Wiskott-Aldrich syndrome: New molecular and biochemical insights

IOURNAL o f t h e

A m e R i c a N A c a D e m Y OF

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1938

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V O L U M E 27

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O C T O B E R 1992 I

Continuing medical education Wiskott-Aldrich syndrome: New molecular and biochemical insights Monica Peacocke, MD, a and Katherine A. Siminovitch, MD b

Boston, Massachusetts, and Toronto, Ontario, Canada The Wiskott-Aldrich syndrome is an uncommon X-linked recessivedisease characterized by eczema, thrombocytopenia, and immunodeficiency. The clinical features begin early in life and include recurrent infections, bleeding, and severe eczema. Unless the condition is treated by bone marrow transplantation, the prognosis of Wiskott-Aldrich syndrome is grave, and premature death caused by sepsis, hemorrhage, or lymphoreticular malignancy is common. Although the biochemical defect responsible for the syndrome is not known, recent investigations with restriction fragment length polymorphisms have mapped the Wiskott-Aldrich syndrome locus to the proximal portion of the short arm of thehuman X chromosome (Xpl I ). The isolation of these DNA markers makes feasible both carrier detection and prenatal diagnosis of Wiskott-Aldrich syndrome and provides an important adjunct to the management of Wiskott-Aldrich syndrome for patients and their families. These genetic data, in conjunction with the recent identification of a specific O-glycosylation defect in lymphocytes from patients with Wiskott-Aldrich syndrome, present an opportunity for the eventual isolation of the Wiskott-Aldrich syndrome gene and identification of the underlying cellular defect. We review the clinical and laboratory features of this syndrome and summarize the new molecular and biochemical approaches that can be used in diagnosis, genetic counseling, and treatment. (J AM ACADDeRMATOL1992;27:507-19.) The first description of the Wiskott-Aldrich synd r o m e ( W A S ) as a distinct clinicopathologic entity was provided in 1937 by a German physician named

O RTHO

The CME artioles are made possible through an educational grant from the Dermatological Division, Ortho Pharmaceutical Corporation. From the Department of Dermatology, New England Medical Center and Tufts University School of Medicine, Boston,a and the Department of Medicine, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto. b Supported in part by Public Health Service grants AG-09927 (M. P.) and MA-9337 (K. A. S.) from the Medical Research Council of Canada. K. A. S. is the recipient of a Career Scientist Award from the Ontario Ministry of Health and a Canadian Life and Health Insurance Agency Medical Scholarship. Reprint requests: Moniea Pcacocke, MD, Department of Dermatology, New England Medical Center, 750 Washington St, No. 114, Boston, MA 02111. 16/2/39048

Wiskott. 1 Aldrich enhanced the understanding of this disorder in 1954 with an elegant description of a large pedigree with a sex-linked recessive condition characterized by draining ears, eczematoid dermatitis, and bloody diarrhea, z We now recognize this syndrome by its characteristic triad of immunodeficiency, thrombocytopenia, and eczema. The underlying biochemical defect in W A S is believed to be expressed primarily by cells derived from the bone marrow; all other WAS manifestations, including the eczematous skin lesions, arise as a result of this defect. It has been estimated from population-based incidence rates that a minimum of six patients with WAS are born each year in the United States. 3 Therefore, as for most primary immunodeficiencies, the incidence of W A S is rare. However, these figures underestimate the true incidence rate because of the

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c o m m o n failure to diagnose W A S in sporadic cases and in patients with the milder, variant forms of the syndrome. A m o n g newly diagnosed patients with WAS, about two thirds will have a positive family history and the other one third reflect new, spontaneous mutations. 4 W A S is usually a lethalcondition; death occurs prematurely becauseofinfection, bleeding, or malignancy) For affected male patients born after 1964, the average life span is 6.5 years, although this average has improved significantly during recent decades) Some patients with WAS have lived long enough to father normal offspring. Analyses of families segregating for WAS have confirmed its restricted expression in male patients, its occurrence in identical male twins, 5 and the lack of any clinical or laboratory abnormalities in obligate female carriers. The latter observation most likely reflects the selective inactivation of the X chromosome bearing the defective gene in female carriers. 69 Because W A S is an X-linked and rare disease, expression in women is highly unlikely, although recently a girl with the classic phenotype of W A S has been reported. 1~ 11 We are currently studying this patient, who is the daughter of an obligate W A S carrier, to determine whether the expression of her disease results from a cytogenetic abnormality or from the nonrandom inactivation of her normal paternal X chromosome. VARIANT FORMS

