Report of five cases of protein-sensitive hypoglycemia with literature review / 中华内分泌代谢杂志

Objective To summarize the clinical feature,gene mutations,diagnosis,treatment,and follow-up data of protein-sensitive hypoglycemia,so as to improve the clinical understanding of the disease.Methods Five patients were diagnosed with protein-sensitive hypoglycemia during June in 2015 and December in 2017 from the Department of Pediatric Endocrinology and Inherited Metabolic Diseases,Children's Hospital of Fudan University.Clinical data of 5 cases were summarized,including clinical manifestations,findings of protein sensitivity test,therapy effect and prognosis.The endocrine and metabolic panel was used to investigate the genetic cause of four patients.Related literatures of protein-sensitive hypoglycemia were reviewed,and the phenotypes,genotypes,and therapy effects were summarized.Results Among the 5 patients diagnosed with positive results of protein-sensitive hypoglycemia,three were found to harbor glutamate dehydrogenase 1 (GLUD 1) mutations (c.965G ＞ A,p.R322H:2 cases;c.943C ＞T,p.H315Y:1 case),and another one had complex heterozygous mutations in L-3-hydroxyacyl-CoA dehydrogenase (HADH,c.29G ＞ C,p.R10P;c.89T＞ A,p.V30E).5 patients were euglycemia without any medical support after low protein diet.In 18 literatures retrieved and this study,there were totally 161 cases of protein-sensitive hypoglycemia (149 cases with GLUD1 mutations and 10 cases with HADH mutations).Conclusions When a child was admitted because of hypoglycemia,the diagnosis of protein-sensitive hypoglycemia should be suspected if he or she also had postprandial hypoglycemia,with or without hyperammonemia.Protein sensitivity test is helpful for us to make the diagnosis of protein-sensitive hypoglycemia.

RecET recombination system driving chromosomal target gene replacement in Zymomonas mobilis

Background: Zymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments. Results: The plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4. Conclusion: This approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.

Clinical features and genetic analysis of 17 cases of glucose-6-phosphate dehydrogenase deficiency in Guizhou province / 中华实用儿科临床杂志

Objective To explore the clinical and genetic characteristics of 17 cases with glucose-6-phosphate dehydrogenase(G6PD) deficiency in Guizhou,China.Methods The clinical features of 17 patients with G6PD deficiency were analyzed,DNA samples were obtained from the patients and some mothers,and the exons and flanking intronic sequences of the G6PD gene were analyzed by the polymerase chain reaction and sequencing.Results The cases had diverse phenotypes,these patients had acute haemolytic anaemia triggered by eating broad beans,infections,ingestion of specific drugs or the neonatal period and chronic nonspherocytic haemolytic anaemia.Three cases of the patients had concomitant diseases for α Mediterranean anemia,acute myeloid leukemia M2 type and neonatal anal membrane stenosis,respectively.G1376T,G1388A and A95G were the commonest G6PD variants in patients in Guizhou,China.G1376T,G1388A and A95G mutations were observed in 82.4％ cases.Two patients had only compound variants(c.1311 C ＞ T,IVS11 nt 93 T ＞ C).One case in the Rongjiang County,Guizhou Province had novel compound variants (c.G1388A,IVS10-10 T ＞ G) in the world.A patient's mother in the Guiyang City,Guizhou Province,China had compound variants (c.1376 G ＞ T,1311 C ＞ T,IVS11 nt 93 T ＞ C) as a carrier.Conclusions G6PD deficiency has a wide range of clinical heterogeneity.A novel G6PD compound variant haplotype c.G1388A,IVS10-10 T ＞ G was first found in the world,and the SNP spectrum of G6PD was enlarged.There may be a G6PD compound variant haplotype c.1376 G ＞ T,1311 C ＞ T,IVS11 nt 93 T ＞ C in Guizhou.

Clinical and genetic analysis of two Chinese patients with isovaleric acidemia and review of literature / 中华实用儿科临床杂志

Objective To discuss the clinical features and treatment of isovaleric academia (IVA) patients,and to gain more comprehensive understanding of isovaleryl-CoA dehydrogenase(IVD) mutation in 2 siblings in order to raise awareness to prevent the occurrence of IVA.Methods The clinical history and laboratory test of 2 cases of children with IVA were carried out.The exons and neighboring introns of IVD gene of the whole family were PCR-amplified for DNA sequencing.The literature review of IVA in China was also conducted.Results Organic acid analysis of urine by GC/MS for both siblings showed extremely elevated concentrations of isovaleric glycine.For the older sibling,an acute episode of IVA caused severe metabolic stress and eventually death in the neonatal period.However,the disease was well-controlled for the younger sibling due to timely treatment and follow-up care for 2 years.The DNA sequencing of the IVD gene in the family revealed a novel c.1016G ＞ A(C339Y) heterozygous mutation in mother and both of the siblings.No IVD mutation was detected in father or in any of the 50 cases of healthy controls.According to literature review,15 cases of IVA were reported in recent 15 years in China,including neonatal onset (11 cases),acute episode (12 cases),odor of sweaty feet (12 cases),pancytopenia (9 cases),hyperammonemia (5 cases),hypocalcemia (6 cases),and 6 cases of death were reported.Additionally,5 cases that received treatment of BCAA-free formula milk showed positive outcome.However,only 2 cases were followed up for more than 2 years.Conclusions Two new IVA patients carrying c.1016G ＞ A(C339Y) mutation were reported in China.The mutation may lead to conformational change and functional deficient of the IVD protein.It is also necessary to point out that using direct DNA sequencing can not identify all patients with IVA due to limitations of this technology,and thus clinicians should be aware of the possibility of genetic misdiagnosis.Moreover,there is a trend of increasing IVA in China in recent years.

