Wiskott-Aldrich syndrome

PURPOSE OF REVIEW: Wiskott-Aldrich syndrome is caused by mutations of the Wiskott-Aldrich syndrome protein gene, which codes for a cytoplasmic protein with multiple functions. This review will focus on recent progress in understanding the molecular basis of Wiskott-Aldrich syndrome and its ramifications for the cure of this lethal disease. RECENT FINDINGS: The discovery of the causative gene has revealed a spectrum of clinical phenotypes demonstrating a strong genotype/phenotype correlation. The discovery of unique functional domains of Wiskott-Aldrich syndrome protein has been instrumental in defining mechanisms that control activation of Wiskott-Aldrich syndrome protein. Long-term follow up of patients undergoing hematopoietic stem cell transplantation has led to important modifications of the procedure. Studies of Wiskott-Aldrich syndrome protein-deficient cell lines and wasp-knockout mice have paved the way for possible gene therapy. SUMMARY: Wiskott-Aldrich syndrome protein gene mutations result in four clinical phenotypes: classic Wiskott-Aldrich syndrome and X-linked thrombocytopenia, intermittent thrombocytopenia and neutropenia. Wiskott-Aldrich syndrome protein is a signaling molecule and instrumental for cognate and innate immunity, cell motility and protection against autoimmune disease. The success of hematopoietic stem cell transplantation is related to the recipient's age, donor selection, the conditioning regimen and the extent of reconstitution. Since Wiskott-Aldrich syndrome protein is expressed exclusively in hematopoietic stem cells, and because Wiskott-Aldrich syndrome protein exerts a strong selective pressure, gene therapy is expected to cure the disease.     

Wiskott-aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by thrombocytopenia with small platelets, eczema, recurrent infections, autoimmune disorders, IgA nephropathy, and an increased incidence of hematopoietic malignancies. The identification of the responsible gene, WASP (Wiskott-Aldrich Syndrome Protein), revealed clinical heterogeneity of the syndrome, and showed that X-linked thrombocytopenia without, or with only mild immunodeficiency and eczema, is also caused by mutations of WASP. The study of WASP and its mutations demonstrates how a single gene defect can cause multiple and complex clinical symptoms.     

X-linked thrombocytopenia and Wiskott-Aldrich syndrome: similar regional assignment but distinct X-inactivation pattern in carriers

While inherited X-linked (XL) isolated thrombocytopenia is a mild condition, the Wiskott-Aldrich syndrome (WAS) associates severe thrombocytopenia with an immunodeficiency component and has a poor prognosis. Whether these conditions correspond to separate genetic entities or to different mutations of the same gene(s) remains unresolved. The Wiskott-Aldrich syndrome locus has been assigned to Xp 11.2 by means of RFLP studies. The X-inactivation pattern in female carriers has been found to follow a skewed pattern in the hematopoietic cells, thus allowing carrier detection. We studied a family with four members affected by XL thrombocytopenia and report the results of genetic segregation analysis, together with the X-inactivation pattern of hematopoietic cells from an obligate female carrier. Although the affected locus mapped to the same region as that of WAS, lymphocytes presented a skewed pattern of X-inactivation, whereas polymorphonuclear lymphocytes (PMN) did not. These results provide further evidence that the Wiskott-Aldrich syndrome and XL thrombocytopenia are different expressions of mutations within a single locus and that the severity of the disease corresponds to distinct hematopoietic cell selections in obligate carriers.     

Atypical presentation of Wiskott-Aldrich syndrome: diagnosis in two unrelated males based on studies of maternal T cell X chromosome inactivation

Congenital thrombocytopenia may occur in isolation or accompanied by eczema and immunodeficiency, as part of the X-linked hereditary Wiskott-Aldrich syndrome (WAS). Because the clinical and immunologic picture of WAS is variable, particularly early in life, definite diagnosis cannot always be made in cases with a negative family history. Two unrelated males with sporadic congenital thrombocytopenia had only questionable immunologic abnormalities as infants, making them clinically indistinguishable from cases of isolated thrombocytopenia, although one developed episodic neutropenia and the other began to manifest a multisystem autoimmune disease at 2 years of age. Evaluation of X chromosome inactivation in the T cells of both patients' mothers showed each of these women to have the same highly skewed X chromosome inactivation pattern seen in carriers of typical familial WAS. A T-cell defect was subsequently directly demonstrated in the second patient, whose lymphocytes failed to proliferate to periodate and anti-CD43. Taken together, these data suggest the presence of T cell immunodeficiency consistent with WAS in these patients. Furthermore, their mothers were found to have a very high likelihood of being carriers, lending support to the diagnosis of a hereditary disease in these boys and making possible genetic prediction in other family members and subsequent pregnancies.     

