Construction of CRISPR/Cas9 system targeting mouse Tsc1 and Tsc2 gene and verification of targeting effectiveness / 中国生物制品学杂志

@#ObjectiveTo design and construct CRISPR/Cas9 gene editing system targeting Tsc1 and Tsc2 genes,and verify the effectiveness of gene editing at cellular level.MethodsThree sgRNA guide sequences were designed for mouse Tsc1 and Tsc2 genes respectively. The sgRNA expression vector was constructed and co-transfected with the Cas9 expression plasmid into mouse N2a cells. After the positive cells were obtained through drug screening,the DNA fragments at the targeting site were amplified by PCR,and the targeting efficiency was verified by TA clone sequencing.ResultsThe five targets of Tsc1-M-sgRNA2 and Tsc1-M-sgRNA3 of Tsc1 gene and Tsc2-M-sgRNA1,Tsc2-M-sgRNA2 and Tsc2-M-sgRNA3 of Tsc2 gene were all edited,and the editing efficiency was 40%,80%,30%,30% and 20%,respectively.ConclusionA CRISPR-Cas9 gene editing system with editing efficiency targeting mouse Tsc1 and Tsc2 genes was successfully constructed.

Nueva mutación en el gen TSC2 protege contra el cáncer colorrectal en población mexicana / A novel mutation in the TSC2 gene protects against colorectal cancer in the Mexican population

Resumen Introducción: El cáncer colorrectal (CCR) es una enfermedad compleja debido al gran número de factores que influyen en su desarrollo, incluyendo variantes en genes supresores de tumores. Objetivo: Estimar las frecuencias alélicas y genotípicas de las variantes c.3915G>A y c.5371G>A del gen TSC2 en una población mexicana con CCR, así como analizar la asociación con el desarrollo de CCR. Métodos: Se incluyeron 126 muestras de sangre periférica de pacientes con diagnóstico de CCR esporádico y 134 de individuos sanos, considerados como grupo de control. La identificación de los genotipos se llevó a cabo mediante PCR tradicional y digestión enzimática. Todos los individuos firmaron una carta de consentimiento informado. Resultados: El alelo A de la variante c.3915G>A (RM = 0.31, IC 95 % = 0.15-0.69, p = 0.004), así como el haplotipo A/G de las variantes c.3915G>A y c.5371G>A (RM = 0.28, IC 95 % = 0.12-0.68, p = 0.005) mostraron un posible efecto protector contra CCR esporádico. El análisis in silico indicó que ambas variantes generan modificaciones en el proceso de corte y empalme. Conclusión: La presencia de la variante c.3915G>A del gen TSC2 sugiere un posible efecto protector contra CCR esporádico en población mexicana; sin embargo, no se observó esta asociación con la variante c.5371G>A.

Abstract Introduction: Colorectal cancer (CRC) is a complex disease due to the large number of factors that influence its development, including variants in tumor suppressor genes. Objective: To estimate allelic and genotypic frequencies of c.3915G>A and c.5371G>A variants of the TSC2 gene in a Mexican population with CRC, as well as to analyze their association with the development of CRC. Methods: 126 peripheral blood samples from patients diagnosed with sporadic CRC and 134 from healthy individuals, regarded as the control group, were included. Identification of genotypes was carried out using traditional PCR and enzymatic digestion. All individuals signed an informed consent letter. Results: The A allele of the c.3915G>A variant (OR = 0.31, 95% CI = 0.15-0.69, p = 0.004), as well as A/G haplotype of the c.3915G>A and c.5371G>A variants (OR = 0.28, 95% CI = 0.12-0.68, p = 0.005) showed a possible protective effect against sporadic CRC. In silico analysis indicated that both variants generate modifications in the splicing process. Conclusion: The presence of TSC2 gene c.3915G>A variant suggests a possible protective effect against sporadic CRC in the Mexican population; however, no association was observed with the c.5371G>A variant.

Clinical phenotype and gene analysis of TSC2/PKD1 adjacency gene syndrome / 中华神经科杂志

Objective:To explore the clinical phenotype and gene characteristics of a case of TSC2/PKD1 adjacency gene syndrome, so as to improve the clinical understanding of the disease.Methods:A case of TSC2/PKD1 adjacency gene syndrome diagnosed in the Department of Neurology of the Children′s Hospital Affiliated to Zhengzhou University was analyzed retrospectively. The clinical data, laboratory examination, imaging characteristics and gene variation characteristics of the child were summarized.Results:The patient was a 17 months old girl, with the main complaint of "intermittent convulsion with 17 months of underdevelopment". The clinical manifestations were epileptic seizures, which were in the form of a series of spastic seizures, absence seizures, focal seizures, and depigmentation spots can be seen in the trunk and neck. Cranial magnetic resonance imaging showed multiple patchy signals in the cortex and subcortical areas of the bilateral cerebral hemispheres, multiple small nodular shadows under the ependyma of the bilateral lateral ventricles, the heart color Doppler ultrasound showed patent foramen ovale and pericardial effusion, and the abdomen color Doppler ultrasound showed polycystic kidney. Ophthalmic color Doppler ultrasound showed that there were localized small swelling lesions around the optic disc of the left eye. The whole exon gene sequencing of the pedigree showed the proband had partial deletion of TSC2 gene (NM_000548) at chromosome position chr16: 2125799-2185690. The real-time quantitative detection system verified that exons 23-42 were deleted, and all exons of PKD1 gene were deleted (NM_001009944), and multiple ligation dependent probe amplification verified that exons 1-46 were deleted, and no downstream gene deletion was found. The overall deletion size was about 60 kb. Both of the girl's father and mother had normal phenotypes and were wild-type.Conclusions:TSC2/PKD1 adjacency gene syndrome is relatively rare. It can have clinical manifestations of tuberous sclerosis/autosomal dominant polycystic kidney disease. Most of the nervous system and kidney are seriously affected, and the prognosis is poor. TSC2/PKD1 gene deletion and variation is the genetic cause of the TSC2/PKD1 adjacency gene syndrome.

Analysis of clinical phenotype and gene variants in a patient with classic tuberous sclerosis complex / 中华皮肤科杂志

Objective:To analyze clinical phenotypes and pathogenic mutations of a patient with classic tuberous sclerosis complex.Methods:Clinical data was collected from a patient with classic tuberous sclerosis complex. Next-generation sequencing was performed to screen pathogenic gene variants, and Sanger sequencing to verify the mutations. Minigene plasmids were constructed and transfected into the human renal epithelial cell line 293T, and RNA was extracted for transcriptional analysis.Results:The patient clinically presented with recurrent epileptic seizures, facial angiofibroma, periungual fibroma, pulmonary lymphangioleiomyomatosis, renal angiomyolipoma and multiple osteosclerosis. Next-generation sequencing revealed a suspected pathogenic variant in the TSC2 gene in the patient. Sanger sequencing identified a heterozygous mutation c.336_336+15delGGTAAGGCCCAGGGCG in exon 4 of the TSC2 gene in the patient, but not in his parents or 100 unrelated healthy controls. Moreover, this mutation had not been previously reported. The minigene experiment showed changed mRNA sequence of the TSC2 gene in this patient with loss of the authentic splice site in exon 4 and insertion of a 74-bp intron, which shifted the splice site 90 bp downstream (r.336delins336+16_336+90) .Conclusion:The novel heterozygous mutation c.336_336+15delGGTAAGGCCCAGGGCG in exon 4 of the TSC2 gene can lead to aberrant splicing, and may contribute to tuberous sclerosis complex in this patient.

Genetic analysis of a mosaic case with low proportion mutation of / 浙江大学学报·医学版

OBJECTIVE@#To perform gene mutation analysis in a patient with atypical clinical manifestations of tuberous sclerosis (TSC) for definite diagnosis.@*METHODS@#Peripheral blood DNA was obtained from a patient with clinically suspected TSC and her parents, and all exons and their flanking sequences of @*RESULTS@#A heterozygous nonsense mutation c.1096G>T (p.E366*) was identified in the exon 11 of the @*CONCLUSIONS@#The somatic mosaic mutation c.1096G>T (p.e366*) may be responsible for the phenotype of TSC in this patient. And the drop digital PCR is expected to be a diagnostic method for somatic cells mosaicism.

