ATG Start Site Research Articles

Effect of SNPs in the Promoter Region on the Expression of Cytochrome P450 2E1 (CYP2E1) in Pig Liver.

Boar taint, an unfavorable odor in the meat of intact male pigs, is caused primarily by the accumulation of two compounds: androstenone and skatole. This multifactorial trait is regulated by numerous dietary, management and genetic factors. At the mechanistic level, there are many genes known to be involved in boar taint metabolism. Cytochrome P450 2E1 (CYP2E1) impacts boar taint through the phase I metabolism of skatole. The aim of this study was to identify single-nucleotide polymorphisms (SNPs) within the CYP2E1 gene promoter and explore their relationship with the expression of CYP2E1 mRNA and protein. Sequencing of the promoter region using pools of genomic DNA identified seven promoter region SNPs at -159, -586, -1693, -1806, -2322, -2369 and -2514 bp upstream of the ATG start site. Genomic DNA was obtained from 65 boars from the three major swine breeds: Duroc, Landrace and Yorkshire, and individual animals were genotyped for the identified SNPs. RNA was isolated from liver tissue and quantitative PCR was performed to measure CYP2E1 gene expression, while levels of CYP2E1 protein in liver were measured by Western blotting. Significant within-breed variation in CYP2E1 protein and mRNA expression was observed, indicating significant differences in gene expression among individuals. However, levels of CYP2E1 mRNA and protein were not significantly correlated. Two SNPs within the promoter were significantly associated with CYP2E1 mRNA expression, but not with protein expression. This study provides evidence of additional mutations affecting the gene expression of CYP2E1 and suggests that factors that affect the differences in translation of CYP2E1 mRNA may also be important in affecting skatole metabolism.

A novel GATA2 distal enhancer mutation results in MonoMAC syndrome in 2 second cousins

AbstractMutations in the transcription factor GATA2 can cause MonoMAC syndrome, a GATA2 deficiency disease characterized by several findings, including disseminated nontuberculous mycobacterial infections, severe deficiencies of monocytes, natural killer cells, and B lymphocytes, and myelodysplastic syndrome. GATA2 mutations are found in ∼90% of patients with a GATA2 deficiency phenotype and are largely missense mutations in the conserved second zinc-finger domain. Mutations in an intron 5 regulatory enhancer element are also well described in GATA2 deficiency. Here, we present a multigeneration kindred with the clinical features of GATA2 deficiency but lacking an apparent GATA2 mutation. Whole genome sequencing revealed a unique adenine-to-thymine variant in the GATA2 –110 enhancer 116,855 bp upstream of the GATA2 ATG start site. The mutation creates a new E-box consensus in position with an existing GATA-box to generate a new hematopoietic regulatory composite element. The mutation segregates with the disease in several generations of the family. Cell type–specific allelic imbalance of GATA2 expression was observed in the bone marrow of a patient with higher expression from the mutant-linked allele. Allele-specific overexpression of GATA2 was observed in CRISPR/Cas9-modified HL-60 cells and in luciferase assays with the enhancer mutation. This study demonstrates overexpression of GATA2 resulting from a single nucleotide change in an upstream enhancer element in patients with MonoMAC syndrome. Patients in this study were enrolled in the National Institute of Allergy and Infectious Diseases clinical trial and the National Cancer Institute clinical trial (both trials were registered at www.clinicaltrials.gov as #NCT01905826 and #NCT01861106, respectively).

Intermittent hypoxia-induced enhancement of sociability and working memory associates with CNTNAP2 upregulation.

Hypoxia is an environmental risk factor for many disorders throughout life. Perinatal hypoxia contributes to autism spectrum disorder (ASD), while hypoxic conditions in the elderly facilitate memory deficits. However, the effects of hypoxia on adolescence remains elusive. CNTNAP2 is a critical molecule in ASD pathogenesis with undefined mechanisms. We investigate hypoxia's impact on adolescence and the underlying mechanism related to CNTNAP2. Three-chamber social approach test, Y maze, Morris Water Maze and Open Field Test were applied to evaluate behavioral alterations. Immunoblotting, 5'- RACE and dual-luciferase reporter assay were performed to examine CNTNAP2 protein expression, transcription start site (TSS) of human CNTNAP2 gene and CNTNAP2 promoter activity, respectively. Intermittent hypoxia treatment improved social behaviors and working memory in adolescent mice. CNTNAP2 was increased in the brains of hypoxia-treated mice. The sequencing results identified the TSS at 518 bp upstream of the translation start site ATG. Hypoxia upregulated CNTNAP2 by interacting with functional hypoxia response elements in CNTNAP2 promoter. Intermittent hypoxia enhanced sociability and working memory associated with CNTNAP2 upregulation. Our study provides novel insights into intermittent hypoxia's impact on development and the interaction between genetic and environmental risk factors in ASD pathogenesis.

