Preimplantation Genetic Testing for Aneuploidy Improves Live Birth Rates with In Vitro Produced Bovine Embryos: A Blind Retrospective Study

Approximately one million in vitro produced (IVP) cattle embryos are transferred worldwide each year as a way to improve the rates of genetic gain. The most advanced programmes also apply genomic selection at the embryonic stage by SNP genotyping and the calculation of genomic estimated breeding values (GEBVs). However, a high proportion of cattle embryos fail to establish a pregnancy. Here, we demonstrate that further interrogation of the SNP data collected for GEBVs can effectively remove aneuploid embryos from the pool, improving live births per embryo transfer (ET). Using three preimplantation genetic testing for aneuploidy (PGT-A) approaches, we assessed 1713 cattle blastocysts in a blind, retrospective analysis. Our findings indicate aneuploid embryos have a 5.8% chance of establishing a pregnancy and a 5.0% chance of given rise to a live birth. This compares to 59.6% and 46.7% for euploid embryos (p < 0.0001). PGT-A improved overall pregnancy and live birth rates by 7.5% and 5.8%, respectively (p < 0.0001). More detailed analyses revealed donor, chromosome, stage, grade, and sex-specific rates of error. Notably, we discovered a significantly higher incidence of aneuploidy in XY embryos and, as in humans, detected a preponderance of maternal meiosis I errors. Our data strongly support the use of PGT-A in cattle IVP programmes.

Jumping Translocation in a Patient with Acute Leukemia and Fatal Evolution

Jumping translocations are uncommon cytogenetic abnormalities in which a segment of a donor chromosome, often 1q, is transferred to two or more receptor chromosomes. We describe the case of a 64-year-old man with a history of acute myeloid leukemia associated with myelodysplastic syndrome, who presented with a relapse of the leukemia and, concomitantly, with the appearance of a jumping translocation involving chromosome 1q. The patient had a poor clinical course without the possibility of performing targeted treatment, and he died 5 months after relapse. Jumping translocations are a reflection of chromosomal instability, and they could be related to epigenetic alterations such as pericentromeric chromatin hypomethylation, telomere shortening, or pathogenic variants of the TP53 gene. The existing data suggests a poor clinical outcome, a high risk of disease progression, and an unfavorable prognosis. More molecular studies are required to gain an in-depth understanding of the genetic mechanism underlying these alterations and their clinical significance and to be able to apply an optimal treatment to patients.

Homologous Recombination within Large Chromosomal Regions Facilitates Acquisition of β-Lactam and Vancomycin Resistance in Enterococcus faecium

ABSTRACTThe transfer of DNA betweenEnterococcus faeciumstrains has been characterized both by the movement of well-defined genetic elements and by the large-scale transfer of genomic DNA fragments. In this work, we report on the whole-genome analysis of transconjugants resulting from mating events between the vancomycin-resistantE. faeciumC68 strain and the vancomycin-susceptible D344RRF strain to discern the mechanism by which the transferred regions enter the recipient chromosome. Vancomycin-resistant transconjugants from five independent matings were analyzed by whole-genome sequencing. In all cases but one, the penicillin binding protein 5 (pbp5) gene and the Tn5382vancomycin resistance transposon were transferred together and replaced the correspondingpbp5region of D344RRF. In one instance, Tn5382inserted independently downstream of the D344RRFpbp5gene. Single nucleotide variant (SNV) analysis suggested that entry of donor DNA into the recipient chromosome occurred by recombination across regions of homology between donor and recipient chromosomes, rather than through insertion sequence-mediated transposition. The transfer of genomic DNA was also associated with the transfer of C68 plasmid pLRM23 and another putative plasmid. Our data are consistent with the initiation of transfer by cointegration of a transferable plasmid with the donor chromosome, with subsequent circularization of the plasmid-chromosome cointegrant in the donor prior to transfer. Entry into the recipient chromosome most commonly occurred across regions of homology between donor and recipient chromosomes.

