Lipids: new biology and therapeutic targets revealed through human genetics

Abstract

This year's Deuel Conference on Lipids focused on the seminal contribution of human genetics to lipid biology and disease. Named in honor of Harry J Deuel Jr, famous single author of the three-volume textbook The Lipids (Interscience Publishers Inc., NY, USA) and established in 1955, the Deuel Conference is considered the longest-standing annual meeting on lipid metabolism in the USA. The 2013 meeting took place at the Silverado Resort of Napa Valley (CA, USA) and was characterized by first-rank scientific contributions and discussions in a friendly and informal setting. Four scientific sessions over 2 and a half days, a poster session and generous breaks allowed for stimulating scientific, as well as social interactions of scientists from academia and industry alike.

Novel pathways regulating lipoprotein metabolism

The scientific sessions were opened by program co-chair Kathryn Moore (New York University Medical Center, NY, USA), who together with co-chair Dan Rader (University of Pennsylvania School of Medicince, PA, USA) is to be credited for putting together an exciting and top-grade scientific agenda. Moore's talk highlighted the use of cholesterol-efflux regulating miRNAs 33a/b as targets for therapeutic intervention. Subcutaneous injection of an anti-miR33 oligonucleotide in monkeys resulted in a sustained elevation of plasma HDL and reduction of VLDL levels [1]. In Ldlr ^-/- mice, anti-miR33 also reduced atherosclerotic plaque size and reached plaque macrophages, where it stimulated ABCA1 expression, and suppressed inflammatory markers. Based on these convincing findings, a Phase I trial may soon be initiated. Alan Tall (Columbia University, NY, USA) reported that the combined deficiency for both Abca1 and Abcg1 , in a subpopulation of bone-marrow derived cells [2], is proatherogenic for three reasons: it stimulates the formation of macrophage precursors in the bone marrow, triggers their mobilization from the spleen and increases macrophage recruitment to atherosclerotic plaques. Similar to ABCG1 , ABCG4 also mediates cholesterol efflux to HDL. Interestingly, Abcg4 ^-/- mice demonstrate increased formation of megakaryocytes and platelets, and with this, a risk for thrombosis and atherosclerosis, consistent with the observation that variants at the human ABCG4 locus are associated with megakaryocyte formation [3].

Further light on hematopoietic lineages was shed by Edward Lynn (St Joseph's Healthcare Hamilton and McMaster University, Ontario, Canada) who reported that deficiency for the macrophage-expressed TDAG51 , a gene implicated in adipocyte differentiation [4], is atheroprotective in ApoE ^-/- mice and acts to reduce lipid-load to foam cells and necrosis within lesions. Edward Fisher (New York University, USA) provided an insightful summary on the mechanisms that control VLDL secretion from the liver, which is regulated less via apoB synthesis, but rather by its degradation [5]. Of particular interest is his finding in primary hepatocytes that insulin inhibits secretion of large VLDL particles by stimulating apoB degradation via autophagy-dependent mechanisms. In a short presentation selected from the abstracts, Jessica Lee (University of California, USA) reported that the Diet1 gene modulates bile acid homeostasis through FGF15/19-dependent signaling. Concluding the first session, Endre Kiss-Toth (University of Sheffield, UK) and Rob Bauer (University of Pennsylvania, USA) reported on the tribbles family of serine-threonine kinase-like proteins. TRIB1 is a well established modulator of MAPK- and Akt-dependent signaling [6], and TRIB1 variants associate with plasma lipids, as well as coronary artery disease (CAD) [7]. Several lines of evidence from macrophages and mice overexpressing or deficient for TRIB1 were presented that indicate this protein as being atheroprotective.

The Havel lecture

The keynote lecture of the conference, in honor of Richard J Havel, the father of liporotein biology [8] and conference organizer for many years, was given by Rick Lifton (Yale University, CT, USA). In his presentation 'From human genetics to validated therapeutic targets', he outlined his numerous and seminal contributions to the understanding of blood pressure regulation in all three technology-driven eras of gene discovery through human genetics:

*The Mendelian disease era, where by sequencing in families with extreme blood pressure phenotypes, he could establish the central importance of salt absorption in the kidney via the rennin-angiotensin system for blood pressure regulation;

*The common disease era, where through array-based analysis of close to 200,000 individuals by genome-wide association studies (GWAS), he contributed to the discovery of, thus far, 29 genetic loci;

*Our current era where sequencing of the whole-genome or protein-coding genome (exome) allows assessment of the contribution of both rare and common genetic variation to complex traits.

