DNA Copy Number Changes Research Articles

Childhood Asthma and Mitochondrial Biomarkers for Exposure-Related Outcomes (CAMERO) study: Time-related Changes in Mitochondrial DNA Copy Number

Air pollution and allergen exposure leads to reactive oxygen species production, causing DNA damage and mitochondrial metabolic activity changes. The Childhood Asthma and Mitochondrial Biomarkers for Exposure Related Outcomes (CAMERO) study hypothesizes that mitochondrial DNA (mtDNA) copy number and methylation patterns change in response to environmental exposures. We hypothesize that changes in biomarkers are associated with disease activity among asthmatic children.

Genomic Adaption and Mutational Patterns in a HaCaT Subline Resistant to Alkylating Agents and Ionizing Radiation.

Sulfur mustard (SM) is a chemical warfare agent that can damage DNA via alkylation and oxidative stress. Because of its genotoxicity, SM is cancerogenic and the progenitor of many chemotherapeutics. Previously, we developed an SM-resistant cell line via chronic exposure of the popular keratinocyte cell line HaCaT to increasing doses of SM over a period of 40 months. In this study, we compared the genomic landscape of the SM-resistant cell line HaCaT/SM to its sensitive parental line HaCaT in order to gain insights into genetic changes associated with continuous alkylation and oxidative stress. We established chromosome numbers by cytogenetics, analyzed DNA copy number changes by means of array Comparative Genomic Hybridization (array CGH), employed the genome-wide chromosome conformation capture technique Hi-C to detect chromosomal translocations, and derived mutational signatures by whole-genome sequencing. We observed that chronic SM exposure eliminated the initially prevailing hypotetraploid cell population in favor of a hyperdiploid one, which contrasts with previous observations that link polyploidization to increased tolerance and adaptability toward genotoxic stress. Furthermore, we observed an accumulation of chromosomal translocations, frequently flanked by DNA copy number changes, which indicates a high rate of DNA double-strand breaks and their misrepair. HaCaT/SM-specific single-nucleotide variants showed enrichment of C > A and T > A transversions and a lower rate of deaminated cytosines in the CpG dinucleotide context. Given the frequent use of HaCaT in toxicology, this study provides a valuable data source with respect to the original genotype of HaCaT and the mutational signatures associated with chronic alkylation and oxidative stress.

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions.

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.

Tos4 mediates gene expression homeostasis through interaction with HDAC complexes independently of H3K56 acetylation

Saccharomyces cerevisiae exhibits gene expression homeostasis, which is defined as the buffering of transcription levels against changes in DNA copy number during the S phase of the cell cycle. It has been suggested that S. cerevisiae employs an active mechanism to maintain gene expression homeostasis through Rtt109-Asf1-dependent acetylation of histone H3 on lysine 56 (H3K56). Here, we show that gene expression homeostasis can be achieved independently of H3K56 acetylation by Tos4 (Target of Swi6-4). Using Nanostring technology, we establish that Tos4-dependent gene expression homeostasis depends on its forkhead-associated (FHA) domain, which is a phosphopeptide recognition domain required to bind histone deacetylases (HDACs). We demonstrate that the mechanism of Tos4-dependent gene expression homeostasis requires its interaction with the Rpd3L HDAC complex. However, this is independent of Rpd3’s well-established roles in both histone deacetylation and controlling the DNA replication timing program, as established by deep sequencing of Fluorescence-Activated Cell Sorted (FACS) S and G2 phase populations. Overall, our data reveals that Tos4 mediates gene expression homeostasis through its FHA domain-dependent interaction with the Rpd3L complex, which is independent of H3K56ac.

Relationships between UBE3A and SNORD116 expression and features of autism in chromosome 15 imprinting disorders

Chromosome 15 (C15) imprinting disorders including Prader–Willi (PWS), Angelman (AS) and chromosome 15 duplication (Dup15q) syndromes are severe neurodevelopmental disorders caused by abnormal expression of genes from the 15q11–q13 region, associated with abnormal DNA methylation and/or copy number changes. This study compared changes in mRNA levels of UBE3A and SNORD116 located within the 15q11–q13 region between these disorders and their subtypes and related these to the clinical phenotypes. The study cohort included 58 participants affected with a C15 imprinting disorder (PWS = 27, AS = 21, Dup15q = 10) and 20 typically developing controls. Semi-quantitative analysis of mRNA from peripheral blood mononuclear cells (PBMCs) was performed using reverse transcription droplet digital polymerase chain reaction (PCR) for UBE3A and SNORD116 normalised to a panel of internal control genes determined using the geNorm approach. Participants completed an intellectual/developmental functioning assessment and the Autism Diagnostic Observation Schedule-2nd Edition. The Dup15q group was the only condition with significantly increased UBE3A mRNA levels when compared to the control group (p < 0.001). Both the AS and Dup15q groups also had significantly elevated SNORD116 mRNA levels compared to controls (AS: p < 0.0001; Dup15q: p = 0.002). Both UBE3A and SNORD116 mRNA levels were positively correlated with all developmental functioning scores in the deletion AS group (p < 0.001), and autism features (p < 0.001) in the non-deletion PWS group. The findings suggest presence of novel interactions between expression of UBE3A and SNORD116 in PBMCs and brain specific processes underlying motor and language impairments and autism features in these disorders.

Genetic ancestry modifies the relationship between fine particulate matter and placental mitochondrial mutational load

Introduction: Exposure to ambient fine particulate matter (PM2.5) is linked to changes in placental mitochondrial DNA (mtDNA) copy number. Whether PM2.5 impacts mitochondrial mutational load in placental tissue-another biomarker of oxidative damage and aging has not been studied. Further, genetic ancestry likely impacts this relationship and may inform health disparities. Methods: We examine the association between PM2.5 and placental mtDNA mutational load in an urban multi-ethnic cohort (N=285). Mothers' daily exposure to PM2.5 over gestation was estimated using a satellite-based spatio-temporally resolved prediction model. Whole mtDNA sequencing was performed and mutations and haplogroups were determined. Bayesian Distributed Lag Interaction regression models (BDLIMs) were used to statistically model and visualize the PM2.5 timing-dependent pattern of associations with mtDNA mutations (total load and gene-specific) and explore effect modification by haplogroup. Results: Overall, increased PM2.5 exposure across pregnancy was not associated with total mutational load. However, results varied by mtDNA haplogroup with increases in PM2.5 being associated with higher total mutational load for African (cumulative effect 1.92, 95%CI 0.46, 3.48) and Asian (cumulative effect: 1.33, 95% CI 0.07, 2.73) haplogroups; a critical window was identified between 29 and 35 weeks gestation for African haplogroups only. Gene-wise analyses suggested that increased PM2.5 exposure during mid pregnancy (20-25 weeks) might have a stronger impact on mutations located in genes coding for ATP synthase subunits regardless of haplogroup. Conclusions: Placental mtDNA mutations, associated with increased PM2.5 exposure mid to late pregnancy, may have consequences on placental energy production, aging, and metabolic regulation that may impact offspring development. Understanding how these associations differ based on ancestry may further elucidate the etiology of environmentally-related disease disparities.

Abstract 1968: Early on-treatment genomic instability level in cell free DNA as a predictive and prognostic marker in metastatic breast cancer patients

