Patients present
with pancreatitis, eruptive xanthomas and heptoslenomegaly. Marcais
et al. [185] identified Q139X mutation in the APOAV gene associated
with chylomicronemia. The patient was a homozygote, severe hypertri-
glyceridemia in the heterozygotes was associated with a second allele of
TG raising minor APOAV haplotype, suggesting that severe hypertriglyc-
eridemia in heterozygotes may be an interplay between apoAV defi-
ciency and other TG raising apoAV haplotypes. The full expression of
the syndrome was characterized by HLP type 5 hyperlipidemia. APOAV
gene mutation found in chylomicronemic subjects are Q97X (with com-
plete ApoAV deficiency) [186,187] and Q145X (predicted to generate a
truncated apoAV) [188]. A mutation that resulted in an 8 amino acid loss
in the signal sequence resulted in a mutated apoAV which was intracel-
lularly missorted to lipid droplets and not secreted. This mutation pre-
sented as severe hypertriglyceridemia in a 11 month old boy, but only
while he was being breastfed [189]. Mutations in the APOAV gene stud-
ied because of severe hypertriglyceridemia in three patients without
mutations in the LPL, APOCII, GPIHBP1 gene were: 12 bp deletion
p.S232_L235del; missense mutation p. L253P and the 4 bp deletion
resulting in a C-terminallly truncated protein, p. D332VfsX4. Structural
and functional analysis suggests that the mutant residues were impor-
tant in apoAV function [190]. One patient who was compound heterozy-
gous for the mutations c.289CN T (p. Q97*) and c.823CN T (p.Q275*) in
the APOAV gene presented with marked hypertriglyceridemia and his-
tory of recurrent pancreatitis [191].
Plasma TG concentrations were found to be associated with poly-
morphisms of the APOAV gene. Three different haplotypes of APOAV
have been described to result in significant differences in TG levels.
Five polymorphisms were found to define three common haplotypes
(denoted APOAV*1, APOAV*2 and APOAV*3). These three haplotypes
represent 82%, 8% and 8% of the APOAV chromosomes examined.
APOAV*2 is distinguished from the common haplotype APOAV*1 by
four nucleotide substitutions (− 1131T N C, c.-3A NG, IVS3 + 476G N T
and c1259TNC). APOAV*3 is distinguished from the common haplotype
by the substitution c56CN G (S19W). Both APOAV*2 and APOAV*3 are
associated with higher TG levels [192].
2.3.6. LPL inhibitors
Brunzell et al. [193] described a mother and her son with HLP type 1
with very low levels of postheparin plasma lipolytic activity, and circu-
lating inhibitor of lipoprotein lipase, who differed from subjects with li-
poprotein lipase deficiency in that the enzyme was present in adipose
tissue at much higher levels than those seen in normal subjects. They
also differed from subjects with deficiency of apoCII in that apoCII was
present in their plasma in normal or elevated amounts. The authors sug-
gest an autosomal dominant status for the carriers of the LPL inhibitor.
Anti LPL antibodies have been described in rheumatic diseases, mainly
systemic lupus erythematosus and systemic sclerosis [194]. Ashraf
et al. [195] describe a patient with Sjogren's syndrome, with no disease
causing mutations in GPIHBP1, APOAV, APOCII and LMF. LPL antibody
was detected by immunoblotting. The patient responded to immu-
nosuppressive therapy for Sjogren's syndrome with resolution of
hypertriglyceridemia,
2.3.7. Polygenic causes of hypertriglyceridemia
In contrast to the rare familial syndromes, together with environ-
mental influences, common and rare variants in multiple genes may
collectively determine an individual's plasma TG concentration. GWAS
test for associations between common genetic variations with SNPs
and either quantitative or discrete traits. Differences in gene structure
that occur infrequently in populations ie less than 1% are called mu-
tations whereas differences that occur more frequently ≥ 1% are
called polymorphisms [196]. A second strategy, to test for gene asso-
ciations with traits, is the linkage approach in which families are
screened for loci linked to hypertriglyceridemia [197]. An emerging
suggestion is that multiple common and rare variants in multiple
genes with different functions contribute jointly to a given pheno-
type [198]. Average triglyceride concentrations in people could be a
balance between harmful and protective alleles.
The recent definitive study by the Global Lipids Genetics consortium
which analyzed N100,000 subjects identified 95 loci that showed
GWAS association with normal variation in lipid traits [199]. The
analysis identified 32 loci which contributed to variations in plasma
TG concentration. The SNPs at each of the 32 loci contributed to 9.6%
of the total variation in plasma TG concentration, corresponding to
25–35% of the total genetic contribution to TG variability. The major-
ity of genetic variation associated with plasma TG remains unattrib-
uted [200].
Wang et al. [201] sequenced DNA from non diabetic hypertriglyc-
eridemia patients and found a substantial proportion of heterozygous
LPL, APOCII and APOAV mutations among these patients. GWAS showed
that APOAV and LPL already known to be involved in monogenic
hypertriglyceridemia were involved in plasma triglyceride concentra-
tion as were the newer players GCKR (glucokinase regulator protein)
and APOB [202]. The insertion of two leucines in the leucine stretch of
the signal peptide of PCSK9 (p.L15_L16ins2L) is found in patients with
FCH as well as FH, suggesting that PCSK9 variants may contribute to
this multigenic disease and that there is a potential overlap between
the two disorders. It is possible that this mutation is in linkage disequi-
librium with other variants within the PCSK9 gene [203]. In the Japanese
population TRIB1 and GALNT2 have been associated with lipid metabo-
lism. TRIB1 encodes tribble-1 which regulates activity of MAPK and
GALNT2 encodes N-acetylgalactosaminyltransferase 2 which is involved
in protein glycosylation [204].
For the TG associated loci, genetic determinants of plasma lipopro-
tein metabolism from the Global Lipids Genetics consortium study
showed association with different Frederickson associated HLP pheno-
types. This suggests that the common variants increase susceptibility
to hypertriglyceridemia and the HLP phenotypes (HLP type 2B (FCH),
3, 4 (FHTG) and 5). The authors reported associations of common vari-
ants with both HDL cholesterol and LDL-cholesterol and suggest that
pleiotropic variants were jointly associated with plasma TG, LDL choles-
terol and HDL cholesterol. Both rare genetic variants and lipid associated
common variants occur across the HLP phenotypes and may partly ex-
plain the phenotypic heterogeneity among hypertriglyceridemic pa-
tients. It is hypothesized that the extremes of a complex genetic trait
such as patients with the Frederickson HLP phenotypes are found as a
cumulative contribution of several common alleles, though some pa-
tients such as most of those with HLP type 1 and a percentage of those
with HLP type 2A and type 5 have a rare loss of function mutation
with a large effect [205,206]. The population prevalence for HLP type
2B is estimated as 1:40; HLP type 4 as 1:20 and HLP type 5 as 1:600
[140].
Further analysis suggests a mechanistic role in lipoprotein me-
tabolism for at least some of the GWAS identified loci. For example,
GCKR encodes glucokinase regulatory protein, an allosteric regulator
that allows mobilization of glucokinase in response to increased
cellular glucose concentrations. The P446L variant in the GCKR
promotes glucose uptake and mobilization, de novo TG synthesis
while attenuating fatty acid oxidation [207]. Mutations in CREBL3
that produced nonfunctional CREBH protein resulted in hypertri-
glyceridemia in humans [208]. Upstream transcription factor
(USF1) targets fatty acid synthase, and can be anticipated to be asso-
ciated with hyperlipidemia [209]. It has been suggested that the indi-
vidual genes identified in hypertriglyceridemia can be incorporated
into models of (i) adipose tissue dysfunction (ii) hepatic fat accumu-
lation and VLDL overproduction (ii) delayed clearance of apoB con-
taining particles [210].
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
2.3.8. Novel rare genetic diseases associated with hypertriglyceridemia
In families with severe transient childhood hypertriglyceridemia
and fatty liver followed by hepatic steatosis and sustained liver dys-
function, a mutation in the GPD1 gene which encodes glycerol-3-
phosphate dehydrogenase was identified. One possible mechanism
for transient hypertriglyceridemia is possibly by limiting the conver-
sion of Glycerol-3-phosphate to dihydroxyacetone phosphate, and
increasing the amount of glycerol-3-phosphate available for TG syn-
thesis [211,212].
HL is an enzyme made primarily by hepatocytes. Evidence from clin-
ical and animal studies supports a role for HL in the metabolism of sev-
eral lipoprotein classes. HL has been reported to augment the uptake of
HDL cholesterol by the liver through a reverse cholesterol transport pro-
cess; be responsible for the hydrolysis of TG in LDL and participate in the
conversion of VLDL to LDL. Missense mutations in the encoding exons
R186H, L334F, S267F, T383M, and A174T have been described and
found to be responsible for HL deficient phenotypes. It is a rare genetic
disorder and appears to be inherited as an autosomal recessive trait.
HL deficient patients present with hypercholesterolemia, hypertriglyc-
eridemia though patients display variable phenotypes. Consistent find-
ings are an elevation in HDL2 cholesterol and marked TG enrichment of
LDL and HDL particles, and some affected individuals have premature
cardiovascular disease [213,214].
Other monogenic conditions that do not primarily affect lipoprotein
metabolism can cause hypertriglyceridemia. Inherited lipodystrophies
are rare autosomal dominant or recessive disorders characterized by se-
lective and variable loss of adipose tissue. Marked hypertriglyceridemia
is a common feature of these disorders and highlights the role of adipose
tissue in lipid homeostasis. Eight different genetic loci, including
1-acylglycerol-3-phosphate-O-acyltransferase 2, Berardinelli-Seip con-
genital lipodystrophy 2, caveolin 1, lamin A/C, PPARγ, v-AKT murine
thymoma oncogene homolog 2, zinc metalloprotease and LMF1 have
been linked to different lipodystrophy syndromes. Mutations in these
genes may cause fat loss and dyslipidemia through multiple mecha-
nisms, which remain to be fully elucidated; however, they may involve
defects in development and differentiation of adipocytes, and prema-
ture death and apoptosis of adipocytes. Hypertriglyceridemia is a conse-
quence of increased VLDL synthesis from the liver, which is loaded by
ectopic TG deposition, reduced clearance of triglyceride-rich lipopro-
teins or both [215,216].
2.4. ApoE
2.4.1. ApoE and lipoprotein metabolism
The apoE gene is located on chromosome 19q13.2 within a gene
cluster with apoCI and apoCII. Human apoE has 299 amino acids. The
gene contains 4 exons. The 18 amino acid signal sequence is removed
prior to secretion. ApoE contains two independently folded functional
domains: a 22 kDa N-terminal domain (residues 1–191) and a 10 kDa
C-terminal domain (residues 222–299). A short hinge region connects
the two domains. The N-terminal domain exists in a lipid free state as
a 4 helix (residues 24–42, 54–81, 87–122, 130–164) bundle of amphi-
pathic α-helices and contains the LDL-receptor binding region (residues
136–150 in helix 4). The C-terminal domain has a high affinity for lipids
and is responsible for lipoprotein binding. ApoE interacts with the
LDLR, LRP and heparan sulfate proteoglycans (HSPG), and serves an
antiatherogenic function by mediating the clearance of VLDL and CM
remnants. The liver is the major source of apoE. ApoE associates with
most lipoproteins except for LDL. ApoE exists in three major isoforms,
apoE2, apoE3 and apoE4. ApoE3 is the most common isoform and has
cysteine and arginine at residues 112 and 158, respectively. ApoE2 con-
tains cysteine and apoE4 contains arginine at both sites. Both apoE4 and
apoE3 bind to the LDLR with high affinity whereas apoE2 exhibits poor
binding to the LDLR and is associated with HLP type 3. Association of
apoE with lipid is required for its high affinity binding to the LDLR [18,
217,218]. The N-terminal domain contains the primary HSPG binding
157
site colocalised with the LDLR binding site. Molecular modeling suggests
that Arg136, Ser139, His140, Arg142, Lys143, Arg145, Lys146 and
Arg147 that line the groove of helix 4 make direct contact with either
the 2-O-sulfo groups of the iduronic acid monosaccharides or the N
and 6-O-sulfo groups of the glucosamine sulfate monosaccharides
[219]. The NMR structure of apoE3 suggests that the C-terminal domain
presents a large exposed hydrophobic surface that likely initiates bind-
ing to lipids. ApoE3 displays salt bridges and hydrogen bonds causing
shielding of the LDLR binding region from binding to receptors, by the
C-terminal [17]. Relative to apoE3 more apoE4 is found in VLDL than
HDL. The distribution of apoE2 was similar to apoE3, indicating that
cysteine-arginine interchange at position 158 does not affect distribu-
tion [220]. Lipid free apoE interacts with ABCA1, and the lipid
translocase activity of ABCA1 generates HDL-apoE particles. The three
apoE isoforms showed similar kinetics in their abilities to induce
ABCA1 dependent cholesterol efflux [221]. Studies on apoE deficient
mice suggest that overexpression of apoE3 in apoE−/− livers stimu-
lates VLDL secretion, indicating a role for apoE in hepatic TG secretion
independent from its role in lipoprotein clearance [222]. The differential
effects of overexpression of human apoE3 on plasma cholesterol and TG
metabolism was investigated in transgenic rabbits. In apoE3 high
expressors, the elevated expression of apoE3 appeared to stimulate
hepatic VLDL production and inhibit VLDL lipolysis. The overexpression
of apoE3 in transgenic rabbits altered the plasma lipid profile to resem-
ble the combined hyperlipidemia phenotype. The VLDL from high
expressors was enriched with apoE3 and decreased in apoCII, which
contributed to a decrease in lipolysis. The authors suggest that the dif-
ferential expression of apoE3 may play a role in modulating plasma cho-
lesterol and TG content [223].
ApoE3 and apoE4 bind similarly to LDLR, but compared to apoE3,
apoE4 reduces plasma cholesterol less in humans. It has been suggested
that the C112R substitution located in the helix region, destabilizes it,
and changes the interaction between the N and C terminal domains,
which changes the organization of segments which play a critical role
in the interaction of lipid surfaces. ApoE4 exhibits a better lipid binding
ability than apoE3, and coupled with the VLDL particle surface being
predominantly covered with lipid (in comparison to HDL) leads to
apoE4 binding better than apoE3 to VLDL. Studies in apoE-null mice
suggest that the expression of apoE4 gave rise to higher VLDL cholester-
ol and lower HDL cholesterol relative to apoE3. It has been suggested
that VLDL remnant clearance is decreased in apoE4 expressing mice
and this decreases the amount of VLDL components available for incor-
poration into HDL accounting for the proatherogenic profile (higher
VLDL cholesterol/HDL cholesterol ratio) [224,225]. Meta-analysis
suggests a linear relationship between apoE genotypes (when or-
dered as E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, E4/E4) with LDL cholester-
ol and with coronary risk [226]. The apoE2 allele is associated with an
anti-atherogenic lipid pattern (lower LDL cholesterol and higher
HDL cholesterol) in children [227]. However, those apoE2 homozy-
gotes who develop HLP type 3, because of secondary risk factors
are at increased risk for both coronary and peripheral artery athero-
sclerosis. Although the cholesterol lowering effect of apoE2 has been
confirmed in several populations the mechanism is not completely
understood. Studies with transgenic mice suggest that apoE2 de-
creases LDL cholesterol by impairing LPL mediated hydrolysis of
VLDL, mostly by displacing apoCII. Effects of apoE2 on cholesterol
and TG in transgenic mice were dose dependent. Plasma TG levels
(mostly in VLDL and IDL) increased as apoE2 levels increased. Simi-
larly human apoE2 homozygotes who are hypolipidemic have low
levels of LDL cholesterol and a slight accumulation of TG. One expla-
nation is that sufficient apoE2 is present to impair lipolysis of TG rich
lipoproteins but sufficient apoE2 is present to mediate remnant
clearance by the LDLR or HSPG/LRP [228]. Overt hypercholesterolemia
and hypertriglyceridemia can be precipitated when other factors
which further stress the already compromised remnant catabolism of
apoE2 homozygotes are present.
158 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
Once internalized triglyceride rich lipoproteins (CM and VLDL) dis-
integrate in peripheral endosomes. Although core lipids and apoB are
targeted to lysosomes, apoE is targeted to recycling endosomes or to
the Golgi secretory pathway. HDL or purified apoAI stimulates the re-
lease of internalized apoE in hepatocytes and fibroblasts. It has been
suggested that HDL derived apoAI is internalized and targeted to pre-
existing apoE/cholesterol containing endosomes to promote apoE
recycling and cholesterol efflux. ApoE4 is less efficient compared with
apoE3 in promoting cholesterol efflux in fibroblasts and astrocytes. It
has been speculated that the inefficient processing of intracellular cho-
lesterol could contribute to the association of apoE4 with atherosclero-
sis. Depending on the apoE isoform different aspects of lipid metabolism
seem to contribute to the development of dyslipidemia [229]. Of inter-
est is that individuals with the apoE4 allele have an increased risk and
earlier onset of Alzheimer disease [230].
