Molecular Analysis of the Pre-BCR Complex in a Large Cohort of Patients Affected by Autosomal-Recessive Agammaglobulinemia

ORIGINAL ARTICLE Molecular analysis of the pre-BCR complex in a large cohort of patients affected by autosomal-recessive agammaglobulinemia

S Ferrari1, R Zuntini1, V Lougaris2, A Soresina2,VSˇourkova´1,12, M Fiorini2, S Martino3, P Rossi4, MC Pietrogrande5, B Martire6, G Spadaro7, F Cardinale8, F Cossu9, P Pierani10, I Quinti11, C Rossi1 and A Plebani2 1Medical Genetics Unit and CRBa, S.Orsola-Malpighi University Hospital, Bologna, Italy; 2Institute of Molecular Medicine ‘Angelo Nocivelli’ and Department of Pediatrics, University of Brescia, Brescia, Italy; 3Department of Pediatrics, University of Torino, Torino, Italy; 4Department of Pediatrics, Ospedale Bambino Gesu`, Roma, Italy; 5Department of Pediatrics, University of Milano, Milano, Italy; 6Department of Pediatrics II, University of Bari, Bari, Italy; 7Department of Immunology, University of Napoli ‘Federico II’, Napoli, Italy; 8Department of Pediatrics I, University of Bari, Bari, Italy; 9Department of Pediatrics, University of Cagliari, Cagliari, Italy; 10Department of Pediatrics, University of Ancona, Ancona, Italy and 11Department of Immunology, University of Roma ‘La Sapienza’, Roma, Italy

Autosomal-recessive agammaglobulinemia is a rare and heterogeneous disorder, characterized by early-onset infections, profound hypogammaglobulinemia of all immunoglobulin isotypes and absence of circulating B lymphocytes. To investigate the molecular basis of the disease, 23 patients with early-onset disease and no mutations in Bruton tyrosine kinase, the gene responsible for X-linked agammaglobulinemia, were selected and analyzed by direct sequencing of candidate genes. Two novel mutations in the m heavy chain (mHC) gene (IGHM) were identified in three patients belonging to two unrelated families. A fourth patient carries a previously described G4A nucleotide substitution at the À1 position of an alternative splice site in IGHM; here, we demonstrate that this mutation is indeed responsible for aberrant splicing. Comparison of bone marrow cytofluorimetric profiles in two patients carrying different mutations in the IGHM gene suggests a genotype–phenotype correlation with the stage at which B-cell development is blocked. Several new single nucleotide polymorphisms (SNPs) both in the mHC and in the l5-like/VpreB-coding genes were identified. Two unrelated patients carry compound heterozygous variations in the VpreB1 gene that may be involved in disease ethiology. Genes and Immunity (2007) 8, 325–333; doi:10.1038/sj.gene.6364391; published online 5 April 2007

Keywords: agammaglobulinemia; pre-BCR; mHC; IGHM mutations; human immunodeficiency

