Chromosomal Copy Number Research Articles

Genome alteration of Leishmania orientalis under Amphotericin B inhibiting conditions.

Amphotericin B (AmB) is a potent antifungal and antiparasitic medication that exerts its action by disrupting the cell membrane of the leishmanial parasite, leading to its death. Understanding the genetic alterations induced by Amphotericin B is crucial for gaining insights into drug resistance mechanisms and developing more effective treatments against Leishmania infections. As a new Leishmania species, the molecular response of Leishmania orientalis to anti-leishmanial drugs has not been fully explored. In this study, Leishmania orientalis strain PCM2 culture was subjected to AmB exposure at a concentration of 0.03 uM over 72 hours compared to the control. The genomic alteration and transcriptomic changes were investigated by utilising the whole genome and RNA sequencing methods, followed by the analysis of single nucleotide polymorphisms (SNPs), differential gene expression, and chromosomal copy number variations (CNVs) assessed using read depth coverage (RDC) values across the entire genome. The chromosomal CNV analysis showed no significant difference between L. orientalis from the control and AmB-treated groups. The distribution of SNPs displayed notable variability, with higher SNP incidence in the control group compared to the AmB-treated group. Gene ontology analysis unveiled functions of the SNPs -associated genes involved in transporter function, genetic precursor synthesis, and purine nucleotide metabolism. Notably, the impact of AmB treatment on the L. orientalis gene expression profiles exhibited diverse expressional alterations, particularly the downregulation of pivotal genes such as the tubulin alpha chain gene. The intricate interplay between SNPs and gene expression alterations might underscore the complex regulatory networks underlying the AmB resistance of L. orientalis strain PCM2.

Preimplantation genetic testing for a Chinese pedigree affected with Primary carnitine deficiency

To investigate the results of preimplantation genetic testing for monogenic diseases (PGT-M) in a Chinese pedigree affected with Primary carnitine deficiency (PCD). A pedigree affected with PCD who visited Hainan Women and Children's Medical Center in April 2023 due to "SLC22A5 gene mutation found in offspring genetic testing and preparing for a second child" was selected as the study subject. Pathogenicity of the proband's variant sites was determined by referring to the Standards and Guidelines for the Interpretation of Sequence Variants established by the American College of Medical Genetics and Genomics (ACMG). Sanger sequencing was used to verify the variant sites of SLC22A5 gene in the proband and her parents, and the single nucleotide polymorphism (SNP) haplotype of the family was constructed by SNP microarray (SNP array) method to determine the carrier status of pathogenic genes. After fertilization via assisted reproductive technology, whole genome amplification (WGA) was performed on the biopsied trophoblastic cells. Sanger sequencing, next-generation sequencing (NGS), and SNP array techniques were then used to detect the variants in the SLC22A5 gene and chromosome copy number variation (CNV) in the embryos. Embryos without the variants were selected for transferring. After the successful pregnancy of the proband's mother, amniocentesis was not performed for prenatal diagnosis due to repeated vaginal bleeding. After delivery, neonatal peripheral blood sample was collected to verify the results of PGT-M, and follow-up was conducted. This study was reviewed and approved by the Medical Ethics Committee of Hainan Women and Children's Medical Center (Ethics No. HNWCMC-2022-178). In this study, the c.338G>A and c.760C>T variants in SLC22A5 gene were evaluated as pathogenic variants. Sanger sequencing results of this family showed that the c.338G>A and c.760C>T variants of the proband were inherited from his father and mother, respectively. Haplotypes of c.338G>A and c.760C>T variants of SLC22A5 gene were successfully constructed. PGT-M results showed that 2 of the 8 blastulas biopsied failed WGA, and the CNV detection results of the remaining 6 blastocysts were all euploid: 2 had no mutations in the SLC22A5 gene, 3 were single heterozygous carriers of paternal or maternal origin, and 1 was compound heterozygous carriers of paternal and maternal origin. Combined with the embryo morphology score, an intrauterine singleton pregnancy was achieved after the successful transfer of an optimal embryo with no CNV abnormalities and no paternal or maternal SLC22A5 gene mutations, resulting in the birth of a healthy female baby at 38+3 weeks of gestation. The results of peripheral blood chromosomal karyotyping analysis, CNV detection and SLC22A5 gene c.338G>A and c.760C>T site variant detection of the infant were consistent with those of PGT-M, and no abnormality was found. PGT-M had helped the couple carrying SLC22A5 gene variant to have a healthy offspring and effectively blocked the transmission of PCD in this family.

