ABSTRACT
OBJECTIVE@#To explore the characteristics of SLC25A13 gene variants in 16 infants with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).@*METHODS@#The infants were subjected to high-throughput DNA sequencing for coding exons and flanking regions of the target genes. Suspected variants were verified by Sanger sequencing and bioinformatic analysis.@*RESULTS@#Among the 16 NICCD cases, 15 were found to harbor pathogenic variants. Among these, IVS14-9A>G, c.1640G>A, c.762T>A, c.736delG, c.1098Tdel and c.851G>A were previously unreported.@*CONCLUSION@#Six novel SLC25A13 variants were found by high-throughput sequencing, which has enriched the spectrum of SLC25A13 gene variants and provided a basis for genetic counseling and prenatal diagnosis.
Subject(s)
Humans , Infant , Infant, Newborn , Calcium-Binding Proteins/genetics , Cholestasis, Intrahepatic/genetics , Citrullinemia/genetics , Mitochondrial Membrane Transport Proteins/genetics , Mutation , Organic Anion Transporters/genetics , Protein DeficiencyABSTRACT
OBJECTIVE@#To determine the carrier rate for 21 inherited metabolic diseases among a Chinese population of childbearing age.@*METHODS@#A total of 897 unrelated healthy individuals (including 143 couples) were recruited, and DNA was extracted from their peripheral blood samples. Whole exome sequencing (WES) was carried out to screen potential variants among 54 genes associated with 21 inherited metabolic diseases. Pathogenic and likely pathogenic variants and unreported loss-of-function variants were analyzed.@*RESULTS@#One hundred fourty types of pathogenic/likely pathogenic variants (with an overall number of 183) and unreported loss-of-function variants were detected, which yield a frequency of 0.20 per capita. A husband and wife were both found to carry pathogenic variants of the SLC25A13 gene and have given birth to a healthy baby with the aid of preimplantation genetic diagnosis. The detected variants have involved 40 genes, with the most common ones including ATP7B, SLC25A13, PAH, CBS and MMACHC. Based on the Hardy-Weinberg equilibrium, the incidence of the 21 inherited metabolic diseases in the population was approximately 1/1100, with the five diseases with higher incidence including citrullinemia, methylmalonic acidemia, Wilson disease, glycogen storage disease, and phenylketonuria.@*CONCLUSION@#This study has preliminarily determined the carrier rate and incidence of 21 inherited metabolic diseases among a Chinese population of childbearing age, which has provided valuable information for the design of neonatal screening program for inherited metabolic diseases. Pre-conception carrier screening can provide an important measure for the prevention of transmission of Mendelian disorders in the population.
Subject(s)
Female , Humans , Infant, Newborn , Asian People/genetics , China , Exome , Metabolic Diseases/genetics , Mitochondrial Membrane Transport Proteins/genetics , Oxidoreductases/genetics , Exome SequencingABSTRACT
Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.
Subject(s)
Animals , Male , Mice , Rats , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , Atractyloside/pharmacology , Peptidyl-Prolyl Isomerase F , Matrix Metalloproteinases , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore , RNA, MessengerABSTRACT
OBJECTIVES@#To study the physical and neuropsychological development of children with Citrin deficiency (CD).@*METHODS@#A total of 93 children, aged 1.9-59.8 months, who were diagnosed with CD by @*RESULTS@#For the 93 children with CD, the incidence rate of failure to thrive was 25% (23 children) and the proportion of small for gestational age was 47% (44 children). For the 100 cases of CD, the incidence rates of growth retardation, underweight, emaciation, overweight, and microcephalus were 23% (23 cases), 14% (14 cases), 4% (4 cases), 8% (8 cases), and 9% (9 cases), respectively. The incidence rate of neuropsychological developmental delay was 25% (25 cases), and the incidence rates of development delay in the five domains of adaptability, gross motor, fine motor, language, and social ability were 7% (7 cases), 15% (15 cases), 7% (7 cases), 9% (9 cases), and 7% (7 cases), respectively.@*CONCLUSIONS@#Physical and neuropsychological developmental delay can be observed in children with CD, and physical and neuropsychological development should be regularly assessed.
