ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2019

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1619

Zebrafish models for large-scalegenetic screens in dyslipidemia andatherosclerosis

Validation and application

MANOJ KUMAR BANDARU

ISSN 1651-6206ISBN 978-91-513-0822-7urn:nbn:se:uu:diva-397715

Dissertation presented at Uppsala University to be publicly examined in Humanistiskateatern, Engelska Parken, Thunbergsv. 3H, Uppsala, Thursday, 16 January 2020 at 09:15for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will beconducted in English. Faculty examiner: PhD Shannon Fisher (Department of Pharmacology& Experimental Therapeutics, Boston University School of Medicine).

AbstractBandaru, M. K. 2019. Zebrafish models for large-scale genetic screens in dyslipidemiaand atherosclerosis. Validation and application. Digital Comprehensive Summaries ofUppsala Dissertations from the Faculty of Medicine 1619. 46 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-513-0822-7.

Hundreds of loci have been robustly associated with circulating lipids, atherosclerosisand coronary artery disease; but for most loci the causal genes and mechanisms remainuncharacterized. The overall aim of my thesis is to develop and validate novel in vivo modelsystems that are suitable for high-throughput, image-based genetic screens in coronary arterydisease and related traits, and use these model systems to systematically characterize positionalcandidate genes.

In Study I, I developed an experimental pipeline to validate the suitability of zebrafish larvaeas a model system for systematic, large-scale characterization of drugs and genes associated withdyslipidemia and atherosclerosis. Using this pipeline, I showed that five days of overfeedingand cholesterol supplementation have independent pro-atherogenic effects in zebrafish larvae,which could be diminished by concomitant treatment with atorvastatin and ezetimibe. CRISPR-Cas9-induced mutations in orthologues of proof-of-concept genes resulted in higher LDLcholesterol levels (apoea), and more early stage atherosclerosis (apobb.1). Finally, the pipelinehelped me to identify putative causal genes for circulating lipids and early-stage atherosclerosis(LPAR2 and GATAD2A).

In Study II, I characterized cardiometabolic traits in apoc2 mutant zebrafish larvae and foundthat, similar to humans, larvae with two non-functional apoc2 alleles have higher whole-bodylevels of triglycerides and total cholesterol, and more vascular lipid deposition than larvaewithout mutations in apoc2. Interestingly, apoc2 mutant larvae also had lower glucose levelsafter adjusting for triglyceride levels, suggesting that therapeutic stimulation of apoc2 to preventhypertriglyceridemia may result in hyperglycemia. Still, zebrafish larvae with mutations inapoc2 can be a useful model to identify and characterize additional causal genes for triglyceridemetabolism.

In Study III, I examined the effects of mutations in pcsk9 on atherosclerosis and diabetes-related traits in nearly 5,000 zebrafish larvae. Similar to the loss-of-function mutations in PCSK9in humans, larvae with mutations in pcsk9 had lower LDLc levels and were protected fromearly-stage atherosclerosis. Interestingly, mutations in pcsk9 also resulted in fewer pancreatic β-cells in 10 days old larvae, which suggests the higher risk of diabetes in humans with mutationsin PCSK9 may result from a direct effect on the beta cell.

Based on these large-scale proof-of-concept studies, my thesis confirms that zebrafishlarvae can be used for large-scale, systematic genetic screens in dyslipidemia and early-stageatherosclerosis.

Keywords: zebrafish, dyslipidemia, atherosclerosis, genetic screens, high-cholesterol diet,APOE, APOB, LDLR, APOC2, PCSK9, LPAR2, GATAD2A

Manoj Kumar Bandaru, Department of Immunology, Genetics and Pathology, Medicinskgenetik och genomik, Rudbecklaboratoriet, Uppsala University, SE-751 85 Uppsala, Sweden.

© Manoj Kumar Bandaru 2019

ISSN 1651-6206ISBN 978-91-513-0822-7urn:nbn:se:uu:diva-397715 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-397715)

To Nannamma.”A proper goodbye from your grandson”.

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Bandaru, MK, Emmanouilidou, A, Ranefall, P, Von der Heyde, B, Mazzaferro, E, Klingström, T, Masiero, M, Dethlefsen, O, Le-din, J, Larsson, A, Brooke, HL, Wählby, C, Ingelsson, E, den Hoed, M. (2019) Zebrafish larvae as a model system for system-atic characterization of drugs and genes in dyslipidemia and ath-erosclerosis. Manuscript in revision

II Bandaru, MK, Emmanouilidou, A, Larsson, A, Ingelsson, E, den Hoed, M. (2019) Apoc2 mutant zebrafish: a model for hyper-triglyceridemia and early-stage atherosclerosis. Manuscript

III Bandaru, MK, Mazzaferro, E, Emmanouilidou, A, Larsson, A, Ingelsson, E, den Hoed, M. (2019) Image-based, in vivo charac-terization of cardiometabolic consequences of mutations in pcsk9. Manuscript

Contents

1. Introduction ............................................................................................... 11 1.1. Coronary artery disease, survey of the field ...................................... 11

1.1.1. Global significance .................................................................... 11 1.1.2. Pathophysiology ........................................................................ 11 1.1.3. Risk factors ................................................................................ 13 1.1.4. Genetics ..................................................................................... 14 1.1.5. Treatment ................................................................................... 17 1.1.6. Mining GWAS loci for novel treatments ................................... 18 1.1.7. Candidate gene prioritization ..................................................... 18

1.2. Zebrafish for translational research ................................................... 19 1.2.1. Need for a new animal model .................................................... 19 1.2.2. Scope for genetic screens using zebrafish model ...................... 20 1.2.3. Previous small-scale studies on using zebrafish to model dyslipidemia and atherosclerosis ......................................................... 20

2. Aims .......................................................................................................... 25 2.1. General aim ....................................................................................... 25 2.2. Specific aims ..................................................................................... 25

Study Ⅰ ............................................................................................... 25 Study Ⅱ ............................................................................................... 25 Study Ⅲ ............................................................................................... 25

3. Study summaries ....................................................................................... 26 Study Ⅰ ............................................................................................... 28 Study Ⅱ ............................................................................................... 30 Study Ⅲ ............................................................................................... 32

4. Concluding remarks .................................................................................. 34

5. Future perspectives ................................................................................... 35

6. References ................................................................................................. 39

Abbreviations

APOB Apolipoprotein B APOC-II Apolipoprotein C-II APOE Apolipoprotein E CAD Coronary artery disease CETP Cholesterol ester transfer protein Chr Chromosome CI Confidence interval CRISPR Clustered regularly interspaced short palindromic repeats DNA Deoxyribonucleic acid dpf Days post-fertilization EGFP Enhanced green fluorescent protein eQTL Expression quantitative trait loci GATAD2A GATA Zinc Finger Domain Containing 2A GMIP GEM Interacting Protein gRNA Guide ribonucleic acid GWAS Genome-wide association studies HCD High-cholesterol diet HDLc High-density lipoprotein cholesterol HMGCR 3-hydroxy-3-methyl-glutaryl-coenzyme A reductaseLD Linkage disequilibriumLDLc Low-density lipoprotein cholesterolLDLR Low-density lipoprotein receptorLPAR2 Lysophosphatidic Acid Receptor 2MAF Minor allele frequencyLPL Lipoprotein lipaseMPEG1 Macrophage Expressed Gene 1MPO MyeloperoxidaseNPC1L1 Niemann-Pick C1-Like Protein 1oxLDL Oxidized low-density lipoproteinPCR Polymerase chain reactionPCSK9 Protein convertase subtilisin/kexin type 9SD Standard deviationSNP Single nucleotide polymorphismTALEN Transcription activator-like effector nucleasesTM6F2 Transmembrane 6 Superfamily Member 2VAST Vertebrate automated screening technology

