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CR1 polymorphisms associated with SLE
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087- MOLECULAR BASIS OF COMPLETE C4 DEFICIENCY IN A SLE PATIENT WITH FOUR MUTANT GENES: A 2- BP INSERTION AT EXON 29 IN C4A AND A l-BP DELETION AT EXON 13 IN C4B. Kristi L. Rupert 1,2,Joann M. Moulds 3, Yan Yang I.‘, Robert Warren 3, John Reveille ‘, Frank C. Arnett ’ and C. Yung Yu I.*. ‘Children’s Research Institute, 2The Ohio State University, Columbus OH; and ‘Rheumatology and Clinical Immunogenetics, University of Texas Houston Health Science Center, Houston, Texas. The complement component C4 protein is encoded by isotypic genes C4A and C4B located in the major histocompatibility complex (MHC). Several studies have linked deficiencies in C4 with SLE, a devastating autoimmune disease. Complete deficiencies of C4A and C4B are rare; only about 20 patients have been described thus far, all affected with SLE or lupus-like symptoms. One of the patients has been followed up clinically in the past 16 years. This patient experienced multiple episodes of otitis media, pneumonia, recurring fevers, rash and developed a non-deforming recurrent polyarthritis. To determine the molecular basis of the complete C4 deficiency, the gene dosage of C4A and C4B in this patient and his parents were determined by RFLP analysis. It revealed the presence of four C4 mutant genes. PCR analysis coniirmed thatboth copies of the non-expressed C4A genes contain a 2-bp msertion (TC) at codon 1213 in exon 29, which is a common mutation in many C4A null genes. However, subsequent analysis did not reveal the identical mutation in C4B. The patient’s C4B gene was selectively cloned and the 41 exons completely sequenced. The sequence revealed a novel I-bp deletion (C) at codon 522 in exon 13 that results in a frame-shift mutation and a premature stop codon. A multiplex PCR in which the known mutations in C4A and C4B can be elucidated has been developed. This multiplex I’CR could have clinical applications.
089- ROLE OF COMPLEMENT RECEPTORS TYPE 1 (CRllCD35) AND TYPE 2 (CR2/CD21) IN MURINE SLE S. A. Boackle, J. M. Brown, and V. M. Holers. University of Colorado Health Sciences Center. Denver, CO. The levels of CR1 and CR2 are decreased by - 50% on B lymphocytes of patients with systemic lupus erythematosus (SLE), as well as in two murine models of human SLE. MRUIpr and (NZBXNZW)Fl mice, but not in C57BU6 or C57BV6/lpr. In MRUlpr and (NZBXNZW)Fl mice, CRllCRL levels are decreased from the predisease state until death, suggesting that these alterations are not due to Immune complex-mediated receptor down-regulation. To determine whether the decrease in receptor levels is important in the pathogenesis of SLE, we are analyzing MRU/pr mice in which the CRlICR2 knockout (KO) genotype has been backcrossed. We are testing the hypothesis that the early introduction of complete or partial deficiency of CRl/CR2 alters the course of the autoimmune disease. In these studies, KO mice were backcrossed 4 generations onto the MRL//pr background, after which +Imice were intercrossed to generate +I+, +/-, and -/cohorts. Twelve mice from each cohort are being analyzed at biweekly intervals for proteinuria and for the development of cryoglobulins and autoantibodies to dsDNA. ssDNA, and IgG (rheumatoid factor). Kidneys will be examined at 14-16 weeks for the development of glomerulonephritis. Preliminary studies reveal a trend toward increased levels of anbdsDNA at 14 weeks of age in -I- and +/- animals compared to +I+ [-I- 0.82+0.58 (n=6), +I- 1.01+0.47 (n=6), +I+ 0.18+0.05 (n=4)] as well as a trend towards the development of proteinuria at an earlier age in the -/- and +I- animals. These ongoing studies should help define the contribution of altered EfRl/CR2 levels to the pathogenesis of SLE and the loss self-tolerance to relevant disease-related autoantigens.
