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Snapshots of Genomic Evolution
Microarrays reveal an ancient pathogen adapting to life in San Francisco
Edward R. Winstead

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A resurgence of tuberculosis in San Francisco in the late 1980s led researchers to create public health registries documenting the geography and circumstances of new cases. During the 1990s, meticulous record keeping and the DNA fingerprinting of hundreds of bacterial strains helped the city monitor and reduce the incidence of disease. Now, researchers are using genomic tools to ask old questions about tuberculosis in new ways.

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In one of the first such studies, a team from Stanford University School of Medicine used DNA microarrays to compare strains of M. tuberculosis based on the content of their genomes. Peter M. Small, of the Division of Infectious Diseases and Geographic Medicine, and colleagues detected DNA deletions in the genomes of a dozen strains, demonstrating that the pathogen is losing genes. The patterns of deletions were virtually identical in bacteria belonging to the same strain; the patterns of deletions differed between strains.

"The strains were selected because they had well-defined and interesting epidemiologic data, and the disease they caused was well characterized," says Small. "We hope ultimately to link several thousand well-characterized isolates to the genomic data, which should allow us to identify associations between specific genes and specific manifestations of disease."

Different strains of M. tuberculosis behave very differently in humans. The severity of the disease and the risk for transmitting an infection vary widely depending on the pathogen. No one knows why some infected individuals remain free of symptoms for a lifetime while others go on to die. Most studies on this subject focus on environmental rather than genetic factors.

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The Stanford team hypothesized that the content of bacterial genomes influences clinical outcomes. "This study was an opportunity to see if there are genomic differences that can help explain the spectrum of clinical disease," says Midori Kato-Maeda, who led the research. "And even with a small number of strains we were able to find genomic differences that influence the risk for pulmonary cavitation, which is a clinical phenotype."

As the amount of genomic deletion increased, the likelihood that a bacterium would cause pulmonary cavitation, a complication of tuberculosis, decreased. "We propose that the accumulation of deletions among clinical isolates generally diminish their virulence," the researchers write in a paper describing the study in Genome Research.

"Not every tuberculosis infection is the same, and it remains to be seen whether the most severe strains have characteristic deletion patterns," says Thomas R. Gingeras, of Affymetrix Inc., in Santa Clara, California, who executed the array aspects of the study.

An analysis of about 100 clinical strains is underway in Small’s laboratory. "Our dream is to be able to take a sputum sample from a patient and tell them if they have a strain that is a particularly nasty bug and should be prioritized for treatment and disease control," says Small.

Circular map of genomic deletions among M. tuberculosis. View larger

During the 1990s, Stanford researchers 'fingerprinted' two thousand isolates identified in San Francisco and parts of Mexico. By the end of the decade, genomics had emerged and DNA fingerprinting was no longer the only molecular tool for monitoring tuberculosis.

"It was really the combination of the sequencing of M. tuberculosis and the development of microarrays that allowed us to switch to a high-throughput system for characterizing genomes," says Small. The Sanger Centre in Great Britain sequenced the H37Rv strain of M. tuberculosis in 1998. Today a second sequenced strain is available from The Institute for Genomic Research (TIGR) in Rockville, Maryland.

Two years ago, Small led a study that compared the genomes of various anti-tuberculosis vaccines using DNA microarrays developed at Stanford. They found a high degree of variability—including genomic deletions—among the attenuated strains, which for decades had propagated in vitro. This was evidence, they said, for the ongoing evolution of the vaccine strains since their original derivation.

The loss of bacterial genes is evolution plain and simple, says Gingeras. "What's new is that we're monitoring it."

Gingeras built the microarray used in this study a few years ago. "I designed the array to interrogate gene expression profiles in M. tuberculosis, but it was amenable to detecting deletions," he says. "I had a method but needed a biological question, and Peter [Small] had that."

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The M. tuberculosis gene array is a glass chip arrayed with oligonucleotide sequences obtained from the H37Rv strain. About a quarter of the genome is interrogated by the oligonucleotide sequences synthesized on specific sites on the array. There are about 200,000 synthesis sites on the chip. Labeled DNA binds to synthesis sites with different levels of intensity depending on how closely the sequences match up.

"A perfect match should be the brightest, the most fluorescent," explains Gingeras. "The intensity of these matches is what we monitor as we walk along the genome. Deletions are seen as blanks on the probe set and that's how they're identified and mapped."

The hybridization experiments generated data ('intensity values') for each of the 200,000 synthesis sites. Hidden in this enormous data set were the genomic deletions. To find them, the researchers ran novel software designed to detect deletions while screening out false positives.

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"The signal for over 100,000 probes is quite noisy," says Hugh Salamon, the Stanford postdoctoral fellow who wrote the software. His algorithm was capable of detecting deletions several hundred base pairs long or longer. The shorter deletions were otherwise undetectable. "We really did need computational tools and informatics to see the small deletions," he says.

"This kind of work is what some people are calling systems biology, and it involves a great deal of collaboration because no one person has expertise in all aspects of the research," says Salamon. "But getting people to cooperate across disciplines is not always easy and requires a kind of flexibility and, eventually, trust."

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Kato-Maeda, M. et al. Comparing genomes within the species Mycobacterium tuberculosis. Genome Res 11, 547-554 (April 2001).
Salamon, H. et al. Detection of deleted genomic DNA using a semiautomated computational analysis of GeneChip data. Genome Res 10, 2044-2054 (December 2000).
Behr, M.A. et al. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284, 1520-1523 (May 28, 1999).
Gingeras, T.R. et al. Simultaneous genotyping and species identification using hybridization pattern recognition analysis of generic mycobacterium DNA arrays. Genome Res 8, 435-448 (May 1998).

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