Proband-Only Exome Sequencing for Intellectual Disability in Iran: Diagnostic Yield and Genetic Insights.

Intellectual disability (ID) is a leading cause for referral to genetic services, with the most severe cases typically attributed to single genetic defects. This study aimed to evaluate the diagnostic yield of cost-effective proband-only exome sequencing for individuals diagnosed with ID within the Iranian population for the first time where a high rate of parental consanguinity exists. A total of 99 unrelated patients with ID were investigated by exome sequencing during 8 years. As a result, 43 pathogenic/likely pathogenic variants were identified in 40 patients, indicating a molecular diagnostic rate of 40.4% (40/99). The inclusion of five chromosomal copy number variations in the subsequent analysis increased the diagnostic rate of proband-only exome sequencing to 45.4% (45/99). Additionally, parental testing revealed five de novo variants. This contributed to a total diagnostic rate of 50.5% (50/99). In our study, proband-only exome sequencing achieved a remarkable diagnostic rate, identifying nearly half of the ID cases. This rate of diagnosis could be primarily attributed to prevalent consanguineous marriage in the Iranian population and the rare identification of de novo variants. With the ongoing advancements in neurogenetics, proband-only exome sequencing demonstrates significant potential as a future cost-effective diagnostic approach in Iran.

Mosaic variegated aneuploidy in development, ageing and cancer.

Mosaic variegated aneuploidy (MVA) is a rare condition in which abnormal chromosome counts (that is, aneuploidies), affecting different chromosomes in each cell (making it variegated) are found only in a certain number of cells (making it mosaic). MVA is characterized by various developmental defects and, despite its rarity, presents a unique clinical scenario to understand the consequences of chromosomal instability and copy number variation in humans. Research from patients with MVA, genetically engineered mouse models and functional cellular studies have found the genetic causes to be mutations in components of the spindle-assembly checkpoint as well as in related proteins involved in centrosome dynamics during mitosis. MVA is accompanied by tumour susceptibility (depending on the genetic basis) as well as cellular and systemic stress, including chronic immune response and the associated clinical implications.

Investigation of chromosomal anomalies and copy number variations in children diagnosed with autism spectrum disorder by array CGH method.

This study aimed to identify the chromosomal anomalies and copy number variations (CNVs) in autism spectrum disorder (ASD) and to provide genotype/phenotype correlations. Fifty-four patients diagnosed with ASD between March 2021 and June 2022 were included in the study. Patients were evaluated by cytogenetic analysis and array comparative genomic hybridisation analysis (aCGH). The structural and numerical chromosomal anomaly was detected in 3.7%, and the CNVs were identified in 18.52% of patients. Of the CNVs detected, 27.3% were identified as pathogenic, 18.2% as likely pathogenic and 54.5% as VUS. The copy number gain rate of the detected CNVs was higher than the copy number losses rate, 70% and 30% respectively. As an important finding in the study, a new pathogenic CNV with a 6.3-mb copy number gain in the 3p22.3p22.2 region, whose gene region had not been previously defined in OMIM, was detected. Identifying a genetic aetiology may provide clinicians with more information about disease prognosis and risk of recurrence.

Genetic analysis of 280 children with unexplained developmental delay or intellectual disability using whole exome sequencing

IntroductionDevelopmental delay (DD) and intellectual disability (ID) are key manifestations of neurodevelopmental disorders (NDDs), characterized by considerable clinical and genetic variability, which complicates genetic diagnosis. Whole exome sequencing (WES) has become an effective method for uncovering genetic causes in patients with unexplained DD/ID.MethodsWe retrospectively analyzed WES data from 280 patients diagnosed with unexplained DD/ID. Demographic information and genetic variants identified through WES were assessed, along with an evaluation of clinical factors that might influence the detection of genetic causes.ResultsPathogenic variants were detected in 73 cases (36.07%), including 25 cases involving pathogenic chromosomal copy number variations. Clinical factors such as age, sex, gestational age, birth weight, anoxia, jaundice, associated symptoms, family history, muscle strength, muscle tone, epilepsy, brain MRI findings, EEG results, and the severity of DD/ID did not significantly impact the WES outcomes. However, a significant correlation was observed between delivery mode and positive WES results, with a higher diagnostic yield among patients delivered via caesarean section.ConclusionsWES is a valuable approach for identifying genetic causes in patients with unexplained DD/ID, providing benefits for patient management, family genetic counseling, and long-term prognosis assessment.Clinical trial numberNot applicable.

