A human severe combined immunodeficiency (SCID) condition with increased sensitivity to ionizing radiations and impaired V(D)J rearrangements defines a new DNA recombination/repair deficiency

Abstract

The products of recombination activating gene (RAG)1 and RAG2 initiate the lymphoid-specific phase of the V(D)J recombination by creating a DNA double-strand break (dsb), leaving hairpin-sealed coding ends. The next step uses the general DNA repair machinery of the cells to resolve this dsb. Several genes involved in both V(D)J recombination and DNA repair have been identified through the analysis of in vitro mutants (Chinese hamster ovary cells) and in vivo situations of murine and equine severe combined immunodeficiency (scid). These studies lead to the description of the Ku-DNA-dependent protein kinase complex and the XRCC4 factor. A human SCID condition is characterized by an absence of B and T lymphocytes. One subset of these patients also demonstrates an increased sensitivity to the ionizing radiation of their fibroblasts and bone marrow precursor cells. This phenotype is accompanied by a profound defect in V(D)J recombination with a lack of coding joint formation, whereas signal joints are normal. Functional and genetic analyses distinguish these patients from the other recombination/repair mutants, and thus define a new group of mutants whose affected gene(s) is involved in sensitivity to ionizing radiation and V(D)J recombination.

Figure 1

(A) Pedigree and biological status of P5, P11, P12, and P15. Peripheral blood counts of mature T, B, and NK cells were determined by FACS^® immunostaining with anti-CD3, anti-CD19, and anti-CD56 mAbs, respectively. (B) Sensitivity to ionizing radiation in CFU-GM from P10 and P15. Plating efficiency of marrow cell cultures was determined after increasing doses (0.5–3 Gray) of γ-radiation. Results are expressed as percentage of survival relative to unirradiated cells. C represents the survival curve obtained with cells from an age-matched control. P10 shows normal radiosensitivity, whereas the survival curve from P15 cells is representative of the group of patients showing increased cell radiosensitivity.

Figure 3

(A) Structure of the V(D)J recombination extrachromosomal substrates. pHRecCJ and pHRecSJ constructs were adapted from pBlueRec (37). They contain the SV40 large T coding sequence and the SV40-Ori for autonomous replication in human cells, and a LacZ gene interrupted by a 310-bp DNA stuffer flanked by RSS on both sides. pHRecCJ is used for studying the coding joint formation. In the pHRecSJ plasmid, the two RSS were inverted to allow for the analysis of signal joint formation. (B) Analysis of signal joints after pHRecSJ recombination. The recovered pHRecSJ reporter plasmids were PCR-amplified with T3 and T7 primers, digested (+) or not (−) by ApalI, and run on a 2% agarose gel. The 500-bp band represents the PCR band obtained from unrecombined pHRecSJ. The 190-bp band represents the fusion of the RSS in the recombined plasmid in the absence of treatment with ApalI. This band is further digested to a 100-bp fragment with ApalI.

Figure 2

(A) DNA end–binding assay using cellular extracts from T⁻B⁻ SCID patients P5, P11, P12, and P15. Whole cell extracts were preincubated for 30 min at 4°C or not with specific mAb for Ku70/80 (lanes 3, 6, 9, and 12) or Rag1 (lanes 4, 7, 10, and 13) as control. (B) DNA-PK activity in cellular extracts from P5, P11, P12, and P15. MO59J and MO59K cell lines (36) lacking and containing active DNA-PKcs, respectively, were used as controls. DNA-PK activity was determined in the absence (−) or presence (+) of linear DNA as specific activator.

Figure 4

Genotype analysis of P5 family members with highly polymorphic microsatellite markers. Lane 1, father; lane 2, mother; lane 3, P5; lane 4, P11; lane 5: P12. For each gene upstream and downstream markers were used. None of the markers that are heterozygous in the parents are found homozygous in the three affected children.