The Wiskott-Aldrich syndrome (WAS) is an X-linked dis-
order that is characterized by thrombocytopenia, eczema,
impaired cellular and humoral immunity, and increased
susceptibility to lymphoid malignancies and autoimmu-
nity (1). The gene responsible for WAS was identified by
positional cloning and encodes the WAS protein (WASP),
a 502–amino acid proline-rich peptide (2). WASP is con-
stitutively expressed in cytoplasm of all nonerythroid
hematopoietic cells (3, 4) and appears to be of central
importance for signal transduction and cytoskeleton
reorganization events of hematopoietic cells (5).
Somatic revertant mosaicism has been described in an
increasing number of genetic disorders. Both back muta-
tions leading to restoration of wild-type sequences (6–15)
and second-site mutations resulting in compensatory
changes (16–18) have been demonstrated in mosaic indi-
viduals. Revertant mosaicism acquires a particular clini-
cal relevance in the case of patients affected with a pri-
mary immunodeficiency because it may lead to selective
growth advantage of the corrected cells and improvement
of disease symptoms. In fact, spontaneous in vivo rever-
sion has been reported in single cases of adenosine deam-
inase deficiency (12), X-linked severe combined immun-
odeficiency (8), and WAS (15), where patients showed
atypical and/or progressively mild clinical course because
of the selective growth advantage of the revertant lym-
phocytes. To date, evidence has been provided for at least
three mechanisms leading to back mutations or second-
site mutations in inherited disorders: intragenic recom-
bination (7, 10), gene conversion (9), and DNA poly-
merase slippage (11, 15, 18). Slippage-type events are
thought to cause insertion and deletion mutations of
small repetitive sequences in a number of human genet-
ic diseases and explain the instability of repetitive
sequences observed in bacterial, yeast, and eukaryotic sys-
The Journal of Clinical Investigation| May 2003| Volume 111| Number 9
Second-site mutation in the Wiskott-Aldrich syndrome
(WAS) protein gene causes somatic mosaicism
in two WAS siblings
Taizo Wada,1Akihiro Konno,1Shepherd H. Schurman,1Elizabeth K. Garabedian,2
Stacie M. Anderson,1Martha Kirby,1David L. Nelson,3and Fabio Candotti1
1Genetics and Molecular Biology Branch,
2Medical Genetics Branch, National Human Genome Research Institute, and
3Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
Revertant mosaicism due to true back mutations or second-site mutations has been identified in sev-
eral inherited disorders. The occurrence of revertants is considered rare, and the underlying genetic
mechanisms remain mostly unknown. Here we describe somatic mosaicism in two brothers affected
with Wiskott-Aldrich syndrome (WAS). The original mutation causing disease in this family is a sin-
gle base insertion (1305insG) in the WAS protein (WASP) gene, which results in frameshift and abro-
gates protein expression. Both patients, however, showed expression of WASP in a fraction of their
T cells that were demonstrated to carry a second-site mutation causing the deletion of 19 nucleotides
from nucleotide 1299 to 1316. This deletion abrogated the effects of the original mutation and
restored the WASP reading frame. In vitro expression studies indicated that mutant protein encod-
ed by the second-site mutation was expressed and functional, since it was able to bind to cellular part-
ners and mediate T cell receptor/CD3 downregulation. These observations were consistent with evi-
dence of in vivo selective advantage of WASP-expressing lymphocytes. Molecular analysis revealed
that the sequence surrounding the deletion contained two 4-bp direct repeats and that a hairpin
structure could be formed by five GC pairs within the deleted fragment. These findings strongly sug-
gest that slipped mispairing was the cause of this second-site mutation and that selective accumula-
tion of WASP-expressing T lymphocytes led to revertant mosaicism in these patients.
J. Clin. Invest. 111:1389–1397 (2003). doi:10.1172/JCI200315485.
Received for publication March 19, 2002, and accepted in revised form
February 4, 2003.
Address correspondence to: Fabio Candotti, Disorders of
Immunity Section, Genetics and Molecular Biology Branch,
National Human Genome Research Institute, National Institutes
of Health, 49 Convent Drive, Building 49, Room 3A20, MSC
4442, Bethesda, Maryland 20892-4442, USA.
Phone: (301) 435-2944; Fax: (301) 480-3678;
Taizo Wada’s present address is: Department of Pediatrics,
Kanazawa University Graduate School of Medical Science,
Taizo Wada and Akihiro Konno contributed equally to this work.
Conflict of interest: The authors have declared that no conflict of
Nonstandard abbreviations used: Wiskott-Aldrich syndrome
(WAS); recombinant IL-2 (rIL-2); polymorphonuclear cell (PMN);
phycoerythrin (PE); glutathione S-transferase (GST); T cell
receptor-β (TCR-β); verprolin homology, cofilin homology,
and acidic (VCA).
tems (19–22). Polymerase slippage is also the most com-
monly invoked mechanism to explain triplet repeat
expansion in human diseases (e.g., Huntington disease,
fragile X syndrome, and Friedreich ataxia) (23).
