Wiskott-Aldrich syndrome in a family with Fanconi anemia

Wiskott-Aldrich syndrome in a family with Fanconi anemia Jurg Rohrer, PhD, Raul C. Ribeiro, MD, Arleen D. A u e r b a c h , PhD, Betty Mirro, MD, a n d Mary Ellen Conley, MD From the Departments of Immunology and Hematology-Oncology, St. Jude Children's Research Hospital,Memphis,Tennessee,the Laboratory of Human Genetics and Hematotogy, Rockefeller University,New York, New York, and the Department of Pediatrics, Universi~of Tennessee College of Medicine, Memphis Thrombocytopenia may be the presenting finding for both Wiskott-Aldrich syndrome and Fanconi anemia. We examined a sibship of four boys who had features of both of these hematologic disorders, Peripheral blood lymphocytes from three of the boys demonstrated DNA instability when cultured with diepoxybutane, confirming the diagnosis of Fanconi anemia in these patients. However, results of linkage analysis and X chromosome inactivation studies were consistent with the diagnosis of Wiskott-Aldrich syndrome in two of the boys, including one of the boys with Fanconi anemia. These findings could be aflributed to the occurrence of two rare genetic disorders in a single family or to an unusual variant of Fanconi anemia. The recent identification of the Wiskott-Aldrich gene permitted us to address this question directly. Epstein-Barr virus-transformed cell lines from the two boys thought to have Wiskott-Aldrich syndrome on the basis of linkage analysis failed to express transcripts for the Wiskott-Aldrich gene. Genomic DNA from these two patients demonstrated a G insertion in the tenth exon of the Wiskott-Aldrich gene, resulting in a frameshift and a premature stop codon. Surprisingly, the patient with Fanconi anemia and a null mutation in the Wiskott-Aldrich gene had typical Fanconi anemia but mild Wiskott-Aldrich syndrome. (J Pediatr 1996; 129:50-5) Wiskott-Aldrich syndrome is an X-linked recessive disorder characterized by thrombocytopenia, eczema, recurrent bacterial infections and an increased susceptibility to both malignancy and autoimmune disease; however, clinical manifestations are highly variable and the most consistent feature is thrombocytopenia with small platelets.l-4 Until recently, there were no widely accepted clinical or laboratory tests that could confirm the diagnosis of Wiskott-Aldrich syndrome. However, in 1994, DelTy et al. 5 used a posifional cloning Supported in part by grants from the National Institutes of Health (AI25129, HL32987, and by National Cancer Institute CORE grams PO1 CA20180 and P30 CA21765); by American Lebanese Syrian Associated Charities; and by funds from the Federal Express Chair of Excellence. Submitted for publication Oct. 24, 1995; accepted Feb. 20, 1996. Reprint requests: Mary Ellen Conley, MD, St. Jude Children's Research Hospital, 332 North Landerdale, Memphis, TN 38105. Copyright © 1996 by Mosby-Year Book, Inc. 0022-3476/96/$5.00 + 0 9¤20¤73090

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approach to isolate the gene that is mutated in Wiskott-A1drich syndrome. Work from this group and others has shown that the WAS gene consists of 12 exons spanning approximately 9 kb of DNA. 5-7 The predicted 502 amino acid sequence of the protein encoded by this gene does not bear homology to any known proteins, and thus its function remains unknown.

See related article, p. 56.

DEB PCR RNA SSCP

Diepoxybutane Polymerasechain reaction Ribonucleicacid Single-strandconformation polymorphism

Thrombocytopenia can also be the presenting finding in patients with Fanconi anemia, an autosomal recessive disorder associated with progressive bone marrow failure, skel-

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+ 4

]2.5 4

II 12 12 4 2.5

9 4

iii 12 2.5

12 2.5

B

4

9 4

Fi9. 1. Pedigree•fthefami•ydescribedinthisartic•e.Squaresden•temales;circlesrepresentfema•es.Thenumberswithin the squares or circles indicate the number of male or female siblings of each parent. The patient numbers are shown above and to the right of the symbol for the appropriate individual. The numbers below the symbols indicate the alleles at the polymorphic loci DXS7 (top) and DXS14 (bottom) inherited by that individual. The matemal X chromosome retained in the somatic cell hybrids, the active X, carried the 12 kb allele at DXS7 and the 2.5 kb alMe at DXS14.