Long non-coding RNA MALAT1 enhances the apoptosis of cardiomyocytes through autophagy inhibition by regulating TSC2-mTOR signaling

BACKGROUND: Our previous study showed that knockdown of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) attenuated myocardial apoptosis in mouse acute myocardial infarction (AMI). This study aims to explore whether MALAT1 enhanced cardiomyocyte apoptosis via autophagy regulation and the underlying mechanisms of MALAT1 regulating autophagy. METHODS: Cardiomyocytes were isolated from neonatal mice and then stimulated with hypoxia/reoxygenation (H/R) injury to mimic AMI. The autophagy level was assessed using GFP-LC3 immunofluorescence and western blot analysis of autophagy-related proteins. RNA pull-down and RNA immunoprecipitation (RIP) was performed to analyze the binding of MALAT1 and EZH2. Chromatin immunoprecipitation (ChIP) assay was performed to analyze the binding of TSC2 promoter and EZH2. The cell apoptosis was evaluated using TUNEL staining and western blot analysis of apoptosis-related proteins. RESULTS: H/R injury increased MALAT1 expression in cardiomyocytes. Furthermore, MALAT1 overexpression inhibited, whereas MALAT1 knockdown enhanced the autophagy of cardiomyocytes. Moreover, MALAT1 overexpression recruited EZH2 to TSC2 promoter regions to elevate H3K27me3 and epigenetically inhibited TSC2 transcription. Importantly, TSC2 overexpression suppressed mTOR signaling and then activated the autophagy. Further results showed that MALAT1 inhibited proliferation and enhanced apoptosis of cardiomyocytes through inhibiting TSC2 and autophagy. CONCLUSION: These findings demonstrate that the increased MALAT1 expression induced by H/R injury enhances cardiomyocyte apoptosis through autophagy inhibition by regulating TSC2-mTOR signaling.

Genetic analysis of a pedigree affected with tuberous sclerosis complex caused by a novel mutation of TSC1 / 中华医学遗传学杂志

Objective@#To analyze the clinical phenotype of a Chinese pedigree affected with Tuberous sclerosis complex(TSC) and explore pathogenic mutations of TSC1 and TSC2 gene.@*Methods@#Unique clinical phenotypes, the results of imaging, examination of the proband and special family history, collectively, made the constellation of features of TSC.Genomic DNA was obtained from six affected and eight unaffected members of the family and potential mutations of the TSC1 and TSC2 genes were detected by PCR-amplification of the exons and exon-intron boundaries and direct sequencing.A total of 150 normal unrelated individuals were used as controls.@*Results@#Genetic analysis documented the presence of a heterozygous mutation, c. 1781_1782delTG (p.Val594GlyfsX11), in the exon 15 of TSC1 gene within all the patients of the family. This mutation was not observed in the eight unaffected family members or in the 150 unrelated control subjects from the same population , or the Human Gene Mutation Database(HGMD)and had completely co-segregated with the disease phenotype in the family.@*Conclusions@#The c. 1781_1782delTG mutation of TSC1 gene may be responsible for the tuberous sclerosis complex in this family. The data presented in the present study are of significance to clinicians, as well as genetic counselors, and may provide new clues for molecular diagnosis of this disease.

Tsc1/Tsc2 complex: A molecular target of capsaicin for protection against testicular torsion induced injury in rats / 中草药·英文版

Objective: The detailed knowledge about protective effects of capsaicin (cap)and involved mechanisms against testicular torsion (TT)is still not available completely. Methods: Male Wistar rats were assigned into four major cohorts: (i)sham, (ii)TT, (iii)three subgroups subjected to TT and different doses of cap (100, 500, and 1000 µg/mL), and (iv)three subgroups of healthy animals subjected to various concentrations of cap. The animals were decapitated at 24 h after reperfusion, and the evaluation of protein expression was performed by Western blotting assay. At 72 h after reperfusion, apoptotic cell death and tissue injury were evaluated by TUNEL nuclear and H&E staining, respectively. Results: The results showed that cap administration following TT significantly increased the expression of tuberous sclerosis proteins 1 and 2 (Tsc1/Tsc2)in a dose-dependent manner (P < 0.05). Cap decreased cell apoptosis at highest dose. Likewise, cap contributed to the preservation of tubular morphology and decreased tissue injury at the highest tested concentration (1000 µg/mL). Conclusion: Collectively, our findings demonstrate the validity of cap as a therapeutic agent against TT through targeting Tsc1/Tsc2 in a dose-dependent manner.

Ginsenoside Rg_1 induces leukemia stem cell senescence via SIRT1/TSC_2 signal axis / 中国中药杂志

The aim of this paper was to investigate the effect of SIRT1/TSC_2 signal axis on leukemia stem cell senescence induced by ginsenoside Rg_1. CD34~+CD38~- leukemia stem cells(CD34~+CD38~-LSCs) was isolated by magnetic cell sorting(MACS) and divided into two groups. The control group cells were routinely cultured, 40 μmol·L~(-1) ginsenoside Rg_1 was added to the control group for co-culture in Rg_1 group. The effect of Rg_l to induce CD34~+CD38~-LSCs senescence were evaluated by senescence-associated β-Galactosidase(SA-β-Gal) staining, cell cycle assay, CCK-8 and Colony-Assay. The expression of senescence associated SIRT1, TSC_2 mRNA and protein was examined by Real-time fluorescence quantitative PCR(FQ-PCR) and Western blot. The results showed that the CD34~+CD38~-LSCs could effectively be isolated by MACS, and the purity of CD34~+CD38~-LSCs is up to(95.86±3.04)%. Compared with the control group, the percentage of positive cells expressed SA-β-Gal in the Rg_1 group is increased, the senescence morphological changes were observed in the CD34~+CD38~-LSCs in the Rg_1 group. The proliferation inhibition rate and the number of cells entered G_0/G_1 phase in the Rg_1 group were increased, but the colony-formed ability was decreased, Rg_1 could significantly inhibit the proliferation and self-renewal ability of CD34~+CD38~-LSCs. The expression of SIRT1 and TSC_2 mRNA and protein were down regulated in the Rg_1 group compared with the control group. Our research implied that Rg_1 may induce the senescence of CD34~+CD38~-LSCs and SIRT1/TSC_2 signal axis plays a significant role in this process.

A case of tuberous sclerosis induced by a somatic mosaic mutation in the TSC2 gene / 中华皮肤科杂志

Objective To investigate clinical features and detect mutations in a case of tuberous sclerosis complex (TSC) caused by a somatic mosaic mutation in the TSC2 gene.Methods Peripheral blood samples were obtained from a patient with suspected TSC,his parents,and 200 unrelated healthy controls.Genomic DNA was extracted from these blood samples,polymerase chain reaction (PCR)and nextgeneration sequencing were performed to amplify all the exons and their flanking sequences of the TSC 1 and TSC2 genes followed by DNA sequencing,so as to identify mutations in the TSC 1 and TSC2 genes.DNA was also extracted from lesional skin tissues of the patient,and PCR was conducted to amplify the target fragment of the TSC2 gene followed by DNA sequencing.Results The patient clinically presented with facial angiofibroma,depigmented patches on the waist,periungual fibroma and angioleio-myolipoma of the kidney,which were consistent with the diagnosis of TSC.A mutation c.5130_5131insT(p.V1711Cfs* 18) was identified in the TSC2 gene in the patient.A higher frequency of the mutation was found in the DNA of the tumor tissue than in that of the peripheral blood.No such a mutation was found in his parents'DNA,unrelated healthy controls or any public database.Conclusion The somatic mosaic mutation c.5130_513 1insT in the TSC2 gene is responsible for the phenotype of TSC in the patient.

