scholarly journals SUN-074 Loss-Of-Function Mutations in GATA4 in Patients with 46,XY Disorders of Sex Development Without Cardiac Defects

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Yena Lee ◽  
Arum Oh ◽  
Han-Wook Yoo ◽  
Jin-Ho Choi
Keyword(s):  
Heart Defects ◽  
Disorders Of Sex Development ◽  
External Genitalia ◽  
Luciferase Reporter ◽  
Molecular Characteristics ◽  
Incomplete Penetrance ◽  
Loss Of Function ◽  
Sex Development ◽  
Wide Range

Abstract Background: Disorders of sex development (DSD) encompass a wide range of conditions associated with numerous causative genes. In about 50-60% of 46,XY DSD individuals, the underlying molecular cause remains uncertain. GATA4 haploinsufficiency has been described in patients with congenital heart defects (CHD), while only a few studies reported mutations related to 46,XY DSD phenotype. This study investigated clinical phenotypes and molecular characteristics of two 46,XY DSD patients with GATA4 mutations. Methods: Mutation analysis was performed in patients with 46,XY DSD by whole exome sequencing (WES) using Illumina NextSeq platform. Clinical and endocrine characteristics were reviewed retrospectively. GATA4 variants identified by WES were verified by Sanger sequencing. Functional activity of GATA4 variants was tested by luciferase reporter assay on the SRY and AMH promoter using two different cell systems including HEK293 and NCI-H295R. Results: Subject 1 presented with micropenis and hypospadias at the age of 5 months. Karyotype was 46,XY. Mullerian duct remnants were not found in pelvic ultrasound. The patient underwent urethroplasty at the age of 10 months and was reared as a male. Subject 2 with complete female external genitalia was referred to our hospital because of 46,XY karyotype on G-scanning. The patient underwent laparoscopic orchiectomy at the age of 1.8 years and was assigned as a female. Both patients were responsive to hCG stimulation tests and did not have CHD. Subject 1 harbored a novel heterozygous variant of c.643A>G (p.R215G)] in GATA4, whereas a previously reported variant of c.1220C>A (p.P407Q) was identified in Subject 2. In vitro luciferase reporter assays using SRY and AMH promoter revealed decreased transcriptional activity of both p.P407Q and p.R215G. Conclusions: This study expanded phenotypic spectrum of mutations in GATA4 in patients with 46,XY DSD without CHD. GATA4 mutations in patients with 46,XY DSD may not be associated with CHD. Possible explanations for phenotypical variability comprise incomplete penetrance, variable expressivity, and oligogenic mechanisms.

Molecular Characteristics of Sequence Variants in GATA4 in Patients with 46,XY Disorders of Sex Development without Cardiac Defects

2019 ◽  
Vol 13 (5-6) ◽  
pp. 240-245
Author(s):  
Jin-Ho Choi ◽  
Yena Lee ◽  
Arum Oh ◽  
Gu-Hwan Kim ◽  
Han-Wook Yoo
Keyword(s):  
Heart Defects ◽  
Disorders Of Sex Development ◽  
External Genitalia ◽  
Pathogenic Mutation ◽  
Luciferase Reporter ◽  
Molecular Characteristics ◽  
Sequence Variants ◽  
Sex Development ◽  
Stimulation Tests

A <i>GATA4</i> haploinsufficiency has been well described in patients with congenital heart defects (CHDs), whilst only a few studies have reported mutations related to a 46,XY disorder of sex development (DSD) phenotype. This study investigated the clinical phenotypes and molecular characteristics of two 46,XY DSD patients harboring <i>GATA4</i> variants. Mutation analysis was performed using a targeted gene panel or whole-exome sequencing. The transactivation activity of each variant protein was examined by in vitro luciferase reporter assay using the <i>AMH</i> and <i>SRY</i> promoters. Subject 1 presented with a micropenis and hypospadias. Subject 2 showed complete female external genitalia with a 46,XY karyotype. Both patients were responsive to hCG stimulation tests and did not manifest CHD. A novel heterozygous variant, c.643A>G (p.R215G), in <i>GATA4</i> was identified in Subject 1, whereas Subject 2 harbored a previously reported variant, c.1220C>A (p.P407Q), in <i>GATA4</i> and a previously known pathogenic mutation, i.e., c.226C>T (p.Q76*) in the <i>AR</i> gene. The reporter assays using the <i>SRY</i> and <i>AMH</i> promoters revealed decreased transcriptional activity of both p.P407Q and p.R215G. However, the <i>GATA4</i> p.P407Q variant was classified as likely benign. In conclusion, it is essential to integrate clinical features and endocrine findings when interpreting sequence variants.

