Proc. Natl. Acad. Sci. USAVol. 92, pp. 4706-4710, May 1995Genetics

Identification of mutations in the Wiskott-Aldrich syndrome geneand characterization of a polymorphic dinucleotide repeat atDXS6940, adjacent to the disease geneSAU-PING KWAN*t, TRACY L. HAGEMANN*, BRIAN E. RADTKE*, R. MICHAEL BLAESEt, AND FRED S. ROSEN§*Department of Immunology, Rush Medical School, Chicago, IL 60612; tNational Center for Human Genome Research, National Institutes of Health, Bethesda,MD 20892; and §Department of Pediatrics, Harvard Medical School, Boston, MA 02115

Communicated by Frederick W. Alt, Children's Hospital, Boston, MA, February 23, 1995 (received for review January 11, 1995)

ABSTRACT The Wiskott-Aldrich syndrome (WAS) is anX-chromosome-linked recessive disease characterized by ec-zema, thrombocytopenia, and immunodeficiency. The diseasegene has been localized to the proximal short arm of the Xchromosome and recently isolated through positional cloning.The function of the encoded protein remains undetermined. Inthis study we have characterized mutations in 12 unrelatedpatients to confirm the identity of the disease gene. We havealso revised the coding sequence and genomic structure for theWAS gene. To analyze further the transmittance of the diseasegene, we have characterized a polymorphic microsatellite atthe DXS6940 locus within 30 kb of the gene and demonstratethe inheritance of the affected alleles in families with a historyof WAS.

The Wiskott-Aldrich syndrome (WAS) is an X-chromosome-linked recessive immunodeficiency disease. Affected malespresent with recurrent infections, eczema, and thrombocyto-penia with deformed microplatelets (1, 2). The immunologicaldisorder is characterized by in vitro defects in T-cell functionand failure of antibody production following immunizationwith polysaccharide antigens (3). By using genetic linkageanalysis, the WAS gene was mapped initially to the pericentricregion of the X chromosome (4) and localized further to theproximal short arm (5-10). The gene has now been placed inthe region between the OATL1 and GATA loci at Xpll.23(unpublished data).The gene that is defective in WAS has recently been

identified (11). The coding and genomic sequences character-ized in this study update those originally reported.¶To confirmthe identity of the gene implicated in this disease, we haveanalyzed the mutations in the WAS gene of 12 unrelatedpatients. Only three mutations have been described thus far(11), two of which were at the same nucleotide, and all werein the same exon. In this report we have found 11 additionalmutations that involved single base changes, small deletions,and an insertion. These data further support the idea that thisgene is responsible for the molecular defect involved in WAS.In addition, we have isolated and characterized a polymorphicCA dinucleotide repeat, DXS6940, which lies within 30 kb ofthe gene.

MATERIALS AND METHODSCloning the WAS Gene. Yeast artificial chromosome (YAC)

clones (12) isolated from the Xpll.23 region between theOATL1 and GATA markers were used to identify cosmidclones from the Imperial Cancer Research Fund (ICRF)library (13) and for direct selection (14) of transcribed se-quences in this region. Cosmids mapping back to the OATL1(15) and GATA (16, 17) loci were utilized in the screening of

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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American Type Culture Collection cDNA library 77442, andseveral clones were identified. Of these clones, the cDNA p427isolated with the cosmid ICRFc104A02167 from OAT YAC2at the OATL1 locus (18) had nearly 100% identity with theWASP (WAS protein) cDNA sequence reported by Derry etal (11). The WAS positive cosmid A02167 is also found in theGATA positive YACs ICRFy905B1026 and ICRFy900C01160.Additional cDNA clones positive for the WAS gene were alsoisolated from two different lymphocyte-specific libraries.Primers from the coding sequence of the WAS gene were usedto sequence the entire p427 cDNA and to determine theexon/intron boundaries of the gene within cosmid A02167.Regions that demonstrated discrepancies with the WASPcDNA (11) were sequenced several times from both strands ofthe cloned and PCR-amplified DNAs.

Patients and Isolation of Genomic DNA. Diagnosis of theWAS in the families used in this study was based on the criteriaof the World Health Organization Committee on Immunode-ficiency (19). All of the patients were included in previouslinkage analyses (5, 9). Genomic DNA was prepared fromperipheral blood or Epstein-Barr virus-transformed B-celllines as described (5).PCR Amplification of Exons for Single-Strand Conforma-

tion Polymorphism (SSCP) Analysis and Sequencing. Theexons listed in Table 1 were amplified with the PCR under thefollowing conditions for 28 cycles: exons 1, 3-5, 11, and 12,95°C for 60 s, 62°C for 45 s, 72°C for 60 s; exons 2, 7, and 8,95°C for 60 s, 58°C for 40 s, 65°C for 60 s; exons 6 and 9 andthe 3' end of exon 10, 95°C for 60 s, 55°C for 50 s, 62°C for 60s; 5' end of exon 10, 95°C for 60 s, 60°C for 60 s, 68°C for 120s. PCR, sequencing reactions, and SSCP analysis (20) werecarried out as described (21). Each mutant exon was sequencedfrom two or more different amplification reaction templatesfrom both strands of the product. The PCR products for eachexon were run in parallel with 20 normal control exons forSSCP analysis to reduce the possibility of polymorphism. PCRproducts from exon 6 were analyzed in 60 normal individuals.No variations were found in the SSCP banding patterns of thenormal exons.PCR Amplification and Analysis of the CA Repeat at

DXS6940. The microsatellite found at the DXS6940 locus wasamplified from 100 ng of genomic DNA with the primers206L (5'-TCA CAT CCT GGA CAT ACA CC-3') and 228R(5'-TGA GTA TGT CAC AGG ATG TG-3'). The PCR wascarried out in Perkin-Elmer/Cetus reaction buffer with 200,tM (each) dGTP, dATP, and dTTP, 25 tuM dCTP, 0.8 ,tCi of[a-32P]dCTP (1 Ci = 37 GBq), and 0.5 unit of Amplitaq(Perkin-Elmer/Cetus) in a 10-tlI reaction mixture under thefollowing conditions: 28 cycles of 95°C for 60 s, 55°C for 60 s,

Abbreviations: WAS, Wiskott-Aldrich syndrome; YAC, yeast artifi-cial chromosome; SSCP, single-strand conformation polymorphism.tTo whom reprint requests should be addressed.¶The sequences reported in this paper have been deposited in theGenBank data base (accession nos. U19824 and U19927).

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Table 1. Primers used in SSCP and sequencing analysisProduct

Exon Forward primer (5' -> 3') Position* Reverse primer (5' -> 3') Position* size, bplt GGTTTTTTGCATTTCCTGTTC -100 AGGAAGAGGAAGAAACGGTG +54 2862t CCTGACCAGACTCCACTGAC -39 CTTGAAGCTATGGACACATATG + 102 2823 CCTCAGTGCCACTGTGCCTC -39 TTCCCATCTCCTCTCCACAC +55 1814 GTGTGGAGAGGAGATGGGAA -65 CACTCACCTCTGCCCAACTT +46 2145 AAGTTGGGCAGAGGTGAGTG -83 AGAGAGTTATCACAGCCCTG +90 2156 GGCTGTGATAACTCTCTACA -36 CCATCCATCCAGAGACACAG +38 1287 TGGTAAGTGGGTCAATGAGC -43 CAGCTGTCCACTTGTTCATG +70 2888 AAGGAAGGGCAGTGAGGATT -45 GGTGGAAGTTTAGTGGAGTC +43 1319 CGCCTTATTCCTCTACTCCT -46 GACTGAGTGACTTAGTGCGT +77 277

10o TCAGTCAGGAGTTGGTCAGT -106 GTCCAGAACGTCCAGTAGCT nt 1162 313CAGCTACTGGACGTTCTGGA nt 1172 CAGTATCCTGACTTAGACGG +69 290

11 GAGAAATGCTCCTTTCCCAG -57 TAGCCCTGGGAGCCAGGTTT +43 21512 CTCCCAGGGCATCTTATCTT -35 AGCACAGGGCAGCAAGTAAC nt 1571 119

*The nucleotide positions for the 5' end of each primer relative to the amplified exon are indicated: negative numbers refer to the position withinthe 3' end of the preceding intron, positive numbers indicate the position with the 5' end of the succeeding intron, and positions designated ntare corrected cDNA positions.tThe forward primer of exon 1 and the reverse primer of exon 2 were taken from ref. 11.tExon 10 was divided into two products due to its size. The first primer set amplifies the 5' end of the exon and the second set amplifies the 3'end.