Genomic Mechanisms of 17p / TP53 Loss in Primary “ultra High-risk” and Refractory Chronic Lymphocytic Leukemia: Results from the CLL2O Trial

Background: Unraveling the cytogenetic background helped to decipher the molecular basis of many hematologic cancers and to develop specific therapies. Recently, using chromosome banding analysis (CBA), jumping translocations were identified as a cause of 17p loss in multiple myeloma, providing new insights into the origin of clonal evolution and copy number alterations (CNA) (Sawyer et al, Blood 2014). In chronic lymphocytic leukemia (CLL) the genomic mechanisms leading to 17p loss are not fully understood. Aims: Characterization of underlying mechanisms of 17p loss using CBA and correlation with other clinicobiological features in “ultra high-risk” CLL. Methods: Samples from 112 patients (pts.) with refractory and/or 17p- CLL enrolled in the multicenter CLL2O trial were screened for CNAs by Affymetrix 6.0 SNP array analysis of CD19 sorted CLL cells and for chromosomal abnormalities by CBA using CpG oligonucleotide and interleukin-2 stimulation. Results: Considering both CBA and SNP data, 728 aberrations resulted in a mean of 6.5/case. 89 (79%) pts. had 17p deletion and 83 (74%) TP53 mutation. Regarding the origin of 17p/TP53 loss, 6 distinct types of rearrangements could be delineated: 1) whole arm translocations (WAT) 2) jumping translocations (JT) 3) dicentric chromosomes (DC) 4) cytogenetically balanced translocations (CBT) 5) other unbalanced translocations and 6) interstitial 17p deletions. WAT were identified in 33/112 (30%) cases and 30/33 (91%) involved chromosome 17 leading to 17p loss. Chromosomes involved ≥ 2 times in an unbalanced WAT were der(17;18)(q10;q10) (8, 24%), der(8;17)(q10;q10) (5, 15%), der(15;17)(q10;q10) (4, 12%), i(17)(q10) (4, 12 %), der(17;22)(q10;q10) (2, 6%). JT were identified in 11 (10 %) cases, 6 showing jumping WAT with 17q as donor chromosome, 1 case with breakpoints located in the pericentromeric regions of chromosome 17p11 (donor chromosome) and the receptor chromosomes 4p14 and 16p11. In 4 cases, initially a WAT involving 17q occurred and subsequently the partner chromosome “jumped off” leaving a 17p deletion behind. DC were detected in 19 pts., 8 with breakpoint in 17p11, 7/8 with TP53 mutation. Of note, all cases had the breakpoint on chromosome 17 in 17p11 indicating a fragile site affecting the pericentromeric region. Interestingly, of a total of 382 translocations observed by CBA, only 32 were CBT and except for those involving the IGH and IGK/L loci (n=6) all were random. 17p involvement in CBT was detected in 4 cases, 3 had TP53 deletion and all were TP53 mutated. Of the unbalanced translocations, der(17)t(8;17) was identified in 5 pts. simultaneously generating 8q gain. Nevertheless, breakpoints on chromosome 17p covered cytobands 17p11-13 and on chromosome 8, 8q11-22, one case having the breakpoint telomeric to the TP53 locus and no TP53 mutation, pointing to other putative candidate genes on 17p. In 36/112 (32%) cases, 17p deletion was induced by random rearrangements. Interstitial 17p deletions were identified in only 9/112 (8 %) cases. According to the inclusion criteria of the trial, 36/112 (32%) pts. had 17p deletion and were treatment-naïve while 76/112 (68%) were relapsed or refractory to fludarabine or bendamustine based therapy, 53/76 (70%) having a 17p deletion. Treatment naïve pts. had a mean of 7.36 aberrations/case and pretreated pts. 6.09/case. Focusing on WAT and JT, 18/33 (54%) pts. with WAT and 7/11 (63%) pts. with JT were pretreated whereas 57/78 (73%) pts. in the other cytogenetic subgroups had prior therapy exposure. Considering other genomic features, WAT and JT occurred almost exclusively within complex karyotypes (≥3 chromosomal aberrations), 31/33 WAT and 10/11 JT, were IGHV unmutated, 30/33 WAT and 11/11 JT and harbored TP53mutations, 29/33 WAT and 10/11 JT. Conclusions: “Ultra high-risk” CLL pts. are characterized by a high genomic complexity as compared to standard risk treatment-naïve CLL pts. (CLL8 trial with 1.8 CNAs/case). Previous genotoxic therapy had no influence on the total number of aberrations or the underlying mechanism, suggesting an intrinsic genomic instability of the tumor cells with TP53 alterations. WAT and JT emerged as nonrandom aberrations involved in 17p loss. Given the strong association of TP53 deletion with TP53 mutations of the remaining allele, one may speculate that TP53 mutations precedes TP53 deletion by disrupting the normal DNA repair mechanisms permitting incorrect recombinations. Disclosures Stilgenbauer: Amgen: Honoraria, Research Funding; Genzyme: Honoraria, Research Funding.

Transferring chromosome DNA fragments from multiple donor cells into a host strain for yeast strain improvement

Based on a common biological phenomenon — homologous recombination — a novel method was developed by transferring chromosome DNA fragments extracted from multiple donor cells into a host strain. Through this method of transferring DNA fragments, foreign DNA fragments are introduced into one host cell and multiple positive traits from multiple strains may be integrated into the host strain. We first confirmed its feasibility in both prokaryotic and eukaryotic cells by selecting reverse mutants to prototrophy from auxotrophic strains through receiving chromosomal DNA fragments of wild-type parental strains. We then applied this method to Saccharomyces cerevisiae to improve its ethanol and temperature tolerance. We introduced donor chromosome DNA fragments from different S. cerevisiae strains with improvements in ethanol or temperature tolerance into a common strain S. cerevisiae and obtained a strain with much superior ethanol and temperature tolerance. The results showed that the Transferring DNA Fragments method provides a new way for strain breeding.

Characterization of the Porphyromonas gingivalis conjugative transposon CTnPg1: determination of the integration site and the genes essential for conjugal transfer

In our previous study, extensive genomic rearrangements were found in two strains of the Gram-negative anaerobic bacterium Porphyromonas (Por.) gingivalis, and most of these rearrangements were associated with mobile genetic elements such as insertion sequences and conjugative transposons (CTns). CTnPg1, identified in Por. gingivalis strain ATCC 33277, was the first complete CTn reported for the genus Porphyromonas. In the present study, we found that CTnPg1 can be transferred from strain ATCC 33277 to another Por. gingivalis strain, W83, at a frequency of 10−7 to 10−6. The excision of CTnPg1 from the chromosome in a donor cell depends on an integrase (Int; PGN_0094) encoded in CTnPg1, whereas CTnPg1 excision is independent of PGN_0084 (a DNA topoisomerase I homologue; Exc) encoded within CTnPg1 and recA (PGN_1057) on the donor chromosome. Intriguingly, however, the transfer of CTnPg1 between Por. gingivalis strains requires RecA function in the recipient. Sequencing analysis of CTnPg1-integrated sites on the chromosomes of transconjugants revealed that the consensus attachment (att) sequence is a 13 bp sequence, TTTTCNNNNAAAA. We further report that CTnPg1 is able to transfer to two other bacterial species, Bacteroides thetaiotaomicron and Prevotella oralis. In addition, CTnPg1-like CTns are located in the genomes of other oral anaerobic bacteria, Porphyromonas endodontalis, Prevotella buccae and Prevotella intermedia, with the same consensus att sequence. These results suggest that CTns in the CTnPg1 family are widely distributed among oral anaerobic Gram-negative bacteria found in humans and play important roles in horizontal gene transfer among these bacteria.