Proc. Nail. Acad. Sci. USAVol. 91, pp. 9062-9066, September 1994Immunology

Genomic organization and structure of Bruton agammaglobulinemiatyrosine kinase: Localization of mutations associated with variedclinical presentations and course in X chromosome-linked agammaglobulinemia

(protein-tyrosine kinase/primary immunodeficiency disease/missense mutation/PCR/DNA sequencing)

YUKO OHTA*, ROBERT N. HAIRE*, RONDA T. LITMAN*, SHU MAN Fut, ROBERT P. NELSON*, JAMIE KRATZ*,STEPHEN J. KORNFELD*, MAITE DE LA MORENA*, ROBERT A. GOOD*, AND GARY W. LITMAN*4*Department of Pediatrics, University of South Florida College of Medicine, All Children's Hospital, St. Petersburg, FL 33701; and tDepartment of InternalMedicine, University of Virginia School of Medicine, Charlottesville, VA 22908

Contributed by Robert A. Good, May 19, 1994

ABSTRACT X chromosome-linked agammaglobulinemiais a life-threatening disease that involves a failure in normaldevelopment ofB lymphocytes and is associated with missensemutations in BTK, a gene encoding a cytoplasmic tyrosinekinase (Bruton agammaglobulinemia tyrosine kinase, EC2.7.1.112), a member of the Tec family of protein-tyrosinekinases. The genomic organization has been determined byusing conventional restriction fragment mapping, extendedDNA sequencing, and PCR fragment-sizing approaches. TheDNA sequences of the 18 coding exons composing BTK andtheir flanking-region sequences are reported; an additionalexon(s) encodes a 5' untranslated segment. Single-base-pairsubstitutions and 4-nt deletions resulted in amino acid re-placement, premature termination, frameshift, and exondeletion in a group of X chromosome-linked agammaglobu-linemia patients exhibiting different clinical presentationsand courses. The nature of the mutations is interpreted interms of the genomic organization of the BTK gene and thedisease course in individual patients. Several examples arefound in which the same mutation occurs in unrelatedpatients, and one of these mutations occurs at the same codonthat is substituted in the murine form of BTK, resulting in Xchromosome-linked immunodeficiency disease. Considerablevariation in presentation and disease course in X chromo-some-linked agammaglobulinemia appears associated withthe nature and position of different missense mutations.

X chromosome-linked aggamaglobulinemia (XLA), alsoknown as Bruton agammaglobulinemia (1), occurs with anaverage frequency of 1/50,000 male births. The disease isassociated with infection by high-grade, encapsulated, extra-cellular pyogenic pathogens such as Streptococcus pneumo-nae and Haemophilus influenzae. Low serum immunoglob-ulin as well as marked deficits in circulating B cells and themore mature pre-B cells in bone marrow (2) are routinelaboratory findings in the disease. Obligate carriers do notexhibit immunological defects. Analyses of nonrandom in-activation of the X chromosome have shown the defect toaffect the B, but not T, lymphoid lineages (3). Before the useof intravenous gammaglobulin therapy, the disease invari-ably proved fatal.

Conventional linkage analyses have localized the gene tochromosome Xq22 (4-7) and have shown the XLA trait tobe closely linked to the marker DXS178 (8). Positionalcloning (9) and DNA cross-hybridization approaches (10)were used independently to localize the gene defect to a

cytoplasmic tyrosine kinase designated ATK (agammaglob-ulinemia tyrosine kinase) (9) or BPK (B-cell progenitortyrosine kinase) (10). The corresponding gene in the mouseis the site of the gene defect in X chromosome-linkedimmunodeficiency disease (XID) and has been termedBruton tyrosine kinase (Btk), which is the currently ac-cepted designation for this protein-tyrosine kinase in human(EC 2.7.1.112) and mouse (11, 12).The complete nucleotide sequence of the mRNA (cDNA)