TSC1 and TSC2 gene mutations and their implications for treatment in Tuberous Sclerosis Complex: a review

Abstract Tuberous sclerosis complex is an autosomal dominant disorder characterized by skin manifestations and formation of multiple tumors in different organs, mainly in the central nervous system. Tuberous sclerosis is caused by the mutation of one of two tumor suppressor genes, TSC1 or TSC2. Currently, the development of novel techniques and great advances in high-throughput genetic analysis made mutation screening of the TSC1 and TSC2 genes more widely available. Extensive studies of the TSC1 and TSC2 genes in patients with TSC worldwide have revealed a wide spectrum of mutations. Consequently, the discovery of the underlying genetic defects in TSC has furthered our understanding of this complex genetic disorder, and genotype-phenotype correlations are becoming possible, although there are still only a few clearly established correlations. This review focuses on the main symptoms and genetic alterations described in TSC patients from 13 countries in three continents, as well as on genotype-phenotype correlations established to date. The determination of genotype-phenotype correlations may contribute to the establishment of successful personalized treatment for TSC.

Tuberous sclerosis complex secondary to fetal heart occupying lesions: a case report and literature review / 临床儿科杂志

Objective To explore the clinical characteristics of tuberous sclerosis complex (TSC). Methods The clinical data of one child with TSC were collected. The clinical features and gene mutation were analyzed. Results A 36-day-old girl had abnormal nodules found by echocardiography, which was considered multiple cardiac rhabdomyomas. There were multiple hypomelanotic macules distributed over the skin surface of the trunk and legs. Cranial MRI showed cortical nodules, subependymal nodules and cerebral white matter radial migration line. A mutation in the TSC2 gene (c.4541-4544delCAAA) was found by second generation high-throughput sequencing technology and tuberous sclerosis complex was confirmed. Conclusion Gene detection is helpful in the early diagnosis of tuberous sclerosis complex.

Effect of TSC2 gene expression downregulation by lentivirus induced RNA interference on U937 cell line and its mechanism / 中华血液学杂志

Objective@#To investigate the effect of biology and mTOR pathway activity of down-regulated TSC2 gene expression on U937 leukemia cells.@*Methods@#Gene expression was down-regulated by lentivirus induced RNA interference on TSC2 high expressed U937 cell line; the proliferation, apoptosis and differentiation were detected by CCK-8 assay, colony formation assay and flow cytometry; the gene expression level and protein kinase activity were detected by qRT-PCR and Western blot.@*Results@#Down-regulated expression of TSC2 gene promoted U937 cell proliferation and colony formation ability (P<0.05) . The proportion in G0/G1 phase of TSC2 down-regulated U937 cell was much lower than that of the control cells [ (52.53±3.75) % vs (75.10±4.33) %, t=6.829, P=0.002], the S phase [ (22.43±1.00) % vs (15.47±1.20) %, t=-5.581, P=0.019] and G2/M phase [ (25.03±4.34) % vs (14.33±0.91) %, t=-5.413, P=0.013] was remarkably higher than that of the control cells (P<0.05) . There were no statistically significant differences in cell apoptosis and differentiation (P>0.05) . Down-regulation of TSC2 led to the increased activity of mTOR, 4EBP1 and S6K1, but did not influence the activity of AKT. The expressions of proliferation related cyclinD1, c-myc and PTEN were also up-regulated after TSC2 silenced, but the expressions of P27KIP and BCL-XL were not changed.@*Conclusion@#Downregulation of TSC2 could promote the proliferation of U937 cells through up-regulation of mTOR activity.

Relationship between gene mutation and clinical phenotype in patients with tuberous sclerosis complex / 中华神经科杂志