Introduction the assembly of the mature B-cell receptor (BCR) – composed of mHC, light chain, and the Iga/Igb hetero- Early B-cell development is a highly regulated process, dimeric signal-transducing elements – and its expression taking place in the bone marrow. The transition from the on the surface of newly formed immature B cells.3 The pro-B-cell to the pre-B-cell stage is defined by sequential correct expression and signaling activity of the BCR are immunoglobulin gene rearrangements and surface ex- required for normal B-cell development. Accordingly, pression of the pre-B-cell receptor (pre-BCR) complex mutations in the genes required for BCR assembly or for containing the m heavy chain (mHC), Iga/Igb hetero- its signaling cascade, severely impair B-cell development 1 dimers and the surrogate light chains (SLC) (VpreB/l5). in the bone marrow, causing X-linked and autosomal- Various studies have investigated the different steps and recessive forms of agammaglobulinemia in humans, the transcription factors involved in this phase of B-cell characterized by low/absent serum immunoglobulin 2 development. The checkpoint at the pre-BCR level is levels and absence of circulating B cells.4 required for the following VJL rearrangement that allows Mutations in the gene coding for the Bruton tyrosine kinase (Btk), a cytoplasmic protein that mediates BCR Correspondence: Dr S Ferrari, Laboratorio di Genetica Medica, signaling, were found to be responsible for X-linked Policlinico S.Orsola-Malpighi – Pad.11, via Massarenti 9, 40138 agammaglobulinemia (XLA).5,6 In XLA, B-cell develop- Bologna, Italy. ment in the bone marrow is blocked at the pro-B- to pre- E-mail: [email protected] B-cell stage.7,8 However, even though XLA accounts for 12Current address: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Praha. 85–90% of all cases of agammablobulinemia, a small Received 6 December 2006; revised and accepted 23 February 2007; group of patients have a block of B-cell development published online 5 April 2007 without evidence of BTK mutations. The family trees of Pre-BCR mutations in autosomal-recessive agammaglobulinemia S Ferrari et al 326 these patients are suggestive of an autosomal-recessive Table 1 Relevant features of the 23 patients included in this study form of agammaglobulinemia. The crucial role of BCR signaling for B-cell development and the phenotype of Patient Sex Age at IgG IgA IgM %B a b b b animal models point to the genes encoding for compo- diagnosis (mg/dl ) (mg/dl ) (mg/dl ) cells nents of the pre-BCR as promising candidate genes for autosomal-recessive forms of agammaglobulinemia. In P1 M 8 months 17 o6.5 1 o1 m P2 F 24 months 15 23 17 o1 fact, a mouse knockout model demonstrates that HC is P3 M 5 years 257 10 31 1–2 essential for B-cell development; lack of mHC causes P4 M 23 years 279 o6.5 12 o1 a specific block at the pro-B- to pre-B-cell stage and a P5 M 14 months 208 8 35 o1 severe reduction of B-cell number in the periphery.9 Four P6 F 10 years 297 13 14 1–2 point mutations, a 2-bp deletion and few large deletions P7 M 9 months 100 7 3 o1 of the human mHC gene (IGHM) have been identified P8 M 9 years 252 12 29 o1 previously by genetic analysis in patients with auto- P9 F 5 months 110 o6.5 11 o1 10,11 P10 M 24 months 150 20 38 1 somal-recessive forms of agammaglobulinemia. An- P11 F 9 months 18 o6.5 3 1–2 other member of the pre-BCR complex – the l5 gene – P12 M 6 months 179 8 11 o1 was also knocked out in the mouse, resulting in a leaky P13 M 5 years 20 o6.5 10 2 phenotype with a specific block at the pre-B-cell stage of P14 M 12 months 11 o6.5 5 1–2 differentiation; however, 4-months-old mice retain 20% P15 F 20 months o5 o6.5 o15 o1 of peripheral B cells and show a normal capability to P16 F 6 years 72 o6.5 4 1–2 P17 F 9 years ND o6.5 9 o1 mount an antibody response to both T-dependent and P18 F 31 years 41 o6.5 10 o1 12 T-independent antigens. In humans, a single case of P19 F 9 years 85 10 28 o1 compound heterozygote mutations in the l5/14.1 gene P20 M 4 months 42 o6.5 61 1–2 was associated with classical autosomal-recessive agam- P21 M 1 month ND o6.5 o18 o1 maglobulinemia,13 emphasizing the fact that mutations P22 F 16 months ND o6.5 o18 o1 in the same gene often affect B-cell development and P23 M 29 years 255 o6.5 25 o1 functionality to different extents in humans and mice. a Finally, knockout models of the last two components of Age at the time of the first immunoglobulin dosage. b the pre-BCR complex, CD79a and CD79b,14,15 provide Immunoglobulin reference ranges vary according to the age. evidence for their requirement in B-cell differentiation in Values used in our centers, 4–12 months: IgG 220–920, IgA 10–85, the bone marrow, in particular in the V-DJ rearrangement IgM 28–200; 13–36 months: IgG 365–1710, IgA 17–180, IgM 48–340; process. Recently, two patients were found to have 3–8 years: IgG 530–1960, IgA 37–315, IgM 49–290; 9–16 years: IgG 640–1920, IgA 60–300, IgM 59–200; 416 years: IgG 690–1500, IgA homozygous defects in Iga:16,17 comparison with mHC- 85–410, IgM 40–240. ND, not available before immunoglobulin deficient patients reveals, in both cases, a block at infusion. the early stages of B-cell development and similarly impaired V-DJ rearrangements.16 In this study, 23 Italian patients diagnosed with agammaglobulinemia (11 females and 12 males for whom mutations in BTK were excluded) were analyzed mia subregistry. The remaining seven male patients were for mutations in each of the genes encoding for the pre- excluded form this study because either deceased or BCR proteins. We identified one previously known untraceable. Patients previously subjected to similar mutation and two novel mutations in the coding region genetic screenings in other laboratories were excluded. of the IGHM gene and several new genetic polymor- Table 1 summarizes the main clinical features of these phisms (single nucleotide polymorphisms (SNPs)) in the patients, showing absent or nearly absent counts of IGHM and VpreB/l5 genes. circulating B cells. The genetic analysis was performed by polymerase chain reaction (PCR) amplification and direct DNA Results sequencing of all exons from the genes encoding the five proteins of the pre-BCR: six exons of mHC gene Patient selection (IGHM), three exons of l5-like gene (IGLL1), two exons of A total of 23 patients, from the Italian Association of VpreB gene (VpreB1), five exons of Iga gene (mb-1) and Pediatric Hematology and Oncology (AIEOP)-Italian six exons of Igb gene (B29). Network of Primary Immunodeficiencies (IPINET) database, were selected for this study. They belong to 22 The mHC gene (IGHM) is mutated in agammaglobulinemic unrelated families where they represent sporadic cases, patients with the exception of patients P21 and P22 that are Four patients (P15, P17, P21 and P22) from three siblings. History of consanguinity has not been reported unrelated families were found to carry homozygous in any of the patients. mutations in the mHC gene (IGHM). In all cases, both Before this study, sequence analysis of the BTK gene healthy parents were heterozygous carriers of the was performed on 123 out of 128 male patients present in mutation, although none of the families had a known the XLA registry: BTK mutations were found in 103 history of consanguinity. Figure 1 summarizes the results patients (comprising all familiar cases and 86 sporadic of the IGHM screening: two novel mutations were cases). identified in three patients belonging to two families. We analyzed 13 out of 20 male patients with no First, P17 is homozygous for a complex 5-bp deletion and mutations in BTK gene and 10 female patients already 3-bp insertion at codon 377 in exon 4, leading to a assigned to the autosomal-recessive agammaglobuline- frameshift and a premature stop codon after seven amino

Genes and Immunity Pre-BCR mutations in autosomal-recessive agammaglobulinemia S Ferrari et al 327 a b c I.1 I.2 I.1 I.2 I.1 I.2

II.1 (P15) II.1 (P17) II.1 (P21) II.2 (P22)

