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Clinical Immunology (2008) 128, 322–328

Identification of a Btk mutation in adysgammaglobulinemic patient with reducedB cells: XLA diagnosis or not?Simona Graziani a,1, Gigliola Di Matteo a,1, Luigi Benini b, Silvia Di Cesare a,Maria Chiriaco a, Loredana Chini a, Marco Chianca a, Fosca De Iorio b,Maria La Rocca a, Roberta Iannini a, Stefania Corrente a,Paolo Rossi a,c, Viviana Moschese a,⁎

a Department of Pediatrics, University of Rome Tor Vergata, Italyb Department of Gastroenterology, University of Verona, Italyc Division of Immunology and Infectious Diseases, Children’s Hospital Bambino Gesù, Rome, Italy

Received 16 April 2008; accepted with revision 28 May 2008

⁎ Corresponding author. DepartmentVergata, Viale Oxford 81, 00133 Rome

E-mail address: moschese@med.un1 SG and GDM equally contributed to

1521-6616/$ – see front matter © 200doi:10.1016/j.clim.2008.05.012

Abstract The identification of a Btk mutation in a male patient with b2% CD19+ B cellswarrants making the diagnosis of X-linked Agammaglobulinemia (XLA).

Herein we report the case of a 31 year-old male with a gradual decline of peripheral Blymphocytes and low IgA and IgM but normal IgG levels. His clinical history revealed recurrentrespiratory and skin infections, sclerosing cholangitis and chronic obstructive pancreatitis.Molecular studies revealed a novel aminoacidic substitution in Btk protein (T316A). His mother,maternal aunts and a maternal female cousin were heterozygotes for the same Btkmutation andwere variably affected with pulmonary emphysema.

This is a puzzling case where the patient's clinical history and laboratory findings divorcemolecular genetics. Either this case confirms the variable expressivity of XLA disease or theT316A change in Btk SH2 domain is a novel non-pathogenic mutation and another unknown genealteration is responsible for the disease.© 2008 Elsevier Inc. All rights reserved.

KEYWORDSXLA;Btk;Sclerosing cholangitis;Chronic obstructivepancreatitis

Introduction

X-Linked or Bruton's Agammaglobulinemia (XLA) is an X-linkedrecessive humoral immunodeficiency disease caused by

of Pediatrics, Policlinico Tor, Italy. Fax: +39 06 20900530.iroma2.it (V. Moschese).this work.

8 Elsevier Inc. All rights reserved

mutations in the gene coding for the Bruton's Tyrosine kinase(Btk), which is implied in the development of B cells.Mutations of Btk gene cause the incomplete differentiationof B cell precursors to mature B cells or the inefficientexpansion of pre-B cells into later B cell stages [1,2]. XLA ischaracterized by a defect in peripheral blood B lymphocytes(CD19+b2%) and very low levels of all classes of immunoglo-bulins, resulting in increased susceptibility to bacterialinfections (sinusitis, pneumonia, pyogenic dermatitis, osteo-myelitis) [3–5]. Patients usually become symptomatic in

.

323Identification of a Btk mutation in a dysgammaglobulinemic patient with reduced B cells

infancy or early childhood after their maternal immunoglo-bulins (Igs) have been lost. In addition to typical XLA, severalatypical cases have been reported with mild or no clinicalsymptoms and/or normal IgG values in the presence of Btkmutations [6–12]. Atypical XLA patients, in the absence of adefinitive diagnosis by Btk sequencing, might commonlyreceive an initial diagnosis of common variable immunode-ficiency (CVID), specific antibody deficiency or IgA deficiency[9,13–16].

Female carriers are usually asymptomatic, except for onereported case of XLA disease caused by extremely skewed X-chromosome inactivation in a 10months old Japanese girl [17].

Interestingly, a novel non-pathogenic mutation in SH3domain of Btk has been proved not to be associated to XLAdisease [18].