Abstract Metastatic breast cancer is mostly incurable and patients are treated with serial chemotherapy regimens to prolong life and decrease symptoms. Treatment efficacy is usually monitored by imaging every 3 months and by the measurement of blood tumor biomarkers despite their poor sensitivity and specificity. Novel and more timely approaches to monitoring treatment response and estimating prognosis are needed to enable more rapid treatment decisions. The Genomic Instability Number (GIN) is a recently published measure of DNA copy number changes across the genome that can be derived from low pass whole genome sequencing of cell free DNA in plasma. As it is genome wide, this method does not require previous tumor sequencing and offers a non-invasive option for liquid biopsy testing. We measured the GIN in plasma from a cohort of metastatic breast cancer patients undergoing therapy and correlated GIN levels with clinical response and outcome data. Methods: Blood samples (n=69) were collected from 27 patients with metastatic breast cancer undergoing standard of care therapy. Blood was collected prior to the beginning of a new line of therapy (T0), and then at T1 within 10 days and T2 within 22 days post-treatment initiation. The GIN was calculated for all 69 samples analyzed and we used a pre-determined detection threshold of GIN&amp;gt;170. Treatment response was assessed at 3 and at 6 months post-treatment. Results: 20 of the 27 metastatic patients analyzed had detectable GIN levels in at least one sample. Pre-treatment baseline GIN levels ranged from 80 to 6599 with undetectable GIN in 33% of patients. Baseline plasma GIN was not significantly associated with clinical characteristics, clinical response, progression free survival (PFS) or overall survival (OS). However, the average GIN level at early on-treatment time points, T1 or T2, was associated with clinical response with lower levels in patients who presented tumor response at 3 months (p&amp;lt;0.05) but not at 6 months. We found that detectable GIN at T1 was associated with significantly lower OS (p=0.014) but not with PFS, while detectable GIN values at T2 were associated with both poorer PFS (P=0.017) and OS (p=0.0085). The dynamic changes in GIN during treatment were also associated with treatment response and outcome. In fact, the degree of fall in GIN from baseline to T1 and from baseline to T2 was associated with early clinical response (at 3 months) (p&amp;lt;0.001 and p=0034 respectively). Interestingly, the fall of detectable GIN at T1 was highest in TNBC patients (average 73%) compared to non TNBCs (average 36%) (p=0.016). Finally, the percentage drop of GIN at T1 but not atT2 was significantly associated with PFS (p=0.016). In conclusion, the results from our study highlight the value of early on-treatment ctDNA measurements as a predictor of therapeutic response in the metastatic setting, and provide an approach that does not need to rely on tumor sequencing, thus making it much more clinically feasible. Citation Format: Adriana Aguilar-Mahecha, Josiane Lafleur, Susie Brousse, Cristiano Ferrario, Kimberly A. Holden, Graham McLennan, Taylor J. Jensen, Mark Basik. Early on-treatment genomic instability level in cell free DNA as a predictive and prognostic marker in metastatic breast cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1968.

Changes in peripheral mitochondrial DNA copy number in metformin-treated women with polycystic ovary syndrome: a longitudinal study

BackgroundPatients with polycystic ovarian syndrome (PCOS) are associated with known alterations in mitochondria DNA copy number (mtDNA-CN). The aim of this study is to study the change in mtDNA-CN in patients with PCOS who were treated with metformin.MethodsThis is a prospective cohort of patients with PCOS, who received metformin for one year. From 2009 to 2015, 88 women diagnosed with PCOS, based on the Rotterdam criteria, were enrolled. Serial measurements of mtDNA-CN, 8-hydroxydeoxyguanosine (8-OHdG), anthropometric, metabolic, endocrine, and inflammatory markers were obtained before and after 3, 6, and 12 months of treatment.ResultsA significant decrease in mtDNA-CN was seen over the course of one year. Other markers, including 8-OHdG, testosterone, free androgen index, blood pressure and liver enzymes, also decreased in the same interval. On regression analysis, there was a significant association between the change in mtDNA-CN and serum total testosterone, and no association between mtDNA-CN and metabolic factors.ConclusionsTreatment with metformin is associated with a time-dependent decrease in mtDNA-CN in patients with PCOS who are treated over the course of one year. This may signify a reduction in mitochondria dysfunction. The change in mtDNA-CN corresponds to a similar change in serum total testosterone, and suggests a possible relationship between mtDNA-CN and testosterone.Trial registrationClinicalTrials.gov, NCT00172523. Registered September 15, 2005

Association between mitochondrial DNA content and opium exposure.

To date, not much study has been done to investigate the mitochondrial DNA (mtDNA) copy number as the potential biomarker for opium exposure. Here, we conducted a cross-sectional study to determine the relative mtDNA content as the potential biomarker for opium exposure. Quantitative real-time PCR was performed to investigate the mtDNA copy number variation across 205 individuals, including blood samples of 45 opium users, 41 cigarette users, 47 dual users, and 72 never users of any product. We found a significantly higher mtDNA content among the opium-only users (adjusted OR: 3.21; 95% CI: [1.34, 7.66]; P = .009) and dual users (adjusted OR: 2.64; 95% CI: [1.15, 6.1]; P = .02) compared to that in never users even after adjustment for confounding factors, age, and sex. Discordantly, analysis of mitochondrial DNA in cigarette smokers revealed an indirect association between cigarette smoking and mtDNA content although it was not statistically significant. The reason behind the increased mitochondrial DNA is unclear. The possible hypothesis is that there might be a way to compensate for the oxidative damage induced by opium consumption. Taken together, our findings indicated that the mtDNA copy number may alter during opium exposure. Since changes in the mitochondrial DNA copy number was associated with the etiology of many diseases including cancer, further investigations on the mtDNA copy number may shed light on the carcinogenicity of opium consumption and means for early detection among the populations who have been exposed to opium and its products.