Subject(s)
Child , Humans , Infant , Citrullinemia , Mitochondrial Membrane Transport Proteins , Retrospective StudiesABSTRACT
OBJECTIVE@#To explore the clinical characteristics and genetic findings of patients with infantile intrahepatic cholestasis.@*METHODS@#The clinical data were collected in children who were admitted to the Department of Gastroenterology in Children's Hospital, Capital Institute of Pediatrics from June 2017 to June 2019 and were suspected of inherited metabolic diseases. Next generation sequencing based on target gene panel was used for gene analysis in these children. Sanger sequencing technology was used to verify the genes of the members in this family.@*RESULTS@#Forty patients were enrolled. Pathogenic gene variants were identified in 13 patients (32%), including @*CONCLUSIONS@#The etiology of infantile intrahepatic cholestasis is complex. Next generation sequencing is helpful in the diagnosis of infantile intrahepatic cholestasis.
Subject(s)
Child , Humans , Alagille Syndrome/genetics , Cholestasis, Intrahepatic/genetics , Citrullinemia , Genetic Testing , High-Throughput Nucleotide Sequencing , Mitochondrial Membrane Transport Proteins , MutationABSTRACT
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.
Subject(s)
Animals , Rats , Adenosine Triphosphate , Animals, Newborn , Caffeic Acids , Pharmacology , Cell Hypoxia , Cells, Cultured , Glycogen Synthase Kinase 3 beta , Physiology , Lactates , Pharmacology , Mitochondria, Heart , Physiology , Mitochondrial Membrane Transport Proteins , Myocytes, Cardiac , Proto-Oncogene Proteins c-akt , Physiology , Rats, Sprague-Dawley , Reactive Oxygen Species , Metabolism , Signal Transduction , PhysiologyABSTRACT
OBJECTIVE@#To study the clinical features and gene mutation spectrum of children with sideroblastic anemia (SA) and the clinical value of targeted next-generation sequencing in the molecular diagnosis of children with SA.@*METHODS@#Clinical data were collected from 36 children with SA. Targeted next-generation sequencing was used to detect mutations in SA-related pathogenic genes and genes associated with heme synthesis and mitochondrial iron metabolism. The association between genotype and clinical phenotype was analyzed.@*RESULTS@#Of the 36 patients, 32 had congenital sideroblastic anemia (CSA) and 4 had myelodysplastic syndrome with ring sideroblasts (MDS-RS). Mutations in CSA-related genes were detected in 19 children (19/36, 53%), among whom 9 (47%) had ALAS2 mutation, 4 (21%) had SLC25A38 mutation, and 6 (32%) had mitochondrial fragment deletion. No pathogenic gene mutation was detected in 4 children with MDS-RS. Among the 19 mutations, 89% (17/19) were known mutations and 11% (2/19) were novel mutations. The novel mutation of the ALAS2 gene c.1153A>T(p.I385F) was rated as "possibly pathogenic" and the novel mutation of the SLC25A38 gene c.175C>T(p.Q59X) was rated as "pathogenic".@*CONCLUSIONS@#ALAS2 and SLC25A38 gene mutations are commonly seen in children with CSA, but mitochondrial gene fragment deletion also accounts for a relatively high proportion. For children with hypoplastic anemia occurring in infancy, mitochondrial disease should be considered.
Subject(s)
Child , Humans , 5-Aminolevulinate Synthetase , Anemia, Sideroblastic , Genetics , Genetic Diseases, X-Linked , Mitochondrial Membrane Transport Proteins , Mutation , Myelodysplastic Syndromes , PhenotypeABSTRACT
OBJECTIVE@#To explore the clinical and genetic features of an infant with citrin deficiency (CD).@*METHODS@#Clinical data of the patient was collected and analyzed. Genomic DNA was extracted from peripheral blood samples collected from the patient and her parents. Targeted exome sequencing was performed to explore the genetic cause, and Sanger sequencing was used to confirm the detected variants. SLC25A13 mRNA was extracted from peripheral blood lymphocytes of the infant. The effect of novel mutation of SLC25A13 was analyzed by reverse transcription-PCR, cDNA cloning and Sanger sequencing.@*RESULTS@#The SLC25A13 genotype of the patient was determined as c.845_c.848+1delG/c.1841+3_1841+4delAA, with the latter having not been reported. The mutation has affected the splicing of the SLC25A13 mRNA, giving rise to an aberrant transcript [r.1841_1842ins1841+1_1841+67; 1841+3_c.1841+4del].@*CONCLUSION@#A novel SLC25A13 mutation c.1841+3_1841+4delAA and the resultant abnormal splicing variant were discovered by combined DNA sequencing and cDNA cloning. The finding has enabled definite diagnosis of CD and enriched the spectrum of SLC25A13 mutations.
