|, proprotein convertase subtilisin/kexin type 9, FH3, HCHOLA3, LDLCQ1, NARC-1, NARC1, PC9, FHCL3|
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme encoded by the PCSK9 gene in humans on chromosome 1. It is the 9th member of the proprotein convertase family of proteins that activate other proteins. Similar genes (orthologs) are found across many species. As with many proteins, PCSK9 is inactive when first synthesized, because a section of peptide chains blocks their activity; proprotein convertases remove that section to activate the enzyme. The PCSK9 gene also contains one of 27 loci associated with increased risk of coronary artery disease.
PCSK9 is ubiquitously expressed in many tissues and cell types. PCSK9 binds to the receptor for low-density lipoprotein particles (LDL), which typically transport 3,000 to 6,000 fat molecules (including cholesterol) per particle, within extracellular fluid. The LDL receptor (LDLR), on liver and other cell membranes, binds and initiates ingestion of LDL-particles from extracellular fluid into cells, thus reducing LDL particle concentrations. If PCSK9 is blocked, more LDLRs are recycled and are present on the surface of cells to remove LDL-particles from the extracellular fluid. Therefore, blocking PCSK9 can lower blood LDL-particle concentrations.
PCSK9 has medical importance because it acts in lipoprotein homeostasis. Agents which block PCSK9 can lower LDL particle concentrations. The first two PCSK9 inhibitors, alirocumab and evolocumab, were approved as once every two week injections, by the U.S. Food and Drug Administration in 2015 for lowering LDL-particle concentrations when statins and other drugs were not sufficiently effective or poorly tolerated. The cost of these new medications, as of 2015[update], was $14,000 per year at full retail; judged of unclear cost effectiveness by some. While these medications are prescribed by many physicians, the payment for prescriptions are often denied by insurance providers. As a result pharmaceutical manufacturers lowered the prices of these drugs.
- 1 History
- 2 Structure
- 3 Function
- 4 PCSK9 Inhibitor Drugs
- 5 References
- 6 Further reading
- 7 External links
In February 2003, Nabil Seidah, a scientist at the Clinical Research Institute of Montreal in Canada, discovered a novel human proprotein convertase, the gene for which was located on the short arm of chromosome 1. Meanwhile, a lab led by Catherine Boileau at the Necker-Enfants Malades Hospital in Paris had been following families with familial hypercholesterolaemia, a genetic condition that, in 90% of cases causes coronary artery disease (FRAMINGHAM study) and in 60% of cases may lead to an early death; they had identified a mutation on chromosome 1 carried by some of these families, but had been unable to identify the relevant gene. The labs got together and by the end of the year published their work, linking mutations in the gene, now identified as PCSK9, to the condition. In their paper, they speculated that the mutations might make the gene overactive. In that same year, investigators at Rockefeller University and University of Texas Southwestern had discovered the same protein in mice, and had worked out the novel pathway that regulates LDL cholesterol in which PCSK9 is involved, and it soon became clear that the mutations identified in France led to excessive PCSK9 activity, and thus excessive removal of the LDL receptor, leaving people carrying the mutations with too much LDL cholesterol. Meanwhile, Dr. Helen H. Hobbs and Dr. Jonathan Cohen at UT-Southwestern had been studying people with very high and very low cholesterol, and had been collecting DNA samples. With the new knowledge about the role of PCSK9 and its location in the genome, they sequenced the relevant region of chromosome 1 in people with very low cholesterol and they found nonsense mutations in the gene, thus validating PCSK9 as a biological target for drug discovery.
The solved structure of PCSK9 reveals four major components in the pre-processed protein: the signal peptide (residues 1-30); the N-terminal prodomain (residues 31-152); the catalytic domain (residues 153-425); and the C-terminal domain (residues 426-692), which is further divided into three modules. The N-terminal prodomain has a flexible crystal structure and is responsible for regulating PCSK9 function by interacting with and blocking the catalytic domain, which otherwise binds the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. While previous studies indicated that the C-terminal domain was uninvolved in binding LDLR, a recent study by Du et al. demonstrated that the C-terminal domain does bind LDLR. The secretion of PCSK9 is largely dependent on the autocleavage of the signal peptide and N-terminal prodomain, though the N-terminal prodomain retains its association with the catalytic domain. In particular, residues 61-70 in the N-terminal prodomain are crucial for its autoprocessing.
Role and regulatory function
This protein plays a major regulatory role in cholesterol homeostasis, mainly by reducing LDLR levels on the plasma membrane. Reduced LDLR levels result in decreased metabolism of LDL-particles, which could lead to hypercholesterolemia. When LDL binds to LDLR, it induces internalization of LDLR-LDL complex within an endosome. The acidity of the endosomal environment induces LDLR to adopt a hairpin conformation. The conformational change causes LDLR to release its LDL ligand, and the receptor is recycled back to the plasma membrane. However, when PCSK9 binds to the LDLR (through the EGF-A domain), PCSK9 prevents the conformational change of the receptor-ligand complex. This inhibition redirects the LDLR to the lysosome instead.
PCSK9 is synthesized as a soluble zymogen that undergoes autocatalytic intramolecular processing in the endoplasmic reticulum. The protein may function as a proprotein convertase. PCSK9 is expressed mainly in the liver, the intestine, the kidney, and the central nervous system. PCSK9 also plays an important role in intestinal triglyceride-rich apoB lipoprotein production in small intestine and postprandial lipemia.
After being processed in the ER, PCSK9 co-localizes with the protein sortilin on its way through the Golgi and trans-Golgi complex. A PCSK9-sortilin interaction is proposed to be required for cellular secretion of PCSK9. In healthy humans, plasma PCSK9 levels directly correlate with plasma sortilin levels, following a diurnal rhythm similar to cholesterol synthesis. The plasma PCSK9 concentration is higher in women compared to men, and the PCSK9 concentrations decrease with age in men but increase in women, suggesting that estrogen level most likely plays a role. PCSK9 gene expression can be regulated by sterol-response element binding proteins (SREBP-1/2), which also controls LDLR expression.
PCSK9 may also have a role in the differentiation of cortical neurons.