2.4.2. Pathophysiology of dysbetalipoproteinemia
Familial dysbetalipoproteinemia or HLP type 3 is characterized by el-
evated plasma triglycerides and cholesterol that predisposes affected
subjects to the development of premature atherosclerosis. The hyperlip-
idemia is caused by accumulation of CM remnants and IDL known col-
lectively as β-VLDL, these abnormal apoE enriched lipoproteins are
the hallmark of the disease. HLP type 3 associated with homozygosity
for apoE2 presents with recessive inheritance. In a Dutch population,
apoE2 homozygosity occurred at a frequency of 0.6%, of whom 18% pre-
sented with HLP type 3 [231]. A study by Feussner et al. [232] shows that
the prevalence of HLP type 3 among apoE2 homozygous relatives of
German HLP type 3 patients is 30%. The condition generally does not
present until adulthood in men and in the post-menopausal years in
women. Most apoE2/E2 subjects are either normolipidemic or even
hypocholesterolemic, onset of dysbetalipoproteinemia requires other
metabolic stressors. ApoE2 is essential but not usually sufficient to
cause HLP type 3. Overproduction of lipoproteins (eg diabetes),
impaired lipolysis of lipoproteins and decreased remnant clearance
(eg hypothyroidism) can precipitate dyslipoproteinemia in patients
who are predisposed to HLP type 3 [233]. Metabolic conditions include
insulin resistance [231], diabetes mellitus, nephrotic syndrome, obesity
and hypothyroidism. Overproduction of VLDL may also be the result of
excess alcohol intake or pregnancy [234]. Certain drugs: anti-psychotic
medications [235,236] and antiretroviral therapy [237] have been asso-
ciated with HLP type 3.
Several groups have studied genetic factors that may contribute to
the expression of HLP type 3. Data from family studies suggest that
one or more genes are additional factors predisposing to HLP type 3.
Hennemann et al. [238] found the frequency of rare alleles APOCIII
3238 G NC and APOAV -1131T NC was significantly higher in the HLP
type 3 patients compared with normolipemic apoE2/E2 patients. In ad-
dition they found that the LPL c.27 GNA mutation was associated with
more severe hyperlipidemia whereas the APOAV c.56 G N C mutation
was weak modifier. The molecular mechanisms underlying the associa-
tion with hypertriglyceridemia were still unclear. Other mutations lo-
cated near the polymorphic site eg within the APOAI/CIII/AIV gene
cluster near the APOCIII 3238 G N C polymorphism, could be candidates
for the causative mutation. The authors conclude that polymorphisms in
lipolysis genes are associated with the severity of HLP type 3 in apoE2/
E2 homozygotes. Other studies suggest that the S19W variant of
apoAV is a factor in determining HLP type 3 in patients with apoE2/E2
genotype [239]. The N291S variant in the LPL gene predisposes to
more severe hypertriglyceridemia [240]. The N291S substitution was
suggested as a predisposing factor contributing to the development hy-
perlipidemia when associated with apoE2 genotype [241]. Other studies
suggest that it is unlikely that the D9N, N291S, and S447X variants in the
LPL gene play an important role in the development of HLP type 3 [242].
HLP type 3 has been associated with the S267F mutation in HL [243].
Rare apoE variants have been identified that have been implicated in
a dominant mode of inheritance ie subjects heterozygous for the
mutation display HLP type 3. ApoE variants associated with a dominant
inheritance HLP type 3 are associated with amino acid substitution in
the LDLR binding region (residues 136, 142, 145, 146, 147). Wardell
et al. [244] describe a patient who was heterozygous for apoE2 and
apoE2 with a further R136S mutation who presented with HLP type 3.
A further heterozygous variant apoE3/E2(R136C) has been associated
with HLP type 3. Two children in the same family were carriers of the
rare mutation and were heterozygous for the apoE4/E2(R136C) and
apoE3/E2(R136C) variant and were hyperlipidemic [245]. ApoE3 has ar-
ginine in position 136. Carriers who were heterozygous for the mutation
apoE3(R136H) displayed an increased concentration of VLDL cholester-
ol and TG. All carriers showed β-VLDL on electrophoresis though they
did not display clinical features [246]. Two siblings with apoE2/
E3(R136S) phenotype presented with HLP type 5. Rare mutations in
the apoE gene and the associated HLP type are summarized by Vialettes
et al. [247]. It is controversial if mutations in the Arg136 site have a dom-
inant mode of inheritance. The presence of the apoE2(R136S) variant
has been linked with incomplete dominance of HLP type 3 [248].
Other studies suggest that ApoE2(R136S) mutation fully expresses
HLP type 3 when associated with a second allele coding for apoE2 and
only partially in association with a second allele coding for apoE4
[249]. Two heterozygous carriers apoE3/E2(R136C) carriers were classi-
fied as HLP type 4. The authors suggest that apoE2(R136C) causes
dysbetalipoproteinemia in a recessive fashion [250]. Double mutants
have been reported in apoE. Homozygosity for apoE (E13K, R145C)
was associated with severe HLP type 3 in a 24 y old female [251].
ApoE variants causing dominant transmission of HLP type 3 differ
from apoE2 in that they not only exhibit decreased binding to LDLRs
but also to HSPG. The recessive nature HLP type 3 and the variable ex-
pression of hyperlipidemia in patients with apoE2 may be explained
by the high capacity of remnants possessing apoE2 to bind to HSPG.
Impaired HSPG interaction may be only one factor responsible for the
remnant accumulation in the patients with dominant transmission of
HLP type 3. Increased defective apoE to apoE3 ratio on the remnant lipo-
protein can overwhelm the normal binding activity of apoE3 and could
result in impaired interaction with HSPG [252].
A single mutation R142L in the heterozygous state apoE2(R142L)/E3
presented with dysbetalipoproteinemia [253]. Some of the mutations
associated with dominant HLP type 3 are R142C [254], K146Q [255]
and K146E [256]. The double mutation K146N/R147W presented at an
early age, with a proband who developed eruptive xanthomas at the
age of 3 [257]. Transgenic mice studies suggested that the mutant was
a dominant negative ligand which prevented receptor mediated rem-
nant clearance and inhibited the biogenesis of HDL [258]. Heterozygous
mutation with a dominant form of mutation ApoE3/ApoE4(R142C)
were described in 1983. Studies with transgenic mice suggest that this
mutation alters the function of LPL, LCAT, decreased lipoprotein clearance
and inhibited the maturation of discoidal HDL into spherical HDL particles
[259]. The apoE2(R145C) variant has been associated with an autosomal
dominant mode of inheritance with incomplete penetrance [260]. A var-
iant of apoE4 which included an inframe repeat of 21 nucleotides (coding
for 7 amino acids) in exon 4 commonly referred to as apoE-Leiden, is
associated with dominantly inherited dysbetalipoproteinemia with a
high degree of penetrance. Age, however, exerts a significant effect on
the severity of the hyperlipidemia. ApoE-Leiden possesses a reduced
LDLR binding activity [261].
A rare form of HLP type 3 associated with apoE deficiency, with un-
detectable plasma apoE was reported in 1981. The deficient apoE gene
resulted from a point mutation which causes aberrant splicing of the
third intron of the apoE gene resulting in low levels of aberrant forms
of mRNA [262]. The genetic pattern of the kindred is consistent with
an autosomal recessive mode of inheritance in which only the homozy-
gotes develop tuboeruptive xanthomas. The proband's brother had
striking xanthomas in unusual locations: ears, palms, elbows and
knees. The concentration of apoE in homozygotes was b0.5% of that ob-
served in normal. Heterozygotes had a mean plasma apoE concentration
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
that was ~50% of normal [263]. ApoE deficiency resulted (i) impaired
catabolism of VLDL/chylomicron and their remnants due to lack of
direct removal (ii) an increased rate of catabolism of LDL apoB100, likely
due to upregulation of LDLR activity due to decreased hepatic cholester-
ol content caused by poor delivery of apoE deficient lipoproteins,
and the inability of apoE deficient VLDL/IDL to compete with LDL for
the receptor (iii) reduced VLDL production (iv) delayed catabolism of
Lp(a) [264].
Several other patients with apoE deficiency have been reported
[265,266]. A mutation that causes a truncated apoE with a high VLDL
cholesterol/TG ratio in the homozygote was reported by Lohse et al.
[267]. Other rare mutants are a 10 bp deletion in exon 4 leading to a pre-
mature stop codon in amino acid 229 [268], a deletion in codon 31 lead-
ing to premature stop in codon 60 [269] and a G insertion in codon 95 or
96 leading to a premature stop at codon 146 [270]. Heterozygosity for
the apoE deficient mutations may be clinically silent, clinical expression
may depend on the other apoE allele. The apoE null allele in combina-
tion with apoE2 resulted in HLP type 3, but when inherited with
apoE4 had no marked effect on plasma lipids [271].
2.4.3. Clinical presentation
Untreated patients can present with cutaneous and tendinous
xanthoma which are not specific for dysbetalipoproteinemia, however,
palmar crease xanthomas are specific for the disease. Other clinical
manifestations are atherosclerosis and when severe hypertriglyc-
eridemia is present, pancreatitis [140].
Electrophoretic demonstration of β-VLDL is considered a gold stan-
dard for the diagnosis of this disorder, though no simple diagnostic
test is available. In the series by Marais et al. [272] the fasting TG ranged
from 1.6–63.4 mmol/L with a median of 4.9 mmol/L, the fasting choles-
terol ranged from 4.8–34.3 mmol/L with a mean of 11.79 mmol/L, the
mean molar ratio of total cholesterol/TG was 2.07 ± 1.03 (median
1.96, range 0.39–7.63). The authors suggest that dyslipoproteinemia
should be considered in all patients with a mixed combined hyperlipid-
emia total cholesterol to TG ratio around 2:1, but should also be consid-
ered in severe hypertriglyceridemia. Cholesterol enriched VLDL is
diagnostic of dysbetalipoproteinemia but sample preparation requires
ultracentrifugation which is not available in routine diagnostic laborato-
ries. Each VLDL, LDL and IDL lipoprotein contains one apoB100 molecule,
larger lipoproteins contain proportionately more lipid and less apoB than
smaller molecules. A VLDL-cholesterol to serum TG molar ratio ≥ 0.69
(≥ 0.30 when using mg/dL) reflects the presence of cholesterol rich
β-VLDL and can be diagnostic of HLP type 3 [273]. Other data suggest a
total cholesterol/apoB ratio N 6.2 and TG/apoB ratio b 10.0 or conversely
the apoB/total cholesterol ratio can be used to diagnose HLP type 3
[274,275].
2.4.4. Lipoprotein glomerulopathy
Lipoprotein glomerulopathy (LPG) was first reported in 1989. Cases
were mainly reported in east Asia including Japan and China though
later cases were reported in Europe and the United States, suggesting
that this disease may be more common in European populations than
originally thought. LPG is a rare inherited renal disease, transmitted as
autosomal dominant with incomplete penetrance. Pathologically LPG
is characterized by dilated glomerular capillaries that are filled with li-
poprotein rich materials called lipoprotein thrombi. In addition to histo-
logical findings clinical manifestations are proteinuria and dyslipidemia.
Proteinuria is sometimes mild but in most cases progresses to nephrotic
syndrome [276].
The hypothesis that abnormal lipoproteins with rare apoE mutants
accumulate in the glomeruli was supported by the discoveries of apoE
Sendai (R145P) and apoE Kyoto(R25C) [277,278]. The virus mediated
transduction of apoE Sendai resulting in the development of LPG in
apoE deficient mice confirmed the etiologic role of apoE mutation in
LPG [279]. The distribution of apoE2 Sendai among lipoprotein classes
was similar to apoE2 and apoE3. ApoE2 Sendai showed impaired
159
binding to the LDLR but only moderately reduced binding to heparin.
Hyperlipidemia has not been consistently observed in LPG patients
without nephrotic syndrome. Patients with LPG do not reveal the char-
acteristic clinical features of HLP type 3, palmar xanthomas or athero-
sclerosis. ApoE concentrations are elevated in LPG. In LPG, lipoprotein
deposition occurs in the lumen of the capillaries. In HLP type 3 lipids
may accumulate in mesangial foam cells [280].
Although evidence suggests a direct association between apoE mu-
tations and LPG, the mechanism that leads to the deposition of the lipo-
proteins in the glomerulus is unknown. A study of LPG linked apoE3
variants, R145P, R147P, R158P showed that the mutations induce signif-
icant thermodynamic, structural, and aggregation-related perturbations
in the molecule of apoE3 that cause unfolding of the N-terminal domain
to a more molten-globule like structure. This may be the result of the
substitution of the helix breaker proline residue in a helical segment
[281]. In another murine experimental study renal lesions specific to
LPG were shown to develop in Fc receptor γ chain (FcRγ) knock out
mice in which chronic graft-versus-host disease had been induced. In
studies with FcRγ/apoE knock out mice the human apoE3 injected
mice showed LPG like changes, suggesting that macrophage dysfunc-
tion contributed to the development of LPG in the presence of apoE ab-
normalities. It has been suggested that dysfunction of lipid clearance in
the kidney may be another mechanism for development of LPG [282].
The report by Zhang et al. [283] of LPG in patients without apoE muta-
tions suggest that multiple factors other than apoE are involved in the
pathogenesis of LPG.
Hu et al. [284] studied 35 patients with ApoE3(R25C) mutation
within 31 unrelated families and 28 (apoE3 (R25C) mutation) asymp-
tomatic relatives. The existence of 28 asymptomatic relatives suggested
an incomplete penetrance for the apoE3(R25C) mutation. An ApoE3 ho-
mozygote, heterozygous for a 54 bp deletion from exon 4 causing a 18
amino acid deletion (Q156-G173) presented with LPG and systemic
atherosclerotic complications. One of his daughters heterozygous for
the mutation did not have proteinuria or atherosclerosis diseases
[285]. A few other mutations associated with LPG are apoE(R150P)
[286], apoE2(R150G) [287], apoE3(R114C) [288] and apoE(R158P)
[289]. More than a 100 cases have been reported since 1989 [290].
Recurrence of LPG in a transplanted kidney suggests that LPG is
caused by extra renal factors [291]. Results from case studies suggest
that fibrates may improve renal pathology and may result in complete
remission of the disease [292].
2.5. Decrease in concentration of lipid or lipoprotein
2.5.1. Familial hypobetalipoproteinemia
Familial hypobetalipoproteinemia (FHL) is characterized by abnor-
mally low levels of apoB containing lipoproteins in the proband, no un-
derlying medical cause (eg malnutrition, hyperthyroidism, chronic liver
disease, intestinal fat malabsorption such as seen in sprue and chronic
pancreatitis) and with parents with the same biochemical trait. FHL
is caused by mutations in apoB, PCSK9 and angiopoietin-like proteins
(ANGPTL3). Additionally as yet unidentified genes may cause the
FHL phenotype, linkage of FHL to loci near 3p21.1–22 and 13q have
been reported [293,294]. The precise prevalence of FHL is not known,
though a prevalence of 1.9% was reported in the Framingham offspring
population [295].
2.5.1.1. Loss of function mutations in PCSK9. If the normal function of
PCSK9 is to reduce the number of LDLRs, one would expect loss of func-
tion mutations in the gene to increase the number of LDLRs and thereby
cause hypocholesterolemia. In line with this notion, livers of mice lack-
ing PCSK9 show increased LDLR protein, increased clearance of circulat-
ing lipoproteins, decreased plasma cholesterol and increased sensitivity
to statins [296]. A search for mutations among subjects with low LDL
cholesterol levels, identified loss of function mutations in PCSK9:
Y142X, C679X, R46L. The mutations are associated with lower LDL
160 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
cholesterol levels resulting in reduced risk of IHD [297]. The reduction in
risk of IHD was larger than predicted by the observed reduction in LDL
cholesterol alone. This could be because genotype is a better predictor
of lifelong exposure to LDL cholesterol than LDL cholesterol measured
in adult life. [298]. The Y142X and C679X mutations were common in
African Americans (2% combined frequency) and rare in European
Americans (b 0.1%) and were associated with a mean lowering of 40%
in LDL cholesterol. The plasma range of LDL cholesterol in subjects
with the nonsense mutation in PCSK9 ranged from first to fiftieth per-
centile suggesting other factors (eg diabetes, weight, age) may contrib-
ute to plasma LDL cholesterol variation. Haplotype studies suggest that
each of the two mutations had a single founder and that both were an-
cient mutations [299]. Screening healthy hypocholesterolemic subjects
for PCSK9 mutations identified subjects heterozygous for the mutations
R46L, G106R and R237W associated with hypocholesterolemia. The
mutation R46L and N157K in the PCSK9 gene were possibly associated
with increased response to statin treatment in a group of FH heterozy-
gotes [300]. The R46L sequence variation was much less common in
African Americans (0.3%) than in European Americans (1.6%) suggest-
ing an ethnic influence on allele frequency [301]. The loss of function
mutations R46L, G106R, N157K, and R237W had increased cell surface
LDLR and increased level of LDL internalization compared to the wild
type PCSK9 [302]. Other variants associated with lower LDL cholesterol
levels in population studies are L253F and A443T variants. Individuals
heterozygous for the nonsense mutations in PCSK9 did not show in-
creased hepatic TG content. The low LDL cholesterol variants were not
associated with increased hepatic TG content suggesting that the rate
of VLDL secretion is not reduced and that impaired VLDL secretion is
not the major mechanism for reducing LDL cholesterol. WT PCSK9
does not significantly affect the production of VLDL apoB [301,303].