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1. Introduction

1.1. Coronary artery disease, survey of the field 1.1.1. Global significance Coronary artery disease (CAD) reached epidemic proportions during mid-20th century1 and still continues to be the most common disease worldwide that causes disability and death. According to the World Health Organization sta-tistics for 2015, an estimated 7.4 million people died from CAD, representing 13% of all deaths globally2. Although age-adjusted mortality due to CAD has gradually decreased in developed countries, it was an underlying cause of 1 of every 7 deaths during 20143. It is projected that by 2030, deaths due to CAD might increase by 14.9% in men and 13.1% in women4. This illustrates the need for implementing more effective measures to identify and prevent the risk factors (primordial prevention) as well as to prevent clinical manifesta-tions of CAD in the high risk group (primary prevention) and the affected group (secondary prevention). While the primordial, primary and secondary prevention of CAD have significantly improved, thanks to contributions from pathological, therapeutic, and epidemiological studies, the pharmaceutical in-dustry has failed to develop conceptually new medication in the last three dec-ades. Such developments are essential to reduce the projected consequences of CAD on morbidity and mortality in the decades to come.

1.1.2. Pathophysiology In CAD, the coronary arteries supplying blood and nutrients to the heart be-come hard and narrow due to accumulation of lipids and immunogenic ele-ments that form plaques in the artery wall (Figure 1). Functional studies in murine model systems and relevant cell types, as well as clinicopathological studies of autopsies5-7 have elucidated that atherosclerosis is the main under-lying pathophysiological process of CAD. Early-stages of atherosclerosis manifests when cholesterol-rich apolipoprotein-B (apoB)-containing lipopro-teins retain in the arterial wall8-10. Normally, lipoproteins <70 nm in diameter flux into and out of the arterial wall11. However, when their concentrations in plasma raise above the normal levels, a fraction of the lipoproteins that enter the arterial wall are retained12, 13. The retained lipoproteins undergo modifica-

12

tions such as lipolysis, proteolysis and oxidization leading to endothelial acti-vation and initiation of inflammatory processes such as recruitment of plate-lets, neutrophils and monocytes to the scene14, 15. Monocytes transmigrate across the arterial wall, where they proliferate and differentiate into macro-phages, and take up the oxidized lipids, forming foam cells16. In advance-stage atherosclerosis, apoptosis and necrosis of the foam cells, together with local accumulation of extracellular lipids, calcium and other debris can lead to fatty streak formation. This in turn triggers the recruitment of smooth muscle cells to form a fibrous cap over the necrotic core17. Over time, intimal calcification, neo-vascularization of growing plaques, and degradation of the fibrous cap by proteases can increase the influx of pro-inflammatory T cells into atheroscle-rotic plaques, making them acute and unstable. Rupturing of such unstable plaques can ultimately lead to thrombosis and myocardial infarction18. Despite some knowledge on how atherosclerosis develops, a precise understanding of its underlying molecular processes is still lacking. For example, we are far from understanding how the lipid loading into macrophages precisely affects macrophage biology and atherosclerotic processes19. Understanding the events at each particular stage will likely shed light onto the process as a whole, and could potentially uncover new drug targets.

Figure 1. Etiology of coronary artery disease. The coronary arteries that normally carry blood and oxygen to the heart become hard and narrow in coronary artery dis-ease, due to the formation of plaques consisting of lipids, macrophages and smooth muscles cells. Advanced plaques upon fissure can completely occlude the coronary arteries. Image was modified from Wikimedia Commons20.

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1.1.3. Risk factors Epidemiological studies have provided insights into the risk factors associated with the development of CAD. Multiple large-scale, longitudinal epidemio-logical studies have highlighted a range of risk factors, including behavioral habits (i.e. smoking, alcohol use, high-fat diet, and a sedentary lifestyle), med-ical conditions (i.e. diabetes, hypertension, dyslipidemia, and obesity), lipid components in blood (i.e. higher cholesterol, higher triglycerides, higher LDL cholesterol and lower high-density lipoprotein cholesterol levels in the circu-lation) and physical traits (i.e. higher age, male gender and short stature) (re-viewed in ref. 21) (Figure 2). All the above risk factors are regarded as conven-tional risk factors of CAD, and all except age and sex are influenced by genetic factors. For example, over 250 genetic loci have now been identified as being robustly associated with diabetes, largely through insulin resistance and beta-cell dysfunction22-24. Our understanding of risk factors has helped develop risk prediction models that allow selection of individuals at risk for preventive strategies. However, up to 20% of CAD patients have no conventional risk factors and 40% have only one25. Therefore, to further improve the sensitivity of risk prediction models, contemporary studies are focusing on identifying molecular biomarkers that characterize pathological signs of the disease26, 27.

Figure 2. Conventional risk factors associated with increased risk for CAD. All these risk factors are influenced by genetic factors and account for part of CAD her-itability.

14

1.1.4. Genetics Several epidemiological studies provided the clues for familial aggregation of CAD28, 29. Subsequent studies in twins have shown that the heritability of CAD is estimated at 57% for men and 38% for women30, 31. In 2004, the INTER-HEART study confirmed that a positive family history of CAD is a risk factor, independently of age, sex, smoking and geographical region. After adjusting for genetically influenced conventional risk factors, the risk due to family his-tory was only slightly attenuated, implying that additional genetic factors that directly influence disease risk play a role in disease susceptibility32. These findings strengthened the arguments in favor of trying to improve our under-standing of the genetic basis of CAD. Numerous genomic and genetic studies have since been employed to dissect the genetic factors underlying CAD.

Genome-wide linkage studies Efforts to understand the genetic basis of CAD began in the 1980s, using a genome-wide linkage approach. Microsatellite markers (short tandem repeat sequences) were genotyped in related individuals, particularly in sibling pairs of CAD or myocardial infarction affected families, to locate the disease sus-ceptibility loci flanked by the markers. There studies were not very success-ful33, and the identified loci varied across families from different geographical regions, e.g. 13q12-13 in Iceland34, 2q21.2-22 and Xq23-26 in Finland35, 14q32.2 in Germany36, 1p34-36, 3q13 and 5q31 in the US37, and 2p11 and 17p11-17q21 in the UK38. Fine mapping of locus 13q12-13 with additional markers led to the identification of arachidonate 5-lipoxygenase activating protein (ALOX5AP), which represents the only gene identified by an early-stage linkage study that we now know is indeed robustly linked to CAD risk34. The lack of success in identifying susceptible loci or genes of statistically un-equivocal linkage can largely be attributed to: 1) the extensive genetic heter-ogeneity of CAD; 2) the inability of the small set of microsatellite markers (~400 markers), which are spaced as far as ~10 centiMorgan apart; 3) inade-quate statistical power due to small sample size; 4) absence of genotypic data for an adequate number of affected and unaffected family members39; and 5) the low penetrance of effect for common variants.

