Identification of genome-wide copy number variation-driven subtypes for the treatment and prognostic prediction of esophageal carcinoma.

Background: Esophageal carcinoma (ESCA) is a frequently detected gastrointestinal cancer. Copy number variants (CNVs) have a dramatic impact on the screening, diagnosis and prognostic prediction of cancers. However, the mechanism of action of CNVs on ESCA occurrence and progression remains unclear. Methods: ESCA samples from The Cancer Genome Atlas (TCGA) were typed by consensus clustering using CNV-associated genes. Weighted Gene Co-Expression Network Analysis (WGCNA) was used to section gene modules closely related to the two clusters, and sub-networks were constructed as hub genes. In addition, seven prognosis-correlated genes were further screened and retained by multivariate Cox regression analysis to develop a prognostic assessment model. The ssGSEA algorithm assessed energy metabolism levels in patients from different clusters and risk groups. Finally, quantitative real-time PCR (qRT-PCR) and live-dead cell staining verified the expression of genes associated with CNV risk scores. Results: ESCA was classified into two subtypes based on CNV values. Compared with cluster 1, cluster 2 had significantly higher level of immune score and tumor-associated immune cell infiltration as well as a noticeably better overall survival. The three modules most associated with the two clusters were identified by WGCNA, and a prognostic model with a strong prediction performance was constructed with their genes. Glycolysis, lactate metabolism, fatty acid synthesis, glutathione, methionine, and tryptophan metabolic pathway enrichment scores were remarkably higher in patients in cluster 1 and the high-risk group than in cluster 2 and the low-risk group. Knockdown PIK3C2A promoted ESCA cells apoptosis and inhibited cell vibiality. Conclusion: The current research maybe provides new understanding for the pathogenesis of ESCA based on CNV, providing an effective guidance for its clinical diagnosis and prognostic evaluation.

Genomic alterations in retinoblastoma tumors of Argentine patients.

INTRODUCTION: Retinoblastoma is initiated by inactivation of RB1 gene, but additional alterations may be required for tumor progression. Substitution and INDEL variants in different genes, aside RB1, are infrequent, while large copy number variants (CNVs) like gains on 1q, 2p, 6p and loss on 16q are common, they include oncogenes or tumor suppressors and are typical of retinoblastoma. AIM: To provide the molecular profile that is useful for prognosis and understanding of retinoblastoma development. METHODS: To identify genomic variants in six retinoblastoma tumors whole exome sequencing and informatic analysis were performed. RESULTS: RB1 was the only gene with nonsense or frameshift mutations. SNVs in other 11 genes were missense and at non-canonical splice-sites, all nonpathogenic. CNVs, similar to those reported, were identified in all retinoblastoma tumors. The most frequent were 1q gain and 16q loss. Additionally, deletions were identified on 13q, including RB1 gene, and on the X chromosome, including BCOR gene, the most frequently mutated, after RB1, in retinoblastoma. The number of CNVs detected in each tumor was between 1 and 7, depending on the age at diagnosis. CONCLUSION: The analysis of genomic alterations in retinoblastoma is useful to understand the severity of tumor progression and to apply appropriate treatments.

Diagnostic yield of exome sequencing-based copy number variation analysis in Mendelian disorders: a clinical application.

Next-generation sequencing (NGS) coupled with bioinformatic tools has revolutionized the detection of copy number variations (CNVs), which are implicated in the emergence of Mendelian disorders. In this study, we evaluated the diagnostic yield of exome sequencing-based CNV analysis in 449 patients with suspected Mendelian disorders. We aimed to assess the diagnostic yield of this recently utilized method and expand the clinical spectrum of intragenic CNVs. The cohort underwent whole exome sequencing (WES) and clinical exome sequencing (CES). Using GATK-gCNV, we identified 12 pathogenic CNVs that correlated with their clinical findings and resulting in a diagnostic yield of 2.67%. Importantly, the study emphasizes the role of CNVs in the etiology of Mendelian disorders and highlights the value of exome sequencing-based CNV analysis in routine diagnostic processes.

Beyond IQ: executive function deficits and their relation to functional, clinical, and neuroimaging outcomes in 3q29 deletion syndrome.