088- ERYTHROCYTE CR1 LEVELS DG NOT CORRELATE WlTH DISEASE ACTIVITY IN HISPANIC SLE PATIENTS. Moulds M. Roberts S, Tew M, Reveille J and Alarcon G for the LUMINA Study Group. University of Texas-Houston Medical School and the University of Alabama at Birmingham.
CR1 POLYMORPHISMS ASSOCIATED WITH SLE. Daniel J. Birmingham, Grace Liang, and Xiao-Ping Shen.The Department of Int Med, The Ohio State University, Columbus, OH. We have recently identified a number of polymorphic sites in the CR1 molecule, including sites at residues 643 (wild-type I or T), 1812 (K or R) and 1928 (A or T), relative to the CR1 A allotype. Here we report updated frequencies in a larger cohort of normals (n = 42) and SLE patients (n = 56,36 with nephritis). The frequency of the 1928-T allele was no different between the normal (19.5%) and SLE patients (20.9%). Nor was the frequency of the 643-T allele different between normals (20.2%) and SLE patients (13.4%, p = 0.242, two-tailed). However, subset analysis showed a tendency of the 643-T allele to be under represented in the SLE patients with nephritis (9.7%, p = 0.079). For the 1812 CR1 variant, only the wild-type 1812-K allele was found in the 42 normals, whereas 5 of 56 SLE patients (3 with nephritis) were found to express one 1812-R allele @ = 0.074 for allele frequency difference). To assess functional differences, binding assays were performed with serumopsonized immune complexes (IC) and erythrocytes (E) from individuals expressing CR1 genotyped as wildtype (643-IY1812KK/ 1928-AA,n=l), 643-IT(n=3), 643-‘lT(n=l), 1928-AT(n=2), or 1812~KR(n=l). Whenopsonizedwith lO%AB serum,the% IC binding was solely dependent on E-CR1 levels (mean of n=4, r =0.959, p =O.OOOZ), regardless of the CR1 genotype. When 10% C3-deficient serum was used, IC binding was dependent on E-CR1 levelsforall(r=0.955,p=O.OO08)exceptthe 1812-KRvariant, which bound less than half the amount predicted based on its level. ELISA-based assays using soluble E-CR1 from individuals genotyped as wild-type, 643-TT, or 1812-KR showed no differences in the ability to bind purified Clq, C4b, C3b, or C3bi. Studies are continuing to determine if defective IC binding activity of the I8 12KR variant is due to aberrant spatial distribution on the E surface.
Several studies have noted reduced levels ofe@rccyte CR1 (ECRI) with disease activity in conditions having increased levels of circulating immune complexes, eg. SLE. CR1 deficiency appears IObe due IOincreased proteolysis of CRI Onlya few serial studies of E-CR1 have been reported aad no definitive mle for E-CR1 quantitation has been established. Therefore, we studied a welldefined population of SLE patients who have been monitored for disease activity over the past five years. A total of 69 Hispanic patients meeting ACR criteria for SLE were enrolled in the LUMINA m, The patients were exam&d by the same group of investigators and disease activity was scored using standardized methods, ie. SLAM. Healthy Hispanic controls (n=43) were drawn from the same geographicarea. E-CR1 was quanrifiedusing a previously described ELISA method and the Hind Ill alleles were assessed using a PCR-RFLP. The frequenciesof the Hand L alleles in controls were similar to those reported in Caucasians. however, the frequency of the L allele was increased in the SLE patients @= 0.043). Although there was some correlation between E-CR1 and the Hind III RFLP, there was considerableoverlap between groups. Twenty-three patients having serial samples covering at least 3 years were chosen for further evaluation. Of these. 9 had no significant change in E-CR1 levels and 14 showed fluchmtions. Ilkteen patients had increased disease activity at some point as defined by the SLAM scores bat this did not correlatewith E-CR1 levels overall (R=O.Ql). When analyzed individually some patients showed loss of E-CR1 during active disease while others had no change at all. We conclude that E-CR1 levels are not an effective melhod for predicting disease activity in SLE patients.
