Re: RE: ADA Gene

Well Shane, I had to do quite a lot of reading but here are the answers to your questions

Question 1:

The ADA gene  encodes an enzyme which is responsible for the degradation of adenosine 
or deoxyadenosine to inosine and deoxyinosine respectively.  Thus both adenosine and 
deoxyadenosine accumulate in the absence of the enzyme.  The adenosine and deoxyadenosine 
nucleotides arise through the activity of kinase enzymes in the cells.  How does SCID 
thus arise in ADA-defective patients? Well, the absence of ADA inhibits the activity of 
an enzyme, ribonucleotide reductase which in turn inhibits the synthesis of DNA.  
B and T cells are unable to proliferate in the presence of deoxyadenosine and thus the 
ADA-defficient patient is unable to build up any kind of immune response.

The expression of the ADA gene varies not only between different tissues but is also 
changes at different stages of development.  In the lymphoid tissues the 'hierachy' of 
expression levels is as follows:  immature cortical thymocytes >medullary thymocytes > 
mature T lymphocytes.  Several potential mechanisms may regulate the levels of ADA mRNA.  
Studies have been carried out on a number of cell lines such as HLA-60 and Molt 4.  
These have suggested that difference in the levels of ADA mRNA arise not through 
alterations in the initiation of transcription but through variations in transcript 
elongation.  A very interesting set of experiments has been carried out using probes 
specific to a number of sites in the gene.  ADA transcripts from tissues with high level 
expression of the ADA gene hybridised to probes distributed across the gene.  However,
with tissues where there was low level expression it was found that the transcripts 
hybridised only to probes that were near the promoter and not those that were at the 
3' end of the gene.  Several so called 'DNAse sensitive sites' have been found 4.3 and 
8.5 Kb downstrema of the first exon and these have enhancer activity (i.e they increase 
transcription of the ADA gene).  

A number of deletions and splicing defects have been observed in patients who are ADA 
deficient as well as several point mutations in the coding region of the gene.  Most 
patients are compound heterozygotes (having a different mutation on each copy of the 
ADA gene).  A 3250 bp deletion, incorporating the promoter sequence and exon 1 has 
been found in both american and belgian patients.  this is thought to have arisen 
through homologous recombination between the Alu sites already described.    


Question 2:
The ADA gene is located on the long arm of chromosome 20 more specifically 20q12-q13.1.  
It is 32040 base pairs long from the transcription start site to the polyadenylation 
site. The gene is composed of 12 exons.  These Exons have the following lengths:

Exon 1: 128 bp, Exon 2:62 bp
Exon 3: 123 bp, Exon 4: 144 bp
Exon 5: 116 bp, Exon 6: 128 bp
Exon 7: 72 bp,  Exon 8: 102 bp
Exon 9: 65 bp,  Exon 10:130 bp
Exon 11: 103 bp,Exon 12:325 bp

So as expected the vast amount of the gene is present as non-coding sequences.  
Perhaps of interest is the presence of 23 Alu repetitive sequences scattered throughout 
the gene.  In addition the Promoter does not carry a TATA box and the CAAT sequence and 
is highly GC rich.


As far as your interest on gene therapy/ genetic engineering is concerned, Shane, I was 
only able to find information specific for ADA which only account for about 25% of all 
SCID patients.  following the discovery that ADA-transduced T cells have a survival 
advantage over non-ADA-expressing T cells, the NIH approved a proposal which involved 
targeting T cells directly rather than stem cells.  The general proceedure is as follows:

(a) blood mononuclear cells are isolated from the patient through leukophoresis.
(b) these cells are cultured for a few days with an anti-CD 3 antibody and interleukin 2.
(c) the cells are then incubated with a LASN amphotropic retroviral vector which carries 
a cDNA copy of the ADA gene.  This construct has the ADA gene under the control of a MMLV 
LTR promoter sequence.
(d) the T cells are then reinfused

The gene transfer proceedure described above has to be repeated continuously over a time 
span ranging from 6 to 8 months.

The NIH has described how this proceedure was carried out on two patients during the 
period 1990-1991.  One of these patients was 2 years old whilst the other was 3 years old.  
Both had been on IVIg treatment and had received prophylactic antibiotics but were unable 
to continue such treatment.  These patients went on to develop persisting lymphopenia.
It was upon these observations that these two patients were then entered into the Gene 
Therapy Study.  Last reports indicate that both patients were doing well clinically since 
there has been an increase in the number of Lymphocytes (Lymphocyte defficiency is the 
fundamental characteristic of an ADA-defficient patient).  In addition an increase in the 
Lymphocyte ADA activity has been observed since commencing the gene therapy treatment.
However the following points must be made about gene therapy:

(a) only 1-10% of the reinfused T cells actually carry the ADA cDNA construct
(b) the level of circulating ADA is much less that an alternative treatment which involves 
the external introduction of PEG-ADA


Here in Europe a different approach has been adopted which involves targeting stem cells.  
The trials that have been carried out involve introducing retroviral constructs into so 
called stem cell-enriched preparations (achieved by using anibodies specific for CD34).  
These patients must first be treated with a number of hematopoietic growth factors, more 
specifically G-CSF, GM-CSF and Il3 all three of which increase the level of target cells.
In Los Angeles stem cell targeting has been tried on three neonates in which the target 
stem cells were obtained from the umbilical cord and placenta.  Thus such a treatment has 
several advantages most notably that it is non-invasive, tretment can begin almost 
immediately after birth and the umbilical chord is a very rich source of stem cells.

Gene therapy in general takes a long time to evaluate since the effects of the treatment 
must be monitored over several years.


Question 3:

Well Shane I searched in the literature for this and i couldn't find any reference on a 
chimpanzee homologue to the human ADA gene.  I tried the Genetics DATABASE at the European 
Molecular Biology Laboratories in Hiedelberg and again no luck.  However I am sure that an 
equivalent does exist since ADA is a housekeeping gene and hence very important!!!!! (the 
understatement of the century)


Well Shane, I hope that this information is what you wanted.  The main reference that I used 
for my study is a book called "Genes in Medicine" by Rasko and Downes which was published 
last year.  Happy studying and Good luck !!!!! 
Robert