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Previous genomewide association studies (GWASs), single nucleotide polymorphisms (SNPs) on cyclin-dependent kinase inhibitor 2 A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and cyclin-dependent kinase inhibitor 2B antisense RNA1 (CDKN2B-AS1) were reported as risk loci for glioma, a subgroup of the brain tumor. To further characterize this association with the risk of brain tumors in a Korean population, we performed a fine-mapping association study of CDKN2A, CDKN2B, and CDKN2B-AS1.A total of 17 SNPs were selected and genotyped in 1,439 subjects which were comprised of 959 patients (pituitary adenoma 335; glioma 324; meningioma 300) and 480 population controls (PCs). We discovered that a 3'untranslated region (3'UTR) variant, rs181031884 of CDKN2B (Asian-specific variant), had significant association with the risk of pituitary adenoma (PA) (Odds ratio = 0.58, P = 0.00003). Also, rs181031884 appeared as an independent causal variant among the significant variants in CDKN2A and CDKN2B, and showed dose-dependent effects on PA.Although further studies are needed to verify the impact of this variant on PA susceptibility, our results may help to understand CDKN2B polymorphism and the risk of PA.

In higher eukaryotes, cell-cycle transitions are regulated by different cyclin-dependent kinases (Cdks) and Cdk inhibitors (CKIs). CKIs include two groups, the Ink4 (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) and the Cip/Kip (p21Cip1, p27Kip1, and p57Kip2) families. The hyperactivity of histone deacetylases (HDACs) is associated with cancer induction. Histone deacetylase inhibitors (HDACIs) such as sodium butyrate (NaBT) can inhibit HDAC activity resulting in apoptosis induction. The present study was designed to investigate the effect of sodium butyrate on p16INK4a, p14ARF, p15INK4b, class I HDACs (HDACs 1, 2, 3), and class II HDACs (HDACs 4, 5, 6), cell growth inhibition, and apoptosis induction in pancreatic cancer AsPC-1 and colon cancer HCT-116 cell lines. In fact, we want to know whether sodium butyrate can reactivate Ink4 and Cip/Kip families by HDACs inhibition.The AsPC-1 and HCT-116 cells were treated with sodium butyrate at different periods. Then, the MTT assay, cell apoptosis assay, and qRT-PCR were done to determine viability, apoptosis, and the relative expression level of the genes respectively.The sodium butyrate increased p16INK4a, p14ARF, and p15INK4b and decreased class I and II HDACs significantly. Besides, HCT-116 cell was more sensitive to sodium butyrate in comparison to AsPC-1 cell.The sodium butyrate can reactivate the p16INK4a, p14ARF, and p15INK4b through inhibition of HDACs in AsPC-1 and HCT-116 cell lines.

Metastases are often the direct cause of death from pancreatic ductal adenocarcinoma (PDAC). The role of genomic alterations (GA) in mediating tropism and metastasis formation by PDAC cells is currently unknown. We aimed to identify GAs predisposing colonization of PDAC cells to the liver and decipher mechanisms enabling this process. In order to reveal specific genes, we studied the frequency of GA in 8,880 local and 7,983 metastatic PDAC samples. We observed differential pattern of GA in the local tumor and specific metastatic sites, with liver metastases characterized by deletion of CDKN2A/B (encoding p16/p15, respectively). The role of CDKN2A/B in promoting liver metastasis was evidenced by enhanced tumorigenic phenotype of p15/p16-deleted PDAC cells when exposed to hepatocytes conditioned media. The liver is characterized by high-ammonia low-glutamine environment and transcriptomic assays indicated unique adaptation of PDAC cells to these conditions, including regulation of genes leading to reduced glutaminolysis, like overexpression of GLUL and reduction in GLS2. Furthermore, metabolic assays indicated an increase in glutamate derived from [U

Expression of miR-154 is upregulated in the diseased heart and was previously shown to be upregulated in the lungs of patients with pulmonary fibrosis. However, the role of miR-154 in a model of sustained pressure overload-induced cardiac hypertrophy and fibrosis had not been assessed. To examine the role of miR-154 in the diseased heart, adult male mice were subjected to transverse aortic constriction for four weeks and echocardiography was performed to confirm left ventricular hypertrophy and cardiac dysfunction. Mice were then subcutaneously administered a locked nucleic acid antimiR-154 or control over three consecutive days (25 mg/kg/day) and cardiac function was assessed 8 weeks later. Here, we demonstrate that therapeutic inhibition of miR-154 in mice with pathological hypertrophy was able to protect against cardiac dysfunction and attenuate adverse cardiac remodelling. The improved cardiac phenotype was associated with attenuation of heart and cardiomyocyte size, less cardiac fibrosis, lower expression of atrial and B-type natriuretic peptide genes, attenuation of profibrotic markers and increased expression of p15 (a miR-154 target and cell cycle inhibitor). In summary, this study suggests that miR-154 may represent a novel target for the treatment of cardiac pathologies associated with cardiac fibrosis, hypertrophy and dysfunction.