62°C for 60 s. The resulting products were run on a 7%polyacrylamide denaturing gel in lx TBE (90 mM Tris/64.6mM boric acid/2.5 mM EDTA, pH 8.3) and analyzed throughautoradiography of the dried gel.

RESULTS

Sequencing of the WAS Gene. Previous to the report of theWAS gene sequence, we had isolated several cDNAs near theWAS locus. Of these cDNAs, clone p427 was very similar insequence to the WAS gene, although we found several dis-crepancies between the published coding sequence of WASP(11) and that of p427. These differences ranged from single

11 GAO AAG ACA AGO GCA GAA AGC ACC ATO AGT OGG GOC CCAi Not oer Gly Gly Pro

50 ATO GOA GOA AG0 CCC G0 GGaC COA GGA GCA CCA GOC GTT CAG CAG AAC6 Met Gly Gly Arg Pro aly aly Arg Gly Ala Pro Ala Vl Gin OlGin Aon

98 ATA CCC TCC ACC CTC CTC CAGO AC CAC GAG AAC CAG cOA CTC TTT GAG22 Ile Pro ser Thr Lou Lou Gin Aop NHi Olu Asn Gin Xrg Lu Phe Glu

en I146 ATO CTT 0GA CGA AAa TG TT AcG CTO GCC ACT OCA GTT OTT CAG CTO38 Not Lou Gly Arg Lys Cyt Lou Twr Lou Ala Thr Ala Val Val Gin Leu

i94 TAC CTGO GC CTO CoC CCT GOA OCT GAG CAC T70 ACCA1 G GAG CAT TGT5s Tyr Lou Ala Lou Po Pro aly Ala Glu His Trp Thr Lys alu NHis Cy

242 0GG GCT 7TG TOC TTC T7G AAG GAT AAC CCC CAG AaG TCC TAC TTC ATC0o aly Ala al Cys Ph. Val Lys Asp Aon Pro lGn Lys 8or Tyr Pho I1e

290 COC CTT TAC GOC CTT cA' GCT GOT c00 cTO cTC T70 GAA AO GAG CT086 Arg Lou Tyr Gly Lou OlGn Ala aly rg Leu Lou Trp Glu Bin alu Leu

338 TAC TCA CAG CTT GTC TAC TCC ACC CCC ACC CCC TTC TTC CACc CC TTC102 Tyr Ner Oln Lou Val Tyr Ner Thr Pro Thr Pro Phe Phe His Thr Ph.

rs 3386 GT GOA GAT AC TGC CAA OCO GG CTG AAC TTT OCA 0AC GAGOAC Aa11s Ala Gly Alp Asp Cy0 Oln Ala Gly Lou Asn Pho Ala Asp Glu Alp Ulu434 occ CAG acc TTCc7 G0 cc CTCaT0 CAG GAG AAG ATA CAA AAA A1G AAT134Xl1 Gin Ala Phe Arg Ala Lou Val Gin Olu Lym Ile Gin Lys Arg Aon

482 CAG AG1 CaA AOT oGA GAC AGA coc CAG CTA CCC CCA CCA CCA ACA CCAiso Oln Arg Oln Ser 0ly Asp Arg Arg Gin Lou Pro Pro Pro Pro Thr Pro

530 OCC AATGA1a Oka AGA AGA oA CTC CCA CCC CTa CCC CTa CAT CCAi66 Ala AIn Olu Glu Arg Arg Gly Gly Lou Pro Pro Leu Pro Lou NHi Pro

