Unique mutational profile associated with a loss of TDG expression in the rectal cancer of a patient with a constitutional PMS2 deficiency

Abstract

Cells with DNA repair defects have increased genomic instability and are more likely to acquire secondary mutations that bring about cellular transformation. We describe the frequency and spectrum of somatic mutations involving several tumor suppressor genes in the rectal carcinoma of a 13-year-old girl harboring biallelic, germline mutations in the DNA mismatch repair gene PMS2. Apart from microsatellite instability, the tumor DNA contained a number of C:G→T:A or G:C→A:T transitions in CpG dinucleotides, which often result through spontaneous deamination of cytosine or 5-methylcytosine. Four DNA glycosylases, UNG2, SMUG1, MBD4 and TDG, are involved in the repair of these deamination events. We identified a heterozygous missense mutation in TDG, which was associated with TDG protein loss in the tumor. The CpGs mutated in this patient's tumor are generally methylated in normal colonic mucosa. Thus, it is highly likely that loss of TDG contributed to the supermutator phenotype and that most of the point mutations were caused by deamination of 5-methylcytosine to thymine, which remained uncorrected owing to the TDG deficiency. This case provides the first in vivo evidence of the key role of TDG in protecting the human genome against the deleterious effects of 5-methylcytosine deamination.

Supplementary Fig. I

Bisulfite genomic sequencing at CpG sites of APC and MLH1 coding regions (A) and at the CpG island encompassing the TDG transcription start site (B). Each row of circles shows the methylation status of a cloned target sequence. Open circles: unmethylated CpG; black or gray filled circles: methylated or mutated CpGs, respectively. In panel A, the positions of the APC and MLH1 CpG dinucleotides that underwent somatic transitions in the tumor of Patient 1 are indicated below the columns of circles. Note that the two APC mutations are found in different clones, indicating that they are located in trans which was also confirmed by allele-specific PCR (see Section 4).

Supplementary Fig. II

Evolutionary conservation of TDG. Alignment of the amino acid sequence of TDG from different organisms flanking the position of the somatic mutation p.D284Y identified in Patient 1. Identical amino acids are in black boxes, similar ones in grey boxes. The site of the mutation is indicated by an arrow.

Fig. 1

Immunohistochemical staining of colon cancers with antibodies against MLH1, PMS2 and TDG. (A–C): Colon cancer of Patient 1; (D): colon cancer from an unrelated CMMR-D patient with a homozygous germline PMS2 mutation (Patient 2). MLH1 expression in the tumor cells was normal (A), while its heterodimeric partner PMS2 was absent in the colon cancer of Patient 1 (B). PMS2 was absent also in normal stromal cells (B) and in the normal mucosa (not shown). (C) In the same cancer, expression of the DNA repair enzyme TDG was markedly reduced or completely absent in the nuclei. (The cytoplasmic staining may either represent background, or residual expression of TDG in this cellular compartment.) (D) TDG was normally expressed in the colon cancer of the unrelated CMMR-D patient (Patient 2) used as positive control. Inset: staining of this cancer with TDG pre-immune serum (negative control).

Fig. 2

Sequencing electropherograms of TDG in the DNA of Patient 1. Upper panel: sequence of the TDG gene from non-neoplastic tissue containing solely the wild type allele. Lower panel: sequence of TDG in tumor DNA, showing the heterozygous somatic mutation p.D284Y.