Complex Chromosome Research Articles

Successful Pregnancy following PGT-SR in a Couple with Complex Chromosome Rearrangement and Recurrent IVF Failures: A Case Report.

Successful Pregnancy following PGT-SR in a Couple with Complex Chromosome Rearrangement and Recurrent IVF Failures: A Case Report.

Haplotype-resolved de novo assembly revealed unique characteristics of alternative lengthening of telomeres in mouse embryonic stem cells.

Telomeres protect chromosome ends from DNA damage responses, and their dysfunction triggers genomic alterations like chromosome fusion and rearrangement, which can lead to cellular death. Certain cells, including specific cancer cells, adopt alternative lengthening of telomere (ALT) to counteract dysfunctional telomeres and proliferate indefinitely. While telomere instability and ALT activity are likely major sources of genomic alteration, the patterns and consequences of such changes at the nucleotide level in ALT cells remainunexplored. Here we generated haplotype-resolved genome assemblies for type I ALT mouse embryonic stem cells, facilitated by highly accurateor ultra-long reads and Hi-C reads. High-quality genome revealed ALT-specific complex chromosome end structures and various genomic alterations including over 1000 structural variants (SVs). The unique sequence (mTALT) used as a template for type I ALT telomeres showed traces of being recruited into the genome, with mTALT being replicated with remarkably high accuracy. Subtelomeric regions exhibited distinct characteristics: resistance to the accumulation of SVs and small variants. We genotyped SVs at allele resolution, identifying genes (Rgs6, Dpf3and Tacc2) crucial for maintaining ALT telomere stability. Our genome assembly-based approach elucidated the unique characteristics of ALT genome, offering insights into the genome evolution of cells surviving telomere-derived crisis.

Chromoanagenesis of chromosome 22 in a subject with obesity and borderline cognitive performance

Chromoanagenesis events consist of complex chromosome rearrangements with multiple breakpoints in one or few chromosomes. Mechanisms of chromoanagenesis are split into three major groups: chromothripsis, chromoanasynthesis and chromoplexy. This study aims to delineate a chromoanagenesis event at the level of chromosome 22 in an individual showing obesity and borderline cognitive performance as major disturbances. The proband and his parents were subjected to conventional karyotyping, CGH array and whole genomic sequencing (WGS). By conventional karyotyping a “de novo” pericentric inversion of chromosome 22 was identified. CGH array identified several imbalances (either deletions or duplications) in the long arm of chromosome 22; the largest is a 4.5 Mb duplication at 22q12.1-22q1.3. The detection of extensive duplications would suggest the occurrence of a chromoanasynthesis event. WGS, in addition to the structural alterations identified by karyotyping and CGH array, revealed two translocations from chromosome 22 to chromosomes 6 and 21 as well as a heterozygous pathogenetic variant of ALMS1 gene; the latter could have contributed to the obesity of our patient. The pericentric inversion induces loss of initial part of TCF20 gene including the 5’ regulatory region and the first, noncoding, exon. Heterozygous loss-of-function mutations of TCF20 gene have been found in patients with autism spectrum disorder or intellectual disability, some of them presenting obesity. It is, therefore, possible that disruption of TCF20 gene structure would contribute to a fraction of the patient’s phenotype.

Characterization of cryptic complex chromosome rearrangements in balanced chromosomal rearrangement carriers and their PGT-SR clinical outcome assessments

Several reports have presented that balanced chromosomal rearrangements (BCRs) carriers with normal phenotypes may be carriers of complex rearrangements. However, the incidence and PGT clinical outcomes of cryptic complex chromosome rearrangements (CCCRs) in individuals with BCRs is remain unknown. We recruited a cohort of 1,264 individuals with BCR carriers from 2016 to 2021 at the Reproductive and Genetic Hospital of CITIC Xiangya. Peripheral blood was collected for karyotyping and genomic DNA extraction and the PGT-SR clinical outcomes of CCCRs carriers were analyzed and compared with those of BCR carriers. Our findings revealed that 3.6% (45/1,264) of BCR carriers had CCCRs, involving 3–25 breakpoints on 1–3 chromosomes. Furthermore, when mate-pair sequencing was employed, 63.3% (19/30) of CCCR carriers were found to have chromosome rearrangements that were different from those identified by the MicroSeq technique. And the transferable embryo rate of CCCR carriers with 3 chromosomes was significantly lower than that of CCCR carriers with only 1–2 chromosomes. In this research, we revealed that some of the BCR carriers were actually CCCR carriers, and the prognosis of PGT in CCCR carriers with one or two chromosomes is better than that of CCCR carriers with three chromosomes.

Secondary myeloid malignancy after CAR T cell therapy for non-Hodgkin lymphoma (NHL).

