Polymorphisms of human CD19 gene: possible association with susceptibility to systemic lupus erythematosus in Japanese

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Abstract

CD19 regulates the signaling for B lymphocyte development, activation and proliferation. In mice, CD19 deficiency and overexpression were shown to result in hypogammaglobulinemia and autoantibody production, respectively. In the present study, we screened for the polymorphisms of CD19 , and examined the detected polymorphisms for the association with rheumatoid arthritis (RA), Crohn’s disease and systemic lupus erythematosus (SLE). Two SNPs, c.705G>T (P235P and IVS14-30C>T, were decreased (P = 0.0096 and P = 0.028, respectively), in SLE. A GT repeat polymorphism, c.*132(GT)12–18, was detected within the 3’-untranslated region, and individuals with 15 times repeat was significantly increased in the independent two groups of Japanese SLE patients (P = 0.011 and P = 0.035, respectively); the overall difference between total SLE and controls was striking (P = 0.0061). No association was observed for RA and Crohn’s disease. In addition, no variations other than the common polymorphisms were detected in four patients with common variable immunodeficiency, the phenotype of which resembles CD19 deficient mice. In Caucasian SLE families, this GT repeat polymorphism was rare. CD19 mRNA level in the isolated peripheral blood B lymphocytes was lower in individuals possessing (GT)15–18 alleles compared with those without these alleles, both in controls and in SLE patients; however, the difference did not reach statistical significance. These results suggested that either the slight reduction in the CD19 mRNA level associated with the elongation of GT repeat, or an allele of another locus in linkage disquilibrium with CD19 (GT)15–18, may be associated with susceptibility to SLE in Japanese.

Introduction

CD19 is a 95 kDa B lymphocyte-specific member of the immunoglobulin superfamily expressed by early pre-B cells from the time of heavy chain rearrangement until plasma cell differentiation.1,2,3 CD19 associates with CD21, CD81 and Leu13 on the B cell surface to form the B cell coreceptor complex, which acts as a modulator of B cell receptor (BCR) signaling.4,5 Because CD21 is a receptor for C3 cleavage fragments, antigen-C3d complexes are capable of binding to the B cells through CD21.6,7 Therefore, activated complement fragments could generate CD19 signaling as a result of C3d binding to CD21. It was also reported that IgM and heparan sulfate binds to CD19 as complement-independent ligands.8,9 CD19 has also been implicated as a signaling partner for several other surface receptors including CD40,10 CD38,11 CD72,10 VLA412 and FcγRIIB.13,14 Altering CD19 density on the cell surface in gene-targeted or transgenic mice has been shown to significantly influence B cell functions. B cells from CD19-deficient (CD19−/−) mice are hyporesponsive to transmembrane signals and generate modest immune responses.15,16,17,18,19,20,21 In contrast, overexpression of CD19 in transgenic mice results in B cells that are hyperresponsive to transmembrane signals and have elevated humoral immune responses. Such mice also demonstrate diminished numbers of B cells in the peripheral pool, presumably caused by the enhanced negative selection in the bone marrow. In addition, production of autoantibodies including anti-double-strand (ds) DNA antibodies and rheumatoid factors are observed.15,17,18,19,20,22,23 Thus, CD19 seems to function as a general rheostat that defines signaling thresholds critical for expansion of the peripheral B cell pool.24

The 240 amino acid cytoplasmic region of CD19 contains nine conserved tyrosine residues,2 the phosphorylation of which following BCR and/or CD19 ligation provides Src homology 2 (SH2) recognition motifs that recruit regulatory molecules to the cell surface. The Lyn, Fyn and Lck protein tyrosine kinases (PTKs) as well as the proto-oncogene Vav interact with CD19. CD19 also interacts with phosphatidylinositol 3-kinase (PI3-kinase).25 PI3-kinase can regulate the membrane localization and activation of numerous downstream effecter molecules.

In accordance with the hypothesis that the development of autoimmune diseases is likely to involve a number of genetic factors, susceptibility loci suggested from the genome-wide screening have been mapped on many chromosomal regions. The candidate regions of rheumatoid arthritis (RA) or Crohn’s disease include 16p11.2, where the gene coding for CD19 is located.26,27 (The susceptibility gene to Crohn’s disease within this region has been identified to be NOD2, located at 16q12, during this study.28,29) In addition, B cells in the patients with systemic lupus erythematosus (SLE) have been shown to be activated.30 Of interest, the level of CD19 expression in B cells was demonstrated to be approximately 20% higher in patients with systemic sclerosis,31 one of the systemic autoimmune diseases associated with antinuclear antibodies.