Objective To study the relationship between gene mutation and clinical phenotype in patients with tuberous sclerosis complex (TSC).Methods The clinical data of 76 patients with TSC diagnosed in Guangdong 999 Brain Hospital were collected between May 2007 and May 2014 and then TSC gene mutation analysis was performed.Genotype-phenotype analyses for all the patients were also carried out.Results Fifty of the 76 (66％) patients were male,and 26 (34％) were female,in which 19 (31％) patients presented with cyst-like cortical tuber,69 (92％) with skin lesions,16 (30％) with renal lesions,50 (69％) with mental retardation and 39 still suffered seizures after a year.In this study,22 (29％) cases showed TSC1 gene mutation,31 (59％) presented TSC2 gene mutation,and 15 (20％)cases had no mutation identified.The mutation ratio of TSC1 ∶ TSC2 was approximately 3 ∶ 5,while the mutation ratio of TSC1 ∶ TSC2 was 1 ∶ 1 for familial TSC patients,and 1 ∶ 2 for sporadic TSC patients.Comparing to those with TSC1 gene mutation and no mutation identified,patients with TSC2 gene mutation exhibited statistical meaning on the aspects of the onset age of seizure (Z =1.688,P =0.007),seizure onset before l-year-old (x2 =10.584,P =0.001),epilepsy duration (x2 =4.996,P =0.025),spasms onset (x2 =10.111,P =0.001),cyst-like cortical tuber (x2 =9.182,P =0.002),skin lesions (x2 =9.016,P =0.003),as well as renal lesions (x2 =6.079,P =0.014).No apparent relation was found between genotype and intelligence outcome.Conclusions The patients with TSC2 gene mutations presented severer symptoms in seizure onset than those with TSC1 gene mutation and no mutation identified.The patients with TSC2 gene mutation were characterized by early onset of seizure,especially before 1-year-old,others like spasms onset,cyst-like cortical tuber,skin lesions,as well as renal lesions being more vulnerable.Therefore,more active treatment should be given to the patients with TSC2 gene mutation.

Association study between polymorphisms of TSC1, TSC2 ,PTEN genes and autism in Chinese Han population / 中华行为医学与脑科学杂志

Objective To explore the association between the polymorphisms of TSC1,TSC2,PTEN genes and autism in Chinese Han population.Methods 274 autism patients and 386 heahh controls were recruited,and SnaPshot technique was used to genotype the 13 tagSNPs of TSC1,TSC2 and PTEN genes.The allele,genotype and haplotype frequencies of the SNPs were compared using SHEsis and SNPStats softwares.Results Mter Bonferroni correction,the allele distribution of rs2809244 (TSC1) (x2 =9.537,P=0.002,adjusted P=0.016),rs1050700 (TSC1) (x2 =9.313,P=0.002,adjusted P=0.016),rs2072314(TSC2) (P＜0.01,adjusted P＜0.01) and rs8063461 (TSC2) (P＜0.01,adjusted P＜0.01)showed significant difference between two groups (P＜0.05).The genotype frequencies of rs2072314(TSC2)and rs8063461(TSC2) showed significant difference between two groups(P＜0.05).Moreover,the frequency of haplotype A-G (OR =14.548,95％ CI =5.450-38.830) in the haplotype block rs2809244-rs3761840 showed significant difference between two groups(P＜0.05),A-G significantly increases the risk of autism.The frequencies of haplotype A-A (OR=0.608,95％ CI =0.409-0.903,P=0.013),G-A (OR=7.812,95％ CI =5.338-11.459,P＜0.01)and G-G (OR=0.356,95％ CI =0.274-0.463,P＜0.01) in the haplotype block rs2074969-rs8063461 were identified,which were significant difference between two groups(P＜0.05),and AA and G-G significantly reduced but G-A increased the risk of autism.Conclusion The polymorphisms of TSC1 and TSC2 genes might associate with autism in Chinese Han population.

Analysis of TSC2 gene mutational in tuberous sclerosis complex / 临床神经病学杂志

Objective To analyse the mutation of pathogenic gene TSC2 in tuberous sclerosis complex (TSC). Methods Using polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP), all the 41 exons of TSC2 gene were analyzed in 4 TSC cases(include 1 suspect case) from one family and 1 sporadic TSC case ,and compared with the kin familial controls and kinless normal controls. Results Missense mutation on exon33 1346S→P (4037T→C) of TSC2 was found in 4 familial cases, and no mutation of TSC2 gene was found in the sporadic case and all the health controls. Conclusion Missense mutation on exon33 (1346S→P,4037T→C)is a new discovery in TSC2 gene of patients with TSC.