Further a role for common PCSK9 variants (both coding and noncoding)
has been suggested for determining plasma levels of LDL cholesterol.
The effects of these variants are much smaller than the effects associated
with rare variants, levels differed by b5% from those without the com-
mon alleles [301]. Population studies identified further novel mutations
associated with hypocholesterolemia: frameshift mutation causing a
premature termination codon A68fsL82X, and amino acid substitutions
T771I, V114A, A522T and P616L [304]. A spectrum of loss of function
mutations has been identified. A study of Japanese population showed
that one missense mutation R93C was statistically associated with low
LDL cholesterol levels [305]. In a Canadian study multiple PCSK9 variants
were reported in the population suggesting that the PCSK9 gene is a
highly polymorphic gene. Specific combinations of PCSK9 variants
may be required to evaluate the effect of multiple variants on LDL cho-
lesterol levels [306].
A loss of function mutation Q152H at the site of autocatalytic cleav-
age was studied in a French Canadian family. The mutation prevents the
cleavage of the PCSK9 and precludes PCSK9 secretion. In cell culture
conditions the mutation has a dominant negative effect and prevents
wild type PCSK9 secretion [307]. In a study of a French family, the
monoallelic double mutant R104C/V114A exhibited a dominant nega-
tive effect on the secretion of wild type PCSK9 secretion [308]. Zhao
et al. [309] report a healthy, fertile compound heterozygote for
an inframe 3 bp deletion (c.290_292delGCC) which deletes an arg at
codon 97 and the Y142X mutation. Expression of ΔR97 mutant showed
that the mutant prevented autocatalytic cleavage and secretion of
PCSK9. To determine the effect of loss of function mutations, the recom-
binant forms of the protein were expressed in cells. The Y142X protein
was predicted to initiate nonsense-mediated RNA decay and no
immunodetectable protein was found in the cell lysate. The mutations
R46L, A443T had no detectable effect on the processing or secretion of
PCSK9. Compared to the WT, the amount of mature protein was de-
creased in the cells that expressed the L253F mutant suggesting
that the mutant interfered with autocatalytic processing. Both the
L253F and C679X mutations interfered with the secretion of PCSK9
[309]. A number of natural and artificial mutants were investigated
for processing and secretion defects. Analysis of the autocatalytic
cleavage residue Gln152, suggested the autocleavage specificity
was Gln N Met N Ala N Ser N Thr ~ Asn; all other residues led to the for-
mation of an unprocessed zymogen that acted as a dominant nega-
tive, retaining the WT zymogen in the cell [310].
The function of PCSK9 in areas other than promoting LDLR degra-
dation is under investigation. Recent data demonstrate that PCSK9
enhances the degradation of LDLR as well as the closely related
receptors, VLDL receptor, LRP1 and apolipoprotein E receptor 2
[311,312]. Studies in mice suggest that PCSK9 limits visceral adipo-
genesis, likely via adipose VLDL receptor regulation. Further, lesions
observed in the regenerating liver of PCSK9−/− mice were effectively
prevented by feeding the mice high cholesterol diet indicating in the
mice loss of PCSK9 leads to critical hypocholesterolemia [313]. The
absence of PCSK9 can be protective against melanoma invasion in
mouse liver [314]. It has been suggested that VLDL receptor and apo-
lipoprotein E receptor 2 exert major effects during brain develop-
ment. However, the brains of PCSK9 −/− mice did not show overt
morphological defects [311]. The only phenotype of PCSK9 deficien-
cy in humans is lower plasma LDL cholesterol levels but it is possible
that aging or other environmental factors may reveal other pheno-
types associated with PCSK9 deficiency. The loss of function PCSK9
SNP rs11591147 which resulted in lowering of cholesterol 10–16%
was not associated with noncardiovascular clinical events in an el-
derly population [315].
PCSK9 is a secreted protein which circulates in serum in a truncated
and intact form [316]. ELISA to measure PCSK9 used antibodies raised
against recombinant full length PCSK9 and recombinant PCSK9 was
used as standards for the assay. It is not known which epitopes these an-
tibodies recognize [317]. Circulating PCSK9 levels have been measured
in healthy middle aged individuals of European descent, in children
and adolescents and ethnically diverse cohorts. The general findings
are that circulating PCSK9 levels vary over a wide range among appar-
ently healthy cohorts and correlate with plasma LDL concentration. It
has been suggested that common and low frequency genetic variants
in PCSK9 locus influence the individual variation among healthy middle
aged individuals [318]. There are diurnal changes in PCSK9 and PCSK9
concentrations are reduced by fasting [319]. Plasma concentrations of
PCSK9 do not appear to be useful for screening for novel PCSK9 variants
[320], but may be useful for monitoring treatments for FH patients
[321].
2.5.1.2. Loss of function mutations in the inducible degrader of LDLR (IDOL).
IDOL is a sterol responsive E3 ubiquitin ligase recently identified as a
novel post-transcriptional regulator of LDLR. Acting as an E3 ubiquitin
ligase IDOL supports ubiquitylation of LDLR on its protruding intracellu-
lar tail thus targeting it for lysosomal degradation. In response to rising
cholesterol levels LXR induces IDOL production thus limiting further up-
take of exogenous cholesterol through the LDLR pathway. The IDOL
mechanism for feedback inhibition is independent of and complemen-
tary to the SREBP pathway [322]. Increasing the expression of IDOL re-
sults in decreased LDLR and LDL uptake into cells, whereas loss of
IDOL expression results in increased LDLR [323]. IDOL gene was se-
quenced in patients with LDL cholesterol below the 20th percentile in
whom mutations in LDLR, APOB, and PCSK9 were ruled out. The IDOL
variant R266X represents a complete loss of IDOL function unable to
promote ubiquitylation and subsequent degradation of LDLR. This is
the first report of a loss of function IDOL variant and will require further
examination in additional populations [324].
2.5.1.3. ApoB specific hypobetalipoproteinemia. Apo B specific familial
hypobetalipoproteinenia (FHLB, OMIM 615558), an autosomal codomi-
nant disorder is associated with mutations in the APOB gene. Most APOB
mutations lead to the formation of truncated apoB protein of various
sizes which to a variable extent lose the capacity to form plasma lipo-
proteins in the liver and intestine and export lipids from these organs.
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
Missense nontruncating mutations of the APOB gene can also cause
FHLB.
A large kindred with a nonsense mutation leading to a stop codon at
residue 2486 of apoB (called apoB 54.8 according to the centile system)
was identified, following population screening for individuals with
≤10th percentile total cholesterol. The proband was an asymptomatic
male, heterozygous for the apoB mutation [325]. A further mutation
with a premature stop codon at 3387 (apoB 75) was identified in a
kindred with hypobetalipoproteinemia. The proband was a 46 y old
healthy male heterozygous for the apoB mutation [326]. A nonsense
mutation that produced a truncated protein that contains 1754 amino
acid (apoB 38.7) was found in 57 y old woman with homozygous
FHLB. The patient presented with retinitis pigmentosa, acanthocytosis,
and loss of deep tendon reflexes. In addition the patient had diabetes
mellitus with nephropathy, anemia, cholelithiasis, hepatic hemangio-
ma, bronchiectasis, and extensive calcification of major arteries [327].
Three splice-site mutations have been linked with FHLB: (i) c.3843-
2A→G in which the joining of exon 24 to exon 26 lead to a premature
termination codon and a protein of 1260 amino acids (apoB 27.77);
(ii) c.904+4A→G in which an exon 7 and exon 9 junction lead to a pre-
mature stop codon at position 248 (apoB 5.44) (iii) c.4217-1G→T which
joined exon 25 to a thymidine at position c. 5397 which lead to a prema-
ture stop codon at position 1381 (apoB 30.42). The truncated apoBs
were not detected in the plasma suggesting that they were not secreted
as lipoprotein constituents. Ultrasound and liver biopsy examination of
heterozygote carriers of these mutations revealed the presence of fatty
liver [328]. Burnett et al. [329,330] reported two missense mutations
R463W and L343V in two FHLB kindred. Despite low lipid concentra-
tions, in affected members, none had neurological problems or malab-
sorption. Compared with unaffected family members liver enzymes
were increased and α-tocopherol concentrations decreased in hetero-
zygotes. Serum α-tocopherol concentrations were lowest in apoB
R463W homozygotes. R463W patients accumulate fat in their intestinal
enterocytes and exhibit a blunted postprandial rise in their blood lipids
[331]. Five nontruncating missense apoB mutations A31P, L324M,
G912D and G945S were identified in heterozygous carriers of FHLB.
All probands and some of the respective family members had fatty
liver documented by ultrasound examination [332].
In the study by Fouchier et al. [333] most FHLB heterozygotes were
asymptomatic, though some presented with mild clinical phenotypes.
Statistically significant differences between affected and unaffected
groups were found for plasma TC, LDL cholesterol, HDL cholesterol, TG
and apoB levels. The proband of the 11712delC (apoB 86) family pre-
sented with mild diarrhea and the proband of the R2507X (apoB 55)
family presented with diabetes mellitus type I and neurological
symptoms. The neurological complaints diminished after vitamin E
administration. The authors identified a total of 9 different apoB mu-
tations associated with the FHLB probands (R412X or apoB 9,
R463W, 1718delAT or apoB 12, 2534delA or apoB 18, 2783delC or
apoB 20, Q1309X or apoB 29, 11548del TT or apoB 84). All carriers of
the mutation were completely free from IHD. One patient inherited
the R3500Q mutation from her father and the 11712delC mutation
from her mother. Her lipid profile suggested compensation of one disor-
der by another.
Tarugi et al. [334] reported on the clinical phenotype of three FHLB
kindred heterozygous for mutations which resulted in truncated
apoBs. The first proband was heterozygous for a C deletion at position
1310/1311 leading to the synthesis of apoB 8.15, which was not detect-
able in the plasma. The proband showed steatorrhea and fatty liver. The
authors suggest that very short truncated apoB without the MTP bind-
ing domain which is part of apoB48, have a reduced capacity to assem-
ble dietary lipids into chylomicrons. In the second kindred a nonsense
mutation leads to the formation of apoB 33.4. The proband had cerebro-
vascular disease and fatty liver. Two other carriers in this kindred
showed fatty liver. In the third kindred, the proband was heterozygous
for a T deletion in exon 26 which resulted in apoB 75.7. The proband
161
who was a heavy drinker had steatohepatitis whereas his teetotaller
daughter, an apoB 75.7 carrier, had no detectable fatty liver.
The following mutations were identified in the FHLB group:
2534delA apoB 18; Q1309X apoB 29, R2507X apoB 55, and 11712delC
apoB 86 by Sankatsing et al. [335]. They observed a decreased level of ar-
terial wall stiffness in FHLB subjects, most pronounced in the absence of
nonlipid risk factors, suggestive of cardiovascular protection in FHLB in-
dividuals. The authors report slightly higher levels of HDL cholesterol in
FHBL subjects. The latter can be a consequence of low levels of TG min-
imizing the exchange of cholesterol esters from HDL cholesterol to apoB
containing particles through the action of CETP.
The structure of the βα1 domain of human apoB has been based on
its homology to lipovitellin. This homology model suggests that the βα1
domain consists of a β barrel and an α helical domain. In vitro experi-
ments suggest that the βα1 contains multiple MTP binding sites
encompassing residues 430–570, 512–721 and probably 2–154 [336,
337,338]. Using transfected cells, expression studies with the mutant
R463W protein suggested that the mutant protein had an increased
binding for MTP and, in addition, the mutant protein was retained with-
in the ER and was secreted poorly compared to the WT protein [329,
330]. In other studies with transfected cell lines the mutant A31P was
least secreted and the secretion of G912D barely affected. Degradation
of the post ER A31P protein was achieved by autophagy. The studies
suggest that feedback inhibition of lipogenesis and degradation of
apoB A31P can contribute to total fat accumulation in the liver. [332].
Early studies suggest that the truncated forms of apoB are secreted at re-
duced rates. The secretion rates of apoB 89, apoB 75, apoB 54.8 and apoB
31 were 92%, 64%, 37% and 12% of apoB100. Additionally the truncated
forms had increased fractional catabolic rates. The linear regression
line of apoB length versus secretion rate has a zero intercept for apoB se-
cretion at apoB 28, suggesting that the smaller forms of apoB are not se-
creted [339,340]. In general, the shorter the truncated apoB form the
lower the lipid to protein ratio in the lipoprotein [341]. Thus low levels
of truncated apoB containing lipoproteins are probably due to quantita-
tive differences in their production, conversion or removal rates. It is
likely that the occurrence of fatty liver in FHLB depends on the balance
between hepatic TG synthesis and VLDL secretion. In FHLB heterozy-
gotes with mutations in apoB specifying short truncations of apoB unde-
tectable in plasma, hepatic secretion of VLDL was decreased. VLDL
apoB100 production rates in FHLB heterozygotes are lower than
would be expected from a functioning allele. The secreted VLDL was,
however, relatively TG enriched. The cellular mechanism underlying
decreased VLDL apoB100 production in FHLB heterozygotes is not un-
derstood. Theoretically the presence of truncated apoB may interfere
with VLDL assembly leading to increased intracellular degradation of
apoB100 [342]. The clearance of truncated apoB which contain the
LDLR binding domain is faster than the clearance of apoB100. It has
been suggested that the enhanced clearance of truncated apoB is pri-
marily mediated by LDLRs. On the basis of negative regulatory effect of
the ‘bow’ structure of the carboxy terminal of apoB, removal of the
carboxy terminal can lead to enhanced receptor binding [343]. Trunca-
tions of apoB shorter than 3200 amino acids (apoB-70), which do not
possess the LDLR region are redirected from the liver to the kidney,
which becomes the major organ of uptake [344]. The phenotype of
apoB dependent hypobetalipoproteinemia is the net result of several
processes including VLDL production rate and rate of removal of LDL
via the LDLR.
The clinical phenotype of heterozygous FHLB is not well defined.
Though often asymptomatic they can develop fatty liver. Several factors
affect the amount of TG accumulated in the liver. Schonfeld et al. [345]
suggest different rates of free fatty acid flux to the liver could not ac-
count for hepatosteatosis in FHLB subjects, and that hepatic steatosis
is secondary to impaired VLDL secretion, irrespective of metabolic
conditions. Genetically engineered mice with truncated apoB, display
fatty livers with compensatory down regulation of hepatic fatty acid
synthesis. Thus the amount of TG accumulated in the liver is probably
162 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
modulated by a number of genes. Both genetic and environmental fac-
tors would affect the degree of fat accumulation. A novel nonsense mu-
tation in apoB (p.K2240X) was responsible for fatty liver in a large
kindred. In this family, carriers of this mutation developed cirrhosis
and liver cancer. The complex relationship responsible for the sequelae
of fatty liver requires further investigation [346]. A case of FHLB who
was heterozygous for c.11040 T N G mutation (apoB 80.5) was in addi-
tion carrying the HFE mutations C282Y and H63D as well as the α1-
antitrypsin PiZ variant. The case had an unusual combination of disor-
ders that could all be contributing to the observed liver dysfunction
and fatty liver [347]. The apoE genotype has been reported to account
for 15–60% of the variability in plasma LDL in FHLB subjects. The physi-
ological basis for this effect is yet to be determined [348].
Clinical features of homozygous FHLB can include acanthocytosis,
deficiencies of fat soluble vitamins secondary to malabsorption, atypical
retinitis pigmentosa and neuromuscular abnormalities. These defects
can also be observed with FHLB heterozygotes. Retinitis pigmentosa
and other neuropathies are primarily the result of deficiencies of fat sol-
uble vitamins, attributable to their impaired transport. A patient identi-
fied as a compound heterozygote (c.1315C NT apoB 39/c.537 + 1G N T
apoB 3.4–4.5) for FHLB was admitted at 4 months for severe fat malab-
sorption. He was treated with medium chain fatty acids. He developed
cirrhosis, portal hypertension, esophageal varices as well as severe
neurological deficiencies by the age of 11 [349]. The clinical status
of homozygous FHLB patients is variable. A 48 year old female homo-
zygous for the mutation c.6240T N A which resulted in a truncated
apoB, apoB 45.2 was asymptomatic with normal levels of vitamin E.