DgD-
087- MOLECULAR BASIS OF COMPLETE C4 DEFICIENCY IN A SLE PATIENT WITH FOUR MUTANT GENES: A 2- BP INSERTION AT EXON 29 IN C4A AND A l-BP DELETION AT EXON 13 IN C4B. Kristi L. Rupert 1,2,Joann M. Moulds 3, Yan Yang I.‘, Robert Warren 3, John Reveille ‘, Frank C. Arnett ’ and C. Yung Yu I.*. ‘Children’s Research Institute, 2The Ohio State University, Columbus OH; and ‘Rheumatology and Clinical Immunogenetics, University of Texas Houston Health Science Center, Houston, Texas. The complement component C4 protein is encoded by isotypic genes C4A and C4B located in the major histocompatibility complex (MHC). Several studies have linked deficiencies in C4 with SLE, a devastating autoimmune disease. Complete deficiencies of C4A and C4B are rare; only about 20 patients have been described thus far, all affected with SLE or lupus-like symptoms. One of the patients has been followed up clinically in the past 16 years. This patient experienced multiple episodes of otitis media, pneumonia, recurring fevers, rash and developed a non-deforming recurrent polyarthritis. To determine the molecular basis of the complete C4 deficiency, the gene dosage of C4A and C4B in this patient and his parents were determined by RFLP analysis. It revealed the presence of four C4 mutant genes. PCR analysis coniirmed thatboth copies of the non-expressed C4A genes contain a 2-bp msertion (TC) at codon 1213 in exon 29, which is a common mutation in many C4A null genes. However, subsequent analysis did not reveal the identical mutation in C4B. The patient’s C4B gene was selectively cloned and the 41 exons completely sequenced. The sequence revealed a novel I-bp deletion (C) at codon 522 in exon 13 that results in a frame-shift mutation and a premature stop codon. A multiplex PCR in which the known mutations in C4A and C4B can be elucidated has been developed. This multiplex I’CR could have clinical applications.
089- ROLE OF COMPLEMENT RECEPTORS TYPE 1 (CRllCD35) AND TYPE 2 (CR2/CD21) IN MURINE SLE S. A. Boackle, J. M. Brown, and V. M. Holers. University of Colorado Health Sciences Center. Denver, CO. The levels of CR1 and CR2 are decreased by - 50% on B lymphocytes of patients with systemic lupus erythematosus (SLE), as well as in two murine models of human SLE. MRUIpr and (NZBXNZW)Fl mice, but not in C57BU6 or C57BV6/lpr. In MRUlpr and (NZBXNZW)Fl mice, CRllCRL levels are decreased from the predisease state until death, suggesting that these alterations are not due to Immune complex-mediated receptor down-regulation. To determine whether the decrease in receptor levels is important in the pathogenesis of SLE, we are analyzing MRU/pr mice in which the CRlICR2 knockout (KO) genotype has been backcrossed. We are testing the hypothesis that the early introduction of complete or partial deficiency of CRl/CR2 alters the course of the autoimmune disease. In these studies, KO mice were backcrossed 4 generations onto the MRL//pr background, after which +Imice were intercrossed to generate +I+, +/-, and -/cohorts. Twelve mice from each cohort are being analyzed at biweekly intervals for proteinuria and for the development of cryoglobulins and autoantibodies to dsDNA. ssDNA, and IgG (rheumatoid factor). Kidneys will be examined at 14-16 weeks for the development of glomerulonephritis. Preliminary studies reveal a trend toward increased levels of anbdsDNA at 14 weeks of age in -I- and +/- animals compared to +I+ [-I- 0.82+0.58 (n=6), +I- 1.01+0.47 (n=6), +I+ 0.18+0.05 (n=4)] as well as a trend towards the development of proteinuria at an earlier age in the -/- and +I- animals. These ongoing studies should help define the contribution of altered EfRl/CR2 levels to the pathogenesis of SLE and the loss self-tolerance to relevant disease-related autoantigens.
