ApoB gene nonsense and splicing mutations in a compound heterozygote for familial hypobetalipoproteinemia.

Two novel apoB gene mutations were identified in a patient (CM) with phenotypic homozygous hypobetalipopro- teinemia. Haplotype analysis of the apoB alleles from this pa- tient and his family members revealed him to be a genetic com- pound for the disease. In contrast to previous studies of other hypobetalipoproteinemic patients, no clues existed as to where in the apoB gene the molecular defects resided. Therefore, it was necessary to characterize the apoB genes of the patient by se- quence analysis. The apoB gene contains 29 exons and is 43 kb in length. The gene encodes a 14.1 kb mRNA and a 4563 amino acid protein. Both apoB alleles from the patient were cloned via 26 sets of polymerase chain reactions (PCR). These clones con- tained a total of approximately 24 kb of apoB gene sequence, in- cluding regions 5' and 3' to the coding region, 29 exons, and the introdexon junctions. Complete DNA sequence analysis of these clones showed that each apoB allele had a mutation. In the paternal apoB allele, there was a splicing mutation. The first base of the dinucleotide consensus sequence (GT) in the 5' splice donor site in intron 5 was replaced by a T. It is likely that this base substitution interferes with proper splicing and results in the observed absence of plasma apoB. In the maternal apoB al- lele, there was a nonsense mutation. The first base of the Arg codon (CGA) at residue 412 in exon 10 was replaced by a T, resulting in a termination codon (%A). The nonsense mutation is likely to terminate translation after residue 411 resulting in a severely truncated protein only 9% of the length of B-100. The inheritance of these defective apoB alleles cosegregated with low total cholesterol levels observed in family members. One of the siblings, MM, who also presented with phenotypic homozygous hypobetalipoproteinemia, had both defective apoB alleles. Of the two other siblings, both of whom were phenotypical hetero- zygotes for the disease, one (GM) had the allele with the splicing mutation and the other OM) had the allele with the nonsense mutation. In summary, a strategy is presented for identify- ing apoB gene mutations by PCR cloning and sequencing. This is useful for analysis of defects in patients where there is no clue as to the location of the mutation. The technique has resulted in the identification of two navel apoB gene mutations.- Huang, L-S., H. Kayden, R. J. Sokol, and J. L. Breslow. ApoB gene nonsense and splicing mutations in a compound heterozygote for familial hypobetalipoproteinemia. J. Lipid Rex. 1991. 32: 1341-1348.