Although W A S typically has a triad of signs, two milder diseases have been cited as W A S variants: an X-linked form of thrombocytopenia 12,j3 and a WAS-like syndrome associated with renal disease.t4, 15 Familial thrombocytopenia has several distinct patterns of inheritance, including an autosomal dominant 162~ and an X-linked recessive form21-23; the X-linked recessive type is more common. The relationship between the X-linked form and W A S is unclear. However, platelets of the X-linked form are usually small and resemble WAS platelets. 24 Recent studies with restriction fragment length polymorphisms (RFLPs) have mapped a form of X-linked thrombocytopenia with small platelets to the same area on the human X chromosome as WAS. 24,26 This finding strengthens the suggestion that W A S and X-linked thrombocytopenia are related. An association between W A S and kidney disease is also well knownJ 4, ts, 26-30However, the spectrum of WAS-associated renal lesions is broad and inconsistent among families. This situation has led

most investigators to believe that the kidney lesions are the result of the underlying immune disorder, of increased risk of infection, and of subsequent therapy with potentially nephrotoxic antibiotics.14 CLINICAL ASPECTS Boys with WAS usually are seen by the physician in the first few months of life with bleeding problems resulting from their hemorrhagic diathesis. 2' 31 In certain cases, circumcision provides the first clue that a disorder of hemostasis exists. Petechiae, epistaxis, and bloody diarrhea are common, and more significant hemorrhagic episodes, in particular intracranial bleeding, are a significant cause of death in boys who have not undergone splenectomy. Episodes of infection begin by about 6 months of age, continue throughout life, and represent a major cause of premature death. 31 Affected boys are unusually prone to both gram-positive and gramnegative bacterial infections and thus may have recurrent bouts of otitis media, sinusitis, and pneumonia. Severe periodontal disease also Occurs. 10,11 Meningitis and overwhelming bacterial septicemia are frequent and may be fatal. Less common, although still potentially lethal, are fulminant viral infections with herpes simplex, varicella (Fig. 1 ), or cytomegalovirus. 3t-33 Eczema also occurs early in life. The eczema associated with W A S behaves nmch like classic atopic eczema and improves with age. Indeed, the morphologic characteristics and pathologic descriptions are identical and indistinguishable. 31 Eczematous lesions occur on the face, scalp, arms, and legs with relative sparing of the abdomen and back. Biopsy specimens reveal acute and chronic inflammation with hemorrhage. 31 Eczema may also represent a serious complication of WAS, in some cases progressing to severe erythroderma. Skin pathogens can superinfect the eczematous lesions, resulting in cellulitis, furunculosis, and abscesses. Many other cutaneous lesions are associated with WAS and usually reflect either the atopic aspect of the disorder or the immunocompromised state of the host. Thus, boys with WAS can have numerous drug eruptions, leukocytoclastic vasculitis, herpes zoster, molluscum contagiosum, and warts. Another complication of W A S is an increased risk for malignancies, 3, 27, 34 which clearly contributes to early mortality. The risk of tumor increases with age and has been estimated to be as high as 100 times greater than normal. 3 Lymphoid tumors predominate and may, at least in some instances, rep-

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Wiskott-Aldrich syndrome 509

resent the sequelae of Epstein-Barr virus-induced polyclonal lymphoproliferation. 35 Non-Hodgkin's lymphoma of the large-cell type is the most common kind of tumor and often occurs initially as a primary lymphoid tumor in the brain. The incidence of other lymphoid malignancy including leukemia and Hodgkin's disease also appears increased in patients with W A S ) In this context, enlarged lymph nodes and hepatosplenomegaly resulting from lymphoid hyperplasia occur commonly in WAS. Thus, early detection of lymphoid tumors can be difficult and requires monitoring of adenopathy by lymph node biopsy. LABORATORY ASPECTS Many laboratory abnormahties, usually platelet and immunologic defects, have been associated with WAS. Much of this information is conflicting and confusing. To some extent, the discrepancies may reflect true differences in the expression of the genetic defect as is consistent with the clinical heterogeneity of WAS. Other reasons for this confusion include the small number of patients st udied, changes in laboratory technology, laboratory values changing with patients' age, intercurrent illness including malignancy confounding laboratory data, the presence or absence of a spleen, and whether the patient has true WAS or a variant form. Thus, the following summary of laboratory abnormalities described in WAS should be regarded as neither all-inclusive nor absolute; many of the defects described will not be found in every patient.

