Somatic mosaicism in Wiskott–Aldrich syndrome suggestsin vivoreversion by a DNA slippage mechanism

Abstract

Somatic mosaicism caused by in vivo reversion of inherited mutations has been described in several human genetic disorders. Back mutations resulting in restoration of wild-type sequences and second-site mutations leading to compensatory changes have been shown in mosaic individuals. In most cases, however, the precise genetic mechanisms underlying the reversion events have remained unclear, except for the few instances where crossing over or gene conversion have been demonstrated. Here, we report a patient affected with Wiskott-Aldrich syndrome (WAS) caused by a 6-bp insertion (ACGAGG) in the WAS protein gene, which abrogates protein expression. Somatic mosaicism was documented in this patient whose majority of T lymphocytes expressed nearly normal levels of WAS protein. These lymphocytes were found to lack the deleterious mutation and showed a selective growth advantage in vivo. Analysis of the sequence surrounding the mutation site showed that the 6-bp insertion followed a tandem repeat of the same six nucleotides. These findings strongly suggest that DNA polymerase slippage was the cause of the original germ-line insertion mutation in this family and that the same mechanism was responsible for its deletion in one of the propositus T cell progenitors, thus leading to reversion mosaicism.

Lentiviral vectors transcriptionally targeted to hematopoietic cells by WASP gene proximal promoter sequences

“…The proximal WASP gene promoter in the context of a SIN lentiviral vector may have useful characteristics for clinical trials of gene therapy for WAS as it achieves hematopoietic-specific and physiologically relevant levels of WASp. Expression of the WASP transgene confers a selective growth advantage to transduced T cells as described in spontaneous reversions of WAS patients [29][30][31] as well as in other models of CD3-activated WASp + cells. 32,33 This observation indicates that the protein expressed in WW-transduced cells is functional.…”

“…[29][30][31][32][33] Therefore, HVS-WAS/1 cells were transduced with the WW lentiviral vector at MOIs of 0.01, 0.1 and 1 and kept in culture for 39 days. The percentage of WASpexpressing cells was analyzed by flow cytometry at days 7, 27 and 39 after transduction.…”

Impaired in vitro regulatory T cell function associated with Wiskott–Aldrich syndrome

“…Mutation analysis of the WAS gene and flow cytometry analysis of WASp expression were performed as described (20). All human studies were approved by the NHGRI Institutional Review Board.…”

Successes and risks of gene therapy in primary immunodeficiencies

“…BMT from an HLA-identical sibling is the treatment of choice and can be curative for WAS, but the risks of BMT from other sources are high. 62 Skewed X-inactivation in female carriers of WAS 63 and the occurrence of spontaneous somatic mutations restoring WASP function associated with clinical improvement and repopulation by the wildtype phenotype 64 indicate a survival advantage of genecorrected cells over WASP-deficient cells. Preclinical studies have shown that retroviral transduction corrects the immunologic defect in WASP knockout mice and in human cell lines, with increased actin polymerization and functional correction of T cells after transduction.…”