S78 GOT GoA GAC CAA GA dac CCT ccA OTa OT CCO CTC TCC CTO OGG CTa12 Gly Gly Asp OlGn Gly ly Pro Pro Val Gly Pro Lou 8er Lou Gly Leu

626 0GC ACA OTO GoC ATC Cao AC CCT oAC ATC ACO AOT TCA COA TAC COTi1e Ala Thr Val Asp I10 Gin aon Pro Asp Ile Thr 8er 8er Arg Tyr Arg674 o000 CTC CA CA CCT GOA CCT AGC CCA GCT OAT AAG AAA CGC TCA G0214 Gly Lou Pro Ala Pro Gly Pro 8er Pro Ala Asp Lys Lys Arg 8er Gly72 AAG AAG AoG ATC AGC AAa G ATAT TT GOT aCA CCC AOT GOA TTC 1Aab230 Lys Lys Lys I1e Ber Lys Ala Asp 1e Gly Ala Pro Ber Gly Phe Lysm0 CAT OTC AGC CAC OT0 GoG 04 GAC CCC CAG AAT 0GA TTT OGAC TG AAC246 His Val Br His Val Gly Trp Amp Pro OlGin ASn aly Pho Asp Val Asn

818 AAC CTC GAC CCA OAT CTO COG AGT CTO TTC TCC AGG GCA G0A ATC AOC262 AIn Lou Asp Pro Asp Leu Arg 8er Lou Phe 8or Arg Ala Gly 7le ser

866 GAG GCC CAG CTC ACC oAC OCC GAG ACC T CT ATCT ACTTC TC AC TTC278 alu Ala lGn Lou Thr Asp Ala Glu Thr Ner Lys Lou Ile Tyr Asp Phe

amino acid changes to insertions that generate a frameshift inthe region between nt 1131 and 1312. Several of these changeshave recently been corrected (11), and p427 and WASP appearto be cDNAs from the same gene. Three added cytosines werefound: one after nt 1131, causing a shift in codon phase, andtwo additional cytosines in exon 10 that bring the codingsequence back in frame with the original termination signal inexon 12. We have also found, contrary to the previous report,the nucleotide sequence at positions 1304-1315 within exon 10to be GGC CTG GCC CCT, whereas the WASP sequenceshows GGC CCT GGC CCT. The same sequence found inp427 was also observed in WAS cDNAs from two independentlibraries, the WAS-positive cosmid clone A02167, and genomic

91 ATT GAG GACCAOG GT7 CT0 GAGG0CT GTO C CAG GAG ATO AGO C0C29 I10 Olu Asp Gin Gly Gly Lou Glu Ala Val Arg Gin alu Not Arg Arg962 CAG GAG CCA CTT CCO CCO CCC CCA CCO CCA TCT COa GOA00 O AAC CAO310 Gin Olu Pro Lou Pro Pro Pro Pro Pro Pro 8er Arg Gly Gly Aon Oln

o1010 CTC CCC CO0 CCC CCT ATT TO 000GO T AAC AA1 GOT COT TCT GOT CCA326 Lou Pro Arg Pro Pro 10e Val Gly Oly Aon Lys aly Arg Ber Oly Pro

o0S8 CTG CCC CCT OTA CCT TTO000 ATT OCC CCA CCC CCA CCA ACA CCC CO342 Lou Pro Pro Val Pro Lou Gly Ie1 Ala Pro Pro Pro Pro Thr Pro Arg1i06o CCC c CCC CCA G0C C1A GGG aac CCT CCA CCA CCA CCC CCT CCA3s5 Gly Pro Pro Pro Pro Gly Arg Gly Gly Pro Pro Pro Pro Pro Pro Pro

115s OCT ACT GA C0T TCT GGA CCA CT0 CCC CCT CA CCCCCC T GA OCT GOT374 Ala Thr Gly Arg ser aly Pro Lou Pro Pro Pro Pro Pro aly Ala alyi202 G0 ccA CCC ATO CCA CCA CCA CCO CCA CCa CCO CCA CCO CC CCC AOC390 Gly Pro Pro Not Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 8er

