An intronic mutation in MLH1 associated with familial colon and breast cancer

Abstract

Single base substitutions can lead to missense mutations, silent mutations or intronic mutations, whose significance is uncertain. Aberrant splicing can occur due to mutations that disrupt or create canonical splice sites or splicing regulatory sequences. The assessment of their pathogenic role may be difficult, and is further complicated by the phenomenon of alternative splicing. We describe an HNPCC patient, with early-onset colorectal cancer and a strong family history of colorectal and breast tumors, who harbours a germ line MLH1 intronic variant (IVS9 c.790 +4A>T). The proband, together with 2 relatives affected by colorectal-cancer and 1 by breast cancer, have been investigated for microsatellite instability, immunohistochemical MMR protein staining, direct sequencing and Multiplex Ligation-dependent Probe Amplification. The effect of the intronic variant was analyzed both by splicing prediction software and by hybrid minigene splicing assay. In this family, we found a novel MLH1 germline intronic variant (IVS9 c.790 +4A>T) in intron 9, consisting of an A to T transversion, in position +4 of the splice donor site of MLH1. The mutation is associated with the lack of expression of the MLH1 protein and MSI in tumour tissues. Furthermore, our results suggest that this substitution leads to a complete skip of both exon 9 and 10 of the mutant allele. Our findings suggest that this intronic variant plays a pathogenic role.

Fig. 1

I-1: died at age 42 of colon cancer. II-1: 2 metachronous colon cancers at age 48 and 82. II-2: died at age 76 of colon cancer (onset age 74). II-3: died at age 62 of colon cancer (onset age 61). II-4: died at age 47 of breast cancer. II-5: died at age 69 of colon cancer (onset age 68). III-2: breast cancer (onset age 48). III-3: leukaemia. III-5: colon cancer (onset age 52). III-6: 2 metachronous colon cancers at age 31 and age 35. IV-1: colon cancer (onset age 24). Arrow indicates the proband. Red borders indicate carrier status

Fig. 2

Immunohistochemical staining for MLH1 (a), and MSH2 (b), proteins showing loss of MLH1 expression in the proband’s colon cancer

Fig. 3

Sequence analysis of the MLH1 IVS9 c.790 +4A>T. The figure a shows the electropherogram of genomic DNA mutation. The arrow indicate the intronic variation. The figures b and c shows the electropherogram of wt cDNA (b), and the electropherogram of cDNA fragment, lacking both exon 9 and 10 (c)

Fig. 4

Minigene Functional Splicing Assay. a The hybrid minigene (pCalfaM) used in transient transfection splicing assays in HeLa cells is shown. Exons are indicated as boxes. Introns are indicated as lines. MLH1 Exon 9 and 10 (white and gray box) were tested for splicing efficiency by using specific primers (arrows). b RT-PCR products from transfection experiments. RNA splicing variants corresponding to wt (2) exon 9 exclusion (3) and both exon 9 and 10 exclusion (4) are shown. (1) indicate a splice variant generated as an artefact of the minigene. M is the 100 bp marker