Clinical course of patients with WASP gene mutations

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.     

Nonrandom inactivation of the X chromosome in early lineage hematopoietic cells in carriers of Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked (Xp11.22) recessive immunodeficiency syndrome characterized by susceptibility to opportunistic and pyogenic infections, thrombocytopenia, and eczema. Previous studies of obligate carriers of WAS documented that nonrandom inactivation of the X chromosome carrying the defective gene is observed in all peripheral blood cells. The existence of both abnormal platelets and lymphocytes is consistent with a defect that affects early hematopoietic precursors. We isolated CD34+ hematopoietic progenitor cells collected from obligate carriers of WAS by apheresis and used polymerase chain reaction analysis of a polymorphic variable number of repeats (VNTR) within the X-linked androgen receptor to document nonrandom inactivation. These data show that nonrandom inactivation of the X-chromosome in WAS-obligate carriers occurs early during hematopoietic differentiation.     

Mutations of the WASP gene in 10 Japanese patients with Wiskott-Aldrich syndrome and X-linked thrombocytopenia

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, immunodeficiency, and eczema. X-linked thrombocytopenia (XLT) is a mild form of WAS with isolated thrombocytopenia. Both phenotypes are caused by mutation of the Wiskott-Aldrich syndrome protein (WASP) gene. In this study, we identified mutations of the WASP gene in 10 Japanese patients from 9 unrelated families with WAS/XLT. All XLT patients (n = 3) and one WAS patient had a missense mutation at the PH domain of WASP. Two WAS patients had nonsense mutations. One WAS patient had exon 8 skipping caused by one nucleotide deletion at the acceptor site of intron 7. Three WAS patients had genomic deletions; one of the three had a large genomic deletion involving exons 3 to 7. Codons 45 and 86 seem to be the hot spots of the WASP mutation, because missense mutations in these codons have been reported previously in several WAS/XLT patients in addition to the patients in this report, and patients with the same mutation show a similar clinical phenotype. All other mutations are novel, indicating that the mutations of WASP are heterogeneous. EB virus-transformed cell lines from XLT patients expressed nearly normal amounts of WASP, whereas those from typical WAS patients expressed almost undetectable amounts of WASP. We conclude that the analysis of gene mutation and protein expression of WASP are useful together in assessing the severity of WAS.     

X-linked thrombocytopenia caused by a mutation in the Wiskott-Aldrich syndrome (WAS) gene that disrupts interaction with the WAS protein (WASP)-interacting protein (WIP)

OBJECTIVE: We studied two adult brothers with severe congenital thrombocytopenia in order to determine the genetic etiology of their inherited disorder. Despite the absence of eczema or immunodeficiency, a mutation of the Wiskott-Aldrich syndrome (WAS) gene was suspected because of the presence of microthrombocytes. MATERIALS AND METHODS: Peripheral blood was obtained for characterization of hematopoietic cells and megakaryocyte progenitors. The coding region of the WAS gene was fully sequenced, and expression of the Wiskott-Aldrich syndrome protein, WASP, was evaluated by immunoblotting. The ability of WASP to physically associate with the WASP-interacting protein, WIP, was tested by yeast and mammalian two-hybrid techniques. RESULTS: In addition to thrombocytopenia, our investigation revealed an increased frequency of peripheral megakaryocyte progenitors (CFU-Mk) and incomplete cytoplasmic maturation by electron microscopy. Sequencing the WAS gene revealed a single base mutation, resulting in substitution of proline for arginine 138 (i.e., Arg138Pro). Immunoblotting demonstrated reduced expression of the mutant WAS protein, and we showed that the Arg138Pro mutation significantly, but incompletely, disrupts WASP-WIP interaction. CONCLUSIONS: In this pedigree, X-linked thrombocytopenia is caused by a rare mutation in the fourth exon of the WAS gene. WASP levels are reduced in lymphocyte cell lines derived from the affected individuals. Furthermore, the mutation significantly but incompletely disrupts WASP-WIP interaction, whereas substitution of alanine or glutamic acid residues at the same position does not. This raises the possibility that protein-protein interaction and WASP stability are related properties.     

