Corresponding author: SONG Guang yao Human umbilical venous endothelial cells (HUVEC) were incubated with free fatty acids (FFAs) of various degrees of saturation. C18:1 and C18:2 inhibited the synthesis of nitric oxide (NO) and endothelin (ET) in HUVEC, and ET synthesis was inhibited much more than NO synthesis, and C18:0 increased ET synthesis in HUVEC, suggesting that the effects of FFAs on HUVEC were related to their degree of saturation.

BACKGROUND: Phenylketonuria is caused by gene mutation of phenylalanine hydroxylasel (PAH), which is mainly induced by permutation, short segments and insertion of base.OBJECTIVE: To evaluate the gene mutation of phenylalanine hydroxylasel in phenylketonuria in Hui nationality.DESIGN: Open study.SETTING: Urumqi General Hospital of Lanzhou Military Area Command of Chinese PLA; Capital Pediatrics Institute.PARTICIPANTS: A boy of Hui nationality in China and aged 3.1 years was selected in this study. The boy had intellect hysteresis in his one year and received medical treatment in his three years, while he was diagnosed as cerebral paralysis. After repeatedly inefficient treatment, he was hospitalized in our hospital on December 13, 2004. Iron sesquichloride in urine was strongly positive and concentration of serum phenylalanine was 1 680 μmol/L; therefore, he was diagnosed as the typical phenylketonuria.METHODS: 5 mL venous blood was selected from the boy and his parents, respectively, and anticoagulated with EDTA-Na2. DNA in gene group was extracted by using typical phenol/chloroform method. In addition, polymerase chain reaction (PCR) primer sequence of extron 7, 6, 11, 3, 12 and 5 of PAH gene was designed based on references. And then, PCR products were detected with 2% agarose gel electrophoresis. 5 μL PCR products were mixed with the same volume of degenerated buffer solution, degenerated at 97 ℃ for 5 minutes, put in iced bath and performed with 80 g/Lnon-degenerated polyacrylamide gel electrophoresis. After that, the products were dealt with sliver staining routinely, and single strand DNA banding patterns were analyzed and recorded. ABI377 automatic sequenator (PE Company) was used to detect PCR sequence and purify PCR product in Shanghai Boya Biotechnology Company.MAIN OUTCOME MEASURES: Iron sesquichloride in urine, concentration of serum phenylalanine and mutant gene types of phenylalanine hydroxylase.RESULTS: Extron 7, 6, 11, 3, 12 and 5 of PAH gene were analyzed in the boy and his parents. The results demonstrated that SSCP electrophoresis in extron 6 was different from that in the normal control group. Site of electrophoresis strip of his father was coincident with that of his mother, but different from that of the boy. Sequencing results indicated that point mutation (cytosine replaced by thymine), which was a R176X mutant heterozygote, occurred at the 526th site of cDNA of phenylalanine hydroxylase gene in his parents; however, two chromosomes of the boy had mutation at the same site, which was R176X mutant homozygote.CONCLUSION: Mutation of R176X homozygote of phenylketonurea is firstly reported in Hui nationality in China.

Objective To perform mutation analysis of survival motor neuron gene 1 (SMN1 in two spinal muscular atrophy (SMA) patients and their parents to evaluate the effects of the two SMN1 gene mutations on the transcript levels of the gene and preliminarily predict their effects on the structure and function of SMN protein.Methods Mutation analysis of SMN1 gene was carried out by multiplex ligationdependent probe amplification,reverse transcript-polymerase chain reaction (RT-PCR) and cloning sequencing.Transmission of the mutations was confirmed by the mutation analysis in patients' parents.The full-length SMN1 (SMN1-fl) transcript levels of the patients carrying these subtle mutations were detected using quantitative RT-PCR.Results The two patients were diagnosed as SMA Ⅱ and SMA Ⅲ.They carried p.Val19GlyfsX21 and p.Ala2Gly SMN1 mutations in SMN1 gene,respectively.Both of the two mutations were originated from their fathers.Compared with the healthy individuals (23.5 ± 4.9),the two patients had a significant reduction in the level of SMN1-fl transcripts (t =3.322,P =0.011 (p.Ala2Gly) ;t =6.964,P =0.000 (p.Val19GlyfsX21)).However,compared with the healthy carriers (14.1 ±4.5),the patient with p.Ala2Gly mutation had no significant reduction in the level of SMN1-fl transcripts (13.9 ±3.6,t =0.058,P =0.955) ; however,the patient with p.Val19GlyfsX21 mutation had a significant reduction (4.9± 2.4,t =3.725,P =0.004).Conclusions Two SMN1 gene mutations are identified in our study.The mutation p.Val19GlyfsX21 is a novel mutation and p.Ala2Gly is firstly reported in Chinese SMA patients.p.Val19GlyfsX21 may possibly lead to decreased SMN1-fl mRNA by nonsense-mediated messenger RNA decay,however,p.Ala2Gly has no obvious effects on the amount of the SMN1-fl transcripts,indicating that its deleterious effect may be occurring at SMN protein level or the function of SMN protein.

