Germline truncating mutations in both MSH2 and BRCA2 in a single kindred

Abstract

There has been interest in the literature in the possible existence of a gene that predisposes to both breast cancer (BC) and colorectal cancer (CRC). We describe the detailed characterisation of one kindred, MON1080, with 10 cases of BC or CRC invasive cancer among 26 first-, second- or third-degree relatives. Linkage analysis suggested that a mutation was present in BRCA2. DNA sequencing from III: 22 (diagnosed with lobular BC) identified a BRCA2 exon 3 542G>T (L105X) mutation. Her sister (III: 25) had BC and endometrial cancer and carries the same mutation. Following immunohistochemical and microsatellite instability studies, mutation analysis by protein truncation test, cDNA sequencing and quantitative real-time PCR revealed a deletion of MSH2 exon 8 in III: 25, confirming her as a double heterozygote for truncating mutations in both BRCA2 and MSH2. The exon 8 deletion was identified as a 14.9 kb deletion occurring between two Alu sequences. The breakpoint lies within a sequence of 45 bp that is identical in both Alu sequences. In this large BC/CRC kindred, MON1080, disease-causing truncating mutations are present in both MSH2 and BRCA2. There appeared to be no increased susceptibility to the development of colorectal tumours in BRCA2 mutation carriers or to the development of breast tumours in MSH2 mutation carriers. Additionally, two double heterozygotes did not appear to have a different phenotype than would be expected from the presence of a mutation in each gene alone.

Figure 1

Hereditary breast and CRC Family MON1080. The numbers immediately under the symbols are individual numbers. The abbreviation ‘ut’ indicates untested. Mutations in brackets are inferred. MSH2=MSH2 exon 8 deletion present. L105X=BRCA2^*L105X mutation present. ENDO=endometrial cancer.

Figure 2

(A) Microsatellite instability at various loci in astrocytoma of patient II: 20. (1) D2S123; (2) BAT 25 and (3) BAT 26. (B) Immunohistochemical staining for MSH2 and MLH1 in the astrocytoma of patient II: 20. Upper panel: lack of staining for the MSH2 antibody indicating MSH2 inactivation in the tumour. Lower panel: normal staining of MLH1 in the astrocytoma (× 400). (C) Protein truncation test shows a truncated protein in patients II: 18 and III: 25. (Lanes 1 and 2) the double-headed arrow indicates the protein product from the normal allele. The single arrow indicates the truncated protein. (D) PCR products of cDNA segment including exons 5–9. Lanes 1–7: the double headed arrrow indicates PCR product of normal allele. Lanes 5 and 6: single arrow indicates PCR product of mutant allele. (E) The first sequence of cDNA shows the junction of exons 7 and 8. The second sequence shows the exon 8 deletion found in patients with HNPCC-related cancers in family MON1080.

Figure 3

Characterisation of the MSH2 exon 8 deletion: the intron 7 Alu Y shares 83% identity with the intron 8 Alu Y. There are 17 Alu family sequences in intron 7 and 18 Alu family sequences in intron 8. The MON1080 deletion is the result of the possible recombination between identical 45 bp sequences found in both AluY sequences.