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The Contemplative Process of Identifying Predisposing Loci and Genes

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 phenotypes—in 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 lupus—a 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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Chromosomal Regions Linked to Autoimmune Diseases. Click to enlarge.















"You want to get at the root cause of the disease,but there's no particular factor that's so overwhelmingly contributory that you can eliminate all the others."

—Dwight Kono