ICOS deficiency in patients with common variable immunodeficiency

Clinical Immunology 113 (2004) 234 – 240 www.elsevier.com/locate/yclim

ICOS deficiency in patients with common variable immunodeficiency Ulrich Salzera, Andrea Maul-Pavicica, Charlotte Cunningham-Rundlesb, Simon Urschelc, Bernd H. Belohradskyc, Jiri Litzmand, Are Holme, Jose´ Luis Francof, Alessandro Plebanig, Lennart Hammarstromh, Andrea Skrabli, Wolfgang Schwingeri, Bodo Grimbachera,* a

Division of Rheumatology and Clinical Immunology, University of Freiburg, 79106 Freiburg, Germany b Pediatrics and Immunobiology, Mount Sinai School of Medicine, New York, 10029, United States c Division of Infectious Diseases and Immunology, University Childrens Hospital, University of Munich, Germany d Department of Clinical Immunology and Allergology, St. Anne University Hospital, Masaryk University, Brno, Czech Republic e Research Institute for Internal Medicine, National Hospital, Oslo, Norway f Grupo de Immunodeficiencias Primarias, Departamento de Microbiologı´a y Parasitologı´a, Facultad de Medicina, Universidad de Antioquia, Medellin, Colombia g Clinica Pediatrica, Universita` di Brescia and Istituto Medicina Molecolare bAngelo NocivelliQ, Spedali Civili, Brescia, Italy h Division of Clinical Immunology, IMPI, Karolinska Institute at Huddinge Hospital, Stockholm, Sweden i Division of Pediatric Hemato-Oncology, Department of Pediatrics, University of Graz, Austria Received 18 April 2004; accepted 6 July 2004 Available online 17 September 2004

Abstract Common variable immunodeficiency (CVID) is the most frequent clinically significant primary antibody deficiency in man, predisposing to recurrent bacterial infections. Recently, we showed that the homozygous loss of the inducible costimulator (ICOS) on activated T cells may result in an adult onset form of CVID with autosomal recessive inheritance (AR-CVID). We screened 181 sporadic CVID patients and 13 CVID patients from nine families with AR-CVID for mutations in ICOS by genomic DNA sequencing. In the AR-CVID families, the genomic integrity of the ligand for ICOS (ICOS-L) was also evaluated. In two of the nine AR-CVID families, we identified five individuals with ICOS deficiency, carrying the identical large genomic deletion of ICOS as previously described. In the remaining seven AR-CVID families, we subsequently sequenced the coding region of the ICOS ligand but found no mutations. The incidence of ICOS deficiency among patients with CVID is less than 5%. Worldwide, there are now a total of nine patients diagnosed with ICOS deficiency most likely due to a common founder. ICOS-L deficiency could not be identified in families with AR-CVID. D 2004 Elsevier Inc. All rights reserved. Keywords: CVID; ICOS; Immunodeficiency

Introduction The diagnosis of common variable immunodeficiency (CVID) is based on markedly reduced serum levels for IgG * Corresponding author. Division of Rheumatology and Clinical Immunology, University Hospital Freiburg, University of Freiburg, Hugstetterstr. 55, 79106 Freiburg, Germany. Fax: +49 761 270 3531. E-mail address: [email protected] (B. Grimbacher). 1521-6616/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2004.07.002

and IgA or IgM, an impaired ability to specific antibody production after vaccination or exposure, and exclusion of secondary causes for antibody deficiency [1]. Although it is a rare disease with an estimated prevalence of 1 in 25,000 in the Western population, it is the second most frequent primary immunodeficiency (PID) after selective IgA deficiency (IgAD) and the most frequent PID requiring medical attention. Most cases of CVID are sporadic, however, about 10% are familial with a predominance of autosomal dominant over autosomal recessive inheritance [2,3]. CVID and IgAD

