X-linked Agammaglobulinemia: Clinical Manifestations and Diagnosis

X-linked Agammaglobulinemia
Lalita Tearprasert ; M.D.
11 September 2015

• Introduction
• Epidemiology
• Pathogenesis
• Clinical manifestations
• Diagnosis
• Management
• Prognosis
Scope

Introduction
• First recognized human immune deficiency
• Discovered in 1952 by Colonel Ogden Bruton; Agammaglobulinemia
- Case report 8-year-old boy, recurrent infections over a 4-year period
- Majority of infections: pneumococcus
- Bruton attempted to vaccinate him against this pathogen
>> no gamma globulin was produced
- Treated with monthly intramuscular injections of human gamma globulin with
significant clinical improvement
- No family history
- Subsequent cases revealed a similar clinical phenotype with
an X-linked pedigree
Thomas A.E. Plattes-Mills. Middleton's Allergy 8th Edition. 1144-74.
Bruton OC. Agammaglobulinemia. Pediatrics 1952; 9:722±7.

Epidemiology
• Incidence and prevalence
- Unknown because general population screening for the
disorder is not done (1/3 new mutation)
- Prevalence of 1/10,000 in the general population
- Varies in many country
ex. 1/379,000 in USA, 1/100,000 in norway
• Only Male
Sigmon J. et al.Clinical and Molecular Allergy 2008, 6:5.
Thomas A.E. Plattes-Mills. Middleton's Allergy 8th Edition.1144-74 .

P. Benjasupattananan. et al. J Clin Immunol (2009) 29:357–364.
In Thailand
• Reviewed medical record
in the past 18 years at Siriraj Hospital
• 67 patients (44 males, 23 females)

Notarangelo D.et al. J Allergy Clin Immunol 2009;124:1161-78.
Notarangelo D.et al. J Allergy Clin Immunol 2010;125:S182-94.
85
%
5
%
• 2 forms genetic in agammaglobulinemia
1.) X-linked agammaglobulinemia (XLA) -- 85%
2.) AR agammaglobulinemia (ARA) -- 10-15%
Agammaglobulinemia

• XLA caused by mutations in the human BTK gene
• In1993, two groups of investigators independently and almost
simultaneously discovered the mutated gene in XLA.
- The European group called atk gene
(agammaglobulinemia tyrosine kinase)
- The American group called bpk gene
(B-cell pro-genitor kinase)
A compromise was reached with the term
"Btk (Bruton's tyrosine kinase)" in honor of Dr. Bruton
Pathophysiology
Conley ME, et al. Immunological Reviews 2005. Vol. 203: 216–234.
Vetrie D, et al. Nature1993;361:226–233.
Tsukada S, et al. Cell 1993;72:279–290.

BTK gene
• Contains 19 exons and spreads over 37 kb of DNA
• Located on Xq 21.33-q22.
• Consists of 659 amino acid residues
• BTK gene provides instructions for making a BTK protein
http://ghr.nlm.nih.gov/ A service of the U.S. National Library of Medicine. February 2012.

• Intracellular signal transduction molecules
• 75 kDa cytoplasmic protein tyrosine kinase
(Member of Tec family)
• Expressed in many hematopoietic lineages
except in T cells and NK cells
- High levels in all B lineage cell except plasma cells
- Myeloid cells and platelets
• Human BTK shares 99% aa sequence identity with
mouse and rat BTK.
BTK protein
Kanegane et al. J Allergy Clin Immunol Volume 108, Number 6.

• BTK protein consists of 5 functional domains
- pleckstrin homology (PH) domain
- Tec homology (TH) domain
- Src homology 3 (SH3) domain
- Src homology 2 (SH2) domain
- Catalytic kinase (SH1) domain
• Catalytic activity resides in the kinase domain while the
other domains are necessary for protein-protein interactions.
• Mutations in all domains of the BTK gene have been shown to cause XLA.
Mutations are scattered throughout the gene.
Hendriks RW. Nature Reviews Cancer 14, 219–232 (2014).
H. B. GASPAR & M. E. CONLEY. Clin Exp Immunol 2000; 119:383±389.