[350]. It is likely that apoB mutations will affect lipid homeostasis
in multiple cell types and multiple mechanisms may be called upon
to maintain lipid homeostasis.
2.5.2. Abetalipoproteinemia
MTP is an ER protein that catalyzes the transfer of neutral lipids and
is essential for the formation of VLDL and CM. The MTP gene found on
chromosome 4q22–24 codes for an 894 amino acid protein which is pri-
marily expressed in enterocytes and hepatocytes. MTP is found as a het-
erodimer with the enzyme, protein disulfide isomerase. The isomerase
is inactive with respect to its enzyme activity in the MTP complex. Pro-
tein disulfide isomerase mutants lacking enzyme activity are fully func-
tional in lipid transfer activity if associated with normal MTP. Based on
the sequence homology of MTP with lipovitellin, MTP is proposed to
have three structural motifs: N-terminal β-barrel (amino acid residues
22–297) which mediates the interaction with apoB, central α-helix
(amino acid residues 298–603) which associates with protein disulfide
isomerase and C-terminal lipid binding domain (amino acid residues
604–894) which is involved in lipid binding and lipid transfer [351].
Abetalipoproteinemia (ABL, OMIM 200100) is an autosomal reces-
sive hypocholesterolemia which presents in its severe form, during in-
fancy with failure to thrive, severe diarrhea and a lipid malabsorption
syndrome. In ABL patients, neurological impairment (ataxia and periph-
eral neuropathy) and retinopathy are a consequence of vitamin A/E de-
ficiency as well as deficiencies in polyunsaturated fatty acids. Clinical
and biochemical features of ABL are generally indistinguishable from
homozygous and compound heterozygous FHLB, though more severe
than heterozygous FHLB. The clinical presentation of ABL can, however,
be heterogeneous and its subsequent downstream complications de-
pend on early diagnosis and treatment [352]. Mutations in the MTP
gene are the principal cause of ABL. In some instances, frameshift, non-
sense and splice site mutations that are located throughout the entire
MTP gene are predicted to encode truncated forms of MTP resulting in
complete loss of function. The others are missense mutations that
cause ABL by reducing the ability of MTP to transfer lipids. Deficient
MTP activity targets apoB for degradation preventing the secretion of
CM and VLDL. MTP knockout mice died during embryonic development
suggesting deficiency of MTP caused lethal embryonic development
[353]. Mice with conditional deletion of the MTP gene in villus
enterocytes, displayed steatorrhea and large TG droplets within the vil-
lus enterocytes. In addition hepatic lipogenesis underwent compensato-
ry induction with increased VLDL secretion in the face of decreased
chylomicron secretion [354].
Pons et al. [355] report on two patients, compound heterozygotes for
mutations within the MTP gene leading to severe ABL. The first genomic
mutation c.619G N T resulted in a deletion of exon 6 and a shift in the
open reading frame leading to a premature stop codon at position 234
resulting in an abnormal truncated protein of 233 amino acids. The sec-
ond genomic mutation c.1237-28A N G induces a deletion of exon 10,
with no shift in reading frame leading to a protein of 858 amino acids.
After expression in HeLa and HepG2 cells both MTP mutants localized
in the ER despite not binding to protein disulfide isomerase. The two
mutants induced a loss of MTP TG transfer activity leading to ABL. The
first patient presented at 13 months with severe malabsorption. His sis-
ter, diagnosed at 6 years had no rotula reflex. Both patients presented
with complete absence of LDL and almost complete absence of VLDL
and low concentrations of HDL particles, raised alanine transaminase
and severe vitamin E deficiency. The patients had severe immunoglob-
ulin deficiency requiring immunoglobulin substitution. An intestinal
endoscopy of the 13 month old patient showed a white aspect of duode-
nal villi characteristic of intestinal fat malabsorption. Both parents had
normal lipid values ruling out a dominant form of inheritance. Other
mutations resulting in a truncated form of the MTP protein and a severe
ABL phenotype are: c398-399delAA [356], c2611delC, c923 GNA [357],
c.419-420insA [358], c.2076-39_2303 + 52del319 [359], c.619-3T N G,
c.923G NA [360]. A patient with subclinical hypothyroidism and ABL
was found to be a compound heterozygote for a splice site mutation
in intron 1 (c.61 + 2T N C) and a single insertion in exon 4 (c.419-
420insA) which resulted in a truncated protein of 140 amino acid
[361].
Di Filippo et al. [362] report a case of ABL with an unusual clinical
phenotype. The four year old girl presented with severe liver injury,
low levels of LDL cholesterol, and subnormal levels of vitamin E,
but only mild fat malabsorption and no retinitis pigmentosa or
acanthocytosis. The subject exhibited compound heterozygosity for
two novel MTP mutations: one missense mutation (p.L435H) and an
intronic deletion (c.619-5_619-2del). Transfected cell lines expressing
the missense mutation exhibited negligible levels of MTP activity. In
contrast the intronic deletion showed an incomplete splicing defect
with 26% of normal splicing being retained. The small amount of MTP
activity resulting from residual normal splicing in the patient is thought
to explain the atypical phenotype.
More than 30 MTP gene mutations causing ABL have been described.
Narcisi et al. [363] report a cohort of eight patients who presented with
classical ABL, fat malabsporption during infancy, low plasma cholester-
ol, TG and apoB levels. In contrast HDL cholesterol was within reference
range in all but two patients. Mutations which were identified in both
alleles of the MTP gene in all eight patients, resulted in truncated form
of MTP with variable number of amino acids at its carboxy terminal
end. Missense mutations R540H, S590I, G746E and N780Y have been
found to cause ABL. The R540H mutation prevents the association of
the protein disulfide isomerase with MTP causing the mutant MTP to
be nonfunctional based upon its inability to reconstitute apoB secretion
in a cell culture system [364]. The predicted tertiary structure suggests
that R540H and S590I are located in the central α helical domain where-
as G746E and N780Y are in the C-terminal β sheet domain. Mutants
S590I, G746E and N780Y interact with protein disulfide isomerase but
showed no phospholipid or TG transfer activity and did not support
apoB secretion [365]. Ohashi et al. [366] describe a 27 year old male ho-
mozygous for the N780Y mutation with none of the manifestations of
classical ABL even though his plasma apoB and vitamin E were virtually
undetectable. A 58 year old male, homozygous for the mutation S590I
with the absence of apoB containing lipoproteins, acanthocytosis was
notable only by the absence of neurological involvement and normal vi-
tamin E concentration. He subsequently developed adenocarcinoma of
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
the ileum. It is likely that environmental and genetic factors can influ-
ence the presentation of the ABL phenotype [367]. One study reported
an association between common MTP polymorphisms and LDL levels
[368].
Two patients with contrasting phenotypes were presented by Miller
et al. [369]. The first patient was a compound heterozygote with one de-
letion (c 103_127del25) and another missense mutation (Y528H) who
presented at 4 months with failure to thrive. The second presented at
37 years with marked hypocholesterolemia, and was found to carry
three missense mutations: G264R, R540C, and N649S. Studies with
transfected cell lines suggested that all missense mutations bound to
apoB48 and protein disulfide isomerase. Mutations Y528H and R540C
displayed negligible levels of MTP lipid transfer activity and N649S
displayed a partial reduction. G264R retained full lipid transfer activity.
The R540C is predicted to disrupt an internal salt bridge that has an ef-
fect on MTP structure. The N649S converts a hydrophilic asparagine to a
smaller still polar serine. The partial activity of N649S may provide a
biochemical explanation for the mild ABL phenotype of the patient.
Diagnosis of FHLB and ABL depend on signs and symptoms,
acanthocytosis on blood smear and virtually absent apoB containing
lipoproteins. Obligate heterozygote parents of ABL patients have
normal lipid levels consistent with autosomal recessive inheritance.
Heterozygous FHLB may variably show some of the clinical features
and have TG and LDL cholesterol levels around 5th-10th percentile
for age and sex, consistent with autosomal co-dominant inheritance,
though a high degree of interindividual variability is seen even with-
in the same kindred. In general, laboratory investigations show a
lipid profile with nearly absent TG, apoB and LDL cholesterol for
ABL and homozygous FHLB patients. Definite diagnosis involves se-
quencing of the MTP and apoB genes. Investigations include lipid
profile, serum transaminases, markers for lipid soluble vitamins,
and periodic assessment of ocular and neurological function. Treat-
ment includes a low fat diet, and supplementation with essential
fatty acids and high doses of fat soluble vitamins. Prognosis is vari-
able and may depend on early diagnosis and adherence to treatment
[370,371,372].
2.5.3. Familial combined hypolipidemia
A family of proteins structurally similar to angiopoietins ANGPTLs
has been identified. To date 8 ANGPTLs have been discovered. In
humans ANGPTL3 is located on chromosome 1 (1p31.1-p22.3) and
encodes a 460 amino acid protein consisting of a signal peptide, an
N-terminal segment containing coiled-coil domains and a C-terminal fi-
brinogen like domain which are connected by a linker region. All
ANGPTLs have a similar structure except ANGPTL8 which lacks the fi-
brinogen domain. ANGPTL3 is activated by cleavage at a proprotein
convertase consensus site to release the N-terminal domain and its ac-
tivity is regulated by ANGPTL8 [373,374]. Both ANGPTL3 and ANGPTL4
inhibit LPL activity and mice lacking Angptl3 and Angptl4 genes have in-
creased LPL activity and reduced levels of TG [375]. ANGPTL3 acts as an
inhibitor of EL, which is recognized as one factor that influences HDL
metabolism [376]. Although ANGPTL3 and ANGPTL4 both inhibit LPL,
their effect on lipoprotein metabolism is divergent and complex. It has
been suggested that ANGPTL3 and ANGPTL4 inhibit LPL through distinct
mechanisms [377,378]. Both irreversible dissociation of LPL dimers or
reversible noncompetive inhibition have been described as the mecha-
nism of inactivation of LPL by ANGPTL4 [379]. Loss of function mutation
in ANGPTL3 has been shown to cause familial combined hypolipidemia
(FCHL, OMIM 605019). In a cohort of subjects with low LDL, TG and HDL,
the prevalence of ANGPTL3 gene mutations was about 10% [380].
In a cohort of patients with loss of function S17X mutation in
ANGPTL3, homozygous patients showed complete loss of ANGPTL3
while heterozygous S17X carriers showed partial deficiency. Homozy-
gotes showed reduced cholesterol levels in VLDL, LDL and HDL and a
marked reduction in TG. Heterozygotes showed similar lipoprotein
levels to noncarriers. Compared with noncarriers, heterozygotes did
163
not show a difference in post heparin LPL mass or activity, whereas ho-
mozygotes showed a 3.5 fold higher levels in these parameters. The
FCHL phenotype is at least in part attributable to deficiency of ANGPTL3
and increased activity of LPL. One explanation for the low levels of HDL
seen in homozygous subjects is that the S17X mutation affects the
secretion of the lipoproteins or that ANGPTL3 acts by inhibiting EL.
The reduced levels of FFA observed in homozygotes can contribute
to decreased rate of VLDL production by the liver [381]. Other muta-
tions in ANGPTL3 associated with FCHL are: pG400VfsX5, pI19LfsX22,
pN147X, E129X [382,383].
Minicocci et al. [384] studied the biochemical clinical characteristics
of 14 homozygous, 8 compound heterozygous and 93 heterozygous
carriers of mutations in ANGPTL3. The authors identified nonsense
mutations, frameshift mutations (eg p.S122Kfs*3), nucleotide deletions
(eg c.361-365delAACTC) and missense mutations (eg p.G56V). Com-
pared with noncarriers, homozygotes as well as heterozygotes had a re-
duction of all plasma lipoproteins and apolipoproteins. Compared to
homozygotes, the presence of a single mutant allele reduced lipoprotein
levels to a lesser extent, suggesting a codominant form of inheritance.
However, there was not a linear gene-dosage dependent effect of
ANGPTL3 mutations on lipoprotein levels. The authors explored the
genotype-phenotype relationship of different mutant alleles on plasma
ANGPTL3 concentration. Though the analysis was hampered by the
small number of patients, mutations predicting truncated proteins had
the most pronounced effect in reducing plasma levels of ANGPTL3.
The functional role of missense mutations can be only be clarified by ex-
pression studies. The authors did not detect fatty liver in their cohort of
FCHL subjects and found that none of the homozygotes were affected by
diabetes. It is possible that ANGPTL3 has a more comprehensive role in
regulation of not only lipid but glucose metabolism. Low plasma
HDL and reduced cholesterol efflux from cells observed by Pisciotta
et al. [382] can contribute to increased atherosclerosis but may be
counterbalanced by lifelong low exposure to low LDL cholesterol, and
merits further investigation.
A common variant of ANGPTL4, E40K, that was present in ~ 3% of
European-Americans was associated with lower levels of TG and LDL
cholesterol and higher levels of HDL cholesterol. Expression studies
with wild type and mutant protein in cells suggested that ANGPTL4
formed dimers and tetramers in cells prior to secretion. Following secre-
tion and cleavage of the protein the oligomeric structure of the N-
terminal domain was retained while the C-terminal dissociated into
monomers. Inhibition of the cleavage did not interfere with the oligo-
merization of ANGPTL4 or its ability to inhibit LPL, whereas mutations
that prevented oligomerisation compromised the ability of the protein
to inhibit LPL. The E40K substitution destabilizes the protein after secre-
tion, preventing the oligomerisation of the protein and abolishing the
ability of the protein to inhibit LPL [385]. Multiple loss of function muta-
tions in ANGPTL3 and ANGPTL4 genes were associated with low plasma
levels of TG [386,387]. ANGPTL4 variants E40K and T266M are associat-
ed with low TG levels in patients with type 2 diabetes [388]. A signifi-
cant association between SNPs in the RXR gamma gene and FCHL was
reported in a further study suggesting that other genes may play a
role in the genetic susceptibility to FCHL [389].
2.5.4. Other candidate genes
GWAS have identified a locus on chromosome 1p13 which was
strongly associated with LDL cholesterol expression [390]. A com-
mon noncoding polymorphism at the 1p13 locus rs12740374 created a
C/EBP transcription factor binding site and altered the expression of
SORT1 gene which modulates VLDL secretion and alters plasma VLDL
levels [391]. Hepatic overexpression of sortilin reduced apoB secretion
and increased LDL catabolism resulting in reduced LDL cholesterol [392].
2.5.5. Chylomicron retention disease
The SAR1B gene encodes the Sar1b protein which is involved in CM
transport from the ER to the Golgi apparatus. The Sar1b protein belongs
164 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
to a family of small GTPases and is a coat protein II (COPII) component.
The COPII coat drives vesicle formation. COPII vesicle formation is initi-
ated by guanine nucleotide exchange factor Sec12, which recruits Sar1b.
Sar1b-GTP in turn recruits Sec23/Sec24 dimer which binds the cargo
molecules. These molecules recruit a heterodimer Sec13/Sec31 which
drives membrane bending and vesicle formation [393,394]. The SARIB
gene is located at chromosome 5q31.1. It is composed of eight exons
and alternative splicing of exon2 is predicted to lead to two transcripts.
A second human protein isoform of Sar1, Sar1a is encoded by SAR1A
gene located in chromosome 10q22.1. In mice the evaluation of Sar1b
mRNA revealed the skeletal muscle as the tissue with the highest
Sar1b expression, followed by the heart and the liver, the organs com-
posing the digestive tract, brain and finally the lung and adipose tissue
[395].
Recently mutations in the SAR1B gene were identified as respon-
sible for chylomicron retention disease (OMIM 246700, CMRD) or
Anderson's disease. When SAR1B is mutated, the pre-chylomicron
transport vesicle cannot fuse with the Golgi apparatus, which then
induces an accumulation of pre-chylomicron transport vesicles in
the cytoplasm of the enterocytes. The condition is characterized by
severe fat malabsorption, usually associated with failure to thrive
in infancy as well as deficiency in fat soluble vitamins, low blood cho-
lesterol levels and the absence of CM from blood. Affected individ-
uals accumulate CM like particles in the enterocytes which contain
large cytosolic droplets. Jones et al. [396] identified coding sequence
variants in SAR1B in affected individuals. In total eight mutations,
two frameshift, one splice site and five missense alleles were identified.
The five missense mutations (G37R, D137N, S179R, S179I, L181P) cause
changes to the GTP binding pocket of Sar1b and the homozygous splice
site mutation is predicted to abolish the production of functional Sar1b.
