Next generation sequencing (NGS) technology is playing an increasingly important role in the diagnosis of genetic diseases. Whole exome sequencing (WES), which targets the coding regions of the genome, has been widely used in the diagnosis of genetic diseases for its low cost and high efficiency. However, compared to conventional methods, the Next Generation Sequencing (NGS) process is intricate, and there is variability in the expertise of data analysts and variant interpreters, which may lead to inconsistencies in the outcomes. To ensure the quality of testing and enhance the diagnostic rate of diseases, this consensus has provided recommendations regarding the laboratory setup, operational procedures, data analysis, result interpretation, and quality control for WES, with an aim to standardize its application in the detection of genetic disorders.

Objective:To investigate the expression, prognosis, and role of G protein-coupled receptor kinase-interacting protein 1 (GIT1) in patients with hepatocellular carcinoma (HCC) tumor micro environments.Methods:Clinical data of 140 cases who underwent complete HCC surgical resection from January 2015 to December 2021 in Subei People's Hospital affiliated to Yangzhou University, Jiangsu Province, were included. Tumor tissue and adjacent tissue samples were collected for immunohistochemical analysis. The patients were divided into a high expression group and a low expression group according to the expression of GIT1. Cox regression was used to analyze the risk factors for prognosis in patients with HCC. Fifteen pairs of cancer tissues and adjacent tissues were randomly matched for quantitative polymerase chain reaction (RT-PCR), western blot (WB), and immunohistochemical analysis. GITI knockout or overexpression cell lines of human hepatoma cell lines HepG2, HuH7 and MHCC97-H, and mouse hepatoma cell line Hepa 1-6 were constructed. The effects on M2 macrophage polarization were analyzed by flow cytometry. A mice tumor model was constructed. The growth curve of tumor tissue overexpressing GIT1 was plotted. Bioinformatics analysis of the Cancer Genome Atlas (TCGA) data was performed using OncoLnc, Kaplan-Meier Plotter, UALCAN, and GEPIA databases to explore the differential expression of GIT1 in HCC patients and its effect on prognosis.Results:Bioinformatics analysis showed that the expression level of GIT1 was significantly higher in HCC tissues than in normal liver tissues ( P<0.05). RT-PCR and WB experiments showed that GIT1 was highly expressed in HCC. The follow-up results showed that high expression of GIT1 was associated with poor prognosis in patients with HCC. The high expression of GIT1 was an independent risk factor for the prognosis in patients with HCC ( HR=2.562, 95% CI: 0.231-0.704, P<0.05). Functional enrichment analysis combined with TIMER database analysis found that GIT1 expression level was associated with multiple immune cell infiltrations in HCC, but the correlation coefficient with macrophage infiltration was the highest ( r=0.545, P<0.001). Mice tumorigenesis experiments showed that the tumor volume of GIT1-overexpressing mice was significantly increased ( P<0.05). Additionally, flow cytometry indicated that after GIT1 overexpression, there was a low degree of M1 infiltration/polarization (wild type: 5.06%±0.11%, overexpression type: 4.09%±0.04%; P<0.05) and a high degree of M2 infiltration/polarization (wild type: 10.20%±0.33%, overexpression type: 14.7%±0.12%; P<0.05). Conclusion:GIT1 serves as a prognostic biomarker in HCC, promoting tumor progression through its high expression and enhances M2 macrophage infiltration.

Next generation sequencing (NGS) technology is playing an increasingly important role in the diagnosis of genetic diseases. Whole exome sequencing (WES), which targets the coding regions of the genome, has been widely used in the diagnosis of genetic diseases for its low cost and high efficiency. However, compared to conventional methods, the Next Generation Sequencing (NGS) process is intricate, and there is variability in the expertise of data analysts and variant interpreters, which may lead to inconsistencies in the outcomes. To ensure the quality of testing and enhance the diagnostic rate of diseases, this consensus has provided recommendations regarding the laboratory setup, operational procedures, data analysis, result interpretation, and quality control for WES, with an aim to standardize its application in the detection of genetic disorders.