Wang LD. et al. Immunology 2003;110411-20.
• BTK required for pre-BCR signaling
• BTK is typically found in the cytoplasm but moves to the
membrane during B-cell activation, where upon it is phosphorylated by a number of different kinases.
• Association of the BTK SH2 domain with the B cell linker protein (BLNK) is required for the
activation of PLC gamma by BTK

• In turn, BTK then phosphorylates a range of downstream targets, ultimately resulting in
the activation of cellular pathways that govern vital B-cell processes.
Hendriks RW. Nature Reviews Cancer 14, 219–232 (2014).
H. B. GASPAR & M. E. CONLEY. Clin Exp Immunol 2000; 119:383±389.

• BTK was found to be already expressed in very early stages of B cell differentiati
chain gene rearrangements
• The loss of btk expression coincides with the loss of cell division capacity
and of several membrane B cell antigens, such as sIg, CD19, CD20,
CD22, CD24, CD40 and CD72
• In very early stages of B cell development the btk gene product may
be present in an inactive form which is activated, e.g. by phosphorylation,
in the pre-B cell stage through interactions with other B-lineage
signalling molecules.
• BTK protein is absent in most patients with XLA
De Weers M. et al. Eur J Immunol. 1993;23:3109-14

• More than 600 different mutations in the BTK gene have been found.
• 80%–90% have identified mutations
- 90% : Single base pair substitution & insertion or deletion < 5 base pair
10%–20% not clear ( have mutations that are not easily detected
or have disorders that are phenotypically similar to XLA but
are caused by different gene defects )
• Mutations in BTK are highly variable.
• No clear correlation has been found between mutation location and
clinical phenotype.
Conley et al. Am. J. Hum. Genet. 62:1034–1043, 1998.
http://ghr.nlm.nih.gov/ A service of the U.S. National Library of Medicine. February 2012.
BTK gene mutation

• 55% of males have no family history of XLA
2 possibilities exist:
1.) De novo causing mutation : 15%-20% of cases
2.) Mother is a carrier of a disease-causing mutation : 80%-85% of cases
• Female carriers of XLA can be identified by the presence of either
nonrandom X chromosome inactivation in their B cells or the
mutated gene (if known in the family)
Pagon RA.,et al. GeneReviews.

Wattanasirichaigoon D.et al. J Hum Genet (2006) 51:1006–1014.
• First report of mutations of BTK from Thailand
• 4 novel and 3 known BTK-mutations (6 Thai and 1 Burmese)
• 6 were sporadic

B cell maturation
Pieper K., et al. J Allergy Clin Immunol 2013;131:959-71.
ARA XLA

• X-linked recessive : Male
• Asymptomatic during the first few months of life because of the
protection by maternally derived IgG antibodies.
• Onset around 4-6 months
• Recurrent bacterial infections, particularly otitis media, sinusitis
and pneumonia, in the first 1 years of life. (60-80%)
Clinical manifestations
Practice parameter for diagnosis and management of primary immune deficiency. AAAI. Volume 94, May 2005.
Thomas A.E. Plattes-Mills. Middleton's Allergy 8th Edition.1144-74.

• May be systemic (e.g., meningitis or septicemia) or involve mucous
membrane surfaces (sinusitis, pneumonia, otitis, conjunctivitis, or
gastrointestinal and urinary tract infections), joints (septic arthritis), or
skin (cellulitis or abscesses)
• Bronchiectasis or persistent enteroviral infections develop
• Growth and development usually are normal
• Autoimmune disorders do not seem to be a frequent problem in
patients with XLA, unlike in patients with CVID.
Ballow M. J Allergy Clin Immunol. April 2002.
Trakultivakorn and Ochs. Asian pacific journal of allergy and immunology (2006) 24: 57-63.

• Majority of patients are diagnosed < 5 years of age
(Mean 35 months)
• 20% diagnosed within the first year of life

Plebani A. Clinical Immunology Vol. 104, No. 3, September; 221–230, 2002.
73 males

• 6 patients with XLA
• Mean age of onset was 2.5 years
• Mean age at diagnosis was 7.3 years
• Pneumonia being the predominant
manifestation
• Bronchiectasis 5/6
• Most H.influenzae
f

• Predisposition for malignancy : less clear
- Filipovich and Shapiro reported that 4.2% of patients in an
immune deficiency cancer registry had XLA. Lymphoreticular
and gastrointestinal malignancies were the most common
Physical examinations
• Tonsils, adenoids, and peripheral lymph nodes are very small because
of the absence of germinal centers and fail to undergo normal
hypertrophy in response to infection.

