OBJECTIVE: To explore the genetic variation of a Chinese family affected with congenital insensitivity to pain with anhidrosis and albinism. METHODS: Whole exome sequencing (WES) was carried out to screen potential variants within genomic DNA extracted from the proband and his parents. Whole genome sequencing (WGS) was applied when variants were not found completely. Suspected variants were validated by Sanger sequencing. RESULTS: WES has identified a heterozygous c.1729G>C (p.G577R) variant of NTRK1 gene and two heterozygous variants of OCA2 gene, namely c.1363A>G (p.R455G) and c.1182+1G>A. WGS has identified two additional heterozygous variants c.(851-798C>T; 851-794C>G) in deep intronic regions of the NTRK1 gene. CONCLUSION The compound heterozygous variants of the NTRK1 gene probably underlay the congenital insensitivity to pain with anhidrosis. And the compound heterozygous variants of the OCA2 gene probably underlay the albinism in the proband. In the case where no variant is detected by WES in the coding region, WGS should be considered to screen potential variants in the whole genome.
Albinism ; Child ; DNA Mutational Analysis ; Hereditary Sensory and Autonomic Neuropathies/genetics* ; Heterozygote ; Humans ; Membrane Transport Proteins ; Mutation ; Pedigree

Proteases are important molecules that are involved in many physiological and pathological processes of the human body, such as growth, apoptosis and metastasis cancer cells. They are potential targets in cancer diagnosis and biotherapy. In this study, we analyzed the salivary protease spectrum of patients with oral squamous cell carcinoma (OSCC), oral benign masses and chronic periodontitis, as well as that of health, using human protease array kits, enzyme-linked immunosorbent assay, western blot and immunofluorescence. The salivary protease spectrum was found to be associated with oral diseases. For example, the saliva of patients with OSCC contained increased numbers of proteases than those of other oral diseases and health. The levels of matrix metalloproteinase (MMP)-1, MMP-2, MMP-10, MMP-12, A disintegrin and metalloprotease (ADAM)9, A disintegrin and metalloprotease with thrombospondin type 13 motifs (ADAMST13), cathepsin V and kallikrein 5 in the saliva of patients with OSCC were significantly increased compared with those of other groups. Taking MMP-1, cathepsin V, kallikrein 5 and ADAM9 as biomarkers of OSCC, cutoff values were199, 11.34, 9.29 and 202.55 pg·mL, respectively. From the area under the curve, sensitivity and specificity, the combination of cathepsin V/kallikrein5/ADAM9 was an optimal biomarker for diagnosing OSCC. Thus, analysis of the salivary protease spectrum may be an innovative and cost-efficient approach to evaluating the health status of the oral cavity. Specifically, increases in cathepsin V, kallikrein 5 and ADAM9 may be useful biomarkers in the screening and diagnosis of OSCC.
ADAM Proteins ; Biomarkers, Tumor ; analysis ; Carcinoma, Squamous Cell ; diagnosis ; metabolism ; Humans ; Matrix Metalloproteinase 9 ; analysis ; Membrane Proteins ; Mouth Neoplasms ; diagnosis ; metabolism ; Saliva ; chemistry

OBJECTIVE: To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role. METHODS: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting. RESULTS: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002. CONCLUSION SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.
Adenosine Triphosphate ; analysis ; Animals ; Animals, Newborn ; Caffeic Acids ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Glycogen Synthase Kinase 3 beta ; physiology ; Lactates ; pharmacology ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocytes, Cardiac ; drug effects ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology

OBJECTIVE: To analyze the clinical presentation and gene of 2 pedigrees with suspected oculocutaneous albinism(OCA), and provide basis for clinical classification, genetic counseling and prenatal diagnosis. METHODS: Variants were identified using next-generation sequencing(NGS) and confirmed by Sanger sequencing in 2 pedigrees with suspected OCA. The pathogenicity of the variants was analyzed according to the American College of Medical Genetics and Genomics (ACMG) standard. RESULTS: Two compound heterozygous mutations of TYR and OCA2 genes were identified respectively in 2 pedigrees with suspected OCA. The mutation of c.819+3insATATGCC in TYR and the mutation of c.1870G>C in OCA2 are first reported in this study. The pathogenicity analysis shows that two novel mutations are likely pathogenic by combination of prediction of SIFT, Polyphen-2 and Human Splicing Finder. CONCLUSION The findings of this study expand the mutational spectrum of OCA. Compound heterozygous mutations in the TYR and OCA2 gene may be responsible for clinical manifestations of 2 pedigrees with suspected OCA.
Albinism, Oculocutaneous ; DNA Mutational Analysis ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Membrane Transport Proteins ; Monophenol Monooxygenase ; Mutation ; Pedigree ; Pregnancy

OBJECTIVE: To determine the frequencies of deafness gene mutations among patients with non-syndromic hearing loss (NSHL) from northern Jiangsu province. METHODS: A total of 117 patients with NSHL were enrolled. The coding region of GJB2 gene, IVS7-2A>G and 2168A>G mutations of SLC26A4 gene, and 1555A>G and 1494C>T mutations of mitochondrial DNA 12S rRNA were subjected to Sanger sequencing. Patients in whom no mutation was detected were further tested by targeted gene capture and high-throughput sequencing. RESULTS: Among the 117 patients, 86 (73.50%) were found to carry mutations. GJB2 gene mutations were found in 61 patients (52.14%), including 22 (18.80%) with homozygous mutations and 39 (33.33%) with heterozygous mutations. SLC26A4 gene mutations were found in 19 patients (16.24%), including 4 (3.42%) with homozygous mutations and 15 with heterozygous mutations (14.53%). Mitochondrial 12S rRNA gene mutation was found in 6 patients (5.13%). Targeted gene capture and high-throughput sequencing of 8 patients identified 4 further cases, including 1 with RDX gene 129_130del and 76_79del compound heterozygous mutations, 1 with OTOF gene 1274G>C homozygous mutation, 1 with SLC26A4 gene 919-2A>G and IVS16-6G>A compound heterozygous mutation, and 1 with SLC26A4 gene 919-2A>G and A1673T compound heterozygous mutation. CONCLUSION The frequency of mutation among patients with NSHL from north Jiangsu was 73.50%, and GJB2 gene was most commonly mutated.
China ; Connexins ; DNA Mutational Analysis ; DNA, Mitochondrial ; Hearing Loss ; genetics ; Humans ; Membrane Proteins ; Mutation ; Sulfate Transporters

This retrospective analysis aimed to investigate the mutation profile of 16 common mutated genes in de novo acute myeloid leukemia (AML) patients. A total of 259 patients who were diagnosed of de novo AML were enrolled in this study. Mutation profiling of 16 candidate genes were performed in bone marrow samples by using Sanger sequencing.We identified at least 1 mutation in 199 of the 259 samples (76.8%), and 2 or more mutations in 31.7% of samples. FLT3-ITD was the most common mutated gene (16.2%, 42/259), followed by CEBPA (15.1%, 39/259), NRAS (14.7%, 38/259), and NPM1 (13.5%, 35/259). Concurrence was observed in 97.1% of the NPM1 mutated cases and in 29.6% of the double mutated CEBPA cases. Distinct patterns of co-occurrence were observed for different hotspot mutations within the IDH2 gene: R140 mutations were associated with NPM1 and/or FLT3-ITD mutations, whereas R172 mutations co-occurred with DNMT3A mutations only. Concurrence was also observed in 86.6% of epigenetic regulation genes, most of which co-occurred with NPM1 mutations. The results showed certain rules in the mutation profiling and concurrence of AML patients, which was related to the function classification of genes. Defining the mutation spectrum and mutation pattern of AML will contribute to the comprehensive assessment of patients and identification of new therapeutic targets.
Adolescent ; Adult ; Aged ; CCAAT-Enhancer-Binding Proteins ; genetics ; Child ; Child, Preschool ; China ; DNA Mutational Analysis ; Female ; GTP Phosphohydrolases ; genetics ; Gene Expression Profiling ; Gene Frequency ; Genetic Predisposition to Disease ; Humans ; Kaplan-Meier Estimate ; Leukemia, Myeloid, Acute ; genetics ; Male ; Membrane Proteins ; genetics ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; Phenotype ; Retrospective Studies ; Young Adult ; fms-Like Tyrosine Kinase 3 ; genetics