XLA, as well as other primary immunodeficiencies (PID),shows an higher incidence of autoimmune diseases than inthe general population. In XLA the most common auto-immune features are arthritis, dermatomyositis, autoim-mune haemolytic anemia (AHA) and sclerodermia [19]. Sofar, sclerosing cholangitis has been commonly observed inHyper-IgM patients [20,21].

Herein we describe the case of a 31 year-old dysgamma-globulinemic male with a reduced number of peripheral Blymphocytes and an atypical clinical phenotype character-ized by sclerosing cholangitis and chronic obstructivepancreatitis in which a Btk mutation was identified.Furthermore, all female carriers of the Btk mutationsuffered from a mild to severe pulmonary emphysema.

Materials and methods

Subjects

All subjects gave their informed consent to performimmunological and genetic analysis. All studies, includingcell count and flow cytometric analysis, were performed atthe Pediatric Immunology and Biotechnology Laboratory, TorVergata University, Rome.

Cell lines

EBV-transformed lymphoblastoid cell lines (LCL) wereobtained by incubating about 5×106 PBMC from the XLApatient and his family (see Fig. 1; II-2, II-4, II-5, III-1), with

Figure 1 The genealogical

2 ml of supernatant from the EBV-secreting cell line B95-8 for1 h at 37 °C. Infected cells were then washed and cultured at1×106 cells/ml in RPMI 1640 containing 25 mM Hepes, 20%foetal bovine serum from U.S., 2 mM L-glutamine and 1 μg/ml of cyclosporin A in 24-well culture plates. Clumps of cellswere observed at the end of the first or the second week andstable EBV-lymphoblastoid cell lines were established by theend of 4 weeks.

Mutation analysis

Genomic DNA, isolated from the PBMC of patients andhealthy controls (QIAamp DNA Blood kit, QIAGEN GmbH,Hilden, Germany), was amplified using primers flanking the19 coding exons and promoter of the Btk gene (seq. X58957).PCR reactions were carried out in a volume of 50 μlcontaining 100 ng of genomic DNA, 200 μM of each dNTP,0.4 μM of each primer and 1.25 U GoTaq DNA polymerase(Promega, Madison WI, USA). The samples were denaturatedat 95 °C for 5 min followed by 40 cycles at 95 °C for 30 s,annealing at exon specific temperature (available uponrequest) for 30 s, extension at 72 °C for 30 s with a finalextension at 72 °C for 5 min.

DHPLC (Denaturing High Performance Liquid Chromato-graphy), used as a screening method to analyze Btk gene, wasperformed on aWAVE analysis system (Transgenomic, Omaha,NE) using different temperature conditions (available uponrequest). Direct sequencing of PCR products with abnormalchromatography was then performed after purifying PCRproducts with QIAquick PCR purification kit (QIAGEN, Hilden,Germany). Both strands were sequenced using the BigDyeTerminator v3.1 Cycle Sequencing Kit (Applied Biosystems,Foster City, CA, USA) and analyzed on an ABI 3130 automatedsequencer (Applied Biosystems, Foster City, CA, USA).

Immunoblot analysis

Total cells were lysed on ice cold JS lysis buffer (50mMTris/HClpH 8, 150mMNaCl, 1.5mMMgCl2, 5mMEGTA, 1% Triton-X, 10%glycerol, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin,10 μg/ml pepstatin, 1 mM DTT) for 20 min on ice and thenclarified by centrifugation at 12000 rpm for 10 min at 4 °C.20 μg of total cell lysate were size-fractionated by SDS-PAGEgel and then transferred to nitrocellulose membrane (Protran,Schleicher & Schuell-Bioscience GmbH, Dassel, Germany).

tree of our XLA patient.

324 S. Graziani et al.

Membranes were blocked in 5% milk for 1 h at roomtemperature and then incubated with a primary antibody(anti-Btk C20, Santa Cruz Biotechnology Inc., CA, USA), 1:300dilution, for 1 h at room temperature. Specific proteins werevisualised by ECL (LiteAblot, Euroclone SpA, Switzerland).

