Simultaneous genotyping for all three known structural mutations in the human mannose-binding lectin gene

We describe a rapid and simple method for genotyping the three known structural mutations within exon 1 of the mannan-binding lectin (MBL) gene. A PCR-amplifiable synthetic DNA (Universal Heteroduplex Generator) was annealed to genomic PCR product from exon 1 to generate unique DNA heteroduplexes for each mutation. Heteroduplexes were then resolved by non-denaturing polyacrylamide gel electrophoresis. The technique was initially validated with previously typed samples and then applied to previously untyped samples with the results confirmed by DNA sequencing. © 1997 Wiley-Liss, Inc.     

A new strategy for mannose-binding lectin gene haplotyping

The mannose-binding lectin 2 (MBL2) gene is polymorphic and codes for a protein with an important role in the innate immune response, whose variants have been associated with a great number of diseases. Point variations have been described in the 5' regulatory region at positions -550 (MBL2*H or *L) and -221 (*X or *Y), in the 5' untranslated sequence at position +4 (*P or *Q), and in the coding sequence of exon 1 at codons 52, 54, and 57 (MBL2*A or D, A or B, and A or C, respectively). These can be in cis or in trans configuration. The different haplotypes influence the immunological phenotype of the individual, which makes MBL2 haplotyping very important. Previously described MBL2-typing methods do not present adequate haplotype resolution or are too complex and costly. We have developed a new MBL2-typing strategy that is economical and renders rapid and reliable results without ambiguities. We typed 202 individuals of European, 32 of African, and 16 of Oriental descent. Only five to six reactions from 10 possible PCR-SSPs (sequence-specific polymerase chain reactions) were sufficient to genotype one individual unambiguously. The reactions were specific for amplification of the variants located upstream of the coding sequence. The results were associated to the results of hybridizations of the amplified products with eight sequence-specific oligonucleotide probes (SSOP). The strategy led to identification of eight alleles: MBL2*HYPA, HYPD, LYPA, LYPB, LYPD, LYQA, LYQC, and LXPA. Their frequencies in each of the groups were similar to those of other populations studied to date, with MBL2*LYPD (g.[-550G>C; -221C>G; 4T>C; 223C>T; 230A>G; 239A>G]) being novel. All samples were found to be in Hardy-Weinberg equilibrium.     

Genotyping of the three major allelic variants of the human mannose‐binding lectin gene by denaturing gradient gel electrophoresis

The three major allelic variants of the mannose‐binding lectin gene are responsible for structural defects leading to immune deficiency. The corresponding mutations are all located within exon 1 and result in amino acid substitutions in the collagenous region of the protein, which is involved in the oligomerization process. We have developed a simple and efficient strategy that permits simultaneous genotyping of these known allelic variants of the MBL gene by means of a single polymerase chain reaction (PCR) reaction followed by a denaturing gradient gel electrophoresis (DGGE). In addition, this procedure also allows for screening novel alleles due to mutations located elsewhere in the analyzed segment of the gene. During this study, we identified a previously undescribed nucleotide change in exon 1 at codon 44. Hum Mutat 14:80–83, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of mannose-binding lectin as a mechanism in progressive immunoglobulin A nephropathy

Immunoglobulin A nephropathy (IgAN), the pathogenesis of which remained still unclear is one of the leading courses of end-stage renal disease in approximately 50% affected patients. On the basis of several researches, the activation of complement mannose-binding lectin (MBL) pathway might be the underlying mechanism in disease progress. In order to investigate the relationship between MBL pathway and IgAN, we discussed the MBL gene polymorphism as well as its expressed level in serum, urine and renal parenchymal, with renal outcome in IgAN patients. The significantly down-regulated expression of MBL was discovered, which may serve as a potential urinary biomarker in progressive IgAN according to the results of difference in gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The single nucleotide polymorphisms of MBL gene in promoter and exon region were found and confirmed relating with the poor prognosis of progressive IgAN patients. As a result, the deficient activation of MBL pathway caused by the mutation of MBL accompanied with low expressed level of MBL in serum might be the potential inspiring regulation in IgAN, and will attract a promising insight in remedy of IgAN to inhibit further progress.     