7033 Background: Prior studies have described toxicities associated with chimeric antigen receptor T cell therapy (CART). Recently, myeloid malignancies were identified in myeloma patients (pt) after receipt of cilta-cel. In this study, we sought to describe the cumulative incidence and characteristics of secondary myeloid malignancies in pt treated with CART for NHL. Methods: We conducted a single institution retrospective study among pt ages ≥18 years (yr) who received commercial CART for NHL at Ohio State between December 2017 to April 2022. Descriptive statistics were used to summarize pt/disease characteristics. Cumulative incidence was calculated from date of infusion with death/relapse as competing risks. Results: For 190 pt treated with CART, median age was 63 yr (23-85 yr). Disease subtypes included diffuse large B-cell lymphoma (BCL)/high grade BCL, Richter transformation, and mantle cell lymphoma (79.5%, 8.4%, and 7.4%). Products included tisa-cel (47.9%), axi-cel (43.1%), brexu-cel (7.4%), and liso-cel (1.6%). Grade 3-4 neutropenia developed in 29% and 18% of pt at 30 days and 90 days post-CART. Median follow-up was 17 mo (0.5-63 mo). 48% were alive at time of analysis. Myeloid malignancy post-CART occurred in 7 pt with a median time to development of 29 mo (18-41 mo). Characteristics (Table) included complex karyotype, TP53 and chromosome 5/7 abnormalities. Cumulative incidence of secondary malignancy was 1%, 5%, and 6% at 2, 3, and 4 yr post-CART. Conclusions: We report on incidence of secondary myeloid malignancy in pt receiving CART for NHL. Mutational characteristics of secondary malignancies were those classically associated with therapy-related myeloid malignancy, thus presumed to be associated with prior exposure to chemotherapy (eg. alkylators/VP-16). Further investigation in larger cohorts with additional observation time, to allow for appropriate risk discussion, is required. [Table: see text]

An Integrated Approach Including CRISPR/Cas9-Mediated Nanopore Sequencing, Mate Pair Sequencing, and Cytogenomic Methods to Characterize Complex Structural Rearrangements in Acute Myeloid Leukemia.

Complex structural chromosome abnormalities such as chromoanagenesis have been reported in acute myeloid leukemia (AML). They are usually not well characterized by conventional genetic methods, and the characterization of chromoanagenesis structural abnormalities from short-read sequencing still presents challenges. Here, we characterized complex structural abnormalities involving chromosomes 2, 3, and 7 in an AML patient using an integrated approach including CRISPR/Cas9-mediated nanopore sequencing, mate pair sequencing (MPseq), and SNP microarray analysis along with cytogenetic methods. SNP microarray analysis revealed chromoanagenesis involving chromosomes 3 and 7, and a pseudotricentric chromosome 7 was revealed by cytogenetic methods. MPseq revealed 138 structural variants (SVs) as putative junctions of complex rearrangements involving chromosomes 2, 3, and 7, which led to 16 novel gene fusions and 33 truncated genes. Thirty CRISPR RNA (crRNA) sequences were designed to map 29 SVs, of which 27 (93.1%) were on-target based on CRISPR/Cas9 crRNA nanopore sequencing. In addition to simple SVs, complex SVs involving over two breakpoints were also revealed. Twenty-one SVs (77.8% of the on-target SVs) were also revealed by MPseq with shared SV breakpoints. Approximately three-quarters of breakpoints were located within genes, especially intronic regions, and one-quarter of breakpoints were intergenic. Alu and LINE repeat elements were frequent among breakpoints. Amplification of the chromosome 7 centromere was also detected by nanopore sequencing. Given the high amplification of the chromosome 7 centromere, extra chromosome 7 centromere sequences (tricentric), and more gains than losses of genomic material, chromoanasynthesis and chromothripsis may be responsible for forming this highly complex structural abnormality. We showed this combination approach's value in characterizing complex structural abnormalities for clinical and research applications. Characterization of these complex structural chromosome abnormalities not only will help understand the molecular mechanisms responsible for the process of chromoanagenesis, but also may identify specific molecular targets and their impact on therapy and overall survival.