088- ERYTHROCYTE CR1 LEVELS DG NOT CORRELATE WlTH DISEASE ACTIVITY IN HISPANIC SLE PATIENTS. Moulds M. Roberts S, Tew M, Reveille J and Alarcon G for the LUMINA Study Group. University of Texas-Houston Medical School and the University of Alabama at Birmingham.
CR1 POLYMORPHISMS ASSOCIATED WITH SLE. Daniel J. Birmingham, Grace Liang, and Xiao-Ping Shen.The Department of Int Med, The Ohio State University, Columbus, OH. We have recently identified a number of polymorphic sites in the CR1 molecule, including sites at residues 643 (wild-type I or T), 1812 (K or R) and 1928 (A or T), relative to the CR1 A allotype. Here we report updated frequencies in a larger cohort of normals (n = 42) and SLE patients (n = 56,36 with nephritis). The frequency of the 1928-T allele was no different between the normal (19.5%) and SLE patients (20.9%). Nor was the frequency of the 643-T allele different between normals (20.2%) and SLE patients (13.4%, p = 0.242, two-tailed). However, subset analysis showed a tendency of the 643-T allele to be under represented in the SLE patients with nephritis (9.7%, p = 0.079). For the 1812 CR1 variant, only the wild-type 1812-K allele was found in the 42 normals, whereas 5 of 56 SLE patients (3 with nephritis) were found to express one 1812-R allele @ = 0.074 for allele frequency difference). To assess functional differences, binding assays were performed with serumopsonized immune complexes (IC) and erythrocytes (E) from individuals expressing CR1 genotyped as wildtype (643-IY1812KK/ 1928-AA,n=l), 643-IT(n=3), 643-‘lT(n=l), 1928-AT(n=2), or 1812~KR(n=l). Whenopsonizedwith lO%AB serum,the% IC binding was solely dependent on E-CR1 levels (mean of n=4, r =0.959, p =O.OOOZ), regardless of the CR1 genotype. When 10% C3-deficient serum was used, IC binding was dependent on E-CR1 levelsforall(r=0.955,p=O.OO08)exceptthe 1812-KRvariant, which bound less than half the amount predicted based on its level. ELISA-based assays using soluble E-CR1 from individuals genotyped as wild-type, 643-TT, or 1812-KR showed no differences in the ability to bind purified Clq, C4b, C3b, or C3bi. Studies are continuing to determine if defective IC binding activity of the I8 12KR variant is due to aberrant spatial distribution on the E surface.
Several studies have noted reduced levels ofe@rccyte CR1 (ECRI) with disease activity in conditions having increased levels of circulating immune complexes, eg. SLE. CR1 deficiency appears IObe due IOincreased proteolysis of CRI Onlya few serial studies of E-CR1 have been reported aad no definitive mle for E-CR1 quantitation has been established. Therefore, we studied a welldefined population of SLE patients who have been monitored for disease activity over the past five years. A total of 69 Hispanic patients meeting ACR criteria for SLE were enrolled in the LUMINA m, The patients were exam&d by the same group of investigators and disease activity was scored using standardized methods, ie. SLAM. Healthy Hispanic controls (n=43) were drawn from the same geographicarea. E-CR1 was quanrifiedusing a previously described ELISA method and the Hind Ill alleles were assessed using a PCR-RFLP. The frequenciesof the Hand L alleles in controls were similar to those reported in Caucasians. however, the frequency of the L allele was increased in the SLE patients @= 0.043). Although there was some correlation between E-CR1 and the Hind III RFLP, there was considerableoverlap between groups. Twenty-three patients having serial samples covering at least 3 years were chosen for further evaluation. Of these. 9 had no significant change in E-CR1 levels and 14 showed fluchmtions. Ilkteen patients had increased disease activity at some point as defined by the SLAM scores bat this did not correlatewith E-CR1 levels overall (R=O.Ql). When analyzed individually some patients showed loss of E-CR1 during active disease while others had no change at all. We conclude that E-CR1 levels are not an effective melhod for predicting disease activity in SLE patients.
DgD-