Cancer susceptibility is a critical factor in the understanding of carcinogenesis. Intraperitoneal (i.p.) injection of an iron chelate, ferric nitrilotriacetate (Fe‐NTA), produces hydroxyl radicals via Fenton reaction to induce ferroptosis in renal proximal tubules. Rats or mice subjected to repeated i.p. injections of Fe‐NTA develop renal cell carcinoma (RCC). To elucidate the molecular mechanisms that cause susceptibility to renal carcinogenesis, we first established an inter‐strain difference in the susceptibility to Fe‐NTA‐induced renal carcinogenesis in mice. Based on a previous observation of a low incidence of RCC with this model in C57BL/6J strain mice, we investigated A/J strain mice here, which demonstrated significantly higher susceptibility to Fe‐NTA‐induced renal carcinogenesis. Homozygous deletion of the Cdkn2a/2b tumor suppressor locus was detected for the first time in A/J strain mice. Focusing on ferroptosis and iron metabolism, we explored the mechanisms involved that lead to the difference in RCC development. We compared the protective responses in the kidney of A/J and C57BL/6J strains after Fe‐NTA treatment. After 3‐week Fe‐NTA treatment, A/J mice maintained higher levels of expression of glutathione peroxidase 4 and xCT (SLC7A11), leading to a lower level of lipid peroxidation. Simultaneously, A/J mice had decreased expression of transferrin receptor and increased expression of ferritin to greater degrees than C57BL/6 mice. After a single Fe‐NTA injection, higher levels of oxidative cell damage and cytosolic catalytic Fe(II) were observed in C57BL/6J mice, accompanied by a greater increase in lipocalin‐2. Lipocalin‐2 deficiency significantly decreased oxidative renal damage. Our results suggest that a genetic trait favoring ferroptosis resistance contributes to high susceptibility to Fe‐NTA‐induced RCC in A/J strain., Cancer susceptibility is an important issue when considering cancer prevention. In this paper, we used an iron‐mediated oxidative stress‐induced renal carcinogenesis model in mice and found that ferroptosis resistance is an important factor determining cancer susceptibility.

Objective The patterns of lncRNA CDKN2B-AS1 in coronary heart disease (CHD) have been extensively studied. This study investigated the competing endogenous RNA (ceRNA) network of CDKN2B-AS1 in coronary atherosclerosis (CAS). Methods Microarray analyses were performed to screen out the CHD-related lncRNAs (CDKN2B-AS1) and the downstream microRNAs (miR-126-5p). The expression of CDKN2B-AS1 in serum of patients with CHD and healthy volunteers was detected. Vascular smooth muscle cells (VSMCs) were treated with oxidized low density lipoprotein (ox-LDL) to establish the cell model. Then pcDNA-CDKN2B-AS1 and/or miR-126-5p mimic were transfected into ox-LDL-treated VSMCs to estimate cell proliferation, apoptosis and inflammation. The ceRNA network of CDKN2B-AS1 along with the possible pathway in CHD was testified. Results CDKN2B-AS1 expression was low in patients with CHD and ox-LDL-treated VSMCs. Upon CDKN2B-AS1 overexpression, TNF-α, NF-κB and IL-1β levels in VSMCs were decreased, the proliferation of VSMCs was inhibited and the apoptosis rate was increased. Overexpression of miR-126-5p could reverse these trends. CDKN2B-AS1 as a ceRNA competitively bound to miR-126-5p to upregulate PTPN7. CDKN2B-AS1 inhibited VSMC proliferation and accelerated apoptosis by inhibiting the PI3K-Akt pathway. Conclusion LncRNA CDKN2B-AS1 upregulates PTPN7 by absorbing miR-126-5p and inhibits the PI3K-Akt pathway, thus hindering the proliferation and accelerating apoptosis of VSMCs induced by ox-LDL, thus being a therapeutic approach for CAS.