Candidate-gene association studies Most progress in understanding the genetic basis of CAD has come from as-sociation studies. Early association studies in the late 1980s attempted to iden-tify genes associated with quantifiable risk factors of CAD (e.g. plasma lipo-proteins, blood glucose) and rare single-gene (Mendelian) disorders that in-volve premature CAD (e.g. due to familial hypercholesterolemia and dyslipidemia). Such studies commenced by selecting putative candidate genes, either by their location in a region of linkage (for example, ALOX5AP at 13q12-13), or on the basis of other evidence that they might affect disease

15

risk- for example, genes involved in lipid metabolism were hypothesized to play a role in atherosclerosis. The selected candidate genes were compared between patients and controls for the allele frequencies of a set of variants within or near the genes. Such efforts demonstrated the importance of variants in genes encoding LDL receptor (LDLR)40, cholesterol ester transfer protein (CETP)41, apolipoprotein B (APOB)42 and apolipoprotein E (APOE)43 for lipid metabolism and CAD pathogenesis. However, few variants have been consist-ently replicated across studies. This is at least in part due to differences in study design and very limited statistical power (i.e. no more than a few hun-dred cases per study) in most studies44, 45. Moreover, the biased approach of examining only a subset of SNPs within a subset of genes using a small sample size increases the likelihood of missing common and rare variants with low to modest effect sizes45. This issue was circumvented to some extent by compre-hensive sequencing of candidate genes in a large population (comprising pa-tients and controls) to identify both common (minor allele frequency – MAF >5%, i.e. 1 in 20) and rare (MAF <1%) variants that have functional conse-quences. Deep sequencing of PCSK9 (encoding proprotein convertase subtil-isin/kexin type 9 serine protease), for example, has led to identification of rare variants that reduce LDLc by 40% and confer protection from CAD46. Another candidate-gene sequencing effort found that individuals with low levels of HDLc were significantly more likely to harbor rare nonsynonymous variants in ABCA1, APOA1, and LCAT, which had previously been implicated in dyslipidemia47. However, hypothesis-driven candidate gene association stud-ies mostly pointed towards obvious disease pathways, thereby limiting the scope for identifying new pathways and developing new therapies, with the exception of PCSK9.

Genome-wide association studies In addition to candidate-gene association studies that screen for a set of genetic variants within pre-selected genes, genetic epidemiologists have also em-ployed comprehensive, hypothesis-free screening for genetic variants across the genome that are associated with CAD or CAD-associated traits. This ap-proach has become known as the genome-wide association (GWA) approach. In GWA studies (GWAS), genomes of tens of thousands of CAD cases and CAD-free controls can now be examined for a few million common SNPs (typically MAF >1%). GWAS are largely based on the accomplishments of the Human Genome Project48, HapMap Project49, and 1000 Genomes Pro-ject50, which provided a reference of 3.2 billion nucleotide base pairs of the human genome and 11 million relatively common single nucleotide polymor-phisms (SNPs) that serve as high density genetic markers for the entire ge-nome51. Also, the significant progress of genotyping arrays52 that simultane-ously type a large number of common variants (now up to 5 million SNPs) in a quick and efficient process have made GWAS more feasible. In 2007, three GWAS in data from up to 23,000 individuals for ~1 million SNPs reported a

16

strong association of SNPs on chromosome 9p21.3 with CAD risk53-55. Fol-lowing these initial publications, numerous studies have confirmed the cur-rently poorly understood role of the chromosome 9p21.3 region on CAD risk. The next round of GWAS for CAD identified 11 more SNPs, where each ad-ditional copy of the risk allele conferred a 30-40% higher risk of CAD56. At the same time, additional GWAS and follow-up studies reported 11 other loci that were robustly associated with risk of CAD57-60. Findings in these initial GWAS studies demonstrated that common susceptibility variants for CAD carried minimal incremental risk, and additional loci would require very large sample sizes - in the order of tens of thousands to hundreds of thousands - to be uncovered61. Recognizing the need for larger samples sizes, researchers that previously published successful results independently started collaborat-ing, to form the CARDIoGRAM consortium (comprising >20,000 cases and >60,000 controls) and the C4D Genetics Consortium (>15,000 cases and >15,000 controls) in 2011. Subsequent efforts confirmed associations for nearly all previously reported loci and uncovered 17 new loci through meta-analysis of all available results across consortia62, 63. Through further collabo-ration between these two consortia, and testing associations for up to 2.5 mil-lion SNPs (85% with MAF >1%), the CARDIoGRAMplusC4D consortium (>60,000 cases and >120,000 control) reported an additional 15 previously unanticipated loci in 201364, and eight more loci in 2015, by interrogating 6.7 million common (MAF >5%) and 2.7 million low-frequency (0.5% < MAF < 5%) variants65. More recently, two separate studies querying additional sam-ples and SNPs have identified a further 20 previously unanticipated loci66, 67. Altogether, these studies have resulted in the identification of 78 CAD-asso-ciated loci in largely European and, to a lesser extent, South Asian popula-tions. Approximately 20% of the loci are located near genes with known roles in metabolism of LDLc (e.g. LDLR, APOB, APOE, PCSK9 and HMGCR) or triglyceride-rich lipoproteins (e.g. APOA1-APOC3-APOA4, APOA3, and APOC5), reinforcing the key roles of these pathways in the development of CAD, and providing internal validation of GWAS findings68. An additional 10-20% of loci are related to blood pressure, inflammation, cellular prolifera-tion and/or vascular remodeling68. For most other loci (~60%), the causal genes and mechanisms in disease susceptibility have not yet been identified69.

Exome sequencing studies Although GWAS for CAD have identified many susceptibility loci harboring variants (MAF >1%) with modest effect sizes, the majority of these variants are located outside the protein-coding regions (exons), making it difficult to pinpoint causal genes and affected pathways68, 70. As exonic variants most of-ten exert functional consequences, and the translation of their association to causal genes would be more straightforward, geneticists started to test associ-ations of rare variants within protein-coding regions of the genome (exome). Such exome association studies typically involve sequencing of exomes from

17

individuals experiencing clinical forms of CAD alongside controls, followed by mapping of protein-altering variants (e.g. nonsynonymous, splice-site, and nonsense single-nucleotide substitutions) and testing their association with CAD risk. In 2014, a collaborative endeavor of many exome sequencing stud-ies as a part of the Exome Sequencing Project has contributed to the develop-ment of an exome genotyping chip that includes a subset of putative functional exonic variants (~240,000) with MAF >0.1%, selected from data in >12,000 individuals71. Since then, many studies employed both exome sequencing - to find novel variants - and genotyping to validate findings. A study by the Ex-ome Sequencing Project sequenced exomes of 18 666 genes in about 4000 individuals and identified three rare loss-of-function variants (0.7% MAF) in APOC3 (apolipoprotein C-III) that were associated with triglyceride levels. Carriers of these rare variants had a 40% lower risk of CAD72. Another exome sequencing effort with data from ~5000 early-onset MI cases and MI-free con-trols identified rare coding variants in LDLR (0.5% MAF) and APOA5 (apolipoprotein A-V) (0.5% MAF). LDLR mutation carriers had higher plasma LDLc, whereas APOA5 mutation carriers had higher plasma triglycer-ide levels, increasing the risk of MI by 4.2- and 2.2-fold respectively73. An-other finding was related to 15 rare loss-of-function variants (0.2% MAF) in NPC1L1 (Niemann–Pick C1-like 1), which were associated with lower LDLc and a 53% relative reduction of CAD risk74. Despite the success of these stud-ies in identifying rare variants, many exclusively pointed to previously estab-lished genes for lipoprotein metabolism. However, additional discoveries from whole-exome sequencing are expected as larger sample sizes become available.

