Rapid genotyping of MBL2 gene mutations using real-time PCR with fluorescent hybridisation probes

Steffensen, Rudi; Hoffmann, Kerstin; Varming, Kim

doi:10.1016/s0022-1759(03)00190-x

Cited by 49 publications

(44 citation statements)

References 30 publications

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“…Multiplex real-time polymerase chain reaction (PCR) was used to genotype for polymorphisms at codons 52, 54 and 57 in exon 1 of MBL2. 26 A new genotyping method, based on the 5 0 nuclease (TaqMan) assay in combination with minor-groove-binder (MGB) probes, was used for screening for the promotor polymorphisms. A variant of this method was previously used by others to discriminate alleles of MBL2.…”

Section: Methodsmentioning

confidence: 99%

MBL2 polymorphism and risk of severe infections in multiple myeloma patients receiving high-dose melphalan and autologous stem cell transplantation

Mølle

Peterslund

Thiel

et al. 2006

Bone Marrow Transplant

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Severe infections related to treatment are common in patients with multiple myeloma (MM). Genetic polymorphisms of the immune system may influence the risk of infections. Mannan-binding lectin (MBL) is part of the innate immune system, and individuals homozygous for wild-type MBL encoding gene (MBL2) have a wellfunctioning MBL pathway of complement activation, in contrast to individuals carrying one or two variant alleles. We evaluated 113 courses of high-dose melphalan and autologous stem cell transplantation (ASCT) in patients with MM. Patients homozygous for wild-type MBL2 had a significantly reduced risk of septicaemia during the ASCT procedure compared with patients carrying variant MBL2: Odds Ratio (OR) 0.19 (95% CI: 0.04-0.77), (P ¼ 0.02) in multivariate analysis. The risk of Common Toxicity Criteria grade 3-4 infections in general was not affected by wild-type MBL2: OR 1.20 (95% CI: 0.52-2.78), (P ¼ 0.67). The findings indicate that MBL to some extent protects against the most severe infections during ASCT.

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Section: Methodsmentioning

confidence: 99%

MBL2 polymorphism and risk of severe infections in multiple myeloma patients receiving high-dose melphalan and autologous stem cell transplantation

Mølle

Peterslund

Thiel

et al. 2006

Bone Marrow Transplant

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“…MBL genotyping. A newly developed real-time PCR technique was used to genotype for polymorphisms in the human MBL (MBL2) gene (21). These comprised two variations in the 5Ј regulatory region at positions Ϫ550 (H/L) and Ϫ221 (X/Y), one in the 5Ј untranslated sequence at position ϩ4 (P/Q), and three structural mutations within exon 1 at codons 52, 54, and 57, also known as the D, B, and C variants, respectively.…”

Section: Methodsmentioning

confidence: 99%

Association Between Mannose-Binding Lectin and Vascular Complications in Type 1 Diabetes

et al. 2004

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Complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. We investigated serum mannose-binding lectin (MBL) levels and polymorphisms in the MBL gene in type 1 diabetic patients with and without diabetic nephropathy and associated macrovascular complications. Polymorphisms in the MBL gene and serum MBL levels were determined in 199 type 1 diabetic patients with overt nephropathy and 192 type 1 diabetic patients with persistent normoalbuminuria matched for age, sex, and duration of diabetes, as well as in 100 healthy control subjects. The frequencies of high-and low-expression MBL genotypes were similar in patients with type 1 diabetic and healthy control subjects. High MBL genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy given a high MBL genotype assessed by odds ratio (OR) was 1.52 (1.02-2.27, P ‫؍‬ 0.04). Median serum MBL concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria: 2,306 g/l (interquartile range [IQR] 753-4,867 g/l) vs. 1,491 g/l (577-2,944 g/l), P ‫؍‬ 0.0003. In addition, even when comparing patients with identical genotypes, serum MBL levels were higher in the nephropathy group than in the normoalbuminuric group. Patients with a history of cardiovascular disease had significantly elevated MBL levels independent of nephropathy status (3,178 g/l [IQR 636 -5,231 g/l] vs. 1,741 g/l [656 -3,149 g/l], P ‫؍‬ 0.02). The differences in MBL levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high MBL genotypes (P < 0.0001). Our findings suggest that MBL may be involved in the pathogenesis of micro-and macrovascular complications in type 1 diabetes, and that determination of MBL status might be used to identify patients at increased risk of developing these complications. Diabetes 53

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“…Assays have been developed for MBL2 genotyping in the past employing amplicon melting curve analysis with SybrGreenI (Hladnik et al, 2002;Arraes et al, 2006) and fluorescent hybridization probes (Steffensen et al, 2003). While fluorescent hybridization probes (Steffensen et al, 2003) in combination with HRMA should be able to resolve all MBL2 variants, this method uses expensive fluorophores. Furthermore, whole amplicon melting to complement the probe data is not possible.…”

Section: Discussionmentioning

confidence: 99%

High-throughput genotyping of mannose-binding lectin variants using high-resolution DNA-melting analysis

et al. 2010

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High Resolution Melting Analysis (HRMA) is a rapid and sensitive method for single nucleotide polymorphism (SNP) analysis. In the present study we present a novel HRMA assay to detect three SNPs in close proximity of each other in the first exon of the gene encoding mannosebinding lectin (MBL), a key molecule of innate immunity. These SNPs have been selected for their known biological and clinical relevance. The three SNPs in MBL2 were simultaneously determined in sixty-nine human DNA samples using HRMA and a single non-fluorescent melting probe, without any post-PCR processing of samples. Combining analyses from amplicon melting and probe melting, we have been able to discriminate ten exon 1 MBL2 genotypes with HRMA, making it a suitable tool for MBL genotyping. A second HRMA assay is presented to detect a relevant polymorphism (Y/X SNP) in the MBL2 promoter region. In conclusion, HRMA is a closed tube assay that is easy to setup and lends itself perfectly for high throughput genotyping of MBL2 variants. The present study thereby facilitates further clinical studies into the role of MBL in inflammatory and infectious disease.

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Rapid genotyping of MBL2 gene mutations using real-time PCR with fluorescent hybridisation probes

Cited by 49 publications

References 30 publications

MBL2 polymorphism and risk of severe infections in multiple myeloma patients receiving high-dose melphalan and autologous stem cell transplantation

MBL2 polymorphism and risk of severe infections in multiple myeloma patients receiving high-dose melphalan and autologous stem cell transplantation

Association Between Mannose-Binding Lectin and Vascular Complications in Type 1 Diabetes

High-throughput genotyping of mannose-binding lectin variants using high-resolution DNA-melting analysis

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