Gene inactivation of the cyclin‑dependent kinase inhibitors p16

Epigenetics is the science of altering gene expression without changing nucleotide sequences and may be induced by various environmental factors, including pesticides. The aim of this study was to investigate certain epigenetic changes including the methylation of CDKN2B, CDKN2A, and MGMT gene promoters and histone modifications of H3K9ac, H4K16ac, H4K20me3, and H3K4me3, as well as their association with the levels of organochlorine pesticides (OCPs) in children with acute lymphoblastic leukemia (ALL). The evaluation of OCP levels, promoter methylation, gene expression, and expression of histone modifications was performed by gas chromatography (GC), methylation-specific polymerase chain reaction (MS-PCR), reverse transcription PCR (RT-PCR), and western blotting, respectively. The results indicated that 76.2 % of CDKN2B promoters and 85.1 % of MGMT promoters were hypermethylated in children with ALL compared to healthy children. In addition, the relative expression of CDKN2B, MGMT, H4K16ac, and H3K4me3 showed a significant decrease in children with ALL compared to healthy children. Levels of OCPs in children with ALL were significantly higher than in healthy children. Furthermore, the results revealed that the rise in the OCP levels was associated with an increase in methylation at the promoter level of CDKN2B and MGMT as well as a decrease in the relative expression of H4K16ac and H3K4me3. Therefore, it can be concluded that exposure to OCPs is associated with the induction of epigenetic changes at the level of DNA and histones, which may lead to leukemia.

Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer., The genomic characterisation of nasopharyngeal carcinoma (NPC) remains crucial. Here, the authors perform whole-genome sequencing for 70 NPCs with EBV gene expression, report the somatic alterations and EBV-mediated effects converging on NF-κB activation and immune escape and identify targetable homozygous MTAP deletions.

MXDs are transcription repressors that antagonize MYC-mediated gene activation. MYC, when associated with MIZ1, acts also as a repressor of a subset of genes, including p15 and p21. A role for MXDs in regulation of MYC-repressed genes is not known. We report that MXDs activate transcription of p15 and p21 in U2OS cells. This activation required DNA binding by MXDs and their interaction with MIZ1. MXD mutants deficient in MIZ1 binding interacted with the MYC-binding partner MAX and were active as repressors of MYC-activated genes but failed to activate MYC-repressed genes. Mutant MXDs with reduced DNA-binding affinity interacted with MAX and MIZ1 but neither repressed nor activated transcription. Our data show that MXDs and MYC have a reciprocally antagonistic potential to regulate transcription of target genes.

Background Minor progress in pancreatic cancer treatment and prognosis implies that more reliable animal models are urgently needed to decipher its molecular mechanisms and preclinical research. We recently reported a genetically engineered adult mouse model where Cdkn2b downregulation was required together with Cdkn2a downregulation to inactivate the Rb pathway. Besides, the role of Smad4, which is mutated more frequently than Cdkn2b in human pancreatic cancer, was determined critical on the development of the pancreas tumor by some reports. However, the impact of Smad4 deficiency in combination with PDAC-relevant mutations, such as Cdkn2a when induced in adult pancreas has not been completely elucidated in mice. Methods Lentiviral delivered oncogene/tumor suppressors in adult pancreas. The development of pancreatic cancer was monitored. Hematoxylin and eosin staining and immunofluorescence were performed for pathological identification of the pancreatic cancer. Real-time polymerase chain reaction, immunofluorescence and western blot were used to test gene expression. Results Loss of Smad4 could cooperate with alterations of KRAS, Trp53, and Cdkn2a to induce pancreatic cancer in adult mice. The role of Smad4 was mainly in downregulating the expression of Cdkn2b and further inducing phosphorylation of the Rb1 protein. Conclusions These findings show an essential role of Smad4 deficiency in pancreatic ductal adenocarcinoma (PDAC) formation. This model better recapitulates the adult onset, clonal origin, and genetic alterations in human PDAC and can be simply generated on a large-scale.

Loss of chromosome 9p21 is observed in one-thirds of clear-cell renal cell carcinoma (ccRCC) and is associated with poorer patient survival. Unexpectedly, 9p21 LOH does not lead to decreased expression of the 9p21 tumor suppressor genes, CDKN2A and CDKN2B, suggesting alternative mechanisms of 9p-mediated tumorigenesis. Concordantly, CRISPR-mediated 9p21 deletion promotes growth of immortalized human embryonic kidney epithelial cells independently of the CDKN2A/B pathway inactivation. The 9p21 locus has a highly accessible chromatin structure, suggesting that 9p21 loss might contribute to kidney cancer progression by dysregulating genes distal to the 9p21 locus. We identified several 9p21 regulatory hubs by assessing which of the 9p21-interacting genes are dysregulated in 9p21-deleted kidney cells and ccRCCs. By focusing on the analysis of the homeobox gene 13 (HOXB13) locus, we found that 9p21 loss relieves the HOXB13 locus, decreasing HOXB13 methylation and promoting its expression. Upregulation of HOXB13 facilitates cell growth and is associated with poorer survival of patients with ccRCC. Implications: The results of our study propose a novel tumor suppressive mechanism on the basis of coordinated expression of physically associated genes, providing a better understanding of the role of chromosomal deletions in cancer.