Objective To investigate the clinical characteristics of methylmalonic academia in adolescence cases. Methods 4 cases were diagnosed methylmalonic academia by gas chromatography- masss pectrogram whose clinical, manifestations and treatment process were analyzed. Results The main clinical manifestations in 4 cases with methylmalonic academia were intellect impairment,epilepsy, pyramid signs; 2 of them suffered with hypopsia and optic atrophy, one of them suffered with papilledema. Symptoms were improved after treated with cobamamide and L-carnitine in all the 4 cases 1 months later. Conclusions The main clinical characteristics of methylmalonic academia in adolescence were intellect impairment, epilepsy and pyramid signs. The symptoms could be improved after treatment.

Objective To analyze the distribution of common chromosomal karyotypes of patients with Turner syndrome (TS), and to explore the correlation between the age and height standard deviation scores (HSDS) on diagnosis.Methods Retrospective investigation was performed for the data of age and HSDS on diagnosis in 273 TS girls(≤ 18.0 years old)diagnosed by chromosomal karyotypes.The main statistical methods were analyzed with t-test and Pearson correlation test by using the SPSS 18.0 statistical software.Results (1) There were 4 kinds of common chromosomal karyotypes in the TS :45, X (87/273 cases,31.9％),46, X, i (Xq) (43/273 cases, 15.7％) ,45, X/46, X, i (Xq) (36/273 cases, 13.2％) and 45, X/46, XX (23/273 cases, 8.4％), respectively, the adolescent TS all had delayed puberty.For the cases with 45, X karyotypes ,3 cases presented mental retardation and 2 cases with organs deformity.(2)The patients with 45 ,X/46,X,i(Xq) karyotypes or with 46,X,i(Xq) karyotypes had the maximum(12.56 age) or the minimum(9.70 age) mean age on diagnosis, respectively, there was a significant difference between 2 groups (t =3.019, P =0.004).The maximum deviation from normal height was found in the patients with karyotypes of 46, X,i (Xq) (HSDS =-4.04), and the minimum deviation was in the patients with karyotypes of 45,X/46, XX (HSDS =-3.16), and there was a significant difference between 2 groups (t =-2.95, P =0.004).(3) More than 75.7％ of TS patients was diagnosed when their heights deviated above 3 SD,and their mean age on diagnosis was 12.10 age,which was 3 years later than those patients within 2 SD.(4) There was a significant negative correlation between the age and HSDS on diagnosis in the groups of common chromosomal karyotypes[45,X、46,X,i(Xq) and 45,X/46,XX] (r =-0.551,-0.560,-0.622,all P ＜ 0.01), except for the group with the 45, X/46, X, i (Xq).Conclusions (1) In this study, the consti-tuent ratios of these 4 common chromosomal karyotypes were different from those in Europe and America's.(2)Patients with 45 ,X may have more severe symptoms than others.(3)The mean age on diagnosis was at least 3.0 years earlier when considered HSDS below-2.00 as an indicator for chromosomal karyotype screening,which would facilitate earlier diagnosis.

OBJECTIVETo establish a hyperphenylalaninemia related genes screening method using Ion Torrent Personal Genome Machine (PGM) for early detection and differential diagnosis of hyperphenylalaninemia (HPA).

METHODSThree children with known HPA mutations and a healthy control were used for setting up the method. Ten children with HPA with known mutations were recruited for validating the method. Ion Ampliseq PCR was used to amplify the 5' and 3' untranslated region, coding sequence, and flanking introns of PAH, GCH1, PTS, QDPR, and PCBD1 genes. After the enrichment with the Ion OneTouch system, the products were sequenced by PGM. Data from the PGM were processed with Torrent Suite v2.2 software package. All variations were confirmed by Sanger sequencing.

