De novo constitutional MLH1 epimutations confer early-onset colorectal cancer in two new sporadic Lynch syndrome cases, with derivation of the epimutation on the paternal allele in one

Abstract

Lynch syndrome is an autosomal dominant cancer predisposition syndrome classically caused by germline mutations of the mismatch repair genes, MLH1, MSH2, MSH6 and PMS2. Constitutional epimutations of the MLH1 gene, characterized by soma-wide methylation of a single allele of the promoter and allelic transcriptional silencing, have been identified in a subset of Lynch syndrome cases lacking a sequence mutation in MLH1. We report two individuals with no family history of colorectal cancer who developed that disease at age 18 and 20 years. In both cases, cancer had arisen because of the de novo occurrence of a constitutional MLH1 epimutation and somatic loss-of-heterozygosity of the functional allele in the tumors. We show for the first time that the epimutation in one case arose on the paternally inherited allele. Analysis of 13 tumors from seven individuals with constitutional MLH1 epimutations showed eight tumors had lost the second MLH1 allele, two tumors had a novel pathogenic missense mutation and three had retained heterozygosity. Only 1 of 12 tumors demonstrated the BRAF V600E mutation and 3 of 11 tumors harbored a mutation in KRAS. The finding that epimutations can originate on the paternal allele provides important new insights into the mechanism of origin of epimutations. It is clear that the second hit in MLH1 epimutation-associated tumors typically has a genetic not epigenetic basis. Individuals with mismatch repair-deficient cancers without the BRAF V600E mutation are candidates for germline screening for sequence or methylation changes in MLH1.

Figure 1

Constitutional MLH1 epimutation in “Proband YT.” (a) Allelic bisulfite sequencing demonstrated that hemiallelic methylation at the “Deng-C” region of the MLH1 promoter was widespread throughout his somatic tissues. Horizontal lines represent individual clones and dots indicate CpG dinucleotides within the sequenced fragment; black is methylated, white is unmethylated. (b) Allelic quantification of genomic DNA and mRNA samples in the proband and parents by pyrosequencing at the benign c.655A>G SNP (rs1799977) within MLH1 exon 8. The yellow-shaded region shows the peaks at the SNP site. Two peaks showing equal levels of the “A” and “G” alleles in the proband’s normal gastric mucosa DNA indicate heterozygosity. In the tumor DNA there is significant reduction of the transcriptionally active “G” allele (indicated by downward arrow), consistent with loss-of-heterozygosity (LOH) of the unmethylated allele. In the mRNA from the proband’s saliva and PBL there was loss of expression of the “A” allele (indicated by downward arrows). Genotyping of this SNP in the parents showed that the silenced “A” allele was maternally inherited. (c) Allelic expression analysis at the C/A SNP (rs9311149) within the 3′UTR of the EPM2AIP1 gene shows allelic transcriptional loss of EPM2AIP1 in proband YT as well. (d) Proband YT pedigree and summary of haplotypes from informative SNPs according to the key provided. Allele colors relate to parental origin of inheritance, with red indicating maternally derived alleles, blue showing paternally derived alleles and black indicating unknown parentage. Transcription of the paternally inherited allele was demonstrated, as indicated by arrows. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2

Constitutional MLH1 methylation in somatic tissues of Proband BF. (a) Bisulfite pyrosequencing in peripheral blood lymphocytes (PBL) and other somatic tissues in BF show soma-wide mosaic methylation of the MLH1 “Deng-C” region. The sequence analyzed is written above the peaks within each pyrogram. Vertical gray bars indicate the position of the C/T (Y) nucleotides within the five CpG sites interrogated for methylation status. The level of methylation C:T at each site is given in the boxes above, with the average methylation score of the five CpG sites provided at the top of each pyrogram. The vertical yellow bar indicates a control for complete bisulphite conversion. (b) Clonal bisulfite sequencing within the MLH1-C region shows the allelic pattern of methylation. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 3

Transcriptional inactivation of expression of the paternal MLH1 and EPM2AIP1 alleles in Proband BF and LOH of the maternal allele in the tumor. (a) Sequence electropherograms across the MLH1 exon 6 c.474C>T (rs4647256) SNP (indicated by arrows) in Proband BF and family members. The genomic DNA and RNA extracted from the proband’s lymphoblastoid cells (LCL) show that she is heterozygous for the SNP, but only the “C” allele is expressed, indicating loss of expression of the T allele. Genotyping in the parents showed obligate inheritance of the active “C” allele from the mother, who is homozygous “C.” Therefore, the inactivated “T” allele was paternally inherited. The tumor shows considerable reduction of the functional “C” allele. (b) Electropherograms spanning a novel G/A SNP site and the C/A rs9311149 SNP in the 3′UTR of EPM2AIP1 in the proband’s genomic DNA (gDNA), mRNA and in the genomic DNA of family members. The proband was found to be heterozygous for both SNPs. The LCL mRNA shows monoallelic expression of the “A” allele of the novel G/A SNP and the “C” allele of SNP rs9311149. Both SNP sites were informative as to the parental origin of the alleles, with the mother heterozygous for the novel SNP and homozygous “C” for the rs9311149 SNP and the father homozygous “G” for the novel SNP and heterozygous for rs9311149. The expressed allele was thus maternally derived, confirming the epimutation arose on the paternal allele. (c) Proband BF pedigree and summary of haplotypes from informative SNPs according to the key provided, with red showing maternal and blue showing paternal inheritance. The expressed maternal allele is indicated by an arrow. The proband’s sibling inherited the opposite parental alleles. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

Point mutations serving as the “second hit” in the colorectal tumors of Patient A. Electropherograms showing the acquired somatic missense mutations identified in the tumors (bottom as labeled), which were not present in the germline DNA (top). The positions of each mutation are indicated by arrows. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]