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Demystifying HDL Cholesterol-A "Human Knockout" to the Rescue?

The substantial cost associated with failure of a therapeutic approach in a large outcomes-based clinical trial, as has occurred recently for multiple compounds targeting HDL cholesterol metabolism, has led to intense interest in improved target selection. Careful study of humans who carry mutations that inactivate a putative drug target leverages naturally occurring genetic variation to study the impact of lifelong deficiency of a specific protein. As such, human genetics research can provide an opportunity to confirm the physiologic relevance of a target in humans before large-scale investments in drug development.

Human Genetics to Inform Novel Therapy, the Case of PCSK9

Naturally occurring human DNA sequence variation can provide the foundation for important insights into disease biology and novel therapeutic strategies. Within the field of lipid metabolism, the rapid clinical development of proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors to reduce LDL cholesterol provides proof-of-concept for the potential of this approach. Following the initial 2003 discovery of a gain-of-function mutation in the gene PCSK9 (proprotein convertase subtilisin/kexin type 9) [3] associated with significantly increased LDL cholesterol, a 2005 report noted that about 2% of black individuals harbor an inactivating (premature stop) mutation in the PCSK9 gene. Carriers of this mutation benefitted from lifelong lower LDL cholesterol concentrations and inborn protection from coronary disease without detectable toxicity (1). Further, the identification of healthy individuals with 2 inactivating mutations, effectively "human knockouts" for PCSK9, further increased optimism that the protein could be targeted without off-target effects.

These observations provided additional support for the hypothesis that pharmacologic inhibition of PCSK9 might similarly lower LDL cholesterol and decrease vascular risk. Translational research studies demonstrated that PCSK9 plays a previously unrecognized role in preventing hepatic uptake of LDL cholesterol by accelerating the catabolism of LDL receptors. These findings led to a pharmaceutical arms race to develop PCSK9 inhibitors using monoclonal antibody or RNA interference approaches. By 2015, less than a decade after the initial identification of the PCSK9 mutations, 2 antibody-based PCSK9 inhibitors were approved by the US Food and Drug Administration primarily for treatment of patients with genetic

forms of hypercholesterolemia. Multiple large clinical trials are underway to ascertain efficacy and safety of these PCSK9 inhibitors in a range of high-risk cardiovascular patients.

Revising the HDL Hypothesis-Is Promoting Reverse Cholesterol Transport the Key?

The strong inverse association between HDL cholesterol concentrations and risk of coronary disease gave rise to the "HDL hypothesis"--namely that a therapy that increased concentrations of HDL might reduce risk of atherosclerotic disease. However, HDL concentrations are linked to other cardiovascular risk factors, including triglyceride-rich lipoproteins and markers of inflammation that may have confounded the initial epidemiologic associations. Second, common genetic variants with a statistically robust (albeit quantitatively modest) impact on HDL cholesterol concentrations have minimal relationship with risk of coronary disease (2). Because genetic variants are distributed largely at random within the population, these analyses may be less subject to confounding. Third, multiple HDL raising compounds (e.g., niacin and cholesteryl ester transfer protein inhibitors) failed to improve clinical outcomes in recent clinical trials. In short, despite major investments in basic and clinical research, the HDL hypothesis as initially put forth has failed to come to fruition.

HDL particles are hypothesized to protect against coronary disease via the reverse cholesterol transport pathway. In this paradigm, HDL serves as the acceptor of cholesterol that is effluxed from cells in the periphery, e.g., the lipid-laden foam cells found in atherosclerotic plaques. Cholesterol within HDL particles is selectively taken up into the liver via the scavenger receptor BI (SRBI) protein and ultimately undergoes biliary excretion and removal from the body. A large body of transgenic mouse experiments has noted that the impact of perturbations on flux through the reverse cholesterol transport pathway is a better predictor of atherosclerotic phenotypes than the effect on static measurements of HDL cholesterol concentrations. A key example of discordance between HDL concentrations and atherosclerosis relates to SRBI--despite markedly increased HDL cholesterol concentrations, mice deficient in SRBI demonstrate an increase in atherosclerosis (3). The proposed mechanism of this increased plaque burden relates to impaired hepatocyte uptake of HDL cholesterol, thus decreasing flux through the reverse cholesterol transport pathway. Although multiple previous investigators have linked human mutations in SCARB1 (scavenger receptor class B member 1, the gene encoding the SRB1 protein) with HDL cholesterol concentrations, the relevance of the animal model finding to human atherosclerosis had remained speculative.

