PCR-restriction fragment length polymorphism method to detect the X/Y polymorphism in the promoter site of the mannose-binding lectin gene.

To the Editor: Mannose-binding lectin (MBL), a pattern-recognition molecule produced by the liver and present in serum, is an important player in the innate immune system. MBL acts by binding various carbohydrate structures on microbial surfaces, after which it activates the complement system via the lectin pathway. In addition, MBL can promote direct opsonophagocytosis of microorganisms and modulate diverse inflam-matory mediators (1). Deficiency of MBL was first identified in association with a common defect of opsonization in children. Additional studies have identified MBL deficiency as a risk factor for diverse infectious diseases (1). In addition , MBL deficiency has been found to be associated with certain autoimmune diseases (1) and, recently , atherosclerosis (2). MBL deficiency is caused by mutations in the coding and promoter regions of the MBL gene, which have a profound effect on plasma concentrations of the MBL protein. Three point mutations have been found in the structural region of the MBL gene: at codons 54, 52, and 57 in exon 1. These mutations cause allelic variants designated B, C, and D, respectively. In addition to these exon 1 mutations, there are three major polymorphisms in the promoter region of the MBL gene, which are expressed as four haplotypes called HYP, LYP, LXP, and LYQ. The X/Y polymorphism at position Ϫ221 is important because the X haplotype is highly prevalent [ϳ24% in Caucasian populations (3)] and is associated with low circulating MBL concentrations , comparable to those of the structural variants B, C, and D (4). Therefore, determination of the exon 1 point mutations and the X/Y promoter polymorphism is adequate to assess MBL deficiency in a certain individual. Several methods have been described for the detection of mutations in the structural region, such as PCR followed by restriction fragment length polymorphism (RFLP) analysis (4). This method is simple and gives unambiguous results, but such a method has not been published for the detection of the X/Y promoter polymorphism mentioned above. Here we describe a PCR-RFLP method for the X/Y polymorphism. The method fits well with the PCR-RFLP method we implemented for the detection of the three point mutations in exon 1. PCR was carried out in a final volume of 50 ␮L containing 1 ␮L (ϳ30 –100 ng) of genomic DNA, 2.5 mM MgCl 2 , 50 ␮M deoxynucleotide triphosphates, 0.4 ␮M upstream primer (5Ј-GTTTCCACTCATTCTC-ATTCCCTAAG-3Ј), 0.4 ␮M downstream primer (5Ј-GAAAACTCAG-GGAAGGTTAATCTCAG-3Ј), and 1 U of AmpliTaq Gold …