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Association of Ficolin 1 promoter polymorphisms with clinical leprosy
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Abstracts / Molecular Immunology 47 (2010) 2198–2294
and undetectable C1r. C3 and C4 protein levels were 2156 g/mL (ref: 1045–1670 g/mL) and 2238 g/mL (ref: 445–740 g/mL), respectively. Immunofixation experiments confirmed high levels of C4A and C4B and elevated C3F and C3S proteins in the patient and his mother. The coding regions of the C1R genes from the patient were PCR-amplified and sequenced. A C to T mutation at position 6392 in exon 10 of the C1R gene changed Arg-380 to a stop codon was revealed. A sequence-specific-primer PCR assay was developed to screen for the novel C1R mutation. The mother is heterozygous for this mutation. However, the R380X mutation was not detectable in 181 unrelated African American SLE patients. Similar to homozygous C1q or C4 deficiencies, C1r-deficient patients develop SLE. Unlike most SLE patients, C4 and C3 protein levels in C1r-deficiency patients are consistently high. Mutations leading to early complement component deficiencies and SLE, with the exception of the 2-bp insertion into codon-1213 of C4A, are observable mainly in probands’ families or isolated populations. Such observations support the concept that genetic variants with large effect for a common disease only exist at very low frequencies in the population. doi:10.1016/j.molimm.2010.05.072 152 Association of Ficolin 1 promoter polymorphisms with clinical leprosy A.B.W. Boldt a , M.I.N. Sanchez a , E.R.S. Stahlke b , I.J.T. MessiasReason a a Laboratório de Imunopatologia Molecular – Hospital de Clínicas, UFPR, Curitiba, PR, Brazil b Departamento Estadual de Saúde do Paraná, Curitiba, PR, Brazil
Background: Mycobacterium leprae exploits complement activation and opsonophagocytosis in order to infect phagocytes. Ficolin 1 (FCN-1, M-ficolin) initiates the lectin pathway on monocyte surfaces and presents high interindividual variability in serum levels. Promoter polymorphisms could be partly responsible for this variability and modulate disease susceptibility. Methods: We genotyped the FCN1 promoter in 227 controls (C) and 227 leprosy patients (L) from Southern Brazil, being 135 lepromatous, 34 borderline, 23 tuberculoid and 14 indeterminate (21 unspecified). We genotyped rs2989727 (−1981G/A), rs10120023 (−542G/A) and rs10117466 (−144C/A) using PCR with sequence-specific primers (SSPs). Results. Genotype distributions were in Hardy–Weinberg equilibrium, but different between C and L (P = 0.034). The frequency of the −542A−144A/−544A−144C genotype was higher among C (3.52% or 8/227 × 0% or 0/2227, P = 0.004), as well as the frequency of the −542A−144C diplotype (3.74% or 17/454 vs. 1.1% or 5/454, P = 0.01, OR = 0.298 [CI95% = 0.109–0.814]). Frequencies did not differ between leprosy conditions. The −542 and −144 polymorphisms were further predicted to modify transcription factor binding sites in silico. Since the −542A−144C diplotype does not favor recognition by oncogene products, it may reduce FCN1 concentration at the membrane and in serum, disincouraging mycobacterial infection. Conclusions: This pioneering work points to the importance of FCN1 promoter polymorphisms in modulating susceptibility to leprosy, as an innate immune receptor. The functional relevance of −542−144 diplotypes was inferred and should be addressed in further functional studies. doi:10.1016/j.molimm.2010.05.073
153 Multiplex sequence-specific PCR reveals association of different masp2 haplotypes with leprosy A.B.W. Boldt a , E.R.S. Stahlke b , I.J.T. Messias-Reason a a
Laboratório de Imunopatologia Molecular – Departamento de Patologia Médica- Hospital de Clínicas, UFPR, Curitiba, PR, Brazil b Departamento Estadual de Saúde do Paraná – Curitiba, PR, Brazil
Leprosy is an ancient disease that comprises a wide range of manifestations. Mannose-binding lectin (MBL) and ficolins (FCN) may enhance the attachment and ingestion of Mycobacterium leprae and other intracellular pathogens that exploit activation of the MBL-associated serine protease 2 (MASP-2) to invade phagocytes. In a previous study, we identified an association of low MBL levels and corresponding MBL2 haplotypes with protection against lepromatous and borderline leprosy. In this work, we investigated MASP2 polymorphisms (SNPs other than p.D120G) in 234 leprosy patients (L) and 234 controls (C) with similar socioeconomic status and ethnic background from Southern Brazil. Patients were diagnosed according to Ridley and Jopling criteria and the study was approved by the local medical ethics committee. We simultaneously genotyped rs7548659 SNP in the MASP2 promoter, p.R99Q and p.P126L in exon 3, rs17409276 in intron 8, p.D371Y and p.V377A in exon 9, p.R439H and rs1782455 (p.S493syn) in exon 11 using a new multiplex PCR strategy with sequence-specific primers. MASP-2 concentration and complement activation were verified by ELISA with selected genotypes. Genotype distributions were in Hardy and Weinberg equilibrium, but significantly different between C and L (P = 0.015). The frequency of MASP2 genotypes producing intermediate MASP-2 levels (CRPCYVRT and haplotypes without p.V377A or p.R99Q) was higher among C (34/234 vs. 16/234, respectively, P = 0.005, OR = 0.43 [95%CI = 0.23-0.81]). Low MASP-2 producing genotypes (with ARPCYVRT and another haplotype containing either the p.V377A or p.R439H alleles) were found associated with lepromatous leprosy, as opposed to tuberculoid and indeterminate forms (13/139 vs. 0/40, respectively, P = 0.033). In contrast to MBL, lower MASP-2 levels could therefore increase susceptibility to more severe forms of leprosy, whereas intermediate levels may protect against the disease. Financial support: CAPES and CNPq. doi:10.1016/j.molimm.2010.05.074 154 Genotypic diversity of complement component C4 and kidney transplantation outcome Markus Wahrmann a , Bernd Döhler d , Andrea Ruthenstroth d , Helmuth Haslacher b , Thomas Perkmann b , Markus Exner b , Andrew J. Rees c , Georg A. Böhmig a a
Department of Medicine III, Medical University of Vienna, Vienna, Austria b Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria c Department of Pathology, Medical University of Vienna, Vienna, Austria d Department of Transplantation Immunology, University of Heidelberg, Heidelberg, Germany Background: Clinical and experimental data, including the observation of tight associations between capillary complement C4 split product deposition (C4d) and transplant injury, have suggested a major pathogenic role of complement as a key effector of organ allograft rejection. As gene copy number (GCN) varia-
Abstracts / Molecular Immunology 47 (2010) 2198–2294
and undetectable C1r. C3 and C4 protein levels were 2156 g/mL (ref: 1045–1670 g/mL) and 2238 g/mL (ref: 445–740 g/mL), respectively. Immunofixation experiments confirmed high levels of C4A and C4B and elevated C3F and C3S proteins in the patient and his mother. The coding regions of the C1R genes from the patient were PCR-amplified and sequenced. A C to T mutation at position 6392 in exon 10 of the C1R gene changed Arg-380 to a stop codon was revealed. A sequence-specific-primer PCR assay was developed to screen for the novel C1R mutation. The mother is heterozygous for this mutation. However, the R380X mutation was not detectable in 181 unrelated African American SLE patients. Similar to homozygous C1q or C4 deficiencies, C1r-deficient patients develop SLE. Unlike most SLE patients, C4 and C3 protein levels in C1r-deficiency patients are consistently high. Mutations leading to early complement component deficiencies and SLE, with the exception of the 2-bp insertion into codon-1213 of C4A, are observable mainly in probands’ families or isolated populations. Such observations support the concept that genetic variants with large effect for a common disease only exist at very low frequencies in the population. doi:10.1016/j.molimm.2010.05.072 152 Association of Ficolin 1 promoter polymorphisms with clinical leprosy A.B.W. Boldt a , M.I.N. Sanchez a , E.R.S. Stahlke b , I.J.T. MessiasReason a a Laboratório de Imunopatologia Molecular – Hospital de Clínicas, UFPR, Curitiba, PR, Brazil b Departamento Estadual de Saúde do Paraná, Curitiba, PR, Brazil
Background: Mycobacterium leprae exploits complement activation and opsonophagocytosis in order to infect phagocytes. Ficolin 1 (FCN-1, M-ficolin) initiates the lectin pathway on monocyte surfaces and presents high interindividual variability in serum levels. Promoter polymorphisms could be partly responsible for this variability and modulate disease susceptibility. Methods: We genotyped the FCN1 promoter in 227 controls (C) and 227 leprosy patients (L) from Southern Brazil, being 135 lepromatous, 34 borderline, 23 tuberculoid and 14 indeterminate (21 unspecified). We genotyped rs2989727 (−1981G/A), rs10120023 (−542G/A) and rs10117466 (−144C/A) using PCR with sequence-specific primers (SSPs). Results. Genotype distributions were in Hardy–Weinberg equilibrium, but different between C and L (P = 0.034). The frequency of the −542A−144A/−544A−144C genotype was higher among C (3.52% or 8/227 × 0% or 0/2227, P = 0.004), as well as the frequency of the −542A−144C diplotype (3.74% or 17/454 vs. 1.1% or 5/454, P = 0.01, OR = 0.298 [CI95% = 0.109–0.814]). Frequencies did not differ between leprosy conditions. The −542 and −144 polymorphisms were further predicted to modify transcription factor binding sites in silico. Since the −542A−144C diplotype does not favor recognition by oncogene products, it may reduce FCN1 concentration at the membrane and in serum, disincouraging mycobacterial infection. Conclusions: This pioneering work points to the importance of FCN1 promoter polymorphisms in modulating susceptibility to leprosy, as an innate immune receptor. The functional relevance of −542−144 diplotypes was inferred and should be addressed in further functional studies. doi:10.1016/j.molimm.2010.05.073
153 Multiplex sequence-specific PCR reveals association of different masp2 haplotypes with leprosy A.B.W. Boldt a , E.R.S. Stahlke b , I.J.T. Messias-Reason a a
Laboratório de Imunopatologia Molecular – Departamento de Patologia Médica- Hospital de Clínicas, UFPR, Curitiba, PR, Brazil b Departamento Estadual de Saúde do Paraná – Curitiba, PR, Brazil
Leprosy is an ancient disease that comprises a wide range of manifestations. Mannose-binding lectin (MBL) and ficolins (FCN) may enhance the attachment and ingestion of Mycobacterium leprae and other intracellular pathogens that exploit activation of the MBL-associated serine protease 2 (MASP-2) to invade phagocytes. In a previous study, we identified an association of low MBL levels and corresponding MBL2 haplotypes with protection against lepromatous and borderline leprosy. In this work, we investigated MASP2 polymorphisms (SNPs other than p.D120G) in 234 leprosy patients (L) and 234 controls (C) with similar socioeconomic status and ethnic background from Southern Brazil. Patients were diagnosed according to Ridley and Jopling criteria and the study was approved by the local medical ethics committee. We simultaneously genotyped rs7548659 SNP in the MASP2 promoter, p.R99Q and p.P126L in exon 3, rs17409276 in intron 8, p.D371Y and p.V377A in exon 9, p.R439H and rs1782455 (p.S493syn) in exon 11 using a new multiplex PCR strategy with sequence-specific primers. MASP-2 concentration and complement activation were verified by ELISA with selected genotypes. Genotype distributions were in Hardy and Weinberg equilibrium, but significantly different between C and L (P = 0.015). The frequency of MASP2 genotypes producing intermediate MASP-2 levels (CRPCYVRT and haplotypes without p.V377A or p.R99Q) was higher among C (34/234 vs. 16/234, respectively, P = 0.005, OR = 0.43 [95%CI = 0.23-0.81]). Low MASP-2 producing genotypes (with ARPCYVRT and another haplotype containing either the p.V377A or p.R439H alleles) were found associated with lepromatous leprosy, as opposed to tuberculoid and indeterminate forms (13/139 vs. 0/40, respectively, P = 0.033). In contrast to MBL, lower MASP-2 levels could therefore increase susceptibility to more severe forms of leprosy, whereas intermediate levels may protect against the disease. Financial support: CAPES and CNPq. doi:10.1016/j.molimm.2010.05.074 154 Genotypic diversity of complement component C4 and kidney transplantation outcome Markus Wahrmann a , Bernd Döhler d , Andrea Ruthenstroth d , Helmuth Haslacher b , Thomas Perkmann b , Markus Exner b , Andrew J. Rees c , Georg A. Böhmig a a
Department of Medicine III, Medical University of Vienna, Vienna, Austria b Department of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria c Department of Pathology, Medical University of Vienna, Vienna, Austria d Department of Transplantation Immunology, University of Heidelberg, Heidelberg, Germany Background: Clinical and experimental data, including the observation of tight associations between capillary complement C4 split product deposition (C4d) and transplant injury, have suggested a major pathogenic role of complement as a key effector of organ allograft rejection. As gene copy number (GCN) varia-