1250 TCC G AAT A CCA CC CCT CCC CCAC TC CCT CCT GCT CTO OTO CCT406 Ser Gly Asn Gly Pro Ala Pro Pro Pro Lou Pro Pro Ala Lou Val Pro

129s 0CC a000 C CT7 OCC CCT GOT 00GOT c0 O 0AOca CTT TTG OAT cAA422 Ala Gly oTy Leu Ala Pro G glOly OlX Arg Sly Ala Lou Leu Asp oln

136 ATC COO CA0 OA ATT CAOCT0 AaC AjkdA ACC CCT 000OCC CCA0aO AG C438 1e Arg olnO ly Ile Oln Lou AIn Lys Thr Pro Oly Ala Pro alu eor1394 TCA OCO CTO CA CCA CCA CCT CAO 1aC TCA A1O OOA CTO OTO 00aOcc4s4 sr Ala Lou oin Pro Pro Pro Gin ner Ser Olu Oly Leu Val Oly Ala1442 CT0 ATO CAC 0 ATO CAG AAO A1A AOC AGA OCC ATC CAC TCC TCC7 dAC470 Lou Not Rio Val Not Gin Lys Arg Nor Arg Ala Ile Hio Ner 8er Asp149 OAA 000 OAO OAC CAO OCT O C GAT GAA GAT GAA GAT GAT GAA TOO OAT486 Olu 0ly alu Asp Gin Ala 0ly Asp olu Asp Olu Alp Asp Olu Trp Asp1538 OAC TOA OTO OCT GAO TTA CTT GCT OCC CTO TOC TCC TCC CCOCA0O ACS02 Alp stop1586 ATO GCT CCC CCT CCA CCT OCTCT0 TOC CCA CCC TCC ACT CTC CTC TTC1634 CAO OCC CCC AAC CCC CCA TTT CTT CCC CAC CAA CCC CTC CAA TOC TOT1682 TAT CCC TOC CTO 0TC CTC ACA CTC ACC CAA CAA TCC CAA GOC CCT TTT1730o TAT ACA AAA ATT CTCA0 T TCT CTT CAC TCAA0G ATT TTT AAA GAA AAAi7 TAA AA1 aAT TOT CTT TC7T TC TCT CTATAA7 Aa aaa Aaa AAA A

FIG. 1. Sequence of the WAS cDNA p427. The complete nucleotide sequence of p427 is indicated with the translated amino acid numberedsequence. Although p427 did not contain the first 10 nt in this figure, they are included here to facilitate comparison and referencing of aminoacid and nucleotide positions, which are indicated on the left. The additional cytosines after nt 1311 are included with the appropriate codons. Theexon boundaries are labeled above the nucleotide sequence and marked with a vertical slashed line.

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DNA from three unrelated individuals. The corrected nucle-otide sequence translates into Leu and Ala residues at aminoacid positions 425 and 426 rather than Pro and Gly (Fig. 1).To study the mutations of the WAS gene, in patients with the

disease, intron placement within the gene was determined andadditional sequence data from the boundaries were generated.Sequence analysis of the cosmid A02167, which contains theentire coding sequence of WAS, revealed an additional 100-bpintron (Fig. 2A) within the interval originally reported as exon3 (11). We have renumbered the exons accordingly (Figs. 1 and2B) and will refer to the revised nomenclature (exons 1-12)throughout this report.Mutation Analysis in Patients with the WAS. To character-

ize further mutations involved in WAS, primers were gener-ated from the introns to analyze the coding sequence directlyfrom genomic DNA. Each of the 12 exons and the flankingintervening sequences were PCR amplified with the primers(Table 1) depicted in Fig. 2B and assayed for alterations in theSSC (20). In the patients where a mutant exon was identified,all 12 exons were scanned but only 1 exon demonstrated anaberrant SSC pattern. The implicated portion of DNA wassubsequently sequenced to define the altered nucleotides.Mutations were identified in 12 unrelated patients with thisstrategy. Eleven of these mutations were unique and one wasduplicated in 2 patients from unrelated families. In the familiesstudied, carrier females and affected male siblings demon-strated the same mutant exon with SSCP and sequencinganalysis, and immunologically normal family members did not.Two of the mutations identified resulted in premature stop

codons. Patient 253 demonstrated a shift in the conformationof exon 1 (Fig. 3). A transition of C134 -> T introduces a TGAcodon at Arg-34. Exon 3 of patient 1120 (Fig. 3) revealed a C329-> T substitution at Gln-99 that leaves a TAG stop codon. Bothof these nonsense mutations truncate the WAS protein earlyin translation, eliminating nearly the entire coding sequencefrom the protein.

