These rare mutations in a human gene called TLR4 lend susceptibility to meningococcal sepsis, which strikes over 2,500 people a year in the United States. About half of those who contract meningococcal sepsis are younger than the age of two, and the disease has an overall case fatality rate of 12 percent.
"It's a very fast-moving, dramatic, and often fatal disease," says TSRI Immunology Professor Bruce Beutler, who led the research, which will be published in an upcoming issue of the journal Proceedings of the National Academy of Sciences.
"We took a large number of people with meningococcal sepsis and compared them to normal controls who were ethnically and geographically matched," says Beutler. "We found that the TLR4 gene had more mutations in the sepsis populations."
Besides demonstrating that the risk of severe sepsis increases with these mutations, which can be passed from parent to child, the study also suggests that it may be possible to protect people who are at risk. While not practical at the moment, eventually patients with mutations to their TLR4 genes might be given prophylactic treatment, for instance, before they undergo surgery or travel somewhere they are likely to be exposed to meningococcal bacteria.
Blinding the Immune System
Scientists had suspected that genetic factors determine who gets the severe form of the disease and who does not. Evidence for this was seen during outbreaks of this disease, when the severe and deadly form of sepsis was more likely to strike related individuals.
Further evidence was seen in mice, and it was known for years that certain strains of mice are more susceptible to infections with bacteria like N. meningitides--but nobody had ever shown this to be true in humans. Now, the TSRI team found a number of rare mutations in one essential gene in the innate immune system called Toll-like receptor 4 (TLR4), which is important in endotoxin recognition, giving people a higher probability of contracting meningococcal sepsis.
TLR4 is part of the mammalian endotoxin receptor and is an important gene because it detects the early stages of a bacterial infection. It is a powerful pro-inflammatory receptor, responsible for activating the immune system to attack invading gram-negative bacteria like N. meningitidis. During a mammalian innate immune response, TLR4 recognizes endotoxins from the bacteria and activates macrophages, which then ingest and destroy the foreign pathogens.
"The TLRs are the eyes of the innate immune system," says Beutler, who had suspected that these eyes may be myopic for some people and that mutations in the TLR4 gene may underlie the genetic component of severe sepsis.
In order to address this, Beutler looked for extremely rare mutations in the gene by obtaining hundreds of samples of DNA from his collaborator Martin L. Hibberd, then a doctor at Imperial College in London (Hibberd has since taken a position at the Genome Institute of Singapore). He then amplified and sequenced the entire TLR4 gene from each patient, looking for all the rare mutations that are present in the gene--rather than taking the traditional approach of looking at one common mutation within that gene.
After the researchers had analyzed all the genetic information, they found a significant excess of low frequency mutations in the TLR4 genes of the people who had contracted sepsis versus the control group.
Instead of asking how many people have one given mutation, Beutler and his colleagues simply asked how many people have any genetic variation. A few individuals had the same mutations, but by and large they were single, isolated mutations. "However," warns Beutler, "we can only account for a certain fraction of the [genetic] risk."
Nevertheless, this is the first time that a comparison of the collective mutations at a given genetic locus has been made in any infectious disease. Infectious diseases are, after all, primarily caused by an invading organism, and only in recent times has a concerted effort been made to find genetic determinants of susceptibility in the patient. Beutler and his team had to write special software to make the comparison of the thousand different sequences possible and find the individual mutations therein. Significantly, the technique of measuring the genetic variation "load" of the entire gene locus could be applied to other sorts of diseases as well: particularly diseases in which both genes and environment play a role.
The article, "Assay Of Locus-Specific Genetic Load Implicates Rare Tlr4 Mutations in Meningococcal Susceptibility," is authored by Irina Smirnova, Navjiwan Mann, Annemiek Dols, H. H. Derkx, Martin L. Hibberd, Michael Levin, and Bruce Beutler. The article will be available online the week of April 28, 2003 at: http://www.
This work was supported by The National Institutes of Health and also by the Meningitis Research Foundation, a United Kingdom charity.