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
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
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.
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
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.
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.
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.
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.
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.