ABSTRACT
OBJECTIVES: To investigate the clinical and genetic features of children with 3-methylcrotonyl-coenzyme A carboxylase deficiency (MCCD). METHODS: A retrospective analysis was conducted on the clinical manifestations and genetic testing results of six children with MCCD who attended Children's Hospital Affiliated to Zhengzhou University from January 2018 to October 2023. RESULTS: Among the six children with MCCD, there were 4 boys and 2 girls, with a mean age of 7 days at the time of attending the hospital and 45 days at the time of confirmed diagnosis. Of all children, one had abnormal urine odor and five had no clinical symptoms. All six children had increases in blood 3-hydroxyisovaleryl carnitine and urinary 3-hydroxyisovaleric acid and 3-methylcrotonoylglycine, and five of them had a reduction in free carnitine. A total of six mutations were identified in the MCCC1 gene, i.e., c.1630del(p.R544Dfs*2), c.269A>G(p.D90G), c.1609T>A(p.F537I), c.639+2T>A, c.761+1G>T, and c.1331G>A(p.R444H), and three mutations were identified in the MCCC2 gene, i.e., c.838G>T(p.D280Y), c.592C>T(p.Q198*,366), and c.1342G>A(p.G448A). Among these mutations, c.269A>G(p.D90G) and c.1609T>A(p.F537I) had not been previously reported in the literature. There was one case of maternal MCCD, and the child carried a heterozygous mutation from her mother. Five children with a reduction in free carnitine were given supplementation of L-carnitine, and free carnitine was restored to the normal level at the last follow-up visit. CONCLUSIONS: This study identifies two new mutations, c.269A>G(p.D90G) and c.1609T>A(p.F537I), thereby expanding the mutation spectrum of the MCCC1 gene. A combination of blood amino acid and acylcarnitine profiles, urine organic acid analysis, and genetic testing can facilitate early diagnosis and treatment of MCCD, and provide essential data for genetic counseling.
Subject(s)
Carnitine , Mutation , Humans , Female , Male , Carnitine/analogs & derivatives , Carnitine/blood , Retrospective Studies , Infant, Newborn , Urea Cycle Disorders, Inborn/genetics , Urea Cycle Disorders, Inborn/diagnosis , Carbon-Carbon Ligases/genetics , Carbon-Carbon Ligases/deficiency , Infant , Carboxy-Lyases/genetics , Carboxy-Lyases/deficiencyABSTRACT
BACKGROUND: Major bleeding occurs annually in 1%-3% of patients on vitamin K antagonists (VKAs), despite close monitoring. Genetic variants in proteins involved in VKA response may affect this risk. AIM: To determine the association of genetic variants (cytochrome P450 enzymes 2C9 [CYP2C9] and 4F2 [CYP4F2], gamma-glutamyl carboxylase [GGCX]) with major bleeding in VKA users, separately and combined, including vitamin K epoxide reductase complex subunit-1 (VKORC1). METHODS: A case-cohort study was established within the BLEEDS cohort, which includes 16,570 patients who initiated VKAs between 2012 and 2014. We selected all 326 major bleeding cases that occurred during 17,613 years of follow-up and a random subcohort of 978 patients. We determined variants in CYP2C9, CYP4F2, GGCX, VKORC1 and evaluated the interaction between variant genotypes. Hazard ratios for major bleeding with 95% confidence intervals (95% CI) were estimated by weighted Cox regression. RESULTS: Genotype was determined in 256 cases and 783 subcohort members. Phenprocoumon was the most prescribed VKA for both cases and the subcohort (78% and 75%, respectively). Patients with major bleeding were slightly older than subcohort patients. CYP4F2-TT carriership was associated with a 1.6-fold (95% CI 0.9-2.8) increased risk of major bleeding compared with CC-alleles, albeit not statistically significant. For the CYP2C9 and GGCX variants instead, the major bleeding risk was around unity. Carrying at least two variant genotypes in CYP2C9 (poor metabolizer), CYP4F2-TT, and VKORC1-AA was associated with a 4.0-fold (95%CI 1.4-11.4) increased risk, while carriers of both CYP4F2-TT and VKORC1-AA had a particularly increased major bleeding risk (hazard ratio 6.7, 95% CI 1.5-29.8) compared with carriers of CC alleles in CYP4F2 and GG in VKORC1. However, the number of major bleeding cases in carriers of multiple variants was few (8 and 5 patients, respectively). CONCLUSIONS: CYP4F2 polymorphism was associated with major bleeding, especially in combination with VKORC1 genetic variants. These variants could be considered to further personalize anticoagulant treatment.