Subject(s)
Female , Humans , Base Sequence , Citrullinemia , Mitochondrial Membrane Transport Proteins , Genetics , Mutation , PedigreeABSTRACT
Polyamines (putrescine, spermidine, and spermine) are essential polycations that play important roles in various physiological and pathophysiological processes in mammalian cells. The study was to investigate their role in cardioprotection against ischemia/reperfusion (I/R) injury and the underlying mechanism. Isolated hearts from male Sprague-Dawley rats were Langendorff-perfused and cardiac I/R was achieved by 30 min of global ischemia followed by 120 min of reperfusion. Different concentrations of polyamines (0.1, 1, 10, and 15 μmol/L of putrescine, spermidine, and spermine), cyclosporin A (0.2 μmol/L), or atractyloside (20 μmol/L) were given 10 min before the onset of reperfusion. The hemodynamics were monitored; the lactate dehydrogenase (LDH) levels in the coronary effluent were measured spectrophotometrically; infarct size was determined by the 2,3,5-triphenyltetrazolium chloride staining method; and mitochondrial permeability transition pore (MPTP) opening was determined spectrophotometrically by the Ca-induced swelling of isolated cardiac mitochondria. The results showed that compared to I/R alone, 0.1 and 1 μmol/L polyamines treatment improved heart function, reduced LDH release, decreased infarct size, and these effects were inhibited by atractyloside (MPTP activator). In isolated mitochondria from normal rats, 0.1 and 1 μmol/L polyamines treatment inhibited MPTP opening. However, 10 and 15 μmol/L polyamines treatment had the opposite effects, and these effects were inhibited by cyclosporin A (MPTP inhibitor). Our findings showed that polyamines may have either protective or damaging effects on hearts suffering from I/R by inhibiting or activating MPTP opening.
Subject(s)
Animals , Male , Rats , Cyclosporine , Pharmacology , Mitochondria, Heart , Physiology , Mitochondrial Membrane Transport Proteins , Physiology , Myocardial Reperfusion Injury , Polyamines , Metabolism , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Humans , Adenosine Triphosphate , Apoptosis , Benzyl Alcohols , Pharmacology , Caspase 3 , Cell Line , Cell Survival , Cyclosporine , Pharmacology , Cytochromes c , Glucosides , Pharmacology , Hydrogen Peroxide , Pharmacology , Membrane Potential, Mitochondrial , Mitochondrial Membrane Transport Proteins , Physiology , Myocytes, Cardiac , Metabolism , Oxidative Stress , Reactive Oxygen SpeciesABSTRACT
Kidney stone disease is a major cause of chronic renal insufficiency. The role of long non-coding RNAs (lncRNAs) in calcium oxalate-induced kidney damage is unclear. Therefore, we aimed to explore the roles of lncRNAs in glyoxylate-exposed and healthy mouse kidneys using microarray technology and bioinformatics analyses. A total 376 mouse lncRNAs were differentially expressed between the two groups. Using BLAST, 15 lncRNA homologs, including AU015836 and CHCHD4P4, were identified in mice and humans. The AU015836 expression in mice exposed to glyoxylate and the CHCHD4P4 expression in human proximal tubular epithelial (HK-2) cells exposed to calcium oxalate monohydrate were analyzed, and both lncRNAs were found to be upregulated in response to calcium oxalate. To further evaluate the effects of CHCHD4P4 on the cell behavior, we constructed stable CHCHD4P4-overexpressing and CHCHD4P4-knockdown HK-2 cells. The results showed that CHCHD4P4 inhibited cell proliferation and promoted the epithelial-mesenchymal transition in kidney damage and fibrosis caused by calcium oxalate crystallization and deposition. The silencing of CHCHD4P4 reduced the kidney damage and fibrosis and may thus be a potential molecular target for the treatment of kidney stones.
