CHRONIC GRANULOMATOUS DISEASE CGD Pathogenesis and Diagnosis

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CHRONIC GRANULOMATOUS DISEASE (CGD)Pathogenesis
and Diagnosis
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Topics to be considered

• Review of NADPH Oxidase Pathway
• Mutations in CGD Patients
• Pathogenesis in CGD and clinical characteristics
• Diagnosis of CGD
• Treatment of CGD
• Case Study

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Chronic Granulomatous Disease

• Rare (1/200,000) inherited disorder of the
  reduced nicotinamide dinucleotide phosphate
  oxidase complex (NADPH) - may be higher
• Phagocytes defective in production of
  microbicidal reactive oxidant superoxide anion
  and metabolites (hydrogen peroxide, hydroxyl
  anion, hypohalous acid
• Patients suffer from life-threatening bacterial
  fungal infections. Dramatic inflamatory response
  leads to granuloma formation

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CGD - History

• First described in 1957 by Good et. al. As fatal
  granulomatous disease of childhood
• CGD granulomas had similarities to other
  granuloma forming diseases (Wegener
  granulomatosis, brucellosis, sarcoidosis,
  Hodgkins disease, etc.)
• Combination of recurrent suppurative and
  inflammatory complications in childhood
  recognized - distinct clinical syndrome
• Originally thought to be X-linked as only
  diagnosed in boys

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History continued

• 1960s CGD established as a disease of phagocytes
• Neutrophils exhibit normal phagocytic activity
• Bacteriocidal activity against S. aureus
  impaired
• 1990s to present - Actual defects elucidated
• CGD genetically heterogeneous, caused by mutation
  in any one of four structural components of NADPH
  oxidase complex
• Mouse knockout models created
• Gene therapy trials now underway

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NADPH Oxidase derived Reactive Oxidant Pathways

NADP
H

NADPH
e-
Cytoplasmic space
NADPH Oxidase Complex
FAD
Phagocytic Cell Membrane
Phagocytic Cell Membrane
Heme
Heme
H2O
Vaculolar and/or Extracelluar space
catalase
O2

e-
Superoxide anion
O-2
SD
H2O2
O2
NO
peroxynitrite anion
Fe2
Cl-
OOON-
Fe3
MPO
Fenton Rxn
hydroxyl anion
hypohalous acid
OH-
HOCl
NO2-
nitryl chloride
NO2Cl
PATHOGENS
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Cytoplasmic space
NORMAL ACTIVITY
Second Activation signal sequence
FAD heme 1 heme 2
RhoGDI
gp91phox
Rac
Phagocytic Cell/Granule Membrane
p22phox
NO2Cl
H2O2
e-
OH-
OOON-
Vaculolar and/or Extracelluar space
HOCl
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Current Cellular Phenotype Genotype in CGD
Genotype Freq Cellular Defect
Phenotype X-linked X910
Flavocytochrome - Not detectable ( missing
gp9l phox ) X91-
Flavocytochrome - Decreased quantity X91
Flavocytochrome - Normal
quantity (defective gp9l phox) Autosomal Recessiv
e A- p22 phox Flavocytochrome
- Not detectable ( missing p22 phox ) A p22
phox Flavocytochrome - Normal quantity
(defective P22 phox) A p47 phox p47 phox -
Not detectable A p67 phox p67 phox
- Not detectable
70
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20
5
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Mutations in CGD

• In X91 103 specific mutations identified in 13
  exons of gp91phox CYBB gene
• 11 deletions
• 24 frameshifts
• 23 nonsensense
• 17 splice region
• 23 missense
• 2 regulatory region
• Approximately 90 come from X linked carriers
• 10 from de novo mutations
• X linked CDG Heterogeneous spectrum of
  mutations
• Similar heterogeneity for the p22 phox gene CYBA
  and p67 phox CGD gene NCF2.
• In contrast, p47 phox CGD - majority due to GT
  deletion (exon two in NCF1 gene) - frameshift
  yielding premature stop codon at amino acid 51.