Translation of SRBI Biology Using Genetics--From a Single (Wo)Man to Mice

An elegant series of experiments recently led by Daniel Rader's laboratory involved gene sequencing to extend the SRBI data in mice to humans ((4); Fig. 1). Candidate genes were sequenced in 328 individuals with very high HDL cholesterol concentrations. One participant, a 67-year old woman with an HDL of 152 mg/dL (about 3 times normal), was homozygous for a P376L missense mutation in SCARB1. To determine the functional significance of this mutation, induced pluripotent stem cells from this individual were isolated and differentiated into primary hepatocytes. As expected for a damaging mutation, the ability of these P376L hepatocytes to selectively uptake cholesterol was almost completely eliminated. Additional in vitro and murine studies confirmed that this variant led to loss-of-function of the SRBI protein, likely via impaired posttranslational modification.

On confirming that the P376L variant severely damaged the SRBI protein, the authors called back the homozygous, human knockout individual as well as 8 identified heterozygotes for additional phenotyping to systematically test hypotheses derived from previous descriptions of SCARB1 knockout mice. Although SRBI plays a role in steroidogenesis and fertility in mice, the study did not detect any differences in hormone concentrations between these individuals and appropriate controls--indeed, the homozygous woman had borne 2 healthy children. Similarly, detailed analyses of platelet counts and function failed to replicate the thrombocytopenia and impaired platelet aggregation noted in SCARB1 knockout mice and a previous report based on a different inactivating SCARB1 in humans. Lastly, despite a very high HDL cholesterol, a scan of the homozygous woman's carotid arteries revealed evidence of substantial subclinical atherosclerosis.

Characterizing rare mutations in highly selected populations, such as those with very high HDL cholesterol concentrations, may lead to ascertainment bias or chance findings. The research team attempted to overcome these limitations by analyzing the relationships between P376L in SCARB1 (as ascertained using a genotyping chip), lipid concentrations, and risk of coronary disease. Because this variant is rare in the population (present in approximately 1 in 1700 individuals), this analysis required a large number of individuals analyzed across multiple studies. With regard to impact on HDL cholesterol, an analysis in >300 000 individuals confirmed that the P376L variant was associated with an 8 mg/dL increase in circulating concentrations. More importantly, a metaanalysis of 16 case control studies for coronary disease demonstrated a seemingly paradoxical 79% increased coronary risk in P376L carriers vs noncarriers, a finding that reached nominal statistical significance (P = 0.018). These data, triggered by the identification of a single individual with an extreme HDL cholesterol phenotype, demonstrate for the first time that a genetic variant associated with higher HDL concentrations is likely to increase risk of coronary disease. This is presumably related to impaired reverse cholesterol transport related to SRBI deficiency and provides key support for the notion that flux through this pathway is an important determinant of human atherosclerosis. It is important to note that this increase in cardiovascular risk for a rare specific genetic variant does not negate the broader epidemiologic relationship of increased HDL and reduced vascular risk. It does, however, underscore the core complexities of HDL metabolism in vascular disease and reinforces current opinions that change in HDL cholesterol concentrations is an unreliable surrogate for therapeutic efficacy.

A Way Forward in Biomedicine--Humans First!