The morphokinetic signature of human blastocysts with mosaicism and the clinical outcomes following transfer of embryos with low-level mosaicism

BackgroundGenetic mosaicism is commonly observed in human blastocysts. Embryos’ morphokinetic feature observed from time-lapse monitoring (TLM) is helpful to predict the embryos’ ploidy status in a non-invasive way. However, morphokinetic research on mosaic embryos is extremely limited. Moreover, transfer of mosaic embryos is a new attempt in reproductive medicine, while studies regarding the clinical and neonatal outcomes following transfer of embryos with different levels and types of mosaicism are needed. This study aimed to investigate the morphokinetic characteristics of mosaic blastocysts, uncover clinical outcomes of mosaic embryos, and evaluate the effect of level and type of mosaicism on transfer outcomes.ResultsA total of 923 blastocysts from 229 preimplantation genetic testing cycles were cultured in TLM incubators in a single fertilization center between July 2016 and July 2021. Multivariate logistic regression models showed mosaic embryos had significantly shorter time to reach morula when compared with euploid (P = 0.002), mosaic with aneuploid (P = 0.005), and aneuploid (P = 0.005) embryos after adjusting the potential confounders. KIDScore is an artificial intelligence scoring program from time lapse incubation system to predict embryo implantation potential. Mosaic with aneuploid embryos had significantly lower KIDScore than euploid (P = 6.47e−4), mosaic (P = 0.005), and aneuploid (P = 0.004) embryos after adjustment. Meanwhile, we compared the clinical outcomes following transfer of low-level (< 50%) mosaic embryos (N = 60) with euploid embryos (N = 1301) matched using propensity scoring collected from September 2020 to January 2023. Mosaic embryos had significantly lower clinical pregnancy rate (41.67% vs. 57.65%, P = 0.015) and live birth rate (38.33% vs. 51.35%, P = 0.048) than the euploid embryos. Subgroup analyses showed the whole, segmental, and complex chromosome mosaic embryos had the similar clinical outcomes.ConclusionsThe shortened time to reach morula in mosaic embryos and the low KIDScore in mosaic with aneuploid embryos revealed innovative clues to embryo selection with the non-invasive TLM and provided new insights into biological mechanism of chromosomal abnormality. The analyses of overall and subgroups of mosaic embryo transfer outcomes helped to optimize embryo transfer scheme for in-vitro fertilization procedures. Multi-center prospective studies with large sample sizes are warranted to validate our results in the future.

M-FISH evaluation of chromosome aberrations to examine for historical exposure to ionising radiation due to participation at British nuclear test sites

Veterans of the British nuclear testing programme represent a population of ex-military personnel who had the potential to be exposed to ionising radiation through their participation at nuclear testing sites in the 1950s and 1960s. In the intervening years, members of this population have raised concerns about the status of their health and that of their descendants, as a consequence. Radiation dose estimates based on film badge measurements of external dose recorded at the time of the tests suggest any exposure to be limited for the majority of personnel, however, only ∼20% of personnel were monitored and no measurement for internalised exposure are on record. Here, to in-part address families concerns, we assay for chromosomal evidence of historical radiation exposure in a group of aged nuclear test (NT) veterans, using multiplex in situ hybridisation (M-FISH), for comparison with a matched group of veterans who were not present at NT sites. In total, we analysed 9379 and 7698 metaphase cells using M-FISH (24-colour karyotyping) from 48 NT and 38 control veteran samples, representing veteran servicemen from the army, Royal Airforce and Royal Navy. We observed stable and unstable simple- and complex-type chromosome aberrations in both NT and control veterans’ samples, however find no significant difference in yield of any chromosome aberration type between the two cohorts. We do observe higher average frequencies of complex chromosome aberrations in a very small subset of veterans previously identified as having a higher potential for radiation exposure, which may be indicative of internalised contamination to long-lived radionuclides from radiation fallout. By utilising recently published whole genome sequence analysis data of a sub-set of the same family groups, we examined for but found no relationship between paternal chromosome aberration burden, germline mutation frequency and self-reported concerns of adverse health in family members, suggesting that the previously reported health issues by participants in this study are unlikely to be associated with historical radiation exposure. We did observe a small number of families, representing both control and NT cohorts, showing a relationship between paternal chromosome aberrations and germline mutation sub-types which should be explored in future studies. In conclusion, we find no cytogenetic evidence of historical radiation exposure in the cohort of nuclear veterans sampled here, offering reassurance that attendance at NTs sites by the veterans sampled here, was not associated with significant levels of exposure to radiation.

DaCSeg: Divide and conquer for accurate overlapping chromosome instance segmentation in metaphase cell images

Chromosome segmentation is essential for karyotyping analysis, which provides a reliable basis for subsequent disease diagnosis. Recent deep segmentation techniques can significantly boost the efficiency of chromosome micrographs processing. However, existing classical Mask R-CNN-based methods cannot deal with chromosome overlapping issues highly susceptible to appearing in clinical data. In our opinion, it is difficult for current computational methods to achieve accurate segmentation without a specific design for complex overlapping. Therefore, we propose a divide-and-conquer automatic framework, which shows better robustness for complex chromosome instance segmentation. Exploiting a series of specially designed modules, our end-to-end model first performs an estimation of whether the chromosomes overlap and then generates precise attention masks for overlapping areas. With the assistance of the shape prior knowledge, it produces final instance segmentation predictions for the chromosomes. The experimental results show that the proposed method achieves mean Average Precision (mAP) of 84.10%, AP50 of 98.61%, AP75 of 97.62%, Precision of 89.32%, Recall of 88.87%, and F1-score of 89.09%, which are superior to other state-of-the-art methods. Code and models are available at https://github.com/labiip/DaCSeg.