Variants of PCSK9 can reduce or increase circulating cholesterol. LDL-particles are removed from the blood when they bind to LDLR on the surface of cells, including liver cells, and are taken inside the cells. When PCSK9 binds to an LDLR, the receptor is destroyed along with the LDL particle. PCSK9 degrades LDLR by preventing the hairpin conformational change of LDLR. If PCSK9 does not bind, the receptor will return to the surface of the cell and can continue to remove LDL-particles from the bloodstream.
Other variants are associated with a rare autosomal dominant familial hypercholesterolemia (HCHOLA3). The mutations increase its protease activity, reducing LDLR levels and preventing the uptake of cholesterol into the cells.
In humans, PCSK9 was initially discovered as a protein expressed in the brain. However, it has also been described in the kidney, the pancreas, liver and small intestine. Recent evidence indicate that PCSK9 is highly expressed in arterial walls such as endothelium, smooth muscle cells, and macrophages, with a local effect that can regulate vascular homeostasis and atherosclerosis. Accordingly, it is now very clear that PCSK9 has pro-atherosclerotic effects and regulates lipoprotein synthesis.
As PCSK9 binds to LDLR, which prevents the removal of LDL-particles from the blood plasma, several studies have determined the potential use of PCSK9 inhibitors in the treatment of hyperlipoproteinemia (commonly called hypercholesterolemia). Furthermore, loss-of-function mutations in the PCSK9 gene result in lower levels of LDL and protection against cardiovascular disease.
In addition to its lipoprotein synthetic and pro-atherosclerotic effects, PCSK9 is involved in glucose metabolism and obesity, regulation of re-absorption of sodium in the kidney which is relevant in hypertension. Furthermore, PCSK9 may be involved in bacterial or viral infections and sepsis. In the brain the role of PCSK9 is still controversial and may be either pro-apoptotic or protective in the development of the nervous system. PCSK9 levels have been detected in the cerebrospinal fluid at a 50-60 times lower level than in serum.
A multi-locus genetic risk score study based on a combination of 27 loci including the PCSK9 gene, identified individuals at increased risk for both incident and recurrent coronary artery disease events, as well as an enhanced clinical benefit from statin therapy. The study was based on a community cohort study (the Malmo Diet and Cancer study) and four additional randomized controlled trials of primary prevention cohorts (JUPITER and ASCOT) and secondary prevention cohorts (CARE and PROVE IT-TIMI 22).
PCSK9 Inhibitor Drugs
Several studies have determined the potential use of PCSK9 inhibitors in the treatment of hyperlipoproteinemia (commonly called hypercholesterolemia). Furthermore, loss-of-function mutations in the PCSK9 gene result in lower levels of LDL and protection against cardiovascular disease.
As a drug target
Drugs can inhibit PCSK9, leading to lowered circulating LDL particle concentrations. Since LDL particle concentrations are thought by many experts to be a driver of cardiovascular disease like heart attacks, it is plausible that these drugs may also reduce the risk of such diseases. Clinical studies, including phase III clinical trials, are now underway to describe the effect of PCSK9 inhibition on cardiovascular disease, and the safety and efficacy profile of the drugs. Among those inhibitors under development in December 2013 were the antibodies alirocumab, evolocumab, 1D05-IgG2 (Merck), RG-7652 and LY3015014, as well as the RNAi therapeutic inclisiran. PCSK9 inhibitors are promising therapeutics for the treatment of people who exhibit statin intolerance, or as a way to bypass frequent dosage of statins for higher LDL concentration reduction.
A review published in 2015 concluded that these agents, when used in patients with high LDL-particle concentrations (thus at greatly elevated risk for cardiovascular disease) seem to be safe and effective at reducing all-cause mortality, cardiovascular mortality, and heart attacks. However more recent reviews conclude that while PCSK9 inhibitor treatment provides additional benefits beyond maximally tolerated statin therapy in high-risk individuals, PCSK9 inhibitor use probably results in little or no difference in mortality.
Regeneron Pharmaceuticals (in collaboration with Sanofi) became the first to market a PCSK9 inhibitor, with a competitor Amgen reaching market slightly later. The drugs are approved by the FDA for treatment of hypercholesterolemia, notably the genetic condition heterozygous familial hypercholesterolemia which causes high cholesterol levels and heart attacks at a young age. More recently these drugs were also approved by the FDA for the reduction of cardiovascular events including a reduction in all cause mortality.
An FDA warning in March 2014 about possible cognitive adverse effects of PCSK9 inhibition caused concern, as the FDA asked companies to include neurocognitive testing into their Phase III clinical trials.
A number of monoclonal antibodies that bind to and inhibit PCSK9 near the catalytic domain were in clinical trials as of 2014[update]. These include evolocumab (Amgen), bococizumab (Pfizer), and alirocumab (Sanofi/Regeneron Pharmaceuticals). As of July 2015[update], the EU approved these drugs including Evolocumab/Amgen according to Medscape news agency report. A meta-analysis of 24 clinical trials has shown that monoclonal antibodies against PCSK9 can reduce cholesterol, cardiac events and all-cause mortality. The most recent guidelines for cholesterol management from the American Heart Association and American College of Cardiology now provide guidance for when PCSK9 inhibitors should be considered, particularly focusing on cases in which maximally tolerated statin and ezetimibe fail to achieve goal LDL reduction.
A possible side effect of the monoclonal antibody might be irritation at the injection site. Before the infusions, participants received oral corticosteroids, histamine receptor blockers, and acetaminophen to reduce the risk of infusion-related reactions, which by themselves will cause several side effects.
Peptides that mimick the EGFA domain of the LDLR that binds to PCSK9 have been developed to inhibit PCSK9.
The PCSK9 antisense oligonucleotide increases expression of the LDLR and decreases circulating total cholesterol levels in mice. A locked nucleic acid reduced PCSK9 mRNA levels in mice. Initial clinical trials showed positive results of ALN-PCS, which acts by means of RNA interference.
