Research Report 2000 - MDC

Genome Research,
Gene Variation, and
Complex Disease
Margret Hoehe
The systematic analysis of DNA
sequence variation in biomedically
relevant genes is the key to a) the
identification of genetic risk factors in
common, complex diseases (‘Medical
Genomics’), and b) the identification
of genetic variation involved in
individually different drug responses
(‘Pharmacogenomics’). To this end,
the Genome Research Group has a)
developed highly efficient
technologies, which allow sequence
comparisons of candidate genes in
large numbers of individuals in the
megabase range (e.g. ‘Multiplex
Sequence Comparison’); b) applied
these technologies to variation
analyses of candidate genes defined
by biology and/or genetic mapping; c)
identified numerous variants in genes
potentially involved in hypertension,
substance dependence, and other
common, complex diseases; d)
predicted numerous individually
different forms of the genes by means
of haplotype analyses; e) developed
bioinformatic approaches to haplotype
classification into functionally related
groups; f) identified variants, or
combinations of variants (pattern),
associated with complex disease.
Thus, combined approaches have been
developed to establish complex
genotype-phenotype-relationships
against a background of high natural
genome sequence variability. This
line of research, development and
production has been supported by the
German Human Genome Project. It
has prepared the background for
functionally characterizing genetic
variations conferring risk of disease; a
‘Functional Genomics’ research
project is presently being established
in this group.
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Gene sequence diversity,
haplotypes, and genotypephenotype-relationships
We have applied the approaches
described above to test the potential
involvement of the human mu opioid
receptor gene (OPRM1) in substance
dependence. All functionally relevant
regions of this candidate gene,
including 6.7 kb regulatory, exonic
and critical intronic sequences, were
analysed by ‘Multiplex Sequence
Comparison’ in 250 subjects and
controls. A total of 43 variants were
identified, and 52 different haplotypes
predicted in the subgroup of 172
African-Americans. These haplotypes
were classified by hierarchical cluster
analysis into two functionally related
categories, one of which was
significantly more frequent in
substance-dependent individuals.
Common to this category was a
characteristic pattern of sequence
variants, which was associated with
several forms of substance
dependence (opioid and cocaine
dependence). This study provides the
first example of the possibility of
establishing genotype-phenotyperelationships
in a situation of
abundant gene sequence variation.
Moreover, to our knowledge, this
work represents the largest body of
sequence data so far on multiple
individuals for the same gene
(manuscripts in review). A large
sample including 250 Israeli
substance-dependent individuals and
controls has also been analysed, and a
global survey has been performed.
Systematic comparative sequence
analysis of the human beta2
adrenergic receptor gene, including its
known regulatory and coding regions
in more than 400 individuals, resulted
in a total of 15 identified variants,
several of which were functionally
significant. An additional 700
individuals were genotyped and these
included hypertensive patients,
individuals characterized by saltsensitivity/resistance,
beta2 receptor
binding, vasodilator response, and a
series of other cardiovascular
parameters including responsiveness
to various forms of experimentally
induced mental and physical stress, as
well as obese patients. Three major
haplotypes of the beta2 adrenergic
receptor gene were identified, and
observed in 80-95% of all subjects
from several independent studies.
Evidence of a genetic risk profile for
essential hypertension has been
obtained. Generally, evidence of the
involvement of beta2 variation in
increased blood pressure, in vivo
vasodilator response to beta2 agonists,
catecholamines, and heart size was
obtained. Beta adrenergic receptor
gene haplotypes are being expressed
and functionally characterized. An
additional technological development
has led to the first application of
MALDI-TOF mass spec for beta2
genotyping.
Systematic analysis of genetic
variation in three chemokine receptor
genes (BLR1, BLR2, and the Fusin
gene) in more than 200 patients
suffering from tumors such as acute
leukemia, Hodgkin- and non-Hodgkin
lymphomas, and controls, again
resulted in numerous variations .
Variants that cause an exchange of
conserved amino acids have been
identified, and are now being
expressed and functionally
characterized. Additional genes
studied include the cannabinoid
receptor gene, the TRHR gene, the
beta1 adrenergic receptor gene, and
the beta myosine heavy chain gene.
These projects have been carried out
in close collaboration with the Max
Planck Institute for Molecular
Genetics (Berlin), Department of
Genetics, Harvard Medical School
(Boston), Department of Genetics,
Yale University (New Haven), Franz
Volhard Clinic and Robert Rössle
Clinic at the MDC, Free University
(Berlin), University of Graz, INSERM
(Paris and Strasbourg), Karolinska
Institute (Stockholm), and
Pennsylvania State University
(Philadelphia).