Intercellular mosaic methylation in fast-growing Mycobacterium tuberculosis clinical isolates

Abstract Orphan DNA adenine methyltransferases (MTases) of Mycobacterium tuberculosis (Mtb) exhibit diversity across clinical isolates, but the forces driving this variation are not entirely clear. Recently, we observed several isolates exhibiting anomalous hypomethylation by Type I MTase Mycobacterial Adenine Methyltransferase C (MamC) despite a wild-type mamC genotype (‘MamC-anomalous’ isolates). Investigating this hypomethylation through multiple analyses revealed three key findings. First, heterogeneity analysis revealed intercellular mosaic methylation (IMM) in MamC-anomalous isolates. While they often exhibit phase-variable heterogeneity, this is the first report of IMM by a prokaryotic Type I MTase. Second, MamC-anomalous isolates exhibited a large, stable difference in chromosome copy number along the replication axis (a proxy for bacterial growth rate), suggesting a distinct growth phase accompanied by MamC hypomethylation. Third, MamC methylation efficiency decreased progressively with distance from origin of replication on both strands, with a marked exaggeration in MamC-anomalous isolates. In contrast, other Mtb MTases (MamA and MamB) exhibited lower methylation levels away from the origin only on the lagging strand, and without exaggeration in MamC-anomalous isolates. We conclude that, among Mtb MTases, MamC is uniquely linked to growth dynamics.

Abstract IA005: Tumor cell-based liquid biopsy to characterize prostate cancer

Abstract Circulating Tumor Cells (CTCs) hold the promise of comprehensive yet noninvasive molecular characterization of cancer cells during the course of tumor evolution and progression. To dissect the epigenetic profile of prostate CTCs, we undertook paired DNA methylation and RNA sequencing of single CTCs isolated from blood specimens of patients with prostate cancer. Across the genome, our study identified 40 core Partial Methylation Domains (PMDs) that arise early in tumorigenesis and are shared by all individual CTCs across multiple patients. Key among these is a single genomic locus harboring multiple interferon-inducible genes along with all the CD1 gene family members, implicated in innate immunity. Based on mouse models, we propose that early biallelic silencing of multiple genes residing within a single PMD may contribute to the escape from immune surveillance in prostate and potentially other cancers. Studies of CTC heterogeneity within individual patients are limited by the very small number of cells that can be isolated from standard 10ml blood volumes. To enable such analyses, we applied a new high throughput microfluidic platform, capable of processing an entire leukapheresis product, interrogating 2-3 L of blood volume equivalent. Applying this technology to patients with metastatic prostate cancer, we successfully enriched thousands of CTCs from individual cases, enabling the characterization of intra-patient heterogeneity at the level of cell morphology and marker expression, chromosome copy number variation and exome-wide genotyping, and transcriptionally-defined tumor cell subpopulations. Such high-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies. Citation Format: Daniel A Haber, Shyamala Maheswaran, Hongshan Guo, Joanna Vuille, Shih-Bo Huang, Ben S Wittner, Lecia V Sequist, Richard J Lee, Patricia AR Brunker, David T Miyamoto, Avanish Mishra, Mehmet Toner. Tumor cell-based liquid biopsy to characterize prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr IA005.

