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The nature of DNA differences

New ROMA technique enables researchers to scan the entire human genome; reveals genetic copy-number differences can be found even among healthy individuals

Conventional wisdom holds that people are more alike than they are different. But a recent study has found that individuals may differ significantly when it comes to how many copies of some of their genes they possess.

The study, led by researchers at Cold Spring Harbor Laboratory and involving Dr. Barbara Trask's lab in the Human Biology Division, used a powerful new DNA-profiling technique originally developed to find differences in the number of copies of genes between healthy cells and cancer cells. Unexpectedly, this method now has revealed several major and previously unknown differences of this kind in DNA samples obtained from 20 unrelated "normal" individuals.

Experts believe that the improved understanding of normal variation in gene copy number afforded by this technique will make it easier to distinguish the DNA differences responsible for the development of cancer and other diseases.

"For example, if we are looking for genome differences between people who have prostate cancer and those who don't, it will now be easier to tell whether those differences are truly cancer-specific," said Dr. Janet Young, a staff scientist in the Trask lab and a co-author of the study.

Trask lab part of team

The results appear in the July 23 issue of Science. Other co-authors at the center include Trask, director of the Human Biology Division, and her research technicians Hillary Massa and Megan Walker. Additional collaborators included colleagues from the Broad Institute in Cambridge, Mass., the Karolinska Institute in Stockholm, Sweden, and Stony Brook University in New York.

Although scientists have long known that all individuals except identical twins differ somewhat in their DNA, a map of variation in gene copy number among healthy individuals has never before been created, Young said.

"Human DNA copy-number variation has previously been studied mainly by looking at individual genes," she said. "The new technique now makes it possible to scan the entire genome to look for this kind of large-scale variation."

The human genome — the complete set of DNA instructions for development and life — consists of 23 pairs of chromosomes that contain a total of roughly 30,000 genes, each of which is a blueprint to make a specific protein. Although all individuals possess nearly the same set of genes, each gene can have multiple forms that differ somewhat in DNA sequence. Until now, scientists have mostly focused on identifying the different gene forms, some of which are linked to disease susceptibility or to other observable traits.

ROMA technique

The new technique, called ROMA (representational oligonucleotide microarray analysis), allows scientists to view a broader landscape of the genome, focusing on large chunks of chromosomes rather than individual genes. Using this method, researchers at Cold Spring Harbor Laboratory last year published the discovery of chromosomal amplifications (excess copies of DNA segments) and deletions (missing DNA segments) associated with a variety of human cancers in individual patients.

Although the basic principles of heredity state that each person inherits two copies of each gene — one from their mother and one from their father — some genes become duplicated so that multiple copies of a gene can exist in tandem on a chromosome or on several different chromosomes. During the course of their earlier work, the researchers detected several gene copy-number differences in human DNA when they carried out "normal to normal" comparisons of DNA from different individuals. Only a handful of such differences in gene copy number among healthy individuals — including some examples studied in the Trask lab that involve genes located near the tips of chromosomes — were already known.

In the new study, the scientists created an extensive map of this type of genetic variation in normal human DNA. The researchers sampled blood and multiple tissues from 20 individuals from a variety of geographic backgrounds. Differences in the chromosomal DNA purified from these samples were detected by ROMA.

The researchers identified 76 places in the genome in which they detected gross differences among individuals in terms of the number of copies of a particular gene or chunk of a chromosome. The investigators found that there was an average of 11 sites in the genome where gene copy number differed between any two individuals. These differences are called copy-number polymorphisms (CNPs). To confirm the differences by a second method, the Trask lab and the group in Stockholm used a technique called FISH (fluorescent in situ hybridization) that makes it possible to light up parts of the chromosome to visualize the number of copies of a particular gene.

"Some of these differences might have no effects on a person's health or outward characteristics and some might have a lot of effect," Young said. "It depends on which genes are within or near these regions."

"Another important concept to take away from this work is that there is no simple way to define'normal.'"

Investigating disease links

Among the genes associated with the newly-identified CNPs that may have implications for health were those involved in Cohen syndrome, an inherited disease that can cause some physical abnormalities and learning disabilities, and others implicated in neurological development, leukemia and drug-resistant forms of breast cancer. In addition, some CNPs identified genes with known influence on "normal" human traits including one — neuropeptide-Y4 receptor — that is directly involved in the regulation of food intake and body weight.

Future work will be aimed at determining whether there is a relationship between CNPs and susceptibility to health problems such as neurological disease, cancer and obesity.

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