A Novel WASP Gene Mutation in a Chinese Boy with Wiskott–Aldrich Syndrome

SummaryTwelve different mutations in the WASP gene were found in twelve unrelated families with Wiskott-Aldrich syndrome (WAS) or X-linked thrombocytopenia (XLT). Four frameshift, one splice, one nonsense mutation, and one 18-base-pair deletion were detected in seven patients with WAS. Only missense mutations were found in five patients diagnosed as having XLT. One of the nucleotide substitutions in exon 2 (codon 86) results in an Arg to Cys replacement. Two other nucleotide substitutions in this codon, R86L and R86H, have been reported previously, both giving rise to typical WAS symptoms, indicating a mutational hot spot in this codon. The finding of mutations in the WASP gene in both WAS and XLT gives further evidence of these syndromes being allelic. The relatively small size of the WASP gene facilitates the detection of mutations and a reliable diagnosis of both carriers and affected fetuses in families with WAS or XLT.

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A Case of Wiskott-Aldrich Syndrome With Dual Mutations in Exon 10 of the WASP Gene: An Additional De Novo One-Base Insertion, Which Restores Frame Shift Due to an Inherent One-Base Deletion, Detected in the Major Population of the Patient's Peripheral Blood Lymphocytes

Blood ◽

10.1182/blood.v92.2.699 ◽

1998 ◽

Vol 92 (2) ◽

pp. 699-701 ◽

Cited By ~ 30

Author(s):

Tadashi Ariga ◽

Masafumi Yamada ◽

Yukio Sakiyama ◽

Osamu Tatsuzawa

Keyword(s):

Peripheral Blood ◽

De Novo ◽

Peripheral Blood Lymphocytes ◽

Blood Lymphocytes ◽

Wiskott Aldrich Syndrome ◽

Frame Shift ◽

Wasp Gene ◽

Exon 10 ◽

Base Insertion

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Developing Lentiviral Vectors for Gene Therapy of Wiskott-Aldrich Syndrome.

Blood ◽

10.1182/blood.v104.11.3170.3170 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3170-3170

Author(s):

Marguerite V. Evans-Galea ◽

Matthew M. Wielgosz ◽

Ted S. Strom ◽

Hideki Hanawa ◽

John M. Cunningham ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Stem Cell ◽

T Cell ◽

Gene Transfer ◽

Hela Cells ◽

Lentiviral Vectors ◽

Proximal Promoter ◽

Wiskott Aldrich Syndrome ◽

Wasp Gene

Abstract The Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive disorder caused by mutations in the WASP gene. WASP is an effector protein in the actin polymerization pathway. Characterized by immunodeficiency, micro-thrombocytopenia and eczema, untreated WAS patients may also suffer hemorrhages, recurring infections and potential malignancies. WAS can be cured by bone marrow (BM) transplant but because many patients lack a suitable donor, stem cell-targeted gene transfer is being developed as an alternative therapeutic approach. We have demonstrated correction of the T-cell proliferation defect in Wasp− mice using MSCV oncoretroviral vectors (Blood102:3108, 2003). However, our competitive repopulation studies in mice with both wild-type (WT) and gene-corrected Wasp− BM, demonstrated only a modest selective advantage for gene modified lymphocytes. Correction of the lymphocytopenia was observed only in animals having high proportions of transduced cells. In addition, variability in the level of gene expression among gene-corrected cells was associated with only partial correction of the T-cell cytokine secretory defects. Thus, the efficiency of stem cell-targeted gene transfer as well as the level and consistency of gene expression are likely to be key factors that determine success in any clinical application of gene transfer for the treatment of WAS. In attempting to improve vector design, we have used lentiviral vectors because of their greater efficiency in transducing repopulating stem cells as we recently demonstrated in a non-human primate model (Blood103:4062, 2004). The WASP gene is regulated by two promoter regions. The proximal promoter lies immediately adjacent to the translation start site with the distal promoter found 6 kb upstream, followed by an alternate first exon. We have developed a series of third generation, self-inactivating lentiviral vectors containing the MSCV, proximal or distal WASP promoters driving GFP in the reverse transcriptional orientation. The WASP promoters were active in lymphocytes but not HeLa cells in vitro. However, expression was low in lymphocytes and granulocytes in mice transplanted with genetically modified stem cells. In an effort to abrogate any position-effect variegation and enhance expression, we generated a new series of vectors with the transcriptional unit in the forward orientation that also contained the woodchuck post-transcriptional regulatory element, the chicken beta-globin 5′ DNase I hypersensitive site 4 (I) and human beta-interferon scaffold attachment region (S) insulator elements. Either GFP or murine Wasp cDNA were included for expression analysis. Despite the complexity of the SI-containing vectors, titers of concentrated preparations ranged from 9x106 TU/ml to 5x107 TU/ml and enabled transduction of both cell lines and murine hematopoietic stem cells. Low-level GFP expression from the proximal promoter was detected in HeLa cells with higher expression found in lymphocytes (NALM6 and Jurkat cells). Expression of GFP under the control of the proximal WASP promoter was detected in vivo in multiple hematopoietic lineages in mice transplanted with transduced stem cells. Future efforts will focus on further characterization and optimization of vector design with the goal of achieving consistent, high level expression.

