Humans accumulate microsatellite instability with acquired loss of MLH1 protein in hematopoietic stem and progenitor cells as a function of age

Abstract

<jats:title>Abstract</jats:title><jats:p>Hematopoietic stem and progenitor cells (HPCs) are necessary for long-term survival. Genomic instability and persistent DNA damage may cause loss of adult stem cell function. The mismatch repair (MMR) pathway increases replication fidelity and defects have been implicated in malignant hematopoietic diseases. Little, however, is known about the role MMR pathway failure plays in the aging process of human HPCs. We hypothesized that loss of MMR occurs in HPCs as a process of human aging. We examined microsatellite instability and expression of the MMR genes MutL homologue 1 (MLH1) and MutS homologue 2 (MSH2) in HPCs and colony-forming cell-derived clones (CFCs) from human donors aged 0 to 86 years. CFCs from donors &gt; 45 years had a greater frequency of microsatellite instability and CD34+ progenitors lacking MLH1 expression and protein than individuals ≤ 45 years. Loss of MSH2 did not correlate with age. Thus, a potentially early event in the normal human aging process is microsatellite instability accumulation in normal human HPCs associated with the loss of MLH1 protein expression.</jats:p>

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

“…While we accept this study does not link loss of MLH1 expression and promoter methylation to tumorigenic mutation; it does represent the identification of a predisposing processes of MLH1 expression loss in the CFC from otherwise normal individuals and is, therefore, a significant finding. We previously reported the appearance of MSI in normal human hematopoietic progenitor cells increases with age [10]. Additionally we found a correlation between CFC with evidence of MSI, loss of MLH1 expression, and increased MLH1 promoter methylation.…”

“…Using Sanger sequencing, we previously identified a correlation between microsatellite instability and MLH1 promoter methylation in a small number of normal HPC [10]. In this large high-throughput bisulfite sequence library of the MLH1 promoter, sequences from each CFC were “tagged” with a unique barcode.…”

“…The mononuclear cell fraction was obtained by ficolldensity gradient separation as described previously [10]. CD34 + cells were isolated from the mononuclear cell fraction by immune-magnetic separation with the CD34 + isolation kit (Miltenyi Biotech, Auburn, CA) according to the manufacturer's protocol.…”

“…Genomic DNA was obtained from one fraction of the cells obtained from an individual CFC by proteinase K digestion as previously described in [10]. Briefly one fraction of the CFC was washed twice with PBS and then incubated at 55°C for 2 hours in 75 ul lysis buffer (10mM Tris-HCl (pH 8.0), 1mM EDTA, 1% Triton-X 100, and 10 mg/mL proteinase K).…”

“…We recently determined MLH1 expression lost occurs as a function of age in human hematopoietic progenitor cells (HPC) [10]. We observed significant MSI accumulation in the HPC and colony forming cells (CFC)s obtained from normal donors as a function of donor age.…”