Differences In Copy Number Research Articles

Pathogenic and transcriptomic differences of emerging SARS-CoV-2 variants in the Syrian golden hamster model.

BackgroundFollowing the discovery of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid spread throughout the world, new viral variants of concern (VOC) have emerged. There is a critical need to understand the impact of the emerging variants on host response and disease dynamics to facilitate the development of vaccines and therapeutics.MethodsSyrian golden hamsters are the leading small animal model that recapitulates key aspects of severe coronavirus disease 2019 (COVID-19). We performed intranasal inoculation of SARS-CoV-2 into hamsters with the ancestral virus (nCoV-WA1-2020) or VOC first identified in the United Kingdom (B.1.1.7, alpha) and South Africa (B.1.351, beta) and analyzed viral loads and host responses.FindingsSimilar gross and histopathologic pulmonary lesions were observed after infection with all three variants. Although differences in viral genomic copy numbers were noted in the lungs and oral swabs of challenged animals, infectious titers in the lungs were comparable between the variants. Antibody neutralization capacities varied, dependent on the original challenge virus and cross-variant protective capacity. Transcriptional profiling of lung samples 4 days post-challenge (DPC) indicated significant induction of antiviral pathways in response to all three challenges with a more robust inflammatory signature in response to B.1.1.7 infection. Furthermore, no additional mutations in the spike protein were detected at 4 DPC.InterpretationsAlthough disease severity and viral shedding were not significantly different, the emerging VOC induced distinct humoral responses and transcriptional profiles compared to the ancestral virus. These observations suggest potential differences in acute early responses or alterations in immune modulation by VOC.FundingIntramural Research Program, NIAID, NIH; National Center for Research Resources, NIH; National Center for Advancing Translational Sciences, NIH.

The Association of EBV and HHV-6 Viral Load with Different NK and CD8+ T Cell Subsets in The Acute Phase of Relapsing-Remitting Multiple Sclerosis.

Objective Epstein-Barr virus (EBV) and Human Herpes virus 6 (HHV-6) are believed to involve in multiple sclerosis (MS) pathogenesis. Natural killer (NK) and CD8+ T cells have essential roles in handling viral infections and their phenotypic and functional properties may be influenced following exposure to viral infections. Here, we investigated the association of NK and CD8+ T cells subpopulations frequency with EBV and HHV-6 viral load in MS patients. Materials and Methods In this case-control study, EBV and HHV-6 viral load were evaluated in plasma of newly diagnosed relapsing-remitting MS (RRMS) patients at relapse phase (n=23), who were not on disease-modifying therapy (DMT), and sex- and age-matched healthy controls (n=19) using real-time polymerase chain reaction (PCR). The frequency of NK and CD8+ T cells subsets were assessed by CD27, CD28, CD45RO, CD56, and CD57 markers using flow cytometry. Results Despite the increased level of EBV viral load in RRMS patients compared to the control group, there was no statistically significant difference in EBV and HHV-6 copy numbers between the studied groups. In addition, a significant decrease was observed in the percentages of CD56brightCD57- and CD56dimCD57+CD8lowCD45RO- NK cells in RRMS patients in comparison to healthy controls. Analysis of CD8+ T cell subsets showed a substantially high proportion of CD27+CD28+CD45RO+CD57-CD8hiT cells in patients at relapse phase compared to controls. The frequency of NK and T cells subtypes was not associated with EBV and HHV6 plasma viral loads. ConclusionThese findings further highlight the variation of NK and CD8+ T cells subsets frequency in clinically active RRMS patients. Since the composition of cells was not associated with EBV and HHV-6 viral load, perhaps other viral infections may be involved in altered NK and CD8+ T cells subpopulation. Larger cohort studies are needed to confirm these results.