Piatelets Circulating platelets are decreased in number and in size (volume), 36 and the bleeding time is prolonged 37,38 (Table I). In general, bone marrow biopsy specimens reveal a normal or slightly increased number of megakaryocytes. 39, 4o Although early studies suggested that WAS platelet survival was normal,39,4o more recent investigations demonstrated that both autologous and allogeneic platelet survival is reduced. 36-38,41 Whether this is reduction specific to WAS or common to all patients with thrombocytopenia remains to be determined.38 Splenectomy seems to increase both platelet number and size, sometimes even into the normal range. 36,42, 43 Together, these data strongly suggest a primary platelet problem in WAS leading to ineffective thrombopoiesis and thrombocytopenia. Other investigators have explored an immunemediated mechanism for platelet destruction in

Fig. 1. Varicella (chickenpox) in a boy with WAS. WAS and report conflicting data. 37-39,42-44 However, patients with WAS are at risk for a variety of autoimmune phenomena. It is therefore possible that although an intrinsic defect is evident in WAS platelets leading to ineffective thrombopoiesis, intermittent immune-mediated platelet destruction may also occur. Platelet function studies have also been performed on W A S platelets, and the data, again, are contq_icting.37-39,45-48 These contradictory functional results have been explained by differences in platelet preparation, platelet number, and platelet size. However, when some of these differences are corrected by splenectomy, at least in one patient reported by Ochs et al., 38 results of the platelet function studies (aggregation) return to normal.

Immunology Abnormalities in both the cellular and humoral arms of the immune system are found in boys with WAS. The immune defect broadly affects antigen processing.30, 38,49 This effect is not surprising because these boys are at risk for a variety of infectious, malignant, autoimmune, and allergic complications.

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Table I. WAS-specific platelet abnormalities

Platelets are decreased in number. Platelets have decreased mean platelet volume (size). Bleeding time is prolonged. Both number and size improve with splenectomy. Functional studies are variable.

In the early investigations of the WAS immune system, one of the most consistent and striking findings was the absence of or low circulating levels of antibodies to the blood group antigens, the isohemagglutinins.29, 30,49 To this day, this observation remains one of the most consistent findings (Table II). When these boys were challenged with polysaccharide blood group antigens, an antibody response was still absent despite a normal antibody response to other nonpolysaccharide antigens. 29, 3s From these data, it was hypothesized that part of the immunedeficiency of WAS resulted from an inability to generate an antibody response to polysaccharide antigens, a function generally believed to be a T-cell-independent phenomenon. This finding suggests a primary B-cell-specific immune defect.29, 30, 38, 49 That most antibodies to polysaccharide antigens were of the IgM type seemed to explain the low levels of circulating IgM characteristic of WAS. Boys with WAS have normal levels of the immunoglobulin (Ig) G2 subclass, also thought to contain high levels of antipolysaccharide antibodies. 5~ Although bone marrow aspirate and biopsy specimens have demonstrated normal levels of plasma ceils, elevated serum levels of IgA and IgE are commonly found. In contrast, IgM levels are usually below normal in WAS, possibly because of increased catabolism &this antibody. 5t Another serologic abnormality is monoclonal gammopathy. 52 Because boys with WAS are also at risk for viral and fungal infections, as well as lymphoreticular malignancies, a B-cell defect was clearly not enough to explain all of the WAS immunodeficiency. Inasmuch as it is primarily the T cell that is responsible for immune protection against fungal and viral diseases, investigations of T-cell functions have demonstrated aberrant delayed-type hypersensitivity and cutaneous anergy to a variety of antigens. 29, 3s, 49 When circulating lymphocyte numbers have been quantified in WAS, they are usually decreased29, 30, 38, 49; the decrease is believed to be in the circulating T-cell population. 38 When the thy-

Table II. WAS-specific immune abnormalities

Low to absent isohemagglutinins No antibody response to polysaccharide antigens Low IgM High IgA and IgE Normal IgG Abnormal size of lymphocyte CD43 Abnormal mitogen responses to anti-CD43 and periodate

mus and lymph nodes of WAS patients have been examined, both before and after death, an absence of tissue lymphocytes was noted) ~ 49 The function of these lymphocytes is normal in vitro as assessed by mitogenic responses. 29, 38 However, responses of WAS lymphocytes in mixed lymphocyte cultures to allogeneic cells are diminished.38 Morphologic examination of WAS lymphocytes by electron microscopy has demonstrated few cell surface microvilli; these microvilli are short and blunted when compared with normal lymphocytes.53 The significance of this finding is unclear. Although not consistently noted, abnormalities in both neutrophil38 and monocyte54function have been described in selected cases. THERAPEUTIC APPROACHES In the past, the therapeutic approach to WAS included mostly palliative measures that dealt with bleeding and infection. An early trial with transfer factor 5y58 was initially promising in reversing some of the infectious complications and some aspects of the immune defect. However, the results of this therapy proved inconsistent, and it is no longer used. Two major advances, splenectomy and bone marrow transplantation, have significantly increased survival and have provided a better quality of life for boys with this disorder. Skin

The skin disorders associated with WAS, including eczema, should be treated aggressively with standard regimens. The immunodeficiency of WAS does not preclude the use of steroids, administered orally or topically. This is also the case for antibiotic-related drug eruptions and leukocytoclastic vaculitis. When there are bacterial infections of the skin, samples should be cultured and the infection treated with antibiotics. Molluscum contagiosum is frequently recalcitrant to therapy.

