The immunophenotypic and immunogenotypic B-cell differentiation arrest in bone marrow of RAG-deficient SCID patients corresponds to residual recombination activities of mutated RAG proteins.
The protein products of the recombination activating genes (RAG1 and RAG2) initiate the formation of immunoglobulin (Ig) and T-cell receptors, which are essential for B- and T-cell development, respectively. Mutations in the RAG genes result in severe combined immunodeficiency disease (SCID), generally characterized by the absence of mature B and T lymphocytes, but presence of natural killer (NK) cells. Biochemically, mutations in the RAG genes result either in nonfunctional proteins or in proteins with partial recombination activity. The mutated RAG genes of 9 patients from 7 families were analyzed for their recombination activity using extrachromosomal recombination substrates, rearrangement of endogenous Ig loci in RAG gene-transfected nonlymphoid cells, or the presence of Ig gene rearrangements in bone marrow (BM). Recombination activity was virtually absent in all 6 patients with mutations in the RAG core domains, but partial activity was present in the other 3 RAG-deficient patients, 2 of them having Omenn syndrome with oligoclonal T lymphocytes. Using 4-color flow cytometry, we could define the exact stage at which B-cell differentiation was arrested in the BM of 5 RAG-deficient SCID patients. In 4 of 5 patients, the absence of recombination activity was associated with a complete B-cell differentiation arrest at the transition from cytoplasmic (Cy) Igmu(-) pre-B-I cells to CyIgmu(+) pre-B-II cells. However, the fifth patient showed low frequencies of precursor B cells with CyIgmu and surface membrane IgM, in line with the partial recombination activity of the patient's mutated RAG gene and the detection of in-frame Ig gene rearrangements in BM.
Noordzij
Jeroen G
JG
Department of Immunology, Erasmus MC, University Medical Center Rotterdam, dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands.
de Bruin-Versteeg
Sandra
S
Verkaik
Nicole S
NS
Vossen
Jaak M J J
JM
de Groot
Ronald
R
Bernatowska
Ewa
E
Langerak
Anton W
AW
van Gent
Dik C
DC
van Dongen
Jacques J M
JJ
D001402:B-Lymphocytes / Q000166:cytology* / Q000187:drug effects; D001854:Bone Marrow Cells / Q000276:immunology*; D016022:Case-Control Studies; D002454:Cell Differentiation / Q000187:drug effects; D002675:Child, Preschool; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology; D005192:Family Health; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D015321:Gene Rearrangement / Q000187:drug effects / Q000235:genetics; D005803:Genes, Immunoglobulin; D005838:Genotype; D018398:Homeodomain Proteins / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology; D006801:Humans; D016130:Immunophenotyping; D007223:Infant; D008297:Male; D009154:Mutation; D009687:Nuclear Proteins; D016511:Severe Combined Immunodeficiency / Q000235:genetics / Q000276:immunology / Q000473:pathology*
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein