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Adam J. Hirst 1, Alicia Zhang 1, Mark Hills 1, Allen C. Eaves 1, 2, Sharon A. Louis 1 and Arwen L. Hunter 1.

1. STEMCELL Technologies Inc., Vancouver, Canada.

2. Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada.

Chromosomal aberrations, including numerical aneuploidy, chromosomal rearrangements and sub-microscopic changes, have been widely reported in cultured human pluripotent stem cells (hPSCs). Genetic variants can affect hPSC growth rates, cell survival and differentiation potential. Recurrent genetic abnormalities observed in hPSCs are also observed in human cancers, an observation that raises concerns for downstream clinical applications. The hPSC Genetic Analysis Kit employs a qPCR-based method designed to rapidly detect the most common genetic abnormalities observed in hPSC cultures. Specifically, primer-probe assays were optimized to detect the minimal critical regions on chromosomes 1q, 8q, 10p, 12p, 17q 18q, 20q and Xp, as well as a control region on chromosome 4p. These regions represent approximately 70% of all reported abnormalities in hPSC cultures. Amplification efficiencies for all primer-probe sets were measured at ≥ 90% (n = 2). Abnormalities were detected in 4 different hPSC lines each containing a 1q duplication, 10p deletion, 12 trisomy or 20q duplication (p < 0.001), with no other genetic abnormalities detected in other regions (p > 0.1). Duplication of 20q11.21 is a submicroscopic abnormality often missed when using G-banding karyotyping. As a case study, we analyzed the WLS-4D1 human induced pluripotent stem cell (hiPSC) line using G-banding, fluorescent in situ hybridization (FISH), and the hPSC Genetic Analysis Kit. Although this hiPSC line was found to be karyotypically normal by G-banding, duplication of 20q11.21 was only detected by using the hPSC Genetic Analysis Kit and confirmed by FISH. To determine assay sensitivity, fluorescently-labelled hPSC lines known to be abnormal for 10p, 12p and 20q were mixed with unlabeled normal diploid hPSC at varying ratios. Results indicate that our qPCR-based approach was able to detect genetically abnormal hPSCs when present at a minimal frequency of 30% (n = 3; p xtagstartz 0.05). In summary, the hPSC Genetic Analysis Kit offers researchers a reliable, fast and cost-effective tool to routinely monitor and pre-screen the hPSC lines in their laboratory for recurrent genetic abnormalities.

Submitting Author must be underlined¹, Co-Author¹, Co Author², Co-Author^1*,

¹Institute or Organization; ²Institute or Organization

Repeated sequences, which are very common in genomes, provide additional opportunities for non-allelic homologous recombination (NAHR). These events can lead to genetic alterations such as deletions, inversions, translocations or copy number variation. As a consequence, many human disorders result from genomic rearrangements involving repeated sequences. For example, the Alu repeats, which are present in ~1 million copies in the human genome, have been found at the deletion breakpoints of a considerable number of genes involved in human pathologies. Despite extensive observations that repeated sequences are predisposed to rearrangements, the events leading to these reactions are not fully understood. Here, we explored the possibility that replication stress, a typical threat to cell survival, could trigger recombination between repeats, and identified the genes regulating this process.

In order to detect and study recombination between repeated sequences, we examined ADE2 recombinants in the yeast genome, which are generated by recombination between inverted repeats of two ade2 mutant alleles separated by a 1.3kb TRP1 gene. It was published previously that spontaneous recombination between the two ade2 copies is highly dependent on Rad52, acting in two independent recombination pathways. First, Rad52 acts as a loader of Rad51 which mediates gene conversion events. In the second pathway, Rad52, in association with Rad59, promotes inversion events which reverse the intervening TRP1 sequence.

We show that NAHR between the two ade2 copies is greatly stimulated by a localized replication barrier (Tus/Ter system) at the precise site of the inverted repeats. Thus, our results strongly suggest that fork stalling near repeated sequences can stimulate NAHR. Unlike spontaneous events, our genetic study reveals that replication associated NAHR reactions rely on a unique and particular pathway dependent on Rad51 catalytic activity, Rad52 and its single-strand annealing partner Rad59. We propose a model implicating a template switch event between the repeated sequences on the two sister chromatids at a stalled replication fork. Our genetic observations suggest that following fork stalling, the fork reversal proteins Rad5 and Mph1, together with the DNA end resection nucleases Mre11 and Exo1, create a ssDNA substrate onto which the Rad57-Rad55 paralogs and the Shu complex facilitate Rad51 filament assembly. The recombinase then catalyses strand invasion of the parental duplex on one of the two repeats, which initiates DNA synthesis by the replicative DNA polymerase Pol δ. Depending on how long DNA synthesis is, the recombination product after resolution of the reversed fork can be gene conversion or inversion.

Repeated sequences, which are very common in genomes, provide additional opportunities for non-allelic homologous recombination (NAHR). These events can lead to genetic alterations such as deletions, inversions, translocations or copy number variation. As a consequence, many human disorders result from genomic rearrangements involving repeated sequences. For example, the Alu repeats, which are present in ~1 million copies in the human genome, have been found at the deletion breakpoints of a considerable number of genes involved in human pathologies. Despite extensive observations that repeated sequences are predisposed to rearrangements, the events leading to these reactions are not fully understood. Here, we explored the possibility that replication stress, a typical threat to cell survival, could trigger recombination between repeats, and identified the genes regulating this process.