Based on these observations, CD19 was considered to be a strong candidate for a susceptibility gene to some of the autoimmune diseases. In the present study, we screened human CD19 for polymorphisms, and examined the possibility that any of the polymorphisms may be associated with the susceptibility to RA, SLE or Crohn’s disease. In addition, since the phenotype of CD19 deficient mice resembles that of human common variable immunodeficiency (CVID), we also made an attempt to detect mutations in the patients with CVID.

Results

Identification of CD19polymorphisms

Systematic screening of the entire CD19coding region, the promoter region ( −1050 bp) and 3’-untranslated region (UTR) was performed on genomic DNA samples from 32 Japanese healthy individuals and 36 patients (16 with SLE, 16 with RA and four with CVID), using PCR-single-strand conformation polymorphism (SSCP). The nucleotide sequences of the detected variations were determined using direct sequencing. Through the screening, three single nucleotide polymorphisms (SNPs) within the coding region, two SNPs within introns flanking the exon-intron junctions, four SNPs within the promoter region and a GT repeat polymorphism within the 3’-UTR were identified (Figure. 1). The designations of the variations are based on references.32 Mutations other than common variations were not detected in the patients with CVID.

Figure 1
figure1

Genomic configuration of human CD19 gene, and variations detected in the Japanese. Boxes indicate exons. The designations of the variations are based on references.3233 In Caucasians, c.*132(GT)10 was also detected.

Association of CD19 variations with SLE in Japanese

Next we examined whether any of the polymorphisms of CD19 is associated with the susceptibility to RA, SLE and Crohn’s disease in the Japanese population, using case-control analysis. For this purpose, genotypes of these polymorphisms were determined in 127 patients with RA, 87 with SLE (SLE1), 156 with Crohn’s disease as well as 247 healthy Japanese individuals. Table 1 summarizes allele carrier frequencies of detected CD19 SNPs in the promoter and in the coding region. c.705T (P235P)within exon 4 (P= 0.0096, Pcorr= 0.12, odds ratio (OR) = 0.49, 95% confidence interval (CI): 0.29–0.84 and IVS14-30Twithin intron 14 (P = 0.028, Pcorr = 0.36, OR = 0.56, 95% CI: 0.33–0.94) showed a negative association with SLE. Significant association was not observed between any of the other SNPs with SLE, nor between any of the SNPs with RA and Crohn’s disease.

Table 1 CD19 SNPs in the patients with RA, SLE, Crohn’s disease and controls

Table 2 shows the frequencies of 3’UTR dinucleotide repeat polymorphism. Carrier frequency of c.*132(GT)18(the allele coding for 18 times GT repeat starting from the 132nd nucleotide counted from the nucleotide 3’ of the termination codon in the cDNA sequence30,31) was significantly higher in SLE (SLE1) (15/87, 17.2%) compared with that in healthy individuals (17/247, 6.9%) (P = 0.005, Pcorr = 0.065, OR = 2.82, 95% CI: 1.37–5.79). The allele frequency of c.*132(GT)182 = 7.6, df = 1, P= 0.006, Pcorr = 0.078), the distributions of genotype (χ2 = 19.0, df = 10, P= 0.04, Pcorr = 0.36) and allele frequencies (χ2= 9.8, df = 4, P= 0.04, Pcorr = 0.36) were also significantly different between SLE1 and controls. To test whether this association can be replicated, an independent group of SLE patients (SLE2) were analyzed for the GT repeat polymorphism. Compared to controls (6.9%), the increased carrier frequency of c.*132(GT)18 in SLE2 (9.1%) was not as remarkable as that of SLE1 (17.2%) as shown in Table 2. However, the carrier frequency of c.*132(GT)15 was significantly higher in SLE2 than controls (6.1% vs1.2%, χ2= 4.8, df = 1, P= 0.03, Pcorr = 0.39)

Table 2 Frequencies of 3′UTR dinucleotide repeat polymorphism in the patients with RA, SLE, Crohn’s disease and Controls

From these results, we considered that this GT repeat within 3’UTR may be associated with SLE when the number of GT repeat was 15. Therefore, carrier frequency and allele frequency of the c.*132(GT)15–18 were compared in SLE1, SLE2 and controls (Table 3). The carrier frequency of c.*132(GT)15–18 was significantly increased both in SLE1 (P= 0.011) and SLE2 (P= 0.035). When the two groups were combined, a more striking difference was observed between total SLE and the controls (P= 0.0061). The frequency of c.*132(GT)15–18 alleles was also significantly higher in SLE (P = 0.012).