U. Salzer et al. / Clinical Immunology 113 (2004) 234–240

may occur in the same family, which suggests a possible common genetic basis for these two disease entities [4]. The age of onset of CVID has two major peaks: the first between 5 and 10 years of age, the second in early adulthood, ages 20–30. The phenotype of CVID is characterized by the sequelae of hypogammaglobulinemia with recurrent upper respiratory infections by encapsulated bacteria. In addition to these infections, the phenotype of CVID varies and may be accompanied by autoimmune phenomena, splenomegaly, granulomas, and the development of certain types of cancer [1,5]. The inducible costimulator (ICOS) belongs to the family of costimulatory T cell molecules such as CD28 and CTLA4 [6]. All three genes were mapped to chromosome 2q33. ICOS spans over 25 kb and is divided into five exons and four introns [7,8]. Fifty SNPs have been reported for ICOS: 16 lie within the promoter region, 13 are intronic, 1 is within exon 5, 1 SNP lies within an exon–intron boundary, and the remaining 19 SNPs are in the 3V UTR [8–10]. ICOS is only expressed on activated T cells and coinduces the secretion of IL-4, IL-5, IL-6, GM-CSF, TNF-a, and IFN-g but is pivotal for the superinduction of IL-10 [11]. Highest expression of ICOS is found within the T cell zones of secondary lymphoid organs and in the apical light zones of germinal centers [6,11]. This expression pattern and the cytokines induced by ICOS point to an important role of ICOS:ICOS-L interaction in mediating T–B cell cooperation and promoting the terminal differentiation of B cells into memory cells and plasma cells. This is further supported by findings in the ICOS and ICOS-L knockout mice, both showing a defect in germinal center formation and impaired humoral immune responses, especially after secondary immunizations [12–14]. ICOS-L is the unique ligand for ICOS and is constitutively expressed on both lymphoid and nonlymphoid tissues. ICOS-L can be up-regulated by inflammatory stimuli such as TNF-a, IFN-g, and lipopolysaccharide (LPS) [15]. ICOS-L shares 20% homology with CD80 and

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Table 2 Clinical features of CVID patients (n = 194) Sex Age

Age of diagnosis of CVID

Clinical presentation

Female Male b10 years 10–30 years 31–50 years N50 years Unknown b10 years 10–30 years 31–50 years N50 years Unknown Recurrent sinusitis Recurrent bronchitis Recurrent pneumonia Bronchiectasis Recurrent gastrointestinal infections Autoimmune phenomenaa splenomegaly Granuloma formation Malignancyb

106 (55%) 88 (45%) 11 (6%) 41 (21%) 96 (49%) 39 (20%) 7 (4%) 34 (17%) 85 (44%) 52 (27%) 11 (6%) 12 (6%) 123 (63%) 130 (67%) 98 (50%) 52 (27%) 36 (18%) 42 74 14 10

(21%) (38%) (7%) (5%)

a

Including idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, pernicious anemia, rheumatoid arthritis, psoriasis, vitiligo, diabetes mellitus, and systemic lupus erythematosus. b Including thymoma, Hodgkin lymphoma, non-Hodgkin lymphoma, glioma, acute lymphatic leucemia, and cutaneous T cell lymphoma.

CD86 (the ligands of CD28 and CTLA4). The gene encoding ICOS-L is located on chromosome 21q22.3 and consists of seven exons [16]. ICOS-L is expressed as two splice variants designated as hGL50 and B7H2, with hGL50 showing an expression pattern restricted to lymphoid organs [17,18]. ICOS-L expression on naRve B cells is down regulated via B cell receptor and ICOS engagement or IL-4 signaling, but it can be sustained by stimulation via CD40 [19]. We recently identified homozygous deletions in ICOS in 4 of 32 patients with CVID [20]. Therefore, the aim of this study was to determine the incidence of ICOS deficiency

Table 1 Amplification and sequencing primers for ICOS and ICOS-L

ICOS

ICOS-L

Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon

1 2 3 4 5 1 2 3 4 5 6 7

Forward primer (5VY3V)

Reverse primer (5VY3V)

Annealing temperature (8C)

TGC CTT TCA TAT AGC CAT TG GCA ACA GAG ATG ACT TTA TGC ATT GA AAG CCC TTT CTA TAA ACC ACA TT TGG GGGATA TTC TTT TTG GTC GTG TAT GAA AGG CAA TGG AGA GG GCG GGA GCG CAG TTA GAG C CAC GGG CAC GCC TGA TGT TCT GGA CCT CAC CAT GAA ATG TC CCA TGT CGG GGC ACA ATG CCT GGC TGT GGT TGG GGT TAT C GGT GTT TAG GGG GCT GCT GAG AGC GGC CGA CCG CAG AAA CGC ACT T

AGT CAT TTT GCC TTT CAT CTT T CTG CAT CTA AGT GAA CTC CAA TGT CTC CCT GTT GGT CAA A TCT AGA ATT AGG CCT TGG AGA TGT T AGA ATG CTG GCC CAT TAA AGA TGA T CTC ACA GGT TCA CCC AGG GG GTG CTG CGG TGC TTC GTC CTT ATT GCT AAC TAA CTC AAG AGG CAA GCG GTG GTT CTC ACT GTG CTT GGC AGT GTC AGG AAT G ACA GGC CGG CCG TGT TTT TAG C GGA ACA GCC GAG GGA CAT TG

