OBJECTIVE: To investigate the molecular pathogenic mechanisms of a family with hereditary factor Ⅶ (FⅦ) deficiency. METHODS: A family (3 generations, 12 members) with hereditary FⅦ deficiency, in which the proband presented with menorrhagia and was admitted to the First Affiliated Hospital of Wenzhou Medical University in April 2023, was selected as the study subject. Clinical data of the family members were collected. Peripheral venous blood samples were collected from all 12 members for routine coagulation tests and genomic DNA extraction. All exons and flanking sequences of the F7 gene were amplified by PCR and analyzed by Sanger sequencing. Thrombin generation assay was performed to evaluate the coagulation potential of the proband and her parents. Multiple online bioinformatics software tools were used to analyze the conservation and pathogenicity of candidate variants identified in the proband. The pathogenicity of variant was classified 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). Homology modeling of the variant FⅦ protein was performed using homology modeling (SWISS-MODEL). Amino acid sequence alignment between wild-type and variant FⅦ proteins was conducted using MEGA v7, and spatial conformational differences were analyzed using PyMOL to assess the potential impact of the F7 gene variants on the structure and function of the FⅦ protein. This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics No.: KY2022-R193). RESULTS: Coagulation tests showed that the proband's prothrombin time (PT) was significantly prolonged to 33.1 s, and both factor Ⅶ activity (FⅦ:C) and antigen (FⅦ:Ag) levels were reduced to 2%. Her parents, eldest sister, second sister, younger brother, and four children all showed mildly prolonged PT, with FⅦ:C and FⅦ:Ag levels approximately 50% of normal. Genetic sequencing identified compound heterozygous variants in the F7 gene of the proband: a heterozygous missense variant c.722C>A (p.Thr241Asn) in exon 7, and a heterozygous deletion variant c.1261_1261delA (p.Ile421Ser*fs75) in exon 8. Retrieval from domestic and international databases found no previous reports of the latter variant, suggesting it is novel. Familial co-segregation analysis confirmed that these variants were inherited from her father and mother, respectively. The thrombin generation assay demonstrated that the proband had a significantly decreased peak thrombin height (peak ratio: 29.5%), significantly increased thrombin lag time ratio and time-to-peak ratio (3.03 and 2.93, respectively), but only a mildly decreased endogenous thrombin potential (ETP) ratio of 90.7%. Online bioinformatics analysis indicated that threonine-241 (p.Thr241) in the FⅦ protein was not conserved, while isoleucine-421 (p.Ile421) was highly conserved. Both the p.Thr241Asn and p.Ile421Serfs*75 variant sites in the proband's F7 gene were predicted to be pathogenic. According to the ACMG guidelines, the p.Thr241Asn (PM3+PP1+PP3+PP4+PP5) and p.Ile421Ser*fs75 (PM2+PM4 +PP1+PP3+PP4) variants were both classified as "likely pathogenic". Structural analysis of the FⅦ protein indicated that the p.Ile421Ser*fs75 frameshift variant led to the substitution of Cysteine-428 by Alanine, preventing the formation of a critical disulfide bond between amino acid residues 400 and 428 present in the wild-type FVII protein. CONCLUSION The compound heterozygous variants p.Thr241Asn and p.Ile421Ser*fs75 in the F7 gene are likely the genetic etiology responsible for the reduced FⅦ levels in this hereditary FⅦ deficiency family.
Adult ; Female ; Humans ; Male ; Middle Aged ; China ; Factor VII/chemistry* ; Factor VII Deficiency/genetics* ; Heterozygote ; Mutation ; Pedigree ; East Asian People/genetics*

OBJECTIVE: To investigate the molecular mechanism for a family with Hereditary coagulation factor Ⅻ (FⅫ) deficiency. METHODS: The proband, a 63-year-old female, was admitted to the First Affiliated Hospital of Wenzhou Medical University in August 2024 for lumbar disc herniation. Coagulation tests, including prothrombin time (PT), activated partial thromboplastin time (APTT), and FⅫ activity (FⅫ:C), were carried out for the proband and her family members (9 individuals from three generations) using a one-stage clotting assay. The level of FⅫ antigen (FⅫ:Ag) was determined with an Enzyme-linked immunosorbent assay (ELISA). Sanger sequencing was conducted to identify potential variants in the F12 gene. Multiple in silico tools were used to predict the conservation, hydrophobicity, and structural impact of the identified variants. Recombinant expression plasmids were constructed and transiently transfected into HEK293T cells. The recombinant FⅫ protein was analyzed using Western blotting (WB) and ELISA. This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics No.: KY2022-R193). RESULTS: The proband showed a markedly prolonged APTT (160.3 s) and significantly decreased FⅫ:C (2%) and FⅫ:Ag (5%) levels. Analysis of the F12 gene sequence revealed a 46C/T genotype in the promoter region, a heterozygous c.1457G>A (p.Cys467Tyr) missense variant in exon 12, and a heterozygous c.1561G>A (p.Glu502Lys) missense variant in exon 13. Bioinformatic analysis showed that the p.Cys467 is highly conserved across various species, and the p.Cys467Tyr variant may affect local structural stability of the FⅫ protein. The p.Cys467Tyr variant had no effect on the transcription of the F12 gene. However, the variant has significantly decreased the FⅫ:Ag levels and FⅫ protein expression in the cell culture supernatant compared to the wild-type expression vector, while in the cell lysate, it is higher than the wild-type expression vector. In other words, the p.Cys467Tyr variant has probably caused a secretion defect of FⅫ protein. CONCLUSION The 46C/T genotype, the heterozygous p.Cys467Tyr missense variant, and the heterozygous p.Glu502Lys missense variant are associated with reduced plasma FⅫ levels in this pedigree. The p.Cys467Tyr variant, which was unreported previously, did not affect the synthesis of FⅫ but may have resulted in a secretion defect.
Humans ; Female ; Middle Aged ; Mutation, Missense ; Pedigree ; HEK293 Cells ; Male ; Factor XII/genetics* ; Adult ; Factor XII Deficiency/genetics*