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Splenectomy One of the most significant contributions to the management of WAS is splenectomy. For patients who are without appropriate siblings or who are reluctant to undertake the increased risk of a T-cell bone marrow transplantation, the morbidity associated with bleeding can be significantly reduced by splenectomy. 42 Although initially described as a life-threatening procedure in children akeady at risk for lethal infections, this procedure is well tolerated by most patients. Splenectomy consistently elevates platelet counts, increases platelet size, and decreases markedly the risk of bleeding complications. 42 The increased risk of serious bacterial infection in boys without a spleen can be lessened by long-term suppressive antibiotic therapy or adjuvant intravenous gamma globulin administration or both, as well as by careful clinical observation.

Bone marrow transplantation Another significant advance in the management of WAS is bone marrow transplantation. 59-61It was first attempted in the late 1960s. 58 Successful correction of the defects associated with WAS by allogeneic bone marrow transplantation from an HLAidentical sibling was reported by Parkman et al. 61 in 1978. Since that time, many boys have undergone successful transplantation with complete reversal of both the platelet and immune dysfunction, as well as disappearance of the eczema. 59"65 Whether the increased risk of lymphoreticular malignancy is affected remains to be determined. The many infectious and hematologic complications of WAS warrant bone marrow transplantation as early as possible in the course of the disease. Children who are in good health at the time of transplantation have a tendency to tolerate bone marrow transplantation better than those who have a history of serious infections. Because of the absence of HLA-identical siblings in many WAS families, the use of T-cell-depleted HLA-haploidentieal bone marrow transplantation has been explored with s o m e s u c c e s s . 66-69 The procedure of T-cell depletion is used to decrease the risk of graft-versus-host disease. In contrast to other primary immunodeficiencystates in which the immune defect is more profound and less specific than in WAS, aggressive prophylaxis against graft-versushost disease is recommended. The success of both types of bone marrow transplantation suggests that WAS may be an ideal candidate disease for human

Wiskott-Aldrich syndrome 511 gene therapy, because the primary WAS defect is present in easily transplantable cells. NEW CONCEPTS Mapping the WAS gene As is true of many single-gene inherited diseases, the biochemical and molecular basis of WAS is unknown. Selective inactivation of the X-chromosome carrying the mutant gene renders WAS female patients phenotypically normal. Therefore, carrier detection and prenatal diagnosis in WAS was not possible until recombinant D N A technology led to the development of genetic markers known as RFLPs.Z5, 70 RFLP markers detect variants, usually single base pair differences, between the genomic DNA nucleotide sequences of different persons. These DNA polymorphisms occur so frequently that two members of a homologous chromosome pair differ in sequence at about one in every 300 nucleotide base pairs. If the variation maps within and alters a given restriction enzyme recognition sequence, cleavage at that site may be lost and the sizes of the restriction fragments so generated will differ between chromosome homologues. Such alleficvariation can be detected by Southern blotting analysis with DNA probes complementary to the region near the restriction enzyme polymorphic site or RFLP marker. Because RFLPs are inherited in a Mendelian codominant fashion, they provide a rich source of markers for linkage studies of families segregating for genetic diseases. 71 Data from such RFLPbased linkage analyses have identified the chromosomal location of a broad spectrum of disease genes, the development of tests for genetic prediction in such conditions, and finally, the framework for identification of the actual mutated genes in cystic fibrosis,72 Duchenne muscular dystrophy, 73and yon Recklinghausen neurofibromatosis.74 In studies aimed at identifying and characterizing the WAS gene, our group has investigated families segregating for WAS with X-chromosome--specific RFLP probes. Data from our initial linkage studies revealed close linkage between the WAS locus and two marker loci, DXS14 and DXS7, both mapping to the proximal short arm of the X-chromosome (Xp). 25 Since this initial observation, these markers have been successfully used for WAS prenatal diagnosis.7~ Localization of the WAS gene to the Xp pericentromeric region was supported by our subsequent demonstration of close linkage between WAS and five other polymorphic loci, including the Xp

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t'eacocke and Siminovitch LOCUS

p21 7

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DXS7 TIMP DXS255 DXS146 "" DXS14 DXZ1 DXB159

PROBE

RECOMBINANT TOTAL MEIOSES

L1.28 pTIMP M27B pTAK8 58.1 pBamX-7 cpx73

0/22 0/24 0/43 1/10 3/36 2/39 2/17

pEAK ESTIM/~T~, LOD F) 5.56 6.62 12.35 1.75 7.02 8.36 2.10

0.00 0.00 0.00 0.06 0.03 0.02 0.01

q13

q21

Fig. 2. Summary of linkage relationships between WAS and seven pericentromeric X-chromosome loci.