Integrative epigenomic and high-throughput functional enhancer profiling reveals determinants of enhancer heterogeneity in gastric cancer

BackgroundEnhancers are distal cis-regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity—however, most predicted enhancer regions remain to be functionally tested.MethodsWe analyzed 132 epigenomic histone modification profiles of 18 primary gastric cancer (GC) samples, 18 normal gastric tissues, and 28 GC cell lines using Nano-ChIP-seq technology. We applied Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to assess functional enhancer activity. An Activity-by-contact (ABC) model was employed to explore the effects of histone acetylation and CapSTARR-seq levels on enhancer-promoter interactions.ResultsWe report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are GC-associated in vivo (> 50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying CapSTARR-seq to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers, even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans-acting TFs involved in GC expression. We identified cancer-relevant genes (ING1, ARL4C) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4α as a master trans-acting factor associated with GC enhancer heterogeneity.ConclusionsOur results indicate that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, providing insights into the relative contribution of genetic (cis) and regulatory (trans) mechanisms to GC enhancer functional heterogeneity.

Correspondence of aCGH and long-read genome assembly for detection of copy number differences: A proof-of-concept with cichlid genomes.

Copy number variation is an important source of genetic variation, yet data are often lacking due to technical limitations for detection given the current genome assemblies. Our goal is to demonstrate the extent to which an array-based platform (aCGH) can identify genomic loci that are collapsed in genome assemblies that were built with short-read technology. Taking advantage of two cichlid species for which genome assemblies based on Illumina and PacBio are available, we show that inter-species aCGH log2 hybridization ratios correlate more strongly with inferred copy number differences based on PacBio-built genome assemblies than based on Illumina-built genome assemblies. With regard to inter-species copy number differences of specific genes identified by each platform, the set identified by aCGH intersects to a greater extent with the set identified by PacBio than with the set identified by Illumina. Gene function, according to Gene Ontology analysis, did not substantially differ among platforms, and platforms converged on functions associated with adaptive phenotypes. The results of the current study further demonstrate that aCGH is an effective platform for identifying copy number variable sequences, particularly those collapsed in short read genome assemblies.

Mitochondrial DNA Copy Number in Rett Syndrome Caused by Methyl-CpG-Binding Protein-2 Variants

To determine changes in mitochondrial DNA (mtDNA) copy number in peripheral blood in Rett syndrome caused by methyl-CpG-binding protein-2 (MECP2) variants and explore the mechanism of mitochondrial dysfunction in Rett syndrome. Female patients who were diagnosed with Rett syndrome and had an MECP2 variant (n=142) were recruited in this study, along with the same number of age- and sex-matched healthy controls. MtDNA copy number was quantified by real-time quantitative polymerase chain reaction with TaqMan probes. The differences in mtDNA copy number between the Rett syndrome group and the control group were analyzed using the independent-samples t test. Linear regression, biserial correlation analysis, and one-way ANOVA were applied for the correlations between mtDNA copy number and age, clinical severity, variant types, functional domains, and hot-spot variants. MtDNA copy number was found to be significantly increased in the patients with Rett syndrome with MECP2 gene variants compared with the control subjects. Age, clinical severity, variant types, functional domains, and hot-spot variants were not related to mtDNA copy number in patients with Rett syndrome. MtDNA copy number is increased significantly in patients with Rett syndrome, suggesting that changes in mitochondrial function in Rett syndrome trigger a compensatory increase in mtDNA copy number and providing new possibilities for treating Rett syndrome, such as mitochondria-targeted therapies.

Soil nitrification response to dairy digestate and inorganic ammonium sources depends on soil pH and nitrifier abundances

AbstractTo reduce CH4 emissions, some dairy operations use manure as feedstock for anaerobic digesters, after which the nonvolatilized portion forms a digestate effluent with high NH4+ content that is used as fertilizer. When applied to soil, N fertilizers are subject to nitrification, where NH3–oxidizing archaea (AOA) and bacteria (AOB) oxidize NH3 to NO2–, NO2––oxidizing bacteria (NOB) oxidize NO2– to NO3−, and N oxides are produced from biotic and abiotic reactions. This study used soil microcosms to evaluate the properties of nitrification in response to the addition of N as digestate vs. NH4Cl in soils from the different climatic regions of the western Oregon coastal plain (CP) and eastern Oregon Columbia River basin (CRB). In each soil, properties of NH3 oxidation or N oxide accumulation did not differ when N was provided via digestate or NH4Cl over the normal temperature range; however, nitrification rates, communities, and products differed between soils. The average nitrification rate in CP soil was almost twofold higher than in CRB soil (P ≤ .001), despite no significant differences in AOB activity or AOB amoA copy numbers. With N addition, AOA activity also increased in CP soil, where AOA amoA copy numbers were fivefold higher than CRB soil (P < .002). Accumulation of NO2– only in CRB soil was likely due to NOB inhibition by high NH3 concentrations (∼560 μM) present at the high pH of CRB soil (8.5). Overall, nitrification was dictated by differences in edaphic properties, temperature, and nitrifier communities more than N source.

