GENETIC MUTATIONS IN LDL-C CLEARANCE AND IMPLICATIONS FOR CV RISK

LDL-C Clearance Pathway

“The LDL receptor studies lend experimental support to the epidemiologists’ suggestion that the levels of plasma cholesterol usually seen in Western industrialized societies are inappropriately high. This support derives from knowledge of the affinity of the LDL receptor for LDL. The receptor binds LDL optimally when the lipoprotein is present at a cholesterol concentration of 2.5 mg/dL. In view of the 10 to 1 gradient between concentrations of LDL in plasma and interstitial fluid, a level of LDL-cholesterol in plasma of 25 mg/dL would be sufficient to nourish body cells with cholesterol. This is roughly one-fifth of the level usually seen in Western societies. Several lines of evidence suggest that plasma levels of LDL-cholesterol in the range of 25–60 mg/dL (total plasma cholesterol of 110 to 150 mg/dL) might indeed be physiologic for human beings.51

MICHAEL BROWN AND JOSEPH GOLDSTEIN

The Nobel Foundation, 1985

As an essential component of cellular membranes and a precursor for the synthesis of all steroid hormones, including bile acids and vitamin D, cholesterol plays a pivotal role in many physiological functions. However, excess levels of cholesterol can become toxic and are directly related to the risk for CV events7,52

Physiological LDL-C levels

Over the course of history, the link between cholesterol and CV risk has evolved significantly. Epidemiological studies have shown that in non-industrialized hunter-gatherer societies evidence of atherosclerosis was rare, even in individuals 70 or 80 years of age.10 The average LDL-C levels in those populations are estimated at around 50 to 75 mg/dL, similar to the LDL-C levels of healthy newborns today (30 mg/dL range).10,53 In the 1980s, “desirable” or “normal” cholesterol was <200 mg/dL.54 “Normal” LDL-C levels by today’s standard are considered to be <100 mg/dL, yet the average total cholesterol level in American adults is 208 mg/dL.10 However, even individuals with lower cholesterol levels carry some risk of CV disease. One study shows that subclinical atherosclerosis is present in nearly half (49.7%) of individuals with ‘normal’ LDL-C levels under the age of 54.23 The top 10% of the population with the highest LDL-C levels accounts for only 20% of the CHD events, leaving many individuals at risk of under-diagnosis and under-treatment for CHD.10,23,55

Cholesterol Synthesis

The liver is the main site for the production of cholesterol as well as for the regulation of serum LDL-C levels through LDL receptor (LDLR)-mediated clearance.9 In addition to LDL produced by the liver, cholesterol is obtained through dietary sources and produced de novo by other tissues (e.g. CNS and steroidal tissues).56,57 While systemic LDL-C levels can be an important source of cholesterol, all mammalian cells have the ability to synthesize it de novo from acetate.8

Circulating LDL-C levels of no more than 25 mg/dL can provide an additional source for cellular metabolism.8

The process of cholesterol biosynthesis begins with the conversion of acetyl-CoA to mevalonate with 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase catalyzing the third, rate limiting step and a major point of regulation in the cholesterol biosynthetic pathway.

Cholesterol in the brain and steroidogenic tissues

In the central nervous system, cholesterol is primarily obtained through de novo synthesis and via HDL-C (Figure 13) while the blood-brain barrier prevents exchange with lipoprotein cholesterol from the circulation.56 The segregation ensures that cholesterol metabolism within the brain is isolated from changes in systemic LDL-C levels due to diet or medication.57

Steroidogenic tissues, derive cholesterol for the production of steroid hormones predominantly from de novo synthesis and via HDL-C.58,59,60 Results from lipid-lowering trials have consistently shown that LDL-C reduction does not affect the production of gonadal or adrenal steroid hormones in a range of patients and treatment settings.61,62,63

Figure 13: In the CNS, cholesterol is predominately obtained by de novo synthesis. LDL-C does not cross the blood-brain barrier56,57,64

Figure 13 Open

Figure 14: Despite reducing LDL-C, statins do not alter gonadal or adrenal steroid hormones61,62,63

Figure 14 Figure 14
LDLR recycling and cholesterol homeostasis

Both dietary and de novo produced cholesterol is transported in the blood by cholesterol rich LDLs including apolipoprotein B containing lipoproteins.7 LDL uptake occurs through binding of apoB-containing lipoproteins to the LDL receptor and internalization of the complex through endocytosis. After internalization the receptors dissociate from their ligands and are recycled back to the surface while LDL is hydrolyzed in lysosomes.7

An estimated 70% of systemic LDL-C is cleared from the circulation through the LDL receptor (LDLR) while the remaining 30% is eliminated through intracellular scavenger receptors. Homeostatic control of intracellular cholesterol levels is achieved through complex regulatory feedback loops that ensure intracellular levels remain constant regardless of changes in circulating LDL-C levels.7Early studies in patients with genetic disorders of lipid metabolism demonstrated how intracellular cholesterol levels regulate both the rate limiting step in the cholesterol biosynthetic pathway and the number of LDL receptors responsible for systemic LDL-C uptake to balance de novo synthesis and uptake.7