1.1.5. Treatment Adherence to a healthy lifestyle is highly recommended to both prevent and manage CAD75, 76. As Elliot Joslin stated: “Genetics loads the gun, but the environment pulls the trigger”. CAD became more manageable after thera-peutic studies had developed a range of medical and surgical procedures. Med-ical therapies to prevent disease progression and recurrent cardiac events pri-marily include medication to lower lipids (e.g. using statins and ezetimibe), lower blood pressure (e.g., atenolol, ramipril, valsartan) and prevent platelet coagulation (e.g. aspirin, clopidogrel, warfarin)77. Surgical revascularization procedures like stent implantation and bypass grafting are commonly em-ployed during clinical manifestations of CAD (i.e. thrombosis, angina, myo-cardial infarction) to prevent sudden cardiac death78. Because of a substantial risk for recurrence of cardiac events, medical therapy is intensified following a surgical procedure79. However, it must be noted that some medical therapies can cause side effects. For example, lipid lowering drugs (statins) were shown to increase the risk of diabetes in recent clinical trials and genetic association studies80, 81, and currently used anticoagulants increase the risk of cerebral

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bleeding82. The combination of an increasing global incidence and undesirable side effects of available therapies presents the need for novel treatment strat-egies for CAD.

1.1.6. Mining GWAS loci for novel treatments As mentioned earlier, the majority of GWAS-identified loci for CAD are not known to be related to conventional risk factors or pathways. For example, loci 9p21.3 and 6p24.1 have been strongly associated with CAD in several independent GWAS (reviewed in 69), but the causal genes and mechanisms that influence disease risk remain incompletely understood for 9p21.383 and unknown for 6p21.469. Identifying and characterizing the causal genes in such loci will increase our understanding of CAD pathophysiology, and may lead to novel drug targets.

In theory, translating GWAS loci to genes and disease mechanisms seems straightforward: first, one identifies promising candidate genes within the lo-cus; next, one characterizes the candidate genes to identify the putative causal genes; and finally, one examines pathways and mechanisms through which putative genes act. However, in practice there are challenges at every stage. Firstly, while candidate genes can be identified using available bioinformatics tools, each tool uses different databases (e.g. expression quantitative trait loci and epigenetic marks) and algorithms to pinpoint promising candidate genes, and hence, multiple genes may be predicted to be causal within each locus. To not throw away the child with the bath water, it is advisable to employ a range of bioinformatics tools and select all top candidate genes for experimental fol-low-up. Secondly, animal models are required to characterize promising can-didate genes, but the traditionally used murine model systems cannot provide the throughput that is required for systematic characterization. Finally, puta-tive causal genes identified using an experimental model system have to be further validated and characterized in higher order vertebrate models to con-firm how perturbation of a gene affects processes at a molecular, cellular, physiological, and whole organismal level in the context of the development of CAD.

1.1.7. Candidate gene prioritization A range of bioinformatics tools permit systematic analysis of GWAS results (i.e. using summary statistics) to identify positional candidate genes. These tools operate by: 1) identifying the putative causal variants; and/or 2) identi-fying the genes that are functionally related to genes in other identified loci. During the former step, both the significantly associated variants and the var-iants that are in high linkage disequilibrium (LD) with the significantly asso-ciated variants are individually evaluated for their functional implications,

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such as amino acid changes, presence in known regulatory regions of the ge-nome, and LD with expression quantitative trait loci using the publicly avail-able genomic data in Encyclopedia of DNA elements (ENCODE), RoadMap Epigenomics and Genotype-Tissue Expression (GTEx) projects84. Variants with possible functional consequences are considered as putative causal vari-ants and are taken forward to next step. Tools like GenomeRunner85, RiVI-ERAbeta86 and FineMap87 facilitate effective assessment of putative causal variants. In the next step, genes affected by the putative causal variants are determined with tools like Sherlock88, RegulomeDB89, and MetaXcan90, which evaluate the possible associations of variants at a transcriptomic and translational level – such as altering protein function, transcript abundance, splice sites, and regulation of expression of genes located further away from lead SNPs91. Some tools (e.g. MetaXcan90, DEPICT92 and Pascal93) addition-ally provide tissues, cell types and pathways by which putative causal variants and candidate genes are anticipated to influence the disease-associated traits. However, as mentioned earlier, it is recommended to employ a range of bio-informatics tools and select top candidate genes across approaches. Since the results from bioinformatic annotation remain prediction, these results should be used to inform further functional studies, e.g. in animal models; not as providing an absolute truth.

1.2. Zebrafish for translational research 1.2.1. Need for a new animal model Traditionally, candidate genes within a disease-associated locus have been in-vestigated by knock-out or over-expression of the corresponding orthologous genes in an appropriate animal model, and subsequent characterization in the context of disease-associated phenotypes. For many years, mouse models have served as the premier vertebrate model system to perform such genetic studies, including in cardiovascular diseases94. The apolipoprotein E (ApoE) and low-density lipoprotein (Ldlr) knockout mice are well-established mouse models that are commonly used in cardiovascular disease research95, 96. How-ever, mouse models do not facilitate the large-scale screening for candidate genes in GWAS-identified loci, for reasons like deficient library of knockout models, difficulties of embryonic manipulations, slow development and life cycles, slow development of atherosclerosis, and expensive maintenance costs.

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1.2.2. Scope for genetic screens using zebrafish model During the last decade, the zebrafish has become increasingly popular to study human diseases, since they resemble humans in their development and metab-olism97, and have a well-annotated genome with orthologues for at least 70% of human genes98. Embryonic genetic manipulation has been challenging for decades, but recent developments in multiplexed mutagenesis using Clustered Regulatory Interspaced, Short Palindromic Repeats (CRISPR) and CRISPR associated systems (Cas9) can now be efficiently applied in zebrafish to intro-duce mutations in up to eight genes simultaneously99. In this context, it is help-ful that zebrafish eggs are fertilized and develop externally, and re-implanta-tion of embryos after mutagenesis is not required. The after-effects of the in-duced mutations and the disease progression at cellular, physiological and whole-organismal levels can be conveniently monitored in zebrafish larvae thanks to their innate optical transparency until ~30 days post-fertilization (dpf). The availability of transgenic lines with fluorescently labelled cells, and the possibility to administer desired reagents (e.g. fluorescent dyes) through bodily infusion are additional strengths of this vertebrate model system. More-over, recent improvements in automated handling, positioning and orienting of zebrafish larvae imply that relevant cellular processes can now be automat-ically imaged and quantitatively analyzed in high-throughput100, making full use of the advantage of the zebrafish’s high fecundity (~300 offspring per week).

1.2.3. Previous small-scale studies on using zebrafish to model dyslipidemia and atherosclerosis Small-scale studies suggested that zebrafish larvae may be used to model dyslipidemia and early-stage atherosclerosis. Firstly, larvae fed on a high-cho-lesterol diet for 10 days become hypercholesterolemic, and accumulate lipids and oxidation-specific epitopes in their vasculature, much like in early-stage atherosclerosis in humans101-103. Secondly, additional suppression of ldlr ex-pression during the first 8 days of larval development drastically increased LDLc levels, indicating that the role of ldlr in LDLc metabolism is conserved in zebrafish104. Thirdly, zebrafish mutants with nonfunctional apolipoprotein CII (APOCII) fed on a normal diet were hypertriglyceridemic, and accumu-lated lipids and lipid-laden macrophages in their vasculature, much like pa-tients with APOCII deficiency105. Fourthly, treating hypercholesterolemic and hypertriglyceridemic zebrafish with commonly prescribed lipid-lowering drugs, i.e. statins and/or ezetimibe, ameliorated cholesterol and triglyceride levels104, 106, 107, and high resolution imaging showed lower levels of vascular lipid deposition in zebrafish larvae107.An overview of the studies performed so far, including the sample size, is shown in Table 1.

Tabl

e 1.

Maj

or d

evel

opm

ents

in ze

braf

ish a

s a m

odel

syst

em fo

r dys

lipid

emia

and

ath

eros

cler

osis

.