Increased α-tocopherol metabolism in horses with equine neuroaxonal dystrophy.

BackgroundEquine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with a vitamin E deficiency within the first year of life. Vitamin E consists of 8 isoforms metabolized by the CYP4F2 enzyme. No antemortem diagnostic test currently exists for eNAD/EDM.Hypothesis/ObjectivesBased on the association of α‐tocopherol deficiency with the development of eNAD/EDM, we hypothesized that the rate of α‐tocopherol, but not γ‐tocopherol or tocotrienol metabolism, would be increased in eNAD/EDM‐affected horses.AnimalsVitamin E metabolism: Proof of concept (POC) study; eNAD/EDM‐affected (n = 5) and control (n = 6) horses. Validation study: eNAD/EDM‐affected Quarter Horses (QHs; n = 6), cervical vertebral compressive myelopathy affected (n = 6) horses and control (n = 29) horses. CYP4F2 expression and copy number: eNAD/EDM‐affected (n = 12) and age‐ and sex‐matched control (n = 11‐12) horses.MethodsThe rates of α‐tocopherol/tocotrienol and γ‐tocopherol/tocotrienol metabolism were assessed in equine serum (POC and validation) and urine (POC only) using liquid chromatography tandem mass spectrometry (LC‐MS/MS). Quantitative reverse‐transcriptase PCR (qRT‐PCR) and droplet digital (dd)‐PCR were used to assay expression and genomic copy number of a CYP4F2 equine ortholog.ResultsMetabolic rate of α‐tocopherol was increased in eNAD/EDM horses (POC,P < .0001; validation, P = .03), with no difference in the metabolic rate of γ‐tocopherol. Horses with eNAD/EDM had increased expression of the CYP4F2 equine orthologue (P = .02) but no differences in copy number.Conclusions and Clinical ImportanceIncreased α‐tocopherol metabolism in eNAD/EDM‐affected QHs provides novel insight into alterations in vitamin E processing in eNAD/EDM and highlights the need for high‐dose supplementation to prevent the clinical phenotype in genetically susceptible horses.

Natural copy number variation of tandemly repeated regulatory SNORD RNAs leads to individual phenotypic differences in mice.

Genic copy number differences can have phenotypic consequences, but so far this has not been studied in detail in natural populations. Here, we analysed the natural variation of two families of tandemly repeated regulatory small nucleolar RNAs (SNORD115 and SNORD116) in the house mouse (Mus musculus). They are encoded within the Prader-Willi Syndrome gene region, known to be involved in behavioural, metabolic, and osteogenic functions in mammals. We determined that the copy numbers of these SNORD RNAs show substantial natural variation, both in wild-derived mice as well as in an inbred mouse strain (C57BL/6J). We show that copy number differences are subject to change across generations, making them highly variable and resulting in individual differences. In transcriptome data from brain samples, we found SNORD copy-number correlated regulation of possible target genes, including Htr2c, a predicted target gene of SNORD115, as well as Ankrd11, a predicted target gene of SNORD116. Ankrd11 is a chromatin regulator, which has previously been implicated in regulating the development of the skull. Based on morphometric shape analysis of the skulls of individual mice of the inbred strain, we show that shape measures correlate with SNORD116 copy numbers in the respective individuals. Our results suggest that the variable dosage of regulatory RNAs can lead to phenotypic variation between individuals that would typically have been ascribed to environmentally induced variation, while it is actually encoded in individual differences of copy numbers of regulatory molecules.