Just as knockout mice provided an opportunity for unprecedented advances in experimental biology, human knockouts may well serve as a key reagent in translational medicine. These individuals may be less rare than previously thought--an exome sequencing study of approximately 7000 Pakistani individuals identified homozygous protein inactivating mutations in 961 genes, several of which relate to targets of drugs currently in development (5).

Moving forward, using human genetics to prove physiologic relevance and help prioritize molecular pathways may become an important component of the biomedical research armamentarium. Investigators trained to functionally characterize the identified mutations and conduct hypothesis-based phenotyping at scale will be essential to realizing the potential for public health gain. Ongoing efforts to reduce genotyping cost, promote data sharing, and develop large biobanks of individuals consented for call-back phenotyping will serve as key catalysts of this work. If successful, this approach will build on the framework provided by the study of SRB1 deficiency to provide key additional insights into human disease.

Author Contributions: AH authors confirmed they have contributed to the intellectual content of this paper and have met the following3 requirements: (a) significant contributions to the conception and design, acquisition Of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: A.V. Khera, ACCF/Merck Cardiovascular Research Fellowship; P.M Ridker, investigator initiated research support from the NHLBI, NCI, Amgen, Novartis, Pfizer, Kowa, and AstraZeneca.

Expert Testimony: None declared.

Patents: P.M Ridker, co-inventor of patents held by the Brigham and Women's Hospital that relate to the use of inflammatory biomarkers in cardiovascular disease and diabetes that have been licensed to AstraZeneca and Siemens.


(1.) Cohen JC, Boerwinkle E, Mosley TH Jr, Hobbs HH. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med 2006; 354:1264-72.

(2.) Voight BF, Peloso GM, Orho-Melander M, Frikke-Schmidt R, Barbalic M, Jensen MK, et al. Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study. Lancet 2012;380:572-80.

(3.) Trigatti B, Rayburn H, Vinals M, Braun A, Miettinen H, Penman M, etal. Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology. Proc Natl Acad Sci U S A 1999;96: 9322-7.

(4.) Zanoni P, Khetarpal SA, Larach DB, Hancock-Cerutti WF, Millar JS, Cuchel M, et al. Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease. Science 2016;351:1166-71.

(5.) Saleheen S, Natarajan P, Zhao W, Rasheed A, Khetarpal S, Wong HH, etal. Human knockouts in a cohort with a high rate of consanguinity. bioRxiv. 10.1101/031518 (Accessed June 2016).

Amit V. Khera [1] and Paul M Ridker [2] *

[1] Center for Human Genetic Research and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Boston, MA; [2] Center for Cardiovascular Disease Prevention and Division of Cardiovascular Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.

* Address correspondence to this author at: Brigham and Women's Hospital, 900 Commonwealth Ave. East, 3rd Floor, Boston, MA, 02215. Fax 617-734-1508;

Received May 2,2016; accepted June 23,2016.

Previously published online at DOI: 10.1373/clinchem.2016.258244

[3] Human genes: PCSK9, proprotein convertasesubtilisin/kexin type 9; SCARB1, scavenger receptor class B member 1.

Caption: Fig. 1. Characterization of a SCARB1 gene mutation causal of high HDL concentrations in humans.

Candidate genes were sequenced in individuals ascertained based on very high HDL cholesterol concentrations. A homozygote for the P376L missense variant in the SCARB1 gene was identified. Stem cell and animal model data confirmed that this variant led to loss of function of the SRB1 protein. Deep phenotyping of the homozygote and 8 heterozygote carriers noted increase HDL cholesterol but authors did not replicate abnormal steroidogenesis or platelet aggregation noted in previous knockout mouse studies. Finally, a metaanalysis of this variant in >300 000 people confirmed an association with an 8-mg/dL increase in HDL as well as a paradoxical 79% increase in risk of coronary disease.
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Title Annotation:Perspective
Author:Khera, Amit V.; Ridker, Paul M.
Publication:Clinical Chemistry
Geographic Code:1USA
Date:Jan 1, 2017
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