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) play an important role in cancer development and progression. However, the mechanism by which they enhance the chemoresistance of ovarian cancer is unknown. METHODS: Conditioned media of BM-MSCs (BM-MSC-CM) were analyzed using a technique based on microRNA arrays. The most highly expressed microRNAs were selected for testing their effects on glycolysis and chemoresistance in SKOV3 and COC1 ovarian cancer cells. The targeted gene and related signaling pathway were investigated using in silico analysis and in vitro cancer cell models. Kaplan-Merier survival analysis was performed on a population of 59 patients enrolled to analyze the clinical significance of microRNA findings in the prognosis of ovarian cancer. RESULTS: MiR-1180 was the most abundant microRNA detected in BM-MSC-CM, which simultaneously induces glycolysis and chemoresistance (against cisplatin) in ovarian cancer cells. The secreted frizzled-related protein 1 (SFRP1) gene was identified as a major target of miR-1180. The overexpression of miR-1180 led to the activation of Wnt signaling and its downstream components, namely Wnt5a, β-catenin, c-Myc, and CyclinD1, which are responsible for glycolysis-induced chemoresistance. The miR-1180 level was inversely correlated with SFRP1 mRNA expression in ovarian cancer tissue. The overexpressed miR-1180 was associated with a poor prognosis for the long-term (96-month) survival of ovarian cancer patients. CONCLUSIONS BM-MSCs enhance the chemoresistance of ovarian cancer by releasing miR-1180. The released miR-1180 activates the Wnt signaling pathway in cancer cells by targeting SFRP1. The enhanced Wnt signaling upregulates the glycolytic level (i.e. Warburg effect), which reinforces the chemoresistance property of ovarian cancer cells.
Adenosine Triphosphate/chemistry* ; Adult ; Aged ; Bone Marrow Cells/cytology* ; Cell Line, Tumor ; Cell Proliferation ; Cells, Cultured ; Drug Resistance, Neoplasm/genetics* ; Female ; Flow Cytometry ; Follow-Up Studies ; Glycolysis ; Humans ; Intercellular Signaling Peptides and Proteins/metabolism* ; Membrane Proteins/metabolism* ; Mesenchymal Stem Cells/cytology* ; MicroRNAs/genetics* ; Middle Aged ; Multivariate Analysis ; Ovarian Neoplasms/genetics* ; Up-Regulation ; Wnt Signaling Pathway

BACKGROUND/AIMS: MicroRNAs (miRNAs) were reported to be responsible for intestinal permeability in diarrhea-predominant irritable bowel syndrome (IBS-D) rats in our previous study. However, whether and how miRNAs regulate visceral hypersensitivity in IBS-D remains largely unknown. METHODS: We established the IBS-D rat model and evaluated it using the nociceptive visceral hypersensitivity test, myeloperoxidase activity assay, restraint stress-induced defecation, and electromyographic (EMG) activity. The distal colon was subjected to miRNA microarray analysis followed by isolation and culture of colonic epithelial cells (CECs). Bioinformatic analysis and further experiments, including dual luciferase assays, quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay, were used to detect the expression of miRNAs and how it regulates visceral hypersensitivity in IBS-D rats. RESULTS: The IBS-D rat model was successfully established. A total of 24 miRNAs were differentially expressed in the distal colon of IBS-D rats; 9 were upregulated and 15 were downregulated. Among them, the most significant upregulation was miR-200a, accompanied by downregulation of cannabinoid receptor 1 (CNR1) and serotonin transporter (SERT). MiR-200a mimic markedly inhibited the expression of CNR1/SERT. Bioinformatic analysis and luciferase assay confirmed that CNR1/SERT are direct targets of miR-200a. Rescue experiments that overexpressed CNR1/SERT significantly abolished the inhibitory effect of miR-200a on the IBS-D rats CECs. CONCLUSIONS: This study suggests that miR-200a could induce visceral hyperalgesia by targeting the downregulation of CNR1 and SERT, aggravating or leading to the development and progression of IBS-D. MiR-200a may be a regulator of visceral hypersensitivity, which provides potential targets for the treatment of IBS-D.
Animals ; Blotting, Western ; Colon ; Computational Biology ; Defecation ; Diarrhea ; Down-Regulation ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells ; Hyperalgesia ; Hypersensitivity ; Irritable Bowel Syndrome ; Luciferases ; Microarray Analysis ; MicroRNAs ; Models, Animal ; Permeability ; Peroxidase ; Rats ; Real-Time Polymerase Chain Reaction ; Receptors, Cannabinoid ; Serotonin Plasma Membrane Transport Proteins ; Serotonin ; Up-Regulation

