Interrogate the mechanism of non-homologous chromosomal translocations in human germ cells
Date Issued
2009
Date
2009
Author(s)
Hsieh, Li-Ging
Abstract
Since the human genome project finished in 2003, many studies focused on the variations in the human genome. From 2002 to 2007, the international HapMap project mapped over 3.1 million single nucleotide polymorphisms (SNPs). Using different approaches, until recently, about 40000 >1kb structural variations (SVs) had been detected between non-disease phenotype individuals. Theses observations reflected the important contribution of SVs to human genome variation.o interrogate which mechanisms involve in SVs formation in human genome, PCR-based approach is the most sensitive one. Previously, an inverse PCR-based approach, RECORD (Restriction Enhanced Capturing of Rearrange DNA), was used to identify MLL and EWSR1 translocations in human germline. MLL and EWSR1 are frequently involved in recurrent translocation associated with infant and childhood leukemia and childhood sarcoma, respectively.he aim of my project is to use human TCF3, which is a known target of recurrent translocation associated with childhood acute lymphoblastic leukemia (ALL), as an anchor gene to examine whether similar event occurs in human sperm and to explore the underlying mechanisms involved. Moreover, we will use RECORD and translocation specific PCR to validate germline translocations which identified previously.o assess the detection limit of RECORD, we used HEK293 spiked with K562 genomic DNA as template, and examined the recovery of BCR-ABL fusion from K562 cells. We found that the detection limit by RECORD is about 5×10-4. By using TCF3 as an anchor gene, we identified 19 non-homologous interchromosomal translocation events from 12 donors. These 19 translocation events include four translocation partners in geneic region and 15 in intergenic region. Sequence analysis of the 2 kb regions flanking both sides of breakpoints excluded the involvement of non-allelic homologous recombination (NAHR). A microhomology of 1-13 base pairs at the breakpoint junction indicates that either non-homologous end joining (NHEJ) or microhomology mediated endjoining (MMEJ) may be responsible for germline translocation. though the core factors, Ku70/Ku80 of NHEJ are not expressed during meiotic recombination.sing MLL as an anchor gene, we identified 20 additional MLL-F2 translocations from the haploid genomes of 12 sperm donors. Eight of which were mapped in the genic region and 12 in intergenic region of partners. At the breakpoint junctions, there are microhomologies of 2-10 nucleotides.hen microhomology of the anchor gene is considered always on the top strand, the translocation partners can be classified into four groups, based on its location on the p or q arm, and its sequence from top or bottom strand. Thus 8 derivatives of translocation products can be defined by using 5’ and 3’ iPCR in our studies. This germline balanced exchange between non-homologous chromosomes is mediated by microhomology. To distinguish from MMEJ reported only in somatic cells, we proposed this putative mechanism as microhomology mediated interchromosomal translocation (MMIT). As the important purpose of meiotic recombination is to repair DNA double-strand breaks (DSBs), we suggest that MMIT may be another meiotic DSBs repair pathway in male germ cells.
Subjects
sperm
microhomology-mediated chromosomal translocation
SDGs
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