encoded by human BTK has been described (9). BTK is a659-amino acid polypeptide consisting of Src homology do-main 3 (SH3)-like, and 2 (SH2)-like, ATP-binding, and sub-strate-specific domains. The gene originally was shown to beexpressed in B cells, pancreas, lung (9), and in cells of themyeloid lineage (10). It has been reported recently that BTKis expressed in hematopoietic cells, except in T lymphocytesand terminally differentiated plasma cells (13). TheBTKgeneis most related to the Tec subfamily of protein-tyrosinekinases. Unlike the src-type genes, which encode intracel-lular (nonreceptor) protein-tyrosine kinases and are bound tothe plasma membrane by myristylation, the tec genes lack amyristylation site and, hence, are not known to be membraneassociated.

Identification of the BTK mRNA (cDNA) has facilitatedidentification in XLA patients of various mutations, includ-ing single-base-pair substitutions and insertions (9, 14, 15);however, this approach is limited in the requirements forsufficient amounts of mRNA to derive cDNA transcriptsthat are then examined in terms of loss or gain of arestriction site(s), cloned, and sequenced or analyzed forsingle-strand conformation polymorphism(s). Further-more, there are inherent complications in ascertainingfemale carrier status by using this approach, and the natureofmany classes of genetic lesions cannot be ascertained. Todevelop approaches for rapidly identifying missense muta-tions in theBTKgene and to better understand relationshipsbetween the structure and function of BTK and relatedgenes, the genomic organization and significant portions ofthe DNA sequence oftheBTKgene have been determined.§This information is used to characterize mutations in aseries of patients with XLA who exhibit various clinicalpresentations and courses.

Abbreviations: BTK, Bruton agammaglobulinemia tyrosine kinase;XID, X chromosome-linked immunodeficiency disease; XLA, Xchromosome-linked agammaglobulinemia; SH2 and SH3, Src ho-mology domains 2 and 3, respectively.1To whom reprint requests should be addressed at: All Children'sHospital, 801 Sixth Street South, St. Petersburg, FL 33701.§The sequences reported in this paper have been deposited in theGenBank data base (accession nos. U10084-U10087).

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Proc. Natl. Acad. Sci. USA 91 (1994) 9063

MATERIALS AND METHODSStandard Methods. DNA extraction, RNA isolation, and

PCR amplification of the BTK gene transcript used standardmethods. The PCR reaction employed BamHI-linkered prim-ers complementing untranslated sequences flanking the cod-ing region of BTK (9, 10): 5'-TCAGGACTGAGCACA-CAGGT-3' and 5'-CCAAGAAGCTTATTGGCGAGC-3'.The opposing strands for DNA sequencing (16) were pro-duced independently from cDNA in separate PCR reactionsto eliminate the possibility of Taq polymerase incorporationerrors. A human genomic DNA library (Novagen) inAGEM-il was screened using standard methods, using dif-ferent PCR-generated probes complementing BTK.PCR Amplification of Genomic DNA and Detection of Spe-

cific Mutations. Primers, including cloning sites, used formutation detection in genomic DNA were as follows: exon10-11 (P18) 5'-TTATGACCAGGAGCCACTCA-3', 5'-GCATCAAGGAGCTATTAGG-3'; exon 16 (P08/13/21) 5'-TTCTACTGGTCAGCAGAAGCTT-3 ' ,5'-TTACTTCTG-GAGGGAAAGATGA-3'. In addition, primers specific forthe normal (N) and mutant (A) alleles were as follows: P17mutation-N primer 5'-CAGGCAGCTCGAAACTGTTT-3',and A primer 5'-CAGGCAGCTCGAAACTGGTA-3',5'-TTACTTCTGGAGGGAAAGATGA-3'; P22 mutation-Nprimer 5'-AGTCTCACTGGTCTCTGTTT-3' and A primer5'-AGTCTCACTGGTCTCTGCAC-3' ,5'-TTACTTCTG-GAGGGAAAGATGA-3'.

