Identification of a point mutation resulting in a heat-labile adenosine deaminase (ADA) in two unrelated children with partial ADA deficiency.

Abstract

We have determined the mutation in a child with partial adenosine deaminase (ADA) deficiency who is phenotypically homozygous for a mutant ADA gene encoding a heat-labile enzyme (Am. J. Hum. . Sequencing of cDNA demonstrated a C to A transversion that results in the replacement of a proline by a glutamine residue at codon 297. As this mutation generated a new recognition site in exon 10 of genomic DNA for the enzyme Alu I, Southern blot analysis was used to establish that this child was indeed homozygous for the mutation. The abnormal restriction fragment generated by this mutation was also found in a second partially ADA-deficient patient who phenotypically is a genetic compound and also expresses a heat-labile ADA (in addition to a more acidic than normal ADA) (Am. J. Hum. Genet. 38:13-25). Sequencing of cDNA clones from the second patient established the identical codon 297 mutation. Transfection of the mutant cDNA into heterologous cells resulted in expression of a heat-labile ADA of normal electrophoretic mobility and isoelectric point, properties exhibited by the ADA in the patients' cells.

Molecular Basis for Paradoxical Carriers of Adenosine Deaminase (ADA) Deficiency That Show Extremely Low Levels of ADA Activity in Peripheral Blood Cells Without Immunodeficiency

“…We note that the residual ADA activity in the PBMC of the presently described paradoxical carriers is only 1-2% of normal, significantly less than the 4 -70% that has been reported for previously identified ADA partials (10,(12)(13)(14)(15)(16)(17). Nevertheless, their overall ability to catabolize dAdo in vivo is apparently sufficient to prevent the metabolic abnormalities responsible for lymphopenia and immune dysfunction.…”

“…Heterogeneity of ADA heat stability has previously been reported in cells from other individuals with partial ADA deficiency (13,32). In one case, the responsible mutation was identified as P297Q (14). Atomic structural and biophysical analysis of these mutant proteins may provide a more precise understanding of the mechanism for thermal instability.…”

Paradoxical expression of adenosine deaminase in T cells cultured from a patient with adenosine deaminase deficiency and combine immunodeficiency.

“…After centrifugation for 2 min in a microcentrifuge, 0.1 ml of supernatant was passed over Sephadex G-25 equilibrated with 25 mM Tris-HCl, pH 7.4, 15 mM KCI, 1 mM EDTA, and I mM dithiothreitol using the "spun-column" technique (24). ADA activity was assayed (25) using 150 MM [14C]adenosine (20,,000 cpm/ nmol) and purine nucleoside phosphorylase (PNP) (26) using 100 IM…”

“…1% of normal activity in blood mononuclear cells and B cell lines (complete deficiency) (6,9,10,13), the latter has 4-70% of normal activity (partial deficiency). Structural gene mutations, which presumably diminish enzyme stability or catalytic activity to different extents, occur in both groups (14)(15)(16)(17)(18)(19)(20)(21). The degree of ADA deficiency determines the severity of toxic effects of deoxyadenosine, including dATP pool expansion and inactivation of S-adenosylhomocysteine hydrolase.…”

Capillary Electrophoresis in Diagnosis and Monitoring of Adenosine Deaminase Deficiency

“…Traces of dAdo in plasma and urine are a characteristic feature of severely affected infants (2 ). Patients affected by SCID can exhibit a considerable heterogeneity in biochemical, clinical, and immunologic findings (3 ); a dramatic reduction of ADA activity and high dATP concentrations in erythrocytes, however, represent a universal finding for homozygous ADA deficiency (4 ).…”