Detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (MBL) gene by polymerase chain reaction with sequence-specific primers

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Abstract

This study describes a new approach to the determination of all known mannan-binding lectin (MBL) mutations. The distribution of known variants of the MBL gene in a population of healthy unrelated Danes was determined and the genotype was correlated with the plasma MBL concentrations. The following genetic polymorphisms were studied: three point mutations in the promoter region at position −550 (H/L variants), −221 (X/Y variants), −70 (nt C or T), one point mutation in the 5′ untranslated (UT) region at position +4 (P/Q variants) and three point mutations located at codons 52, 54 and 57 in exon 1 of the MBL gene, at nucleotide positions 223, 230 and 239, respectively. To perform genotyping, we designed sequence specific primers for a polymerase chain reaction (PCR–SSP). PCR–SSP is a powerful technique for the discrimination of alleles resulting from single base substitutions and is a widely used technique. Another major advantage of the PCR–SSP method is its ability to determine whether sequence motifs are in cis or trans. The frequencies of variants in exon 1 obtained by PCR–SSP were completely comparable to results obtained by previously described PCR methods, restriction fragment length polymorphism (RFLP) and site-directed mutagenesis (SDM). This PCR–SSP method is performed with standard laboratory equipment and has the capacity to detect all genetic variants in 100 samples in 2 days at an estimated total cost of GBP 11 per sample. Analysing the correlation between MBL haplotype and plasma MBL levels, we confirmed that three different structural variants, B, C and D and the promoter haplotypes HY, LY and LX have a dominant effect on the concentration of MBL. The HY haplotype is associated with the highest plasma concentration, the LY haplotype with intermediate levels and the LX haplotype with the lowest levels. The LX haplotype was found to be associated with very low levels of MBL similar to those found in association with the structural B genotype. The gene frequencies of variants in the MBL gene in the Danish population studied correspond to previous reports on Caucasian populations.

Introduction

Mannan-binding lectin (MBL) is a calcium-dependent plasma lectin secreted by the liver. In vitro it acts as a defence molecule and clinical evidence suggests that it plays an important role in innate immune defence (Turner, 1996). MBL may possibly act directly as an opsonin in the clearance of various mannose-rich microorganisms by phagocytes (Nepomuceno et al., 1997), but its main activity is thought to be mediated through the activation of the complement system via the MBL pathway. This pathway is similar to the classical pathway. MBL binds to carbohydrates on microorganisms and the MBL-associated serine proteases MASP-1 and MASP-2 are activated to cleave C4 and C2 (Matsushita and Fujita, 1992, Matsushita and Fujita, 1995, Thiel et al., 1997). Low concentrations of MBL cause defective opsonisation and phagocytosis in association with recurrent infections in infants as well as in children (Sumiya et al., 1991, Summerfield et al., 1997). Mutations in the coding region of the MBL gene, which is located on chromosome 10 at q11.2–q21, affect the concentration of MBL in serum (Sastry et al., 1989). Three mutations have been found in the structural region of the molecule (codons 52, 54 and 57) giving rise to three allelic variants called D, B and C, respectively, while the wild type is called A (Lipscombe et al., 1992, Lipscombe et al., 1996, Madsen et al., 1994). The three point mutations occur at nucleotides 223 (C to T), 230 (G to A) and 239 (G to A) of exon 1 for the D, B and C alleles, respectively. This causes the substitution of arginine by cysteine at codon 52, the substitution of glycine by aspartic acid at codon 54 and the substitution of glycine by glutamic acid at codon 57. These amino acid substitutions are thought to affect the tertiary structure of the collagenous region of the protein. Among Caucasians, low MBL concentrations are largely explained by the dominant effect of the point mutation at codon 54 (the B allele) of the MBL gene (Garred et al., 1992). The gene frequency in Caucasians of this allotype is about 13%. Additional polymorphisms are found in the promoter region of the gene (Madsen et al., 1995). Two promoter variants, H and L at position 550 are in linkage disequilibrium with the X and the Y variant at position 221 and are found as three haplotypes e.g. HY, LY and LX. The HY is associated with the highest plasma levels of MBL, the LY haplotype with intermediate levels and the LX haplotype is associated with the lowest circulating plasma levels of MBL (Madsen et al., 1995).