TERT Promoter Mutations and Telomerase in Melanoma.

Malignant melanoma is an extremely malignant tumor with a high mortality rate and an increasing incidence with a high mutation load. The frequency of mutations in the TERT promoter exceeds the frequency of any known noncoding mutations in melanoma. A growing number of recent studies suggest that the most common mutations in the TERT promoter (ATG start site −124C>T and −146C>T) are associated with increased TERT mRNA expression, telomerase activity, telomere length, and poor prognosis. Recently, it has been shown that TERT promoter mutations are more correlated with the occurrence, development, invasion, and metastasis of melanoma, as well as emerging approaches such as the therapeutic potential of chemical inhibition of TERT promoter mutations, direct telomerase inhibitors, combined targeted therapy, and immunotherapies. In this review, we describe the latest advances in the role of TERT promoter mutations and telomerase in promoting the occurrence, development, and poor prognosis of melanoma and discuss the clinical significance of the TERT promoter and telomerase in the treatment of melanoma.

An Anthocyanin-Related Glutathione S-Transferase, MrGST1, Plays an Essential Role in Fruit Coloration in Chinese Bayberry (Morella rubra).

Chinese bayberry (Morella rubra) is a fruit tree economically important in China and accumulates abundant amounts of anthocyanins in fruit as it ripens. Owing to the fact that all anthocyanin containing fruit tissues in Chinese bayberry are edible and anthocyanins can provide various health benefits in human body, the mechanisms underpinning anthocyanin accumulation in this fruit are worthy of investigation. It has been known that in plants anthocyanins are synthesized in the cytoplasmic surface of the endoplasmic reticulum and subsequently transported into the vacuole for storage, and glutathione S-transferases (GSTs) have been verified to be involved in this process. But the characterization and functionalization of the GST counterpart in Chinese bayberry is not available. The GST anthocyanin transporter MrGST1 was discovered to be related with anthocyanin accumulation in fruit from distinct developmental stages of “Biqi,” a staple cultivar that accumulates over 1 mg/g anthocyanins in ripe fruit. The expression of MrGST1 was well associated with anthocyanin accumulation either in fruit collected at six developmental stages or in ripe fruit from 12 cultivars. MrGST1 was found to be responsible for the transport of anthocyanins but not proanthocyanidins when the Arabidopsis tt19 mutant was functionally complemented. Transient ectopic expression of MrGST1 in combination with MrMYB1.1 and MrbHLH1 dramatically boosted pigmentation in Nicotiana tabacum leaves in contrast to MrMYB1.1 and MrbHLH1. The promoter of MrGST1 comprised eight MYB binding sites (MBSs) according to cis-element analysis. Data from yeast one-hybrid assay and dual-luciferase tests demonstrated that MrMYB1.1 exerted considerable transactivation effect on the MrGST1 promoter by recognizing the MBS4, the fourth MBS from the ATG start site. Our results together provided molecular evidence for the contribution of MrGST1 in regulating anthocyanin accumulation in Chinese bayberry fruit.

Genomic and Computational Analysis of Novel SNPs in TNP1 Gene Promoter Region of Bos indicus Breeding Bulls.

