- Email: [email protected]
Correlation between genetic profile, serum levels and functionality of the polymorphic complement protein C4
3954
Abstracts / Molecular Immunology 44 (2007) 3909–3994
P47 Antibodies to C1q in sudden deafness Lillemor Skattum a , Sara Axelsson b , Anna StjernquistDesatnik b , Sven Lindberg b , Lennart Truedsson a a
Department of Laboratory Medicine, Section of Microbiology, Immunology and Glycobiology, Lund University, Lund, Sweden b Department of Oto-rhino-laryngology, Lund University Hospital, Lund, Sweden Antibodies to (a-C1q) are autoantibodies directed against the collagenous part of C1q. A-C1q are found in many clinical conditions, including systemic lupus erythematosus, hypocomplementemic urticarial vasculitis and membranoproliferative glomerulonephritis. Unexplained sudden hearing loss, termed sudden deafness (SD), is in a proportion of cases presumed to have an autoimmune cause, and is often associated with other autoimmune diseases. Several autoantigens have been suspected to play a role in the pathogenesis of SD, and autoantibodyscreening is standard in these patients. Still, in most SD cases, no associated disease or signs of autoantibody are found. Considering that two inner ear-proteins with partly collagenlike structure have been described in fish, we speculated that autoantibodies in SD might cross-react with C1q. We investigated sera from 92 patients with SD for presence of a-C1q by an ELISA. A-C1q was detected in 12 patients (13%). Western blot analysis was negative in all but 2 cases showing reactivity with the A chain of the C1q molecule. The C1q concentrations in the SD patients were normal and unrelated to presence of a-C1q. SD patients with a-C1q did not have other diagnoses associated with a-C1q. In the same 92 patients, other autoantibodies, many of which have been previously described in association with SD, were analysed. Increased concentrations of autoantibodies were found in the following number of patients: anti-nuclear antibody: 10 (11%), anti-cardiolipin antibody: 8 (9%), anti-HSP-70 antibody: 6 (7%), rheumatoid factors: 6 (7%), anti-neutrophil cytoplasmatic antibody: 1 (1%). In conclusion, reactivity to C1q is present in SD. This likely represents a cross-reactivity with collagen-like inner ear proteins which suggests that a type II autoimmune reaction might be involved in the pathogenesis. In our SD patients, a-C1q was the most frequently detected autoantibody suggesting that analysis of a-C1q should be performed in suspected SD. doi:10.1016/j.molimm.2007.06.107
P48 Correlation between genetic profile, serum levels and functionality of the polymorphic complement protein C4 Diana Wouters a,c , Anneke van der Horst a , Margreet Hart a , Irma Rensink a , Martin de Boer b , Dennis Schooneman b , Pauline van Schouwenburg b , Judy Geissler b , Dirk Roos b , Lucien Aarden a , D¨orte Hamann c a
Department of Immunopathology, Sanquin Research at CLB, The Netherlands b Department of Blood Cell Research, Sanquin Research at CLB, The Netherlands c Department of Autoimmune Diseases, Sanquin Diagnostics, Amsterdam, The Netherlands Complement C4 exists in two isotypes, C4A and C4B. The two genes, with 99% sequence homology, occur in multiple copies and many allelic forms. C4A binds more efficiently to amino-groups and C4B to hydroxyl-groups. C4A deficiency is associated with autoimmune disease and C4B deficiency with increased susceptibility for bacterial infections. To analyse the relation of C4-polymorphism with disease, a combination of geno- and phenotyping is most accurate. We developed assays to study the C4-polymorphism on both protein and genetic level. By ELISA we quantified C4A and C4B levels in serum. In addition, we developed a multiplex ligationdependent probe amplification (MLPA) to determine gene copy number of C4A and C4B and detect the presence of silencing mutations and retroviral transposons. Finally, we set up a functional assay which makes use of the binding preferences of C4A and C4B. This assay may be used to quantify C4A and C4B or to investigate whether both proteins are functionally active. In serum of 105 healthy volunteers we determined protein concentration and functionality of C4A and C4B and correlated these to the genetic profile. MLPA analysis revealed a frequency of complete C4A and C4B deficiency of 6.7% and 4.8%, respectively. The variation in gene copy number was comparable to literature data, consisting of 5.7% of individuals with two genes, 25.7% with three genes, 61% with four genes, 7.6% with five genes and none with six genes. Comparing MLPA results with total C4, C4A and C4B protein levels as determined by ELISA and functional assay, we found a full agreement between protein data and genetic profile. The functional assay appears to correlate even better with C4 genetic profile than the ELISA. In conclusion, we developed assays to identify the complement C4-polymorphism on both protein and genetic level. Although C4 serum protein concentration varies widely, C4A and C4B protein level correlated well to gene copy number. These new assays enable us to study the C4 polymorphism in patients with autoimmune diseases or bacterial infections. doi:10.1016/j.molimm.2007.06.108
