OBJECTIVE: To explore the genotype-phenotype relationship in a child with Opitz G/BBB syndrome (OS) with mild clinical phenotype. METHODS: A child with motor developmental delay as the initial symptom admitted to Xi'an Children's Hospital on June 10, 2021 was selected for this study. Clinical data were collected, and peripheral blood samples were obtained from the child and his mother. Whole exome sequencing (WES) was performed to identify genetic variant in the child. Candidate variant were verified by Sanger sequencing to assess inheritance patterns and pathogenicity. Real-time fluorescence quantitative PCR (RT-qPCR) and Western blot (WB) analyses were conducted to evaluate the effects of the variant on mRNA and protein expression, respectively, using recombinant expression plasmids generated in vitro. This study was approved by the Medical Ethics Committee of Xi'an Children's Hospital (Ethics No. 20240045). RESULTS: The child, a 9-month-and-7-day-old boy, presented with a low nasal bridge, hypertelorism, and difficulty sitting independently. Echocardiography revealed an atrial septal defect. WES identified a homozygous variant in the MIDI gene, c.1483C>T (p.R495X), which was confirmed by Sanger sequencing and found to be inherited from the mother.Recombinant expression plasmids were successfully constructed. RT-qPCR analysis showed that the variant significantly reduced MIDI gene mRNA expression, while WB results indicated that the variant led to the production of a truncated protein. CONCLUSION The mild clinical phenotype of OS in this child may be attributed to the mRNA degradation escape mechanism induced by the nonsense variant c.1483C>T (p.R495X) in the MIDI gene. These findings provide valuable diagnostic insights for this pedigree and contribute to the understanding of the genotype-phenotype correlation in OS.
Humans ; Male ; Infant ; Transcription Factors/metabolism* ; Microtubule Proteins/genetics* ; Craniosynostoses/genetics* ; Hypospadias/genetics* ; Codon, Nonsense/genetics* ; RNA, Messenger/metabolism* ; Female ; RNA Stability/genetics* ; Phenotype ; Nuclear Proteins/genetics* ; Ubiquitin-Protein Ligases ; Esophagus/abnormalities* ; Hypertelorism

The variable domain of heavy-chain antibody (VHH) has been developed widely in drug therapy, diagnosis, and research. Escherichia coli is the most popular expression system for VHH production, whereas low bioactivity occurs sometimes. Mammalian cells are one of the most ideal hosts for VHH expression at present. To improve the yield of VHH in Expi293F cells, we optimized the signal peptide (SP) and codons of VHH. Firstly, the fusion protein VHH1-Fc was used to screen SPs. The SP IFN-α2 showed the highest secretion as quantified by enzyme-linked immunosorbent assay (ELISA). Subsequently, codon optimization by improving GC3 and GC content doubled the yield of VHH1 and kept its binding activity to Senecavirus A (SVA). Finally, the mean yields of other 5 VHHs that fused with SP IFN-α2 and codon-optimized were over 191.6 mg/L, and these VHHs had high recovery and high purity in the culture supernatant. This study confirms that SP IFN-α2 and codon optimization could produce VHHs in Expi293F cells efficiently, which provides a reference for the large-scale production of VHHs.
Codon/genetics* ; Protein Sorting Signals/genetics* ; Escherichia coli/metabolism* ; Humans ; Recombinant Fusion Proteins/biosynthesis* ; Interferon-alpha/metabolism* ; Immunoglobulin Heavy Chains/immunology* ; Cell Line ; Immunoglobulin Variable Region/immunology*

OBJECTIVE: To analyze FOXC2 gene variant in a family affected with lymphodema-distichiasis syndrome (LDS). METHODS: Peripheral blood samples were collected for the extraction of DNA and protein. Whole-exome sequencing was carried out to detect variants in the proband. Suspected variant was validated by Sanger sequencing. Western blotting was used to detect changes in protein expression. RESULTS: The proband and his mother were both found to carry a heterozygous nonsense variant c.177C>G (p.Tyr59X) of the FOXC2 gene, which was previously unreported. Down-regulated expression of FOXC2 was detected by Western blotting. Prenatal ultrasonography of the fetus indicated increased nuchal thickness. Amniocentesis was performed at 21+1 weeks of pregnancy, genetic testing suggested that the fetus also carried the c.177C>G variant. CONCLUSION The patients' condition may be attributed to the heterozygous nonsense variant c.177C>G of the FOXC2 gene, which resulted in a significant decrease in FOXC2 expression. Increased nuchal thickness may also be related with decreased FOXC2 expression. Above finding has expanded the variant spectrum of the FOXC2 gene.
Codon, Nonsense ; Eyelashes ; abnormalities ; Female ; Forkhead Transcription Factors ; genetics ; metabolism ; Gene Expression ; Genetic Testing ; Genetic Variation ; Humans ; Lymphedema ; genetics ; Pedigree ; Pregnancy ; Prenatal Diagnosis

