Integrated Proteogenomic Characterization of Clear Cell Renal Cell Carcinoma.

To elucidate the deregulated functional modules that drive clear cell renal cell carcinoma (ccRCC), we performed comprehensive genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic characterization of treatment-naive ccRCC and paired normal adjacent tissue samples. Genomic analyses identified a distinct molecular subgroup associated with genomic instability. Integration of proteogenomic measurements uniquely identified protein dysregulation of cellular mechanisms impacted by genomic alterations, including oxidative phosphorylation-related metabolism, protein translation processes, and phospho-signaling modules. To assess the degree of immune infiltration in individual tumors, we identified microenvironment cell signatures that delineated four immune-based ccRCC subtypes characterized by distinct cellular pathways. This study reports a large-scale proteogenomic analysis of ccRCC to discern the functional impact of genomic alterations and provides evidence for rational treatment selection stemming from ccRCC pathobiology.

AID and Reactive Oxygen Species Can Induce DNA Breaks within Human Chromosomal Translocation Fragile Zones.

DNA double-strand breaks (DSBs) occurring within fragile zones of less than 200 base pairs account for the formation of the most common human chromosomal translocations in lymphoid malignancies, yet the mechanism of how breaks occur remains unknown. Here, we have transferred human fragile zones into S. cerevisiae in the context of a genetic assay to understand the mechanism leading to DSBs at these sites. Our findings indicate that a combination of factors is required to sensitize these regions. Foremost, DNA strand separation by transcription or increased torsional stress can expose these DNA regions to damage from either the expression of human AID or increased oxidative stress. This damage causes DNA lesions that, if not repaired quickly, are prone to nuclease cleavage, resulting in DSBs. Our results provide mechanistic insight into why human neoplastic translocation fragile DNA sequences are more prone to enzymes or agents that cause longer-lived DNA lesions.

Targeted genomic translocations and inversions generated using a paired prime editing strategy.

A variety of cancers have been found to have chromosomal rearrangements, and the genomic abnormalities often induced expression of fusion oncogenes. To date, a pair of engineered nucleases including ZFNs, TALENs, and CRISPR-Cas9 nucleases have been used to generate chromosomal rearrangement in living cells and organisms for disease modeling. However, these methods induce unwanted indel mutations at the DNA break junctions, resulting in incomplete disease modeling. Here, we developed prime editor nuclease-mediated translocation and inversion (PETI), a method for programmable chromosomal translocation and inversion using prime editor 2 nuclease (PE2 nuclease) and paired pegRNA. Using PETI method, we successfully introduced DNA recombination in episomal fluorescence reporters as well as precise chromosomal translocations in human cells. We applied PETI to create cancer-associated translocations and inversions such as NPM1-ALK and EML4-ALK in human cells. Our findings show that PETI generated chromosomal translocation and inversion in a programmable manner with efficiencies comparable of Cas9. PETI methods, we believe, could be used to create disease models or for gene therapy.

ZAP70: A Key Gene Identified by Differential Expression Analysis for Early Diagnosis of Fetuses with Emanuel Syndrome.

Emanuel syndrome is a rare autosomal disorder characterized by microcephaly, heart defects, cleft palate and developmental delay. However, there is a lack of specific prenatal screening for Emanuel syndrome. To screen for early diagnostic marker genes in fetuses with karyotype+der[22]t(11;22)(q23;q11) of Emanuel syndrome. Transcriptome sequencing and clinical trait data of t(11;22)(q23;q11) translocation samples were screened from the GEO database. The differentially expressed genes (DEGs) were screened by principal component analysis of gene expression by R package, and intersections were taken with balanced and unbalanced DEGs. Then, the correlation with clinical traits was determined by WGCNA analysis, GO and KEGG enrichment analysis, and then univariate Cox analysis and Lasso analysis were performed to obtain the key genes. The core regulatory genes were obtained after protein-protein interaction (PPI) network analysis. A total of 50 DEGs were obtained after differential analysis. WGCNA analysis showed that DEG was associated with the chromosomal imbalance and age module. GO and KEGG enrichment analyses showed candidate genes were associated with exocytic vesicle membrane, synaptic vesicle membranes, glycoprotein complex, dystrophin-associated glycoprotein complex and malaria. COX and Lasso analyses yielded 5 hub genes, including ZBED9, RGS20, SGCB, ETV5, and ZAP70. The results of PPI identified the key regulatory gene associated with chromosomal imbalance as the ZAP70 gene. ZAP70 may be a key gene for early diagnosis of Emanuel syndrome in fetuses with+der[22]t(11;22)(q23;q11) karyotype.