Subject(s)
Humans , Animals , Rabbits , Kidney Calculi/genetics , Mitochondrial Membrane Transport Proteins/physiology , Cell Proliferation/genetics , Epithelial-Mesenchymal Transition/genetics , RNA, Long Noncoding/physiology , Fibrosis , Calcium Oxalate , Kidney Calculi/physiopathology , Up-Regulation , Cell Fractionation , Cell Line , Blotting, Western , Microarray Analysis , Cell Proliferation/physiology , Epithelial-Mesenchymal Transition/physiology , Real-Time Polymerase Chain ReactionABSTRACT
Infantile liver failure syndrome type 1 (ILFS1) is a Mendelian disease due to biallelic mutations in the cytoplasmic leucyl-tRNA synthetase gene (LARS). This study aimed to report the clinical and molecular features of the first non-caucasian ILFS1 patient, providing reliable evidences for the definite diagnosis of ILFS1. The 2 years and 9 months old male patient was referred to the hospital with hepatosplenomegaly over 1 year. At age 17 months, he was found to have hepatosplenomegaly and anemia. Since then, he had been managed in different hospitals. The laboratory tests showed liver dysfunction, hypoproteinemia, coagulopathy and anemia, along with histologically-confirmed cirrhosis and fatty liver; however, the etiology remained undetermined. The subsequent SLC25A13 mutation analysis by means of prevalent mutation screening and Sanger sequencing only revealed a paternally-inherited mutation c.1658G>A, and no aberrant SLC25A13 transcripts could be detected from the maternal allele on cDNA cloning analysis, ruling out the possibility of citrin deficiency. Further target exome high-throughout sequencing of genes relevant to genetic liver diseases detected a paternal c.2133_2135del (p.L712del) and a maternal c.1183G>A (p.D395N) mutation in LARS gene. This finding was then confirmed by Sanger sequencing, and ILFS1 was thus definitely diagnosed. The child has been followed up till age 4 years, and his condition became stabilized.
Subject(s)
Child, Preschool , Humans , Male , High-Throughput Nucleotide Sequencing , Leucine-tRNA Ligase , Genetics , Liver Failure , Diagnosis , Genetics , Mitochondrial Membrane Transport Proteins , Genetics , MutationABSTRACT
<p><b>OBJECTIVE</b>To detect potential mutations in six patients with citrullinemia.</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral blood samples from the patients. Mutations of the ASS1, ASL and SLC25A13 genes were screened using microarray genotyping combined with direct sequencing.</p><p><b>RESULTS</b>One patient was diagnosed with argininosuccinate lyase deficiency, and has carried a homozygous c.1311T>G (p.Y437*) mutation of the ASL gene. The remaining five patients were diagnosed with neonatal intrahepatic cholestasis due to citrin deficiency, and have respectively carried mutations of the SLC25A13 gene including [c.851-854delGTAT+c.851-854delGTAT], [c.851-854delGTAT+IVS6+5G>A], [c.851-854delGTAT+IVS16ins3kb], [c.851-854delGTAT+IVS6-11A>G] and [c.851-854delGTAT+c.1638-1660dup23]. Among these, the c.1311T>G mutation was first identified in the Chinese population, and the IVS6-11A>G mutation was a novel variation which may affect the splicing, as predicted by Human Splicing Finder software.</p><p><b>CONCLUSION</b>This study has confirmed the molecular diagnosis of citrullinemia in six patients and expanded the mutational spectrum underlying citrullinemia.</p>
Subject(s)
Female , Humans , Infant , Infant, Newborn , Male , Argininosuccinate Lyase , Genetics , Argininosuccinate Synthase , Genetics , Citrullinemia , Genetics , DNA Mutational Analysis , Mitochondrial Membrane Transport Proteins , Genetics , MutationABSTRACT