Introduction: Follicular thyroid carcinoma (FTC) is more aggressive than the most common papillary thyroid carcinoma (PTC). However, the current research on FTC is less than PTC. Here, we investigated the effects of long noncoding RNA (lncRNA) GAS5 and miR-221-3p in FTC. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to detect GAS5 and miR-221-3p expression in the FTC tissues and cells. Cell proliferation was assessed by CCK8 and EdU assays. Flow cytometry was performed to determine the cell cycle. The dual-luciferase reporter assay was employed to validate the binding relationship of GAS5/miR-221-3p and miR-221-3p/cyclin-dependent kinase inhibitor 2B (CDKN2B). Western blot was conducted to measure the protein level of CDKN2B. Results: Our results displayed that GAS5 was downregulated, while miR-221-3p was upregulated in FTC tissues and cells. What’s more, overexpression of GAS5 or miR-221-3p inhibition induced G0/G1 phase arrest and inhibited cell proliferation of FTC cells. GAS5 acted as a sponge of miR-221-3p, and CDKN2B was a target gene of miR-221-3p. Additionally, GAS5 inhibited cell cycle and proliferation of FTC cells via reducing miR-221-3p expression to enhance CDKN2B expression. Conclusion: GAS5 induced G0/G1 phase arrest and inhibited cell proliferation via targeting miR-221-3p/CDKN2B axis in FTC. Thus, GAS5 may be a potential therapeutic target for the treatment of FTC.

Background: In view of the roles of long non-coding RNA CDKN2B antisense RNA 1 (CDKN2BAS1) in various human diseases, we investigated the function of CDKN2B-AS1 and explored its therapeutic and prognostic target value in multiple biological processes. The aim of this review was to explore the molecular mechanism and clinical significance of CDKN2B-AS1 in various types of diseases. Materials and Methods: In this review, the biological functions and mechanisms of lncRNA CDKN2B-AS1 in a variety of pathophysiological processes were summarized and analyzed. The correlated studies were collected via a systematic search of PubMed, Wiley Online Library, and ScienceDirect. Results: CDKN2B-AS1 is a potential long non-coding RNA that has been shown to be aberrantly expressed in various malignancies, containing hepatocellular carcinoma, intrahepatic cholangiocarcinoma, esophageal squamous cell carcinoma, gastric cancer, colonic adenocarcinoma, cervical cancer, ovarian cancer, breast cancer, glioma, lung cancer, laryngeal squamous cell carcinoma and osteosarcoma, involving in the processes of tumor cells proliferation, migration, invasion and inhibition of tumor cells apoptosis. Besides, CDKN2B-AS1 has been proved implicated in numerous non-malignant diseases, such as idiopathic pulmonary fibrosis, endometriosis, inflammatory bowel disease, intracranial aneurysm, diabetes mellitus and its complications, primary open angle glaucoma, ischemic stroke, atherosclerosis, coronary artery diseases, hypertension and heart failure, participating in the procession of lipid, carbohydrate metabolism and inflammation regulation. Conclusions: Long non-coding RNA CDKN2B-AS1 likely serves as a promising therapeutic target or prognosis biomarker in multiple human diseases.

Development of coronary artery disease (CAD) is primarily due to the process of atherosclerosis, however the prognosis of CAD depends on pleiotropic effects of the genes located at 9p21.3 region. Genome wide association studies revealed association of variants in this region with CAD pathology. However, specific marker in predicting CAD development or progression is not yet identified. In the present study, 35 SNPs at 9p21.3 region, located in the cyclin dependent kinase inhibitor (CDKN2A/CDKN2B) genes, were genotyped among 350 CAD cases and 480 controls from the southern Indian population of Hyderabad using fluidigm nanofluidic SNP genotyping system and the data were analyzed using PLINK and R softwares. Of the 35 SNPs analysed, only one SNP, rs7865618, was found to be highly significantly associated with CAD, even after correction for multiple testing (p = 0.008). The AG and GG genotypes of this SNP conferred 3.08 and 1.93 folds increased risk for CAD respectively. In particular, this SNP was significantly associated with severe anatomic (triple vessel disease p = 0.023) and phenotypic (acute coronary syndrome p = 0.007) categories of CAD. Pair wise SNP interaction analysis between the SNPs of 9p21.3 and 11q23.3 regions revealed significantly increased risk of three SNPs of 11q23.3 region that were not associated individually, in conjunction with rs7865618 of 9p21.3.

