Somatic revertant mosaicism due to back mutations or second-site mutations has been reported recently in several genetic diseases: tyrosinemia type 1 (6
), Bloom syndrome (7
), epidermolysis bullosa (9
), Fanconi anemia (10
), and primary immunodeficiencies, including adenosine deaminase deficiency (12
), X-linked severe combined immunodeficiency (8
), and WAS (14
). The molecular mechanism of such events remains mostly unclear, except for the few cases where intragenic recombination (7
), gene conversion (9
), or DNA polymerase slippage (11
) has been shown. The real frequency of the occurrence of revertant mosaicism also remains to be determined, although it is assumed to be extremely low regardless of the mutagenic mechanism. Here we describe the unusual case of two WAS siblings presenting with somatic mosaicism due to the same second-site mutation, a deletion of 19 bp, including the original single nucleotide insertion. The Δ19bp mutation was observed only in the patients’ T lymphocytes and led to reconstitution of WASP expression. Sequencing analysis of the WASP
gene in the general population excluded the possibility that the Δ19bp mutation could be a functional polymorphism. The Δ19bp mutation was not detectable in PBMC samples from the patients’ mother, although somatic mosaicism would be difficult to detect in women due to lack of selective advantage. Both patients, however, had the normal 46 XY male karyotype, thus ruling out the possibility that the WASP+
T lymphocytes could have arisen from maternal T cell engraftment. It is also extremely unlikely that both patients acquired the same mutant T cells postnatally from infusions of blood products. We therefore conclude that the T lymphocytes carrying the Δ19bp mutation originated in both patients in vivo due to a second-site mutation.
Revertant mosaicism due to a second-site mutation has been reported previously (16
). In particular, the occurrence of the same second-site mutation in two brothers with Fanconi anemia has been reported by Waisfisz et al. (17
), who proposed the presence of the same second-site mutation having resulted from the same specific molecular mechanism in the two subjects. Similarly, our observations strongly suggest that a common underlying mechanism resulted in the occurrence of the same second-site mutation in the two brothers with WAS, described here. Slipped mispairing is a well-accepted mechanism for genomic instability, resulting in insertions and deletions of repetitive sequences in many genetic disorders (19
). Analysis of the sequence surrounding the Δ19bp second-site mutation (Figure c) leads us to propose that slipped mispairing is at the basis of our findings. We argue that the presence of two 4-bp direct sequences (CCTG) and the hairpin structure created by five GC pairs within the deleted fragment are able to induce slippage and mispairing of the nascent and template strands during DNA replication.
The slipped mispairing event leading to the Δ19bp deletion may have occurred in a primitive pluripotent hematopoietic progenitor or in a committed T cell precursor. The latter possibility is supported by the notion that WAS-carrier women preferentially inactivate the X chromosome carrying the mutated WASP
gene in hematopoietic progenitors (33
) and all mature hematopoietic cells (35
cells deriving from a revertant pluripotent hematopoietic progenitor would therefore be expected to show selective advantage among all lineages, whereas we could only detect WASP+
cells within the CD3+
T lymphocytes from our two patients.
On the other hand, the occurrence of the revertant mosaicism in both patients suggests that the second-site mutation is not an extremely rare event. It seems unlikely, therefore, that such occurrence would coincidentally target only the same cell-differentiation stage in both patients. On the contrary, slipped mispairing could be a relatively common event with respect to nucleotide 1299–1316 of the WASP sequence and, therefore, that the Δ19bp deletion may occur in a variety of cell types, but become apparent only in cells characterized by a long life span that can accumulate over time. To the detection limits of our genotyping assay, we could clearly demonstrate the presence of the Δ19bp deletion only among patients’ purified T lymphocytes, however. These findings, taken together with the detection of WASP+ cells among both CD4+ and CD8+ T lymphocytes and the observation of polyclonal TCR-β gene rearrangements in WASP+ T cells, allow placement of the second-site mutation event at the level of a T cell progenitor cell before CD4/CD8 lineage commitment and TCR rearrangement.
Consistent with our previous observations (15
), there is evidence of in vivo selective advantage of WASP+
T lymphocytes in these two patients. Flow-cytometric studies showed that 2.4–6.2% WASP+
cells appeared among CD45RA+
naive T cells, while more than half of the CD45R0+
memory T cells were WASP+
in both patients. These findings clearly indicate WASP+
T cells had selective growth advantage over WASP–
counterparts during the differentiation and proliferation steps leading to the acquisition of the immunological memory. The reasons why the patients showed higher proportion of revertant CD4+
T lymphocytes than CD8+
T cells are presently unclear. Immunophenotyping of T lymphocyte subsets demonstrated an increased number of activated memory CD4+
T lymphocytes in both patients (Table ). It is therefore possible that peripheral expansion of these CD4+
T lymphocytes had favored the selective advantage of revertant cells within this subset of lymphocytes.
Our data also suggest that the expression of the internally deleted WASP mutant generated by the Δ19bp mutation was responsible for the in vivo selective advantage of revertant T cells through reconstitution of WASP function. It has been recognized recently that WASP function is regulated through an autoinhibitory conformation involving intramolecular interaction between the GTPase-binding domain and the verprolin homology, cofilin homology, and acidic (VCA) region of WASP (37
). The original insG mutation destroys the VCA region, thus likely interfering with the protein tertiary structure and resulting in instability. On the contrary, the Δ19bp mutation results in the deletion of 6-AA at the end of the proline-rich region but restores the sequence of the VCA domain, which can regenerate its ability to interact to the GTPase-binding domain, thus resulting in a more stable protein (38
). Accordingly, our in vitro reconstitution experiments showed that WASP expression, binding to SH3 partners, and TCR/CD3 downregulation in T cells carrying the Δ19bp mutant were compatible with those observed in the presence of wild-type WASP and indicated that the Δ19bp mutant is functional.
The availability of archived PBMC samples from patient II-1 offered us a unique opportunity to study the accumulation of WASP+
T lymphocytes over time in this patient. We were able to detect WASP expression in his lymphocytes at age 8, and the percentage of WASP+
cells increased with age, suggesting that the accumulation of WASP+
cells in peripheral blood had started more than 10 years ago. Neither of the two patients in this study, however, have yet shown improvement of their clinical phenotype. This is in contrast to a previously described case of somatic mosaicism in a WAS patient carrying more than 80% of revertant T lymphocytes (15
), but is consistent with the finding of Ariga et al. who did not observe clinical improvement in another case of WAS with revertant mosaicism (14
). One explanation for the lack of clinical benefit from the occurrence of the second-site mutation in the patients described here is that the absolute numbers of WASP+
T lymphocytes, and especially revertant naive T lymphocytes, were simply not high enough to correct the WAS phenotype. If this is the case, it is possible that an improvement will be observed in the future because of the selective accumulation of WASP+
T lymphocytes. Alternatively, the function of the mutant WASP carrying the Δ19bp mutation may not be as effective in vivo as the wild-type WASP.
In summary, our studies provide evidence for potential beneficial outcomes of slipped mispairing events and tie this genetic mechanism to the generation of somatic mosaicism. Although our current knowledge regards back mutations or second-site mutations as extremely rare events, the observation of the present two cases raises the possibility that genetic reversions may take place more often than is commonly accepted, but remain undetected because they do not necessarily result in modification of the clinical phenotype. Because revertant mosaicism is a natural form of gene therapy, the identification of revertants and characterization of mechanism underlying back mutations or second-site mutations is obviously of great importance for the development of new therapeutic strategies for genetic disorders and deserve active investigation.