Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency Kerstin Felgentreff, MD,

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Presentation transcript:

Functional analysis of naturally occurring DCLRE1C mutations and correlation with the clinical phenotype of ARTEMIS deficiency Kerstin Felgentreff, MD, Yu Nee Lee, PhD, Francesco Frugoni, PhD, Likun Du, PhD, Mirjam van der Burg, PhD, Silvia Giliani, PhD, Ilhan Tezcan, MD, Ismail Reisli, MD, Ester Mejstrikova, MD, Jean-Pierre de Villartay, PhD, Barry P. Sleckman, MD, PhD, John Manis, MD, PhD, Luigi D. Notarangelo, MD Journal of Allergy and Clinical Immunology Volume 136, Issue 1, Pages 140-150.e7 (July 2015) DOI: 10.1016/j.jaci.2015.03.005 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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 2 RSSs 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 10 Gy, and γH2AX dephosphorylation was quantified by means of flow cytometry at 1, 8, 24, and 36 hours in cells expressing the DCLRE1C-hCD2 vector. The difference in GFP expression and downregulation of γH2AX is shown for Dclre1c−/− abl pro-B cells transduced with WTa DCLRE1C or a mock plasmid, respectively. LTR, Long terminal repeat. Journal of Allergy and Clinical Immunology 2015 136, 140-150.e7DOI: (10.1016/j.jaci.2015.03.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 DNA repair activities were analyzed in Dclre1c−/− abl pro-B cells blocked in G0/G1 cell-cycle phases. MFIs for γH2AX at 1, 8, 24, and 36 hours after irradiation with 10 Gy are shown for one representative experiment testing 6 ARTEMIS mutants compared with WTa and mock-transduced Dclre1c-/- abl pro-B cells. Error bars indicate SDs. *P ≤ .05 and **P ≤ .01. Journal of Allergy and Clinical Immunology 2015 136, 140-150.e7DOI: (10.1016/j.jaci.2015.03.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Correlation of V(D)J recombination with DNA repair activities. A and B, Recombination and DNA repair activity levels of 41 genetic DCLRE1C variants were studied in 23 missense mutations and in-frame deletions (Fig 3, A) and 18 frameshift mutations (Fig 3, B) affecting various domains: the metallo-β-lactamase domain (amino acids 1-155), the β-CASP domain (amino acids 156-385), and the C-terminal region (amino acids 386-692).5 C-E, V(D)J recombination activity levels of mutations affecting the β-lactamase (Fig 3, C), β-CASP (Fig 3, D), and C-terminal (Fig 3, E) domains of the protein are plotted against DNA repair activities. Activity levels are expressed as percentages of WTa performance. Error bars indicate SDs. ***P ≤ .001. Journal of Allergy and Clinical Immunology 2015 136, 140-150.e7DOI: (10.1016/j.jaci.2015.03.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 A and B, Activity levels for recombination (Fig 4, A) and DNA repair (Fig 4, 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 < .0001 and P = .0001). C, Percentages of patients heterozygous or homozygous for a loss-of-function or hypomorphic DCLRE1C allele are shown for patients with SCID and those with leaky SCID. LOF, Loss of function. Journal of Allergy and Clinical Immunology 2015 136, 140-150.e7DOI: (10.1016/j.jaci.2015.03.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Recombination and DNA repair activity levels of hypomorphic DCLRE1C variants with various vector copy numbers. Expression levels of 2 hypomorphic DCLRE1C variants and 1 polymorphism were titrated by varying vector copy numbers to 6 or greater or 2. A, Expression of human DCLRE1C was assessed by means of quantitative PCR in cDNA samples and normalized to WTa (indicated by dashed line). B and C, Activity levels for V(D)J recombination (Fig 5, B) and DNA repair (Fig 5, C) were compared between 6 or more or 2 vector copies or the pool containing variable copy numbers per cell. Error bars indicate SDs. *P ≤ .05, **P ≤ .01, and ***P ≤ .001. Journal of Allergy and Clinical Immunology 2015 136, 140-150.e7DOI: (10.1016/j.jaci.2015.03.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Characterization of Dclre1c−/− abl pro-B cells. A, The lack of Dclre1c expression (isoforms a, b, and c) in the abl pro-B cells used was confirmed by means of PCR. B, Dclre1c−/− abl pro-B cells were engineered with a retroviral vector containing an inverted GFP cassette and the human CD4 sequence. Single-cell clones were selected, and a single vector copy integration was confirmed by means of southern blotting after digesting genomic DNA with EcoRI, NcoI, and HindIII. Journal of Allergy and Clinical Immunology 2015 136, 140-150.e7DOI: (10.1016/j.jaci.2015.03.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Cell-cycle block with imatinib. For analysis of recombination and DNA repair activities, Dclre1c−/− abl pro-B cells were blocked in the G0/G1 cell-cycle phase by means of treatment with 3 μmol/LO of the tyrosine kinase inhibitor imatinib. Journal of Allergy and Clinical Immunology 2015 136, 140-150.e7DOI: (10.1016/j.jaci.2015.03.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Functional activities of DCLRE1C and Dclre1c isoforms. A, To compare recombination activities of different isoforms, sequences of the human DCLRE1C isoforms a, b, and c and the murine Dclre1c isoform a were introduced into Dclre1c−/− abl pro-B cells engineered with the invGFP-hCD4 construct. GFP expression was assessed by means of flow cytometry in cells enriched for expression of both constructs (CD4+CD2+). B, Expression of murine Dclre1c and human DCLRE1C transcripts was confirmed by means of PCR with cDNA as templates. DAPI, 4′-6-Diamidino-2-phenylindole dihydrochloride. Journal of Allergy and Clinical Immunology 2015 136, 140-150.e7DOI: (10.1016/j.jaci.2015.03.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Quantification of WT and mutated DCLRE1C expression. Expression of DCLRE1C and CD2 was assessed by means of quantitative PCR (ΔΔCT method) in cDNA from all mutant clones analyzed. β2-Microglobulin was used as a housekeeping gene, and expression levels were normalized to WTa. Journal of Allergy and Clinical Immunology 2015 136, 140-150.e7DOI: (10.1016/j.jaci.2015.03.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Three Artemis variants reported in patients presenting with leaky SCID were selected for further study of recombination and DNA repair activity levels in cells containing 6 or more or 2 vector copy numbers. The number of vector copy integrations was analyzed by means of Southern blotting of genomic DNA digested with EcoRI. Journal of Allergy and Clinical Immunology 2015 136, 140-150.e7DOI: (10.1016/j.jaci.2015.03.005) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions