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The Contemplative Process of Identifying Predisposing Loci
In order to identify the genetic components of any disease
when there are no obvious candidates, one must identify the
chromosomal regions containing the genes and then screen virtually
all the genes in the region for their involvement. This long
and complicated process is known as positional cloning.
The process starts with one of the murine lupus models that
Theofilopoulos has characterized. These mice develop lupus-like
symptoms spontaneously, in much the same way as the disease
is manifested in afflicted people. One of these lupus strains
is crossed with a nonautoimmune strain and then those offspring
are interbred to generate "F2" offspring that contain diverse
random combinations of genetic material from the original
parents. The general chromosomal locations of predisposing
genes can be then deduced by identifying, in a large number
of afflicted F2 models, what chromosomal regions are inherited
from the lupus-prone strain with a greater frequency than
chance alone. These intervals or loci are generally resolved
to around 20 to 40 cMorgans or about 40 to 80 million base
pairs, and can contain hundreds of genes, a size generally
too large to screen for specific genetic alterations.
To narrow the interval, a new model, called a congenic,
is developed that consists of one background strain containing
only the locus region from the other strain. This permits
direct testing of the effect of a single locus on the development
of lupus, either through the replacement of the predisposing
locus from the lupus-prone strain with the normal counterpart,
or by the addition of the predisposing interval to the normal
strain. Breeding of this initial congenic is a time-consuming
process requiring backcrossing of the interval for several
generations, on average requiring two years or more. Further
narrowing of the region down to about a million base pairs
can then be accomplished by generating additional congenics
containing smaller and smaller intervals.
"Once you accomplish that, then you can mine the data from
the genome projects and start to make intelligent conclusions
about genes that are known," says Theofilopoulos. A significant
effort, however, is still needed to screen candidate genes,
a process that involves extensive cloning, sequencing and
analysis of gene expression.
A general problem, however, with positional cloning of genes
involved in lupus is that the disease is made up of many phenotypesin
fact, lupus is diagnosed clinically when a patient has four
or more of 11 possible defining conditions. Mathematically,
that gives several thousand possible combinations of symptoms
that can be defined as lupus, although, practically, the number
of common clinical scenarios are much fewer.
However, there are only three model strains that develop
three specific types of lupus, which is more like having three
individuals with the disease. The three strains are not a
perfect representation of all the different manifestations
of the disease in the 1.4 million people believed to be afflicted
with lupus in the United States.
"Because of the diversity of the disease in the human population,"
says Theofilopoulos, "it is appropriate to focus our initial
studies on these models."
Another complication is the fact that lupus involves an
array of interacting genes. So identifying one gene may give
you some of the answers, but certainly not all.
"You want to get at the root cause of the disease," says
Kono. "But there's no particular factor that's so overwhelmingly
contributory that you can eliminate all the others."
The two remain positive that, through this approach and
concurrent studies in humans, at least some of the predisposing
genes will be identified.
Another approach they are taking to find therapeutic targets
is to identify genes that may be normal and unmutated, but
which encode products that may be involved in the disease
pathogenesis. These genes can be identified by deleting them
in the lupus models.
"We have already selected genes that we believe will have
an important effect on the disease process," says Theofilopoulos.
For instance, they have identified one possible target for
therapy, an inhibitor of cyclin-dependent kinases that is
overexpressed in T cells during lupus that may be responsible
for one symptom of advanced lupusa flood of helper T
cells that are resistant to proliferation and apoptosis. Targeting
this inhibitor and promoting cell proliferation might make
severe lupus mild.
By deleting the genes encoding this inhibitor, Theofilopoulos
and Kono have prevented the helper T cells from accumulating
and reduced all the serologic and histologic characteristics
of the disease.
Another class of genes that have been found to contribute
to the disease are the Type 1 and Type 2 interferons, pro-inflammatory
molecules referred to as IFN-a
and IFN-g respectively. Based
on these findings, Theofilopoulos and Kono have reported that
using naked cDNA encoding the receptor for IFN-g
could block the activity of the IFN-g
and cure the lupus in these models.
Developing recombinant receptors for IFN-a
and IFN-g should provide better
means to reduce the severity of the disease.
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