Age Related Expression of hMLH1 but Not hMSH2 in Human Hematopoietic Stem Cells Predicts Genomic Instability.

Abstract 3235

Poster Board III-172

DNA damage repair pathways have been shown to be important for hematopoietic function and longevity. A number of studies focusing on the loss of DNA damage repair genes have reported age related loss of hematopoietic stem cells, reduced hematopoietic function, and increased rates of hematologic cancer. A poorly studied pathway with respect to its effect on hematopoietic function is the mismatch repair (MMR) pathway. Microsatellite instability (MSI), one marker of MMR failure, has been observed in acute lymphoblastic leukemia, myelodysplastic syndrome, and lymphocytes obtained from the peripheral blood of the elderly. Further, studies investigating MMR pathway disruption in mice show an increased incidence of age related hematologic disorders and failure to repopulate in competitive transplant experiments. These reports led to the formation of the hypothesis that age related loss of MMR in a subset hematopoietic stem/progenitor cells (HSC) leads to a progenitor population with a mutator phenotype potentially leading to cell death or leukemogenesis.

To test this hypothesis we clonally expanded HSC from cord blood, bone marrow, and bone core samples from various aged individuals most of whom had normal CBCs. These colony forming units (CFU) were then tested for MSI at five microsatellite loci previously used in the diagnosis of the MMR defective disease HNPCC (BAT 25, BAT 26, D2S123, D5S346, and D17S250). These results showed a statistically significant increase in high grade instability (measured as MSI observed at >20% of loci tested) in CFU obtained from individuals over the age of 50 compared to those in the age range between 0 and 50 years (p=0.0034). Next we sought to determine if loss of gene expression in MMR genes could account for the observed MSI. Gene expression of two genes known to be transcriptionally downregulated in MMR failure, hMLH1 and hMSH2, were assessed by qRT-PCR in CFU obtained from pure CD34⁺ immunomagnetic separations of HSC from various aged donors. Further, individual CD34⁺ cells from these same donors were fixed to slides and immunostained for hMLH1 and hMSH2. We observed a significant loss of hMLH1 gene expression in a subset of HSC obtained from older donors above age 42, but no pattern of loss of hMSH2 gene expression. The age related loss of MMR in HSC suggests that MMR pathway proficiency is important for hematopoiesis. These results suggest that loss of MMR might precede myelodysplastic syndromes and leukemia in older individuals.