Hot Topic

Chronic Granulomatous Disease (CGD)

Clinical Features and Laboratory Testing

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Published: January 2010

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Dr. Abraham and Dr. Joshi provide an overview of primary immunodeficiency (PID) and describe the clinical features and molecular pathogenesis of chronic granulomatous disease (CDG) and appropriate diagnostic laboratory tests.

Presenters: Roshini S. Abraham, PhD

  • Consultant in theDivision of Clinical Biochemistry & Immunology
  • Director of the Cellular & Molecular Immunology Laboratory
  • Director of the Jeffrey Modell Foundation Diagnostic Center for Primary Immunodeficiencies at Mayo Clinic

Presenters: Avni Y. Joshi, MD

  • Instructor in Pediatrics and Medicine, Division of Pediatric Infectious Disease and Allergy/Immunology, Dept. of Pediatric and Adolescent Medicine

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Transcript

Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice.

Our presenters for this program are Dr. Avni Joshi and Dr. Roshini Abraham. During this program, we will be discussing general concepts of primary immunodeficiencies and their incidence before moving into details on the pathogenesis, common clinical features and treatment, and laboratory evaluation for Chronic Granulomatous Disease (CGD).

Before we begin our discussion on CGD, we will provide an introductory overview on primary immunodeficiencies and the immune system.

Primary Immunodeficiencies (PIDs): What Are They?

PIDs or primary immunodeficiencies are, typically speaking, congenital, inherited disorder of immune function. They can also arise as de novo or sporadic mutations in genes that regulate the immune response or components of the immune system.

PIDs are very diverse in their clinical manifestations; however, a common theme is the presence of recurrent infections, which can be life-threatening. Recurrent infections are not the only complication of PIDs - there can be other manifestations of immune dysregulation, such as autoimmunity, and hematological anomalies including cytopenias and lympho-proliferative disorders.

In certain cases, the PID can present as part of complex genetic syndrome where the immunological aberration is one feature in a constellation of other anomalies including skeletal and facial dysmorphisms. PIDs can be a challenge to diagnose because of the extreme clinical heterogeneity in presentation, which is related to the underlying complexity of the immune system and the specific immune component(s) that are affected.

There are over 120 distinct genetic defects that are associated with over 150 PIDs and this number continues to increase every year with new discoveries of genetic mutations that explain previously known clinical phenotypes. In this context, it may be appropriate to mention that immunology is one of the fastest growing disciplines of medicine with a complexity that is mind-boggling and where paradigm shifts occur almost on a daily basis.

Relative Distribution of the PIDs

The PIDs can be classified based on the component of the immune system that are affected - these include humoral or B cell defects, T cell or cellular defects, phagocytic defects and complement defects. The humoral or antibody deficiencies account for the majority of the PIDs, ~65%, while "pure" cellular deficiencies account for only 5% of the total cases of PIDs. The combined cellular and humoral immune deficiencies account for another 15%, while the phagocytic and complement deficiencies account for the remaining 10% and 5% respectively.

Components and Kinetics of the Immune Response

This is a graphical representation or cartoon of both the components and the kinetics of the immune response. The innate immune compartment, which includes neutrophils, monocytes, macrophages, dendritic cells, complement and even NK cells, is generally activated within a few minutes to hours after encounter with a pathogen and is typically antigen non-specific. Pathogen recognition includes the PAMPs - pathogen-associated molecular patterns. On the other hand, the adaptive immune response, which includes B cells, T cells and again NK cells (which can play a dual role in both compartments of immunity) is antigen-specific and requires more time for activation and typically takes 96 hours or greater. However, the antigen-specificity gives it greater "potency" in combating pathogenic targets.

Mechanisms of Innate Immunity

As discussed in the previous slide, the phagocytes and complement are integral components of the innate immune response. The phagocytes can be subdivided into 2 groups - macrophages and monocytes, and neutrophils. Neutrophils typically can be considered the "first responders or EMTs" of the innate immune compartment. However, in submucosal tissue macrophages are the first to encounter pathogens and are later reinforced by neutrophils. Again as mentioned in the previous slide, both macrophages and neutrophils recognize pathogens by means of cell-surface receptors that can distinguish between surface molecules displayed by the host and the pathogen.

Defect in the Innate Immune System: Chronic Granulomatous Disease

Chronic Granulomatous Disease (CGD) is a classic example of a primary immunodeficiency affecting the innate immune system. This disease is genetically heterogeneous and caused by defects or mutations in the genes that encode the various subunits of the respiratory enzyme - NADPH oxidase, which on activation leads to the generation of hydrogen peroxide and superoxide that is bactericidal.

NADPH oxidase is composed of 6 subunits, 2 of which are membrane-bound - gp91phox or cytochrome B, encoded by the CYBB gene, and the gp22phox, encoded by the NCF2 gene. There are 3 cytosolic components - the p67phox, the p47phox and the p40phox encoded by the CYBA, NCF1 and NCF4 genes respectively. The G-protein, Rac-2 is also associated with the NADPH oxidase complex (and defects in the Rac-2 gene lead to a form of leukocyte-adhesion deficiency). The p40phox has not been shown to be associated with the pathogenesis of CGD and is thought to play a regulatory role in the NADPH oxidase activity, while all the remaining components are considered essential for the generation of the superoxide burst.