3

4

1::3---O DXS7 TIMP

OAT DXS255 DXS146 DXS14 DXZ1 DXS1

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DXS7 TIMP OAT DXS255 DXS146 DXS14 DXZl DXS1

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Fig. 3. Segregation pattern of eight RFLP marker loci in a WAS kindred. Probe alleles are designated by a, b, or c. O, Obligate carrier; I , affected male; 1:3,unaffected male. Recombinations between WAS and DXS146 and between WAS and DXS 14 are apparent in individuals 7 and 10, respectively. loci DXS255 and TIMP, the Xq loci DXS1 and PGK1, and the X-chromosome centromeric locus DXZ1 75, 76 The relative position of some of these loci on the X chromosome and a summary of our

current data pertaining to their linkage relationships with WAS are shown in Fig. 2. Results of multipoint linkage analysis place the WAS locus between DXS7 and DXS14, a localization since confirmed by data from several groups. 77' 78 Our recent finding of recombination between the DXS 146 and WAS loci suggests that the site of the WAS locus can be further refined to the chromosome region bracketed by DXS7 and DXS146. This crossover event, as well as a recombination between WAS and DXS 14, was detected in the representative kindred shown in Fig. 3. Most recently, the WAS locus was placed between markers TIMP and DXS255, 79 paving the way for physical mapping of the Xpl 1.22-Xpl 1.3 area and the identification of the abnormal gene in WAS. Among the various markers listed in Fig. 2, the DXS255 locus, which maps to Xcen-Xpl 1.22, is the most tightly linked to WAS; the maximal lod score for WAS-DXS255 is 12.35 at a recombination fraction (0) of 0.00. DXS255 is a variable locus, and the probe defining this locus (M27/~) is informative in the majority of women. Similarly, D X Z I , a second locus closely linked to WAS, is composed of highly polymorphic alpha satellite sequences, 8~ and heterozygosity at this locus can be detected in virtually all women. The highly polymorphic information content of these two marker loci and the identification of a number of other marker loci linked to the WAS locus have rendered predictive genetic testing feasible in the majority of WAS families. Analysis

of X-chromosome

inactivation

patterns Although R F L P linkage analysis permits assessment of carrier status in the female members of famih'es segregating for WAS, 7~this approach can-

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Wiskott-Aldrich syndrome 513

PGK

HPRT

---t"

--4"

-- +:

N

C

N

im

--+

DXS255 --

C

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Fig. 4. RFLP-methylation analysis of Epstein-Barr virus B cells from earner (C) and normal (N) woman. RFLP patterns at PGK, HPRT, and DXS255 loci are shown before ( - ) and after (+) HpaII digestion. In all controls, both RFLP allelic bands remain after HpaH cleavage, but band intensities are reduced by about 50% (random X-inactivation pattern). In contrast, in WAS carriers, HpaII cleavage results in complete elimination of one allelic fragment, leaving the other intact (nonrandom X-chromosome inactivation pattern). not be used in situations in which WAS arises de novo. However, even in the latter context, detection of W A S carriers has recently become possible with the availability of new techniques for assessing patterns of X-chromosome inactivation. This approach to carrier state detection is based on information suggesting that, at least in some tissues, WAS carriers do not manifest the random pattern of X-chromosome inactivation consistent with normal lyonization, 81 but instead appear to inactivate preferentiaUy the X chromosome bearing the mutant gene.8, 9 Detection of a nonrandom X-chromosome inactivation pattern has also been reported in other X-linked diseases, 82 including several immunodeficiency diseases, 83,g4 and provides a means for distinguishing obligate carriers from noncarrier women. The initial demonstration of nonrandom X inactivation in WAS carriers was achieved by analysis of women who were double heterozygous for the WAS and glucose-6-phosphate dehydrogenase enzymes.6, 7 We have recently confirmed this finding by RFLP-methylation analysis, 8' 9 a technique in which the methylation patterns of specific X-linked genes are compared between the two X chromosomes. This method capitalizes on the differential methylation of some constitutively expressed or socalled housekeeping genes on the active and inactive X chromosomes. 85, 86 RFLP heterozygosity is used to distinguish the two X-chromosomes, and methylation-sensitive enzymes such as HpalI are used to assess methylation patterns. Accordingly, the activation state of each X chromosome in specific cell populations can be ascertained. To examine the value of RFLP-methylation analysis in WAS carrier detection, we initially used