Preimplantation genetic testing for complex chromosomal rearrangements: clinical outcomes and potential risk factors.

Complex chromosome rearrangements (CCR) are rare structural abnormalities involving at least three breakpoints, categorized into three types based on their structure: type A (three-way rearrangements), type B (double two-way translocations), and type C (exceptional CCR). However, thus far, limited data exists on preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) in CCR carriers. This study aims to evaluate the clinical outcomes and influencing factors of PGT-SR in couples with CCR. Fifteen couples with unique CCR recruited from 793 couples following PGT-SR between January 2017 and May 2023. In addition, a total of 54 CCR cases, 39 previously reported as well as 15 newly added, were included in the analysis of factors associate with normal/balanced embryos. A total of 100 blastocysts were biopsied and analyzed in 15 CCR couples after 17 PGT-SR cycles, with 16.0% being euploid, 78.0% aneuploid and 6.0% mosaic. 11 normal/balanced embryos and one mosaic embryo were transferred, resulting in eight live births. Furthermore, based on the combined data from 54 CCR carriers, the proportion of normal/balanced embryos was 10.8%, with a significant decrease observed among female carriers compared to male heterozygotes (6.5% vs. 15.5%, p = 0.002). Type B exhibited the lowest rate of euploid embryos at only 6.7%, followed by type A at 11.6% and type C at 14.0%, although the differences were not significant (p = 0.182). After completing the multivariate generalized estimating equation (GEE) analysis, type B (p = 0.014) and female carrier (p = 0.002) were identified as independent risk factors for fewer euploid embryos. The occurrence of balanced CCR in patients with reproductive abnormalities may be more frequent than we expected. Despite the proportion of normal/balanced embryos being significantly low, which can be influenced by CCR type and carrier's sex, PGT-SR may improve the reproductive outcomes among CCR cases. These findings can optimize the clinical management and genetic counseling of CCR carriers seeking assisted reproductive technology (ART).

A combination of long- and short-read genomics reveals frequent p-arm breakpoints within chromosome 21 complex genomic rearrangements

PurposeAlthough chromosome 21 is the smallest human chromosome, it is highly relevant in the pathogenicity of both cancer and congenital diseases, including Alzheimer disease and trisomy 21 (Down syndrome). In addition, cases with rare structural variants (SVs) of chromosome 21 have been reported. These events vary in size and include large chromosomal events, such as ring chromosomes and small partial aneuploidies. The p-arm of the acrocentric chromosome 21 was devoid of reference genomic sequence in GRCh37 and GRCh38, which hampered our ability to solve genomic rearrangements and find the mechanism of formation of disease-causing SVs. We hypothesize that conserved satellite structures and segmental duplications located on the p-arm play an important role in the formation of complex SVs involving chromosome 21. MethodsThree cases with complex chromosome 21 rearrangements were studied with a combination of short-read and long-read genome sequencing, as well as optical genome mapping. The data were aligned to the T2T-CHM13 assembly. ResultsWe were able to resolve all 3 complex chromosome 21 rearrangements in which 15, 8, and 26 breakpoints were identified, respectively. By comparing the identified SV breakpoints, we were able to pinpoint a region between 21p13 and 21p12 that appears to be frequently involved in chromosome 21 rearrangements. Importantly, we observed acrocentric satellite DNA at several breakpoint junctions suggesting an important role for those elements in the formation of complex SVs. ConclusionTaken together, our results provide further insights into the architecture and underlying mechanisms of complex rearrangements on acrocentric chromosomes.

Transcription induces context-dependent remodeling of chromatin architecture during differentiation.

Metazoan chromosomes are organized into discrete spatial domains (TADs), believed to contribute to the regulation of transcriptional programs. Despite extensive correlation between domain organization and gene activity, a direct mechanistic link is unclear, with perturbation studies often showing little effect. To follow chromatin architecture changes during development, we used Capture Hi-C to interrogate the domains around key differentially expressed genes during mouse thymocyte maturation, uncovering specific remodeling events. Notably, one TAD boundary was broadened to accommodate RNA polymerase elongation past the border, and subdomains were formed around some activated genes without changes in CTCF binding. The ectopic induction of some genes was sufficient to recapitulate domain formation in embryonic stem cells, providing strong evidence that transcription can directly remodel chromatin structure. These results suggest that transcriptional processes drive complex chromosome folding patterns that can be important in certain genomic contexts.

