Researchers Put Linkage Disequilibrium on the Map

Image © Nature MUTATION AND TRUNCATION: These DNA sequence electropherograms show a patient from "family 7" who is homozygous for a cytosine c insertion, as indicated by the arrow. The mutation encodes a truncated NOD2 protein. (Reprinted with permission from Nature, 411:603-6, 2001.) After years of failed promises that researchers would find genes linked to cancers, heart disease, and other complex human ailments, two independent research teams, using different approaches, localized a gene on human chromosome 16 that increases susceptibility to Crohn disease. One team was led by Gilles Thomas, Fondation Jean Dausset-CEPH, Paris; the other was led by Gabriel Nuñez, University of Michigan Medical School, and Judy Cho, University of Chicago Hospitals. The scientists described these 2001 discoveries in back-to-back Nature papers, two of this issue's three Hot Papers.1,2 The Nuñez team, using a classical candidate gene approach, discerned that the NOD2 gene, which plays a role in recognizing bacteria, is faulty in patients with Crohn disease. This discovery suggests that the disease is partially caused by an innate immune response to bacteria. The Thomas team's findings championed positional, or linkage disequilibrium (LD) mapping, a statistical approach to finding disease genes. Overall, the findings "are a great success for complex traits," says John Rioux, Whitehead Institute Center for Genome Research. NOD2 is the first common, complex disease gene that scientists have been able to identify. A common disease is defined as occurring in more than one case per 1,000 people; a complex one involves more than one gene. Data derived from the Science Watch/Hot Papers database and 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. J.-P. Hugot et al., "Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease," Nature, 411:599-603, May 31, 2001. (Cited in 258 papers) Y. Ogura et al., "A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease," Nature, 411:603-6, May 31, 2001. (Cited in 250 papers) D.E. Reich et al., "Linkage disequilibrium in the human genome," Nature, 411:199-204, May 11, 2001. (Cited in 187 papers) "The overwhelming significance of these findings is the genuine discovery of a susceptibility gene for a complex disease. The field of complex diseases is literally littered with irreproducible results," says Christopher Matthew of Guy's, King's and St. Thomas' School of Medicine, London. Matthew led a team that replicated the Nuñez team's results in a different patient population, just one month later.3 The French team, using a positional (LD) approach, focused on a particular DNA region that had already been linked to disease. The researchers pinpointed the suspect gene by identifying correlations between it and known genetic markers. Although LD mapping had been used in the past to find genes for single-gene diseases, nobody before had used it to identify genes for complex diseases, says Rioux. "This is one of the successes in terms of using positional cloning for complex disorders," says Thomas' coauthor Jean-Pierre Hugot, a pediatrician at Hopital Robert Debre, Paris. Since then, a handful of research groups worldwide have used LD mapping to identify genes for other complex disorders, including diabetes, schizophrenia, and stroke. "There is always a lot of optimism in science, but there is a fair amount of pessimism as well," says Rioux. "Linkage disequilibrium had yet to prove a successful approach [in searching for genes for complex diseases]. Hugot's paper provided a huge level of optimism that it really can be done." Rioux says that the Nuñez team's discovery was also remarkable. They not only found the NOD2 gene, but also made important headway into understanding its function. "The two approaches were complementary, and in fact it was really nice to see the papers back-to-back," says Hugot. "They confirmed our results, and vice versa." Meanwhile, researchers from the Whitehead Institute had been learning things about LD that had important implications for the use of LD mapping in the search for complex disease genes. Three weeks before the NOD2 finding appeared in Nature, a paper by Whitehead's David Reich, Eric Lander, their colleagues, and those from the University of Oxford also appeared in Nature.4 This third Hot Paper "was the first publication of an assessment of the extent of linkage disequilibrium in multiple regions of the genome," explains Rioux. Courtesy of Jean-Pierre Hugot Main steps of IBD1/NOD2/CARD15 positional cloning for Crohn disease MAKING LD MAPPING BETTER Reich says that he and his colleagues were surprised to discover that LD extended about eight times longer than the expected 5,000 to 8,000 base pairs predicted by theory. As it turns out, the average LD extent, at least among people of North European descent, was 60,000 base pairs. This was good news for LD cartographers. It meant that LD mapping, especially large-scale and perhaps even genome-wide mapping, might be easier than anticipated. The news got