Journal of Infection (2014) xx, 1e4

Recent advances in chronic granulomatous disease David Goldblatt Institute of Child Health, University College London and The Great Ormond Street Hospital for Sick Children NHS Foundation Trust, 30 Guilford Street, London WC1N 1EH, UK Accepted 18 July 2014 Available online - - -

KEYWORDS Primary immunodeficiency; Immunocompromise; Haematopoietic stem cell transplant; Gene therapy

Summary Chronic Granulomatous Disease (CGD) is a primary immunodeficiency caused by abnormities in the NADPH Oxidase that is involved in the respiratory burst responsible for initiating the killing of microbes ingested by phagocytic cells. The hallmark of CGD is recurrent infection but the inflammatory complications can prove difficult to treat. New insights into the mechanisms responsible for the inflammatory complications have led to new therapies. The treatment of CGD colitis with an anti-tumour necrosis alpha agent has been shown to be successful but associated with significant infectious complications. Haematopoietic stem cell transplants offer the possibility of cure for those with ether a matched or unrelated donor transplant, with results of the latter improving significantly over recent years. Gene Therapy offers the promise of cure without the need for a transplant but better vectors are required. ª 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Introduction CGD is a primary immunodeficiency disorder with a recent reported incidence in Europe of approximately 1/250,0001 but as low as 1/120,000 in the United Kingdom and Ireland.2 CGD is caused by an inherited defect in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex present in a variety of cells including phagocytes. The NADPH oxidase enzyme complex consists of two membrane-spanning subunits, gp91phox and p22phox, as well as three cytosolic components p47phox, p67phox, and

p40phox. (Reviewed by Segal3) Rac1 and Rac2, lowmolecular-weight guanosine triphosphate-binding proteins, also associate with the complex and are involved in the regulation of the NADPH oxidase.4,5 Approximately, 66% of all CGD cases result from mutations within the X-linked gp91phox gene (CYBB), followed by the autosomal recessive forms of CGD, with defects in the gene coding for p47phox (NCF-1) accounting for 30% of all CGD cases. Only 5% of the cases are due to mutations in CYBA, NCF-2 or NCF-4 which encode for p22phox, p67phox and p40phox respectively.6,7

E-mail address: [email protected] 0163-4453/ª 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Goldblatt D, Recent advances in chronic granulomatous disease, J Infect (2014), 10.1016/j.jinf.2014.07.013


Molecular basis of CGD NADPH oxidase is required for the ‘respiratory burst’ and has a critical role in microbial killing. It reduces molecular oxygen to superoxide, which subsequently reacts to form reactive oxygen species (ROS) such as hydrogen peroxide, hypochlorous acid, and hydroxyl radicals. Superoxides and their derivatives were initially thought to be the effector molecules responsible for microbial killing but new insights suggest that that this may be over simplistic.8 For example recent work exploring the mechanisms underlying NADPH oxidase function suggest that the influx of cations into and pH changes within the phagocytic vacuoles activate neutrophil proteases (neutrophil elastase, cathepsin G) that are critical for the intracellular antimicrobial activity of phagocytes.9 Activated neutrophils also kill by extracellular mechanisms, including neutrophil extracellular traps (NETs), which are composed of chromatin decorated with granular proteins.10 NETs bind bacteria and fungi and expose antimicrobial molecules. Generation of NETs is NADPH dependent and thus impaired in CGD.11 Sufferers of CGD therefor have impaired phagocytic function and are susceptible to recurrent bacterial and fungal infections. Patients are particularly susceptible to fungal infection, typically from Aspergillus species, but also catalase positive bacteria including Staphylococcus aureus and Burkholderia cepacia.12 Most patients present with infections, typically lymph node abscesses, but also recurrent respiratory infection, deep-seated abscesses and septicaemia.13 A major clinical complication of CGD is however due to uncontrolled inflammation in various organs including the GI tract, bladder, skin and the eye.14 The molecular basis for this inflammation and its associated trigger(s) remain poorly understood but a source of considerable interest with the focus on pathways that disrupt normal T cell regulatory control of inflammatory mechanisms.15,16 The consequences are the predominant cause of poor quality of life for those living with CGD.

Diagnosing CGD Making the diagnosis of CGD is not technically difficult, and historically is based on the use of the “gold standard” nitroblue tetrazolium assay.17 More recently the description and subsequently wide availability of a flow cytometric based assay18 has facilitated the diagnosis of CGD in nonspecialist laboratories. This assay is based on the reduction of dihydrorhodamine-123 (DHR) by phorbal myristate acetate stimulated phagocytic cells and is particularly useful as it can demonstrate two populations of cells in carriers. However, care must be taken in the interpretation of a negative test as acutely ill patients may show abnormal stimulation indices which normalise on recovery.19 Once abnormal NADPH oxidase has been discovered a variety of techniques can be used to assess the presence of absence of individual NADPH proteins.20 More difficult is to know whom to test for CGD as some of the clinical manifestations are not uncommon. For example, The National Laboratory for Leukocyte Functions in Israel evaluated 1296 patients over a period of 24 years, referred because of recurrent, severe pyogenic infections.21 CGD was diagnosed in 51

D. Goldblatt patients (3.9%) illustrating that even one of the defining clinical sign of CGD, recurrent bacterial infection, is not a sensitive or specific sign for CGD. As a result of the nonspecificity of the clinical symptoms late diagnosis is not uncommon and is not restricted to those with milder disease due to residual NADPH oxidase.22

Colitis Colitis is an important gastrointestinal manifestation of CGD, typically seen more commonly, but not exclusively in the X-linked form of the disease.23 In a recent survey 15/94 patients in UK and Ireland had colitic symptoms prior to diagnosis and on follow up as many as 40% of patients2,12,13 manifest with colitis. The clinical presentation of colitis is similar to that of inflammatory bowel disease, either Ulcerative Colitis (UC) or Crohns disease,24,25 and includes diarrhoea, abdominal pain and rectal bleeding. As occasionally the diagnosis of CGD is made by gastroenterologists who have previously labelled a patient as having a primary diagnosis of inflammatory bowel disease (IBD), Jaggi and colleagues screened 120 individuals aged 12 months to 21 years with a diagnosis of Chrohn’s disease, Ulcerative colitis or indeterminate IBD for CGD.26 Thirty percent of the patients had also reported a history of infection, yet using DHR not a single patient was shown to have CGD. The mainstay of therapy to date for the colitic complications of CGD has been T cell modulating agents such as azathioprine, sulfasalazine and thalidomide combined with or replacing the use of steroids.27,28 More recently, following the success of anti-tumour necrosis factor alpha (TNF-a) treatment in IBD, antibodies to TNF-a have been used in CGD. Recent experience from the NIH indicates that while the treatment is effective and can induce remission of the colitis, the immunosuppressive properties associated with antagonising TNF on a background of immune deficiency can prove problematic.29 Uzel and colleagues describe 5 CGD patients (3 XL, 2AR) who developed infectious complications while on Infliximab therapy. Three of the patients developed Burkholderia infections, one a candida infection and one multiple liver abscesses presumed to be due to Staphylococcus aureus. Neutralising TNF-a thus appears to enhance susceptibility of CGD patients to infections suggesting that TNF-a has an important role in host immunity where NADPH oxidase is absent.

Haematopoietic stem cell transplant in CGD The only curative procedure for CGD currently is a haematopoietic stem cell transplant (HSCT). The uncertain long term outcomes for CGD led the Working Party Inborn Errors of the European Blood and Marrow Transplantation (EBMT) group toward a pragmatic clinical approach to indications for HSCT in CGD.30 The group considered standard risk patients to be those in good health but with a history of one life threatening infection in the past or severe granulomatous disease and/or the non-availability of specialist care and/or non-compliance with antibiotic prophylaxis. In this clinical setting patients generally have an excellent outcome after HSCT if a HLA matched donor sibling (or matched unrelated donor (MUD), see below) is available.

Please cite this article in press as: Goldblatt D, Recent advances in chronic granulomatous disease, J Infect (2014), 10.1016/j.jinf.2014.07.013

Recent advances in chronic granulomatous disease Experience in Europe has shown excellent results with HLA identical sibling HSCT’s with survival in excess of 90% and the reversal of complications such as colitis.30 Recently a multinational prospective study of reduced intensity conditioning (RIC) and HLA matched HSCT was published.31 21 HLA matched related donors and 35 HLA MUD’s were recruited to the study. Overall 2 year probability of survival was 96% with graft failure in only 3/56 patients similar numbers with Graft versus host disease. The overall survival for MUD’s was similar with an overall survival of 91%, a significant improvement on earlier studies and outcomes. In centres with experience, HSCT’s should be considered the treatment of choice for a child of standard risk with any genetic form of CGD. Post HSCT children have improved clinical outcomes including growth32 and demonstrate improved psychological outcomes.33


3. 4.


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8. 9.

Gene therapy for CGD 10.

The first human stem cell (HSC) gene therapy trial for CGD disease was initiated in the US without chemotherapy, but only achieved short-term engraftment with no long-lasting benefit for patients.34 This suggested that it was probably necessary to condition patients prior to gene therapy, as CGD-corrected cells are not likely to have any selective growth or survival advantage when re-infused. More recently, five early phase clinical studies, involving a total of 12 patients have been conducted in several centres worldwide incorporating conditioning with low intensity alkylating agent.35 The experience of Grez and colleagues demonstrated biologically restoration of NADPH-oxidase activity in circulating neutrophils for a short time after engraftment, and some clinical benefit allowing the majority of patients to clear pre-existing infections. However, clinical benefit was usually only transient, and gene marking rapidly decreased with only few patients having significant marking after three months, indicating an absence of long-term engraftment.36 Two patients treated with a specific gammaretroviral vector developed myelodysplasia with monosomy 7 as a result of insertional transactivation of myeloproliferative genes.37 One subject died 27 months after gene therapy from overwhelming sepsis while the other underwent a successful allogeneic HSC transplant. To improve the safety and efficacy of gene therapy for CGD, a new lentiviral vector has been designed that directs transgene expression primarily in myeloid cells38 and holds out the promise for safer gene therapy and long term cure of CGD.










Conflict of interest The author has no conflict of interest to report.




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Recent advances in chronic granulomatous disease.

Chronic Granulomatous Disease (CGD) is a primary immunodeficiency caused by abnormities in the NADPH Oxidase that is involved in the respiratory burst...
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