Five missense mutations were identified. Patient 278 dem-onstrated a C -> G transversion at nucleotide position 163 thatreplaces Cys-43 with a Trp codon in exon 1. A transition of C168-> T in exon 2 (Fig. 3) translates into a Met codon rather thanThr-45 in patient 1153. A second missense substitution, Pro-58-> Leu, in exon 2 was also caused by a C -> T transition inpatient 1146 at nt 207. Two unrelated patients demonstratedthe same G ->A transition in exon 4 at nt 431 generating a Lyscodon at Glu-133 in individuals 1173 and 251. A third patient(1090) exhibited a G434 -> A transition in the next codon,resulting in an Ala-134 -> Thr substitution. Although the WASprotein has not been well characterized, all of these mutations

1 2 3 4 6 10(a e' c 0 C 0 0CX q 0O C4 N-O 0 0O

In N -__o r- W,_I 0 V T cOMO%0v- w- '-0 0v- 0v-C%iw-mo 0w- OCm

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FIG. 3. SSCP analysis of the mutated exons found in WAS patients.Each panel shows an autoradiogram with the SSCP pattern of theexons indicated above the bars. Amplified exons from normal controls(C) were run in parallel with the aberrant patient exons as indicatedabove each lane with the patient identity numbers. Each exon wasanalyzed for each of the patients; however, only the mutant exons areshown.

involve a nonconserved amino acid exchange that could pro-vide the rationale to explain deleterious effects on the encodedprotein's structure and function.Two patients each had single nucleotide deletions. In patient

1068, G248 was missing from exon 2 (Fig. 3), which brings aTGA stop codon in phase almost immediately at nt 258-260.Patient 238 demonstrated a deletion of G1305 causing a frame-shift in exon 10 (Fig. 3), which brings a stop codon in phase atnt 1365-1367 before the end of the exon.Exon 4 exhibited an altered SSCP pattern in patient 344. A

6-bp insertion of ACGAGG at nt 434 introduces a thirdAsp-Glu set to a series of repeats in this region of the codingsequence. The effect of the added amino acid residues to thisregion of the translated peptide is unclear but could conceiv-ably alter the conformation or function of the protein. Thisarea may be frequently mutated since patients 1173, 251, and1090 also had base substitutions in the same area (nt 431 and434).

Patient 1100 exhibited an altered conformation of the PCRproduct amplified from exon 6 (Fig. 3). A substitution in thedonor splice site of intron 6 at position +5 was revealed aftersequencing. Although the mutation does not break theGT/AG boundary rule (22), it does alter the donor splice siteconsensus sequence (GTAAGT), where the +5 guanine is themost highly conserved position beyond the initial invariant GT(23), with a nucleotide frequency of 0.84 (24). Similar muta-tions have caused skipping of the preceding exon and other

A exon 3 394 intron 3

GGAGATgtaagtgatcaaccagccctcgggcctcacttggggtgtggagaggagatintron 3 395 exon 4

gggaaagtttcgggggacctgggaggcggctgaccccaaggtatgttcagGACTGC1 kb

3 4 56* m lI

7 8 9* * m * m

II lI

I

12

{}-FIG. 2. Sequence of intron 3 and the genomic structure of the WAS gene. (A) A previously unreported intron was found within the coding

sequence of the WAS gene, and the sequence and position within the gene are indicated. (B) The revised genomic structure of the WAS gene isshown with the horizontal lines indicating the intervening sequences and the blocks positioned along the line representing the exons. The updatedexon nomenclature is indicated above the exons. The relative positions of the primers listed in Table 1 and used for amplication of exons in patientmutation analysis are depicted as arrowheads flanking each exon. Exon 10 was split into two PCR products for SSCP due to its size, and two setsof primers are shown.