A vaccine that targets PCSK9 has been developed to treat high LDL-particle concentrations. The vaccine uses a VLP (virus-like particle) as an immunogenic carrier of an antigenic PCSK9 peptide. VLP's are viruses that have had their DNA removed so that they retain their external structure for antigen display but are unable to replicate; they can induce an immune response without causing infection. Mice and macaques vaccinated with bacteriophage VLPs displaying PCSK9-derived peptides developed high-titer IgG antibodies that bound to circulating PCSK9. Vaccination was associated with significant reductions in total cholesterol, free cholesterol, phospholipids, and triglycerides.
Naturally occurring inhibitors
The plant alkaloid berberine inhibits the transcription of the PCSK9 gene in immortalized human hepatocytes in vitro, and lowers serum PCSK9 in mice and hamsters in vivo. It has been speculated that this action contributes to the ability of berberine to lower serum cholesterol. Annexin A2, an endogenous protein, is a natural inhibitor of PCSK9 activity.
- GRCh38: Ensembl release 89: ENSG00000169174 - Ensembl, May 2017
- GRCm38: Ensembl release 89: ENSMUSG00000044254 - Ensembl, May 2017
- "Human PubMed Reference:". National Center for Biotechnology Information, U.S. National Library of Medicine.
- "Mouse PubMed Reference:". National Center for Biotechnology Information, U.S. National Library of Medicine.
- Seidah NG, Benjannet S, Wickham L, Marcinkiewicz J, Jasmin SB, Stifani S, Basak A, Prat A, Chretien M (February 2003). "The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation". Proc. Natl. Acad. Sci. U.S.A. 100 (3): 928–33. Bibcode:2003PNAS..100..928S. doi:10.1073/pnas.0335507100. PMC 298703. PMID 12552133.
- Zhang L, Song K, Zhu M, Shi J, Zhang H, Xu L, Chen Y (2016). "Proprotein convertase subtilisin/kexin type 9 (PCSK9) in lipid metabolism, atherosclerosis and ischemic stroke". International Journal of Neuroscience. 126 (6): 675–680. doi:10.3109/00207454.2015.1057636. PMID 26040332.
- Lagace TA (October 2014). "PCSK9 and LDLR degradation: regulatory mechanisms in circulation and in cells". Current Opinion in Lipidology. 25 (5): 387–93. doi:10.1097/MOL.0000000000000114. PMC 4166010. PMID 25110901.
- Mega JL, Stitziel NO, Smith JG, Chasman DI, Caulfield MJ, Devlin JJ, Nordio F, Hyde CL, Cannon CP, Sacks FM, Poulter NR, Sever PS, Ridker PM, Braunwald E, Melander O, Kathiresan S, Sabatine MS (June 2015). "Genetic risk, coronary heart disease events, and the clinical benefit of statin therapy: an analysis of primary and secondary prevention trials". Lancet. 385 (9984): 2264–71. doi:10.1016/S0140-6736(14)61730-X. PMC 4608367. PMID 25748612.
- "BioGPS - your Gene Portal System". biogps.org. Retrieved 2016-08-19.
- Weinreich M, Frishman WH (2014). "Antihyperlipidemic therapies targeting PCSK9". Cardiology in Review. 22 (3): 140–6. doi:10.1097/CRD.0000000000000014. PMID 24407047.
- Gearing ME (2015-05-18). "A potential new weapon against heart disease: PCSK9 inhibitors". Science in the News. Harvard University.
- Joseph L, Robinson JG (2015). "Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibition and the Future of Lipid Lowering Therapy". Progress in Cardiovascular Diseases. 58 (1): 19–31. doi:10.1016/j.pcad.2015.04.004. PMID 25936907.
- Hlatky MA, Kazi DS (2017). "PCSK9 Inhibitors: Economics and Policy". Journal of the American College of Cardiology. 70 (21): 2677–2687. doi:10.1016/j.jacc.2017.10.001. PMID 29169476.
- Gina Kolata, "These Cholesterol-Reducers May Save Lives. So Why Aren’t Heart Patients Getting Them?", The New York Times, Oct. 2, 2018. Retrieved 5 October 2018.
- Baum SJ, Toth PP, Underberg JA, Jellinger P, Ross J, Wilemon K (2017). "PCSK9 inhibitor access barriers-issues and recommendations: Improving the access process for patients, clinicians and payers". Clinical Cardiology. 40 (4): 243–254. doi:10.1002/clc.22713. PMC 5412679. PMID 28328015.
- Navar AM, Taylor B, Mulder H, Fievitz E, Monda KL, Fievitz A, Maya JF, López JA, Peterson ED (2017). "Association of Prior Authorization and Out-of-pocket Costs With Patient Access to PCSK9 Inhibitor Therapy". JAMA Cardiology. 2 (11): 1217–1225. doi:10.1001/jamacardio.2017.3451. PMC 5963012. PMID 28973087. Lay summary – Thomson Reuters.
- "PCSK9 price-cut matchup is on, as Regeneron and Sanofi slash Praluent list tag 60%". FiercePharma. Retrieved 2019-05-18.
- Hall SS (April 2013). "Genetics: a gene of rare effect". Nature. 496 (7444): 152–5. Bibcode:2013Natur.496..152H. doi:10.1038/496152a. PMID 23579660.
- Sijbrands EJ, Westendorp RG, Defesche JC, de Meier PH, Smelt AH, Kastelein JJ (2001). "Mortality over two centuries in large pedigree with familial hypercholesterolaemia: family tree mortality study". BMJ (Clinical Research Ed.). 322 (7293): 1019–23. doi:10.1136/bmj.322.7293.1019. PMC 31037. PMID 11325764.
- Abifadel M, Varret M, Rabès JP, Allard D, Ouguerram K, Devillers M, Cruaud C, Benjannet S, Wickham L, Erlich D, Derré A, Villéger L, Farnier M, Beucler I, Bruckert E, Chambaz J, Chanu B, Lecerf JM, Luc G, Moulin P, Weissenbach J, Prat A, Krempf M, Junien C, Seidah NG, Boileau C (June 2003). "Mutations in PCSK9 cause autosomal dominant hypercholesterolemia". Nat. Genet. 34 (2): 154–6. doi:10.1038/ng1161. PMID 12730697.
