The low density lipoprotein (LDL) receptors in fibroblasts from 132 subjects with the clinical syndrome of homozygous familial hypercholesterolemia were analyzed by immunoprecipitation with an anti-LDL receptor monoclonal antibody. 16 of the 132 cell strains (12%) synthesized no immunodetectable LDL receptor protein, indicating the presence of two mutant genes that failed to produce cross-reacting material (crm- mutations). DNA and mRNA from 15 of the 16 crm- patients, representing 30 crm- genes, were available for further study. Haplotype analysis based on 10 restriction fragment length polymorphisms (RFLPs) suggested that the 30 crm- genes represent 13 mutant alleles. Four of the alleles produced no mRNA. Three of these four mRNA- alleles had large deletions ranging from 6 to 20 kb that eliminated the promoter region of the gene. The fourth mRNA- allele did not contain any deletion or alteration in the promoter sequence; the reason for the mRNA- phenotype was not apparent. Nine alleles were positive for mRNAs, of which three encoded mRNAs of abnormal size. One of the abnormal mRNAs was produced by a gene harboring a deletion, and another was produced by a gene with a complex rearrangement. The third abnormal-sized mRNA (3.1 kb larger than normal) was produced by an allele that had no detectable alterations as judged by Southern blotting. The other six mRNA+ alleles appeared normal by Southern blotting and produced normal-sized mRNA but no receptor protein. The current studies demonstrate that mRNA analysis coupled with haplotype determination by Southern blot analysis can be used to classify crm- mutations at a genetic locus where multiple alleles exist.
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