Chronic granulomatous disease

Chronic Granulomatous disease
Jintana Chataroopwijit
10 Feb 2017

Content
• Introduction
• Function of NADPH oxidase
• Molecular genetic aspect
• Clinical manifestation
• Diagnosis and investigation
• Treatment

Introduction
• First described in the 1950s
• Disorder of phagocytic cells leading to recurrent
infections
• Deficient superoxide (O2–) generation via the
phagocyte nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase system
• Incidence : 1/200,000–250,000 live births in USA
and Europe
Clinical Immunology: Principles and practices 2008
Pediatr Allergy Immunol 2016: 27: 242–253.

Introduction
• Inherited in X-linked and autosomal recessive
patterns
• Common organisms : catalase-positive bacteria
and fungi
• Presentation : pneumonia, liver abscess, skin
infections, lymphadenitis or osteomyelitis,
bacteremia(uncommon)

NADPH oxidase : function and disease
• NADPH oxidase : multicomponent enzyme complex
containing both cytosolic and membrane-bound
proteins
• To produce reactive oxygen species (ROS) in
activated phagocytes (neutrophils, monocytes, and
macrophages)

Schematic representation of the NADPH oxidase system. Chemoattractants interact with their receptors on the neutrophil surface, leading to an
increase in intracellular calcium concentration. This activation results in the assembly of the NADPH oxidase complex following phosphorylation of
cytosolic factors. This is turn leads to superoxide production. DAG, diacylglycerol; PIP2, phosphatidylinositol bisphosphate; IP3, inositol
triphosphate; α, β, γ, subunits of the GTP-coupled receptors.

Phagocytosis and killing of microorganisms by phagocytic leukocytes
British Medical Bulletin, 2016, 118:53–66

• Catalytic core of phagocytic NADPH oxidase consists
of flavo-cytochrome b558 (transmembrane protein)
1. Catalytic glycosylated gp91phox : cell membrane
was stabilized by p22phox
2. Nonglycosylated p22phox
• Attachment of cytosolic components of the NADPH
oxidase: p47phox, p67phox, p40phox, and GTPase-
Rac

• During the phagocytic NADPH oxidase activation
• phosphorylation of p47phox leads to conformational
changes --> allowing interaction with p22phox
• resultant membrane translocation of p47phox with the
activation of GTPase-Rac assembles the other
cytoplasmic subunits p40phox and p67phox to form the
active oxidase complex

• Extracellular superoxide anions (O2 ) and
hydrogen peroxide (H2O2) are produced in the
phagolysosome (respiratory burst)
• ROS and proteins from granules mediate together
the killing of microorganisms

• Underlying mechanisms of hyperinflammation
• defective neutrophil apoptosis
• skewed nuclear factor-kB signaling
• upregulation of tumor necrosis factor alfa, interleukin (IL)-17, IL-6,
and granulocyte colony-stimulating factor
• prolonged IL-8 messenger RNA activation
• impaired activation of Nrf2
• decreased autophagy
• increased inflammasome activation

Hyperinflammation
• Deficient autophagic antimicrobial defence --> excessive
IL-1beta release
• Deficient regulation of autoreactive T cells
Brazilian Journal of Medical and Biological Research (2014) 47(11): 924-928,

Hyperinflammation : Autophagia
Step of autophagia

Hyperinflammation : Autophagia
Reciprocal effects of autophagy on inflammasome activity

T cell activation
• Defect in regulation of T-lymphocyte activity
• P47phox knock-out mice develop more severe arthritis
than wild type animals when challenge with collagen-
specific T cells

• Neutrophil extracellular traps (NETs)
• externalized chromatin (DNA and histones)
• cytosolic/granular proteins and may
• Bind bacteria and fungi and expose antimicrobial
molecule --> control bacterial and fungal infection
• No direct link between ROS produced by NADPH
oxidase and NET formation
Journal of infection 2014: 69:s32-s35

• Bustamante et al. : germline mutations in the gp91phox-
encoding gene (CYBB) conferring the phenotype of X-
linked recessive mendelian susceptibility to
mycobacterial disease type 2 (XRMSMD) syndrome, but
not XR-CGD
• XR-MSMD selectively affects the monocyte derived
macrophages and B cells, but not monocytes or
granulocytes

• May be some relation between the gp91phox protein and MSMD,
indicating that the macrophage respiratory burst is involved in
protection against mycobacterial infections
• Low levels of proinflammatory cytokines, particularly interferon
gamma (INF-c), probably induce a defect in the IL-12/INF-c axis,
which has a crucial role in protective immunity to mycobacteria

Molecular genetic aspects of CGD
• Reports from Europe, USA and Japan : XR-CGD most common form
(60%)
• 30% of AR-CGD : p47phox deficiency
• 10% p22phox and p67phox deficiency (each about 5%)
• Iranian and Turkish cohorts : AR predominance (consanguineous
marriages)