汉族儿童MBL基因启动子-221位点、外显子I区SNP和基因单体型分析

研究浙江省汉族儿童MBL基因启动子-221位点和外显子I区单核苷酸多态性(SNP)、基因单体型与血浆MBL水平的关系。血浆MBL浓度的检测采用ELISA法,MBL基因SNP分析采用序列分析法,基因单体型分析采用SHEsis软件,统计分析采用SPSS软件11.0版。结果105例汉族儿童中MBL基因启动子区-221位点X/Y和Y/Y基因型分别占19%和81%,该位点变异频率为0.095;外显子I基因型A/A、A/B和B/B分别占69.5%、27.6%和2.9%,B型变异频率为0.167。基因单体型有YA、YB、XA三种,其中以YA最多见,频率为0.741,其次是YB,频率为0.164。血浆MBL浓度范围为3~6 025 ng/ml,中位数为1057 ng/ml,外显子A/A型的MBL浓度显著高于A/B型,后者又显著高于B/B型;而启动子Y/Y型的MBL浓度比X/Y型高。本研究中汉族儿童MBL基因外显子I区+230位点的变异频率为0.167,外显子I区+230位点变异可导致血浆MBL水平明显下降。     

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

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 mannose‐binding 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. © 2010 Wiley‐Liss, Inc.     

Differential recognition of obligate anaerobic bacteria by human mannose-binding lectin.

Deficiency of the innate, humoral immune component mannose-binding lectin (MBL) predisposes individuals to a variety of infections, but the importance of MBL in infection by anaerobes has not been addressed. The attachment of MBL to a wide range of anaerobic bacteria associated with human disease and colonization was surveyed. The results suggest that for the species we examined, resistance to MBL binding may be associated with organisms that are more commonly pathogenic and that MBL binding to some bacteria may be phase variable.     

From gels to chips: “Minisequencing” primer extension for analysis of point mutations and single nucleotide polymorphisms

In the minisequencing primer extension reaction, a DNA polymerase is used specifically to extend a primer that anneals immediately adjacent to the nucleotide position to be analyzed with a single labeled nucleoside triphospate complementary to the nucleotide at the variant site. The reaction allows highly specific detection of point mutations and single nucleotide polymorphisms (SNPs). Because all SNPs can be analyzed with high specificty at the same reaction conditions, minisequencing is a promising reaction principle for multiplex high‐throughput genotyping assays. It is also a useful tool for accurate quantitative PCR‐based analysis. This review discusses the different approaches, ranging from traditional gel‐based formats to multiplex detection on microarrays that have been developed and applied to minisequencing assays. Hum Mutat 13:1–10, 1999. © 1999 Wiley‐Liss, Inc.     

幼年特发性关节炎患儿甘露糖结合凝集素基因启动子区SNP研究

目的探讨甘露糖结合凝集素(MBL)基因启动子区单核苷酸多态性(SNP)与幼年特发性关节炎(JIA)易感性的关系。方法对50例JIA患儿和48名正常健康儿童MBL基因启动子区SNP位点-550(G/C,称H/L等位基因)和-221(G/C,称X/Y等位基因)采用等位基因特异性PCR法(PCR-SSP)检测,并分析其单元型及基因型频率。结果共检出HY、LY和LX三种单元型,在JIA患儿中的频率依次为0.540、0.270和0.190,而在正常儿童中频率分别为0.594、0.292和0.114;两组间各单元型比较均无显著性差异。结论MBL基因启动子区单核苷酸多态性与JIA无相关性。     

Design of a complement mannose-binding lectin pathway-specific activation system applicable at low serum dilutions

Recently we showed that alternative pathway (AP) amplification was responsible for more than 80% of specific classical pathway-induced terminal pathway activation under physiological conditions. The present study aimed to design a system for specific lectin pathway (LP) activation applicable at low serum dilutions with a fully functional AP. Comparison between activation of normal human serum (NHS), a mannose-binding lectin (MBL) homozygous D/D-deficient serum, and sera deficient in C1q and C2, all diluted 1 : 2, was essential to document optimal conditions for LP specificity. Mannan on the solid phase of enzyme-linked immunosorbent assay (ELISA) plates was used for activation, showing 0.5 microg mannan/well to give optimal conditions because at this concentration a good signal was preserved for C4 and TCC deposition in NHS, whereas the C3 deposition observed in C2-deficient serum at higher mannan concentrations reached nadir at 0.5 microg/well, indicating a lack of direct AP activation under these conditions. Pooled NHS and C1q-deficient serum gave the same degree of C4 and terminal complement complex (TCC) deposition, whereas deposition of these products was not obtained with MBL-deficient serum. Reconstitution with purified MBL, however, restored the depositions. A blocking anti-MBL monoclonal antibody (mAb) completely abolished the complement deposition, in contrast to a non-inhibiting anti-MBL mAb. Activation of C2-deficient serum induced C4 deposition similar to NHS, but negligible deposition of C3 and TCC, confirming the lack of direct activation of AP. Thus, this assay is unique in being LP-specific at low serum dilution and thus particularly suitable to study LP activation mechanisms and the role of AP amplification under physiological conditions.     

