Severe combined immune deficiency due to a homozygous 3.2-kb deletion spanning the promoter and first exon of the adenosine deaminase gene

We have investigated the structural gene for adenosine deaminase (ADA) in a female infant with ADA deficiency associated severe combined immune deficiency (ADA-SCID) disease and her family by DNA restriction-fragment-length analysis. In this family a new ADA-specific restriction-fragment-length variant was detected, which involves a 3.2-kb deletion spanning the ADA promoter as well as the first exon. It was found that the patient, who was born to a consanguineous couple, was homozygous and both her parents and her brother were heterozygous for the deletion. No ADA-specific mRNA could be detected by hybridization in fibroblasts derived from this patient. Thus the patient was established to be homozygous for a true null ADA allele. In the light of the apparently normal development of most tissues except the lymphoid tissue the above finding directly questions the classification of ADA as a 'housekeeping' enzyme.     

Localization of human adenosine deaminase (ADA) gene sequences to the q12----q13.11 region of chromosome 20 by in situ hybridization

The gene for human adenosine deaminase (ADA), an enzyme constitutively expressed in all tissues investigated so far and deficient in some cases of severe combined immune deficiency, was previously assigned to chromosome 20 by syntenic analysis, using somatic cell hybrids and quantitative enzyme studies on patients with chromosome abnormalities. Attempts at regional localization of ADA through indirect approaches have so far resulted in uncertainties, as well as apparent inconsistencies. In situ hybridization of high-resolution somatic and pachytene chromosomes using a 3H-labeled cDNA probe of the ADA gene localized the gene to 20q12----q13.11. Rearrangements involving this region have been reported in various human hematological malignancies; in this regard, possible implications of the physical proximity of the ADA gene locus to that of SRC, an oncogene previously localized to the same region of chromosome 20, are briefly discussed.     

One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity.

We have cloned and sequenced an adenosine deaminase (ADA) gene from a patient with severe combined immunodeficiency (SCID) caused by inherited ADA deficiency. Two point mutations were found, resulting in amino acid substitutions at positions 80 (Lys to Arg) and 304 (Leu to Arg) of the protein. Hybridization experiments with synthetic oligonucleotide probes showed that the determined mutations are present in both DNA and RNA from the ADA-SCID patient. In addition, wild-type sequences could be detected at the same positions, indicating a compound heterozygosity. Studies with ADA expression clones mutagenized in vitro showed that the mutation at position 304 is responsible for ADA inactivation.     

Isolation of cDNA clones for human adenosine deaminase

Clones encoding human adenosine deaminase (ADA) were isolated from a cDNA library made from the lymphoblastoid cell line MOLT-4. The isolation procedure was based on the selection of clones hybridizing with a radioactive probe complementary to an RNA preparation, which had been highly enriched in ADA-specific mRNA. The latter RNA preparation was obtained by size-fractionating MOLT-4 RNA and selecting fractions that were translatable into ADA. The assay for the presence of ADA in the in vitro translation products, was based on immunoprecipitation with a specific anti-ADA serum. The antiserum used was shown to precipitate a 42-kDal protein with the properties of ADA. Positive clones were further screened by means of hybrid-released in vitro translation assays. Two clones were obtained which were able to select mRNA that could be translated into a 42-kDal protein immunoprecipitable with the ADA-antiserum. By use of Southern blots containing DNA from somatic cell hybrids, one of these ADA cDNA clones was assigned to the human chromosome 20 known to contain the ADA gene.     