The NADPH oxidase components primarily function in electron transfer or translocation to, or stabilization of membrane components.

Gp91phox and p67phox are involved in electron transfer while p40phox and p47phox are associated with stabilization and translocation to the membrane.

Molecular Pathogenesis

The molecular pathogenesis of CGD includes mutations in 4 of the genes that encode the NADPH oxidase complex.

Of these, the CYBB gene encoding the gp91phox is an X-linked gene and therefore, these mutations are inherited in an X-linked manner. Mutations in this gene account for the majority of the CGD cases (65-70%). Mutations in the p47phox, p67phox and p22phox are all inherited in an autosomal recessive manner and the accounts for 25%, 5% and 5% cases respectively.

The pathogenesis of CGD spans the spectrum of possible mutations including missense, nonsense, deletions, frameshift, splice site, intronic and regulatory variations.

Clinical Features

The characteristic clinical features of CGD include recurrent infections, especially those involving the skin, lungs, lymph nodes and liver. In particular, osteomyelitis, gingivitis and perianal and perirectal abscesses are common.

It is quite remarkable that the infectious etiology in CGD is largely confined to 5 pathogens -Staphylococcus aureus, Burkholderia.(Pseudomonas) cepacia, Serratia sp., Nocardia sp., Aspergillus sp. Not surprisingly, CGD patients are most commonly infected with catalase+ microorganisms. This is due to the fact that most microbes spontaneously generate their own hydrogen peroxide, but, catalase + microbes degrade their own hydrogen peroxide into water and oxygen which is not possible with catalase negative microbes.

Infections with catalase negative microbes allow the generation of hydrogen peroxide which is harnessed by host phagocytes and can compensate for the defective NADPH oxidase enzyme in CGD patients. Treatment of CGD is typically managed using a triple regimen of bactrim, itraconazole, and IFN-gamma. Other therapeutic approaches may be used depending on the clinical context.

Laboratory Diagnosis of Neutrophil Oxidative Burst

There are a few different methods available for the laboratory testing of neutrophil function as a diagnostic test for CGD.

The primary endpoint of lab testing in CGD is the assessment of the neutrophil oxidative burst since the molecular defects are confined to this enzyme complex.

The 3 major tests that have been used for clinical diagnostic purposes in CGD include the:

  • Reduction of Nitro Blue-Tetrazolium (NBT) – performed on slides
  • Measurement of superoxide production by visible spectrophotometry
  • Measurement of Reactive oxygen species (ROS) production by flow cytometry, fluorometry or chemiluminescence

Nitro Blue-Tetrazolium Test (NBT)

The NBT test is a semiquantitative method for evaluating neutrophil oxidative burst dysfunction in CGD patients.

The panel on the right demonstrates that neutrophils ingest the dye, nitroblue tetrazolium, and in the presence of reactive oxygen species, the yellow colored NBT compound is converted to the purple-blue formazan compound.

The panel on the left demonstrates that neutrophils from CGD patients fail to generate reactive oxygen species and therefore, cannot reduce the yellow NBT dye and thus there is no change in the color produced.

Dihydrorhodamine (DHR) Flow Cytometric Assay for Diagnosis of CGD

The dihydrorhodamine flow cytometry based assay is the more commonly used diagnostic screening test for CGD in reference laboratories and larger medical centers.

This test is based on the principle that nonfluorescent DHR (dihydrorhodamine) 123 when phagocytosed by normal activated neutrophils (after stimulation with PMA – phorbol myristate acetate) can be oxidized by hydrogen peroxide, produced during the activated neutrophil respiratory oxidative burst, to rhodamine 123, a green fluorescent compound, which can be detected by flow cytometry.

Therefore, the detected fluorescence is an indirect measure of neutrophil function and oxidative burst. The top left-hand panel demonstrates normal neutrophil activation resulting in a robust oxidative burst and a complete shift of the peak (containing the cells) to the right indicating DHR oxidation to rhodamine. This shift can potentially be reported as a stimulation index (SI), which is the ratio of the mean fluorescent intensity of stimulated cells over unstimulated cells. In other words, the SI is the ratio of cells with rhodamine to the cells with DHR.

The top right panel depicts an X-linked CGD female carrier demonstrating two populations – one with no fluorescent shift, ie no oxidative burst and conversion of DHR to rhodamine, and the second normal peak, showing a typical oxidative burst. This would be expected in carrier females of X-linked diseases, since one allele is mutant and the other allele is normal. CGD is one of the PIDs where there is significantly skewed lyonization (random X-chromosome inactivation) resulting in female carriers with a clinical phenotype of disease. While it has been reported that even the presence of ~10% neutrophils with normal oxidative burst is sufficient to prevent a clinical phenotype, sometimes that is not always the case and therefore, clinical correlations should be performed with the DHR assay results. Further, it has been reported that there can be age-related changes in lyonization, with skewing more apparent with increasing age, resulting in clinically symptomatic CGD in older carrier females.