55 58 55 58 62 58 63 63 63 58 58 63

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Table 3 Genotype frequencies of SNPs at the ICOS-locus SNPa

Genotype frequency, n/(%)

p1203

CVID HDb CVID HDb CVID HDb CVID HDb

T-repeat p3990 P4031 a b

T/T 5 (3) T/T 0 (0) T[13] 130 (91) T[13] 99 (93) T/T 32 (20) T/T 18 (16) C/C 15 (10) C/C 3 (3)

C/T 28 (17) C/T 15 (14) T[14] 13 (9) T[14] 5 (5) G/T 63 (41) G/T 55 (50) A/C 34 (22) A/C 35 (32)

C/C 128 (80) C/C 95 (86) T[15] 0 (0) T[15] 2 (2) G/G 62 (39) G/G 37 (34) A/A 106 (68) A/A 72 (65)

n n n n n n n n

= = = = = = = =

161 110 145 106 157 110 155 110

Single nucleotide polymorphism. HD = healthy donor, refers to Ref. [10].

(OMIM #607594) among patients with CVID. In addition, we screened autosomal recessive CVID (AR-CVID) families for disease causing mutations in the ICOS-L gene.

Patients and methods Patients Patients were diagnosed with CVID according to the ESID criteria (www.esid.org). One hundred and twentythree patients were recruited from central Europe, 16 patients were from Scandinavia, 48 patients were from the United States, and 7 patients were from Colombia. Informed written consent was obtained from each individual before participation under the internal ethics review board-approved clinical study protocol (#239/99). Ten milliliters of heparinized blood was obtained from each participant of the study for isolation of genomic DNA using DNA Blood isolation kit reagents (Puregene DNA isolation kit; Gentra Systems, Minneapolis, USA).

Sequencing of ICOS and ICOS-L The coding sequence of the five exons and the adjacent intron–exon boundaries of ICOS was amplified from genomic DNA using primer pairs and conditions as indicated in Table 1. Long-range PCR from genomic DNA was performed using Takara LA Taq (Cambrex Bioscience Rockland Inc., Rockland, USA) with the following primers: LRfw (5VY3V) TGG GGC TTT ATC TTT ATT ATC AGG, LRrv (5VY3V) TGG GCG CTA TCT CAT TCT CT. The coding sequence of seven exons and adjacent intron–exon boundaries of ICOS-L was amplified from genomic DNA using the primer pairs and conditions as indicated in Table 1. PCR products were sequenced with the PCR amplification primers. After gel electrophoresis on an ABI Prismk 377 DNA Sequencer (PE Applied Biosystems, Foster City, USA), the data were analyzed with aid of the DNA Sequencing Analysis software, version 3.4 (PE Applied Biosystems) and Sequenchertk version 3.4.1 (Gene Codes Corporation, Ann Arbor, USA).

Fig. 1. Pedigrees of newly identified patients with ICOS deficiency; circles, female; squares, male; slashed rhombuses, deceased; filled symbols, affected; open symbols, unaffected. Individuals II.1 and II.2 died due to complications at birth as premature neonates. No autopsies were performed. Long-range PCR analysis of ICOS. Genomic DNA from members of family C and D was amplified with primers covering ICOS exons 2 and 3. From DNA of a healthy donor, a 3-kb PCR fragment could be amplified, in the heterozygote parents (family D) both a 3-kb and a shortened 1.2-kb fragment was present, and in all affected individuals only a 1.2-kb fragment could be detected, indicating a genomic deletion.

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Table 4 T and B cell numbers and IgG, IgA, and IgM levels in ICOS deficiency Family

Individual

Normal range Age N16 years Age 3–5 years C II.3 II.4 D II.1 II.3 II.4

Age of onset

CD3+ T cells (cells/Al)

CD19+ B cells (cells/Al)

IgG (g/l)

IgA (g/l)

IgM (g/l)

20 18 15 4 3

700–2100 900–4500 629 816 2683 2727 2923

100–500 200–2100 127 28 184 686 882

7–16 5–13 1.01 0.57 2.55 2.13 0.54

0.7–4.0 0.4–1.8 b0.06 b0.06 b0.25 0.58 0.2

0.4–2.3 0.4–1.8 0.38 1.8 0.26 0.57 0.43

Pathologic values are printed bold.

Genotyping of the ICOS locus Patients showing a genomic deletion of ICOS were genotyped at the ICOS locus using the polymorphic markers D2S115 and D2S2289. Genotyping PCR was performed at the recommended conditions. PCR results were analyzed on an ABI 377 sequencer (PE Applied Biosystems) with the Collection and Analysis software packages (PE Applied Biosystems). Allele sizes were determined with the help of GENOTYPER (PE Applied Biosystems).

