Wiskott-Aldrich syndrome: no strict genotype-phenotype correlations but clustering of missense mutations in the amino-terminal part of the WASP gene product

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

Blood ◽

10.1182/blood.v90.7.2680.2680_2680_2689 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2680-2689 ◽

Cited By ~ 7

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

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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Clinical course of patients with WASP gene mutations

Blood ◽

10.1182/blood-2003-05-1480 ◽

2004 ◽

Vol 103 (2) ◽

pp. 456-464 ◽

Cited By ~ 227

Author(s):

Kohsuke Imai ◽

Tomohiro Morio ◽

Yi Zhu ◽

Yinzhu Jin ◽

Sukeyuki Itoh ◽

...

Keyword(s):

Clinical Phenotype ◽

Pneumocystis Carinii ◽

Gene Mutations ◽

Japanese Patients ◽

Severe Form ◽

Missense Mutations ◽

Hematopoietic Stem ◽

Wiskott Aldrich Syndrome ◽

Wasp Gene ◽

Genotype Correlation

Abstract Mutations of the Wiskott-Aldrich syndrome protein (WASP) gene result either in the classic Wiskott-Aldrich syndrome (WAS) or in a less severe form, X-linked thrombocytopenia (XLT). A phenotype-genotype correlation has been reported by some but not by other investigators. In this study, we characterized WASP gene mutations in 50 Japanese patients and analyzed the clinical phenotype and course of each. All patients with missense mutations were WASP-positive. In contrast, patients with nonsense mutations, large deletions, small deletions, and small insertions were WASP-negative. Patients with splice anomalies were either WASP-positive or WASP-negative. The clinical phenotype of each patient was correlated with the presence or absence of WASP. Lack of WASP expression was associated with susceptibility to bacterial, viral, fungal, and Pneumocystis carinii infections and with severe eczema, intestinal hemorrhage, death from intracranial bleeding, and malignancies. Rates for overall survival and survival without intracranial hemorrhage or other serious complications were significantly lower in WASP-negative patients. This analysis provides evidence for a strong phenotype-genotype correlation and demonstrates that WAS protein expression is a useful tool for predicting long-term prognosis for patients with WAS/XLT. Based on data presented here, hematopoietic stem cell transplantation should be considered, especially for WASP-negative patients, while the patients are young to improve prognosis.

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SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2154 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2154-2164 ◽

Cited By ~ 50

Author(s):

D J DeMarini ◽

M Winey ◽

D Ursic ◽

F Webb ◽

M R Culbertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Amino Acid ◽

Gene Product ◽

Signal Sequence ◽

Null Alleles ◽

Nucleoside Triphosphate ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

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The Wiskott-Aldrich syndrome and X-linked congenital thrombocytopenia are caused by mutations of the same gene

Blood ◽

10.1182/blood.v86.10.3797.bloodjournal86103797 ◽

1995 ◽

Vol 86 (10) ◽

pp. 3797-3804 ◽

Cited By ~ 150

Author(s):

Q Zhu ◽

M Zhang ◽

RM Blaese ◽

JM Derry ◽

A Junker ◽

...

Keyword(s):

Clinical Symptoms ◽

Point Mutations ◽

Gene Mutations ◽

Exon Skipping ◽

Splice Site Mutation ◽

Missense Mutations ◽

Site Mutation ◽

Exon 2 ◽

Wiskott Aldrich Syndrome ◽

Was Gene

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, small platelets, eczema, recurrent infections, and immunodeficiency. Besides the classic WAS phenotype, there is a group of patients with congenital X-linked thrombocytopenia (XLT) who have small platelets but only transient eczema, if any, and minimal immune deficiency. Because the gene responsible for WAS has been sequenced, it was possible to correlate the WAS phenotypes with WAS gene mutations. Using a fingerprinting screening technique, we determined the approximate location of the mutation in 13 unrelated WAS patients with mild to severe clinical symptoms. Direct sequence analysis of cDNA and genomic DNA obtained from patient-derived cell lines showed 12 unique mutations distributed throughout the WAS gene, including insertions, deletions, and point mutations resulting in amino acid substitutions, termination, exon skipping, or splicing defects. Of 4 unrelated patients with the XLT phenotype, 3 had missense mutations affecting exon 2 and 1 had a splice-site mutation affecting exon 9. Patients with classic WAS had more complex mutations, resulting in termination codons, frameshift, and early termination. These findings provide direct evidence that XLT and WAS are caused by mutations of the same gene and suggest that severe clinical phenotypes are associated with complex mutations.

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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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A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness

Microbiology ◽

10.1099/0022-1317-81-1-67 ◽

2000 ◽

Vol 81 (1) ◽

pp. 67-74 ◽

Cited By ~ 590

Author(s):

Lieven Stuyver ◽

Sija De Gendt ◽

Caroline Van Geyt ◽

Fabien Zoulim ◽

Michael Fried ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Complete Genome ◽

Core Antigen ◽

Terminal Part ◽

Hbv Genotype ◽

Amino Terminal ◽

B Virus ◽

The Us ◽

Genotype A

The hepatitis B virus (HBV) genotype was determined in a total of 121 plasma samples collected in France and the US from patients chronically infected with HBV. HBV genotype A was predominant in this collection, appearing in 66 samples (54%), while genotypes B, C, D, E and F occurred in 4 (3%), 14 (12%), 23 (19%), 1 (1%) and 0 (0%) of samples, respectively. However, the genotype of a total of 13 (11%) samples (2 from France, 11 from the US) could not be determined with the methodology used. Sequence analysis, and subsequent phylogenetic analysis of the complete genome and the individual open reading frames, showed that the virus isolate from these samples was 3248 bp long and, phylogenetically, did not cluster with any of the known genotypes. This strain was provisionally called HBV genotype G. Virus isolates that were obtained from geographically separated regions like France and the US were closely related to each other. All virus strains analysed contained some characteristic differences when compared to genotype A: a translational stop codon at aa 2 and 28 of the preCore region; a 36 nt (12 aa) insert in the amino-terminal part of the Core antigen (HBcAg); a 2 aa deletion in the carboxy-terminal part of HBcAg; and a 1 aa deletion in the preS1 open reading frame. The deduced amino acid sequence of HBsAg suggests that this newly discovered genotype G strain belongs to serological group adw2.

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Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas.

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2659 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2659-2663 ◽

Cited By ~ 5

Author(s):

M Hahn ◽

W S Hayward

Keyword(s):

B Cell ◽

Gene Product ◽

Primary Structure ◽

Avian Leukosis Virus ◽

Nucleotide Sequences ◽

Missense Mutations ◽

B Cell Lymphomas ◽

Altered Expression

We have determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myc genes contained missense mutations. This strongly supports the notion that the c-myc proto-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

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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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