SCREENING FOR MUTATIONS IN THE EXON-26 OF THE APOLIPOPROTEIN-B GENE IN HYPERCHOLESTEROLEMIC FINNISH FAMILIES BY THE SINGLE-STRAND CONFORMATION POLYMORPHISM METHOD

To date, the only known apolipoprotein B (ape B) mutation causing hype rcholesterolemia is the apo B 3500 Arg --> Gin or the familial defecti ve apo B (FDB) mutation. This mutation has not been detected in the Fi nnish population. We have set up a systematic single strand conformati on polymorphism (SSCP) analysis-based screening method to search for o ther mutations in the exon 26 of the apo B gene in 21 Finnish hypercho lesterolemic probands. The 7572-bp exon 26 covers half of the coding r egion of the gene including the DNA sequence coding for the putative l ow-density lipoprotein (LDL) receptor binding site on the apo B protei n. Exon 26 was amplified as six 1190- to 1435-bp fragments, each of wh ich was further split into three smaller 213 to 579-bp segments by res triction enzymes. These digestion products were run on nondenaturing p olyacrylamide gels using at least three different electrophoretic cond itions and autoradiographed. Ah previously known genetic variants in t he exon 26 were detected by the SSCP method. A C-->T change at nucleot ide 7064, in complete association with the Xbal site, was characterize d by direct sequencing. This variant did not affect the amino acid seq uence of the ape B protein. The SSCP based procedure appears suitable for systematic screening for DNA sequence changes in large coding regi ons. (C) 1994 Wiley-Liss, Inc.