Results A cohort of 194 CVID patients was collected for analysis of mutations in ICOS. All patients had a typical clinical history of recurrent respiratory infections and were diagnosed with CVID according to the ESID criteria (www.esid.org). A summary of their clinical presentation is given in Table 2. An early onset of the disease (b10 years) was observed in 34 patients. One hundred and eighty-one patients were sporadic cases with a negative family history for CVID, and 13 patients originated from nine families with AR-CVID. The parents were consanguineous in five of these nine families. In the 181 sporadic CVID patients, the sequence of the five exons and the adjacent exon–intron boundaries of ICOS revealed no mutations. Since the expression of ICOS may be influenced by genetic polymorphisms outside the coding region, we evaluated the single nucleotide polymorphisms (SNP) p1203, p2412, p3990, p4031 (names refer to position in GenBank accession number AF488347) and one T repeat of the ICOS gene (NCBI SNP id: rs5837889–90). However, the frequency estimation of these five SNPs showed no significant differences in CVID patients when compared to a healthy control population [10] (see Table 3). By contrast, exons 2 and 3 could not be amplified from the DNA of five patients from two of the nine families with familial CVID. These findings suggested a homozygous deletion. Therefore, we performed a long-range PCR that spans 3 kb of genomic DNA amplifying both exons 2 and 3 of ICOS (Fig. 1). DNA from healthy controls yielded a 3-kb DNA fragment. In contrast, all affected individuals in the

two families were homozygous for a shortened DNA fragment of 1.2 kb. The PCR result in parent I.1 and I.2 of family D displays a clear heterozygote state with both DNA fragments present (Fig. 1). The 1.2-kb DNA fragment in the five affected individuals was subjected to DNA sequencing (Fig. 2A). In all five newly diagnosed patients, we identified the same 1815 bp deletion as has been reported in families A and B of the original description of ICOS deficiency [20] (Fig. 2B, shaded sequence). The homozygous loss of 1815 bp genomic DNA results in an mRNA frameshift with a premature stop codon leading to a truncated ICOS protein, which putatively consists of the signal peptide and nine dnonsenseT amino acids. The absence of ICOS expression on activated T cells was verified for individuals II.3 and II.4 of family C by FACS analysis (data not shown). As all patients with ICOS deficiency carry the same large deletion, a common founder for all four families was anticipated. To prove this, we examined the polymorphic marker D2S2289 that is adjacent to the ICOS locus and two SNPs (NCBI SNP id: rs4675378 and rs4270326), flanking the deleted region. The five newly diagnosed patients as well as the four originally published cases [20] were homozygous for the same 203 bp allele at D2S2289 and had identical SNPs (data not shown). Thus, a common founder for the mutations in all four families with ICOS deficiency is very likely. The eight patients from the remaining seven AR-CVID families had a normal ICOS sequence. Since we evaluated ICOS in 194 CVID patients, we can now calculate the incidence of ICOS deficiency in this cohort to four out of 190 unrelated families or nine out of 194 CVID patients (approximately 5%). Because the ICOS-L knockout mice display the very same phenotype as the ICOS knockout mice [14], ICOS-L was a plausible disease causing candidate gene in the remaining seven AR-CVID families. Therefore, ICOS-L was sequenced in these patients. In two patients from two consanguineous families, a previously nondescribed polymorphism was detected in exon 3 of ICOS-L. Individual AR6 was heterozygous and AR4 was homozygous for a G to A transition at position 490 of the ICOS-L mRNA ref. #NM_015259 (see Fig. 2C). The alteration results in an amino acid change from Valine to Isoleucine at position 128 (Val128Ile) in a nonconserved part of the protein. However,

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Fig. 2. (A) Sequencing results of long-range PCR products of affected individuals in families C and D and a healthy donor (HD). The PCR fragments shown in Fig. 1 were subjected to DNA sequencing. The site of the deletion is indicated by an arrow. The starting and ending points of the deletion are located within highly homologous regions of DNA, so that the wt and mutated sequences differ only in two single bases (indicated by asterisks) in the segment shown. (B) Deletion of 1815 bp in ICOS. Sequencing of the shortened PCR product of 1.2 kb revealed a 1815 bp deletion in ICOS from all five newly identified ICOSdeficient patients, including parts of intron 1, exon 2, intron 2, exon 3, and parts of intron 3. The shaded area designates the deleted part of ICOS. (C) Single nucleotide polymorphism in exon 3 of ICOS-L. Exon 3 of ICOS-L was amplified by PCR and subjected to DNA sequencing. A previously undescribed polymorphism resulting in a heterozygous (AR6) or homozygous (AR4) G to A transition is shown.