* * HD HD HD CCA CCGGTAAA GTGACCA GGTC A C A G A CCT

II.1 (P15) II.1 (P17) II.1, II.2 (P21, P22) CCA CCA G TAAA GCGGA GGTCCA G A CCT

I.1 I.1 I.1 CCA CCNG TAAA GTATGYGRMSRGCSC GGTC M M RRM C T

I.2 I.2 I.2 CCA CCNGTAAA GTATGYGRMSRG CSC GGTC M M RRM C T

1956G >A 1789_1793 delTGACCinsCGG 1217delA G433S V377AfsX7 T246Q fsX3 Figure 1 Mutation analysis of IGHM in patients with autosomal-recessive agammaglobulinemia. (a) Electropherograms from a portion of exon 4 showing the 1956 G4A mutation at the base corresponding to position –1 of the alternative splice site in P15 compared to the wild- type (wt) sequence from a healthy donor (HD) and to the sequence obtained from heterozygous parents (I.1 and I.2). (b) Electropherograms from a portion of exon 4 showing the 1789–1793 delTGACC insCGG mutation in P17 compared to the wt sequence (HD) and to the sequence obtained from heterozygous parents (I.1 and I.2). (c) Electropherograms from a portion of exon 3 showing the 1217 delA mutation in P21 and P22 compared to the wt sequence (HD) and to the sequence obtained from heterozygous parents (I.1 and I.2).

acids. Second, P21 and P22 are siblings carrying a tion block (determined by cytofluorimetric analysis of homozygous 1-bp deletion in exon 3, an ‘A’ deletion at the bone marrow, data not shown) due to an unknown codon 246. This mutation causes frameshift and pre- genetic defect, and a healthy donor. mature termination after three amino acids. Reverse transcription was performed on total RNA In these three patients, both the secreted and mem- extracted from bone marrow cells. Semi-quantitative brane forms of the mHC protein are predicted to be PCR was performed using primers specific for the missing. secreted and the membrane form respectively, as shown The G4A single base transition at codon 433 within schematically in Figure 2a. The amount of mRNA in each exon 4 found in P15 is a previously known mutation sample was normalized by reverse transcriptase-PCR described by Yel et al.10 and Granados et al.11 It affects (RT-PCR) of b-actin mRNA (Figure 2b), and confirmed the À1 position of the alternative splice site required for by precise quantitation of the amount of b-actin mRNA the production of the membrane-bound isoform of the using Real-time PCR (data not shown). protein and, in addition, it causes a glycine to serine Figure 2 shows that the mRNA corresponding to the substitution in the secreted isoform. It has been assumed secreted isoform of mHC is present in all the patients that the mutation impairs the alternative splicing analyzed, even though its amount is generally lower required to encode the membrane isoform of the mHC, than that in healthy controls. The different levels of mHC but its actual effect on RNA splicing has not yet been expression may reflect the smaller number of CD19- demonstrated experimentally. positive B-cell precursors in the bone marrow of these To establish the effect of the G4A (Gly433Ser) subjects; a subsequent assessment of the expression mutation, we analyzed the expression of the alternatively levels of the CD19 marker by Real-time PCR proved spliced IGHM mRNA species in bone marrow cells that this is indeed the case (Figure 2c). obtained from patients P15 (carrying the Gly433Ser The mRNA corresponding to the membrane isoform is mutation), P17 carrying the new IGHM mutation present in samples P9 and P17. In sample P15, on the described above, P9 who shows a pro-B-cell differentia- other hand, the only product amplified by the specific

Genes and Immunity Pre-BCR mutations in autosomal-recessive agammaglobulinemia S Ferrari et al 328 a Alternative splice site Cryptic donor site in P15 ExM1 Ex 3 Ex 4

wt secreted µHC SF SR wt membrane µHC MF MR P15 mutant MF MR

bcsecreted µHC membrane µHC wt P9 P15 P17 wt P9 P15 P17 6 CD19 expression (%) 5

500 3 400 bp 250 in P15 2

β-actin 0 mRNA P9 P15 P17 Figure 2 Altered spicing due to the 1956 G 4A substitution at codon 433. (a) The schematic diagram shows part of the IGHM gene (exon 3, exon 4 and exon M1) with the positions of the canonical donor site for generation of the membrane-specific mRNA from the wt gene (alternative splice site) and the donor site identified in the mutated IGHM (‘cryptic donor site’, activated in patient P15 carrying the 1956 G4A mutation). The different mRNA species produced after spicing of the wt and P15 transcripts and the primers used for amplification of the secreted (SF and SR) and membrane (MF and MR) isoforms are also outlined. (b) Semi-quantitative RT-PCR performed on total RNA extracted from bone marrow cells of three patients (P9, P15 and P17) and a healthy donor (wt). Primers specific for the secreted (left part of the picture) and the membrane (right part of the picture) form were used in parallel on the same samples. b-Actin mRNA was measured as reference gene. The only product amplified in P15 by the membrane-specific couple of primers MF/MR is about 400 bp long (indicated by the arrow) and derives from the usage of the cryptic donor site within intron 4, as outlined in (a). (c) Real-time PCR analysis performed on total RNA extracted from bone marrow cells. All cDNA were assayed in triplicate. The expression levels of CD19 were normalized using b-actin as endogenous control and compared to the expression of CD19 in the healthy donors. The CD19 expression levels in patients are reported as the percentage of expression levels in healthy controls (set at 100%).

