BACKGROUND:During diabetic wound healing,the sustained activation of M1 macrophages exacerbates the inflammatory response and hinders wound repair.Auranofin,an anti-inflammatory drug,has not been clearly studied for its effects on M1 macrophages and its potential role in diabetic wound healing.OBJECTIVE:To investigate the effects of different concentrations of auranofin on the biological function of M1 macrophages and evaluate its potential application in diabetic wound healing.METHODS:RAW264.7 and THP-1 cells were used as research models.M1 polarization was induced using different concentrations of interferon-γ and lipopolysaccharide.M1 macrophages were treated with 1 and 2 μmol/L auranofin.Cell counting kit-8 assay was used to evaluate the effect of auranofin on cell viability.Quantitative real-time PCR was performed to detect mRNA expression of interleukin-1β,interleukin-6,and tumor necrosis factor-α.ELISA was employed to measure the levels of interleukin-1β,interleukin-6,and tumor necrosis factor-α in the supernatant.Western blot analysis was used to assess the expression of nuclear factor-κB(p65),phosphorylated mitogen-activated protein kinases(MAPK),and total MAPK proteins.Additionally,6-8-week-old male C57BL/6J and db/db diabetic mice were used for wound healing experiments,with the mice divided into C57 control,db/db control and auranofin treatment groups,each containing six animals.Dorsal skin defect modeling and treatment with intraperitoneal injection of auranofin were performed to observe wound healing in mice.RESULTS AND CONCLUSION:(1)Cell experiments showed that co-treatment with interferon-y(10 ng/mL)and lipopolysaccharide(100 ng/mL)significantly induced M1 polarization in RAW264.7 and THP-1 cells,resulting in increased mRNA expression of interleukin-1β,interleukin-6,and tumor necrosis factor-α.Treatment with auranofin(1 and 2 μmol/L)reduced the mRNA expression of these inflammatory factors in the cells and inhibited the secretion of inflammatory factors in the cell supernatant.(2)Auranofin treatment significantly suppressed the activation of nuclear factor-κB(p65)and phosphorylated MAPK signaling pathways.(3)Animal experiments showed that auranofin promoted wound healing in db/db diabetic mice,suggesting that auranofin has strong anti-inflammatory effects and may facilitate the healing of wounds in diabetic mice.

BACKGROUND:During diabetic wound healing,the sustained activation of M1 macrophages exacerbates the inflammatory response and hinders wound repair.Auranofin,an anti-inflammatory drug,has not been clearly studied for its effects on M1 macrophages and its potential role in diabetic wound healing.OBJECTIVE:To investigate the effects of different concentrations of auranofin on the biological function of M1 macrophages and evaluate its potential application in diabetic wound healing.METHODS:RAW264.7 and THP-1 cells were used as research models.M1 polarization was induced using different concentrations of interferon-γ and lipopolysaccharide.M1 macrophages were treated with 1 and 2 μmol/L auranofin.Cell counting kit-8 assay was used to evaluate the effect of auranofin on cell viability.Quantitative real-time PCR was performed to detect mRNA expression of interleukin-1β,interleukin-6,and tumor necrosis factor-α.ELISA was employed to measure the levels of interleukin-1β,interleukin-6,and tumor necrosis factor-α in the supernatant.Western blot analysis was used to assess the expression of nuclear factor-κB(p65),phosphorylated mitogen-activated protein kinases(MAPK),and total MAPK proteins.Additionally,6-8-week-old male C57BL/6J and db/db diabetic mice were used for wound healing experiments,with the mice divided into C57 control,db/db control and auranofin treatment groups,each containing six animals.Dorsal skin defect modeling and treatment with intraperitoneal injection of auranofin were performed to observe wound healing in mice.RESULTS AND CONCLUSION:(1)Cell experiments showed that co-treatment with interferon-y(10 ng/mL)and lipopolysaccharide(100 ng/mL)significantly induced M1 polarization in RAW264.7 and THP-1 cells,resulting in increased mRNA expression of interleukin-1β,interleukin-6,and tumor necrosis factor-α.Treatment with auranofin(1 and 2 μmol/L)reduced the mRNA expression of these inflammatory factors in the cells and inhibited the secretion of inflammatory factors in the cell supernatant.(2)Auranofin treatment significantly suppressed the activation of nuclear factor-κB(p65)and phosphorylated MAPK signaling pathways.(3)Animal experiments showed that auranofin promoted wound healing in db/db diabetic mice,suggesting that auranofin has strong anti-inflammatory effects and may facilitate the healing of wounds in diabetic mice.

OBJECTIVES: According to the World Health Organization (WHO), infertility rates have been steadily rising worldwide. Identifying risk factors for contrast agent reflux into the myometrium during hysterosalpingo-contrast sonography (HyCoSy) is of clinical significance in reducing this complication and improving infertility treatment. However, there is currently no standardized pre-evaluation method for predicting intra-myometrial contrast reflux, with clinical assessment often relying on physician experience and patient symptoms. This study aims to identify imaging risk factors for contrast agent reflux into the myometrium using four-dimensional (4D) HyCoSy and to construct a nomogram-based predictive model to assist in clinical decision-making. METHODS: A retrospective analysis was conducted on 1 274 infertile women who underwent 4D HyCoSy at the Women and Children's Hospital of Hunan and the the Third Xiangya Hospital of Central South University from January 1, 2020, to December 15, 2022. Patients were divided into a reflux group (n=234) and a non-reflux group (n=1 040) based on the presence of intra-myometrial contrast reflux. Univariate and multivariable Logistic regression analyses were used to identify significant predictors, which were then used to construct a nomogram model. Internal validation was performed using 500 bootstrap resamples. RESULTS: The age of the reflux group was significantly higher than that of the non-reflux group [(31.82±5.27) years vs (30.66±4.83) years, P=0.001 1]. Primary infertility was more common in the non-reflux group (50.96%), while secondary infertility dominated in the reflux group (76.50%), with 72.65% having a history of gynecological surgery (P<0.001). Abnormal menstrual volume and discomfort during the procedure were more common in the reflux group, while the non-reflux group tolerated higher contrast agent doses (P<0.001). Imaging differences included endometrial thickness, tubal wall smoothness, and peritoneal contrast dispersion, with the non-reflux group showing thicker endometrium and smoother, more patent tubes. The nomogram model yielded an area under the curve (AUC) of 0.854, indicating good predictive performance. The AUC of the decision curve analysis (DCA) for internal validation of the model was 0.737. When the threshold probability for contrast agent reflux into the myometrium ranged from 0.05 to 0.95, the maximum net benefit reached 0.18. The net benefit of applying the nomogram predictive model exceeded that of either full intervention or no intervention, indicating that the model demonstrates good clinical predictive performance. CONCLUSIONS The nomogram model, based on infertility type, endometrial thickness, contrast agent dose, and discomfort symptoms, effectively predicts intra-myometrial contrast agent reflux after 4D HyCoSy. It provides a valuable tool for clinicians to implement early preventive measures and reduce the risk of contrast leakage and associated complications.