Chromoanagenesis in Haematological Malignancy: Review of Samples from Patients with Acute Leukemia and MDS

Background: Complex chromosome rearrangements (CCRs) include a variety of structural aberrations grouped as chromothripsis, chromoanasynthesis and chromoplexy. Although the underlying mechanisms for these phenomena are unknown and likely to be different, they appear to originate from a single event. While chromothripsis and chromoanasynthesis affect limited genome sites their role and genome associations in haematological malignancy remain to be elucidated. Aims: This study aimed to provide clarity on the location, incidence and association with TP53 gene profile of chromothripsis (cth) and chromoanasynthesis (cha) events in MDS/AML. Methods: We analysed data from routine investigations carried out by haematological malignancy diagnostic service (HMDS) at University College London Hospital (UCLH) on 2,506 samples of acute myeloid leukaemia (AML) and myelodysplasia (MDS), including G-banding, FISH (Cytocell), chromosome microarray analysis (CMA, Agilent) and target NGS(Illumina). Analysis of 87 samples with complex bone marrow karyotypes were reported following the ISCN 2020 as: (i) cth(alternating disomy and heterozygous loss along a chromosome or chromosomal segment) or (ii) cha (deletions and one or two copy number gains in a single chromosome or chromosome region including copy number variants without the clustered breakpoints of chromothripsis). Statistical analysis used RStudio (v 1.4.1106) to carry Pearson's chi-squared (χ²) and Fisher's exact (2-sided) tests. Results: Complex karyotypes harbouring cth and cha type aberrations were detected in 65 out of 727 AML and 22 out of 1779 MDS samples. The cth events were seen alongside cha in 67% of cases, rarely presenting as a sole abnormality (2%), while cha alone was found in 31% of samples. Most frequently cth was mapped at chromosome 21 (17%), chromosome 7 (15%), chromosome 17 and 19 (13%) and chromosome 5 (11%) (Fig.1a). In this cohort of 87cases, chromothripsis was found to be associated with TP53 deletions ( TP53Del/WT) in 7 (7%) and with TP53 mutations ( TP53Dpl/mut) in 22 (25%), while concurrent TP53 deletions and mutations ( TP53Del/mut) were detected in 29 (33%) ( p = 0.0005). Samples with TP53Del/mut harboured cth in chromosomes 7 (7%), 17 (6%), 8, 9, 19 and 21 (5%) whilst cases with TP53Dpl/mut had cth in chromosomes 21 (9%), 19 (8%), 5 (7%) and 7 (5%). (Fig 1 b). The cha aberrations generally followed the genome location of cth events with some exception. Firstly, chromosomes X,13 and 21 appear to have only cth and is seen at slightly higher level on chromosome 19 (cth 13% vs cha 9%). Secondly, cha was most frequently seen on 5q (61 %), chromosome 17 (40%) and 7q (34%). However, the same chromosomes also had the greatest number of cases presenting with concurrent cth and cha, seen in 63/87 (72%) for chromosome 5, 46/87 (53%) for chromosome 17 and 43/87 (49%) for chromosome 7. Importantly, only cases harbouring 5q deletions showed a statistically significant association with TP53 status ( p = 0.0011) in this cohort irrespective of cth and cha presence. Notably, 24/29 cases (83%) with TP53Del/mut were cth positive and had 5q31 deletion. This finding is an important observation but not in keeping with reports of exclusive association of TP53 mutations with concurrent 5q and 7q loss (1, 2 and 3). The concurrent recording of cth and cha performed by this study could explain the discrepancy, most likely due to high frequency of cha events in 5q. Summary This study highlights issues pertinent to risk stratification in routine diagnostic settings: (i) MDS/AML cases with complex genome harbouring either or both cth and cha share common TP53 aberrations (del/mut) and (ii) deletion of 5q31 is positively associated with TP53 aberrations and presence of cth events. (iii) CMA offers a comprehensive, sensitive, fast and financially superior way to detect cth, cha events along with genome complexity compared to G banding complemented by FISH. Overall, combining CMA and TP53 mutation screening offers a fast, reliable way to detect high risk disease in MDS/AML. Keywords: Complex genome, CMA, chromothripsis, TP53 mutations

T Cell Selection Is Not Helpful for Therapeutic Efficacy of CART19 in Relapsed or Refractory B-ALL Patients with Peripheral Blood Leukemia Burden at Time of Apheresis

