Chromosome 22 Provides Human Genome Preview

For this article, Laura DeFrancesco interviewed Ian Dunham, senior research fellow at the Sanger Centre, Welcome Trust, UK; Bruce Roe, a research professor of chemistry and biochemistry at the University of Oklahoma; and Michio Hirano, Florence Irving assistant professor of neurology at Columbia University College of Physicians and Surgeons. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. I. Dunham et al., "The DNA sequence of human chromosome 22," Nature, 402:489-95, 1999. (Cited in 358 papers) I. Nishino et al., "Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder," Science, 283:689-92, 1999. (Cited in 75 papers) It was but a mere 30 million bases of the 3 billion that make up the human genome, but at the time, it represented a major breakthrough. The sequence of chromosome 22 published in December 1999 was the largest contiguous DNA sequence to be completed. It took hundreds of researchers--217, to be exact--scattered in four laboratories worldwide, working for several years to accomplish the task. And it was the first time that researchers could see the organization of an entire chromosome at the base pair level. The chromosome 22 sequence turned out to be a good preview of what the whole genome had in store. The spacing of repeated sequences along the chromosome and the presence of large-scale duplications were features that have turned up throughout the genome. The sequence also revealed a large number of pseudogenes, roughly 20 percent of the total gene population. "To see the entire sequence of a human chromosome for the first time is like seeing an ocean liner emerge out of the fog, when all you've ever seen before were rowboats," Francis Collins, director of the National Human Genome Research Institute of the National Institutes of Health, said at the time. The NHGRI supported the US contribution to the sequencing of chromosome 22. Technology was a key component, and not just in the laboratory. The Internet provided an online meeting place for the researchers, who met face to face only a few times during the course of the work, says study leader Ian Dunham, senior research fellow at the Sanger Centre, wellcome Trust, UK and first author on the Hot Paper. Joining the Sanger Centre in this effort were researchers at the University of Oklahoma, the Keio University School of Medicine in Tokyo, and Washington University in St. Louis. This work also validated the approach to sequencing that had been adopted by the publicly funded human genome project that used the mapped clone approach as opposed to the whole-genome shotgun sequencing technique used by Celera Genomics Group, which published its chromosome 22 sequence 18 months later when the human genome was published.1,2 The group also predicted the total number of genes that comprise the human genome, based on the number of genes they found on chromosome 22. At the time, anyone's best guess was that there were upwards of 100,000 human genes; this group came up with a prescient 40,000. Courtesy of University of Oklahoma Bruce Roe Keeping such a large group on the same page might have been difficult but for the fact that they all realized they were making history. Bruce Roe, a research professor of chemistry and biochemistry at the University of Oklahoma says that once they realized that this remarkable accomplishment was within their grasp, they worked together to get it done. "The idea of doing the first human chromosome and getting it finished was much more important than any individual. ... No arguments, no politics. If we misinterpreted what one group said, we bit the bullet and went on." The Right Choice The reason for choosing chromosome 22 was scientific, according to Roe, though its small size, representing just under 2 percent of the human genome, was a factor. But, Roe says, "The four groups had a deep and abiding scientific interest in specific regions of the chromosome that were disease-related." With its 545 genes, chromosome 22 holds particular interest to Roe because it is home to genes involved with two birth defects (cat eye syndrome and DiGeorge's syndrome) as well as the breakpoint for a translocation that is associated with chronic myeloid leukemia. Also residing on chromosome 22 is an immunoglobulin region of interest to the Japanese group and a collection of cancer-related genes, neurofibromatosis among them, which both the Sanger Centre and Washington University scientists study. Also present are 54 known disease-related genes, including schizophrenia and amyotrophic lateral sclerosis. At a chromosome 22 meeting in 1995 in Cambridge, the four groups compared notes on their sequencing progress and realized that without even trying, they already had amassed half of it. "We said, 'Heavens to Mergatroid,' we could