RESULTSIdentification ofBTK Exons. Four positive A clones (AGEM

16.8, AGEM 13.7, AGEM 16.1, and AGEM 13.3) were iden-tified and plaque-purified. Restriction-digestion mappingshowed these clones to be overlapping, encompassing =36 kbof genomic DNA, including BTK. Individual A clones weredigested with various restriction endonucleases, a series offragments of various length, which hybridized with the BTKcDNA probe, were identified, cloned, and sequenced; thesequences were then compared with the BTK cDNA se-quence. A physical map encompassing the 18 individualcoding exons of BTK was constructed (Fig. 1); the overalllength (initiation codon to stop codon) ofBTK is -26 kb. Anadditional 5' exon(s) was inferred from the sequences of theBTK cDNA (9).Genomic Sequence. Table 1 shows the 5' and 3' sequences,

overall lengths of 18 BTK-coding exons, and the 5' and 3'intron sequences. The complete exon sequences are found inthe GenBank submissions and are identified as correspondingto cDNA sequence described in ref. 9. The putative splicedonor and acceptor sites are identified and are consistent

with those of other eukaryotic genes. Also indicated are theabsolute lengths of the intervening sequences determined byDNA sequencing and/or estimated by inter-exon fragmentlength measurements, which correspond to those illustratedin Fig. 1. Sequences representing a total of =26 kb oftheBTKgene are deposited in GenBank.XLA Patients. The clinical (infection) history, age at diag-

nosis, immunoglobulin levels, and B-cell marker values foreight XLA patients presently under care in the All Children'sHospital clinics are summarized in Table 2. Several trendsare evident in this patient group: they were either 8 yr or olderor 2 yr or younger at diagnosis. Patients P05 and P22 were notclinically diagnosed with hypogammaglobulinemia until 14and 16 yr of age, respectively; P22 was referred with adiagnosis of common variable immunodeficiency disease;however, genetic testing identified a mutation in the BTKgene. This patient produced antibody to diphtheria andtetanus toxoid vaccines. P08, P13, and P21 are cousins; P08presented with recurrent pneumonias and panhypogamma-globulinemia. P13 had recurrent infections and panhypogam-maglobulinemia but at 23 mo had a normal immunologicresponse to tetanus toxoid. P21 had normal IgG, normalantibody responses to diphtheria and tetanus toxoid vaccinesbut had low IgA, low IgG2 and IgG3 (not illustrated), andslightly low IgM levels. The universal finding in these XLApatients is decreased serum IgM levels compared with age-matched normals and low-to-absent IgA levels (except forP22). To some degree, the infection histories of the patientsreflect the extent ofclinical workup and the use ofantibiotics,without a definitive diagnosis, before referral. For example,in P21, early therapeutic intervention was based on a clinicaldiagnosis of XLA in family members P08 and P13 and aconfirmed genetic diagnosis in this patient, even though thechild responded to diphtheria and tetanus toxoid vaccines(with production of specific antibody). Thus, three patientswith the same mutation had variable phenotypic expressionat different ages, although the final outcome of their diseasemay be similar. The findings in this family and P22 areparticularly noteworthy in that the hallmark of humoralimmunodeficiency, the inability to make specific antibody inresponse to immunization, was not found.

Mutations. Missense mutations were demonstrated in alleight patients in this series (Table 3). Single-base-replace-ment substitutions were detected in six individuals, includingthe three members of the same family, P08, P13, and P21.Three substitutions result in amino acid replacements, andone substitution results in a stop codon (P09). The mutationin P18 affects a splice junction at the 5' of intron 10. In thiscase, the complete nucleotide sequence of the amplifiedcDNA transcript shows the absence of sequence correspond-

1 kbH-H

FIG. 1. The relative exon/intron organization of the human BTK gene as related to the known sequence of the BTK cDNA. UT, untranslated;ATP, binding site for ATP; KIN, kinase domain, from ref. 9. Intron length is indicated by lines between boxes; see Table 1.