even better when, following Reich's paper, another Whitehead Institute scientist, Mark Daly, and his colleagues characterized these long stretches of LD in a way that made it possible to use LD in a systematic search for complex disease genes.5 Rioux and collaborators were the first to use the new and improved haplotype-based LD mapping approach to identify a complex disease gene.5 Again, the disease was Crohn's, but the gene was on chromosome 5, not 16. Before these Whitehead studies became public, researchers worked on LD somewhat in the dark, says Rioux. Although the Hugot team used LD mapping, they did not necessarily do so in a comprehensive way. But, admits Rioux, "they moved ahead and found an association signal. It may not have been done with the complete knowledge of the structure of LD, but it was the first." The Reich team's data confirm the out-of-Africa expansion hypothesis, an event that supposedly happened 50,000 to 100,000 years ago. Moreover, they provide some intriguing insights into how this occurred. "Effectively, fewer than five DNA samples gave rise to most of the modern European gene pool--a really, really dramatic event," says Reich. This does not mean that five people left home and crossed the desert, but it could mean that Northern Europeans descended from as few as 100 people who interbred for several generations. THE HUNT FOR NOD2 Because it was a novel approach, the use of LD mapping to find a complex disease gene was a long, risky endeavor. "We took a chance of course," says Hugot, "but it is always necessary to take a chance in science." The hunt for NOD2 began in the early 1990s when Thomas, Hugot, and colleagues used linkage analysis to show that a region on human chromosome 16 was linked to the Crohn disease phenotype. Although many scientists had suspected that genetics played a role in Crohn disease, it wasn't until these early results appeared in 19966 that the hunt for Crohn disease genes really took off. Several groups followed suit, confirming the French team's findings. But nobody could identify a gene. Not until nine years after they first set out to recruit families for their study, did Thomas and his collaborators finally locate NOD2. The American team, on the other hand, homed in on NOD2 in a matter of months. Unlike Hugot's team, Nuñez, who had been studying the function of the NOD2 protein, had a hunch that the NOD2 gene, which was located on chromosome 16 in the region that Thomas' team had implicated in 1996, was the culprit. So he called up his geneticist colleague Judy Cho and said, "Look, we have this gene but no DNA samples." Cho provided them. The scientists sequenced NOD2 in families with Crohn disease and showed that a defective NOD2 gene was associated with susceptibility to disease. "It was very remarkable," says Nuñez. "We could do the work very quickly because we suspected that NOD2 was the gene." Even though Crohn disease was first described in the medical literature in 1932, its etiology was, and still is, a mystery. But, because Nuñez approached NOD2 from a functional perspective, one of the strengths of his finding was the functional--and etiological--insight it provided regarding its role in the recognition of bacteria. "This has shifted the way we think about Crohn's disease. It could be a clue to understanding [it]," says Nuñez. It also explains why Nuñez believes his Hot Paper has been just as interesting to immunologists as it has to geneticists. "It covers many fields," he says. The identification of NOD2 will not lead directly to a cure for Crohn disease, says Nuñez, but the results "suggest that there may be ways to minimize or prevent disease." Identifying the bacteria that bind with NOD2 will be one of the next important steps and could lead to the development of an antibiotic. Leslie Pray (lpray@nasw.org) is a freelance writer in Northampton, Mass. References 1. J.-P. Hugot et al., "Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease," Nature, 411:599-603, May 31, 2001. (Cited in 258 papers) 2. Y. Ogura et al., "A frameshift mutation in NOD2 associated with susceptibility to Crohn' s disease," Nature, 411:603-6, May 31, 2001. (Cited in 250 papers) 3. J. Hampe et al., "Association between insertion mutation in NOD2 gene and Crohn's disease in German and British populations," Lancet, 357:1925-1928, 2001. 4. D.E. Reich et al., "Linkage disequilibrium in the human genome," Nature, 411:199-204, May 11, 2001. (Cited in 187 papers) 5. L. Pray, "The promise that haplotypes hold," The Scientist, 15[23]:21, Nov. 26, 2001. 6. J.-P. Hugot et al., "Mapping of a susceptibility locus for Crohn's disease on chromosome 16," Nature, 379:821-3, 1996.

Researchers Put Linkage Disequilibrium on the Map

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	Image © Nature
	MUTATION AND TRUNCATION: These DNA sequence electropherograms show a patient from "family 7" who is homozygous for a cytosine c insertion, as indicated by the arrow. The mutation encodes a truncated NOD2 protein. (Reprinted with permission from Nature, 411:603-6, 2001.)