Two patients homozygous for the mutation (p. D48TfsX17) which gave
rise to a truncated protein and admitted at b 1 year of age with diarrhea,
acquired tolerance to a fat diet in later years. A further patient homozy-
gous for a frameshift stop codon (p.L28RfsX7) was a heterozygous
carrier of a PCSK9 (p.L21dup) variant [397]. Cefalu et al. [398] describe
a proband, a five month old baby with mutation in the SAR1B gene
(c. 75–76 del TG-L28fsX34) who presented with failure to thrive
and malabsorption. His father was heterozygous for the mutation
but his mother who was homozygous for the mutation was symptom
free of CMRD. It is likely that that CMRD is a more complex disorder
and that further modifier genes may be involved in the ER-to Golgi
transport. Charcosset et al. [399] describe three further unique homozy-
gous mutations in French families who originated from Turkey, Algeria
and Portugal: stop codon in exon 6 (p.E122X), a whole deletion of exon
2 (c. 1-4482_58+1406 del 5946 ins15bp) and a missense mutation in
exon 7 (p.G185V). Phenotypical data for missense mutations were not
different from nonsense mutation, conversely the same missense
mutation was associated with different profiles between families
and even among the same family. The authors observed a lack of
genotype-phenotype correlation among patients with CMRD carry-
ing mutations in the SAR1B gene and suggest that mutations and
polymorphism in modifier genes eg transcriptional factors might af-
fect the functionality of the network of proteins involved in ER-to-
Golgi transport.
Sar1b also promotes hepatic VLDL secretion which would explain
the observation that some CMRD patients develop hepatic steatosis. It
has been suggested that Sar1b modulates the expression of genes
encoding cholesterol biosynthesis which may explain the severe
hypocholesterolemia phenotype found in CMRD [400]. Patients with
CMRD have a lower rate of production of apoB100 and apoAI from the
liver [401]. It is likely that a network of modifier genes are involved
the mechanisms that determine the phenotype of the disease in any
given individual and that CMRD is a more complex disease than a simple
autosomal recessive disorder. Okada et al. [402] describe a patient with
CMRD phenotype, a normal SAR1B gene coding sequence and maternal
uniparental disomy of chromosome 7.
Descriptions of cardiac and muscular abnormalities may be related
to tissue specific expression of the abnormal Sar1b protein. Creatine ki-
nase levels were increased in patients homozygous for a mutation in the
seventh codon which resulted in a change in Glutamic acid to a stop
codon, resulting in a truncated protein of 166 amino acids as well as in
a patient with the G37R mutation [403].
One study describes the phenotypic expression of CMRD and its evo-
lution over time in a French Canadian cohort and a French cohort of
Turkish, Algerian and Portugese stock. All 16 CMRD patients presented
within the first few months of life with diarrhea and failure to thrive. Se-
vere hypocholesterolemia coupled with normal TG was associated with
low LDL and HDL cholesterol and low apoAI and apoB. The late diagnosis
in the French cohort and the later introduction of a low fat diet with a
high polyunsaturated fatty acid/saturated fatty acid ratio and supple-
ments of fat soluble vitamins A, D and E in doses recommended for
ABL was thought to account for the delayed catchup growth in the
French cohort compared to the Canadian patients. Clinically only one
case had a neurological handicap and one patient suffered from hepato-
megaly [404]. Neurologic and ophthalmologic complications are less se-
vere than FHLB and ABL. [405]. The similarity of CMRD to other types of
hypocholesterolemia can lead to the wrong diagnosis unless molecular
analysis is performed. Sequencing of SAR1B provides an accurate diag-
nosis of CMRD.
2.5.6. ApoCIII deficiency
The APOC3 gene resides within the APOA5–APOA4–APOC3–APOA1
multigene human cluster on human chromosome 11q23. The locus pro-
vides a strong association signal in GWAS of plasma TG concentration,
although there is some ambiguity of the genes that underlie this signal,
both APOC3 and APOA5 have been associated with plasma TG concentra-
tion [406]. ApoCIII is a 79 amino acid O-linked glycoprotein composed of
6 amphipathic α helices and is expressed in the liver and intestine [407].
On entry into the plasma, apoCIII is rapidly exchanged among TG rich li-
poproteins and HDL, which acts as a reservoir for this apolipoprotein.
The distribution of apoCIII is however, asymmetrical, with different
apoCIII molecules per particle within HDL and VLDL pools [16]. Recent ev-
idence suggests that apoCIII and apoAV are opposing modulators of plas-
ma TG metabolism [408]. Plasma apoCIII concentration varies from
~10 mg/dL in normal subjects to N 30 mg/dL in hypertriglyceridemic sub-
jects. Increased apoCIII production is a characteristic feature of patients
with hypertriglyceridemia [409].
ApoCIII expression is down regulated in part by the insulin response
element (IRE) on the APOC3 gene. A variant of APOC3 promoter within
the IRE results in loss of insulin regulation. The variant APOC3 promoter
is common in human population and can contribute to the development
of hypertriglyceridemia [410]. The transcription of APOC3 is mediated
by several other transcription factors including PPAR [411].
Several functions have been proposed for the role of apoCIII in regu-
lating TG metabolism. ApoCIII is known to inhibit LPL activity. It has
been suggested that when bound to TG rich lipoproteins apoCIII pre-
vents the binding of LPL to the lipid surface. TG rich lipoproteins stabi-
lize LPL and protect the enzyme from inhibitors like ANGPTL4. The
addition of apoCIII decreased the protective effect of TG rich particles
on LPL and increased their susceptibility to factors such as ANGPTL4
[412]. ApoCIII also impairs the hepatic uptake of triglyceride rich lipo-
proteins, possibly by interfering with the interaction of apoE on the lipo-
proteins with LDL and LRP receptors [413]. The metabolism of VLDL is
influenced by its content of apoE and apoCIII [414]. An intrahepatic
role of human apoCIII in promoting VLDL secretion was suggested fol-
lowing in vitro studies with transfected cell lines [415]. ApoCIII or
apoCIII rich VLDL may contribute directly to atherogenesis by activating
endothelial cells and recruiting monocytes to them [416].
GWAS identified heterozygous carriers of the null mutation (R19X)
in the gene encoding apoCIII in an isolated Amish population. The
carriers expressed half the amount of apoCIII present in noncarriers.
Mutation carriers compared with noncarriers had lower fasting and
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
postprandial TG, higher levels of HDL cholesterol and lower levels of
LDL-cholesterol. Subclinical atherosclerosis, as measured by coronary
artery calcification, was less common in carriers than noncarriers, sug-
gesting that lifelong deficiency of apoCIII may have a cardioprotective ef-
fect [417]. In isolated European populations the frequency of the rare
allele (R19X) has been shown to be increased [418]. A further mutation
A23T was found in Mayan Indian subjects with hypotriglyceridemia.
The mutant displayed reduced affinity to phospholipid liposomes with
normal inhibitory effect on LPL activity. The in vitro studies suggest that
the less efficient binding of the A23T mutant might lead to faster catab-
olism of the protein and reduced plasma apoCIII levels [419]. Other
studies suggest that the presence of the mutant can prevent VLDL as-
sembly and secretion from hepatocytes with TG accumulation within
the microsomal lumen independent of MTP activity [15]. The naturally
occurring mutation K58E abolished the stimulatory effect of apoCIII on
the assembly and secretion of VLDL; suggesting that the C-terminal
lipid binding domain of apoCIII plays a role in VLDL assembly [420].
Whole genome sequencing identified a rare variant of APOC3 gene
(rs138326449) associated with plasma TG levels [421].
Confirmation of the role of apoCIII in the clearance of TG rich lipopro-
teins comes from transgenic mouse studies. Disruption of the APOC3 gene
results in protection from postprandial hypertriglyceridemia, while the
overexpression of human APOC3 gene results in hypertriglyceridemia
[422,423]. The mutation apoCIII Q38K resulted in a moderate but signifi-
cant elevation in TG in a Mexican family. The elevated plasma levels of TG
further supports a role for ApoCIII in the regulation of TG metabolism
[424].
3. Disorders of HDL metabolism
3.1. HDL and cardiovascular disease
Numerous population studies have shown that an inverse relation-
ship exists between plasma HDL and IHD risk. Two HDL subclasses can
be separated by ultracentrifugation: HDL2 is less dense and relatively
lipid rich, HDL3 is more dense and relatively protein rich. On agarose
gel the HDL separates into α migrating particles which represent the
majority of circulating HDL and preβ particles which represent poorly
lipidated HDL. Further resolution can be achieved by a 2-dimensional
electrophoretic method [425]. A 0.026 mM (10 mg/L) increment in
HDL cholesterol was associated with a risk decrease in IHD of 2% in
men [426,427]. Later studies suggest that smaller dense HDL3 choles-
terol drove the inverse association with HDL and IHD [428]. The in-
verse relationship of HDL cholesterol with IHD risk decreased in
patients with coronary artery disease, suggesting that HDL choles-
terol has limited use in cardiovascular risk stratification in such
patients [429]. HDLs display a wide spectrum of activities which include
in addition to cholesterol efflux, anti-inflammatory and anti-oxidative
action and maintenance of endothelial function [430]. HDL consists of
several subpopulations of particles who do not all inhibit atherosclero-
sis to the same extent [431]. A plateau effect in terms of IHD risk reduc-
tion was observed for men and women with high levels of HDL [432].
However, a causal relationship between low HDL and IHD risk is not un-
disputed. Many different factors affect both HDL levels and IHD risk, and
statistical adjustments do not guarantee the absence of other confound-
ing factors. It is likely that HDL subspecies or individual HDL compo-
nents will better predict atherosclerosis [433]. One view is that it is
not HDL concentration but HDL function that has a causal relationship
with atheroprotection [434].
3.2. Decrease in HDL concentration
Familial hypoalphalipoproteinemia is defined as an HDL cholesterol
level below the 10th percentile without a secondary cause and asso-
ciated with a family history of low HDL levels. When considering
hypoalphalipoproteinemia single gene, Mendelian disorders are
165
mutations in APOAI, LCAT, and ABCA1. Rare and common variants
can contribute to genetic variations in HDL. Mutations in apoAI,
LCAT, PLTP and ABCA1 were found in 12% of subjects with HDL deficien-
cy, leaving 88% of HDL deficient subjects without a genetic diagnosis
[435]. In a further study, one of six individuals with HDL cholesterol
below the fifth percentile had rare mutation in APOA1 or ABCA1 [436].
Recently data have emerged that suggests extreme levels of HDL choles-
terol in families have a polygenic origin. Greater than 40 different genes
are currently reported to affect HDL levels [437].
3.2.1. Monogenic disorders associated with decreased HDL levels
3.2.1.1. ApoAI deficiency. The gene for ApoAI is located on chromosome
11q23 and consists of 4 exons encoding the primary transcript of 267
amino acids, subsequent cleavage of both the intracellular and the se-
creted protein produces a 243 amino acid protein necessary for the as-
sembly of HDL Residues 44–243, are composed of eight 22 amino acid
and two 11 amino acid repeats that are predicted to form amphipathic
α-helices [438,439]. The frequency of hypoalphalipoproteinemia due
to mutant apoAI (OMIM 604091) was estimated as 6% in Japanese sub-
jects with low HDL and 0.3% in the Japanese population [440]. Absence
of apoAI in a patient resulted in low HDL, premature coronary artery dis-
ease and corneal opacification. The patient's defect was a deletion of the
entire apoAI/C3/A4 gene complex [441]. Two sisters with HDL deficien-
cy, undetectable plasma AI and apoCIII with premature IHD and coro-
nary bypass by the age of 30 were described by Norum et al. [442].
The defect was a DNA rearrangement affecting the adjacent apoAI and
apoC3 genes. Two siblings with HDL deficiency and no plasma ApoAI
were found to be homozygous for the deletion in exon 3 of the apoAI
gene (c.85 delC, Q5FsXII). This mutation causes a frameshift leading to
a premature stop codon and abolished the synthesis of apoAI. Although
both siblings had corneal opacification and planar xanthomas only one
of them had premature coronary artery disease, probably as a result of
mildly elevated LDL levels. The carriers had HDL cholesterol values
below the 10th percentile of the population, though none had planar
xanthomas or coronary artery disease. In two further kindred HDL
deficiency was due to nucleotide substitution in exon 4 (L141R) of
the apoAI gene. This mutation was reported to hamper the ability
of apoAI to recruit cellular cholesterol and to activate LCAT. In addi-
tion the L141R mutation affects the stability of apoAI in plasma
[443]. A further mutation S36A was identified in a subject with se-
vere hypoalphalipoproteinemia. Although the ability of the S36A
mutation to bind to lipoprotein surfaces was not altered, the muta-
tion significantly impaired LCAT activation [444]. A 67 year old
male homozygous for the V156E substitution in apoAI presented
with HDL deficiency, corneal opacity and coronary artery disease.
The son who was heterozygous for the mutation showed normal
HDL levels. In vivo studies in rabbits suggested that the variant was
rapidly cleared from plasma compared to normal apoAI [445]. Deletion
in two successive adenine residues in codon 138 of the apoAI gene
resulting in a frameshift mutation at amino acid residues 138–178 was
identified in a patient with marked HDL cholesterol and apoAI deficiency,
myocardial infarction and moderate corneal opacities. His brother who
was homozygous for the mutation with marked HDL cholesterol and
apo AI deficiency had no clinical evidence of coronary heart disease. Het-
erozygotes had 50% of normal plasma HDL cholesterol with mean apoAI
concentrations decreased in subpopulations (large α1 and small preβ)
of HDL particles [446]. DNA sequencing of a 51 year old patient revealed
homozygosity for a frameshift mutation in exon 4 of the apoAI gene lead-
ing to a stop codon at residue 178. The patient exhibited corneal opacities,
planar xanthomas and extensive coronary heart disease [447]. A deletion
in exon 4 of the apoAI gene leading to frameshift mutation and early ter-
mination at codon 200 was identified in a 69 year old patient with
marked apoAI deficiency, corneal opacity but neither xanthomas or
symptoms of coronary heart disease [448]. HDL subpopulations in homo-
zygotes and heterozygotes in patients with a nonsense mutation in apoAI
166 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
codon −2,Q[−2]X, displayed altered chemical composition with deple-
tion in apoAI, cholesteryl ester, and glycerphospholipid. The authors sug-
gest that the defective atheroprotective properties of HDL were related to
altered lipid and apolipoprotein composition [449].
Mutants apoAIMilano (R173C) and apoAIParis (R151C) are natural var-
iants of the apoAI that cause no deleterious health effects despite low
levels of plasma apoAI and HDL and high levels of HDL triglycerides in
heterozygote mutation carriers [450,451]. While some studies report
an enhanced ability of apoAI(R173C) to recruit cellular cholesterol
others a report a similar effect of apoAI(R173C) to the wild type protein
[452,453]. It has been suggested that the arginine is potentially involved
in an intrahelical salt bridge with E169 and that the loss of the arginine
residue destabilizes the helix domain of the apoAI molecule altering its
lipid binding characteristic [454]. It has been proposed that disulfide
bonds involving C173 or C151 can influence protein conformation and
facilitate dissociation of the C-terminal from HDL to recruit additional
lipids [455].
Naturally occurring mutations in human apoAI are known to be as-
sociated with low plasma HDL levels and hereditary amyloidosis. The
hereditary amyloidogenic mutations are clustered in the amino termi-
nus and in residues 170–180 of the protein. In most hereditary
amyloidogenic mutations, the N-terminal fragment of mutated apoAI
is the predominant form of the protein found in amyloid fibril deposits,
suggesting that the N-terminal region is critical to the amyloid fibril for-
mation of apoAI. The first reported amyloidogenic apoAI variant G26R is
hypothesized to allow the β-strand of residues 20 to 25 to extend to po-
sition 26 and beyond, thereby increasing the likelihood for more global
β-strand interactions [456,457]. The L178H variant is associated with
severe cardiac and laryngeal amyloidosis as well as skin lesions. Howev-
er in contrast to G26R, L178H exhibits increased helical content [458].
Genetic analysis of the apoAI gene revealed three missense mutations
A175P, F71Y, E34K and a frameshift mutation H155MfsX46 which
were associated with amyloidosis [459].
3.2.1.2. Tangier disease. The ABCA1 gene is localized on chromosome
9q31. ABCA1 gene contains 50 exons and codes for a 2261 amino acid in-
tegral membrane transporter protein that mediates the export of cho-
lesterol and phospholipids to lipid poor lipoprotein. ABCA1 consists of
two transmembrane domains, each formed by six alpha helices and
two intracellular nucleotide binding domains. Upregulation of ABCA1
is dependent on the enhanced expression of PPARγ and LXRα [460].
Identification of genetic defects in ABCA1 causing Tangier disease
(OMIM 205400) has revealed a key role for ABCA1 in cholesterol trans-
port. Mutated ABCA1 fails to mediate cholesterol efflux to lipid poor
preβ HDL or its major apolipoprotein apoAI. However, the precise
mechanisms by which ABCA1 mediates cholesterol efflux remain
elusive. Recent studies have implicated several signaling mechanisms
(eg protein kinase A (PKA), PKC) in ABCA1 function and apoAI lipidation
[461]. Several groups have reported that mutations in the ABCA1 gene
are responsible for Tangier disease. Defective removal of cellular choles-
terol and phospholipids and a deficiency in HDL-mediated efflux of
intracellular cholesterol has been demonstrated in Tangier disease fi-
broblasts [462].