Tumor necrosis factor alpha promoter polymorphisms and liver abnormalities of homozygotes for the 845G>A(C282Y) hereditary hemochromatosis mutation

Mutations of the WASP gene have been previously shown to be responsible for classical Wiskott-Aldrich syndrome, isolated X-linked thrombocytopenia, and severe, congenital X-linked neutropenia. We report herewith 2 families in which affected males had a history of intermittent thrombocytopenia with consistently reduced platelet volume, in the absence of other major clinical features, and carried missense mutations of the WASP gene that allowed substantial protein expression. This observation broadens the spectrum of clinical phenotypes associated with WASP gene defects, and it indicates the need for molecular analysis in males with reduced platelet volume, regardless of the platelet number.     

Bone marrow transplantation for the treatment of immune deficiency states

The first successful allogeneic bone marrow transplants were performed in children with severe combined immune deficiency (SCID). Bone marrow transplants for patients with SCID have been in the forefront of clinical bone marrow transplantation including the first successful use of T lymphocyte-depleted haploidentical bone marrow and matched unrelated donors. Successful bone marrow transplantation for most forms of SCID requires only the engraftment of donor lymphoid stem cells; donor hematopoietic stem cell engraftment is usually not required. The Wiskott-Aldrich syndrome was the first genetic disease involving the hematopoietic stem cell to be completely corrected by allogeneic bone marrow transplantation. The successful transplantation of Wiskott-Aldrich syndrome patients demonstrated that agents with adequate anti-lymphoid and hematopoietic stem cell activity were necessary in order to achieve complete donor lymphoid and hematopoietic stem cell engraftment. Initially, total body irradiation and now busulfan are used to ablate recipient hematopoietic stem cells, while cyclophosphamide is used to ablate recipient lymphoid stem cells. No single agent/drug is capable of eliminating both stem cell populations. Histocompatible bone marrow transplantation has a role in the treatment of patients with immune deficiency due to primary defects of the hematopoietic stem cell. The recent introduction of cytokines (gamma-interferon and granulocyte colony stimulating factor) may reduce the need for bone marrow transplantation for myeloid immune deficiency states. Initial attempts to treat patients with the acquired immune deficiency syndrome by bone marrow transplantation were limited by the lack of effective concomitant anti-viral therapy. Bone marrow transplantation for immune deficiency states continues to be in the forefront of human bone marrow transplantation.     

Hereditary X-linked thrombocytopenia maps to the same chromosomal region as the Wiskott-Aldrich syndrome

Hereditary X-linked thrombocytopenia occurs either as isolated thrombocytopenia or as a part of the Wiskott-Aldrich syndrome (WAS). We studied X-linked thrombocytopenia in a family with eight affected male members, none of whom exhibited the increased susceptibility to infection that occurs in WAS. We found a significant linkage between thrombocytopenia and DXS 146, a marker on the proximal part of the short arm of the X-chromosome. WAS has previously been mapped to the same chromosomal region. The present findings indicate that X-linked thrombocytopenia and WAS are closely related and may even be caused by different mutations of the same gene. This view is supported by our findings of atopic symptoms and minor deviations in immunologic variables among some of the affected subjects.     

Mechanisms of WASp-mediated hematologic and immunologic disease

The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin polymerization in hematopoietic cells. The dynamic nature of cytoskeletal changes during a variety of cellular processes demands complex mechanisms for coordinated integration of input signals, precise localization within the cell, and regulated activation of the Arp2/3 complex. Mutations in the Wiskott-Aldrich syndrome gene either inhibit or dysregulate normal WASp function, resulting in clinical diseases with complex and disparate phenotypes. This review highlights recent advances that have enhanced our understanding of the mechanisms by which these molecular defects cause hematologic and immunologic disease.     