Flow cytometric analysis of Btk phosphorylation

Lymphoblastoid cell lines (10×106 cells) were collected bycentrifugation and stimulated by hydrogen peroxide (10 mM)at 37 °C for 5 min [22]. Collected cells were fixed for 10 minat 37 °C and then permeabilized by adding 1 ml of BDPhosflow buffer III for 30 min on ice according to BD Phosflowprotocol 3 (BD Biosciences Pharmigen, San Diego, CA,USA).Pelleted cells were resuspended in BD Pharmigen Stain buffercontaining PE conjugated anti-phosphoY551-Btk (BD Bios-ciences Pharmigen, San Diego, CA, USA) at room tempera-ture and incubated a minimum of 1 h. The analysis wascarried out on a FACScan using CellQuest software (BDBiosciences Pharmigen, San Diego, CA, USA).

Results

Case presentation

We report the case of a 31 year-old male with a history ofrecurrent infections that was referred to our hospital forimmunological evaluation. As reported in the genealogicaltree (Fig. 1) two maternal uncles (II-1; II-3) had died forunknown infections in the first year of life. In addition, hismother, two maternal aunts and one maternal female cousinwere affected by mild (II-2, II-4, III-1) to severe (II-5)pulmonary emphysema. His medical history revealed recur-rent episodes of enteritis associated to failure to thrive sincehis first months of life. At the age of 3 months he developedmeasles complicated with pneumonia and pulmonaryabscess. In the following years he suffered from recurrentepisodes of Staphylococcus aureus' pneumonia, urinary tract

Table 1 Immunological data of our male patient and female car

III-3 II-2

Lymphocytes 1230/μl 140CD3+ (%) 89 82CD4+ (%) 39 55CD8+ (%) 46 28CD19+ (%) 2 4.7CD19+CD27+IgD+IgM+ (%) 10 19CD19+CD27+IgD−IgM− (%) 13 30CD24highCD38high (%) 6 2.7IgM (mg/dl) 6 65IgG (mg/dl) 833 856IgA (mg/dl) 32 99Antibody response to Pneumococcus (1) Low NorAntibody response to Tetanus (2) Normal NorAntibody response to Haemophilus (3) Normal Nor

nt=not tested.(1) Protective Level : 3 fold increase.(2) Protective Level : N0.1 UI/ml.(3) Protective Level : N0.15 mg/L.

infections and diffuse pyodermitis with cutaneous abscessesthat led the clinicians to an immunological investigation.Retrospective data of the first decade of his life reported achemotactic defect and a dysgammaglobulinemia (normalIgG with low IgA and IgM serum concentrations). Lymphocytesubsets were available at 12 years of age and showed normaltotal lymphocyte count with an inverted CD4+/CD8+ ratio andB lymphocyte percentage of 8%. At that time his clinical andimmunological features led to the diagnosis of commonvariable immunodeficiency and he was treated with i.v.gammaglobulin substitution. However, replacement therapywas not performed on a consistent basis since the patient hadshifted several referral centres in the second decade of life.

At that time severe chronic obstructive lung disease wasobserved and pulmonary Computed Tomography (CT) provedthe presence of pulmonary interstitial fibrosis with absces-sual cavity in the right lung. The sweat test was negative.Pulmonary function tests confirmed the obstructive respira-tory deficiency.

At 28 years of age he was hospitalized for weight loss,jaundice and abdominal pain. Laboratory data revealed: VES65 mm/H, PCR 229 mg/l, ALT 141 IU/l, AST 165 IU/l, GGT230 IU/l, ALP 358 IU/l, total bilirubin 5.05 mg/dl, amylase202 IU/l, lipase 400 IU/l. Abdominal CT showed stenosis inthe distal tract and dilatation in the proximal-medium tractof Wirsung's Duct. An Endoscopic Retrograde Cholangio-Pancreatography (ERCP) confirmed dilatation of both intra-hepatic bile ducts and Wirsung's Duct (type IV according toCremer's Classification) with a clinical diagnosis of sclerosingcholangitis and of chronic obstructive pancreatitis. Torelieve the obstructed pancreatic flow a stent was insertedin the main pancreatic duct. A fewmonths later, when he wasfirst admitted to our division, his physical exam revealed anunderweight male with multiple cutaneous scars, mildhepatosplenomegaly, prolonged expiration and wheezing.His immunological data are shown in Table 1. At this timedysgammaglobulinemia due to low IgA and IgM with normalIgG, low values of CD19+ B cells (percentage values in repeatstudies ranging from 1.98 to 2.8%) and inverted CD4+/CD8+