BACKGROUND: 3q29 deletion syndrome (3q29del) is a rare (~1:30 000) genomic disorder associated with a wide array of neurodevelopmental and psychiatric phenotypes. Prior work by our team identified clinically significant executive function (EF) deficits in 47% of individuals with 3q29del; however, the nuances of EF in this population have not been described. METHODS: We used the Behavior Rating Inventory of Executive Function (BRIEF) to perform the first in-depth assessment of real-world EF in a cohort of 32 individuals with 3q29del (62.5% male, mean age = 14.5 ± 8.3 years). All participants were also evaluated with gold-standard neuropsychiatric and cognitive assessments. High-resolution structural magnetic resonance imaging was performed on a subset of participants (n = 24). RESULTS: We found global deficits in EF; individuals with 3q29del scored higher than the population mean on the BRIEF global executive composite (GEC) and all subscales. In total, 81.3% of study subjects (n = 26) scored in the clinical range on at least one BRIEF subscale. BRIEF GEC T scores were higher among 3q29del participants with a diagnosis of attention deficit/hyperactivity disorder (ADHD), and BRIEF GEC T scores were associated with schizophrenia spectrum symptoms as measured by the Structured Interview for Psychosis-Risk Syndromes. BRIEF GEC T scores were not associated with cognitive ability. The BRIEF-2 ADHD form accurately (sensitivity = 86.7%) classified individuals with 3q29del based on ADHD diagnosis status. BRIEF GEC T scores were correlated with cerebellar white matter and subregional cerebellar cortex volumes. CONCLUSIONS: Together, these data expand our understanding of the phenotypic spectrum of 3q29del and identify EF as a core feature linked to both psychiatric and neuroanatomical features of the syndrome.

Biallelic Deletion of PEX26 Exon 4 in a Boy with Phenotypic Features of both Zellweger Syndrome and Infantile Refsum Disease.

Introduction: Peroxisome biogenesis disorders (PBDs) encompass a group of diseases marked by clinical and genetic heterogeneity. Phenotypes linked to PBDs include Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease (IRD), rhizomelic chondrodysplasia punctata type 1, and Heimler syndrome. PBD phenotypes manifest through hypotonia, developmental delay, facial dysmorphism, seizures, liver dysfunction, sensorineural hearing loss, and retinal dystrophy. Methods: The proband underwent comprehensive clinical evaluation, followed by whole-exome sequencing (WES) coupled with copy number analysis (CNV), aimed at identifying potential disease-causing variants aligning with the observed phenotype. Results: Our findings detail an individual exhibiting developmental delay, hearing loss, visual impairment, hepatomegaly, and splenomegaly, attributed to a biallelic deletion of exon 4 in the PEX26 gene. The WES analysis of the index case did not uncover any pathogenic/likely pathogenic single-nucleotide variations that could account for the observed clinical findings. However, the CNV data derived from WES revealed a homozygous deletion in exon 4 of the PEX26 gene (NM_001127649.3), providing a plausible explanation for the patient's clinical features. The exon 4 region of PEX26 encodes the transmembrane domain of the protein. The transmembrane domain plays a crucial role in anchoring the protein within lipid bilayers, and its absence can disrupt proper localization and functioning. As a result, this structural alteration may impact the protein's ability to facilitate essential cellular processes related to peroxisome biogenesis and function. Conclusion: The index patient, which presented with hearing loss, retinal involvement and hepatic dysfunction in adolescence age, has atypical clinical course that can be considered unusual for Zellweger syndrome (ZS) and IRD phenotypes, and its rare genotypic data (in-frame single exon deletion) expands the PBD disease spectrum. This study revealed for the first time that PEX26 protein transmembrane domain loss exhibits an unusual course with clinical findings of IRD and ZS phenotypes. WES studies, incorporating CNV analyses, empower the identification of novel genetic alterations in genes seldom associated with gross deletion/duplication variations, such as those in the PEX26 gene. This not only enhances diagnostic rates in rare diseases but also contributes to broadening the spectrum of causal mutations.

FANCD2 genome binding is nonrandom and is enriched at large transcriptionally active neural genes prone to copy number variation.