primers is about 150 bp larger than the expected 250-bp- calculated in our laboratory from a control group of long fragment. Direct sequence analysis of the mutant healthy individuals (indicated with a in Table 2). Two of cDNA product from P15 shows an insertion of 137 the novel SNPs (variants 8 and 9 in Table 2) are also nucleotides (from nucleotide 1956 to 2092 of the reference found in control individuals. This is generally true for all sequence X57331). The insertion seen in the mutant is the SNPs observed in the patients’ population, where due to highly efficient usage of a cryptic GT-donor site their allelic frequencies do not significantly differ from within intron 4 that causes the inclusion in the mRNA of the expected frequencies calculated in normal indivi- the 50 portion of intron 4. The novel transcript contains duals. Two notable exceptions are variant 1 that is the sequence specific for the secreted isoform of mHC and significantly more frequent in the patients’ population an extra portion of intron 4 (Figure 2). Thus, the net effect (Po0.01), and variant 7 that was not found in 184 on mHC of the mutation at the alternative splice site of chromosomes from healthy Italian donors. intron 4 in P15, is that only the secreted isoform with a It is noteworthy that patient P5, in addition to variants Gly433Ser missense substitution is present in the patient, 8 and 9, carries eight rare SNPs simultaneously. Two of while synthesis of the membrane isoform is completely them, S190G and V214G, are present in homozygous abolished. state. The same 10 SNPs are also present in the patients’ mother, with the main difference that she is heterozygous Single nucleotide polymorphisms of IGHM gene for both S190G and V214G. Due to the lack of a bone Previous studies have established that the IGHM locus is marrow biopsy from this patient, we could not assess if highly polymorphic.11 In our study, we identified 16 this unusual SNPs pattern has any consequence on the intragenic SNPs, including three novel SNPs and 13 expression of the mHC. SNPs described already in the NCBI SNPs database and/ or in Granados et al.11 (Table 2). These nucleotide l5 (IGLL1) and VpreB (VpreB1) genes variations occurred either outside or inside the coding The four patients (P15, P17, P21 and P22) with mutations region; the latter contains both silent and missense in the IGHM gene were excluded from further screening variants. Unless stated otherwise, all SNPs were hetero- of the other pre-BCR genes, while the remaining 19 zygous. The frequency of the IGHM gene polymorph- patients were analyzed for the presence of mutations in isms found in our group of patients were compared to the l5- and VpreB-coding genes (IGLL1 and VpreB1). the caucasian population frequencies published in the The so-called SLCs l5-like and VpreB are key NCBI database, when available, or to the frequencies components of the pre-BCR protein complex: the human

Genes and Immunity Pre-BCR mutations in autosomal-recessive agammaglobulinemia S Ferrari et al 329 Table 2 Single nucleotide polymorphisms (SNPs) in mHC

Site in the gene Change Effect Frequency (patients) Frequency (NCBI) Patient ID

a 1_Ex 1 codon 20 ACG4ACA fA ¼ 3/46 2/192 P7, P11, P16 a 2_Ex 1 codon 76 GAC4GAG D76E fG ¼ 1/46 3/192 P5

3_Ex 2 codon 106 GCT4GCC fC ¼ 21/46 0.48

4_Ex 2 codon 190 AGC4GGC S190G fG ¼ 3/46 0.12 P5 (Ho), P10

5_Ex 2 codon 214 GTC4GGC V214G fG ¼ 2/46 NG P5 (Ho)

6_Ex 3 codon 226 ATC4ATT fT ¼ 1/46 NG P5 a 7_Ex 3 codon 280 ACT4ACC fC ¼ 1/46 0/184 P5 b a 8_Int 1 +22 C4T fT ¼ 2/46 3/192 P5, P11 b a 9_Int 2 +214 C4T fT ¼ 1/46 1/178 P5

10_Int 5 +30 T4C fC ¼ 1/46 0.072 P5

11_Int 5 +40 G4A fA ¼ 1/46 0.072 P5 b 12_Int 5 +62 G4T fG ¼ 1/46 NG P11

13_Int 5 +138 G4A fA ¼ 23/46 NG 0 14_5 int –52 C4T fT ¼ 1/46 NG P5 0 15_5 int –66 C4T fT ¼ 16/44 NG 0 16_3 UTR +50 T4A fA ¼ 22/46 0.37 aIndicates the frequencies calculated in our laboratory from healthy subjects. bIndicates the novel single nucleotide polymorphisms. NG, not genotyped. SNPs found in normal controls by Granados et al.11

Table 3 Single nucleotide polymorphisms (SNPs) in l5 and VpreB

Site in the gene Change Effect Frequency (patients) Frequency (NCBI) Patient ID

a l5 Exon 1 codon 33 GTC4ATG V33M fA ¼ 2/38 3/188 P12, P23 a Exon 2 codon 92 AAC4AAT fT ¼ 1/38 1/186 P23 a Exon 2 codon 94 ACG4AAG T95L fT ¼ 1/38 1/186 P23 a Exon 3 codone148 ACC4ATC T148I fT ¼ 2/38 14/186 P9, P10 a Exon 3 codon 159 GGC4AGC G159S fA ¼ 2/38 4/186 P9, P10 a Exon 3 codon 162 ATG4AAG M162K fA ¼ 1/38 5/186 P3 a Exon 3 codon 183 CCC4CCT fT ¼ 1/38 0/186 P23 a Exon 3 codon 189 CGC4CAC R189H fA ¼ 1/38 1/186 P18 0 VpreB 5 –97 G4A fA ¼ 1/38 0.025 P14 0 a 5 –62 G4A fA ¼ 2/38 0/192 P1, P18

