Question

Genetic Mapping of Innate Immune Defects.

Septic shock is a very dangerous medical condition that is described in your textbook on page 135. It is essentially an acute hyper-activation of the macrophage innate immune response, resulting in systemic (instead of localized) production of cytokines, with deleterious effects. Normally, local cytokine production at an infection causes “leakiness” in blood vessels, so that leukocytes can exit and migrate to the infection site (erythrocytes also leak out in the process, leading to the “redness” characteristic of inflammation). However, when cytokines are released throughout the body (“systemically”), then all the blood vessels become leaky (“vascular permeability”), leading to precipitous drops in blood pressure, and frequently organ failure and death. Septic shock has a 50% mortality rate, and is the leading cause of death in intensive care units (ICU’s) in hospitals.

One of the most common triggers of septic shock is LPS. In normal Gram-negative infections, this outer-membrane molecule is only present at the site of infection; however, if the bacteria reach the blood-stream, then LPS can quickly disseminate and trigger massive cytokine release throughout the body. This dangerous property of LPS led to its original name, “endotoxin”. (“endo” was meant to distinguish it from secreted “exo” bacterial toxins; unlike exotoxin virulence factors like cholera toxin, LPS/endotoxin is not a virulence factor evolved to exploit host cells).

Mice were often used to study septic shock, and a mouse strain that was resistant to septic shock was discovered in the 1960s. Normally, mice injected with LPS would succumb quickly to septic shock and death, but these mutant mice (C3H/HeJ strain) showed no effect upon LPS injection. On the other hand, when infected with live Gram-negative bacteria, the C3H/HeJ mice were unusually susceptible to infection and death.

In some ways, the C3H/HeJ mice were analogous to the boys diagnosed with CGD (chronic granulomatous disease) in the 1960s, in that both had heritable conditions that impaired immunity. A heritable defect implies the existence of a mutated gene that normally contributes to the affected process.

2a. The typical process for identifying an unknown gene that has a “phenotype” of interest is to “map” it to a particular chromosome, and then narrow its location to a smaller and smaller portion of this chromosome. Using animal models, this involves many generations, and tests of each generation to see which regions of which chromosome are always inherited in the individuals that exhibit the trait. This implies that chromosomes are not inherited intact from generation to generation: if chromosomes are strands of nucleotides covalently linked to each other, how is it that they are not inherited intact from parent to child?

2b. When mapping a trait in humans, instead of following inheritance through multiple generations (even if the disease status of ancestors is recorded, mapping is not feasible within family trees since DNA samples of ancestors were not preserved), researchers instead work with multiple independent families that seem to exhibit the same genetic disorder. For CGD genetic mapping, it was fortuitous that in the 1960s the disorder was identified in multiple unrelated patients. What feature of the CGD disease made identifying the affected gene a little easier by allowing the researchers to narrow their focus to a single chromosome?

2c. Is CGD a recessive or dominant genetic disorder?

2d. What traits or defects were observed in patients with CGD?

2e. Identifying the gene affected in CGD patients led to the discovery of an enzyme complex important in innate immunity. What is the name of this enzyme complex, and what does it do?

2f. The C3H/HeJ mice had a genetic syndrome with both beneficial and detrimental traits. In your own words, describe the beneficial vs detrimental aspects.

Answer 1

2a. Chromosomes are not inherited intact from parent to child because of the process of genetic recombination which occurs during meiosis in the process of gamete formation. In eukaryotes it is achieved by chromosomal crossover.

2b. Most cases of chronic granulomatous disease are transmitted as a mutation on the X chromosome. Therefore, we can say that CGD is a X-linked trait and thus can be easily identified through pedigree analysis. This feature of the CGD disease made identifying the affected gene a little easier

2c. CGD is a recessive disorder.

2d. Weakened immune system leading to characteristic specific infections such as pneumonia, funginemia, cellulitis etc in a decreasing order of frequency. Sometimes people with CGD can get infected with atypical conditions such as candidiasis etc.

2e. The enzyme complex is named PHOX or phagocyte NADPH oxidase. This enzyme oxidizes NADPH and reduces molecular oxygen to produce superoxide anions, a reactive oxygen species. Superoxide is then disproportionated into peroxide and molecular oxygen by superoxide dismutase. Finally, peroxide is used by myeloperoxidase to oxidize chloride ions into hypochlorite (the active component of bleach), which is toxic to bacteria. Thus, NADPH oxidase is critical for phagocyte killing of bacteria through reactive oxygen species.