PENK Genes Research Articles

Integrating genome-wide association and transcriptome analysis to provide molecular insights into growth rates in sheep1

Investigating genetic markers and key genes associated with sheep growth rate using integrated multi-omics approaches could provide valuable insights for the sheep industry. Based on the average daily gain (ADG), fast-growing (Ncase=70) and slow-growing (Ncontrol=70) Hu sheep were selected for a genome-wide association study (GWAS). Ten Hu sheep (fast-growing, n = 5; slow-growing, n = 5) and ten Dorper sheep (fast-growing, n = 5; slow-growing, n=5) were selected for comparative transcriptome analysis. We identified hub genes and tissue-specific genes (TSGs) using weighted gene co-expression network analysis (WGCNA) and RNA sequencing (RNA-Seq) data from ten tissues, respectively. Ten genes were found within 50 kb distances of the significant single nucleotide polymorphisms (SNPs). Based on a comparative transcriptomic analysis, a total of 501 and 441 differentially expressed genes (DEGs) were identified in the HF vs. HS and DF vs. DS comparisons, respectively. We found some important signaling pathways closely associated with fat metabolism and energy metabolism, such as “regulation of lipolysis in adipocytes”, “Oxidative phosphorylation”, and “Thermogenesis”. Several DEGs play a crucial role in fat deposition (such as ADRB3, PDE3B, FABP4, SERPINE1, PLIN1, and FOXO6) and muscle development (MYL3). Using the WGCNA analysis, 15 genes were considered as hub genes associated with ADG. Integration of GWAS and RNA-Seq data indicates that BRINP3 and PENK may further influence the growth rate by regulating feeding behavior in sheep. Association analysis of 1,071 Hu sheep populations revealed that mutations in the BRINP3 (BRINP3 g.16903465 T>C) and PENK (PENK g.39289926 T>C) genes were significantly related to the growth traits (P < 0.05). Our research provides novel insights into understanding the molecular mechanisms underlying growth traits in sheep. BRINP3 and PENK genes may be potential key candidate genes related to sheep growth rate.

Evaluation of Kynu, Defb2, Camp, and Penk Expression Levels as Psoriasis Marker in the Imiquimod-Induced Psoriasis Model.

Psoriasis is a noncontagious auto-inflammatory chronic skin disease. So far, some of the inflammatory genes were upregulated in mouse model of psoriasis. This study examined changes in skin mRNA expression of L-kynureninase (Kynu), cathelicidin antimicrobial peptide (Camp), beta-defensin 2 (Defb2), and proenkephalin (Penk) in a mouse model of imiquimod-induced psoriasis. Tree groups of C57BL/6 female mice were allocated. The imiquimod (IMQ) cream was administered to the mice dorsal skin of the two groups to induce psoriatic inflammation. In the treatment group, IMQ was administered 10 min after hydrogel-containing M7 anti-IL-17A aptamer treatment. Vaseline (Vas) was administered to the negative control group. The psoriatic skin lesions were evaluated based on the psoriasis area severity index (PASI) score, histopathology, and mRNA expression levels of Kynu, Camp, Defb2, and Penk using real-time PCR. In order to assess the systemic response, the spleen and lymph node indexes were also evaluated. The PASI and epidermal thickness scores were 6.01 and 1.96, respectively, in the IMQ group, and they significantly decreased after aptamer administration to 1.15 and 0.90, respectively (P < 0.05). Spleen and lymph node indexes showed an increase in the IMQ group, followed by a slight decrease after aptamer treatment (P > 0.05). Additionally, the mRNA expression levels of Kynu, Defb2, Camp, and Penk genes in the IMQ-treated region showed a significant 2.70, 4.56, 3.29, and 2.61-fold increase relative to the Vas mice, respectively (P < 0.05). The aptamer-treated region exhibited a significant decrease in these gene expression levels (P < 0.05). A positive correlation was found between Kynu, Penk, and Camp expression levels and erythema, as well as Camp expression with PASI, scaling, and thickness (P < 0.05). According to our results, it seems that Kynu, Camp, and Penk can be considered appropriate markers for the evaluation of psoriasis in IMQ-induced psoriasis. Also, the anti-IL-17 aptamer downregulated these important genes in this mouse model.

Short Tandem Repeat Variation in the CNR1 Gene Associated With Analgesic Requirements of Opioids in Postoperative Pain Management.