A role for mannose-binding lectin, a component of the innate immune system in pre-eclampsia

Problem Mannose‐binding lectin (MBL) is a pattern‐recognition receptor that activates complement and modulates inflammation. Homozygosity for the most common allele of the MBL2 gene that is associated with high MBL serum concentrations is more prevalent among patients with pre‐eclampsia. The objective of this study was to determine maternal plasma MBL concentrations in normal pregnant women and patients with pre‐eclampsia. Method of study This cross‐sectional study included normal pregnant women (n = 187) and patients with pre‐eclampsia (n = 99). Maternal plasma MBL concentrations were determined by ELISA. Results Women with pre‐eclampsia had a higher median maternal plasma MBL concentration than normal pregnant women. MBL concentration distribution curves were three‐modal, the subintervals in normal pregnancy were low (<143.7), intermediate (143.7–1898.9) and high (>1898.9 ng/mL). The proportion of normal pregnant women was larger in the low subinterval, while the proportion of patients with pre‐eclampsia was larger in the high subinterval (P = 0.02). Normal pregnant women in the high subinterval had a larger rate of placental underperfusion than those in the low and intermediate subintervals (P = 0.02). Conclusions The median maternal plasma MBL concentration is elevated in patients with pre‐eclampsia and a larger proportion of these patients are in the high subinterval than normal pregnant women, suggesting that this component of the innate immune system is involved in the mechanisms of disease in pre‐eclampsia.     

Significantly increased levels of mannose-binding lectin (MBL) in rheumatic heart disease: a beneficial role for MBL deficiency

Although mannose-binding lectin (MBL) is known to be involved in the primary defense against microorganisms, there are emerging lines of evidence for an active proinflammatory role for MBL in different chronic diseases. In this study we determined the circulating levels of MBL in patients with rheumatic heart disease (RHD). A total of 100 patients (77 women, 23 men; mean age 45.8 +/- 11 years, range 19-76 years) with chronic RHD, and a previous diagnosis of rheumatic fever, were studied. Transthoracic echocardiography was performed in all patients to evaluate valvular heart disease. Ninety-nine healthy individuals matched for age, sex and ethnic origin were included as controls. MBL concentration was measured by enzyme-linked immunosorbent assay and C3 and C4 levels by turbidimetry. MBL levels were significantly higher in patients with RHD than in healthy subjects (mean +/- SEM: 3036.2 +/- 298.9 ng/ml versus 1942.6 +/- 185.5 ng/ml, P <0.003). In addition, MBL deficiency was more prevalent in controls (17.1%) than in patients (9% P <0.09). Concentrations of C4 were within the normal range (22.7 +/- 0.8 mg/dl, normal: 10.0-40.0 mg/dl), while C3 concentrations were found to be elevated (109.2 +/- 3.6 mg/dl, normal: 50.0-90.0 mg/dl). No correlation was observed between serum MBL levels and valve area or the type of surgical procedure. The significantly elevated circulating MBL levels in patients with RHD together with the greater prevalence of MBL deficiency in controls suggest that MBL may cause undesirable complement activation contributing to the pathogenesis of RHD.     

Validation of the use of DNA pools and primer extension in association studies of sporadic colorectal cancer for selection of candidate SNPs

Colorectal cancer (CRC) is a multifactorial disease that involves both lifestyle and genetic factors. To identify single nucleotide polymorphisms (SNPs) associated with sporadic CRC, we used pooled DNA samples representing 230 cases with sporadic CRC and 540 controls. The allele frequency of the SNPs was estimated in the two pools using a genotyping method based on primer extension and capillary electrophoresis (CE). The sensitivity of the method was high, which permitted the detection of an odds ratio (OR) of 1.5. Validation of the method showed that it is robust, linear, sensitive, and reproducible. Of the 224 SNPs investigated, 20 potential candidates associated with CRC were identified, including IL6 -174G>C (g.22062318G>C), XRCC1 c.685 C>T (p.Arg194Trp), PPARGC1A g.92945042C>T (3'UTR 96516), GSTP1 c.342A>C (p.Ile105Val), GSTM1 c.573C>G (p.Lys173Asn), and SULT1A1 g.19934792G>A (p.Arg213His). All were borderline significant, and none were significant at the 5% level. A high number of the SNPs (40%) were not polymorphic in our population. We conclude that instead of looking for single risk factors, investigators should examine individual combinations of potential risk factors to clarify the genetic predisposition to CRC.     

