Three cases of carnitine-acylcarnitine transposase deficiency and literature review / 中国新生儿科杂志

Objective To study the clinical features,biochemical characteristics and gene mutations of patients with carnitine-acylcarnitine translocase deficiency (CACTD).Method The clinical data,biochemical markers and gene mutations of three cases with CACTD admitted our hospital in 2017 were retrospectively analyzed.The related literatures were searched from China national knowledge infrastructure,wanfang database,PubMed,national center for biotechnology information and Embase using keywords "neonate","infant","carnitine-acylcarnitine deficiency","carnitine-acylcarnitine translocase",and SLC25A20"(up to April 2018).Result (1) Three cases (2 boys and 1 girl) with CACTD were full-term infants without asphyxia after birth.The mothers had no abnormal pregnancy,and the parents had no consanguinity.All the patients had poor response and severely hypoglycemia 15～20 hours after birth.Hyperammonemia,elevated liver enzymes and creatine kinase,severe dicarboxylic aciduria,significantly increased level of long-chain acylcarnitine,and significantly decreased concentration of free carnitine were observed in all 3 patients.Significantly decreased serum ketone body was observed in 2 cases.All of them had recurrent atrioventricular block and ventricular tachycardia requiring repeated electrocardioversion,lidocaine,and amiodarone treatment.Arginine,carnitine and special formula with low fat and high medium-chain-triglyceride were given to two infants.Two infants died of cardiorespiratory failure at 3-day and 8-day of life,respectively.The other infant's clinical condition improved significantly.However,he was discharged from our NICU at the request of his parents.Gene analysis revealed that compound heterozygous mutations c.199-10T＞G and IVS7-9_16 ins (a possible novel mutation) were detected in the SLC25A20 gene of case 2.Homozygous mutation c.199-10T＞G was identified in the SLC25A20 gene of case 3 whose parents both carried this mutation.(2) A total of 17 articles and 50 cases were retrieved and analyzed.A total of 40 mutations were found in the SLC25A20 gene.Homozygous mutations were found in 23 cases,and compound heterozygous mutations were found in 27 cases.The mutation of c.199-10T＞G was the most common mutation and occurred 22 times in the patients from Asia population.Other mutations were found less than 6 times.The review showed that the most common clinical features included hypoketotic hypoglycemia,hyperammonemia,elevated liver enzymes and creatine kinase,remarkable dicarboxylic aciduria,significantly increased level of long-chain acylcarnitine,significantly decreased free carnitine,arrhythmia and cardiomyopathy.Mostly,the onset of symptoms was within 1 week after birth (88％,44/50).The mortality was 69.8％ (30/43).Most patients died within the first year of their life.Conclusion Early recognition,early diagnosis and prompt treatment are crucial for CACTD patients.Gene analysis is a reliable diagnostic method.The mutation of c.199-10T＞G is the most common SLC25A20 mutation reported in Asia population.Hypoketotic hypoglycemia is an early sign of this disease.Families with a proband need prenatal diagnosis during the second pregnancy.

Detection of promoter and 3′ UTR mutation in A20 gene of a case with T cell lymphoma cell leukemia / 中华血液学杂志

Objective@#To clarify the characteristics of the A20 regulatory changes by analyzing mutations in the non-coding region of the A20 gene in patients with T-cell lymphoma leukemia (T-LCL) .@*Methods@#PCR and nucleotide sequence analysis were used to detect mutations in the non-coding region of the A20 gene, and DNA samples from PBMCs of 52 cases of T-LCL and 99 healthy controls.@*Results@#A missense mutation (c.-672T>G) was detected in the A20 gene promoter from one T-LCL patient, which has been registered as a SNP (rs139054966) in gene bank. Meanwhile, a new mutation was detected in the 3′ UTR mRNA (3916 (C>G) ) . These two mutations were absent in other T-LCL samples and controls.@*Conclusion@#The rs139054966 (c.-672T>G) and 3916 (C>G) mutations in the A20 gene were detected in T-LCL patients for the first time. There was also rs139054966 located on the binding region of the transcription factor P53, and its significance remained to be further clarified.

Detection of promoter and 3' UTR mutation in A20 gene of a case with T cell lymphoma cell leukemia / 中华血液学杂志

Objective: To clarify the characteristics of the A20 regulatory changes by analyzing mutations in the non-coding region of the A20 gene in patients with T-cell lymphoma leukemia (T-LCL) . Methods: PCR and nucleotide sequence analysis were used to detect mutations in the non-coding region of the A20 gene, and DNA samples from PBMCs of 52 cases of T-LCL and 99 healthy controls. Results: A missense mutation (c.-672T>G) was detected in the A20 gene promoter from one T-LCL patient, which has been registered as a SNP (rs139054966) in gene bank. Meanwhile, a new mutation was detected in the 3' UTR mRNA (3916 (C>G) ) . These two mutations were absent in other T-LCL samples and controls. Conclusion: The rs139054966 (c.-672T>G) and 3916 (C>G) mutations in the A20 gene were detected in T-LCL patients for the first time. There was also rs139054966 located on the binding region of the transcription factor P53, and its significance remained to be further clarified.

