ENU Mutagenesis as a Strategy to Identify Novel Genes Involved in the Regulation of HDL Cholesterol Levels

Cardiovascular disease is one of the largest causes of death and disability in the industrialized world. HDL cholesterol is one of the factors used by clinicians to assess the risk of developing cardiovascular diseases. Because raising HDL cholesterol levels has been identified as a preventative strategy for disease management, recent research has been shifted towards the development of novel therapeutics targeting increasing HDL cholesterol levels, which in itself requires an understanding of HDL cholesterol metabolism. Despite considerable progress in understanding genes that affect the HDL particle, its function, and cholesterol content, genes identified to date explain only a small percentage of the genetic variation. We used a phenotype-driven approach to discover novel genes that affect HDL cholesterol levels. Using N-ethyl-N-nitrosourea mutagenesis in mice, 4 mutant lines (Hlb218, Hlb320, Hlb280, and Hlb446) with abnormal HDL cholesterol levels were established. Causal mutations in these lines were identified using a combination of linkage analysis in a cross between mutant and C57L/J and high- throughput technologies (microarray, RNASeq, and exome sequencing). The mutation in Hlb218 mapped within a 12 Mbp region on Chr 10. High-throughput sequencing of Hlb218 liver RNA identified a transition of G to A in Pla2gl2b, which leads to a cysteine to tyrosine change in the protein and most likely causes a loss of a disulfide bridge. The mutation in Hlb320 mapped within a 17 Mbp region on Chr 7. Microarray analysis of Hlb320 liver RNA showed a 7-fold downregulation of Hpn; sequencing identified a transition of T to C in the 3\u27 splice site of exon 8. Northern blot confirmed lower mRNA expression level in Hlb320 and showed no change in splicing, suggesting that the mutation most likely affects the splicing rate and leads to a lower protein level. Comparison of additional phenotypes in Hlb218 and Hlb320 to phenotypes in the corresponding knockout mouse models further supported the causality of the ENU mutations in the identified genes. The mutation in Hlb280 mapped within a 34 Mbp region on Chr 12. Exome sequencing of DNA identified a transversion of T to A in exon 7 of Ylpm1. The mutation in Hlb446 mapped within a 6 Mbp region on Chr 1. Exome sequencing of DNA identified a transition of A to G in intron 8 of Lamc1. The causality of ENU mutation in Hlb280 and Hlb446 on HDL cholesterol levels remain to be tested but embryonic lethality of complete knockouts of both Ylpml and Lamcl makes it challenging. At the same time, lack of viable complete knockout mouse models make the ENU mutants valuable tools to further study the role of these genes, their effect on HDL cholesterol levels, and their role in metabolism. The work presented in this thesis demonstrates that ENU mutagenesis, although it has its challenges, is a method that can be used to identify genes involved in the phenotype under the investigation

Detection of early myocardial ischemia in sudden cardiac death by advanced molecular technologies

Sudden cardiac death remains a major cause of mortality around the world. Early myocardial ischemia is often a plausible explanation for cardiac arrest in many sudden cardiac death cases but an established postmortem method for its diagnostics is currently lacking. The use of contemporary methods was proposed as a potential diagnostics strategy, specifically evaluation of a panel of molecular tissue markers. Mass spectrometry immunohistochemistry (MS-IHC) and matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI IMS) were investigated as advanced technologies to simultaneously measure multiple molecules in a single tissue section and directly from slide. Both MS-IHC and MALDI IMS successfully detected ischemic areas and allowed not only simultaneous testing of multiple markers but also visualization of molecules’ distribution within the tissue by image reconstruction and quantification of the signal. MALDI IMS was also used to characterize molecular changes in response to ischemia by an untargeted approach. Both technologies are powerful mass spectrometry-based tools with a great potential for investigating molecular mechanisms in pathology-related research, accelerating tissue marker discovery, and integrating in routine diagnostics

Detection of early myocardial ischemia in sudden cardiac death by advanced molecular technologies

Sudden cardiac death remains a major cause of mortality around the world. Early myocardial ischemia is often a plausible explanation for cardiac arrest in many sudden cardiac death cases but an established postmortem method for its diagnostics is currently lacking. The use of contemporary methods was proposed as a potential diagnostics strategy, specifically evaluation of a panel of molecular tissue markers. Mass spectrometry immunohistochemistry (MS-IHC) and matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI IMS) were investigated as advanced technologies to simultaneously measure multiple molecules in a single tissue section and directly from slide. Both MS-IHC and MALDI IMS successfully detected ischemic areas and allowed not only simultaneous testing of multiple markers but also visualization of molecules’ distribution within the tissue by image reconstruction and quantification of the signal. MALDI IMS was also used to characterize molecular changes in response to ischemia by an untargeted approach. Both technologies are powerful mass spectrometry-based tools with a great potential for investigating molecular mechanisms in pathology-related research, accelerating tissue marker discovery, and integrating in routine diagnostics