Increased expression of one of two adenosine deaminase alleles in a human choriocarcinoma cell line following selection with adenine nucleosides

JEG-3 is a human choriocarcinoma cell line characterized by low levels of adenosine deaminase expression. For the purpose of studying adenosine deaminase gene regulation in the JEG-3 cells, we attempted to select variant cells having increased adenosine deaminase expression. This was accomplished by selecting cells for resistance to the cytotoxic adenosine analogs 9-beta-D-arabinofuranosyl adenine (ara-A) or 9-beta-D-xylofuranosyl adenine (xyl-A), both of which could presumably be detoxified by the action of adenosine deaminase. Single step high dose selection was ineffective in obtaining cells with increased adenosine deaminase. However, multistep selection using either ara-A or xyl-A resulted in cell populations with increased adenosine deaminase activity. Removal of selective pressure resulted in decreased adenosine deaminase levels. Subclones of xyl-A-resistant cells belonged to one of three phenotypic classes characterized by either elevated adenosine deaminase levels, decreased adenosine kinase levels, or both of these features. One subclone (A3-1A7) with unaltered adenosine kinase expression showed a 20-fold increase in adenosine deaminase expression. Further selection of this subclone for increasing xyl-A resistance resulted in an additional 2-fold increase in adenosine deaminase expression, followed by loss of adenosine kinase expression. These adenosine kinase-deficient cells showed no subsequent increase in adenosine deaminase expression in response to further xyl-A selection pressure. These results confirmed that xyl-A toxicity was mediated through its phosphorylated form and indicated that resistance may result from increased adenosine deaminase levels and/or adenosine kinase deficiency. The increased adenosine deaminase expression of the A3-1A7 subclone was exclusively in the ADA 2 allelic form. However, cell fusion experiments between A3-1A7 cells and mouse C1-1D cells established the existence of functional copies of both ADA 1 and ADA 2 allelic genes in the A3-1A7 cells. The increased expression of only one of the two functional ADA alleles, the requirement for a stepwise selection protocol to obtain cells with increased adenosine deaminase, and the instability of the adenosine deaminase phenotype in the absence of selective pressure suggest that the alteration of adenosine deaminase phenotype in the drug-resistant cells was the result of adenosine deaminase gene amplification.     

Use of the integrated steady state rate equation to investigate product inhibition of human red cell adenosine deaminase and its relevance to immune dysfunction

The analysis of progress curves using the integrated rate equation was applied to the adenosine deaminase-catalyzed conversion of adenosine to inosine. Adenosine deaminase was purified from human red blood cells of phenotypes ADA 1, ADA 2, and ADA 2-1. For all three types, no measurable product inhibition by inosine was observed. These results do not confirm the hypothesis that inosine accumulation in purine nucleoside phosphorylase deficiency causes adenosine deaminase inhibition, resulting in a common mechanism for the immune defects related to these two enzyme deficiencies.     

Identification of a deletion in the adenosine deaminase gene in a child with severe combined immunodeficiency

A patient with adenosine deaminase-deficient severe combined immunodeficiency is described whose defect is secondary to deletion of a portion of the ADA structural gene. In Southern analyses, DNA from this patient does not hybridize to a genomic probe that includes the 3' end of exon 1. This implies that both his parents are heterozygous for deletions of exon 1 sequences. Consistent with this finding, the patient has no detectable adenosine deaminase mRNA by Northern analysis. This is the first report of a deletion mutation as the cause of adenosine deaminase deficiency.     

Disruption of the adenosine deaminase (ADA) gene using a dicistronic promoterless construct: Production of an ADA-deficient homozygote ES cell line

In man, deficiency of ADA activity is associated with an autosomal recessive form of severe combined immunodeficiency (SCID), a disease with profound defects of both cellular and humoral immunity. Current treatments of ADA deficient patients include bone marrow transplantation, enzyme replancement and somatic gene therapy. The mechanism of the selective immune cell pathogenesis in ADA-SCIDS is, however, still poorly understood. Thus, the generation of an ADA deficient mouse model will be of considerable benefit to understand better the pathophysiology of the disorder and to improve the gene therapy treatments.We have disrupted the adenosime deaminase (ADA) gene in embryonic stem cells using a new efficient promoter trap gene-targeting approach. To this end, a dicistronic targeting construct containing a promoterless IRES geo cassette was used. This cassette allows, via the internal ribosomal entry site (IRES), the direct cap-independent translation of the geo reporter gene which encodes a protein with both -galactosidase and neomycin activities. After indentification of targeted clones by Southern blot, successful inactivation of the ADA gene was first confirmed by producing, from our heterozygote clones, an homozygote cell line. This line shows no ADA activity as judged by zymogram analysis. Second, we have been able to detect in the targeted clones, a specific galactosidase activity using a sensitive fluorogenic assay. The targeted ES cell clones are currently being injected into blastocysts to create an ADA deficient mouse model.     