Tangier disease manifests with symptoms and signs resulting from
the deposition of cholesteryl esters in nonadipose tissue; in peripheral
nerves leading to neuropathy and in reticulo-endothelial organs such
as liver, spleen, lymph nodes and tonsils causing their enlargement
and discoloration. An association with early cardiovascular disease can
be variable. The biochemical signs of Tangier disease are low plasma
HDL, apoAI, cholesterol and normal or high plasma TG. Hematological
findings in patients with Tangier disease are thrombocytopenia and
stomatocytosis. Clinical signs combine differently in each patient. The
clinical phenotype is inherited as an autosomal recessive trait and the
biochemical phenotype is inherited as an autosomal co-dominant trait
[463]. ABCA1 deficiency impairs the efflux of free cholesterol from
cells, leading to intracellular accumulation of cholesteryl esters and
prevents the conversion of lipid poor apoAI particles into preβ-HDL
and causes rapid catabolism of the poorly lipidated HDL mainly by the
kidney [464]. Hepatocyte specific ABCA1 knockout mice have a similar
lipid phenotype to Tangier disease, with a twofold elevation of plasma
VLDL TG, 50% lower LDL and 80% reduction in HDL concentration. The
lipid phenotype arises from an increased secretion of hepatic VLDL, in-
creased uptake of LDL by the LDLR, elimination of nascent HDL particle
assembly by the liver and hypercatabolism of apoAI by the kidney
[465]. In the absence of ABCA1 newly secreted apoAI do not form
preβ-HDL particles, which leads to decreased PI3K activation and in-
creased lipid mobilization [466].
A proband was a 6 year old Caucasian female born from consanguin-
eous marriage. Her medical history included hepatosplenomegaly, ane-
mia and thrombocytopenia. At the bone marrow biopsy a high number
of lipid laden macrophages were observed. Her plasma profile showed
an HDL cholesterol of 0.06 mmol/L and undetectable apoAI levels.
This patient was found to be homozygous for an intronic mutation
(c.4465-34AN G) predicted to encode a truncated protein. The authors
describe five patients (aged 6 months to 76 years) who presented
with Tangier disease, and shared two clinical manifestations of Tangier
disease, thrombocytopenia and splenomegaly. In four of them bone
marrow biopsy revealed the presence of lipid loaded macrophages,
one patient had peripheral sensory neuropathy, and clinical manifesta-
tions of premature coronary artery disease were documented in one
adult patient and his twin brother. Four patients without classical man-
ifestations of Tangier disease were found to be heterozygous for ABCA1
mutation and were classified as familial HDL deficiency [467]. A patient
homozygous for the R1270X in the ABCA1 gene mutation presented
with a bleeding tendency and anemia secondary to spontaneous splenic
hematoma [468]. A homozygous G–C splice site mutation adjacent to
exon 33 at the − 1 position, in the ABCA1 gene was associated with a
multiple sclerosis like neurologic presentation and the presence of sub-
clinical atherosclerosis [469].
The diagnosis of Tangier disease requires the demonstration of mo-
lecular defects in the gene encoding ABCA1. More than a 150 missense,
nonsense, frameshift ABCA1 mutations have been described in patients
with familial HDL deficiency and Tangier disease. [http://www.hgmd.
cf.ac.uk/ac/gene.php?gene=ABCA1]. Severe HDL deficiency in a patient
was caused by two autosomal recessive mutations of ABCA1, one pre-
dicted to lie in the transmembrane segment (V1704D) and the other
(L1379F) in the extracellular loop. Both mutations prevent normal traf-
ficking of ABCA1, thereby explaining their inability to mediate apoAI
dependent lipid efflux [470]. The mutant W590S has a near-normal
apolipoprotein binding activity but defective lipid transport [471].
Brunham et al. [472] report three new kindreds with Tangier disease.
One patient was identified to be homozygous for a nonsense mutation
p. Q1038X, a further kindred showed compound heterozygosity for mis-
sense variants p. R937V and p.T940M, and a third pedigree was com-
pound heterozygous for a frameshift variant p.I1200Hfs*4 and an
intronic variant that lead to the creation of a cryptic splice site and pre-
mature truncation p. S1392Rfs*6. Cases of non classical Tangier disease
have been described. A patient who did not have any corneal opacities
or organomegaly on a CT scan of the abdomen was found to have
three mutations in the ABCA1 gene. All three mutations p.A1046D, p.
Y1532C and p. W1699C were reported to be deleterious in functional
studies [473].
3.2.1.3. LCAT deficiency. The human LCAT gene encompasses 4.2 kb and is
localized on chromosome16q21-22. LCAT is synthesized primarily by
the liver and undergoes N- and O-linked glycosylation to become enzy-
matically active. The enzyme has both phospholipase A2 and an acyl-
transferase activity as it catalyzes the transacylation of the sn-2 fatty
acid of lecithin to the free 3-β hydroxyl group of cholesterol forming ly-
solecithin and cholestery esters. The enzyme has an esterase activity on
monomeric substrates. LCAT is active both on LDL and HDL and is pri-
marily associated with HDL which contains apoAI the major activator
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
of LCAT. Using site directed mutagenesis the catalytic triad of LCAT was
shown to include S181, D345 and H377. Residues F103 and L182 were
thought to form an oxyanion hole and residues C50–C74 contributed
to enzyme-substrate interaction [474]. Familial LCAT deficiency (FLD,
OMIM 245900) is characterized by the absence of LCAT activity and re-
duced HDL cholesterol in plasma. In FLD plasma LCAT is either absent or
completely lacks LCAT activity. In fish-eye disease (FED, OMIM 136120)
the mutant LCAT lacks activity primarily on HDL lipids but preserves
LCAT activity on apoB containing lipoproteins. FED mutations specifical-
ly affect phospholipase A2 activity on HDL, whereas esterase activity
on the monomeric substrate and acyltransferase activity on LDL are
retained. Some mutants have decreased acyltransferase on both LDL
and HDL, though the acyltransferase activity on HDL decreased more
than the acyltransferase activity on LDL. Classic FLD mutations impair
activity on all substrates. A structural basis for the difference between
FED and FLD phenotypes may depend on whether the mutations impair
the catalytic triad, impair the accessibility to the catalytic residues or
affect the fold of the enzyme [475,476].
Thirteen families carrying 17 different mutations were identified by
Calabresi et al. [477]. Seven of the ten probands carrying two mutant
LCAT alleles had FLD and three had FED, reflecting a milder clinical phe-
notype. All carriers of two mutant LCAT alleles had low plasma HDL cho-
lesterol, with at least a 6 fold variation in HDL cholesterol level. The
variability was unrelated with inherited defects in LCAT function as
both FLD and FED had overlapping HDL cholesterol levels. Plasma total
and LDL cholesterol levels in carriers of 2 mutant LCAT alleles showed
a wide interindividual variability. Elevated plasma TG were a fre-
quent findings among cases and LCAT mutations had a gene-dose de-
pendent effect on plasma TG and VLDL. The common clinical findings
of carriers of 2 mutant LCAT alleles were bilateral corneal opacity and
normochromic anemia of varying severity. Six of the 15 carriers of 2
mutant LCAT alleles had end-stage renal failure requiring hemodial-
ysis. In this study, 34/44 of heterozygotes were apparently healthy.
The low HDL levels in carriers of two mutant alleles of LCAT is associ-
ated with alterations in HDL structure and particle distribution,
which likely reflect the accumulation in plasma of cholesterol ester
poor apoAI containing discoidal HDL which cannot mature into spheri-
cal HDL because of the lack of LCAT activity [478]. In homozygotes the
majority of apoAI was found in small poorly lipidated small disk shaped
preβ1-HDL particles and some apoAI is found in larger, lipid poor discoi-
dal HDL particles with α mobility. Heterozygotes had lipid profiles sim-
ilar to controls except that apoCIII was found only in preβ-HDL [479]. In
the absence of LCAT abnormal lipid particles have been observed
throughout lipoprotein fractions. Lipoprotein X (LpX) is an abnormal li-
poprotein that appears in cholestasis and LCAT deficiency. Electron mi-
croscopic studies suggest that LpX are spherical particles which contain
primarily free cholesterol and phoshatidylcholine [480]. Lipoprotein
fractions specific to LCAT deficiency has been identified by high perfor-
mance liquid chromatography with a gel filtration column. Four specific
fractions specific to LCAT deficiency corresponding to (i) large lipopro-
teins (N80 nm) (ii) large LDL (iii) small HDL to large LDL and (iv) small
HDL were identified. Large LDL and HDL fractions had compositions
specific to FLD and not FED. The authors suggest that abnormal lipopro-
teins may play a causal role in renal pathology [481]. LCAT deficiency re-
sult in low levels of HDL through preferential hypercatabolism of apoAII
and apoAI and HDL particles compared to normal controls [482].
Renal disease is a cause of mortality and morbidity in patients with
FLD. The kidney disease presents as proteinuria in its early stage and
increases in severity by the 4th and 5th decade in life, sometimes
progressing to nephrotic syndrome and end stage renal disease. Homo-
zygosity for the mutation p.W99R at position 99 of the LCAT protein was
reported in a 33 year old female with a history of proteinuria. Renal bi-
opsy revealed mesangial expansion with the presence of mesangial
foam cells and glomerular basement membrane thickening. Electron
microscopy confirmed the presence of marked basement membrane
abnormalities, with the inclusion of lipid droplets in paramesangial
167
and mesangial locations [483]. Genetic analysis of a further patient
with nephrotic syndrome revealed two mutations in the LCAT gene:
(i) deletion of 5 nucleotides in exon 1, which caused a frameshift after
R23 with the introduction of 52 amino acids and a termination codon
at position 76 (ii) S181N amino acid substitution which converted the
S181 amino acid which is a component of the catalytic triad conserved
in all examined animal species [484]. A patient with inhibitory anti-
LCAT antibody presented with nephrotic syndrome and glomerular le-
sions similar to FLD. Treatment with steroids resulted in complete re-
mission of the nephrotic syndrome with normalization of the serum
LCAT activity and HDL level and disappearance of foam cell accumula-
tion in renal tissue [485].
Intriguingly, premature IHD is not a consistent feature of FLD or FED
and the role of LCAT mutations in atherosclerosis has been disputed. The
effects of LCAT deficiency on carotid artery intima-media thickness, a
surrogate marker for IHD have been reported in several studies. In a
study by Hovingh and his colleagues [486] heterozygosity for the LCAT
gene defects was associated with low HDL cholesterol levels, increased
levels of TG, C-reactive protein and increased intima-media thickness.
In contrast in the study by Calabresi and colleagues [487] carotid intima
thickness was decreased in both FLD and FED patients. Both studies
used carotid ultrasound to measure arterial wall thickness. A further
study which used magnetic resonance imaging to localize the carotid ar-
tery showed that carriers of LCAT gene mutations exhibited an in-
creased risk of carotid atherosclerosis [488]. Serum from carriers of
LCAT deficiency has the same capacity as normal serum for ABCA1 me-
diated cholesterol efflux, probably due to increased content of preβ-
HDL [489]. LCAT deficiency has been shown be associated with defective
HDL antioxidant/anti-inflammatory properties [490] but, surprisingly,
was not associated with parameters of increased lipid peroxidation
[491]. Population studies provided inconclusive data on the association
between reduced levels of LCAT activity and cardiovascular risk [492].
Results have been equally contradictory in the experimental world
using animal models, as reviewed by Ng [493]. In animal models the
presence of other pro-atherogenic apoB containing lipoproteins and ad-
ditional proteins, CETP and LDLR, appear to play a role. Studies on
mouse models suggest that LCAT may modulate obesity and insulin sen-
sitizing pathways, though further studies on underlying mechanisms
are warranted [493,494]. Two case presentations further illustrate the
paradoxical relationship between LCAT deficiency, low HDL levels and
cardiovascular risk. Genetic analysis identified two mutations Y83X,
R99C in the LCAT gene in a patient with no trace of subclinical athero-
sclerosis. The patient had corneal opacity, no cardiovascular symptoms
and low HDL [495]. A second patient with LCAT deficiency presented
with corneal opacity, undetectable LCAT levels, and renal failure. He
had severe vascular disease, lower limb peripheral arterial obstruction
with necrosis of two left toes and angina with trivasal occlusive coro-
nary artery disease [496].
3.3. Increased HDL concentration
3.3.1. Monogenic disorders associated with increased HDL levels
3.3.1.1. CETP deficiency. The human CETP gene is mapped to chromosome
16q12-21 near the LCAT gene, spans 25 kb pairs and encodes a 476
amino acid protein [497]. In humans CETP is expressed predominantly
in the liver, spleen and adipose tissue [498]. The evidence that CETP is
essential for HDL metabolism came from the discovery of CETP deficien-
cy (OMIM 143470). Most cases of CETP mutations have been described
in Japanese families. Genetic CETP deficiency has been described as the
most frequent cause of hyperalphalipoproteinemia (HALP) in the
Japanese. Mutations described have been splice site mutations (muta-
tions in the 5′ splice donor site of intron 14 (Int14+1G→A), missense
mutations (D442G) and nonsense mutations (G309X). Plasma CETP
mass and activity were not detected in homozygous subjects with the
Int14+1G→A, G181X, Q182X or G309X mutation. The Int14+1G→A
168 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
mutation causes exon 14 skipping which introduces premature termi-
nation at codon 403. The truncated protein is rapidly degraded intracel-
lularly in transfected COS-1 cells. The subjects with the heterozygous
Int14+1G→A mutation had about half the CETP mass of normal con-
trols. Homozygous subjects with the Int14+1G→A mutation had mark-
edly elevated HDL cholesterol levels, while the heterozygous subjects
had moderately increased HDL cholesterol levels [499]. A promoter mu-
tation (G to A substitution at the − 69 nucleotide of the promoter re-
gion) is thought to affect transcription of the CETP gene. A reporter
gene assay showed that this mutation markedly decreased transcription
in HepG2 cells [500]. CETP deficiency contributes to approximately 50%
of HALP in the Japanese population [501]. Only a few Caucasian individ-
uals with CETP deficiency have been identified in case reports. A single
base substitution resulting in a R268X codon in both alleles was identi-
fied in a Caucasian subject [502]. The serum of a patient with Swedish
ancestry and a homozygous mutation R37X in the CETP gene, showed
a normal or enhanced cholesterol efflux with via ABCG1/SR-BI, and de-
creased cholesterol efflux with ABCA1. The patient's plasma HDL was
characterized by large preβ-HDL known to be better acceptors through
the former pathways [503]. Genetic CETP deficiency was rare among
subjects with HALP in The Netherlands. Heterozygous CETP deficiency
was identified in only 1 of 95 HALP families. This genetic defect was
shown to cause skipping of exon7 and is predicted to result in a prema-
ture truncation of the CETP protein [504]. To date several common poly-
morphisms and rare variants of the CETP which cause depletion of CETP
activity have been described across populations [505].
One of these common polymorphisms is TaqIB, a silent base change
affecting the 277th nucleotide of the first intron of the CETP gene. The
B2 allele, absence of the TaqI restriction site, has been found to be asso-
ciated with elevated plasma HDL cholesterol. Since it is not clear how
this SNP can modulate CETP expression it has long been thought this
SNP is linked to another change that more directly affects CETP expres-
sion. A large meta-analysis using a Mendelian randomization approach
showed that the B2 allele yielded a significant risk reduction in coronary
artery disease compared to the B1 allele. However, the elevation in HDL
cholesterol levels due to TaqIB polymorphism was marginally associat-
ed with reduced coronary artery disease, challenging the conventional
view that raising HDL cholesterol levels may result into protection
from IHD risk [506]. In a further meta-analysis three CETP genotypes
(TaqIB, I405V, −629CNA) that were associated with moderate inhibi-
tion of CETP activity, and modestly high HDL cholesterol levels, show a
weak inverse association with coronary risk [507]. A definite relation-
ship between CETP and coronary artery disease is inconsistent and
there is considerable dispute among genotype-phenotype studies
[508]. In one study, common CETP polymorphisms which beneficially
contributed to higher HDL cholesterol were paradoxically associated
with increased incidence of coronary disease [509]. The role of CETP
gene variations in longer life expectancy is complex and needs further
elucidation [510].
Mechanisms responsible for the variations in coronary risk with in-
creased HDL cholesterol as a result of genetic variations in CETP remain
putative. It has been suggested that HDL cholesterol formed under con-
ditions of decreased CETP activity may be dysfunctional. Teh et al. [502]
report a raised HDL cholesterol, normal LDL cholesterol and mild
hypertriglyceridemia in a patient who was homozygous for the R268X
codon in the CETP gene. The patient's VLDL and LDL were cholesteryl
ester poor and triacylglycerol rich while her HDL was cholesteryl ester
rich and triacylglycerol poor. HDL particles from CETP deficient subjects
displayed normal anti-oxidative capacity [511] and decreased ability to
protect endothelial cells from the development of endothelial dysfunc-
tion [512]. It is likely that any increase in HDL cholesterol levels is offset
by reduced apoAI mediated removal of cholesterol from macrophages.