Transition nuclear proteins (TNPs), the principal proteins identified in the condensing spermatids chromatin, have been found to play a key role in histone displacement and chromatin condensation during mammalian spermatogenesis. One such gene belonging to the TNP family called TNP1 gene is abundantly expressed in the regulation of spermatogenesis, and its sequence is remarkably well conserved among mammals. Genomic analysis, by sequencing and computational approach, was used to identify the novel polymorphisms and to evaluate the molecular regulation of TNP1 gene expression in Sahiwal cattle breeding bulls. DNA samples were sequenced to identify novel single nucleotide polymorphisms (SNPs) in the TNP1 gene. Modern computational tools were used to predict putative transcription factor binding in the TNP1 promoter and CpG islands in the TNP1 promoter region. In the TNP1 gene, four SNPs, three TATA boxes, and one CAAT box were identified. One CAAT box was discovered at 89 bp upstream of start site ATG. The computational analyses indicated that the polymorphisms inside the promoter sequence results in an added HNF-1 transcription factor binding site. In contrast, the other variations may remove the naturally occurring SRF transcription factor binding site. The CpG islands in the TNP1 promoter region were predicted to be absent by the MethPrimer program before and after SNP site mutations. These findings pave the way for more research into the TNP1 gene's promoter activity and the links between these SNPs and reproductive attributes in the Sahiwal breeding bulls.

Frequency of somatic mutations in TERT promoter, TP53 and CTNNB1 genes in patients with hepatocellular carcinoma from Southern Italy.

Somatic mutations in the TERT promoter and in the TP53 and CTNNB1 genes are considered drivers for hepatocellular carcinoma (HCC) development. They show variable frequencies in different geographic areas, possibly depending on liver disease etiology and environmental factors. TP53, CTNNB1 and TERT genetic mutations were investigated in tumor and non-tumor liver tissues from 67 patients with HCC and liver tissue specimens from 41 control obese subjects from Southern Italy. Furthermore, TERT expression was assessed by reverse transcription-quantitative PCR. Neither CTNNB1 mutations or TP53 R249S substitution were detected in any case. The TP53 R72P polymorphism was found in 10/67 (14.9%) tumors, but was not found in either non-tumor tissues (P=0.001) or controls (P=0.009). TERT gene promoter mutations were found in 29/67 (43.3%) tumor tissues but were not found in either non-tumor (P<0.0001) or control liver specimens (P<0.0001). The most frequent mutation in the tumors was the known hot spot at −124 bp from the TERT ATG start site (−124G>A, 28 cases, 41.8%; P<0.0001). A new previously never reported TERT promoter mutation (at −297 bp from the ATG, −297C>T) was found in 5/67 (7.5%) tumors, in 0/67 (0%) non-tumor (P<0.0001), and in 0/41 (0%) controls (P=0.07). This mutation creates an AP2 consensus sequence, and was found alone (1 case) or in combination (4 cases) with the −124 bp mutation. The mutation at −124 and −297 bp induced a 33-fold (P<0.0001) and 40-fold increase of TERT expression levels, respectively. When both mutations were present, TERT expression levels were increased >300-fold (P=0.001). A new TERT promoter mutation was identified, which generates a de novo binding motif for AP2 transcription factors, and which significantly increases TERT promoter transcriptional activity.

The Study of the Impact of Genetic Polymorphism of Leptin Gene G2548A on Obesity and Its Related Traits

BackgroundLeptin is a neuroendocrine protein hormone with 167 amino acids mainly produced by adipose tissue that regulates food intake and energy expenditure to maintain relative stability of body weight and energy storage over long period of time. A single nucleotide polymorphism in leptin, consisting of G to A substitution at nucleotide −2548 upstream from ATG start site (LEP G‐2548A, rs7799039) has been found to influence leptin expression, possibly at the transcriptional level and was associated with increased leptin secretion in adipocytes. However, these high levels of leptin do not serve their function in controlling the obesity of these individuals possibly due to a condition known as ‘leptin resistance’.AimAssess the risk of the genetic polymorphism of leptin gene G‐2548A on obesity.Material and MethodA case‐control study which consisted of 66 individuals of different BMI (46 normal, 16 overweight and 4 obese). The different genotypes of leptin G‐2548A were determined by restriction fragment length polymorphism polymerase reaction after DNA extraction from saliva samples.ResultsA high risk of obesity was seen among carrier of AA genotype compared to other genotypes (GG&amp;GA) (OR=2.6, 95% CI= 0.9–7.5). Furthermore AA genotype was found to be a risk factor for abdominal obesity (OR=1.3, 95%CI= 0.5–3.7).ConclusionCarrier of AA genotype of leptin G‐2548A polymorphism are at risk of obesity.Support or Funding InformationRak Medical and Health Sciences UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

204 - Mitochondria-specific SQR deficiency in mice causes lethal impairment of sulfur respiration