Fanconi anemia (FA) is a rare genetic disease characterized by congenital abnormalities and increased risk for bone marrow failure and cancer. Central nervous system defects, including acute and irreversible loss of neurological function and white matter lesions with calcifications, have become increasingly recognized among FA patients, and are collectively referred to as Fanconi Anemia Neurological Syndrome or FANS. The molecular etiology of FANS is poorly understood. In this study, we have used a functional integrative genomics approach to further define the function of the FANCD2 protein and FA pathway. Combined analysis of new and existing FANCD2 ChIP-seq datasets demonstrates that FANCD2 binds nonrandomly throughout the genome with binding enriched at transcription start sites and in broad regions spanning protein-coding gene bodies. FANCD2 demonstrates a strong preference for large neural genes involved in neuronal differentiation, synapse function, and cell adhesion, with many of these genes implicated in neurodevelopmental and neuropsychiatric disorders. Furthermore, FANCD2 binds to regions of the genome that replicate late, undergo mitotic DNA synthesis (MiDAS) under conditions of replication stress, and are hotspots for copy number variation. Our analysis describes an important targeted role for FANCD2 and the FA pathway in the maintenance of large neural gene stability.

The fetal origins of metabolic health: exploring the association between newborn biological age and metabolism hormones in childhood.

BACKGROUND: Telomere length (TL), mitochondrial DNA copy number (mtDNAcn), and DNA methylation age (DNAmAge) are common aging biomarkers. However, research on the associations between these three markers at birth and subsequent metabolic status was limited. This study aimed to evaluate the association between TL, mtDNAcn, and DNAmAge in newborns and the variation in metabolic hormones of children at 3 years old. METHODS: This research involved 895 mother-child pairs from a birth cohort in China, with TL and mtDNAcn measured using quantitative real-time PCR, DNA methylation (DNAm) assessed using Infinium MethylationEPIC Beadchip, and DNAm age (DNAmAge) determined using Horvath's epigenetic clock. Insulin and leptin levels were measured via electrochemiluminescence assay. Multivariable adjusted linear regression and restricted cubic spline (RCS) analysis were utilized to examine the association between aging markers and metabolic hormones. RESULTS: The linear regression analysis indicated the percentage change of metabolism hormones for per doubling of aging biomarkers alterations and found significant associations between DNAmAge and insulin levels (adjusted percent change (95% CI), - 13.22 (- 23.21 to - 1.94)), TL and leptin levels (adjusted percent change (95% CI), 15.32 (1.32 to 31.24)), and mtDNAcn and leptin levels (adjusted percent change (95% CI), - 14.13 (- 21.59 to - 5.95)). The RCS analysis revealed significant non-linear associations between TL (Ln transformed) and insulin (Ln transformed) (P = 0.024 for nonlinearity), as well as DNAmAge (Ln transformed) and leptin (Ln transformed) (P = 0.043 for nonlinearity). Specifically, for TL and insulin, a positive association was observed when TL (Ln transformed) was less than - 0.05, which transitioned to an inverse association when TL (Ln transformed) was greater than - 0.05. Regarding DNAmAge and leptin, there was a sharp decline when DNAmAge (Ln transformed) was less than - 1.35, followed by a plateau between - 1.35 and - 0.67 and then a further decline when DNAmAge (Ln transformed) was greater than - 0.67. CONCLUSIONS: In this prospective birth cohort study, variation in metabolic hormones of children at 3 years old was associated with TL, mtDNAcn, and DNAmAge at birth. These findings suggested that TL, mtDNAcn, and DNAmAge might play a role in the biological programming of metabolic health from birth.

Nanopore sequencing with unique molecular identifiers enables accurate mutation analysis and haplotyping in the complex lipoprotein(a) KIV-2 VNTR.

