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Complement receptor 2 polymorphisms associated with systemic lupus erythematosus modulate alternative splicing.

Douglas KB, Windels DC, Zhao J, Gadeliya AV, Wu H, Kaufman KM, Harley JB, Merrill J, Kimberly RP, Alarcón GS, Brown EE, Edberg JC, Ramsey-Goldman R, Petri M, Reveille JD, Vilá LM, Gaffney PM, James JA, Moser KL, Alarcón-Riquelme ME, Vyse TJ, Gilkeson GS, Jacob CO, Ziegler JT, Langefeld CD, Ulgiati D, Tsao BP, Boackle SA - Genes Immun. (2009)

Bottom Line: Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls.Two of these SNPs are in exon 10, directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon.These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.

View Article: PubMed Central - PubMed

Affiliation: University of Colorado Denver School of Medicine, Aurora, CO 80045, USA.

ABSTRACT
Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971, rs17615, rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs 32.6% in controls, P=0.016, OR=0.90 (0.82-0.98)). Two of these SNPs are in exon 10, directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon. Effects of these SNPs and a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.

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Analysis of CR2 exon trapping in Raji and HK cells. (A) The CR2 minigene containing the minor or major alleles at the SNPs in exons 10 and 11 (shown by arrows) was cloned into the pL53In vector so that the CR2 exons are flanked by the second and third exons of the rat preproinsulin gene (in grey). Transcription of the minigene after transfection is driven by the RSV LTR (90° arrow). (B) Quantitative RT-PCR strategy to measure relative levels of vector-derived trapped CR2 exons. The primers to detect trapping of the long isoform are located in exon 11 of CR2 and exon 3 of the rat preproinsulin gene, and the probe spans exons 11 and 12 of CR2. The primers to detect trapping of the short isoform are located in exon 10 of CR2 and exon 3 of the rat preproinsulin gene, and the probe spans exons 10 and 12 of CR2. (C) Relative amounts of vector-derived isoform mRNA in Raji and HK cells. After 24 hours, RNA was prepared from transfected cells and subjected to RT-PCR. In both cell lines, the amount of vector-derived long isoform mRNA relative to short isoform mRNA was decreased in cells transfected with the minor allele construct. Data shown are the results of two independent experiments.
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Figure 3: Analysis of CR2 exon trapping in Raji and HK cells. (A) The CR2 minigene containing the minor or major alleles at the SNPs in exons 10 and 11 (shown by arrows) was cloned into the pL53In vector so that the CR2 exons are flanked by the second and third exons of the rat preproinsulin gene (in grey). Transcription of the minigene after transfection is driven by the RSV LTR (90° arrow). (B) Quantitative RT-PCR strategy to measure relative levels of vector-derived trapped CR2 exons. The primers to detect trapping of the long isoform are located in exon 11 of CR2 and exon 3 of the rat preproinsulin gene, and the probe spans exons 11 and 12 of CR2. The primers to detect trapping of the short isoform are located in exon 10 of CR2 and exon 3 of the rat preproinsulin gene, and the probe spans exons 10 and 12 of CR2. (C) Relative amounts of vector-derived isoform mRNA in Raji and HK cells. After 24 hours, RNA was prepared from transfected cells and subjected to RT-PCR. In both cell lines, the amount of vector-derived long isoform mRNA relative to short isoform mRNA was decreased in cells transfected with the minor allele construct. Data shown are the results of two independent experiments.

Mentions: To evaluate an effect of these four SNPs on the splicing efficiency of exon 11, an exon trapping system was used. As the genomic DNA segment extending from exon 10 to exon 12 encompasses only 1114 nucleotides (Figure 2A), we amplified this region including flanking DNA from intron 9 and intron 12 from a BAC clone containing human CR2 (RP11-35C1) and cloned it into the exon trapping vector pL53In.20 The unique KpnI site in pL53In used for cloning is located within the second intron of the rat preproinsulin gene and is flanked by the second exon of the gene containing a 5′ splice donor site and the third exon containing a 3′ splice acceptor site and polyadenylation site, allowing splice variants to be generated that contain the rat 5′ and 3′ preproinsulin exons surrounding the trapped CR2 exons (Figure 3A). Vectors containing either the major or the minor alleles at all four SNPs in exons 10 and 11, representing the two haplotypes found in >95% of controls (G1-1 and G1-2, Table 2), were transiently transfected into the Raji lymphoblastoid B cell line and the HK FDC cell line 21 to allow splicing to occur in the two cellular environments in which CR2 is expressed, since tissue-specific factors are known to influence alternative splicing.22, 23


Complement receptor 2 polymorphisms associated with systemic lupus erythematosus modulate alternative splicing.