Immunology: Ohta et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

Table 1. Sequences flanking BTK exons

Exon Intron Exon Exonno. 3' 5' splice size, bp 3' splice 5' no. size, bp

1 CAGGACTGAG TTCATTATCAACA" CACACAGGTG 2 1712 TGAACGTGGG fIAAGTTTCTCG 509 ATTCTACTTCTCAG AGAAGAGGCA 3 993 ACAGATTCCG fIAAGAAGAGAC 2700* TTATTTCATTACA" AGAAGAGGTG 4 694 TCCCTTCCAG faGAGTTATTTT 1553 TTTCATCGACCCA" GTTGTATATG 5 82S CTCAAAAACG fIGAGAATTATT 7300* CTTTCCTGCTACA" TAATCCGGTA 6 1296 AGGAATGGAA UIAAGCAATCTG 319 TTCTTGTTTTGCA" GCTTAAAACC 7 687 GGAGGACCAG fIATACAGGGAA 1414 TCCTAACTACATA" ATCTTGAAAA 8 1888 ATAAAAATGG faGAGTCCACAC 418 TGTTTCCTTCACA" GCAGGAAGGC 9 639 AAATGTATGA MIAAGTATGTTT 739 TTCTCCCTTCACA" GTGGTATTCC 10 5510 AAAGCAAGAG UIAAGTGTGGAA 591 TTTCGTTGTTTCA" GGGAAAGAAG 11 8011 AATCCACAGG GIGAGTGCTACT 179 ATCCACTTCTTCAG GGACCCTCAA 12 12812 AACTCTGCAG UTGAGTACCAGG 726 TCTCTCTGTTTCA" GACTCATATC 13 7513 CTGGGATACG fIAACTCCTTAT 551 GACTTTATTGTTA" GATCATGGGA 14 17214 AAGTCATGAT GIGAGTTATAGC 515 TGCCTTTCCTGTA" GAATCTTTCC 15 21715 CCGAGACCTG UIGGGACCTTAG 1357 TGTTTGCACTACAG GCAGCTCGAA 16 6516 GCCTGTCCAG UIGAGTGTGGCT 640 TGAATCCCTTGCA" GTATGTCCTG 17 11917 TGGGCTTTTG GIAAGGTGGAAA 517 TTTTCACCTTCTAG GGGTTTTGAT 18 15818 CTGGCATGAG GIAAGTGCTTTA 3232 TTCTTTGGTTTTA" AAAGCAGATG 19 50319 TAGAAAGCTT GAAAGTCTTTGG

Exon lengths are based on DNA sequence. The lengths of introns are based on DNA sequences and/or fragment length estimates (*). Splicedonor and acceptor sequences are underlined. Numbering of exon positions corresponds to sequence presented in ref. 9. Exon 1 sequence isfrom ref. 9.

ing to exons 10 and 11. Nucleotide deletions were identifiedin two other patients. In P17, a 4-nt deletion, GTTT (orTTTG) involving nt 1712-1715 (or 1713-1716) results in anamino acid deletion, reading-frame shift, and prematuretermination within exon 16. Another 4-nt deletion, GTTT orTTTG in P22, occurs at -13 to -10 or -12 to -9 at the 3'end of intron 15 and results in a failure to correctly processexon 16, as well as a subsequent exon-17 frameshift andpremature stop. Size determinations of total BTK cDNA inthese patients correspond to the deletions observed in thesequenced cDNAs.

Carrier Detection Using PCR Amplification. Carrier statuswas examined in 16 members from the family ofP08, P13, andP21 that involves a G1706- A substitution resulting in the lossof a Taq I site. In this case, a PCR amplification was designedto yield a 181-bp fragment representing exon 16 and portionsof the flanking introns 15 and 16. After Taq I digestion, thenormal chromosome yields 95- and 86-bp fragments, thecarrier female yields a 181-bp fragment as well as 95- and86-bp fragments, and the affected male (P08) yields only the181-bp fragment (Fig. 2). A pair of mutation-specific PCRprimers were synthesized to detect the GTTT/TTTG in-