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A 5′ Regulatory Sequence Containing Two Ets Motifs Controls the Expression of the Wiskott-Aldrich Syndrome Protein (WASP) Gene in Human Hematopoietic Cells

Blood ◽

10.1182/blood.v91.12.4554.412k26_4554_4560 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4554-4560 ◽

Cited By ~ 1

Author(s):

A. Petrella ◽

I. Doti ◽

V. Agosti ◽

P. Carandente Giarrusso ◽

D. Vitale ◽

...

Keyword(s):

Transcription Factors ◽

Jurkat Cells ◽

Regulatory Sequence ◽

Regulatory Sequences ◽

Specific Expression ◽

Wiskott Aldrich Syndrome ◽

Nuclear Extracts ◽

Ets Family ◽

Wasp Gene ◽

Syndrome Protein

The recently-identified Wiskott-Aldrich syndrome protein gene (WASP) is responsible for the Wiskott-Aldrich X-linked immunodeficiency as well as for isolated X-linked thrombocytopenia (XLT). To characterize the regulatory sequences of the WASP gene, we have isolated, sequenced and functionally analyzed a 1.6-Kb DNA fragment upstream of the WASP coding sequence. Transfection experiments showed that this fragment is capable of directing efficient expression of the reporter chloramphenicol acetyltransferase (CAT) gene in all human hematopoietic cell lines tested. Progressive 5′ deletions showed that the minimal sequence required for hematopoietic-specific expression consists of 137 bp upstream of the transcription start site. This contains potential binding sites for several hematopoietic transcription factors and, in particular, two Ets-1 consensus that proved able to specifically bind to proteins present in nuclear extracts of Jurkat cells. Overexpression of Ets-1 in HeLa resulted in transactivation of the CAT reporter gene under the control of WASP regulatory sequences. Disruption of the Ets-binding sequences by side-directed mutagenesis abolished CAT expression in Jurkat cells, indicating that transcription factors of the Ets family play a key role in the control of WASP transcription.

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The Mouse Homolog of the Wiskott–Aldrich Syndrome Protein (WASP) Gene Is Highly Conserved and Maps near the Scurfy (sf) Mutation on the X Chromosome

Genomics ◽

10.1006/geno.1995.9979 ◽

1995 ◽

Vol 29 (2) ◽

pp. 471-477 ◽

Cited By ~ 32

Author(s):

JONATHAN M.J. DERRY ◽

PHILIPP WIEDEMANN ◽

PATRICK BLAIR ◽

YUKER WANG ◽

JULIE A. KERNS ◽

...

Keyword(s):

X Chromosome ◽

Mouse Homolog ◽

Wiskott Aldrich Syndrome ◽

Wasp Gene ◽

Syndrome Protein

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Wiskott-Aldrich Syndrome/X-Linked Thrombocytopenia: WASP Gene Mutations, Protein Expression, and Phenotype

Blood ◽

10.1182/blood.v90.7.2680 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2680-2689 ◽

Cited By ~ 169

Author(s):

Qili Zhu ◽

Chiaki Watanabe ◽

Ting Liu ◽

Diane Hollenbaugh ◽

R. Michael Blaese ◽

...

Keyword(s):

Splice Site ◽

Gene Mutations ◽

Missense Mutations ◽

Ph Domain ◽

Glutathione S Transferase ◽

Mild Phenotype ◽

Wiskott Aldrich Syndrome ◽

Mild Disease ◽

Exon 4 ◽

Wasp Gene

Abstract Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), caused by mutations of the WAS protein (WASP) gene, represent different phenotypes of the same disease. To demonstrate a phenotype/genotype correlation, we determined WASP gene mutations in 48 unrelated WAS families. Mutations included missense (20 families) and nonsense (eight) mutations located mostly in exons 1 to 4, and splice-site mutations (seven) and deletions and insertions (13) located preferentially in exons 7 to 11. Both genomic DNA and cDNA were sequenced and WASP expression was measured in cell lysates using peptide-specific rabbit anti-WASP antibodies. WASP was expressed in hematopoietic cell lines including bone marrow–derived CD34+ cells. Missense mutations located in exons 1 to 3 caused mild disease in all but one family and permitted WASP expression, although frequently at decreased concentration. Missense mutations affecting exon 4 were associated with classic WAS and, with one exception, barely detectable WASP. Nonsense mutations caused classic WAS and lack of protein. Insertions, deletions, and splice-site mutations resulted in classic WAS and absent, unstable, truncated, or multiply spliced protein. Using affinity precipitation, WASP was found to bind to Src SH3-containing proteins Fyn, Lck, PLC-γ, and Grb2, and mutated WASP, if expressed, was able to bind to Fyn-glutathione S-transferase (GST) fusion protein. We conclude that missense mutations affecting the PH domain (exons 1 to 3) of WASP inhibit less important functions of the protein and result in a mild phenotype, and that missense mutations affecting exon 4 and complex mutations affecting the 3′ portion of WASP interfere with crucial functions of the protein and cause classic WAS.

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