Antibody-mediated activation of the classical pathway of complement may compensate for mannose-binding lectin deficiency

Deficiency of mannose-binding lectin (MBL), a recognition molecule of the lectin pathway of complement, is associated with increased susceptibility to infections. The high frequency of MBL deficiency suggests that defective MBL-mediated innate immunity can be compensated by alternative defense strategies. To examine this hypothesis, complement activation by MBL-binding ligands was studied. The results show that the prototypic MBL ligand mannan can induce complement activation via both the lectin pathway and the classical pathway. Furthermore, antibody binding to mannan restored complement activation in MBL-deficient serum in a C1q-dependent manner. Cooperation between the classical pathway and the lectin pathway was also observed for complement activation by protein 60 from Listeria monocytogenes. MBL pathway analysis at the levels of C4 and C5b-9 in the presence of classical pathway inhibition revealed a large variation of MBL pathway activity, depending on mbl2 gene polymorphisms. MBL pathway dysfunction in variant allele carriers is associated with reduced MBL ligand binding and a relative increase of low-molecular-mass MBL. These findings indicate that antibody-mediated classical pathway activation can compensate for impaired target opsonization via the MBL pathway in MBL-deficient individuals, and imply that MBL deficiency may become clinically relevant in absence of a concomitant adaptive immune response.     

Dry-reagent disposable biosensor for visual genotyping of single nucleotide polymorphisms by oligonucleotide ligation reaction: application to pharmacogenetic analysis

Most genotyping methods for known single-nucleotide polymorphisms (SNPs) are based on hybridization with allele-specific probes, oligonucleotide ligation reaction (OLR), primer extension or invasive cleavage. OLR offers superior specificity because it involves two recognition events; namely, the hybridization of an allele-specific probe and a common probe to adjacent positions on target DNA. OLR products can be detected by microtiter well-based colorimetric, time-resolved fluorimetric or chemiluminometric assays, electrophoresis, microarrays, microspheres, and homogeneous fluorimetric or colorimetric assays. We have developed a simple, robust, and low-cost disposable biosensor in dry-reagent format, which allows visual genotyping with no need for instrumentation. The OLR mixture contains a biotinylated common probe and an allele-specific probe with a (dA)(20) segment at the 3'-end. OLR products are denatured and applied to the biosensor next to gold nanoparticles that are decorated with oligo(dT) strands. The sensor is immersed in the appropriate buffer and all components migrate by capillary action. The OLR product is captured by immobilized streptavidin at the test zone (TZ) of the sensor and hybridizes with the oligo(dT) strands of the nanoparticles. A characteristic red line is generated due to the accumulation of nanoparticles. The excess nanoparticles are captured by immobilized oligo(dA) at the control zone of the strip, giving a second red line. We have applied successfully the proposed OLR-dipstick assay to the genotyping of four SNPs in the drug-metabolizing enzyme genes CYP2D6 ((*)3 and (*)4) and CYP2C19 ((*)2 and (*)3). The results were in agreement with direct sequencing.     

New mutations,polymorphisms, and rare variants in the ATM gene detected by a novel SSCP strategy

The gene for ataxia‐telangiectasia, ATM, spans about 150 kb of genomic DNA. ATM mutations are found along the entire gene, with no evidence of a mutational hot spot. Using DNA as the starting material, we screened the ATM gene in 92 A‐T patients, using an optimized single‐strand conformation polymorphism (SSCP) technique that detected all previously known mutations in the polymerase chain reaction (PCR) segments being analyzed. To expedite screening, we sequentially loaded the SSCP gels with three different sets of PCR products that were pretested to avoid overlapping patterns. Many of the DNA changes we detected were intragenic polymorphisms. Of an expected 177 unknown mutations, we detected ∼70%, mostly protein truncating mutations (that would have been detectable by protein truncation testing if RNA starting material had been available). Mutations have now been defined for every exon of the ATM gene. Herein, we present 35 new mutations and 34 new intragenic polymorphisms or rare variants within the ATM gene. This is the most comprehensive compilation of ATM polymorphisms assembled to date. Defining polymorphic sites as well as mutations in the ATM gene will be of great importance in designing automated methods for detecting mutations. Hum Mutat 14:156–162, 1999. © 1999 Wiley‐Liss, Inc.     