Find

ings

In

terv

entio

ns

Sam

ples

R

efer

ence

s D

=day

s; M

=mon

ths;

W=w

eeks

Hig

h ch

oles

tero

l die

t (H

CD)-i

nduc

ed d

yslip

idem

ia

HCD

(3 M

) 11

adu

lts

101

HCD

(14

D)

Hom

ogen

ates

of 2

0 la

rvae

(n=3

) 10

8 H

CD (1

4 D

) H

omog

enat

es o

f 20

larv

ae (n

=6)

106

Hyp

erch

oles

tero

lem

ia-in

duce

d va

scul

ar li

pid

depo

sitio

n

HCD

+BO

DIP

Y-C

11 (1

0 D

) 4

larv

ae

101

HCD

(3 M

) 9

adul

ts 10

1 H

CD+B

OD

IPY

-C11

(14

D)

4 la

rvae

10

2

HCD

-indu

ced

oxid

atio

n of

lipo

prot

eins

HCD

(3 M

) 8

adul

ts 10

1 H

CD (1

4 D

) H

omog

enat

es o

f 50

larv

ae (n

=3)

108

HCD

(14

D)

6 la

rvae

10

2

Recr

uitm

ent o

f mon

ocyt

es to

the

vasc

ular

lipi

d de

posit

s H

CD (1

0 D

) 12

larv

ae

101

HCD

(14

D)

Hom

ogen

ate

of 5

0 la

rvae

10

8

Lipi

d-lo

wer

ing

drug

s red

uce

HCD

-indu

ced

dysli

pide

mia

HCD

+Sim

vasta

tin (1

4 D

) H

omog

enat

es o

f 20

larv

ae (n

=2)

106

HCD

+Eze

timib

e (1

4 D

) H

omog

enat

es o

f 20

larv

ae (n

=3)

HCD

(10

D),

HCD

+Ato

rvas

tatin

(5 D

) N

ot a

vaila

ble

107

HCD

(10

D),

HCD

+Eze

timib

e (5

D)

Not

ava

ilabl

e

apoc

2 m

utan

t zeb

rafis

h as

a m

odel

for h

yper

trigl

ycer

idem

ia

apoc

2 kn

ocko

ut; N

orm

al d

iet (

10 D

) 3

larv

ae

105

apoc

2 kn

ocko

ut; N

orm

al d

iet (

3 M

) 6

adul

ts

Hyp

ertri

glyc

erid

emia

-indu

ced

early

-sta

ge a

ther

oscl

eros

is ap

oc2

knoc

kout

; HCD

+BO

DIP

Y (1

0 D

) 8

larv

ae

105

Role

of d

isrup

ted

ldlr

in h

yper

lipid

emia

ld

lr k

nock

dow

n; H

CD (2

D)

Hom

ogen

ates

of 1

00 la

rvae

(n=2

) 10

4 ld

lr k

nock

out

4 la

rvae

10

9

cont

inue

d Ta

ble

1.

Find

ings

In

terv

entio

ns

Sam

ples

R

efer

ence

s D

=day

s; M

=mon

ths;

W=w

eeks

Role

of d

isrup

ted

ldlr

in v

ascu

lar l

ipid

dep

ositi

on

ldlr

kno

ckdo

wn;

HCD

(2 D

) 45

larv

ae

104

ldlr

kno

ckou

t; H

CD+B

OD

IPY

(5 D

) 15

larv

ae

109

Lxra

mut

ant z

ebra

fish

as m

odel

for h

yper

chol

este

role

mia

nr

1h3

knoc

kout

; HCD

(21

D)

3 to

5 a

dults

11

0

Act

ivity

of c

etp

incr

ease

s in

hype

rcho

leste

role

mic

zeb

rafis

h H

DC+

Turm

eric

ext

ract

(5 W

) 80

adu

lts

111

HCD

+Gra

pe e

xtra

ct (5

W)

70 a

dults

11

2 Ex

pres

sion

anal

ysis

of a

polip

opro

tein

s- A

, B, &

E in

zeb

rafis

h In

-situ

hyb

ridiz

atio

n fro

m 1

to 6

D

3 la

rvae

11

3 A

bbre

viat

ions

: HCD

- hig

h ch

oles

tero

l die

t; ap

occ2

- apo

lipop

rote

in C

II; ld

lr- lo

w-d

ensit

y lip

opro

tein

rece

ptor

; Lxr

a- li

ver x

rece

ptor

alp

ha; n

r1h3

- nuc

lear

re

cept

or su

bfam

ily 1

, gro

up H

, mem

ber 3

; cet

p- c

hole

stero

l este

r tra

nsfe

r pro

tein

23

Most of the studies highlighted in Table 1 took advantage of available reporter lines (Table 2), in conjunction with using lipid labelling dyes to characterize early-stage atherogenic processes. Advancing further, Fang et. al. engineered a transgenic zebrafish that conditionally expresses a fluorescently labelled hu-man monoclonal antibody IK17, which specifically binds to and labels oxi-dized LDL102. The studies listed in Table 1 also used various enzymatic as-says, immunoassays, and chromatography techniques on homogenates of 25 to 100 larvae per sample to characterize dyslipidemia and atherosclerosis in zebrafish larvae. So far, gene expression or function have been altered using morpholino-based downregulation104 or TALEN-based mutagenesis tech-niques105, 110.

On the need for further validation and method development and refinement Like humans, zebrafish are genetically heterogeneous. As a result, data from a large number of larvae is needed to draw meaningful conclusions. Although the aforementioned proof-of-principle studies have so far provided promising results, they were based on observations in fewer than 25 larvae per condition (Table 1). This was at least in part because mounting larvae in low melting agarose for imaging is time-consuming. In addition, whole-body cholesterol, triglyceride and protein levels were usually quantified in homogenates of 20-100 pooled larvae (Table 1). While suitable and efficient for dietary and drug treatment interventions, pooling larvae for phenotypic characterization is not optimal in genetic interventions, where – depending on the approach - se-quencing of individual larvae may be required. Next, results from downregu-lation of gene expression using morpholinos have to be interpreted with cau-tion as in many studies, morphant phenotypes could not be confirmed in mu-tants114, 115. While TALEN-based mutagenesis is a better alternative to mor-pholinos in this aspect, it lacks in the ability to edit multiple genes simultaneously116. This might be necessary when there are multiple zebrafish orthologues for a human gene, and all of them have to be targeted together to ensure that no genetic compensation occurs for the loss of one or the other.

With the recent developments in imaging and image-quantification tech-nologies, it is now possible to acquire and quantify image-based traits in a large number of larvae. For example, with a Vertebrate Automated Screening Technology (VAST) Bioimager coupled to a fluorescence microscope, larvae can automatically be positioned and oriented within the microscope’s field of view, and fluorescent images of larvae can be acquired in a high-through-put100. The acquired images can subsequently be quantified in batch mode us-ing image-analysis software like CellProfiler117. Furthermore, lipid-analyzers such as Mindray BS-380 that are designed to analyze lipid fractions in human plasma samples can be used to quantify whole-body levels of LDLc, HDLc, triglycerides, total cholesterol and glucose at single larval level. Finally, using the state-of-the art multiplexed CRISPR-Cas9 mutagenesis, it is now possible

24

to target up to at least nine genes simultaneously99, 118, 119. By combining these technical advances in imaging, image-analysis and mutagenesis, it is now pos-sible to target multiple zebrafish orthologues together and examine their ef-fects in a large number of larvae.

Hence, confirmation of initial findings, an improved resolution of quanti-tative readouts, and a higher throughput are desirable before we can conclude if zebrafish larvae can be used as a model system for large-scale characteriza-tion of candidate genes for dyslipidemia, atherosclerosis and CAD.