Sulfide quinone reductase (SQR) protein is a mitochondrial enzyme highly conserved among species and has been considered as an important enzyme mediating proton transfer in mitochondria during sulfur metabolism (respiration), however, the detailed mechanism and physiological impacts of SQR function are still unknown. We generated SQR genome-edited mice to investigate functions of SQR in sulfur respiration. Deletion of translational start site ATG in mouse Sqr locus caused illegitimate translation and resulted in lack of mitochondrial localization signal in N-terminal region of SQR, we have named this mutant allele as SQRΔN. In SQRΔN mutant mice, SQRΔN protein is expressed at the same level as wild-type mice, but the SQR∆N protein is not localized in mitochondria and remains in cytosol. Pups from SQR∆N heterozygous parents showed mendelian distribution of the genotypes, indicating that the ablation of SQR in mitochondria is not associated with reduced viability in utero. However, SQRΔN homozygous mice showed growth arrest after the third postnatal weeks and they die within tenth week after birth. We quantified the sulfide metabolites in several organs (liver, lung, heart and muscle) of SQRΔN mice and their wild-type littermates via LC-MS/MS analysis and as we expected, SQRΔN mice showed a sulfur metabolite profile different from that of the wild-type littermates. Immortalized embryonic fibroblast derived from SQRΔN homozygous mice showed the decreased mitochondrial membrane potential formation. Mitochondria in soleus muscle and liver of SQRΔN showed markedly altered mitochondrial morphology in transmission electron microscopy analysis. These results indicate that SQR in mitochondria is indeed contribute to the membrane potential formation and energy metabolisms.

Abstract 4798: Targeting hTERT for treatment of glioblastoma (GBM)

Abstract Approximately 86% of GBM tumors exhibit a mutation at -124 or -146 bp upstream from the ATG start site in the transcription activating promoter region of Human telomerase reverse transcriptase (hTERT). Mutations in the hTERT promoter are known to impair repression, leading to overexpression of hTERT and tumor maintenance. Yet, complete understanding of mechanistic implications is still necessary. While overexpressed hTERT is associated with oncogenesis and resistance to apoptosis, we also observe phenotypes unrelated to the reverse transcription function. We characterized long-term glioma cell lines and glioma PDX models by hTERT promoter mutation status, hTERT mRNA expression, and hTERT protein expression in subcellular fractions. The -124 and -146 mutations are located in the major 5-12 G-quadruplex and result in misfolding of the silencer element, thus causing over-expression of hTERT. Using a diverse small molecule library, we identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, TG-4260, which binds to the 26 mer base-pair heteroduplex loop, which is the nucleation site for cooperative folding of the major 5-12 G-quadruplex. The chaperone effect of TG-4260 corrects DNA hTERT G-quadruplex misfolding resulting from the promoter mutations and restores the silencer function of the G-quadruplex. TG-4260 directly decreases the transcription activity of the −124, −124/125, −138/139, and −146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. This is the first example of the use of a pharmacoperone molecule to correct the misfolding of a DNA G-quadruplex element resulting from mutations in an early folding intermediate. We screened GBM cell models against this novel small molecule inhibitor that interferes with mutated hTERT promoter and demonstrated that TG-4260 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. Finally, telomere phenotypes from treated cells indicate non-canonical functions of telomerase may also contribute to glioma pathogenesis. Citation Format: Saumya Reddy Bollam, Hyun-Jin Kang, Sen Peng, Vijay Gokhale, Laurence Hurley, Michael Berens, Harshil Dhruv. Targeting hTERT for treatment of glioblastoma (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4798.

Hydroxysteroid dehydrogenase HSD1L is localised to the pituitary-gonadal axis of primates.