- Parag H. Joshi, Seth S. Martin, and Roger S. Blumenthal, "The fascinating story of PCSK9 inhibition: Insights and perspective from ACC", Cardiology Today, May 2014. Retrieved 5 October 2018.
- Abifadel M, Elbitar S, El Khoury P, Ghaleb Y, Chémaly M, Moussalli ML, Rabès JP, Varret M, Boileau C (September 2014). "Living the PCSK9 adventure: from the identification of a new gene in familial hypercholesterolemia towards a potential new class of anticholesterol drugs". Current Atherosclerosis Reports. 16 (9): 439. doi:10.1007/s11883-014-0439-8. PMID 25052769.
- "FDA approves Praluent to treat certain patients with high cholesterol". www.fda.gov. Retrieved 2015-07-26.
- PCSK9 gene - Genetics Home Reference
- "PCSK9 proprotein convertase subtilisin/kexin type 9 [Homo sapiens (human)] - Gene - NCBI". www.ncbi.nlm.nih.gov. Retrieved 2016-08-19.
- "PCSK9 - Proprotein convertase subtilisin/kexin type 9 precursor - Homo sapiens (Human) - PCSK9 gene & protein". www.uniprot.org. Retrieved 2016-08-19.
- Du F, Hui Y, Zhang M, Linton MF, Fazio S, Fan D (December 2011). "Novel domain interaction regulates secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein". The Journal of Biological Chemistry. 286 (50): 43054–61. doi:10.1074/jbc.M111.273474. PMC 3234880. PMID 22027821.
- Lo Surdo P, Bottomley MJ, Calzetta A, Settembre EC, Cirillo A, Pandit S, Ni YG, Hubbard B, Sitlani A, Carfí A (December 2011). "Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH". EMBO Reports. 12 (12): 1300–5. doi:10.1038/embor.2011.205. PMC 3245695. PMID 22081141.
- Piper DE, Jackson S, Liu Q, Romanow WG, Shetterly S, Thibault ST, Shan B, Walker NP (May 2007). "The crystal structure of PCSK9: a regulator of plasma LDL-cholesterol". Structure. 15 (5): 545–52. doi:10.1016/j.str.2007.04.004. PMID 17502100.
- Bottomley MJ, Cirillo A, Orsatti L, Ruggeri L, Fisher TS, Santoro JC, Cummings RT, Cubbon RM, Lo Surdo P, Calzetta A, Noto A, Baysarowich J, Mattu M, Talamo F, De Francesco R, Sparrow CP, Sitlani A, Carfí A (January 2009). "Structural and biochemical characterization of the wild type PCSK9-EGF(AB) complex and natural familial hypercholesterolemia mutants" (PDF). The Journal of Biological Chemistry. 284 (2): 1313–23. doi:10.1074/jbc.M808363200. PMID 19001363.
- Kwon HJ, Lagace TA, McNutt MC, Horton JD, Deisenhofer J (February 2008). "Molecular basis for LDL receptor recognition by PCSK9". Proceedings of the National Academy of Sciences of the United States of America. 105 (6): 1820–5. Bibcode:2008PNAS..105.1820K. doi:10.1073/pnas.0712064105. PMC 2538846. PMID 18250299.
- doi:10.1038/nsmb1235. PMID 17435765. Cunningham D, Danley DE, Geoghegan KF, Griffor MC, Hawkins JL, Subashi TA, Varghese AH, Ammirati MJ, Culp JS, Hoth LR, Mansour MN, McGrath KM, Seddon AP, Shenolikar S, Stutzman-Engwall KJ, Warren LC, Xia D, Qiu X (2007). "Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia". Nat. Struct. Mol. Biol. 14 (5): 413–9.
- doi:10.1016/j.str.2007.04.004. PMID 17502100. Piper DE, Jackson S, Liu Q, Romanow WG, Shetterly S, Thibault ST, Shan B, Walker NP (2007). "The crystal structure of PCSK9: a regulator of plasma LDL-cholesterol". Structure. 15 (5): 545–52.
- "The Evolving Role of PCSK9 Modulation in the Regulation of LDL-Cholesterol". Archived from the original on 18 May 2015. Retrieved 13 May 2015.
- Zhang DW, et al. (June 2007). "Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation". J Biol Chem. 282 (25): 18602–18612. doi:10.1074/jbc.M702027200. PMID 17452316.
- Norata GD, Tibolla G, Catapano AL (2014-01-01). "Targeting PCSK9 for hypercholesterolemia". Annual Review of Pharmacology and Toxicology. 54: 273–93. doi:10.1146/annurev-pharmtox-011613-140025. PMID 24160703.
- Bergeron N, Phan BA, Ding Y, Fong A, Krauss RM (October 2015). "Proprotein convertase subtilisin/kexin type 9 inhibition: a new therapeutic mechanism for reducing cardiovascular disease risk". Circulation. 132 (17): 1648–66. doi:10.1161/CIRCULATIONAHA.115.016080. PMID 26503748.
- Le May C, Kourimate S, Langhi C, Chétiveaux M, Jarry A, Comera C, Collet X, Kuipers F, Krempf M, Cariou B, Costet P (May 2009). "Proprotein convertase subtilisin kexin type 9 null mice are protected from postprandial triglyceridemia". Arteriosclerosis, Thrombosis, and Vascular Biology. 29 (5): 684–90. doi:10.1161/ATVBAHA.108.181586. PMID 19265033.
- Rashid S, Tavori H, Brown PE, Linton MF, He J, Giunzioni I, Fazio S (July 2014). "Proprotein convertase subtilisin kexin type 9 promotes intestinal overproduction of triglyceride-rich apolipoprotein B lipoproteins through both low-density lipoprotein receptor-dependent and -independent mechanisms". Circulation. 130 (5): 431–41. doi:10.1161/CIRCULATIONAHA.113.006720. PMC 4115295. PMID 25070550.
- Gustafsen C, Kjolby M, Nyegaard M, Mattheisen M, Lundhede J, Buttenschøn H, Mors O, Bentzon JF, Madsen P, Nykjaer A, Glerup S (February 2014). "The hypercholesterolemia-risk gene SORT1 facilitates PCSK9 secretion". Cell Metabolism. 19 (2): 310–8. doi:10.1016/j.cmet.2013.12.006. PMID 24506872.
