Am. J. Hum. Genet. 58:477-483, 1996

Discordant Phenotype in Siblings with X-Linked AgammaglobulinemiaMichael J. Bykowsky,1 Robert N. Haire,2 Yuko Ohta,2 Huayang Tang,3 Sun-Sang J. Sung,3Ekaterina S. Veksler,' Jeffrey M. Greene,1 Shu Man Fu,3 Gary W. Litman,2 andKathleen E. Sullivan'1Department of Pediatrics, Division of Allergy, Immunology, and Infectious Diseases, Children's Hospital of Philadelphia, Philadelphia;2Department of Pediatrics, University of South Florida College of Medicine, St. Petersburg; and 3Department of Internal Medicine, Universityof Virginia Health Sciences Center, Charlottesville

Summary

X-linked agammaglobulinemia (XLA) is a congenitalhumoral immunodeficiency caused by a defect in a B-cell-specific signaling molecule, Btk. There has been littleconcordance of phenotype with genotype in this disor-der, and defects in Btk cause immunodeficiencies thatrange from mild impairment to complete inability toproduce antibodies. The factors modifying the pheno-type of XLA are not understood. The current study isthe first description of two male siblings with identicalT134-+C mutations in the translation initiation ATG ofBtk who have different clinical phenotypes as well asdifferent laboratory phenotypes. The proband lacks im-munoglobulins and B cells and has recurrent infections,while the elder, affected brother has normal levels ofIgG and IgM and very few infections. Both have un-detectable levels of Btk kinase activity in circulatingmononuclear cells. Complete sequencing of Btk genetranscripts in both brothers revealed no additional mu-tations to account for the discordant phenotypes. Thisdescription provides unequivocal evidence that the phe-notype of XLA is influenced by factors additional to theBtk gene.

Introduction

Bruton type or X-linked agammaglobulinemia (XLA)was first described in 1952 (Bruton 1952) and is theprototypical humoral immune deficiency with normalcellular immunity. It occurs in - 1/50,000 live malebirths and is typically characterized by absent or ex-tremely low numbers of circulating B lymphocytes, ab-sence of B-dependent lymphoid tissue, reduced concen-

Received September 8, 1995; accepted for publication November27, 1995.

Address for correspondence and reprints: Dr. Kathleen Sullivan,Division of Allergy, Immunology, and Infectious Diseases, The Chil-dren's Hospital of Philadelphia, 34th Street and Civic Center Boule-vard, Philadelphia, PA 19104. E-mail: sullivak@mail.med.upenn.eduC 1996 by The American Society of Human Genetics. All rights reserved.0002-9297/96/5803-0005$02.00

trations of immunoglobulins of all classes, and inabilityto make functional antibody responses. Patients withXLA are susceptible to recurrent bacterial infectionswith encapsulated organisms and are at risk of dissemin-ated infections with enteroviruses (Wilfert et al. 1977;Lederman and Winkelstein 1985). The gene defect inXLA has been mapped to the long arm of the X chromo-some at Xq22 by conventional linkage analysis (Kwanet al. 1986; Guiolo et al. 1989). The protein encodedby this gene has recently been identified as a cytoplasmictyrosine kinase (Bruton agammaglobulinemia tyrosinekinase [Btk]; E.C.2.7.1.112) (Tsukada et al. 1993; Vetrieet al. 1993) that is activated through stimulation of sur-face immunoglobulin (de Weers et al. 1994a).Not all XLA patients conform to the classic phenotype

(Lederman and Winkelstein 1985; Leickly and Buckley1986; Kornfeld et al. 1995). Btk gene analysis has facili-tated the identification of various mutations in patientswith XLA (Tsukada et al. 1993; Vetrie et al. 1993; Ohtaet al. 1994; Saffran et al. 1994) expressing distinct phe-notypes. Although variable phenotypes have been re-ported in these studies, the phenotype of all membersof a family have been similar. We report the first caseof XLA in which brothers with the same mutations intheir Btk gene exhibit different phenotypic expressionof the disease.