probes for the X chromosome-specific housekeeping genes phosphoglycerate kinase (PGK) and hypoxanthine phosphoribosyltransferase (HPRT). These two genes contain sequences that are differentially methylated on the active and inactive X chromosome.S, 9 As shown in Fig. 4, our data indicate that nonrandom inactivation of the X chromosome distinguishes obligate WAS carriers from noncarriers and represents a reliable marker for the W A S carrier state. However, the clinical applicability of this assay is limited by the relatively low frequency of R F L P heterozygosity at the P G K and H P R T loci. Accordingly, we and others have carried out similar analyses at the DXS255 locus, 87 an X-linked locus that also manifests both differential methylation on the two X chromosomes and high frequency of heterozygosity. Although RFLP-methylation analysis at this locus does permit the assessment of the X-chromosome inactivation pattern (see Fig. 4), our cumulative data indicate an unreliable correlation between the methylation pattern of DXS 255 and X-chromosome activation state (unpublished observations). The reasons for this remain unclear at this time. Therefore we do not recommend this marker as the sole method for determination of carrier state. Nonrandom X-chromosome inactivation in X-linked diseases is thought to reflect selection against cells expressing the defective gene. Its detection is useful not only for carrier detection but also for determining which cell types express the defect and might be relevant to the aberrant phenotype. Therefore the finding of selective X-chromosome inactivation in B cells, T cells, monocytes, 9 platelets, and granulocytes 8 of W A S carriers suggests that cells of most peripheral blood lineages express the

514 Peacocke and Siminovitch gene defect and are selected against at an early stage of hematopoietic development. X-inactivation analyses have also been used to show that germline mosaicism occurs at the WAS locus and may account for our observations and those of others of disease transmission by phenotypicaUy normal men.88 Thus the finding of nonrandom inactivation of the paternal rather than the maternal X chromosome in three obligate WAS earners from one kindred confirmed RFLP segregation data suggesting that the disease originated from the male germ cell X-chromosomal mosaicism. Germline mosaieism also has been described in other X-linked recessive conditionssg' 90 such as X-linked agammaglobulinemia.91 Its recognition is of major importance in genetic counseling. The increased capacity for detection of this phenomenon should permit a more accurate estimation of its frequency and the identification of its occurrence in additional X-linked diseases.

Cytogenetic studies Identification of frank chromosomal abnormalities (deletions, translocations, rearrangements) associated with expression of a genetic disorder can greatly facilitate localization and ultimately isolation of the disease gene. Translocations, for example, were key to the isolation of the genes for both Duchenne muscular dystrophy71 and von Recklinghausen neurofibromatosis.73 Although X-chromosome deletions have been detected in association with several immunodeficiencystates, to date the only karyotypic abnormality associated with WAS 92 has been an inversion within the short arm of chromosome 6. This anomaly was described in a single sporadic case, and its relevance, if any, to the clinical phenotype is unclear. Moreover, flow cytometry-karyotypic analysis in 10 patients with WAS revealed no evidence of deletions in the X chromosome (unpublished observations). These data do not preclude the presence, in at least some patients, of X chromosome microdeletions that are smaller than the limits of detection of this assay system.

Defective O-glyeosylation In the course of examining the cell surface glycoprotein profile of lymphocytes from patients with WAS, our group identified a WAS-specific defect in O-glyean biosynthesis. 93 These studies were prompted by previous data from other groups documenting the abnormal expression on WAS peripheral blood mononuclear cells and lymphocytes of a

