Copy Number Abnormalities Research Articles

Oncomine panel testing in the clinical management of colorectal cancer: An Irish experience.

212 Background: Early onset colorectal cancer (EOCRC) is defined as colorectal cancer (CRC) in those under the age of 50. It has been hypothesized that the molecular signatures of EOCRC could differ from those of late onset CRC (LOCRC). Next Generation Sequencing (NGS) using the Oncomine Precision Assay is a useful tool to guide treatment decisions based on the presence or absence of oncogene drivers. While not all of these mutations currently influence the clinical management of CRC patients, ongoing research on the function of these genes and the development of new drugs could offer insights into patient prognosis, treatment choices and prediction of therapy resistance. Methods: Of >1500 CRC identified between 2010 and 2024, we investigated the clinical and genetic characteristics of 38 EOCRC and 41 LOCRC cases. All cases were microsatellite stable and staged I-III. NGS was performed with the Ion Torrent Genexus integrated platform using the Oncomine 45-gene Precision Assay panel. DNA hotspot mutations and copy number variations (CNVs) were assessed, followed by the classification of clinically relevant mutations using the genetic variant OncoKB database. Results: There were 38 EOCRC and 41 LOCRC cases. The age range was 27 to 49 and 50 to 91, with median ages of 44 and 72, in the EOCRC and LOCRC groups, respectively. Based on the stratification of NGS mutation levels as per the OncoKB classification, all patients in both groups (100%) had level 1 actionable mutations. NRAS wild type (WT) status was present in 100% of patients in both groups. However, differences were observed in KRAS status: 84% (32/38) of EOCRC were KRAS WT, while only 65% (27/41) of LOCRC were KRAS WT. While 3 % (n=1) of the EOCRC cohort had an ERBB2 amplification, none were detected in the LOCRC group. BRAF V600E mutations were present in 11% (4/38) of EOCRC and 12% (5/41) of LOCRC. Level 4 mutations were detected in 18% (n=7) of EOCRC, all of which were PIK3CA mutations, while these were seen in 24% (n=10) of the LOCRC group, including 9 PIK3CA mutations and 1 FGFR1 mutation. Regarding concurrent mutations, 2 patients in the EOCRC group had two mutations each: one with concurrent KRAS and PIK3CA mutations, and another with concurrent JAK2 and KRAS mutations. The LOCRC group had a higher incidence of concurrent mutations, with 8 patients affected: 6 had concurrent KRAS and PIK3CA mutations, and 2 had concurrent BRAF and PIK3CA mutations. Conclusions: Our study demonstrates that EOCRC have higher KRAS WT status and fewer concurrent mutations compared to LOCRC, who exhibit more frequent KRAS non-G12C oncogenic mutations. It also underscores the importance of routine NGS mutation panel analysis in CRC management. Additionally, identifying specific mutations in patients that relapse can guide targeted therapies, potentially improving outcomes and offering personalized treatment options.

Classification of Fibro-osseous Tumors in the Craniofacial Bones using DNA Methylation and Copy Number Alterations.

Fibro-osseous tumors of the craniofacial bones are a heterogeneous group of lesions comprising cemento-osseous dysplasia (COD), cemento-ossifying fibroma (COF), juvenile trabecular ossifying fibroma (JTOF), psammomatoid ossifying fibroma (PsOF), fibrous dysplasia (FD), and low-grade osteosarcoma (LGOS) with overlapping clinicopathological features. However, their clinical behavior and treatment differ significantly, underlining the need for accurate diagnosis. Molecular diagnostic markers exist for subsets of these tumors, including GNAS mutations in FD, SATB2 fusions in PsOF, mutations involving the RAS-MAPK signaling pathway in COD, and MDM2 amplification in LGOS. Since DNA methylation and copy number profiling are well established for the classification of central nervous system tumors, our aim was to investigate whether this tool might be used as well for classifying fibro-osseous tumors in the craniofacial bones. We collected a well-characterized, multicenter cohort with available molecular data, including COD (n = 20), COF (n = 13), JTOF (n = 10), PsOF (n = 25), FD (n = 23), LGOS (n = 4), and high-grade osteosarcoma (HGOS; n = 11). Genome-wide DNA methylation and copy number variation data were generated using the Illumina Infinium Methylation EPIC array interrogating >850 000 CpG sites. DNA methylation profiling yielded evaluable results in 73/106 tumors, including 6 CODs, 12 COFs, 6 JTOFs, 19 PsOFs, 18 FDs, 2 LGOSs, and 10 HGOSs. Unsupervised clustering and dimensionality reduction (Uniform Manifold Approximation and Projection) revealed that FD, extragnatic PsOF, and HGOS formed distinct clusters. Surprisingly, COD, COF, JTOF, and mandibular PsOF clustered together, apart from other craniofacial bone tumors. LGOS did not form a distinct cluster, likely due to the low number of cases. Copy number analysis revealed that FD, COD, COF, JTOF, and PsOF were typically characterized by flat copy number profiles compared to LGOS with gains of chromosome 12 and HGOS with multiple heterogeneous copy number alterations. In conclusion, using DNA methylation and copy number profiles, benign fibro-osseous tumors can be separated from low-grade and high-grade osteosarcomas in the craniofacial bones, which is of diagnostic value in challenging cases with overlapping clinicopathological features.