• Rarely, patients with XLA also have a short stature caused by
growth hormone deficiency.
• A newly discovered mutation in myeloid elf-1–like (MEF gene)
• Only known to occur in a single family, shares many features with XLA
• These patients must be distinguished from patients with XLA who have poor
growth secondary to malnutrition.
• 2/4 patients (II-4 and III-3)
- recurrent otitis media, rhinitis,
and conjunctivitis
• 1 pateint (II-3) monoarticular arthritis
• The youngest (III-4) asymtopmatic
• pan-hypogammaglobulinemia and
isolated growth hormone deficiency
Stewart M.et al. Immunol Res (2008) 40:262–270.

• Mild or "leaky" phenotype (Rare : Late onset)
- less severe disease, lack repeated infection in childhood
- The block in B-cell differentiation may be leaky, resulting in
some Ig synthesis (B cells > 2%)
- Ddx : CVID may also present with profound hypogammaglobulinemia
and markedly reduced numbers of B cells.
Molecular analysis for mutations in BTK is necessary to distinguish
CVID from patients with XLA.
Usui K, Sasahara Y, Tazawa R, et al. Respir Res 2001.

• The case presented here is the only adult-onset case of the 107 cases
in the XLA registry of the Ministry of Health and Welfare, Japan
• patient's BTK gene was sequenced
• found to have a 2 base pair (AG)
deletion in the codon for Glu605 (AGT)
in exon 18 that encodes a part
of the kinase domain of the BTK protein.
Usui K, Sasahara Y, Tazawa R, et al. Respir Res 2001.

• A 51-year-old male presenting with chronic nasal congestion,
recurrent sinusitis,sporadic pneumonia,and pronounced B cell
deficiency.
• A family history suggestive of an XLA was noted.
• A mutation(Cys 145 --> StoP)was identified in Btk
cDNA and was confirmed in amplified exon6 of genomic DNA
from both the proband and an affected nephew.
MolecularMedicine,Volume2, Number5, September1996.

• 10-month-old Japanese girl
• Frequent respiratory infections and otitis media during the last 2 months.
• Nonconsanguineous parents
• Her father was diagnosed XLA at the age of 3 years , Healthy mother
• Low serum immunoglobulin G (IgG), IgA, and IgM
• Found a mutation in the first single base pair of intron 11 (G >A)
of BTK gene in her father and heterozygous mutation in the patient
at the same site
• Patient was diagnosed as having XLA associated with a defect of BTK
caused by heterozygous abnormality of the BTK gene and nonrandom X
inactivation of maternally derived X chromosome in which normal BTK gene
is located
Takada H. et al. Blood 2004;103:185-7.

- Encapsulated organisms
(pneumococci, streptococci, and Haemophilus influenzae)
- High-grade pathogens
(meningococci, staphylococci, Pseudomonas organisms,
and various species of Mycoplasma)
- Resistance to viral infection is usually normal, except
>> enteroviruses (coxsackie, echovirus) that may result in
vaccine-related paralytic poliomyelitis or a dermatomyositis-
meningoencephalitis syndrome
>> hepatitis viruses
>> poliovirus
- Giardia, Cryptosporidium cause diarrhea
- Ureaplasma and Mycoplasma cause jount infections, urogenital tract
Pathogens

Diagnosis
• Family history
• Clinical manifestations
• Laboratory
- Intermittent neutropenia can occur, particularly at
onset of an acute infection.
- Low serum IgG, IgM and IgA level
- Peripheral blood CD19 B-cell counts < 2%
Thomas A.E. Plattes-Mills. Middleton's Allergy 8th Edition.1144-74.

