Activity of Endogenous L1 Retrotransposons in Human Embryonal Cells

Recent high throughput studies have led to the discovery of de novo L1 retrotransposition in malignant somatic cells, as well as large numbers of novel insertions, many of which are highly active in cell culture assays. These data suggest that L1 elements are robustly active, undergoing extensive diversification in contemporary human genomes. Despite this there is little direct evidence of endogenous L1 retrotransposition in the human germline or early embryogenesis: data from very rare disease causing insertions is indirect, subject to strong acquisition bias, and is often equivocal with respect to the origin of the insertions. For L1s to be evolutionarily successful they must retrotranspose during early human development or in the germline, in order to transmit copies to the next generation. The purpose of this thesis was to develop sensitive and yet robust methods to screen human embryos and embryonic cell models for de novo full-length endogenous L1 insertions. We developed a new high throughput sequencing technique, which was able to recover single molecule retrotransposition events. Based on this technique we identified 172 candidate novel L1 insertions in a total of three human embryos, represented by whole-genome amplified DNA of individually dissected blastomeres and the remaining blastocyst tissue. 57 of these insertions are potentially genuine de novo endogenous L1 insertions. Moreover, we have identified a candidate germline specific L1 insertion from a healthy adult donor. Therefore, this study has detected candidate de novo L1 retrotransposition events in human embryos and germlines, using an approach that enables complete validation and characterization of the insertions, despite operating at the single molecule and single cell level. We consider this technical innovation will be most significant in the ongoing dissection of how L1, the dominant human transposon, is actively driving the evolution of modern human genomes

A novel L1 retrotransposon marker for HeLa cell line identification

The HeLa cell line is the oldest, most widely distributed, permanent human cell line. As a nearly ubiquitous inhabitant of laboratories using tissue culture techniques, its aggressive growth characteristics make it a problematic contaminant that can overgrow less robust cell lines. Consequently, HeLa contamination is common in both the research laboratory and cell line repository contexts, and its detection is hampered by the lack of a rapid, sensitive and robust assay. Here we report the development of a HeLa-specific DNA diagnostic test: a single duplex detection PCR assay targeting an L1 retrotransposon insertion. All HeLa clones from a geographically diverse panel were positive by this assay, and the particular L1 insertion we identified appears to be unique to the HeLa cell line. The assay can detect very low levels of HeLa contamination (<1%), and can be performed on un-purified cell pellets, allowing rapid routine screening

Understanding the Genomic Structure of Copy-Number Variation of the Low-Affinity Fcγ Receptor Region Allows Confirmation of the Association of FCGR3B Deletion with Rheumatoid Arthritis.

Fcγ receptors are a family of cell-surface receptors that are expressed by a host of different innate and adaptive immune cells, and mediate inflammatory responses by binding the Fc portion of immunoglobulin G. In humans, five low-affinity receptors are encoded by the genes FCGR2A, FCGR2B, FCGR2C, FCGR3A, and FCGR3B, which are located in an 82.5-kb segmental tandem duplication on chromosome 1q23.3, which shows extensive copy-number variation (CNV). Deletions of FCGR3B have been suggested to increase the risk of inflammatory diseases such as systemic lupus erythematosus and rheumatoid arthritis (RA). In this study, we identify the deletion breakpoints of FCGR3B deletion alleles in the UK population and endogamous native American population, and show that some but not all alleles are likely to be identical-by-descent. We also localize a duplication breakpoint, confirming that the mechanism of CNV generation is nonallelic homologous recombination, and identify several alleles with gene conversion events using fosmid sequencing data. We use information on the structure of the deletion alleles to distinguish FCGR3B deletions from FCGR3A deletions in whole-genome array comparative genomic hybridization (aCGH) data. Reanalysis of published aCGH data using this approach supports association of FCGR3B deletion with increased risk of RA in a large cohort of 1,982 cases and 3,271 controls (odds ratio 1.61, P = 2.9×10(-3) )