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splicing anomalies (25). No variations were observed in theSSCP banding pattern of the PCR product from exon 6 in >60normal individuals.A Polymorphic Microsatellite at the WAS Locus. A (CA)2

dinucleotide repeat was isolated and characterized from a 5-kbEcoRI fragment of the cosmid A02167 at the DXS6940 locus.The microsatellite was found to be polymorphic with threealleles of 166 (Al), 168 (A2), and 170 (A3) bp with the flankingprimers 206L and 228R. The frequencies of the alleles were0.33, 0.56, and 0.11 for Al, A2, and A3, respectively, with anobserved heterozygosity of 0.57 in 82 X chromosomes from 61unrelated individuals. Sex-linked codominant inheritance wasdemonstrated as shown with the family in Fig. 4.The pedigree illustrated in Fig. 4 exhibits all three of the

DXS6940 alleles and sex-linked inheritance with the WASphenotype. As shown by the affected brothers (IV-5 and IV-6)in this pedigree, the disease gene is associated with allele A3.Their mother (III-7) is homozygous for A3; however, heraffected X chromosome appears to be maternal since she hasa deceased brother (III-8), who died of WAS. The grand-mother 11-4 (A1/A3) is most likely the source of the mutationsince two of her brothers (II-1 and 11-3) share the same A3allele. Individual II-2 demonstrates the A2 allele in thispedigree indicating that the great grandmother should beheterozygous with A3/A2. The great grandfather most likelycarried the Al allele inherited by his daughter (II-4). The twonormal brothers, III-3 and III-4, have inherited the nonaf-fected paternal Al allele from their mother. The sisters, III-1and III-5, have also inherited this allele and passed it on totheir sons, IV-1, IV-2, and IV-4. Although IV-3 is heterozy-gous for the A3 allele, she is not a carrier in that therepresented maternal X chromosome can be traced to hernormal grandfather, 11-5.

DISCUSSIONIn this study we have cloned the WAS gene from YACs at theOATL1 and GATA loci. The map generated from these YACsplaces WAS between these two loci on the short arm of the Xchromosome at p11.23. Here we report the updated codingsequence ofWAS (from several sources ofcDNA and genomicDNA) and also the genomic structure. The sequence datagenerated from the intron boundaries were used to develop aseries of PCR primers to directly analyze the DNA of affected

11 !S112s--.ll l2 3 4 5 6 7 8_

IV

FIG. 4. Pedigree analysis of a WAS family with the markerDXS6940. To show the association of the DXS6940 locus with the WASgene and to demonstrate the alleles of the polymorphism, each of themembers of the pedigree was analyzed with the marker. Black boxesindicate affected males, open boxes indicate immunologically normalmales, half-black circles indicate possible carrier females as implicatedthrough the inheritance of the DXS6940 alleles, and open circlesindicate noncarrier females. The alleles Al, A2, and A3 are 166, 168,and 170 bp each (bands from bottom to top, respectively). Thepedigree shows all three alleles with the disease gene following theA3allele. The mutation in the WAS gene of the affected individuals in thisfamily has not been identified. The pedigree shows the portion of thefamily for which DNA was available and affected males who have diedof WAS to establish obligate carriers.

Table 2. Mutations in patients with the WAS

Patient Exon Mutation Amino acid changeNonsense 253 1 CGA -* TGA Arg-34 -* stop

1120 3 CAG > TAG Gln-99 - stopMissense 278 1 TGC > TGG Cys-43 - Trp

1153 2 ACG -ATG Thr-45 - Met1146 2 CCC - CTC Pro-58 --Leu1173 4 GAG -AAG Glu-133 - Lys251 4 GAG - AAG Glu-133 - Lys1090 4 GCC ACC Ala-134 - Thr

