Gene-edited pseudogene resurrection corrects p47phox-deficient chronic granulomatous disease

2016-12-28 18:37:14

Blood Advances; December 28, 2016: /DOI:10.1182/bloodadvances.2016001214

Randall K. Merling, Douglas B. Kuhns, Colin L. Sweeney, Xiaolin Wu, Sandra Burkett, Jessica Chu, Janet Lee, Sherry Koontz, Giovanni Di Pasquale, Sandra A. Afione, John A. Chiorini, Elizabeth M. Kang, Uimook Choi, Suk See De Ravin and Harry L. Malech


Pseudogenes are duplicated genes with mutations rendering them nonfunctional. For single-gene disorders with homologous pseudogenes, the pseudogene might be a target for genetic correction. Autosomal-recessive p47phox-deficient chronic granulomatous disease (p47-CGD) is a life-threatening immune deficiency caused by mutations in NCF1, a gene with 2 pseudogenes, NCF1B and NCF1C. The most common NCF1 mutation, a GT deletion (ΔGT) at the start of exon 2 (>90% of alleles), is constitutive to NCF1B and NCF1C. NCF1 ΔGT results in premature termination, undetectable protein expression, and defective production of antimicrobial superoxide in neutrophils. We examined strategies for p47-CGD gene correction using engineered zinc-finger nucleases targeting the exon 2 ΔGT in induced pluripotent stem cells or CD34+ hematopoietic stem cells derived from p47-CGD patients. Correction of ΔGT in NCF1 pseudogenes restores oxidase function in p47-CGD, providing the first demonstration that targeted restoration of pseudogene function can correct a monogenic disorder.


Chronic granulomatous disease (CGD) is a primary immune deficiency resulting from defects in any 1 of 5 protein subunits that comprise the phagocyte nicotinamide adenine dinucleotide phosphate oxidase (phox) complex, resulting in a failure to generate microbicidal reactive oxygen species. Phenotypically, CGD is characterized by recurrent bacterial and fungal infections, granuloma formation, hyperinflammation, and autoimmunity. Mutations in neutrophil cytosolic factor 1 (NCF1) are responsible for autosomal-recessive p47phox-deficient CGD (p47-CGD), comprising ?25% of CGD cases. Notably, while mutations span across the genes for all other CGD patient phox genes, >80% of p47-CGD patients are homozygous for a 2-nt deletion (ΔGT) from the GTGT start of exon 2, resulting in a codon frame shift and premature termination, abrogating p47phox expression. The same ΔGT is constitutive to the start of presumptive exon 2 in the NCF1 pseudogenes NCF1B and NCF1C. Gene conversion resulting from close proximity of NCF1, NCF1B, and NCF1C at chromosome 7q11.23 8 likely accounts for the disproportionately high rates of the ΔGT mutation–mediated p47phox-deficient CGD relative to the other 3 genetic types of autosomal-recessive CGD. Recombination events between NCF1 and its pseudogenes may be responsible for the ΔGT mutation defect acquired by the resulting product at the NCF1 locus

Evolutionarily, pseudogenes are nonessential but persistent copies of genes that are dysfunctional through mutation(s) resulting in defective expression or nonfunctional products. By one report,9 at least 19?724 regions in the human genome may encode pseudogenes, although the total number of pseudogenes is expected to be far greater. These changes in the chromosomes due to mutations, insertions, deletions, duplications, and chromosomal rearrangements are driving forces for genome evolution and can be beneficial, detrimental, or inconsequential.

Here, we examined strategies for p47-CGD gene correction using engineered zinc-finger nucleases (ZFNs) targeting the exon 2 ΔGT in induced pluripotent stem cells (iPSCs) derived from p47-CGD patients. We show that production of p47phox protein in association with restoration of oxidase activity can occur following gene-targeted correction of the exon 2 ?GT at the NCF1 locus or its pseudogenes. This is a first report of functional correction of a monogenic disorder mediated by gene-editing resurrection of pseudogene function.

Empire Genomic's NCF1 FISH probe was used in this publication.

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