In addition, Lifton also highlighted several examples whereby human genetics has led to highly promising therapeutic targets, among them ROMK (KCNJ1 ) for blood pressure reduction [9].

Approaches to new pathway discovery

The power of exome sequencing to identify disease genes was further underlined by several talks in a session launched by Debbie Nickerson (University of Washington, USA). In addition to giving further examples of new genes underlying a variety of Mendelian disorders, Nickerson also discussed the challenges of current exome analysis pipelines (e.g., shortcomings in detecting copy-number variants). She strongly advertised data available through the National Heart, Lung, and Blood Institute Exome Sequencing Project [101] and the Centers for Mendelian Genomics [102] as an unprecedented resource for human genetic discoveries. How such data sets can be successfully leveraged was presented by Cristin Willer (University of Michigan, USA) who, by exome analysis of >2000 individuals, demonstrated several known and novel genes associated with plasma LDL at genome-wide significance. Increased sample sizes are likely to lead to the identification of more genes, which is again evidenced by the latest GWAS that, by almost doubling the number of individuals genotyped (to >180,000), revealed 62 novel lipid loci, thereby increasing the previously reported number from 95 [7] to 157. The idea that human genetics enables solid conclusions on the causality of findings and reliable risk predictions was convincingly demonstrated by Sek Kathiresan (Massachusetts General Hospital, USA). Through regression modeling using 185 variants associated with TG, LDL and HDL, he assessed the strength of how these parameters are interelated and individually linked to CAD. As CAD associated tightly with LDL and TG, but considerably less with HDL, the findings presented strongly suggest that covariance of HDL is a consequence rather than a cause of lipid imbalances leading to CAD. Kathiresan also presented examples for several novel genes, in which rare variants influence TG levels and the risk of myocardial infarction.

Refined statistical tools are likely to reveal additional disease genes in the near future; however, functional studies are needed in order to derive mechanistic information as a basis for therapy development. In a systematic manner, this can be achieved by analyzing cells, either by quantifying functional readouts upon candidate gene knockdown and overexpression, as discussed in my own presentation [10], or by comparing functional insights obtained from patient-derived cells with their respective genotypes. Following the latter strategy in lymphoblasts, Ron Krauss (Children's Hospital, Oakland Research Institute, CA, USA) and Marisa Wang Medina (Children's Hospital, Oakland Research Institute) reported several novel genes for which expression correlates with that of HMGCR and is induced by statins [11], among them GATM , RHOA and HNRNPA1. Interestingly, for RHOA and HNRNPA1, distinct isoforms appear to differentially affect cellular cholesterol levels and the expression of lipid-regulatory genes, which hints at a little-explored layer of lipid metabolism regulation.

New pathways in atherosclerosis & CAD

A second key lecture of the meeting, sponsored by the Journal of Lipid Research , was given by Shaun Coughlin, Director of Cardiovascular Genomics Institute (University of California, USA). Coughlin presented novel insights on S1P-dependent signaling. Mice deficient for both S1P-generating sphingosine kinases in hematopoetic lineages demonstrate considerably reduced S1P levels and activity in plasma, suggesting the bone marrow as the main source of circulating S1P. Mutant mice demonstrated increased vascular permeability and when exposed to leakage-inducing agents, die from volume deficiency and blood pressure dysregulation [12]. One likely explanation for this is that S1P controls blood vessel integrity via its endothelial receptor S1PR1, which, upon binding, recruits actin and actinin to the plasma membrane. Interestingly, S1PR1 deficiency in zebrafish and mice prevent formation of functional cardiomyocytes, possibly due to perturbation of F-actin-dependent sarcomere formation.