RESULTSFor the 4 samples, the PGM output was 94.22 Mb, with approximately 99.5% of reads mapping to the target regions. Among these samples, we detected 74 variations (28 positions) including 6 known mutations. Compared with database and results of Sanger sequencing, 55 (18 positions) polymorphisms and 13 (4 positions) false positive calls were confirmed. For the 10 samples, all the known mutations were successfully identified.

CONCLUSIONIon Torrent PGM sequencing is suitable for screening genetic mutation underlying HPA from the perspective of metabolic pathways, which can meet the clinical demand for individualized diagnosis and treatment.

High-Throughput Nucleotide Sequencing ; methods ; Humans ; Mutation ; Phenylketonurias ; genetics

OBJECTIVETo understand the pathogenic effect and the correlation between the genotype and phenotype of the 4 novel missense mutations (G247S, E280G, P362T and A434D) of phenylalanine hydroxylase gene (PAH).

METHODS(1) The enzyme activity of the 4 mutants was assessed by using transient protein expression in mammalian cells. (2) The PAH amino acid sequences among different animal species were alignmented. (3) The effects of the 4 missense mutations on the protein structure were analyzed. (4) The clinical phenotype of the patients with PKU were analyzed, according to their blood Phe levels prior to treatment and the Phe tolerance.

RESULTS(1) The residual enzyme activity expressed in vitro of G247S, E280G, P362T and A434D were 3.1%, 0.4%, 8.2% and 21.7% of the wild-type PAH respectively; (2)Gly247, Glu280 and Pro362 were among the highly conserved amino acids, while Ala434 was only moderately conserved; (3) As revealed by 3D structural analysis, G247S and E280G, being located at the active center of the enzyme, interfered with the binding of PAH to BH4 and ferrousion respectively, while P362T and A434D affected the formation and stability of the dimer and the tetramer of PAH; (4) As shown by clinical phenotypic analysis, classical PKU were observed in patients carrying G247S and E280G, moderate PKU were observed in patients carrying A434D, whereas both classical and moderate PKU were observed in patients carrying P362T.

CONCLUSION(1) The E280G, G247S, P362T and A434D are all disease-causing mutations, with those located at the center of the enzyme displaying the most marked pathogenic effect; (2)The results of the structural analysis of the 3D molecule are consistent with the activity assessment of the enzyme expressed in vitro; (3) The consistency is observed between the genotype, the enzymatic activity expressed in vitro and the clinical phenotype.

Amino Acid Sequence ; Animals ; Child ; Child, Preschool ; Female ; Gene Expression ; Genotype ; Humans ; Infant ; Infant, Newborn ; Male ; Models, Molecular ; Molecular Sequence Data ; Mutant Proteins ; chemistry ; genetics ; metabolism ; Mutation, Missense ; Phenotype ; Phenylalanine Hydroxylase ; chemistry ; genetics ; metabolism ; Phenylketonurias ; enzymology ; genetics ; Protein Conformation ; Sequence Alignment ; Structure-Activity Relationship

Objective To study the mutation characteristics in phenylalanine hydroxylase gene of Xinjiang minority nationality phenylketonuria (PKU) patients and provide a scientific basis for PKU prevention and cure strategy.Methods Mutations in phenylalanine hydroxylase gene were detected by Dolymerase chain reaction-single strand comformation polymorphism (PCR/SSCP) and gene sequencing in 12 minoritv nationality patients.Results Thirteen different mutations,including 8 missense mutations,1 nonsense mutation and 3 splice mutations were found in 24 alleles.The moat common mutations were EX696A＞G and P281 L.which were respectively prevalent in Asia and Europe populations.The common mutations were R243Q,R111X,R176X and F161S.The mutation frequency of R243Q was the highest and R111X was the third highest in Northern China.R176X and F161S were two rare mutations world wide.Especially.F161S was a Chinese-specific mutation because it was for the second time that it was found in China.The mutations detected in this study were first reported in these 3 minority nationality populations,which showed a distinct ethical characteristic.Condusions There is not only a consanguineous relation but also a distinct difference in PAH gene distribution between Xinjiang minority nationality population and yellow race and Latin-American.The results suggest that Xinjiang could probably be a special PAH gene distribution region.

