N59: Highly Sensitive MLH1 Methylation Analysis In Blood Allows The Identification Of Low-Level Epigenetic Mosaicism

E. Dámaso1, J. Canet1, G. Vargas-Parra1, À. Velasco2, E. Darder2, A. Fernández1, F. Marin1, À.Izquierdo2, V. Piñol3, H. Uchima3, J. Luis Soto4, 5, M. Hitchins6, C. Lázaro1, 7, B. Queralt3, J. Brunet2, 7, M. Pineda1, 7, G. Capellá1, 7

1 – Hereditary Cancer Program, Catalan Institute of Oncology, Insititut d’Investigació Biomèdica de Bellvitge (IDIBELL), ONCOBELL Program, L’Hospitalet de Llobregat, Barcelona, Spain. 2 – Hereditary Cancer Program, Catalan Institute of Oncology,  Institut d’Investigació Biomèdica de Girona (IDIBGI), Girona, Spain. 3 – Hospital Universitari Josep Trueta, Girona, Spain. 4 – Hereditary Cancer Program Valencian Region, Molecular Genetics Laboratory, Elche University Hospital, Elche, Alicante, Spain. 5 – Alicante Institute for Health and Biomedical Research (ISABIAL-FISABIO Foundation), Alicante, Spain. 6 – Department of Medicine, Division of Oncology, Stanford University, Stanford, California, United States. 7 – Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Spain.


Aim: Constitutional MLH1 epimutations are a rare cause of Lynch syndrome. Low methylation levels (<10%) have been occasionally described. The aim of this study was the identification of patients with low levels of epigenetic mosaicism in MLH1 gene.

Method: Eighteen patients with MLH1 hypermethylated tumors and undetectable methylation in blood as assessed by Methylation-Specific (MS) Multiplex Ligation-Dependent Probe Amplification were included. Highly sensitive MS-Melting Curve Analysis (MS-MCA) at MLH1 promoter was used to screen for epigenetic mosaicism. Constitutional methylation was confirmed by other methods. Mutational analysis of hereditary cancer genes including MLH1 was performed.

Results: MS-MCA analysis identified one case (5.6%) with low levels of methylation (1-2%) in blood DNA. The patient had developed 3 gastrointestinal tumors at ages 22, 24 and 25, sharing MLH1 promoter hypermethylation and loss of heterozygosity associated with c.655A allele. The presence of low MLH1 methylation levels was confirmed by clonal bisulfite sequencing, evidencing the association with c.-93G allele. The extension of the hypermethylated region overlaps with the reported in constitutional MLH1 epimutation carriers. No rare germline variants were identified.

Conclusion: The use of highly sensitive techniques such as MS-MCA has demonstrated to be useful for the detection of low levels of methylation in blood.