Carrier detection in the Wiskott Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by immunodeficiency and severe thrombocytopenia in affected males, but no demonstrable clinical abnormalities in carrier females. Through analysis of the methylation patterns of X-linked genes that display restriction fragment length polymorphisms (RFLPs), we studied the pattern of X-chromosome inactivation in various cell populations from female relatives of patients with WAS. The peripheral blood T cells, granulocytes, and B cells of eight obligate WAS carriers were found to display specific patterns of X-chromosome inactivation clearly different from these of normal controls. Thus, carriers of WAS could be accurately identified using this analysis.     

Defects in T-cell-mediated immunity to influenza virus in murine Wiskott-Aldrich syndrome are corrected by oncoretroviral vector-mediated gene transfer into repopulating hematopoietic cells

The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by immune dysfunction, thrombocytopenia, and eczema. We used a murine model created by knockout of the WAS protein gene (WASP) to evaluate the potential of gene therapy for WAS. Lethally irradiated, male WASP- animals that received transplants of mixtures of wild type (WT) and WASP- bone marrow cells demonstrated enrichment of WT cells in the lymphoid and myeloid lineages with a progressive increase in the proportion of WT T-lymphoid and B-lymphoid cells. WASP- mice had a defective secondary T-cell response to influenza virus which was normalized in animals that received transplants of 35% or more WT cells. The WASP gene was inserted into WASP- bone marrow cells with a bicistronic oncoretroviral vector also encoding green fluorescent protein (GFP), followed by transplantation into irradiated male WASP- recipients. There was a selective advantage for gene-corrected cells in multiple lineages. Animals with higher proportions of GFP+ T cells showed normalization of their lymphocyte counts. Gene-corrected, blood T cells exhibited full and partial correction, respectively, of their defective proliferative and cytokine secretory responses to in vitro T-cell-receptor stimulation. The defective secondary T-cell response to influenza virus was also improved in gene-corrected animals.     

Two novel activating mutations in the Wiskott-Aldrich syndrome protein result in congenital neutropenia

Severe congenital neutropenia (SCN) is characterized by neutropenia, recurrent bacterial infections, and maturation arrest in the bone marrow. Although many cases have mutations in the ELA2 gene encoding neutrophil elastase, a significant proportion remain undefined at a molecular level. A mutation (Leu270Pro) in the gene encoding the Wiskott-Aldrich syndrome protein (WASp) resulting in an X-linked SCN kindred has been reported. We therefore screened the WAS gene in 14 young SCN males with wild-type ELA2 and identified 2 with novel mutations, one who presented with myelodysplasia (Ile294Thr) and the other with classic SCN (Ser270Pro). Both patients had defects of immunologic function including a generalized reduction of lymphoid and natural killer cell numbers, reduced lymphocyte proliferation, and abrogated phagocyte activity. In vitro culture of bone marrow progenitors demonstrated a profound reduction in neutrophil production and increased levels of apoptosis, consistent with an intrinsic disturbance of normal myeloid differentiation as the cause of the neutropenia. Both mutations resulted in increased WASp activity and produced marked abnormalities of cytoskeletal structure and dynamics. Furthermore, these results also suggest a novel cause of myelodysplasia and that male children with myelodysplasia and disturbance of immunologic function should be screened for such mutations.     

X-linked thrombocytopenia in a girl

We report X-linked thrombocytopenia (XLT) in a 6-year-old girl with petechiae and thrombocytopenia from the age of 3 months. Her 2-year-old brother was also diagnosed with XLT. The Wiskott-Aldrich syndrome protein (WASP) gene was detected as a replacement of +5th G to Aon intron 6 using sequence analysis, and the WASP expression levels in this patient were one-third those of a healthy control. The X-inactivation analysis of the patients lymphocytes showed a random pattern of X-chromosome inactivation. To our knowledge, this is the first confirmed report of XLT in a female.     

