Objective To evaluate the value of targeted second-generation sequencing (NGS) in the genetic diagnosis of tuberous sclerosis complex (TSC) associated with renal complications.Methods The clinical data of 43 patients (with 33 patients of definite diagnosis and 10 patients of possible diagnosis)with tuberous sclerosis complex associated with renal complications were analyzed.There were 26 females and 17 males with a mean age of 28 (10 ～ 48) years ranging from 10 to 48 years old.All patients had renal complications,including 39 renal angiomyolipomas,3 renal cysts and 1 renal cell carcinoma.Written informed consents were signed,and 5ml peripheral blood was drawn for DNA extraction.Mutations of TSC1 and TSC2 genes were detected by the NGS technology,and then confirmed by Sanger sequencing.Results TSC1 or TSC2 pathogenetic mutants were identified in 39 patients (90.7％),which were consistent with clinical phenotypes and two non-significant mutations were identified in two individuals,while no mutation was detected in the other two cases.Fourteen novel mutations were reported for the first time,including 8 frameshift mutation,3 deletion mutation,2 nonsense mutation and 1 splicing mutation.Conclusion NGS is an accurate and less time-consuming technology in detecting TSC1 or TSC2 gene mutation,which has great value in genetic diagnosis of tuberous sclerosis complex associated with renal complications.

Objective To study the correlation of genotype and phenotype in patients of tuberous sclerosis complex-associated renal angiomyolipomas (TSC-RAML).Methods The clinical and gene mutation data from 39 patients with TSC-RAML diagnosed in Peking Union Medical College Hospital were collected from January 2014 to December 2015.The clinical phenotype and genotype correlation associated with TSC-RAML was analyzed.A total of 39 patients (15 male,24 female) were enrolled,with mean age of 28.4 years (range,13 ～48).Results Five cases showed TSC1 gene mutation,thirty-two patients presented with TSC2 gene mutation and two cases didn't find any mutations.The mutations ratio of TSC1:TSC2 was 1∶6.4.Comparing to those with TSC1 gene mutation and no mutation identified,patients with TSC2 gene mutation had significantly higher frequency with hypomelanotic macules and dental enamel pits.The maximum diameter and clinical stage of patients with TSC2 gene mutation was significantly larger than patients with non-TSC2 gene mutations,along with higher frequency of AML bleeding risk and accepted invasive treatment.Conclusions The patients with TSC2 gene mutations presented higher frequency with hypomelanotic macules and dental enamel pits than those with non-TSC2 gene mutations.Comparing to those with TSC1 gene mutation and no mutation identified,the frequency of AML bleeding risk and loss of renal function was higher in patients with TSC2 gene mutations.Therefore,more active surveillance and treatment should be given to the patients of TSC-RAML with TSC2 gene mutations.

Objective: Tuberous sclerosis complex (TSC) is a multisystem genetic disorder caused by mutations in the TSC1 and TSC2 genes, but the molecular events contributing to TSC are not well understood.However, little is known about the role of microRNAs in TSC.To explore the microRNA differential expression profile between tuberous sclerosis complex cell line TSC2-/-MEFs and normal type cell line TSC2+/+ MEFs, and to provide new clues to study the mechanism of microRNA function in tuberous sclerosis complex.Methods: TSC2-/-MEFs and TSC2+/+ MEFs cell lines were cultured in vitro, each with three samples chosen as the experimental group and the control group respectively.Total RNA was isolated using TRizol and purified with RNeasy mini kit according to manufacturer''s instructions.RNA quality and quantity were measured by using nanodrop spectrophotometer and RNA integrity was determined by gel electrophoresis.Total RNAs were extracted by TRizol, followed by RNA quantification and quality control.MicroRNA profiles were analyzed by microarray and the threshold value used to screen up-regulated more than 2-fold change or down-regulated less than 0.5-fold change compared with controls.Real-time PCR was used to validate the reliability of microarray.Cell counting kit-8 (CCK-8) assay was performed to evaluate the proliferation.Results: Fourteen microRNAs, including miR-18a-5p, miR-376c-3p, miR-136-5p, miR-467c-5p, miR-467b-5p, miR-5104, miR-3098-3p, miR-30a-3p, miR-302b-3p, miR-18a-3p, miR-19b-1-5p, miR-19a-5p, miR-20a-5p, miR-155-5p, were up-regulated, while twenty-six microRNAs, including miR-200b-3p, miR-450a-1-3p, miR-542-5p, miR-199b-5p, miR-10a-5p, miR-466c-5p, miR-450a-5p, miR-450b-5p, miR-542-3p, miR-351-5p, miR-322-3p, miR-199a-3p, miR-335-5p, miR-10b-5p, miR-351-3p, miR-155-3p, miR-497a-5p, miR-503-5p, miR-148a-3p, miR-1843a-5p, miR-199a-5p, miR-490-5p, miR-450a-2-3p, miR-322-5p, miR-214-3p, miR-450b-3p, were down-regulated in tuberous sclerosis complex cell line TSC2-/-MEFs compared with normal type cell line TSC2+/+ MEFs (P<0.05).Real-time PCR confirmed the expressions of miR-136-5p, miR-30a-3p, miR-302b-3p, miR-10b-5p, miR-148a-3p, miR-199a-5p consistent with the microarray data (P<0.05).Furthermore, the overexpression of miR-199a-5p significantly inhibited cell proli-feration (P<0.05).Conclusion: There are differences in the expression of miRNA between the tube-rous sclerosis complex cell line TSC2-/-MEFs and normal cell line TSC2+/+ MEFs.MiRNA-199a-5p plays an important role in tuberous sclerosis complex, which may be developed as an important molecular target for the treatment of tuberous sclerosis complex.

Objective To evaluate the efficacy and safety of 131I-metaiodobenzylguanidine (131 I-MIBG) in treatment of malignant pheochromocytoma/malignant paraganglioma (MPHEO/MPGL).Methods The clinical data of 96 cases of MPHEO/MPGL (60/36) treated with 131I-MIBG between December 1998 and April 2014 were retrospectively reviewed.Among them,the malignant pheochromocytoma was found in 60 cases and malignant paraganglioma was found in 36 cases.Seventy-eight patients (81.2％)presented initially with hypertension,whereas 18 patients (18.8％) presented adrenal incidentaloma.Before 131I-MIBG treatment,24 h urinary norepinephrine was (409.5± 127.2) nmol,24 h urinary dopamine was (99.3±41.1) nmol,24 h urine adrenaline was (1 409.9±336.0) nmol.Before treatment,the compound iodine solution was given to each one.Then,all patients were given an initial course of 131I-MIBG therapy (5.55,7.40 GBq).Subsequent 131I-MIBG treatment (5.55,7.40 GBq) was undertaken every three to six months.The patients got symptomatic,hormonal or radiological response underwent sbsequent 131I-MIBG therapy (3.70,5.55 GBq) every year.All patients underwent clinical symptoms (headache,palpitate,sweating,hypertension),biochemical (24 h urine catecholamin) and radiological evaluation (CT/MRI) within 6 months to evaluate the efficacy and safety of 131I-MIBG treatment.Results After one to eleven sessions of 131 I-MIBG treatment,in total,266 doses of 131 I-MIBG were administered,average dose was 6.49 GBq.22.9％ of patients demonstrated radiological partial response (≥ 50％ reduction in tumor size) after first or repeated 131 I-MIBG treatment.Eleven cases (11.5％) achieved clinical complete response,41 cases (42.7％) achieved clinical partial response and 23 cases (24.0％) maintained the stable clinic symptoms.After treatment,24 h urinary norepinephrine (164.3±71.6) nmol and dopamine (49.7±24.7) nmol showed significantly decline,compared with those before treatment (P＜ 0.05).While,24 h urinary epinephrine (1 354.7±433.4) nmol had no obvious change (P＞0.05).No life-threatening adverse events were reported,but 2 MPGL patients developed transient leucopenia or thrombocytopenia after four and five times 131 I-MIBG treatment,respectively.Conclusions Treatment with repeated low dose 131I-MIBG is well tolerated and effective in controlling the progression and alleviating the clinical symptoms.The 131I-MIBG therapy is an effective and safe treatment modality for MPHEO/MPGL.

Skull deformity is common ailment of infants,and maybe cause developmental deformity,with high incidence in China.In this article,we reviewed the related factors,prevention and treatment for skull deformity,aimed at providing references for its further application.