BACKGROUND: Repetitive genome regions, such as variable number of tandem repeats (VNTR) or short tandem repeats (STR), are major constituents of the uncharted dark genome and evade conventional sequencing approaches. The protein-coding LPA kringle IV type-2 (KIV-2) VNTR (5.6 kb per unit, 1-40 units per allele) is a medically highly relevant example with a particularly intricate structure, multiple haplotypes, intragenic homologies, and an intra-VNTR STR. It is the primary regulator of plasma lipoprotein(a) [Lp(a)] concentrations, an important cardiovascular risk factor. Lp(a) concentrations vary widely between individuals and ancestries. Multiple variants and functional haplotypes in the LPA gene and especially in the KIV-2 VNTR strongly contribute to this variance. METHODS: We evaluated the performance of amplicon-based nanopore sequencing with unique molecular identifiers (UMI-ONT-Seq) for SNP detection, haplotype mapping, VNTR unit consensus sequence generation, and copy number estimation via coverage-corrected haplotypes quantification in the KIV-2 VNTR. We used 15 human samples and low-level mixtures (0.5 to 5%) of KIV-2 plasmids as a validation set. We then applied UMI-ONT-Seq to extract KIV-2 VNTR haplotypes in 48 multi-ancestry 1000 Genome samples and analyzed at scale a poorly characterized STR within the KIV-2 VNTR. RESULTS: UMI-ONT-Seq detected KIV-2 SNPs down to 1% variant level with high sensitivity, specificity, and precision (0.977 ± 0.018; 1.000 ± 0.0005; 0.993 ± 0.02) and accurately retrieved the full-length haplotype of each VNTR unit. Human variant levels were highly correlated with next-generation sequencing (R2 = 0.983) without bias across the whole variant level range. Six reads per UMI produced sequences of each KIV-2 unit with Q40 quality. The KIV-2 repeat number determined by coverage-corrected unique haplotype counting was in close agreement with droplet digital PCR (ddPCR), with 70% of the samples falling even within the narrow confidence interval of ddPCR. We then analyzed 62,679 intra-KIV-2 STR sequences and explored KIV-2 SNP haplotype patterns across five ancestries. CONCLUSIONS: UMI-ONT-Seq accurately retrieves the SNP haplotype and precisely quantifies the VNTR copy number of each repeat unit of the complex KIV-2 VNTR region across multiple ancestries. This study utilizes the KIV-2 VNTR, presenting a novel and potent tool for comprehensive characterization of medically relevant complex genome regions at scale.

Efficiency of Non-Invasive Prenatal Testing in Detecting Fetal Copy Number Variation: A Retrospective Cohort Study.

Purpose: Screening of pathological copy number variations (CNVs) is important for early-diagnosis of hereditary disease. This study was designed to investigate the efficiency of non-invasive prenatal testing (NIPT) in detecting fetal CNVs. Methods: This retrospective analysis included fetuses with CNVs between January 2018 and December 2020. Karyotype analysis and CNV sequencing (CNV-seq) were performed. We then analyzed the positive predictive values of the subchromosomal microdeletions and microduplications. Results: Fifty-eight subjects with aberrant CNVs were screened after NIPT, among which 44 finally underwent amniocentesis. CNV-seq confirmed the presence of CNVs in 24 cases. This indicated that false positivity rate of NIPT was 45.5%. Among 24 cases with CNVs after CNV-seq, only 4 showed consistent findings with karyotype analysis, which showed that karyotyping analysis yielded a missed diagnosis rate of 83.3% for the genome CNV. Positive predictive value (PPV) was 50.0% for CNVs with a length of <5 Mb after NIPT screening. PPV for CNVs with a length of 5 Mb-10 Mb was 33.3%, while that for CNVs with a length of ≥10Mb was 60%. For CNVs duplication after NIPT, the PPV was 65.2%, while that for deletion was 36.4%. Conclusion: For CNVs detected after NIPT, it should be combined with ultrasonographic findings, karyotype analysis, CNV-seq or CMA to determine the pregnancy outcome. Expanding NIPT may increase the risk of unnecessary invasive surgery and unintended selective termination of pregnancy.

Microbial rrn copy number is associated with soil C: N ratio and pH under long-term fertilization.

Soil microbial life-history strategies, as indicated by rRNA operon (rrn) copy numbers, strongly influence agro-ecosystem functioning. Long-term N fertilization causes strong and lasting changes in soil properties, yet its impact on microbial strategies remains largely unexplored. Using long-term field experiments across three agro-ecosystems, we consistently found that N fertilization strongly decreased soil C: N ratio and pH, further increasing the community-level rrn copy number, including both average rrn copy number and total 16S rRNA copy number. Soil C: N stoichiometry balanced by N supplement favored the growth of N-dependent copiotrophic species containing high rrn copy numbers (an average of 2.5) and increased their network connections, predominantly contributing to community-level rrn copy number increase. Decreased soil pH caused by N fertilization also favored the growth of some species whose abundances negatively correlated with pH, partially contributing to the community-level rrn copy number increase. By examining the genomes of two dominant species, we found that microorganisms with a higher rrn copy number (6), e.g., Streptomyces scabiei, possessed more genes related to C and N transport and metabolism. In contrast, the Mycobacterium simiae with a lower rrn copy number (1) has more genes associated with secondary metabolite biosynthesis and lipid transport and metabolism. Our finding challenges the concept of microbial life-strategy regulation solely by nutrient availability, highlighting the important contributions of soil stoichiometric balance and pH to microbial strategies in agro-ecosystems under long-term N inputs.

Landscape of epithelial cell subpopulations in the human esophageal squamous cell carcinoma microenvironment.