Steroid hormones play clinically important and specific regulatory roles in the development, growth, metabolism, reproduction and brain function in human. The type 1 and 2 11-beta hydroxysteroid dehydrogenase enzymes (11β-HSD1 and 2) have key roles in the pre-receptor modification of glucocorticoids allowing aldosterone regulation of blood pressure, control of systemic fluid and electrolyte homeostasis and modulation of integrated metabolism and brain function. Although the activity and function of 11β-HSDs is thought to be understood, there exists an open reading frame for a distinct 11βHSD-like gene; HSD11B1L, which is present in human, non-human primate, sheep, pig and many other higher organisms, whereas an orthologue is absent in the genomes of mouse, rat and rabbit. We have now characterised this novel HSD11B1L gene as encoded by 9 exons and analysis of EST library transcripts indicated the use of two alternate ATG start sites in exons 2 and 3, and alternate splicing in exon 9. Relatively strong HSD11B1L gene expression was detected in human, non-human primate and sheep tissue samples from the brain, ovary and testis. Analysis in non-human primates and sheep by immunohistochemistry localised HSD11B1L protein to the cytoplasm of ovarian granulosa cells, testis Leydig cells, and gonadatroph cells in the anterior pituitary. Intracellular localisation analysis in transfected human HEK293 cells showed HSD1L protein within the endoplasmic reticulum and sequence analysis suggests that similar to 11βHSD1 it is membrane bound. The endogenous substrate of this third HSD enzyme remains elusive with localisation and expression data suggesting a reproductive hormone as a likely substrate.

Abstract 756: Molecular characterization of triploid and tetraploid urine FISH samples

Abstract Urothelial carcinomas (UC) are characterized, among other aberrations, by chromosome 3, 7, 17 gains and 9p21 (p16) loss. Since UC cells readily exfoliate into the urine, molecular cytogenetics techniques have been used to detect cells consistent with UC diagnosis and follow-up: a multitarget Fluorescence In Situ Hybridization (FISH) test, composed of 3, 7 and 17 centromeres and 9p21 locus multicolor probes, has been proved useful for atypical cells identification in both void urine and lower and upper urinary tract samples before morphological changes are apparent through cytological and histological examination. To date, there are no uniform criteria for urine FISH scoring though the test is commonly considered positive following the UroVysion Bladder Cancer Kit criteria (Abbott Molecular): 4 or more cells with a gain of 2 or more chromosomes (polysomy), and/or 12 or more cells with 9p21 homozygous deletion. This definition of polysomy includes trisomy and tetrasomy classes, but, as tetraploidy may represent cells at S/G2 phase, this criterion may lead to false-positives; moreover it has been described that also inflammation induces tetraploidy, and that small percentages of normal urine cells show trisomy/tetrasomy. On the other hand, triploidy and tetraploidy may have a direct role in tumorigenesis, as an intermediate state in the development of cancer aneuploidy. At the moment little is reported about the clinical correlations of urine FISH cases showing only trisomy/tetrasomy and more data are needed to clarify the diagnostic value of triploid and tetraploid urothelial cells. Aiming to better classify patients with a trisomy/tetrasomy urine FISH result, we extracted genomic DNA from 50 fixed urine cell suspensions, previously tested by urine FISH, and investigated them for the presence of fibroblast growth factor receptor 3 (FGFR3) and telomerase reverse transcriptase (TERT) promoter mutations, which constitute the most recurrent somatic alterations in BC. In particular frequent mutations in exons 7, 10 and 15 of FGFR3 characterize non-muscle invasive UC while mutations in the core promoter region of TERT gene, mainly at -124 and -146 positions from the ATG start site, have emerged as the most frequent somatic genetic alteration in both non-muscle invasive and muscle invasive UC. All specimens were subjected to PCR amplification and Sanger sequencing for FGFR3 exon 7 and 10, and for the C228T and C250T TERT promoter mutations. A preliminary analysis of 20 urine FISH cases showed 4 samples with trisomy/tetrasomy, and 1 of these 4 had a mutation of the TERT promotor. Complete results of the FGFR3 and TERT promoter molecular analysis will be presented and correlated to both FISH results and patients clinical data, in order to evaluate if the presence of FGFR3 and/or TERT promoter mutations in urine samples with FISH detected trisomy/tetrasomy may help to identify patients with a higher risk of recurrence and progression. Citation Format: Lorenza Pecciarini, Valeria De Pascali, Ilaria Francaviglia, Anna Talarico, Chiara Iacona, Massimo Freschi, Maria Giulia Cangi, Claudio Doglioni. Molecular characterization of triploid and tetraploid urine FISH samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 756. doi:10.1158/1538-7445.AM2017-756