Impacts of A20 gene deletion on clinicopathological features and prognosis of diffuse large B cell lymphoma and relative molecular mechanism / 重庆医学

Objective To detect the A20 gene deletion,investigate the impacts of A20 gene deletion on clinicopathological features and prognosis of DLBCL,and relationship between activation of NF-κB pathway and relative molecular pathogenesis.Methods A20 gene deletion was detected by fluorescence in situ hybridization (FISH).The expression of A20,Survivin,P65 and Ki-67 were detected by immunohistochemistry stain.Apoptosis was assayed by TUNEL.Follow-up and statistical analysis were done.Results The deletion rate of A20 gene was 21.7％.The deletion rate of A20 gene was obviously higher in ABC-like DLBCL than that in GCB-like DLBCL (30.6％ vs.8.3％,P＜0.05).It was observed that there was a negative correlation between A20 protein expression and A20 gene deletion (r=-0.259,P =0.023).The expression of P65 and Survivin protein was positively correlated with the A20 gene deletion (r=0.280,P =0.015;r =0.313,P =0.007).Apoptosis rate was significantly reduced in DLBCL patients with A20 gene deletion.The apoptosis rate was higher in cases with positive expression of A20 protein,while that was lower in cases with positive expression of p65 and Survivin protein than those with negative expression of corresponding protein.There was no statistically significant difference in apoptosis rate between ABC-like and GCB-like DLBCL patients (P＞0.05).COX regression analysis indicated that age,A20 gene deletion,types of DLBCL and Ki67 expression were independent factors associated with survival status.Log-rank test showed that there was a statistical difference in survival status between the cases with and without A20 gene deletion (P=0.015).Conclusion A20 gene deletion may associate with the attenuation of A20 protein expression.The latter weakens negative feedback regulation of A20 protein for NF-κB pathway.An up-regulated expression of Survivin and abnormal proliferation and apoptosis may be result from the abnormal activation of NF-κB.A20 gene deletion brings certain influence on clinical course and prognosis of DLBCL.

Changes of A20 expression in mesangial cells of LPS-induced diabetic nephropathy rat model / 天津医药

Objective To observe the changes of A20 in mesangial cells of diabetic nephropathy (DN) rat model in?duced by lipopolysaccharide (LPS)-rat, and to explore its possible mechanism. Methods (1)Thirty health male Wistar rats were randomly divided into two group. Model rats were given streptozotocin (STZ) at a dose of 60 mg/kg by intraperitoneal in?jection. Rats in the control group received the same volume of citrate buffer in the same way. Levels of blood glucose and uri?nary microalbumin were detected in two groups at the 6th and the 8th week. Changes of renal pathology were observed by HE staining. Changes of protein A20 were observed by immunohistochemistry. (2) Expression changes of gene and proteins A20, nuclear factor (NF)-κB, IκB, IKKγand MCP-1 in renal cells treated with LPS were determined after treatment with different time points (0, 2, 4, 6, 12, 24, 48 and 72 h) and different concentrations (0.1, 1 and 10μg/L). Results (1) Levels of blood glucose and urinary microalbumin were significantly increased in model group compared with those of control group ( P <0.01). HE stainig showed that hyaline degeneration in tubular epithelial cells was found in model group, especially at the 8th week. Results of immunohistochemistry showed that expression of protein A20 significantly decreased in kidney tubules and nearly disappeared in glomerulus in model group compared with that of control group, which expressed less at the 8th week. (2) There was no significant difference in the expression of IKKγbetween different concentrations and different times. Com?pared with 0 h, the expression of A20 protein was increased at 2 h and 4 h, except that the expression of A20 protein in?creased after 6 h (P<0.05). Meanwhile NF-κB expression increased and IκB expression decreased in different time points (P<0.05). In addition, the expressions of A20 and IκB were decreased concentration-dependently (P<0.05). The expres?sion levels of NF-κB and MCP-1 were increased concentration-dependently (P<0.05). Conclusion A20 may involve in the development of diabetic nephropathy by regulating the NF-κB pathway.

The effect of knockdown A20 expression on the proliferation, apoptosis and migration of MCF-7 cells / 安徽医科大学学报

Objective To study the effect of knockdown A20 expression on the proliferation, apoptosis and migra-tion of MCF-7 cells and to evaluate the potential value of the A20 gene as the therapeutic target of breast cancer. Methods Synthesized siRNA targeted to A20 gene or negative control siRNA were transfected into MCF-7 cells by using lipofectamine 2000. CCK8 assay, Annexin V and 7-AAD double staining cytometry, Transwell assay were performed to investigate the effect of knockdown A20 mRNA expression on the proliferation, apoptosis, migration of MCF-7 cells, respectively. Results It can inhibit the proliferation and migration as well as promote the apoptosis in MCF-7 cells by knockdown A20 mRNA expression. Conclusion A20 gene plays an important role in the prolif-eration, apoptosis and migration of MCF-7 cells and it could be a potential therapeutic target of breast cancer.