Exon 2 codon 76 GAC4AAC D76N fA ¼ 1/38 0.027 P1

Exon 2 codon 121 AGC4AGT fT ¼ 6/38 0.15

Exon 2 codon 122 TCG4TTG S122L fT ¼ 1/38 0.037 P14

Exon 2 codon 132 GAG4AAG E132K fA ¼ 6/38 0.15 0 3 UTR +15 A4G fG ¼ 7/38 0.25 0 3 UTR +59 C4T fT ¼ 5/38 0.16 aIndicates the frequencies of the newly identified variants, calculated in our laboratory from healthy subjects. l5-like coding gene was mutated in one instance of Iga and Igb genes autosomal-recessive agammaglobulinemia.13 Further- Sequence analysis of the Iga-coding gene (mb-1) did not more, mice lacking the VpreB proteins show a block of show the presence of nucleotide variations in any of the the B-cell differentiation at the pre-B-cell stage.18,19 patients. Table 3 summarizes the SNPs identified in our Analysis of the Igb gene (B29) showed that all patients patients’ population. We found several new variants in are carriers, in one or both alleles, of the haplotype the VpreB1 and IGLL1 genes that are also present in represented by the two following SNPs: a T4C silent healthy subjects with frequencies not significantly change at codon 122 in exon 3, and a 30-UTR þ 89 T4C different form that found in patients. transition. Patients P1 and P14 are compound heterozygous for 50 The allelic frequencies of these polymorphisms in the À62/D76N and 50 À97/S122L mutations in VpreB1;in patients’ population were similar to those reported in the addition, patient P23 carries the V33M variation on one NCBI SNPs database. allele of IGLL1 gene and the three polimorphisms at codon 92 and 94 in exon 2 and at codon 183 in exon 3 on Cytofluorimetric analysis of bone marrow B-cell precursors the other allele. Since the frequency of all these alleles in The prevailing view of B-cell lineage commitment the general population is rather low, it cannot be ruled identifies three phenotypes based on the expression of out that these variations contribute to the disease cell-surface markers: cells go through a CD34 þ / ethiology. CD19À/CD10 þ early pro-B-cell stage to become

Genes and Immunity Pre-BCR mutations in autosomal-recessive agammaglobulinemia S Ferrari et al 330 a HD XLA µ HC-/- (P15) µ HC-/- (P17)

2.5 2 2.1 5 0.18 1.15 2 0.11 CD34

23 0.5 0.26 0.08

CD19 b 2.2 2.3 1.8 5.3 0.14 1.19 1.75 0.35 CD34

10 0.2 0.16 0.1

CD10 c 9.5 12.5 0.5 5 0.18 1.13 0.05 0.13 CD19

1.4 0.5 0.22 0.3

CD10 Figure 3 Cytofluorimetric analysis of B-cell progenitors. Bone marrow mononuclear cells from a control (HD), a XLA patient and two mHC- deficient patients (P15 and P17) were stained with antibodies to CD34, CD19 and CD10 and analyzed by FACSCalibur Flow Cytometer. The same number of gated lymphoid cells have been collected for each sample. The dot plot distributions of CD34 versus CD19 (a), CD34 versus CD10 (b) and CD19 versus CD10 (c) are shown. The B-cell precursors in the mHC-deficient patient P15, as well as in the XLA individual used as control, show a typical late pro-B cells profile with the majority of CD34 þ cells being CD19 þ /CD10 þ . The immunological profile of the pro-B cell precursors in patient P17 is different, showing an earlier pro-B cells profile (CD34 þ /CD19À/CD10 þ /À).

CD34 þ /CD19 þ /CD10 þ late pro-B cells, followed by impaired than in P15 (Figure 2c). In both mHC-deficient the CD34À/CD19 þ /CD10 þ pre-B-cell stage of devel- patients, as well as in the XLA patient, the CD34 þ / opment. The use of surface markers to define the lineage CD19 þ pro-B-cell precursors are also CD10 þ . The affiliation and the differentiation stage of CD34 þ evidence of different pro-B-cell profiles in patients P15 progenitors provides a preliminary characterization of (CD34 þ /CD19 þ /CD10 þ typical of late pro-B cells) eventual blocks of B-cell development. and P17 (CD34 þ /CD19À/CD10 þ /À typical of earlier We compared the bone marrow of an XLA patient with maturation stages) suggests a link between the pheno- the bone marrow obtained from two patients carrying type of B-cell precursors and the severity of the mutation mutations in the IGHM gene (P15 and P17). in the IGHM gene (Figure 3b and c). Three-color staining of gated lymphoid cells with antibodies to CD34, CD19 and CD10 showed that in the bone marrow of the mHC-deficient patient P15, as well as Discussion in the bone marrow of the XLA subject, the majority of CD34 þ cells are CD19 positive, indicating that in these This study reports the comprehensive genetic analysis of patients the late pro-B-cell precursors are present and the pre-BCR in 23 Italian patients affected by autosomal- accumulate (Figure 3a). In contrast, the immunological recessive agammaglobulinemia. This mode of inheri- profile of the mHC-deficient patient P17 shows that most tance is rare compared to the more frequent X-linked CD34 þ cells are CD19 negative, while only few pro-B- agammaglobulinemia; in fact, our patients constitute the cell precursors are present (0.11%). Accordingly, Real- second largest reported population after the collection of time PCR analysis performed on mRNA extracted from patients described in Granados et al.11 total bone marrow cells, confirmed that expression of Among the five genes screened by complete sequence CD19 in the bone marrow of P17 is more severely analysis of exons and exon/intron boundaries, two novel