Draft Genome Assembly and Transcriptome Dataset for European Turnip (Brassica rapa L. ssp. rapifera), ECD4 Carrying Clubroot Resistance.

Draft Genome Assembly and Transcriptome Dataset for European Turnip (Brassica rapa L. ssp. rapifera), ECD4 Carrying Clubroot Resistance.

Strong correlation between urine and vaginal swab samples for bacterial vaginosis.

BackgroundVaginal swabs have been traditionally used for the diagnosis of bacterial vaginosis (BV). Currently, there are limited studies that have investigated the use of other sample types other than vaginal swabs for the detection of BV from South African populations. This study investigated whether urine can be used for the detection of BV-associated microorganisms in South African pregnant women.MethodsOne-hundred self-collected vaginal swabs and urine samples were obtained from women presenting for antenatal care at King Edward VIII Hospital in Durban. The BD MAX™ vaginal panel assay was used for diagnosing BV and droplet digital polymerase chain reaction was used to quantify Gardnerella vaginalis, Prevotella bivia, Atopobium vaginae and Lactobacillus crispatus. The absolute counts were determined on the QX200 Droplet Reader (Bio-Rad) using the QuantaSoft Software. Data analysis was performed with statistical computing software called R, version 3.6.1.ResultsMedian copy numbers obtained for G. vaginalis and P. bivia across urine and swabs in BV-positive samples were not significantly different (p = 0.134 and p = 0.652, respectively). This was confirmed by the correlation analysis that showed a good correlation between the two sample types (G. vaginalis [r = 0.63] and P. bivia [r = 0.50]). However, the data obtained for A. vaginae differed, and a weak correlation between urine and swabs was observed (r = 0.21). Bacterial vaginosis-negative samples had no significant difference in median copy numbers for L. crispatus across the urine and swabs (p = 0.062), and a good correlation between the sample types was noted (r = 0.71).ConclusionThis study highlights the appropriateness of urine for the detection of microorganisms associated with BV.

Mutations in BRCA-related breast and ovarian cancer in the South African Indian population: A descriptive study

Knowledge of the genetic landscape of a specific population group is vital for population-specific diagnosis and treatment of familial breast cancer. Although BRCA-related diagnostic testing has long been implemented in South Africa, the genotyping approach previously failed for the SA Indian population as it was based on other SA population groups. Because this population is uniquely admixed, the lack of population-specific data resulted in the implementation of comprehensive mutation screens for BRCA1/2. A total of 223 female patients were screened for clinically actionable variants. High-resolution melting analysis (HRMA) was used to screen 88 patients for DNA alterations in the coding and splice site boundaries of BRCA1 exons 2–9, BRCA1 exons 11–23, BRCA2 exons 2–9 and BRCA2 exons 12–27. The protein truncation test (PTT) was used to screen the three larger exons (BRCA1 exon 10 and BRCA2 exons 10 and 11) for protein termination changes. Multiplex ligation-dependent probe amplification (MLPA) was used to determine the presence of larger indels and possible copy number differences. Next Generation Sequencing (NGS) was performed on the remaining 135 samples. All potential variants were confirmed by performing Sanger DNA sequencing. The search revealed 28 different pathogenic heterozygotic variants, together with nine variants of unknown significance (VUS). The results suggested that the SA Indian population represents a different genetic admixture compared to that of mainland India, as only five pathogenic variants corresponded to those reported for mainland India. Familial breast cancer testing for SA Indian patients should therefore be performed as comprehensively as possible as the pathogenic variants seem to be family- rather than population-specific. Furthermore, predictive testing of family members will contribute to relieve the financial burden on the country's healthcare system, as increased surveillance and appropriate management could prevent disease.

Differential Expression and Copy Number Variation of Gasdermin (GSDM) Family Members, Pore-Forming Proteins in Pyroptosis, in Normal and Malignant Serous Ovarian Tissue.