Abstract 1169: mtTERT promoter as a target for treatment of glioblastoma

Abstract Approximately 86% of GBM tumors exhibit mutation at -124 or -146 bases upstream of the ATG start site in the transcription activating promoter region of Human telomerase reverse transcriptase (hTERT). Mutation in the promoter region of hTERT impairs repression, leading to overexpression of hTERT; inappropriate hTERT is associated with oncogenesis, tumor maintenance, and resistance to apoptosis. We surveyed long-term glioma cell lines and glioma PDX models for mt-hTERT; mRNA and protein expression of hTERT were assessed by qPCR and western blot. The -124 and -146 mutations are located in the major 5-12 G-quadruplex and result in misfolding of the silencer element and consequent over-expression of hTERT. Using a diverse small molecule library, we identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, TG-4260, which binds to the 26 mer base-pair heteroduplex loop, which is the nucleation site for cooperative folding of the major 5-12 G-quadruplex. The chaperone effect of TG-4260 corrects DNA hTERT G-quadruplex misfolding resulting from the somatic mutations and restores the silencer function of the G-quadruplex thereby reducing hTERT activity. TG-4260 directly decreases the transcription activity of the WT and the −124, −124/125, −138/139, and −146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. Finally, TG-4260 significantly inhibits telomerase and shortens telomere length after five days of treatment and induces a senescence-like phenotype. This is the first example of the use of a pharmacoperone molecule to correct the misfolding of a DNA G-quadruplex element resulting from mutations in an early folding intermediate. Finally, we screened GBM cell models against a novel small molecule inhibitor that interferes with mutated hTERT promoter and demonstrated that TG-4260 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. Citation Format: Saumya R. Bollam, Harshil D. Dhruv, Hyun-Jin Kang, Sen Peng, Vijay Gokhale, Laurence Hurley, Michael Berens. mtTERT promoter as a target for treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1169. doi:10.1158/1538-7445.AM2017-1169

OS01.3 mtTERT promoter as a target for treatment of Glioblastoma

AbstractApproximately 86% of GBM tumors exhibit mutation at -124 or -146 bases upstream of the ATG start site in the transcription activating promoter region of Human telomerase reverse transcriptase (hTERT). Mutation in the promoter region of hTERT impairs repression, leading to overexpression of hTERT; inappropriate hTERT is associated with oncogenesis, tumor maintenance, and resistance to apoptosis. We surveyed long-term glioma cell lines and glioma PDX models for mt-hTERT; mRNA and protein expression of hTERT were assessed by qPCR and western blot. The -124 and -146 mutations are located in the major 5–12 G-quadruplex and result in misfolding of the silencer element and consequent over-expression of hTERT. Using a diverse small molecule library, we identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, TG-4260, which binds to the 26 mer base-pair heteroduplex loop, which is the nucleation site for cooperative folding of the major 5–12 G-quadruplex. The chaperone effect of TG-4260 corrects DNA hTERT G-quadruplex misfolding resulting from the somatic mutations and restores the silencer function of the G-quadruplex thereby reducing hTERT activity. TG-4260 directly decreases the transcription activity of the WT and the −124, −124/125, −138/139, and −146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. Finally, TG-4260 significantly inhibits telomerase and shortens telomere length after five days of treatment and induces a senescence-like phenotype. This is the first example of the use of a pharmacoperone molecule to correct the misfolding of a DNA G-quadruplex element resulting from mutations in an early folding intermediate. Finally, we screened GBM cell models against a novel small molecule inhibitor that interferes with mutated hTERT promoter and demonstrated that TG-4260 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes.

Mutation affecting the proximal promoter of Endoglin as the origin of hereditary hemorrhagic telangiectasia type 1

