PRKDC mutations in a SCID patient with profound neurological abnormalities

Abstract

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded by PRKDC) functions in DNA non-homologous end-joining (NHEJ), the major DNA double strand break (DSB) rejoining pathway. NHEJ also functions during lymphocyte development, joining V(D)J recombination intermediates during antigen receptor gene assembly. Here, we describe a patient with compound heterozygous mutations in PRKDC, low DNA-PKcs expression, barely detectable DNA-PK kinase activity, and impaired DSB repair. In a heterologous expression system, we found that one of the PRKDC mutations inactivated DNA-PKcs, while the other resulted in dramatically diminished but detectable residual function. The patient suffered SCID with reduced or absent T and B cells, as predicted from PRKDC-deficient animal models. Unexpectedly, the patient was also dysmorphic; showed severe growth failure, microcephaly, and seizures; and had profound, globally impaired neurological function. MRI scans revealed microcephaly-associated cortical and hippocampal dysplasia and progressive atrophy over 2 years of life. These neurological features were markedly more severe than those observed in patients with deficiencies in other NHEJ proteins. Although loss of DNA-PKcs in mice, dogs, and horses was previously shown not to impair neuronal development, our findings demonstrate a stringent requirement for DNA-PKcs during human neuronal development and suggest that high DNA-PK protein expression is required to sustain efficient pre- and postnatal neurogenesis.

Figure 1. MRI scan images of patient NM720.

Brain MRI in a normal 3-year-old child (A–E) and in patient NM720 at 3 months (F–J) and 2 years (K–O). In the patient, midline sagittal T1-weighted images (F and K) showed an absent pituitary bright spot (pit), thin and short corpus callosum (cc), and small uprotated cerebellar vermis (cbv). Parasagittal (G and L) and axial T2-weighted (H, I, M, and N) images showed reduced number of gyri, and mildly thick cortex with blurred gray-white border (ctx). Coronal images (J and O) showed the same changes in the cortex, as well as mildly small hippocampi. Striking progressive atrophy was seen involving all brain regions on the second scan (asterisks in L–N).

Figure 2. Defective DSB repair in patient fibroblasts.

(A) Control (48BR), Artemis null (F02/385), LIGIV syndrome (180BR and 495BR), patient (NM720), and mother (709BR) confluent G0 phase cells were exposed to 3 Gy γ-rays, and the number of γH2AX foci was determined at the indicated times. BG, background (no irradiation). (B) 1BR3 control and patient confluent G0 phase cells were exposed to 3 Gy γ-rays in the presence or absence of 5 μM DNA-PKi, and the number of γH2AX foci was enumerated at the indicated times.

Figure 3. Reduced DNA-PKcs expression and activity in patient cells.

(A) Western blotting analysis shows reduced DNA-PKcs protein in patient cells. Whole cell extracts (50 μg) from 1BR3 control or patient cells were processed for Western blotting using the indicated antibodies. (B) Western blotting analysis confirmed that residual DNA-PKcs protein was validated against human tumor cells, M059K and M059J. M059J cells are null for DNA-PKcs (53). (C) DNA-PK–dependent kinase activity was examined using whole cell lysates from 1BR3 control, mother, and patient immortalized primary hTERT fibroblasts.

Figure 4. Identification of mutational changes in PRKDC cDNA.

(A) Dye-terminator sequence figures illustrating the c.10721C>T mutational change in 1 allele of the patient and mother. (B) Dye-terminator sequence figures illustrating a double sequence commencing at c.1624, the bp at the boundary between exons 15 and 16. (C) RT-PCR amplification products using primers located in exons 15 and 17. Control cells yielded a single product of the size anticipated for the presence of exon 16. Patient cells yielded 2 bands, the smaller of which was the size expected for a product lacking exon 16. The greater signal could be the result of enhanced amplification of a smaller fragment. (D) mRNA transcript levels, determined using primers specific for HPRT, the WT p.A3574 allele (PRKDC c.10712C), or the mutant p.A3574V allele (PRKDC c.10712C>T). All cell lines showed equal expression of HPRT. cDNA of 48BR control cells had 2-fold greater levels of the WT allele compared with cDNA from mother or patient cells, which expressed the same level of the p.A3574 and p.A3574V mutant alleles. (E) Conservation of the A3574 residue between species and location of the identified mutational changes in PRKDC in relation to important domains.

Figure 5. DNA-PKcs cDNA complements the DSB repair defect observed in patient cells.

GFP-tagged full-length DNA-PKcs cDNA (GFP FL-PK) or GFP empty vector (GFP) was transfected into 1BR3 control, patient, 180BR LIGIV syndrome, and mother hTERT cells. 24 hours after transfection, cells were irradiated with 3 Gy γ-rays, and 53BP1 foci in GFP⁺ cells were enumerated at 8 and 16 hours. The identification of GFP⁺ cells was critical, since the transfection frequency using the large DNA-PKcs cDNA and human fibroblasts was low (<1%). Substantial correction of the DSB repair defect of patient cells was observed upon expression of full-length DNA-PKcs cDNA. No correction was observed after transfection of full-length DNA-PKcs cDNA into LIGIV syndrome cells.

Figure 6. A3574V and exon 16–deleted DNA-PKcs impair DNA-PKcs kinase activity and DNA-PKcs function in the response to DNA damage and during V(D)J recombination.

(A) Full-length, A3574V, or exon 16–deleted DNA-PKcs or empty vector was transiently transfected into DNA-PKcs–defective hamster V3 cells. 48 hours after transfection, cells were exposed to zeocin as indicated, and survival was estimated 7 days later. Expression of DNA-PKcs following Western blotting is also shown. Note that the exon 16–deleted DNA-PKcs cDNA was cloned into a distinct vector (RMCE) compared with A3574V DNA-PKcs cDNA (pCMV); the former is an improved vector for large inserts and was used in later studies. Survival analyses using these 2 vectors are presented separately. (B) Estimated percent recombination of coding junctions in V3 cells after expression of full-length, A3574V, or exon 16–deleted DNA-PKcs or empty vector together with plasmids encoding the RAG recombinases and a recombination substrate that assesses coding end-joining. (C and D) Estimation of DNA-PK activity in whole cell extracts from V3 cells expressing full-length, A3574V, or exon 16–deleted DNA-PKcs or empty vector. DNA-PK was extracted using DNA–cellulose beads, and activity was assessed using a biotinylated p53 peptide as a substrate (C) or by examining autophosphorylation after Western blotting and analysis using DNA-PKcs or phospho-2056–DNA-PKcs antibodies (D).