【Objective】 To analyze the genetic variation characteristics and clinical phenotypes of a family with primary microcephaly (MCPH) caused by RTTN gene variation, and to provide reference for genetic counseling and prenatal diagnosis. 【Methods】 Clinical data of the three patients (including 2 fetuses and 2-year-old proband,and one fetus with clinical diagnosis) and their parents were collected and analyzed. Two of the children and their parents were tested by trio whole exome sequencing (trio-WES), sanger sequencing validation sites, and the hazard of their compound heterozygous variants was predicted. Literature review was conducted through domestic and international databases to collect reported RTTN gene mutation cases. 【Results】 Three patients in this family had anomalies of the septum pellucidum, hypoplasia of the corpus callosum and other brain malformations during fetal period. The proband (G2) and fetus (G3) showed intrauterine growth retardation and MCPH in late pregnancy; besides, G2 was born with global developmental delay. Trio-WES detected a c.2101(exon16)C>T(p.Arg701Ter,1526) nonsense and a c.2863(exon22)G>A(p.Glu955Lys)missense in the RTTN gene of G2 and G3, which were inherited from their father and mother, forming a compound heterozygous variant. According to the American College of Medical Genetics and Genomics (ACMG) variant classification guidelines, two variants were likely to be pathogenic (LP) and uncertain significance (VUS). Among them, c.2863(exon22)G>A was a newly discovered missense, which was predicted by the software to be harmful to the gene product. 【Conclusions】 Complex heterozygous variations of RTTN gene (c.2101C>T and c.2863G>A) are the genetic cause of MCPH in this family. This report has enriched the variation spectrum of RTTN gene, provided guidance for prenatal diagnosis and reproduction of this family, as well as material and reference for further understanding of the diseases caused by this gene mutation.

OBJECTIVE: To explore the genetic basis for a child with multiple malformations. METHODS: A child who had presented at Shanxi Provincial Children's Hospital in February 2021 was selected as the study subject. Clinical data of the patient was collected, and whole exome sequencing (WES) was carried out to screen pathogenic variants associated with the phenotype. Candidate variant was validated by Sanger sequencing of her family members. RESULTS: The child had normal skin, but right ear defect, hemivertebral deformity, ventricular septal defect, arterial duct and patent foramen ovale, and separation of collecting system of the left kidney. Cranial MRI showed irregular enlargement of bilateral ventricles and widening of the distance between the cerebral cortex and temporal meninges. Genetic testing revealed that she has harbored a heterozygous variant of NM_178014.4: c.217A>G (p.Met73Val) in the TUBB gene, which was unreported previously and predicted to be likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). The child was diagnosed with Complex cortical dysplasia with other brain malformations 6 (CDCBM6). CONCLUSION CDCBM is a rare and serious disease with great genetic heterogeneity, and CDCBM6 caused by mutations of the TUBB gene is even rarer. Above finding has enriched the variant and phenotypic spectrum of the TUBB gene, and provided important reference for summarizing the genotype-phenotype correlation of the CDCBM6.
Humans ; Child ; Female ; Abnormalities, Multiple ; Blood Group Antigens ; Family ; Malformations of Cortical Development/genetics* ; Brain ; Mutation