[Abstract ] Objective To investigate the growth and development of children after liver transplantation.Methods Seventy children who underwent liver transplantation in Beijing Friendship Hospital and Tianjin First Central Hospital from September 2006 to May 2014 were included in the study.Height Z-scores (ZH )and weight Z-scores (ZW )of each child were respectively calculated at transplantation and 6 months,1 year,2 years,3 years,4 years and 5 years after transplantation.The change trends of ZH and ZW at each time point were analyzed.Moreover,ZH and ZW at each time point were compared with the standardized mean of growth 0 (standardized mean ) formulated according to the survey data of children's physical development in nine provinces and municipalities in 2005.Children were divided into Z≥ -2 group and Z <-2 group according to ZH and ZW at transplantation.And the change trend of ZH and ZW of each group was compared.Results The mean of ZH and ZW at the time of liver transplantation was -1.63 ± 1.35 and-1.53 ±1.52 respectively.After liver transplantation,ZH and ZW increased gradually.ZH and ZW of children before transplantation and 6 months after transplantation were significantly lower than standardized mean (all in P <0.01).ZH and ZW in other postoperative periods had no statistical significance with standardized mean (all in P >0.05).There were significant difference between ZH≥ -2 group and ZH <-2 group at transplantation and 6 months and 1 year after transplantation (both in P <0.05).Zw of Zw≥ -2 group and Zw <-2 group at transplantation and 6 months,1 year and 2 years after transplantation had difference with statistical significance (all in P <0.05).Conclusions The growth and development of children with liver transplantation recover gradually and increase significantly at 1 year after transplantation.

Objective:To analyze the MRI characteristics of surgical resected renal angiomyolipoma (AML) smaller than 4 cm.Methods:A total of 112 patients with surgical pathology confirmed renal AML of which the maximum diameter was smaller than 4 cm were analyzed retrospectively in the First Medical Centre, Chinese PLA General Hospital from January 2014 to November 2020, 5 of which were epithelioid angiomyolipoma (EAML) patients. According to the presence or absence of visible fat in lesions on MRI, the lesions were divided into AML with fat group and AML without visible fat (AML wovf) group. The MRI features were evaluated, including maximum diameter, location, growth pattern, shape, beak sign, angular interface with renal parenchyma, pseudo-capsule, hemorrhage, cystic degeneration, coagulative necrosis, flowing void in the tumor, signal intensity and homogeneity on T 2WI and diffusion weighter imaging (DWI), the peak enhanced phase. The differences of maximum diameter of AML with fat and AML wovf were analyzed using Mann-Whitney U test, and the differences of MRI features were analyzed using χ 2 test or Fisher′s exact probability test. Results:There were 123 lesions found in 112 patients, and 96 lesions contained fat and 27 lesions were AML wovf. 82 lesions showed round and round-like shapes, 112 lesions showed exophytic growth pattern, 71 lesions with peak enhancement in corticomedullary phase. And the numbers of lesions with angular interface with renal parenchyma, beak sign, cystic degeneration, pseudo-capsule, hemorrhage were 30, 49, 1, 1, 1, respectively. There was no coagulative necrosis in all lesions. Compared with AML with fat, AML wovf was single lesion. The diameters of AML with fat and AML wovf were 2.5 (1.7, 3.5) and 1.8 (1.4, 2.3) cm respectively, with statistically significant difference ( Z=-2.80, P=0.005). In the AML with fat and AML wovf, 65 and 12 cases were heterogeneous in T 2WI, 44 and 5 lesions showed beak sign, 26 and 4 lesions showed angular interface with renal parenchyma, 57 and 10 cases were heterogeneous in DWI. And there were 5 and 6 lesions showed the endophytic, 44 and 8 lesions showed partly exophytic, 47 and 13 lesions showed exophytic in patterns of tumor growth respectively. The beak sign, homogeneous in T 2WI and DWI, patterns of tumor growth showed statistical differences in AML with fat and AML wovf (all P<0.05), and there was no significant difference in other features ( P>0.05). A total of 5 EAML patients were with 8 lesions. One patient had 4 lesions with fat, other patients had single lesion in which 2 lesions with fat, 2 lesions without visible fat. One lesion without visible fat showed hemorrhage. Conclusions:Surgical resected AML smaller than 4 cm is often exophytic round and round-like, enhanced in corticomedullary phase, showing angular interface with renal parenchyma and beak sign, with rare cystic degeneration, pseudo-capsule, hemorrhage and improbable coagulation necrosis. AML wovf is single smaller lesion which often shows endophytic growth pattern, and beak sign is infrequent. EAML seems to be present in two modes, multiple lesions with fat and AML wovf with hemorrhage.