Mahirah Binti Mohd Norazinan

22352821

Outline
INTRODUCTION
What is Adenosine Deaminase?
ADA deficiency

ADA-DEFICIENT IN MOUSE MODEL

PEG-ADA REPLACEMENT THERAPY
CONCLUSION
REFERENCES

What is Adenosine
Deaminase?
An enzyme involve in purine metabolism

Aminohydrolases, catalyze conversion of adenosine to

inosine and 2-deoxyadenosine to 2-deoxyinosine

Source: Sauer et al. 2012
Mostly important in human for lymphocyte
development,

ADA Deficiency
Caused by mutation in a gene that encodes for ADA

enzyme on chromosome 20
Autosomal recessive disease
Very rare, but very dangerous
Main features ADA deficiency: Causes accumulation
of ADA cytotoxic metabolites (Adenosine & 2Deoxyadenosine) in plasma, lymphoid tissues, and
red blood cells
Associated with severe depletion of three major
categories of lymphocytes, T-cells, B-cells and NK
cells that play vital role in immune system (Aldrich et
al. 2003)
Could result in severe combined immunodeficiency
(SCID)

ADA-Deficient Mouse
Model

Using two-stage genetic engineering strategy

ADA expression in trophoblast cells of placenta critical for

fetal development in mouse (Blackburn et al. 1998)

In order to generate completely ADA-deficient postnatal

mice, ADA minigene which targeted expression specifically

to the trophoblast lineage was introduced onto ADAdeficient background
Accomplished by intercrossing mice carrying the

trophoblast-specific ADA minigene with mice heterozygous

for the null ADA allele

Figure 1. Comparison between ADA +/+ and ADA -/- phenotype

Source : Apasov et al. 2001

 Using histological analysis

Tissues of interest were dissected from control and ADA-

deficient mice on postnatal day 15

Tissues were rinsed in ice-cold phosphate-buffered saline
and then fixed overnight in 4% paraformaldehyde in
phosphate-buffered saline at 4 C
Stained with hematoxylin and eosin using a Rapid Chrome
staining kit

Figure 2. Reduction in the

size of thymus and spleen
of ADA deficient mice

Source: Blackburn et al. 1998

 Immunology
For xamination of peripheral lymphocytes, blood was

collected from the tail vein of mice directly into EDTA-coated

microcentrifuge tubes
Complete blood counts were determined using an H1
analyzer (Technicon Instruments Corp.)
For determination of serum immunoglobulins, whole blood
was collected by cardiac puncture, allowed to set on ice for
15 min and then centrifuged
Serum immunoglobulin levels were determined using a single
radioimmunodiffusion assay

Figure 3. ADA deficient

mice exhibit lymphopenia
and immunodeficiency
Source: Blackburn et al. 1998

 The level of ADA substrates, Adenosine and 2-

deoxyadenosine in ADA deficient mice detection by

nucleoside analysis
accumulation of adenosine and 2-deoxyadenosine found in

the serum of ADA deficient mice

Adenosine levels were found to be elevated in all tissues
examined with the greatest accumulation occurring in the
liver, kidney, and lung
2-Deoxyadenosine was also detected in all ADA deficient
tissues examined with the greatest accumulation
occurring in the lymphoid organs

Figure 4. Levels of Adenosine

and 2-deoxyadenosine in
ADA deficient mice
Source: Blackburn et al. 1998

 In situ analysis of apoptosis in thymocytes

Frozen tissue preparations and apoptosis analysis in the spleen,

thymus,and lymph nodes of ADA+/+ and ADA/ mice were

performed
Side scatter versus forward scatter in thymi from ADA/ and

ADA+/+ littermates revealed an increase in the number of

dead cells among immature T cells from ADA/ thymi
The proportions of dead cells among mature T cells found in

the peripheral lymphoid organs (spleen and lymph nodes) were

similar between ADA+/+ and ADA/ mice

Figure 5. Increased apoptosis in ADA -/- mice

Source : Apasov et al. 2001

Pegylated-Bovine ADA (PEG-ADA)

Replacement Therapy
Livesaving, but not curative treatment
Modified enzyme remains in the extracellular

fluid where it can degrade toxic purines

Almost normalizes the metabolic
abnormalities in purine metabolism
Does not completely correct immune
function, but immunoprotection is restored,
dramatic clinical improvements occurs
Require weekly intramuscular injections with
PEG-ADA
Source: Sauer et al. 2012

Figure 6. Immune reconstitution and development of autoimmunity after PEG-ADA treatment

Source: Sauer et al. 2012

Conclusions
ADA deficiency cause by mutation in ADA

gene at chromosome number 20

Lack of ADA enzyme affect the development
of lymphocytes in immune system
Accumulation of ADA metabolites: Adenosine
& 2-deoxyadenosine due to ADA deficiency
PEG-ADA replacement therapy act as an
alternative treatment for ADA deficiency
Further research involving the mechanisms
of ADA metabolites with the development of
lymphocytes in immune system

References
Aldrich, M.B., Chen, W., Blackburn, M.R., Valdez, H.M., Datta, S.K. &
Kellems, R.E. (2003), Impaired germinal center maturation in
adenosine deaminase deficency, The Journal of Immunology, vol.
171, no. 10, pp. 5562-5570.
Apasov, S.G., Blackburn, M.R., Kellems, R.E., Smith, P.T. &
Sitkovsky, M.V. (2001). Adenosine deaminase deficiency increases
thymic apoptosis and causes defective T cell receptor signaling,
The Journal of Clinical Investigation, vol. 108, no.1, pp. 131-141.
Blackburn, M.R., Datta, S.K. & Kellems, R.E. (1998). Adenosine
Deaminase-deficient Mice Generated Using a Two-stage Genetic
Engineering Strategy Exhibit a Combined Immunodeficiency, The
Journal of Biological Chemistry, vol. 273, no.9, pp. 5093-5100.
Sauer, A.V., Brigida, I., Carriglio, N. & Aiuti, A. (2012), Adenosine
Deaminase-deficient Mice Generated Using a Two-stage Genetic
Engineering Strategy Exhibit a Combined Immunodeficiency,
Frontiers in Immunology, vol. 3, no. 265, pp. 1-19.

THANK YOU.