tron-15 and exon-16 deletions in P22 and P17, respectively. InP17, primers complementing the GTTT1712-1715 site and anintron-16 primer were used. The PCR product formed withthe normal-specific primer pair was 104 bp, whereas a 100-bpproduct formed with the P17 mutation-specific primer pair;the carrier of the P17 mutation formed products with bothnormal-specific and mutation-specific primer pairs (Fig. 2). Asimilar result was achieved with P22, in which the 4-bpdeletion occurs in intron 15. In this case, normal primingyields a 130-bp product, whereas mutation-specific primingof P22 yields a 126-bp product. P22 carrier DNA amplifiedwith both mutated and unmutated primer pairs.

DISCUSSION

The physical map ofBTK presented here permits identifica-tion of relationships between the BTK cDNA sequences andspecific exons. In humans, this will permit the developmentof rapid assays for missense mutation detection and, in thecase of the mouse, may facilitate efforts to derive constructsfor obtaining different types of null-mutant animals and for

Table 2. Patient clinical data

Age at Immunoglobulin levels at diagnosis PeripheralPatient Clinical history diagnosis IgG, mg/dl IgM, mg/dl IgA, mg/dl blood CD20, %P05 Pneumonia, joint swelling 14 yr n/a 56 (60-250) ND (85-385) <1P08* Otitis media, URI,

pulmonary abscesses 8 yr 100 (725-1938) 50 (60-226) 6 (52-256) <1P09 Pseudomonas sepsis,

Pneumocystis carinii 4 mo 20 (X) 22 (31-131) ND (3-42) 1.3P13* Otitis media, FUO 23 mo 320 (538-1400) 37 (54-206) 1 (16-75) 1P17 Pneumonia, Pseudomonas,

Staphylococcus aureus 17 mo ND (450-1188) 40 (44-175) <2.5 (14-85) <1P18 Otitis media, sinusitis 6 mo ND (X) 14 (38-50) 13 (8-54) <1P21* Otitis media, URI, FUO 20 mo 700 (538-1400) 48 (54-206) 7 (16-75) 2P22 Recurrent URI 17 mo 408t 42t 17* 10*Peripheral blood CD20 percentage normal value is 7-19%o; n/a, not applicable (patient receiving IgG); ND, not detectable; normal age matched

values are shown in parentheses. URI, upper respiratory infection; (X), not applicable due to maternal contribution; FUO, fever of unknownorigin.*Related family members.*Normal age-matched values not available; single determination from outside facility.

9064 Immunology: Ohta et al.

Proc. Natl. Acad. Sci. USA 91 (1994) 9065

Table 3. Classification of six different mutations in eightpatients reported

Patient Exon1-bp substitutionG215- A Arg28 His P05 2G-36 T Gln78 Stop P09 3G1706- A Arg525 - Gln P08/13/21 16

4-nt deletionA'708 ACTGTTTGGTA1719 P17 16A1708 ACTGGTA1719

CTGTTTGCACTACAGG1699 P22* 16CTGCACTAGAfG1699

Splice-junction mutationG I026'lI G'026AT P18* 10-11

Nucleotide and peptide sequence designations are from ref. 9;exon locations are from Fig. 1 and Table 1. The 4-nt deletions forpatients P17 and P22 are shown below the normal sequence. Thesplice junctions for patients P18 and P22 are underlined.*atient P22 has a 4-nt deletion and P18 has a single-base-pairsubstitution, both of which affect splice junction.