Humans ; Female ; Nomograms ; Contrast Media/adverse effects* ; Retrospective Studies ; Adult ; Ultrasonography/methods* ; Hysterosalpingography/methods* ; Infertility, Female/diagnostic imaging* ; Myometrium/diagnostic imaging* ; Risk Factors

Objective:To explore the clinical phenotypes and genetic characteristics of a pedigree with Distal arthrogryposis type 5D (DA5D) caused by compound heterozygous variants in the ECEL1 gene. Methods:A child (proband) diagnosed with DA5D and his family members (proband′s parents and sister) who was admitted to the Department of Rehabilitation Medicine of Henan Children′s Hospital in July 2022 due to " multiplex distal arthrogryposis" were enrolled into this study. Clinical data of the proband were collected and peripheral blood samples were obtained from the proband and members of his family about 3 mL. Trio-whole genome sequencing (trio-WGS) was carried out to detected the genetic variations of the proband and his family members. The candidate′s pathogenic gene variants were screened and analyzed by Genome Aggregation Database (gnomAD) and other databases. The screened variants wer annotated for clinical phenotypes using databases like the Online Mendelian Inheritance in Man (OMIM). The pathogenicity of the candidate variants was predicted by bioinformatics tools such as Provean. Based on the guidelines of the American College of Medical Genetics and Genomics (ACMG), pathogenicity ratings were conducted for variant sites. The protein conservation and mutation structure prediction of ECEL1 protein among species were carried out though MEGA-X and PyMOL. The research protocol of this study was reviewed by the Ethics Committee of Henan Provincial Children′s Hospital (Approval No. 2023-H-H01), and informed consent for clinical research was obtained from the guardians of the probands.Results:The proband had multiplex distal arthrogryposis involving hands, feet, knees, and ankles, and had right ptosis, micrognathia, low auricular position, and upturned nose. The parents and sister both had normal phenotypes. Trio-WGS and Sanger sequencing revealed that the child had compound heterozygous variants of paternal c. 1742_c.1743insT and maternal c. 2314T>G, for which the father and sister were carriers of the c. 1742_c.1743insT heterozygous variant and the mother was carrier of c. 2314T>A. Neither mutation site has been reported. According to guidelines of ACMG, the c. 1742_c.1743insT variant was classified as likely pathogenic (PSV1+ PM2_Supporting), and c. 2314T>G was classified as uncertain (PM2_Supporting+ PM3+ PP3). The results of conserved analysis of amino acid residue sequences of ECEL1 protein showed that the missense mutation of the maternal c. 2314T>G(p.Cys772Gly) was highly conserved among humans and other seven species. The protein structure prediction revealed that the c.1742_c.1743insT frameshift mutation led to the protein truncation, and the c. 2314T>G missense mutation resulted in the failure of forming 1 disulfide bond.Conclusion:The compound heterozygous variants of ECEL1 gene were considered to be pathogenic for this DA5D patient, which have expanded the mutational spectrum of the ECEL1 gene and provided a reference for clinical diagnosis as well as genetic counseling for this family.

Objective:To explore the clinical phenotype and genetic characteristics of a child with neurodevelopmental disorder caused by homozygous frameshift variant of the TRAPPC6B gene, and to provide reference for the diagnosis of the disease. Methods:A child with neurodevelopmental disorder caused by homozygous variant of TRAPPC6B gene who was admitted to the Children′s Hospital Affiliated to Zhengzhou University in March 2023 due to " inability to stand and walk independently at 1 year and 3 months old" was selected as the study object. The clinical data were collected by retrospective analysis method. Target region high-throughput sequencing was carried out on the child and parental peripheral blood samples, and candidate variant was verified by Sanger sequencing and bioinformatic analysis. The pathogenicity of variant was rated according to the Standards and Guidelines for the Interpretation of Sequence Variants released by American College of Medical Genetics and Genomics (ACMG) (hereinafter referred to as ACMG guidelines). The study has been approved by the Medical Ethics Committee of the Children′s Hospital Affiliated to Zhengzhou University (Ethics No.2022-K-L025). Results:The child was a 1-year-and-3-months-old boy whose parents were sib mating. The child presented with global developmental delay, microcephaly and short stature. MRI showed poor white matter myelination, abnormal signals of bilateral periventricular white matter and bilateral external sac, thin corpus callosum, and widening of the third ventricle. Genetic testing revealed that the TRAPPC6B gene of the child had a homozygous variant of c. 240_241delAA (p.Q80Hfs*34), which was inherited from his parents. According to the ACMG guidelines, this variant was judged to be potentially pathogenic (PVS1_Strong+ PM2_Supporting+ PM3_Supporting), resulting in premature occurrence of terminator codons and a change in the three-dimensional structure of protein. The variant was located in the functional domain, which may directly affect the functional domain of the protein, resulting in functional domain defects. Conclusion:The frameshift variation of TRAPPC6B gene c. 240_241delAA (p.Q80Hfs*34) has not been reported, which may be the genetic cause of neurodevelopmental disorders in child in this study. These findings expand the variation spectrum of TRAPPC6B gene and provid basis for genetic counseling and prenatal diagnosis of this family.

