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