Abstract Introduction: We have demonstrated peripheral blood (PB) blasts negatively affected chimeric antigen receptor T cells targeting CD19 (CART19) production and the clinical efficacy of CART19 therapy in patients (pts) with refractory/relapsed B cell acute lymphoblastic leukemia (r/r B-ALL) (published in Molecular Therapy: Methods &amp; Clinical Development ,2021). Whether sorting T cells before activation might be helpful for ex-vivo CART19 quality and in-vivo treatment efficacy requires further investigation. Here, we retrospectively analyzed 40 pts with or without upfront T cell purification to evaluate these indexes. Patients and Methods: From July 2021 to June 2023, 40 r/r B-ALL with PB blasts at time of apheresis experienced CART19 therapy. After leukapheresis, all pts' PBMCs were isolated using lymphocyte separation liquid. 30 pts didn't perform T cells selection (group 1) and 10 pts performed T cells selection (group 2). All of cells were activated with CD3 and CD28 antibodies for 24h, then transduced with the lentivirus encoding anti-CD19-CD3zeta-4-1BB CAR and cultured for 5-6 days in serum-free media containing IL2, IL7, IL15, IL21. The baseline characteristics, including age, sex, previous therapy period, blasts in BM, EMD distribution, complex chromosome aberration, poor fusion genes and gene mutations, did not differ between the two groups (Table 1). Cell viability, expanding fold, percentage of CD3 +T and CD3 + CD8 +T cells, transfection efficiency, B cells were all examined pre CART19 infusion. All pts received cyclophosphamide 250 mg/m2/d X 3d, and fludarabine 30 mg/m2/d X 3d, followed by CART19 infusion with a median number of 9.7(0.17-60) X10 5cells/kg in group 1 and 10 (4-16.3) X10 5cells/kg in group 2 ( P=0.99). PB and BM blasts was determined by flow cytometry (FCM). All pts underwent bone marrow (BM) biopsy examination and FCM studies on day 15 or day 30 to determine the response and remission status. Results: At time of apheresis, group 1 and group 2 had a median leukemia cell of 18% (2%-71%) and 19.4% (7%-54.4%) in PB ( P=0.5), respectively. In group 1 and group 2, the median percent of CD3 +T and CD3 + CD8 +T cells were 97.7% (70% - 99.5%) vs. 97.7% (94.8%-99.2%) ( P=0.46) and 66% (24.3% - 88.3%) vs. 64.2% (28% - 84.4%) ( P=0.45), respectively. The median cell viability, transfection efficiency and cell expanding fold in the final products was 82.9%（44.4%-92.2%) vs. 88.6%（76.4%-95.2%）( P=0.09), 31.4%（3.1%-65.5%) vs. 23.6% (12.4%-35.5%) ( P=0.08) and 3.18-fold（0.24-13.6) vs. 2.32-fold（0.48-8.13) ( P=0.83), respectively. Both of normal B and leukemia cells were undetectable pre CART19 infusion in two groups. After CART19 infusion, the median peak CART19/ lymphocyte cells, expanding fold and absolute CART19 count in vivo was 45% (0-79.4%) vs. 57.9% (0-75%) ( P=0.86), 16.7-fold (0-397.3) vs.13.2-fold (0-406)( P=0.96) and 229 (0-4810) X10 6/L blood vs. 242 (0-3220) X10 6/L blood ( P=0.83) in two groups, respectively. In group 1, 28/30 cases were evaluable and 25/28 (89.3%) obtained complete remission (CR)/ incomplete count recovery CR (CRi) and 24/28 (85.7%) obtained minimal residual disease (MRD) - CR. 1/2 patient unevaluable died from pneumonia due to severe and persisted neutropenia. One no-responder was infused at &amp;lt;1 X10 5 cells/kg and his culture failed to expand in vivo, and the other no-responder was infused at 1.16 X10 5 cells/kg (with TEL-AML1 positive). In group 2, 8/10 cases were evaluable and 6/8 (75%) obtained CR/CRi with all MRD - CR (75%). The two non-responders had no detectable CART19 in PB after infusion for one month. Results were shown in Table2. Conclusion: We have demonstrated pts with PB blasts without sorting T cells had poorer ex-vivo CART19 quality and in-vivo treatment efficacy compared to pts without PB blasts (published). Sorting T cells before activation is expected to remove the adverse culture conditions in the system. However, in the present study, upfront T cell purification didn't improve ex vivo cell characteristics and in vivo cell dynamics in r/r B-ALL pts with PB blasts, showing that the negative influence of PB blasts at the time of apheresis could not be reversed by upfront T cell purification. Therefore, leukemia burden in PB is suggested to be reduced before leukapheresis. Absolutely, one of the limitations of our study is that a relatively small number of products was evaluated, and larger samples need to be included to further demonstrated.

Development of a Set of Wheat-Rye Derivative Lines from Hexaploid Triticale with Complex Chromosomal Rearrangements to Improve Disease Resistance, Agronomic and Quality Traits of Wheat.