Exposure to the Heavy-Metal Lead Induces DNA Copy Number Alterations in Zebrafish Cells.

DNA copy number variants are associated with the development of complex neurological diseases and disorders including autism spectrum disorder, schizophrenia, Alzheimer's disease, and Parkinson's disease. Exposure to multiple environmental chemicals including various heavy metals is suggested as a risk factor in these neurological diseases and disorders, but few studies have addressed if heavy-metal exposure can result in de novo DNA copy number changes as a genetic mechanism contributing to these disease outcomes. In this study to further investigate the relationship between heavy-metal exposure and de novo copy number alterations (CNAs), zebrafish fibroblast cells were exposed to the neurotoxicant lead (Pb). A crystal violet assay was first used to determine exposure concentrations with >80% cell confluency. Then a zebrafish-specific array comparative genomic hybridization platform was used to detect CNAs following a 72 h Pb exposure (0.24, 2.4, or 24 μM). The Pb exposure resulted in 72 CNA amplifications ranging in size from 5 to 329 kb. No deletions were detected. CNAs resulted in 15 CNA regions (CNARs), leaving 7 singlet CNAs. Two of the singlets were within high repeat genomic locations. The number of CNAs tended to increase in a concentration-dependent manner. Several CNARs encompassed genes previously reported to have altered expression with Pb exposure, suggesting a mechanistic link. In addition, almost all genes are associated within a molecular network with amyloid precursor protein, a key molecular target associated with the pathophysiology of Alzheimer's disease. Overall, these findings show that Pb exposure results in de novo CNAs that could serve as a mechanism driving adverse health outcomes associated with Pb toxicity including neurological disease pathogenesis for further study.

Mitochondrial Dysfunction Is an Early Consequence of Partial or Complete Dystrophin Loss in mdx Mice.

Duchenne muscular dystrophy (DMD) is characterized by rapid wasting of skeletal muscle. Mitochondrial dysfunction is a well-known pathological feature of DMD. However, whether mitochondrial dysfunction occurs before muscle fiber damage in DMD pathology is not well known. Furthermore, the impact upon heterozygous female mdx carriers (mdx/+), who display dystrophin mosaicism, has received little attention. We hypothesized that dystrophin deletion leads to mitochondrial dysfunction, and that this may occur before myofiber necrosis. As a secondary complication to mitochondrial dysfunction, we also hypothesized metabolic abnormalities prior to the onset of muscle damage. In this study, we detected aberrant mitochondrial morphology, reduced cristae number, and large mitochondrial vacuoles from both male and female mdx mice prior to the onset of muscle damage. Furthermore, we systematically characterized mitochondria during disease progression starting before the onset of muscle damage, noting additional changes in mitochondrial DNA copy number and regulators of mitochondrial size. We further detected mild metabolic and mitochondrial impairments in female mdx carrier mice that were exacerbated with high-fat diet feeding. Lastly, inhibition of the strong autophagic program observed in adolescent mdx male mice via administration of the autophagy inhibitor leupeptin did not improve skeletal muscle pathology. These results are in line with previous data and suggest that before the onset of myofiber necrosis, mitochondrial and metabolic abnormalities are present within the mdx mouse.

Frequent copy number gains of SLC2A3 and ETV1 in testicular embryonal carcinomas.

Testicular germ cell tumours (TGCTs) appear as different histological subtypes or mixtures of these. They show similar, multiple DNA copy number changes, where gain of 12p is pathognomonic. However, few high-resolution analyses have been performed and focal DNA copy number changes with corresponding candidate target genes remain poorly described for individual subtypes. We present the first high-resolution DNA copy number aberration (CNA) analysis on the subtype embryonal carcinomas (ECs), including 13 primary ECs and 5 EC cell lines. We identified recurrent gains and losses and allele-specific CNAs. Within these regions, we nominate 30 genes that may be of interest to the EC subtype. By in silico analysis of data from 150 TGCTs from The Cancer Genome Atlas (TCGA), we further investigated CNAs, RNA expression, somatic mutations and fusion transcripts of these genes. Among primary ECs, ploidy ranged between 2.3 and 5.0, and the most common aberrations were DNA copy number gains at chromosome (arm) 7, 8, 12p, and 17, losses at 4, 10, 11, and 18, replicating known TGCT genome characteristics. Gain of whole or parts of 12p was found in all samples, including a highly amplified 100 kbp segment at 12p13.31, containing SLC2A3. Gain at 7p21, encompassing ETV1, was the second most frequent aberration. In conclusion, we present novel CNAs and the genes located within these regions, where the copy number gain of SLC2A3 and ETV1 are of interest, and which copy number levels also correlate with expression in TGCTs.