Deletion 1068 2 G248 deletion Frameshift238 10 G1305 deletion Frameshift

Insertion 344 4 6-bp insert Introducesat nt 434 Asp-Glu

Splice site 1100 6 G -> A intron 6, Alters donorposition +5 splice site

individuals for SSCP in each of the exons and flankingintervening sequences of the gene.We have identified mutations in the WAS gene of 12

unrelated patients, confirming that the sequences isolated hereare responsible for the defect. A summary of the characterizedmutations is found in Table 2. The altered sequences werevaried; however, exons 1, 2, and 4 carried the mutations in 9of the 12 patients. Most of the mutated sequences were foundnear the 5' end of the gene, and none was found to alter thepredicted nuclear localization signal or the acidic C terminus.

Mutations responsible for the WAS phenotype could not beassigned in three other individuals. The 5' untranslated regionof the WAS gene has not yet been characterized, and promotermutations may be responsible for the molecular defect in thesepatients. Other sequences such as the 3' untranslated region,including the poly(A) signal, or intervening sequences (beyondthe boundaries of the exons) that could affect splicing may alsobe involved in generating aberrant transcripts. A larger mo-lecular weight transcript of -4.2 kb has been observed inthymus tissue and lymphoblast cell lines (11). If this transcriptis the result of alternative splicing, then the possibility remainsthat the gene could extend further upstream or additionalcoding sequences could be found within the introns. Theseregions may provide additional sources of mutation, or the4.2-kb transcript may simply represent heterogeneous nuclearRNA that has not been completely processed rather than analternately spliced message. The amplified PCR productsassayed for SSCP are in the size range appropriate for thistechnique; however, it is still possible that some mutations mayescape detection with the conditions and primer sets utilized.The WAS appears to affect only the white blood cells and

platelets and no other cells. This supposition is affirmed by thefact that only these formed elements of the blood exhibitnonrandom X chromosome inactivation in female obligateheterozygous carriers of the defective gene (26-28). In fact,white blood cells early in lineage commitment exhibit nonran-dom inactivation of the X chromosome in carrier females (29).The T cells and platelets of affected males exhibit disorgani-zation of the cytoskeleton and loss of microvilli, as shown byscanning and transmission electron microscopy (30, 31).Takeuchi et aL (32) have induced similar cytoskeletal pertur-bations in mouse thymoma cells by inserting antisense oligo-nucleotides to the three related actin binding proteins, ezrin,radixin, and moesin. It has been hypothesized by Derry et aL(11) that the WAS protein may be a transcription factor duethe acidic C terminus and putative nuclear localization signal.However, the protein is extremely proline rich, suggesting thatit may interact with Src homology 3 (SH3) domains (33), whichhave been found in several proteins involved in cytoskeletalorganization (34). Whether the defects observed in the archi-tecture of WAS affected cells are the direct result of a flaw in

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the cytoskeletal structure or an indirect result of inappropriateexpression of other cell-specific transcripts under the regula-tion of a WAS transcription factor remains to be elucidated.Further analysis of the mutations causing a loss of function inthis protein, including those described here, may eventuallyreveal the nature of the protein's role in T-cell and plateletfunction and morphology.The isolation and characterization of the polymorphic

marker DXS6940 will facilitate the study of WAS and otherdisease genes such as X-linked retinitis pigmentosa type 2,Aland Island eye disease, and congenital stationary nightblindness, all of which have been genetically linked to thisregion. Since DXS6940 is within 30 kb of the WAS gene,crossover events are not likely to occur between the two loci,making the polymorphism found here a powerful tool forlinkage analysis, especially in families where the WAS mutationproves difficult to identify.

This research was supported in part by National Institutes of HealthGrants AI31587 to S.-P.K. and AI31541 and RR02172 to F.S.R.; agrant from the March of Dimes Birth Defects Foundation to S.-P.K.,who is a recipient of National Institutes of Health Research CareerDevelopment Award K04-01044; and the WAS fund.

1. Wiskott, A. (1937) Monatsschr. Kinderheilkd. 68, 212-216.2. Aldrich, R. A., Steinberg, A. G. & Campbell, D. C. (1954) Pedi-

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