Subjects and Methods

The proband (I1l) is 7.5 years old and was well until2 years of age, when he began developing recurrentpneumonia, sinusitis, and otitis media. His height andweight are in the 25th percentile for age. He has nopalpable lymphoid tissue and scarred tympanic mem-branes. Laboratory evaluation revealed markedly re-duced immunoglobulins and B cells, and failure to makespecific antibodies, all of which were consistent with adiagnosis of XLA (tables 1-3). Informed consent wasobtained for the use of peripheral blood from bothbrothers for the studies described below.The elder brother (112) of the proband was diagnosed

with a clinical pneumonia at 2 years of age but hassubsequently been well. He has never been hospitalized

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Am. J. Hum. Genet. 58:477-483, 1996

Table 1

Serum Immunoglobins

Serum Proband (M1) Affected Brother (112)Immunoglobulins (5 years of age) (12 years of age)

IgG <.10 g/liter 14.50 g/literIgA <.08 g/liter <.08 g/literIgM .34 g/liter .89 g/literIgE 8 IU/ml 13 IU/mlIgG1 None detected 7.45 g/literIgG2 None detected .17 g/literIgG3 None detected 2.18 g/literIgG4 None detected .01 g/liter

NOTE.-Laboratory values were obtained at the time of diagnosis,prior to IVIG therapy.

and has not required nor received frequent courses ofantibiotics. His height and weight are in the 50th-75thpercentile for age. He has absent tonsillar and lymphoidtissue and scarred tympanic membranes. Laboratoryevaluation revealed normal IgG and IgM levels. IgA wasnot detectable, and levels of IgG2 and IgG4 subclasseswere markedly reduced (tables 1-3). He failed to re-spond adequately to immunizations, although he had aminimal response to tetanus reimmunization.

Sequence AnalysisMononuclear cells were purified from peripheral

blood, and total RNA was isolated using RNazol B (Tel-Test). Single-stranded cDNA was prepared from totalRNA (Haire et al. 1994). Btk cDNA was produced byPCR in two segments. The 5' portion extended from nt90 to nt 1623 by using primers 5'-TCAGGACTGAG-CACACAGG-3' and 5'-ACCGCTFCCAGACTCAG-CAG-3'. The 3' portion extended from nt 1326 to nt2193 by using primers 5'-CCAAAGGACCTGACCTT-CTI7G-3' and 5'-GCTCGAAAATAAGCTTCTIrGG-3'.The primers derived from the 5' and 3' UTR were

BamHI-linkered. The Btk DNA was cloned into M13by EcoRIIBamHI digestion by using the natural EcoRIsite in the Btk at nt 1453. The complete nucleotide se-

quences of Btk transcripts from both brothers were de-termined, and the presence of a point mutation in exon2 was confirmed by sequencing exon 2, primed fromgenomic DNA (Ohta et al. 1994). The primers used toamplify the second exon were upstream: 5'-GAGCATT-CCATTGCCTT-3'; and downstream: 5'-GCCAAGTC-CTTGATATC-3'. The mutation described in this reportintroduces an EagI restriction site into exon 2 and serves

as a basis for characterizing the mutation in other familymembers.

In Vitro Kinase Assays and Western BlotsPurified monocytes, B cells, and T cells were obtained

by fractionating peripheral blood mononuclear cells(PBMC). Monocytes were purified from PBMC by cen-

trifugation in a continuous Percoll gradient (Sung et al.1992), followed by magnetic bead depletion (Mini-MACS) of contaminating B, T, and NK cells by usingbiotinylated monoclonal antibodies against CD20(KoBl), CD3 (A32), and CD56 (B159.5.2), and strep-tavidin-conjugated magnetic beads. Lymphocytes fromPBMC depleted of monocytes and NK cells by leucyl-leucine methyl ester lysis (Thiele and Lipsky 1986) were

separated into T and B cell fractions by E-rosetting.These fractions were subjected to additional purificationby a magnetic depletion step using biotinylated mono-

clonal antibodies against CD14, CD56, and either CD20or CD3, for T and B cell populations, respectively. Thefinal cell purities by FACScan analysis were as follows:monocytes, 95%; T cells, 98%; and B cells, 90%.