Increase of mtDNA number and its mutant copies in rat brain after exposure to 150 MeV protons

Proton beam therapy is widely used for treating brain tumor. Despite the efficacy of treatment, the use of this therapy has met some limitations associated with possible damage to normal brain tissues located beyond the tumor site. In this context, the exploration of the harmful effects of protons on the normal brain tissues is of particular interest. We have investigated changes in the total mitochondrial DNA (mtDNA) copy number and identified mtDNA mutant copies in three brain regions (the hippocampus, cortex and cerebellum) of rats after irradiation their whole-head with 150MeV protons at doses of 3 and 5Gy. The study was performed in 2-months old male Spraque Dawley rats (n = 5 each group). The mtDNA copy numbers were determined by real-time PCR. The level of mtDNA heteroplasmy was estimated using Surveyor nuclease technology. Our results show that after head exposure to protons, levels of mtDNA copy number in three rat brain regions increase significantly as the levels of mtDNA mutant copies increase. The most significant elevation is observed in the hippocampus. In conclusion, an increase in mtDNA mutant copies may contribute to mitochondrial dysfunction accompanied by increased oxidative stress in different brain regions and promote the development of neurodegenerative diseases and the induction of carcinogenesis.

Role of the mitochondrial stress response in human cancer progression.

Dysregulated mitochondria often occurred in cancers. Mitochondrial dysfunction might contribute to cancer progression. We reviewed several mitochondrial stresses in cancers. Mitochondrial stress responses might contribute to cancer progression. Several mitochondrion-derived molecules (ROS, Ca2+, oncometabolites, exported mtDNA, mitochondrial double-stranded RNA, humanin, and MOTS-c), integrated stress response, and mitochondrial unfolded protein response act as retrograde signaling pathways and might be critical in the development and progression of cancer. Targeting these mitochondrial stress responses may be an important strategy for cancer treatment.

The associations between prenatal exposure to polycyclic aromatic hydrocarbon metabolites, umbilical cord blood mitochondrial DNA copy number, and children’s neurobehavioral development

Exposure to polycyclic aromatic hydrocarbons (PAHs) during pregnancy is a risk factor for adverse neurobehavioral development outcomes. Mitochondrial DNA are sensitive to environmental toxicants due to the limited ability of repairing. The change of mitochondrial DNA copy number (mtDNAcn) might be a biologically mechanism linking PAH exposure and children’s neurobehavioral impairment. Our aims are to explore whether PAH metabolites in maternal urine were associated with children’s neurobehavioral development at 2 years old and umbilical cord blood mtDNAcn, and whether mtDNAcn was a mediator of PAH-related neurobehavioral development. We included 158 non-smoking pregnant women from Taiyuan City, Shanxi Province. Maternal urinary eleven PAH metabolites were detected by high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). MtDNAcn in cord blood was detected by real time quantitative polymerase chain reaction (RT-PCR). Children’s neurodevelopment was measured by Gesell Developmental Schedules (GDS) when children were two years age. Generalized linear models and restricted cubic spline models were applied to assess the relationships between PAH metabolites in maternal urine and GDS scores and mtDNAcn. A mediation analysis was also conducted. Generalized linear models showed the relationships of sum of PAH metabolites (Σ-OHPAHs) in maternal urine with decreased motor score, and Σ-OHPAHs with increased mtDNAcn (p for trend < 0.05). Urinary levels of Ln (Σ-OHPAHs) increased one unit was related to a 2.08 decreased in motor scores, and Ln (Σ-OHPAHs) increased one unit was related to 0.15 increased in mtDNAcn. Mediation analysis did not find mtDNAcn can be a mediator between PAH metabolites and neurobehavioral development. Our results suggest that prenatal exposure to PAH decreased children’s neurobehavioral development scores and increased mtDNAcn. And reducing exposure to PAH during pregnancy will benefit to improving neurobehavioral development in children.In our present cohort study, sum of PAH metabolites in urine of pregnant women were related with motor score and were positively associated with umbilical cord blood mtDNA copy number.