Pagon RA.,et al. GeneReviews.
• Gene mutation
1.) Prenatal diagnosis also has been accomplished by detection
of the mutated gene in chorionic villus or amniocentesis samples
2.) Confirmed by demonstrating
- absence of BTK protein in monocytes or platelets
- detection of a mutation in BTK in DNA

• 10% to 15% of agammaglobulinemic patients
• Clinically indistinguishable from XLA
• Either sex
• Suspected in
- Female patients with agammaglobulinemia
- Families with an autosomal recessive pattern of inheritance
- Consanguinity
- Agammaglobulinemic male patients in whom BTK mutations
cannot be identified
AR agammaglobulinemia

• Mutations in the genes encoding immunoglobulin heavy or light chains
or their associated signaling molecules lead to agammaglobulinemia
or hypogammaglobulinemia
• Most defects in the μ heavy chain (IGHM) gene (chromosome 14)
• Early onset infections, tend to be younger at the age of diagnosis and
and more complications than XLA
• B cells are absent in their peripheral blood, while most XLA
patients have a small number of circulating B cells
Yel L.et al. N Engl J Med 1996; 335:1486-93.
Lopez Granados E. et al. J Clin Invest. 2002;110:1029-35.

• Defining the genetic etiology of antibody deficiencies is
important for subsequent genetic counseling, carrier detection,
and prenatal diagnosis.
• Early diagnosis and treatment would improve the survival.
• Intravenous immunoglobulin (IVIG) and antibiotic prophylaxis
were the conventional treatments which resulted in an increasing
survival rate.
• Gene therapy of XLA could be considered as a paradigm for future.
Treatment
P. Benjasupattananan. et al. J Clin Immunol (2009) 29:357–364.
Moreau T.et al.Current Gene Therapy.Volume 7, Issue 4;284 - 294.

• 1950s, 1960s, and 1970
- Treated with plasma therapy or intramuscular gamma globulin,
therapies that provided only low concentrations of serum IgG.
- The majority of patients died with an acute infection,
chronic lung disease or enteroviral encephalitis before they
reached adulthood.
• Mid1980s, IVIG came into common use.
- Achieve normal or near normal concentrations of IgG.
- When coupled with aggressive use of antibiotics, IVIG has markedly
improved the outcome of patients with XLA.
IVIG

• IVIG at a dose of 400-800 mg/kg every 3 to 4 weeks.
• Keep trough IgG > 500 mg./dl.
• Trough serum IgG levels > 800 mg/dL may be necessary
to fully prevent chronic sinusitis, bronchiectasis, and enteroviral
infections.
• Despite receiving IVIG, some patients develop persistent
enteroviral infections or crippling sinopulmonary disease,
because no effective means exists for replacing secretory IgA
at the mucosal surface.

• Patients may benefit from prophylaxis, although there are no controlled
studies that provide evidence for their use and wide variations in
practice are observed
• Typically SMX-TMP
(United States used in approximately 50%, Europe in 14%)
Antibiotic
prophylaxis
Kuruvilla and De la Morena. J Allergy Clin Immunol Pract 2013.
• Chronic antibiotic therapy in addition to IVIG infusions can be
effective for management of pansinusitis or bronchiectasis

Vaccination
Immune deficiency foundation. Volume 1 Issue 1 October 1998.

Prognosis
• Good
(if early IVIG replacement therapy, and in the absence of polio,
other persistent enteroviral infections, arthritis, or lymphoreticular
malignancy)
• A full active lifestyle is to be encouraged and expected.
• Long-term follow up
- Bronchiectasis and gastroenteritis may occur despite IVIG treatment.
- Close monitoring and aggressive treatment of acute or chronic
infections are essential

• 73 males XLA
• Delayed diagnosis >> risk of developing CLD increased
• Respiratory tract infections remained the most prominent clinical problem
observed also during follow-up despite immunoglobulin substitution treatment.
Plebani A. Clinical Immunology Vol. 104, No. 3, September; 221–230, 2002.

Cause of death
• Chronic enteroviral meningoencephalitis may cause serious
morbidity or mortality in XLA patients. This complication is usually
caused by ECHO viruses. Treatment with high doses of IVIG and
with the antiviral drug pleconaril.
• Its occurrence has dropped considerably since IVIG has been
routinely administered to patients but does occur rarely,
even with replacement.

• 87 adults with a definitive diagnosis of XLA.
• 10 had died at greater than 21 years of age.
• Enterovirus was acquired before the patient was treated with
intravenous gammaglobulin.
V . Howard et al. / Clinical Immunology 118 (2006) 201–208.

Take home message
• Early diagnosis and treatment
- IVIG, ATB prophylaxis
• Defining genetic etiology
• Long-term follow-up : Infection

X-linked Agammaglobulinemia: Clinical Manifestations and Diagnosis

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X-linked Agammaglobulinemia: Clinical Manifestations and Diagnosis