scholarly journals Long non-coding RNA OIP5-AS1 aggravates acute lung injury by promoting inflammation and cell apoptosis via regulating the miR-26a-5p/TLR4 axis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Qingsong Sun ◽  
Man Luo ◽  
Zhiwei Gao ◽  
Xiang Han ◽  
Weiqin Wu ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Inflammatory Response ◽  
Cell Apoptosis ◽  
Quantitative Polymerase Chain Reaction ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Interacting Protein ◽  
Wide Range

Abstract Background Acute lung injury (ALI) is a pulmonary disorder that leads to acute respiration failure and thereby results in a high mortality worldwide. Increasing studies have indicated that toll-like receptor 4 (TLR4) is a promoter in ALI, and we aimed to explore the underlying upstream mechanism of TLR4 in ALI. Methods We used lipopolysaccharide (LPS) to induce an acute inflammatory response in vitro model and a murine mouse model. A wide range of experiments including reverse transcription quantitative polymerase chain reaction, western blot, enzyme linked immunosorbent assay, flow cytometry, hematoxylin–eosin staining, RNA immunoprecipitation, luciferase activity and caspase-3 activity detection assays were conducted to figure out the expression status, specific role and potential upstream mechanism of TLR4 in ALI. Result TLR4 expression was upregulated in ALI mice and LPS-treated primary bronchial/tracheal epithelial cells. Moreover, miR-26a-5p was confirmed to target TLR4 according to results of luciferase reporter assay. In addition, miR-26a-5p overexpression decreased the contents of proinflammatory factors and inhibited cell apoptosis, while upregulation of TLR4 reversed these effects of miR-26a-5p mimics, implying that miR-26a-5p alleviated ALI by regulating TLR4. Afterwards, OPA interacting protein 5 antisense RNA 1 (OIP5-AS1) was identified to bind with miR-26a-5p. Functionally, OIP5-AS1 upregulation promoted the inflammation and miR-26a-5p overexpression counteracted the influence of OIP5-AS1 upregulation on cell inflammatory response and apoptosis. Conclusion OIP5-AS1 promotes ALI by regulating the miR-26a-5p/TLR4 axis in ALI mice and LPS-treated cells, which indicates a promising insight into diagnostics and therapeutics in ALI.

scholarly journals LINC00680 enhances hepatocellular carcinoma stemness behavior and chemoresistance by sponging miR-568 to upregulate AKT3

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Gege Shu ◽  
Huizhao Su ◽  
Zhiqian Wang ◽  
Shihui Lai ◽  
Yan Wang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Xenograft Model ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Mechanism Study ◽  
Downstream Signaling ◽  
Mouse Xenograft Model ◽  
High Level

Abstract Background Hepatocellular carcinoma (HCC) has an extremely poor prognosis due to the development of chemoresistance, coupled with inherently increased stemness properties. Long non-coding RNAs (LncRNAs) are key regulators for tumor cell stemness and chemosensitivity. Currently the relevance between LINC00680 and tumor progression was still largely unknown, with only one study showing its significance in glioblastoma. The study herein was aimed at identifying the role of LINC00680 in the regulation HCC stemness and chemosensitivity. Methods QRT-PCR was used to detect the expression of LINC00680, miR-568 and AKT3 in tissue specimen and cell lines. Gain- or loss-of function assays were applied to access the function of LINC00680 in HCC cells, including cell proliferation and stemness properties. HCC stemness and chemosensitivity were determined by sphere formation, cell viability and colony formation. Luciferase reporter, RNA immunoprecipitation (RIP), and RNA pull-down assays were performed to examine the interaction between LINC00680 and miR-568 as well as that between miR-568 and AKT3. A nude mouse xenograft model was established for the in vivo study. Results We found that LINC00680 was remarkably upregulated in HCC tissues. Patients with high level of LINC00680 had poorer prognosis. LINC00680 overexpression significantly enhanced HCC cell stemness and decreased in vitro and in vivo chemosensitivity to 5-fluorouracil (5-Fu), whereas LINC00680 knockdown led to opposite results. Mechanism study revealed that LINC00680 regulated HCC stemness and chemosensitivity through sponging miR-568, thereby expediting the expression of AKT3, which further activated its downstream signaling molecules, including mTOR, elF4EBP1, and p70S6K. Conclusion LINC00680 promotes HCC stemness properties and decreases chemosensitivity through sponging miR-568 to activate AKT3, suggesting that LINC00680 might be a potentially important HCC diagnosis marker and therapeutic target.

