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Case Reports
. 2019 Nov 28;11(1):171.
doi: 10.1186/s13148-019-0762-6.

Highly sensitive MLH1 methylation analysis in blood identifies a cancer patient with low-level mosaic MLH1 epimutation

Estela Dámaso  1 , Júlia Canet-Hermida  1 , Gardenia Vargas-Parra  1   2 , Àngela Velasco  2   3 , Fátima Marín  1   2 , Esther Darder  3 , Jesús Del Valle  1   2 , Anna Fernández  1 , Àngel Izquierdo  3 , Gemma Mateu  4 , Glòria Oliveras  4 , Carmen Escribano  5 , Virgínia Piñol  6 , Hugo-Ikuo Uchima  6 , José Luis Soto  7 , Megan Hitchins  8 , Ramon Farrés  2 , Conxi Lázaro  1   9 , Bernat Queralt  10 , Joan Brunet  1   3   9   11 , Gabriel Capellá  12   13 , Marta Pineda  14   15
Affiliations
Case Reports

Highly sensitive MLH1 methylation analysis in blood identifies a cancer patient with low-level mosaic MLH1 epimutation

Estela Dámaso et al. Clin Epigenetics. .

Abstract

Constitutional MLH1 methylation (epimutation) is a rare cause of Lynch syndrome. Low-level methylation (≤ 10%) has occasionally been described. This study aimed to identify low-level constitutional MLH1 epimutations and determine its causal role in patients with MLH1-hypermethylated colorectal cancer.Eighteen patients with MLH1-hypermethylated colorectal tumors in whom MLH1 methylation was previously undetected in blood by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were screened for MLH1 methylation using highly sensitive MS-melting curve analysis (MS-MCA). Constitutional methylation was characterized by different approaches.MS-MCA identified one patient (5.6%) with low-level MLH1 methylation (~ 1%) in blood and other normal tissues, which was confirmed by clonal bisulfite sequencing in blood. The patient had developed three clonally related gastrointestinal MLH1-methylated tumor lesions at 22, 24, and 25 years of age. The methylated region in normal tissues overlapped with that reported for other carriers of constitutional MLH1 epimutations. Low-level MLH1 methylation and reduced allelic expression were linked to the same genetic haplotype, whereas the opposite allele was lost in patient's tumors. Mutation screening of MLH1 and other hereditary cancer genes was negative.Herein, a highly sensitive MS-MCA-based approach has demonstrated its utility for the identification of low-level constitutional MLH1 epigenetic mosaicism. The eventual identification and characterization of additional cases will be critical to ascertain the cancer risks associated with constitutional MLH1 epigenetic mosaicism.

Keywords: Constitutional MLH1 epimutation; Epigenetic mosaicism; Highly sensitive methodologies; Lynch syndrome; Methylation.

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Conflict of interest statement

GC and JdV have received personal fees from VCN Biosciences and AstraZeneca, respectively, outside the submitted work. There are no other relationships or activities that could appear to have influenced the submitted work. All other authors declare that they have no competing interest.

Figures

Fig. 1
Fig. 1
Identification and characterization of constitutional MLH1 epigenetic mosaicism in case 29. a Methylation analysis by MS-MCA of the MLH1 promoter C-region in blood DNA from case 29. The patient shows levels of methylation around 1%. b Family pedigree from case 29. The epimutation carrier is indicated by an arrow. Circles, females; squares, males; filled, cancer affected; vertical line at center, not otherwise specified. Cancer location and age at diagnosis are indicated. Generations are indicated on the left margin in Roman numerals. c Methylation analysis by pyosequencing of the MLH1 promoter C-region in blood DNA from case 29 and one healthy control. Each CpG is numbered according its position relative to the translation initiation codon. d Clonal bisulfite sequencing of the MLH1 promoter in PBL DNA from case 29. Each horizontal line represents a specific allele. CpG dinucleotides are depicted by circles. Black and white circles indicate methylated and unmethylated CpG sites, respectively. The allele at rs1800734 (c.-93G>A) is indicated as A or G. Methylation in case 29 is confined to the G allele. Each CpG analyzed is numbered according to its position relative to the translation initiation codon
Fig. 2
Fig. 2
Somatic mutation profile of the different tumor lesions. a Venn diagram representing the total number of somatic mutations that are unique to each tumor lesion or shared at least between two tumor lesions. b Type of somatic mutations identified in each tumor sample. Only coding mutations have been considered. CR, colorectal; SB, small bowel; G, gastric
Fig. 3
Fig. 3
Analysis of the dose of MLH1 alleles at rs1799977 (c.655A>G). a Schematic representation of the MLH1 epimutation found in case 29. Promoter methylation is associated with the c.-93G allele (in approximately 2% of cells), located in cis with exonic c.655G allele, which showed subtle reduced transcriptional activity in blood. In concordance, the MLH1 c.655A allele showed LOH in the three tumor lesions developed by the proband. b Allele-specific expression (ASE) analysis at MLH1 c.655G>A in cDNA derived from leukocytes of case 29 and two heterozygous controls. A slightly diminished expression of the c.655G allele was observed. c Loss of heterozygosity (LOH) analysis in tumors from case 29. Allele specific values show loss of the c.655A allele in all three tumors
Fig. 4
Fig. 4
Representation of the differentially methylated region across the MLH1 locus in blood and colorectal mucosa from case 29, MLH1 epimutation carriers (n = 12), Lynch syndrome genetic mutation carriers (n = 61), and healthy controls (n = 41). Representation of the differentially methylated region across the MLH1 locus in blood DNA (a) and in normal colorectal mucosa (b). β-values obtained from Infinium 450k Human Methylation array analysis are displayed as a log (mean β-value × 100) against the genomic coordinate for each CpG site interrogated. The relative locations of the CpG sites are not drawn to scale. CpG sites are located between Chr3:36,985,516-37,219,077 coordinates. Above, CpG islands (CI) are represented as black rectangles and their shores are represented in gray. The location of the Deng regions (DR) A, B, C and D of the MLH1 promoter are indicated by white rectangles. Genes (G) containing the displayed CpG sites are represented as gray rectangles, using the Ensembl GRCh37 database as the reference for gene coordinates
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