Effects of prenatal exposure to particulate air pollution on newborn mitochondrial DNA copy number

Prenatal exposure to particulate matter (PM) in ambient air has been linked to changes in newborn mitochondrial DNA copy number (mtDNAcn), but the effects of exposure are inconsistent. We aimed to investigate the effect of weekly PM exposure during pregnancy on newborn mtDNAcn. The present study included 762 mother-infant pairs who were recruited in a birth cohort established between November 2013 and March 2015 in Wuhan, China. Mother’s prenatal daily exposure to PM2.5 and PM10 was calculated using a spatial-temporal land use regression model. Relative mtDNAcn in cord blood leukocytes was determined by quantitative real-time polymerase chain reaction. Distributive lag regression models (DLMs) were applied to estimate the association between PM exposure and newborn mtDNAcn. In the adjusted models, prenatal PM2.5 exposure during 25–32 weeks and PM10 exposure during 25–31weeks were significantly associated with decreased cord blood mtDNAcn. PM2.5 exposure during the third trimester was related to decreased mtDNAcn (cumulative percent change: −8.55%, 95% CI: −13.32%, −3.51%). We also identified other exposure windows (17–22 and 11–22 weeks) in which PM exposure was positively associated with mtDNAcn. Overall, exposure to particulate air pollution during mid-to-late gestation is significantly associated with alterations in newborn mtDNAcn, potentially suggesting an enhanced sensitivity to PM exposure during this period.

Landscape of copy number aberrations in esophageal squamous cell carcinoma from a high endemic region of South Africa

BackgroundEsophageal squamous cell carcinoma (ESCC) is an aggressive cancer with one of the highest world incidences in the Eastern Cape region of South Africa. Several genome wide studies have been performed on ESCC cohorts from Asian countries, North America, Malawi and other parts of the world but none have been conducted on ESCC tumors from South Africa to date, where the molecular pathology and etiology of this disease remains unclear. We report here tumor associated copy number changes observed in 51 ESCC patients’ samples from the Eastern Cape province of South Africa.MethodsWe extracted tumor DNA from 51 archived ESCC specimens and interrogated tumor associated DNA copy number changes using Affymetrix® 500 K SNP array technology. The Genomic Identification of Significant Targets in Cancer (GISTIC 2.0) algorithm was applied to identify significant focal regions of gains and losses. Gains of the top recurrent cancer genes were validated by fluorescence in situ hybridization and their protein expression assessed by immunohistochemistry.ResultsTwenty-three significant focal gains were identified across samples. Gains involving the CCND1, MYC, EGFR and JAG1 loci recapitulated those described in studies on Asian and Malawian cohorts. The two most significant gains involved the chromosomal sub-bands 3q28, encompassing the TPRG1 gene and 11q13.3 including the CTTN, PPFIA1and SHANK2 genes. There was no significant homozygous loss and the most recurrent hemizygous deletion involved the B3GAT1 gene on chromosome 11q25. Focal gains on 11q13.3 in 37% of cases (19/51), consistently involved CTTN and SHANK2 genes. Twelve of these cases (23,5%), had a broader region of gain that also included the CCND1, FGF19, FGF4 and FGF3 genes. SHANK2 and CTTN are co-amplified in several cancers, these proteins interact functionally together and are involved in cell motility. Immunohistochemistry confirmed both Shank2 (79%) and cortactin (69%) protein overexpression in samples with gains of these genes. In contrast, cyclin D1 (65%) was moderately expressed in samples with CCND1 DNA gain.ConclusionsThis study reports copy number changes in a South African ESCC cohort and highlights similarities and differences with cohorts from Asia and Malawi. Our results strongly suggest a role for CTTN and SHANK2 in the pathogenesis of ESCC in South Africa.

Retrotransposition Bursts in the Adult Brain: Stress, Running, and Instrumental Learning Increase the Number of L1 Retrotransposon DNA Copies in the Mouse Nervous System