Steroid-induced glaucoma (SIG) is the most common adverse steroid-related effect on the eyes. SIG patients can suffer from trabecular meshwork (TM) dysfunction, intraocular pressure (IOP) elevation, and irreversible vision loss. Previous studies have mainly focused on the role of extracellular matrix turnover in TM dysfunction; however, whether the cellular effects of TM cells are involved in the pathogenesis of SIG remains unclear. Here, we found that the induction of cellular senescence was associated with TM dysfunction, causing SIG in cultured cells and mouse models. Especially, we established the transcriptome landscape in the TM tissue of SIG mice via microarray screening and identified ANRIL as the most differentially expressed long non-coding RNA, with a 5.4-fold change. The expression level of ANRIL was closely related to ocular manifestations (IOP elevation, cup/disc ratio, and retinal nerve fiber layer thickness). Furthermore, p15, the molecular target of ANRIL, was significantly upregulated in SIG and was correlated with ocular manifestations in an opposite direction to ANRIL. The reciprocal regulation between ANRIL and p15 was validated using luciferase reporter assay. Through depletion in cultured cells and a mouse model, ANRIL/p15 signaling was confirmed in cellular senescence via cyclin-dependent kinase activity and, subsequently, by phosphorylation of the retinoblastoma protein. ANRIL depletion imitated the SIG phenotype, most importantly IOP elevation. ANRIL depletion-induced IOP elevation in mice can be effectively suppressed by p15 depletion. Analyses of the single-cell atlas and transcriptome dynamics of human TM tissue showed that ANRIL/p15 expression is spatially enriched in human TM cells and is correlated with TM dysfunction. Moreover, ANRIL is colocalized with a GWAS risk variant (rs944800) of glaucoma, suggesting its potential role underlying genetic susceptibility of glaucoma. Together, our findings suggested that steroid treatment promoted cellular senescence, which caused TM dysfunction, IOP elevation, and irreversible vision loss. Molecular therapy targeting the ANRIL/p15 signal exerted a protective effect against steroid treatment and shed new light on glaucoma management.

Homozygous deletion (HD) of CDKN2A and CDKN2B (CDKN2A/B

Mucinous tubular and spindle cell carcinoma (MTSCC) is a rare subtype of renal cell carcinoma with characteristic histologic features and chromosomal alterations. Although typically indolent, a small subset of cases has been reported to exhibit aggressive clinical behavior. We retrospectively identified 33 patients with MTSCC, consisting of 10 cases of locally advanced / metastatic-MTSCC (pT3 or N1 or M1) and 23 kidney confined-MTSCC (pT1/T2) without disease recurrence or progression. Utilizing a single nucleotide polymorphism array and a targeted next-generation sequencing platform, we examined genome-wide molecular alterations in 24 cases, including 11 available samples from 8 patients with locally advanced / metastatic-MTSCC. Ten patients with locally advanced / metastatic-MTSCC were 8 females (80%) and 2 males (20%). At nephrectomy, 7 of these 10 cases (70%) were pT3 or pN1 while the remaining 3 (30%) were pT1/T2. Eight patients (80%) developed metastases and common sites included lymph node (4, 40%), bone (4, 40%), and retroperitoneum (3, 30%). Four patients died of disease (40%) during follow-up. Locally advanced / metastatic-MTSCCs shared typical MTSCC genomic profiles with loss of chromosomes 1, 4, 6, 8, 9, 13, 14, 15, and 22, while some exhibited additional complex genomic alterations, most frequently a relative gain of 1q (7/8). Homozygous loss of CDKN2A/B was observed in 3 (38%) locally advanced / metastatic-MTSCCs. Tumor necrosis, solid nested/sheet pattern, irregular trabecular/single-file infiltration in a desmoplastic stroma, lymphovascular space invasion, and increased mitotic activity were associated with locally advanced / metastatic-MTSCCs (all p < 0.05). Our findings reveal that MTSCCs with aggressive clinical behavior have progressed through clonal evolution; CDKN2A/B deletion and additional complex genomic abnormalities may contribute to this process. Recognizing the morphologic presentation of high grade MTSCC and evaluating adverse histologic features seen in these tumors can help establish a definitive diagnosis and stratify patients for treatment and prognostication.

Depletion of S-adenosyl methionine and 5-methyltetrahydrofolate; and elevation of total plasma homocysteine were documented in CAD patients, which might modulate the gene-specific methylation status and alter their expression. In this study, we have aimed to delineate CAD-specific epigenetic signatures by investigating the methylation and expression of 11 candidate genes i.e. ABCG1, LIPC, PLTP, IL-6, TNF-α, CDKN2A, CDKN2B, F2RL3, FGF2, P66 and TGFBR3. The methylation-specific PCR and qRT-PCR were used to assess the methylation status and the expression of candidate genes, respectively. CAD patients showed the upregulation of IL-6, TNF-α, CDKN2A, CDKN2B, F2RL3, FGF2, P66, and TGFBR3. Hypomethylation of CDKN2A loci was shown to increase risk for CAD by 1.79-folds (95% CI 1.22–2.63). Classification and regression tree (CART) model of gene expression showed increased risk for CAD with F2RL3 > 3.4-fold, while demonstrating risk reduction with F2RL3