ObjectiveTo investigate the structural and functional characteristics of gut microbiota in common traditional Chinese medicine （TCM） syndromes of irritable bowel syndrome with diarrhea （IBS-D）. MethodsIBS-D patients who visited the Hospital of Chengdu University of Traditional Chinese Medicine， and healthy participants from the Physical Examination Centre of the same hospital were recruited from 1st January 2020 to 31st March 2021.The IBS-D patients were classified into syndrome of liver constraint and spleen deficiency， and syndrome of spleen deficiency and dampness exuberance； together with the recruited healthy participants， there were liver-constraint group， dampness-exuberance group， and healthy group. General information， including age， gender and body mass index （BMI）， were collected， and Irritable Bowel Syndrome Symptom Severity Scale （IBS-SSS） as well as Irritable Bowel Syndrome Quality of Life Scale （IBS-QOL） scores were additionally collected from IBS-D patients. Fresh fecal samples were also collected and tested by macro-genome sequencing technology for abundance statistical display， PCoA， Anosim， LEfSe bioinformatic analysis of the annotated gut microbiota structure and function. ResultsThere was no statistically significant difference in the general information of the participants in the three groups （P＞0.05）； the difference in the IBS-SSS and IBS-QOL scores between liver-constraint group and dampness-exuberance group were not statistically significant （P＞0.05）. The study included 28 cases each in liver-constraint group， dampness-exuberance group， and healthy group. The number of specific genes to patients in liver-constraint group was 269 135， with 216 156 in dampness-exuberance group and 249 759 in healthy group， accounting of total 1 784 036 in the three groups. There were differences in the relative abundance distribution of the top ten species of gut microbiota among the three groups， with smaller differences at the phylum， class and order levels， and larger differences at the family， genus and species levels. There were differences in the relative abundance of structure and function of the gut microbiota among the three groups. Species PCoA and Anosim analyses at the species level showed significant differences in the composition of the microbiota among the three groups. Further LEfSe analyses showed that patients in liver-constraint group were screened for 14 dominant strains， of which Clostridium sp. CAG 217， Lachnospira pectinoschiza， Anaerotruncus sp. CAG 528， Paeniclostridium sordellii， Eubecterium sp. CAG 76， Bacillus cereus were affected to a greater extent in abundance differences； dampness-exuberance group screened 24 species of dominant bacteria， of which Roseburia inulinivorans， Eubacterium sp. CAG 251， Roseburia hominis， Unclassified Eubacterium rectale， Roseburia intestinalis， and Megamonas funiformis were affected to a greater extent in abundance differences； no dominant functional genes were screened for patients in liver-constraint group， and dampness-exuberance group was screened for flagellum assembly （ko02040）， porphyrin metabolism （ ko00860）， salmonella infection （ko05132）， and benzoic acid degradation （ko00362）. The differentially dominant functional genes in liver-constraint group and dampness-exuberance group may mainly focus on metabolism （including biodegradation and metabolism of exogenous substances， energy metabolism， lipid metabolism， etc.）. ConclusionIBS-D with syndrome of liver constraint and spleen deficiency is characterized by the enrichment of 14 gut microbiota， such as Clostridium sp. CAG 217， while IBS-D with syndrome of spleen deficiency and dampness exuberance is characterized by the enrichment of 24 gut microbiota， such as Roseburia inulinivorans， and 4 functional enrichments， such as flagellum assembly. Clostridium sp. CAG 217 and Roseburia inulinivorans are expected to be biomarkers for IBS-D patients in the two syndromes， respectively.

Objective To investigate the effect of continued nursing on postoperative outcomes of discharged patients with chronic suppurative otitis media.Methods Toally 198 discharged patients were randomized into the control group (n=98) and the observation group (n=100).The patients in both groups received normal health education and additionally the continued nursing was used in the observation group for health education by way of phone call follow-up,on-the-spot consultation and QQ consultation.The mastery of related knowledge and skills,incidence of secondary tympanic membrane and readmission rate were compared.Results Six months after discharge,the observation group was significantly better than those the control group in the mastery of related knowledge and skills (P＜0.05).The incidence of secondary tympanic membrane perforation was not statistically significant between the groups(P＞ 0.05).The readmission rate at the second week and the third week between the two groups had no statistically significant difference (P＞ 0.05).However,the readmission rate at the first,second,third and sixth month were statistically different (P＜0.05).Conclusion Continued nursing to the postoperative discharged patients with chronic suppurative otitis media can improve the follow-up rate after discharge and self-nursing capacity,and as a result promote their recovery.