- Schulz R, Schlüter KD, Laufs U (March 2015). "Molecular and cellular function of the proprotein convertase subtilisin/kexin type 9 (PCSK9)". Basic Research in Cardiology. 110 (2): 4. doi:10.1007/s00395-015-0463-z. PMC 4298671. PMID 25600226.
- Cariou B, Langhi C, Le Bras M, Bortolotti M, Lê KA, Theytaz F, Le May C, Guyomarc'h-Delasalle B, Zaïr Y, Kreis R, Boesch C, Krempf M, Tappy L, Costet P (2013-01-01). "Plasma PCSK9 concentrations during an oral fat load and after short term high-fat, high-fat high-protein and high-fructose diets". Nutrition & Metabolism. 10 (1): 4. doi:10.1186/1743-7075-10-4. PMC 3548771. PMID 23298392.
- Lakoski SG, Lagace TA, Cohen JC, Horton JD, Hobbs HH (July 2009). "Genetic and metabolic determinants of plasma PCSK9 levels". The Journal of Clinical Endocrinology and Metabolism. 94 (7): 2537–43. doi:10.1210/jc.2009-0141. PMC 2708952. PMID 19351729.
- Baass A, Dubuc G, Tremblay M, Delvin EE, O'Loughlin J, Levy E, Davignon J, Lambert M (September 2009). "Plasma PCSK9 is associated with age, sex, and multiple metabolic markers in a population-based sample of children and adolescents". Clinical Chemistry. 55 (9): 1637–45. doi:10.1373/clinchem.2009.126987. PMID 19628659.
- Zhang DW, Garuti R, Tang WJ, Cohen JC, Hobbs HH (September 2008). "Structural requirements for PCSK9-mediated degradation of the low-density lipoprotein receptor". Proceedings of the National Academy of Sciences of the United States of America. 105 (35): 13045–50. Bibcode:2008PNAS..10513045Z. doi:10.1073/pnas.0806312105. PMC 2526098. PMID 18753623.
- Pollack A (5 November 2012). "New Drugs for Lipids Set Off Race". The New York Times.
- "Entrez Gene: PCSK9 proprotein convertase subtilisin/kexin type 9".
- Dubuc G, Chamberland A, Wassef H, Davignon J, Seidah NG, Bernier L, Prat A (August 2004). "Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia". Arterioscler. Thromb. Vasc. Biol. 24 (8): 1454–9. doi:10.1161/01.ATV.0000134621.14315.43. PMID 15178557.
- Norata GD, Tavori H, Pirillo A, Fazio S, Catapano AL (August 2016). "Biology of PCSK9: beyond LDL cholesterol lowering". Cardiovascular Research. 112 (1): 429–42. doi:10.1093/cvr/cvw194. PMC 5031950. PMID 27496869.
- Ferri N, Tibolla G, Pirillo A, Cipollone F, Mezzetti A, Pacia S, Corsini A, Catapano AL (February 2012). "Proprotein convertase subtilisin kexin type 9 (PCSK9) secreted by cultured smooth muscle cells reduces macrophages LDLR levels". Atherosclerosis. 220 (2): 381–6. doi:10.1016/j.atherosclerosis.2011.11.026. PMID 22176652.
- Wu CY, Tang ZH, Jiang L, Li XF, Jiang ZS, Liu LS (January 2012). "PCSK9 siRNA inhibits HUVEC apoptosis induced by ox-LDL via Bcl/Bax-caspase9-caspase3 pathway". Molecular and Cellular Biochemistry. 359 (1–2): 347–58. doi:10.1007/s11010-011-1028-6. PMID 21847580.
- Giunzioni I, Tavori H, Covarrubias R, Major AS, Ding L, Zhang Y, DeVay RM, Hong L, Fan D, Predazzi IM, Rashid S, Linton MF, Fazio S (January 2016). "Local effects of human PCSK9 on the atherosclerotic lesion". The Journal of Pathology. 238 (1): 52–62. doi:10.1002/path.4630. PMC 5346023. PMID 26333678.
- Cohen JC, Boerwinkle E, Mosley TH, Hobbs HH (March 2006). "Sequence variations in PCSK9, low LDL, and protection against coronary heart disease". The New England Journal of Medicine. 354 (12): 1264–72. doi:10.1056/NEJMoa054013. PMID 16554528.
- Groves C, Shetty C, Strange RC, Waldron J, Ramachandran S (August 2016). "A study in high-risk, maximally pretreated patients to determine the potential use of PCSK9 inhibitors at various thresholds of total and LDL cholesterol levels" (PDF). Postgraduate Medical Journal. 93 (1098): postgradmedj–2016–134062. doi:10.1136/postgradmedj-2016-134062. PMID 27531965.
- Robinson JG (August 2016). "Nonstatins and Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitors: Role in Non-Familial Hypercholesterolemia". Progress in Cardiovascular Diseases. 59 (2): 165–171. doi:10.1016/j.pcad.2016.07.011. PMID 27498088.
- Rosenson RS, Jacobson TA, Preiss D, Djedjos SC, Dent R, Bridges I, Miller M (August 2016). "Erratum to: Efficacy and Safety of the PCSK9 Inhibitor Evolocumab in Patients with Mixed Hyperlipidemia". Cardiovascular Drugs and Therapy. 30 (5): 537. doi:10.1007/s10557-016-6684-z. PMC 6828239. PMID 27497929.
- Peng W, Qiang F, Peng W, Qian Z, Ke Z, Yi L, Jian Z, Chongrong Q (July 2016). "Therapeutic efficacy of PCSK9 monoclonal antibodies in statin-nonresponsive patients with hypercholesterolemia and dyslipidemia: A systematic review and meta-analysis". International Journal of Cardiology. 222: 119–129. doi:10.1016/j.ijcard.2016.07.239. PMID 27494723.