Objective:To comprehensively analyze the expression profile of circular RNA (circRNA) and construct competing endogenous RNA (ceRNA) regulatory networks in tuberous sclerosis complex related renal angiomyolipoma (TSC-RAML).Methods:According to the diagnostic criteria of TSC determined by the international consensus group on tuberous sclerosis in 2012, tumor tissues and paired normal renal tissues of 3 patients with TSC-RAML who were diagnosed in our hospital from January 2017 to January 2019 were collected. The circRNA, miRNA and mRNA of 3 paired samples were detected by circRNA, miRNA chip technology and next generation sequencing respectively, and the differential molecules were determined. Gene Ontology(GO) functional enrichment analysis and Kyoto Encyclopedia of genes and genomes(KEGG) pathway enrichment analysis were performed based on differential mRNA molecules and host genes of circRNA. Based on differential circRNA, miRNA and mRNA, up-regulated and down-regulated ceRNA regulatory networks were established.Results:A total of 330 up-regulated and 336 down-regulated differential circRNA, 8 up-regulated and 7 down-regulated miRNA, 800 up-regulated and 1130 down-regulated mRNA were screened. Through GO and KEGG enrichment analysis, many pathways including lipid metabolism, focal adhesion and mineral absorption were abnormally altered. Finally, the up-regualted ceRNA network led by hsa_circ_0092022, hsa_circ_0076859 and hsa_circ_0033388 and down-regulated network led by hsa_circ_0000374, hsa_circ_0000141, hsa_circ_0072665, hsa_circ_0009503 and hsa_circ_0000009 were constructed.Conclusions:There were many differentially expressed circRNA between TSC-RAML and paired normal renal tissues. ceRNA regulatory networks may be involved in the occurrence and development of TSC-RAML.

Objective: The traditional Chinese medicine Anluohuaxianwan (ALHXW) has been used to treat liver fibrosis induced by chronic hepatitis B virus (HBV) infection. However, the anti-fibrosis mechanisms of ALHXW remain to be investigated. This study used a rat model of carbon tetrachloride (CCl₄)-induced liver fibrosis to explore the potential antifibrogenic mechanisms of ALHXW. Methods: Twenty-seven male Wistar rats were randomly assigned to control group, model group, and treatment group (n = 9 per group). Rats in the model and treatment group were injected intraperitoneally with 40% CCl₄(2 ml/kg), and rats in the control group were administered saline twice a week for 6 weeks. Starting at week 4 following model construction, rats in the treatment group received daily gavages with ALHXW solution (concentration 0.15 g/ml) daily, while rats in the control and model groups were given saline for a total of 6 weeks. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured from blood samples collected at the end of weeks 3, 6 and 9. Histopathological examination of liver tissue was performed to evaluate liver fibrosis at week 9. At the same time, the mRNA expression of TGF-β1 and Smads in liver tissues was quantified by real-time reverse transcription polymerase chain reaction (RT-PCR), and TGF-β1 protein level in the liver was measured by Western blot. Inter-group comparison was performed using analysis of variance (ANOVA) when the continuous data were normally distributed and satisfied the homogeneity of variance; otherwise, nonparametric tests were used. Categorical data were compared between groups using nonparametric tests. Results: ALHXW markedly alleviated liver injury in the treatment group after 3 weeks of therapy as indicated by a significantly reduced level of ALT compared with the model group [(162.98 ± 73.14)U/L vs (322.52 ± 131.76)U/L, P = 0.047], and a 39.8% reduction in AST level compared with the model group[ (537.56 ± 306.06)U/L vs (892.98 ± 358.19)U/L, P = 0.053]. Moreover, at the end of the 6-week therapy, histopathological diagnosis showed that liver fibrosis was significantly reduced in the ALHXW-treated group compared with that in the model group (P = 0.002). The relative expression of TGF-β1 mRNA and protein in the liver were significantly lower in ALHXW-treated rats than that in model rats (1.34 ± 0.31 vs 1.78 ± 0.45, P = 0.025; 0.39 ± 0.02 vs 0.57 ± 0.04, P = 0.003). Conclusion ALHXW treatment can reverse CCl₄-induced liver fibrosis in rats. Its mechanisms of anti-fibrosis may occur through the inhibition of TGF-β1 synthesis and TGF-β1/Smads signaling pathway, which in turn suppress the activation of hepatic stellate cells and thereby reverses fibrosis.