Rabbit polyclonal and mouse monoclonal anti-Btkantibodies were generated against the PH domain(amino acids 1-175) of human Btk using a glutathioneS-transferase (GST)-Btk fusion protein as the immuno-gen. The antibodies identify the 77-kD Btk in both west-

ern blot and immunoprecipitation experiments. The rab-

Table 2

Specific Antibody Formation

PROBAND (I1l) AFFECTED BROTHER (112)

SPECIFIC ANTIBODY Preimmune Postimmune Preimmune Postimmune

FORMATIONa Titers Titers Titers Titers

Tetanus <.1 IU/ml <.1 IU/ml <.1 IU/mi 25 IU/mlDiphtheria <.01 IU/ml <.01 IU/ml <.01 IU/ml <.01 IU/mlH. influenza <.55 mg/ml <.55 mg/ml <.55 mg/ml <.55 mg/mlAnti-A None 1:8Anti-B None None

NoTE.-See note to table 1.

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Bykowsky et al.: Discordant XLA Phenotype in Siblings

Table 3

Lymphocyte Subsets

LymphocyteSubsets Proband (111) Affected Brother (112)

CD3 2,360 cells/mm3 1,857 cells/mm3CD4 1,260 cells/mm3 1,225 cells/mm3CD8 1,060 cells/mm3 494 cells/mm3CD19 0 cells/mm3 4 cells/mm3CD20 8 cells/mm3 6 cells/mm3

NOTE.-See note to table 1.

bit anti-Btk antibody was depleted of anti-GSTreactivities by passage of the antiserum over GST-Sepha-rose columns, followed by affinity column chromatogra-phy using GST-Btk-conjugated Sepharose beads. Therabbit anti-Syk polyclonal antibody was generatedagainst the region of human Syk between the SH2 andkinase domains (amino acids 274-371) as a GST-Sykfusion protein and recognized the 72-kD Syk proteinin western blots and immunoprecipitations. The rabbitpolyclonal and mouse monoclonal anti-Lyn antibodieswere from UBI and Transduction Laboratories, respec-tively.

Cells lysates were prepared in 10 mM Tris HCI (pH8.0), 150 mM NaCl, 1% NP-40, 1% glycerol, 1 mMEDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfo-nyfluoride, and 1 jg/ml each of aprotinin, trypsin inhibi-tor, and leupeptin. The supernatants (after centrifuga-tion at 14,000 x g) were used in immunoprecipitationsusing rabbit Ab and protein A-Sepharose beads. Thekinase reaction was performed at room temperature for15 min by using 10 iCi of [32P]-ATP (3,000 Ci/mmol;DuPont NEN). The samples were separated on 8% SDS-PAGE and transferred to nitrocellulose membrane. Thelabeled proteins were visualized by autoradiography.Proteins were detected on the same blots by using west-ern blots and enhanced chemiluminescence (Amersham).Band intensities were quantitated by scanning densitom-etry using a flatbed scanner (Color One Scanner; Apple)and a Biosoft analysis program (Scan Analysis).

Results

The proband (I11) had clinical and laboratory findingscompatible with XLA. In contrast, his elder brother (112)had clinical and laboratory features most compatiblewith common variable immunodeficiency or IgA defi-ciency with IgG2 and IgG4 subclass deficiencies. Theelder brother not only had normal levels of IgG andIgM, he was also able to produce isohemagglutinins andantitetanus responses (tables 1 and 2). In an effort toestablish the diagnosis more clearly in these patients,

Btk transcripts were amplified from PBMC mRNA. AT134-+C mutation in the translation initiation ATG (Ve-trie et al. 1993) located in the second exon (Ohta et al.1994) of the Btk gene was identified in both brothers(fig. 1). This mutation, which creates a site for the re-striction endonuclease EagI, would be expected to im-pair translation of the Btk mRNA. In order to rule outthe unlikely possibility that an additional mutation(s)was present, the complete nucleotide sequence of thecDNA was resolved in both patients. No additional mu-tations were identified.