Retrotransposons, the DNA elements which proliferate by reverse transcription of their RNA intermediates, comprise a major fraction of mammalian genomes. In the human genome a particularly abundant retrotransposon ‐ long interspersed nuclear element‐1 (LINE‐1 or L1) has 5x105 copies 80–100 of which are active and competent for retrotransposition. Recent findings indicate that LINE‐1 is active in neuronal precursor cells during differentiation, and its de novo insertions can change expression of neuron‐specific genes and cause somatic mosaicism in the adult brain. However, there has been limited understanding whether such retrotranspositions happen in the adult brain, when and how they occur, and what is their importance for the CNS function. In our study we tested whether prolonged stress, wheel running, or operant learning can lead to changes in L1 DNA copy number in the mouse brain. For this purpose, we subjected mice to immobilization stress (2 hours a day, 10 days), wheel running (unlimited access, 10 days) or nose‐poke operant training (1 session a day, 10 days). We collected the samples of hippocampus, frontal cortex, cerebellum, muscle and testicle tissues from experimental and home cage control mice. We performed qPCR for analysis of 3′‐UTR and ORF‐1 copy number. We observed significant increase in the 3′‐UTR and ORF‐1 copy number in all brain structures of the stress group compared to the home cage group. Nose‐poke training was associated with specific increase in 3′‐UTR copy number in the hippocampus. We also observed a small increase in 3′‐UTR and ORF‐1 copy number in all investigated brain structures in wheel running group. Altogether our results show that retrotranspositions is a common event in the adult brain under different conditions of novel experience.Support or Funding InformationRFBR # 19‐015‐00550

Vascular endothelial growth factor alleviates mitochondrial dysfunction and suppression of mitochondrial biogenesis in models of Alzheimer’s disease

Purpose Mitochondrial dysfunction is a prominent feature of Alzheimer’s disease (AD). As vascular endothelial growth factor (VEGF) has been shown to be protective in AD, the aim of this study was to investigate the effects of VEGF on mitochondrial function in models of AD. Materials and methods Adeno associated virus (AAV)-VEGF was injected into the hippocampus of APP/PS1 mice. Cognitive function was assessed in these mice with use of the Morris water maze (MWM) and β-amyloid (Aβ) levels in the hippocampus were also measured. Cell viability and reactive oxygen species (ROS) levels were determined in the SH-SY5Y cells treated with Aβ25–35 which served as a cell model of AD. Transmission electron microscopy (TEM) was used to evaluate structural changes in mitochondria and mitochondrial DNA (mtDNA) copy number and mitochondrial membrane potential (MMP) were also recorded. Finally, we investigated the effects of VEGF upon mitochondrial biogenesis, autophagy and mitochondrial autophagy (mitophagy) as determined both in vivo and in vitro with western blots. Results VEGF treated mice showed improvements in spatial learning and memory along with reduced Aβ levels. VEGF protected SH-SY5Y cells against Aβ25–35 induced neurotoxicity as demonstrated by increased cell viability and decreased ROS production. Associated with these effects were improvements in mitochondrial structure and function, and increased numbers of mitochondria resulting from stimulation of mitochondrial biogenesis. Conclusions VEGF alleviates Aβ related patholoy in models of AD. In part, these beneficial effects of VEGF result from protection of mitochondria and stimulation of mitochondrial biogenesis.

DNA copy number changes correlate with clinical behavior in melanocytic neoplasms: proposal of an algorithmic approach.

Increasingly, molecular methods are being utilized in the workup of melanocytic neoplasms. To this end, we sought to correlate data from a single nucleotide polymorphism (SNP) array platform based on molecular inversion probes with clinical data. Copy number variation (CNV) data were obtained on 95 melanocytic tumors (6 ordinary nevi, 15 atypical nevi, 34 ambiguous neoplasms, and 40 melanomas) from 92 patients. The average number of significant CNVs was 0 for nevi, 0.6 for atypical nevi (range 0-3), 2.8 for ambiguous neoplasms (range 0-17), and 18.1 for melanomas (range 0-69). Clinical follow-up data were available in 57 of 95 lesions (56 of 92 patients). Tumors from patients with adverse events demonstrated an average number of CNVs of 24.5 (range 6-69) as compared with 7.9 (range 0-35) among tumors without an associated adverse event (p ≤ 0.001). No adverse events were observed in nevi including atypical nevi. Adverse events were found in 2 of 19 ambiguous neoplasms and 10 of 32 melanomas with follow up. In these two latter groups of neoplasms, the correlation between adverse events and the average number of CNVs remained statistically significant even when controlled for Breslow depth (21.5 versus 8.7, p value = 0.036). No neoplasm with adverse events had ≤3 CNVs. These results provide further evidence that SNP array testing for CNVs may be helpful in the classification and prognostication of ambiguous neoplasms. Based on these results, an algorithmic approach to challenging melanocytic neoplasms using CNV data is suggested, using as cutoff of >3 CNVs with some caveats, as the threshold for a positive result. Future clinical validation, using a larger cohort of relevant tumors, will be necessary.