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International Atherosclerosis
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January 2008

 


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IAS Visiting Fellowship Award - Final Report

Dr. Vivienne Homer - 2006 IAS Visiting Fellowship Award Winner
Laboratory of Biochemical Genetics and Metabolism, Rockefeller University
Laboratory of Dr. Jan Breslow, The Rockefeller University

By: Dr. Vivienne Homer
Canterbury Health Laboratories
Christchurch
New Zealand

Prior to receiving the International Atherosclerosis Society visiting fellowship, we had screened a group of 188 local patients (Christchurch, New Zealand) with clinical familial hypercholesterolemia (FH) for mutations in the LDLR and the APOB genes, using dHPLC, DNA sequencing and MLPA (multiplex ligation probe assay). From this group, 29 LDLR mutations were detected, many of which were novel. While 3 patients were found to have common FDB mutations. Based on clinical data, 57 of the remaining patients were screened for gain of function proprotein convertase subtilisin kexin type 9 (PCSK9) mutations. Fourteen South African patients with FH, where LDLR and APOB mutations had been excluded, were also analysed for PCSK9 mutations.

Sequencing of the PCSK9 gene revealed two novel mutations (D129G and A168E) in two New Zealand patients, while the previously reported mutations S127R and R237W were detected in two South African patients. All four patients were heterozygous for their respective missense mutations and these mutations were not found in any of the other FH patients screened.

After identifying these PCSK9 mutants the aim was to determine the functional significance of the A168E and D129G mutations on PCSK9 and LDLR function and investigate more general aspects of the role of PCSK9 in hyperlipidemia and cellular lipoprotein metabolism.

From September 26th –December 23rd 2006, I visited Dr. Breslow and colleagues at the Laboratory of Biochemical Genetics and Metabolism, at The Rockefeller University, New York, USA. Dr. Breslow’s laboratory has been at the forefront of studies on PCSK9, identifying the gene in 2003 and subsequently showing that it can induce hypercholesterolemia by degrading the LDL receptor in the hepatocyte secretory pathway (Maxwell and Breslow, 2004) (Maxwell et al, 2005). Dr. Breslow’s research group also investigates other complex hereditary and environmental factors involved in the development of atherosclerosis, including the role of macrophage cholesterol transporter StARD5 and the cholesterol regulated liver gene ADAM11. Therefore, it was of great interest and relevance to visit Dr. Breslow’s laboratory to expand my skill base and further my current research.

The original objectives of my research at the Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, were to:

1. Investigate the effect of the identified PCSK9 mutants on protein function.
2. Determine further details of the PCSK9 synthesis and functional pathways.

This work was to be undertaken with the help of another post-doctoral fellow that was to be employed at the Breslow Laboratory. Due to unforeseen circumstances that were out of my control, two months before leaving for New York I was informed that I would be unable to carry out the planned research. Instead, the focus of my visit was changed to fit in with projects that were currently being undertaken in the laboratory. Nevertheless, the proposed work was at the forefront of current research and involved new techniques that I had no experience of.

Dr. Breslow and his colleagues are currently conducting a genome wide association study on a population from a small island in Micronesia. This population has a high incidence of heart disease, obesity and diabetes, and is a good population to study,due to their longer regions of linkage disequilibrium and decreased allelic diversity (Bonnen et al, 2006). Thousands of SNPs have been genotyped on 100 K affymetrix arrays and later 500 K arrays. The data is currently being mined for associations to various traits. Currently approximately 15-30% of people with classical FH are negative for mutations in the LDLR, PCSK9 and APOB genes, which are associated with FH. This suggests that new genes involved in this disease process have yet to be discovered and that hypercholesterolemia may be caused by the interaction of multiple loci. Whole genome association studies have the ability to yield important insights into the unknown genes involved in FH, and their interactions.