The Diurnal Variation in Mitochondrial Gene in Human Type 2 Diabetic Mesenchymal Stem Cell Grafts.

The application of regenerative therapy through stem cell transplantation has emerged as a promising avenue for the treatment of diabetes mellitus (DM). Transplanted tissue homeostasis is affected by disturbances in the clock genes of stem cells. The aim of this study is to investigate the diurnal variation in mitochondrial genes and function after transplantation of adipose-derived mesenchymal stem cells (T2DM-ADSCs) from type 2 diabetic patients into immunodeficient mice. Diurnal variation in mitochondrial genes was assessed by next-generation sequencing. As a result, the diurnal variation in mitochondrial genes showing troughs at ZT10 and ZT22 was observed in the group transplanted with adipose-derived mesenchymal stem cells derived from healthy individuals (N-ADSC). On the other hand, in the group transplanted with T2DM-ADSCs, diurnal variation indicative of troughs was observed at ZT18, with a large phase and amplitude deviation between the two groups. To evaluate the diurnal variation in mitochondrial function, we quantified mitochondrial DNA copy number using the Human mtDNA Monitoring Primer Set, measured mitochondrial membrane potential using JC-1, and evaluated mitophagy staining. The results showed a diurnal variation in mitochondrial DNA copy number, mitophagy, mitochondrial membrane potential, and NF-kB signaling in the N-ADSC transplant group. In contrast, no diurnal variation was observed in T2DM-ADSC transplants. The diurnal variation in mitochondrial function revealed in this study may be a new marker for the efficiency of T2DM-ADSC transplantation.

Mitochondrial DNA copy number and risk of cardiovascular disease and all-cause mortality: a systematic review and meta-analysis of observational studies.

Increasing studies have explored the correlation of mitochondrial DNA copy number (mtDNA-CN) abnormalities with cardiovascular disease (CVD) and all-cause mortality; however, their findings are contradictory. This systematic review and meta-analysis sought to quantitatively summarize current studies to elucidate the impact of mtDNA-CN on CVD outcomes and all-cause mortality. Relevant studies were searched for in PubMed, Embase, and Web of Science databases, up to October 23, 2023. Summary relative risks (RRs) and 95% confidence intervals (CIs) were calculated with the random-effects model. In total, 22 articles were included in the systematic review, 13 of which were included in the meta-analysis of CVD outcomes and 8 in all-cause mortality. Compared to the highest mtDNA-CN level, the summary RR (95% CI) for the lowest mtDNA-CN level was 2.09 (95% CI 1.59-2.75) for CVD, 1.70 (95% CI 1.29-2.24) for coronary heart disease (CHD), 1.43 (95% CI 1.15-1.79) for heart failure (HF), 1.88 (95% CI 1.08-3.28) for stroke, and 1.33 (95% CI 1.21-1.47) for all-cause mortality. Lower mtDNA-CN may increase the risk of CVD, including CHD, HF, and stroke, as well as all-cause mortality. MtDNA-CN is a potential predictor of CVD and all-cause mortality.

Bootstrap-based inference for multiple mean-variance changepoint models

Identifying multiple change points in the mean and/or variance is crucial across various fields, including finance and quality control. We introduce a novel technique that detects change points for the mean and/or variance of a noisy sequence and constructs confidence intervals for both the mean and variance of the sequence. This method integrates the weighted bootstrap with the Sequential Binary Segmentation (SBS) algorithm. Not only does our technique pinpoint the location and number of change points, but it also determines the type of change for each estimated point, specifying whether the change occurred in the mean, variance, or both. Our simulations show that our method outperforms other approaches in most scenarios, clearly demonstrating its superiority. Finally, we apply our technique to three datasets, including DNA copy number variation, stock volume, and traffic flow data, further validating its practical utility and wide-ranging applicability.