riers

II-4 II-5 Range

0/μl 1380/μl 1380/μl80 84 61–8460 54 32–6021 29 13–407.9 4.2 10–3129 nt 13.7 (6.1–32) [43]28 nt 13.8 (6.1–24) [43]2.6 nt (2.06±0.27%) [44]71 62 40–230987 843 700–1600118 80 70–400

mal Normal nt –mal Normal nt –mal Normal nt –

Figure 2 (a) MRCP shows the presence of a stenotic segmentat the level of the pancreatic head with marked dilatation of themain duct and of its main branches in the pancreatic body andtail. (b) ERCP shows the long narrowing of the extrahepatic bileduct and the alternation of dilated and stenotic tracts inintrahepatic ducts typical of sclerosing cholangitis. Note thepresence of the pancreatic stent.

325Identification of a Btk mutation in a dysgammaglobulinemic patient with reduced B cells

ratio were confirmed. CD3+ lymphocyte count and prolif-erative lymphocyte response to mitogens (data not shown)were normal. Post-vaccination antibody titers to Tetanustoxoid and Haemophilus influenzae were normal but he hada low response to Streptococcus pneumoniae after two dosesof 23 polyvalent vaccine (Pneumo 23, Aventis Pasteur MSD)(Table 1).

About 1 year later the patient suffered from threeepisodes of acute cholangitis always associated with high

Figure 3 Molecular analysis of Btk gene in the patient (III-3) and h11 of Btk gene detected by DHPLC and (b) sequencing analysis.

levels of pancreatic enzymes. During these episodes, hisblood cultures tested positive for Klebsiella and Pseudomo-nas Aeruginosa. Abdominal CT confirmed the presence ofsmall hepatic abscesses, the dilatation of the biliary systemand an enlarged pancreas. The magnetic resonance cholan-giopancreatography (MRCP) showed a long stenosis of themain pancreatic duct at the head level, with dilation of themain duct and of its branches above that segment (Fig. 2a).The extrahepatic bile duct was stenotic for 9 cm with dilatedand narrowed segments of the intrahepatic ducts. SinceERCP showed plugging of the pancreatic stent this wasremoved. Thus, after sphincterotomy, a 9 cm long biliarystent was advanced to the hylar region (Fig. 2b). Cytologicalanalysis of the brushing had ruled out a cholangiocarcinoma.Enteral nutrition, antibiotics and substitutive pancreaticenzymes together with steroid therapy were started with apartial clinical response. A few months later, he sufferedfrom a new severe cholangitic episode and the hepaticoje-junostomy was done. To date, the option for the hepatictransplant has been delayed.

Since his family history was strongly suggestive of an X-linked immunodeficiency, a clinical and immunologicalinvestigation was extended to maternal family members.His mother (II-4), two maternal aunts (II-2; II-5) and onefemale cousin (III-1) reported recurrent respiratory infec-tions during childhood and suffered from pulmonary emphy-sema which was very severe in II-5. His brother and othermaternal cousins were healthy apart from seasonal allergicsymptoms. Alpha-1 anti-trypsin status was investigated infemale carriers: alpha-1 anti-trypsin blood levels ranged145–164 mg/dl (normal values 83–199 mg/dl) and isoelectricfocusing showed a normal MM genotype. His mother andmaternal aunts were available to perform immunologicalstudies as reported in Table 1. Immunoglobulin values werenormal, circulating B cells ranged from 4% to 7.9% with anormal distribution of transitional, mature and memory Bcells. Post-vaccination antibody titers were adequate as wellas cellular immunity studies.

