V Strumba (1),T Blackwell (1), E Sliwerska (1), J Li (2), AJ Schatzberg (2), EG Jones (3), WE Bunney
(4), RM Myers (2), H Akil (1) SJ Watson (1), M Burmeister (1) R Bernard (1)
(1) University of Michigan
(2) Stanford University
(3) UC Davis
(4) UC Irvine
Effect of functional variants on gene expression in human brain.
Short abstract (600 character limit):
Many polymorphisms have been found associated with behavior or psychiatric disorders. However, the
mechanism of how genetic variants lead to phenotypic differences is usually not known. Towards this
goal, we test for association between functional variants in candidate genes and expression levels of
thousands of genes. Specifically, we evaluated variants in the SLC6A4 (5-HTT), COMT and MAOA
genes and expression levels measured by Affymetrix microarrays performed on mRNAs isolated from
postmortem brains. Genes identified as changed are then analyzed for the biological pathways involved.
Long abstract (6000 character limit):
Since the completion of the Human Genome Project, major efforts such as HapMap project now
concentrate on identifying and cataloging human polymorphisms such as SNPs and deletions. Even if an
association between a phenotype and a SNP is found, the mechanism of how these variations change the
phenotype remains a mystery. Here, we address this question by searching for a downstream molecular
phenotype of common genetic variants that have been shown to be functional (either affecting mRNA
levels or protein function). We chose a number of polymorphisms that have been previously found
associated with behavior or psychiatric disorders, and tested for association of these genetic variants with
expression levels of thousands of genes in human postmortem brain samples, as measured on Affymetrix
expression microarrays. Brain samples were from subjects that died relatively suddenly (i.e. did not spend
extended time in “agonal” state in the hospital), and were either controls without any psychiatric illness,
or had a diagnosis of depression, bipolar disorder or schizophrenia. mRNA was isolated from eight brain
regions that were dissected either anatomically or by laser capture microscoscopy.
We used linear regression as the principal statistical tool to identify an association. We
incorporated technical (e.g. batch effects) as well as demographic co-factors (e.g. age) in the analysis
when appropriate. Three genetic variants were the focus of the study reported here. The first is 5HTTLPR, the serotonin or 5-hydroxytryptamine transporter (5-HTT) length polymorphic repeat. 5-HTT is
of particular interest because it is one of the primary targets for various antidepressants, such as tricyclics
and selective serotonin re-uptake inhibitors (SSRIs). The gene encoding this transporter, SLC6A4,
contains a common (allele frequencies of 0.45/0.55) functional length polymorphism in its promoter,
which has long(l) and short(s) alleles. The s allele is associated with higher levels of anxiety (Lesch et al
1996; Schinka et al 2004; Sen et al 2004) and in interaction with significant life events leads to increased
risk of depression (Caspi et al 2003). It was previously found to result in lower 5-HTT expression than the
l allele when studied in tissue culture cells or in transformed blood cell lines (Lesch et al 1996). We
postulated that production of lower amount of this neurotransmitter transporter would have significant
effects on other genes downstream of 5-HTT. Towards this aim, we first tested whether the previously
reported association with mRNA expression of the target mRNA is also true in brain. While not
significantly different in many brain regions, where the expression level is relatively low, we found that
indeed individuals with one or two s alleles (s/s or s/l) had significantly (p=0.028, n=27 samples) lower
expression levels than homozygous l/l individudals in the raphe region of the brain, the region where most
serotonergic neurons originate. Comparing s/s and s/l with l/l individuals in this brain region, we
identified a list of >100 genes whose expression level appeared significantly affected by the 5-HTTLPR
genotype. Functional analysis of this list of genes may help shed light on the exact mechanism of 5-HTT
action.
The second variant of interest is an exonic common (0.6/0.4) variant, Val108/158Met, in the
catechol-O-methyl-transferase (COMT) gene. The Met allele is known to result in lower enzymatic
activity (Lachman et al 1996). This variation has been found in an often replicated study to be associated
with cognitive processing ability (Egan et al 2001). We postulated that change in enzymatic activity
would also have downstream effects on the expression of other genes, but did not find any statistically
significant changes in our data so far. The third variant to be tested is a functional promoter variation in
the X-linked gene for the mono-amine-oxidase A, MAO-A, which has been shown to affect mRNA levels
in tissue culture (Sabol et al 1998; Samochowiec et al 1999), and has been found associated with risk for
conduct disorder and impulsive violent behavior when considered in conjunction with early childhood
treatment (Caspi et al 2002).
Since the effect of genotype downstream on expression levels of genes is relatively small, it turned
out to be important to carefully match or control for other factors. These included technical factors such as
batch of chips or hybridizations, as well as demographic factors (e.g. age). In addition, we carefully
excluded subjects who were under prolonged agony before death, since this has previously been shown by
our group to affect expression of many genes (Li et al 2004).
Conflict of interest statement:
The authors are members of the Pritzker Neuropsychiatric Disorders Research Consortium, which
is supported by the Pritzker Neuropsychiatric Disorders Research Fund L.L.C. A shared intellectual
property agreement exists between this philanthropic fund and the University of Michigan, Stanford
University, the Weill Medical College of Cornell University, the Universities of California at Davis, and
at Irvine, to encourage the development of appropriate findings for research and clinical applications.
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