Immunodeficiency is a tough nut to CRAC: the importance of calcium flux in T cell activation

Severe Combined Immunodeficiency (SCID) is a rare primary immunodeficiency disease often characterized by a block in T cell development, which may also affect the normal development of B cells and NK cells. Several different mutations are known to give rise to SCID, and multiple genes are involved. Consequently, there are several different forms of SCID, which can be classified according to the metabolic and cellular defects that impede normal lymphocyte function. The two most prevalent forms of SCID are X-linked SCID and adenosine deaminase (ADA) deficiency SCID, together accounting for approximately 70-80% of disease cases. Other genetic abnormalities associated with this syndrome range from defective T cell receptor rearrangement to non-functional signaling molecules. Recently, a new genetic defect has been described in which mutations in a key component of Ca(2+) release activated-channels (CRAC) result in T lymphocyte malfunction.     

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.     

Basic defect in the expression of adenosine deaminase in ADA- SCID disease investigated through the cells of an obligate heterozygote

Summary The nature of the defect of a female baby who died of severe combined immunodeficiency (SCID) disease associated with adenosine deaminase deficiency (ADA^-) was investigated. Since tissue or tissue culture material was not available for subsequent studies, the expression of ADA in her cells was investigated in the somatic cell hybrid clones derived from a fusion between the lymphocytes from one of her two obligate heterozygote parents and thymidine kinase deficient Chinese hamster (a3) fibroblasts. The results of analyses of the human chromosomes and biochemical markers in 12 independent clones and 27 subclones indicated that the ADA deficiency in the patient is determined probably by a mutation in the structural gene for ADA in chromosome 20 leading either to the production of catalytically defective molecules or to the cessation of the production of ADA. Incidentally, the involvement of chromosome 2, which carries a gene for adenosine deaminase complexing protein (ADCP), in the causation of ADA deficiency was excluded. The in vitro approach through the cells from an obligate heterozygote described in this paper may have a general application in pursuing studies on other cases of inborn errors of metabolism whenever the material from the affected individuals (i.e., the homozygotes) is not available or not suitable for direct investigations.A part of this work was presented at the New York State Department of Health, Birth Defects Institute Symposium IX (Inborn Errors of Specific Immunity), Albany, October 16–18, 1978 and reported as an abstract in the proceedings of the Fifth International Conference on Human Gene Mapping, Edinburgh, July 1979. Cytogenet Cell Genet 22:164     

A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution.

In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation in exon 11. This resulted in the amino acid substitution of a valine for an alanine at position 329 of the ADA protein. Sequence analysis revealed that this mutation created a new BalI restriction site. Using Southern analyses, we were able to directly screen individuals to determine the frequency of this mutation. By combining data on eight families followed at our institution with data on five other families reported in the literature, we established that five of 13 patients (seven of 22 alleles) with known or suspected point mutations have this defect. This mutation was found to be associated with three different ADA haplotypes. This argues against a founder effect and suggests that the mutation is very old. In summary, a conservative amino acid substitution is found in a high proportion of patients with ADA deficiency; this can easily be detected by Southern analysis.     

Adenosine deaminase deficiency: genotype-phenotype correlations based on expressed activity of 29 mutant alleles.

Adenosine deaminase (ADA) deficiency causes lymphopenia and immunodeficiency due to toxic effects of its substrates. Most patients are infants with severe combined immunodeficiency disease (SCID), but others are diagnosed later in childhood (delayed onset) or as adults (late onset); healthy individuals with "partial" ADA deficiency have been identified. More than 50 ADA mutations are known; most patients are heteroallelic, and most alleles are rare. To analyze the relationship of genotype to phenotype, we quantitated the expression of 29 amino acid sequence-altering alleles in the ADA-deleted Escherichia coli strain SO3834. Expressed ADA activity of wild-type and mutant alleles ranged over five orders of magnitude. The 26 disease-associated alleles expressed 0.001%-0.6% of wild-type activity, versus 5%-28% for 3 alleles from "partials." We related these data to the clinical phenotypes and erythrocyte deoxyadenosine nucleotide (dAXP) levels of 52 patients (49 immunodeficient and 3 with partial deficiency) who had 43 genotypes derived from 42 different mutations, including 28 of the expressed alleles. We reduced this complexity to 13 "genotype categories," ranked according to the potential of their constituent alleles to provide ADA activity. Of 31 SCID patients, 28 fell into 3 genotype categories that could express <=0.05% of wild-type ADA activity. Only 2 of 21 patients with delayed, late-onset, or partial phenotypes had one of these "severe" genotypes. Among 37 patients for whom pretreatment metabolic data were available, we found a strong inverse correlation between red-cell dAXP level and total ADA activity expressed by each patient's alleles in SO3834. Our system provides a quantitative framework and ranking system for relating genotype to phenotype.     