etal and pigmentary abnormalities, and an increased incidence of malignancy. 8-1° Fanconi anemia, which is also clinically quite variable, is characterized by D N A instability.9, ]0 Diagnosis of this disorder is confirmed by in vitro studies showing increased sensitivity to D N A cross-linking agents, such as diepoxybutane.ll At least foür subtypes of Fanconi anemia have been identified by complementation studies. 12 The gene responsible for group C Fanconi anemia (the F A C gene) was cloned in 1992 by complementation studies in which a cell line from an affected patient was transfected with a complementary-DNA library, and selection was used to isolate a corrected cell line. 1~ The predicted amino acid sequence of the F A C protein does not provide any clues to its function. Approximately 15% of patients with Fanconi anemia belong to complementation group C; however, the percentage varies with ethnic background, and no reported black patients with Fanconi anemia belong to this group. 14 The genes that are defective in other complementation groups have not yet been identified. We examined a sibship of six black children (Fig. 1) in which three of the four boys had hematologic abnormalities but some had features of Wiskott-Aldrich syndrome, whereas others fulfilled the diagnostic criteria of Fanconi anemia. This finding could be explained either by the occurrence of two rare genetic disorders within the same family (both Wiskott-Aldrich syndrome and Fanconi anemia occur with a frequency <1/100,0003, 15) or by the inheritance of an unrelated genetic disorder with features of both Wiskott-Aldrich syndrome and Fanconi anemia. The recent cloning of the gene that is abnormal in Wiskott-Aldrich syndrome made it possible to answer this question directly. The results indicated that one of the affected brothers had Wiskott-Aldrich syndrome; two brothers, including the symptom-free brother,

had Fanconi anemia; and the youngest brother had both Wiskott-Aldrich syndrome and Fanconi anemia.

CASE REPORTS Patient l ¢ame to St. Jude Children's Research Hospital at 17 months of age, in 1980, for diagnosis and treatment of lymphadenopathy of 6 weeks' duration. His height was at the 90th percentile and weight at the 60th percentile, and physical examination findings were unremarkable except for generalized lymphadenopathy. Subsequent lymph node biopsy was nondiagnostic, showing reactive hyperplasia, and lymphadenopathy remained a chronic problem throughout the patient's clinical course. During the initial evahiation, the patient was found to have microcytosis with an elevated percentage of hemoglobin A2, consistent with a diagnosis of [3thalassemia trait. A platelet count of 30,000 was also noted. The thrombocytopenia was not responsive to treatment with high intravenous doses of immune globulin, high doses of steroids, or vincristine. The patient underwent splenectomy at 5 years of age, which did result in an increased platelet count. Dufing the next 3 years the patient had 6 episodes of pneumococcal sepsis, two associated with shock. At 8 years of age he was started on a regimen of long-term intravenous immune globulin therapy because of failure to make antibody to pneumococcal antigens. When he was 12 years of age, severe hyperviscosity syndrome developed in association with IgM and IgG paraproteins. He was treated with exchange transfusions and plasmapheresis, after which he had two episodes of sepsis. At 14 years of age, pancytopenia developed; it was initially responsive to steroid therapy but became resistant within 1 year. Hemorrhagic cystitis caused by adenoviral infection occurred when the patient was 15 years of age. Massive abdominal lymphadenopathy caused ureteral obstruction. The patient died of an intracranial hemorrhage in 1995 at 15 years of age. Patient 2 was referred for evaluation of pancytopenia at 9 years of age, in March 1990. He was at the 75th percentile for height and the 40th percentile for weight and had no skeletal or pigmentary abnormalities. The medical history was unremarkable except for

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Table I. Complete blood cell counts in the affected boys

WBC (/mm3) Hemoglobin (grrddl) MCV Platelets (/mm3) MPV Neutrophils (%) Lymphocytes (%) Monocytes (%) Basophils (%)

Patient I

Patient 2

Patient 3

Patient 4

7000 11.5 73 176,000 6.1 50 37 11

2000 4.7 105 18,000 7.8 18 74 8

6300 10.9 88 188,000 7.2 35 52 5 1

9500 11.8 83 24,000 4.5 33 66 1

Normal control values 5-17,000 11-15.0 74-89 200-470,000 7.4-10.4 10-45 38-84 0-14 0-4

WBC, White blood cell count; MCV, mean corpuscular volume; MPV, mean platelet volume.

