Hepatitis B virus (HBV) infects hepatocytes that are in the G0/G1 phase with intact nuclear membrane and organized chromosome architecture. In the nucleus of the infected cells, HBV covalently closed circular (ccc) DNA, an episomal minichromosome, serves as the template for all viral transcripts and the reservoir of persistent infection. Nuclear positioning of cccDNA can be assessed by the spatial distance between viral DNA and host chromosomal DNA through Circular Chromosome Conformation Capture (4C) combined with high-throughput sequencing (4C-seq). The 4C-seq analysis relies on proximity ligation and is commonly used for mapping genomic DNA regions that communicate within a host chromosome. The method has been tailored for studying nuclear localization of HBV episomal cccDNA in relation to the host chromosomes. In this study, we present a step-by-step protocol for 4C-seq analysis of HBV infection, including sample collection and fixation, 4C DNA library preparation, sequence library preparation, and data analysis. Although limited by proximity ligation of DNA fragments, 4C-seq analysis provides useful information of HBV localization in 3D genome, and aids the understanding of viral transcription in light of host chromatin conformation.

Duplication is a foundation of molecular evolution and a driver of genomic and complex diseases. Here, we develop a genome editing tool named Amplification Editing (AE) that enables programmable DNA duplication with precision at chromosomal scale. AE can duplicate human genomes ranging from 20 bp to 100 Mb, a size comparable to human chromosomes. AE exhibits activity across various cell types, encompassing diploid, haploid, and primary cells. AE exhibited up to 73.0% efficiency for 1 Mb and 3.4% for 100 Mb duplications, respectively. Whole-genome sequencing and deep sequencing of the junctions of edited sequences confirm the precision of duplication. AE can create chromosomal microduplications within disease-relevant regions in embryonic stem cells, indicating its potential for generating cellular and animal models. AE is a precise and efficient tool for chromosomal engineering and DNA duplication, broadening the landscape of precision genome editing from an individual genetic locus to the chromosomal scale.

Chromosome banding can be defined as the lengthwise variation in staining properties along a chromosome stained with a dye. Chromosome banding became more practical in the early 1970s and is an essential technique used in karyotyping to identify human chromosomes for both clinical and research purposes. Most importantly, karyotyping is now considered a mandatory investigation of all newly diagnosed leukemias. Some banding methods, such as Giemsa (G)-, reverse (R)-, and centromere (C)-banding, still contribute greatly by being used as a routine procedure in clinical cytogenetic laboratory nowadays. Each chromosome has a unique sequence of bar code-like stripes, allowing the identification of individual homologues and the recognition of structural abnormalities through analyzing the disruption of the normal banding pattern at specific landmarks, regions, and bands as described in the ideogram. Since the quality of metaphases obtained from malignant cells is generally inferior to normal constitutional cells for karyotyping, a practical and accurate chromosome identification training guide is indispensable for a trainee or newly employed cytogenetic technologist in a cancer cytogenetic laboratory. The most common and currently used banding methods and chromosome recognition guide for distinguishable bands of each chromosome are described in detail in this chapter with an aim to facilitate quick and accurate karyotyping in cancer cells.

Automated karyotyping is of great importance for cytogenetic research, as it speeds up the process for cytogeneticists through incorporating AI-driven automated segmentation and classification techniques. Existing frameworks confront two primary issues: Firstly the necessity for instance-level data annotation with either detection bounding boxes or semantic masks for training, and secondly, its poor robustness particularly when confronted with domain shifts. In this work, we first propose an accurate segmentation framework, namely KaryoXpert. This framework leverages the strengths of both morphology algorithms and deep learning models, allowing for efficient training that breaks the limit for the acquirement of manually labeled ground-truth mask annotations. Additionally, we present an accurate classification model based on metric learning, designed to overcome the challenges posed by inter-class similarity and batch effects. Our framework exhibits state-of-the-art performance with exceptional robustness in both chromosome segmentation and classification. The proposed KaryoXpert framework showcases its capacity for instance-level chromosome segmentation even in the absence of annotated data, offering novel insights into the research for automated chromosome segmentation. The proposed method has been successfully deployed to support clinical karyotype diagnosis.

Glioblastoma multiforme (GBM) encompasses brain malignancies marked by phenotypic and transcriptional heterogeneity thought to render these tumors aggressive, resistant to therapy, and inevitably recurrent. However, little is known about how the spatial organization of GBM genomes underlies this heterogeneity and its effects. Here, we compile a cohort of 28 patient-derived glioblastoma stem cell-like lines (GSCs) known to reflect the properties of their tumor-of-origin; six of these were primary-relapse tumor pairs from the same patient. We generate and analyze 5 kbp-resolution chromosome conformation capture (Hi-C) data from all GSCs to systematically map thousands of standalone and complex structural variants (SVs) and the multitude of neoloops arising as a result. By combining Hi-C, histone modification, and gene expression data with chromatin folding simulations, we explain how the pervasive, uneven, and idiosyncratic occurrence of neoloops sustains tumor-specific transcriptional programs via the formation of new enhancer-promoter contacts. We also show how even moderately recurrent neoloops can relate to patient-specific vulnerabilities. Together, our data provide a resource for dissecting GBM biology and heterogeneity, as well as for informing therapeutic approaches.