other types of transgenesis important in understanding thefunction ofBTK in B-cell development.The overall organizational features of the BTK gene indi-

cate some similarity to that of the Src kinases; however, thestrong conservation of exon/intron boundaries from SH3through the C terminus seen among the Src kinases (17) is notentirely consistent with BTK organization. Nevertheless,based on the localization of specific mutations in this andother studies, it is possible to draw analogies between knownfunctional domains of the Src-like genes and BTK. Prelimi-nary evidence from another Tec family kinase, TXK (18) (andunpublished data), indicates that TXK, BTK, and, by impli-cation, possibly other Tec family genes also may exhibitstrong conservation of exon/intron boundaries.The most immediate use for the information reported here

is in analyzing missense mutations in patients with XLA (Fig.3), determining carrier status, ruling out XLA, and reclassi-fying atypically presenting primary immunodeficiencies. Themost profound clinical immunodeficiency in this study,marked by two life-threatening infections in the first 4 mo oflife, occurs in patient P09 and is consistent with the Gln78Stop in the 5' portion of-the coding region. A more N-terminalsingle-base-pair substitution in patient P05 G215 A resultsin an Arg28 -+ His replacement of the same amino acid thatis mutated in murine XID. However, in XID, the mutationoccurs at the first position in the conserved (mammalian)arginine codon (C214 - T) resulting in an Arg28 -- Cyssubstitution (11, 12). In the mouse, XID is associated with a

1 2 3 4 5 6 7 8 9._~ ~bp PL

310=234 -194 -1810-118-957_72- 860-

milder immunodeficiency (compared with XLA), marked bythe inability to form antibodies to polysaccharide antigensand haptenated polysaccharide and by a B-cell proliferativedefect upon stimulation with antiimmunoglobulin (19). Whilethese studies were in progress, an identical mutation wasdetected in an unrelated individual (14). The clinical courseof patient P18, in which exons 10-11 are deleted, has beenrelatively less severe. Although this deletion does not causea reading-frame shift, the loss of half of SH2, which involves}3A, aA, SIB, and SC (Src) residues, would result in anincomplete phosphotyrosine-recognition domain (20, 21).Exon 16, which encodes the kinase activity ofBTK, is the

site of three different types of mutations in this study andaffords an opportunity to examine the relationships betweenspecific mutations and physiological consequences. Familymembers P08 and P13 had hypogammaglobulinemia andantibody deficiency during their early years, which worsenedwith advancing age. The BTK mutation G1706 - A, whichresults in Arg525 -- Gln, also has been reported in anunrelated family (9). These patients, including family memberP21, have low levels of circulating B cells at an early age withmildly decreased IgM and variable IgG levels, although allare IgA deficient. P21 had demonstrated ability to makespecific antibody upon immunization with diphtheria andtetanus toxoid vaccines. In earlier studies from this labora-tory, it was demonstrated that comprehensive analyses ofimmunoglobulin gene transcripts are highly informative incharacterizing immunodeficiency (22). Immunoglobulinheavy-chain mRNA (cDNA) transcripts have been isolatedand sequenced from P08 at 15 yr of age. All six variable-region heavy-chain (VH) families, a large number of differentdiversity-region (D) segments, and four of six joining-regionheavy-chain (JH) gene segments were found to be expressedand showed evidence for extensive junctional diversity andsomatic mutation ofrearranged immunoglobulin heavy-chaingenes (not illustrated). Within the spectrum of clinical pre-sentation and course in XLA, P08, P13 and P21 are consid-ered less severe. Without the remarkable family history andcapability to detect mutations in BTK, these patients wouldlikely have been classified as one spectrum of commonvariable immunodeficiency disease, and patient P21 wouldnot be diagnosed. The clinical course ofpatient P22, in whichexon 16 is deleted owing to a 4-nt deletion at the 3' of intron15, that results in the loss of a splice donor site, frameshift,and premature termination also has not been particularlysevere. Originally, this patient was diagnosed with commonvariable immunodeficiency disease. However, the clinicalhistory of patient P17, in which a deletion, frameshift, andpremature termination occur within exon 16, was-associatedwith two life-threatening infections within the first 17 mo of

