A Novel Insertional Mutation and Differentially Spliced mRNAs in the Human BRCA1 Gene

Abstract

In familial cases of mammary and ovarian carcinomas, particularly in those occurring at an early age, numerous mutations have been described in the human BRCA1 and BRCA2 genes located on chromosomes 17q21 and 13q12.3, respectively. We have identified a Caucasian family with several members suffering from mammary and/or ovarian carcinomas, most of them before the age of 50. The joint occurrence of several other carcinomas in this family, e.g., gastric carcinoma or hypernephromas, alerted our interest to study possible molecular causes. The index patient had breast cancer at age 26, bilateral cancer of the ovaries and Fallopian tubes at age 37, and hypernephroma at 46 years. From DNA of the index patient and her mother, we PCR-amplified and determined the nucleotide sequence of all 22 exons coding for the protein and parts of the flanking introns of the BRCA1 gene. Six polymorphic, probably non-consequential nucleotide exchanges were found in exons 11, 13, and 16. In both women we detected a 10 nucleotide pair insertion in exon 6 of the BRCA1 gene. This insertion led to the truncation of the gene product beyond amino acid 82. RT-PCR experiments using oligodeoxyribonucleotide primers located in exons 2 and 10 revealed biallelic expression of the BRCA1 gene in peripheral white blood cells (PWBCs) of the index patient and of normal individuals. The results of a protein truncation test performed with either mRNA from the index patient’s PWBCs or with subcloned cDNA fragments confirmed the biallelic expression of the normal and the truncated BRCA1 alleles. When studying transcription patterns of the BRCA1 gene, we found several splicing variants in the 5′-part of the gene involving exons 3, 5, 6, 7, the first codon of exon 8, and exons 9 and 10. In some of these alternate splice products, the RING finger motif encoded by exons 3 and 5 was obliterated. This observation was also made with mRNAs from PWBCs of healthy individuals or from different human cell lines. This alternate splicing pattern is not directly relevant in eliciting the oncogenic phenotype, but may contribute to a reduction in the amount of full length BRCA1 protein below a critical level.