Table 2. Transgenic zebrafish and fluorescent tracers

Tools Labels Applications Refe-rences

Transgenic zebrafish fli1:EGFP* Endothelial cells Angiogenesis, vascular lipid

accumulation and inflamma-tion

120,101

lyz:EGFP, lyz:DsRed2 Myeloid cells Vascular inflammation 121, 101 mpeg1:EGFP mpeg1:mCherry*

Macrophages Vascular inflammation 102, 122

mpo:EGFP* Neutrophils Vascular inflammation 123 hsp70-IK17:EGFP* Oxidized LDL Vascular accumulation of oxi-

dized lipids 102

Lipoprotein/lipid tracers and enzyme substrates Dil-LDL LDL in circulation LDL distribution in flow 124 cholesterol BODIPY-C11

CE trafficking, LDL, lipid deposits

Vascular lipid accumulation 125, 101

NBD-cholesterol Lipid deposits Intestinal cholesterol absorp-tion and trafficking

125

BODIPY-C12 Fatty-acid trafficking, lipid droplets

Intestinal fatty acid absorption and trafficking

126

PED6 PLA2 activity Digestive tract and vascular PLA2 activity

127

Monodansylpentane* Lipid deposits Vascular lipid deposition 128, 129

Table adapted from 103 Abbreviations: hsp- heat-shock protein; CE- cholesterol ester; PLA2- phospholipase A2 *Tools used in this thesis

25

2. Aims

2.1. General aim My thesis is based at the intersection of the challenges for functional valida-tion of candidate genes for coronary artery disease, that is, to develop and validate novel in vivo model systems that are suitable for high-throughput, image-based genetic screens in coronary artery disease and related traits, and use these model systems to systematically characterize positional candidate genes.

2.2. Specific aims Study I To validate zebrafish as a model system for dyslipidemia, atherosclerosis and coronary artery disease using a dietary intervention, a treatment intervention with lipid lowering drugs, and a multiplexed, CRISPR-Cas9-based genetic in-tervention of proof-of-concept genes. Further, to characterize four candidate genes in a GWAS-identified, pleiotropic locus on chr 19p13.11. (Manuscript in revision)

Study II To confirm or refute the role of apolipoprotein CII in hypertriglyceridemia and early-stage atherosclerosis in zebrafish larvae; and to examine if apolipo-protein CII represents a promising target for therapeutic intervention. (Manu-script)

Study III To examine the effects of mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) on dyslipidemia, atherosclerosis and diabetes-related traits in zebrafish larvae, and assess if these effects are consistent with loss-function mutations in PCSK9 in humans and with PCSK9 inhibitors in humans. (Man-uscript)

26

3. Study summaries

Study design, methods and main results An overview of each of the Studies, I-III, is presented in Table 3.

Tabl

e 3.

Ove

rvie

w of

Stu

dies

I, II

and

III

Stud

y I

II

III

Inte

rven

tions

an

d sa

mpl

e si-

zes

•D

iet –

ove

rfeed

ing,

cho

leste

rol s

uppl

emen

-ta

tion;

n=2

366

Zebr

afish

orth

olog

ue o

f apo

lipop

rote

in C

II (a

poc2

); n=

384

Zebr

afish

orth

olog

ue o

f pro

prot

ein

con-

verta

se su

btili

sin/k

inas

e ty

pe 9

(pcs

k9);

n=11

21

•D

rugs

– a

torv

asta

tin p

lus e

zetim

ibe;

n=1

063

•Ze

braf

ish o

rthol

ogue

s of p

roof

-of-c

once

ptge

nes

– A

POE,

APO

B, L

DLR

; n=3

83•

Zebr

afish

orth

olog

ues o

f can

dida

te g

enes

–LP

AR2A

, GAT

AD2A

, TM

6SF2

, GM

IP;

n=54

7

Trai

ts q

uant

i-fie

d

•W

hole

-bod

y lip

id a

nd g

luco

se le

vels

•W

hole

-bod

y lip

id a

nd g

luco

se le

vels

•W

hole

-bod

y lip

id a

nd g

luco

se le

vels

•V

ascu

lar e

arly

-sta

ge a

ther

ogen

ic tr

aits

(incl

.ac

cum

ulat

ion

of li

pids

, mac

roph

ages

, neu

-tro

phils

and

oxi

dize

d LD

L)

•V

ascu

lar e

arly

-sta

ge a

ther

ogen

ic tr

aits

(incl

.ac

cum

ulat

ion

of li

pids

, mac

roph

ages

and

ne

utro

phils

)

•V

ascu

lar e

arly

-sta

ge a

ther

ogen

ic tr

aits

(incl

. acc

umul

atio

n of

lipi

ds, m

acro

-ph

ages

and

neu

troph

ils)

• Bod

y siz

e•

Body

size

•Bo

dy si

ze•

Lipi

d de

posit

ion

in li

ver

•Li

ver s

ize

and

lipid

dep

ositi

on in

live

r

Ana

lysis

•O

verfe

edin

g vs

. Nor

mal

feed

ing

•2

vs. 0

mut

ated

apo

c2 a

llele

s (w

ith a

ndw

ithou

t inc

ludi

ng a

dditi

onal

wild

type

larv

aefro

m o

ther

scre

ens)

•Ef

fect

of e

ach

addi

tiona

l mut

ated

pcs

k9al

lele

by

incl

udin

g ad

ditio

nal w

ildty

pela

rvae

from

oth

er sc

reen

s•

Chol

este

rol s

uppl

emen

tatio

n vs

. No

chol

es-

tero

l•

Dru

g tre

atm

ent v

s. N

o tre

atm

ent

•Ef

fect

of e

ach

addi

tiona

l mut

ated

apo

c2 a

l-le

le (w

ith a

nd w

ithou

t inc

ludi

ng a

dditi

onal

wild

type

larv

ae fr

om o

ther

scre

ens)

•Fo

r eac

h ta

rget

ed g

ene,

2 v

s.0 m

utat

ed a

l-le

les

•Fo

r eac

h ta

rget

ed g

ene,

effe

ct o

f eac

h ad

di-

tiona

l mut

ated

alle

le•

Effe

ct o

f eac

h ad

ditio

nal m

utat

ed a

llele

acro

ss a

ll ta

rget

ed g

enes

28

Study I Zebrafish larvae as a model system for systematic characterization of drugs and genes in dyslipidemia and atherosclerosis In this study, I performed a large-scale validation of the zebrafish model sys-tem for dyslipidemia and atherosclerosis using 1) a dietary intervention; 2) a treatment intervention with lipid lowering drugs; and 3) genetic intervention of proof-of-concept genes. Finally, I used the model system to characterize four candidate genes in GWAS-identified pleotropic locus on chr 19p13.11.

In the dietary intervention, atherogenic potential of overfeeding and dietary cholesterol supplementation was examined in nearly 2000 transgenic larvae with fluorescently labelled macrophages, neutrophils, oxidized LDL and/or vascular endothelium. In the treatment intervention with the LDLc lowering drugs atorvastatin and ezetimibe, nearly 1000 transgenic larvae were overfed on a cholesterol supplemented diet with or without the drug treatment. In the genetic intervention of proof-concept genes- APOE, APOB and LDLR were targeted together in zebrafish with labelled macrophages and neutrophils us-ing a multiplexed CRISPR-Cas9 approach. Similarly, the four candidate genes – LPAR2, GATAD2A, TM6SF2 and GMIP in the GWAS-identified locus were genetically intervened together. In both genetic interventions, offspring (F1 generation) from the CRISPR founders were examined. All larvae were first visualized and quantified for early-stage atherosclerotic traits at 10 days post-fertilization using a semi-automated imaging set up (i.e. A Vertebrate Auto-mated Screening Technology [VAST] BioImager and fluorescent microscope) and objective image quantification pipelines. Subsequently, whole-body lipid and glucose levels were quantified at the single larval level using enzymatic assays, and CRISPR-Cas9 target sites in each larva were sequenced using paired-end sequencing. Finally, associations between the exposures (i.e. diet, drugs, or genes) and effects on the quantified traits were analyzed.