- Urban D, Pöss J, Böhm M, Laufs U (October 2013). "Targeting the proprotein convertase subtilisin/kexin type 9 for the treatment of dyslipidemia and atherosclerosis". Journal of the American College of Cardiology. 62 (16): 1401–8. doi:10.1016/j.jacc.2013.07.056. PMID 23973703.
- Norata GD, Tibolla G, Catapano AL (August 2014). "PCSK9 inhibition for the treatment of hypercholesterolemia: promises and emerging challenges". Vascular Pharmacology. 62 (2): 103–11. doi:10.1016/j.vph.2014.05.011. PMID 24924410.
- Cohen J, Pertsemlidis A, Kotowski IK, Graham R, Garcia CK, Hobbs HH (February 2005). "Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9". Nature Genetics. 37 (2): 161–5. doi:10.1038/ng1509. PMID 15654334.
- Kathiresan S (May 2008). "A PCSK9 missense variant associated with a reduced risk of early-onset myocardial infarction". The New England Journal of Medicine. 358 (21): 2299–300. doi:10.1056/NEJMc0707445. PMID 18499582.
- Ridker PM, Pradhan A, MacFadyen JG, Libby P, Glynn RJ (August 2012). "Cardiovascular benefits and diabetes risks of statin therapy in primary prevention: an analysis from the JUPITER trial". Lancet. 380 (9841): 565–71. doi:10.1016/S0140-6736(12)61190-8. PMC 3774022. PMID 22883507.
- Berger JM, Vaillant N, Le May C, Calderon C, Brégeon J, Prieur X, Hadchouel J, Loirand G, Cariou B (March 2015). "PCSK9-deficiency does not alter blood pressure and sodium balance in mouse models of hypertension". Atherosclerosis. 239 (1): 252–9. doi:10.1016/j.atherosclerosis.2015.01.012. PMID 25621930.
- Sharotri V, Collier DM, Olson DR, Zhou R, Snyder PM (June 2012). "Regulation of epithelial sodium channel trafficking by proprotein convertase subtilisin/kexin type 9 (PCSK9)". The Journal of Biological Chemistry. 287 (23): 19266–74. doi:10.1074/jbc.M112.363382. PMC 3365958. PMID 22493497.
- Norata GD, Pirillo A, Ammirati E, Catapano AL (January 2012). "Emerging role of high density lipoproteins as a player in the immune system". Atherosclerosis. 220 (1): 11–21. doi:10.1016/j.atherosclerosis.2011.06.045. PMID 21783193.
- Diedrich G (September 2006). "How does hepatitis C virus enter cells?". The FEBS Journal. 273 (17): 3871–85. doi:10.1111/j.1742-4658.2006.05379.x. PMID 16934030.
- Chen YQ, Troutt JS, Konrad RJ (May 2014). "PCSK9 is present in human cerebrospinal fluid and is maintained at remarkably constant concentrations throughout the course of the day". Lipids. 49 (5): 445–55. doi:10.1007/s11745-014-3895-6. PMID 24659111.
- Lopez D (2008). "Inhibition of PCSK9 as a novel strategy for the treatment of hypercholesterolemia". Drug News Perspect. 21 (6): 323–30. doi:10.1358/dnp.2008.21.6.1246795. PMID 18836590.
- Steinberg D, Witztum JL (June 2009). "Inhibition of PCSK9: a powerful weapon for achieving ideal LDL cholesterol levels". Proc. Natl. Acad. Sci. U.S.A. 106 (24): 9546–7. Bibcode:2009PNAS..106.9546S. doi:10.1073/pnas.0904560106. PMC 2701045. PMID 19506257.
- Mayer G, Poirier S, Seidah NG (November 2008). "Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels". J. Biol. Chem. 283 (46): 31791–801. doi:10.1074/jbc.M805971200. PMID 18799458.
- "Bristol-Myers Squibb selects Isis drug targeting PCSK9 as development candidate for prevention and treatment of cardiovascular disease". Press Release. FierceBiotech. 2008-04-08. Retrieved 2010-09-18.
- Fitzgerald, Kevin; White, Suellen; Borodovsky, Anna; Bettencourt, Brian R.; Strahs, Andrew; Clausen, Valerie; Wijngaard, Peter; Horton, Jay D.; Taubel, Jorg; Brooks, Ashley; Fernando, Chamikara; Kauffman, Robert S.; Kallend, David; Vaishnaw, Akshay; Simon, Amy (2017). "A Highly Durable RNAi Therapeutic Inhibitor of PCSK9". New England Journal of Medicine. 376 (1): 41–51. doi:10.1056/NEJMoa1609243. ISSN 0028-4793. PMC 5778873. PMID 27959715.
- Sheridan C (December 2013). "Phase 3 data for PCSK9 inhibitor wows". Nature Biotechnology. 31 (12): 1057–8. doi:10.1038/nbt1213-1057. PMID 24316621.
- Stein EA, Raal FJ (December 2014). "New therapies for reducing low-density lipoprotein cholesterol". Endocrinology and Metabolism Clinics of North America. 43 (4): 1007–33. doi:10.1016/j.ecl.2014.08.008. PMID 25432394.
- Vogel RA (June 2012). "PCSK9 inhibition: the next statin?". Journal of the American College of Cardiology. 59 (25): 2354–5. doi:10.1016/j.jacc.2012.03.011. PMID 22465426.
- Navarese EP, Kolodziejczak M, Schulze V, Gurbel PA, Tantry U, Lin Y, Brockmeyer M, Kandzari DE, Kubica JM, D'Agostino RB, Kubica J, Volpe M, Agewall S, Kereiakes DJ, Kelm M (July 2015). "Effects of Proprotein Convertase Subtilisin/Kexin Type 9 Antibodies in Adults With Hypercholesterolemia: A Systematic Review and Meta-analysis". Annals of Internal Medicine. 163 (1): 40–51. doi:10.7326/M14-2957. PMID 25915661.
- Durairaj A, Sabates A, Nieves J, Moraes B, Baum S (August 2017). "Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Its Inhibitors: a Review of Physiology, Biology, and Clinical Data". Current Treatment Options in Cardiovascular Medicine. 19 (8): 58. doi:10.1007/s11936-017-0556-0. PMID 28639183.