Adenosine deaminase deficiency due to heterozygous abnormality consisting of a deletion of exon 7 and the absence of enzyme mRNA

An adenosine deaminase (ADA;EC 3.5.4.4)-deficient B lymphoblastoid cell line BADO5 derived from a Japanese patient with severe combined immunodeficiency disease and two B lymphoblastoid cell lines, BAMO5 from his mother and BAFO5 from his father, were characterized. To identify mutations affecting ADA activity, we prepared cDNAs to ADA mRNAs of the BADO5 cell line for nucleotide sequencing. Sequence analysis of one of the BADO5 ADA cDNA clones revealed deletion of exon 7, and one point mutation of base 629 from G to A that did not affect the amino acid sequence. All clones of the BADO5 cell line so far examined showed the absence of exon 7 by Southern blotting analysis. Ribonuclease protection assay with an RNA probe spanning from exon 5 to exon 11 showed that the BADO5 ADA mRNA had a deletion of exon 7, the BAMO5 mRNA had normal length, and the BAFO5 mRNA had two species with a deletion of exon 7 and with normal length. Consequently, the patient's ADA genes resulted from one allele of the BAMO5 ADA gene that did not produce a detectable mRNA, and the other allele of the BAFO5 ADA gene producing an aberrant mRNA without exon 7.     

A point mutation in the 5′ splice region of intron 7 causes a deletion of exon 7 in adenosine deaminase mRNA

An adenosine deaminase (ADA;EC 3.5.4.4)-deficient B lymphoblastoid cell line BAD05 derived from a Japanese patient with severe combined immunodeficiency was characterized. As previously reported, one allele of BAD05 expresses undetectable ADA mRNA, and the other allele produces an aberrant mRNA without exon 7. Genomic ADA DNA of BAD05 spanning from a portion of exon 6 to a portion of exon 8 was amplified by PCR. The amplified fragments were cloned into a vector, and 8 clones were isolated and sequenced. The analytical result showed a single base change of G to A at the invariant 5′ GT of intron 7 of ADA gene in one allele of BAD05, which accounts for the elimination of exon 7 during splicing. © 1993 Wiley-Liss, Inc.     

Adenosine deaminase activity in recipients of bone marrow from immunodeficient mice homozygous for the wasted mutation

Mice homozygous for the mutation wasted (wst/wst) have been postulated to be a model for the form of human severe combined immunodeficiency disease (SCID) that is secondary to a genetic deficiency of adenosine deaminase (ADA). To test this hypothesis more critically, we transplanted marrow from wst/wst and littermate control mice into lethally irradiated normal recipients. The Vmax and Km values for ADA in recipient's hematologic and non-hematologic tissues did not differ significantly. These results indicate that the wasted mouse is not a model for ADA deficiency and SCID.     

The effects of an induced adenosine deaminase deficiency on T-cell differentiation in the rat