Sentinel lymph node (SLN) biopsy is typically offered to patients with primary cutaneous melanomas (PCMs) of ≥ 1 mm depth, but not all SLNs are positive using this cutoff. To ascertain whether positivity is genetically regulated, genetic analysis was performed using an augmented enrichment-based next-generation DNA and RNA sequencing assay in SLN-negative (Group 1, n = 8, mean depth 1.3 mm) and SLN-positive PCMs (controls, Group 2, n = 4, mean depth 1.4 mm). In Group 1, the mean number of mutations was 21 (range 3-48) with the most frequent mutations occurring in NF1 (75%) followed by TP53 (63%), CDKN2A and BRAF (38%), and NRAS (25%), while in Group 2, the ean number of mutations was 9.5 (range 5-18) with mutations in NRAS and BRAF being the most frequent (50%) followed by those in ATM, CDKN2A, CDKN2B, and NOTCH1 (25%). Increased frequency of NF1-inactivating mutations in Group 1 provides provocative early data that the presence of NF1 mutations might confer a less aggressive phenotype.

Purpose Family-based genetic linkage analysis and genome-wide association studies (GWASs) have identified many genomic loci associated with primary open-angle glaucoma (POAG). Several causative genes of POAG have been intensively analyzed by sequencing in different populations. However, few investigations have been conducted on the identification of variants of coding region in the genes identified in GWASs. Therefore, further research is needed to investigate whether they harbor pathogenically relevant rare coding variants and account for the observed association. Methods To identify the potentially disease-relevant variants (PDVs) in POAG-associated genes in Chinese patients, we applied molecular inversion probe (MIP)-based panel sequencing to analyze 26 candidate genes in 235 patients with POAG and 241 control subjects. Results The analysis identified 82 PDVs in 66 individuals across 235 patients with POAG. By comparison, only 18 PDVs in 19 control subjects were found, indicating an enrichment of PDVs in the POAG cohort (28.1% versus 7.9%, p = 8.629e-09). Among 26 candidate genes, the prevalence rate of PDVs in five genes showed a statistically significant difference between patients and controls (33 out of 235 versus 1 out of 241, p = 4.533e-10), including ATXN2 (p = 0.0033), TXNRD2 (p = 0.0190), MYOC (p = 0.0140), FOXC1 (p = 0.0140), and CDKN2B (p = 0.0287). Furthermore, two sisters harboring a stop-loss mutation EFEMP1 p.Ter494Glu were found in the POAG cohort, and further analysis of the family strongly suggested that EFEMP1 p.Ter494Glu was a potentially disease-causing mutation for POAG. A statistically significant difference in age at diagnosis between patients with PDVs and those without PDVs was found, implying that some of the identified PDVs may have a role in promoting the early onset of POAG disease. Conclusions The results suggest that some of the associations identified in POAG risk loci can be ascribed to rare coding variants with likely functional effects that modify POAG risk.

Lysine specific demethylase 2B, KDM2B, regulates genes that participate in cellular development, morphogenesis, differentiation and metabolism as a component of the polycomb repressive complex 1 (PRC1). The CxxC finger of KDM2B is responsible for the DNA binding capacity of this epigenetic regulator, acting as a sampling mechanism across chromatin for gene repression OBJECTIVES: The molecular determinants of the CxxC-DNA interaction remain largely unknown, revealing a significant knowledge gap to be explored. Our goal was to elucidate the key residues of the CxxC domain that contribute to its function as well as to further elaborate on the significance of this domain in the KDM2B role METHODS: By using electrophoresis mobility swift assay, we identified structural elements of CxxC domain that participate in the DNA recognition. We created mouse embryonic fibroblasts overexpressing different truncated and point-mutated mouse KDM2B variants to examine the contribution of the KDM2B domains in replicative senescence bypass RESULTS: In this study, we show that only the CxxC finger is essential for the ability of mKDM2B to bypass replicative senescence in primary cells by ink4A-Arf-ink4B locus repression, and that this is mediated by specific interactions of residues R585, K608 and K616 with non-methylated CpG containing DNA CONCLUSIONS: These results provide new structural insights into the molecular interactions of CxxC and could serve as a stepping-stone for developing domain-specific inhibitors for KDM2B.

Background: Head and neck squamous cell carcinoma (HNSCC) is a significant global burden. The development of a diagnostic or recurrence monitoring test could evolve from the exploitation of molecul...