The results show that five days of overfeeding and cholesterol supplemen-tation have independent pro-atherogenic effects, including elevated total cho-lesterol and triglyceride levels, more vascular deposition of lipids and oxi-dized LDL, and more co-localization of lipids with macrophages and neutro-phils. Combined treatment with the lipid lowering drugs for five days pro-tected larvae from the pro-atherogenic effects of overfeeding and cholesterol supplementation. CRISPR-Cas9-induced mutations in orthologues of proof-of-concept genes resulted in higher LDLc levels (apoea), and more early stage atherosclerosis (apobb.1). Finally, our pipeline helped to identify putative causal genes for circulating lipids and early-stage atherosclerosis (LPAR2 and GATAD2A) (Figure 3).

29

In conclusion, our pipeline facilitates systematic, in vivo characterization of drugs and candidate genes to increase our understanding of disease etiol-ogy, and can likely help identify novel targets for therapeutic intervention.

Figure 3. The effect of overfeeding and cholesterol supplementation (n>2000), treat-ment with atorvastatin and ezetimibe (n>1000), and mutations in apoea, apobb.1, lpar2a and gatad2ab on whole-body lipid and glucose levels, vascular atherogenic traits and body size. Across all forest plots, circles represent the effect sizes and whiskers represent 95% confidence intervals. In the forest plots for apoea and apobb.1, open circles and dotted lines represent the effect of two functionally knocked-out alleles vs. two unmodified alleles, and full circles and filled lines repre-sent the additive per mutated allele effect.

30

Study II Apoc2 mutant zebrafish: a model for hypertriglyceridemia and early-stage atherosclerosis A zebrafish model system for hypertriglyceridemia can be useful to systemat-ically characterize positional candidate genes for triglyceride metabolism. Re-cent studies performed elsewhere suggested that zebrafish with mutations in apolipoprotein CII (apoc2) have elevated triglyceride levels and more early-stage atherosclerosis. However, results were based on data from only three wildtype and three mutant larvae. Hence, in Study II, I generated apoc2 found-ers using CRISPR-Cas9, and phenotypically characterized their offspring to: 1) further validate our pipeline for systematic characterization of candidate genes for dyslipidemia; 2) confirm or refute a role for apoc2 in hypertriglyc-eridemia and early-stage atherosclerosis in zebrafish larvae; and 3) examine if apolipoprotein CII represents a promising target for therapeutic intervention.

I targeted apoc2 in transgenic zebrafish with fluorescently labelled macro-phages and neutrophils and examined the larvae (F1 generation) from an in-cross of founders. Larvae were fed on regular dry food enriched with 4% extra cholesterol from the age of 5 days post-fertilization (dpf) until 9 dpf. At 10 dpf, 384 larvae were imaged to visualize body size, vascular atherogenic traits and lipid accumulation in the liver, followed by semi-automated quantifica-tion of whole-body lipoprotein and glucose levels and paired-end sequencing of the CRISPR-targeted site.

Of the 384 sequenced F1 larvae, 174, 119 and 61 larvae carried 0, 1 and 2 mutated apoc2 alleles. Compared with the 174 larvae free of mutations in apoc2, the 35 larvae with anticipated complete loss of apoc2 had higher whole-body levels of triglycerides, HDLc and total cholesterol, and a tended to have lower whole-body glucose levels. Mutant larvae also tended to have more vascular lipid deposition. Interestingly, the trends for lower whole-body glucose levels and more vascular lipid deposition in larvae with anticipated loss of functional apoc2 reached significance when larvae (n=3812) from other screens, in which apoc2 was not experimentally perturbed were included as additional wildtype controls (Figure 4).

Thus, our large-scale study confirms the role of apoc2 in hypertriglycer-idemia and early-stage atherosclerosis. While apoc2 mutant zebrafish model can be used as a genetic background to identify and characterize causal genes for triglyceride metabolism, independent and opposite effects on triglycerides and glucose suggest that APOCII may not be suitable as a target for prevention and treatment of coronary artery disease.

31

Figure 4. The effect of mutations in apoc2 (n=384) on whole-body lipid and glucose levels, vascular atherogenic traits, metabolic traits and body size. Forest plot on the left includes 174 wildtype larvae identified within the apoc2 genetic screen. Forest plot on the right includes additional wildtype larvae from other screens in which apoc2 was not experimentally perturbed. In both forest plots, open circles and the dotted lines represent the effect of two functionally knocked-out alleles vs. two un-modified alleles, and full circles and filled lines represent the additive per mutated allele effect. Circles represent the effect sizes and whiskers represent 95% confi-dence intervals.

32

Study III Image-based, in vivo characterization of cardiometabolic consequences of mutations in pcsk9 Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) have emerged as a new class of drugs, reducing LDLc by 50–60% in several clinical settings, i.e. more than the maximal dose of statins plus ezetimibe combined typically accomplish. However, Mendelian randomization studies and clinical studies suggest that the LDLc lowering effect of PCSK9 inhibition may be accompanied by an elevated risk of incident diabetes. In this study, we mim-icked the mechanistic action of PCSK9 inhibitors in humans by inducing mu-tations in pcsk9 in zebrafish and examined the effects of such mutations on dyslipidemia, early-stage atherosclerosis and diabetes-related traits in nearly 5000 zebrafish larvae.

We targeted pcsk9 using CRISPR-Cas9 mutagenesis to generate founder fish (F0 generation) in two reporter backgrounds: 1) with fluorescently la-belled macrophages and neutrophils; and 2) with fluorescently labelled insulin expressing cells and hepatocytes. At 10 dpf, 2x384 F1 larvae in the former background were imaged for vascular atherogenic traits, and another 353 F1 larvae in the later background were imaged for diabetes-related traits. Further-more, body size and whole-body lipid and glucose levels were quantified in all larvae. Larvae were finally sequenced at the CRISPR-targeted site in pcsk9 using paired-end sequencing.

Of the total 1121 screened F1 larvae, 15, 24 and 1019 larvae carried 0, 1 and 2 mutated pcsk9 alleles respectively. Given the small number of larvae with 0 or 1 mutated alleles, we used comparable data from other screens in which pcsk9 was not experimentally perturbed as wildtype controls, that is, larvae from a dietary intervention (n=2366), drug treatment intervention with atorvastatin and ezetimibe (n=1063), genetic screen for APOE, APOB and LDLR (n=383), and a genetic screen for APOC2 (n=384), i.e., larvae charac-terized in Studies I and II. Compared with nearly 2500 larvae free from muta-tions in pcsk9 that were included in Studies I and II, each additional mutated allele in pcsk9 was associated with lower LDLc levels, on average. Each ad-ditional mutated allele in pcsk9 was also associated with less vascular lipid deposition, less co-localization of macrophages with lipids, more co-localiza-tion of macrophages with neutrophils and fewer pancreatic β-cells (Figure 5).

In conclusion, our findings in pcsk9 mutant larvae are in line with results from people carrying loss-of-function PCSK9 mutations, and are also in line with the effects of PCSK9 inhibitors in humans. Furthermore, the direct effect of mutations in pcsk9 on the number of pancreatic β-cells, suggests that the higher risk of diabetes in humans with mutations in PCSK9 may result from a direct effect on the beta cell.