Loss-of-function mutation in Pcsk1 increases serum APOA1 level and LCAT activity in mice

Abstract Background The convertase subtilisin/kexin family 1 gene (PCSK1) has been associated in various human genetics studies with a wide spectrum of metabolic phenotypes, including early-onset obesity, hyperphagia, diabetes insipidus, and others. Despite the evident influence of PCSK1 on obesity and the known functions of other PCSKs in lipid metabolism, the role of PCSK1 specifically in lipid and cholesterol metabolism remains unclear. This study evaluated the effect of loss of PCSK1 function on high-density lipoprotein (HDL) metabolism in mice. Results HDL cholesterol, apolipoprotein A1 (APOA1) levels in serum and liver, and the activities of two enzymes (lecithin-cholesterol acyltransferase, LCAT and phospholipid transfer protein, PLTP) were evaluated in 8-week-old mice with a non-synonymous single nucleotide mutation leading to an amino acid substitution in PCSK1, which results in a loss of protein’s function. Mutant mice had similar serum HDL cholesterol concentration but increased levels of serum total and mature APOA1, and LCAT activity in comparison to controls. Conclusions This study presents the first evaluation of the role of PCSK1 in HDL metabolism using a loss-of-function mutant mouse model. Further investigations will be needed to determine the underlying molecular mechanism. </jats:sec

Loss-of-function mutation in Pcsk1 increases serum APOA1 level and LCAT activity in mice.

BACKGROUND: The convertase subtilisin/kexin family 1 gene (PCSK1) has been associated in various human genetics studies with a wide spectrum of metabolic phenotypes, including early-onset obesity, hyperphagia, diabetes insipidus, and others. Despite the evident influence of PCSK1 on obesity and the known functions of other PCSKs in lipid metabolism, the role of PCSK1 specifically in lipid and cholesterol metabolism remains unclear. This study evaluated the effect of loss of PCSK1 function on high-density lipoprotein (HDL) metabolism in mice. RESULTS: HDL cholesterol, apolipoprotein A1 (APOA1) levels in serum and liver, and the activities of two enzymes (lecithin-cholesterol acyltransferase, LCAT and phospholipid transfer protein, PLTP) were evaluated in 8-week-old mice with a non-synonymous single nucleotide mutation leading to an amino acid substitution in PCSK1, which results in a loss of protein\u27s function. Mutant mice had similar serum HDL cholesterol concentration but increased levels of serum total and mature APOA1, and LCAT activity in comparison to controls. CONCLUSIONS: This study presents the first evaluation of the role of PCSK1 in HDL metabolism using a loss-of-function mutant mouse model. Further investigations will be needed to determine the underlying molecular mechanism

Abstract 498: Decreased High-Density Lipoprotein Cholesterol Levels and Macrophage Cholesterol Efflux Capacity in Mice Lacking Proprotein Convertase Subtilisin/Kexin Type 9

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low-density lipoprotein cholesterol catabolism and has been proposed as a promising therapeutic target for lowering low-density lipoprotein cholesterol levels in hypercholesterolemia patients. Several studies have shown that PCSK9 influences HDL cholesterol levels, however the molecular mechanism and potential consequence of targeting PCSK9 needs further study. Methods and Results: We isolated serum from Pcsk9 knockout (KO) mice to determine HDL cholesterol levels, HDL subunits and macrophage cholesterol efflux capacity. HDL cholesterol levels in Pcsk9 KO mice were significantly decreased in both sexes fed a normal chow and an atherogenic diet. HDL subunits were separated by gradient gel electrophoresis and HDL proteins were identified by mass spectrometry. Apolipoprotein E (APOE) was found in large HDL subunits in C57BL6/J (B6) mice, while it was absent in Pcsk9 KO mice. We found that Pcsk9 KO mice have decreased levels of serum APOE and increased LDLR in livers. We demonstrated that LDLR regulates serum APOE level by measuring this in mice containing gain- and loss-of-function in LDLR. APOE levels were decreased in Pcsk9 KO and human LDLR overexpressing transgenic mice, and increased in mouse containing non-functional LDLR. We tested the hypothesis that absence of APOE in HDL of Pcsk9 KO mice decreases macrophage cholesterol efflux capacity. Decreased fluorescently-labeled cholesterol efflux of Pcsk9 KO serum was demonstrated using both human THP-1 and mouse J774A.1 macrophage foam cells in vitro. We isolated hearts from Pcsk9 KO mice fed an atherogenic diet for 10 weeks until 34 weeks of age and determined atherosclerosis susceptibility by measuring atherosclerotic lesion size. The average volume of atherosclerotic lesions in the first 120 um of the ascending aorta was not significantly different between B6 and Pcsk9 KO mice. Conclusion: PCSK9 plays a critical role in regulating not only LDL, but also HDL in mice. The results demonstrate that increased LDLR level in Pcsk9 KO mouse liver likely decreases APOE in large HDL, which impairs macrophage cholesterol efflux capacity of Pcsk9 KO mouse serum. </jats:p