Aims: We sought to reveal the landscape of epithelial cell subpopulations in the human esophageal squamous cell carcinoma microenvironment and investigate their parts on esophageal squamous carcinoma (ESCC) development. Background: Epithelial cells play an important role in the occurrence and development of ESCC through multiple mechanisms. While the landscape of epithelial cell subpopulations in ESCC, remains unclear. Objective: Exploring the role of epithelial cell subpopulations in ESCC progression. Methods: Seurat R package was used for single-cell RNA sequencing (scRNA-seq) data filtering, dimensionality reduction, clustering and differentially expressed genes analysis. Cellmarker database was adopted for cell cluster annotation. Functional enrichment analysis was carried out by Gene Ontology (GO) analysis. InferCNV package was conducted for copy number variation (CNV) of epithelial cell subpopulations in all chromosomal regions. Pseudotime trajectory analysis was implemented for exploring differentiation trajectory of epithelial cells subgroups during the cancer progression. CellChat analysis was used for probing the interactions between epithelial cells and NK/T cells. cellular experiments were performed using Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR), Wound-Healing Assay and transwell. Results: 11 major cell subpopulations were identified in ESCC and adjunct tissues. Further reclassification of epithelial cells uncovered 4 subpopulations. Enrichment analysis revealed that highly expressed genes in 4 epithelial cell subpopulations were related to cell proliferation, immune response and angiogenesis. CNV analysis found that UBD + epithelial cells and GAS2L3+ epithelial cells had a higher proportion of CNV. Cell differentiation trajectories disclosed that KRT6C+ and GSTA1+ epithelial cells were in an intermediate state of differentiation, while UBD+ and GAS2L3+ epithelial cells are in an end state of differentiation during ESCC progression. Finally, we found that four epithelial cell subpopulations all inhibited NK/T cells through NECTIN2-TIGIT and CLEC2B-KLRB1. Low ATF3 and DDIT3 mRNA expression inhibited ESCC cell migration and invasion. Conclusion: Here, we obtained a through epithelial cell atlas of ESCC at single-cell resolution, explored the role of epithelial cell in ESCC progression, and unveiled immunosuppressive signals to NK/T cells in promoting ESCC. Our findings expand the comprehension of epithelial cells and offer a theoretical guidance for future anti-epithelial cell treatment of ESCC.

Aligning genotyping and copy number data in single trophectoderm biopsies for aneuploidy prediction: uncovering incomplete concordance.