Subject(s)
Anticoagulants , Hemorrhage , Polymorphism, Genetic , Vitamin K Epoxide Reductases , Vitamin K , Humans , Vitamin K/antagonists & inhibitors , Hemorrhage/chemically induced , Hemorrhage/genetics , Hemorrhage/epidemiology , Female , Male , Aged , Vitamin K Epoxide Reductases/genetics , Cohort Studies , Anticoagulants/adverse effects , Middle Aged , Cytochrome P-450 CYP2C9/genetics , Genotype , Cytochrome P450 Family 4/genetics , Aged, 80 and over , Carbon-Carbon Ligases/genetics , Case-Control StudiesABSTRACT
3-Methylcrotonyl-CoA carboxylase (MCC) catalyzes the two-step, biotin-dependent production of 3-methylglutaconyl-CoA, an essential intermediate in leucine catabolism. Given the critical metabolic role of MCC, deficiencies in this enzyme lead to organic aciduria, while its overexpression is linked to tumor development. MCC is a dodecameric enzyme composed of six copies of each α- and ß-subunit. We present the cryo-EM structure of the endogenous MCC holoenzyme from Trypanosoma brucei in a non-filamentous state at 2.4 Å resolution. Biotin is covalently bound to the biotin carboxyl carrier protein domain of α-subunits and positioned in a non-canonical pocket near the active site of neighboring ß-subunit dimers. Moreover, flexibility of key residues at α-subunit interfaces and loops enables pivoting of α-subunit trimers to partly reduce the distance between α- and ß-subunit active sites, required for MCC catalysis. Our results provide a structural framework to understand the enzymatic mechanism of eukaryotic MCCs and to assist drug discovery against trypanosome infections.
Subject(s)
Carbon-Carbon Ligases , Catalytic Domain , Cryoelectron Microscopy , Protozoan Proteins , Trypanosoma brucei brucei , Acetyl-CoA Carboxylase , Carbon-Carbon Ligases/metabolism , Carbon-Carbon Ligases/chemistry , Carbon-Carbon Ligases/genetics , Fatty Acid Synthase, Type II , Holoenzymes/chemistry , Holoenzymes/metabolism , Models, Molecular , Protein Binding , Protein Multimerization , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Protozoan Proteins/genetics , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/metabolismABSTRACT
Identifying genes whose expression is associated with schizophrenia (SCZ) risk by transcriptome-wide association studies (TWAS) facilitates downstream experimental studies. Here, we integrated multiple published datasets of TWAS, gene coexpression, and differential gene expression analysis to prioritize SCZ candidate genes for functional study. Convergent evidence prioritized Propionyl-CoA Carboxylase Subunit Beta (PCCB), a nuclear-encoded mitochondrial gene, as an SCZ risk gene. However, the PCCB's contribution to SCZ risk has not been investigated before. Using dual luciferase reporter assay, we identified that SCZ-associated SNPs rs6791142 and rs35874192, two eQTL SNPs for PCCB, showed differential allelic effects on transcriptional activities. PCCB knockdown in human forebrain organoids (hFOs) followed by RNA sequencing analysis revealed dysregulation of genes enriched with multiple neuronal functions including gamma-aminobutyric acid (GABA)-ergic synapse. The metabolomic and mitochondrial function analyses confirmed the decreased GABA levels resulted from inhibited tricarboxylic acid cycle in PCCB knockdown hFOs. Multielectrode array recording analysis showed that PCCB knockdown in hFOs resulted into SCZ-related phenotypes including hyper-neuroactivities and decreased synchronization of neural network. In summary, this study utilized hFOs-based multi-omics analyses and revealed that PCCB downregulation may contribute to SCZ risk through regulating GABAergic pathways, highlighting the mitochondrial function in SCZ.