Short tandem repeats (STRs) and variable number of tandem repeats (VNTRs) that have been identified at approximately 0.7 and 0.5 million loci in the human genome, respectively, are highly multi-allelic variations rather than single-nucleotide polymorphisms. The number of repeats of more than a few thousand STRs was associated with the expression of nearby genes, indicating that STRs are influential genetic variations in human traits. Analgesics act on the central nervous system via their intrinsic receptors to produce analgesic effects. In the present study, we focused on STRs and VNTRs in the CNR1, GRIN2A, PENK, and PDYN genes and analyzed two peripheral pain sensation-related traits and seven analgesia-related traits in postoperative pain management. A total of 192 volunteers who underwent the peripheral pain sensation tests and 139 and 252 patients who underwent open abdominal and orthognathic cosmetic surgeries, respectively, were included in the study. None of the four STRs or VNTRs were associated with peripheral pain sensation. Short tandem repeats in the CNR1, GRIN2A, and PENK genes were associated with the frequency of fentanyl use, fentanyl dose, and visual analog scale pain scores 3 h after orthognathic cosmetic surgery (Spearman’s rank correlation coefficient ρ = 0.199, p = 0.002, ρ = 0.174, p = 0.006, and ρ = 0.135, p = 0.033, respectively), analgesic dose, including epidural analgesics after open abdominal surgery (ρ = −0.200, p = 0.018), and visual analog scale pain scores 24 h after orthognathic cosmetic surgery (ρ = 0.143, p = 0.023), respectively. The associations between STRs in the CNR1 gene and the frequency of fentanyl use and fentanyl dose after orthognathic cosmetic surgery were confirmed by Holm’s multiple-testing correction. These findings indicate that STRs in the CNR1 gene influence analgesia in the orofacial region.

Hypermethylated circulating DNA detection using picoliter droplet-based PCR in colorectal cancer.

622 Background: Circulating tumor DNA (ctDNA) is thoroughly investigated as a surrogate biomarker of tumor follow-up, in different cancer types, such as colorectal cancer (CRC). Droplet-based digital PCR (ddPCR) is a highly sensitive and also quantitative method for detection of very low amount of ctDNA. Since many different genes can be mutated within a specific tumor type and also wide mutation spectrum can occur within a specific gene, procedures for ctDNA monitoring can be time consuming and need to be improved for a routinely use. To overcome these drawbacks, we characterized the methylation status of 3 genes frequently hypermethylated in CRC to identify universal markers for tumor follow-up. Methods: The characterization of the methylated status of the WIF, NPY and PENK genes in the tumor DNA was performed in 56 CRC of different stages and 45 corresponding plasma samples using droplet-based dPCR, after DNA bisulfite conversion. A two-panels assay (with albumin as a reference) was developed. Methylation level of these 3 genes in tumor tissues was compared to corresponding normal tissues (n = 22) and plasma samples (MetctDNA). To validate, plasma samples of additional 91 patients were analyzed for the presence of ctDNA both by the characterization of KRAS, BRAF, TP53 and PIK3CA mutations (MutctDNA) and of MetctDNA, at various stages of their follow-up, and 9 of them had MetctDNA assessment during treatment follow-up. Results: All tumor samples were positive for WIF1 and/or NPY markers. Hypermethylation of these two genes was significantly higher in tumor tissue compared to normal, independently of the tumor stage (p &lt; 0.0001). MetctDNA could be detected in 75% of metastatic CRC patients and 24% of localized CRC patients (stage 1 to 3). MetctDNA and MutctDNA fractions were strongly correlated (R2 &gt; 0.9, p &lt; 0.0001). During follow-up, MetctDNA levels changes allowed monitoring of tumor evolution in different stages CRC patients. Conclusions: These results indicate that determination of MetctDNA by droplet-based dPCR can reach same efficiency than MutctDNA for ctDNA assessment, using only 2 markers, and thus could be considered as a universal surrogate marker of tumor follow-up in CRC patients.

Inferring chronological age from DNA methylation patterns of human teeth.