Genes and Immunity Pre-BCR mutations in autosomal-recessive agammaglobulinemia S Ferrari et al 331 mutations in the mHC gene were found in three patients described. These elements can act both by stimulating belonging to two different families. P17 is homozygous (enhancers) or by repressing (silencers) splicing. Exonic for a complex 5-bp deletion À3-bp insertion in exon 4. splicing enhancers (ESEs), in particular, are crucial for P21 and P22 are siblings with a homozygous 1-bp efficient recognition of intron–exon junctions. The ESE deletion in exon 3. Both mutations cause frameshifts Finder algorithm (http://rulai.cshl.edu/tools/ESE/) and premature termination and, as a consequence, both predicts a difference in the pattern of ESEs distribution the secreted and the membrane isoforms of mHC are in exon 1 of the mHC gene depending on the presence of predicted to be absent. The healthy parents of the the polymorphism at codon 20 (variant 1). Notably, patients in these two families are not aware of any according to the SNPs frequencies calculated in our degree of consanguinity; in each case, the mutated alleles laboratory, this variant is significantly more frequent in show an identical haplotype, indicating that a founder the patients’ population than in healthy individuals. effect may be involved. Three patients (P7, P11 and P16) carry this SNP on one A third homozygous mutation in the mHC gene, allele and P11 also carries variant 8. Owing to the corresponding to a single base pair substitution at the – unavailability of bone marrow samples from P11, we do 1 position of the alternative splice site in exon 4, was not know if the presence of these two SNPs influences detected in patient P15. This mutation is recurrent in the efficiency of mRNA splicing. patients from different countries and immunoglobulin Finally, the mutation frequency of mHC, as well as of haplotypes.11 In the present study, we have directly the other genes of the pre-BCR, may be underestimated analyzed how this mutation interferes with the splicing owing to our sequencing strategy that addresses exons process, showing that usage of a cryptic GT-donor site and adjacent intronic and UTR regions but would not within intron 4 of the mutant mHC gene generates a detect additional variations in the promoter and/or novel transcript bearing an insertion of 137 nucleotides introns of the gene. derived from intronic sequences. In principle, if the Several SNPs have also been detected in the VpreB1 cryptic site had occurred upstream of the secreted gene. Two of them (50 À62 and 50 À97) map in the isoform STOP codon, it would generate an aberrant promoter region of the gene, but fall outside the known mHC isoform (fusing the secreted and membrane isoform transcription factor-binding sites.21 SNP (50 À97) is sequences). Our data provide formal proof that the present in P14 together with the S122L amino-acid G4A mutation at codon 433 completely abolishes change that is predicted to have an impact on the protein the synthesis of the membrane isoform of the mHC, as structure. SNP (50 À62) is present in two patients (P1 and further confirmed by cytofluorimetric analysis of the P18). P1 also carries an amino acidic change in exon 1 bone marrow from P15, where mHC is absent from (D76N), which should be neutral with respect to the the surface of B-cell precursors (data not shown), and protein structure as determined by the PolyPhen soft- define the molecular mechanism of this disease causing ware. Further work will be required to understand if any mutation. of these SNPs is actually a disease-causing mutation. The frequency of mHC mutations in our cohort (4/23) Once again, the lack of bone marrow biopsies, as in the is similar to that reported by Granados et al.11 (20–30%), case of P5 and P11, hampers the analysis of mRNAs and taking into account that two families registered in the proteins. Italian database were excluded from this study because Assembly of a functional pre-BCR is considered a they had been described previously as carriers of a major checkpoint for B-cell differentiation taking place in Gly433Ser mutation (F11 and F12 in Granados et al.11). the bone marrow. An early block of this process occurs in It should be noted that a source of uncertainty and patients with mutations in BTK, mHC, Iga and l5-like. possibly underestimation of the mutation rate arises in To explore if different mutations in mHC could give rare cases, such as in patient P5, where the simultaneous rise to subtle differences at the level of the B-cell presence of 10 rare SNPs on one allele and two rare SNPs development in the bone marrow, we analyzed two on the other, may alter the expression of functional mHC patients (P15 and P17). Specifically, we asked if the cell at the level of mRNA or protein. Unfortunately the phenotype from complete knock out of mHC (P17) differs compound effect of polymorphic variants is difficult to from the one where the mHC secreted isoform, bearing a assess, especially when a bone marrow biopsy is not S4G missense mutation, is still present (P15). We available, such as in this case. In total, 16 SNPs were performed a side-by-side analysis of B-cell populations identified within the mHC gene, three of which are novel in P15 and P17, compared to an XLA patient. We (indicated with a in Table 2). Three SNPs cause an amino confirmed that the pre-B-cell subset (CD34-CD19 þ )is acid substitution (D76E, S190G and V214G) within the nearly absent in all patients. This finding was expected loops linking the b strands of the immunoglobulin and further reinforces the notion that B-cell development domain. It is unlikely that any of these substitutions does not progress beyond the pre-BCR checkpoint in has deleterious consequences on the protein structure both forms of agammaglobulinemia. Interestingly, the since the loops are poorly conserved either in humans or precursor profiles differ between P15 and P17, with an mice. As mentioned above, the outcome of the simulta- increased number of CD34 þ CD19ÀCD10 þ /À precur- neous occurrence of two or three of these amino-acid sor cells in P17, suggesting that a block at an earlier pro- changes – patient P5 is heterozygous for D76E and B-cell stage may occur in this patient. It is possible that homozygous for S190G and V214G – is harder to predict. the different phenotype observed in P15 and P17 may be In addition, it is well known that variations within due to factors unrelated to the IGHM mutation, such as exons, either missense or silent, may interfere with the genetic background, age and immunological status of mRNA processing.20 Several examples of exonic cis- the patients that are hard to estimate due to the rarity of elements important for correct splice-site identification patients and bone marrow biopsies. Nevertheless, and distinct from the classical splicing signals have been further functional studies of the mutated proteins may

Genes and Immunity Pre-BCR mutations in autosomal-recessive agammaglobulinemia S Ferrari et al 332 confirm the specificity of this developmental block and (Assay_on_Demand Hs00221068_m1) and standard soft- elucidate the underlying molecular mechanism. ware for quantification as recommended by the manu- After completion of this screen focused on the pre-BCR facturer. CD19 gene expression levels were normalized to genes, we also analyzed the patients’ DNA for mutations the expression level of b-actin mRNA determined for in the BNLK gene, based on the finding that a defect in each sample. Comparative quantitation between patients BNLK was identified in a single patient with agamma- and healthy donor was carried out using the 2ÀDDCt globulinemia.22 BLNK is normal in all patients of our method (ABI User Bulletin #2). The relative values of cohort, therefore, the causative genetic defect must CD19 were expressed as the percentage of the expression involve novel genes in about 80% of the recessive levels of CD19 in patients with respect to the expression agammaglobulinemia cases. level of CD19 in the healthy donor, set at 100%.