Table II. DEB sensitivity in phytohemagglutininstimulated peripheral blood lymphocytes

Father Mother Sister Patient 1 Patient 2 Patient 3 Sister Patient 4 Control range FA range

Spontaneous

DEB induced

ND ND ND ND 0.06 0.04 ND 0.14

0.06 0.06 0.02 0.09 5.44 3.24 0.06 1.47

(0-0.06) (0.02-0.80)

(0-0.10) (1.06-23.9)

ND, Not done; FA, Fanconi anemia.

two episodes of fever in the preceding month. Bone marrow biopsy showed marked hypocellularity, consistent with severe aplastic anemia. Transfnsions were reqnired to maintain the patient's hemoglobin level at greater than 5 gm/dl for the remaindèr of bis clinical course. During the next 2 years the patient had seven hospital admissions for fever and neutropenia. In 1992, at 11 years of age, be died of complications of transplantation of bone marrow from an HLA-matched sister. Patient 3 was examined in 1990 at 7 years of age, when his brothers were noted to have hematologic abnormalities. He was at the 60th percentile for height and weight and had normal findings on physical examination. He remains clinically well, with a hemoglobin level of 11.5 grrddl and a platelet count of 175,000/mm 3 in 1995 at 12 years of age. Patient 4 was referred at 12 months of age, in November 1989, for thrombocytopenia noted during an evaluation for a respiratory tract infection. At that time his height, weight, and head circumference were at the 5th percentile but he bad no skeletal er pigmentary abnormalities. His growth has continued at the 5th percentile. The patient was hospitalized at 2 years of age for pneumococcal sepsis, coincident with an episode of pneumococcal sepsis in his oldest brother. Two months later he was hospitalized for observation after head trauma with thrombocytopenia. When he was 4 yem's of age a macrocytic anemia and an elevated hemoglobin F value were

noted. Transfusion-dependent anemia developed at 5 years of age. At 6 years of age the patient was admitted to the hospital for fever and neutropenia. Except for several bouts of otitis, recurrent infections have not been a clinical problem.

METHODS Chromosomal breakage studies. Sensitivity to DEB was assayed as described) 1 Whole blood cultures were stimulated with phytohemagglutinin for 72 to 96 hours. DEB was added 24 hours after the initiation of the cultures. The mean number of chromatid breaks in each metaphase was calculated. X c h r o m o s o m e inactivation studies. Peripheral blood lymphocytes that had been stimulated with phytohemagglutinin and interleukin-2 were fused to a Chinese hamster cell line that is deficient in the X-linked enzyme hypoxanthine phosphoribosyl transferase, as previously described) 6 Somatäc cell hybrids that were resistant to selecfive media were isolated and grown to confluence. D N A from each hybrid was analyzed by Southem Not with a probe that detected the polymorphism at the DXS178 locus on flae X chromosome. N o r t h e r n biet analysis. Ribonucleic acid was extracted from Epstein-Barr virus-transformed B cell lines with the use of guanidine thiocyanate as previously descfibed.17 The R N A samples were electrophoresed through 1% agarose in the presence of formaldehyde. R N A was then transferred ente nylon filters and analyzed with phosphorus 32-1abeled probes, as described. 17 Single-strand eonformation polymorphism analysis. To obtain sufficient D N A sequence to design polymerase chain reaction primers that would flank each of the 12 exons of the WAS gene, a complementary D N A probe defived by PCR was used to select a cosmid clone from the Lawrence Livermore X-chromosome library. Coding sequences were subcloned into pBluescriptSK (Stratagene, La Jolla, Calif.) and complementary D N A primers were used to sequence across intron/exon borders. The following primers were used to identify the mutation in the family: 5 ' - A G T C A G G A G T -

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TGGTCAGT (starting 104 bp 5' to the start of exon 10), and 3'-GGTCCAGAACGTCCAGTA (ending at nucleotide 1173 of the coding sequence). PCR conditions and SSCP were performed as previously described. 17 DNA sequeneing. The same PCR primers that were employed in the reaction demonstrating an abnormal SSCP band were used to produce two independent PCR products that were cloned and sequenced as previously reported. 17