Alterations in Genome Organization in Lymphoma Cell Nuclei due to the Presence of the t(14;18) Translocation.

Chromosomal rearrangements have been shown to alter genome organization, consequently having an impact on gene expression. Studies on certain types of leukemia have shown that gene expression can be exacerbated by the altered nuclear positioning of fusion genes arising from chromosomal translocations. However, studies on lymphoma have been, so far, very limited. The scope of this study was to explore genome organization in lymphoma cells carrying the t(14;18)(q32;q21) rearrangement known to results in over-expression of the BCL2 gene. In order to achieve this aim, we used fluorescence in situ hybridization to carefully map the positioning of whole chromosome territories and individual genes involved in translocation in the lymphoma-derived cell line Pfeiffer. Our data show that, although there is no obvious alteration in the positioning of the whole chromosome territories, the translocated genes may take the nuclear positioning of either of the wild-type genes. Furthermore, the BCL2 gene was looping out in a proportion of nuclei with the t(14;18) translocation but not in control nuclei without the translocation, indicating that chromosome looping may be an essential mechanism for BCL2 expression in lymphoma cells.

[Clinical, histological and genetic correlations in melanocytic tumours with chromosomal rearrangements]. / Corrélations cliniques, morphologiques et génétiques dans les tumeurs mélanocytaires avec translocations chromosomiques.

In some tumoral subtypes chromosomal translocations lead to an oncogenic chimeric protein acting as a tumorigenesis driver event. The main fusion model combines the promoter swapping of an inactivated tumor suppressor gene and a functional kinase that evades its regulatory system. The range of described fusions keeps growing in the 2023 WHO classification of melanocytic tumours. It is not limited to the group of Spitz tumours as previously but now extends to blue tumours and dermal tumours with a melanocytic phenotype. Molecular pathology helps detect these anomalies using clinical and morphological features. This analysis is essential as this strongly conditions the adapted local treatment of such tumours who are often overtreated.

Autopolyploid inheritance and a heterozygous reciprocal translocation shape chromosome genetic behavior in tetraploid blueberry (Vaccinium corymbosum).

Understanding chromosome recombination behavior in polyploidy species is key to advancing genetic discoveries. In blueberry, a tetraploid species, the line of evidences about its genetic behavior still remain poorly understood, owing to the inter-specific, and inter-ploidy admixture of its genome and lack of in depth genome-wide inheritance and comparative structural studies. Here we describe a new high-quality, phased, chromosome-scale genome of a diploid blueberry, clone W85. The genome was integrated with cytogenetics and high-density, genetic maps representing six tetraploid blueberry cultivars, harboring different levels of wild genome admixture, to uncover recombination behavior and structural genome divergence across tetraploid and wild diploid species. Analysis of chromosome inheritance and pairing demonstrated that tetraploid blueberry behaves as an autotetraploid with tetrasomic inheritance. Comparative analysis demonstrated the presence of a reciprocal, heterozygous, translocation spanning one homolog of chr-6 and one of chr-10 in the cultivar Draper. The translocation affects pairing and recombination of chromosomes 6 and 10. Besides the translocation detected in Draper, no other structural genomic divergences were detected across tetraploid cultivars and highly inter-crossable wild diploid species. These findings and resources will facilitate new genetic and comparative genomic studies in Vaccinium and the development of genomic assisted selection strategy for this crop.

Temporal and structural patterns of hepatitis B virus integrations in hepatocellular carcinoma.