Correlation between polymorphism of rs5029924 in A20 promoter region and posttraumatic susceptibility to sepsis / 中华创伤杂志

Objective To investigate relationship of single nucleotide polymorphism (SNP)at rs5029924 locus in A20 promoter region and posttraumatic sepsis.Methods PCR-DNA sequencing was used to analyze different gene distribution at rs5029924 locus of 103 trauma patients with sepsis (Group A),120 trauma patients without sepsis (Group B) and 135 healthy peoples (control group).Relation of different genotypes at rs5029924 locus to sepsis susceptibility was analyzed.Peripheral blood cells of healthy peoples of different genotypes were stimulated using lipopolysaccharides (LPS) in vitro.Expression of A20 mRNA was measured by fluorescent quantitative PCR,expression of A20 protein by flow cytometry,and levels of TNF-α and IL-1β by ELISA method.Results Frequency of rs5029924 genotypes CC,CT and TT was respective 77.8％,20.0％ and 2.2％ in control group； 63.1％,34.0％ and 2.9％ in Group A； 83.3％,15.0％ and 1.7％ in Group B.Significantly lower frequency of CC genotype was observed in Group A when compared to Group B and control group (P ＜0.05),but no statistical differences were recorded between Group B and control group (P ＞ 0.05).CT/TT genotype increased risk coefficient of sepsis to 2.397-fold higher level when compared to CC genotype.Allele T increased prevalence of sepsis significantly as well (OR =2.056) when compared to allele C.After LPS treatment in vitro,CC genotype individuals revealed significantly higher levels of A20 mRNA and protein in peripheral blood leukocytes,but significantly lower levels of TNF-α and IL-1 β when compared to CT/TT genotype individuals.Conclusion Polymorphism of rs5029924 locus is associated with sepsis susceptibility and the reason may be that mutant genes affect promoter activity and down-regulate A20 expression,which fails to suppress inflammation.

Zinc finger protein gene A20 inhibits lipopolysaccharide-induced IL-8 expression of endothelial cells / 第三军医大学学报

Objective To investigate the effects of zinc finger protein gene A20 on the inhibition of lipopolysaccharide (LPS) induced interleukin 8 (IL 8) expression in endothelial cells. Methods Plasmid pcDNA3.1EHA20 was transfected into human umbilical vein endothelial cells (HUVECs) by DOTAP method. The positive cell clones were screened with G418. The stable transfection and expression of A20 in HUVECs were determined by immunofluorescent analysis. IL 8 expression was detected by sandwich ELISA with double monoclonal antibodies. Results High expression of A20 gene in HUVECs transfected with pCDNA3.1EHA20 was confirmed by immunofluorescent analysis. IL 8 expression increased in LPS induced endothelial cells. A20 gene could inhibit more than 70% LPS induced IL 8 expression ( P

OVEREXPRESSION OF ZINC FINGER PROTEIN INHIBITS LPS-INDUCED EXPRESSION OF E-SELECTIN IN ENDOTHELIAL CELLS / 解剖学报

Objective Previous studies have demonstrated that LPS can induce endothelial cell activation and the expression of E-selectin. In this study, we examined whether A20 gene could inhibit the expression of E-selectin in endothelial cells induced by LPS. Methods With the help of DOTAP, endothelial cells were transfected with pCDNA3.1 EHA20. The postive cell clones were selected with G418.The stable transfection and expression of A20 in the endothelial cells were determined by immunofluorescence analysis. The E-selectin expression was checked by immunofluorescence, Western blot and in situ hybridization. Results Abundant A20 stable expression in endothelial cells transfected with pCDNA3.1 EHA20 was confirmed by immunofluorescence analysis. E-selectin expression increased in LPS-inducible endothelial cells. A20 gene inhibited 90% LPS-inducible E-selectin expression(P

TRANSFECTION OF ENDOTHELIAL CELLS WITH pCAGGSEHA20 AND ITS STABLE EXPRESSION / 解剖学报

Objective To observe the effectiveness of transferring human A20 gene into endothelial cells. Methods The shuttle plasmid pCAGGSEHA20 was constructed using gene cloning and recombined technique. Endothelial cells were transfected with pCAGGSEHA20 and pMAMneo by DOTAP. The postive cell clones were selected with G418. The stable transfection and expression of A20 in the endothelial cells were determined by in situ hybridization and immunohistochemical analysis. Results The two fragments digested from pCAGGSEHA20 by EcoRⅠ represented 4 6?kb and 2 3?kb by electrophoresis, which were confirmed to be the carrier and the A20 gene fragments inserted originally. The above results indicate that the construction of pCAGGSEHA20 was successful. Abundant A20 stable expression in endothelial cells transfected with pCAGGSEHA20 was confirmed by in situ hybridization and immunohistochemical analysis.Conclusion By means of the DOTAP, hA20 gene can be transferred and stably expressed in endothelial cells.