Subject(s)
Carbon-Carbon Ligases , Multiomics , Schizophrenia , Humans , Metabolomics , Organoids , Prosencephalon , Schizophrenia/genetics , Carbon-Carbon Ligases/geneticsABSTRACT
γ-glutamyl carboxylase (GGCX), also known as vitamin K-dependent glutamyl carboxylase, catalyzes the posttranslational modification of specific glutamate residues in vitamin K-dependent proteins (VKDPs), and participates multiple biological functions including blood coagulation, bone metabolism, vascular calcification, and cell proliferation. It has been reported originally that GGCX pathogenic variation causes blood coagulation deficiency, which is called as vitamin K-dependent coagulation factor deficiency 1 (VKCFD1). Recently, it has been found that GGCX gene variation results in multiple clinical phenotypes, including dermatological, ophthalmological, skeletal or cardiac abnormalities. Among them, dermatological phenotype is the most common, which is known as pseudoxanthoma elasticum-like syndrome. This paper has reviewed the GGCX pathogenic variation associated phenotypes, in order to increase the recognition of GGCX-related genetic diseases and to help its diagnosis and treatment.
Subject(s)
Blood Coagulation Disorders, Inherited , Carbon-Carbon Ligases , Blood Coagulation Disorders, Inherited/diagnosis , Blood Coagulation Disorders, Inherited/enzymology , Blood Coagulation Disorders, Inherited/genetics , Blood Coagulation Disorders, Inherited/metabolism , Carbon-Carbon Ligases/genetics , Carbon-Carbon Ligases/metabolism , Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Humans , Phenotype , Vitamin K/metabolism , Vitamin K 1ABSTRACT
γ-Glutamyl carboxylase (GGCX) generates multiple carboxylated Glus (Glas) in vitamin K-dependent (VKD) proteins that are required for their functions. GGCX is processive, remaining bound to VKD proteins throughout multiple Glu carboxylations, and this study reveals the essentiality of processivity to VKD protein function. GGCX mutants (V255M and S300F) whose combined heterozygosity in a patient causes defective clotting and calcification were studied using a novel assay that mimics in vivo carboxylation. Complexes between variant carboxylases and VKD proteins important to hemostasis (factor IX [FIX]) or calcification (matrix Gla protein [MGP]) were reacted in the presence of a challenge VKD protein that could potentially interfere with carboxylation of the VKD protein in the complex. The VKD protein in the complex with wild-type carboxylase was carboxylated before challenge protein carboxylation occurred and became fully carboxylated. In contrast, the V255M mutant carboxylated both forms at the same time and did not completely carboxylate FIX in the complex. S300F carboxylation was poor with both FIX and MGP. Additional studies analyzed FIX- and MGP-derived peptides containing the Gla domain linked to sequences that mediate carboxylase binding. The total amount of carboxylated peptide generated by the V255M mutant was higher than that of wild-type GGCX; however, the individual peptides were partially carboxylated. Analysis of the V255M mutant in FIX HEK293 cells lacking endogenous GGCX revealed poor FIX clotting activity. This study shows that disrupted processivity causes disease and explains the defect in the patient. Kinetic analyses also suggest that disrupted processivity may occur in wild-type carboxylase under some conditions (eg, warfarin therapy or vitamin K deficiency).