An elite hexaploid triticale Yukuri from Australia was used as a bridge for transferring valuable genes from Secale cereale L. into common wheat for enriching the genetic variability of cultivated wheat. Non-denaturing-fluorescence in situ hybridization (ND-FISH) identified that Yukuri was a secondary triticale with a complete set of rye chromosomes and a 6D(6A) substitution. Seed protein electrophoresis showed that Yukuri had a unique composition of glutenin subunits. A set of Yukuri-derived wheat-rye introgression lines were created from a Yukuri x wheat population, and all lines were identified by ND-FISH with multiple probes and validated by diagnostic molecular marker analysis. A total of 59 wheat-rye introgression lines including modified chromosome structural variations of wheat, and new complex recombinant chromosomes of rye were detected through ND-FISH and Oligo-FISH painting based on oligonucleotide pools derived from wheat-barley genome collinear regions. Wheat lines carrying the 1R chromosome from Yukuri displayed resistance to both stripe rust and powdery mildew, while the lines carrying the 3RL and 7RL chromosome arms showed stripe rust resistance. The chromosome 1R-derived lines were found to exhibit a significant effect on most of the dough-related parameters, and chromosome 5R was clearly associated with increased grain weight. The development of the wheat-rye cytogenetic stocks carrying disease resistances and superior agronomic traits, as well as the molecular markers and FISH probes will promote the introgression of abundant variation from rye into wheat improvement programs.

Scrambling the genome in cancer: causes and consequences of complex chromosome rearrangements.

Complex chromosome rearrangements, known as chromoanagenesis, are widespread in cancer. Based on large-scale DNA sequencing of human tumours, the most frequent type of complex chromosome rearrangement is chromothripsis, a massive, localized and clustered rearrangement of one (or a few) chromosomes seemingly acquired in a single event. Chromothripsis can be initiated by mitotic errors that produce a micronucleus encapsulating a single chromosome or chromosomal fragment. Rupture of the unstable micronuclear envelope exposes its chromatin to cytosolic nucleases and induces chromothriptic shattering. Found in up to half of tumours included in pan-cancer genomic analyses, chromothriptic rearrangements can contribute to tumorigenesis through inactivation of tumour suppressor genes, activation of proto-oncogenes, or gene amplification through the production of self-propagating extrachromosomal circular DNAs encoding oncogenes or genes conferring anticancer drug resistance. Here, we discuss what has been learned about the mechanisms that enable these complex genomic rearrangements and their consequences in cancer.

QuarTeT: a telomere-to-telomere toolkit for gap-free genome assembly and centromeric repeat identification.

A high-quality genome is the basis for studies on functional, evolutionary, and comparative genomics. The majority of attention has been paid to the solution of complex chromosome structures and highly repetitive sequences, along with the emergence of a new 'telomere-to-telomere (T2T) assembly' era. However, the bioinformatic tools for the automatic construction and/or characterization of T2T genome are limited. Here, we developed a user-friendly web toolkit, quarTeT, which currently includes four modules: AssemblyMapper, GapFiller, TeloExplorer, and CentroMiner. First, AssemblyMapper is designed to assemble phased contigs into the chromosome-level genome by referring to a closely related genome. Then, GapFiller would endeavor to fill all unclosed gaps in a given genome with the aid of additional ultra-long sequences. Finally, TeloExplorer and CentroMiner are applied to identify candidate telomere and centromere as well as their localizations on each chromosome. These four modules can be used alone or in combination with each other for T2T genome assembly and characterization. As a case study, by adopting the entire modular functions of quarTeT, we have achieved the Actinidia chinensis genome assembly that is of a quality comparable to the reported genome Hongyang v4.0, which was assembled with the addition of manual handling. Further evaluation of CentroMiner by searching centromeres in Arabidopsis thaliana and Oryza sativa genomes showed that quarTeT is capable of identifying all the centromeric regions that have been previously detected by experimental methods. Collectively, quarTeT is an efficient toolkit for studies of large-scale T2T genomes and can be accessed at http://www.atcgn.com:8080/quarTeT/home.html without registration.

Effects of sexual polyploidization on reproductive development in a full-sib poplar family