Here, we described two WAS siblings with revertant
mosaicism due to the same second-site mutation, like-
ly caused by slipped mispairing. The original mutation
responsible for the disease in this family is single base G
insertion predicted to cause frameshift and premature
termination of the protein. A second-site mutation was
found in T lymphocytes from both brothers that carried
a deletion of a 19 bp fragment, including the original
mutation. This deletion resulted in restoration of the
reading frame and the generation of an internally trun-
cated, yet functional, WASP protein. Revertant T lym-
phocytes showed WASP expression and selective growth
advantage in vivo over the WASP-negative (WASP–) cells.
The occurrence of the same second-site mutation in two
subjects from the same family strongly suggests a com-
mon underlying mechanism, and the characteristics of
nucleotide sequence involved in the mutation are clear-
ly compatible with slipped strand mispairing. From our
observations we hypothesize that slipped mispairing is
commonly occurring in the contest of this particular
sequence, but only recognized in the rare cases in which
selective advantage permits the accumulation of rever-
tant cells above the detection threshold.
Case presentation. Figure 1 shows the pedigree of the
patients’ family. Patient II-1 is an 18-year-old male who
developed petechiae early after birth when his platelet
count was in the range 7,000–20,000/µl. In the first year
of life, he had eczema, otitis media, and conjunctivitis.
After a clinical diagnosis of WAS was made at the age of
16 months, he underwent a splenectomy because of per-
sistent thrombocytopenia. Between the age of 2 and 3
years, he had multiple episodes of pneumonia and
recurrent leukocytoclastic vasculitis. Frequent ear infec-
tions and one episode of bronchitis are described until
the age of 6, when he suffered from streptococcal pneu-
monia and sepsis. Between the ages of 12 and 16 years,
he had documented episodes of medium vessel vasculi-
tis leading to recurrent skin ulcers.
Patient II-1’s younger brother (II-2) also developed
thrombocytopenia early after birth and underwent a
splenectomy at the age of 6 months. The postoperative
clinical history is remarkable for occasional otitis
media and continued severe eczema until the age of 9
years, when he developed molluscum contagiosum,
which progressed unremittingly for 3 years until all
extremities and truncal surfaces accessible to hands
were affected. At age 13, he developed non-Hodgkin B
cell lymphoma, which relapsed after chemotherapy at
the age of 15. Both clinical histories are consistent with
severe WAS phenotype (score of 5) (24). No clinical
improvement with age has yet been observed in either
patient. No history of other affected males was noted
in the maternal side of the family.
Cell preparation. PBMCs were isolated by Ficoll-
Hypaque (Mediatech Inc., Washington, DC, USA) gra-
dient centrifugation from the patients and healthy
controls. For T lymphocyte cultures, PBMCs were
maintained in the presence of 100 ng/ml of anti-CD3
(OKT3; Ortho Diagnostic Systems Inc., Raritan, New
Jersey, USA), 5 µg/ml of anti-CD28 mAb (PharMingen,
San Diego, California, USA), and 100 IU/ml of recom-
binant IL-2 (rIL-2) (gift of C. Reynolds, National Can-
cer Institute, Frederick, Maryland, USA) for 3 days in
RPMI-1640 medium (Life Technologies Inc., Rockville,
Maryland, USA) containing 10% FBS (Gemini Bio-
Products, Woodland, California, USA) and antibiotics.
Cells were then cultured in RPMI-1640 plus 10% FBS
medium supplemented with 100 IU/ml of rIL-2 for 12
days. Human T lymphotropic virus type 1–transformed
WAS T cell lines carrying the 57delG “null” mutation
(25) were maintained in the same medium. MOLT-4
cells were obtained from American Type Culture Col-
lection (Manassas, Virginia, USA).
Flow-cytometric analysis of WASP and cell sorting. Flow
cytometric analysis of WASP was performed as described
previously (15) with minor modifications. PBMCs were
stained for cell surface antigens prior to cell membrane
permeabilization using the following mAb’s: CyChrome-
conjugated anti-CD3 or anti-CD20 (PharMingen); TRI-
color–labeled anti-CD8 (Caltag Laboratories Inc.,
Burlingame, California, USA); FITC-conjugated anti-
CD4 (Caltag Laboratories Inc.); FITC-conjugated anti-
CD45RA and anti-CD45R0 (PharMingen); and FITC-
conjugated anti-CD14 or anti-CD56 (Becton Dickinson
Immunocytometry Systems, San Jose, California, USA).