Subject(s)
Carbon-Carbon Ligases , Vitamin K , Blood Coagulation , Carbon-Carbon Ligases/chemistry , Carbon-Carbon Ligases/genetics , Factor IX/metabolism , HEK293 Cells , Humans , Peptides , Proteins , Vitamin K/metabolism , WarfarinABSTRACT
γ-Glutamyl carboxylase (GGCX) catalyzes the γ-carboxylation of 15 different vitamin K dependent (VKD) proteins. Pathogenic variants in GGCX cause a rare hereditary bleeding disorder called Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1). In addition to bleedings, some VKCFD1 patients develop skin laxity and skeletal dysmorphologies. However, the pathophysiological mechanisms underlying these non-hemorrhagic phenotypes remain elusive. Therefore, we have analyzed 20 pathogenic GGCX variants on their ability to γ-carboxylate six non-hemostatic VKD proteins in an in vitro assay, where GGCX variants were expressed in GGCX-/- cells and levels of γ-carboxylated co-expressed VKD proteins were detected by a functional ELISA. We observed that GGCX variants causing markedly reduced γ-carboxylation of Gla rich protein (GRP) in vitro were reported in patients with skin laxity. Reduced levels of γ-carboxylated Matrix gla protein (MGP) are not exclusive for causing skeletal dysmorphologies in VKCFD1 patients. In silico docking of vitamin K hydroquinone on a GGCX model revealed a binding site, which was validated by in vitro assays. GGCX variants affecting this site result in disability to γ-carboxylate VKD proteins and hence are involved in the most severe phenotypes. This genotype-phenotype analysis will help to understand the development of non-hemorrhagic phenotypes and hence improve treatment in VKCFD1 patients.
Subject(s)
Blood Coagulation Disorders, Inherited , Carbon-Carbon Ligases , Blood Coagulation Disorders, Inherited/genetics , Carbon-Carbon Ligases/chemistry , Carbon-Carbon Ligases/genetics , Carbon-Carbon Ligases/metabolism , Carboxy-Lyases , Humans , MutationABSTRACT
Congenital combined vitamin K-dependent clotting factors deficiency (VKCFD) is a rare autosomal recessive disease resulting in hemorrhagic symptoms usually associated with developmental disorders and bone abnormalities. Pathogenic variants in two genes encoding enzymes of the vitamin K cycle, GGCX and VKORC1, can lead to this disorder. We present the case of a male fetus with a brachytelephalangic chondrodysplasia punctata (CDP), absence of nasal bone, growth restriction, and bilateral ventriculomegaly at 18 weeks of gestation. Pathological examination showed a Binder phenotype, hypoplastic distal phalanges, stippled epiphyses, and brain abnormalities suggestive of a brain hemorrhage. Two GGCX pathogenic variants inherited respectively from the mother and the father were identified. To our knowledge, this is the first prenatal description of VKCFD. Even if it remains a rare etiology, which is mostly described in children or adult patients, VKCFD should be considered in fetuses with CDP.
Subject(s)
Carbon-Carbon Ligases , Chondrodysplasia Punctata , Blood Coagulation Factors , Carbon-Carbon Ligases/genetics , Chondrodysplasia Punctata/diagnosis , Chondrodysplasia Punctata/genetics , Female , Fetus , Humans , Male , Pregnancy , Vitamin K , Vitamin K 1 , Vitamin K Epoxide Reductases/geneticsABSTRACT
Identifying multiple ultra-rare genetic syndromes with overlapping phenotypes is a diagnostic conundrum in clinical genetics. This study investigated the pathogenicity of a homozygous missense variant in GNB5 (GNB5L; NM_016194.4: c.920T > G (p. Leu307Arg); GNB5S; NM_006578.4: c.794T > G (p. Leu265Arg)) identified through exome sequencing in a female child who also had 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency (newborn screening positive) and hemoglobin E trait. The proband presented with early-onset intellectual disability, the severity of which was more in keeping with GNB5-related disorder than 3-MCC deficiency. She later developed bradycardia and cardiac arrest, and upon re-phenotyping showed cone photo-transduction recovery deficit, all known only to GNB5-related disorders. Patient-derived fibroblast assays showed preserved GNB5S expression, but bioluminescence resonance energy transfer assay showed abolished function of the variant reconstituted Gß5S containing RGS complexes for deactivation of D2 dopamine receptor activity, confirming variant pathogenicity. This study highlights the need for precise phenotyping and functional assays to facilitate variant classification and clinical diagnosis in patients with complex medical conditions.