Figure 15: Recycling of LDLRs enables efficient clearance of LDL particles7,65,66

Figure 15 Open
Regulation by PCSK9

Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) is a member of the mammalian proprotein convertase family of secretory serine endoproteases that functions as a molecular chaperone, binding to LDLR and targeting it for lysosomal degradation.67,68 PCSK9 directs the degradation of LDLR via two separate pathways: in the intracellular pathway nascent PCSK9 binds to LDLR and directs it from the trans-Golgi to the lysosome.67

In the extracellular pathway, secreted PCSK9 binds to LDLR and upon internalization directs it for degradation by the lysosome thus inhibiting further LDLR recycling and LDL-C clearance.67

Genetically induced low LDL-C levels and implications for cardiovascular risk

Consistent with data from clinical trials Mendelian randomization studies have demonstrated that variants in over 50 genes associated with lower LDL-C levels, including variants in HMG-CoA reductase, NPC1L1, and the LDLR confer a lower risk for coronary heart disease (CHD).3 The effect of each variant on the risk for CHD is proportional to the magnitude of absolute change in LDL-C and all variants have the same effect on CHD risk per unit reduction in LDL-C levels again indicating that the reduction in the risk for CHD is independent of the mechanism by which LDL-C is lowered.3

Figure 16: Genetic studies and pharmacologically lowered LDL-C shows causality with reduced risk for CHD3

Figure 16 Open
Genetic variations in PCSK9: Genetic inhibition of PCSK9 function reduces circulating LDL-C levels67,69

Gain-of-function mutations in PCSK9 were first identified in French and shortly after in Norwegian families with clinical familial hypercholesterolemia (FH) and shown to be responsible for the hyperlipidemia phenotype.70,71 In contrast, loss of function mutations in PCSK9 were associated with significant reductions in circulating LDL-C levels.70

Case studies of individuals with loss of function PCSK9 mutations reveal that long-term exposure to very low LDL-C levels (14-16 mg/dL) is not associated with any obvious adverse effects.72,73,74,75

Figure 17: Genetic variants of PCSK9 demonstrate its importance in regulating LDL levels65,69,76

Figure 17 Open
Familial Hypercholesterolemia

Studies in patients with inherited disorders of lipid metabolism provided the first evidence of a causal relationship between cholesterol and coronary heart disease.14 Familial hypercholesterolemia (FH) is an autosomal co-dominant disorder caused by mutations that affect LDL-C clearance. FH is most often caused by loss-of-function (LOF) mutations in the LDL receptor (LDLR) gene, LOF mutations in the apoB gene that reduce the ability of apoB-containing lipoproteins to bind to the LDL receptor, or gain-of-function (GOF) mutations in the PCSK9 gene that result in reduced levels of LDLR in the liver.3,77 Heterozygous FH affects between 1 in 200 and 250 patients and is characterized by marked hyperlipidemia (LDL-C levels typically >190 mg/dL ) and premature development of atherosclerosis and coronary artery disease.78,79 An estimated 50% of men and 30% of women with heterozygous FH will experience a coronary event before the age of 60.80Without effective treatment individuals with FH reach the cumulative LDL-C burden sufficient for the development of CHD on average 20 years earlier compared to patients without FH.79

In patients with FH the risk of CV events is proportional to the magnitude and duration of exposure to elevated LDL-C levels.3 Commonly used assessment tools such as the US Framingham Risk Score are therefore not appropriate in these patients as they do not account for lifelong exposure to hyperlipidemia. FH is often not diagnosed until after the occurrence of a major coronary event and undertreated with fewer than 1 in 20, i.e. only 5%, of patients achieving the recommended LDL-C levels.78,79 Patients with homozygous FH (HoFH) present with a much more severe phenotype compared to patients with heterozygous FH (HeFH) with untreated LDL-C levels that can exceed 500 mg/dL at birth and almost universal development of advanced CVD before the age of 10 years old.3,7 Genetic and biochemical studies in patients with FH led to several landmark discoveries in the field, including the role of the LDLR in LDL-C clearance, the discovery of LDLR endocytocis and recycling and the description of feedback loops regulating cholesterol biosynthesis and LDLR number.7

In patients with FH, the risk of CV events and the extent of underlying atherosclerosis is proportional to the absolute magnitude and duration of exposure to elevated LDL-C levels.3

Genetic variations in other genes that regulate cholesterol metabolism

Niemann-Pick C1-like 1 (NPC1L1) protein is a transmembrane protein mainly expressed in the small intestine where it is thought to mediate cholesterol transport across the apical membrane of enterocytes.81 NPC1L1 is a homologue of Niemann-Pick C1 (NPC1), a protein with an established role in intracellular cholesterol transport mutations which are associated with the lipid storage disorder Niemann-Pick disease type C1.81 Individuals carrying inactivating mutations in NPC1L1 have significantly reduced LDL-C levels (12 mg/dL LDL-C reduction) and a 53% lower risk of coronary heart disease compared to non-carriers.82