<p><b>OBJECTIVE</b>To identify pathogenic mutations in a Chinese pedigree affected with methylmalonic academia for genetic counseling and prenatal diagnosis.</p><p><b>METHODS</b>Molecular analysis of the MUT, MMACHC, MMAA and MMAB genes was performed for the proband with methylmalonic academia by Ion Torrent semiconductor sequencing. Candidate mutations were validated by Sanger sequencing. The couple was offered prenatal diagnosis via analyzing of the fetal DNA through amniocentesis.</p><p><b>RESULTS</b>The proband was found to be compound heterozygous for c.609G>A (p.Trp203X) and c.658-660del AAG (p.Lys220del) mutations, which were inherited respectively from each of his parents. Prenatal diagnosis showed that the fetus has inherited two wild-type parental alleles.</p><p><b>CONCLUSION</b>The targeted Ion Torrent PGM sequencing has detected pathogenic mutations in the Chinese pedigree affected with methylmalonic academia, which has provided molecular evidence for clinical diagnosis, genetic counseling and prenatal diagnosis for the family.</p>
Subject(s)
Adult , Female , Humans , Infant , Male , Pregnancy , Alkyl and Aryl Transferases , Genetics , Amino Acid Metabolism, Inborn Errors , Embryology , Genetics , Asian People , Genetics , Base Sequence , Carrier Proteins , Genetics , China , High-Throughput Nucleotide Sequencing , Methods , Methylmalonyl-CoA Mutase , Genetics , Mitochondrial Membrane Transport Proteins , Genetics , Molecular Sequence Data , Mutation , Pedigree , Prenatal Diagnosis , MethodsABSTRACT
<p><b>Objective</b>To investigate the protective effect of Wuziyanzong Pills (WYP) in the rat model of oligoasthenospermia (OAS) and its action mechanism.</p><p><b>METHODS</b>Sixty male SD rats were equally randomized into six groups: normal control, OAS model, Shengjing Capsules (1.6 g per kg of the body weight), low-dose WYP (1 g per kg of the body weight), medium-dose WYP (2 g per kg of the body weight), and high-dose WYP (4 g per kg of the body weight). The OAS model was established by intragastric administration of Tripterygium glucoside at 30 mg per g per d for 6 weeks. From the 3rd week of modeling, the rats of the medication groups were treated intragastrically with corresponding drugs for 4 weeks. Then all the rats were sacrificed for measurement of the testicular and epididymal organ coefficients, examination of epididymal sperm quality and apoptosis, and detection of the openness of the sperm mitochondrial permeability transition pore (MPTP). Histopathological changes in the testis were observed by HE staining and the apoptosis of spermatogenic cells determined by Hochest staining.</p><p><b>RESULTS</b>WYP obviously improved the organ coefficients of the testis and epididymis, increased sperm concentration, motility and viability, decreased the apoptosis of spermatogenic cells, and inhibited the abnormal openness of MPTP in the OAS model rats. HE staining showed that the number and levels of spermatogenic cells were significantly increased while Hochest staining manifested that the apoptosis of spermatogenic cells was remarkably inhibited in the seminiferous tubules of the testis in the WYP-treated rats.</p><p><b>CONCLUSIONS</b>WYP can improve sperm quality and reduce the apoptosis of spermatogenic cells (including sperm) in OAS model rats, which may be related with its inhibitory effect on the abnormal openness of MPTP.</p>
Subject(s)
Animals , Male , Rats , Apoptosis , Asthenozoospermia , Drug Therapy , Drugs, Chinese Herbal , Pharmacology , Epididymis , Mitochondrial Membrane Transport Proteins , Oligospermia , Drug Therapy , Random Allocation , Rats, Sprague-Dawley , Sperm Count , Sperm Motility , Spermatozoa , Cell Biology , Testis , TripterygiumABSTRACT
<p><b>OBJECTIVE</b>To analyze the clinical features and potential mutations of the SLC25A13 gene in a boy affected with neonatal intrahepatic cholestasis.</p><p><b>METHODS</b>Clinical data and peripheral venous blood sample of the child, and peripheral venous blood samples of both parents, were collected. All coding exons of the SLC25A13 gene were amplified with PCR and subjected to direct DNA sequencing.</p><p><b>RESULTS</b>The boy was found to be a compound heterozygote carrying c.851_854delGTAT and IVS16ins3kb mutations of the SLC25A13 gene, which were respectively inherited from his mother and father.</p><p><b>CONCLUSION</b>Based on its clinical and genetic features, the patient was diagnosed with neonatal intrahepatic cholestasis caused by citrin deficiency.</p>