During my time in the Breslow laboratory, I learnt about single nucleotide polymorphism (SNP) microarrays, which ones were the best to use, problems associated with them and quality control. I learnt about the different ways to design genome association studies, but most importantly how to analyze the data and mine for significant genetic associations. Various software programs have been used in previous genome association studies and the Breslow laboratory was comparing the different types in order to determine the most sensitive and specific.

I gained experience in using HaploView, Plink, and Family Based Association Test (FBAT) software. I spent some of my time looking for correlations between known restriction fragment length polymorphisms and cholesterol levels in the study population using an ABI DNA sequencer and gene mapper software. Unfortunately no correlations were determined. I also resequenced the PCSK9 gene of individuals from the study population with cholesterol levels in the 5th percentile in an effort to detect novel mutants that lower LDL-cholesterol. The genome association study knowledge that I have gained is invaluable for the planning and eventual implementation of a similar type of study to be conducted in Christchurch.

One of the benefits of being at Rockefeller University was the access to the large number of meetings/presentations, which were of extremely high quality. In particular I enjoyed the presentations at The New York Lipid and Vascular Biology Research Club meeting, where they talked on the topic of Macrophages and Atherosclerosis, and a presentation by Dr. Jurg Ott on computational biology, at an Advanced Linkage workshop. The CME Genome-Wide Association Studies: Design and Analysis meeting, held at Yale, was also brilliant. There I learnt about the latest methods for designing and analyzing genome-wide studies. The presentations by experts in the field of association studies, Dr. Abecasis and Dr. Boehnke were particularly informative. I also attended the American Heart Association meeting, held in Chicago, where the highlights for me were presentations by Dr. Philippe Costet, who spoke on PSCK9 repression by fenofibric acid and a presentation by Dr. Gustav Schonfeld, who spoke on Familial Hypobetalipoproteinemia and fatty liver.

This visiting fellowship was a major eye-opener for me. I was able to see how large laboratories function, what makes them successful, and experience scientific research on a different level. I learnt the basics of how to conduct a whole genome association study and can apply these skills to similar studies that will be performed on our Christchurch diabetic and dyslipidemia populations.

As my original objectives could not be achieved during my visit to New York, we collaborated with Dr. Gilles Lambert and his colleagues at the Heart Research Institute in Sydney, Australia. The mutant PCSK9 expression studies outlined in objective 1 were performed. Subsequently, a manuscript on this work has been published in Atherosclerosis (Homer et al, 2006). These protein expression methods will be set-up in our laboratory in Christchurch and used in future analyses.

I would like to extend my thanks and appreciation to the International Atherosclerosis Society for supporting my travel to The Rockefeller University. I would also like to thank Dr. Breslow and colleagues for allowing me to visit their laboratory.

References:

Maxwell, K. N. and Breslow, J. L.
Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype. Proc.Natl.Acad.Sci.U.S.A 101.18 (2004): 7100-05.

Maxwell, K. N., Fisher, E. A., and Breslow, J. L.
Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment. Proc.Natl.Acad.Sci.U.S.A 102.6 (2005): 2069-74.

Bonnen, P.E.; Pe'er, I.; Plenge, R.M.; Salit, J.; Lowe, J.K.; Shapero, M.H.; Lifton, R.P.; Breslow, J.L.; Daly, M.J.; Reich, D.E.; Jones, K.W.; Stoffel, M.; Altshuler, D.; Friedman, J.M.
Evaluating potential for whole-genome studies in Kosrae, an isolated population in Micronesia. Nat.Genet. 38.2 (2006): 214-17.

Homer, V. M.; Marais, A.D.; Charlton, F.; Laurie, A.D.; Hurndell, N.; Scott, R.; Mangili, F.; Sullivan, D.R.; Barter, P.J.; Rye, K.A.; George, P.M.; Lambert, G.
Identification and characterization of two non-secreted PCSK9 mutants associated with familial hypercholesterolemia in cohorts from New Zealand and South Africa. Atherosclerosis (2007).