scholarly journals p53 upregulated by HIF-1α promotes hypoxia-induced G2/M arrest and renal fibrosis in vitro and in vivo

2018 ◽  
Vol 11 (5) ◽  
pp. 371-382 ◽  
Author(s):  
Limin Liu ◽  
Peng Zhang ◽  
Ming Bai ◽  
Lijie He ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Renal Fibrosis ◽  
Intraperitoneal Administration ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
P53 Expression ◽  
Tubular Cells

Abstract Hypoxia plays an important role in the genesis and progression of renal fibrosis. The underlying mechanisms, however, have not been sufficiently elucidated. We examined the role of p53 in hypoxia-induced renal fibrosis in cell culture (human and rat renal tubular epithelial cells) and a mouse unilateral ureteral obstruction (UUO) model. Cell cycle of tubular cells was determined by flow cytometry, and the expression of profibrogenic factors was determined by RT-PCR, immunohistochemistry, and western blotting. Chromatin immunoprecipitation and luciferase reporter experiments were performed to explore the effect of HIF-1α on p53 expression. We showed that, in hypoxic tubular cells, p53 upregulation suppressed the expression of CDK1 and cyclins B1 and D1, leading to cell cycle (G2/M) arrest (or delay) and higher expression of TGF-β, CTGF, collagens, and fibronectin. p53 suppression by siRNA or by a specific p53 inhibitor (PIF-α) triggered opposite effects preventing the G2/M arrest and profibrotic changes. In vivo experiments in the UUO model revealed similar antifibrotic results following intraperitoneal administration of PIF-α (2.2 mg/kg). Using gain-of-function, loss-of-function, and luciferase assays, we further identified an HRE3 region on the p53 promoter as the HIF-1α-binding site. The HIF-1α–HRE3 binding resulted in a sharp transcriptional activation of p53. Collectively, we show the presence of a hypoxia-activated, p53-responsive profibrogenic pathway in the kidney. During hypoxia, p53 upregulation induced by HIF-1α suppresses cell cycle progression, leading to the accumulation of G2/M cells, and activates profibrotic TGF-β and CTGF-mediated signaling pathways, causing extracellular matrix production and renal fibrosis.

Silencing of hsa_circ_0000515 Inhibits Proliferation, Migration and Invasion of Gastric Cancer Cells by Sponging miR-615-5p In Vitro

2021 ◽  
Vol 11 (8) ◽  
pp. 1466-1476
Author(s):  
Xuli Wang ◽  
Aiping Wang
Keyword(s):  
Gastric Cancer ◽  
Underlying Mechanism ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Pcr Analysis ◽  
Circular Rnas ◽  
Loss Of Function ◽  
Gastric Cancer Cells ◽  
Novel Target

Circular RNAs (circRNAs) have been reported to participate in the molecular mechanism of human cancers. This study investigates the role of circRNA hsa_circ_0000515 in gastric cancer (GC) cells and the underlying mechanism associated with microRNA-615-5p (miR-615-5p). qRT-PCR analysis showed the upregulation of hsa_circ_0000515 and downregulation of miR-615-5p in GC cell lines. Loss-of-function experiments indicated that suppression of hsa_circ_0000515 inhibited cell proliferation, migration, and invasion. Dual-luciferase reporter assay highlighted that hsa_circ_0000515 was able to act as a ceRNA of miR-615-5p. Furthermore, hsa_circ_0000515 could interact with splicing factors and bind miR-615-5p to regulate progression of GC cells. Deficiency of miR-615-5p reverses the inhibitory roles of si-hsa_circ_0000515 on the proliferation, migration, and invasion of GC cells. The findings highlighted the promising uses of hsa_circ_0000515 as a likely novel target for gastric cancer treatment.

scholarly journals LncRNA GAPLINC Promotes Renal Cell Cancer Tumorigenesis by Targeting the miR-135b-5p/CSF1 Axis

2021 ◽  
Vol 11 ◽  
Author(s):  
Siyuan Wang ◽  
Xiaorong Yang ◽  
Wenjie Xie ◽  
Shengqiang Fu ◽  
Qiang Chen ◽  
...  
Keyword(s):  
Renal Cell ◽  
Noncoding Rna ◽  
Cell Cancer ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Long Intergenic Noncoding Rna ◽  
And Migration ◽  
Molecular Therapeutic