Subject(s)
Female , Humans , Infant , Infant, Newborn , Male , Base Sequence , Cholestasis, Intrahepatic , Genetics , Citrullinemia , DNA Mutational Analysis , Family Health , Heterozygote , Mitochondrial Membrane Transport Proteins , Genetics , Mutagenesis, Insertional , Mutation , Sequence DeletionABSTRACT
BACKGROUND: A newborn screening (NBS) program has been utilized to detect asymptomatic newborns with inherited metabolic diseases (IMDs). There have been some bottlenecks such as false-positives and imprecision in the current NBS tests. To overcome these issues, we developed a multigene panel for IMD testing and investigated the utility of our integrated screening model in a routine NBS environment. We also evaluated the genetic epidemiologic characteristics of IMDs in a Korean population. METHODS: In total, 269 dried blood spots with positive results from current NBS tests were collected from 120,700 consecutive newborns. We screened 97 genes related to NBS in Korea and detected IMDs, using an integrated screening model based on biochemical tests and next-generation sequencing (NGS) called NewbornSeq. Haplotype analysis was conducted to detect founder effects. RESULTS: The overall positive rate of IMDs was 20%. We identified 10 additional newborns with preventable IMDs that would not have been detected prior to the implementation of our NGS-based platform NewbornSeq. The incidence of IMDs was approximately 1 in 2,235 births. Haplotype analysis demonstrated founder effects in p.Y138X in DUOXA2, p.R885Q in DUOX2, p.Y439C in PCCB, p.R285Pfs*2 in SLC25A13, and p.R224Q in GALT. CONCLUSIONS: Through a population-based study in the NBS environment, we highlight the screening and epidemiological implications of NGS. The integrated screening model will effectively contribute to public health by enabling faster and more accurate IMD detection through NBS. This study suggested founder mutations as an explanation for recurrent IMD-causing mutations in the Korean population.
Subject(s)
Humans , Infant, Newborn , Computational Biology , DNA/chemistry , Dried Blood Spot Testing , Galactokinase , Genomics , Haplotypes , High-Throughput Nucleotide Sequencing , Incidence , Membrane Proteins/genetics , Metabolic Diseases/diagnosis , Metabolism, Inborn Errors/diagnosis , Mitochondrial Membrane Transport Proteins/genetics , Neonatal Screening , Polymorphism, Genetic , Republic of Korea/epidemiology , Sequence Analysis, DNAABSTRACT
The opening of mitochondrial permeability transition pore (MPTP) plays a critical role in platelet activation. However, the potential trigger of the MPTP opening in platelet activation remains unknown. Inflammation is the crucial trigger of platelet activation. In this study, we aimed to explore whether and how the important inflammatory cytokine IL-17 is associated with MPTP opening in platelets activation by using MPTP inhibitor cyclosporine-A (CsA). The mitochondrial membrane potential (ΔΨm) was detected to reflect MPTP opening levels. And the platelet aggregation, activation, and the primary signaling pathway were also tested. The results showed that the MPTP opening levels were increased and Δψm reduced in platelets administrated with IL-17. Moreover, the levels of aggregation, CD62P, PAC-1, P53 and the phosphorylation of ERK2 were enhanced along with the MPTP opening in platelets pre-stimulated with IL-17. However, CsA attenuated these effects triggered by IL-17. It was suggested that IL-17 could induce MPTP opening through ERK2 and P53 signaling pathway in platelet activation and aggregation.