Repeated sequences, which are very common in genomes, provide additional opportunities for non-allelic homologous recombination (NAHR). These events can lead to genetic alterations such as deletions, inversions, translocations or copy number variation. As a consequence, many human disorders result from genomic rearrangements involving repeated sequences. For example, the Alu repeats, which are present in ~1 million copies in the human genome, have been found at the deletion breakpoints of a considerable number of genes involved in human pathologies. Despite extensive observations that repeated sequences are predisposed to rearrangements, the events leading to these reactions are not fully understood. Here, we explored the possibility that replication stress, a typical threat to cell survival, could trigger recombination between repeats, and identified the genes regulating this process.

In order to detect and study recombination between repeated sequences, we examined ADE2 recombinants in the yeast genome, which are generated by recombination between inverted repeats of two ade2 mutant alleles separated by a 1.3kb TRP1 gene. It was published previously that spontaneous recombination between the two ade2 copies is highly dependent on Rad52, acting in two independent recombination pathways. First, Rad52 acts as a loader of Rad51 which mediates gene conversion events. In the second pathway, Rad52, in association with Rad59, promotes inversion events which reverse the intervening TRP1 sequence.

We show that NAHR between the two ade2 copies is greatly stimulated by a localized replication barrier (Tus/Ter system) at the precise site of the inverted repeats. Thus, our results strongly suggest that fork stalling near repeated sequences can stimulate NAHR. Unlike spontaneous events, our genetic study reveals that replication associated NAHR reactions rely on a unique and particular pathway dependent on Rad51 catalytic activity, Rad52 and its single-strand annealing partner Rad59. We propose a model implicating a template switch event between the repeated sequences on the two sister chromatids at a stalled replication fork. Our genetic observations suggest that following fork stalling, the fork reversal proteins Rad5 and Mph1, together with the DNA end resection nucleases Mre11 and Exo1, create a ssDNA substrate onto which the Rad57-Rad55 paralogs and the Shu complex facilitate Rad51 filament assembly. The recombinase then catalyses strand invasion of the parental duplex on one of the two repeats, which initiates DNA synthesis by the replicative DNA polymerase Pol δ. Depending on how long DNA synthesis is, the recombination product after resolution of the reversed fork can be gene conversion or inversion.

Repeated sequences, which are very common in genomes, provide additional opportunities for non-allelic homologous recombination (NAHR). These events can lead to genetic alterations such as deletions, inversions, translocations or copy number variation. As a consequence, many human disorders result from genomic rearrangements involving repeated sequences. For example, the Alu repeats, which are present in ~1 million copies in the human genome, have been found at the deletion breakpoints of a considerable number of genes involved in human pathologies. Despite extensive observations that repeated sequences are predisposed to rearrangements, the events leading to these reactions are not fully understood. Here, we explored the possibility that replication stress, a typical threat to cell survival, could trigger recombination between repeats, and identified the genes regulating this process.

In order to detect and study recombination between repeated sequences, we examined ADE2 recombinants in the yeast genome, which are generated by recombination between inverted repeats of two ade2 mutant alleles separated by a 1.3kb TRP1 gene. It was published previously that spontaneous recombination between the two ade2 copies is highly dependent on Rad52, acting in two independent recombination pathways. First, Rad52 acts as a loader of Rad51 which mediates gene conversion events. In the second pathway, Rad52, in association with Rad59, promotes inversion events which reverse the intervening TRP1 sequence.

We show that NAHR between the two ade2 copies is greatly stimulated by a localized replication barrier (Tus/Ter system) at the precise site of the inverted repeats. Thus, our results strongly suggest that fork stalling near repeated sequences can stimulate NAHR. Unlike spontaneous events, our genetic study reveals that replication associated NAHR reactions rely on a unique and particular pathway dependent on Rad51 catalytic activity, Rad52 and its single-strand annealing partner Rad59. We propose a model implicating a template switch event between the repeated sequences on the two sister chromatids at a stalled replication fork. Our genetic observations suggest that following fork stalling, the fork reversal proteins Rad5 and Mph1, together with the DNA end resection nucleases Mre11 and Exo1, create a ssDNA substrate onto which the Rad57-Rad55 paralogs and the Shu complex facilitate Rad51 filament assembly. The recombinase then catalyses strand invasion of the parental duplex on one of the two repeats, which initiates DNA synthesis by the replicative DNA polymerase Pol δ. Depending on how long DNA synthesis is, the recombination product after resolution of the reversed fork can be gene conversion or inversion.

ΑΡΘΡΟ 1 Όλοι οι άνθρωποι γεννιούνται ελεύθεροι και ίσοι στην αξιοπρέπεια και τα δικαιώματα. Είναι προικισμένοι με λογική και συνείδηση, και οφείλουν να συμπεριφέρονται μεταξύ τους με πνεύμα αδελφοσύνης.

ΑΡΘΡΟ 2 Κάθε άνθρωπος δικαιούται να επικαλείται όλα τα δικαιώματα και όλες τις ελευθερίες που προκηρύσσει η παρούσα Διακήρυξη, χωρίς καμία απολύτως διάκριση, ειδικότερα ως προς τη φυλή, το χρώμα, το φύλο, τη γλώσσα, τις θρησκείες, τις πολιτικές ή οποιεσδήποτε άλλες πεποιθήσεις, την εθνική ή κοινωνική καταγωγή, την περιουσία, τη γέννηση ή οποιαδήποτε άλλη κατάσταση. Δεν θα μπορεί ακόμα να γίνεται καμία διάκριση εξαιτίας του πολιτικού, νομικού ή διεθνούς καθεστώτος της χώρας από την οποία προέρχεται κανείς, είτε πρόκειται για χώρα ή εδαφική περιοχή ανεξάρτητη, υπό κηδεμονία ή υπεξουσία, ή που βρίσκεται υπό οποιονδήποτε άλλον περιορισμό κυριαρχίας.