Animals ; Codon, Nonsense ; Disease ; genetics ; Drosophila ; genetics ; metabolism ; Drosophila Proteins ; genetics ; Humans ; Pseudogenes ; Receptors, Odorant ; genetics

Research of the FLG mutation in various ethnic groups revealed non-overlapping mutation patterns. In addition, Japanese and Chinese atopic patients showed somewhat different mutations. These ethnic differences make the research on Korean patients mandatory; however, no systematic research on Korean atopic dermatitis (AD) patients has been performed. This study aims to investigate the genetic polymorphism of FLG in Korean atopic dermatitis patients. The study was made up of three groups including 9 Ichthyosis vulgaris (IV) patients, 50 AD patients and 55 normal controls: the ichthyosis group was incorporated due to the reported association between the FLG mutation and IV. In comparison to other sequencing methods, the overlapping long-range PCR was used. We revealed the genetic polymorphism of filaggrin in Koreans, and at the same time, we discovered nonsense mutations in p.Y1767X and p.K4022X in Korean AD patients. By using FLG sequencing techniques confirmed in this study, new mutations or genetic polymorphisms with ethnic characteristics would be detected and further larger studies of repeat number polymorphisms could be performed.
Adult ; Alleles ; Asian Continental Ancestry Group/*genetics ; Base Sequence ; Codon, Nonsense ; DNA/blood/chemistry/metabolism ; DNA Mutational Analysis ; Dermatitis, Atopic/*genetics ; Female ; Genotype ; Heterozygote ; Humans ; Ichthyosis Vulgaris/genetics ; Intermediate Filament Proteins/*genetics ; Male ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

OBJECTIVETo explore the function of a novel nonsense mutation c.1476G>A of ITGB3 gene using an in vitro expression system.

METHODSAn eukaryotic expression vector containing ITGB3 c.1476G>A cDNA was generated by site-directed mutagenesis and transformed into E.coli. Plasmid DNA was extracted and sequenced to confirm the target mutations. Wild-type and mutant recombination plasmids were transfected into Chinese hamster ovarian cancer (CHO) cells by nonliposome method, and the stable expression cells were harvested by G418 screening. The ITGB3 gene mRNA transcription and GPIIIa expression level in CHO cells were detected with real-time quantitative PCR, Western blotting and flow cytometry, respectively.

RESULTSThe eukaryotic expression vectors of wild ITGB3 cDNA and c.1476G>A mutant were successfully constructed. CHO cells with stable expression were obtained after transfection and screening. Compared with the wild-type transfected cells, the amount of CD61 antigen expression was 37% and mRNA transcription level was only 6% in the mutant-transfected cells. Full length GPIIIa protein was found only in the stably wild-type-transfected cells, but not in mutant-transfected cells by Western blotting analysis.

CONCLUSIONThe ITGB3 c.1476G>A mutation can decrease the transcription level and further affect GPIIIa synthesis and CD61 antigen expression.

Animals ; Base Sequence ; Blood Platelets ; cytology ; metabolism ; CHO Cells ; Cloning, Molecular ; Codon, Nonsense ; genetics ; Cricetinae ; Cricetulus ; Humans ; Integrin beta3 ; genetics ; metabolism ; Molecular Sequence Data ; Plasmids ; genetics ; metabolism ; Point Mutation