Subject(s)
Carbon-Carbon Ligases/genetics , GTP-Binding Protein beta Subunits/chemistry , GTP-Binding Protein beta Subunits/genetics , Genetic Diseases, Inborn/diagnosis , Genetic Diseases, Inborn/etiology , Bioluminescence Resonance Energy Transfer Techniques , Carbon-Carbon Ligases/deficiency , Child , Eye Diseases/etiology , Eye Diseases/genetics , Female , GTP-Binding Protein beta Subunits/metabolism , Genetic Diseases, Inborn/genetics , Genetic Variation , HEK293 Cells , Humans , Infant, Newborn , Intellectual Disability/genetics , Male , Neonatal Screening , Phenotype , Reproducibility of Results , Urea Cycle Disorders, Inborn/etiology , Exome SequencingABSTRACT
It is estimated that up to one-third of all variants causing inherited diseases affect splicing; however, their deleterious effects and roles in disease pathogenesis are often not fully characterized. Given their prevalence and the development of various antisense-based splice-modulating approaches, pathogenic splicing variants have become an important object of genomic medicine. To improve the accuracy of variant interpretation in public mutation repositories, we applied the minigene splicing assay to study the effects of 24 variants that were predicted to affect normal splicing in the genes associated with propionic acidemia (PA)-PCCA and PCCB. As a result, 13 variants (including one missense and two synonymous variants) demonstrated a significant alteration of splicing with the predicted deleterious effect at the protein level and were characterized as spliceogenic loss-of-function variants. The analysis of the available data for the studied variants and application of the American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP) guidelines allowed us to precisely classify five of the variants and change the pathogenic status of nine. Using the example of the PA genes, we demonstrated the utility of the minigene splicing assay in the fast and effective assessment of the spliceogenic effect for identified variants and highlight the necessity of their standardized classification.
Subject(s)
Carbon-Carbon Ligases/genetics , Propionic Acidemia/genetics , RNA Splicing , Carbon-Carbon Ligases/metabolism , Cell Line , Humans , Loss of Function MutationABSTRACT
BACKGROUND AND AIMS: Propionic acidemia (PA) is an autosomal recessive metabolic disorder caused by a deficiency of propionyl-CoA carboxylase and mutations in the PCCA and PCCB genes. In this study, we investigated the clinical characteristics of individuals with PA and conducted genetic analyses to provide new genetic evidence for the diagnosis of PA. MATERIALS AND METHODS: We conducted whole-exome sequencing and Sanger sequencing in four individuals with PA from three unrelated Chinese families. We also performed a structural analysis of the PCCB protein variants. Couples from the three families included in our study underwent in vitro fertilization with preimplantation genetic testing. RESULTS: We found five variants of PCCB. These biallelic variants were inherited from heterozygous parental carriers and were located in the functional domain, absent in human population genome datasets, and predicted to be deleterious. These findings indicate that the variants might be responsible for the clinical features observed in these particular patients with PA. Through successful embryo transfer and implantation, one of the couples fortunately gave birth to a healthy child. CONCLUSION: Overall, our study can expand the mutation spectrum of PCCB and provide useful information for the prenatal diagnosis of PA and genetic counseling for affected individuals.