The association between CETP deficiency and IHD can be influenced
by functional abnormalities and changes in HDL and LDL, the genetic
mutation/polymorphism causing the CETP deficiency as well as envi-
ronmental factors, obesity, alcohol consumption and smoking [499].
3.3.1.2. Loss of function variants in EL. EL is coded by the LIPG gene is a
68 kb glycoprotein secreted by endothelial cells. The amino acid se-
quence of EL is 41% homologous to HL. EL exhibits more phospholipase
activity than TG lipase activity. EL hydrolyses HDL most efficiently of all
lipoprotein fractions [513]. A rare variant N396S in EL is associated with
elevated HDL cholesterol concentration and substantially reduced lipo-
lytic activity [514]. The G26S N-terminal variant had significantly re-
duced plasma levels of EL protein while its catalytic activity was
similar to wild type EL, leading to elevated HDL cholesterol levels
[515]. More work is required to determine whether the resulting in-
crease in HDL cholesterol due to a decrease in EL activity will have an
impact on cardiovascular disease [516]. An association has been report-
ed between rare and common noncoding regulatory variants of the LIPG
gene and HDL cholesterol levels [517].
3.4. Polygenic causes of HDL variation
Numerous genes are suggested to play a role in HDL metabolism.
Strategies for identifying complex polygenic traits that influence HDL
metabolism fall into (i) linkage analysis for the co-segregation of a chro-
mosomal region and a trait of interest using polymorphic markers in
families (ii) genetic association studies which measure an association
between a genetic variant and HDL outcome (iii) GWAS which identify
new genes that influence HDL levels. Eligible candidate genes from
studies were summarized by Boes et al. [518] and include the following
genes: CETP, LPL, ABCA1 and the APOA1C3A4A5 gene cluster. Other
candidate genes include GALNT2, which needs further functional char-
acterization. Weisglass-Volkov et al. [519] summarize the genetic poly-
morphisms in (eg CETP, HL, LPL, EL, LCAT, ABCA1, APOAI, APOCIII,
APOAV, APOE, SR-BI and PON1 (an antioxidative enzyme present on
the HDL particle)) genes described in more than one study as implicated
in HDL cholesterol variation. Both an intronic SNP (rs2294213) [520]
and missense mutation (R235W) [521] in the PLTP gene may contribute
to variation in plasma HDL. GWAS have provided empirical evidence for
polygenic inheritance of dyslipidemia with many genes, each with a
small effect, contributing to variation in lipid levels. However so far
only about a third of the expected genetic heritability of lipid levels
can be explained [522]. The explained heritability of lipids may increase
as more common variants are identified. Studies suggest that very ex-
treme HDL cholesterol phenotypes can have a polygenic origin and
that multiple rare variants can contribute to extreme HDL cholesterol
levels [523]. Gene-gene interactions between variants may further in-
fluence HDL cholesterol concentration [524]. The effects of common
variants on HDL cholesterol may be modified by lifestyle factors. Genetic
factors may modify lipid responses to randomized lifestyle intervention
[525,526]. As a full collection of DNA sequence variants become avail-
able, an allelic risk score may allow for early identification of individuals
at risk for cardiovascular disease and to better target preventive inter-
ventions [527].
4. Secondary dyslipidemias
Secondary non-genetic factors associated with dyslipidemias include
obesity, metabolic syndrome, alcohol consumption, diabetes, renal
disease, pregnancy (mainly in the third trimester), paraproteinemia, sys-
temic lupus erythematosus and medications that include corticosteroids,
oral estrogen, tamoxifen, thiazides, non-cardioselective beta blockers,
bile acid sequestrants, cyclophosphamide, antiretroviral drugs and sec-
ond generation antipsychotic reagents. People who develop secondary
dyslipidemia may have a subtle genetic defect that increases susceptibil-
ity to dyslipidemia.
4.1. Obesity, metabolic syndrome and diabetes
Depending on the degree of duration and weight gain, obesity (ele-
vated body mass index, particularly abdominal or upper body obesity)
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
can progressively cause and/or exacerbate a wide spectrum of morbid-
ities including dyslipidemia, metabolic syndrome and type II diabetes.
The typical dyslipidemia of obesity consists of raised fasting and post-
prandial TG and free FA, decreased HDL cholesterol and normal or
slightly increased LDL cholesterol with a predominance of small dense
LDL—the so called atherogenic triad. However, some individuals seem
to be protected against the more deleterious metabolic effects of obesity
[528].
Reactive oxygen species are generated in vivo under certain condi-
tions. Lipid peroxidation can occur through interactions of lipids and
free radicals. Hyperlipidemia is also associated with oxidant stress
resulting in the formation of oxidized LDL, which contains many athero-
genic oxidized lipids [529].
Abnormalities in lipoprotein are more pronounced in individuals
with central obesity [530]. Intra-abdominal or visceral adipose tissue
as opposed to subcutaneous adipose tissue was found to be more relat-
ed to atherogenic dyslipidemia. Studies on genetic determinants of obe-
sity suggest that the heritability rates of body fats are approximately
50% [531].
Obese men also show a decreased uptake of dietary fat by adipose
tissue, which results in higher delivery of CM remnants to the liver
with enhanced VLDL-TG being delivered to peripheral adipocytes and
ectopic fat deposition [532]. Increased free FA flux to the liver in obesity
leads to hepatic accumulation of TG and increased hepatic synthesis of
VLDL. In insulin resistant individuals the larger VLDL1 containing a
higher TG content is secreted in excess compared to the smaller, dense
VLDL2 [533]. It has been suggested that should correct lipid storage in
adipose tissue fail, free FA will be taken up by non-adipose tissue leading
to metabolic complications. Morbidly obese patients were shown to
have lower gene expression of factors related to lipid uptake and pro-
cessing in visceral adipose tissue. LPL, VLDL receptor, LRP1 and adipocyte
FABP (fatty acid binding protein) expression in visceral adipose tissue
was higher in lean controls rather than in obese patients. Adipose tissue
secretes acylation stimulating protein (ASP), the result of a post transla-
tional modification of the complement factor C3, which stimulates TG
synthesis. ASP expression was higher in the visceral adipose tissue of
lean controls than in obese patients [534]. A further hypothesis is that
the rate of lipolysis of TG rich lipoproteins can be partly regulated by ac-
cumulating free FA that dissociate LPL from the endothelial binding sites
[535]. Both overproduction and decreased fractional catabolic rate of
VLDL was found to be related to hypertriglyceridemia in obese subjects.
An inhibitory effect on LPL of the over production of VLDL apoCIII can
further decrease LPL activity [536]. Lipolysis of TG rich lipoproteins is im-
paired in obesity leading to increased levels of postprandial and fasting
levels of TG. The delayed clearance of TG rich lipoproteins facilitates
the CETP mediated exchange between cholesterol esters in HDL and
TG in VLDL. The CETP transfer process results in lower HDL cholesterol
and indirectly reduces HDL size. In insulin resistant states a decreased
post heparin LPL activity and increased HL is a determinant of low HDL
cholesterol. The increased exchange facilitated by CETP produces LDL
particles enriched in TG that are rapidly hydrolyzed by HL leaving small-
er denser LDL particles [537,538].
The increased incidence of obesity is recognized as a major cause
of non-alcoholic fatty liver disease (NAFLD), the accumulation of
lipid droplets within hepatocytes. NAFLD can lead to non-alcoholic
steatohepatitis and hepatic cirrhosis. A number of factors contribute
to the deposition of lipid droplets within the liver. Glucose and insulin
promote lipogenesis by activation of carbohydrate response element
binding protein (ChREBP) and SREBP1c respectively. These are master
transcription factors that promote the transcription of lipogenic genes.
ChREBP mediates the effects of carbohydrate (nutrients) on lipogenic
gene transcription [539,540]. Sanyal et al. [541] found elevated insulin
concentrations failed to suppress adipose fatty acid flux in NAFLD.
Liver TG in NAFLD was 59% from free FA, 26% from de novo lipogenesis
(DNL) and 15% from diet. During fasting the hepatic use of dietary fatty
acids became reduced and the deficit was made up by adipose tissue
169
derived FA. There was a significant similarity between the contributions
of FA sources found in the liver TG pools and the sources used for TG se-
cretion by the liver via lipoproteins [542]. In NAFLD cholesterol absorp-
tion is decreased and synthesis is increased. These changes were
independent of BMI [543]. While metabolic adaptations occur to com-
pensate for increased fat load, mitochondrial dysfunction eventually oc-
curs. Mitochondrial functional decline is thought to further exacerbate
insulin resistance and lead to the progression of NAFLD [544].
Studies on people with obesity and insulin resistance are essentially
similar. In both fasting and postprandial states, obese subjects require
insulin levels that are several times higher than non-obese subjects to
maintain normal glucose tolerance [545]. Hepatic over production of
VLDL is seen in both obesity and insulin resistant states. Alterations of
insulin sensitive pathways, increased concentrations of free fatty acids
and low grade inflammation all play a role and result in an overproduc-
tion and decreased catabolism of TG rich lipoproteins of intestinal and
hepatic origin.
Plasma free FA are elevated in obese people and various FA are cyto-
toxic. Saturated FA, arachidonic acid and linoleic acid can stimulate the
synthesis of pro-inflammatory cytokines like IL-1, IL-6 and TNF-α,
whereas eicosapentenoic acid (EPA), a fish oil, has anti-inflammatory
properties. FA can regulate complex intracellular signaling systems
thereby modulating cellular metabolism. Several transcription factors
including the PPAR and LXR families are linked in these pathways. Loca-
tion in the body may be a factor in determining whether lipids promote
or suppress inflammation. Elevated plasma lipids may not be inflamma-
tory alone, as the hyperlipidemic state in obesity is indicative of redistri-
bution of lipids from adipose tissue to muscles and liver. The amount
and the kind of FA can regulate cellular signaling and are reviewed in
detail elsewhere [546]. Obesity enhances both lipogenesis and adipo-
genesis within fat depots and the secretion of pro-inflammatory
adipokines and chemokines into the plasma. Macrophages infiltration
of the adipose tissue has been described in obese conditions in humans.
Both adipocytes and macrophages within fat secrete numerous cyto-
kines and hormones that contribute to local inflammation [547]. It can
be hypothesized that the inciting event that causes adipose tissue to be-
come inflamed may have a genetic component, to account for the differ-
ences in metabolic risk between equivalently obese individuals [548].
A reduction in sensitivity to insulin can occur through a genetic de-
fect or can be acquired as a consequence of obesity [549]. Insulin affects
cells through binding to its insulin receptor on the surface of insulin re-
sponsive cells. The stimulated insulin receptor phosphorylates itself and
several substrates including members of the insulin receptor subsrate
(IRS) family, thus initiating downstream signaling [550]. Proinflamma-
tory cytokines like TNFα promotes inhibitory phosphorylation of serine
residues of IRS-1. This phosphorylation inhibits tyrosine phosphoryla-
tion of IRS-1 in response to insulin and prevents downstream signaling
and insulin action [551,552]. Additional pathways linking inflammation
to insulin resistance have been described and it is likely that other path-
ways remain to be discovered [553]. Heat shock proteins appear to have
a potential role in inhibiting inflammatory kinases [554]. In humans, in-
sulin resistance in muscle develops within hours of an acute increase in
plasma free FA [555]. The mechanisms involving free FA induced skele-
tal muscle insulin resistance have not been fully elucidated [556]. It has
been suggested that FA induce insulin resistance in rat muscle through
inhibition of insulin signaling which occurs through activation of a ser-
ine kinase cascade involving PKC-θ [557]. Recent work in rats suggests
that oxidative stress plays a causal role in FA induced hepatic insulin re-
sistance [558]. A further hypothesis is that ectopic lipid induced intra-
cellular increase in diacyl glycerol results in the activation of PKC-θ in
muscle and PKC-ε in the liver and subsequent inhibition of insulin sig-
naling in these tissues [559].
Although free FA are required for normal insulin release chronic ex-
posure to free FA both in vivo and in vitro is associated with marked im-
pairment in glucose stimulated β-cell insulin secretion and decrease in
insulin gene expression. In subjects with a family history of type 2
170 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
diabetes, a sustained increase in plasma free FA impaired β-cell insulin
response to glucose [560]. The molecular mechanisms by which exces-
sive levels of FA and glucose alter β-cell function are beginning to be
unraveled but a number of questions remain [561].
The definition of the metabolic syndrome links apparently unrelated
biological events into a single pathophysiological phenotype. The meta-
bolic syndrome has been proposed as a means of identifying people at
increased risk of cardiovascular disease and diabetes. The risk factors
for cardiovascular disease, which together are known as the metabolic
syndrome are raised blood pressure, dyslipidemia (raised TG and
lowered HDL), raised fasting glucose and central obesity. Three abnor-
mal findings out of 5 would qualify for the metabolic syndrome [562].
Both obesity and type 2 diabetes are associated with insulin resis-
tance. Diabetic dyslipidemia is characterized by high TG levels, reduced
HDL and small dense LDL. Lipid abnormalities observed in type 2 dia-
betics are quantitative, qualitative and kinetic in nature and exist before
the onset of diabetes as part of the insulin resistance syndrome. In type 2
diabetes a central paradox is the apparent selective nature of hepatic in-
sulin resistance, where insulin fails to suppress hepatic glucose produc-
tion yet continues to stimulate lipogenesis. Selective insulin resistance
can contribute to the clinical phenotype of hyperglycemia, dyslipidemia
and NAFLD. To address this paradox investigators have hypothesized
the existence of a branch point in the insulin signaling cascade to sepa-
rate insulin's anabolic signal into discrete pathways controlling gluco-
neogenesis and DNL [563,564,565]. Other studies suggest that the
increased delivery of substrate FA to the liver can explain the insulin
stimulation of hepatic lipogenesis [566,567].
Compared to controls, patients with type 2 diabetes and insulin re-
sistant subjects without diabetes had a higher production rate of intes-
tinal apoB48 containing lipoproteins. Type 2 diabetics had a higher VLDL
apoB100 production rate and decreased fractional catabolic rate com-
pared to controls [568]. Several mechanisms seem to be involved in
the overproduction of VLDL and chylomicrons in type 2 diabetes. In-
creased expression of MTP [569] and reduced inhibition of hormone
sensitive lipase (HSL) by insulin leading to enhanced free FA flux to
the intestine and liver may further drive TG rich lipoprotein secretion
[570,571]. The several pathological factors responsible for increased
TG rich lipoprotein secretion are reviewed by Xiao et al. [572] and
Verges [571]. The mean LDL-cholesterol level was comparable or slight-
ly elevated compared to non-diabetic controls in patients with type 2
diabetes [573]. In kinetic studies LDL fractional catabolic rate is de-
creased. The catabolic defect may be due to the glycosylation of LDL
with reduced affinity for LDLR [574] or decreased LDLR expression
[575]. HDL microheterogeneity is compounded by type 2 diabetes. Gor-
don et al. [576] investigated the alterations of HDL subclasses with type
2 diabetes and reported changes in phospholipid and protein composi-
tions in the larger HDL fractions. Alterations in HDL structure and com-
position have been associated with loss of potentially atheroprotective
functions.
4.2. Hypothyroidism
Thyroid hormones influence diverse metabolic pathways in lipid
and glucose metabolism. Thyroid hormones influence the synthesis,
mobilization and degradation of lipids. The causative mechanisms for
the cholesterol lowering effect of thyroid hormones consist of
(i) increased expression of SREBP2 [577] and a direct effect of 3,5,3′-
triiodo-L-thyronine (T3) on the promoter of the LDLR [578] (ii) in-
creased activity of HL, CETP and LCAT [579]. T3 has been shown to in-
crease apoAV mRNA and protein levels in hepatocytes [580].
T3 negatively regulates the mouse SREBP-1c gene expression in the
liver [581]. Thyroid hormone action is initiated by the binding of T3 to
receptors (TR), which bind to DNA sequences termed the T3 response
elements. Thyroid hormones directly regulate SREBPs but also co-
regulate genes with thyroid hormone response element and sterol re-
sponse element (the nonpalindromic recognition DNA element for
SREBPs) located in close proximity [582]. Human LXR-α mRNA and pro-
moter activity are positively regulated by thyroid hormone. Recent data
suggest a possibility of cross talk between TR and LXR [583]. Nuclear
hormone receptor crosstalk is becoming increasingly recognized as sig-
nificant in thyroid hormone action. Thyroid hormones activate ChREB in
liver and white adipose tissue. In mice TRβ but not TRα is required for
activity in vivo. ChREB has been shown to be a LXR target. In mice
there is crosstalk between LXR and TRβ signaling on the ChREB promot-
er in the liver but not in white adipose tissue [584]. In chicken hepato-
cytes, thyroid hormone receptor and LXR play a role in fatty acid
synthesis by regulating the expression of acetyl CoA carboxylase [585].
