OBJECTIVE: To explore the genetic basis for a child with clinically suspected 3-methylcrotonyl-coenzyme A carboxylase deficiency (MCCD). METHODS: Genomic DNA was extracted from peripheral blood samples of the proband and her parents. Whole exome sequencing was used to screen pathogenic variant in the proband. Suspected variant was verified by Sanger sequencing. Impact of the variant on the structure and function of protein product was analyzed by using bioinformatic software. RESULTS: Sanger sequencing showed that the proband has carried homozygous missense c.1342G>A (p.Gly448Ala) variant of the MCCC2 gene, for which her mother was a heterozygous carrier. The same variant was not detected in her father. The variant was predicted to be pathogenic by PolyPhen-2 and Mutation Taster software, and the site was highly conserved among various species. Based on the American College of Medical Genetics and Genomics standards and guidelines, the c.1342G>A (p.Gly448Ala) variant of MCCC2 gene was predicted to be likely pathogenic(PM2+PP2-PP5). CONCLUSION The homozygous missense variant of the MCCC2 gene c.1342G>A (p.Gly448Ala) probably underlay the molecular pathogenesis of the proband. Genetic testing has confirmed the clinical diagnosis.
Carbon-Carbon Ligases/genetics* ; Child ; Female ; Humans ; Male ; Mutation, Missense/genetics* ; Pedigree ; Urea Cycle Disorders, Inborn/genetics*

Recombinant strains expressing enzymes for ATP regeneration and L-theanine production were constructed and used for the synthesis of L-theanine. The ppk gene encoding polyphosphate kinase (PPK) from Rhodobacter sphaeroides and gmas gene encoding γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays were synthesized, and two recombinant plasmids, pETDuet-ppk+gmas and pET21a-ppk+gmas were constructed for co-expression of PPK and GMAS in Escherichia coli BL21(DE3). SDS-PAGE analysis showed that PPK and GMAS were overexpressed in soluble form in both recombinant strains. GMAS-PPK obtained from the recombinant strain containing pET21a-ppk+gmas was more efficient to synthesize L-theanine. After 24 h at 37 ℃ and pH at 7.0, 86.0% yield of L-theanine was achieved with catalytic amount of ATP. This study extends the application of enzymatic ATP regeneration system. In addition, it provides an efficient method for the biosynthesis of L-theanine.
Carbon-Nitrogen Ligases ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Glutamates ; biosynthesis ; Ligases ; Phosphotransferases (Phosphate Group Acceptor) ; genetics

OBJECTIVE: To investigate the genetic characterization of 3-hydroxyisovalerylcarnitine (C5-OH) metabolic abnormality in neonates. METHODS: Fifty two newborns with increased C5-OH, C5-OH/C3 and C5-OH/C8 detected by tandem mass spectrometry during neonatal screening were enrolled in the study. Genomic DNA was extracted from the whole blood samples of 52 cases and their parents. Seventy-nine genes associated with genetic and metabolic diseases including , were targeted by liquid capture technique. Variation information of these genes was examined by high-throughput sequencing and bioinformatic analysis, and then was classified based on the American College of Medical Genetics and Genomics (ACMG) standards and guidelines. The genetic types were classified as wild-type, -maternal-mutation, -paternal-mutation and -mutation. Wilcoxon rank-sum test was performed for the increased multiples of C5-OH calculated in neonatal screening. RESULTS: Twenty one variants (14 novel) were identified in 37 cases, 6 variants (5 novel) in 4 cases. The increased multiple of C5-OH calculated in -maternal-mutation and -mutation groups were significantly higher than that in wild-type group (all <0.05), while there was no significant difference between MCCC1-paternal-mutation group and wild-type group (>0.05). CONCLUSIONS Mutations on and genes are the major genetic causes for the increased C5-OH in neonates, and maternal single heterozygous mutation can contribute to the moderately to severely increased C5-OH.
Carbon-Carbon Ligases ; genetics ; Carnitine ; analogs & derivatives ; metabolism ; Female ; Genetic Testing ; Genetic Variation ; Humans ; Infant, Newborn ; Male ; Mutation ; Neonatal Screening ; Urea Cycle Disorders, Inborn ; genetics

OBJECTIVEMultiple carboxylase deficiency (MCD) is an autosomal recessive disorder. MCD is characterized by skin rash, metabolic acidosis, vomiting and psychomotor retardation. Depending on deficiency of the enzyme, MCD includes two different forms, biotinidase deficiency (BTD, OMIM 253260) and holocarboxylase synthetase deficiency (HLCSD, OMIM 253270). In this study, we analyzed gene mutations of four Chinese MCD patients and to explore the mutation spectrum and possibility of a molecular diagnosis.

METHODSAll exons and their flanking introns of biotinidase gene and HLCS gene were screened by polymerase chain reaction combined with DNA direct sequencing in four Chinese MCD patients. Genomic DNA was extracted using a kit from the peripheral blood leukocytes of each patient. PCR amplification products were checked by 2% agarose gel electrophoresis and were subsequently sequenced with both the forward and reverse primers.

RESULTSAll patients showed mutations in HLCS gene, whereas no mutation was found in biotinidase gene, proving that all the four patients had HLCS deficiency. Four previously reported mutations in HLCS gene were detected (Y456C, R508W, D634N and 780delG). A missense mutation of 1522C > T in exon 11 of HLCS gene, which was a homozygotic mutation, was identified in patient 1; a mutation of 1522C > T in exon 11 combined with a mutation of 1367A > G in exon 9, which was a compound heterozygotic mutation, was identified in patient 2; a mutation of 1522C > T in exon 11 combined with a mutation of 1900G > A in exon 13, which was a compound heterozygotic mutation, was identified in patient 3; a mutation of 1522C > T in exon 11 combined with a mutation of 780delG in exon 7, which was a compound heterozygotic mutation, was identified in patient 4. All the parents were carriers of mutations. No additional carrier of this four mutations was identified from 50 samples of Chinese controls.

CONCLUSIONThe 1522C > T (R508W) mutation probably represents a mutational hot-spot in Chinese HLCS deficiency patients while the 780delG mutation which was reported only in Japanese patients was found firstly in Chinese patients.

Asian Continental Ancestry Group ; genetics ; Biotinidase ; genetics ; Carbon-Nitrogen Ligases ; genetics ; DNA Mutational Analysis ; Exons ; Female ; Holocarboxylase Synthetase Deficiency ; enzymology ; genetics ; Humans ; Infant ; Infant, Newborn ; Introns ; Male

An increase in vancomycin-resistant enterococcal (VRE) bacteremia in hemato-oncological patients (n=19) in our institution from 2000 through 2001 led us to analyze the molecular epidemiologic patterns and clinical features unique to our cases. The pulsed field gel electrophoresis of the isolates revealed that the bacteremia was not originated from a single clone but rather showed endemic pattern of diverse clones with small clusters. A different DNA pattern of blood and stool isolates from one patient suggested exogenous rather than endogenous route of infection. Enterococcus faecium carrying vanA gene was the causative pathogen in all cases. Patients with VRE bacteremia showed similar clinical courses compared with those with vancomycin-susceptible enterococcal (VSE) bacteremia. Vancomycin resistance did not seem to be a poor prognostic factor because of similar mortality (5/8, 62.5%) noted in VSE bacteremia. Initial disease severity and neutropenic status may be major determinants of prognosis in patients with VRE bacteraemia.
Adolescent ; Adult ; Bacteremia/*drug therapy/microbiology ; Bacterial Proteins/genetics ; Carbon-Oxygen Ligases/genetics ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus/*drug effects/genetics ; Female ; Humans ; Male ; Middle Aged ; *Vancomycin Resistance

Adolescent ; Adult ; Aged ; Carbon-Nitrogen Ligases ; genetics ; Female ; Gene Expression ; Humans ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Nasopharynx ; pathology ; Young Adult

OBJECTIVE: To explore the genetic basis for a patient featuring multiple carboxylase deficiency (MCD). METHODS: PCR and Sanger sequencing were used to detect variant in the coding region of BT and HLCS genes in the patient. Suspected variants were verified in her parents and 80 unrelated healthy controls by a PCR-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The patient was found to carry compound heterozygous variants of the HLCS gene, namely c.286delG (p.Val96Leufs*162) and c.1648G>A (p.Val550Met). The c.286delG (p.Val96Leufs*162) was verified to be novel variant based on the result of PCR-RFLP analysis. No variant was found in the coding regions of BT gene in the patient. CONCLUSION The compound c.286delG (p.Val96Leufs*162) and c.1648G>A (p.Val550Met) variants probably underlie the MCD disorder in this patient. Above results have enriched the variant spectrum of MCA.
Carbon-Nitrogen Ligases ; genetics ; Exons ; Female ; Humans ; Multiple Carboxylase Deficiency ; genetics ; Mutation ; Open Reading Frames ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Sequence Analysis, DNA

OBJECTIVETo explore the molecular mechanism for a boy suspected with 3-methylcrotonyl-CoA carboxylase deficiency by neonatal screening.