- Schmidt AF, Pearce LS, Wilkins JT, Overington JP, Hingorani AD, Casas JP (April 2017). "PCSK9 monoclonal antibodies for the primary and secondary prevention of cardiovascular disease". The Cochrane Database of Systematic Reviews. 4: CD011748. doi:10.1002/14651858.CD011748.pub2. PMC 6478267. PMID 28453187.
- "Medscape". medscape.com. Retrieved 2019-05-18.
- Carroll J (7 March 2014). "Regeneron, Sanofi and Amgen shares suffer on FDA's frets about PCSK9 class". FierceBiotech.
- Lambert G, Sjouke B, Choque B, Kastelein JJ, Hovingh GK (December 2012). "The PCSK9 decade". J. Lipid Res. 53 (12): 2515–24. doi:10.1194/jlr.R026658. PMC 3494258. PMID 22811413.
- Alenghat, Francis J.; Davis, Andrew M. (2019). "Management of Blood Cholesterol". JAMA. 321 (8): 800–801. doi:10.1001/jama.2019.0015. ISSN 0098-7484. PMC 6679800. PMID 30715135.
- Fitzgerald K, Frank-Kamenetsky M, Shulga-Morskaya S, Liebow A, Bettencourt BR, Sutherland JE, Hutabarat RM, Clausen VA, Karsten V, Cehelsky J, Nochur SV, Kotelianski V, Horton J, Mant T, Chiesa J, Ritter J, Munisamy M, Vaishnaw AK, Gollob JA, Simon A (January 2014). "Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial". Lancet. 383 (9911): 60–8. doi:10.1016/S0140-6736(13)61914-5. PMC 4387547. PMID 24094767.
- Shan L, Pang L, Zhang R, Murgolo NJ, Lan H, Hedrick JA (October 2008). "PCSK9 binds to multiple receptors and can be functionally inhibited by an EGF-A peptide". Biochem. Biophys. Res. Commun. 375 (1): 69–73. doi:10.1016/j.bbrc.2008.07.106. PMID 18675252.
- Graham MJ, Lemonidis KM, Whipple CP, Subramaniam A, Monia BP, Crooke ST, Crooke RM (April 2007). "Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice". J. Lipid Res. 48 (4): 763–7. doi:10.1194/jlr.C600025-JLR200. PMID 17242417.
- Gupta N, Fisker N, Asselin MC, Lindholm M, Rosenbohm C, Ørum H, Elmén J, Seidah NG, Straarup EM (2010). Deb S (ed.). "A locked nucleic acid antisense oligonucleotide (LNA) silences PCSK9 and enhances LDLR expression in vitro and in vivo". PLoS ONE. 5 (5): e10682. Bibcode:2010PLoSO...510682G. doi:10.1371/journal.pone.0010682. PMC 2871785. PMID 20498851.
- Lindholm MW, Elmén J, Fisker N, Hansen HF, Persson R, Møller MR, Rosenbohm C, Ørum H, Straarup EM, Koch T (February 2012). "PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman primates". Mol. Ther. 20 (2): 376–81. doi:10.1038/mt.2011.260. PMC 3277239. PMID 22108858.
- "Alnylam Reports Positive Preliminary Clinical Results for ALN-PCS, an RNAi Therapeutic Targeting PCSK9 for the Treatment of Severe Hypercholesterolemia". Press Release. BusinessWire. 2011-01-04. Archived from the original on 2013-02-21. Retrieved 2011-01-04.
- Frank-Kamenetsky M, Grefhorst A, Anderson NN, Racie TS, Bramlage B, Akinc A, Butler D, Charisse K, Dorkin R, Fan Y, Gamba-Vitalo C, Hadwiger P, Jayaraman M, John M, Jayaprakash KN, Maier M, Nechev L, Rajeev KG, Read T, Röhl I, Soutschek J, Tan P, Wong J, Wang G, Zimmermann T, de Fougerolles A, Vornlocher HP, Langer R, Anderson DG, Manoharan M, Koteliansky V, Horton JD, Fitzgerald K (August 2008). "Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates". Proc. Natl. Acad. Sci. U.S.A. 105 (33): 11915–20. Bibcode:2008PNAS..10511915F. doi:10.1073/pnas.0805434105. PMC 2575310. PMID 18695239.
- Crossey E, Amar MJ, Sampson M, Peabody J, Schiller JT, Chackerian B, Remaley AT (October 2015). "A cholesterol-lowering VLP vaccine that targets PCSK9". Vaccine. 33 (43): 5747–55. doi:10.1016/j.vaccine.2015.09.044. PMC 4609631. PMID 26413878.
- Li H, Dong B, Park SW, Lee HS, Chen W, Liu J (August 2009). "HNF1α plays a critical role in PCSK9 gene transcription and regulation by a natural hypocholesterolemic compound berberine". The Journal of Biological Chemistry. 284 (42): 28885–95. doi:10.1074/jbc.M109.052407. PMC 2781434. PMID 19687008.
- Dong B, Li H, Singh AB, Cao A, Liu J (February 2015). "Inhibition of PCSK9 transcription by berberine involves down-regulation of hepatic HNF1α protein expression through the ubiquitin-proteasome degradation pathway". The Journal of Biological Chemistry. 290 (7): 4047–58. doi:10.1074/jbc.M114.597229. PMC 4326815. PMID 25540198.
- Dong H, Zhao Y, Zhao L, Lu F (April 2013). "The effects of berberine on blood lipids: a systemic review and meta-analysis of randomized controlled trials". Planta Medica. 79 (6): 437–46. doi:10.1055/s-0032-1328321. PMID 23512497.
- Seidah NG, Poirier S, Denis M, Parker R, Miao B, Mapelli C, Prat A, Wassef H, Davignon J, Hajjar KA, Mayer G (2012). "Annexin A2 is a natural extrahepatic inhibitor of the PCSK9-induced LDL receptor degradation". PLoS ONE. 7 (7): e41865. Bibcode:2012PLoSO...741865S. doi:10.1371/journal.pone.0041865. PMC 3407131. PMID 22848640.