Inherited deficiency of the enzyme adenosine deaminase (ADA) has been found in a significant proportion of patients with severe combined immunodeficiency disease and inherited defect generally characterized by a deficiency of both B and T cells. Two questions are central to understanding the pathophysiology of this disease: (1) at what stage or stages in lymphocyte development are the effects of the enzyme deficiency manifested; (2) what are the biochemical mechanisms responsible for the selective pathogenicity of the lymphoid system. We have examined the stage or stages of rat T-cell development in vivo which are affected by an induced adenosine deaminase deficiency using the ADA inhibitors, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and 2'-deoxycoformycin (DCF). In normal rats given daily administration of an ADA inhibitor, cortical thymocytes were markedly depleted; peripheral lymphocytes and pluripotent hemopoietic stem cells (CFU-S) all were relatively unaffected. Since a deficiency of ADA affects lymphocyte development, the regeneration of cortical and medullary thymocytes and their precursors after sublethal irradiation was used as a model of lymphoid development. By Day 5 after irradiation the thymus was reduced to 0.10-0.5% of its normal size; whereas at Days 9 and 14 the thymus was 20-40% and 60-80% regenerated, respectively. When irradiated rats were given daily parenteral injections of the ADA inhibitor plus adenosine or deoxyadenosine, thymus regeneration at Days 9 and 14 was markedly inhibited, whereas the regeneration of thymocyte precursors was essentially unaffected. Thymus regeneration was at least 40-fold lower than in rats given adenosine or deoxyadenosine alone. Virtually identical results were obtained with both ADA inhibitors, EHNA and DCF. The majority of thymocytes present at Day 9 and at Day 14 in inhibitor-treated rats had the characteristics of subcapsular cortical thymocytes which are probably the most ancestral of the thymocytes. Thus, an induced ADA deficiency blocked the proliferation and differentiation of subcapsular cortical thymocytes which are the precursors of cortical and medullary thymocytes.     

Correct splicing despite mutation of the invariant first nucleotide of a 5' splice site: a possible basis for disparate clinical phenotypes in siblings with adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency usually causes severe combined immune deficiency in infancy. Milder phenotypes, with delayed or late onset and gradual decline in immune function, also occur and are associated with less severely impaired deoxyadenosine (dAdo) catabolism. We have characterized the mutations responsible for ADA deficiency in siblings with striking disparity in clinical phenotype. Erythrocyte dAdo nucleotide pool size, which reflects total residual ADA activity, was lower in the older, more mildly affected sib (RG) than in her younger, more severely affected sister (EG). Cultured T cells, fibroblasts, and B lymphoblasts of RG had detectable residual ADA activity, while cells of EG did not. ADA mRNA was undetectable by northern analysis in these cells of both patients. Both sibs were found to be compound heterozygotes for the following novel splicing defects: (1) a G+1-->A substitution at the 5' splice site of IVS 2 and (2) a complex 17-bp rearrangement of the 3' splice site of IVS 8, which inserted a run of seven purines into the polypyrimidine tract and altered the reading frame of exon 9. PCR-amplified ADA cDNA clones with premature translation stop codons arising from aberrant pre-mRNA splicing were identified, which were consistent with these mutations. However, some cDNA clones from T cells of both patients and from fibroblasts and Epstein-Barr virus (EBV)-transformed B cells of RG, were normally spliced at both the exon 2/3 and exon 8/9 junctions. A normal coding sequence was documented for clones from both sibs. The normal cDNA clones did not appear to arise from either contamination or PCR artifact, and mosaicism seems unlikely to have been involved. These findings suggest (1) that a low level of normal pre-mRNA splicing may occur despite mutation of the invariant first nucleotide of the 5' splice donor sequence and (2) that differences in efficiency of such splicing may account for the difference in residual ADA activity, immune dysfunction, and clinical severity in these siblings.     

Lack of adenosine deaminase deficiency in the mutant mouse wasted

The possibility that the mutant mouse wasted (wst/wst) may serve as an animal model for studies of severe combined immunodeficiency disease (SCID) and the role of adenosine deaminase (ADA, EC 3.5.4.4) in adenosine metabolism were investigated. The specific activity of ADA in wst/wst compared with control mice was significantly lower by 26% in thymus, but significantly higher by 18% in spleen and 32% in cerebellum. Vmax values of ADA in spleens were 43% higher in wst/wst mice and no changes were observed in Km values. In contrast, the Vmax of ADA was unchanged in erythrocytes from wst/wst mice, but the Km for adenosine was significantly elevated. Thus, based on ADA measurements alone, it may be premature to consider wst/wst mice as a model for ADA deficiency and SCID in humans.     