BackgroundHereditary hemorrhagic telangiectasia (HHT) is a vascular multi-organ system disorder. Its diagnostic criteria include epistaxis, telangiectases in mucocutaneous sites, arteriovenous malformations (AVMs), and familial inheritance. HHT is transmitted as an autosomal dominant condition, caused in 85% of cases by mutations in either Endoglin (ENG) or Activin receptor-like kinase (ACVRL1/ACVRL1/ALK1) genes. Pathogenic mutations have been described in exons, splice junctions and, in a few cases with ENG mutations, in the proximal promoter, which creates a new ATG start site. However, no mutations affecting transcription regulation have been described to date in HHT, and this type of mutation is rarely identified in the literature on rare diseases.MethodsSequencing data from a family with HHT lead to single nucleotide change, c.-58G > A. The functionality and pathogenicity of this change was analyzed by in vitro mutagenesis, quantitative PCR and Gel shift assay. Student t test was used for statistical significance.ResultsA single nucleotide change, c.-58G > A, in the proximal ENG promoter co-segregated with HHT clinical features in an HHT family. This mutation was present in the proband and in 2 other symptomatic members, whereas 2 asymptomatic relatives did not harbor the mutation. Analysis of RNA from activated monocytes from the probands and the healthy brother revealed reduced ENG mRNA expression in the HHT patient (p = 0.005). Site-directed mutagenesis of the ENG promoter resulted in a three-fold decrease in luciferase activity of the mutant c.-58A allele compared to wild type (p = 0.005). Finally, gel shift assay identified a DNA-protein specific complex.ConclusionsThe novel ENG c.-58G > A substitution in the ENG promoter co-segregates with HHT symptoms in a family and appears to affect the transcriptional regulation of the gene, resulting in reduced ENG expression. ENG c.-58G > A may therefore be a pathogenic HHT mutation leading to haploinsufficiency of Endoglin and HHT symptoms. To the best of our knowledge, this is the first report of a pathogenic mutation in HHT involving the binding site for a transcription factor in the promoter of ENG.

MNGO-12. CLONAL SELECTION TOWARD TERT PROMOTER MUTATIONS IN PROGRESSIVE RECURRENT MENINGIOMAS

Tumor recurrence after therapy is not infrequent in meningioma, and recurrent meningiomas often undergo a grade-progression to become atypical or malignant upon recurrence. To date, few are known about the clonal selection toward specific mutations in progressive meningiomas. Here, we determined the TERT promoter mutations in a large series of recurrent progressive meningiomas. We screened a cohort of 57 recurrent progressive meningiomas of 24 patients for TERT promoter mutations. We further sequenced the TERT promoter in a second cohort consisting of 13 patients with radiation-induced meningiomas. The TERT promoter was amplified using Sanger sequencing performed using ABI Prism 3730 DNA Analyzer. Mutations at the residues at −124 (1295228) and −146 (1295250) bp from the ATG start site in the TERT promoter were scored. Somatic TERT mutations were detected in seven samples from four patients (16.6%) with recurrent meningiomas. Three of four TERT mutations were detected in secondary progressed Grade II to Grade III meningiomas. Intriguingly, two different patients with initially TERT wild-type Grade II meningiomas, who underwent standard therapy with surgery and radiation, then developed a recurrent meningioma with de novo TERT mutation. Conversely, in the cohort of radiation-induced meningiomas, the TERT mutation was detected only in one Grade I meningioma (7.7%). Patients with TERT mutations exhibited a significantly shorter overall survival than patients harboring TERT wild-type meningiomas (3.9 vs 10.9 years, p= 0.008). We have identified the emergence of TERT promoter mutation in recurrent progressive meningiomas, indicating the presence of ongoing and potentially therapy-related clonal selection impacting the natural history of these tumors. Patients with TERT-mutant meningiomas exhibited significantly shorter overall survival than their TERT wild-type counterparts.

High prevalence of TERT promoter mutations in micropapillary urothelial carcinoma.

Somatic activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the most common genetic alterations in urothelial carcinoma (UC) of the bladder and upper urinary tract. Little is known, however, about TERT-mutation status in the relatively uncommon but clinically aggressive micropapillary (MPC) variant. We evaluated the presence of TERT promoter mutations in MPC of the bladder and upper urinary tract. A retrospective search of our archives for MPC and UC with micropapillary features (2005-2014) was performed. All slides were reviewed to confirm the histologic diagnosis. Thirty-three specimens from 31 patients had FFPE blocks available for DNA analysis and were included in the study. Intratumoral areas of non-micropapillary histology were also evaluated when present. Samples were analyzed with Safe-SeqS, a sequencing error reduction technology, and sequenced using the Illumina MiSeq platform. TERT promoter mutations were detected in all specimens with pure MPC (18 of 18) and UC with focal micropapillary features (15 of 15). Similar to conventional UC, the predominant mutations identified occurred at positions -124 (C228T) (85%) and -146 (C250T) (12%) bp upstream of the TERT ATG start site. In heterogeneous tumors with focal variant histology, intratumoral concordant mutations were found in variant (MPC and non-MPC) and corresponding conventional UC. We found TERT promoter mutations, commonly found in conventional UC, to be frequently present in MPC. Our finding of concordant intratumoral mutational alterations in cases with focal variant histology lends support to the common oncogenesis origin of UC and its variant histology.