Douglas KB, Windels DC, Zhao J, Gadeliya AV, Wu H, Kaufman KM, Harley JB, Merrill J, Kimberly RP, Alarcón GS, Brown EE, Edberg JC, Ramsey-Goldman R, Petri M, Reveille JD, Vilá LM, Gaffney PM, James JA, Moser KL, Alarcón-Riquelme ME, Vyse TJ, Gilkeson GS, Jacob CO, Ziegler JT, Langefeld CD, Ulgiati D, Tsao BP, Boackle SA - Genes Immun. (2009)

Analysis of CR2 exon trapping in Raji and HK cells. (A) The CR2 minigene containing the minor or major alleles at the SNPs in exons 10 and 11 (shown by arrows) was cloned into the pL53In vector so that the CR2 exons are flanked by the second and third exons of the rat preproinsulin gene (in grey). Transcription of the minigene after transfection is driven by the RSV LTR (90° arrow). (B) Quantitative RT-PCR strategy to measure relative levels of vector-derived trapped CR2 exons. The primers to detect trapping of the long isoform are located in exon 11 of CR2 and exon 3 of the rat preproinsulin gene, and the probe spans exons 11 and 12 of CR2. The primers to detect trapping of the short isoform are located in exon 10 of CR2 and exon 3 of the rat preproinsulin gene, and the probe spans exons 10 and 12 of CR2. (C) Relative amounts of vector-derived isoform mRNA in Raji and HK cells. After 24 hours, RNA was prepared from transfected cells and subjected to RT-PCR. In both cell lines, the amount of vector-derived long isoform mRNA relative to short isoform mRNA was decreased in cells transfected with the minor allele construct. Data shown are the results of two independent experiments.
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Related In: Results  -  Collection

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Figure 3: Analysis of CR2 exon trapping in Raji and HK cells. (A) The CR2 minigene containing the minor or major alleles at the SNPs in exons 10 and 11 (shown by arrows) was cloned into the pL53In vector so that the CR2 exons are flanked by the second and third exons of the rat preproinsulin gene (in grey). Transcription of the minigene after transfection is driven by the RSV LTR (90° arrow). (B) Quantitative RT-PCR strategy to measure relative levels of vector-derived trapped CR2 exons. The primers to detect trapping of the long isoform are located in exon 11 of CR2 and exon 3 of the rat preproinsulin gene, and the probe spans exons 11 and 12 of CR2. The primers to detect trapping of the short isoform are located in exon 10 of CR2 and exon 3 of the rat preproinsulin gene, and the probe spans exons 10 and 12 of CR2. (C) Relative amounts of vector-derived isoform mRNA in Raji and HK cells. After 24 hours, RNA was prepared from transfected cells and subjected to RT-PCR. In both cell lines, the amount of vector-derived long isoform mRNA relative to short isoform mRNA was decreased in cells transfected with the minor allele construct. Data shown are the results of two independent experiments.
Mentions: To evaluate an effect of these four SNPs on the splicing efficiency of exon 11, an exon trapping system was used. As the genomic DNA segment extending from exon 10 to exon 12 encompasses only 1114 nucleotides (Figure 2A), we amplified this region including flanking DNA from intron 9 and intron 12 from a BAC clone containing human CR2 (RP11-35C1) and cloned it into the exon trapping vector pL53In.20 The unique KpnI site in pL53In used for cloning is located within the second intron of the rat preproinsulin gene and is flanked by the second exon of the gene containing a 5′ splice donor site and the third exon containing a 3′ splice acceptor site and polyadenylation site, allowing splice variants to be generated that contain the rat 5′ and 3′ preproinsulin exons surrounding the trapped CR2 exons (Figure 3A). Vectors containing either the major or the minor alleles at all four SNPs in exons 10 and 11, representing the two haplotypes found in >95% of controls (G1-1 and G1-2, Table 2), were transiently transfected into the Raji lymphoblastoid B cell line and the HK FDC cell line 21 to allow splicing to occur in the two cellular environments in which CR2 is expressed, since tissue-specific factors are known to influence alternative splicing.22, 23

Bottom Line: Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls.Two of these SNPs are in exon 10, directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon.These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.

View Article: PubMed Central - PubMed

Affiliation: University of Colorado Denver School of Medicine, Aurora, CO 80045, USA.

ABSTRACT
Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971, rs17615, rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs 32.6% in controls, P=0.016, OR=0.90 (0.82-0.98)). Two of these SNPs are in exon 10, directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC), and the third is in the alternatively spliced exon. Effects of these SNPs and a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact.

Show MeSH
Related in: MedlinePlus