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we could detect this polymorphism also in control individuals at a similar frequency for both heterozygotes and homozygotes. Therefore, we concluded that neither ICOS nor ICOS-L is the disease causing gene in those remaining seven CVID families.

Discussion The CVID phenotype appears to origin from genetic conditions resulting in the cardinal symptoms of primary antibody deficiency. In the past, patients with other monogenic primary immunodeficiencies such as X-linked Agammaglobulinemia (XLA), X-linked lymphoproliferative Syndrome (XLP), or hyper-IgM-syndrome (HIgM) have been misdiagnosed as CVID since these disorders can mimic the phenotype of CVID [21–23]. ICOS deficiency represents the first single gene defect identified to solely cause the CVID phenotype [20]. Thus, we screened a cohort of 194 CVID patients for mutations of ICOS to determine how frequent a defect in ICOS causes CVID. The originally described four ICOS deficiency patients were homozygous for a large genomic deletion in ICOS [20]. The patients came from two CVID families with an autosomal recessive trait. Heterozygote parents and siblings had no clinical signs of immunodeficiency. The immunologic laboratory findings in the heterozygote carriers were also normal, despite a slightly weaker surface staining for ICOS on their activated T cells [20]. We therefore concluded that both alleles of ICOS had to be affected by mutations to cause the phenotype of CVID. In a previous study [24], ICOS was evaluated in 47 unrelated CVID patients by SSCP analysis and no mutations were found. However, SSCP has a sensitivity of about 80% and thus possible mutations in this cohort may have been missed. In a group of 181 sporadic CVID patients, we could not detect any mutations in the coding regions of ICOS. The frequency of a known polymorphism (p4031 AYC) located in the located in the 3VUTR two bp after the stop codon was determined to be comparable to that in control populations [10]. Lee et al. [25] found that there was no difference in the binding affinity to B7RP-1 between the different allelic variants of this polymorphism. However, we cannot exclude alterations or deficiency in ICOS expression due to mutations in the promoter region of ICOS or putative regulatory intronic regions in the CVID patients we analyzed since protein expression of ICOS on activated T cells could only be assessed by flow cytometry in 28 patients. Among nine CVID families most likely affected by an autosomal recessive trait, we identified two new unrelated families with five individuals carrying the identical large genomic deletion in ICOS as previously published [20]. Further genetic analysis of all nine ICOS deficiency patients revealed that they share identical homozygous haplotypes in

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the ICOS locus, indicating that the mutation was passed on to them deriving from a common founder. This is further supported by the fact that three of the four families with ICOS deficiency originate from the same village, which is linked to the origin of the fourth family by the long river of Danube. The Danube was a frequently used route of emigration in the 18th century. The observation that the deleted region is flanked by highly homologous stretches of DNA including a 13-bp identical segment suggests that the deletion resulted from an aberrant recombination event. Family C showed a childhood onset of the disease, which was not observed in the other three families. The flow cytometry analysis of T cells and their proliferative response to mitogens gave similar results as in the originally described patients [20]. The B cell compartment showed a reduction in memory B cell subsets (Table 4), which confirms the results obtained in the previously described ICOS-deficient patients, a phenotype also found in about three quarters of CVID patients. After exclusion of mutations in ICOS, we subsequently evaluated the ICOS-L in the remaining seven AR-CVID families. In two patients, we could identify a previously undescribed polymorphism located in exon 3 of ICOS-L giving rise to an amino acid substitution from Valine to Isoleucine. However, as both amino acids are aliphatic and the exchange is located within a nonconserved part of the protein [16], the structure or function of ICOS-L are unlikely to be impaired, which is further supported by the observation of the same polymorphism in healthy controls at a similar frequency. We suggest mutations of ICOS be sought in patients with an recessive pattern of inheritance of CVID. While we did not identify this mutation in the sporadic cases investigated here, additional cases arising from the same or other ICOS mutations may be identified.

Acknowledgments We thank Dfrte Thiel, Judith Deimel, and Cristina Wfllner for excellent technical assistance and the patients’ physicians Dr. Mezger and Prof. Dr. Vaith. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grant GR 1617/3 and SFB 620/project C2 to B.G and by grants from the National Institutes of Health, AI-467320, AI-48693, and contract N01-AI-30070, NIH-NIAID-DAIT 03-22 to C.C.

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