STUDY QUESTION: To what extent can genotype analysis aid in the classification of (mosaic) aneuploid embryos diagnosed through copy number analysis of a trophectoderm (TE) biopsy? SUMMARY ANSWER: In a small portion of embryos, genotype analysis revealed signatures of meiotic or uniform aneuploidy in those diagnosed with intermediate copy number changes, and signatures of presumed mitotic or putative mosaic aneuploidy in those diagnosed with full copy number changes. WHAT IS KNOWN ALREADY: Comprehensive chromosome screening (CCS) for preimplantation genetic testing has provided valuable insights into the prevalence of (mosaic) chromosomal aneuploidy at the blastocyst stage. However, diagnosis of (mosaic) aneuploidy often relies solely on (intermediate) copy number analysis of a single TE biopsy. Integrating genotype information allows for independent assessment of the origin and degree of aneuploidy. Yet, studies aligning both datasets to predict (putative mosaic) aneuploidy in embryos remain scarce. STUDY DESIGN SIZE DURATION: A single TE biopsy was collected from 1560 embryos derived from 221 couples tested for a monogenic disorder (n = 218) or microdeletion-/microduplication syndrome (n = 3). TE samples were subjected to both copy number and genotyping analysis. PARTICIPANTS/MATERIALS SETTING METHODS: Copy number and SNP genotyping analysis were conducted using GENType. Unbalanced chromosomal anomalies ≥10 Mb (or ≥20 Mb for copy number calls <50%) were classified by degree, based on low-range intermediate (LR, 30-50%), high-range intermediate (HR, 50-70%) or full (>70%) copy number changes. These categories were further subjected to genotyping analysis to ascertain the origin (and/or degree) of aneuploidy. For chromosomal gains, the meiotic division of origin (meiotic I/II versus non-meiotic or presumed mitotic) was established by studying the haplotypes. The level of monosomy (uniform versus putative mosaic) in the biopsy could be ascertained from the B-allele frequencies. For segmental aneuploidies, genotyping was restricted to deletions. MAIN RESULTS AND THE ROLE OF CHANCE: Of 1479 analysed embryos, 24% (n = 356) exhibited a whole-chromosome aneuploidy, with 19% (n = 280) showing full copy number changes suggestive of uniform aneuploidy. Among 258 embryos further investigated by genotyping, 95% of trisomies with full copy number changes were identified to be of meiotic origin. For monosomies, a complete loss of heterozygosity (LOH) in the biopsy was observed in 97% of cases, yielding a 96% concordance rate at the embryo level (n = 248/258). Interestingly, 4% of embryos (n = 10/258) showed SNP signatures of non-meiotic gain or putative mosaic loss instead. Meanwhile, 5% of embryos (n = 76/1479) solely displayed HR (2.5%; n = 37) or LR (2.6%; n = 39) intermediate copy number changes, with an additional 2% showing both intermediate and full copy number changes. Among embryos with HR intermediate copy number changes where genotyping was feasible (n = 25/37), 92% (n = 23/25) showed SNP signatures consistent with putative mosaic aneuploidy. However, 8% (n = 2/25) exhibited evidence of meiotic trisomy (9%) or complete LOH in the biopsy (7%). In the LR intermediate group, 1 of 33 (3%) genotyped embryos displayed complete LOH. Furthermore, segmental aneuploidy was detected in 7% of embryos (n = 108/1479) (or 9% (n = 139) with added whole-chromosome aneuploidy). These errors were often (52%) characterized by intermediate copy number values, which closely aligned with genotyping data when examined (94-100%). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The findings were based on single TE biopsies and the true extent of mosaicism was not validated through embryo dissection. Moreover, evidence of absence of a meiotic origin for a trisomy should not be construed as definitive proof of a mitotic error. Additionally, a genotyping diagnosis was not always attainable due to the absence of a recombination event necessary to discern between meiotic II and non-meiotic trisomy, or the unavailability of DNA from both parents. WIDER IMPLICATIONS OF THE FINDINGS: Interpreting (intermediate) copy number changes of a single TE biopsy alone as evidence for (mosaic) aneuploidy in the embryo remains suboptimal. Integrating genotype information alongside the copy number status could provide a more comprehensive assessment of the embryo's genetic makeup, within and beyond the single TE biopsy. By identifying meiotic aberrations, especially in presumed mosaic embryos, we underscore the potential value of genotyping analysis as a deselection tool, ultimately striving to reduce adverse clinical outcomes. STUDY FUNDING/COMPETING INTERESTS: L.D.W. was supported by the Research Foundation Flanders (FWO; 1S74621N). M.B., K.T., F.V.M., S.J., A.V.T., V.S., D.S., A.D., and S.S. are supported by Ghent University Hospital. B.M. was funded by Ghent University. The authors have no conflicts of interest.

Distinct copy number signatures between residual benign and transformed areas of carcinoma ex pleomorphic adenoma.

The mechanisms involved with the pathogenesis of carcinoma ex pleomorphic adenoma (CXPA) seem to be associated with the accumulation of molecular alterations in the pleomorphic adenoma (PA). In this sense, using array-based comparative genomic hybridization (aCGH) a rare series of 27 cases of CXPA and 14 residual PA (rPA) adjacent to the transformation area, we investigated the profile of the copy number alterations (CNAs) comparing benign residual and transformed areas. The main findings were correlated with the histopathological classification by histologic subtype and degree of invasion. The distribution of losses (p = 0.187) and amplifications (p = 0.172) was not statistically different between rPA and CXPA. The number of gains was increased in the transformed areas compared to the benign residual areas (p = 0.005). PLAG1 gain was maintained along the malignant transformation, as it was observed in both residual PA and CXPA samples, likely being an earlier event during transformation. The amplification of GRB7 and ERBB2 may also be an initial step in the malignant transformation of PA to CXPA (salivary duct carcinoma subtype). Furthermore, the amplification of HMGA2 and RPSAP52 were the most prevalent alterations among the studied samples. It was noteworthy that amplified genes in the transformed areas of the tumors were enriched for biological processes related to immune signaling. In conclusion, our results underscored for the first-time crucial CNAs in CXPA, some of them shared with the residual benign area adjacent to the transformation site. These CNAs included PLAG1 gain, as well as amplification of GRB7, ERBB2, HMGA2, and RPSAP52.

Chromosome aberrations cause tumorigenesis through chromosomal rearrangements in a hepatocarcinogenesis rat model.