Human papillomavirus (HPV) integration is a critical step in cervical cancer development; however, the oncogenic mechanism at the genome-wide transcriptional level is still poorly understood. In this study, we employed integrative analysis on multi-omics data of six HPV-positive and three HPV-negative cell lines. Through HPV integration detection, super-enhancer (SE) identification, SE-associated gene expression and extrachromosomal DNA (ecDNA) investigation, we aimed to explore the genome-wide transcriptional influence of HPV integration. We identified seven high-ranking cellular SEs generated by HPV integration in total (the HPV breakpoint-induced cellular SEs, BP-cSEs), leading to intra-chromosomal and inter-chromosomal regulation of chromosomal genes. The pathway analysis revealed that the dysregulated chromosomal genes were correlated to cancer-related pathways. Importantly, we demonstrated that BP-cSEs existed in the HPV-human hybrid ecDNAs, explaining the above transcriptional alterations. Our results suggest that HPV integration generates cellular SEs that function as ecDNA to regulate unconstrained transcription, expanding the tumorigenic mechanism of HPV integration and providing insights for developing new diagnostic and therapeutic strategies.

Objective: The clinical characteristics and prognosis of 20 patients with small B-lymphocyte proliferative disease with t (14;19) (q32; q13) were analyzed to improve the understanding of such rare cases. Methods: The clinical data of 20 patients with t (14; 19) (q32; q13) small B lymphocyte proliferative disease treated in the First Affiliated Hospital of Nanjing Medical University from April 2013 to December 2020 were retrospectively collected and analyzed. Among them, 10 cases were chronic lymphocytic leukemia (CLL) and 10 cases were other small B-cell malignancies. Results: Among the 20 cases, 10 were male and 10 were female, and the median age at diagnosis was 53.5 (35-88) years old. All patients had absolute lymphocytosis, 19 patients had lymphadenopathy, and 10 patients had splenomegaly. With a median follow-up of 36 (4-163) months, three patients died, and 11 patients had a time to treatment (TTT) ≤12 months. Ten patients (50%) were accompanied by +12, two patients (2/17, 12%) were accompanied by 13q-. Moreover, we found that t (14;19) was associated with unmutated immunoglobulin heavy-chain variable (IGHV) somatic mutation (17/19, 89%) and a biased use of IGHV4-39 (7/17, 41%) was observed. Next-generation sequencing detected one or more gene mutations in 14 (14/17, 82%) cases and a total of 25 gene mutations had been revealed, of which the most frequent were NOTCH1 (35%) , followed by SF3B1 (24%) and KMT2D (18%) . For 10 CLL patients, five (50%) were defined as Rai Ⅲ/Binet C. It is noteworthy that among the 20 cases, two cases actually involved Richter transformation. Conclusions: Small B-cell malignant tumors with abnormal t (14; 19) show unique clinical biological characteristics, often accompanied by a variety of adverse prognostic factors, and tend to have an aggressive clinical course.
目的： 分析20例伴t（14;19）（q32;q13）的小B淋巴细胞增殖性疾病患者的临床特征及预后，以提高对此类少见病例的认识。 方法： 回顾性收集并分析2013年4月至2020年12月南京医科大学第一附属医院收治的20例伴t（14;19）（q32;q13）小B淋巴细胞增殖性疾病患者的临床资料，其中10例为慢性淋巴细胞白血病（CLL），10例为其他小B细胞恶性肿瘤。 结果： 男10例，女10例，中位年龄53.5（35~88）岁。所有患者均出现淋巴细胞绝对计数增多，19例出现淋巴结肿大，10例脾肿大。中位随访36（4~163）个月，3例死亡，11例患者至开始治疗时间（TTT）≤12个月。10例（50%）患者伴+12，2例（2/17,12%）伴13q-。t（14;19）与免疫球蛋白重链可变区（IGHV）无突变相关（17/19，89%），且存在IGHV4-39的偏向使用（7/17，41%）。应用二代测序在14例（14/17，82%）患者中检出一种或多种基因突变，共涉及25种基因异常，其中发生频率最高的是NOTCH1（35%），其次是SF3B1（24%）和KMT2D（18%）。对10例CLL患者分析发现，5例（50%）分期为Rai Ⅲ/Binet C期。在20例患者中，2例发生Richter转化。 结论： 伴t（14;19）异常的小B细胞恶性肿瘤显示出独特的临床生物学特征，常伴多种不良预后因素，倾向于具有侵袭性临床病程。.