Cytofluorimetric analysis Materials and methods Bone marrow mononuclear cells isolated by Ficoll– Hypaque gradient centrifugation were stained with the Registry of patients with agammaglobulinemia following conjugated anti-human monoclonal antibo- The AIEOP-IPINET, was established in 1999 with the aim dies: FITC-labelled anti-human CD10, PE-labelled anti- to provide all patients on the national territory with human CD34 and PerCP-labelled anti-human CD19 clinical assistance based on shared updated diagnostic (Becton–Dickinson). Cells were resuspended in phos- and therapeutical recommendations. Furthermore, the phate buffer saline (PBS), 0.1% bovine serum albumin AIEOP maintains an electronic database (registry) of all (BSA) at the concentration of 5 Â 105–5 Â 106 cells/ml and patients, organized according to their clinical features incubated at 41C for 30 min with 10 ml of each conjugated and genetic diagnosis. In the case of agammaglobuline- antibody. Three-color stained cells were washed twice mia, male patients are initially assigned to the XLA with PBS/BSA and analyzed on a FACSCalibur flow registry. Female patients and the male patients from the cytometer (Becton–Dickinson). XLA registry that do not have mutations in the BTK gene are grouped as putative autosomal-recessive agammaglobulinemia cases. Acknowledgements Mutational analysis We acknowledge the patients and families for their Genomic DNA was extracted from peripheral blood generous cooperation and the following centers partici- leukocytes using standard techniques. Complete se- pating to the AIEOP Network for Primary Immuno- quence analysis of the five genes coding for the pre- deficiencies: Ancona (GV Coppa, P Pierani), Bari (D BCR was performed by PCR amplification of exons and DeMattia, B Martire), Bari (L Armenio, F Cardinale), exon/intron junctions (FastStart Taq DNA Polymerase, Bari (F Dammacco, M Prete), Bologna (G Paolucci, M Roche Diagnostics, GmbH, Mannheim, Germany) under Masi, A Miniaci), Bologna Centro Operativo AIEOP (A standard conditions. PCR products were purified (PCR Pession, R Rondelli), Bologna (G Ambrosioni, P Alvisi), Cleanup Plates, Millipore, Carrigtwohill, Co. Cork, Ire- Brescia (A Plebani, LD Notarangelo, A Soresina), Cagliari land) and sequenced on both strands with Big Dye (Cao, F Cossu), Cagliari (S Del Giacco, P Manconi), Terminator v1.1 Cycle Sequencing Kit (Applied Biosys- Campobasso (I Evangelista), Catanzaro (S Magro, S tems, Warrington, Cheshire, UK) and run on an ABI 3730 Morgione), Catanzaro (P Strisciuglio E Anastasio), Genetic Analyzer (Applied Biosystems). Each new Catania (G Schilliro`, A Sciotto), Chieti (R Paganelli), polymorphism was confirmed by sequencing of at least Como (M Sticca), Cosenza (M Candusso, L Carpino), 89 healthy unrelated controls. The sequence information Firenze (G Bernini, C Azzari), Genova (E Castagnola, for primers used in this study is available upon request. M Gattorno), Mantova (G Pastorelli, S Fasoli), Messina The reference sequences used in this study are X57331 (C Sampietro), Milano (MC Pietrogrande, RM Delle (IGHM gene), M27749 (IGLL1), S74019 (VpreB1), M80462 Piane, C Panisi) Milano (G Cambiaghi), Milano (M (mb-1) and M89957 (B29 gene). Pietrogrande), Milano (MG Roncarolo, A Aiuti), Monza (G Masera, A Biondi, A Sala), Napoli (C Pignata), Napoli Transcripts analysis (V Poggi, G Menna), Napoli (R Di Nardo), Napoli (A Total RNA was isolated from bone marrow mononuclear D’Apuzzo), Napoli (A Pelliccia), Napoli (A Correra), cells, using RNeasy Mini Kit (Qiagen, GmbH, Hilden, Napoli (G Marone, G Spadaro), Padova (L Zanesco, Germany). A total of 1–2 mg RNA was reverse-tran- G Basso, MC Putti), Padova (G Semenzato, C Agostini), scribed into cDNA using Oligo-dT primers and Super- Palermo (GM Amato), Palermo (M Arico`, A Trizzino), Script II (Invitrogen, Carlsbad, CA, USA). PCR for the Parma (G Izzi, P Bertolini), Pavia (F Locatelli, M Zecca), secreted isoform of mHC was performed with primers Pavia (G Rondini, GL Marseglia, R Maccario, G Bossi), specific forward (SF) (50-GCCACTGAAGCAGAC Pesaro (L Felici), Pisa (P Macchia, R Consolini, C Favre), CATCT-30) and specific reverse (SR) (50-ATGACCAGG Rimini (V Vecchi, P Sacchini, G Rinaldi), Roma GACACGTTGTA-30). The membrane mHC isoform was (AG Ugazio, P Rossi, S Livadiotti) Roma (A Stabile), amplified by MF 50-GTGCAGTGGATGCAGAGG-30 and Roma (M Duse), Roma (I Quinti), Roma (V Moschese), MR 50-TCTCAAAGCCCTCCTCGT-30. PCR products Siena (G Morgese, Acquaviva), Treviso (G De Zan), were analyzed by standard gel electrophoresis. b-actin Trieste (P Tamaro, M Rabusin), Torino (PA Tovo, was used as internal control. S Martino), Varese (L Nespoli, M Marinoni), Venezia Real-time PCR analysis was performed with iCycler iQ (A Porcellini), Verona (GA Cazzola). detection system (Biorad, Richmond, CA, USA) using We thank Monica Franzoni for her helpful support in the TaqManR Gene Expression assay for CD19 mRNA the bone marrow analysis.

