Somatic mosaicism in Wiskott--Aldrich syndrome suggests in vivo reversion by a DNA slippage mechanism - PubMed (original) (raw)

Case Reports

. 2001 Jul 17;98(15):8697-702.

doi: 10.1073/pnas.151260498. Epub 2001 Jul 10.

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Case Reports

Somatic mosaicism in Wiskott--Aldrich syndrome suggests in vivo reversion by a DNA slippage mechanism

T Wada et al. Proc Natl Acad Sci U S A. 2001.

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.

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Figures

Figure 1

Figure 1

Simplified pedigree of the proband's family. Solid squares represent affected individuals; diagonal lines indicate deceased subjects. Carrier status of female subjects is indicated by a dot.

Figure 2

Figure 2

Analysis of WASP expression. (a) Western blot analysis of WASP was performed using lysates of PBMC obtained from normal (lane 1), the proband (lane 2), and affected individual III-6 (lane 3). The arrow indicates the position of the WASP band. (b–d) Flow cytometric analysis of WASP. PBMC were stained with mAbs for cell-surface antigens, fixed, and permeabilized. Cells were then reacted with anti-WASP mAb (solid histogram) or a control Ab (open histogram) and further incubated with biotin-conjugated anti-mouse IgG1 followed by streptavidin-phycoerythrin. WASP expression in lymphocytes and monocytes obtained from normal control and the proband is presented (b). WASP expression in CD3+ T, CD20+ B, CD56+ NK, CD4+ T, and CD8+ T cell subsets from the proband is shown (c). The dot plot of a two-color immunofluorescence profile of CD45 isoforms and WASP expression in the proband's CD3+ T cells is presented (d, Left). A second gate is set for the upper quadrants to include CD45RA+CD3+ or CD45R0+CD3+ cells, and WASP expression in each cell population is shown (d, Right).

Figure 3

Figure 3

Mutational analysis of WASP gene. The sequence of exon 4 of the WASP gene was amplified from DNA extracted from normal PBMC and the proband's granulocytes, PBMC, cultured T cells, and skin. Direct sequencing was performed using an automated sequencer. A bar highlights the 6-bp repeat sequence.

Figure 4

Figure 4

CDR3 size distribution of TCR Vβ. Each TCR Vβ fragment was amplified from cDNA with one of the 24 Vβ-specific primers. The size distribution of PCR products was determined by an automated sequencer and GENESCAN software. (a) Normal control; (b) the proband.

Figure 5

Figure 5

(a) Analysis of TCR/CD3 down-regulation. PBMC from normal control, the proband, and affected individuals III-6 and IV-1 were incubated with anti-CD3 mAb, washed, and further reacted with biotinylated goat anti-mouse Ab at 37°C for 60 min. Cells were then washed and stained with streptavidin and anti-CD2 mAb. After fixation, CD3 expression on CD2+ T cells was analyzed by FACS. Solid and open histogram represent CD3 expression in cells unstimulated or treated for 60 min, respectively. (b) Cell proliferation assay. PBMC from normal control, the proband, and WA8, a patient with typical WAS and with a clinical score of 5, were cultured for 72 h with anti-CD3 mAb alone or together with anti-CD28 mAb. DNA incorporation of [3H]thymidine was evaluated. Each bar represents the mean ± SD of triplicate cultures.

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