BRCA2 T2722R is a deleterious allele that causes exon skipping

Abstract

Patients with a strong family history of breast cancer are often counseled to receive genetic screening for BRCA1 and BRCA2 mutations, the strongest known predictors of breast cancer. A major limitation of genetic testing is the number of inconclusive results due to unclassified BRCA1 and BRCA2 sequence variants. Many known deleterious BRCA1 and BRCA2 mutations affect splicing, and these typically lie near intron/exon boundaries. However, there are also potential internal exonic mutations that disrupt functional exonic splicing enhancer (ESE) sequences, resulting in exon skipping. Using previously established sequence matrices for the scoring of putative ESE motifs, we have systematically examined several BRCA2 mutations for potential ESE disruption mutations. These predictions revealed that BRCA2 T2722R (8393C-->G), which segregates with affected individuals in a family with breast cancer, disrupts three potential ESE sites. Reverse-transcriptase polymerase chain reaction analysis confirms that this mutation causes exon skipping, leading to an out-of-frame fusion of BRCA2 exons 17 and 19. This represents the first BRCA2 missense mutation shown to be a predicted deleterious protein-truncating mutation and suggests a potentially useful method for determining the clinical significance of a subset of the many unclassified variants in BRCA1 and BRCA2.

Figure 1

The sequence surrounding the C→G transversion at BRCA2 base 8393 (gray square) in humans, and the homologous regions in mouse, rat, and chicken. BRCA2 T2722R results from a C→G transversion at this base. The top line (italics) in each panel represents the amino acid sequence, and the second line represents the base sequence. The brackets show high-scoring protein-binding motifs for the SR proteins listed on the left. Values in parentheses are the actual scores for the wild-type (left) and mutant (right) sequences for each motif. Note that there are three overlapping high-scoring SF2/ASF motifs for the mouse/rat sequence. The C→G transversion has been detected only in the present study.

Figure 2

Family 98-11. Circles denote female individuals, squares denote male individuals, and diamonds denote individuals for whom sex was not reported. Slash marks indicate individuals who have died. The arrow indicates the proband. Blackened shapes denote affected individuals, unblackened shapes denote unaffected individuals, and half-blackened shapes denote individuals in whom the disease was reported but not confirmed. Causes of death are as follows: individual II-1, stage 4 non–small cell lung cancer; individual II-3, liver cancer; individual I-1, acute myocardial infarction; individual I-2, ovarian cancer; and individual I-7, unknown.

Figure 3

RT-PCR products from patient mRNA with wild-type BRCA2 (lane 2) or heterozygous for BRCA2 T2722R (lane 3).

Figure 4

Sequence of the exon 17/exon 18 junction in wild-type BRCA2 (top) and of the exon17/exon 19 junction from the cloned 375-bp RT-PCR product from the BRCA2 T2722R heterozygote (bottom).