Specific Aim 1: To define the spectrum of DNA sequence
variation in genes controlling lipid metabolism.
In this Project we will provide a comprehensive assessment of
sequence variation in genes participating in the critical metabolic
pathways involved in lipid homeostasis. In the previous cycle
of this Program, we found that individuals with a wide range of
extreme phenotypes, for example very low plasma levels of high
density lipoproteins (HDL) were much more likely to have functional
mutations in candidate genes for those traits than were healthy
individuals. Numerous deleterious mutations were identified in
candidate genes for low plasma levels of HDL. Each mutation was
found in only a small number of subjects but collectively the
number of deleterious mutations was greater in those with low
plasma levels of HDL compared with those with either normal or
high plasma levels of HDL. Thus, we are going to continue to test
the hypothesis that rare sequence variations with large effects
contribute importantly to the variability in the population of
some complex traits. To test this hypothesis we will resequence
the coding sequences and evolutionarily conserved noncoding regions
in 96 individuals with very high (>95th percentile) or low
lipoprotein levels (<5th percentile) from a large, multiethnic
population based sample. Each gene will be resequenced in equal
numbers of African-American and White men and women. This strategy
will reveal both common and rare sequence variants and will provide
an initial indication of which variants are associated with a
specific phenotype.
Specific Aim 2: To define the spectrum of DNA sequence
variation in noncoding sequences that have been highly conserved
through vertebrate evolution.
Comparison of the completed genome sequences from human, mouse,
and pufferfish revealed approximately 2000 noncoding sequence
elements that are highly conserved among these species. The strong
conservation of these regions suggests the sequences contained
in these regions play important roles in the regulation of gene
expression, which is supported by the studies performed by the
Berkeley PGA. We plan to screen these highly conserved noncoding
sequences for sequence variation. All noncoding regions that are
highly conserved between human, mouse, and pufferfish will be
sequenced in 32 individuals with the phenotypes of interest (16
African-Americans and 16 Whites).
Specific Aim 3: To assess the functional sequelae of polymorphisms
and mutations that alter conserved nucleotides in transcription
factors controlling cardiac development and lipid metabolism.
The
functional significance of the sequence variations identified
in Specific Aims 1 and 2 will be determined by testing for association
between the sequence variations and phenotypes in a large number
(n=3500) of ethnically diverse, densely phenotyped participants
from the recently completed Dallas Heart Study. For those polymophisms
that reveal convincing phenotypic associations, we will perform
studies in cells and in genetically-modified animals to determine
the functional effects of variability in the level of expression
or the sequence of the implicated gene. These studies will be
performed in collaboration with investigators in other PGAs with
expertise pertinent to the genes implicated in these studies.