Genome-wide detection and analysis of copy number variations in Sal sheep using a high-density SNP array

Purpose: to identify CNV regions (CNVRs) and identify potentially specific CNVs in Sal sheep.Materials and methods. The research was carried out on 48 Salsk breed sheep. Genotyping was performed using high-density microarrays Ovine SNP 600 K BeadChip array (Illumina, USA). DNA copy number variation (CNV) searches were performed using PennCNV software based on the Oar_v3.1 reference genome.Results. We identified 4585 autosomal CNVs in 48 Sal breed sheep. The average CNV length was 92,3 Kb. 21,1 % of the total number of CNVs are less than 50 Kb in size. On average, from 65 to 138 CNVs were identified per animal. 527 autosomal CNVRs were obtained, representing 54,77 Mb of the entire sheep genome. The proportion of chromosomes covered by CNVR varies by chromosome, from 0,5 % on OAR25 to 9 % on OAR24. Analysis of CNVR in the ENCEMBL system revealed 42 genes in which CNVRs are localized; the remaining CNVRs are located in intergenic regions. Comparison of the identified CNVRs with sheep QTLs from the Animal QTL database showed that 39,24 % of the genes were responsible for reproductive traits, and 20,25 % were responsible for wool-related traits.Conclusions. Functional annotation of CNVRs localized in genes and intergenic spaces was carried out and the total number of detected CNVRs in the QTL database was analyzed. The results showed that CNVs are localized in genes associated with selection-significant traits in sheep.

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

BackgroundSomatotroph neuroendocrine pituitary tumors (sPitNET) are a subtype of pituitary tumors that commonly cause acromegaly. Our study aimed to determine the spectrum of DNA copy number abnormalities (CNAs) in sPitNETs and their relevance.MethodsA landscape of CNAs in sPitNETs was determined using combined whole-genome approaches involving low-pass whole genome sequencing and SNP microarrays. Fluorescent in situ hybridization (FISH) was used for microscopic validation of CNAs. The tumors were also subjected to transcriptome and DNA methylation analyses with RNAseq and microarrays, respectively.ResultsWe observed a wide spectrum of cytogenetic changes ranging from multiple deletions, recurrent chromosome 11 loss, stable genomes, to duplication of the majority of the chromosomes. The identified CNAs were confirmed with FISH. sPitNETs with multiple duplications were characterized by intratumoral heterogeneity in chromosome number variation in individual tumor cells, as determined with FISH. These tumors were separate CNA-related sPitNET subtype in clustering analyses with CNA signature specific for whole genome doubling-related etiology. This subtype encompassed GNAS-wild type, mostly densely granulated tumors with favorable expression level of known prognosis-related genes, notably enriched with POUF1/NR5A1-double positive PitNETs. Chromosomal deletions in sPitNETs are functionally relevant. They occurred in gene-dense DNA regions and were related to genes downregulation and increased DNA methylation in the CpG island and promoter regions in the affected regions. Recurrent loss of chromosome 11 was reflected by lowered MEN1 and AIP. No such unequivocal relevance was found for chromosomal gains. Comparisons of transcriptomes of selected most cytogenetically stable sPitNETs with tumors with recurrent loss of chromosome 11 showed upregulation of processes related to gene dosage compensation mechanism in tumors with deletion. Comparison of stable tumors with those with multiple duplications showed upregulation of processes related to mitotic spindle, DNA repair, and chromatin organization. Both comparisons showed upregulation of the processes related to immune infiltration in cytogenetically stable tumors and deconvolution of DNA methylation data indicated a higher content of specified immune cells and lower tumor purity in these tumors.ConclusionssPitNETs fall into three relevant cytogenetic groups: highly aneuploid tumors characterized by known prognostically favorable features and low aneuploidy tumors including specific subtype with chromosome 11 loss.

The molecular history of IDH-mutant astrocytomas without adjuvant treatment.

Hypermutation and malignant transformation are potential complications arising from temozolomide treatment of IDH-mutant gliomas. However, the natural history of IDH-mutant low-grade gliomas without temozolomide treatment is actually under-studied. We retrieved retrospectively from our hospitals paired tumors from 19 patients with IDH-mutant, 1p19q non-codeleted Grade 2 astrocytomas where no interim adjuvant treatment with either temozolomide or radiotherapy was given between primary resections and first recurrences. Tissues from multiple recurrences were available from two patients and radiotherapy but not temozolomide was given before the last specimens were resected. We studied the natural molecular history of these low-grade IDH-mutant astrocytomas without pressure of temozolomide with DNA methylation profiling and copy number variation (CNV) analyses, targeted DNA sequencing, TERTp sequencing, FISH for ALT and selected biomarkers. Recurrences were mostly higher grades (15/19 patients) and characterized by new CNVs not present in the primary tumors (17/19 cases). Few novel mutations were identified in recurrences. Tumors from 17/19 (89.5%) patients showed either CDKN2A homozygous deletion, MYC or PDGFRA focal and non-focal gains at recurrences. There was no case of hypermutation. Phylogenetic trees constructed for tumors for the two patients with multiple recurrences suggested a lack of subclone development in their evolution when under no pressure from temozolomide. In summary, our studies demonstrated, in contrast to the phenomenon of temozolomide-induced hypermutation, IDH-mutant, 1p19q non-codeleted Grade 2 astrocytomas which had not been treated by temozolomide, acquired new CNVs at tumor recurrences. These findings improve our understanding of the molecular life history of IDH-mutant astrocytomas.

Multi-omics PGT: re-evaluation of euploid blastocysts for implantation potential based on RNA sequencing.

In addition to chromosomal euploidy, can the transcriptome of blastocysts be used as a novel predictor of embryo implantation potential? This retrospective analysis showed that based on differentially expressed genes (DEGs) between euploid blastocysts which resulted and did not result in a clinical pregnancy, machine learning models could help improve implantation rates by blastocyst optimization. Embryo implantation is a multifaceted process, with implantation loss and pregnancy failure related not only to blastocyst euploidy but also to the intricate dialog between blastocyst and endometrium. Although in vitro studies have revealed the characteristics of trophectoderm (TE) differentiation in implanted blastocysts and the function of TE placentation at the implantation site, the precise molecular mechanisms of embryo implantation and their clinical application remain to be fully elucidated. This study involved 102 patients who underwent 111 cycles for preimplantation genetic testing for aneuploidies (PGT-A) between March 2022 and July 2023. The study included 412 blastocysts biopsied at Day 5 [D5] or Day 6 [D6] for patients who underwent PGT-A. The biopsy lysates were split and subjected to DNA and RNA sequencing (DNA- and RNA-seq). One part was used for PGT-A to detect DNA copy number variations, whereas the other part was assessed simultaneously by RNA-seq to determine the transcriptome characteristics. To validate the reliability and accuracy of RNA-seq obtained from this strategy, we initially analyzed the transcriptome of blastocysts with chromosomal aneuploidies. Subsequently, we compared the transcriptomic features of blastocysts with different rates of formation (D5 vs D6) and investigated the network of interactions between key blastulation genes and the receptive endometrium. Then to evaluate the implantation potential of euploid blastocysts, we identified DEGs between euploid blastocysts that resulted in clinical pregnancy (defined as the presence of a gestational sac detected by ultrasound after 5 weeks) and those that did not. These DEGs were then employed to construct a predictive model for optimizing blastocyst selection. The successful detection rate of PGT-A was remarkably high at 99.8%. The RNA data may infer aneuploidy for both trisomy and monosomy. Between the euploid blastocysts that formed on D5 and D6, 187 DEGs were predominantly involved in cell differentiation for embryonic placenta development, the PPAR signaling pathway, and the Notch signaling pathway. These D5/D6 DEGs also exhibited a functional dialog with the receptive phase endometrium-specific genes through protein-protein interaction networks, indicating that the embryo undergoes further differentiation for post-implantation development. Furthermore, a modeling strategy using 280 DEGs between blastocysts leading to successful clinical pregnancies or failing to produce clinical pregnancies was implemented to refine the euploid embryo optimization, achieving areas under the curves of 0.88, 0.71, and 0.84 for the random forest (RF), support vector machine, and linear discriminant analysis models, respectively. Finally, a retrospective analysis of 83 transferred euploid blastocysts using the RF model identified three types of euploid embryos with a decreasing trend in implantation potential. Notably, the implantation rate of the good group was significantly higher than that of the moderate group (88.6% vs 50.0% P = 0.001) and that of the moderate group was higher than that of the poor group (50.0% vs 20.8%, P = 0.035). The sample size was insufficient; thus, a prospective study is needed to verify the clinical effectiveness of the above model. Because we did not analyze blastocysts that led only to biochemical pregnancies but failed clinical pregnancies separately, our classification system still must be modified to screen these embryos. Transcriptomic analysis of blastocysts offers a novel approach for predicting embryo implantation potential, which can be utilized to optimize clinical embryo selection. The ranking system may be effective in reducing the times and costs involved in achieving a clinical pregnancy. This study was funded by the "Pioneer" and "Leading Goose" R&D Program of Zhejiang (No. 2023C03034), the National Natural Science Foundation of China (82101709), and the National Key Research and Development Program for Young Scientists of China (No. 2022YFC2702300). The authors state no competing interests. N/A.

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.

Uterine mesenchymal tumours harboring the KAT6B/A::KANSL1 gene fusion represent a distinct type of uterine sarcoma based on DNA methylation profiles

Uterine mesenchymal tumours harboring KAT6B/A::KANSL1 gene fusions typically exhibit histological and immunophenotypic overlap with endometrial stromal and smooth muscle tumours. To date it remains uncertain whether such neoplasms should be regarded as variants of smooth muscle or endometrial stromal neoplasm, or if they constitute a distinct tumour type. In this study we investigated DNA methylation patterns and copy number variations (CNVs) in a series of uterine tumours harboring KAT6B/A::KANSL1 gene fusions in comparison to other mesenchymal neoplasms of the gynecological tract. Unsupervised hierarchical clustering and t-SNE analysis of DNA methylation data (Illumina EPIC array) identified a distinct cluster for 8/13 KAT6B/A::KANSL1 tumours (herein referred to as core cluster). The other 5 tumours (herein referred to as outliers) did not assign to the core cluster but clustered near various other tumour types. CNV analysis did not identify significant alterations in the core cluster. In contrast, various alterations, including deletions at the CDKN2A/B and NF1 loci were identified in the outlier group. Analysis of the DNA methylation clusters in relation to histological features revealed that while tumours in the core KAT6B/A::KANSL1 cluster were histologically bland, outlier tumours frequently exhibited “high-grade” histologic features in the form of significant nuclear atypia, increased mitotic activity and necrosis. Three of the five patients with outlier tumours died from their disease while clinical follow-up in the remaining two patients was limited (less than 12 months). In comparison, none of the 7 out of 8 patients with tumors in the core KAT6B/A::KANSL1 sarcoma cluster, where follow-up was available, died from disease. Furthermore, only 1 out of 7 patients recurred (mean follow-up of 30 months). In conclusion, KAT6B/A::KANSL1 uterine sarcoma is a molecularly unique type of uterine tumour that should be recognized as a distinct entity. These tumors typically exhibit low-grade histologic features but are occasionally morphologically high-grade; the latter have a DNA methylation profile different from the typical low-grade neoplasms and may be associated with aggressive behaviour.

CNVs in Patients with Neurodevelopmental Disorders: Meta-Analysis

This review is devoted to a comprehensive analysis of DNA copy number variations (CNVs) identified in patients with neurodevelopmental disorders (NDDs) from the literature. The selection of publications was conducted using specifically developed criteria. CNVs were characterized based on their clinical significance, type of copy number alteration (microdeletion/microduplication), size, origin, and gene content. The study sample comprised 3,375 patients with NDDs, among whom pathogenic and likely pathogenic CNVs, as well as variants of uncertain clinical significance, were identified in 395 individuals (12%). Chromosomal variations from each category were identified in 89 (3%), 56 (2%), and 241 (7%) patients, respectively. Nine individuals exhibited combinations of CNVs with varying clinical significance. The number of microduplications slightly exceeded the number of deletions (250 and 204, respectively). The size of most CNVs ranged from 193 bp to 400 kb and from 1 to 3 Mb (237 and 96, respectively). Seventy-two variants originated de novo, while 165 were inherited. Eighty-six CNVs were associated with 33 known microdeletion/microduplication syndromes. The most prevalent syndromes included 1q21.1 microduplication (7/395, 1.8%) (OMIM: 612475), 2p16.3 microdeletion (9/395, 2.3%) (OMIM: 614332), 15q13.3 microdeletion (7/395, 1.8%) (OMIM: 612001), 16p11.2 microdeletion (9/395, 2.3%) (OMIM: 611913), and 22q11.2 microduplications (7/395, 1.8%) (OMIM: 608363). Enrichment analysis revealed that pathogenic CNVs, as well as variants of uncertain clinical significance, are enriched in genes associated with abnormal behavioral/neurological phenotypes. Likely pathogenic CNVs included genes linked to disorders of the nervous system and homeostasis/metabolism.

Chromosomal alteration patterns in PitNETs: massive losses in aggressive tumors.

The molecular biology of pituitary neuroendocrine tumors (PitNETs) revealed few recurrent mutations and extensive chromosomal alterations, with the latter being the driving force in a subset of these lesions. Addressing the need for an easily applicable diagnostic tool, we conducted a retrospective study of 61 PitNETs operated at a tertiary care center. All cases were subtyped according to the 2022 WHO Classification of Endocrine Tumors. A genome-wide next-generation sequencing panel targeting 1500 single nucleotide polymorphisms (SNPs) was used to classify chromosomal imbalances, loss of heterozygosity, and copy number variations in DNA from formalin-fixed paraffin-embedded tissues. We identified four distinct chromosomal patterns, with varying distribution among different tumor lineages. Forty-two of 61 (69%) PitNETs showed chromosomal alterations. Gonadotroph PitNETs showed mostly quiet genomes. The majority of lactotroph PitNETs (19/20, 95%) were altered, exhibiting a gained genome and a remarkably low recurrence rate. Nine of ten (90%) corticotroph PitNETs harbored chromosomal alterations, of which two aggressive corticotroph tumors and one metastatic corticotroph PitNET showed massive chromosomal losses, leading to near-haploid/near-homozygous genomes. The comparison of the molecular profile of primary and recurrent PitNETs of five patients showed no significant accumulation of alterations over time. A simple genome-wide 1500-SNP test can be used in the identification of outspoken aggressive subsets of PitNETs by the occurrence of a near-haploid/near-homozygous genome. Furthermore, the presence of neoplastic tissue in the resected material can be potentially confirmed for non-gonadotroph PitNETs under suboptimal histological assessment conditions.

Odontogenic Myxomas Harbor Recurrent Copy Number Alterations and a Distinct Methylation Signature.

Odontogenic myxoma is a rare, benign, and locally aggressive tumor that develops in the tooth-bearing areas of the jaw. The molecular mechanisms underlying odontogenic myxomas are unknown and no diagnostic markers are available to date. The aim of this study was to analyze DNA methylation and copy number variations in odontogenic myxomas to identify new molecular signatures for diagnostic decision-making. We collected a cohort of 16 odontogenic myxomas from 2006 to 2021 located in the mandible (n = 10) and maxilla (n = 6) with available formalin-fixed paraffin-embedded or fresh frozen tumor tissue from a biopsy or resection material. Genome-wide DNA methylation and copy number variation data were generated from 12 odontogenic myxomas using the Illumina Infinium Methylation EPIC array, interrogating >850,000 CpG sites. Unsupervised clustering and dimensionality reduction (Uniform Manifold Approximation and Projection) revealed that odontogenic myxomas formed a distinct DNA methylation class. Copy number profiling showed recurrent whole-chromosome gains (trisomies) of chromosomes 5, 8, and 20 in all cases, and of chromosomes 10, 12, and 17 in all except one case. In conclusion, odontogenic myxomas harbor recurrent copy number patterns and a distinct DNA methylation profile, which can be used as an additional diagnostic tool in the appropriate clinical and radiologic context. Further research is needed to explain the genetic mechanisms caused by these alterations that drive these locally aggressive neoplasms.

Potential tumor-specific antigens and immune landscapes identification for mRNA vaccine in thyroid cancer.