The present study aimed to explore whether chromosomal polymorphisms (CPs) have negative effects on reproductive outcomes of in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET)/frozen-thawing embryo transfer (FET)? We conducted a retrospective study consisting of 21,867 assisted reproductive technology treatment cycles, among which, fresh embryo transfer cycles accounted for 10,400, and the rest were FET cycles. According to karyotype of CPs, the former was grouped as: group 1 (male carrier, n = 425), group 2 (female carrier, n = 262), and group 3 (couple without CPs, n = 9713). Accordingly, FET cycles were divided into 3 groups: group 4 (male carrier, n = 298), group 5 (female carrier, n = 311), and group 6 (couple without CPs, n = 10,858). The embryo implantation rate (IR), clinical pregnancy rate (CPR), live birth rate (LBR), and early miscarriage rate (EMR) were compared among the groups. In fresh embryo transfer cycles after IVF/ICSI, there were no significant differences in the infertility duration, BMI, basal FSH, no. of oocyte, no. of 2PN, endometrial thickness on trigger day, serum E2, P, and LH level on trigger day (P > 0.05). The female age, no. of 2PN embryo cleavage, top-quality embryo, and no. of embryo transferred were significantly different among groups (P < 0.05). The IR was 38.8%, 36.2%, and 34.0% in groups 1, 2, and 3, respectively. The CPR was 55.1%, 52.3%, and 49.7%, respectively. The LBR was 36.9%, 37.4%, and 36.4%, respectively. The CPR and LBR showed no significant differences among groups. The IR was lower and the EMR was higher in group 3 than those of groups 1 and 2. Binary logistic regression analysis indicated that female age, no. of embryo transferred, EMT, LH, and P on the trigger day were independently factors associated with CPR. Besides, no. of embryo transferred, and EMT on trigger day were associated with LBR, while the CPs was not related with CPR and LBR after IVF/ICSI-ET. In FET cycles, the infertility duration was similar (P > 0.05), but the female age, BMI, no. of embryo transferred were significantly different among groups (P > 0.05). The IR was 24.3%, 23.6% and 22.3% in group 4, 5, and 6, receptivity. The CPR was 31.8%, 30.9%, and 30.0%, the LBR was 23.8%,26.3%, and 23.8%, while the EMR was 12.6%, 13.1%, 14.4%, respectively. The IR, CPR, EMR, and LBR showed no significant differences among groups (P > 0.05). Binary logistic regression analysis indicated that female age, infertility duration, and no. of embryo transferred were independently factors affecting CPR and LBR after FET. The CPs were not associated with CPR and LBR after FET. The results suggested that uniparental carrying of CPs have no effects on the reproductive outcomes after IVF/ICSI-ET/FET. However, it is not clear whether both parents carrying CPs would affect pregnancy outcome.

The current human reference genome, GRCh38, represents over 20 years of effort to generate a high-quality assembly, which has benefitted society1,2. However, it still has many gaps and errors, and does not represent a biological genome as it is a blend of multiple individuals3,4. Recently, a high-quality telomere-to-telomere reference, CHM13, was generated with the latest long-read technologies, but it was derived from a hydatidiform mole cell line with a nearly homozygous genome5. To address these limitations, the Human Pangenome Reference Consortium formed with the goal of creating high-quality, cost-effective, diploid genome assemblies for a pangenome reference that represents human genetic diversity6. Here, in our first scientific report, we determined which combination of current genome sequencing and assembly approaches yield the most complete and accurate diploid genome assembly with minimal manual curation. Approaches that used highly accurate long reads and parent-child data with graph-based haplotype phasing during assembly outperformed those that did not. Developing a combination of the top-performing methods, we generated our first high-quality diploid reference assembly, containing only approximately four gaps per chromosome on average, with most chromosomes within ±1% of the length of CHM13. Nearly 48% of protein-coding genes have non-synonymous amino acid changes between haplotypes, and centromeric regions showed the highest diversity. Our findings serve as a foundation for assembling near-complete diploid human genomes at scale for a pangenome reference to capture global genetic variation from single nucleotides to structural rearrangements.

Male infertility is a multifactorial condition that is closely associated with chromosomal abnormalities. Reciprocal chromosomal translocation (RCT) is a significant structural genetic abnormality. The specific mechanisms of forms of RCT affecting male infertility include the product of chromosomally unbalanced gametes, thereby disrupting the structure and function of important genes responsible for spermatogenesis. RCT breakpoints have been found to disrupt gene structure and function in many medical fields However, the relationship between RCT breakpoints and male infertility remains to be determined. The purpose of this study is to describe 2 male carriers of RCTs 46,XY,t(8;22)(q13;q13) and 46,XY,t(8;14)(q13;q22). Both patients were collected from the second hospital of Jilin University. Semen parameters were detected using the computer-aided semen analysis system. Cytogenetic analysis was performed using standard operating procedure. Related genes on chromosomal breakpoints were searched using Online Mendelian Inheritance in Man. One man had semen parameters within the normal range, but the couple was infertile after 5 years of marriage. The other man showed normal semen parameters, and his wife had experienced 2 spontaneous miscarriages. Using a literature search, the association between chromosome 22q13 breakpoint and fertility were investigated. The results suggest that physicians should focus on the clinical phenotype of the patients and the breakpoints of RCT in genetic counseling. An important gene related to human male infertility is clearly located in chromosome region 22q13, and its function is worthy of further study.