Family studies were performed to determine whetherother male family members had inherited the Btk muta-tion but were asymptomatic. DNA was prepared fromeach available family member; exon 2 was amplified byPCR and digested with EagI to detect the mutation (fig.2). The T134-+C mutation introduces an EagI site, whichallows for restriction digest detection of the mutation.The nephew (II11) of 111 and 112 was found to haveinherited the same Btk mutation as his uncles and hada phenotype at 3 mo of age that resembled that of theproband. He had 0.4% B cells and immunoglobulin lev-els which were markedly low: IgG 197 mg/dl, IgA <7mg/dl, and IgM 8 mg/dl. An 18-year-old male sibling

GCATC--A3

GCCAT

CAA

IT

c

a

EXON2134

..G A CAAMTT Cc3CTGATT..CTTTCTTCTI I3ATACCGc3GTCGcTM

MA AV I

Figure 1 Alterations in the translation initiation site, caused bythe Btk mutation. The Btk genes from both affected brothers weresequenced completely in both directions in both cDNA and genomicDNA, revealing a T-C mutation in the translation initiation codonin exon 2. The relevant sequence from the proband is shown in bothdirections. This mutation introduces a cleavage site for EagI into exontwo.

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Am. J. Hum. Genet. 58:477-483, 1996

'I

I :L211 II2E

1 2 3 4 ; 6 7 8

4~~~~~~~~~~~~~~~~~~1

11 lII 112 113 114 115 116 117 I11 1 C

480bp _

291bp

189bp

Figure 2 EagI restriction digest detection of the Btk mutationin family members. PCR amplification of exon 2 in Btk results in a

480-bp band. The mutation T-*C in Btk exon 2 introduces a cleavagesite for EagI, resulting in 291-bp and 189-bp fragments. Unaffectedfamily members II3 and IIS and the unrelated control (C) show onlythe 480-bp band. The affected male patients-1i, 112, and III1yield the 291-bp and 189-bp bands. The obligate female carriers, I1and II7, yield a normal 480-bp band as well as 291-bp and 189-bpbands.

(115) lacks the mutation, has normal immunoglobulinlevels, and has been clinically well.To determine whether the discordant phenotype could

be explained on the basis of Btk expression, in vitrokinase and western blot analysis were performed. Exper-iments with fractionated PBMC from normal controlsubjects showed that monocytes and B cells are the pre-

dominant cell types expressing Btk. On a per-cell basis,monocytes had three to four times the Btk activity andabout twice as much of the protein as did B cells. PurifiedT cells expressed no detectable Btk activity or protein(fig. 3A). These T cells did express Syk, another proteintyrosine kinase expressed predominantly in B cells andmonocytes. T cells contained levels of Syk activity simi-lar to B cells but only 25% of the amount of Syk proteinas B cells.

In vitro kinase analysis of total PBMC lysates fromthe two brothers showed no detectable Btk activity,whereas Btk activity was readily detected in 1/16 thenumber of normal cells (fig. 3B; compare lane 5 withlanes 7 and 9). Western blots showed that a faint proteinband, <10% of that of control, was detected in II1, but

no Btk could be detected in II2 PBMC (fig. 3B, lanes 7and 9). Because Btk activity and protein expression inPBMC were primarily due to their presence in mono-cytes (fig. 3A) and the PBMC of Ill and I12 contained17% and 8% of monocytes, respectively, these resultsshowed that Btk expression was severely depressed inthe patients, and this depression is not due solely to theabsence of circulating B cells. There is a cross-reactivekinase activity with different mobility in the Btk lanes(marked by an asterisk [*] in fig. 3A and B). This activityhas also been observed by other investigators (Tsukadaet al. 1993; Aoki et al. 1994). This band is of the sameintensity in the two brothers as in normals. The func-tional significance of this kinase is uncertain. To exam-ine whether the phenotypic differences in the two broth-ers were due to differences in the expression of B celland monocyte kinases other than Btk, the activities andprotein levels of two major tyrosine kinases (Syk andLyn) responsible for signal transduction in these cellswere examined (Perlmutter et al. 1993). Syk activity inIll and II2 was 70% and 30%, respectively, of that incontrol cells, although PBMC Syk protein content wasnear normal (fig. 3B). These results suggest that Syk isless activated in patient cells. The kinase activity andprotein level of Lyn were also found to be normal in thepatient PBMC (fig. 3B).