Molecular studies

Genomic DNA was analysed for mutations in SAP, CD40L andBtk genes. Direct sequencing of SAP and CD40L genesdetected no mutations. Conversely, Btk gene molecularanalysis, performed by DHPLC and sequencing, revealed a

is mother (II-4). (a) Chromatographic profile of mutation in exon

326 S. Graziani et al.

new missense substitution in exon 11 (c.1078 aNg, seq.X58957) causing threonine change to alanine in position 316of SH2 protein domain (Fig. 3). This alteration, not reportedin literature yet, was excluded in 100 healthy donors.

As reported in the genealogical tree (Fig. 1) a molecularanalysis was performed in his mother, brother, two maternalaunts and all maternal cousins. The same heterozygous Btkmutation was identified in all females, except for onematernal cousin (III-2).

By immunoblotting analysis, the 77 kD Btk protein wasdetected in total lymphoblastoid cell line (LCL) lysates frompatient (III-3) and his mother (II-4). The intensities of theobserved bands were similar to healthy controls. As shown inFig. 4a the presence of Btk protein was compared to twoother XLA patients (XLA1 and XLA2) characterized by a splicemutation in the PH protein domain (c.441+1GNA) and asubstitution in the kinase protein domain (c.1706GNC),respectively. The detection of normal amount of Btk proteinin the patient's LCL was in contrast with the lack of itsexpression in XLA1 or its strong decrease in XLA2. Phosphor-ylation has been shown to be a critical regulatory mechanismcontrolling Btk function. The presence of a Btk phosphory-lated active form was evaluated by FACS analysis. The use ofa PE conjugated anti-phosphoY551-Btk antibody allowed usto test Btk ability to be phosphorylated after treatment ofLCL with hydrogen peroxide. Fig. 4b shows that LCL from thepatient (III-3) preserved the ability to phosphorylate Btkafter hydrogen peroxide stimulation compared to LCL from a

Figure 4 (a) Btk expression and (b) FACS analysis of Btkphosphorylation in EBV-transformed LCL stimulated by H2O2.

healthy control. Conversely, the Btk phosphorylated formwas absent in LCL from XLA1 and XLA2 patients.

Discussion

Herein we report the case of a PID patient with dysgamma-globulinemia, a gradual decrease of peripheral B lympho-cytes and a clinical history of recurrent infectionscomplicated with a severe sclerosing cholangitis and chronicobstructive pancreatitis. Moreover, this patient, who wasinitially classified as having CVID, due to the number of CD19+

B cells, showed a Btk mutation which is the genetic hallmarkof XLA.

In PID patients gastrointestinal and liver complicationsrange from mild biochemical abnormalities, sclerosingcholangitis, portal hypertension and malignancies [21,23].The development of these diseases has been related tochronic infections caused by pathogens unaffected bystandard immunoglobulins, antibiotic and antifungal prophy-laxis. Frequently, but not in our patient, Cryptosporidiumparvum is identified as a cause of sclerosing cholangitis in HIVpatients [24,25] and in those with X-linked Hyper-IgMsyndrome [21,26]. Recently, liver involvement in primaryhypogammaglobulinemia has been reported mainly to consistof nodular regenerative hyperplasia with an autoimmuneorigin not yet elucidated [23]. In our patient, the lack ofpancreatic clinical response to steroid treatment seems tosupport an obstructive pancreatitis rather than an auto-immune one [27].