Abstracts / Molecular Immunology 44 (2007) 3909–3994
P47 Antibodies to C1q in sudden deafness Lillemor Skattum a , Sara Axelsson b , Anna StjernquistDesatnik b , Sven Lindberg b , Lennart Truedsson a a
Department of Laboratory Medicine, Section of Microbiology, Immunology and Glycobiology, Lund University, Lund, Sweden b Department of Oto-rhino-laryngology, Lund University Hospital, Lund, Sweden Antibodies to (a-C1q) are autoantibodies directed against the collagenous part of C1q. A-C1q are found in many clinical conditions, including systemic lupus erythematosus, hypocomplementemic urticarial vasculitis and membranoproliferative glomerulonephritis. Unexplained sudden hearing loss, termed sudden deafness (SD), is in a proportion of cases presumed to have an autoimmune cause, and is often associated with other autoimmune diseases. Several autoantigens have been suspected to play a role in the pathogenesis of SD, and autoantibodyscreening is standard in these patients. Still, in most SD cases, no associated disease or signs of autoantibody are found. Considering that two inner ear-proteins with partly collagenlike structure have been described in fish, we speculated that autoantibodies in SD might cross-react with C1q. We investigated sera from 92 patients with SD for presence of a-C1q by an ELISA. A-C1q was detected in 12 patients (13%). Western blot analysis was negative in all but 2 cases showing reactivity with the A chain of the C1q molecule. The C1q concentrations in the SD patients were normal and unrelated to presence of a-C1q. SD patients with a-C1q did not have other diagnoses associated with a-C1q. In the same 92 patients, other autoantibodies, many of which have been previously described in association with SD, were analysed. Increased concentrations of autoantibodies were found in the following number of patients: anti-nuclear antibody: 10 (11%), anti-cardiolipin antibody: 8 (9%), anti-HSP-70 antibody: 6 (7%), rheumatoid factors: 6 (7%), anti-neutrophil cytoplasmatic antibody: 1 (1%). In conclusion, reactivity to C1q is present in SD. This likely represents a cross-reactivity with collagen-like inner ear proteins which suggests that a type II autoimmune reaction might be involved in the pathogenesis. In our SD patients, a-C1q was the most frequently detected autoantibody suggesting that analysis of a-C1q should be performed in suspected SD. doi:10.1016/j.molimm.2007.06.107
P48 Correlation between genetic profile, serum levels and functionality of the polymorphic complement protein C4 Diana Wouters a,c , Anneke van der Horst a , Margreet Hart a , Irma Rensink a , Martin de Boer b , Dennis Schooneman b , Pauline van Schouwenburg b , Judy Geissler b , Dirk Roos b , Lucien Aarden a , D¨orte Hamann c a
Department of Immunopathology, Sanquin Research at CLB, The Netherlands b Department of Blood Cell Research, Sanquin Research at CLB, The Netherlands c Department of Autoimmune Diseases, Sanquin Diagnostics, Amsterdam, The Netherlands Complement C4 exists in two isotypes, C4A and C4B. The two genes, with 99% sequence homology, occur in multiple copies and many allelic forms. C4A binds more efficiently to amino-groups and C4B to hydroxyl-groups. C4A deficiency is associated with autoimmune disease and C4B deficiency with increased susceptibility for bacterial infections. To analyse the relation of C4-polymorphism with disease, a combination of geno- and phenotyping is most accurate. We developed assays to study the C4-polymorphism on both protein and genetic level. By ELISA we quantified C4A and C4B levels in serum. In addition, we developed a multiplex ligationdependent probe amplification (MLPA) to determine gene copy number of C4A and C4B and detect the presence of silencing mutations and retroviral transposons. Finally, we set up a functional assay which makes use of the binding preferences of C4A and C4B. This assay may be used to quantify C4A and C4B or to investigate whether both proteins are functionally active. In serum of 105 healthy volunteers we determined protein concentration and functionality of C4A and C4B and correlated these to the genetic profile. MLPA analysis revealed a frequency of complete C4A and C4B deficiency of 6.7% and 4.8%, respectively. The variation in gene copy number was comparable to literature data, consisting of 5.7% of individuals with two genes, 25.7% with three genes, 61% with four genes, 7.6% with five genes and none with six genes. Comparing MLPA results with total C4, C4A and C4B protein levels as determined by ELISA and functional assay, we found a full agreement between protein data and genetic profile. The functional assay appears to correlate even better with C4 genetic profile than the ELISA. In conclusion, we developed assays to identify the complement C4-polymorphism on both protein and genetic level. Although C4 serum protein concentration varies widely, C4A and C4B protein level correlated well to gene copy number. These new assays enable us to study the C4 polymorphism in patients with autoimmune diseases or bacterial infections. doi:10.1016/j.molimm.2007.06.108