Objective:To investigate the expression, prognosis, and role of G protein-coupled receptor kinase-interacting protein 1 (GIT1) in patients with hepatocellular carcinoma (HCC) tumor micro environments.Methods:Clinical data of 140 cases who underwent complete HCC surgical resection from January 2015 to December 2021 in Subei People's Hospital affiliated to Yangzhou University, Jiangsu Province, were included. Tumor tissue and adjacent tissue samples were collected for immunohistochemical analysis. The patients were divided into a high expression group and a low expression group according to the expression of GIT1. Cox regression was used to analyze the risk factors for prognosis in patients with HCC. Fifteen pairs of cancer tissues and adjacent tissues were randomly matched for quantitative polymerase chain reaction (RT-PCR), western blot (WB), and immunohistochemical analysis. GITI knockout or overexpression cell lines of human hepatoma cell lines HepG2, HuH7 and MHCC97-H, and mouse hepatoma cell line Hepa 1-6 were constructed. The effects on M2 macrophage polarization were analyzed by flow cytometry. A mice tumor model was constructed. The growth curve of tumor tissue overexpressing GIT1 was plotted. Bioinformatics analysis of the Cancer Genome Atlas (TCGA) data was performed using OncoLnc, Kaplan-Meier Plotter, UALCAN, and GEPIA databases to explore the differential expression of GIT1 in HCC patients and its effect on prognosis.Results:Bioinformatics analysis showed that the expression level of GIT1 was significantly higher in HCC tissues than in normal liver tissues ( P<0.05). RT-PCR and WB experiments showed that GIT1 was highly expressed in HCC. The follow-up results showed that high expression of GIT1 was associated with poor prognosis in patients with HCC. The high expression of GIT1 was an independent risk factor for the prognosis in patients with HCC ( HR=2.562, 95% CI: 0.231-0.704, P<0.05). Functional enrichment analysis combined with TIMER database analysis found that GIT1 expression level was associated with multiple immune cell infiltrations in HCC, but the correlation coefficient with macrophage infiltration was the highest ( r=0.545, P<0.001). Mice tumorigenesis experiments showed that the tumor volume of GIT1-overexpressing mice was significantly increased ( P<0.05). Additionally, flow cytometry indicated that after GIT1 overexpression, there was a low degree of M1 infiltration/polarization (wild type: 5.06%±0.11%, overexpression type: 4.09%±0.04%; P<0.05) and a high degree of M2 infiltration/polarization (wild type: 10.20%±0.33%, overexpression type: 14.7%±0.12%; P<0.05). Conclusion:GIT1 serves as a prognostic biomarker in HCC, promoting tumor progression through its high expression and enhances M2 macrophage infiltration.

Objective:To discuss the effect of downregulating microRNA-208a(miR-208a)on the resistance of the colorectal cancer cells to 5-fluorouracil(5-FU),and to clarify its related molecular mechanism.Methods:Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of miR-208a and secreted frizzled-related protein 1(SFRP1)mRNA in the 5-FU-resistant colorectal cancer cell line HT-29/5-FU and its parent HT-29 cells.The HT-29/5-FU cells were transfected with miR-208a inhibitor plasmid and its negative control plasmid(inhibitor-NC),and SFRP1 small interfering RNA(si-SFRP1)and its negative control plasmid(si-NC),either separately or in combination,followed by treatment with 5-FU.The cells were divided into inhibitor-NC group,miR-208a inhibitor group,miR-208a inhibitor+si-NC group,and miR-208a inhibitor+si-SFRP1 group.MTT assay was used to detect the proliferation activities of the cells and the resistance indexes were calculated;Annexin V-FITC/PI double staining and flow cytometry were used to detect the apoptotic rates of the cells after treated with different concentrations of 5-FU;Western blotting method was used to detect the expression levels of SFRP1,β-catenin,P-glycoprotein(P-gp),and ATP-binding cassette subfamily B member 1(ABCB1)proteins in the cells in various groups;dual-luciferase reporter gene assay was used to validate the targeting relationship between miR-208a and SFRP1.Results:Compared with HT-29 cells,the expression level of miR-208a in the HT-29/5-FU cells was increased(P＜0.05),and the expression level of SFRP1 mRNA was decreased(P＜0.05).Compared with inhibitor-NC group,the proliferation activity of the cells in miR-208a inhibitor group was decreased(P＜0.05),the resistance index was decreased,the apoptotic rate was increased(P＜0.05),and the expression levels of β-catenin,P-gp,and ABCB1 proteins in the cells were decreased(P＜0.05).The dual-luciferase reporter gene assay results showed that SFRP1 was a target gene of miR-208a and miR-208a could negatively regulate the expression of SFRP1.Compared with miR-208a inhibitor+si-NC group,the proliferation activity of the cells in miR-208a inhibitor+si-SFRP1 group was increased(P＜0.05),the resistance index was increased,the apoptotic rate was decreased(P＜0.05),and the expression levels of β-catenin,P-gp,and ABCB1 proteins in the cells were increased(P＜0.05).Conclusion:Downregulation of miR-208a can improve the resistance of the HT-29/5-FU cells to 5-FU by targeting and upregulating the SFRP1 expression,thereby inhibiting the transmission of the Wnt signaling pathway.