The onset of malignant mesothelioma (MM) is linked to exposure to asbestos fibers. Asbestos fibers are classified as serpentine (chrysotile) or amphibole, which includes the crocidolite, amosite, anthophyllite, tremolite, and actinolite types. Although few studies have been undertaken, anthophyllite has been shown to be associated with mesothelioma, and tremolite, a contaminant in talc and chrysotile, is a risk factor for carcinogenicity. Here, after characterizing the length and width of these fibers by scanning electron microscopy, we explored the cytotoxicity induced by tremolite and anthophyllite in cells from an immortalized human mesothelial cell line (MeT5A), murine macrophages (RAW264.7), and in a rat model. Tremolite and short anthophyllite fibers were phagocytosed and localized to vacuoles, whereas the long anthophyllite fibers were caught on the pseudopod of the MeT5A and Raw 264.7 cells, according to transmission electron microscopy. The results from a 2‐day time‐lapse study revealed that tremolite was engulfed and damaged the MeT5A and RAW264.7 cells, but anthophyllite was not cytotoxic to these cells. Intraperitoneal injection of tremolite in rats induced diffuse serosal thickening, whereas anthophyllite formed focal fibrosis and granulomas on peritoneal serosal surfaces. Furthermore, the loss of Cdkn2a/2b, which are the most frequently lost foci in human MM, were observed in 8 cases of rat MM (homozygous deletion [5/8] and loss of heterozygosity [3/8]) by array‐based comparative genomic hybridization techniques. These results indicate that tremolite initiates mesothelial injury and persistently frustrates phagocytes, causing subsequent peritoneal fibrosis and MM. The possible mechanisms of carcinogenicity based on fiber diameter/length are discussed., MeT5A (immortalized mesothelial cells) and RAW264.7 (macrophage lineage cells) were damaged by tremolite but not anthophyllite. Tremolite induced diffuse peritoneal thickening, whereas anthophyllite induced focal fibrosis. Furthermore, tremolite‐induced malignant mesothelioma frequently lost Cdkn2a/2b.

The

There is a growing body of data reporting the association of genetic alterations in chromosome 9P21 with the risk of developing cancer. In the current study, we studied the association of a genetic variant in CDKN2A/B, rs1333049, with the risk of developing breast cancer. A total of 339 participants with and without breast cancer entered to the study. Genotyping was done by the TaqMan real-time polymerase chain reaction (RT-PCR) method and gene expression analysis was ran by RT-PCR. Our data showed that the minor allele homozygote in the total population was 10%, whereas for heterozygote was 38%. The dominant genetic model demonstrated that individuals with breast cancer had advanced TNM classification. Moreover, the logistic regression revealed that individuals who had CC/CG genotypes might have an enhanced risk of developing breast cancer when compared to the holders of GG genotype (e.g., OR = 2.8; 95% CI,1.4-5.4; p = .001), after regulated for confounders; age and body mass index. Furthermore, our analysis showed that the CDKN2A/B gene was downregulated in patients (p < .001). We showed a meaningful relationship of CDKN2A/B with the risk of breast cancer, cancer, showing the importance of studies in great sample size and several centers for studying the value of the marker as a risk classification in the management of patients with breast cancer.

Background: Chromosomal region 9p21.3 is most robustly associated with coronary artery disease (CAD) in western European populations. However, heterogeneity in CAD phenotypes leads to uncertainty whether 9p21.3 is associated with stable and/or acute clinical presentations of CAD. 9p21.3 is rich in regulatory elements, but the underlying mechanisms of its actions in CAD remain unclear. We investigate the association of 9p21.3 two haplotype blocks lead variants (rs10757278 and rs518394) with first-ever non-fatal myocardial infarction (MI) in CAD patients and their association with CDKN2B mRNA expression in peripheral blood mononuclear cells 6 months after the event. Methods: We included CAD patients with sustained first MI (n = 523) and controls (n = 583). Gene expression was assessed in 72 patients 6 months after MI and 43 healthy controls. TaqMan® technology was used for the gene expression and genotyping analysis. Results: CDKN2B mRNA was significantly lower in MI patients compared with the controls (p = 0.002) and in patients carrying the rs10757278 G risk allele versus AA homozygotes (p = 0.012) 6 months after the event. While we confirmed the association of rs10757278 with CDKN2B expression in MI patients, we failed to find an association between the investigated variants and MI or disease burden. Conclusions: We suggest a dysregulation of gene expression in the 9p21.3 region six months after acute MI, which is affected by a genetic variant in patients. The rs10757278 rare allele is one factor that might lead to prolonged risk for proatherogenic complications. © 2019 The Canadian Society of Clinical Chemists