Deoxyadenosine metabolism and cytotoxicity in cultured mouse T lymphoma cells: a model for immunodeficiency disease.

The inherited absence of either adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with severe immunological impairment. We have developed a cell culture model using a mouse T cell lymphoma to simulate ADA deficiency and to study the relationship between purine salvage enzymes and immune function. 2′-deoxyadenosine triphosphate (deoxyATP) levels have been shown to be greatly elevated in erythrocytes of immunodeficient, ADA-deficient patients, suggesting that deoxyadenosine is the potentially toxic substrate in ADA deficiency. Using a potent ADA inhibitor, we have demonstrated that deoxyadenosine is growth-inhibitory and cytotoxic to S49 cells, and that deoxyATP accumulates in these cells. Cell variants, unable to transport or phosphorylate deoxyadenosine, are much less sensitive to deoxyadenosine, indicating that intracellular phosphorylation of deoxyadenosine is required for the lethal effects.We have partially reversed the cytotoxic effects of deoxyadenosine with deoxycytidine in wild-type cells, but we cannot show any reversal in cell lines lacking deoxycytidine kinase. Adenosine (ado) kinase-deficient cells are extremely resistant to deoxyadenosine in the presence of deoxycytidine. This deoxycytidine reversal of deoxyadenosine toxicity is consistent with an inhibition of ribonucleotide reductase by deoxyATP, and we have shown that incubation of S49 cells with deoxyadenosine markedly reduces intracellular levels of deoxyCTP, deoxyGTP and TTP.Kinetics data in wild-type cells and in cell variants are consistent with the presence of two deoxyadenosine-phosphorylating activities — one associated with ado kinase and another associated with deoxycytidine kinase.The S49 cells appear to be a valid model for the simulation of ADA deficiency in cell culture, and from our results, we can suggest administration of deoxycytidine as a pharmacological regimen to circumvent the clinicopathologic symptoms in ADA deficiency.     

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.     

The use of enzyme therapy to regulate the metabolic and phenotypic consequences of adenosine deaminase deficiency in mice. Differential impact on pulmonary and immunologic abnormalities

Adenosine deaminase (ADA) deficiency results in a combined immunodeficiency brought about by the immunotoxic properties of elevated ADA substrates. Additional non-lymphoid abnormalities are associated with ADA deficiency, however, little is known about how these relate to the metabolic consequences of ADA deficiency. ADA-deficient mice develop a combined immunodeficiency as well as severe pulmonary insufficiency. ADA enzyme therapy was used to examine the relative impact of ADA substrate elevations on these phenotypes. A "low-dose" enzyme therapy protocol prevented the pulmonary phenotype seen in ADA-deficient mice, but did little to improve their immune status. This treatment protocol reduced metabolic disturbances in the circulation and lung, but not in the thymus and spleen. A "high-dose" enzyme therapy protocol resulted in decreased metabolic disturbances in the thymus and spleen and was associated with improvement in immune status. These findings suggest that the pulmonary and immune phenotypes are separable and are related to the severity of metabolic disturbances in these tissues. This model will be useful in examining the efficacy of ADA enzyme therapy and studying the mechanisms underlying the immunodeficiency and pulmonary phenotypes associated with ADA deficiency.     

Genetic heterogeneity in adenosine deaminase (ADA) deficiency: five different mutations in five new patients with partial ADA deficiency.

