Multiplex PCR assay for screening deletions in the low density lipoproteinreceptor gene

Background: In different populations of the world, more than 150 genetic al terations of the LDL receptor gene have been identified; each of which can result in hypercholesterolaemia, but no hot spots in the gene were detected so far. Because of the existence of very variable genetic alterations in d ifferent ethnic communities, none of the assays developed for screening mut ations/deletions in a population defined can be adapted to study the possib le genetic defects. The present study was designed to develop a new, multip lex PCR-based, molecular biological method to screen the whole coding regio n of the LDL receptor gene. Methods: Using primer pairs completely flanking the promoter and the entire exonal region, in the PCR reactions 83-386-bp long, DNA sequences were synthesised in seven different reaction mixtures. The reaction conditions of the multiplex PCR system were optimised in order to synthesise all exons and the promoter region of the gene using only two annealing temperatures. The products could be visualised separately by aga rose gel electrophoresis/ethidium bromide staining. Results: A rapid, effec tive test enabling the screening of DNA alterations in the entire LDL recep tor gene was developed. Using this simple multiplex PCR assay, deletions af fecting more than 10 bp in any part of the gene can be easily detected by a single agarose gel electrophoresis. Conclusions: The simplicity, specifici ty and versatility of the assay make it suitable system for routine screeni ng of LDL receptor gene mutations in large population samples. This PCR ass ay can be recommended for screening of LDL-RG deletions in populations or g roups at high risk for cardiovascular diseases. (C) 2001 Elsevier Science B ,V. All rights reserved.