Objective. DNA methylation played a vital role in the progression of diabetic retinopathy. In this study, we aimed to explore the role of DNA cytosine-5-methyltransferase 1 (DNMT1) in the development of early diabetic retinopathy and its potential underlying mechanism. Methods. Eight-week-old healthy Mongolian gerbils were used to establish type 1 diabetes using streptozotocin (STZ). Alteration of weight, fasting blood glucose, density of RGCs (Tuj1-labeled), and H&E-stained retinal cross sections were applied to evaluate the diabetic retinopathy mouse model. The global DNA methylation level of the retina at different time points after STZ injection was measured using the global methylation assay. Western blot was used to detect the protein expression of DNMT1, DNA methyltransferase 3A (DNMT3A), and 3B (DNMT3B). Quantitative reverse transcription-polymerase chain reactions (qRT-PCR) and western blot were used to determine the expression of CDKN2B. Cell proliferation and cell cycle were evaluated by the MTS assay and flow cytometry. Results. STZ injection caused the increased global DNA methylation level, which reached a maximum at 6 weeks after injection. Moreover, STZ injection caused the damage of RGCs. At 6 weeks after STZ injection, the expression levels of DNMT1 and DNMT3B were significantly increased in the STZ group. DNMT1-induced DNA hypermethylation inhibited the expression of CDKN2B (a negative regulator of cell cycle). DNMT1-mediated DNA methylation facilitated RGC proliferation via regulating the expression of CDKN2B. Conclusion. DNMT1-mediated DNA methylation played an important role in STZ-induced diabetic retinopathy via modulating CDKN2B expression.

Dogs represent a unique spontaneous cancer model. Osteosarcoma (OSA) is the most common primary bone tumor in dogs (OMIA 001441-9615), and strongly resembles human forms of OSA. Several large- to giant-sized dog breeds, including the Leonberger, have a greatly increased risk of developing OSA. We performed genome-wide association analysis with high-density imputed SNP genotype data from 273 Leonberger cases with a median age of 8.1 [3.1–13.5] years and 365 controls older than eight years. This analysis revealed significant associations at the CDKN2A/B gene locus on canine chromosome 11, mirroring previous findings in other dog breeds, such as the greyhound, that also show an elevated risk for OSA. Heritability (h2SNP) was determined to be 20.6% (SE = 0.08; p-value = 5.7 × 10−4) based on a breed prevalence of 20%. The 2563 SNPs across the genome accounted for nearly all the h2SNP of OSA, with 2183 SNPs of small effect, 316 SNPs of moderate effect, and 64 SNPs of large effect. As with many other cancers it is likely that regulatory, non-coding variants underlie the increased risk for cancer development. Our findings confirm a complex genetic basis of OSA, moderate heritability, and the crucial role of the CDKN2A/B locus leading to strong cancer predisposition in dogs. It will ultimately be interesting to study and compare the known genetic loci associated with canine OSA in human OSA.

Background Although cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) is upregulated in glioma, its function and potential mechanism in glioma remain unclear. Methods CDKN2B-AS1 level in glioma tissues and cell lines LN229, U251, and U87 was measured by qRT-PCR. Loss of function assays using short hairpin RNA for CDKN2B-AS1 (sh-CDKN2B-AS1) were performed to evaluate the effect of CDKN2B-AS1 on cell invasion, migration, proliferation, and apoptosis. The relationship among CDKN2B-AS1, miR-199a-5p, and DDR1 was determined by bioinformatics analysis and luciferase reporter assay. Rescue experiments were conducted to explore the function of CDKN2B-AS1 and miR-199a-5p in glioma. An in vivo animal model of lentivirally transduced U87 glioma xenografts in mice was established to confirm the role of CDKN2B-AS1. Results CDKN2B-AS1 is significantly upregulated in glioma tissues and cell lines. CDKN2B-AS1 knockdown significantly inhibits cell proliferation, invasion, and migration, while promotes apoptosis of glioma cell lines U251 and U87. Further, a miR-199a-5p inhibitor attenuates the inhibitory effects of sh-CDKN2B-AS1 on these cell phenotypes. CDKN2B-AS1 positively regulates DDR1 expression by directly sponging miR-199a-5p. Moreover, CDKN2B-AS1 knockdown efficiently inhibits U87 tumor xenograft growth in mice. Conclusion Our study reveals that CDKN2B-AS1 promotes glioma development by regulating the miR-199a-5p/DDR1 axis, suggesting that this lncRNA might be a potential therapeutic target.

Long-term complications such as radiation-induced second malignancies occur in a subset of patients following radiation-therapy, particularly relevant in pediatric patients due to the long follow-up period in case of survival. Radiation-induced gliomas (RIGs) have been reported in patients after treatment with cranial irradiation for various primary malignancies such as acute lymphoblastic leukemia (ALL) and medulloblastoma (MB). We perform comprehensive (epi-) genetic and expression profiling of RIGs arising after cranial irradiation for MB (n = 23) and ALL (n = 9). Our study reveals a unifying molecular signature for the majority of RIGs, with recurrent PDGFRA amplification and loss of CDKN2A/B and an absence of somatic hotspot mutations in genes encoding histone 3 variants or IDH1/2, uncovering diagnostic markers and potentially actionable targets., Radiation-induced gliomas (RIGs) have been reported in patients after treatment with cranial irradiation for various primary malignancies but their origin are still unclear. Here, the authors define the genomic, epigenetic and transcriptional landscape of 32 RIGs cases.