Complete genetic deficiency of adenosine deaminase (ADA) results in a fatal syndrome of severe combined immunodeficiency (SCID). Genetic partial deficiency of ADA, with no detectable enzyme activity in erythrocytes but with variable amounts of enzyme activity detectable in other cells, is usually associated with normal immunologic function but can give rise to a late-onset, cellular immunodeficiency syndrome. We have previously described four different mutant alleles in four such partially ADA-deficient children. We have now examined ADA in lymphoid cells from five additional newly ascertained children with partial ADA deficiency with respect to electrophoretic mobility in starch gel, isoelectric point, heat-stability, and apparent Km and Vmax. These techniques identify at least five different abnormal alleles in these five additional unrelated subjects. Three of these abnormal alleles result in expression of abnormal allelic isozymes (allozymes) different from those previously described. These are: (1) an acidic allozyme that is less acidic than the acidic allozyme we have previously reported; (2) an allozyme that is even less acidic than (1); and (3) an allozyme with apparently normal charge but which is so heat sensitive that the lability to heat can easily be detected at physiologic to febrile temperatures. Two abnormal alleles detected in these five children could correspond with previously reported mutants. These are (4) a basic allozyme that could (but probably doesn't) correspond to the basic allozyme we have previously reported and (5) a "null" allele that cannot be differentiated by these methods from any other "null" allele seen in complete ADA- -SCIDs. Three of the five new patients are genetic compounds, identified either by the presence of two electrophoretically distinguishable allozymes or by family studies that demonstrate presence of a "null" allele in addition to an electrophoretically abnormal allozyme. In three patients, one or both allozymes are phenotypically indistinguishable from an abnormal allozyme also seen in a different individual. Determination of the nucleotide sequence will be required to determine whether or not the phenotypically indistinguishable mutations are indeed genotypically identical. The newly ascertained individuals appear to share a common ethnic West Indian background, out of proportion to the frequency of this ethnic background in the newborn population from which they were ascertained, suggesting that partial ADA deficiency may confer a selective advantage to the homozygous or heterozygous phenotype.     

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.     

Two newly identified mutations (Thr233Ile and Leu152Met) in partially adenosine deaminase-deficient (ADA–) individuals that result in differing biochemical and metabolic phenotypes

Deficiency of adenosine deaminase (ADA^–) results in autosomal recessive immunodeficiency disease of varying severity. Partial ADA^– [ADA deficiency in erythrocytes (RBCs) but substantial ADA in non-RBCs] has also been identified, primarily by population screening of healthy adults in Africa and newborns in New York State. Normal immune function and/or minimal elevations of toxic metabolites in childhood suggested that partial ADA deficiency was benign and therefore that six mutations identified in partially ADA-deficient newborns and expressing 8–80% of normal ADA in non-RBCs were not pathogenic. However, the lowest activity mutation (Arg211Cys) has now been reported in patients with adult-onset immunodeficiency. We have now molecularly and biochemically studied two additional individuals whom we found to represent opposite ends of the spectrum of partial ADA deficiency as to biochemical abnormalities and age of ascertainment. Homozygosity for a newly identified Leu152Met mutation expressing considerably less activity than the pathogenic Arg211Cys mutation was found in a currently healthy 10-year-old Afghanistani child (ascertained at birth). He had the highest accumulation of the metabolite dATP among 13 partially ADA-deficient patients studied, but considerably lower than in those with immunodeficiency. Homozygosity for a newly identified Thr233Ile mutation expressing somewhat greater ADA activity than Arg211Cys was found in a healthy young adult Kung individual, associated with very low metabolite concentrations. Biochemical findings and a family history suggestive of immunodeficiency in prior offspring support the idea that the Leu152Met mutation could result in disease in homozygous individuals challenged by severe environmental insult or in heterozygosity with a null mutation. The pathogenicity of the Thr233Ile mutation, as well as a previously described Ala215Thr mutation with relatively lower activity is less likely but will only be determined by long-term observation of individuals carrying these mutations. Although, in contrast to other partial mutations, neither of these two mutations are at CpG hot spots, the frequency of CpG mutations remains high for partial mutations but is also similarly high in ADA^– immunodeficient patients (5/8 vs 12/21). Received: 7 August 1996 / Accepted: 29 November 1996     

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.     