Objective:To explore the relationship and underlying mechanism between exosomes derived from doxorubicin-resistant osteosarcoma cells and MDR1 and miRNAs. Methods:MG63 and U2OS cell lines were selected to construct doxorubicin-resistant strains, and the 50% inhibitory concentration (half maximal inhibitory concentration, IC 50) of drug-resistant and sensitive strains was detected by MTT, and fluorescence staining was performed at intervals of 15 min between 15 and 120 min to detect the change of fluorescence intensity. RT-PCR and Western Blot were used to detect the expression levels of MDR1 P-gp to verify the drug resistance of osteosarcoma cells. Exosomes were identified by particle size analysis and Western Bolt detection. The endocytosis of PKH26-labeled exosomes from doxorubicin-resistant cells was observed, and the proliferation level and migration of exosomes from doxorubicin-resistant cells co-cultured with osteosarcoma cells were detected by MTT assay and cell scratch assay. The differential expression levels of miRNAs in osteosarcoma-sensitive and drug-resistant cells were verified by sequencing and bioinformatics analysis and RT-PCR assay. Tumor growth, serum exosome identification and mRNA expression level of miR-21-5p in tumor-bearing nude mice between normal osteosarcoma cell group and drug-resistant group, drug-resistant+normal exosome group, drug-resistant+drug-resistant+drug-resistant exosome group were observed. MDR1 expression level in tumor tissue was detected by RT-PCR, Western Blot and immunohistochemistry. Results:The IC 50 of two adriamycin resistant strains were 2.21 vs. 11.81 μg/ml and 0.93 vs. 11.81 μg/ml, respectively, and the fluorescence intensity decreased faster than that of normal strains. The relative mRNA expression levels of MDR1 in two cell lines were normal 1.12±0.16, 1.02±0.11 and drug-resistant 2.15±0.10, 2.127±0.12, respectively. The relative protein expression of P-gp was normal 0.92±0.11, 0.73±0.10 and drug-resistant 0.46±0.03, 0.30±0.04, the differences were statistically significant ( P<0.05). Drug-resistant exosomes can enter osteosarcoma cells through endocytosis and concentrate in the cytoplasm when co-cultured with normal strains. Osteosarcoma cells were co-cultured with drug-resistant exosomes at 2, 4, 6, and 8 μg/ml adriamycin, respectively. Compared with normal group, the proliferation level in drug-resistant group was significantly increased. Compared with the normal cell group 35.95±3.92, 6.72±3.55 and the normal exosome group 51.22±5.55, 19.31±1.93, the drug-resistant cell group 54.20±9.32, 19.24±2.88 and drug-resistant exosome group 76.40±5.41, 30.26±4.87, all had significantly higher cell mobility, the difference was statistically significant ( P<0.05). Exosome sequencing and biogenic analysis of 10 highly upregated miRNAs to validate mRNA expression differences between normal and drug-resistant strains by RT-PCR, showing a significant increase in miR-21-5p expression level of drug-resistant strains (5.89±0.26 vs. 0.99±0.06; 1.05±0.07 vs. 8.80±0.93, P<0.05), the difference was statistically significant ( P<0.05). In MG63 and U2OS, the normal cell group and drug-resistant cell group, and the normal exosome group and drug-resistant exosome group were compared, the tumor volume and the terminal tumor weight of nude mice were increased to varying degrees. MRNA relative expression levels of miR-21-5p in serum exosomes of nude mice after drug intervention were 0.86±0.07 and 0.86±0.05 in normal cell group, respectively. The values were 1.13±0.12, 1.14±0.12 in drug-resistant cell group, 0.71±0.05, 0.75±0.03 in normal exosome group, and 0.90±0.07, 0.93±0.04 in drug-resistant exosome group. Compared with normal and drug-resistant strains, the expression levels of normal and drug-resistant exosome groups were increased, with statistical significance ( P<0.05). Conclusion:The exosomes of drug-resistant cells in osteosarcoma could enhance the proliferation level and migration ability of cells through intercellular transfer of MDR1 and miRNAs. The expression of MDR1 and miR-21-5p in drug-resistant cells and tumor-forming nude mouse serum and tumor tissues were up-regulated which suggested that it might be involved in regulating the drug resistance process of osteosarcoma.