METHODSPCR and Sanger sequencing were used to identify potential mutations of MCCC1 and MCCC2 genes. SIFT and Polyphen-2 software was used to predict the effect of variant on the protein function and conservation of the variant across various species. Human Splicing Finder and Swiss-PdbViewer4.1.0 were applied to analyze the possible mechanism of the variant.

RESULTSFor the proband, a compound heterozygous mutation was discovered in the MCCC1 gene, namely c.539G>T (p.G180V) and c.704_711del (p.A235Vfs*4), which were inherited from his father and mother, respectively. The two mutations have disrupted the protein conformation, which in turn may impact the function of MCC protein.

CONCLUSIONThe compound heterozygous mutations of the MCCC1 gene may contribute to the 3-methylcrotonyl-CoA carboxylase deficiency manifested by the patient.

Amino Acid Sequence ; Base Sequence ; Carbon-Carbon Ligases ; chemistry ; deficiency ; genetics ; DNA Mutational Analysis ; Heterozygote ; Humans ; Infant, Newborn ; Male ; Models, Molecular ; Mutation ; Neonatal Screening ; methods ; Protein Conformation ; Sequence Homology, Amino Acid ; Urea Cycle Disorders, Inborn ; diagnosis ; genetics

Biotin is an important micronutrient that serves as an essential enzyme cofactor. Bacteria obtain biotin either through de novo synthesis or by active uptake from exogenous sources. Mycobacteria are unusual amongst bacteria in that their primary source of biotin is through de novo synthesis. Here we review the importance of biotin biosynthesis in the lifecycle of Mycobacteria. Genetic screens designed to identify key metabolic processes have highlighted a role for the biotin biosynthesis in bacilli growth, infection and survival during the latency phase. These studies help to establish the biotin biosynthetic pathway as a potential drug target for new anti-tuberculosis agents.
Biotin ; biosynthesis ; Carbon-Carbon Ligases ; metabolism ; Carrier Proteins ; metabolism ; Cell Membrane ; metabolism ; Coenzymes ; metabolism ; Fatty Acids ; biosynthesis ; Genes, Bacterial ; Genome, Bacterial ; Metabolic Networks and Pathways ; Molecular Structure ; Mycobacterium Infections ; microbiology ; Mycobacterium tuberculosis ; genetics ; metabolism ; pathogenicity ; physiology ; Virulence

OBJECTIVETo report the clinical diagnosis, treatment and follow-up of children with holocarboxylase synthetas(HCS) deficiency and explore the gene mutation spectrum of the disease.

METHODSEleven children with HCS deficiency were enrolled. Mass spectrometry analysis and biotinidase activity determination were used for diagnosis of HCS deficiency. HCS gene mutations were analyzed by PCR directed sequencing methods. Ten patients received oral biotin treatment (10-40 mg/d). Clinical effects of biotin treatment were observed.

RESULTSAll 11 cases developed apathetic, lethargy and metabolic acidosis at different degrees, and 10 cases presented with skin lesions. The average blood 3-hydroxyisovaleryl-carnitine concentrations and urinary 3-methylcrontonylglycine and methylcitrate concentrations increased significantly. The biotinidase activity increased, being higher over 30% of the normal reference value. Four mutations in HCS gene were identified, and they were c.1522C>T (R508W), c.1088T>A (V363D), c.126G>T (E42D) and c.1994G>C (R665P) (a new variant) and the frequency was 50%, 29%, 7% and 14% respectively. The symptoms disappeared in 10 cases 1-2 weeks after biotin treatment, and blood and urinary abnormal metabolites were gradually reduced to normal 2-6 months after treatment.

CONCLUSIONSHCS deficiency is characterized by nervous system damage, skin lesions and metabolic acidosis. Mass spectrometry analysis, biotinidase activity determination and gene mutation analysis may be helpful in the definite diagnosis of this disorder. The effect of early biotin treatment is satisfactory. The mutations R508W and V363D might be hot-spots in Chinese children with HCS deficiency.

Biotin ; therapeutic use ; Biotinidase ; metabolism ; Carbon-Nitrogen Ligases ; genetics ; Child, Preschool ; Female ; Holocarboxylase Synthetase Deficiency ; diagnosis ; therapy ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation