AIM:To investigate the role of Epac/Rap1 signaling pathway in hypoxia-reoxygenation injury in H9c2 cells,and to explore the mechanism of vitexin regulating the Epac/Rap1 signaling path-way to protect cardiomyocytes from hypoxia-reoxy-genation injury.METHODS:The oxygen glucose de-privation(OGD)model was established using H9c2 cardiomyocytes to simulate hypoxia-reoxygenation injury.The experiment was randomly divided into 7 groups:Normal control group,OGD group,OGD+VT group,OGD+8-CPT+VT group,OGD+ESI-09+VT group,OGD+8-CPT+VT+H-89 group,OGD+ESI-09+VT+H-89 group.Cell viability was measured by MTT.LDH was used to detect cell damage.The ex-pression levels of Epac1 and its downstream Rap1-GTP,CaMK Ⅱ and ERK proteins in H9c2 cells were detected by Western blot.The expression of Epac1 and Rap1 proteins in H9c2 cardiomyocytes was de-tected by immunofluorescence.The mRNA expres-sion of Rap1 and Epac1 in H9c2 cardiomyocytes was quantitatively determined by real-time PCR.Calcium ion fluorescence probe(Fluo-3 AM)was used to detect intracellular[Ca2+]i content.The in-teraction between Epac1 and Rap1 in cells were de-tected by Co-IP.RESULTS:Compared with the nor-mal control group,after hypoxia for 5 h and reoxy-genation for 1 h,the release of LDH,cell viability,Epac1 protein expression,Rap1 activation and RAP1-GTP up-regulation of H9c2 cardiomyocytes in OGD group were significantly increased.VT(10μmol/L)significantly inhibited the activation of Epac1 in H9c2 cardiomyocytes after OGD,and then inhibited the expression of downstream Rap1 ac-tive form Rap1-GTP.In addition,the expression of CaMK Ⅱ protein was down-regulated,but ERK phosphorylation was increased,and intracellular calcium overload was alleviated.Epac1 agonist 8-CPT could counteract the effect of VT,and Epac1 in-hibitor(ESI-09)combined with VT had synergistic effect.PKA inhibitor(Hmur89)had no effect on the expression of Epac1 and its downstream related proteins in cardiomyocytes.CONCLUSION:Hypoxia-reoxygenation can mediate the activation of Epac1/Rap1 signal pathway in cardiomyocytes.VT can pro-tect cardiomyocytes from hypoxia-reoxygenation in-jury by inhibiting Epac1/Rap1 signal pathway,down-regulating CaMK Ⅱ protein expression and promoting ERK phosphorylation.

OBJECTIVE: To explore the clinical and genetic characteristics of two children with developmental delay. METHODS: Two children who had presented at the Children's Hospital Affiliated to Shandong University on August 18, 2021 were enrolled as the study subjects. Clinical and laboratory examination, chromosomal karyotyping and high-throughput sequencing were carried out for both children. RESULTS: Both children had a 46,XX karyotype. High-throughput sequencing showed that they have respectively carried a c.489delG (p.Q165Rfs*14) and a c.1157_1158delAT (p.Y386Cfs*22) frameshifting variant of the CTCF gene, both had a de novo origin and were unreported previously. CONCLUSION The CTCF gene variants probably underlay the development delay in the two children. Above discovery has enriched the mutational spectrum of the CTCF gene and has important implications for revealing the genotype-phenotype correlation for similar patients.
Child ; Humans ; Developmental Disabilities/genetics* ; High-Throughput Nucleotide Sequencing ; Intellectual Disability/genetics* ; Karyotyping ; Mutation

OBJECTIVE: To analyze the clinical and genetic characteristics of three Chinese pedigrees affected with Citrullinemia type I (CTLN1). METHODS: Three children diagnosed at the Children's Hospital Affiliated to Shandong University from 2017 to 2020 were selected as the study subjects. Genomic DNA was extracted from peripheral blood samples of the probands and their parents. Next generation sequencing (NGS) was carried out to detect pathological variants of the probands. Sanger sequencing was used for validating the candidate variant among the pedigrees. RESULTS: The probands have respectively carried compound heterozygous variants of c.207_209delGGA and c.1168G>A, c.349G>A and c.364-1G>A, c.470G>A and c.970G>A of the ASS1 gene, which were respectively inherited from their parents. CONCLUSION The newly discovered c.207_209delGGA and c.364-1G>A variants have enriched the mutational spectrum of the ASS1 gene. And the mutation spectrum of Chinese CTLN1 patients is heterogeneous.
Child ; Humans ; Argininosuccinate Synthase/genetics* ; Citrullinemia/genetics* ; East Asian People ; Mutation ; Pedigree

OBJECTIVE: To explore the clinical characteristics and genetic basis of two brothers featuring X-linked alpha thalassemia mental retardation (ATR-X) syndrome. METHODS: An infant who had presented at the Qilu Children's Hospital in 2020 for unstable upright head and inability to roll over and his family were selected as the study subjects. The clinical features of the child and one of his brothers were summarized, and their genomic DNA was subjected to targeted capture and next generation sequencing (NGS). RESULTS: The brothers had presented with mental retardation and facial dysmorphisms. NGS revealed that they had both harbored a hemizygous c.5275C>A variant of the ATRX gene located on the X chromosome, which was inherited from their mother. CONCLUSION The siblings were diagnosed with ATR-X syndrome. The discovery of the c.5275C>A variant has enriched the mutational spectrum of the ATRX gene.
Humans ; Infant ; Male ; alpha-Thalassemia/diagnosis* ; Ataxia Telangiectasia Mutated Proteins/genetics* ; East Asian People ; Intellectual Disability/genetics* ; Mental Retardation, X-Linked/diagnosis* ; Pedigree ; X-linked Nuclear Protein/genetics*

Objective : To investigate the role of Epac1 / CaMK Ⅱ signaling pathway in myocardial ischemia reper- fusion injury (MIRI) in mice，and to investigate the protective effect of vitexin ( VT) on acute MIRI． Methods: C57 / BL mice were randomly divided into 5 groups : Sham surgery group ( Sham) ，ischemia reperfusion group ( I / R) ，and ischemia reperfusion + vitexin group ( function 3，6，12 mg / kg groups) ．Ligation of the left anterior descending coronary artery (LAD coronary artery) in mice resulted in ischemia of part of the heart tissue for 30min and reperfusion of the blood for 120min．Mouse myocardial ischemia reperfusion injury ( MIRI) model was established．In the sham operation group，only the LAD was not ligated．Serum LDH levels of mice were detected．Hema- toxylin-eosin (H＆E) staining was performed on the left ventricular myocardium of mice to observe the histopatho- logical changes．The expression level of Epac1 in myocardial tissue was observed by immunohistochemistry．The protein expressions of Epac1，Rap1，CaMK Ⅱ and ERK / p-ERK were determined by Western Blot. Results : Compared with Sham group，serum LDH level of mice in I / R group was significantly increased，protein expressions of Epac1， Rap1 and CaMK Ⅱ in myocardial tissue were significantly up-regulated，and ERK1 /2 phosphorylation level was decreased．Compared with I / R group，vitexin (3，6，12 mg / kg) pretreatment group decreased serum LDH level，inhib- ited Epac1，Rap1 and CaMK Ⅱ protein expression in mouse myocardial tissue，and promoted ERK1 /2 phosphoryla- tion(P＜0. 05 or P＜0. 01) ．The histopathological results showed that the myocardial fibers in the I / R group were disordered and broken，with increased gaps and obvious inflammatory cell infiltration．In the vitexin treatment group，the myocardial fibers were arranged more neatly and inflammatory cells were infiltrated less. Conclusion Vitexin may regulate Epac1 / CaMK Ⅱ signaling pathway，down-regulate CaMK Ⅱ protein expression，increase ERK phosphorylation，and effectively reduce MIRI.

OBJECTIVE: To explore the genetic basis for a neonate featuring developmental delay. METHODS: Clinical examination and laboratory tests were carried out for the patient. Peripheral venous blood samples of the proband and his parents were extracted and subjected to target capture next generation sequencing. Candidate variant was verified by Sanger sequencing. RESULTS: The patient, a four-month-old male, has presented with developmental delay and weakness of limbs. Genetic testing revealed that he had harbored a novel c.1432C>T variant of the TNPO3 gene, which was inherited from his mother. The nonsense variant has resulted in premature termination of protein translation and was predicted to be pathogenic by bioinformatics analysis. CONCLUSION The heterozygous c.1432C>T variant of the TNPO3 gene probably underlay the limb-girdle muscular dystrophies form 1F in this patient. Above finding has enriched the variation spectrum of the TNPO3 gene.
Genetic Testing ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Humans ; Infant ; Male ; Muscular Dystrophies, Limb-Girdle/genetics* ; Mutation ; Phenotype ; beta Karyopherins/genetics*

OBJECTIVE: To carry out clinical and genetic analysis for an infant manifesting perianal lesions, diarrhea and multiple intestinal perforations. METHODS: Genomic DNA of the infant was extracted and subjected to targeted capture exome sequencing. Candidate variants were verified by Sanger sequencing of his family members. RESULTS: The patient was found to harbor c.301C>T and c.188+1G>A compound heterozygous variants of the IL10RA gene, which has suggested the diagnosis of IL10RA-related very early-onset inflammatory bowel disease (VEOIBD). CONCLUSION The patient was diagnosed with IL10RA-related VEOIBD. The newly discovered c.188+1G>A variant has enriched the spectrum of IL10RA gene variations.
Genetic Testing ; Humans ; Infant ; Inflammatory Bowel Diseases/pathology* ; Mutation ; Exome Sequencing

OBJECTIVE: To explore the genetic etiology for an infant featuring convulsive status epilepticus, developmental delay and elevated plasma lactate. METHODS: Whole exome sequencing and mitochondrial D-loop sequencing were carried out for the infant. Candidate variants were verified by Sanger sequencing. Previously reported FARS2 gene variants were searched from the PubMed, Wanfang and CNKI databases. RESULTS: The infant was found to harbor compound heterozygous variants of the FARS2 gene, namely c.925G>A (p.G309S) and c.405C>A (p.H135Q), which were inherited from its mother and father, respectively. The former has been recorded by the HGMD as a pathogenic variant, whilst the latter was predicted to be likely pathogenic based on the guidelines of the American College of Medical Genetics and Genomics. A total of 30 COXPD14 cases were retrieved from the literature, with common mutations including missense variants, in-frame deletions, splice-site variants and large deletions. CONCLUSION The common manifestations of COXPD14 have included developmental delay (96%), status epilepticus (97%) and increased lactic acid (96%). The compound heterozygous variants of the FARS2 gene probably underlay the disorder in this child.
Female ; Humans ; Infant ; Genetic Testing ; Mitochondrial Diseases ; Mitochondrial Proteins/genetics* ; Phenylalanine-tRNA Ligase ; Status Epilepticus ; Exome Sequencing

OBJECTIVE: To explore the genetic basis for a child manifesting with intellectual disability, language delay and autism spectrum disorder. METHODS: Genomic DNA was extracted from peripheral blood samples of the child and his family members, and subjected to whole exome sequencing. Candidate variants were verified by Sanger sequencing and interpreted according to the guidelines of the American College of Medical Genetics and Genomics. RESULTS: The child was found to harbor a heterozygous c.568C>T (p.Q190X) nonsense variant of the ADNP gene, which was not detected in either parent by Sanger sequencing. CONCLUSION The clinical and genetic testing both suggested that the child has Helsmoortel-van der Aa syndrome due to ADNP gene mutation, which is extremely rare in China.
Abnormalities, Multiple/genetics* ; Autism Spectrum Disorder/genetics* ; Autistic Disorder/genetics* ; Child ; Heterozygote ; Homeodomain Proteins/genetics* ; Humans ; Intellectual Disability/genetics* ; Mutation ; Nerve Tissue Proteins/genetics* ; Rare Diseases

Objective : To investigate the effect of (exchange protein directly activated by cAMP⁃1) on inner ear hair cell injury with noise⁃induced hearing loss and its potential mechanism in rats. Methods : Twenty Specific pathogen⁃free (SPF) Sprague⁃Dawley (SD) rats were randomly divided into normal control group and noise exposure group. The rats of noise exposure were exposed to 4 kHz at 101 dB sound pressure level (SPL) for 8 h. Auditory brainstem responses(ABR) were measured in animals before noise exposure and 24 h after noise exposure. Surface preparation , transmission electron microscopy and immunohistochemistry were performed on cochlea tissuesto elucidate changes in Epac expression in rat after noise exposure. The expression levels of Epac1、Rap1、CaMK⁃ Ⅱ、Bax、Bcl⁃2、cleaved caspase3(CC3) and cleaved caspase9(CC9) were analyzed using Western blot. Results : There was found a stable temporary threshold shift after noise exposure( P < 0. 05) . The missing of outer hair cells occurred after noise exposure(P < 0. 05) . Transmission electron microscopy indicated that the epidermis plate of HCs was partially dissolved , with loss or fusion of stereocilia , some HC organelles showed serious injuries after noise exposure. Epac1 immunostaining intensities were substantially enhanced in OHCs after noise exposure( P < 0. 05) . The expression levels of Epac1 , CaMK⁃ Ⅱ and Rap1 protein were significantly up⁃regulated after noise exposure(P < 0. 05) . The expression level of Bcl⁃2 was significantly down⁃regulated after noise exposure(P < 0. 05) . The expression levels of Bax , CC3 and CC9 were significantly up⁃regulated after noise exposure(P < 0. 05) . Conclusion Epac1 ⁃Rap1 signaling pathway mediates the early pathological damage in noise⁃exposed cochlea , and participates in the regulation of inner ear hair cells apoptosis. Epac1 ⁃Rap1 pathway is expected to become a new target for intervention in noise⁃induced hearing loss.