Journal of the American Academy of Dermatology highly O-glycosylated integral membrane sialoglycoprotein, CD43. 47, 94This molecule is present on all nonerythroid hernatopoietic cells95,96 but shows cell type-specific and developmental stage-specific variations in its molecular weight because of modifications in the associated O-glycan structure. 97 We postulated that the observed alterations in the expression of CD43 on WAS lymphocytes reflected an abnormality in posttranslational modification of the cell surface molecule rather than an intrinsic defect in the gene encoding the CD43 protein itself. This interpretation was consistent with the lack of any difference in the size or amount of CD43 messenger R N A in WAS compared with normal lymphocytes and the localization of the CD43 gene to chromosome 16 and not the X chromosome.98 Moreover, the finding that proteins that are glycosylated through N-linked structures (such as the H L A class I and LFA-II3 chain molecules) are normally expressed on WAS lymphocytes suggested that impaired CD43 expression might be caused by a specific defect in O-linked glycosylation.93,99 Thus the levels of glycosyltransferase activities in peripheral blood T lymphocytes and Epstein-Barr virus-immortalized B-cell lines of patients with WAS and healthy, age-matched controls were measured. Although patients and control individuals did not differ in nine enzyme activities, they exhibited striking differences in the levels of two O-glycosyltransferase activities, ~- 1,6-N-acetylglueosaminyl transferase (core 2 GlcNAc-T) and a-2,6-sialyltransferase (a6SA-TII). 9a, 99 Altered expression of these particular glycosyltransferase activities is of interest in light of data indicating that these specific activities are differentially expressed depending on the state of cellular activation or differentiation.l~176 Therefore normal resting T cells have negligible levels of core 2 GlcNAc-T and high levels of a6SATII activity, but when they are activated by rnitogens or growth factors (anti-CD3 antibody or interleukin-2), activities of core 2 GlcNAc-T are increased and those of a6SA-TII are decreased. The significance of these activation-associated changes in transferase levels, and particularly in core 2 GlcNAc-T activities, relates to concomitant changes in O-glycan biosynthesis and in the final core structure of the cellular O-linked oligosaccharides. As shown in Fig. 5, the O-glycans associated with lymphocyte membrane glycoproteins are based on two core structures, the small disialotetrasaccharide or core 1 structure and the more complex

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Wiskott-A1drich syndrome 515 SAe2-3GalI31-4GIcNAc~1-6

[-es~q SAm?.-3GalIM -3GalNAc~-Ser/Thr

antI-CD3 + IL-2

b SAot-2-3GalI31-3GaiN Aca- Ser/Thr

Activated T celia

Resting T cell,=

Relative core 2 GIcNAc - transferase activity (nmole/mg/h)

Resting T cells

Activated T cells

Healthy donors

low

h~h

WAS patients

high

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Fig. 5. O-glycosylation in WAS and normal T cells. Modification of O-glycan core structure on surface glycoproteins of normal T cells occurs during anti-CD3/interleukin 2 (IL2)-induced cell activation and reflects a relative increase in activity of core 2 GIcNAc-T activity. By contrast, T-cell activation in WAS is associated with a loss of this enzyme activity. trisaccharide designated as core 2. In the absence of core 2 GlcNAc-T activity (as in resting T ceils) core 1 represents the predominant core structure and, when acted on by ~6SA-TII, yields the disialotetrasaccharide shown on the left in Fig. 5. However, an increase in core 2 GIcNAc-T and a reduction of oe6SA-TII activity (as occurs in activated T cells) are associated with a shift in the O-glycan biosynthetic pathway to the expression of core 2 O-linked oligosaccharides, which can be extended with polylactosamine sequences to yield larger, more complex glycan structures. These activation-associated changes in oligosaccharide core structure account for observed variations in the apparent molecular weight of O-glycosylated molecules such as CD43 on resting versus activated T cells and, as described below, on W A S versus healthy lymphocytes. In addition to differences in basal transferase levels, W A S patients also manifest T-cell activationassociated abnormalities in core 2 GlcNAc-T and c~6SA-TII activities. W A S cells have relative levels of these enzyme activities that are opposite to those found in normal T cells. Core 2 GlcNAc-T activity is high in resting T cells from patients with WAS but decreases markedly in W A S T cells treated with anti-CD3 antibody-interleukin-2 (see Fig. 5). Similarly, Epstein-Barr virus-transformed, activated B cells from patients with WAS display minimal activity of core 2 GlcNAc-T, an activity that is high in Epstein-Barr virus B cells from healthy controls. On the basis of the pattern of glycosyltransferase activities, the O-linked oligosaccharides on WAS resting T cells would contain mainly core 2 structure, and those on activated W A S T cells would have the

smaller core 1 structure. This prediction is borne out by the immunoprecipitation of CD43 from activated WAS T cells, which are smaller than normal. 93 Moreover, these data strongly suggest that although CD43 is an abnormality associated with W A S lymphocytes, it most likely not the only abnormality and that other functionally significant O-glycosylated cell surface molecules are altered in WAS. In view of the prominent platelet abnormalities in WAS, platelet core 2 GlcNAc-T activities were also measured and, as in resting lymphocytes, were found to be much higher in patients with WAS than in controls. 99 Overall, these data indicate that aberrant O-glycosylation occurs in W A S and may reflect a defect in the developmental program for the regulated expression of core 2 GlcNAc-T and a6SA-TII. The observation of impaired O-glycosylation in both lymphocytes and platelets from these patients suggests that this defect contributes to the clinical phenotype, but its relevance to the primary gene defect in W A S remains to be determined. Impaired T-cell function is a major contributing factor to the morbidity in patients with WAS, but the basis for the T-cell dysfunction is unknown. T h e association of W A S with impaired expression of CD43] ~ a cell surface receptor implicated in antigen-independcnt T-cell activation, suggests a possible relationship between defective O-glycosylation and diminished T-cell function in this condition. T o address this possibility, we compared lymphocytes from patients with W A S with control lymphocytes as to their respective proliferative responses to various T-cell mitogens, including two that activate T cells by interactions with CD4393, 102 the oxidizing