The number of MBL haplotypes is further increased by a polymorphism at position +4 in the 5′-untranslated region (P/Q variants) and five additional base substitutions/deletions in the promoter region (including the variants at position −70 where C (cytosine) is replaced by T (thymine) in the gene (Madsen et al., 1994, Madsen et al., 1998). A strong linkage disequilibrium exists between the promotor region and exon 1 of the MBL gene and only a few of the theoretically possible MBL haplotypes have been described (HYPA, HXPA, LYPA, LYQA, LXPA, LYPB, LYQC and HYPD). Of these, the HXPA haplotype has only been described once in three black African systemic lupus erythematosus (SLE) patients (Sullivan et al., 1996). There is also, a strong linkage disequilibrium between the Q allele and the five other substitutions/deletions in the promoter region since these variations are only present in the LYQA and the LYQC haplotypes (Madsen et al., 1995, Madsen et al., 1998). The described polymorphisms in the promoter and exon 1 regions of the human MBL gene are illustrated schematically in Fig. 1.

Previously, MBL genotypes have been investigated by various PCR-based methods, e.g. restriction fragment length polymorphism (RFLP), site-directed mutagenesis (SDM), sequence-specific oligonucleotide (SSO) hybridization, nested primer or DNA hetero duplexes (Madsen et al., 1994, Madsen et al., 1995, Madsen et al., 1998, Jack et al., 1997, Ip et al., 1998). The MBL polymorphism for mutations in codons 54 and 57 has previously been investigated by allele specific PCR, e.g. by amplification refractory mutation system–polymerase chain reaction (PCR–ARMS) (Sullivan et al., 1996, Davies et al., 1998). The aim of this study was to develop a rapid, simple and reliable method for genotyping individuals for all known mutations within the promoter and exon 1, including determination of the cis/trans location of alleles H, L, Y and X in those individuals carrying all four promoter alleles using as few PCR reactions as possible. With this method the problems encountered by previous workers using PCR–SSP for the codon 52 variants in exon 1 and the H, P and Q alleles in the promoter region have been solved by improved primer design and very refined PCR conditions according to previous observations on primer design in general (Kwok et al., 1990). The method developed is characterized by high capacity and low cost and permits the determination of a complete genotype for each individual. In this study, the method was used to determine the influence of the genetic variants on the MBL plasma concentration. To validate the test, we compared the genotype derived by PCR–SSP with the results obtained by previously described PCR–RFLP and PCR–SDM methods for the three mutations in exon 1 (Madsen et al., 1994).

Section snippets

Samples

Blood samples from 100 randomly selected Danish blood donors were collected into EDTA and genomic DNA was extracted by a simple salting-out method (Miller et al., 1988). In brief, nucleated cells were first lysed and digested using a protease K solution. Cellular proteins were then precipitated with saturated NaCl, before the DNA was precipitated with absolute ethanol.

Assay for plasma levels of MBL

Plasma levels of MBL were determined by a time-resolved immunofluorometric assay (TRIMA) as previously described (Christiansen

Results

The gene frequencies of each of the known polymorphic variants in the MBL gene were determined for the Danish population using the PCR–SSP technique and a total of 17 different PCR reactions with two different sets of control primers. However, for routine screening 12 PCR reactions are sufficient since C, T, cis/trans HY, cis/trans LY cis/trans LX may be eliminated as a result of the strong linkage. Fig. 2 shows representative results of agarose gel electrophoresis of the amplified products.

Discussion

In this study, MBL genotypes and plasma concentrations were determined in 100 normal, unrelated Danes. MBL genotyping was performed by PCR with newly developed sequence-specific primers (SSP) which has proven to be a powerful technique for discrimination between alleles arising from single or multiple-base substitutions. In particular, the requirements for restriction endonuclease digestion are circumvented. The method relies on the fact that the Taq DNA polymerase used in the PCR cannot repair

Acknowledgements

This work was supported by grants from Aalborg Frivillige Blodonoreres Fond, Det Obelske Familiefond and Nordjyllands Amts Forskningsfond.

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