Instability in maintenance of X-chromosome inactivation in female human iPSCs: implications for treatment of X-CGD female carriers

Thursday, October 11, 2012 — Poster Session IV
2:00 p.m. – 4:00 p.m.	Natcher Conference Center, Building 45	NIAID	STEMCELL-5

* FARE Award Winner

Authors

• CL Sweeney
• RK Merling
• U Choi
• H Wang
• D Kuhns
• SM Holland
• HL Malech

Abstract

X-linked chronic granulomatous disease (X-CGD) is characterized by defective production of microbicidal reactive oxygen species (ROS) by neutrophils, caused by CYBB mutations. Due to random X-chromosome inactivation during embryonic development, female carriers of CYBB mutations are mosaic for both functional (ROS+) and non-functional (ROS-) neutrophils. In some carriers, extreme skewing of X-inactivation results in clinical X-CGD. To study X-inactivation, we established iPSCs from peripheral blood CD34+ cells of X-CGD female carriers who exhibit extreme skewing of the inactive X-chromosome (Xi) towards silencing of their normal CYBB allele (0.04-6% ROS+ neutrophils by DHR assay, resulting in severe to mild clinical X-CGD). Normal female and X-CGD carrier iPSCs exhibited clonal Xi by SNP expression assays. Upon in vitro neutrophil differentiation, iPSCs exhibited >10-fold increased % ROS+ neutrophils (18-72% ROS+ by DHR; p<0.03) relative to carriers’ peripheral blood, to potentially curative levels. In X-chromosome SNP expression assays, iPSC-derived neutrophils from female carriers and normal clones expressed both X-chromosomes equally, indicating reversal of X-inactivation. Our results demonstrate that Xi instability during female iPSC differentiation enables production of ROS+ neutrophils without gene therapy for extremely lyonized carriers of X-CGD, a finding with implications for treatment of female carriers of X-linked diseases manifesting severe clinical phenotypes.

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