Objective:To investigate the radiosensitizing effect of silencing breast cancer susceptibility gene 1 (shBRCA1) expression on MDA-MB-231 breast cancer bearing nude mice by 3′-deoxy-3′- 18F-fluorothymidine ( 18F-FLT) microPET/CT imaging. Methods:Twenty-four BALB/c nude mice were divided into 4 groups ( n=6 in each group) according to the random number table method, namely negative control (NC) group, NC+ radiotherapy group, shBRCA1 group and shBRCA1+ radiotherapy group. 18F-FLT microPET/CT imaging was performed before and 24 h after radiotherapy. The changes of SUV max before and after radiotherapy were compared among 4 groups, and the total proliferative volume (TPV) of tumors in each group after treatment was also analyzed. The expression of Ki-67 in tumor tissues was analyzed by immunohistochemistry. Data were analyzed by paired t test, one-way analysis of variance, least significant difference t test and Pearson correlation analysis. Results:Breast cancer cells targeting the BRCA1 were constructed. Before radiotherapy, the differences of SUV max among the NC group, NC+ radiotherapy group, shBRCA1 group and shBRCA1+ radiotherapy group were not statistically significant (1.034±0.137, 1.031±0.152, 1.028±0.169 and 1.026±0.156; F=0.00, P=0.999). Twenty-four hours after the end of the four times of radiotherapy, the differences of SUV max among the 4 groups were statistically significant (1.367±0.100, 0.781±0.079, 1.306±0.213 and 0.597±0.129; F=44.77, P<0.001), with lower SUV max in the shBRCA1+ radiotherapy group compared with the NC+ radiotherapy group ( t=2.98, P=0.014). The SUV max of the NC+ radiotherapy group and shBRCA1+ radiotherapy group were reduced compared with those before radiotherapy ( t values: 5.82, 5.44, P values: 0.002, 0.003), while SUV max of the NC group and shBRCA1 group increased compared with those before radiotherapy ( t values: -4.47, -3.98, P values: 0.007, 0.011). TPV was smaller in the shBRCA1+ radiotherapy group compared with that in the NC+ radiotherapy group (0.48±0.03 vs 0.61±0.07; F=25.36, t=3.82, P=0.003). Immunohistochemical assays showed that Ki-67 was less expressed in the shBRCA1+ radiotherapy group than that in the NC+ radiotherapy group (0.286±0.072 vs 0.476±0.093; F=15.73, t=3.61, P=0.007). Correlation analysis showed a positive correlation between Ki-67 expression and SUV max ( r=0.83, P<0.001). Conclusion:18F-FLT microPET/CT imaging can evaluate the radiosensitizing effect of shBRCA1 expression on MDA-MB-231 breast cancer bearing nude mice.

Objective:To evaluate the short-term efficacy and the improvement of quality of life of enzyme replacement therapy (ERT) with Imiglucerase on children with Gaucher disease(GD) through the same time monitoring.Methods:Six children diagnosed as GD who were treated by ERT with Imiglucerase in the Department of Hematology of the Children′s Hospital of Shanxi Province from May 2019 to May 2020 were recruited.Every 3 months, the sizes of the liver and spleen was palpated, the change of bone pain was recorded, and the haematological index was examed.The volumes of the liver and spleen at 1-year treatment were measured by CT.Bone involvement was examined by magnetic resonance imaging (MRI). In addition, the body weight, height, and the 36-Item Short Form Survey (SF-36) were measured and compared with pre-treatment levels.These data were analyzed statistically by SPSS 25.0 and the difference between pretherapy and post-treatment was compared by paired t test. Results:Six children diagnosed as GD received ERT with Imiglucerase.No adverse events were reported.Decreased volumes of the liver and spleen, and increased hemoglobin level and platelet count were detected after 3-6 months of ERT.After 1 year of ERT, hemoglobin level significantly increased compared with pre-treatment level ( t=4.200, P=0.008). Although the platelet count increased at 1-year ERT, it was comparable with pre-treatment level ( t=2.260, P=0.073). The volumes of liver and spleen decreased by (22.10±15.28)% ( t=2.725, P=0.042) and (47.10±18.42)% ( t=3.162, P=0.034) after 1 year of ERT, respectively.During the first year of ERT, the height and weight increased (6.17±2.86) cm ( t=5.286, P=0.003) and (4.08±2.01) kg ( t=4.975, P=0.004), respectively.SF-36 score increased significantly from (489.35±103.99) points to (632.75±73.34) points ( t=5.740, P=0.002). After 1 year of ERT, 1 patient still had bone pain, and 2 cases were worse in bone MRI, which may be attributed to the short period of follow-up and insufficient dose, and another 3 had no change in bone MRI. Conclusions:ERT ameliorates GD-associated anemia, organomegaly and growth retardation, and improves the growth rate of body mass and height and the quality of life in the short period.However, short-term ERT does not improve the bone disease.