p53 gene plays an important role in apoptosis, which is necessary for successful invasion of trophoblast cells. The change from an arginine (Arg) to a proline (Pro) at codon 72 can influence the biological activity of p53, which predisposes to an increased risk of recurrent spontaneous abortion (RSA). In order to investigate the association between p53 polymorphism at codon 72 and RSA, we conducted this meta-analysis. Pubmed, Embase and Web of science were used to identify the eligible studies. Odds ratio (OR) with 95% confidence interval (CI) was used to evaluate the strength of the association. Six studies containing 937 cases of RSA and 830 controls were included, and there was one study deviated from Hardy-Weinberg equilibrium (HWE). There was a significant association between p53 polymorphism at codon 72 and RSA in recessive model (Pro/Pro vs. Pro/Arg+Arg/Arg; OR=1.60, 95% CI: 1.14-2.24) and co-dominant model (Pro/Pro vs. Arg/Arg; OR=1.47, 95% CI: 1.02-2.12) whether the study that was deviated from HWE was eliminated or not. A significant association was observed in allelic model (Pro vs. Arg; OR=1.28, 95% CI: 1.04-1.57) after exclusion of the study that was deviated from HWE. No association was noted in recessive model (Pro/Pro+Pro/Arg vs. Arg/Arg; OR=1.05, 95% CI: 0.86-1.30) and co-dominant model (Pro/Arg vs. Arg/Arg; OR=0.96, 95% CI: 0.77-1.19). Subgroup analysis by ethnicity also indicated a significant association between p53 polymorphism at codon 72 and RSA in Caucasian group. No heterogeneity and publication bias were found. Our meta-analysis implied that p53 polymorphism at codon 72 carries high maternal risk of RSA.
Abortion, Spontaneous ; diagnosis ; ethnology ; genetics ; physiopathology ; Adult ; Alleles ; Asian Continental Ancestry Group ; Case-Control Studies ; Codon ; European Continental Ancestry Group ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Humans ; Odds Ratio ; Polymorphism, Single Nucleotide ; Pregnancy ; Recurrence ; Risk Factors ; Tumor Suppressor Protein p53 ; genetics ; metabolism

The present study was designed to investigate the effects of start codon of nosM on the biosynthesis of nosiheptide. Target genes were amplified by overlap PCR. After homologous recombination to construct engineered strains, nosiheptide production was analyzed by HPLC. Three mutants with different start codon of nosM were constructed, and nosiheptide production of each mutant was analyzed and compared. Replacement of the start codon of nosM significantly decreased the production of nosiheptide. In conclusion, start codon usage could greatly affect the biosynthetic efficiency in the biosynthetic gene cluster of nosiheptide.
Anti-Bacterial Agents ; biosynthesis ; Chromatography, High Pressure Liquid ; Codon, Initiator ; Escherichia coli ; Genes, Bacterial ; Mutation ; Streptomyces ; genetics ; metabolism ; Thiazoles ; metabolism

To express recombinant carboxypeptidase from Thermus aquaticus (Cpase Taq) in Pichia pastosis, the open reading frame coding thermostable Cpase Taq was optimized based on the preference of P. pastoris codon usage and synthesized in vitro. The novel gene was cloned into P. pastoris expression vector pHBM905A and the sequence coding 6xHis tag was fused with the ORF of Cpase Taq gene. The recombinant plasmid was named pHBM905A-Cpase Taq and transformed into P. pastoris GS 115. Transformants were induced with 1% methanol for 72 h until the enzyme yield reached 0.1 mg/ml. The enzyme was purified and its enzymatic properties were analyzed. The results showed that the specific enzyme activity reached maximum at 75 °C and pH 7.5, which was about 80 U/mg. It was the first report about the secretory expression of Cpase Taq in P. pastoris GS115. Because of its large-scale preparation, this enzyme may be applied in industrial hydrolysis of peptides into amino acids in the future.
Bacterial Proteins ; biosynthesis ; genetics ; Carboxypeptidases ; biosynthesis ; genetics ; Cloning, Molecular ; Codon ; Hydrolysis ; Open Reading Frames ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Thermus ; enzymology

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.
Animals ; Antigens, Viral/genetics/metabolism ; Caliciviridae Infections/prevention & control/*veterinary/virology ; Capsid Proteins/*genetics/metabolism ; Cell Culture Techniques/*methods ; Codon/genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/veterinary ; *Gene Expression Regulation, Viral ; Hemorrhagic Disease Virus, Rabbit/*genetics/immunology ; *Rabbits ; Recombinant Proteins/genetics/metabolism ; Sf9 Cells ; Spodoptera ; Viral Structural Proteins/*genetics/metabolism ; Viral Vaccines/genetics/immunology