Current methods to determine chronological age from modern and ancient remains rely on both morphological and molecular approaches. However, low accuracy and the lack of standardized protocols make the development of alternative methods for the estimation of individual's age even more urgent for several research fields, such as biological anthropology, biodemography, forensics, evolutionary genetics, and ancient DNA studies. Therefore, the aim of this study is to identify genomic regions whose DNA methylation level correlates with age in modern teeth. We used MALDI-TOF mass spectrometry to analyze DNA methylation levels of specific CpGs located in the ELOVL2, FHL2, and PENK genes. We considered methylation data from cementum, dentin and pulp of 21 modern teeth (from 17 to 77 years old) to construct a mathematical model able to exploit DNA methylation values to predict age of the individuals. The median difference between the real age and that estimated using DNA methylation values is 1.20 years (SD = 1.9) if DNA is recovered from both cementum and pulp of the same modern teeth, 2.25 years (SD = 2.5) if DNA is recovered from dental pulp, 2.45 years (SD = 3.3) if DNA is extracted from cementum and 7.07 years (SD = 7.0) when DNA is recovered from dentin only. We propose for the first time the evaluation of DNA methylation at ELOVL2, FHL2, and PENK genes as a powerful tool to predict age in modern teeth for anthropological applications. Future studies are needed to apply this method also to historical and relatively ancient human teeth.

Opioid-dependent regulation of high and low fear responses in two inbred mouse strains

The molecular mechanisms underlying the susceptibility or resilience to trauma-related disorders remain incompletely understood. Opioids modulate emotional learning, but the roles of specific receptors are unclear. Here, we aimed to analyze the contribution of the opioid system to fear responses in two inbred mouse strains exhibiting distinct behavioral phenotypes. SWR/J and C57BL/6J mice were subjected to five consecutive electric footshocks (1mA each), and the contextual freezing time was measured. Stress-induced alterations in gene expression were analyzed in the amygdala and the hippocampus. In both strains, the fear response was modulated using pharmacological tools. SWR/J mice did not develop conditioned fear but exhibited increased transcriptional expression of Pdyn and Penk in the amygdala region. Blocking opioid receptors prior to the footshocks using naltrexone (2mg/kg) or naltrindole (5mg/kg) increased the freezing responses in these animals. The C57BL/6J strain displayed high conditioned fear, although no alteration in the mRNA abundance of genes encoding opioid precursors was observed. Double-injection of morphine (20mg/kg) following stress and upon context re-exposure prevented the enhancement of freezing. Moreover, selective delta and kappa agonists caused a reduction in conditioned fear responses. To summarize, the increased expression of the Pdyn and Penk genes corresponded to reduced intensity of fear responses. Blockade of the endogenous opioid system restored freezing behavior in stress-resistant animals. The pharmacological stimulation of the kappa and delta opioid receptors in stress-susceptible individuals may alleviate fear. Thus, subtype-selective opioid receptor agonists may protect against the development of trauma-related disorders.

Abstract 2298: Methylated gene markers discovered in nasopharyngeal carcinoma by differential methylation hybridization (DMH) in high density CpG island DNA chips

Abstract Nasopharyngeal carcinoma (NPC) is a prevalent cancer in southern China and south east Asian countries. A variety of tumor suppressor (TS) genes including p16, RASSF1A, EDNRB, DAP-Kinase, RIZ1, E-Cadherin were found to be hypermethylated in NPC. This, however, could only be the tip of an iceberg. We made use of the high throughput differential methylation hybridization (DMH) technique in screening for methylated DNA markers in 244K CpG island DNA chip in three NPC xenografts (C15, C17, 99-186), a NPC tumor cell line (C666) versus an immortalized NP non-cancer cell line (NP69). This DMH screening discovered 100, 213, 372 and 977 genes respectively in 99-186, C17, C15 and C666 with ≥ 5 CpG sites in their gene promotors being methylated. Among these genes, 28 of them were found to have DNA methylation in all the 4 NPC xenografts/ tumor cell line. Bisulphite sequencing was performed in 17 pairs of tumor and normal biopsy samples from 17 NPC patients. This confirmed tumor specific methylation in the promotor regions of 8 gene markers, namely, NRP3, PCDH10, PENK, ADHFE1, IRX4, GSC, PFN2 &amp; GFI1 with positive methylation ratios (+ve MR) in tumor biopsies of 86.7%, 85.7%, 80%, 72.7%, 54.5%, 50%, 41.7% &amp;23.1% respectively and +ve MR of 18.2%, 0%, 0%, 0%, 0%, 37.5%, 12.5% &amp; 0% respectively in normal biopsies. Methylation of CpG sites appears to occur either all or none with 15 to 45 consecutive CpG sites in these genes either all methylated or non-methylated in tumor biopsies probably indicating the need of methylation of a full cluster of CpG sites in order to affect the gene transcription of the discovered genes. Functions of the DMH identified genes includes cell adhesion (PCDH10), signal transduction (PENK), chloride ion binding (NPR3), regulation of DNA dependent transcription (IRX4) and xenobiotic metabolism (ADHFE1). In fact, PCDH10, PENK and GFI1 genes have been reported to be closely associated with NPC/esophageal carcinoma, pancreatic adenocarinoma and T-cell lymphoma respectively. Further analyses in these methylated markers could be useful in the diagnosis and study of etiology of NPC. Citation Format: Wai Wai Cheng, Roger K.C. Ngan, Dora L.W. Kwong, Tim H.M. Huang, Victor W.S. Ma, Stephen C.K. Law, Loretta Tse, Pierre Busson, George S.W. Tsao, Maria Li Lung, Timothy T.C. Yip. Methylated gene markers discovered in nasopharyngeal carcinoma by differential methylation hybridization (DMH) in high density CpG island DNA chips. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2298. doi:10.1158/1538-7445.AM2014-2298