The lower left panel demonstrates the typical pattern of oxidative burst seen in patients with the autosomal recessive forms of CGD. This tends to be more challenging to interpret since there is not a complete absence of oxidative burst as is seen in the classic X-linked CGD patient (lower right panel). Rather, the autosomal recessive CGD patient shows at least a population of neutrophils with significantly reduced oxidative burst. The X-linked CGD patient does not usually have any neutrophils capable of mounting oxidative burst resulting in a SI that is almost close to one.

Neutrophil Oxidative Burst: Normal Individual

Here is another example of the flow cytometric pattern associated with DHR oxidation by neutrophils to measure oxidative burst. In the laboratory test done at Mayo, the neutrophils are identified based on their size and granularity, using forward and side scatter measurements on the flow cytometer. The data in the 3 upper panels shows the identification of neutrophils in a whole blood sample along with the absence of any fluorescence in the unstimulated neutrophil population, which is in stark contrast to the lower panels, where the neutrophils on stimulation with PMA mount a strong oxidative burst with a complete right shift in fluorescence.

X-linked CGD

This slide provides a typical example of flow data for neutrophil oxidative burst from a patient with X-linked CGD. In contrast to the previous slide, where the lower panel demonstrated a right shift of the neutrophils undergoing oxidative burst, there is absolutely no shift with the activated neutrophils indicating a complete lack of oxidative burst as would be expected from patients with the X-linked form of the disease. The 19 year-old male patient whose results are shown here had both the clinical features of CGD as well as a confirmed de novo mutation in the CYBB gene encoding the gp91phox protein by gene sequencing.

It should also be kept in mind that if there is a de novo mutation in an X-linked CGD patient, the mother will appear completely normal and will not demonstrate the usual carrier flow phenotype since this was not a germline mutation in the mother but arose spontaneously in this male offspring.

Symptomatic Female Carrier with CGD

This slide represents the DHR flow assay data from a 23 year-old female patient with a known family history of X-linked CGD and a personal clinical history of CGD and probably ulcerative colitis with recurrent abscesses and granulomas. The flow results reveal a slightly unusual carrier phenotype with two populations of neutrophils – one negative and the other positive for oxidative burst. In general, for carriers, the distribution of the positive and negative populations is 50% each, however, in this patient it was more of 30% to 70% positive to negative neutrophils for oxidative burst. However, the clinical history combined with the flow data clearly indicates a carrier female who also has a clinical phenotype due to skewed lyonization of the X-chromosome. Gene sequencing revealed the presence of a single copy (heterozygous) of the familial X-linked CYBB mutation.

Autosomal Recessive CGD

This slide reveals the neutrophil oxidative burst pattern for a young male patient with autosomal recessive CGD. As discussed previously, patients with AR-CGD, have a small population of neutrophils with partial oxidative burst as can be visualized in the lower right panel. The positive population on the right of the dividing line in that panel shows rather dim fluorescence on oxidation characteristic of AR-CGD. Further, there is also a negative population of neutrophils, for oxidative burst to the left of the dividing line in the lower right panel. This pattern can be tricky to recognize and should be correlated with clinical history and again confirmed by genetic testing.

It would be apropos to indicate here that there can be a number of confounding factors when interpreting the flow results for the DHR assay for neutrophil oxidative burst – age of sample (particularly if it is >48 hours old from the time of blood collection), transport conditions (since neutrophils are exceedingly labile in their activation status and proneness to early cell death, a shipping control is essential for any sample sent from outside the location of the testing laboratory), and presence of intrinsic neutropenia (this test should not be ordered in patients with profound neutropenia and rather other tests to evaluate for congenital or acquired neutropenias should be ordered).

This flow result for neutrophil oxidative burst on a young female patient reveals normal neutrophil function (ie oxidative burst) as evidenced by the fluorescence on activation in the lower right panel. The patient has a sister with a confirmed p47phox (NCF1 gene) mutation and autosomal recessive CGD. Genetic testing for the NCF1 gene in this patient revealed the presence of a heterozygous mutation, yet the neutrophil oxidative burst by flow was normal. This is very important to bear in mind – carriers of AR-CGD cannot be diagnosed by the flow cytometry method like carriers of X-linked CGD. Carriers of AR-CGD have one perfectly normal allele, which results in normal neutrophil oxidative burst and since 2 copies of the mutant allele are required for the clinical phenotype and failure in neutrophil function there is no evidence by flow cytometry of an aberrant population of neutrophils. Therefore, carriers of AR-CGD can only be identified by genetic testing of the target gene.

Laboratory Test Ordering Information

This slide provides information on ordering the tests described in this presentation including the DHR flow assay which is performed at Mayo Clinic. Genetic testing for all the 4 genes associated with CGD is available through Gene Dx Labs, while there are other academic and reference labs that provide testing for the CYBB gene, which accounts for 70% of CGD cases.


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