Objective:To observe any effect of repetitive transcranial magnetic stimulation (rTMS) on sleep disorders among children with cerebral palsy (CP).Methods:A total of 102 children with CP and disordered sleep were randomly divided into an experimental group and a control group, each of 51. All were given routine rehabilitation and sleep health education, but the experimental group additionally received rTMS for two weeks. The polysomnography (PSG) results of the two groups were recorded and analyzed.Results:The PSG parameters had improved greatly in both groups after the treatment. The percentage of N2 sleep (depth of sleep during light sleep) in the severe cerebral palsy group and of N3 sleep (depth of sleep during deep sleep) in the moderate cerebral palsy group had increased significantly more than in the mild cerebral palsy group, on average. After the intervention the percentages of N2 and N3 in those with mixed cerebral palsy and of N3 in those with involuntary motor cerebral palsy had increased significantly more than in those with spastic cerebral palsy, on average.Conclusion:rTMS treatment can improve the sleep disorders of children with cerebral palsy, especially N2 sleep among children with moderate to severe cerebral palsy, N3 sleep in cases of mixed or dyskinetic CP.

Objective:To investigate the effects of oocyte maturation trigger using follicle-stimulating hormone (FSH) plus human chorionic gonadotropin (hCG) on clinical outcomes of in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET) in gonadotropin-releasing hormone agonist (GnRH-a) cycles. Methods:The retrospective cohort study included 682 patients aged up to 40 years with normal ovarian response who underwent IVF/ICSI-ET at Department of Reproductive Medicine, Women and Children's Hospital, Xiamen University School of Medicine between Feburary 2016 and April 2017. Patients were grouped by whether oocyte maturation was triggered with 250 μg recombinant hCG (r-hCG) plus 300 U urinary FSH (uFSH, dual trigger group, n=439) or 250 μg r-hCG alone (r-hCG group, n=243). The main observation indexes were the clinical pregnancy rate and the live birth rate, and the secondary observation indexes were the high-quality embryo rate, the implantation rate, the biochemical pregnancy rate, the abortion rate, etc. Results:There were no statistically significant differences between the two groups in age, infertility duration, body mass index (BMI), anti-Müllerian hormone (AMH), total dosage and duration of gonadotropin (Gn) used, number of embryos transferred (all P>0.05). The live birth rate, the clinical pregnancy rate, the miscarriage rate, the normal fertilization rate, the cleavage rate, the embryo formation rate and the high-quality embryo rate were not significantly different between the two groups (all P>0.05). The implantation rate [40.47% (191/472)] and the biochemical pregnancy rate [64.20% (156/243)] were higher in dual trigger group than in r-hCG group [32.42% (272/893), P=0.003; 55.35% (272/893), P=0.025]. Conclusion:Dual trigger of oocyte maturation with 250 μg r-hCG plus 300 U uFSH has no benefit on the clinical pregnancy rate and the live birth rate, but could improve the implantation rate and the biochemical pregnancy rate in women undergoing short-acting GnRH-a protocol in IVF/ICSI-ET.