10 11 12 13 14 15 16 17 18 19 20_M rig*~-- s " " PL

130-S 126-104100

FIG. 2. Detection of mutations involving exon 16 of the BTK gene. Restriction-site-loss mutation in patient P08 (also found in family membersP13 and P21) and identical to that reported (9). PCR amplification of a portion of a genomic sequence including exon 16 not digested (lanes 1,3, and 5) or digested (lanes 2, 4, and 6) with Taq 1. Lanes: 1 and 2, normal control; 3 and 4, patient P08; 5 and 6, mother of P08. Specific primingof exon 16 deletion in P17. Normal (N) and deletion (A)-specific priming are compared for normal (lanes 8 and 9), patient P17 (lanes 10 and 11),and mother of P17 (lanes 12 and 13). Specific priming of intron-15 deletion in patient P22. Normal (N) and deletion (4)-specific priming arecompared for normal (lanes 15 and 16), patient P22 (lanes 17 and 18), and mother ofP22 (lanes 19 and 20). HaeIII-digested bX174 standard (lanes7 and 14) fragments are indicated in bp. In addition, PCR product length (PL) for certain bands is indicated at left for analyses of patient P08and at right for analyses of patients P17 and P22.

Immunology: Ohta et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

©o 2 @0 *A\ @©@I ~~A|5UT |SH3 | SH2 T KIN|3UT

AAA A At AtAFIG. 3. Distribution of known mutations and identification of affected regions of the BTK cDNA. The nature of the missense mutations is

indicated as follows: (o), single-base-pair substitution; A, four coding nucleotide deletion; , exon deletion (relative length is indicated); o,insertion. 9, Identical mutations detected in unrelated individuals are described in text. The 5' mutation reported in this series has been describedin ref. 14, and the kinase domain mutation has been described in ref. 9. A, Stop codons resulting from either the single-nucleotide mutationsor deletion and insertion frameshifts. Numbers refer to references as follows: 1 (9); 2 (15); 3 (14); and 4, these studies.

life (Table 2). While the comparison is limited only to thesepatients, the conclusions are reminiscent of the variousclinical courses of muscular dystrophy associated with dif-ferent frameshift changes (23). Splice-junction mutationsoccur in patients P18 and P22. Mutations of this type mayexplain insertion mutations reported elsewhere (14, 15), andin some instances, these mutations now can be definitivelycharacterized by using intron-sequence information that hasbeen obtained in the course of these studies. A significantnumber of large gene deletions have been deduced fromgenomic DNA restriction fragment length polymorphismanalyses in an earlier report (9); however, no mutations ofthis type have been observed in this study.The two instances of identical mutations in unrelated

families described in this report and the identification of areplacement mutation at the same site in human XLA as inmouse XID suggest that a limited number of deleteriouschanges in BTK produce clinically recognizable XLA. Onlycertain findings described here suggest a correlation betweenclinical-presentation course and structural changes in thegene product. However, it is important to recognize thatcompensatory mechanisms encoded at separate loci mayproduce phenotypic variation; adaptive and natural defensesare redundant. Specifically, XLA patients have been classi-fied in two general groups: (i) those presenting at early agewith particularly severe infections and (ii) those with lesssevere disease in which production of immunoglobulin issustained at low-to-normal levels well into the first decade oflife. It is therefore hypothesized that an ontogenetic changemay occur in which the defective tyrosine kinase no longercan sustain the B-cell population, and a progressive reductionin immunoglobulin production occurs. Continued mapping ofmutations as well as deletions in XLA and relating themutation sites to pre-B and B-cell phenotypes will further theunderstanding of the relationship to BTK structure to B-celldifferentiation and proliferation. It also is reasonable topredict that other changes in theBTK gene may result in otherforms of less severe immunodeficiency and, in this regard,examination of a group of patients with particular commonvariable immunodeficiency disease presentations may proveto be of clinical relevance. The identification ofthe TXK gene(18), a homolog of the BTK gene, which is expressed primar-ily in T cells, further emphasizes the significance ofTec-typeprotein-tyrosine kinase genes in the development and func-tional regulation of the immune system.

We thank Barbara Pryor for editorial assistance. This work wassupported by a grant from the Eleanor Naylor Dana Foundation toG.W.L. and by a grant from the National institutes of Health(CA34546) to S.M.F.

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