Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency

Abstract

Background: The endonuclease ARTEMIS, which is encoded by the DCLRE1C gene, is a component of the nonhomologous end-joining pathway and participates in hairpin opening during the V(D)J recombination process and repair of a subset of DNA double-strand breaks. Patients with ARTEMIS deficiency usually present with severe combined immunodeficiency (SCID) and cellular radiosensitivity, but hypomorphic mutations can cause milder phenotypes (leaky SCID).

Objective: We sought to correlate the functional effect of human DCLRE1C mutations on phenotypic presentation in patients with ARTEMIS deficiency.

Methods: We studied the recombination and DNA repair activity of 41 human DCLRE1C mutations in Dclre1c(-/-) v-abl kinase-transformed pro-B cells retrovirally engineered with a construct that allows quantification of recombination activity by means of flow cytometry. For assessment of DNA repair efficacy, resolution of γH2AX accumulation was studied after ionizing radiation.

Results: Low or absent activity was detected for mutations causing a typical SCID phenotype. Most of the patients with leaky SCID were compound heterozygous for 1 loss-of-function and 1 hypomorphic allele, with significant residual levels of recombination and DNA repair activity. Deletions disrupting the C-terminus result in truncated but partially functional proteins and are often associated with leaky SCID. Overexpression of hypomorphic mutants might improve the functional defect.

Conclusions: Correlation between the nature and location of DCLRE1C mutations, functional activity, and the clinical phenotype has been observed. Hypomorphic variants that have been reported in the general population can be disease causing if combined in trans with a loss-of-function allele. Therapeutic strategies aimed at inducing overexpression of hypomorphic alleles might be beneficial.

Keywords: ARTEMIS deficiency; DCLRE1C mutations; DNA repair; V(D)J recombination; nonhomologous end-joining; severe combined immunodeficiency.

FIG. 1. Schematic representation of the experimental outline and readout

A, Dclre1c^−/− abl pro-B cells were retrovirally engineered with a single construct containing an inverted GFP cassette flanked by two RSS and the hCD4 sequence for positive selection. Subsequently, cells were transduced with a second construct containing either WT or mutated hDCLRE1C (isoform a) and the hCD2 sequence. Sufficient targeting of RSS by the Rag1/Rag2 complex and completion of the recombination process, which involves Artemis, allows the GFP cassette to flip into sense orientation. B, GFP expression can be used for quantification of V(D)J recombination activity in cells expressing both vectors (CD4⁺CD2⁺). C, To assess DNA repair activity, cells were irradiated with 10Gy and γH2AX de-phosphorylation was quantified by flow-cytometry at 1h, 8h, 24h, and 36h in cells expressing the DCLRE1C-hCD2 vector. The difference in GFP expression and down-regulation of γH2AX is shown for Dclre1c^−/− abl pro B-cells transduced with WTa DCLRE1C or a mock plasmid, respectively.

FIG. 2

DNA repair activities were analyzed in Dclre1c^−/− abl pro-B cells blocked in G0/G1 cell cycle phases. Mean fluorescence intensities (MFIs) for γH2AX at 1h, 8h, 24h and 36h after irradiation with 10Gy are shown for one representative experiment testing 6 ARTEMIS mutants compared to WTa and mock (A–G). Error bars indicate standard deviations, * p≤0.05; **p≤0.01; ***p≤0.001.

FIG. 3. Correlation of V(D)J recombination with DNA repair activities

Recombination and DNA repair activity levels of 41 genetic DCLRE1C variants were studied in 23 missense mutations and in frame deletions (A) and 18 frameshift mutations (B) affecting various domains: Metallo-β-lactamase domain (amino acids 1–155), β-CASP domain (amino acids 156–385) and the C-terminal region (amino acids 386–692) . V(D)J recombination activity levels of mutations affecting the β-lactamase (C), β-CASP (D) and C-terminal domain (E) of the protein are plotted against DNA repair activities. Activity levels are expressed as percentages of WTa performance. Error bars indicate standard deviations, ***p≤0.001. r, Pearson correlation coefficient.

FIG. 4

The activity levels for recombination (A) and DNA repair (B) of the mutations with the highest activity (in case of compound heterozygosity) found in patients presenting with SCID and leaky SCID are shown (p< 0.0001, and p=0.0001). The percentages of patients being heterozygous or homozygous for a LOF or a hypomorphic DCLRE1C allele are shown for SCID and leaky SCID patients (C). LOF, loss of function.

FIG. 5. Recombination and DNA repair activity levels of hypomorphic DCLRE1C variants with various vector copy numbers

Expression levels of two hypomorphic DCLRE1C variants and one polymorphism were titrated by varying vector copy numbers to ≥6 or 2. Expression of human DCLRE1C was assessed by quantitative PCR in cDNA samples and was normalized to WTa (indicated by dashed line) (A). Activity levels for V(D)J recombination (B) and DNA repair (C) were compared between ≥6 or 2 vector copies, or the pool containing variable copy numbers per cell. Error bars indicate standard deviations. * p≤0.05; **p≤0.01; ***p≤0.001.