Populus is an important source of woody biomass in the Northern Hemisphere. Sexual polyploidization plays a valuable role in the genetic improvement of Populus, resulting in tremendous variation in phenotypic traits. Most studies have focused on the variations in the vegetative growth of allopolyploid Populus; however, variations in the reproductive development of different polyploid genotypes of the progeny remain unclear. In this study, catkin traits, pollen size and viability, meiotic development, and reproductive ability were analyzed based on a full-sib family including diploid and triploid hybrids of (Populus pseudo-simonii × P. nigra ‘Zheyin3#’) × (P. × beijingensis) to understand the effects of sexual polyploidization on reproductive development in Populus hybrids. Catkin traits varied among the genotypes in the full-sib family. A ploidy change resulted in significant variation in male catkin length and male flower number per catkin but not in female catkin traits, suggesting that Populus catkin development may be more sensitive to respond to a change in ploidy level in male plants than in female plants. The male diploid hybrids had significantly smaller pollen grains, but higher pollen germination rates than the triploid hybrids. Giant pollen grains with large diameters were recorded in all-male diploid and triploid hybrids, which were speculated to be unreduced gametes. Premature cytokinesis, misorientation of the spindles, and aberrant cytokinesis were observed in a cytological analysis, which explained the production of unreduced pollen. Premature cytokinesis was only detected in triploid hybrids, which might be attributed to a ploidy change. The frequencies of meiotic abnormalities, such as precocious chromosomal migration, chromosome bridges, and micronuclei in the triploid hybrids were higher than those in diploid hybrids, suggesting that the unbalanced chromosomes in the triploid hybrids caused more complex meiotic chromosome behaviors. Backcrosses with the triploid hybrids resulted in low seed sets and seed germination rates, suggesting that polyploidization reduced pollen viability. Our findings provide new insight into the effect of sexual polyploidization on reproductive development in Populus and could be used to guide a Populus polyploid breeding program.

Revision and analysis of the chromosome variability in the speciose genus Akodon (Rodentia, Sigmodontinae), including new data from Argentina.

Rodentia has a high species number and chromosomal variability. The South American genus Akodon is one of the most speciose muroids, with more than 40 species included in several species groups. Here, we characterize cytogenetically specimens of Akodon from central-western Argentina. Subsequently, we reviewed and analyzed the cytogenetic data for this genus, build a phylogeny and mapped chromosome changes to interpret its evolution. Specimens of A. dolores from central-western Argentina have 2n=42-44/FNa=44 (46, 48) due to a Robertsonian rearrangement. Our data expand the distribution range known for this polymorphism and confirm its geographic structure. Other specimens had 2n=40/FNa=40, representing populations of A. oenos, A. polopi, and A. spegazzinii. All karyotypes have a low amount of heterochromatin, concentrated in centromeres and sex chromosomes, as in other rodents. The complement with 2n=40/FNa=40 is the most frequent in Akodon and is shared by most species in some groups. Chromosome numbers are very diverse. The FNa shows less variability; FNa=42 was recovered as ancestral, excluding A. mimus, which was connected at the base of the Akodon tree and has FNa=44. This indicates a complex chromosome evolution in Akodon, and suggests that reductions and increases in the 2n and FNa evolved independently in some lineages.

A treatment-refractory aggressive MDS-MLD with multiple highly complex chromosome 5 intrachromosomal rearrangements: a case report

BackgroundA patient with a myelodysplastic neoplasm exhibited a karyotype with multiple complex chromosome 5 rearrangements. This patient appeared to have a catastrophic cytogenetic event that manifested as a treatment-refractory aggressive form of disease, which lead to patient demise within one year. Both the clinical presentation and disease course were unusual based on the medical history and morphologic findings. Such cases of myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) with complex abnormalities are not reported in the literature.Case presentationThe patient was a 62-year-old female who presented with pancytopenia and dyspnea. The morphologic appearance of the peripheral blood smear and bone marrow biopsy, along with flow cytometric findings, favored the diagnosis of MDS-MLD unclassifiable. Myelodysplastic syndrome (MDS) with multilineage dysplasia (MDS-MLD), is an MDS characterized by one or more cytopenias and dysplastic changes in two or more of the myeloid lineages (i.e., erythroid, granulocytic, and megakaryocytic). The bone marrow, in particular, showed prominent dysplasia, including the presence of atypical megakaryocytes with small hypolobated morphology reminiscent of those typically seen in MDS with isolated 5q deletion. Cytogenetic analysis, including interphase and metaphase FISH, karyotype and SNP chromosomal microarray were performed, as well as DNA sequencing studies. Cytogenetic analysis showed a very complex karyotype featuring multiple 5q intrachromosomal rearrangements including a pericentric inversion with multiple interspersed deletions and monosomy 7. FISH studies showed a partial deletion of the PDGFRβ gene, and SNP chromosomal microarray and targeted panel-based sequencing identified biallelic loss of function of the TP53 gene. Based on the pathologic findings, the patient was treated for MDS but did not respond to either lenalidomide or azacitidine.ConclusionThe genetic changes described, in particular, the complex intrachromosomal rearrangements of chromosome 5, suggest the occurrence of a sudden catastrophic event that led to an aggressive course in the patient’s disease. Conventional karyotyping, metaphase and interphase FISH, SNP chromosomal microarray and NGS helped to identify the complex genetic changes seen in this case. This highlights the importance of utilizing a multimodality approach to fully characterize complex chromosomal events that may significantly impact disease progression, treatment and survival.