Subject(s)
Carbon-Carbon Ligases/genetics , Propionic Acidemia , China , Female , Heterozygote , Humans , Methylmalonyl-CoA Decarboxylase/genetics , Mutation , Pregnancy , Propionic Acidemia/geneticsABSTRACT
ABSTRACT: Propionic acidemia is associated with pathogenic variants in PCCA or PCCB gene. We investigated the potential pathogenic variants in PCCA or PCCB genes in Fujian Han population.Two probands and their families of Han ethnicity containing two generations were subject to newborn screening using tandem mass spectrometry, followed by diagnosis using urine gas chromatography mass spectrometry. Sanger sequencing was used to identify potential mutations in PCCA and PCCB genes.Compound heterozygous variants were identified in PCCB gene in two siblings of the first family, the youngest girl showed a novel missense variant c.1381G>C (p.Ala461Pro) in exon 13 and a heterozygous missense variant c.1301C>T (p.Ala434Val) in exon 13, which were inherited respectively from their parents. The oldest boy is a carrier with a novel missense variant c.1381G>C (p.Ala461Pro) in exon 13 which were inherited from his father. In the second family, c.1535G>A homozygous mutations were identified in the baby girl, which were inherited respectively from their parents. In silico analysis, several different types of bioinformatic software were utilized, which predicted that the novel variant c.1381G>C in PCCB gene was damaged. According to ACMG principle, the missense variant c.1381G>C (p.Ala461Pro) in exon 13 was a Variant of Undetermined Significance (VUS).One novel missense variant and two missense variants in PCCB gene were identified in the study. The novel variant of PCCB gene identified VUS was identified for the first time in the Chinese population, which enriched the mutational spectrum of PCCB gene.
Subject(s)
Carbon-Carbon Ligases/genetics , Methylmalonyl-CoA Decarboxylase/genetics , Propionic Acidemia/genetics , Asian People/genetics , DNA Mutational Analysis , Female , Genetic Testing , Heterozygote , Humans , Infant , Infant, Newborn , Mutation, Missense , Neonatal Screening , Pedigree , Propionic Acidemia/blood , Propionic Acidemia/diagnosis , Tandem Mass SpectrometryABSTRACT
BACKGROUND: Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in γ-glutamyl carboxylase (GGCX). VKCFD1 patients are treated life-long with high doses of vitamin K in order to correct the bleeding phenotype. However, normalization of clotting factor activities cannot be achieved for all VKCFD1 patients. OBJECTIVE: The current study aims to investigate the responsiveness to vitamin K for all reported GGCX mutations with respect to clotting factors in order to optimize treatment. METHODS: This study developed an assay using genetically engineered GGCX-/- cells, in which GGCX mutations were analyzed with respect to their ability to γ-carboxylate vitamin K dependent pro-coagulatory and anti-coagulatory clotting factors by ELISA. Additionally, factor VII activity was measured in order to proof protein functionality. For specific GGCX mutations immunofluorescent staining was performed to assess the intracellular localization of clotting factors with respect to GGCX wild-type and mutations. RESULTS: All GGCX mutations were categorized into responder and low responder mutations, thereby determining the efficiency of vitamin K supplementation. Most VKCFD1 patients have at least one vitamin K responsive GGCX allele that is able to γ-carboxylate clotting factors. In few patients, the hemorrhagic phenotype cannot be reversed by vitamin K administration because GGCX mutations on both alleles affect either structural or catalytically important sites thereby resulting in residual ability to γ-carboxylate clotting factors. CONCLUSION: With these new functional data we can predict the hemorrhagic outcome of each VKCFD1 genotype, thus recommending treatments with either vitamin K or prothrombin complex concentrate.
