Jayne C. Boyer

Relative rates of insertion and deletion mutations in dinucleotide repeats of various lengths in mismatch repair proficient mouse and mismatch repair deficient human cells.

Microsatellites are DNA elements composed of short tandem repeats of 1-5bp. These sequences are particularly prone to frameshift mutation by insertion-deletion loop formation during replication. The mismatch repair system is responsible for correcting these replication errors, and microsatellite mutation rates are significantly elevated in the absence of mismatch repair. We have investigated the effect of varying the number of repeats in a (CA)n microsatellite on mutation rates in cultured mammalian cells proficient or deficient in mismatch repair. We have also compared the relative rates of single-repeat insertions and deletions in these cells. Two plasmid vectors were constructed for each repeat unit number (n=8, 17, and 30), such that the microsatellites, placed upstream of a bacterial neomycin resistance gene (neo), disrupted the reading frame of the gene in the (-1) or (+1) direction. Plasmids were introduced separately into the cells, where they integrated into the cellular genome. Mutation rates were determined by selection of clones with frameshift mutations in the microsatellite that restored the reading frame of the neo gene. We found that mutation rates were significantly higher for (CA)17 and (CA)30 tracts than for (CA)8 tracts in both mismatch repair proficient (mouse) and deficient (human) cells. A mutational bias favoring insertions was generally observed. In both (CA)17 and (CA)30 tracts, single-repeat insertion rates were higher than single-repeat deletion rates with or without mismatch repair; deletions of multiple repeat units (> or =8bp) were observed in these tracts, where as deletions this large were not found in the (CA)8 tract. Single-repeat mutations of both types were made at similar rates in (CA)8 tracts in human mismatch repair deficient (MMR-) cells, but single-repeat insertion rates were higher than single-repeat deletion rates in mouse mismatch repair proficient (MMR+) cells. Results of these direct studies on microsatellite mutations in cultured cells should be useful for refinement of mathematical models for microsatellite evolution.

Stability of microsatellites in myeloid neoplasias.

Microsatellites are short, repeated DNA sequences that exist throughout the genome. Instability of these sequences, associated with defects in the DNA mismatch repair system, is the hallmark of hereditary non-polyposis colorectal cancer (HNPCC), and is also found in many sporadic cancers. Although many types of solid tumors exhibit this type of genetic instability, its involvement in hematologic cancers is less evident. We have investigated whether microstatellite instability (MSI) is involved in the transformation of myeloid cells to myelodysplastic syndrome (MDS) and/or acute myelogenous leukemia (AML). Both de novo and treatment-associated neoplasias were studied. Only one example of MSI was found in 48 patients, using a panel of 14 different microsatellite loci consisting of repeats of one to four base pairs. These results suggest that the genes responsible for MSI are not involved in the transformation of normal myeloid cells to MDS or AML.

The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts

Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration ClinicalTrials.gov NCT01087307

DNA repair and replication in human fibroblasts treated with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene .

DNA repair and replication were examined in diploid human fibroblasts after treatment with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 microM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4-6 h after treatment with 0.7 microM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 microM) were found to inhibit replicon initiation by up to 50% within 30-60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1 X 10(7) Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 microM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 microM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.

DNA methylation modifies urine biomarker levels in 1,6-hexamethylene diisocyanate exposed workers: A pilot study

DNA methylation may mediate inter-individual responses to chemical exposure and, thus, modify biomarker levels of exposure and effects. We analyzed inter-individual differences in inhalation and skin exposure to 1,6-hexamethylene diisocyanate (HDI) and urine biomarker 1,6-hexamethylene diamine (HDA) levels in 20 automotive spray-painters. Genome-wide 5-methyl cytosine (CpG) DNA methylation was assessed in each individuals peripheral blood mononuclear cells (PBMC) DNA using the Illumina 450K CpG array. Mediation analysis using linear regression models adjusted for age, ethnicity, and smoking was conducted to identify and assess the association between HDI exposure, CpG methylation, and urine HDA biomarker levels. We did not identify any CpGs common to HDI exposure and biomarker level suggesting that CpG methylation is a mediator that only partially explains the phenotype. Functional significance of genic- and intergenic-CpG methylation status was tested using protein-protein or protein-DNA interactions and gene-ontology enrichment to infer networks. Combined, the results suggest that methylation has the potential to affect HDI mass transport, permeation, and HDI metabolism. We demonstrate the potential use of PBMC methylation along with quantitative exposure and biomarker data to guide further investigation into the mediators of occupational exposure and biomarkers and its role in risk assessment.