Tumor mRNA vaccines have been identified as a promising technology for cancer therapy in multiple cancer types, while their efficacy in thyroid cancer (THCA) is unclear. Immunotyping is strongly associated with the immune microenvironment and immune status in cancer, thus it is important in vaccination and therapeutic response. This study is to identify potential valuable antigens and novel immune subtypes of THCA for immune landscape construction, thus screening patients suitable for mRNA vaccination. The clinical information and gene expression files of 568 THCA cases were obtained from the TCGA dataset. The DNA copy number variation and the somatic mutation of THCA were visualized by the cBioPortal database. TIMER was used to investigate the immune infiltrating correlation with candidate antigens. Consensus clustering analysis was conducted to cluster data using the ConsensusClusterPlus package. The immune landscapes of THCA patients were visualized using the Monocle package. The critical hub genes for THCA mRNA vaccines were identified by WGCNA package. To validate, the immunohistochemistry and real-time quantitative PCR (RT-qPCR) were performed to detect the expression level of potential antigen for mRNA vaccine in tissue and cell lines in THCA. Thymidine kinase 1 (TK1) was identified as a potential biomarker of mRNA vaccine against THCA. It was confirmed to be significantly upregulated in THCA tissues and cells lines. Moreover, three novel immune subtypes of THCA were obtained based on the expression consistency of immune-associated genes. The S2 subtype was characterized as an immunological "cold" phenotype with a high expression of immunogenic cell death modulators. S1 and S3 subtypes were immunological "hot" phenotypes with immune checkpoints upregulation. Further, the immune landscape of THCA patients was visualized and ten hub genes for mRNA vaccines were identified. TK1 was a tumor-specific antigen of mRNA vaccines. The patients belonging to the S2 subtype ("cold" tumor) were suitable for mRNA vaccine therapy in THCA. Notably, ten hub genes were conducted as potential biomarkers for identifying suitable patients for mRNA vaccination. These findings provided novel insights into mRNA vaccine development against THCA.

Mechanistic target of rapamycin (mTOR) pathway in Sertoli cells regulates age-dependent changes in sperm DNA methylation

Over the past several decades, a trend toward delayed childbirth has led to increases in parental age at the time of conception. Sperm epigenome undergoes age-dependent changes increasing risks of adverse conditions in offspring conceived by fathers of advanced age. The mechanism(s) linking paternal age with epigenetic changes in sperm remain unknown. The sperm epigenome is shaped in a compartment protected by the blood-testes barrier (BTB) known to deteriorate with age. Permeability of the BTB is regulated by the balance of two mTOR complexes in Sertoli cells where mTOR complex 1 (mTORC1) promotes the opening of the BTB and mTOR complex 2 (mTORC2) promotes its integrity. We hypothesized that this balance is also responsible for age-dependent changes in the sperm epigenome. To test this hypothesis, we analyzed reproductive outcomes, including sperm DNA methylation in transgenic mice with Sertoli cell-specific suppression of mTORC1 (Rptor KO) or mTORC2 (Rictor KO). mTORC2 suppression accelerated aging of the sperm DNA methylome and resulted in a reproductive phenotype concordant with older age, including decreased testes weight and sperm counts, and increased percent of morphologically abnormal spermatozoa and mitochondrial DNA copy number. Suppression of mTORC1 resulted in the shift of DNA methylome in sperm opposite to the shift associated with physiological aging – sperm DNA methylome rejuvenation and mild changes in sperm parameters. These results demonstrate for the first time that the balance of mTOR complexes in Sertoli cells regulates the rate of sperm epigenetic aging. Thus, mTOR pathway in Sertoli cells may be used as a novel target of therapeutic interventions to rejuvenate the sperm epigenome in advanced-age fathers.

Mechanistic target of rapamycin (mTOR) pathway in Sertoli cells regulates age-dependent changes in sperm DNA methylation.

Over the past several decades, a trend toward delayed childbirth has led to increases in parental age at the time of conception. Sperm epigenome undergoes age-dependent changes increasing risks of adverse conditions in offspring conceived by fathers of advanced age. The mechanism(s) linking paternal age with epigenetic changes in sperm remain unknown. The sperm epigenome is shaped in a compartment protected by the blood-testes barrier (BTB) known to deteriorate with age. Permeability of the BTB is regulated by the balance of two mTOR complexes in Sertoli cells where mTOR complex 1 (mTORC1) promotes the opening of the BTB and mTOR complex 2 (mTORC2) promotes its integrity. We hypothesized that this balance is also responsible for age-dependent changes in the sperm epigenome. To test this hypothesis, we analyzed reproductive outcomes, including sperm DNA methylation in transgenic mice with Sertoli cell-specific suppression of mTORC1 (Rptor KO) or mTORC2 (Rictor KO). mTORC2 suppression accelerated aging of the sperm DNA methylome and resulted in a reproductive phenotype concordant with older age, including decreased testes weight and sperm counts, and increased percent of morphologically abnormal spermatozoa and mitochondrial DNA copy number. Suppression of mTORC1 resulted in the shift of DNA methylome in sperm opposite to the shift associated with physiological aging - sperm DNA methylome rejuvenation and mild changes in sperm parameters. These results demonstrate for the first time that the balance of mTOR complexes in Sertoli cells regulates the rate of sperm epigenetic aging. Thus, mTOR pathway in Sertoli cells may be used as a novel target of therapeutic interventions to rejuvenate the sperm epigenome in advanced-age fathers.