Btk gene molecular analysis revealed a novel missensesubstitution in exon 11 (c.1078 ANG) causing threoninechange to alanine in position 316 of SH2 protein domain. TheBtk protein consists of five functional domains, the N-terminal pleckstrin homology (PH) domain, the proline-richTec homology (TH) domain, the Src homology 3 (SH3)domain, the Src homology 2 (SH2) domain and the catalytictyrosine kinase domain (SH1) [28]. Mutations in all 5 domainsof the Btk gene were identified in XLA [29]. The SH3 and SH2domains are small binding modules known to affect theassociation of many proteins involved in intracellular signaltransduction. Although the relevance of the PH and SH3domains of Btk and their functional roles in B cell develop-ment have already been demonstrated, little is known aboutits SH2 domain. Btk SH2 domain seems to be essential forphospholipase C-γ phosphorylation and for interaction with Bcell linker protein (BLNK), whose association is required forPLC-γ activation and intracellular calcium mobilization inresponse to BCR engagement [30,31]. While the majority ofBtk mutations results in deficient expression of the Btkprotein, presumably due to a reduction in Btk mRNA or aninstability of the protein product, in our patient Btk SH2mutation did not affect protein expression and kinaseactivity. However, impairment of phosphotyrosine-bindingcapacity cannot be excluded, as previously reported forother Btk SH2 mutations [32,33].

Our Btk mutated patient had normal levels of IgG, normaltiters to Tetanus and Haemophilus Influenzae vaccines, butlow IgA, low IgM and poor response to Streptococcuspneumoniae immunization. These laboratory findings mightbe consistent with a less severe form of XLA, but this is incontrast with his poor clinical outcome. Some genotype/

327Identification of a Btk mutation in a dysgammaglobulinemic patient with reduced B cells

phenotype correlation was suggested in XLA [4,34,35], butontogenetic deleterious changes that can no longer sustainBtk function in B cells, pancreas and lung or the role ofmodifier genes should at least be considered as potentiallyimpacting the severity of the disease. Considerable variationin XLA presentation and disease course was reported, evenwithin the same family [5,12,13,34]. Recently, Perez deDiego et al. described a novel non-pathogenic mutation inSH3 domain of Btk in a male patient who had normal numberof B cells and no signs of immunodeficiency [18]. Further-more, Conley et al. identified a 58 year-old healthy man withan aminoacid substitution in Btk and a reduced number of Bcells [12]. These findings indicate that a mutation in Btk isnot always associated to a clinical disease.

In our family all female members, except for a maternalcousin, carried the same Btk missense mutation and showeda considerable variation in respiratory disease expressivity.To date, carriers have been demonstrated to be healthy andwith normal immunity [5]. Only one Japanese girl wasreported with extremely skewed X-chromosome inactiva-tion, low levels of IgG and absence of peripheral B cells whocarried a Btk mutation [17]. In XLA female carriers apreferential selection of the non-mutated X-chromosomeas the active allele in B cell precursors does occur. Skewedpattern of X-chromosome inactivation was analyzed at theHUMARA locus and PGK-1 gene, but in the tested femalecarriers these loci were not informative [36,37]. Thecomplexity of genetics is cumbersome [38–42]. So far,there is no evidence that carriers of a Btk mutation haveany increased risk for pulmonary disease; thus, any discus-sion may remain speculative.

To conclude, critical gaps need to be filled for thedefinition of this case. Our patient's clinical and laboratoryhistory is not totally inconsistent with an atypical variant ofXLA. However, being this true, more functional studies arerequired to elucidate the effects of this Btkmutation and themechanisms responsible for the clinical and immunologicalphenotype. Conversely, the occurrence that the T316Achange in Btk SH2 domain is a novel non-pathogenic mutationand another gene alteration causes the disease cannot beexcluded.

Another challenge for clinical immunologists is open.

Acknowledgments

The authors would like to thank Dr. A. Durandy (Inserm Unité768, Hopital Necker-Enfants Malades, Paris) for fruitfuldiscussion; Dr. P. Rogliani (Department of Internal Medicine,University of Rome Tor Vergata) for pulmonary investigations;Dr. A. Filareto (Department of Biopathology and DiagnosticImaging, Human Genetic Section, University of Rome TorVergata), Dr. M. L. Romiti (Department of Pediatrics,University of Rome Tor Vergata) for technical assistance.

This study was supported by Programmi di Ricerca diInteresse Nazionale (PRIN)-Ministero dell'Università e dellaRicerca (MIUR).

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