Objective:To investigate the value of 18F-FDG total-body PET/CT dynamic imaging in evaluating the efficacy of early radiotherapy for MDA-MB-231 breast cancer bearing nude mice. Methods:MDA-MB-231 breast cancer bearing nude mice were established and divided into control group and radiotherapy group based on the random number table method ( n=10 for each group). 18F-FDG total-body PET/CT dynamic imaging was performed before and after the radiotherapy. The SUV max and the maximum tracer uptake net influx constant ( Kimax) of tumors, and the target-to-background ratio (TBR) were calculated. The change of tumor volume was recorded. The value of 18F-FDG total-body PET/CT dynamic imaging in evaluating the efficacy of radiotherapy was accessed using pathological findings as the reference. Paired t test, independent-sample t test and Pearson correlation analysis were used for data analysis. Results:After radiotherapy, SUV max and Kimax(4.66±0.46 and 0.14±0.03) were reduced in the radiotherapy group compared with those before radiotherapy (5.30±0.52 and 0.19±0.03; t values: 4.61, 8.31, P values: 0.001, <0.001), while the SUV max (5.94±0.74 vs 5.24±0.50) and Kimax (0.23±0.03 vs 0.19±0.02) were increased compared with baseline in the control group ( t values: 4.77, 6.87, P values: 0.001, <0.001). TBR Ki was significantly higher than TBR suv based on all images of the 2 groups (14.11±5.58 vs 5.91±1.60; t=8.92, P<0.001). The tumor volume in the radiotherapy group decreased compared with that before radiotherapy, but the difference was not statistically significant ((0.74±0.12) vs (0.81±0.08) cm 3; t=2.24, P=0.052). The results of immunohistochemistry showed that glucose transport protein (Glut)1 was highly expressed in tumors, and the Glut1 positive cell percentage of the radiotherapy group was significantly lower than that of the control group ((38.30±6.18)% vs (69.78±5.37)%; t=12.17, P<0.001). The expression of Glut1 was significantly positively correlated with SUV max and Kimax(the control group: rsuv=0.75, P=0.012; rKi=0.77, P=0.010; the radiotherapy group: rsuv=0.67, P=0.035; rKi=0.77, P=0.010). The apoptosis index (AI) of tumor cells in the radiotherapy group was higher than that in the control group ((24.15±4.00)% vs (10.15±3.05)%; t=8.85, P<0.001). Conclusion:18F-FDG total-body PET/CT dynamic imaging can sensitively monitor the effect of early radiotherapy in MDA-MB-231 breast cancer bearing nude mice.

Abstract: To investigate the value of whole-body dynamic18F-flurodeoxygluse positron emission tomography with computed tomography(18F-FDG PET/CT) Patlak imaging for evaluating the radiosensitization early efficacy in C6 rat glioma. Methods: Twenty-four C6 rat glioma models were established and randomly divided into control group, Oleanolic acid(OA) group, radiotherapy group and OA+radiotherapy group, with 6 rats in each group. 75 min whole-body dynamic18F-FDG PET/CT Patlak imaging was performed before and 48 h after treatment. The left ventricular time-activity curve(TAC) of tumor-bearing rats was used as input function for Patlak analysis to obtain Ki images. The last frame of the dynamic PET scan was selected for static analysis, and the changes of maximum standardized uptake value(SUVmax), Ki values and tumor volume were observed, and the survival time of tumor-bearing rats was recorded. Then, the tumor samples were dissected for Ki-67 and glucose transporter-1(GLUT-1) immunohistochemical staining. Results: Before treatment, there were no significant differences in SUVmaxand Ki values among the 4 groups(F=0.909, 0.858,P>0.05). 48 h after treatment, SUVmaxand Ki values of control group and OA group increased significantly(t=-10.697,-17.516,P<0.01;t=-19.321,-9.887,P<0.01), SUVmaxand Ki values of radiotherapy group and OA+radiotherapy group decreased(t=5.629, 6.916,P<0.01;t=7.241, 10.490,P<0.01), there was no significant difference in SUVmaxbetween radiotherapy group and OA+radiotherapy group 48 h after treatment(t=1.233,P=0.246), while the Ki values in OA+radiotherapy group was significantly lower than that in radiotherapy group, the difference was statistically significant(t=3.051,P<0.05). The immunohistochemistry staining results suggested that the expression of Ki-67 and GLUT-1 in OA+radiotherapy group was lower than that in the other 3 groups(F=63.887, 40.720,P<0.05). In addition, Ki values showed positive correlations with the expression of Ki-67 and GLUT-1(r=0.931, 0.936,P<0.01). Kaplan-Meier analysis showed that the survival time of tumor-bearing rat in OA+radiotherapy group was longer than that in the other three groups, and the difference was statistically significant(χ2=19.817,P<0.01). Conclusion OA has radiosensitization effect on C6 rat glioma. Whole-body dynamic18F-FDG PET/CT Patlak multi-parameter imaging can evaluate the radiosensitization early efficacy in C6 rat glioma, which is more sensitive than PET/CT static imaging.