Table 3 Comparison of the frequency of c.*132(GT)15–18 in the patients with SLE and controls

As for RA and Crohn’s disease, significant association with this GT repeat polymorphism was not observed.

Lack of association in Caucasian SLE patients

We next examined whether the association with SLE is observed in a different population. For such a purpose, 107 Caucasian SLE families recruited in southern California were tested using transmission disequilibrium test (TDT). Significant association was not observed for c.705Tallele (transmitted 29, non-transmitted 43). c.*132(GT)15–18 alleles were found to be rare in Caucasians: only two families possessed c.*132(GT)18 allele, and c.*132(GT)15was not observed. However, of interest, in both families possessing c.*132(GT)18, this allele was transmitted to the patients (χ2= 2.0, df = 1, NS), although the difference did not reach statistical significance, because of the small number of c.*132(GT)18 positive families. Incidentally, c.*132(GT)10, which was not observed in Japanese, was found in two Caucasian families.

Linkage disequilibrium between polymorphic sites

In order to address the relationships among positions c.705, IVS14-30 and c.*132, the polymorphisms of which exhibited skewed distribution in SLE, the allelic associations were analyzed based on the genotyping results of the healthy Japanese individuals. Significant association was observed between CD19 c.705T and IVS14-30T (relative linkage disequilibrium (RLD) = 0.83, χ2 = 93.8, P < 10−10). In addition, tight association was present between c.705G, the common allele, and c.*132(GT)18 (RLD = 1.0, χ2= 8.2, P= 0.004) and c.*132(GT)15 (RLD = 0.84, χ2 = 1.12, P = NS), although the latter did not reach statistical significance due to the small number of individuals with this allele.

Association with clinical characteristics

We analyzed whether GT repeat polymorphism is associated with clinical characteristics of SLE. Interestingly, the patients with c.*132(GT)15 or c.*132(GT)18 were less likely to have anti-Sm antibody compared with those who do not have c.*132(GT)15–18 alleles (P= 0.02). No association was observed for parameters such as age at onset, presence or absence of nephropathy, neurological disorders (seizures or psychosis), serositis, hypo-complementemia, anti-dsDNA, anti-RNP, anti-SSA or anti-SSB antibodies (Table 4).

Table 4 Comparison of the frequency of the clinical characteristics with and without the CD19c.*132 (GT) 15–18 allele in SLE patients

CD19 mRNA expression in B cells

One of the plausible hypotheses on the association of GT repeat polymorphism in the 3’UTR with SLE was that the polymorphism may be directly associated with the expression level of CD19mRNA.34,35 To test this hypothesis, CD19 mRNA level in the peripheral blood B cells isolated from healthy individuals and patients with different GT repeat alleles was semi-quantitatively analyzed using real-time RT-PCR. As shown in Figure 2, the average CD19mRNA level in individuals possessing (GT)15–18 alleles was lower compared with those without these alleles, both in controls (35% reduction) and in SLE patients (20% reduction); however, the difference did not reach statistical significance. The dose of steroids was not significantly different between the patients with (GT)15–18 alleles (11.3 ± 2.1 mg/day prednisolone) and those without (8.3 ± 5.8 mg/day prednisolone), and there was no correlation between the dose of prednisolone and CD19mRNA level (data not shown).

Figure 2
figure2

Relative mRNA levels of CD19 in the peripheral blood B lymphocytes from healthy individuals (a) and from patients with SLE (b). The mRNA level of CD19 was standardized with the concentration of β-actin mRNA. This experiment was repeated twice with similar results. Mean ± s.d. is shown under each column. Although a tendency of low expression level was observed for individuals with (GT)15–18 , the difference did not reach statistical significance (Mann-Whitney’s U-test). No correlation was observed between the dose of steroids and CD19 mRNA level in the patients, and the average dose of prednisolone was not significantly different between the two groups of patients (see text for details).

Discussion

In the present study, we identified a number of nucleotide sequence variations of human CD19, among which three were shown to be significantly associated with SLE. There are three possible reasons for the observed association: false positive (type 1 error), secondary association because of linkage disequilibrium with an allele of another locus, and true primary association of CD19. At this stage, all of these possibilities cannot be excluded.