The effect of the nucleolar protein ZNF385A on the ribosomal DNA copy number variation in response to Cr(VI)-induced DNA damage

Hexavalent chromium [Cr(VI)] is a widely distributed carcinogen in industrial contexts and general environmental contexts. Emerging research highlights the central role of ribosomal DNA (rDNA) in DNA Damage Responses (DDRs). However, there remains a lack of investigation into the potential dose-dependent relationship between exposure to Cr(VI) and alterations in rDNA copy number (CN), as well as the related mechanisms underlying these effects. A molecular epidemiological investigation involving 67 workers exposed to Cr(VI) and 75 unexposed controls was conducted. There was a notable increase in ZNF385A expression, variations in rDNA CN, and elevated γH2AX levels in the peripheral blood of Cr(VI)-exposed workers. Restricted cubic spline (RCS) models showed that blood Cr levels in the exposed population exhibited non-linear dose-dependent relationships with γH2AX, rDNA CN, and ZNF385A. Of considerable interest, there were robust and positive associations between ZNF385A and both γH2AX and rDNA CN. Further in vitro experiments provided concrete evidence that Cr(VI) simultaneously caused an increase in ZNF385A expression and variations in rDNA CN. ZNF385A-depleted cells showed increased sensitivity to Cr(VI)-mediated DDRs and alterations in rDNA CN. This study indicated that ZNF385A played a highly significant role in the rDNA CN variation in response to Cr(VI)-induced DNA damage.

Differential Cellular Response to Mercury in Non-Farmed Fish Species Based on Mitochondrial DNA Copy Number Variation Analysis.

Mercury (Hg) pro-oxidant role on biological systems and its biogeochemical cycle represent a serious threat due to its persistence in marine environment. As the mitochondrial genome is exposed to reactive oxygen species (ROS), the aim of the present study is the validation of the variation in the number of mitochondrial DNA copies (mtDNAcn) as biomarker of oxidative stress in aquatic environment. During summer 2021, three selected fish species (Mullus barbatus, Diplodus annularis and Pagellus erythrinus) were collected in Augusta Bay, one of the most Mediterranean contaminated areas remarkable by past Hg inputs, and in a control area, both in the south-east of Sicily. The relative mtDNAcn was evaluated by qPCR on specimens of each species from both sites, characterized respectively by higher and lower Hg bioaccumulation. M. barbatus and P. erythrinus collected in Augusta showed a dramatic mtDNAcn reduction compared to their control groups while D. annularis showed an incredible mtDNAcn rising suggesting a higher resilience of this species. These results align with the mitochondrial dynamics of fission and fusion triggered by environmental toxicants. In conclusion, we suggest the implementation of the mtDNAcn variation as a valid tool for the early warning stress-related impacts in aquatic system.

Association between mitochondrial DNA copy number and neurodevelopmental outcomes among black and white preterm infants up to two years of age.

Mitochondrial DNA copy number (mtDNAcn) is associated with mitochondrial function, with abnormal copy numbers having been linked to various disease states. Our study aims to understand the association between infant mtDNAcn and infant neurodevelopment, as well as the association with racial disparities. A longitudinal study was conducted with 55 preterm infants from whom a single blood sample was collected during their Neonatal intensive care unit (NICU) stay and used to analyze mtDNAcn. In addition, the NICU Network Neurobehavioral Scale at 36-38 postmenstrual age (PMA) and the Bayley Scale of Infant and Toddler Development (Bayley) Edition III at 1 and 2 years of corrected age were both conducted. Linear regression models were performed to investigate the relationship between infant clinical characteristics, infant neurobehavioral outcomes, and mtDNAcn. The majority of infants studied were white (72.73%), non-Hispanic (70.91%), males (54.55%), delivered through C-section (72.73%), and without preterm premature rupture of membrane (76.36%). Increased mtDNAcn was associated with younger birth gestational age (<30.57 wk, P < 0.001). In addition, the opposite associations between mtDNAcn and neurodevelopmental outcomes were observed between white and black infants up to 1 year of gestational age. Increased mtDNAcn in white infants, and decreased mtDNAcn in black infants may be considered significant predictors of poor early-life neurodevelopmental outcomes in infants. A better understanding of the underlying mechanisms contributing to infant disparity in mtDNAcn and how low or high copy number impacts infant outcomes is essential.