Chronic infection of hepatitis B virus (HBV) is the major cause of hepatocellular carcinoma (HCC). Notably, 90% of HBV-positive HCC cases exhibit detectable HBV integrations, hinting at the potential early entanglement of these viral integrations in tumorigenesis and their subsequent oncogenic implications. Nevertheless, the precise chronology of integration events during HCC tumorigenesis, alongside their sequential structural patterns, has remained elusive thus far. In this study, we applied whole-genome sequencing to multiple biopsies extracted from six HBV-positive HCC cases. Through this approach, we identified point mutations and viral integrations, offering a blueprint for the intricate tumor phylogeny of these samples. The emergent narrative paints a rich tapestry of diverse evolutionary trajectories characterizing the analyzed tumors. We uncovered oncogenic integration events in some samples that appear to happen before and during the initiation stage of tumor development based on their locations in reconstituted trajectories. Furthermore, we conducted additional long-read sequencing of selected samples and unveiled integration-bridged chromosome rearrangements and tandem repeats of the HBV sequence within integrations. In summary, this study revealed premalignant oncogenic and sequential complex integrations and highlighted the contributions of HBV integrations to HCC development and genome instability.

The Recurrent Atypical e8a2 BCR::ABL1 Transcript with Insertion of an Inverted 55 Base Pair ABL1 Intron 1b Sequence: A Detailed Molecular Analysis.

Atypical BCR::ABL1 transcripts are found in approximately 2% of cases of chronic myeloid leukemia. It is important to detect them since affected patients also benefit from tyrosine kinase inhibitor therapy. In the rare e8a2 atypical BCR::ABL1 transcript, two out-of-frame exons are fused, thus, interposed nucleotides are usually found at the fusion site to restore the reading frame. In approximately half of previously reported e8a2 BCR::ABL1 cases, an inserted 55 bp sequence homologous to an inverted sequence from ABL1 intron 1b was detected. The generation of this recurrent transcript variant is not obvious. This work describes the molecular analysis of such an e8a2 BCR::ABL1 translocation from a CML patient. The genomic chromosomal breakpoint is identified, and the formation of this transcript is theoretically explained. The clinical course of the patient is reported, and recommendations are provided for the molecular analysis of future e8a2 BCR::ABL1 cases.

A New Case of Translocation T(2;7)(p23;q35) in Recurrent Pregnancy Loss.

Recurrent pregnancy loss is a phenomenon caused by many etiologies. The majority of these causes are chromosomal anomalies. In this case report, cytogenetic analysis was performed on the family who consulted our department with the complaint of recurrent pregnancy loss. A normal karyotype was found in the female (46, XX); however, t(2;7)(p23;q35) translocation was detected in the male. Reciprocal translocations are a common class of chromosomal abnormalities, and we anticipate this case of translocation will be a new cause for recurrent pregnancy loss. In the analysis, preparations at the level of 500 bands were examined, and at least 20 metaphase areas were evaluated. From the results of cytogenetic and FISH (fluorescence in situ hybridization) analysis, we determined the male had t(2;7)(p23;q35) chromosomal anomaly. The probe binding the patient's 2p23 region signaled at the q-terminal of chromosome 7; however, the other two chromosomes (2 and 7) were normal. There is no report of such a case in the literature for recurrent pregnancy loss complaints. With this case, it will be reported for the first time that an embryo formed with the gametes carrying unbalanced genetic material of an individual with the karyotype 46, XY, t(2;7)(p23;q35) is incompatible with life.

Genomic analysis reveals a KIT-related chromosomal translocation associated with the white coat phenotype in yak.

White coat pigmentation is a striking phenotype of many domesticated species and has various genetic controls. The Tianzhu White yak, an indigenous breed with a complete white coat, has fascinated Tibetans for centuries. However, the genetic basis of this trait remains unknown. Here, we conducted population genomics analysis and genome-wide association study based on the whole-genome sequencing data of 38 white and 59 non-white-coated yak. The results revealed the presence of KIT-linked Cs alleles characterized by the translocations between chromosomes 6 and 29 in all-white yak. Furthermore, structural variations showed additional duplications of the Cs alleles in white yak compared with colour-sidedness cattle. Interestingly, the Cs alleles associated with the white coat phenotype in yak were found to have introgressed from taurine cattle. Our findings unveil the shared genetic control of the white coat phenotype and its evolution in closely related bovine species.