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.     

dATP accumulation and ATP depletion in platelets in adenosine deaminase deficiency: Significance for the immune response?

High levels of dATP and dADP, accompanied by ATP depletion, were found in the platelets of two ADA-deficient children with severe combined immunodeficiency (SCID). In vitro studies demonstrated that even normal platelets had the ability to make dATP from deoxyadenosine (dAR) under physiological conditions. This capability was greatly enhanced by conditions simulating ADA deficiency. These results question whether the platelet has a specific role in the normal immune response.     

Transient expression of human adenosine deaminase cDNAs: identification of a nonfunctional clone resulting from a single amino acid substitution.

Human adenosine deaminase (ADA) is an important purine catabolic enzyme which irreversibly deaminates adenosine and deoxyadenosine. Severe genetic deficiency of ADA leads to an immunological deficiency state in which T-lymphoid cells are selectively destroyed by the accumulation of toxic levels of deoxyadenosine and deoxy-ATP. In preparation for transfer of ADA sequences into a variety of cell types, we explored expression of ADA cDNAs transfected into cultured cells within a simian virus 40-based expression vector. After transfection into monkey kidney (COS) cells, ADA cDNA encompassing the entire coding region of the protein generated human ADA activity. An unexpected finding, however, was the identification of a cDNA clone that failed to produce either human enzyme activity or immunoreactive ADA protein. As this pattern is typical of many naturally occurring mutant ADA alleles, we characterized the molecular defect in this clone. DNA sequence analysis revealed a single nucleotide substitution in amino acid position 50 (glycine-valine). Northern blotting with a unique 17-mer oligonucleotide demonstrated the absence of the mutant sequence in the mRNA from which the cDNA library giving rise to the mutant cDNA was constructed. Therefore, the substitution in the variant cDNA was created during cloning. These data define one critical region of the human ADA protein molecule and suggest a convenient strategy for characterization of the phenotypes associated with naturally occurring mutant alleles.     

A fatal syndrome associating a micromelic dwarfism, an ichthyosiform skin disorder and a severe combined immunologic deficiency. Report of a case and survey of the literature (author's transl)]

Congenital immunologic deficiencies and congenital dwarfisms represent two seemingly unrelated disorders. Here is reported the tenth case of a definite congenital and fatal syndrome associating a severe combined immunologic deficiency and a micromelic dwarfism, affecting mainly the proximal limbs, as well as an ichtyosiform and furrowed skin disorder. Although the adenosine deaminase activity has not been determined in this patient, a 4-month old boy, this syndrome seems to be different from cases of ADA negative SCID. The associated impairment of growth and immunity emphasizes once more the close genetic linkage existing between the development of the skeleton and the lymphoid tissue.     

The potential importance of soluble deoxynucleotidase activity in mediating deoxyadenosine toxicity in human lymphoblasts

Deoxyadenosine and its nucleotides have been implicated in the pathogenesis of the immune dysfunction associated with a genetic deficiency of adenosine deaminase (ADA). We have previously shown that when ADA is blocked with a synthetic inhibitor, human T lymphoblastoid cell lines are more sensitive to deoxyadenosine toxicity, dephosphorylate deoxyadenosine nucleotides at a slower rate, and have much lower levels of ecto-5'-nucleotidase than most B cell lines. It seemed unlikely, however, that an enzyme on the outer surface of the lymphocyte plasma membrane could regulate intracellular deoxynucleotide catabolism. We now report that human lymphoblasts also contain a soluble deoxynucleotidase activity that is distinguishable from the plasma membrane enzyme by several criteria. In multiple human lymphoblastoid cell lines of varying origin and phenotype. soluble deoxynucleotidase correlated significantly (rs = 0.80, p < 0.001) with sensitivity to deoxyadenosine toxicity.     

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.