Objective:To explore the clinical characteristics and genetic etiology of a child with Osteopathia striata with cranial sclerosis (OSCS) due to variant of AMER1 gene. Methods:A child presented at the Affiliated Children′s Hospital of Zhengzhou University in July 2024 due to growth and development retardation was selected as the study subject. A retrospective study was conducted to collect the child′s clinical data. Peripheral blood samples (2 mL each) were collected from the child and her parents, and genomic DNA was extracted for whole exome sequencing (WES). Sanger sequencing was used for the verification of candidate variants. The pathogenicity of variant was rated according to the guidelines from American College of Medical Genetics and Genomics (ACMG). The study has been approved by the Medical Ethics Committee of the Children′s Hospital Affiliated to Zhengzhou University (Ethics No.: 2024-108-001).Results:The patient, a 4-year-and-10-month-old girl, presented with global developmental delay, short stature, cleft palate, distinct facial features, and hearing impairment. WES revealed that she has harbored a heterozygous c. 790_794dup (p.Cys265Trpfs*19) variant of the AMER1 gene, which was not detected in either parent. Based on the guidelines from ACMG, the gene variant was classified as pathogenic (PVS1 + PS2 + PM2_supporting). As the result of a non-triplet base insertion in the coding region of the AMER1 gene, it has converted a codon originally encoding an amino acid into a stop codon, and led to a truncated protein, causing severe alteration and dysfunction of the protein. Conclusion:The child was diagnosed with OSCS for clinical features such as global developmental delay, short stature, cleft palate, distinctive facial features, and hearing impairment, for which the de novo heterozygous frameshift variant AMER1: c. 790_794dup (p.Cys265Trpfs*19) may be accountable. Above finding has expanded the mutational spectrum of OSCS and provided a basis for genetic counseling and prenatal diagnosis for the family.

OBJECTIVE: To explore the clinical phenotype and genetic characteristics of a child with neurodevelopmental disorder caused by homozygous frameshift variant of the TRAPPC6B gene, and to provide reference for the diagnosis of the disease. METHODS: A child with neurodevelopmental disorder caused by homozygous variant of TRAPPC6B gene who was admitted to the Children's Hospital Affiliated to Zhengzhou University in March 2023 due to "inability to stand and walk independently at 1 year and 3 months old" was selected as the study object. The clinical data were collected by retrospective analysis method. Target region high-throughput sequencing was carried out on the child and parental peripheral blood samples, and candidate variant was verified by Sanger sequencing and bioinformatic analysis. The pathogenicity of variant was rated according to the Standards and Guidelines for the Interpretation of Sequence Variants released by American College of Medical Genetics and Genomics (ACMG) (hereinafter referred to as ACMG guidelines). The study has been approved by the Medical Ethics Committee of the Children's Hospital Affiliated to Zhengzhou University (Ethic No.2022-K-L025). RESULTS: The child was a 1-year-and-3-months-old boy whose parents were sib mating. The child presented with global developmental delay, microcephaly and short stature. MRI showed poor white matter myelination, abnormal signals of bilateral periventricular white matter and bilateral external sac, thin corpus callosum, and widening of the third ventricle. Genetic testing revealed that the TRAPPC6B gene of the child had a homozygous variant of c.240_241delAA (p.Q80Hfs*34), which was inherited from his parents. According to the ACMG guidelines, this variant was judged to be potentially pathogenic (PVS1_Strong+PM2_Supporting+PM3_Supporting), resulting in premature occurrence of terminator codons and a change in the three-dimensional structure of protein. The variant was located in the functional domain, which may directly affect the functional domain of the protein, resulting in functional domain defects. CONCLUSION The frameshift variation of TRAPPC6B gene c.240_241delAA (p.Q80Hfs*34) has not been reported, which may be the genetic cause of neurodevelopmental disorders in child in this study. These findings expand the variation spectrum of TRAPPC6B gene and provide basis for genetic counseling and prenatal diagnosis of this family.
Humans ; Infant ; Male ; Frameshift Mutation ; Homozygote ; Neurodevelopmental Disorders/genetics* ; Phenotype

OBJECTIVE: To explore the clinical phenotypes and genetic characteristics of a pedigree with Distal arthrogryposis type 5D (DA5D) caused by compound heterozygous variants in the ECEL1 gene. METHODS: A child (proband) diagnosed with DA5D and his family members (proband's parents and sister) who was admitted to the Department of Rehabilitation Medicine of Henan Children's Hospital in July 2022 due to "multiplex distal arthrogryposis" were enrolled into this study. Clinical data of the proband were collected and peripheral blood samples were obtained from the proband and members of his family about 3 mL. Trio-whole genome sequencing (trio-WGS) was carried out to detected the genetic variations of the proband and his family members. The candidate's pathogenic gene variants were screened and analyzed by Genome Aggregation Database (gnomAD) and other databases. The screened variants were annotated for clinical phenotypes using databases like the Online Mendelian Inheritance in Man (OMIM). The pathogenicity of the candidate variants was predicted by bioinformatics tools such as Provean. Based on the guidelines of the American College of Medical Genetics and Genomics (ACMG), pathogenicity ratings were conducted for variant sites. The protein conservation and mutation structure prediction of ECEL1 protein among species were carried out though MEGA-X and PyMOL. The research protocol of this study was reviewed by the Ethics Committee of Henan Provincial Children's Hospital (Approval No. 2023-H-H01), and informed consent for clinical research was obtained from the guardians of the probands. RESULTS: The proband had multiplex distal arthrogryposis involving hands, feet, knees, and ankles, and had right ptosis, micrognathia, low auricular position, and upturned nose. The parents and sister both had normal phenotypes. Trio-WGS and Sanger sequencing revealed that the child had compound heterozygous variants of paternal c.1742_c.1743insT and maternal c.2314T>G, for which the father and sister were carriers of the c.1742_c.1743insT heterozygous variant and the mother was carrier of c.2314T>A. Neither mutation site has been reported. According to guidelines of ACMG, the c.1742_c.1743insT variant was classified as likely pathogenic (PSV1+PM2_Supporting), and c.2314T>G was classified as uncertain (PM2_Supporting+PM3+PP3). The results of conserved analysis of amino acid residue sequences of ECEL1 protein showed that the missense mutation of the maternal c.2314T>G (p.Cys772Gly) was highly conserved among humans and other seven species. The protein structure prediction revealed that the c.1742_c.1743insT frameshift mutation led to the protein truncation, and the c.2314T>G missense mutation resulted in the failure of forming 1 disulfide bond. CONCLUSION The compound heterozygous variants of ECEL1 gene were considered to be pathogenic for this DA5D patient, which have expanded the mutational spectrum of the ECEL1 gene and provided a reference for clinical diagnosis as well as genetic counseling for this family.