Subject(s)
Carbon-Carbon Ligases , Vitamin K , Carbon-Carbon Ligases/genetics , Humans , Mutation , Phenotype , Vitamin K 1 , Vitamin K Epoxide Reductases/geneticsABSTRACT
γ-Glutamyl carboxylase (GGCX) is an integral membrane protein that catalyzes posttranslational carboxylation of a number of vitamin K-dependent (VKD) proteins involved in a wide variety of physiologic processes, including blood coagulation, vascular calcification, and bone metabolism. Naturally occurring GGCX mutations are associated with multiple distinct clinical phenotypes. However, the genotype-phenotype correlation of GGCX remains elusive. Here, we systematically examined the effect of all naturally occurring GGCX mutations on the carboxylation of 3 structure-function distinct VKD proteins in a cellular environment. GGCX mutations were transiently introduced into GGCX-deficient human embryonic kidney 293 cells stably expressing chimeric coagulation factor, matrix Gla protein (MGP), or osteocalcin as VKD reporter proteins, and then the carboxylation efficiency of these reporter proteins was evaluated. Our results show that GGCX mutations differentially affect the carboxylation of these reporter proteins and the efficiency of using vitamin K as a cofactor. Carboxylation of these reporter proteins by a C-terminal truncation mutation (R704X) implies that GGCX's C terminus plays a critical role in the binding of osteocalcin but not in the binding of coagulation factors and MGP. This has been confirmed by probing the protein-protein interaction between GGCX and its protein substrates in live cells using bimolecular fluorescence complementation and chemical cross-linking assays. Additionally, using a minigene splicing assay, we demonstrated that several GGCX missense mutations affect GGCX's pre-messenger RNA splicing rather than altering the corresponding amino acid residues. Results from this study interpreted the correlation of GGCX's genotype and its clinical phenotypes and clarified why vitamin K administration rectified bleeding disorders but not nonbleeding disorders.
Subject(s)
Carbon-Carbon Ligases/genetics , Carboxy-Lyases/genetics , Protein Processing, Post-Translational/genetics , Amino Acid Sequence , Base Sequence , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Carbon-Carbon Ligases/chemistry , Carboxy-Lyases/chemistry , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Genes, Reporter , Genetic Association Studies , Genetic Pleiotropy , HEK293 Cells , Hemorrhagic Disorders/drug therapy , Hemorrhagic Disorders/genetics , Humans , Mutation , Mutation, Missense , Osteocalcin/genetics , Osteocalcin/metabolism , Protein C/genetics , Protein C/metabolism , Protein Domains , Protein Interaction Mapping , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Precursors/metabolism , RNA Splicing , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Structure-Activity Relationship , Vitamin K/physiology , Vitamin K/therapeutic use , Matrix Gla ProteinABSTRACT
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.
Subject(s)
Carbon-Carbon Ligases/deficiency , Mutation, Missense , Urea Cycle Disorders, Inborn , Carbon-Carbon Ligases/genetics , Child , Female , Humans , Male , Mutation, Missense/genetics , Pedigree , Urea Cycle Disorders, Inborn/geneticsABSTRACT
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.
Subject(s)
Child , Female , Humans , Male , Carbon-Carbon Ligases/genetics , Mutation, Missense/genetics , Pedigree , Urea Cycle Disorders, Inborn/geneticsABSTRACT
Benzophenone synthase (BPS) catalyzes the production of 2,4,6-trihydroxybenzophenone via the condensation of benzoyl-CoA and three units of malonyl-CoA. The biosynthetic pathway proceeds with the formation of the prenylated xanthone α-mangostin from 2,4,6-trihydroxybenzophenone. Structural elucidation was performed to gain a better understanding of the structural basis of the function of Garcinia mangostana L. (mangosteen) BPS (GmBPS). The structure reveals the common core consisting of a five-layer αßαßα fold as found in other type III polyketide synthase enzymes. The three residues Met264, Tyr266 and Gly339 are proposed to have a significant impact on the substrate-binding specificity of the active site. Crystallographic and docking studies indicate why benzoyl-CoA is preferred over 4-coumaroyl-CoA as the substrate for GmBPS. Met264 and Tyr266 in GmBPS are properly oriented for accommodation of the 2,4,6-trihydroxybenzophenone product but not of naringenin. Gly339 offers a minimal steric hindrance to accommodate the extended substrate. Moreover, the structural arrangement of Thr133 provides the elongation activity and consequently facilitates extension of the polyketide chain. In addition to its impact on the substrate selectivity, Ala257 expands the horizontal cavity and might serve to facilitate the initiation/cyclization reaction. The detailed structure of GmBPS explains its catalytic function, facilitating further structure-based engineering to alter its substrate specificity and obtain the desired products.