RESULTS The finding in 1990 of aplastic anemia with an elevated mean corpuscular volume in patient 2 suggested the diagnosis of Fanconi anemia not only in patient 2 but in bis two brothers with thrombocytopenia. Complete blood cell counts from all four brothers were examined (Table I). Peripheral blood lymphocyte cultures from all members of the family were tested for DEB sensitivity. Marked increase in chromosomal breakage was seen in cells from patients 2, 3, and 4, confirming the diagnosis of Fanconi anemia in these patients. However, chromosomal breakage was normal in cells from patient 1 and other family members (Table II). Later studies, using both linkage analysis and screening of genomic DNA for mutations, showed that the family did not belong to Fanconi anemia complementation group C. ~4 Thrombocytopenia with a markedly reduced mean platelet volume in patient 4, as well as the slightly reduced platelet volume in patient 1, who had undergone splenectomy in 1985, suggested the diagnosis of Wiskott-Aldrich syndrome in these two patients. Eczema was not seen in any of the family members. Quantitative immunoglobulin values in the three patients with hematologic abnormalities were usually within normal limits, although all three brothers had at least one serum IgM concentration between 40 and 55 mg/dl, with the normal range being 54 to 254 mg/dl. Anti-blood group antibodies were decreased in patient 1 (blood group B, anti-A of 1:2 at 12 years of age), elevated in patient 2 (blood group O, anti-A of 1:1024, and anti-B of 1:64 at 10 years of age), and normal in patient 4 (blood group B, anti-A of 1:4 at 3 years of age). Antibody titers to pneumococcal serotypes were reduced in patients 1, 2, and 4. Carriers of Wiskott-Aldrich syndrome demonstrate preferential use of the normal, nonmutant X as the active X in platelets, myeloid cells, and lymphocytes. 18, 19 In patients with atypical disease, analysis ofX chromosome inactivation pattems in hematopoietic cells from the mother can be helpful in confirming the diagnosis of Wiskott-Aldrich syndrome in the affected child. 16 Somatic cell hybrids that selectively retain the active X chromosome were derived from T cells from the patients' mother. All 21 T-cell hybrids retained the same X as the active X, providing Support for the diagnosis of Wiskott-Aldrich syndrome in this family. Genetic linkage studies, using probes that identify restriction fragment length

Rohrer et aL

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C124P WAS

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Fig. 2. Northern blot analysis of RNA from Epstein-Barr virustransformed cell lines from a healthy control subject (lane C), from patients 1, 2, and 4, and from another, unrelated patient with Wiskott-Aldrich syndrome (lane P). The filter was stripped and reanalyzed with a [3-actinprobe to demonstrate equal loading of RNA in each lane.

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Fig. 3. SSCP analysis of the 5' part of exon 10 of the WAS gene. DNA from a control subject (C), the mother (M), the father (F), the older sister (S), and the four patients (1 to 4) is shown. polymorphisms that flank the locus for Wiskott-Aldrich syndrome, DXS7 and DXS14, 2°, 21 showed that patients 1 and 4 inherited the matemal alleles on the inactive X chromosome, the chromosome presumed to carry the mutation, whereas the other children in the family inherited the alMes from the active X (Fig. 1). When the WAS gene, responsible for Wiskott-Aldrich syndrome, was identified in 1994, Epstein-B arr vims-transformed B-cell lines from the three brothers with hematologic abnormalities were examined for expression of WAS transcripts by Northern blot analysis. As shown in Fig. 2, transcripts for WAS were markedly decreased or absent in the cell lines from patients 1 and 4. To identify mutations in the WAS gene, we designed oligonucleotide primers to flank each of the 12 exons of the WAS gene; for exons longer than 200 bp, two or more overlapping PCR products were produced. The PCR products were analyzed by SSCP, a technique in which PCR products are separated by electrophoresis on a nondenaturing gel. Changes in sequence, even single base-pair substitutions, usually result in altered migration of DNA.

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The Journal of Pediatrics July 1996

Fig. 4. DNA sequence of the WAS gene in the region spanning base pairs 1021 to 1058 from a control subject and from patient 1. The DNA and protein sequences are read from bottom to top and show a G insertion resulting in a frameshift and a premature stop codon in the DNA from the patient.

The results demonstrated the loss of the normal bands and the gain of aberrant bands in DNA from the 5' part of exon 10 from patients 1 and 4 (Fig. 3). The mother was heterozygous for the normal and the abnormal bands. This region of genomic DNA was cloned and sequenced, and a G insertion in a stretch of 6 Gs between base pairs 1030 and 1035 was found in patients 1 and 4 but not in patient 2 (Fig. 4). The G insertion resulted in a frameshift and a premature stop codon immediately after the insertion at codon 335. Analysis of the other exons of the WAS gene by S SCP did not show any unusual bands--although, as reported by Kwan et al.,7 the amino acid sequence at codons 425 and 426 in exon 10 differed from that reported by Derry et al. 5 and consisted of leucine and alanine, respectively. DISCUSSION Identification of the gene responsible for an inherited disorder often permits a reassessment or redefinition of that disorder. The patients on whom the diagnosis of WiskottAldrich syndrome was based had severe eczema and infections at an early age. 1, 2 Subsequent studies have suggested that patients with thrombocytopenia and small platelets, but few other findings, might also have defects in the WAS gene.22. 23 Patient 4, and patient 1 before splenectomy, would have been considered to have mild Wiskott-Aldrich syndrome. They had no eczema and no significant hemorrhage or infections. Diagnosis was further complicated by the fact that patients 2, 3, and 4 had clinical and laboratory findings of Fanconi anemia, a disorder that shares some features with Wiskott-Aldrich syndrome.