Multi-omics analysis reveals multiple mechanisms causing Prader-Willi like syndrome in a family with a X;15 translocation.

Prader-Willi syndrome (PWS; MIM# 176270) is a neurodevelopmental disorder caused by the loss of expression of paternally imprinted genes within the PWS region located on 15q11.2. It is usually caused by either maternal uniparental disomy of chromosome 15 (UPD15) or 15q11.2 recurrent deletion(s). Here, we report a healthy carrier of a balanced X;15 translocation and her two daughters, both with the karyotype 45,X,der(X)t(X;15)(p22;q11.2),-15. Both daughters display symptoms consistent with haploinsufficiency of the SHOX gene and PWS. We explored the architecture of the derivative chromosomes and investigated effects on gene expression in patient-derived neural cells. First, a multiplex ligation-dependent probe amplification methylation assay was used to determine the methylation status of the PWS-region revealing maternal UPD15 in daughter 2, explaining her clinical symptoms. Next, short read whole genome sequencing and 10X genomics linked read sequencing was used to pinpoint the exact breakpoints of the translocation. Finally, we performed transcriptome sequencing on neuroepithelial stem cells from the mother and from daughter 1 and observed biallelic expression of genes in the PWS region (including SNRPN) in daughter 1. In summary, our multi-omics analysis highlights two different PWS mechanisms in one family and provide an example of how structural variation can affect imprinting through long-range interactions.

Optical Genome Mapping for a Patient with a Congenital Disorder and Chromosomal Translocation.

We performed optical genome mapping (OGM), a newly developed cytogenetic technique, for a patient with a disorder of sex development (DSD) and a 46,XX,t(9;11)(p22;p13) karyotype. The results of OGM were validated using other methods. OGM detected a 9;11 reciprocal translocation and successfully mapped its breakpoints to small regions of 0.9-12.3 kb. OGM identified 46 additional small structural variants, only three of which were detected by array-based comparative genomic hybridization. OGM suggested the presence of complex rearrangements on chromosome 10; however, these variants appeared to be artifacts. The 9;11 translocation was unlikely to be associated with DSD, while the pathogenicity of the other structural variants remained unknown. These results indicate that OGM is a powerful tool for detecting and characterizing chromosomal structural variations, although the current methods of OGM data analyses need to be improved.

Targeted Long-Read Sequencing Reveals Comprehensive Architecture, Burden, and Transcriptional Signatures from Hepatitis B Virus-Associated Integrations and Translocations in Hepatocellular Carcinoma Cell Lines.

Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, creating potentially oncogenic lesions that can lead to hepatocellular carcinoma (HCC). However, our current understanding of integrated HBV DNA architecture, burden, and transcriptional activity is incomplete due to technical limitations. A combination of genomics approaches was used to describe HBV integrations and corresponding transcriptional signatures in three HCC cell lines: huH-1, PLC/PRF/5, and Hep3B. To generate high-coverage, long-read sequencing data, a custom panel of HBV-targeting biotinylated oligonucleotide probes was designed. Targeted long-read DNA sequencing captured entire HBV integration events within individual reads, revealing that integrations may include deletions and inversions of viral sequences. Surprisingly, all three HCC cell lines contain integrations that are associated with host chromosomal translocations. In addition, targeted long-read RNA sequencing allowed for the assignment of transcriptional activity to specific integrations and resolved the contribution of overlapping HBV transcripts. HBV transcripts chimeric with host sequences were resolved in their entirety and often included >1,000 bp of host sequence. This study provides the first comprehensive description of HBV integrations and associated transcriptional activity in three commonly utilized HCC-derived cell lines. The application of novel methods sheds new light on the complexity of these integrations, including HBV bidirectional transcription, nested transcripts, silent integrations, and host genomic rearrangements. The observation of multiple HBV-associated chromosomal translocations gives rise to the hypothesis that HBV is a driver of genetic instability and provides a potential new mechanism for HCC development. IMPORTANCE HCC-derived cell lines have served as practical models to study HBV biology for decades. These cell lines harbor multiple HBV integrations and express only HBV surface antigen (HBsAg). To date, an accurate description of the integration burden, architecture, and transcriptional profile of these cell lines has been limited due to technical constraints. We have developed a targeted long-read sequencing assay that reveals the entire architecture of integrations in these cell lines. In addition, we identified five chromosomal translocations with integrated HBV DNA at the interchromosomal junctions. Incorporation of long-read transcriptome sequencing (RNA-Seq) data indicated that many integrations and translocations were transcriptionally silent. The observation of multiple HBV-associated translocations has strong implications regarding the potential mechanisms for the development of HBV-associated HCC.