BackgroundLong noncoding RNAs (lncRNAs) are closely related to the occurrence and development of cancer. Gastric adenocarcinoma-associated, positive CD44 regulator, long intergenic noncoding RNA (GAPLINC) is a recently identified lncRNA that can actively participate in the tumorigenesis of various cancers. Here, we investigated the functional roles and mechanism of GAPLINC in renal cell carcinoma (RCC) development.MethodsDifferentially expressed lncRNAs between RCC tissues and normal kidney tissues were detected by using a microarray technique. RNA sequencing was applied to explore the mRNA expression profile changes after GAPLINC silencing. After gain- and loss-of-function approaches were implemented, the effect of GAPLINC on RCC in vitro and in vivo was assessed by cell proliferation and migration assays. Moreover, rescue experiments and luciferase reporter assays were used to study the interactions between GAPLINC, miR-135b-5p and CSF1.ResultsGAPLINC was significantly upregulated in RCC tissues and cell lines and was associated with a poor prognosis in RCC patients. Knockdown of GAPLINC repressed RCC growth in vitro and in vivo, while overexpression of GAPLINC exhibited the opposite effect. Mechanistically, we found that GAPLINC upregulates oncogene CSF1 expression by acting as a sponge of miR-135b-5p.ConclusionTaken together, our results suggest that GAPLINC is a novel prognostic marker and molecular therapeutic target for RCC.

scholarly journals A combined in silico, in vitro and clinical approach to characterise novel pathogenic missense variants in PRPF31 in retinitis pigmentosa

2018 ◽  
Author(s):  
Gabrielle Wheway ◽  
Liliya Nazlamova ◽  
Nervine Meshad ◽  
Samantha Hunt ◽  
Nicola Jackson ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
In Silico ◽  
Autosomal Dominant ◽  
Missense Variant ◽  
Incomplete Penetrance ◽  
Clinical Approach ◽  
Loss Of Function ◽  
Autosomal Dominant Retinitis Pigmentosa ◽  
Missense Variants

AbstractAt least six different proteins of the spliceosome, including PRPF3, PRPF4, PRPF6, PRPF8, PRPF31 and SNRNP200, are mutated in autosomal dominant retinitis pigmentosa (adRP). These proteins have recently been shown to localise to the base of the connecting cilium of the retinal photoreceptor cells, elucidating this form of RP as a retinal ciliopathy. In the case of loss-of-function variants in these genes, pathogenicity can easily be ascribed. In the case of missense variants, this is more challenging. Furthermore, the exact molecular mechanism of disease in this form of RP remains poorly understood.In this paper we take advantage of the recently published cryo EM-resolved structure of the entire human spliceosome, to predict the effect of a novel missense variant in one component of the spliceosome; PRPF31, found in a patient attending the genetics eye clinic at Bristol Eye Hospital. Monoallelic variants in PRPF31 are a common cause of autosomal dominant retinitis pigmentosa (adRP) with incomplete penetrance. We use in vitro studies to confirm pathogenicity of this novel variant PRPF31 c.341T>A, p.Ile114Asn.This work demonstrates how in silico modelling of structural effects of missense variants on cryo-EM resolved protein complexes can contribute to predicting pathogenicity of novel variants, in combination with in vitro and clinical studies. It is currently a considerable challenge to assign pathogenic status to missense variants in these proteins.

scholarly journals A Survey of Compound Heterozygous Variants in Pediatric Cancers and Structural Birth Defects

2021 ◽  
Vol 12 ◽  
Author(s):  
Dustin B. Miller ◽  
Stephen R. Piccolo
Keyword(s):  
Heart Defects ◽  
Disorders Of Sex Development ◽  
Compound Heterozygous ◽  
Healthy Population ◽  
Alternative Mechanism ◽  
Disease Development ◽  
Data Resource ◽  
Sex Development ◽  
Gene Copies ◽  
Compound Heterozygous Variants