Humans ; Pedigree ; Arthrogryposis/genetics* ; Male ; Female ; Heterozygote ; Phenotype ; Mutation ; Child ; Metalloendopeptidases

OBJECTIVE: To explore the clinical characteristics and genetic etiology of a child with Osteopathia striata with cranial sclerosis (OSCS) due to variant of AMER1 gene. METHODS: A child presented at the Affiliated Children's Hospital of Zhengzhou University in July 2024 due to growth and development retardation was selected as the study subject. A retrospective study was conducted to collect the child's clinical data. Peripheral blood samples (2 mL each) were collected from the child and her parents, and genomic DNA was extracted for whole exome sequencing (WES). Sanger sequencing was used for the verification of candidate variants. The pathogenicity of variant was rated according to the guidelines from American College of Medical Genetics and Genomics (ACMG). The study has been approved by the Medical Ethics Committee of the Children's Hospital Affiliated to Zhengzhou University (Ethics No.: 2024-108-001). RESULTS: The patient, a 4-year-and-10-month-old girl, presented with global developmental delay, short stature, cleft palate, distinct facial features, and hearing impairment. WES revealed that she has harbored a heterozygous c.790_794dup (p.Cys265Trpfs*19) variant of the AMER1 gene, which was not detected in either parent. Based on the guidelines from ACMG, the gene variant was classified as pathogenic (PVS1 + PS2 + PM2_supporting). As the result of a non-triplet base insertion in the coding region of the AMER1 gene, it has converted a codon originally encoding an amino acid into a stop codon, and led to a truncated protein, causing severe alteration and dysfunction of the protein. CONCLUSION The child was diagnosed with OSCS for clinical features such as global developmental delay, short stature, cleft palate, distinctive facial features, and hearing impairment, for which the de novo heterozygous frameshift variant AMER1: c.790_794dup (p.Cys265Trpfs*19) may be accountable. Above finding has expanded the mutational spectrum of OSCS and provided a basis for genetic counseling and prenatal diagnosis for the family.
Humans ; Female ; Child, Preschool ; Osteosclerosis/genetics* ; Adaptor Proteins, Signal Transducing/genetics* ; Mutation ; Exome Sequencing ; Retrospective Studies ; Tumor Suppressor Proteins

Objective:To explore the clinical phenotypes and genetic characteristics of a pedigree with Distal arthrogryposis type 5D (DA5D) caused by compound heterozygous variants in the ECEL1 gene. Methods:A child (proband) diagnosed with DA5D and his family members (proband′s parents and sister) who was admitted to the Department of Rehabilitation Medicine of Henan Children′s Hospital in July 2022 due to " multiplex distal arthrogryposis" were enrolled into this study. Clinical data of the proband were collected and peripheral blood samples were obtained from the proband and members of his family about 3 mL. Trio-whole genome sequencing (trio-WGS) was carried out to detected the genetic variations of the proband and his family members. The candidate′s pathogenic gene variants were screened and analyzed by Genome Aggregation Database (gnomAD) and other databases. The screened variants wer annotated for clinical phenotypes using databases like the Online Mendelian Inheritance in Man (OMIM). The pathogenicity of the candidate variants was predicted by bioinformatics tools such as Provean. Based on the guidelines of the American College of Medical Genetics and Genomics (ACMG), pathogenicity ratings were conducted for variant sites. The protein conservation and mutation structure prediction of ECEL1 protein among species were carried out though MEGA-X and PyMOL. The research protocol of this study was reviewed by the Ethics Committee of Henan Provincial Children′s Hospital (Approval No. 2023-H-H01), and informed consent for clinical research was obtained from the guardians of the probands.Results:The proband had multiplex distal arthrogryposis involving hands, feet, knees, and ankles, and had right ptosis, micrognathia, low auricular position, and upturned nose. The parents and sister both had normal phenotypes. Trio-WGS and Sanger sequencing revealed that the child had compound heterozygous variants of paternal c. 1742_c.1743insT and maternal c. 2314T>G, for which the father and sister were carriers of the c. 1742_c.1743insT heterozygous variant and the mother was carrier of c. 2314T>A. Neither mutation site has been reported. According to guidelines of ACMG, the c. 1742_c.1743insT variant was classified as likely pathogenic (PSV1+ PM2_Supporting), and c. 2314T>G was classified as uncertain (PM2_Supporting+ PM3+ PP3). The results of conserved analysis of amino acid residue sequences of ECEL1 protein showed that the missense mutation of the maternal c. 2314T>G(p.Cys772Gly) was highly conserved among humans and other seven species. The protein structure prediction revealed that the c.1742_c.1743insT frameshift mutation led to the protein truncation, and the c. 2314T>G missense mutation resulted in the failure of forming 1 disulfide bond.Conclusion:The compound heterozygous variants of ECEL1 gene were considered to be pathogenic for this DA5D patient, which have expanded the mutational spectrum of the ECEL1 gene and provided a reference for clinical diagnosis as well as genetic counseling for this family.