Gasdermins (GSDM) are members of a family of pore-forming effector proteins which lead to membrane permeabilization and pyroptosis, a lytic cell death with pro-inflammatory characteristics. Recently, two members of the gasdermin family, gasdermin B (GSDMB) and gasdermin E (GSDME), were shown to suppress tumor growth, through the involvement of cytotoxic lymphocytes. Other studies also reported the important functions of gasdermins in various cancer types including gastric cancer, hepatocarcinoma, and cervix and breast cancer. However, gasdermins have not been previously studied in the context of serous ovarian cancer. Here, we showed that gasdermin D (GSDMD) and gasdermin C (GSDMC) expression increases in serous ovarian cancer; in contrast, the expression of GSDME and PJVK (Pejvakin, DFNB59) is downregulated, compared to healthy ovaries, in multiple independent gene expression datasets. We found that copy number gains are highly frequent (present in approximately 50% of patients) in genes encoding GSDMD and GSDMC in ovarian cancer, in line with their upregulated expression in serous ovarian cancer. Moreover, we observed that the expression of GSDMB and GSDMD, but not of GSDME, is different among several histotypes of epithelial ovarian cancer. Therefore, we propose that differential expression and copy number variations of certain gasdermins might be associated with the development of serous ovarian cancer, in which different members of the family have distinct functions; however, further research is required in in vivo models to understand how changes in gasdermin family members mechanistically contribute to serous ovarian cancer.

Genetic instabilities as potential biomarkers for pregnancy-associated breast cancer.

e12559 Background: Pregnancy associated breast cancer (PABC), defined as breast cancer diagnosed during or within one year after pregnancy, occurs approximately once in every 3,000 pregnancies and accounts for up to 6.9% of all breast cancers in women ≤ 45 years. Compared to age-matched non-PABC patients, PABC is generally characterized by a particularly aggressive histopathologic profile (more ER-, PR- and HER2 negative tumors and tumors of higher grade) with a higher mortality rate. Whether these cancers arise before or during pregnancy, and whether their genetic profile differs from non-PABC tumors, is currently unknown. This study assesses the genetic background of PABC by detection of specific DNA copy number alterations (CNA). Methods: We assembled 29 triple-negative PABC patients from the Dutch nationwide pathology database (PALGA). From their formalin-fixed and paraffin embedded (FFPE) tumor tissue blocks, DNA was extracted. Multiplex Ligation-Dependent probe Amplification (MLPA) analysis was performed for detection of CNAs in 20 oncogenes and 2 tumor suppressor genes using the P078-D2 kit (MRC Holland). Individual gene copy number loss (MLPA ratio &lt; 0.7), gain (ratio 1.3-2.0) and amplification (ratio &gt; 2.0) was determined and unsupervised hierarchical cluster analysis was performed to identify differences in copy number (CN) patterns between PABC patients. Chi square statistics were used to interrogate associations with clinicopathologic characteristics. Log-Rank test was used to compare Kaplan Meier survival curves between CN subgroups. Results: Triple negative PABC tumors showed frequent copy number loss on chromosome 17q ( CPD, MED1, TOP2A, MAPT, PPM1D) and 8p ( ZNF703 and ADAM9), as well as frequent copy number gain/amplification of MYC (8q) and CCND1 (11q). Cluster analysis identified 2 clusters of PABC patients based on their tumor copy number patterns: a CN-neutral cluster and a CN-high cluster, the latter demonstrating significantly lower chromosome 17 copy numbers than the former (for PPM1D, ERBB2, CPD, MED1, TOP2A, CDC6 and MAPT). Patients with CN-neutral tumors more often showed lymph node metastases (p = 0.028), were of significantly higher stage (p = 0.031), and demonstrated a worse overall survival (p = 0.036) compared with PABC patients harboring CN-high tumors. Conclusions: We demonstrate different tumor CN patterns within a small group of triple negative PABC-patients, associated with distinct aggressiveness. This will form the basis for further in-depth genetic profiling within a larger Dutch PABC cohort, rendering insight in the disease progression and contributing to the development of more targeted and personalized treatments.

The Impaired Bioenergetics of Diabetic Cardiac Microvascular Endothelial Cells.