The mutation in the WAS gene in this family, a G insertion in the 3' half of the gene, resulted in a frameshift and a premature stop codon. Premature stop codons in some genes, including ~-globin 24 and Btk 17 (the defective gene in X-linked agammaglobulinemia), are associated with failure of transcripts to accumulate in the cytoplasm, as seen in these patients. It is interesting to note that this "null" mutation was associated with relatively mild Wiskott-Aldrich syndrome in patients 1 and 4. Villa et al. 6 also described a frameshift mutation in the WAS gene associated with mild disease. Some, although not all, of the other patients that we have identified as having null mutations in the WAS gene have had mild disease. This finding is difficult to interpret at this time because the function of the protein encoded by the WAS gene is not yet known. The variable phenotype in patients with null mutations could be explained by modifying genetic or environmental factors. A potential modifying genetic factor in patient 4 was a second genetic disorder, Fanconi anemia. Although the functions of the proteins that are abnormal in Wiskott-Aldrich syndrome and Fanconi anemia are not yet known; it is clear that the mutations in the genes responsible for both disorders cause defects in production or survival of hematopoietically derived cells. In Wiskott-Aldrich syndrome this effect is easily seen in heterozygous females in whom the majority of platelets, T cells, and to a lesser extent B cells and granulocytes, contain the nonmutant X as the active X.16, 18, 19,23 This is explained by the preferential proliferation or survival of ceUs bearing the normal, nonmutant X as the active X chromosome. Bone marrow from patients with

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Fanconi anemia demonstrates reduced colony formation for erythrocytes and granulocytes. 25 In addition, patients with Wiskott-Aldrich syndrome have progressive defects in both T- and B-cell function. Lymphocyte function is highly dependent on D N A rearrangements of antigen receptor genes and rapid D N A replication in proliferating cells. Thus one might expect these cells to be particularly sensitive to defects in D N A stability, such as those seen in Fanconi anemia. The absence of a synergistic effect of defects in the Wiskott-A1drich syndrome and Fanconi anemia genes suggests that the pathways in which the defective proteins are used may not intersect. Alternatively, it is possible that as a result of the Fanconi anemia gene defect, fewer lymphoid cells from patient 4 reach the stage of differentiation at which the WAS gene has its deleterious effects. When several members of a single family demonstrate clinical findings that are consistent with more than one rare genetic disorder affecting the same cell lineage, most often they have an atypical form of one of those disorders. The fantily described in this article represents an exception to this paradigm. Until the gene for Wiskott-Aldrich syndrome was identified, it could have been argued that the affected members of the family reported here had an uncommon variant of Fanconi anemia with the occurrence of skewed X chromosome inactivation in the mother by chance. The identification of a mutation in the WAS gene refutes this argument. Clarification of the function of the Fanconi anemia proteins and the protein that is abnormal in Wiskott-Aldrich syndrome will help explain the clinical findings in these and other patients. REFERENCES

1. Wiskott A. Familiarer, angeborener Morbus Werlhofii? Monatsschrift Kinderheilkunde 1937;68:212-6. 2. Aldrich P,A, Steinberg AG, Campbell DC. Pedigree demonstrating a sex-linked recessive condition characterized by draining ears, eczematoid dermatitis and bloody diarrhea. Pediatrics 1954;73:133-9. 3. Perry GS, Spector BD, Schuman LM, et al. The Wiskott-Aldrich syndrome in the United States and Canada (1892-1979). J Pediatr 1980;97:72-8. 4. Sutlivan KE, Mullen CA, Blaese RM, Winkelstein JA. A multiinstitutional survey of the Wiskott-Aldrich syndrome. J Pediatr 1994;125:876-85. 5. Derry JM, Ochs HD, Francke U. Isolation of a novel gene mutated in Wiskott-Aldrich syndrome. Cell 1994;78:635-44. 6. Villa A, Notarangelo L, Macchi P, Mantuano E, Cavagni G, Brugnoni D, et al. X-tinked thrombocytopenia and WiskottAldrich syndrome are allelic diseases with mutations in the WASP gene. Nature Geriet 1995;9:414-7. 7. Kwan S-P, Hagemarm TL, Radtke BE, Blaese RM, Rosen FS. Identification of mutations in the Wiskott-Aldrich syndrome gene and characterization of a polymorphic dinucleotide repeat at DXS6940, adjacent to the disease gene. Proc Natl Acad Sci USA 1995;92:4706-10. 8. Fanconi G; Familial constitutional panmyelocytopathy, Fan-