After washing, cells were fixed and permeabilized with
CytoStain kit (PharMingen) and were incubated with
1:200 diluted anti-WASP (3F3-A5) mAb (3) or purified
mouse IgG-1 (PharMingen) at 4°C for 20 min. Cells were
then reacted with biotin-conjugated anti-mouse IgG-1
at 4°C for 20 min, followed by further incubation with
phycoerythrin-conjugated (PE-conjugated) streptavidin
(both from PharMingen). Stained cells were analyzed
with a FACScan flow cytometer and the CellQuest soft-
ware (Becton Dickinson Immunocytometry Systems).
For sequencing analysis, WASP-expressing (WASP+)
and WASP–cells were purified from cultured T lym-
phocytes using a FACSVantage cell sorter (Becton
Dickinson Immunocytometry Systems). The purity of
The Journal of Clinical Investigation| May 2003| Volume 111|Number 9
Pedigree of the study family. Solid symbols represent affected individ-
uals, and the carrier status of subject I-1 is indicated by the central dot.
who did not observe clinical improvement in another
case of WAS with revertant mosaicism (14). One expla-
nation for the lack of clinical benefit from the occur-
rence of the second-site mutation in the patients
described here is that the absolute numbers of WASP+T
lymphocytes, and especially revertant naive T lympho-
cytes, 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 carry-
ing the ∆19bp mutation may not be as effective in vivo
as the wild-type WASP.
In summary, our studies provide evidence for poten-
tial 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 character-
ization 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.
The authors are thankful to R. Michael Blaese for pro-
viding historical samples from patient II-1 and to Andy
Baxevanis for help with sequence analysis. We also
thank Jacqueline Keller for secretarial assistance. This
work was supported in part by Japan Society for the Pro-
motion of Science Research Fellowships for Japanese
Biomedical and Behavioral Researchers at the NIH.
1.Ochs, H.D. 1998. The Wiskott-Aldrich syndrome. Springer Semin.
2.Derry, J.M., Ochs, H.D., and Francke, U. 1994. Isolation of a novel gene
mutated in Wiskott-Aldrich syndrome. Cell.78:635–644.
3.Stewart, D.M., et al. 1996. Studies of the expression of the Wiskott-Aldrich
syndrome protein. J. Clin. Invest.97:2627–2634.
4.Parolini, O., et al. 1997. Expression of Wiskott-Aldrich syndrome protein
(WASP) gene during hematopoietic differentiation. Blood.90:70–75.
5.Snapper, S.B., and Rosen, F.S. 1999. The Wiskott-Aldrich syndrome pro-
tein (WASP): roles in signaling and cytoskeletal organization. Annu. Rev.
6.Kvittingen, E.A., Rootwelt, H., Berger, R., and Brandtzaeg, P. 1994. Self-
induced correction of the genetic defect in tyrosinemia type I. J. Clin. Invest.
7.Ellis, N.A., et al. 1995. Somatic intragenic recombination within the
mutated locus BLM can correct the high sister-chromatid exchange phe-
notype of Bloom syndrome cells. Am. J. Hum. Genet.57:1019–1027.
8.Stephan, V., et al. 1996. Atypical X-linked severe combined immunodefi-
ciency due to possible spontaneous reversion of the genetic defect in
T cells. N. Engl. J. Med.335:1563–1567.
9.Jonkman, M.F., et al. 1997. Revertant mosaicism in epidermolysis bullosa
caused by mitotic gene conversion. Cell.88:543–551.
10.Lo Ten Foe, J.R., et al. 1997. Somatic mosaicism in Fanconi anemia: molec-
ular basis and clinical significance. Eur. J. Hum. Genet.5:137–148.
11.Gregory, J.J., Jr., et al. 2001. Somatic mosaicism in Fanconi anemia: evi-
dence of genotypic reversion in lymphohematopoietic stem cells. Proc. Natl.
Acad. Sci. U. S. A. 98:2532–2537.
12.Hirschhorn, R., et al. 1996. Spontaneous in vivo reversion to normal of an
inherited mutation in a patient with adenosine deaminase deficiency. Nat.
13.Ariga, T., et al. 2001. T-cell lines from 2 patients with adenosine deaminase
(ADA) deficiency showed the restoration of ADA activity resulted from the
reversion of an inherited mutation. Blood.97:2896–2899.
14.Ariga, T., et al. 2001. Spontaneous in vivo reversion of an inherited muta-
tion in the Wiskott-Aldrich syndrome. J. Immunol.166:5245–5249.
15.Wada, T., et al. 2001. Somatic mosaicism in Wiskott-Aldrich syndrome
suggests in vivo reversion by a DNA slippage mechanism. Proc. Natl. Acad.
Sci. U. S. A. 98:8697–8702.
16.Darling, T.N., Yee, C., Bauer, J.W., Hintner, H., and Yancey, K.B. 1999.