Regulation of SET Gene Expression by NFkB.

SET is elevated and mislocalized in the neuronal cytoplasm in brains of Alzheimer's disease (AD) and Down syndrome (DS) patients. Cytoplasm SET leads to inhibition of protein phosphatase 2A and is involved in the tau pathology. However, the regulation of SET gene expression remains elusive. In the present study, we cloned a 1399-bp segment of the 5' flanking region of the human SET gene and identified that the transcription start site (TSS) of SET transcript 1 is located at 123bp upstream of the translation start site ATG in exon 1. Sequence analysis reveals several putative regulatory elements including NFkB, Sp1, and HSE. Luciferase assay and electrophoretic mobility shift assay (EMSA) identified a functional cis-acting NFkB-responsive element in the SET gene promoter. Overexpression and activation of NFkB upregulate transcription of SET isoform 1 but not isoform 2, indicating that the expression of these two isoforms is differentially regulated. The results demonstrate that NFkB plays an important role in regulation of the human SET gene expression. Our findings suggest that oxidative stress and inflammatory responses could result in abnormal SET gene expression, contributing to the tauopathy in AD pathogenesis.

Functional characterization of a defense-related class-III chitinase promoter from Lupinus albus, active in legume and monocot tissues

A class-III chitinase promoter was isolated from Lupinus albus. The region 5′ to the coding sequence of the IF3 gene was amplified by gene walking and sequenced. The proximal 2.0 kb sequence contains a predicted promoter site, including a TATA box, near the ATG start site. To test for minimal sequences needed for promoter activity, the region was restricted into fragments of 1.81, 1.51 and 1.13 kb and cloned into the pDM327 vector, upstream from the bar-gus fusion gene for Biolistic™ transformation. Transformation of lupin embryos, bean callus tissue, maize embryos and Ornithogalum callus demonstrated promoter activity for all fragments. In silico analysis identified putative cis-acting elements in the 1.81 kb fragment that could be important in controlling gene expression. Fungal elicitor activated-, wound-inducible- and ethylene responsive elements were present in the 1.51 kb fragment. Myb elements and CAAT boxes that regulate responses to environmental factors and modulate promoter efficiency were identified in the 1.81 kb fragment. The 1.51 and 1.81 kb fragments were inserted upstream of the gus gene into the pBI121 vector for Agrobacterium tumefaciens transformation of tobacco. Quantitative GUS assays indicated that the promoter fragments are functional in planta and inducible by defense-related signals, wounding, as well as chemical elicitation. All important elements essential for Bion inducibility are present on the shorter (1.51 kb) promoter fragment, but both 5′ distal and proximal cis-elements are required for full functionality. The IF3 promoter is, thus, suitable for use in defense gene constructs prepared for the production of anthracnose resistant lupin.

A study on the anomalies associated with the human sterna in south Indian population

Chondrocalcinosis (CC) is a common cause of joint pain and arthritis that is caused by the deposition of calcium-containing crystals within articular cartilage. Although most cases are sporadic, rare familial forms have been linked to human chromosomes 8 (CCAL1) or 5p (CCAL2) (Baldwin et al. 1995; Hughes et al. 1995; Andrew et al. 1999). Here, we show that two previously described families with CCAL2 have mutations in the human homolog of the mouse progressive ankylosis gene (ANKH). One of the human mutations results in the substitution of a highly conserved amino acid residue within a predicted transmembrane segment. The other creates a new ATG start site that adds four additional residues to the ANKH protein. Both mutations segregate completely with disease status and are not found in control subjects. In addition, 1 of 95 U.K. patients with sporadic CC showed a deletion of a single codon in the ANKH gene. The same change was found in a sister who had bilateral knee replacement for osteoarthritis. Each of the three human mutations was reconstructed in a full-length ANK expression construct previously shown to regulate pyrophosphate levels in cultured cells in vitro. All three of the human mutations showed significantly more activity than a previously described nonsense mutation that causes severe hydroxyapatite mineral deposition and widespread joint ankylosis in mice. These results suggest that small sequence changes in ANKH are one cause of CC and joint disease in humans. Increased ANK activity may explain the different types of crystals commonly deposited in human CCAL2 families and mutant mice and may provide a useful pharmacological target for treating some forms of human CC.