Objective:To investigate the effects of oocyte maturation trigger using follicle-stimulating hormone (FSH) plus human chorionic gonadotropin (hCG) on clinical outcomes of in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET) in gonadotropin-releasing hormone agonist (GnRH-a) cycles. Methods:The retrospective cohort study included 682 patients aged up to 40 years with normal ovarian response who underwent IVF/ICSI-ET at Department of Reproductive Medicine, Women and Children's Hospital, Xiamen University School of Medicine between Feburary 2016 and April 2017. Patients were grouped by whether oocyte maturation was triggered with 250 μg recombinant hCG (r-hCG) plus 300 U urinary FSH (uFSH, dual trigger group, n=439) or 250 μg r-hCG alone (r-hCG group, n=243). The main observation indexes were the clinical pregnancy rate and the live birth rate, and the secondary observation indexes were the high-quality embryo rate, the implantation rate, the biochemical pregnancy rate, the abortion rate, etc. Results:There were no statistically significant differences between the two groups in age, infertility duration, body mass index (BMI), anti-Müllerian hormone (AMH), total dosage and duration of gonadotropin (Gn) used, number of embryos transferred (all P>0.05). The live birth rate, the clinical pregnancy rate, the miscarriage rate, the normal fertilization rate, the cleavage rate, the embryo formation rate and the high-quality embryo rate were not significantly different between the two groups (all P>0.05). The implantation rate [40.47% (191/472)] and the biochemical pregnancy rate [64.20% (156/243)] were higher in dual trigger group than in r-hCG group [32.42% (272/893), P=0.003; 55.35% (272/893), P=0.025]. Conclusion:Dual trigger of oocyte maturation with 250 μg r-hCG plus 300 U uFSH has no benefit on the clinical pregnancy rate and the live birth rate, but could improve the implantation rate and the biochemical pregnancy rate in women undergoing short-acting GnRH-a protocol in IVF/ICSI-ET.

Objective:To document the clinical features of children with cerebral palsy (CP) using magnetic resonance imaging (MRI).Methods:The gross motor functioning of 325 children diagnosed as having CP was graded using the gross motor function classification system (GMFCS). The GMFCS grades were correlated with MRI results in univariate and multivariate logistic regression analyses. The significance of any relationship between the MRI results and co-morbidities was tested using chi-squared tests.Results:Cerebral dysplasia, cerebroventricular enlargement, periventricular leukomalacia (PVL), abnormal signals in the thalami, and morphological changes after hypoxic ischemic encephalopathy were all found to be significantly correlated with GMFCS grading. Moreover, the chi-squared tests indicated that PVL children, children with thinning of the corpus callosum and/or abnormal signals in the thalami were significantly more likely to have visual, auditory or speech impairment complications and/or mental retardation.Conclusions:The findings from MRI correlate well with types of CP, GMFCS grades and co-morbidities among CP children. MRI can be an effective tool for early diagnosis and prognosis of CP in children, indicating needs for clinical rehabilitation.

Objective:To study the complications of irreversible electroporation (nano-knife) ablation on locally advanced pancreatic cancer, and to analyse the causes of complications and related treatment.Methods:The clinical data of 36 patients with locally advanced pancreatic cancer treated with nano-knife ablation at the Fifth Affiliated Hospital of Zhengzhou University from January 2016 to March 2019 were studied retrospectively. The types and incidence of postoperative complications were analyzed. The complications were classified according to the Clavien-Dindo classification, and the severity of the complications was evaluated.Results:There were 15 patients (41.7%) who developed various degrees of complications, including splenic infarction, atrial fibrillation, portal vein thrombosis, pancreatic fistula, pseudoaneurysm, gastrointestinal bleeding, liver abscess and severe pancreatitis. Among them, 6 patients (16.7%) had grade III complication or above. Three (8.3%) patients with grade Ⅲ complications died of upper gastrointestinal bleeding 3 months after operation.Conclusions:Various complications might occur after nano-knife ablation, with postoperative gastrointestinal and abdominal bleeding being the main complications which resulted in death. Measures which can effectively reduce occurrence of complications need to be studied.