PATH-21. SINGEL CELL TRANSCRIPTOMICS OF SPINAL EPENDYMOMA SUBTYPES IDENTIFIES INTRATUMORAL HETEROGENEITY

Abstract Spinal ependymomas comprise glial neoplasms that are classified by histological and molecular features. Two of the respective types, myxopapillary ependymomas (MPE) and spinal ependymomas (SP-EPN) are largely understudied. For each of these, our group identified clinically relevant subtypes with poorer (MPE-A; SP-EPN-A) and more favorable progression-free survival (MPE-B; SP-EPN-B) employing global methylation and transcriptomic profiling. As cellular origin and pathogenesis of these tumors are largely unknown, therapeutic options for patients with unfavorable subtypes are unavailable. We performed single cell transcriptomic sequencing of 12 formalin-fixed and paraffin-embedded spinal ependymomas including all MPE and SP-EPN subtypes and obtained a median of 10,091 cells per tumor after quality control. Uniform Manifold Approximation and Projection (UMAP) analysis revealed intertumoral heterogeneity as most samples formed separate clusters with tumors of the same subtype located in proximity. The expression of literature-defined neoplastic consensus cell states was examined for each cell of the data set and revealed intratumoral heterogeneity. Tumors expressed predominantly two programs, being a ciliated (ependymal-like) cell state (61% of all neoplastic cells) and the astrocytic state (31 % of all neoplastic cells). Interestingly, third and fourth most abundant states (mesenchymal and hypoxia) were almost exclusively expressed in MPE-A and SP-EPN-A. Furthermore, distinct clones of chromosomal copy number variations were identified across all tumors. These clones can be distinguished in UMAP representation of MPE single cell transcriptomes and to a lesser extend in SP-EPN. Next, we sought to characterize the resemblance of integrated tumor subtypes to normal spinal cord cell populations and observed a high similarity to human spinal cord ependymal cells. All spinal ependymoma subtypes display intratumoral heterogeneity with respect to the transcriptomic cell state resemblance of the neoplastic cells. Together with the transcriptomic similarity to ependymal and astrocytic cells, our data suggest the tumor origin within the astro-ependymal lineage.

Neurofibroma-like Desmoplastic Melanoma: A Series of Five Cases Exploring the Role of Molecular Testing as a Diagnostic Adjunct and Highlighting the Differential Diagnosis With Diffuse-type Neurofibroma.

A subset of desmoplastic melanomas (DMs) can show extensive morphologic and immunohistochemical overlap with cutaneous diffuse-type neurofibroma. Neurofibroma-like desmoplastic melanoma (NFLDM) thus poses a significant diagnostic pitfall because the clinical implications of these 2 entities differ dramatically. A series of 17 DMs, including 5 cases of NFLDM, were compared with a cohort of 53 cutaneous diffuse-type neurofibromas to explore the utility of molecular testing in the differential diagnosis between NFLDM and neurofibroma and to determine potentially useful morphologic features in this differential diagnosis. Unlike NFLDM, cutaneous diffuse-type neurofibromas: (1) rarely feature intratumoral or peritumoral lymphoid aggregates, (2) consistently harbor an intrinsic stromal support vasculature composed of evenly spaced capillary-sized vessels, and (3) infiltrate adjacent adipose tissue in a dermatofibrosarcoma protuberans-like manner with a complete lack of chronic inflammation or fat necrosis at the leading edge of the tumor. Conversely, DMs, including NFLDM: (1) do not contain Wagner-Meissner bodies, (2) often induce fat necrosis and/or chronic inflammation at the interface with adjacent fibroadipose tissue, (3) lack the intrinsic capillary-sized stromal vasculature observed in most neurofibromas, and (4) may harbor foci of perineuriomatous differentiation, mimicking a hybrid nerve sheath tumor. Any deviation from the expected clinical or morphologic features of cutaneous diffuse-type neurofibroma should raise suspicion for NFLDM. Although not entirely sensitive or specific, molecular testing can help to support the diagnosis of NFLDM by demonstrating genetic abnormalities associated with melanoma, including a UV-light-induced mutational signature, high tumor mutational burden, and/or chromosomal copy number alterations typical of melanoma.

Prenatal diagnosis and postnatal follow-up of 15 fetuses with 16p13.11 microduplication syndrome.