Subject(s)
Humans , Blood Platelets , Cell Biology , Metabolism , Cell Separation , Cyclosporine , Pharmacology , Dual Specificity Phosphatase 2 , Genetics , Metabolism , Gene Expression Regulation , Interleukin-17 , Metabolism , Pharmacology , Membrane Potential, Mitochondrial , Mitochondria , Metabolism , Mitochondrial Membrane Transport Proteins , Genetics , Metabolism , Mitogen-Activated Protein Kinase 1 , Genetics , Metabolism , P-Selectin , Genetics , Metabolism , Phosphorylation , Platelet Activation , Platelet Aggregation , Primary Cell Culture , Signal Transduction , Tumor Suppressor Protein p53 , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To explore the effects of Panax Quinquefolium Saponin (PQS) on phosphatidylinositol 3-kinase/serine threonine kinase (PI3K/Akt) pathway of neonatal rat myocardial cells subjected to hypoxia.</p><p><b>METHODS</b>Neonatal rat myocardial cells were cultured in vitro. After the myocardial cell injury was induced by hypoxia, the cells were randomized into 5 groups: the normal group, the model group, the positive control group (Ciclosporin A, 2 µ mol/L), the low-dose PQS group (PQSL, 25mg/L), and the high-dose PQS group (PQSH, 50 mg/L). Morphology and behavior of myocardial cells were observed under an inverted microscope. Apoptosis rate and lactate dehydrogenase (LDH) leakage rate of myocardial cells were determined by colorimetry. Mitochondrial transmembrane potential was assessed using a fluorexon laser. Phospho-glycogen synthase kinase (GSK)-3β and phospho-Akt as well as cytochrome C were determined by Western blot</p><p><b>RESULTS</b>LDH leakage in the Ciclosporin A group, PQSH group and PQSL group reduced progressively compared with the model group (P<0.05). Akt and GSK-3β was strongly phosphorylated after treatment with Ciclosporin A and PQS compared with the model group (P<0.05, P<0.01). Compared with the model group (16.41±1.74; 35.28±6.30), both the integrated optical density of mitochondrial permeability transition pore (MPTP) and the mitochondrial transmembrane potential significantly increased in the PQSH group (42.74±2.12; 71.36±6.54) and the PQSL group (39.58±1.49; 66.99±5.45; P<0.05, P<0.01). However, the protein of cytochrome C outside the mitochondrion decreased in the PQSH group (273.66±14.61) and the PQSL group (259.62±17.31) compared with the model group (502.41±17.76; P<0.05).</p><p><b>CONCLUSION</b>Through activation of the PI3K/Akt pathway and inhibition of the MPTP, PQS might protect the heart against ischemia injury and apoptosis of myocardial cells.</p>
Subject(s)
Animals , Animals, Newborn , Cell Hypoxia , Cell Shape , Cell Survival , Cells, Cultured , Glycogen Synthase Kinase 3 , Metabolism , Glycogen Synthase Kinase 3 beta , L-Lactate Dehydrogenase , Metabolism , Membrane Potential, Mitochondrial , Mitochondria , Metabolism , Mitochondrial Membrane Transport Proteins , Metabolism , Myocytes, Cardiac , Cell Biology , Phosphatidylinositol 3-Kinases , Metabolism , Phosphorylation , Protein Serine-Threonine Kinases , Metabolism , Rats, Sprague-Dawley , Saponins , Pharmacology , Signal TransductionABSTRACT
<p><b>OBJECTIVE</b>To detect potential mutations of Y9ASS1, ASL and SLC25A13 genes in four patients manifesting citrullinemia.</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral blood leukocytes. Exons and their flanking sequences of the three genes were amplified with polymerase chain reaction and subjected to direct DNA sequencing.</p><p><b>RESULTS</b>Based on DNA sequence analysis, one case was diagnosed with argininosuccinate synthetase deficiency, and the mutation type (ASS1 gene) was c.236C>T (p.S79F) + c.431C>G (p.P144R). Two cases were diagnosed with argininosuccinic aciduria (ASL gene), and their gene mutations were c.434A>G (p.D145G) + c.1366C>T (p.R456W) and c.331C>T (p.R111W) + IVS8+2insT, respectively. A thirteen months boy who carried a heterozygous 851del4 mutation (SLC25A13 gene) was diagnosed with citrullinemia adult-onset type II.</p><p><b>CONCLUSION</b>Through analysis of relevant pathogenic genes, four patients have been diagnosed.</p>