Among the three polymorphic sites that were shown to be significantly associated with SLE, two SNPs showed negative association with SLE, while the remainder, a GT repeat polymorphism within 3’UTR, showed a significant positive association. The former two were in negative linkage disequilibrium with the latter. If one assumes a primary association of CD19 gene itself, the GT repeat polymorphism is considered most likely to have a primary functional significance, since the SNPs coding for a synonymous substitution and a nucleotide substitution in the intron are unlikely to be associated with any functional changes. It has been shown in a number of other genes that 3’UTR sequences are involved in the regulation of translation or stability of mRNA.34,35

It was recently reported that the CA repeat polymorphism within the 3’UTR of CD19, probably the same polymorphism that we studied, was not associated with SLE in a case-control study in Italy.36 In their data, alleles other than the common allele were rare, consistent with our results in the Caucasian families from the United States. These findings indicate that the frequency of polymorphisms is different among populations, and suggest the possibility that this GT repeat polymorphism might be a risk factor in Japanese, but not in Caucasians. Such an interpretation could explain why the chromosomal region of CD19 was not detected in the genome-wide linkage analyses, in which the majority of the analyzed families were Caucasians, African-Americans and Mexican-Americans, but not Asians.37,38,39,40,41,42,43 It will be interesting to examine this association in the Asian populations other than Japanese. It should also be noted that, although the number was small, c.*132(GT)18 was preferentially transmitted from parents to the SLE patients in the only two families positive for this allele. Thus, CD19 might possibly be relevant in a small proportion of Caucasian SLE patients as well.

At this point, how the elongated GT repeat alleles are associated with SLE remains speculative. Since statistically significant difference was not detected in the CD19 mRNA level between individuals with and without (GT)15–18 alleles, a likely explanation is that this association was caused by linkage disequilibrium with an allele of another locus that has a primary significance for SLE. There are some candidate genes such as IL4R and IL21R in the proximity of CD19; however, the distance between these genes and CD19 is larger than 1 Mb, which is usually beyond the limit of linkage disequilibrium. On the other hand, although statistically not significant, the average CD19 mRNA levels were approximately 35% (in healthy individuals) and 20% (in SLE) lower in individuals possessing (GT)15–18 alleles. It is important to note that such a subtle difference in the expression level of CD19 has been clearly shown to lead to defective regulation of autoantibody production in mice.31 Indeed, the expression level of CD19 in peripheral B cells was reported to be slightly decreased in patients with SLE.31 In addition, a proportion of CD19 deficient mice exhibit antinuclear antibodies (Sato S, personal communication). It is thus speculated that the reduction of CD19 expression might lead to a defect in the tolerance induction to autoantigens probably because of the decreased BCR signaling in the immature B cells,19,44 resulting in the persistence of autoreactive B cells. Therefore, we think that our current results do not exclude both of the above the two possibilities, namely, linkage disequilibrium with another allele and reduced expression of CD19 itself, at this stage.

The third possibility, namely, the association may merely represent a false positive phenomenon, should also be considered. Because a number of polymorphic sites were detected and statistical analysis was done for each of the all detected alleles, the P values reported here do not reach statistical significance after the correction for multiple comparisons. However, the fact that significant association of the GT repeat polymorphism was observed in two independent groups of SLE but not in RA and Crohn’s disease suggests that this association may not represents a false positive phenomenon. Case-control studies are often subject to false-positives derived from population stratification. However, the central part of Japan has been shown to be relatively homogeneous with respect to the genetic background.45 It should also be pointed out that the lack of significant association in Caucasian TDT samples was caused by the low frequency of alleles other than the common c.*132(GT)12 allele in this population, and does not entirely exclude possible association in a small proportion of Caucasian patients, as described above.

In this study, it was observed that the SLE patients with c.*132(GT)15–18 allele were significantly less likely to have anti-Sm antibody. Of particular interest, it was recently reported that the expression level of CD19 regulates the differentiation of potentially autoreactive B cells to B-1 and B-2, as well as autoantibody production, in mice expressing anti-Sm heavy chain transgene.46 Thus, it may be possible that CD19 expression level is associated with the regulation of the differentiation and activation of B cells specific for nuclear autoantigens.