Revertant mosaicism: partial correction of a germ-line mutation in
COL17A1 by a frame-restoring mutation. J. Clin. Invest. 103:1371–1377.
17.Waisfisz, Q., et al. 1999. Spontaneous functional correction of homozy-
gous Fanconi anaemia alleles reveals novel mechanistic basis for reverse
mosaicism. Nat. Genet.22:379–383.
18.Arredondo-Vega, F.X., et al. 2002. Adenosine deaminase deficiency with
mosaicism for a “second-site suppressor” of a splicing mutation: decline
in revertant T lymphocytes during enzyme replacement therapy. Blood.
19.Levinson, G., and Gutman, G.A. 1987. Slipped-strand mispairing: a major
mechanism for DNA sequence evolution. Mol. Biol. Evol.4:203–221.
20.Darvasi, A., and Kerem, B. 1995. Deletion and insertion mutations in short
tandem repeats in the coding regions of human genes. Eur. J. Hum. Genet.
21.Oron-Karni, V., Filon, D., Rund, D., and Oppenheim, A. 1997. A novel
mechanism generating short deletion/insertions following slippage is sug-
gested by a mutation in the human alpha2-globin gene. Hum. Mol. Genet.
22.Bzymek, M., and Lovett, S.T. 2001. Instability of repetitive DNA sequences:
the role of replication in multiple mechanisms. Proc. Natl. Acad. Sci. U. S. A.
23.Pearson, C.E., and Sinden, R.R. 1998. Trinucleotide repeat DNA structures:
dynamic mutations from dynamic DNA. Curr. Opin. Struct. Biol.8:321–330.
24.Zhu, Q., et al. 1995. The Wiskott-Aldrich syndrome and X-linked congen-
ital thrombocytopenia are caused by mutations of the same gene. Blood.
25.Wada, T., Jagadeesh, G.J., Nelson, D.L., and Candotti, F. 2002. Retrovirus-
mediated WASP gene transfer corrects Wiskott-Aldrich syndrome T cell
dysfunction. Hum. Gene Ther.13:1039–1046.
26.Candotti, F., et al. 1999. Retrovirus-mediated WASP gene transfer corrects
defective actin polymerization in B cell lines from Wiskott-Aldrich syn-
drome patients carrying ‘null’ mutations. Gene Ther.6:1170–1174.
27.Candotti, F., et al. 1997. Structural and functional basis for JAK3-deficient
severe combined immunodeficiency. Blood.90:3996–4003.
28.Assaf, C., et al. 2000. High detection rate of T-cell receptor beta chain
rearrangements in T-cell lymphoproliferations by family specific poly-
merase chain reaction in combination with the GeneScan technique and
DNA sequencing. Blood.96:640–646.
29.Miller, A.D., et al. 1991. Construction and properties of retrovirus pack-
aging cells based on gibbon ape leukemia virus. J. Virol.65:2220–2224.
30.Treisman, J., et al. 1995. Interleukin-2-transduced lymphocytes grow in an
autocrine fashion and remain responsive to antigen. Blood. 85:139–145.
31.Onodera, M., et al. 1998. Development of improved adenosine deaminase
retroviral vectors. J. Virol.72:1769–1774.
32.Kolluri, R., et al. 1995. Identification of WASP mutations in patients with
Wiskott-Aldrich syndrome and isolated thrombocytopenia reveals allelic
heterogeneity at the WAS locus. Hum. Mol. Genet.4:1119–1126.
33.Mantuano, E., et al. 1993. Analysis of X-chromosome inactivation in bone
marrow precursors from carriers of Wiskott-Aldrich syndrome and
X-linked severe combined immunodeficiency: evidence that the Wiskott-
Aldrich gene is expressed prior to granulocyte-macrophage colony-form-
34.Wengler, G., Gorlin, J.B., Williamson, J.M., Rosen, F.S., and Bing, D.H.
1995. Nonrandom inactivation of the X chromosome in early lineage
hematopoietic cells in carriers of Wiskott-Aldrich syndrome. Blood.
35.Fearon, E.R., Kohn, D.B., Winkelstein, J.A., Vogelstein, B., and Blaese, R.M.
1988. Carrier detection in the Wiskott-Aldrich syndrome. Blood.
36.Greer, W.L., et al. 1989. X-chromosome inactivation in the Wiskott-Aldrich
syndrome: a marker for detection of the carrier state and identification of
cell lineages expressing the gene defect. Genomics.4:60–67.
37.Kim, A.S., Kakalis, L.T., Abdul-Manan, N., Liu, G.A., and Rosen, M.K. 2000.
Autoinhibition and activation mechanisms of the Wiskott-Aldrich syn-
drome protein. Nature.404:151–158.
38.Devriendt, K.J., et al. 2001. Constitutively activating mutation in WASP
causes X-linked severe congenital neutropenia. Nat. Genet. 27:313–317.
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