OBJECTIVE: To explore the role of mitochondrial permeability transition pore (mPTP) in mediating the protective effect of gastrodin against oxidative stress damage in H9c2 cardiac myocytes. METHODS: H9c2 cardiac myocytes were treated with HO, gastrodin, gastrodin+HO, cyclosporin A (CsA), or CsA+gas+HO group. MTT assay was used to detect the survival ratio of H9c2 cells, and flow cytometry with Annexin V-FITC/PI double staining was used to analyze the early apoptosis rate after the treatments. The concentration of ATP and level of reactive oxygen species (ROS) in the cells were detected using commercial kits. The mitochondrial membrane potential of the cells was detected with laser confocal microscopy. The expression of cytochrome C was detected with Western blotting, and the activity of caspase-3 was also assessed in the cells. RESULTS: Gastrodin pretreatment could prevent oxidative stress-induced reduction of mitochondrial membrane potential, and this effect was inhibited by the application of CsA. Gastrodin significantly lowered the levels of ROS and apoptosis-related factors in HO-exposed cells, and such effects were reversed by CsA. CsA significantly antagonized the protective effect of gastrodin against apoptosis in HO-exposed cells. CONCLUSIONS Gastrodin prevents oxidative stress-induced injury in H9c2 cells by inhibiting mPTP opening to reduce the cell apoptosis.
Adenosine Triphosphate ; analysis ; Apoptosis ; drug effects ; Benzyl Alcohols ; antagonists & inhibitors ; pharmacology ; Caspase 3 ; analysis ; Cell Line ; Cell Survival ; drug effects ; Cyclosporine ; pharmacology ; Cytochromes c ; analysis ; Glucosides ; antagonists & inhibitors ; pharmacology ; Humans ; Hydrogen Peroxide ; antagonists & inhibitors ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondrial Membrane Transport Proteins ; physiology ; Myocytes, Cardiac ; drug effects ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; analysis

OBJECTIVE: To detect potential mutation in a pedigree affected with autosomal recessive non-syndromic deafness. METHODS: Mutation analysis was carried out by next generation sequencing, and suspected mutations were verified by Sanger sequencing. RESULTS: A heterozygous c.235delC mutation of the GJB2 gene, together with compound heterozygous mutations of the OTOF gene [c.1194T>A (p.D398E) and c.2180A>G (p.N727S)] were detected in the proband. The sister of the proband (also had hearing loss) has carried a heterozygous c.235delC mutation in the GJB2 gene, in addition with a heterozygous c.2180A>G(p.N727S) mutation of the OTOF gene. By Sanger sequencing, a heterozygous IVS1+2T>A mutation was further detected in the non-coding region of the GJB2 gene in both sisters. CONCLUSION The compound heterozygous c.235delC and IVS1+2T>A mutations of the GJB2 gene probably account for the hearing loss in the two sisters, among which IVS1+2T>A is considered as a novel pathogenic mutation of the GJB2 gene.
Connexins ; genetics ; DNA Mutational Analysis ; Female ; Hearing Loss, Sensorineural ; genetics ; High-Throughput Nucleotide Sequencing ; Humans ; Membrane Proteins ; genetics ; Mutation ; Pedigree