We previously reported association of HLA-DRB1*1501,47,48 TNFR2-196R49 and FCGR2B-232T/T50 with SLE. In addition, association analysis has been done for CD22 polymorphisms.51 Since these molecules, especially those involved in B cell signaling, can functionally interact with CD19, presence of multiplicative effects between CD19and these molecules was tested. At present, clear multiplicative effect was not observed (data not shown). A substantially larger number of patients and controls will be necessary to evaluate interactions between multiple gene polymorphisms in the future.

Although three of four patients with CVID showed marked decrease in the peripheral CD19+ lymphocyte proportion, it is considered that such deficiency in B cells results from gene(s) other than CD19 itself. Four SNPs were detected in the promoter region, but these sites are not considered to be binding sites for the important transcription factors such as B cell lineage-specific antigen promoter (BSAP, Pax5).52

In conclusion, this study demonstrated a number of polymorphisms within human CD19. Association of CD19 3’UTR dinucleotide repeat polymorphism, or another gene in linkage disequilibrium with it, with susceptibility to SLE was suggested. Further population studies, polymorphism screening of adjacent genes, and functional studies will be necessary to distinguish the two possibilities.

Materials and methods

Patients and healthy individuals

One hundred and twenty-seven patients with RA, 87 patients with SLE (the first SLE group: SLE1), 156 patients with Crohn’s disease as well as 247 healthy individuals were initially examined for the case-control study. To examine whether the associations detected in SLE1 group can be replicated, an independent group of 99 patients with SLE (the second SLE group: SLE2) were collected later; thus the total number of the patients with SLE was 186. To test whether the associations detected in the Japanese patients is also observed in other populations, 107 Caucasian SLE families with affected offsprings were also recruited at UCLA. In addition, four patients with CVID were analyzed for variations.

The healthy individuals, 145 males and 102 females (average age: 36.6 years), consisted of the researchers, laboratory workers and students of the University of Tokyo and Japanese Red Cross Central Blood Center. The patients with RA, 18 males and 168 females (average age: 57.8 years), were diagnosed according to the classification criteria of American College of Rheumatology.53 SLE1 and SLE2 consisted of 12 males and 75 females (average age: 40.3 years), and six males and 93 females (average age: 40.0 years), respectively. The patients with SLE were classified according to the criteria of American College of Rheumatology.54 The patients with Crohn’s disease, 124 males and 32 females (average age: 31.1 years), were diagnosed clinically as well as by radiographic, endoscopic, and histological examination of biopsy specimens. The patients with CVID were three males and one female. The proportion of CD19 positive B cells among the peripheral blood mononuclear cells from the patients with CVID was 10%, 3.3%, 3.0% and 0%, respectively.

All healthy individuals and patients used for the case-control study were unrelated Japanese living in the Tokyo area.

The clinical characteristics of the patients were obtained by reviewing their medical records. When sufficient information was not available, such patients were excluded from the particular analysis. Nephritis (persistent proteinuria greater than 0.5g/day or 3+, or presence of the cellular casts) and central nervous system disease (convulsion or psychosis) were defined according to ACR criteria.54

This study was approved by the Research Ethics Review Committees of the University of Tokyo, Juntendo University and Human Subject Protection Committee of UCLA.

Genomic DNA

Genomic DNA was purified from the peripheral blood leukocytes from the patients and healthy individuals, using the QIAamp®DNA Blood Mini kit (Qiagen, Hilden, Germany).

PCR-SSCP

The primers used for PCR and the annealing temperatures are shown in Table 5. These were designed according to the genomic DNA sequence of human CD19 (GenBank accession no. M84371). Each exon was amplified using flanking intronic primers. Exon 2 and 4 were divided into two segments and exon 11, 12 and 13, 14 were amplified together as one fragment, because the size of the fragment optimal for this method was determined to be 200–400 bp in previous studies.55,56 The promoter region was analyzed using nested PCR, because of the low amplification efficiency of the single-step PCR. The first primer set was designed to amplify −1351136 using long PCR. The amplification condition consisted of initial denaturation at 96°C for 10 min, followed by 35 cycles of denaturation at 96°C for 1 min, annealing at 58°C for 30 sec, and extension at 72°C for 3 min, using a thermal cycler (Thermal cycler MP; Takara, Kyoto, Japan). Subsequently, this fragment was divided into three overlapping segments of 300–450 bp for SSCP analysis. The amplification condition consisted of initial denaturation at 96°C for 10 min, followed by 35 cycles of denaturation at 96°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec, using a thermal cycler (Thermal cycler MP; Takara or GeneAmp PCR system 9600; Perkin-Elmer Applied Biosystems; Foster City, CA, USA). In such an approach, promoter region up to −1050 bp was analyzed.

