Response of Human Hematopoietic Cells to DNA Double-strand Breaks

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.MAS

Insertion of an Alu‐like element in MLH1 intron 7 as a novel cause of Lynch syndrome

BackgroundLynch Syndrome (LS) is caused by germline mutations in the DNA mismatch repair (MMR) genes with mutations in MLH1 accounting for ~40% of LS-related alterations.MethodsMSK-IMPACT analysis was performed on peripheral blood from a patient with early- onset colorectal cancer. Subsequently PCR and sequencing was performed to characterize the insertion. Immunohistochemistry for MMR genes and MLH1 promoter methylation were analyzed on patient's tumor.ResultsMSK-IMPACT germline testing revealed an insertion into c.588+8_588+9 of MLH1 intron 7. The insertion was further characterized as an AluSx-like element with ~115 bp in length. Functional studies demonstrated that the AluSx-like element led to complete disruption of mRNA splicing and probably resulted in transcriptional termination at the poly (A) region of the AluSx-like insertion.ConclusionsThe insertion of a truncated AluSx like element into MLH1 intron 7 results in aberrant splicing and transcription, thereby causing Lynch syndrome. This study confirms that retrotransposon insertions may be an important mechanism for cancer predisposition

Insertion of an SVA element in MSH2 as a novel cause of Lynch syndrome.

Germline mutations in the DNA mismatch repair (MMR) genes cause Lynch syndrome (LS). In this study, we identified and characterized a novel SINE-VNTR-Alu (SVA) insertion in exon 12 of MSH2 in an individual with early-onset colorectal cancer and a very strong LS family history. RT-PCR analysis indicated a larger aberrant MSH2 transcript in one of the family members. MSK-IMPACT next-generation sequencing and long-range PCR analyses revealed an insertion in MSH2 exon 12 at the c.1972 position in an antisense orientation. The insertion was further characterized as an SVA element approximately 3 kb in length, belonging to the SVA_F1 family of retrotransposons. This variant also segregated with LS related cancers in four affected family members in this family. Based on this evidence, this MSH2 SVA insertion is considered pathogenic

Attenuated DNA damage responses and increased apoptosis characterize human hematopoietic stem cells exposed to irradiation

Abstract Failure to precisely repair DNA damage in self-renewing Hematopoietic Stem and early Progenitor Cells (HSPCs) can disrupt normal hematopoiesis and promote leukemogenesis. Although HSPCs are widely considered a target of ionizing radiation (IR)-induced hematopoietic injury, definitive data regarding cell death, DNA repair, and genomic stability in these rare quiescent cells are scarce. We found that irradiated HSPCs, but not lineage-committed progenitors (CPs), undergo rapid ATM-dependent apoptosis, which is suppressed upon interaction with bone-marrow stroma cells. Using DNA repair reporters to quantify mutagenic Non-Homologous End Joining (NHEJ) processes, we found that HSPCs exhibit reduced NHEJ activities in comparison with CPs. HSPC-stroma interactions did not affect the NHEJ capacity of HSPCs, emphasizing its cell autonomous regulation. We noted diminished expression of multiple double strand break (DSB) repair transcripts along with more persistent 53BP1 foci in irradiated HSPCs in comparison with CPs, which can account for low NHEJ activity and its distinct control in HSPCs. Finally, we documented clonal chromosomal aberrations in 10% of IR-surviving HSPCs. Taken together, our results revealed potential mechanisms contributing to the inherent susceptibility of human HSPC to the cytotoxic and mutagenic effects of DNA damage