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Scientists at The Scripps Research Institute Develop New Technology to Map Spread of Malarial Drug Resistance

La Jolla, CA. October 1, 2002 - Scientists at The Scripps Research Institute (TSRI), Harvard University and the Genomics Institute of the Novartis Research Foundation have found a way to use a relatively new but readily available technology to quickly detect markers in the DNA of the most deadly type of malaria pathogen.

The technology could enable scientists and public health workers to identify the particular strain of malaria during an outbreak and determine if it is drug resistant or not.

"One of the reasons for the resurgence of malaria in Africa and in other parts of the world is the spread of drug resistance," says Assistant Professor Elizabeth Winzeler, Ph.D., who is in the Department of Cell Biology at TSRI and the lead author of the study described in the latest issue of the journal Science.

The work should make it easier to follow the spread of drug resistance around the world and assist health ministries in countries where malaria is a problem to come up with strategies to thwart this spread.

Malaria is a nasty and often fatal disease, which may lead to kidney failure, seizures, permanent neurological damage, coma, and death. There are four types of Plasmodium parasites that cause the disease, of which falciparum is the most deadly.

Despite a century of effort to globally control malaria, the disease remains endemic in many parts of the world. With some 40 percent of the world's population living in these areas, the need for more effective vaccines is profound. Worse, strains of Plasmodium falciparum resistant to drugs used to treat malaria have evolved over the last few decades.

The specter of drug resistance is particularly worrisome because drug resistance can spread through the mating of Plasmodium parasites. And drug-resistant Plasmodium falciparum is more deadly and more expensive to treat. Worse, a drug-resistant strain could lead to the re-emergence of malaria in parts of the world where it no longer exists - except for the occasional imported case - such as the United States.

One of the best tools for fighting any infectious disease is to track it and fight it where it occurs. And one of the best ways to determine the origin of a particular malaria infection and to map the spread of infection is to identify what are called single nucleotide polymorphisms (SNPs).

Polymorphisms, the genetic variability among various isolates of one organism, are responsible for drug resistance in malaria pathogens. In order to follow the spread of drug resistance around the world, one needs to look at how these markers spread as well.

In the past, if scientists wanted to detect SNPs, they would pick one particular gene and sequence it, a time- consuming process. For instance, finding enough polymorphisms to map the gene mutation responsible for resistance to the drug chloroquine, one of the traditional drugs given to patients with malaria, took several years and millions of dollars to determine.

"Now," says Winzeler, "we have demonstrated that you can detect thousands of SNPs all at the same time by doing a simple reaction."

The reaction involves taking DNA from the malaria parasite, chopping it into fragments, and plopping the mixture of fragmented DNA on a "gene chip" - a glass or silicon wafer that has thousands of short pieces of DNA attached to it.

DNA chips have become a standard tool for genomics research in the last couple of years, and scientists can quite easily put a large number of different oligonucleotide pieces - even all the known genes in an organism - on a single chip. When applying a sample that contains DNA to the chip, genes that are present in the sample will "hybridize" or bind to complementary oligonucleotides on the chip. By looking to see which chip oligonucleotides have DNA bound, scientists know which genes were being expressed in the sample.

But Winzeler used this technology in a novel way. She compared the DNA of Plasmodium falciparum parasites that were resistant to drugs to those that were not and used the differences in the readouts of the gene chips to determine where the SNPs were. Nobody had ever used a gene chip in this way before.

Nor did such a chip exist. Winzeler worked with researchers at the Genomics Institute of the Novartis Research Foundation to create one just for this purpose.

Using putative malaria genes that were identified in the international malaria genome effort, Winzeler took sequences representing 4,000 distinct pieces of these genes on chromosome 2 of the Plasmodium falciparum genome and had a gene chip constructed.

"Having this type of technology and the genome sequenced allows us to look at the genome in a whole new way," says Winzeler. "If you start doing longitudinal studies after you introduce a new drug, you might be able to identify the drug targets or the mechanisms of resistance. If you can start finding the mutations that are associated with drug resistance, then that tells you how to treat patients in the field."

The new technology should also make it possible to do similar research with other organisms, characterizing genetic variability and perhaps conducting population genetics as well. With population genetics, scientists could quickly determine how similar different genomes are to each other and generate estimates of a pathogen's age or its pattern of spread.

Winzeler found that most of the SNPs were in the DNA of genes that coded for membrane-associated proteins, which is to be expected, since these are the proteins that are on the outer surface of the cell and will endure the greatest selective pressure exerted by host immune systems and drugs.

Significantly, she also found that a number of genes of unknown function were also high in SNPs, which could mean that these unknown genes are also under selective pressure.

"These could represent genes that have important functions in parasite viability or virulence and that warrant further functional characterization," she concludes.

The article, "Excess Polymorphisms in Genes for Membrane Proteins in Plasmodium falciparum" was authored by Sarah K. Volkman, Daniel L. Hartl, Dyann F. Wirth, Kaare M. Nielsen, Mehee Choi, Serge Batalov, Yingyao Zhou, David Plouffe, Karine Le Roch, Ruben Abagyan, and Elizabeth A. Winzeler and appears in the October 4, 2002 issue of the journal Science.

This work was supported by the National Institutes of Health; the Burroughs Wellcome Fund New Initiatives in Malaria Research Award; the Ellison Medical Foundation, Program in Career Development, Research and Training in Global Infectious Diseases; the ExxonMobil Program on Malaria in Africa; and a new scholars award from the Ellison Medical Foundation.

The authors of the paper are affiliated with the following institutionsHarvard University; University of Tromso, Norway; the Genomics Institute of the Novartis Research Foundation; and The Scripps Research Institute.

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