Table 5 Primers and PCR-SSCP conditions used in this study

The amplified DNA was analyzed using SSCP. One μl of solution containing the PCR product was mixed with 7 μl of denaturing solution (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol FF). The mixtures were denatured at 96°C for 5 min and immediately cooled on ice. One μl of the mixtures was applied to 7.5% polyacrylamide gel (acrylamide: bisacrylamide = 49:1) for the analysis of exon 15, and to 10% polyacrylamide gel (acrylamide: bisacrylamide = 49:1) for the analyses of others. Ten percent glycerol was included in the gel for the analyses of exon 2B and 4A because of better separation. Electrophoresis was carried out in 0.5 ×TBE (45 mM Tris-borate (pH 8.0), 1 mM EDTA) under constant current of 20 mA / gel, using a minigel electrophoresis apparatus with a constant temperature control system (90 × 80 × 1 mm, AE-6410 and AE-6370; ATTO, Tokyo, Japan). The optimal temperature and the duration of electrophoresis were determined by preliminary experiments. Single-strand DNA fragments in the gel were visualized by silver staining (Daiichi Pure Chemicals, Tokyo, Japan).

PCR-restriction fragment length polymorphism (PCR-RFLP)

The genotyping of the IVS14-30C>T polymorphism within the intron 14 was performed using PCR-RFLP method. A 256 base pair fragment from the CD19 gene containing the polymorphic site was amplified using a specific primer set (CD19-3’UTRF: 5’-AGAGGGAA CAGGGTTCCTAG-3’, CD19ex15R: 5’-AGGAATACAAAGGGGACTGG-3’). The amplification condition was the same as that for the SSCP analysis. After the PCR reaction, the amplified products were digested with BamHI for 2 h and analyzed on a 10% polyacrylamide gel with SYBR Gold (Molecular Probes, Eugene, OR, USA) staining.

Direct sequencing

PCR products were amplified and both sense and antisense strands were directly sequenced using the same primers as those for the SSCP analysis. Fluorescene-based automated cycle sequencing of PCR products was performed using ABI310 or ABI377 sequencer (ABI PRISM, PE Biosystems) with the dye-terminator method, according to the manufacture’s instructions (ABI PRISM™ dRhodamine Terminator Cycle Sequencing-Ready Reaction Kit).

Dinucleotide repeat polymorphism genotyping

The genotyping of the dinucleotide repeat polymorphism within the 3’UTR was performed using PCR followed by use of sequencer and GENESCANTM software. A 256 base pair fragment containing the polymorphic site was amplified using a Fam or Hex labeled CD19-3’UTRF primer and a CD19ex15R primer. The amplification condition was the same as that for the SSCP analysis. The amplified product was run on a 4% polyacrylamide gel (acrylamide: bisacrylamide = 19:1) using the ABI377 sequencer (ABI-PRISM, PE Biosystems). One and a half μl of solution containing the PCR product was mixed with 2.5 μl of 99.5% formamide, 0.5 μl of loading buffer (50 mg/ml blue dextran, 25 mM EDTA) and 1.0 μl of Gene Scan-500 [ROX] Size Standard (ABI-PRISM, PE Biosystems). The mixtures were denatured at 96°C for 2 min and immediately cooled on ice. Two μl of the mixtures was applied to 4% polyacrylamide gel. Data were collected with ABI377 collection software and size analysis was done using Genescan-500 ROX as a size standard (ABI-PRISM).

Preparation of cDNA from peripheral blood B lymphocytes

B lymphocytes were isolated from peripheral blood mononuclear cells of 11 healthy individuals and 15 patients with SLE using Dynabeads M-450 CD19 (Dynal ASA, Oslo, Norway) according to the manufacturer’s instructions. Total RNA was prepared from peripheral blood B cells using RNeasy Mini kit (Qiagen). cDNA was synthesized using MMLV reverse transcriptase (ReverTra Ace, TOYOBO, Osaka, Japan) and oligo dT primer according to the manufacturer’s instructions. The synthesis condition was at 42°C for 45 min, followed by 99°C for 10 min.