Discussion

Btk is a cytoplasmic protein tyrosine kinase that isexpressed in most hematopoietic tissues (Aoki et al.1994). Although direct demonstration of protein-pro-tein interactions with B cell surface receptors has notbeen shown, it has been inferred that Btk functions ina manner analogous to the other protein tyrosine ki-nases, on the basis of structural homologies. The Techomology region of the molecule, conserved in all Btkfamily members (Smith et al. 1994), interacts with theSH3 region of the Src family proteins Fyn, Lyn, and Hck(Cheng et al. 1994). A second domain with homologyto pleckstrin may be responsible for recruiting Btk tothe cell membrane (Musacchio et al. 1993). The require-ment of Btk for the normal developmental program ofB cells has been demonstrated in vivo in XLA patientswho lack normal numbers of mature B cells (Grey et al.1971). XLA patients can have normal levels of pre-Bcells in their marrow (Pearl et al. 1978) implying that thecritical requirement for Btk occurs during the transitionfrom pre-B cell to B cell. The demonstration of raremature B cells in the periphery (Mensink et al. 1986)suggests that the maturational signals may be redun-dant, allowing occasional cells to mature through a sepa-rate pathway.

It has become increasingly evident that different muta-tions within the same gene in single-gene-defect diseases

480

Bykowsky et al.: Discordant XLA Phenotype in Siblings

BTKPBMC IU T B

-66c-i - 6 6-0

SYKPBMC M T B

1.- P9 * 1.0 0-Ow - 0o.

A_~~~~~~~~~~~a1 2 3 4 6 6 7 6 9 i0t11 13 14

BTKNormal

i .o 6o 6o4l== 6

SYKD1 02 Normal IIl

1 2 3 4 5 6 7 8 9 10 II 12 13 14

LYN32 Normal la U2

CN - -* N-O0O O cN- c-* cv o 66 o . Nr

___________.~~~~~~~~~~~~a

Western 1 6-

1 2 3 4 58 r7 Jo1

_- _

I 2 3 4 5 6 7 8 9 1 2 4 56 7 8 9 10

Figure 3 Expression of Btk in normal and patient PBMC. A, The kinase activities and protein levels of Btk and Syk in total PBMC andfractionated cell populations were examined by in vitro kinase and western blotting. PBMC and purified monocytes (M), T cells (T), and Bcells (B) from the same buffy coat were used. B, Total PBMC from a normal control individual and from the patients II1 and 112 were usedin studying the expression of Btk, Syk, and Lyn. The numbers above the lanes indicate the number of cells x 106 used in each assay. Arrowsindicate the position of the tyrosine kinases. The asterisk (*) in the Btk lanes indicate the position of a cross-reactive kinase.

result in a range of phenotypes (Dean and Santis 1994;Jabs et al. 1994; Muenke et al. 1994; Park et al. 1995;Rutland et al. 1995; Wilkie et al. 1995). Accordingly,mutations within Btk have been associated with a range

of XLA phenotypes, although the phenotype associatedwith each individual mutation is usually similar. Themutations have included single-base-pair substitutions,insertions or deletions (Vetrie et al. 1993; de Weers et al.1994b) resulting in amino acid substitutions, deletions,aberrant stop codons, frameshifts, abnormal splicing, or

combinations of these events (Ohta et al. 1994). Thepatients exhibit a wide variety of phenotypes, includingdelayed or mild presentation with normal antibody re-

sponses to immunizations. The phenotypes in these pa-

tients were not predictable on the basis of the mutation,although all members of one particular kindred were

affected equally.Mutations involving the initiation codon of the Btk

gene have been described elsewhere. In one study (Brad-ley et al. 1994) a patient was found to have the same