Subject(s)
Carbon-Carbon Ligases/chemistry , Carbon-Carbon Ligases/metabolism , Garcinia/enzymology , Plant Proteins/chemistry , Plant Proteins/metabolism , Acyl Coenzyme A/metabolism , Amino Acid Substitution , Carbon-Carbon Ligases/genetics , Catalysis , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Molecular Docking Simulation , Mutation , Plant Proteins/genetics , Structure-Activity Relationship , Substrate Specificity , Tyrosine/chemistryABSTRACT
BACKGROUND: Propionic acidemia (PA) is a severe monogenic disorder characterized by a deficiency of the mitochondrial protein propionyl-CoA carboxylase (PCC) enzyme, which is caused by mutations in the PCCA or PCCB gene. Preconception carrier screening could provide couples with meaningful information for their reproductive options; however, it is not widely performed in China. CASE PRESENTATION: This report describes a case of dizygotic twin siblings conceived by in vitro fertilization (IVF) and diagnosed with propionic acidemia (PA). Their parents had no history of PA. Tandem mass spectrometry and urine gas chromatography/mass spectrometry (GC/MS) of the twin siblings revealed markedly elevated propionyl carnitine (C3), C3/C2, and 3-hydroxypropionate in the plasma and urine. Whole-exome sequencing was performed for the twin siblings. A homozygous missense mutation, c.2002G > A (p.Gly668Arg) in PCCA, was identified in the twin siblings. Sanger sequencing confirmed the homozygous mutation in the twin siblings and identified their parents as heterozygous carriers of the c.2002G > A mutation in PCCA. Both neonates in this case died. This is an emotionally and financially devastating outcome that could have been avoided with genetic carrier screening before conception. If couples are screened before IVF and found to be silent carriers, then reproductive options (such as preimplantation genetic diagnosis or prenatal diagnosis) can be offered to achieve a healthy newborn. CONCLUSION: This case is a reminder to infertile couples seeking IVF that it is beneficial to clarify whether they are silent carriers before undergoing IVF.
Subject(s)
Carbon-Carbon Ligases/genetics , Methylmalonyl-CoA Decarboxylase/genetics , Mutation , Propionic Acidemia/genetics , Adult , China , Female , Fertilization in Vitro , Humans , Infant, Newborn , Male , Parents , Pregnancy , Siblings , Twins, DizygoticABSTRACT
BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive hereditary disease associated with severe muscle atrophy and weakness in the limbs and trunk. The discovery of mutated genes is helpful in diagnosis and treatment for SMA. METHODS: Eighty-three whole blood samples were collected from 28 core families of clinically suspected SMA, and multiplex ligation probe amplification (MLPA) was performed. Afterwards, the complete gene sequence of SMN1 gene was detected. Furthermore, 20 SMA patients were selected from the 28 probands, and 5 non SMA children as controls. The Life Technologies SOLiD™ technology with mate-pair chemistry was utilized to conduct the whole exome high-throughput sequencing. RESULTS: Twenty-two probands were SMA patients, 3 probands carriers, and 3 probands normal individuals. Moreover, 2 parents from 2 SMA families were with 3 SMN1 exon7 copies. Six SMN1 single nucleotide variants (SNVs) were identified in the 83 samples, and c.[84C > T], c.[271C > T], c.[-39A > G] and g.[70240639G > C] were novel. Compared with control group, 9102 mutation were selected out in SMA patients. SPTA1 mutation c.[-41_-40insCTCT], FUT5 SNV c.[1001A > G], and MCCC2 SNV c.[-117A > G] were the 3 most frequent mutations in SMA group (95, 85 and 75%, respectively). CONCLUSIONS: We identified some mutations in both SMN1 and other genes, and c.[271C > T], c.[-41_-40insCTCT], c.[1001A > G] and c.[-117A > G] might be associated with the onset of SMA.