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agent periodate and anti-CD43. Stimulation of WAS lymphocytes with phytohemagglutinin, concanavalin A, and neuraminidase or galactose oxidase elicits proliferative responses in both normal and WAS cells. In striking contrast, however, periodate and anti-CD43, although stimulating DNA synthesis in normal lymphocytes, fail to induce proliferative responses in WAS lymphocytes. Cells from patients with lymphoid malignancies, rheumatoid arthritis, systemic lupus erythematosus, severe combined imrnunodeficiency, ataxia-telangiectasia, X-linked agammaglobulinemia, and Fanconi's anemia have been examined in the periodate assay, and all consistently respond normally (Siminovitch KA, Greer WA, Axelsson B, et al. The Wiskott-Aldrich syndrome: specific diagnosis by selective impairment of CD43 mediated T cell activation. Submitted for publication). Indeed, because the percentage of circulating CD43-positive cells is the same in patients with WAS and in controls, it would appear that the selective nonresponsiveness of WAS cells to mitogens that activate T cells by interactions with CD43 is in some manner related to modification of the carbohydrate structures on the CD43 molecule. This pattern of impaired mitogen responsiveness appears to be not only specifically associated with WAS but also manifested by at least some patients with the attenuated form of WAS, in whom the diagnosis of WAS may be otherwise obscure. 1~ io4 This situation is well illustrated by one young boy with thrombocytopenia, in whom absence of antiCD43 antibody or periodate responses helped elucidate the diagnosis of a variant form of WAS, a diagnosis subsequently substantiated by development of Pneumocystis carinii pneumonia, m3 Similarly, selective impairment of CD43-mediated T-cell activation was found in a young man who had had a "mild" form of WAS since childhood and was seen at age 24 years with both large-cell lymphoma and Kaposi's sarcoma.I~ Therefore, although the relevance of impaired glycosylation to the defect in cellular immunity in WAS remains to be determined, the association of WAS with selective impairment of CD43-mediated T-cell activation may be of significant value in the diagnosis of both WAS and the milder variant forms of this disease. CONCLUSION Among the inherited forms of human immunodeficiency disease, WAS has proved to be particularly challenging from both the clinical and pathophysiologic point of view. The clinical and ira-

munologic heterogeneity of WAS has frustrated attempts to establish a definitive diagnostic marker for the syndrome, and despite extensive study the biochemical and cellular defects underlying this syndrome have remained obscure. This situation has interfered with early diagnosis and with identification of the full phenotypic spectrum of WAS, issues that are pertinent to decisions regarding therapeutic options for patients with WAS. During the past few years, however, our ability to address these diagnostic and etiologic concerns has been dramatically increased by advances in the knowledge of genetics and biochemistry of the disorder. Identification of the subchromosomal location of the WAS gene has paved the way for its eventual isolation and has elucidated markers that render genetic prediction feasible for virtually all WAS families with a positive family history of the disease. The demonstration of nonrandom X-chromosome inactivation in obligate WAS carriers has provided a diagnostic test for carrier status in nonfamilial WAS, as well as information about tissue-specific expression of the gene defect and that germline mosaicism accounts for some cases of WAS. In addition, identification in patients with WAS of a defect in the regulated expression of two glycosyltransferase activities gives additional credence to our data associating WAS with a defect in hematopoietic maturation or differentiation. Altered O-glycosylation also provides a unifying explanation for the distinct phenotype of cellular immune and platelet dysfunction observed in WAS. Finally, the demonstrated association of WAS with a selective defect in CD43-mediated T-cell activation may provide a diagnostic marker for WAS and its variant forms. Such distinctions are particularly relevant in light of data suggesting that patients with milder forms of WAS are still at increased risk for malignancy. Overall, these new perspectives set the stage for delineating the molecular basis for WAS and determining the mechanism whereby expression of this genetic defect interferes with lymphocyte and platelet dysfunction, impairs O-glycosylation, and predisposes patients to lymphoid oncogenesis.

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