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coni's anemia (F.A.). I. Clinical aspects. Semin Hemato11967; 4:233-40. 9. Butturini A, Gale RP, Verlander PC, Adler-Brecher B, Gillio AP, Auerbach AD. Hemätologic abnormalities in Fanconi anemia: an International Fanconi Anemia Registry study. Blood 1994;84:1650-5. 10. Giampietro PF, Adler-Brecher B, Verlander PC, Pavlakis SG, Davis JG, Auerbach AD. The need for more accurate and timely diagnosis in Fanconi anemia: a report from the International Fanconi Anemia Registry. Pediatrics 1993;91:1116-20. 11. Auerbach AD, Rogatko A, Schroeder-Kurth TM. International Fanconi Anemia Registry: relation of clinical symptoms to diepoxybutane sensitivity. Blood 1989;73:391-6. 12. Strathdee CA, Duncan AMV, Buchwald M. Evidence for at least four Fanconi anaemia genes including FACC on chromosome 9. Nature Genet 1992;1:196-8. 13. Strathdee CA, Gavish H, Shannon WR, Buchwald M. Cloning of cDNAs for Fanconi's anaemia by functional complementation. Nature 1992;356:763-7. 14. Verlander PC, Lin JD, Udono MU, Zhang Q, Gibson RA, Mathew CG, et al. Mutation analysis of the Fanconi anemia gene FACC. Am J Hum Genet 1994;54:595-601. 15. Swift M. Fanconi's anaemia in the genetics of neoplasia. Nature 1971;230:370-3. 16. Puck JM, Siminovitch KA, Poncz M, Greenberg CR, Rottem M, Conley ME. Atypical presentation of Wiskott-Aldrich syndrome: diagnosis in two unrelated males based on studies of maternal T cell X chromosome inactivation. Blood 1990; 75:2369-74. 17. Conley ME, Fitch-Hilgenberg ME, Cleveland JL, Parolini O, Rohrer J. Screening of genomic DNA to identify mutations in the gene for Bruton's tyrosine kinase. Hum Mol Genet 1994;3: 1751-6. 18. Gealy WJ, Dwyer JM, Harley JB. Allelic exclusion of glucose6-phosphate dehydrogenase in platelets and T lymphocytes from a Wiskott-Aldrich syndrome carrier. Lancet 1980; 1:63-5. 19. Prchal JT, Carroll AJ, Prchal JF, Crist WM, Skalka HW, Gealy WJ, et al. Wiskott-Aldrich syndrome: cellular impairments and their implication for carrier detection. Blood 1980;56:104854. 20. Peacocke M, Siminovitch KA. Linkage of the Wiskott-Aldrich syndrome with polymorphic DNA sequences from the human X chromosome. Proc Natl Acad Sci USA 1987;84:3430-3. 21. Kwan S, Lehner T, Hagemann T, et al. Localization of the gene for the Wiskott-Aldrich syndrome between two flanking markers, TIMP and DXS255, on Xpl 1.22-Xpl 1.3. Genomics 1991;10:29-33. 22. Donner M, Schwartz M, Carlsson KU, Holmberg L. Hereditary X-linked thrombocytopenia maps to the same chromosomal region as the Wiskott-Atdrich syndrome. Blood 1988;72:184953. 23. de Saint-Basile G, Schlegel N, Caniglia M, et al. X-linked thrombocytopenia and Wiskott-Aldrich syndrome: similar regional assignment but distinct X-inactivation pattern in carriers. Ann Hematol 1991;63:107-10. 24. Baserga SJ, Benz EJ. [3-Globin nonsense mutation: deficient accumulation of mRNA occurs despite normal cytoplasmic stability. Proc Natl Acad Sci USA 1992;89:2935-9. 25. Segal GM, Magenis RE, Brown M, Keeble W, Smith TD, Heinlich MC, et al. Repression of Fanconi anemia gene (FACC) expression inhibits growth of hematopoietic progenitor cells. J Clin Invest 1994;94:846-52.