Compound heterozygous (CH) variants occur when two recessive alleles are inherited and the variants are located at different loci within the same gene in a given individual. CH variants are important contributors to many different types of recessively inherited diseases. However, many studies overlook CH variants because identification of this type of variant requires knowing the parent of origin for each nucleotide. Using computational methods, haplotypes can be inferred using a process called “phasing,” which estimates the chromosomal origin of most nucleotides. In this paper, we used germline, phased, whole-genome sequencing (WGS) data to identify CH variants across seven pediatric diseases (adolescent idiopathic scoliosis: n = 16, congenital heart defects: n = 709, disorders of sex development: n = 79, ewing sarcoma: n = 287, neuroblastoma: n = 259, orofacial cleft: n = 107, and syndromic cranial dysinnervation: n = 172), available as parent-child trios in the Gabriella Miller Kids First Data Resource Center. Relatively little is understood about the genetic underpinnings of these diseases. We classified CH variants as “potentially damaging” based on minor allele frequencies (MAF), Combined Annotation Dependent Depletion scores, variant impact on transcription or translation, and gene-level frequencies in the disease group compared to a healthy population. For comparison, we also identified homozygous alternate (HA) variants, which affect both gene copies at a single locus; HA variants represent an alternative mechanism of recessive disease development and do not require phasing. Across all diseases, 2.6% of the samples had a potentially damaging CH variant and 16.2% had a potentially damaging HA variant. Of these samples with potentially damaging variants, the average number of genes per sample was 1 with a CH variant and 1.25 with a HA variant. Across all samples, 5.1 genes per disease had a CH variant, while 35.6 genes per disease had a HA variant; on average, only 4.3% of these variants affected common genes. Therefore, when seeking to identify potentially damaging variants of a putatively recessive disease, CH variants should be considered as potential contributors to disease development. If CH variants are excluded from analysis, important candidate genes may be overlooked.

scholarly journals Streptomyces sp.: Characterization, Identification and Its Potential as a Ralstonia solanacearum Biological Control Agent in vitro

2019 ◽  
Vol 2 (3) ◽  
pp. 89-96 ◽  
Author(s):  
Rachmad Saputra ◽  
Triwidodo Arwiyanto ◽  
Arif Wibowo
Keyword(s):  
Biological Control ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Biological Control Agent ◽  
Wilt Disease ◽  
Molecular Characteristics ◽  
Streptomyces Sp ◽  
Bacterial Wilt Disease ◽  
Wide Range

Streptomyces sp. bacteria have the potential to produce antibiotic compounds, which are one of the mechanisms that are widely used in biological control. However, in general, biological control mechanisms also occur through competition, cell wall degradation and induced resistance. This study was aimed to determine the physiological, biochemical and molecular characteristics of two isolates of Streptomyces sp. (S-4 and S16 isolates) isolated from the tomatoes roots, and to find out their ability to control Ralstonia solanacearum, which causes bacterial wilt disease on a wide range of hosts. The results showed both Streptomyces sp. isolates had several different physiological and biochemical characteristics and had a different ability to inhibit R. solanacearum in vitro. Streptomyces sp. S-16 isolate had a high similarity with Streptomyces diastaticus subsp. ardesiacus strain NRRL B-1773T based on the molecular identification results. Further research needs to be done to see the potential inhibition of the two Streptomyces isolates in inhibiting the development of bacterial wilt disease in tomato plants caused by R. solanacearum.

CircRNA circ-ERBB2 elevates Warburg effect and facilitates triple-negative breast cancer growth by the miR-136-5p/PDK4 axis

2021 ◽  
Author(s):  
Yihong Huang ◽  
Shuo Zheng ◽  
Ying Lin ◽  
Liming Ke
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Warburg Effect ◽  
Triple Negative ◽  
Pyruvate Dehydrogenase Kinase ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Loss Of Function

Triple negative breast cancer (TNBC) is an aggressive histological subtype of breast cancer. It has been reported that that circRNA circ-ERBB2 (circBase ID: hsa_circ_0007766) is mainly distributed in the cytoplasm of TNBC cells and promotes the proliferation and invasion of TNBC cells. This study aimed to explore the molecular mechanism of circ-ERBB2 regulating the progression of TNBC. Expression of circ-ERBB2 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to investigate the function of circ-ERBB2 in TNBC cells in vitro and in vivo . The regulatory mechanism of circ-ERBB2 was surveyed by bioinformatics analysis, dual-luciferase reporter and RNA immunoprecipitation (RIP) or RNA pull-down assays. We observed that Circ-ERBB2 was overexpressed in TNBC, and TNBC patients with high circ-ERBB2 expression had a poor prognosis. Functionally, circ-ERBB2 knockdown constrained TNBC growth in vivo and reduced Warburg effect, accelerated apoptosis, repressed proliferation, migration, and invasion of TNBC cell in vitro . Mechanically, circ-ERBB2 sponged miR-136-5p to elevate pyruvate dehydrogenase kinase 4 (PDK4) expression. In conclusion, circ-ERBB2 facilitated Warburg effect and malignancy of TNBC cells by the miR-136-5p/PDK4 pathway, at least in part. This study supported circ-ERBB2 as a prognostic indicator for TNBC.

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