Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing.

Jonathan Kenyon

doi:10.23937/2469-570x/1410031

Abstract

Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1, an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylation upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from -938 to -337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1. We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34+ selected hematopoietic stem and progenitor cells.

Highlights

Complex genetic, epigenetic, and phenotypic changes in normal hematopoietic cells are required during leukemogenesis and marrow failure
As has been previously described for many tumor cell types, we report for the first time a correlation between the loss of mut-like-homologue 1 (MLH1) expression and increased MLH1 promoter methylation in colony forming cells (CFC) derived from CD34+ selected hematopoietic stem and progenitor cells
To determine if CpG promoter methylation was associated with loss of MLH1 expression in individual human hematopoietic stem and progenitor cells (HPC), we first identified CFC from four normal donors as having or lacking MLH1 expression by quantitative real time PCR (QRT-PCR)

Summary

Introduction

Epigenetic, and phenotypic changes in normal hematopoietic cells are required during leukemogenesis and marrow failure. While numerous individual mutations are observed, the underlying mutagenic process leading to an increased rate of mutations in hematologic malignancies and to loss of hematopoietic function is unclear. Understanding these events is critical to provide a basis for preventing underlying genomic instability and thereby reduce the risk of marrow failure and malignant transformation. Loss of MMR and increased MSI is a characteristic of myelodysplastic syndrome, commonly culminating in secondary leukemia and other hematologic malignancies. A relationship between loss of MLH1 expression, independent of mutation, and CpG methylation of the 5′ MLH1 promoter is observed in MMR defective tumors and cell lines [4,5,6,7,8,9]

Methods

Results

Conclusion