33

Figure 5. The effect of each additional mutated pcsk9 allele (n=1121) on whole-body lipid and glucose levels, vascular atherogenic traits, metabolic traits and body size. Given the small number of wildtype larvae (n=15) within the pcsk9 genetic screen, wildtype larvae from other screens in which pcsk9 was not experimentally perturbed were include in the association analysis. Full circles and filled lines repre-sent the additive per mutated allele effect.

34

4. Concluding remarks

The three large-scale proof-of-concept studies described in this thesis confirm that zebrafish larvae can be used to model dyslipidemia and early-stage ather-osclerosis. These studies also highlight the necessity to screen a large number of zebrafish larvae in order to draw firm conclusions on the role of genes in complex disease-related traits. Further, I demonstrated how the model system can be used to characterize candidate genes in GWAS-identified loci. This approach presents an opportunity to increase our understanding of the molec-ular causes of cardiometabolic diseases, and reduce the hundreds of candidate genes in GWAS-identified loci to a more feasible number for: 1) further in-depth characterization using higher order animal models; 2) targeted associa-tion studies and genotype-based recall efforts using exome-sequencing data in humans; and 3) adequately powered, target-based small molecule screens.

35

5. Future perspectives

Further characterization of the putative causal genes- LPAR2 and GA-TAD2A. In Study I, the preliminary characterization of candidate genes in a pleiotropic locus on chr 19p13.11 highlighted a role for LPAR2 in cholesterol metabolism and early-stage atherosclerosis, and for GATD2A in cholesterol metabolism. However, these findings were based on a small number of mutant larvae (11 heterozygous mutants for lpar2a and 32 for gatad2ab). To draw firm conclusions on the role of these genes, founders for these genes were generated again, and the screening of their offspring is currently being final-ized. Perhaps, as demonstrated in Study III, the combined phenotyping for early-stage atherosclerosis and diabetes related traits may also shed light on the potential of these genes for being useful therapeutic targets.

Optimizing mutagenesis efficiency for an adequate distribution of geno-types. In genetic screen for two of the genes in Study I (apoba and apobb.1), and for pcsk9 in Study III, there were many larvae with two mutated alleles, and few wildtype controls, reflecting the high mutagenic activity of the CRISPR guide RNAs used for the mutagenesis, in spite of pre-testing and se-lecting the CRISPR guide RNAs that are more likely to induce mutations in one of the two alleles of a gene (heterozygous mutations) in the founder fish. In order to have an adequate number of wildtype controls in future genetic screens, alongside embryos injected with highly efficient guide RNAs, some non-injected siblings will be raised to adulthood in separate tanks. Then, phe-notypic characterization on F1 larvae from in-crosses of injected and non-in-jected fish should result in a higher statistical power.

Genetic screens in apoc2 or apobb.1 knockout background at 5 days post fertilization (dpf). As larvae with mutations in apobb.1 (Study I) and apoc2 (Study II) developed hypertriglyceridemia and early-stage atherosclerosis af-ter 4 days of feeding on high-cholesterol diet, it would be interesting to exam-ine if these traits manifest already at 5 dpf, i.e. without a dietary intervention. If so, then performing genetic screens in one of these mutant backgrounds would allow phenotypic characterization of larvae at 5 dpf without having to feed them on high-cholesterol diet. This would eliminate any batch effects that may prevail during the feeding or maintenance of larval tanks, as well as speed up the screening process.

36

Acknowledgements

This work has been accomplished with the tireless efforts of so many. I would like to take this opportunity to express my gratitude to:

My Main Supervisor Marcel den Hoed, for the opportunities, endless support and help. Your enthusiastic guidance, persistent encouragement and amazing patience through my Master’s thesis to Ph.D. thesis helped me to get this far. Thanks for bringing out the best in me almost always.

My co-supervisor Anastasia Emmanouilidou, thanks for setting up the lab and making it a pleasant playground. I am truly grateful for your guidance throughout the experimental part of this work. Thanks for being supportive all these years and correcting me when necessary.

My co-supervisor Erik Ingelsson, I am sincerely thankful for your feedback on the work and availability despite your busy schedule.

Carolina Wählby and Petter Ranefall, thanks for your help in establishing the image analysis pipelines and constructive feedback on manuscripts. Anders Larsson, thanks for kindly analyzing all the samples in this work. Your efforts are highly appreciated. Collaborating with all of you was a pleasant experi-ence. To Hannah Brooke, a special thanks for your input with the statistical analysis and data visualization.

Benedikt, thanks for programming the pipetting robot and making the sample preparation easy. Also, thanks for being that friend with whom I can talk be-yond work. I thoroughly enjoyed your company in- and out of the office. Eu-genia, you have been a very big helping hand in my projects. Many thanks for sharing the work load, and at times, for taking the entire load on you. Also, thanks for developing the variant-calling pipeline. I feel lucky to have such understanding fellow Ph.D. students.

Tiffany, the CRISPR-mutant lines would not come to life without you. Thanks for the help in generating them and also for teaching me to inject. Joao, thanks for your help in setting up our own fish room. Your DIY ideas are impressive! Ci and Natalie, ex-members in the group, thanks for your intellectual input during our weekly group meetings. Sharing office-space with all of you has been fun.

37

A big thank you goes to the whole Zebrafish Facility at EBC for their support during the beginning of this work: Johan, Katarina, Judith, Beata and everyone else I had the privilege to meet.

To the Epidemiology Group- Tove, Mwenya, Chris, Markus and Stefan; and to the Cell Group- Susanne, Casimiro and Naomi, I am grateful for the many hours of scientific and social engagement.

The many students that joined the group to conduct their thesis projects kindly helped in fish husbandry and other laboratory work. Thanks to each one of you- Lisa, Mauro, Sitaf, Silvia, Chrissy, Mehran, David, Adriana, Shreya, Aida, Taya, Anne, Marta, Endrina, Tiarne, Azizah, Bobby, Tessa and Amine. Also, thanks to Lynsey for taking care of the fish when there were only a few people in the group.

Departments –IMV and IGP have provided an excellent infrastructure for my research and professional development. I particularly thank Anna Foyer, Elin Ekberg, Ulrica Bergström and Helene Norlin for the administrative support.

My extended gratitude to those who balanced the work with the other aspects of life:

To friends in Uppsala who have made life funnier with gatherings 💬💭, sports , festivals 🎆🎄 and parties🎉🍸. Thanks to each one of you- Sunil, Sethu,Srinivas, Sudharsan, Sandeep, Hari, Javeed, Brajesh, Srinu T, Raghu, Mahesh, Varun, Gopi, Ram, Phani, Prakash anna, Chandu anna, Kiran anna, Ramesh anna, Srisailam anna, Soundarya, Anusha, Christine, Saritha, Ammulu, Prathusha, Indu and Sowmya. Huda, Sharanya, Sony, Sandhya and Geetha akka, special thanks for serving delicious food always 😋.

To my buddies from Bachelors and School- Abhishek, Charan and Rupa thanks for being in touch despite your busy lives and for your concern on my wellbeing. Naresh, I will forever be grateful for your help in getting the ad-mission in Uppsala University, thanks.

Finally, to my family. Dad and Mum, thanks for pushing your boundaries throughout your life to give me a comfortable life 🙏. Souji, Pranay, Raja,Pinne and Babaye, heartfelt thanks for filling the son’s shoes and taking care of Dad and Mum in times when I am supposed to be there 👏. Kanaka, mybetter half full, I can’t thank you enough for your love, support and sacrifice above and beyond the call of duty 😘. Thanks for taking care and for alwayshaving my back 💐. And to our 👶, Vismai for being such an easy kid to han-dle.

39

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