Diabetes causes hyperglycemia, which can create a stressful environment for cardiac microvascular endothelial cells (CMECs). To investigate the impact of diabetes on the cellular metabolism of CMECs, we assessed glycolysis by quantifying the extracellular acidification rate (ECAR), and mitochondrial oxidative phosphorylation (OXPHOS) by measuring cellular oxygen consumption rate (OCR), in isolated CMECs from wild-type (WT) hearts and diabetic hearts (db/db) using an extracellular flux analyzer. Diabetic CMECs exhibited a higher level of intracellular reactive oxygen species (ROS), and significantly reduced glycolytic reserve and non-glycolytic acidification, as compared to WT CMECs. In addition, OCR assay showed that diabetic CMECs had increased maximal respiration, and significantly reduced non-mitochondrial oxygen consumption and proton leak. Quantitative PCR (qPCR) showed no difference in copy number of mitochondrial DNA (mtDNA) between diabetic and WT CMECs. In addition, gene expression profiling analysis showed an overall decrease in the expression of essential genes related to β-oxidation (Sirt1, Acox1, Acox3, Hadha, and Hadhb), tricarboxylic acid cycle (TCA) (Idh-3a and Ogdh), and electron transport chain (ETC) (Sdhd and Uqcrq) in diabetic CMECs compared to WT CMECs. Western blot confirmed that the protein expression of Hadha, Acox1, and Uqcrq was decreased in diabetic CMECs. Although lectin staining demonstrated no significant difference in capillary density between the hearts of WT mice and db/db mice, diabetic CMECs showed a lower percentage of cell proliferation by Ki67 staining, and a higher percentage of cellular apoptosis by TUNEL staining, compared with WT CMECs. In conclusion, excessive ROS caused by hyperglycemia is associated with impaired glycolysis and mitochondrial function in diabetic CMECs, which in turn may reduce proliferation and promote CMEC apoptosis.

Tracking COVID-19 with wastewater to understand asymptomatic transmission

IntroductionSARS-CoV-2 RNA is excreted in feces of most patients, therefore viral load in wastewater can be used as a surveillance tool to develop an early warning system to help and manage future pandemics. MethodsWe collected wastewater from 24 random locations at Bangkok city center and 26 nearby suburbs from July to December 2020. SARS-CoV-2 RNA copy numbers were measured using real-time polymerase chain reaction (PCR). ResultsSARS-CoV-2 RNA was detected in wastewater from both the city center and suburbs. Except for July, there were no significant differences in copy numbers between the city center and suburbs. Between October and November, a sharp rise in copy number was observed in both places followed by two to three times increase in December, related to SARS-CoV-2 cases reported for same month. ConclusionsOur study provided the first dataset related to SARS-CoV-2 viral RNA in the wastewater of Bangkok. Our results suggest that wastewater could be used as a complementary source for detecting viral RNA and predicting upcoming outbreaks and waves.

Detailed Reanalysis of 500 Breast Cancers With Equivocal HER2 Immunohistochemistry and Borderline ERBB2 Fluorescence In Situ Hybridization Results.

We investigated the impact of our laboratory's reflex testing process for resolving ERBB2 (HER2) status on breast cancer samples that require additional workup after fluorescence in situ hybridization (FISH), per guideline recommendations published in 2018 by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP). In total, 500 breast cancer specimens with ERBB2 FISH results in groups 2 through 4 (all reported as immunohistochemistry [IHC] equivocal [2+] at external laboratories) were resubmitted for IHC testing in our laboratory. Per the ASCO/CAP guideline, FISH was rescored when internal IHC was also equivocal (2+), targeted to tumor areas demonstrating more intense IHC staining, if observed. Reflex IHC/FISH testing changed the final reported ERBB2 status in 185 of 500 (37.0%) samples. Result changes included discordant IHC (n = 4 score 0, n = 132 score 1+, and n = 16 score 3+) and discordant FISH (n = 33). Numerical differences in FISH scores were comparable for targeted vs nontargeted FISH rescoring (P = .086 for ERBB2 copy number; P = .49 for ERBB2 ratio). Two cases showed larger differences in FISH scores, suggesting heterogeneity. Retesting of breast cancer samples with equivocal IHC frequently changes IHC results, but targeted reanalysis of borderline FISH results rarely identifies significant differences in ERBB2 copy number or ratio.