Chromosome aberrations (CAs), a genotoxic potential of carcinogens, are believed to contribute to tumorigenesis by chromosomal rearrangements through micronucleus formation. However, there is no direct evidence that proves the involvement of CAs in tumorigenesis in vivo. In the current study, we sought to clarify the involvement of CAs in chemical carcinogenesis using a rat model with a pure CA-inducer hepatocarcinogen, acetamide. Whole-genome analysis indicated that hepatic tumors induced by acetamide treatment for 26-30 weeks showed a broad range of copy number alterations in various chromosomes. In contrast, hepatic tumors induced by a typical mutagen (diethylnitrosamine) followed by a nonmutagen (phenobarbital) did not show such mutational patterns. Additionally, structural alterations such as translocations were observed more frequently in the acetamide-induced tumors. Moreover, most of the acetamide-induced tumors expressed c-Myc and/or MDM2 protein due to the copy number gain of each oncogene. These results suggest the occurrence of chromosomal rearrangements and subsequent oncogene amplification in the acetamide-induced tumors. Taken together, the results indicate that CAs are directly involved in tumorigenesis through chromosomal rearrangements in an acetamide-induced hepatocarcinogenesis rat model.

The dysregulation of mitochondrial homeostasis-related genes could be involved in the decrease of mtDNA copy number in systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is a chronic multifactorial autoimmune disease. It is now widely demonstrated that oxidative stress (OS) plays an important role in the modulation of the pathogenesis of this disease. Mitochondrial DNA (mtDNA) is highly vulnerable to OS and it is known a decrease of mtDNA copy number in SLE patients. However, to date, it has not been investigated if this decrease is associated with a dysregulation of mitochondrial homeostasis genes. Our aim is to evaluate the amount of mtDNA copy number and the expression of the genes more involved in the mitochondrial homeostasis pathways, in peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy controls. We analysed the amount of mtDNA in PBMCs of 72 SLE patients and 61 healthy controls by qPCR. Then, we investigated the expression variability of TFAM and SIRT1 (biogenesis), MFN1 and MFF (fusion/fission) and PRKN2 (mitophagy) genes in a subgroup of SLE patients and healthy controls. Interestingly, we have observed a highly significant decrease in mtDNA copies in SLE patients compared to healthy controls (P < 0.0001). In addition, we have shown that the expression levels of SIRT1, MFN1 and PRKN2 genes were significantly decreased in SLE patients with respect to healthy controls (P = 0.00001 for SIRT1, P = 0.0150 for MFN1 and P = 0.0009 for PRKN2). Lastly, we have reported a positive correlation between PRKN2 expression level and mtDNA copy number (P = 0.019, r = 0.475). In conclusion, our data confirm the impairment of mtDNA copy number in the disease and show for the first time a dysregulation of the mitochondrial homeostasis genes. These results could provide additional support to the important role of mitochondria in SLE development.

Modeling antisense oligonucleotide therapy in MECP2 duplication syndrome human iPSC-derived neurons reveals gene expression programs responsive to MeCP2 levels.

Genomic copy-number variations (CNVs) that can cause neurodevelopmental disorders often encompass many genes, which complicates our understanding of how individual genes within a CNV contribute to pathology. MECP2 duplication syndrome (MDS or MRXSL in OMIM; OMIM#300260) is one such CNV disorder caused by duplications spanning methyl CpG-binding protein 2 (MECP2) and other genes on Xq28. Using an antisense oligonucleotide (ASO) to normalize MECP2 dosage is sufficient to rescue abnormal neurological phenotypes in mouse models overexpressing MECP2 alone, implicating the importance of increased MECP2 dosage within CNVs of Xq28. However, because MDS CNVs span MECP2 and additional genes, we generated human neurons from multiple MDS patient-derived induced pluripotent cells (iPSCs) to evaluate the benefit of using an ASO against MECP2 in a MDS human neuronal context. Importantly, we identified a signature of genes that is partially and qualitatively modulated upon ASO treatment, pinpointed genes sensitive to MeCP2 function, and altered in a model of Rett syndrome, a neurological disorder caused by loss of MeCP2 function. Furthermore, the signature contained genes that are aberrantly altered in unaffected control human neurons upon MeCP2 depletion, revealing gene expression programs qualitatively sensitive to MeCP2 levels in human neurons. Lastly, ASO treatment led to a partial rescue of abnormal neuronal morphology in MDS neurons. All together, these data demonstrate that ASOs targeting MECP2 benefit human MDS neurons. Moreover, our study establishes a paradigm by which to evaluate the contribution of individual genes within a CNV to pathogenesis and to assess their potential as a therapeutic target.