Diagnosis of soft tissue tumors using immunohistochemistry as a surrogate for recurrent fusion oncoproteins.

Soft tissue neoplasms encompass a broad spectrum of clinicopathologic manifestations. In a subset of soft tissue tumors, spanning a wide range of clinical behavior from indolent to highly aggressive, recurrent genetic translocations yield oncogenic fusion proteins that drive neoplastic growth. Beyond functioning as primary mechanisms of tumorigenesis, recurrent translocations represent key diagnostic features insofar as the presence of a particular oncogenic gene fusion generally points to specific tumor entities. In addition to more direct methods for identifying recurrent translocations, such as conventional cytogenetics or fluorescence in situ hybridization, immunohistochemistry for a component of the fusion oncoprotein increasingly is being used as a surrogate marker, exploiting the tendency of these fusion components to be distinctively overexpressed by translocation-bearing tumor cells. Diagnostic immunohistochemistry can also be used to identify the characteristic gene expression changes that occur downstream of oncogenic fusions. Here, we review the use of immunohistochemistry to detect surrogate markers of recurrent translocations in soft tissue tumors, focusing on the practical applications and limitations of this diagnostic approach.

Alternative end-joining in BCR gene rearrangements and translocations.

Programmed DNA double-strand breaks (DSBs) occur during antigen receptor gene recombination, namely V(D)J recombination in developing B lymphocytes and class switch recombination (CSR) in mature B cells. Repair of these DSBs by classical end-joining (c-NHEJ) enables the generation of diverse BCR repertoires for efficient humoral immunity. Deletion of or mutation in c-NHEJ genes in mice and humans confer various degrees of primary immune deficiency and predisposition to lymphoid malignancies that often harbor oncogenic chromosomal translocations. In the absence of c-NHEJ, alternative end-joining (A-EJ) catalyzes robust CSR and to a much lesser extent, V(D)J recombination, but the mechanisms of A-EJ are only poorly defined. In this review, we introduce recent advances in the understanding of A-EJ in the context of V(D)J recombination and CSR with emphases on DSB end processing, DNA polymerases and ligases, and discuss the implications of A-EJ to lymphoid development and chromosomal translocations.

The origin of unwanted editing byproducts in gene editing.

The rapid development of CRISPR-Cas genome editing tools has greatly changed the way to conduct research and holds tremendous promise for clinical applications. During genome editing, CRISPR-Cas enzymes induce DNA breaks at the target sites and subsequently the DNA repair pathways are recruited to generate diverse editing outcomes. Besides off-target cleavage, unwanted editing outcomes including chromosomal structural variations and exogenous DNA integrations have recently raised concerns for clinical safety. To eliminate these unwanted editing byproducts, we need to explore the underlying mechanisms for the formation of diverse editing outcomes from the perspective of DNA repair. Here, we describe the involved DNA repair pathways in sealing Cas enzyme-induced DNA double-stranded breaks and discuss the origins and effects of unwanted editing byproducts on genome stability. Furthermore, we propose the potential risk of inhibiting DNA repair pathways to enhance gene editing. The recent combined studies of DNA repair and CRISPR-Cas editing provide a framework for further optimizing genome editing to enhance editing safety.

Reciprocal translocation and Robertsonian translocation in relation to semen parameters: A retrospective study and systematic review.