finish it," recalls Roe. So they divided up the remaining sequence and went back to their laboratories. The group reached consensus on which parts of the chromosome each group would sequence, says Dunham. "Deciding the boundaries as a group effectively stopped any squabbles and left us to concentrate on our own issues." Surprising discoveries ensued. Although processed pseudogenes had been observed before, their sheer number amazed the group. The observation of two genes located entirely within the intron of other genes also hit a chord, according to Dunham. "This had been seen before in mammals but was thought to be exceptional, so seeing two on this small chromosome had an impact," he says. But most surprising was the group's forecast regarding the total number of genes that make up the human genome. "We tried several different ways of predicting ... and we came up with roughly the same number. The data doesn't lie," comments Roe, although he admits that he thought they were way off, by as much as a factor of three. Following the terms of the Bermuda agreement,3 the chromosome 22 group put its data into a public repository on a daily basis. Roe explains: "It's really important. Only a finite number of groups have large grants and can sequence large amounts of DNA. Because there's so much information to be gained, it's absolutely critical that the data is released within 24 to 48 hours." Capital Gains One group that capitalized on the chromosome 22 sequence before the paper was even published was Michio Hirano, Florence Irving assistant professor of neurology at Columbia University College of Physicians and Surgeons. Hirano's group studies a rare genetic disease known as mitochondrial neurogastrointestinal encephalomyopathy, or MNGIE for short. MNGIE is caused by autosomal recessive mutations that map to the tip of the long arm of chromosome 22. While not a mitochondrial mutation itself, it is associated with deletions and depletion of mitochondrial DNA, presumably caused by defective communication between the nuclear and mitochondrial genomes. What that means in molecular terms had not been clear, until Hirano, with the chromosome 22 sequence at hand, found the gene that is mutated in MNGIE. After sequencing only a handful of the 30 to 40 genes that were in the region that MNGIE mapped to, Hirano hit pay dirt. He found that patients with MNGIE had mutations in the gene for thymidine phosphorylase (TP), an enzyme that breaks down the nucleoside thymidine. Hence, if TP activity is lost, excess nucleoside can enter the thymidine salvage pathway. Here's where the mitochondrial connection may lie, as mitochondrial DNA synthesis relies on the thymidine salvage pathway, whereas nuclear DNA synthesis relies on the de novo biosynthetic pathway. Hirano believes that a defective TP could cause an imbalance in nucleotide pools that would have deleterious effects on mitochondrial DNA replication. This discovery has already led to better diagnostic tools for MNGIE and may lead to better treatments as well. Hirano found that MNGIE patients have dramatically elevated levels of thymidine in their plasma, which can be easily checked. In addition, he was able to reduce plasma thymidine levels in MNGIE patients using hemodialysis, though the effects are transient so far. This work has achieved some notoriety as it represents the first instance where a defect in intergenomic communication has been defined at the molecular level. It also may provide some insights into the poorly understood area of mitochondrial nucleotide metabolism. Two years later, the group is no closer to filling in all the gaps, of which at least seven remain, Roe says. It's not for lack of trying--these gaps are unstable, making cloning them impossible. For his part, Roe is looking at regions of the chromosome in other species, including mice, baboons, and zebra fish. He wants to find out just how chromosome 22 evolved. Large chunks of the chromosome, he says, have stayed together throughout evolution, but they have gotten dispersed among the different chromosomes. Says Roe: "It's like a deck of cards being shuffled." Laura DeFrancesco (defrancesco1@earthlink.net) is a freelance writer in Pasadena, Calif. References 1. J.C. Venter et al., "The sequence of the human genome," Science, 291:104-51, 2001. (Cited in 367 papers, according to the Web of Science, ISI, Philadelphia) 2. E.S. Lander, etal., "Initial sequencing and analysis of the human genome," Nature, 409:860-921, Feb. 15, 2001. (Cited in 456 papers) 3. The Bermuda Agreement, forged in 1996 at the International Strategy Meeting on Human Genome Sequencing, calls for the immediate release of human genome sequences into a public database.

Chromosome 22 Provides Human Genome Preview

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