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

This study describes a new approach to the determination of all known manna n-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 r egion 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 exo n 1 of the MBL gene, at nucleotide positions 223, 230 and 239, respectively . To perform genotyping, we designed sequence specific primers for a polyme rase chain reaction (PCR-SSP). PCR-SSP is a powerful technique for the disc rimination of alleles resulting from single base substitutions and is a wid ely used technique. Another major advantage of the PCR-SSP method is its ab ility to determine whether sequence motifs are in cis or trans. The frequen cies of variants in exon I obtained by PCR-SSP were completely comparable t o results obtained by previously described PCR methods, restriction fragmen t length polymorphism (RFLP) and site-directed mutagenesis (SDM). This PCR- SSP method is performed with standard laboratory equipment and has the capa city to detect all genetic variants in 100 samples in 2 days at an estimate d total cast of GBP 11 per sample. Analysing the correlation between MBL ha plotype 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 domi nant effect on the concentration of MEL. The HY haplotype is associated wit h the highest plasma concentration, the LY haplotype with intermediate leve ls 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 asso ciation with the structural B genotype. The gene frequencies of variants in the MBL gene in the Danish population studied correspond to previous repor ts on Caucasian populations. (C) 2000 Elsevier Science B.V. All rights rese rved.