Genes and Immunity Pre-BCR mutations in autosomal-recessive agammaglobulinemia S Ferrari et al 333 This work was supported by grants from Associazione patients with agammaglobulinemia. N Engl J Med 1996; 335: Immunodeficienze Primitive (AIP), Fondazione Golgi, 1486–1493. and Centro Immunodeficienze M. Di Martino-Brescia to 11 Lopez Granados E, Porpiglia AS, Hogan MB, Matamoros N, AP, and by Fondazione Telethon to SF. Krasovec S, Pignata C et al. Clinical and molecular analysis of patients with defects in micro heavy chain gene. J Clin Invest 2002; 110: 1029–1035. 12 Kitamura D, Kudo A, Schaal S, Muller W, Melchers F, References Rajewsky K. A critical role of lambda 5 protein in B cell development. Cell 1992; 69: 823–831. 1 Karasuyama H, Nakamura T, Nagata K, Kuramochi T, 13 Minegishi Y, Coustan-Smith E, Wang YH, Cooper MD, Kitamura F, Kuida K. The roles of preB cell receptor in early Campana D, Conley ME. Mutations in the human lambda5/ B cell development and its signal transduction. Immunol Cell 14.1 gene result in B cell deficiency and agammaglobulinemia. Biol 1997; 75: 209–216. J Exp Med 1998; 187: 71–77. 2 Conley ME, Rohrer J, Rapalus L, Boylin EC, Minegishi Y. 14 Torres RM, Flaswinkel H, Reth M, Rajewsky K. Aberrant B cell Defects in early B-cell development: comparing the conse- development and immune response in mice with a compro- quences of abnormalities in pre-BCR signaling in the human mised BCR complex. Science 1996; 272: 1804–1808. and the mouse. Immunol Rev 2000; 178: 75–90. 15 Gong S, Nussenzweig MC. Regulation of an early develop- 3 Melchers F, ten Boekel E, Seidl T, Kong XC, Yamagami T, mental checkpoint in the B cell pathway by Ig beta. Science Onishi K et al. Repertoire selection by pre-B-cell receptors and 1996; 272: 411–414. B-cell receptors, and genetic control of B-cell development 16 Minegishi Y, Coustan-Smith E, Rapalus L, Ersoy F, Campana from immature to mature B cells. Immunol Rev 2000; 175: D, Conley ME. Mutations in Ig alpha (CD79a) result in a 33–46. complete block in B-cell development. J Clin Invest 1999; 104: 4 Conley ME, Cooper MD. Genetic basis of abnormal B cell 1115–1121. development. Curr Opin Immunol 1998; 10: 399–406. 17 Wang Y, Kanegane H, Sanal O, Tezcan I, Ersoy F, Futatani T 5 Tsukada S, Saffran DC, Rawlings DJ, Parolini O, Allen RC, et al. Novel Ig alpha (CD79a) gene mutation in a Turkish Klisak I et al. Deficient expression of a B cell cytoplasmic patient with B cell-deficient agammaglobulinemia. Am J Med tyrosine kinase in human X-linked agammaglobulinemia. Cell Genet 2002; 108: 333–336. 1993; 72: 279–290. 18 Ma˚rtensson A, Argon Y, Melchers F, Dul JL, Ma˚rtensson I-L. 6 Vetrie D, Vorechovsky I, Sideras P, Holland J, Davies A, Flinter Partial block in B lymphocyte development at the transition F et al. The gene involved in X-linked agammaglobulinaemia into the pre-B cell receptor stage in VpreB1-deficient mice. is a member of the src family of protein-tyrosine kinases. Int Immunol 1999; 11: 453. Nature 1993; 361: 226–233. 19 Mundt C, Licence S, Shimizu T, Melchers F, Ma˚rtensson I-L. 7 Conley ME, Mathias D, Treadaway J, Minegishi Y, Rohrer J. Loss of precursor B cell expansion but not allelic exclusion in Mutations in BTK in patients with presumed X-linked VpreB1/VpreB2 double-deficient mice. J Exp Med 2001; 193: agammaglobulinemia. Am J Hum Genet 1998; 62: 1034–1043. 435. 8 Ochs HD, Smith CI. X-linked agammaglobulinemia. 20 Cartegni L, Chew SL, Krainer AR. Listening to silence and A clinical and molecular analysis. Medicine (Baltimore) 1996; understanding nonsense: exonic mutations that affect splicing. 75: 287–299. Nat Rev Genet 2002; 3: 285–298. 9 Kitamura D, Roes J, Kuhn R, Rajewsky K. A B cell-deficient 21 Gisler R, sigvardsson M. The human VpreB promoter is a mouse by targeted disruption of the membrane exon target for coordinated activation by early B cell factor and E47. of the immunoglobulin mu chain gene. Nature 1991; 350: J Immunol 2002; 168: 5130–5138. 423–426. 22 Minegishi Y, Rohrer J, Coustan-Smith E, Lederman HM, 10 Yel L, Minegishi Y, Coustan-Smith E, Buckley RH, Trubel H, Pappu R, Campana D et al. An essential role for BLNK in Pachman LM et al. Mutations in the mu heavy-chain gene in human B cell development. Science 1999; 286: 1954–1957.

Genes and Immunity