- Abifadel M, Rabès JP, Boileau C, Varret M (June 2007). "[After the LDL receptor and apolipoprotein B, autosomal dominant hypercholesterolemia reveals its third protagonist: PCSK9]". Ann. Endocrinol. (in French). 68 (2–3): 138–46. doi:10.1016/j.ando.2007.02.002. PMID 17391637.
- Allard D, Amsellem S, Abifadel M, Trillard M, Devillers M, Luc G, Krempf M, Reznik Y, Girardet JP, Fredenrich A, Junien C, Varret M, Boileau C, Benlian P, Rabès JP (November 2005). "Novel mutations of the PCSK9 gene cause variable phenotype of autosomal dominant hypercholesterolemia". Hum. Mutat. 26 (5): 497. doi:10.1002/humu.9383. PMID 16211558.
- Benjannet S, Rhainds D, Essalmani R, Mayne J, Wickham L, Jin W, Asselin MC, Hamelin J, Varret M, Allard D, Trillard M, Abifadel M, Tebon A, Attie AD, Rader DJ, Boileau C, Brissette L, Chrétien M, Prat A, Seidah NG (November 2004). "NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol". J. Biol. Chem. 279 (47): 48865–75. doi:10.1074/jbc.M409699200. PMID 15358785.
- Lalanne F, Lambert G, Amar MJ, Chétiveaux M, Zaïr Y, Jarnoux AL, Ouguerram K, Friburg J, Seidah NG, Brewer HB, Krempf M, Costet P (June 2005). "Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells". J. Lipid Res. 46 (6): 1312–9. doi:10.1194/jlr.M400396-JLR200. PMID 15741654.
- Lambert G (June 2007). "Unravelling the functional significance of PCSK9". Curr. Opin. Lipidol. 18 (3): 304–9. doi:10.1097/MOL.0b013e3281338531. PMID 17495605.
- Leren TP (May 2004). "Mutations in the PCSK9 gene in Norwegian subjects with autosomal dominant hypercholesterolemia". Clin. Genet. 65 (5): 419–22. doi:10.1111/j.0009-9163.2004.0238.x. PMID 15099351.
- Maxwell KN, Breslow JL (May 2004). "Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype". Proc. Natl. Acad. Sci. U.S.A. 101 (18): 7100–5. Bibcode:2004PNAS..101.7100M. doi:10.1073/pnas.0402133101. PMC 406472. PMID 15118091.
- Maxwell KN, Soccio RE, Duncan EM, Sehayek E, Breslow JL (November 2003). "Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice". J. Lipid Res. 44 (11): 2109–19. doi:10.1194/jlr.M300203-JLR200. PMID 12897189.
- Naoumova RP, Tosi I, Patel D, Neuwirth C, Horswell SD, Marais AD, van Heyningen C, Soutar AK (December 2005). "Severe hypercholesterolemia in four British families with the D374Y mutation in the PCSK9 gene: long-term follow-up and treatment response". Arterioscler. Thromb. Vasc. Biol. 25 (12): 2654–60. doi:10.1161/01.ATV.0000190668.94752.ab. PMID 16224054.
- Naureckiene S, Ma L, Sreekumar K, Purandare U, Lo CF, Huang Y, Chiang LW, Grenier JM, Ozenberger BA, Jacobsen JS, Kennedy JD, DiStefano PS, Wood A, Bingham B (December 2003). "Functional characterization of Narc 1, a novel proteinase related to proteinase K". Arch. Biochem. Biophys. 420 (1): 55–67. doi:10.1016/j.abb.2003.09.011. PMID 14622975.
- Ouguerram K, Chetiveaux M, Zair Y, Costet P, Abifadel M, Varret M, Boileau C, Magot T, Krempf M (August 2004). "Apolipoprotein B100 metabolism in autosomal-dominant hypercholesterolemia related to mutations in PCSK9". Arterioscler. Thromb. Vasc. Biol. 24 (8): 1448–53. doi:10.1161/01.ATV.0000133684.77013.88. PMID 15166014.
- Pisciotta L, Priore Oliva C, Cefalù AB, Noto D, Bellocchio A, Fresa R, Cantafora A, Patel D, Averna M, Tarugi P, Calandra S, Bertolini S (June 2006). "Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia". Atherosclerosis. 186 (2): 433–40. doi:10.1016/j.atherosclerosis.2005.08.015. PMID 16183066.
- Shibata N, Ohnuma T, Higashi S, Higashi M, Usui C, Ohkubo T, Watanabe T, Kawashima R, Kitajima A, Ueki A, Nagao M, Arai H (December 2005). "No genetic association between PCSK9 polymorphisms and Alzheimer's disease and plasma cholesterol level in Japanese patients". Psychiatr. Genet. 15 (4): 239. doi:10.1097/00041444-200512000-00004. PMID 16314752.
- Sun XM, Eden ER, Tosi I, Neuwirth CK, Wile D, Naoumova RP, Soutar AK (May 2005). "Evidence for effect of mutant PCSK9 on apolipoprotein B secretion as the cause of unusually severe dominant hypercholesterolaemia". Hum. Mol. Genet. 14 (9): 1161–9. doi:10.1093/hmg/ddi128. PMID 15772090.
- Timms KM, Wagner S, Samuels ME, Forbey K, Goldfine H, Jammulapati S, Skolnick MH, Hopkins PN, Hunt SC, Shattuck DM (March 2004). "A mutation in PCSK9 causing autosomal-dominant hypercholesterolemia in a Utah pedigree". Hum. Genet. 114 (4): 349–53. doi:10.1007/s00439-003-1071-9. PMID 14727179.
- Varret M, Rabès JP, Saint-Jore B, Cenarro A, Marinoni JC, Civeira F, Devillers M, Krempf M, Coulon M, Thiart R, Kotze MJ, Schmidt H, Buzzi JC, Kostner GM, Bertolini S, Pocovi M, Rosa A, Farnier M, Martinez M, Junien C, Boileau C (May 1999). "A third major locus for autosomal dominant hypercholesterolemia maps to 1p34.1-p32". Am. J. Hum. Genet. 64 (5): 1378–87. doi:10.1086/302370. PMC 1377874. PMID 10205269.