An insight into how noncoding variants identified by GWAS could regulate gene function and be implicated in disease came from Tom Quertermous (Stanford University, CA, USA). Following up on TCF21 , a tumor suppressor gene with roles in cardiac development and highly associated with CAD [13], he provided compelling evidence for upstream conserved regulatory elements that in smooth muscle cells act as binding sites for transcription factors with roles in blood vessel formation. Also TCF21 binds to cardiovascular-relevant binding sites and downstream effectors of TCF21 include pathways implicated in atherosclerosis, inflammatory response and cell proliferation. A further noncoding, intronic single nucleotide polymorphism in the mucoglycan protein family member ADAMTS7 strongly associates with CAD and myocardial infarction [14]. Muredach Reilly (University of Pennsylvania) described this protein as localizing to the neo-intima of coronary artery vessels and of possible relevance to mediating response to vascular injury. The session was completed by three short presentations discussing the role of apoM as a S1P carrier (Mingxia Liu; Wake Forest School of Medicine, NC, USA), the atheroprotective potential of HDAC9 isoform-specific inhibitors (Nilamadhab Mishra; Wake Forest School of Medicine) and the efficiency of the CXCR7 ligand PF-708 (Pfizer) to reduce triglyceride and atherosclerotic lesion formation in vivo (Miao Wang; Pfizer, MA, USA).

Translating biology into therapeutics

The concluding session of the conference had a strong emphasis on those biological insights that have already been translated to the clinic or are believed to provide a solid basis for future therapies. Deborah Keefe (Novartis, NJ, USA) reported that inhibitors of DGAT1, a membrane-bound O -acyltransferase with roles in absorbing fatty acids from the intestine, reduced postprandial hypertriglyceremia and improved insulin tolerance in several animal species. A first single-dose study in humans launched by AstraZeneca demonstrated consistent results. Likewise, application of the corresponding Novartis compound to a large cohort of patients with Familial Hyperchylocronomia Syndrome markedly lowered TG. Rosanne Crooke (Isis Pharmaceuticals, CA, USA) reported on recent successes of mipomersen (Kynamro(TM)), an antisense apoB-inhibitor that was approved by the US FDA in January 2013 for the treatment of severe hypercholesterolemia. A novel target for antisense-based therapies is APOC3 . In a Phase I trial, subcutaneous injections of ISIS-apoCIII_RX , an APOC3 inhibitor that reduced plasma TG in mice and monkeys, resulted in prolonged reduction of plasma TG and was well-tolerated. Phase II trials in 100 patients with extreme TG levels, as well as a diabetic cohort with moderately elevated TG, are under preparation. As a potential alternative strategy to reduce severely elevated TG in LPL deficiency, Colin Ross (University of British Columbia, Canada) described the long way to success of a gene therapy-based approach [15]. After Phase II/III trials demonstrated that intramuscular injections of adeno-associated vectors encoding the beneficial LPL-variant S447X considerably reduced TG levels and the incidence of pancreatitis, the EMA approved this drug named Glybera ^® in July 2012 as the first gene therapy in Europe. Sam Wright (CSL Ltd, PA, USA) presented encouraging work on how the infusion of CSL II2, in vitro reconstituted HDL analogs, may favorably modify plasma lipids and be atheroprotective. Short talks by Stefan Nilsson (Umea University, Sweden) on the lipoprotein-lipase inhibitor LP071, Bart Duell (Oregon Health and Science University, USA) on the application of the PCSK9 antibody RN316 in familial hypercholesterolemia and Xuchu Que (University of California, USA) on antiatherogenic effects of innate natural antibody E06 targeting oxidized phospholipids, supplemented a session of significant clinical relevance.

Acknowledgements

Limited by the scope and format of this article, the author apologizes to all the speakers and poster presenters whose excellent contributions to the conference could not be considered or needed to be considerably abbreviated. D Rader is acknowledged for helpful comments on the manuscript. The notion, that "you come to this meeting and feel at home"would not have been possible without the outstanding engagement of M Véniant, Chief Organizer and Deuel Chair and B Gordon from the American Society for Biochemistry and Molecular Biology who cared for all local arrangements. Once again, the Deuel Treasurer S Young distinguished himself by attracting generous sponsers who contributed to also make this Deuel meeting 2013 a great success.

Next year's Deuel conference will be held on 4-7 March 2014 in Coronado/San Diego, CA, USA. The program will be available soon (see [103] ).

Financial & competing interests disclosure

This work was supported by a Career Development Award and the Transatlantic Networks of Excellence in Cardiovascular Research Program from Fondation Leducq (grants 12CDA04 and 10CVD03). The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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