Quantitative RT-PCR

Real-time RT-PCR for CD19 and β-actin mRNA was performed using the LightCycler thermal cycler system (Roche Diagnostics GmbH, Mannheim, Germany) and LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics GmbH) according to the manufacturer’s instructions. The primers for CD19 were Forward: 5’-GTCTTATGAGAACGAGGATG-3’, Reverse: 5’-AGGAA TACAAAGGGGACTGG-3’ and for β-actin Forward: 5’-TCCTGTGGCATCCACGAAACT-3’, Reverse: 5’-GAA GCATTTGCGGTGGACGAT-3’. The PCR condition for CD19 consisted of initial denatulation at 96°C for 10 min, followed by 45 cycles of denaturation at 96°C for 5 sec, annealing at 60°C for 10 sec, and extension at 72°C for 10 sec. For β-actin, the annealing was performed at 62°C for 10 sec. The specificity of amplification was checked by the melting curve analysis. Namely, after the amplification, the temperature was slowly elevated and fluorescence intensity was monitored. Specific and nonspecific products have different melting temperatures dependent on their nucleotide compositions. This temperature was 91°C for CD19 and 90°C for β-actin. The quantification data were analyzed by Fit Points Method using the LightCycler™ analysis software. In brief, the concentration of the target mRNA in each sample was estimated from the comparison of the number of cycles necessary for achieving a given fluorescence intensity within a log-linear range of amplification with those of serially diluted reference samples that were run at the same time. The concentrations of the target mRNA were standardized using the concentration of β-actin mRNA. The differences were statistically analyzed using Mann-Whitney’s U-test.

Statistical analysis

The Japanese patients and controls were compared using case-control association analyses. Allele carrier frequency was defined as the percentage of the individuals carrying the allele (homozygotes and heterozygotes) among the total number of the individuals. χ2 test and Fisher’s exact test were used to analyze the association of CD19polymorphisms with susceptibility to each disease or with the clinical features in the Japanese population. The P values for the comparison of presence or absence of individual alleles were corrected for the number of comparisons (Pcorr) by multiplying by 13, since nine biallelic SNPs and one dinucleotide repeat polymorphisms with five alleles were compared (9 + 5 − 1). Those for the distribution of genotypes, allele frequencies or the allele carrier frequency of a set of alleles were corrected by multiplying by 9, since 10 polymorphic sites were detected (10 − 1).

The Caucasian SLE families were analyzed using TDT, as previously described.57 For the families with two or three affected children, only the first child was chosen for the analysis. The significance of TDT was examined using NcNemar’s test. The χ2 value was calculated as follows:

χ2 = (T–NT)2 / (T+NT)

where T and NT is the number of transmitted and non-transmitted allele, respectively. Haplotype frequencies and linkage disequilibrium parameters were estimated from the typing results using EH program.58 The RLD value is defined as the ratio of linkage disequilibrium (LD) to the absolute value of possible maximum (when LD > 0) or possible minimum (when LD < 0) of LD.59 Consequently, RLD is variable in the range between −1 and 1.

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Acknowledgements

The authors are indebted to Dr Shinichi Sato and Dr Makoto Inaoki (Department of Dermatology, Kanazawa University), Dr Manabu Fujimoto (International Medical Center of Japan), Mr Satoshi Makino and Mr Koichi Okamoto (Department of Genetic Information, Tokai University) for helpful discussions, to Dr Jun Ohashi (Department of Human Genetics, The University of Tokyo) for statistical analysis, to Dr Nelson Tsuno (Department of Transfusion Medicine, The University of Tokyo), Michiko Shiota, Tae Komata, Minae Kawashima, Aya Kawasaki and Chieko Kyogoku (Department of Human Genetics, The University of Tokyo) for helpful assistance and discussions.

Author information

Correspondence to N Tsuchiya.

Additional information

This study was supported by Grant-in-Aid for Scientific Research on Priority Areas (C), Grants-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology, and a grant from the Ministry of Health, Labour and Welfare. New polymorphisms described in this study have been deposited in GenBank/ EMBL/ DDBJ accession No. AB052799, AB052814, AB052815, AB052816, AB052817 and AB052818.

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Kuroki, K., Tsuchiya, N., Tsao, B. et al. Polymorphisms of human CD19 gene: possible association with susceptibility to systemic lupus erythematosus in Japanese. Genes Immun 3, S21–S30 (2002) doi:10.1038/sj.gene.6363906

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Keywords

  • CD19
  • polymorphism
  • systemic lupus erythematosus
  • genetics
  • association
  • rheumatoid arthritis

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