Usually leads to X910
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Cytoplasmic space
Second Activation signal sequence
RhoGDI
Defective gp91phox
FAD heme 1
Rac
Phagocytic Cell Membrane
e-

p22phox
NO2Cl
NO2Cl
Insufficient Inhibition
Insufficient Inhibition
H2O2
H2O2
OH-
OH-
OOON-
OOON-
HOCl
Vaculolar and/or Extracelluar space
HOCl
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Pathogens in CGD

• Recurrent infections with catalase producing
  pathogens (Staph. Aureus, Aspergillus sp. )
• Catalase negative pathogens can not degrade their
  own H2O2 providing exogenous source hypohalous
  acid formation
• Other factors besides catalase
• Not susceptible to all catalase organisms
• CGD knockout mice - Catalase-deletant strains of
  Aspergillus nidulans as virulent as wild type

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Pathogens in CGD
Bacterial Presentation Staphylococcus
aureus Soft tissue infection, lymphadenitis,
liver abscess, osteomyelitis, pneumonia,
sepsis Burkholderia (Pseudomonas)
species Pneumonia, sepsis Serratia
marcescens Pneumonia, osteomyelitis, sepsis,
soft tissue infection Nocardia
species Pneumonia, osteomyelitis, brain
abscess Chromobacterium violaceum Soft tissue
infection, sepsis
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Pathogens in CGD
Fungal Presentation Aspergillus
species Pneumonia, osteomyelitis, brain
abscess Paecilomyces species Pneumonia, soft
tissue infection, osteomyelitis Phaeohyphom
ycete species Pneumonia, soft tissue
infection (dark-walled fungi) Penicillium
species Pneumonia, soft tissue
infection Miscellaneous filamentous
fungi Sepsis, soft tissue infection, liver and
yeasts abscess (Zygomycete, Acremonium, Candid
a )
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Infection in CGD

• Often do not have typical signs of infection
• Life threatening infections - asymptomatic (33)
  or mild nonspecific symptoms (20 febrile)
• Leukocytosis in lt50
• Elevated Sedimentation rate in lt50
• Pulmonary infiltrate on routine screening chest
  X-ray - often 1st sign

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Inflammatory Complications in CGD

• Chronic inflammation
• skin ulceration, pneumonitis, inflammatory bowel
  disease.
• Exuberant persistent tissue granuloma
  formation
• Mechanism of abnormal inflammation - largely
  unknown
• Inability of CGD neutrophils to inactivate
  proinflammatory chemoattractants via reactive
  oxidants

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Laboratory Diagnosis
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CGD by Flow Cytometry

• Small whole blood sample is stimulated to undergo
  the oxidative burst with phorbol myristate
  acetate in the presence of Dye.
• Dihydrorhodamine123 when oxidized is fluorescent.
  
• Fluorescence is quantitated. Results expressed as
  ratio of Fl. Stimulated over Fl. Unstimulated.
• CGD test by Flow Cytometry can detect CGD
  patients, carriers, and can suggest the genotype
  of the CGD patient.

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Case Study
Healthy Control
Patient (AD) Twin 1
Patient (JD) Twin 2
Unstimulated
Stimulated
NOI 1,614
NOI 1
NOI 2
Neutrophil oxidative index (NOI) gt 100 Normal
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Case Study -Family
Healthy Control
Patients Sibling (male)
Patients Mother
Unstimulated
Stimulated
NOI 930
NOI 0.9
NOI 16, 900
Neutrophil oxidative index (NOI) gt 100 Normal
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Treatment of CGD

• Prophylaxis with antibiotics
• trimethoprim-sulfamethoxazole
• itraconazole
• Interferon-gamma
• does not increase NADPH oxidase (1st yes, 2nd
  large NIH study - no)
• Augmentation of oxidant-independent antimicrobial
  pathways
• TNF, granule protein synthesis, MHC II
  expression, FC gamma receptors - all increased

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Treatment of CGD

• Granulocyte transfusion
• Has been used in life threatening infections
• No prospective studies
• Many possible adverse reactions
• Bone marrow transplantation
• Curative 60 of time
• Due to morbidity and mortality - not routine use
• May be revisited with stem cell transplantation

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Treatment of CGD

• Gene Therapy
• Ideal candidate for hematopoietic stem cell gene
  therapy
• engraftment of stable normal myeloid stem cell is
  curative
• only 3-10 normal neutrophils required for normal
  protection

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Treatment of CGD

• NIH Gene Therapy Clinical Trial
• 5 patients with p47phox deficient CGD
• Autologous CD34 peripheral blood stem cells
• transduced in vitro with retrovirus vector
  containing p47phox cDNA
• Infused without bone marrow conditioning
• .004 to .05 circulating corrected granulocytes
• 2 of 5 patients had detectable corrected
  granulocytes after 6 months

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