Chromosome anomalies associated with amplification of the adenosine deaminase gene (ADA) in rat hepatoma cells

Two independently selected series of rat hepatoma cell lines resistant to the drug deoxycoformycin (dCF) were analyzed karyotypically. Several forms of homogeneously staining regions (HSRs) were present on metaphase chromosomes of these cells. In some instances HSRs comprised nearly an entire chromosome, which are among the largest chromosomes in the karyotype. Stable resistance to dCF is acquired in rat cells by overproduction of the enzyme adenosine deaminase (ADA) as a result of amplification of ADA gene sequences. We have localized the amplified ADA gene sequences to HSRs on metaphase chromosomes from both series of dCF-resistant cell lines by in situ hybridization. Based upon the number of ADA gene sequences present and the lengths of the HSRs, we have estimated the size of the amplified unit to range from 450 to 1,000 kb.     

Adenosine deaminase and immunodeficiency: An in vitro model

We have examined the effect of adenosine and EHNA, a competitive inhibitor of adenosine deaminase (ADA), upon the ability of human peripheral blood lymphocytes to respond to mitogen. Addition of adenosine at concentrations greater than 10 μm (10^?5m) resulted in inhibition of lymphocyte proliferation at 48 hr of culture, provided that the culture medium was relatively free of ADA activity. The actual concentrations of adenosine remaining in inhibited cultures at the time of harvest were considerably lower than those added initially. EHNA alone also inhibited PHA response (and to a lesser extent PWM and Con A responses), but only at high concentrations. Noninhibitory concentrations of EHNA and adenosine together acted synergistically to produce profound inhibition of lymphocyte proliferation. This may provide an in vitro model to explore further the mechanism of the immunodeficiency associated with deficiency of ADA. Adenosine deaminase activity in stimulated cultures did not differ significantly from that found in unstimulated cultures, and the activity per protein or per DNA actually decreased in stimulated versus unstimulated cultures.     

Analysis of normal and mutant forms of human adenosine deaminase — A review

Summary A deficiency of the enzyme adenosine deaminase is associated with an autosomal recessive form of severe combined immunodeficiency disease in man. The molecular forms of the normal human enzyme have now been well characterized in an effort to better understand the nature of the enzyme defect in affected patients.In some human tissues adenosine deaminase exists predominantly as a small molecular form while in other tissues a large form composed of adenosine deaminase (small form) and an adenosine deaminase-binding protein predominates. The small form of the enzyme purified to homogeneity by antibody affinity chromatography is a monomer of native molecular weight of 37,600. The adenosine deaminase-binding protein, purified by adenosine deaminase affinity chromatography, appears to be a dimer of native molecular weight 213,000 and contains carbohydrate. Based on direct binding measurements, chemical cross-linking studies and sedimentation equilibrium analyses, small form adenosine deaminase has been shown to combine with purified binding protein in a molar ratio of 2:1 respectively to produce the large form adenosine deaminase.Reduced, but widely ranging levels of adenosine deaminating activity, have been reported in various tissues of adenosine deaminase deficient patients. Further, the characteristics of this residual enzyme activity have been analyzed immunochemically to substantiate genetic heterogeneity in this disorder.While many types of immunodeficiency are currently recognized in man, in most cases the molecular defect is unknown. The discovery of a deficiency of the enzyme, adenosine deaminase, ADA, (EC 3.5.4.4), in some patients with severe combined immunodeficiency disease represented an early clue to the pathogenesis of immune dysfunction at the molecular level^1-4. Affected patients with markedly reduced levels of ADA exhibit a defect of both cellular and humoral immunity characterized clinically by severe recurrent infections with a fatal outcome if untreated. Attempts to elucidate the nature of the genetic mutation(s) leading to the reduction of ADA activity in these immunodeficient patients have been complicated in part by an incomplete understanding of the nature of ADA in normal tissues. In this review we will consider the structural characteristics of the normal and mutant forms of ADA as they are currently understood.