A new noninvasive blood-test for the early detection of colorectal cancer.

348 Background: Colorectal cancer (CRC) remains a major medical problem in the world. Its early detection impacts the prognosis and the cost of treatment. Fecal occult blood test is a current noninvasive screening method for CRC detection in asymptomatic average risk individuals. It detects less than 50% of cancers and yields a high number of “normal” colonoscopies. The dysregulation of DNA methylation has been recognized as playing a crucial role during CRC development. The aim of the study was to develop a CRC-specific methylated DNA test in serum. Methods: First, the GoldenGate Methylation microarray containing 1,505 CpG loci within 807 cancer-related genes was used to study methylation patterns in CRC patients. We performed such assays on 9 tissues (4 CRC, 4 normal autologous tissues, 1 polyp), 17 blood and urine pooled samples (7 CRC, 6 polyps, 4 normal) and 20 stool samples (7 CRC, 3 polyps, 10 normal). The clinical status of each sample was assessed by colonoscopy and/or pathology findings. Among methylated genes detected in tumour tissue, stools, and blood, NPY, PENK and WIF1 genes were selected taking into account their high degree of methylation. We then carried out a clinical validation by quantitative multiplex methylation-specific PCR (QM-MSP) in two phases: firstly on 30 tissues (15 CRC, 15 homologous normal tissues) and secondly on 193 serum samples (32 CRC, 161 normal). Results: For to obtain the best accuracy of QM-MSP test, we defined for both biological sources a cumulative methylation index (CMI) by adding of the methylation percentage of each gene. At the CMI of 20.0% on tissues, our QM-MSP test allows to diagnose the CRC with the highest accuracy (100% of specificity and 100% of sensitivity). From sera, the CMI has been standardized at 2.0% giving a sensitivity and specificity for detecting CRC of 94.4% and 59.4% respectively (NPV of 92.1% and PPV of 67.8%). Conclusions: We developed a QM-MSP-based analysis of NPY, PENK, and WIF1 methylated genes in serum. This test classified correctly and with a high accuracy the healthy individuals and colon cancer patients. We propose here new sensitive serum-based epigenetic biomarkers as an effective and simple new noninvasive test for CRC screening in the average and high risk populations.

Methylation of ELOVL2 gene as a new epigenetic marker of age

The discovery of biomarkers able to predict biological age of individuals is a crucial goal in aging research. Recently, researchers' attention has turn toward epigenetic markers of aging. Using the Illumina Infinium HumanMethylation450 BeadChip on whole blood DNA from a small cohort of 64 subjects of different ages, we identified 3 regions, the CpG islands of ELOVL2, FHL2, and PENK genes, whose methylation level strongly correlates with age. These results were confirmed by the Sequenom's EpiTYPER assay on a larger cohort of 501 subjects from 9 to 99 years, including 7 cord blood samples. Among the 3 genes, ELOVL2 shows a progressive increase in methylation that begins since the very first stage of life (Spearman's correlation coefficient = 0.92) and appears to be a very promising biomarker of aging.