Multiplex quantitative imaging of human myocardial infarction by mass spectrometry-immunohistochemistry

Simultaneous assessment of a panel of protein markers is becoming essential in order to enhance biomarker research and improve diagnostics. Specifically, postmortem diagnostics of early myocardial ischemia in sudden cardiac death cases could benefit from a multiplex marker assessment in the same tissue section. Current analytical antibody-based techniques (immunohistochemistry and immunofluorescence) limit multiplex analysis usually to not more than three antibodies. In this study, mass spectrometry-immunohistochemistry (MS-IHC) was performed by combining laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) with rare-metal-isotope-tagged antibodies as a technique for multiplex analysis of human postmortem myocardial tissue samples. Tissue sections with myocardial infarction were simultaneously analyzed for seven primary, rare-metal-isotope-tagged antibodies (troponin T, myoglobin, fibronectin, C5b-9, unphosphorylated connexin 43, VEGF-B, and JunB). Comparison between the MS-IHC approach and chromogenic IHC showed similar patterns in ionic and optical images. In addition, absolute quantification was performed by MS-IHC, providing a proportional relationship between the signal intensity and the local marker concentration in tissue sections. These data demonstrated that LA-ICP-MS combined with rare-metal-isotope-tagged antibodies is an efficient strategy for simultaneous testing of multiple markers and allows not only visualization of molecules within the tissue but also quantification of the signal. Such imaging approach has a great potential in both diagnostics and pathology-related research

Pla2g12b and Hpn Are Genes Identified by Mouse ENU Mutagenesis That Affect HDL Cholesterol.

Despite considerable progress understanding genes that affect the HDL particle, its function, and cholesterol content, genes identified to date explain only a small percentage of the genetic variation. We used N-ethyl-N-nitrosourea mutagenesis in mice to discover novel genes that affect HDL cholesterol levels. Two mutant lines (Hlb218 and Hlb320) with low HDL cholesterol levels were established. Causal mutations in these lines were mapped using linkage analysis: for line Hlb218 within a 12 Mbp region on Chr 10; and for line Hlb320 within a 21 Mbp region on Chr 7. High-throughput sequencing of Hlb218 liver RNA identified a mutation in Pla2g12b. The transition of G to A leads to a cysteine to tyrosine change and most likely causes a loss of a disulfide bridge. Microarray analysis of Hlb320 liver RNA showed a 7-fold downregulation of Hpn; sequencing identified a mutation in the 3\u27 splice site of exon 8. Northern blot confirmed lower mRNA expression level in Hlb320 and did not show a difference in splicing, suggesting that the mutation only affects the splicing rate. In addition to affecting HDL cholesterol, the mutated genes also lead to reduction in serum non-HDL cholesterol and triglyceride levels. Despite low HDL cholesterol levels, the mice from both mutant lines show similar atherosclerotic lesion sizes compared to control mice. These new mutant mouse models are valuable tools to further study the role of these genes, their affect on HDL cholesterol levels, and metabolism

Pla2g12b and Hpn Are Genes Identified by Mouse ENU Mutagenesis That Affect HDL Cholesterol

Despite considerable progress understanding genes that affect the HDL particle, its function, and cholesterol content, genes identified to date explain only a small percentage of the genetic variation. We used N-ethyl-N-nitrosourea mutagenesis in mice to discover novel genes that affect HDL cholesterol levels. Two mutant lines (Hlb218 and Hlb320) with low HDL cholesterol levels were established. Causal mutations in these lines were mapped using linkage analysis: for line Hlb218 within a 12 Mbp region on Chr 10; and for line Hlb320 within a 21 Mbp region on Chr 7. High-throughput sequencing of Hlb218 liver RNA identified a mutation in Pla2g12b. The transition of G to A leads to a cysteine to tyrosine change and most likely causes a loss of a disulfide bridge. Microarray analysis of Hlb320 liver RNA showed a 7-fold downregulation of Hpn; sequencing identified a mutation in the 3′ splice site of exon 8. Northern blot confirmed lower mRNA expression level in Hlb320 and did not show a difference in splicing, suggesting that the mutation only affects the splicing rate. In addition to affecting HDL cholesterol, the mutated genes also lead to reduction in serum non-HDL cholesterol and triglyceride levels. Despite low HDL cholesterol levels, the mice from both mutant lines show similar atherosclerotic lesion sizes compared to control mice. These new mutant mouse models are valuable tools to further study the role of these genes, their affect on HDL cholesterol levels, and metabolism