WASP is involved in proliferation and differentiation of human haemopoietic progenitors in vitro

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, immunodeficiency and eczema. X-linked thrombocytopenia (XLT) is a mild form of WAS with isolated thrombocytopenia. Both phenotypes are caused by mutation of the Wiskott-Aldrich syndrome protein (WASP) gene. In this study we investigated the role of WASP in the differentiation of CD34-positive (CD34+) cells isolated from the bone marrow of patients with WAS (n = 5) or with XLT (n = 4). Megakaryocyte colony formation was significantly decreased in patients with WAS when compared with normal controls. The formation of granulocyte-macrophage colonies and erythroid bursts were also decreased in WAS patinets. In contrast, in XLT patients, formation of all these colonies was normal. However, in vitro proplatelet formation of megakaryocytes induced by thrombopoietin was markedly decreased in both XLT and WAS. Electron microscopic examination revealed that megakaryocytes obtained from WAS or XLT patients grown in vitro had abnormal morphologic features, which seemed to be caused by defective actin cytoskeletal organization, including labyrinth-like structures of the demarcation membrane system and deviated distribution of the alpha-granules and demarcation membrane system. These observations indicate that WASP is involved in the proliferation and differentiation of CD34+ haemopoietic progenitor cells probably by its participation in signal transduction and in the regulation of the cytoskeleton.     

Gene therapy for Wiskott-Aldrich syndrome: rescue of T-cell signaling and amelioration of colitis upon transplantation of retrovirally transduced hematopoietic stem cells in mice

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency that is caused by mutations in the recently identified WASP gene. WASP plays an important role in T-cell receptor-mediated signaling to the actin cytoskeleton. In these studies we assessed the feasibility of using retroviral gene transfer into WASP-deficient hematopoietic stem cells (HSCs) to rescue the T-cell signaling defect that is characteristic of WAS. Upon transplantation of WASP-deficient (WKO) HSCs that have been transduced with WASP-expressing retroviruses, mature B and T cells developed in normal numbers. Most importantly, the defect in antigen receptor-induced proliferation was significantly improved in T cells. Moreover, the susceptibility of colitis by WKO HSCs was prevented or ameliorated in recipient bone marrow chimeras by retrovirus-mediated expression of WASP. A partial reversal of the T-cell signaling defect could also be achieved following transplantation of WASP-deficient HSCs expressing the WASP-homologous protein N-WASP. Furthermore, we have documented a selective advantage of WT over WKO cells in lymphoid tissue using competitive repopulation experiments and Southern blot analysis. Our results provide proof of principle that the WAS-associated T-cell signaling defects can be improved upon transplantation of retrovirally transduced HSCs without overt toxicity and may encourage clinical gene therapy trials.     

A novel protocol to identify mutations in patients with wiskott-Aldrich syndrome

Mutations of WASP (Wiskott-Aldrich syndrome protein) underlie the severe immunodeficiency/platelet disorder Wiskott-Aldrich syndrome (WAS) and its milder variant X-linked thrombocytopenia (XLT). The affected gene, a 12-exon structure on the X-chromosome, is expressed exclusively in blood cells. The encoded product WASP is a 502-amino-acid scaffolding protein that functions in stimulus-induced nucleation of actin filaments to form dynamic cell surface projections. To date, more than 150 mutations have been identified in 300 WAS/XLT kindred worldwide, generally through methodologies that include sophisticated exon screening steps such as single-strand conformation analysis. We report here a simpler protocol, which was designed for use in clinical settings to identify the mutations of newly diagnosed patients. The approach relies on directly sequencing amplified exons according to a staggered schedule based on statistical evaluation of previous cases. In a 2 1/2-year trial, samples from 28 consecutive patients were analyzed; these included 3 "blindly labeled" previously studied cases. The mutations that were identified include a broad spectrum (8 missense, 3 nonsense, 5 splice site mutations, 11 small insertion/deletions, 1 large deletion) and were broadly distributed (in 10 of the 12 exons). All mutations were verified and no discrepancies were encountered. Per patient, a mean of six DNA sequencing reactions and 6-7 h of staff effort sufficed for mutation identification and verification, indicating that the protocol is cost-effective. This cumulative experience demonstrates the suitability, reliability, and versatility of the new protocol.