Elevated levels of mannan-binding lectin [corrected] (MBL) and eosinophilia in patients of bronchial asthma with allergic rhinitis and allergic bronchopulmonary aspergillosis associate with a novel intronic polymorphism in MBL

Mannan-binding lectin (MBL), an important component of innate immunity, binds to a range of foreign antigens and initiates the lectin complement pathway. Earlier studies have reported high plasma MBL levels in allergic patients in comparison to healthy controls. In view of varied plasma MBL levels being determined by genetic polymorphisms in its collagen region, we investigated the association of single nucleotide polymorphisms (SNPs) in the collagen region of human MBL with respiratory allergic diseases. The study groups comprised patients of bronchial asthma with allergic rhinitis (n = 49) and allergic bronchopulmonary aspergillosis (APBA) (n = 11) and unrelated age-matched healthy controls of Indian origin (n = 84). A novel intronic SNP, G1011A of MBL, showed a significant association with both the patient groups in comparison to the controls (P < 0.01). Patients homozygous for the 1011A allele showed significantly higher plasma MBL levels and activity than those homozygous for the 1011G allele (P < 0.05). The 1011A allele also showed a significant correlation with high peripheral blood eosinophilia (P < 0.05) and low forced expiratory volume in 1 s (FEV(1)) (P < 0.05) of the patients. We conclude that the 1011A allele of MBL may contribute to elevated plasma MBL levels and activity and to increased severity of the disease markers in patients of bronchial asthma with allergic rhinitis and ABPA.     

Plasma levels of MASP‐1, MASP‐3 and MAp44 in patients with type 2 diabetes: influence of glycaemic control,body composition and polymorphisms in the MASP1 gene

Mounting evidence indicates that adverse activation of the complement system plays a role in the development of diabetic vascular complications. Plasma levels of the complement proteins mannan‐binding lectin (MBL) and its associated serine proteases (MASP‐1 and MASP‐2) are elevated in diabetes. We hypothesized that single nucleotide polymorphisms (SNPs) in the MASP1 gene may contribute to altered plasma levels of the belonging gene products; MASP‐1, MASP‐3 and mannan‐binding lectin‐associated protein of 44 kDa (MAp44) in patients with type 2 diabetes. To investigate this, we compared plasma levels of MASP‐1, MASP‐3 and MAp44 in 100 patients with type 2 diabetes and 100 sex‐ and age‐matched controls. Ten carefully selected SNPs were analysed using TaqMan^® genotyping assay. Additionally, we included a streptozotocin‐induced diabetes mouse model to directly examine the effect of inducing diabetes on MASP‐1 levels. MASP‐1 levels were significantly higher among patients with type 2 diabetes compared with healthy controls (P = 0·017). Five SNPs (rs874603, rs72549254, rs3774275, rs67143992, rs850312) in the MASP1 gene were associated with plasma levels of MASP‐1, MASP‐3 and MAp44. In the diabetes mouse model, diabetic mice had significantly higher MASP‐1 levels than control mice (P = 0·003). In conclusion, MASP‐1 levels were higher among patients with type 2 diabetes and diabetic mice. The mechanism behind this increase remains elusive.     

三维聚丙烯酰胺凝胶DNA芯片用于大样本单核苷酸多态分型的方法研究

目的 建立三维聚丙烯酰胺凝胶DNA芯片用于大样本单核苷酸多态(single nucleotide polyrnorphisms,SNP)分型的方法.方法 丙烯酰胺基团修饰的PCR产物与丙烯酰胺单体混合后,在丙烯酰胺基团修饰的玻片上点样进行共聚合,建成三维凝胶DNA阵列;芯片与一对特异探针和一对分别标记了Cy3或Cy5的通用序列标签(Tag1和Tag2)进行杂交;杂交后用施加电场的方法去除非特异吸附和错配,最后通过双色荧光共聚焦扫描进行SNP分型.结果 3-D凝胶芯片不但具有很高的固定效率,而且可以提供高效的杂交环境;通用序列标签的使用木需对每个位点都标记荧光,使检测成本大幅降低;外加电场使得单碱基错配容易识别,且能显著降低芯片的信噪比.结论 基于3-D凝胶的基因芯片技术用于大样本SNPs分型简单易操作,且高通量、高特异性、低成本,该方法将可以更广泛地应用于不同需求的DNA检测中.