The clinical phenotypes of 16p13.11 microduplication syndrome have been extensively reported in previous studies, mostly about adults and children, with limited information available on fetal cases. This study aims to explore the genotype-phenotype correlation of fetuses with 16p13.11 microduplication syndrome and analyze the characteristics of prenatal diagnosis indications and provide clinical information for prenatal and postnatal genetic counseling. We conducted a retrospective analysis of 3,451 pregnant women who underwent invasive prenatal diagnosis for SNP array between January 2018 and December 2022 at the Jinan Maternal and Child Health Hospital. Descriptive statistical analysis was performed on the prenatal diagnosis indications, pedigree analysis, pregnancy outcomes and postnatal follow-up of 15 fetuses with 16p13.11 microduplication syndrome. SNP array revealed that 15 fetuses had duplications in the 16p13.11 region with varying prenatal diagnosis indications. Among the cases, 6/15 exhibited ultrasound abnormalities, 5/15 had abnormal chromosomal copy number variations as indicated by non-invasive prenatal testing (NIPT), one case involved advanced maternal age, and 3/15 had other abnormalities. 16p13.11 microduplication syndrome was closely related to ultrasound abnormalities, especially structural abnormalities and soft marker anomalies (abnormal ultrasonic soft indicators), while the indication of NIPT could improve the detection rate of copy number variations (CNVs) in this region. Only 7/15 fetuses underwent pedigree verification, with one case of de novo 16p13.11 microduplication, and the others inherited from one parent. Pregnancy was terminated in 2/15 cases and the outcome of one case is unknown due to loss to follow-up. Among the remaining cases, only one case exhibited a ventricular septal defect, while another presented with omphalocele. No other obvious abnormalities were reported postnatally. The prenatal phenotypes of fetuses with 16p13.11 microduplication were highly associated with ultrasound abnormalities but lacked specificity. Comprehensive genetic tracing, outcome analysis, and follow-up are essential for providing accurate prenatal and postnatal genetic counseling.

Structural alterations of the EGFR gene in glioblastoma samples as a prognostic factor and molecular target for therapy

Introduction. Epidermal growth factor receptor (EGFR) is a transmembrane protein of the receptor tyrosine kinase family that is activated in various cancers (non-small cell lung cancer, colorectal cancer, head and neck tumors). In glial brain tumors, increased EGFR expression levels are characteristic of the most aggressive subtype, glioblastoma. Frequent structural changes of EGFR in glioblastoma are amplification of the chromosome region where the EGFR gene is located, point mutations, as well as deletion of exons 2–7 of the EGFR gene leading to the formation of EGFRvIII transcript.Aim. To determine structural changes of the EGFR gene (point mutations and amplification of the EGFR gene, EGFRvIII transcript) in tumor samples using different methods and to evaluate their potential clinical significance.Materials and methods. The study included 75 patients with brain gliomas (70 of them glioblastoma) aged 34 to 78 years (mean age 56 years). DNA and RNA isolation was performed from fresh frozen tumor tissue, as well as from peripheral blood leukocytes. EGFR gene mutations were determined by next-generation sequencing (NGS), and β allele frequency (BAF) comparative analysis (normal-tumor) was performed to determine the copy number of chromosome 7 regions. Quantitative polymerase chain reaction was used to confirm the EGFR gene amplification in tumor samples, and reverse transcription-PCR was used to detect EGFRvIII variant.Results. The NGS method revealed 11/70 (16 %) mutations in coding regions of EGFR gene in glioblastoma samples, the EGFR gene amplification was detected in 26/70 (37 %) cases; no structural changes of the EGFR gene were detected in 5 glioma samples (astrocytoma, oligodendroglioma). All cases of EGFR gene amplification detected by NGS were confirmed by quantitative polymerase chain reaction. To search for EGFRvIII transcript, 31 tumor RNA samples were examined, of which EGFR amplification was present in 12 samples. EGFRvIII transcript was detected only in samples with EGFR gene amplification – 4/12 (33 %). To assess the clinical significance of structural gene alterations, the frequency of occurrence in primary and recurrent glioblastoma samples was compared.Conclusion. The NGS method allows to detect both point mutations and amplification of the EGFR gene. The EGFR gene amplification was associated with EGFRvIII mutation in 33 % of cases. No statistically significant differences in the frequency of structural changes in the EGFR gene between primary and relapsed glioblastomas were found.

Bovine aortic arch: a potential indicator that may not serve in prenatal diagnosis - a study based on fetal anatomy, genetics, and postnatal clinical outcomes