Prenatal detection of copy number variants.

Prenatal detection of copy number variants (CNVs) plays an important role in the diagnosis of fetal genetic abnormalities. Understanding the methods used for prenatal CNV detection and their clinical significance contributes to the implementation of advanced genetic screening techniques in prenatal care; facilitating early identification and management of genetic disorders in fetuses. Some CNVs impose significant genetic counselling challenges; especially those which are associated with uncertain clinical significance, in the context of variable penetrance and/or expressivity or when identified incidentally. This chapter focuses on the different techniques used for detecting CNVs, including Single Nucleotide Polymorphism (SNP) arrays, comparative genomic hybridization (CGH) arrays, Non-Invasive Prenatal Testing (NIPT), Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS) as well as their advantages and limitations. The tools needed for the classification of CNVs and their clinical relevance are also explored, emphasising the importance of accurate interpretation for appropriate clinical management and genetic counselling.

Resolution of Optimal Mitochondrial and Nuclear DNA Enrichment in Target-Panel Sequencing and Physiological Mitochondrial DNA Copy Number Estimation in Liver Cancer and Non-Liver Cancer Subjects.

Mitochondria generate energy to support cells. They are important organelles that engage in key biological pathways. The dysfunction of mitochondria can be linked to hepatocarcinogenesis, which has been actively explored in recent years. To investigate the mitochondrial dysfunction caused by genetic variations, target-panel sequencing is a flexible and promising strategy. However, the copy number of mitochondria generally exceeds nuclear DNA, which raises a concern that uneven target enrichment of mitochondrial DNA (mtDNA) and nuclear DNA (ncDNA) in target-panel sequencing would lead to an undesirably biased representation of them. To resolve this issue, we evaluated the optimal pooling of mtDNA probes and ncDNA probes by a series of dilutions of mtDNA probes in both genomic DNA (gDNA) and cell-free DNA (cfDNA) samples. The evaluation was based on read count, average sequencing depth and coverage of targeted regions. We determined that an mtDNA:ncDNA probe ratio of around 1:10 would offer a good balance of sequencing performance and cost effectiveness. Moreover, we estimated the median physiological mtDNA:ncDNA copy ratio as 38.1 and 2.9 in cfDNA and gDNA samples of non-liver cancer subjects, respectively, whereas they were 20.0 and 2.1 in the liver cancer patients. Taken together, this study revealed the appropriate pooling strategy of mtDNA probes and ncDNA probes in target-panel sequencing and suggested the normal range of physiological variation of the mtDNA:ncDNA copy ratio in non-liver cancer individuals. This can serve as a useful reference for future target-panel sequencing investigations of the mitochondrial genome in liver cancer.

Concomitant presence of a novel ARPP21 variant and CNVs in Chinese familial amyotrophic lateral sclerosis-frontotemporal dementia patients.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder marked by the degeneration of motor neurons and progressive muscle weakness. Heredity plays an important part in the pathogenesis of ALS. Recently, with the emergence of the oligogenic pathogenic mechanism in ALS and the ongoing discovery of new mutated genes and genomic variants, there is an emerging need for larger-scale and more comprehensive genetic screenings in higher resolution. In this study, we performed whole-genome sequencing (WGS) on 34 familial ALS probands lacking the most common disease-causing mutations to explore the genetic landscape of Chinese ALS patients further. Among them, we identified a novel ARPP21 c.1231G > A (p.Glu411Lys) variant and two copy number variations (CNVs) affecting the PFN1 and RBCK1 genes in a patient with ALS-frontotemporal dementia (FTD). This marks the first report of an ARPP21 variant in Chinese ALS-FTD patients, providing fresh evidence for the association between ARPP21 and ALS. Our findings also underscore the potential role of CNVs in ALS-FTD, suggesting that the cumulative effect of multiple rare variants may contribute to disease onset. Furthermore, compared to the averages in our cohort and the reported Chinese ALS population, this patient displayed a shorter survival time and more rapid disease progression, suggesting the possibility of an oligogenic mechanism in disease pathogenesis. Further research will contribute to a deeper understanding of the rare mutations and their interactions, thus advancing our understanding of the genetic mechanisms underlying ALS and ALS-FTD.