Acetyl CoA carboxylase catalyzes the ATP dependent carboxylation of
acetyl CoA to malonyl-CoA which is the rate limiting step for the fatty
acid synthesis pathway. The liver isoform of acetyl CoA carboxylase is
subject to nutritional and hormonal regulation. Feeding low fat, high
carbohydrate diet stimulates the enzyme 8–20 fold [585]. Among
other transcription factors SREBP-1c plays a role in controlling acetyl
CoA carboxylase [586]. Carnitine palmitoyl transferase 1 (CPT-1) cata-
lyzes the rate controlling step in the pathway of mitochondrial fatty
acid oxidation. It has been shown that thyroid hormones can induce
the expression of the liver isoform of CPT-1 [587].
The conversion of cholesterol to bile acids is required to maintain
cholesterol homeostasis. T3 plays a role in the regulation of cholesterol
7-hydroxylase, the rate limiting step in bile acid synthesis [588]. Tran-
scriptional regulation of the ABCA1 gene involves multiple transcription
sites and binding of several different transcription factors to the ABCA1
gene. A network of transcriptional controls leads to a balanced expres-
sion of ABCA1. Electrophoretic mobility assays demonstrate that
TR/RXR heterodimer is able to bind to the ABCA1 promoter. [589].
The alterations in lipid profile during hypothyroidism are variable. In
one study HLP type 2A was the most common lipid abnormality in pa-
tients with primary hypothyroidism and HLP type 2B the most common
in secondary hypothyroidism. Of a group with hypothyroidism 8.6% had
normal values, 56.3% had HLP type 2A, 33.6% HLP type 2B, and 1.5% HLP
type 4 [590]. Serum cholesterol and LDL cholesterol are increased by ap-
proximately 30% in patients with overt hypothyroidism. Evidence main-
ly suggests normal or slightly elevated levels HDL cholesterol resulting
in unfavorable total cholesterol/HDL ratios [591]. The effects of subclin-
ical hypothyroidism on serum lipid levels are less clear. Although some
studies have demonstrated that total cholesterol and LDL cholesterol are
elevated in subclinical hypothyroidism, others have not shown any
effect of subclinical hypothyroidism on lipid levels. Other factors eg in-
sulin resistance may modify the effects of subclinical hypothyroidism
on serum lipid levels [592].
4.3. Renal disease
In nephrotic syndrome apoB containing lipoproteins, VLDL, IDL and
LDL (hypertriglyceridemia and hypercholesterolemia) are elevated.
HDL is reported to be unchanged or reduced. In addition to these quan-
titative changes, the lipoprotein composition is markedly changed.
Patients with nephrotic syndrome showed a marked elevation in
lipoprotein(a) (Lp(a)) serum concentrations [593]. Most of the work re-
garding the disturbed lipoprotein metabolism in nephrotic syndrome
comes from animal studies.
A study designed to examine the gene profile in the livers of ne-
phrotic rats suggested that genes involved in lipid metabolism were dif-
ferentially regulated in nephrotic rats. Increased expression of hepatic
enzymes in biosynthetic pathways of cholesterol, FA and TG was associ-
ated with enhanced SREBP-1 expression at both transcriptional and
post-translational levels. Genes involved in FA oxidation were down
regulated in the nephrotic liver [594]. Further studies suggest that
upregulation of enzymes involved in FA synthesis is driven by sterol
responsive pathways but not ChREBP dependent pathways [595].
Clement et al. [596] found marked elevation of ANGPTL4 in nephrotic
syndrome and demonstrated that the level correlated with serum TG
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
levels in humans and animals with nephrotic syndrome of diverse
causes. An increase in the production of ANGPTL4 by extra renal organs
in nephrotic syndrome was driven by an elevated plasma FFA/albumin
ratio. Increase in circulating levels of ANGPTL4 has a causal role for the
pathogenesis of hypertriglyceridemia by inhibition of LPL in nephrotic
syndrome. In a series of in vivo and in vitro experiments the authors
demonstrated that by interacting with glomerular endothelial αvβ5
integrin, circulating ANGPTL4 can reduce proteinuria, indicating
that an increase in circulating ANGPTL4 in nephrotic syndrome is a
biological response designed to decrease proteinuria. Deficiencies
in LPL, HL, VLDL receptor, LDLR, LCAT, SR-BI, upregulation of CETP,
hepatic acyl coenzyme A cholesterol acyltransferase, HMGCR, DGAT
(diacyl glycerol acyl transferase) and Lp(a) contribute to nephrotic
dyslipidemia [597,598,599].
End stage renal disease is accompanied by a complex pattern of
alterations in lipoprotein metabolism and structure. A feature is an in-
crease in serum TG due to elevated VLDL, IDL and low HDL. LDL choles-
terol is often normal. Several compositional changes in lipoprotein have
been shown to occur in end stage renal disease, including oxidation,
carbamylation and glycation of apoB. Modified lipoproteins are not eas-
ily recognized by their receptors. An increase in half life in turn favors
modification, leading further to the accumulation of lipoproteins [600].
4.4. Pregnancy
Women, with uncomplicated pregnancy, displayed increased total
cholesterol, LDL cholesterol, HDL cholesterol and lipoprotein(a), in the
second and third trimester. The prominent change was a 2–3 fold in-
crease in TG in the third trimester [601]. During pregnancy, values
N95th percentile are considered supraphysiologal rise in plasma lipids
[602]. Hyperphagia and increased lipid synthesis contribute to maternal
fat accumulation, typically associated with the first two thirds of the
gestation period. Changes in lipid metabolism promote the accumula-
tion of maternal fat stores in early and mid pregnancy and increase fat
mobilization during late pregnancy. In addition fat cells from different
regions of the body show a differential response to pregnancy [603].
During late gestation maternal lipid metabolism changes to a catabolic
condition which promotes the use of lipid as the energy source. Nondi-
abetic pregnant women develop insulin resistance in the third trimester
of pregnancy. Insulin resistance is more marked in gestational diabetes
[604]. The higher concentration of estrogen and insulin resistance are
thought to be responsible for hypertriglyceridemia of pregnancy. The
mechanism by which estrogen and insulin interact is unknown. Recent
studies suggest that in hyperestrogenemic syndromes, estrogen may in-
terfere with insulin binding to its receptor [605] or decrease GLUT4
(Glucose transporter type4) expression in muscle [606] and as a result
decrease insulin sensitivity.
In the last trimester increased adipose tissue lipolysis results in the re-
lease of NEFA (nonesterified fatty acids). The primary destination of NEFA
is the liver, where following reesterification for the synthesis of TG they
are released as VLDL. Both LPL and HL activities in postheparin plasma
have been shown to be decreased in pregnancy [607]. Total cholesterol
and VLDL, LDL and HDL cholesterol increased with gestational time.
Total TG and VLDL, LDL and HDL TG followed a similar trend. VLDL TG/
cholesterol ratio declined in the third trimester whereas in both LDL
and HDL the TG/cholesterol ratio but not the cholesterol/phospholipid
ratio increased during gestation [608]. CETP is highest in the second tri-
mester of gestation and declined at the third trimester. HDL TG increased
during the second and third trimester. CETP activity correlated with HDL
TG levels [609]. Changes in CETP activity can be one of the factors that de-
termine LDL and HDL TG levels. Increased transfer of TG to HDL is due to
reduced LPL activity resulting in higher levels of VLDL and increased
length of exposure to CETP. [610]. The reduction in HL can decrease the
conversion of TG containing HDL2 to the lipid poor HDL3 particles and in-
crements in HDL2 rather than HDL3 seem responsible for the increase in
HDL cholesterol level seen during gestation in women. [611]. Increase in
171
plasma TG during pregnancy can lead to the appearance of atherogenic
dense LDL particles in a small group of women [612]. Patients with eleva-
tions in lipoprotein levels over the accepted range of adaptation may de-
velop hyperlipoproteinemia in their later life. The percentage of women
who will translate to hyperlipoproteinemia remains unknown and
inherited causes may underlie the increase in lipoprotein levels [234].
Studies in rat during gestation indicate that changes in adipose tis-
sue HSL/LPL mRNA levels could be a mechanism for adipose tissue
lipid breakdown during late gestation, suggesting transcriptional regu-
lation of both enzymes during gestation [613]. One possible hypothesis
is that increased HSL activity and lowered LPL activity in adipose tissue
can contribute to changes in maternal lipoprotein profile during preg-
nancy. The increased lipolytic activity in adipose tissue during late ges-
tation enhances the production of VLDL by the liver, which together
with decreased removal of VLDL from the circulation as a result of de-
creased LPL activity in adipose tissue can augment circulating levels of
VLDL. During late pregnancy in rat, tissue LPL varies in different direc-
tions. It decreases in the adipose tissue, but increases in placental and
mammary gland tissue. These changes may divert TG from uptake by
adipose tissue to uptake by the placenta for hydrolysis and transfer of
NEFA to the fetus [614].
5. Treatment of dyslipidemias
The possibility of secondary dyslipidemias needs to be considered
before initiating therapy. The mechanism of action of common choles-
terol lowering drugs in routine use is summarized below.
(i) Statins: Statins competitively inhibit HMGCR reductase activity.
The reduction in intracellular cholesterol increases LDLR expression on
the hepatocyte cell surface and increases extraction of LDL cholesterol
from blood.
(ii) Bile acid sequestrants: By binding to bile acids the drugs remove
the bile acids from the enterohepatic circulation. The liver depleted of
bile synthesizes more from the hepatic stores of cholesterol. The in-
crease in cholesterol catabolism leads to a compensatory increase in
LDLR activity, and to increased clearance of LDL cholesterol from the
circulation.
(iii) Cholesterol absorption inhibitors: By inhibiting intestinal cho-
lesterol at the level of the brush border of the intestine (most probably
by interacting with the NPC1L1 protein) they reduce the amount of cho-
lesterol circulated to the liver.
(iv) Niacin has a broad lipid modulating action, it raises HDL choles-
terol and reduces both LDL cholesterol and TG. There are several mech-
anism by which niacin may affect plasma lipoproteins. Recently the
GPR109A receptor also named the hydroxyl-carboxylic acid receptor
2, expressed in adipocytes and immune cells, that binds niacin has
been identified. Activation of GPR109A in adipocytes results in the in-
hibitory G protein, Gi mediated reduction in adenylate cyclase, decreas-
ing cAMP accumulation and protein kinase A activity. This results in
decreased phosphorylation of HSL and TG hydrolysis, decreased FA
flux to the liver and reduced substrate availability for hepatic VLDL syn-
thesis. Niacin noncompetitively inhibits hepatocyte DGAT resulting in
decreased TG synthesis. It has been suggested that there is an accompa-
nying decrease in CETP mediated exchange of TG for cholesteryl ester
between VLDL and HDL particles leading to a net rise in HDL particles.
A further suggestion is that niacin promotes cholesterol efflux from ad-
ipocytes to apoAI by activation of the PPARγ–LXRα–ABCA1 pathway.
Niacin is thought to exert pleiotropic effects which are lipoprotein inde-
pendent through direct anti-inflammatory effects on cell types involved
in the progression of atherosclerosis [615,616,617].
Both fibrates and n-3 polyunsaturated fatty acids are the available
pharmacological interventions to decrease elevated plasma TG levels.
Fibrates are agonists of PPARα and act through various transcription
factors to regulate lipoprotein metabolism to lower fasting and post-
prandial TG levels. A summary of PPARα mediated gene regulation by
fibrates is shown in Fig. 8. [618].
172 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
Fig. 8. Mechanism of action of fibrates.
Fish contain high levels of two omega-3 FA, EPA (C20:5 n-3) and
docosahexanoic acid (DHA; C22:6 n-3). The effects of n-3 fatty acids are
now well-established. Total cholesterol is not materially affected by n-3
fatty acid consumption, LDL cholesterol concentrations tend to rise by
5–10% and HDL cholesterol by 1–3%, and serum TG concentrations de-
crease by 25–30% [619]. The effects are more likely to be due to decreased
hepatic secretion of VLDL. This effect is probably due to multiple mecha-
nisms: omega-3 FA may (i) not be the preferred substrate for the enzyme
DGAT and thus decrease TG synthesis (ii) interact with nuclear transcrip-
tion factors (PPARα, LXR and farnesol X receptor, HNF-4α) and decrease
TG synthesis and increase fatty acid oxidation and (iii) promote apoB deg-
radation in the liver through stimulation of an autophagic process (iv) in-
duce LPL activity and increase CM and VLDL clearance [620,621].
Statins today are the treatment of choice for lowering LDL cholester-
ol. Patients who fail to achieve adequate reduction of LDL cholesterol
can be considered statin resistant. Resistance to statins can be due to dif-
ferences in pharmacokinetics as well as in target pathways. Target path-
ways include HMGCR and various points along the cholesterol synthesis
and lipoprotein metabolic pathways [622]. There are various genetic
variations that are associated with diversity in the drugs response
[623]. In addition high risk patients do not always achieve sufficient re-
duction on LDL cholesterol, even with high intensity statin treatment.
New novel LDL cholesterol lowering drugs are in development to en-
hance the efficacy of statins. These approaches are based on the inhibi-
tion of PCSK9, apoB or MTP.
Inhibition of PCSK9 may be achieved by a number of approaches, in-
cluding the use of monoclonal antibodies (mAb), antisense nucleotides
(ASO) and small interfering RNAs (siRNA). Phases I, II and III reveal the
efficacy of monoclonal PCSK9 inhibitors to reduce high LDL levels [624].
Mipomersen is an apoB targeted ASO tested against humans. It is ap-
proved by the US FDA for use in homozygous FH patients as an adjunct
to diet and statins. The most studied MTP inhibitor, lomitapide has been
granted FDA approval as an orphan drug to reduce LDL cholesterol in
patients with homozygous FH. Other MTP inhibitors have been studied
in experimental animals. Both lomitapide and mipomersen are associat-
ed with side effects, such as hepatic steatosis and injection site reactions
with mipomersen, that do not allow extended clinical use. Newer forms
of PPARα/γ agonists, RNA based treatments that target apoCIII, and in-
hibitors of IDOL are also in development [625].
The available evidence for elevating low HDL cholesterol levels are
relatively few. The most efficacious pharmacological approach to eleva-
tion of low HDL cholesterol has involved inhibition of CETP. Inhibiting
CETP led to a substantial increase in HDL cholesterol but this has not
translated to beneficial effects on cardiovascular outcomes. The devel-
opment of torcetrapib, the first CETP inhibitor was halted after the
Investigation of Lipid Level Mangement to Understand its Impact in
Atherosclerotic Events (ILLUMINATE) trial revealed an increase in
cardiovascular events in the torcetrapib treated group. It has been sug-
gested that efforts should focus on improving HDL quality in addition to
raising HDL levels when developing HDL raising therapies. Other novel
therapies aimed at raising HDL include apoAI mimetics, reconstituted
HDL, PPARα/γ agonists and LXR agonists [626].
Somatic cell gene therapy is a promising approach for the treatment
of several dyslipidemias [627]. In a clinical trial using adeno-associated
 I. Ramasamy / Clinica Chimica Acta 454 (2016) 143–185
virus serotype 1 (AAV-1) mediated gene transfer of the naturally occur-
ring gain of function LPL mutant S447X, to muscle in subjects with LPL
deficiency, median plasma TG decreased initially. However, long term
follow up of TG showed a return to baseline levels. No subjects devel-
oped an immune response to the LPL transgene product though 4 of
the 8 subjects developed a T-cell response to the vector capsid. Potential
solutions, such as immunomodulation at the time of gene transfer may
need to be explored to achieve sustained persistent expression of the
transgene product [628].
6. Future directions
Severe dyslipidemia, a decrease or increase in serum lipoproteins is
usually a simple monogenic disorder showing a Mendelian inheritance.
More frequently dyslipidemia results from the cumulative effects of
several genes combined with environmental and metabolic stressors
such as high calorie intake, metabolic syndrome, diabetes and certain
drugs. Despite our increased understanding of molecular basis of
dyslipidemias, much remains to be learned. Genetic profiles are far
from complete and there is further room for characteristation of genetic
changes influencing serum lipid levels. Emerging technologies such as
whole genome sequencing may produce more discriminating ‘risk al-
lele’ scores. Although a plethora of antidyslipidemic pharamacological
agents are available, they can be ineffective in the more severe lipid dis-
orders. The results of existing pharmacological treatments must be
viewed alongside newer treatments that are being tested. Future direc-
tions will be to define the missing genetic variation which will open
possibilities for targeted individualized treatment for dyslipidemias.
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