• Mutations : small intragenic deletions/insertions, missense,
non-sense, and splice-site mutations  easily detected by
sequencing
• Uncommon genetic mutation : large deletions and duplications
(difficulty to detect multiple exon deletions/insertions with
conventional methods )

• Conventional methods : sequencing, PCR-SSCP, Southern blot
• Recently
• Array Comparative Genomic Hybridization (array CGH)
• Multiplex ligation dependent probe amplification

• Stasia et al : 2 interesting cases of male XR-CGD
• 5.7-kb duplication spanning exons 6 and 8 of CYBB gene
• deletion of the same region
• Both mutations : probably caused by a single event of nonhomologous
meiotic or mitotic crossing-over between two high-similarity short-tandem-
gene therapy (GT) repeats of introns 5 and 8

X-linked CGD
• The most common : mutations in the gene encoding
gp91phox (CYBB) at Xp21.1
• Mutation : frame shifts, nonsense mutations, missense
mutations, splice region mutations, large and small
deletions and regulatory region mutations
• Patients with large interstitial deletions, adjacent genes
are affected —> McLeod syndrome, Duchenne muscular
dystrophy, or X-linked retinitis pigmentosa

X-linked CGD
• Clinical phenotype of McLeod syndrome
• Compensated hemolysis
• Acanthosis
• Progressive neurodegenerative symptoms : areflexia,
dystonia and choreiform movements
• Absent erythrocyte Kx protein and diminished levels of
Kell blood group antigens  all X-linked CGD
patients should be carefully screened for Kell blood
groups

Autosomal dominant CGD
• 35% of all cases
• 25% Mutations in gene for p47phox (NCF1, located at 7q11.23)
• 6% Mutations in the gene encoding p22phox (CYBA), located at
16q24
• <5% Mutation in the gene for p67phox (NCF2, located at 1q25)

Gene involve in CGD and related CGD
Indian J Pediatr (April 2016) 83(4):345–353

Properties of the NADPH oxidase components
and mutations in CGD patients

Carrier and genetic counseling
• Prenatal diagnosis : sequencing on fetus mutation
• Recently, preimplantation genetic diagnosis combined
with HLA typing

Clinical manifestation
• Infection
• Inflammatory complication

Clinical manifestation : infection
• Bacterial infection : catalase positive
• Staphylococcus aereus
• Burkholderia cepacia
• Serratia marcescensu
• Chromobacterium violaceum
• Granulobacter bethesdensis
• Fungal infection : Aspergillosis
• Tuberculosis and atypical mycobacterial infection

catalase positive organism and
respiratory burst
• Bacteria can produce H₂O₂ by itself
• H₂O₂ can then be converted in to HOCl by MPO arm to
kill catalase +ve organisms
• NADPH oxidase & Myeloperoxidase function : get rid of
H2O2 which categorized under reactive oxidative
species (ROS)
• Catalase degrades H2O2 into H2O and O2

Mechanism of respiratory burst
NADPH oxidase
• O2 ----------------------> O2-( superoxide inoin)
Superixide dismutase(SOS)
• O2- ---------------------------------> H2O2 (Hydrogen peroxide)
Myeloperoxidase
• H2O2 -----------------------> HOCL (highly toxic to bacteria)

Clinical manifestation :
fungal infection
• Aspergillus spp
• A. fumigatus
• A. flavus
• A. niger
• A. nidulans
• Norcardia spp.

• Site of infection
• Pneumonia
• Liver : abscess
• Skin infections/abcess
• Lymph node
• Bone : osteomyelitis
• Bacteremia : uncommon
Clinical manifestation : infection

Clinical manifestation
USA
2000
European
2009
Indian
2014
Pneumonia 79% 66% 82%
Lymphadinitis
Lymphadinitis 53% 50%
Cutaneous
infection
52%
Subcutaneous
abcess
53% 47%
GI involvement 48% 23%
Hepatic abcess

Clinical manifestation : autoimmunity
and inflammatory complication
• Hyperinflammatory condition
• Granulomas : obstruction
• Colitis/enteritis : abdominal pain, diarrhea, rectal bleeding
• Non-infectious arthritis : Rheumatoid arthritis
• Other autoimmune disease

Clinical manifestation : autoimmunity
and inflammatory complication
• Other autoimmune disease
• Discoid lupus
• SLE
• Dermatomyositis
• Sacroiliitis
• Idiopathic thrombocytopenia
• Autoimmune hepatitis
• Hemophagocytic lymphohistiocytosis (HLH) : Burkholderia
cepasia and Leishmania

Diagnosis
• History of severe infection
• Absence of respiratory burst
• Inability of neutrophils to reduce nitroblue tetrazolium
dye or to oxidize dihydrorhodamine

NBT Reduction Test
• In 1967, Baehner and Nathan : leukocytes in patients with CGD
failed to reduce NBT dye due to defective oxidase --> unable to
utilize oxygen and produce reactive species
• NBT : yellow dye ----- > blue formazan
• CGD patients fail to reduce NBT : remain yellow
• X-linked carriers : demonstrate both colors (two populations of cells)
are present
Reduction