ObjectiveTo investigate the structural abnormalities, genetic results, and postnatal clinical outcomes of fetuses with bovine aortic arch (Bovine Aortic Arch, BAA) to provide a basis for prenatal counseling and management.MethodsA retrospective analysis was conducted on 216 fetuses diagnosed with bovine aortic arch through prenatal ultrasound screening at the First Affiliated Hospital of Anhui Medical University and the No.901 Hospital of the Joint Service of the People’s Liberation Army from January 2019 to February 2023. Their family history of genetic diseases, prenatal screening results, and postnatal follow-up data were collected. The fetuses were divided into an isolated BAA group (n = 192) and a non-isolated BAA group (n = 24). Chromosomal karyotyping and copy number variation (CNV) testing were conducted, and statistical analysis was performed using SPSS 22.0 software.ResultsOf the 216 fetuses with BAA, 192 were isolated BAA (88.89%), and 24 were non-isolated BAA (11.11%). Among the isolated BAA fetuses, only 1 case (0.52%) had chromosomal karyotype and pathogenic CNV abnormalities. Among the non-isolated BAA fetuses, 4 cases (16.67%) had chromosomal or CNV abnormalities, but the overall risk was low. The postnatal outcomes of isolated BAA fetuses were good (99.48%), while 79.17% of non-isolated BAA fetuses had good postnatal outcomes.ConclusionMost BAA fetuses are isolated, with a very low incidence of chromosomal abnormalities and pathogenic CNVs, and have good postnatal outcomes. The clinical value of isolated BAA is limited, and invasive prenatal diagnosis is not recommended for low-risk populations. Prenatal screening should focus on the risk of concurrent severe structural anomalies and chromosomal abnormalities.

B-213 Including Turner Syndrome Screening in Newborn Sequencing by Using NGS Read Counts to Infer Sex Chromosomes Copy Number: Leveraging Existing Data to Enhance Early Diagnosis

Abstract Background Turner Syndrome (TS) can lead to heart defects, short stature, and premature ovarian failure. Early diagnosis can improve outcomes, but routine screening is not standard, often delaying diagnosis until symptoms prompt karyotype analysis. Newborn NGS sequencing offers potential for early detection of genetic disorders and targets numerous gene regions on autosomes and sex chromosomes. It could be utilized to deduce sexual chromosomal copy number variations, including not only chrX monosomy but also its partial deletions and structural alterations, and chrY, which increases the risk of gonadoblastoma in TS patients. Thus, this study aims to validate a TS screening approach using NGS read count data from newborn sequencing to develop a strategy for early detection. Methods DNA samples with established chromosomal microarray ChrX and ChrY copy-number assessments were used. The cohort included 13 males (46,XY), 10 females (46,XX), 5 TS cases (45,X), 5 mosaic TS cases (2 with chrY, 3 without), 1 TS case with Isochromosome Xq, 8 Klinefelter Syndrome cases (47,XXY), and one case (48,XXYY). For NGS, a focused newborn sequencing panel targeting 388 genes derived from the larger xGEN Inherited Disease Panel (Integrated DNA Technologies) was employed. The larger panel covers 2,968 chrX regions, 53,455 autosomal regions, and 135 chrY regions. Sample processing utilized the xGEN DNA Library Prep and Hybridization Capture (Integrated DNA Technologies) and sequencing on NextSeq-500 (Illumina). The alignment was conducted with Dragen Enrichment v.3.9.5 and read count analysis with Samtools v.1.19.2. ChrX and ChrY copy numbers were assessed by comparing chrX and chrY read count ratios to autosomal read counts. Hierarchical clustering grouped samples based on ratio similarities, aiding result attribution. To identify structural alterations in each arm of chrX, every tested chrX region was analyzed for read count ratio calculation and subjected to circular binary segmentation using the DNAcopy R package. The observed sex chromosome counts were compared to expected results to validate the approach. Results Observed chrX/autosome and chrY/autosome ratio ranges matched the expected sex chromosome counts: 0.99-1.03 and 0.82-1.02 for 46,XY; 1.92-1.97 and 0.00-0.01 for 46,XX; 1.00-1.02 and 0.00-0.00 for 45,X; 1.15-1.46 and 0.00-0.00 for mosaic TS without chrY; 0.98-1.06 and 0.27-0.32 for mosaic TS with chrY; 2.45 and 0.0 for TS with Isochromosome Xq; 1.82-2.01 and 0.87-0.95 for 47,XXY; and 2.02 and 1.96 for 48,XXYY. Hierarchical cluster analysis grouped these values into three branches: one for 48,XXYY, one for 47,XXY, 46,XX, and Isochromosome Xq, and one for all other TS samples and 46,XY. TS and 47,XXY samples were further divided into subbranches from 46,XX and 46,XY. Circular binary segmentation of chrX read counts provided a visual profile of the copy number for each arm of chrX, explaining structural alterations in out-of-pattern chrX dosages, such as 2.45, 1.15, and 1.46. Conclusions There was complete agreement between the NGS read count-based assessment of sex chromosome copy number and the expected results for 46,XY, 46,XX, 45,X, TS with mosaic and structural variants, 47,XXY, and 48,XXYY samples. This suggests that TS can be effectively screened using newborn sequencing NGS data.