T134-*C mutation reported here. A second patient in thatstudy and one reported in another study (Conley et al.1994) had a different initiation codon mutation,A133-G. All three patients appeared to have the classicXLA phenotype. The ACG codon has been reported to

have some translation initiation function in eukaryotes

(Kozak 1991). Two alternative ATG codons at -23 and+89 do not allow for translation of full-length Btk prod-uct, suggesting that if any functional Btk is produced,the mutant initiation site must be functional.

Variable phenotypic expression in patients with iden-tical mutations is difficult to explain. This is the firstdescription of brothers with identical Btk mutations anddiscordant phenotypes. The younger brother was firstseen at 5 years of age with recurrent sinopulmonaryinfections, including five episodes of pneumonia, andlaboratory values consistent with XLA, including absentimmunoglobulins, absent functional antibodies, andmarkedly diminished peripheral B lymphocytes. His el-der brother had few infections and was initially diag-nosed with IgA and IgG2JIgG4 subclass deficiency. Theelder brother routinely had IgG levels in the 1,200-1,400 mg/dl range, prior to initiation of intravenousimmunoglobulin (IVIG) therapy, although he producedlittle functional antibody in response to immunization.A reservoir of mature B lymphocytes in his spleen, lymphnodes, or bone marrow may have produced the immu-noglobulins. Serum immunoelectrophoresis did not

demonstrate oligoclonal bands, suggesting that the basisfor the persistently elevated serum IgG level is not an

oligoclonal gammopathy. This is further supported bythe fact that he produced both IgM and measurable

A

Lake

Wetern

B

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1.%tk

482 Am. J. Hum. Genet. 58:477-483, 1996

quantities of all four subclasses of IgG prior to the initia-tion of IVIG therapy.The mutation described in this kinship is the result of

a single nucleotide change T134-+C, resulting in an aminoacid substitution of Met-+Thr affecting the translationinitiation codon (Kozak 1991). The Btk cDNA se-quences were fully resolved in both directions in bothbrothers, and no additional mutations were found; how-ever, it is possible that one of the brothers could havea second mutation in an upstream or intronic regulatoryregion of the Btk gene that is affecting the expressionof the first mutation. Arguing against that possibility isthe finding that Btk activity was absent in both brothers.One explanation for the discordant phenotypes in thiskindred could be the influence of other genes or geneproducts regulating the translation of the Btk message(Romeo and McKusick 1994). A second possibility isthat different kinases may be substituting for Btk in theless severely affected sibling.

Variable expression in kindreds with XLA has beenpreviously reported. "Classic" XLA and CVID were di-agnosed in two male members in two different kindreds(Wedgewood and Ochs 1980). Since XLA and CVIDare not commonly seen within a family, variable expres-sion of the X-linked trait was postulated. XLA associ-ated with dysgammaglobulinemia in the same kindredhas been reported (Buckley and Sidbury 1968; Gold-blum et al. 1974), although a definitive molecular diag-nosis of XLA was not made. This is the first reportof discordant XLA phenotype within the same kindredwhere sequencing has confirmed the inheritance of theidentical mutant Btk gene.

It has become increasingly clear that the phenotypein XLA cannot be predicted by the site or type of themutation within the Btk gene (Kornfeld et al. 1994).The establishment of a Btk database (Vihinen et al.1995) will facilitate attempts to predict natural historyon the basis of genotype, but it is now evident thatother factors are involved that may affect the phenotypeexpressed in patients with identical Btk mutations.

AcknowledgmentsWe thank Steven D. Douglas for his helpful discussions and

critical review. This work was supported in part by a MAPS-CHRC award, HD28815, (K.E.S.), an award from the EleanorNaylor Dana Foundation (G.W.L.), and NIH grant R01-CA34546 (S.M.F.).

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