We are living in a time that will probably be recalled as a golden age of discovery in human genetics. Much has been learned from the study of rare single-gene disorders.
Although largely unnoticed by most physicians, these rare diseases impose an enormous burden psychologically and economically on affected patients and their families. Characterization of the gene associated with a disorder provides the key to understanding the relevant pathophysiology of these disorders, and the insight often leads to treatment that can ameliorate the patients burden. Often, insights into the biologic mechanism of rare disorders can be applied to more common disorders as well, with potential benefits for a much larger population.
Without question, the understanding of the biology of chronic granulomatous disease has provided valuable insight into the role of phagocytic blood cells in the inflammatory process. Since the initial description by Good et al in 1957 as a fatal granulomatous disease of childhood, early data from various studies established CGD as a disease of phagocytes. Neutrophils from CGD patients demonstrate normal phagocytic activity in vitro, but bactericidal activity against Staphylococcus aureus is markedly impaired. The diagnosis of CGD is based on a compatible clinical history and demonstration of a defective respiratory burst. The primary defect in CGD resides in the inability of phagocytes to generate superoxide anion due to the absence of some of the components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system.
The NADPH oxidase is composed, in part, of four key proteins that account for the genetic basis of the disorder. These include: a membrane-bound cytochrome b558; a 47kDa cytosolic protein p47phox (47 phagocyte oxidase protein), and a 67kDa cytosolic protein (p67phox). In turn, cytochrome b558 consists of two heme containing subunits, p22phox and glycoprotein 91phox. Upon activation of the phagocyte, the cytosolic proteins along with a lowmolecular-weight guanine triphosphate Rac2 translocate to the membrane and interact with the cytoplasmic domains of the transmembrane cytochrome b558 to form the active oxidase. Oxidase activity depends on p22phox to maintain stability of gp91phox. The latter protein is required for electron transport through its binding of NADPH, flavin and heme.
About 65% of children affected with CGD have a mutation in the CYBB gene located on the X-chromosome, which encodes for gp91phox. The remainders of the patients inherit CGD in an autosomal recessive fashion. Mutations in the genes, comprising components of the NADPH mentioned above, compromise the ability of the phagocyte to generate superoxide and hydrogen peroxide for molecular oxygen following phagocytosis. The failure to activate the NADPH oxidase in CGD predisposes the host to infection with catalase positive microorganisms.
Normal neutrophils generate hydrogen peroxide in the phagosome and use myeloperoxidase, which is delivered into the phagosome from primary granules (azurophil) fusing with the membrane of the phagosome to generate hypochlorous acid from chloride to kill microbes. The hydrogen peroxide produced by normal neutrophils can exceed the capacity ofcatalase, hydrogen peroxide-catabolizing enzymes of many aerobic microorganisms, including S. aureus, most gram-negative bacteria, Candida albicans and Aspergillus, killing the offending microorganism.
In contrast, the CGD neutrophils, which are unable to produce hydrogen peroxide, are unable to kill the catalase microorganisms but are able to dispose of catalase-negative organisms because the microorganisms produce sufficient amounts of hydrogen peroxide to result in a microbicidal effect. Catalase-positive microorganisms can survive for long periods in the CGD phagosome. Eventually the bacteria are killed but are not digested properly in macrophages and can, thereby, contribute to the formation of granulomata.
Exuberant inflammation is typical of CGD and is manifested as microgranulomata at sites of infection that can lead to obstruction of the gastrointestinal and urinary tract, delayed healing of surgical wounds or inflammatory bowel disease. Data from recent studies indicate that CGD neutrophils produce more interleukin-8 than normal neutrophils, indicating an essential feedback loop in which oxidants can attenuate inflammation by limiting IL-8 production.
Additionally CGD neutrophils exhibit delayed apoptosis, which is the normal physiological process that ameliorates tissue damage at sites of inflammation from necrotic lysis and release of proteases from dying neutrophils. These observations link oxidant production to the augmentation of normal neutrophil apoptosis as an important mechanism by which phagocyte-generated oxidants mediate feedback diminution of inflammation by limiting neutrophil necrosis.
Data from recent studies regarding phox led to the discovery of a homologue of gp91phox in gut epithelium; then a series of additional homologues of gp91phox were found in other tissues. There is evidence now emerging for a functional role for the oxidase and its regulator proteins in modulating the function of nonphagocytic cells. For instance, phox is present at low levels in T-lymphocytes and its absence causes a strong shift of T-lymphocytes to a Th1 cytokine pattern upon activation. It has become apparent that CGD patients appear to be vulnerable to developing a variety of Th1-type specific autoimmune diseases including sarcoidosis, inflammatory bowel disease and both adult and juvenile rheumatoid arthritis.
The treatment of CGD by ex vivo gene therapy may provide a model system for correction of other bone marrow hemalogic disorders. Ott et al, reported long-term high-level clinically beneficial correction by ex vivo gene therapy of X-linked CGD in two adult patients. Nonablative busulfan conditioning was used to assist gene therapy mediated correction in that study. The long-term stability and safety of gene therapy mediated correction remains to be determined. For instance, there are concerns about gene insertion predisposing patients to acute myelogenous leukemia. Evidence of the potential genotoxicity of retroviral integration in clinical trials has been seen in a French trial leading to genetic correction in severe combined immunodeficiency patients (SCID). Leukemia occurred in 4 children out of 35 in the French Trial secondary to vector-mediated insertional mutagenesis. In any event, it is exciting to witness the potential of gene therapy in correcting hemalogical disorders but caution needs to temper enthusiasm for the modality of gene correction in patients.
For more information:
- Berendes H, Bridges RA, Good RA. A fatal granulomatosis of childhood: the clinical study of a new syndrome. Minn Med. 1957;40:309-312.
- Ott MG, Schmidt M, Schwarzwaelder K, et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVl1, PRDM16 or SETBP1. Nat Med. 2006;12:401-409.
- Nienhuis AW. Development of gene therapy for blood disorders. Blood. 2008;111:4431-4444.
Laurence Boxer, MD, is Director of Pediatric Hematology/Oncology at the University of Michigan Health System and is editor of HemOnc Todays Pediatric Hematology & Leukocyte Disorders Section.