OBJECTIVE: To explore the genetic basis for a child with unexplained global developmental delay (GDD), seizure, and facial deformity. METHODS: Whole exome sequencing (WES) was carried out for the patient. Candidate variants were verified by Sanger sequencing of the patient and his parents. RESULTS: WES revealed that the patient has carried a previously unreported de novo heterozygous nonsense c.4906C>T (p.Arg1636Ter) variant of the KMT2A gene, Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.4906C>T variant of KMT2A gene was predicted to be pathogenic (PVS1+ PS2+ PM2+PP3). CONCLUSION The heterozygous nonsense c.4906C>T (p.Arg1636Ter) variant of the KMT2A gene probably underlay the disease in the child. Above finding has enriched the spectrum of pathogenic variants of the KMT2A gene.
Abnormalities, Multiple/genetics* ; Child ; Histone-Lysine N-Methyltransferase/genetics* ; Humans ; Intellectual Disability/genetics* ; Male ; Myeloid-Lymphoid Leukemia Protein/genetics* ; Syndrome

Objective:To understand the epidemiological characteristics of infectious diarrhea pathogens in Pudong New Areas of Shanghai from 2013 to 2017 to provide evidence for control and prevention of the disease.Methods:From Jan 2013 to Dec 2017, active surveillance program on diarrhea was conducted in 14 sentinel hospitals (three tertiary-level and nine secondary-level, and two primary-level hospitals) in Pudong New Areas of Shanghai, based on location, catchment areas and number of patients. All recruited outpatients were interviewed in hospitals, using a standard questionnaire. Stool specimens were collected and tested for five viral and eight bacterial pathogens.Results:A total of 9 301 cases with infectious diarrhea were included, and the overall positive rate was 55.7 % (5 179). Positive rates of single virus, single bacteria and mixed infections were 26.7 % (2 481), 17.0 % (1 579) and 12.0 % (1 119), respectively. For single infection, the most commonly detected viruses appeared as norovirus (15.4 %, 1 428/9 301) and rotavirus (7.2 %, 667/9 301). The most commonly detected bacteria were diarrheagenic Escherichia coli (6.7 %, 619/9 301) and non-typhoid Salmonella (3.3 %, 305/9 301). The most common mixed infections were caused by virus-bacteria (4.9 %, 459/9 301). Norovirus (17.0 %, 838/4 938) showed the highest positive rates, followed by Escherichia coli (7.2 %, 354/4 938), both seen in the age group of 20-59 years old group. Rotavirus (9.4 %, 178/1 896) and non-typhoid Salmonella (4.9 %, 93/1 896) were the most common pathogens found in the age group of 0-4 years old. The prevalence of norovirus peaked both in spring and autumn. The other peaks were seen as: Rotavirus in winter, diarrheagenic Escherichia coli in summer and non-typhoid Salmonella in summer. Conclusions:Our data showed that the positive rates of infectious diarrhea pathogens were high in Pudong New Areas of Shanghai from 2013 to 2017. The dominant pathogens would include norovirus, rotavirus and diarrheagenic Escherichia coli but with differenct distributions in age groups. Obvious seasonal patterns were also observed.

Objective To analyze the key factors influencing the number of diseases or conditions in which clinical pathways ( CPs) were implemented at public hospitals of China. Methods Based on the questionnaire survey of 51 public hospitals that had implemented CPs in Shanghai, Hubei province, and Gansu province, a multivariate logistic model was used to analyze the factors that influenced the number of CPs implemented in hospitals. Results In terms of the 14 issues and difficulties found in CPs′implementation, the overall acceptance rate was 38. 8% on average among surveyed hospitals, yet with a great variation(from 7. 1% to 100. 0%). A multivariate logistic model showed that the recognition of public hospitals on the issues and difficulties in CPs′implementation did not affect the number of implemented CPs, and tertiary public hospitals had significantly higher number of CPs implemented than secondary hospitals. Conclusions The resources and management capabilities of public hospitals in China are the determinants of CPs′promotion.