Assessment of circulating microRNA specific for patients with familial adenomatous polyposis.

Circulating microRNAs (miRNAs) are considered promising biomarkers for diagnosis, prognosis, and treatment efficacy of diseases. However, usefulness of circulating miRNAs as biomarkers for hereditary gastrointestinal diseases have not been confirmed yet. We explored circulating miRNAs specific for patients with familial adenomatous polyposis (FAP) as a representative hereditary gastrointestinal disease. Next-generation sequencing (NGS) indicated that plasma miR-143-3p, miR-183-5p, and miR-885-5p were candidate biomarkers for five FAP patients compared to three healthy donors due to moderate copy number and significant difference. MiR-16-5p was considered as an internal control due to minimum difference in expression across FAP patients and healthy donors. Validation studies by real-time PCR showed that mean ratios of maximum expression and minimum expression were 2.2 for miR-143-3p/miR-16-5p, 3.4 for miR-143-3p/miR-103a-3p, 5.1 for miR-183-5p/miR-16-5p, and 4.9 for miR-885-5p/miR-16-5p by using the samples collected at different time points of eight FAP patients. MiR-143-3p/16-5p was further assessed using specimens from 16 FAP patients and 7 healthy donors. MiR-143-3p was upregulated in FAP patients compared to healthy donors (P = 0.04), but not significantly influenced by clinicopathological features. However, miR-143-3p expression in colonic tumors was rare for upregulation, although there was a significant difference by existence of desmoid tumors. MiR-143-3p transfection significantly inhibited colorectal cancer cell proliferation compared to control microRNA transfection. Our data suggested regulation of miR-143-3p expression differed by samples (plasma or colonic tumors) in most FAP patients. Upregulation of plasma miR-143-3p expression may be helpful for diagnosis of FAP, although suppressive effect on tumorigenesis seemed insufficient in FAP patients.

Differences in net fat oxidation, heat production, and liver mitochondrial DNA copy numbers between high and low feed-efficient dairy cows

Improving feed utilization efficiency in dairy cattle could have positive economic and environmental effects that would support the sustainability of the dairy industry. Identifying key differences in metabolism between high and low feed-efficient animals is vital to enhancing feed conversion efficiency. Therefore, our objectives were (1) to determine whether cows grouped by either high or low feed efficiency have measurable differences in net fat and carbohydrate metabolism that account for differences in heat production (HP), and if so, whether these differences also exists under conditions of feed withdrawal when the effect of feeding on HP is minimized, and (2) to determine whether the abundance of mitochondria in the liver can be related to the high or low feed-efficient groups. Ten dairy cows from a herd of 15 (parity = 2) were retrospectively grouped into either a high (H) or a low (L) feed-efficient group (n = 5 per group) based on weekly energy-corrected milk (ECM) divided by dry mater intake (DMI) from wk 4 through 30 of lactation. Livers were biopsied at wk -4, 2, and 12, and blood was sampled weekly from wk -3 to 12 relative to parturition. Blood was subset to be analyzed for the transition period (wk -3 to 3) and from wk 4 to 12. In wk 5.70 ± 0.82 (mean ± SD) postpartum (PP), cows spent 2 d in respiration chambers (RC), in which CO2, O2, and CH4 gases were measured every 6 min for 24 h. Fatty acid oxidation (FOX), carbohydrate oxidation (COX), metabolic respiratory quotient (RQ), and HP were calculated from gas measurements for 23 h. Cows were fed ad libitum (AD-LIB) on d 1 and had feed withdrawn (RES, restricted diet) on d 2. Additional blood samples were taken at the end of the AD-LIB and RES feeding periods in the RC. During wk 4 to 30 PP, H had greater DMI/kg of metabolic body weight (BW0.75), ECM per kilogram of BW0.75 yield, and ECM/DMI ratio, compared with L, but a lower body condition score between wk 4 and 12 PP. In the RC period, we detected no differences in BW, DMI, or milk yield between groups. We also detected no significant group or group by feeding period interactions for plasma metabolites except for Revised Quantitative Insulin Sensitivity Check Index, which tended to have a group by feeding period interaction. The H group had lower HP and HP per kilogram of BW0.75 compared with L. Additionally, H had lower FOX and FOX per kilogram of BW0.75 compared with L during the AD-LIB period. Methane, CH4 per kilogram of BW0.75, and CH4 per kilogram of milk yield were lower in H compared with L, but, when adjusted for DMI, CH4/DMI did not differ between groups, nor did HP/DMI. Relative mitochondrial DNA copy numbers in the liver were lower in the L than in the H group. These results suggest that lower feed efficiency in dairy cows may result from fewer mitochondria per liver cell as well as a greater whole-body HP, which likely partially results from higher net fat oxidation.

Luminal A Breast Cancer Co-expression Network: Structural and Functional Alterations.

Luminal A is the most common breast cancer molecular subtype in women worldwide. These tumors have characteristic yet heterogeneous alterations at the genomic and transcriptomic level. Gene co-expression networks (GCNs) have contributed to better characterize the cancerous phenotype. We have previously shown an imbalance in the proportion of intra-chromosomal (cis-) over inter-chromosomal (trans-) interactions when comparing cancer and healthy tissue GCNs. In particular, for breast cancer molecular subtypes (Luminal A included), the majority of high co-expression interactions connect gene-pairs in the same chromosome, a phenomenon that we have called loss of trans- co-expression. Despite this phenomenon has been described, the functional implication of this specific network topology has not been studied yet. To understand the biological role that communities of co-expressed genes may have, we constructed GCNs for healthy and Luminal A phenotypes. Network modules were obtained based on their connectivity patterns and they were classified according to their chromosomal homophily (proportion of cis-/trans- interactions). A functional overrepresentation analysis was performed on communities in both networks to observe the significantly enriched processes for each community. We also investigated possible mechanisms for which the loss of trans- co-expression emerges in cancer GCN. To this end we evaluated transcription factor binding sites, CTCF binding sites, differential gene expression and copy number alterations (CNAs) in the cancer GCN. We found that trans- communities in Luminal A present more significantly enriched categories than cis- ones. Processes, such as angiogenesis, cell proliferation, or cell adhesion were found in trans- modules. The differential expression analysis showed that FOXM1, CENPA, and CIITA transcription factors, exert a major regulatory role on their communities by regulating expression of their target genes in other chromosomes. Finally, identification of CNAs, displayed a high enrichment of deletion peaks in cis- communities. With this approach, we demonstrate that network topology determine, to at certain extent, the function in Luminal A breast cancer network. Furthermore, several mechanisms seem to be acting together to avoid trans- co-expression. Since this phenomenon has been observed in other cancer tissues, a remaining question is whether the loss of long distance co-expression is a novel hallmark of cancer.

Copy number variation underlies complex phenotypes in domestic dog breeds and other canids.

Extreme phenotypic diversity, a history of artificial selection, and socioeconomic value make domestic dog breeds a compelling subject for genomic research. Copy number variation (CNV) is known to account for a significant part of inter-individual genomic diversity in other systems. However, a comprehensive genome-wide study of structural variation as it relates to breed-specific phenotypes is lacking. We have generated whole genome CNV maps for more than 300 canids. Our data set extends the canine structural variation landscape to more than 100 dog breeds, including novel variants that cannot be assessed using microarray technologies. We have taken advantage of this data set to perform the first CNV-based genome-wide association study (GWAS) in canids. We identify 96 loci that display copy number differences across breeds, which are statistically associated with a previously compiled set of breed-specific morphometrics and disease susceptibilities. Among these, we highlight the discovery of a long-range interaction involving a CNV near MED13L and TBX3, which could influence breed standard height. Integration of the CNVs with chromatin interactions, long noncoding RNA expression, and single nucleotide variation highlights a subset of specific loci and genes with potential functional relevance and the prospect to explain trait variation between dog breeds.