Reciprocal translocation and Robertsonian translocation are known to be causative factors of male infertility. However, the association between autosomal reciprocal translocation, Robertsonian translocation and semen parameters remains controversial. We performed a retrospective study and systematic review to investigate semen parameters in patients with autosomal reciprocal translocation or Robertsonian translocation. We recruited a total of 1,033 controls, 723 reciprocal translocation carriers and 326 Robertsonian translocation carriers. Men in the control, reciprocal translocation and Robertsonian translocation groups had a median age of 32.0 (95% CI, 32.0-33.0), 32.0 (95% CI, 32.0-33.0) and 33.0 (95% CI, 32.0-33.0) years respectively. Results showed that sperm concentration, total number per ejaculate, total motility, progressive motility of autosomal reciprocal translocation and Robertsonian translocation carriers were statistically lower than controls (p < .001). Eleven studies featuring 794 patients were enrolled in this systematic review. Compared with controls, autosomal reciprocal translocation and Robertsonian translocation carriers showed lower sperm concentration, total motility, progressive motility and normal morphology. Our results support the conclusion that sperm concentration, total number per ejaculate, total motility and progressive motility are significantly lower in autosomal reciprocal translocation and Robertsonian translocation carriers than in controls.

Recurrent Translocations in Topoisomerase Inhibitor-Related Leukemia Are Determined by the Features of DNA Breaks Rather Than by the Proximity of the Translocating Genes.

Topoisomerase inhibitors are widely used in cancer chemotherapy. However, one of the potential long-term adverse effects of such therapy is acute leukemia. A key feature of such therapy-induced acute myeloid leukemia (t-AML) is recurrent chromosomal translocations involving AML1 (RUNX1) or MLL (KMT2A) genes. The formation of chromosomal translocation depends on the spatial proximity of translocation partners and the mobility of the DNA ends. It is unclear which of these two factors might be decisive for recurrent t-AML translocations. Here, we used fluorescence in situ hybridization (FISH) and chromosome conformation capture followed by sequencing (4C-seq) to investigate double-strand DNA break formation and the mobility of broken ends upon etoposide treatment, as well as contacts between translocation partner genes. We detected the separation of the parts of the broken AML1 gene, as well as the increased mobility of these separated parts. 4C-seq analysis showed no evident contacts of AML1 and MLL with loci, implicated in recurrent t-AML translocations, either before or after etoposide treatment. We suggest that separation of the break ends and their increased non-targeted mobility-but not spatial predisposition of the rearrangement partners-plays a major role in the formation of these translocations.

Finding underlying genetic mechanisms of two patients with autism spectrum disorder carrying familial apparently balanced chromosomal translocations.

BACKGROUND: Genetic etiologies of autism spectrum disorders (ASD) are complex, and the genetic factors identified so far are very diverse. In complex genetic diseases such as ASD, de novo or inherited chromosomal abnormalities are valuable findings for researchers with respect to identifying the underlying genetic risk factors. With gene mapping studies on these chromosomal abnormalities, dozens of genes have been associated with ASD and other neurodevelopmental genetic diseases. In the present study, we aimed to idenitfy the causative genetic factors in patients with ASD who have an apparently balanced chromosomal translocation in their karyotypes. METHODS: For mapping the broken genes as a result of chromosomal translocations, we performed whole genome DNA sequencing. Chromosomal breakpoints and large DNA copy number variations (CNV) were determined after genome alignment. Identified CNVs and single nucleotide variations (SNV) were evaluated with VCF-BED intersect and Gemini tools, respectively. A targeted resequencing approach was performed on the JMJD1C gene in all of the ASD cohorts (220 patients). For molecular modeling, we used a homology modeling approach via the SWISS-MODEL. RESULTS: We found that there was no contribution of the broken genes or regulator DNA sequences to ASD, whereas the SNVs on the JMJD1C, CNKSR2 and DDX11 genes were the most convincing genetic risk factors for underlying ASD phenotypes. CONCLUSIONS: Genetic etiologies of ASD should be analyzed comprehensively by taking into account of the all chromosomal structural abnormalities and de novo or inherited CNV/SNVs with all possible inheritance patterns.