Cloning and sequence analysis of glyceraldehyde-3-phosphate dehyrogenase gene from Conyza blinii / 中草药

Objective: To clone and analyze glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from Conyza blinii and detect whether it can be the reference gene for C. blinii. Methods: Full length GAPDH was cloned by RACE. DNAMAN, BLAST, and MEGA bioinformatic tools were used to analyze its open reading frame (ORF), homology, and phylogenetic tree. The sequence was acted as internal control gene for semi-quantitative RT-PCR. Results: The gene designated GLGAPDH was 1 418 bp in length. It contained a 1 020 bp ORF encoding 340 amino acids. It shared 96% similarity with Gynura bicolor GAPDH and shared 93% similarity with Mikania micrantha GAPDH. GLGAPDH had closer relationship with GAPDH in G. bicolor. When the sequence acted as internal control gene, the semi-quantitative RT-PCR had benign amplification and good reproducibility. Conclusion: GAPDH is cloned from C. blinii for the first time. The semi-quantitative RT-PCR results prove that GAPDH gene is able to be the reference gene for gene expression analysis. The result of this study will provide the basis for the key enzyme expression and regulate the mechanism analysis in C. blinii effective components biosynthesis pathway.

Association of gene polymorphism of SDH gene with type 2 diabetic retinopathy / 中华实验眼科杂志

Background Some aboard studies showed that polyatomic pathway play an important role in the microsvasculopathy of type 2 diabetes,and sorbitol dehydrogenase (SDH) gene is involved in the pathogenesis of diabetic retinopathy(DR).There is no relevant research at home up to now.Objective This study was to investigate the correlation of sorbitol dehydrogenase (SDH) G-888C gene polymorphism with diabetic retinopathy (DR) in type 2 diabetes mellitus.Methods The SDH genotypes were determined by polymerase chain reactionreaction fragment length polymorphism (PCR-RFLP) in 187 patients with diabetes and 123 normal contrels.Patients were divided into two groups depending on the presence or absence of DR(DR:n=118;NDR:n=69).The frequencies of SDH genotype and allele were assayed and compared among these groups.This study was approved by Ethic Committee of Guangdong General Hospital.Written informed consent was obtained from each individual prior to any relevant medical procedure.Results The disease course in the DR group was significantly longer than that in NDR group(t=2.070,P=0.042),and other clinical features in both groups were non-significant (all P>0.05).The genotype frequencies in the DR group,NDR group and normal control group were 24.6%,8.7% and 8.1%,respectively,and frequencies of the G allele were 42.4%,25.4% and 29.7%,showing statistically significant differences among these three groups.The GG genotype and G allele frequencies were significantly higher in the DR group than those in the NDR group and normal control group (GG:P=0.007,P=0.001;G:P=0.001,P=0.004).There were no significant differences in the frequencies of the GG genotype and G allele between the NDB group and normal control group ( P>0. 05) as well as the proliferative DR group and non-proliferative DR group (P>0.05).Conclusion SDH G-888C gene polymorphism is associated with the development of diabetic retinopathy in southern Chinese.

The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthora perniciosa, the causal agent of witches' broom disease of Theobroma cacao

This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts.

Cloning, Sequence Analysis and Expression of Glutamate Dehydrogenase in Brevibacterium flavum GDK-9 / 微生物学通报

The glutamate dehydrogenase (EC.1.4.1.4) gene which amplified from the genome of Brevibacterium flavum GDK-9 by polymerase chain reaction was linked with pUCm-T for sequence alignment. Analysis of gdh sequences revealed that the whole sequence is 1927 bp, only one ORF existed, which used ATG as the initiation codon and coded a peptide of 448 amino acids with a calculated molecular weight of 48 kD. The comparability between the cloned gdh sequence to the reported sequence is high to 99.55%. Only the 1190th base mutation (C→A) lead to the change of amino acid sequence (Thr→Asn), the others are not. The recombinant plasmid pXG was then transformed into E. coli XL-Blue and Brevibacterium flavum GDK-9 which was induced by IPTG. SDS-PAGE analysis revealed that there was a clear induced protein band with molecular mass of 48.7 kD on expected position. Standard glutamate fermentations indicated that although the level of GDH increases the intracellular glutamate pool, the level of GDH has no influence on glutamate secretion.