KaryoTap Enables Aneuploidy Detection in Thousands of Single Human Cells.

Investigating chromosomal instability and aneuploidy within tumors is essential for understanding tumorigenesis and developing diagnostic and therapeutic strategies. Single-cell DNA sequencing technologies have enabled such analyses, revealing aneuploidies specific to individual cells within the same tumor. However, it has been difficult to scale the throughput of these methods to detect rare aneuploidies while maintaining high sensitivity. To overcome this deficit, we developed KaryoTap, a method combining custom targeted DNA sequencing panels for the Tapestri platform with a computational framework to enable detection of chromosome- and chromosome arm-scale aneuploidy (gains or losses) and copy number neutral loss of heterozygosity in all human chromosomes across thousands of single cells simultaneously. KaryoTap allows detecting gains and losses with an average accuracy of 83% for arm events and 91% for chromosome events. Importantly, together with chromosomal copy number, our system allows us to detect barcodes and gRNAs integrated into the cells' genome, thus enabling pooled CRISPR- or ORF-based functional screens in single cells. As a proof of principle, we performed a small screen to expand the chromosomes that can be targeted by our recently described CRISPR-based KaryoCreate system for engineering aneuploidy in human cells. KaryoTap will prove a powerful and flexible approach for the study of aneuploidy and chromosomal instability in both tumors and normal tissues.

An elevated rate of whole-genome duplications in cancers from Black patients

In the United States, Black individuals have higher rates of cancer mortality than any other racial group. Here, we examine chromosome copy number changes in cancers from more than 1800 self-reported Black patients. We find that tumors from self-reported Black patients are significantly more likely to exhibit whole-genome duplications (WGDs), a genomic event that enhances metastasis and aggressive disease, compared to tumors from self-reported white patients. This increase in WGD frequency is observed across multiple cancer types, including breast, endometrial, and lung cancer, and is associated with shorter patient survival. We further demonstrate that combustion byproducts are capable of inducing WGDs in cell culture, and cancers from self-reported Black patients exhibit mutational signatures consistent with exposure to these carcinogens. In total, these findings identify a type of genomic alteration that is associated with environmental exposures and that may influence racial disparities in cancer outcomes.

Ploidy levels in diverse picocyanobacteria from the Baltic Sea.

In nature, the number of genome or chromosome copies within cells (ploidy) can vary between species and environmental conditions, potentially influencing how organisms adapt to changing environments. Although ploidy levels cannot be easily determined by standard genome sequencing, understanding ploidy is crucial for the quantitative interpretation of molecular data. Cyanobacteria are known to contain haploid, oligoploid, and polyploid species. The smallest cyanobacteria, picocyanobacteria (less than 2 μm in diameter), have a widespread distribution ranging from marine to freshwater environments, contributing significantly to global primary production. In this study, we determined the ploidy level of genetically and physiologically diverse brackish picocyanobacteria isolated from the Baltic Sea using a qPCR assay targeting the rbcL gene. The strains contained one to four genome copies per cell. The ploidy level was not linked with phylogeny based on the identity of the 16S rRNA gene. The variation of ploidy among the brackish strains was lower compared to what has been reported for freshwater strains and was more similar to what has been reported for marine strains. The potential ecological advantage of polyploidy among picocyanobacteria has yet to be described. Our study highlights the importance of considering ploidy to interpret the abundance and adaptation of brackish picocyanobacteria.

Genetic and clinical analysis of two children with microcephaly and mental retardation due to a frameshifting variant of CASK gene

To explore the clinical and genetic characteristics of two children with mental retardation and microcephaly. Two children who had visited the Anhui Children's Hospital respectively on March 12 and June 22, 2021 were selected as the study subjects. Peripheral venous blood samples were collected from them and their parents, and subjected to chromosomal karyotyping and whole exome sequencing analyses. Candidate variants were verified by Sanger sequencing and pathogenicity analysis. Chromosomal karyotyping and copy number detection of the two children had found no abnormality. Whole exome sequencing revealed that child 1 has harbored a c.471delT (p.Pro157Profs*9) frameshifting variant of the CASK gene, whilst child 2 has harbored a c.1259_1269delCTGAGAATAAC (p.Pro420fs*27) frameshifting variant of the CASK gene. Sanger sequencing confirmed that both variants were de novo in origin. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP), both variants were rated as pathogenic (PVS1+PS2+PP3). The de novo variants of the CASK gene probably underlay the pathogenesis of mental retardation and microcephaly in both children.

