A Single Point Mutation in the Splice Donor Site of the Low‐Density‐Lipoprotein‐Receptor Gene Produces Intron Read‐Through, Exon‐Skipped and Cryptic‐Site‐Utilized Transcripts

Abstract

Familial hypercholesterolemia is a genetic disorder caused by niutations of the low-density-lipoprotein (LDL) receptor gene. We characterized the structures of LDL receptor mRNA transcripts in the fibroblasts of a homozygous patient carrying a single base substitution (T-C) at the 5' splice donor site of intron 12 of the LDL receptor gene. We identified three abei~ant transcripts as a consequence of intron-I 2 readthrough, exon-12 skipping and utilization of a cryptic splice donor site. Only a point mutation at the 5' splice donor site caused the production of three alternatively spliced products. None of these transcripts produced a functional LDL receptor protein in this patient.Keywords: low-density-lipoprotein receptor ; familial hypercholesterolemia ; splicing mutation ; read through. Hobbs et al. [4J described a Japanese homozygous FH patient who expressed two different-sized transcripts of the LDL receptor gene; the patient was proved to be a true homozygote carrying a point mutation at the splice donor site of intron 12 of the LDL receptor gene 131. The sizes of the transcripts of the LDL receptor gene in this mutation were about 5.3 kb and 8.4 kb [3, 41. Although the patient expressed an apparently normalsized 5.3 kb transcript, she did not produce the LDL receptor protein that cross-reacted with the monoclonal antibody against the LDL receptor protein [4].We have frequently found the identical mutation in Japanese FH patients. The patients, KIK, YS and MY 151, were true homozygotes for this mutation, whose phenotype belonged to the so-called 'null-allele' phenotype ; neither LDL binding nor LDL receptor protein synthesis was detected in their fibroblasts.According to Robberson et al. 161, the splice acceptor and donor sites of an exon are recognized and defined as a unit by the splicing machinery. Thus, a point mutation at a splice site leads to exon skipping and/or utilization of a cryptic splice site close to the site of mutation [6, 71.As the patient with the mutation at the splice donor site of intron 12 did not produce the functional LDL receptor, the nor- ~~ mal-sized transcript of 5.3 kb found in this patient 13, 41 is expected to result from skipping of exon 12 and/or utilization of a cryptic donor site in intron 1 2 close to the 5' splice site. The production of the 8.4-kb transcript, however, is hard to explain on the basis of the described rule [6,7]. Therefore, in the present study, we analyzed the structure of the transcripts in the fibroblasts of this patient. We identified three aberrantly spliced transcription products; one contained the entire intron 12, one skipped exon 12, and one utilized n cryptic splice site in intron 12. MATERIALS AND METHODSPatient. The proband, KIK, was a patient who visited our lipid clinic in the National Cardiovascular Center for precise examination of hypercholesterolemia. She was 4 years old and had severe hypercholesterolemia. As she was born from consanguineous marriage and her cultured skin fibroblasts showed a negligible amount of LDL receptor activity...