Characterization of zebrafish proenkephalin reveals novel opioid sequences

Cloning and molecular characterization of an homologous gene to proenkephalin in a teleost, the zebrafish Danio rerio is presented in this paper. The new zebrafish proenkephalin (zfPENK) encodes a 249 amino acid polypeptide that displays an identity of 40% to mammalian PENKs and which contains the consensus sequences for four Met-enkephalins, one Leu-enkephalin and one Met-enkephalin-Gly-Tyr, described for the first time in teleosts. Expression studies indicate that zfPENK is selectively expressed in zebrafish brain. Our findings support the concept that PENK genes might have been highly conserved throughout evolution and that its origins might be placed more than 400 million years ago. Moreover, we present evidence that the heptapeptide Met-enkephalin-Gly-Tyr present in fish might be anterior in evolution to the heptapeptide Met-enkephalin-Arg-Phe present in tetrapods. Also another homologous sequence to proenkephalin in zebrafish genome is presented. This sequence might stand for the third exon of a possible duplicate gene of zfPENK. Our findings not only present new data in relation to the evolution of opioid peptides in vertebrates, but also we present a new heptapeptide with putative differential activity than the other peptides derived from the mammalian proenkephalins. Future research will define the functional role of this new heptapeptide in the mechanism that describes opioid activity.

Oxidative stress induces proorphanin FQ and proenkephalin gene expression in astrocytes through p38- and ERK-MAP kinases and NF-kappaB.

Oxidative stress has been implicated in the pathogenesis of stroke, traumatic brain injuries, and neurodegenerative diseases affecting both neuronal and glial cells in the CNS. In this study we have demonstrated that reactive oxygen species (ROS) dramatically induce the expression of two neuropeptide genes, the opioid proenkephalin (pENK) and the opioid-related proorphanin FQ (pOFQ; also known as pronociceptin) in primary astrocytes. Hydrogen peroxide (H2O2) treatment dose-dependently increased pENK and pOFQ mRNA levels with a maximal effect ( approximately 15-fold increase) being detected at 50 microM concentration. Exposing the astrocyte cultures to hypoxia and subsequent re-oxygenation also led to a profound elevation of pOFQ and pENK mRNA levels. Western blot analysis and immunocytochemistry revealed that H2O2 treatment elicited the phosphorylation and nuclear translocation of ERK 1/2 and p38 MAP kinases. Blockade of the p38 or the ERK MAP kinase pathways (by SB202190 and PD98059, respectively) prevented the H2O2-induced increase in pENK and pOFQ mRNA levels indicating a central role for these cascades in the regulation of pOFQ and pENK genes in response to oxidative stress. Regulation of pOFQ and pENK gene expression by ERK and p38 activation may be mediated through the transcription factor cAMP-response element binding protein (CREB). We observed CREB phosphorylation in response to H2O2, which was also prevented by SB202190 and PD98059. The nuclear factor-kappaB (NF-kappaB) pathway appears to be involved exclusively in the induction of pOFQ transcription by H2O2, as NF-kappaB inhibitors antagonized the effect of oxidative stress on pOFQ, but not on pENK expression. The profound induction of these genes by oxidative stress and these other factors may suggest a role for orphanin FQ and enkephalin in injury and stress responses of the CNS and neuropathophysiological conditions involving reactive oxygen species.

Cloning and Characterization of Hamster Proenkephalin Gene

Our previous studies have shown that the hamster adrenal, like the human, contains high levels of preproenkephalin (PPenk) mRNA and enkephalin peptides, and may serve as a mammalian model for the in vivo study of proenkephalin (Penk) gene expression, peptide biosynthesis, and release. To define further the factors that may regulate hamster Penk gene expression, the hamster Penk gene was isolated from a genomic library prepared from Syrian hamster liver. The hamster Penk gene contains four exons and three introns and encodes 268 amino acids including six copies of Met-enkephalin containing peptides and one copy of Leu-enkephalin. In the 5' upstream region, there are TATA and GC boxes and multiple putative regulatory elements including the cAMP response element, AP-1, AP-2, AP-4, and the glucocorticoid response element (GRE). Possible GREs are also present in the introns. A comparison with the human and the rat Penk genes indicates that both the human and hamster Penk gene contain three introns, while the rat Penk gene has two introns. The intron missing from the rat Penk gene is short and separates the first and second exons of the hamster and human genes. In addition, the hamster and human genes share a region (100 bases) in the 5' upstream sequence that is 98% homologous. It is of interest that Penk gene expression is high in the adrenal medulla of both human and hamster, but is much lower in the rat. These homologous regions and the extra intron may contain regulatory features responsible for a high level of expression in the human and hamster adrenal medulla.