NBT reduction test

Dihydrorhodamine (DHR) Test
• Neutrophils are stimulated with Phorbol-12-Myristate-13 Acetate (PMA)
to produce reactive oxygen species such as hydrogen peroxide
superoxide
• Dihydrorhodamine ———————-> rhodamine 123
• Change in fluorescence intensity : flow cytometer

• Reliable diagnosis : cases and carriers of X-linked
• Carriers of autosomal recessive forms of CGD cannot be detected by DHR
as one normal allele is sufficient for adequate function
• Expression of protein subunits of NADPH oxidase on neutrophils can be
studied by flow cytometry : classifying the type of CGD
• Accepted worldwide as the screening test of choice for CGD

histogram, stimulation index
Indian J Pediatr (April 2016) 83(4):345–353X : fluorescence intensity
Y : number of cell

Treatment

Curative treatment
• Hematopoietic stem cell transplantation
• Gene therapy

Hematopoietic stem cell transplantation
• First bone marrow transplant was performed in CGD in 1973
• Since the beginning of the 21st century : HSCT can cure CGD and reverse
organ dysfunction
• Keypoint
1)Age and indication : NADPH oxidase activity
2)Kind of donor to select
3)Conditioning regimen : myeloablative conditioning, reduced-intensity
conditioning regimens

• Indication
• one or more life-threatening infections
• non-compliance with antimicrobial prophylaxis
• steroid-dependent autoinflammation
• In adolescensce: no organ dysfunction

• In 2014, Gungor et al.
• Large prospective multicenter study : 56 patients, aged 0–40 years
(median age: 13 years)
• 42/56 high risk patients, not be candidates for myeloablative HSCT
• Using a reduced conditioning protocol
• Results : 2-year overall survival of 96% and event-free survival of 91%
• The cumulative incidence
• severe acute GVHD grades III–IV : 4%
• chronic GVHD was 7%
• Stable myeloid donor chimerism was found in 93% of surviving patients
• 2 adult patients able to have children : preserve fertility

• Best donors : matched sibling bone marrow (MSD) but low
incidence
• Other : perfectly HLA-matched unrelated donors (MUD) are
almost comparable with those using HLA-MSDs
• Umbilical cord blood could be an option for these patients but
available results concerning CGD patients are limited
• At present : all patients with XR-CGD or AR-CGD with no
residual oxidase activity should be offered HSCT as early as
possible, with either an MSD or MUD

Autologous gene therapy
Curr Allergy Asthma Rep (2016) 16: 39

Gene Therapy for X-Linked Chronic
Granulomatous Disease (X-CGD)
• First clinical trial : National Institutes of Health and Indiana
University
• Gamma retroviral vectors to deliver normal human gp91phox
• No severe adverse events and gene-marked cells were detectable
at low levels for months after treatment
• Superoxide levels were not sufficient to produce significant clinical
improvement

• Three patients were treated at the NIH using reduced intensity
conditioning prior to infusion of gp91phox vector-treated HSCs
• Functionally corrected cells dropped from 24 to 1.1 % at 34 months post
therapy
• Resolution of infections at this low level of transgene expression
• Small amount of normal superoxide production may have protective
clinical outcomes
• Levels were 0.03 % in a second patient who had partial control of
infections at 11 months follow-up and undetectable in the third who
died of invasive fungal infection 6 months post treatment

• 9 X-CGD patients (Frankfurt, Zurich, London, and Seoul)with similar
gamma-retroviral vectors and reduced intensity conditioning
• Clinical benefit in all of these patients
• Over expression of EVI1 led to the development of myelodysplasia with
monosomy seven in two patients
• Gradual loss of functional gene-marked cells at around 8 months after gene
therapy due to epigenetic inactivation of the retroviral promoter with no
leukemogenic events, there was also no significant engraftment after 3
months post gene therapy

• Two parallel gene therapy trials in Europe and the USA based on
these rationales with safer lentiviral vectors
• 1 child with significant disease burden (invasive liver, brain,
abdominal, and pulmonary infections, and inflammatory
complications) treated in Europe
• He was stable until around 3 months post gene therapy, but then
died from respiratory complicationsdue to pre-existing
conditions
• The US trial is currently open and is anticipating treatment of the
first patient by the end of 2015

Ongoing gene therapy clinical trials
HUMAN GENE THERAPY 26:210–219 (April 2015)

Gene therapy

Gene therapy
• Several researchers are involved in gene therapy trials in CGD
world over and this is an active field of research
• Therapy in CGD patients as genes involved in NADPH oxidase
are metabolic genes and are not involved in cellular proliferation
• At present, gene therapy continues to be in an experimental
phase

Survival rate : from past to present
• Change over last two decades : 90% reaching well into adult
• Better recognition, improvement of awareness, effectiveness of
antimicrobial and HSC transplantation
• Major cause of morbidity and mortality : Infection
• Median age of the patient
Before 1991 : 15.53 years
2001 : 20.17 years
2012 :28.12 years

Chronic granulomatous disease

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