Single-cell RNA sequencing explored potential therapeutic targets by revealing the tumor microenvironment of neuroblastoma and its expression in cell death

BackgroundNeuroblastoma (NB) is the most common extracranial solid tumor in childhood and is closely related to the early development and differentiation of neuroendocrine (NE) cells. The disease is mainly represented by high-risk NB, which has the characteristics of high mortality and difficult treatment. The survival rate of high-risk NB patients is not ideal. In this article, we not only conducted a comprehensive study of NB through single-cell RNA sequencing (scRNA-seq) but also further analyzed cuproptosis, a new cell death pathway, in order to find clinical treatment targets from a new perspective.Materials and MethodsThe Seurat software was employed to process the scRNA-seq data. This was followed by the utilization of GO enrichment analysis and GSEA to unveil pertinent enriched pathways. The inferCNV software package was harnessed to investigate chromosomal copy number variations. pseudotime analyses involved the use of Monocle 2, CytoTRACE, and Slingshot software. CellChat was employed to analyze the intercellular communication network for NB. Furthermore, PySCENIC was deployed to review the profile of transcription factors.ResultUsing scRNA-seq, we studied cells from patients with NB. NE cells exhibited superior specificity in contrast to other cell types. Among NE cells, C1 PCLAF + NE cells showed a close correlation with the genesis and advancement of NB. The key marker genes, cognate receptor pairing, developmental trajectories, metabolic pathways, transcription factors, and enrichment pathways in C1 PCLAF + NE cells, as well as the expression of cuproptosis in C1 PCLAF + NE cells, provided new ideas for exploring new therapeutic targets for NB.ConclusionThe results revealed the specificity of malignant NE cells in NB, especially the key subset of C1 PCLAF + NE cells, which enhanced our understanding of the key role of the tumor microenvironment in the complexity of cancer progression. Of course, cell death played an important role in the progression of NB, which also promoted our research on new targets. The scrutiny of these findings proved advantageous in uncovering innovative therapeutic targets, thereby bolstering clinical interventions.

Prenatal diagnosis of fetuses with 15q11.2 BP1-BP2 microdeletion in the Chinese population: a seven-year single-center retrospective study

BackgroundThe 15q11.2 BP1-BP2 microdeletion syndrome is associated with developmental delays, language impairments, neurobehavioral disorders, and psychiatric complications. The aim of the present study was to provide prenatal and postnatal clinical data for 16 additional fetuses diagnosed with the 15q11.2 BP1-BP2 microdeletion syndrome in the Chinese population.MethodsA total of 5,789 pregnancy women that underwent amniocentesis were enrolled in the present study. Both karyotype analysis and chromosomal microarray analysis (CMA) were conducted on these subjects to detect chromosomal abnormalities and copy number variants (CNVs). Whole exome sequencing (WES) was performed to investigate sequence variants in subjects with clinical abnormalities after birth.ResultsSixteen fetuses with 15q11.2 BP1-BP2 microdeletion were identified in the present study, with a detection rate of 0.28% (16/5,789). The 15q11.2 BP1-BP2 microdeletion fragments ranged from 311.8 kb to 849.7 kb, encompassing the NIPA1, NIPA2, CYFIP1, and TUBGCP5 genes. The follow-up results regarding pregnancy outcomes showed that five cases opted for pregnancy termination, while the remaining cases continued with their pregnancies. Subsequent postnatal follow-up indicated that only one case with the 15q11.2 BP1-BP2 microdeletion displayed neurodevelopmental disorders, demonstrating an incomplete penetrance rate of 9.09% (1/11).ConclusionThe majority of fetuses with the 15q11.2 microdeletion exhibit typical features during early childhood, indicating a low penetrance and mild impact. Nonetheless, pregnancies involving fetuses with the 15q11.2 microdeletion require thorough prenatal counseling. Additionally, enhanced supervision and extended postnatal monitoring are warranted for those who choose to proceed with their pregnancies.