Antithrombin deficiency caused by SERPINC1 gene mutations predispose to venous thrombotic events / 中华检验医学杂志

Objective:To analyze 12 antithrombins (AT) gene mutations that cause AT deficiency and discuss the relationship between the SERPINC1 gene. mutations and venous thrombotic events.Methods:This study belongs to case series of observational studies. Collected the clinical data of 12 AT deficiency cases in the First Affiliated Hospital of Wenzhou Medical University from April 2014 to April 2021 and collected the blood samples before treatment. The AT activity (AT: A) and AT antigen (AT: Ag) was detected by chromogenic substrate and immunoturbidimetry, respectively. The 7 exons and flanking sequences of the SERPINC1 gene were sequenced directly by PCR, the suspected mutations were validated by reverse sequencing. Analyzed the correlation between the SERPINC1 gene. mutations and venous thrombotic events and figured out the proportion.Results:The AT: A of the 12 patients all decreased significantly, ranging from 30% to 66%, and the AT: Ag of the 7 patients decreased accordingly, showing type Ⅰ AT deficiency, and the AT: Ag of the other 5 patients were normal, presented type Ⅱ AT deficiency. 12 mutations were found including 6 heterozygous mutations which were discovered for the first time: c.456_458delCTT(p.phe121del), c.318_319insT(p.Asn75stop), c.922G>T(p.Gly276Cys), c.938T>C (p.Met281Thr), c.1346T>A(p.Leu417Gln)and c.851T>C(p.Met252Thr). All 12 patients had venous thrombosis, and 3 cases including 2 compound heterozygotes and 1 single heterozygote all suffered from deep venous thrombosis (DVT) when they were younger without obvious triggers. The other 9 patients all combined with the other thrombotic factors including old age, hypertensive, smoking, pregnancy, and prolonged immobilization.Conclusion:Patients with AT deficiency caused by SERPINC1 gene defects are prone to venous thrombosis, especially combined with other thrombotic factors.

The level of mindfulness and fear of recurrence in patients after radical gastrectomy for gastric cancer: the chain mediating effect of illness uncertainty and negative coping styles / 中国实用护理杂志

Objective:To explore the relationship between the level of mindfulness, illness uncertainty, negative coping style and fear of recurrence in patients after radical resection of gastric cancer, and to understand the internal mechanism of how mindfulness affects the fear of recurrence.Methods:This was a cross sectional survey. From January 2019 to March 2021, the convenience sampling method was used to select 227 patients undergoing radical gastric cancer surgery in the Affiliated Hospital of Naval Medial University of Chinese PLA as the research objects. The general information questionnaire, Mindfulness Attention and Awareness Scale, Fear of Disease Progression Simplified Scale, Mishel′s Illness Uncertainty Scale and Simplified Coping Style Questionnaire were used for questionnaire surveys. The relationship between the level of mindfulness, illness uncertainty, negative coping style and fear of recurrence was explored and the model was tested.Results:The returned questionnaires were 207 with a recovery rate of 91.19%(207/227). Pearson correlation analysis showed that the level of mindfulness was negatively correlated with illness uncertainty, negative coping style, and fear of recurrence ( r=-0.176, -0.269, -0.480, all P<0.01). Illness uncertainty, negative coping styles were positively correlated with fear of recurrence ( r=0.433, 0.420, both P<0.01). The mediation model test showed that mindfulness had a significant direct effect on fear of relapse (effect value was -0.220), illness uncertainty and negative coping styles had significant partial mediating effect between mindfulness level and fear of recurrence (effect value were -0.036, -0.030). And the chain mediating effect of illness uncertainty and negative coping style was also significant (effect value was -0.006). Conclusions:The level of mindfulness can not only have a direct impact on the fear of recurrence in patients after radical gastrectomy, but also indirectly affect the fear of recurrence through the chain mediating effect of illness uncertainty, negative coping style, and disease uncertainty→negative coping style.

Phenotypic and genotypic analysis of a pedigree affected with hereditary protein C deficiency due to heterozygous deletional mutation of PROC gene / 中华医学遗传学杂志

OBJECTIVE@#To analyze the phenotype and genetic variants of a pedigree affected with hereditary protein C (PC) deficiency.@*METHODS@#The protein C activity (PC:A) of the proband and her family members (a four-generation pedigree including 11 individuals) were tested by chromogenic substrate method, and the protein C antigen (PC:Ag) was detected with an enzyme-linked immunosorbent assay(ELISA). The 9 exons and flanking sequences of the protein C (PROC) gene were amplified by PCR and directly sequenced. Suspected mutation was validated by clone sequencing and in other members of the family. MutationTaster and ClustalX-2.1-win was used to analyze the pathogenicity and conservation of the mutation site,respectively. Three-dimentional protein model and amino acids interaction were analyzed with Swiss-PdbViewer software.@*RESULTS@#The PC: A and PC: Ag of the proband were decreased to 46% (reference range: 70%-130%) and 50% (referencerange:70%-140%), respectively. Her grandmother,aunt, cousin and younger brother also showed declined PC:A and PC:Ag by approximately 50%. Genetic analysis revealed that the above individuals have all carried a deletional mutation c.1212-1212delG (p.Met364TrpfsX15) in exon 9 of the PROC gene which can cause replacement of Methionine at position 364 by Tryptophan and alteration of 15 downstream amino acids, and produce a premature stop codon at position 378. The score of MutationTaster was 1.000, indicating that the variant is pathogenic. Conservative analysis showed that the 15 altered amino acids are located in a conserved region across nine homologous species. Protein model analysis showed that the mutation has disrupted a catalytic domain of protein C thereby affected its function.@*CONCLUSION@#The heterozygous c.1212-1212delG deletional mutation in exon 9 of the PROC gene, which was unreported previously,probably accounts for the decrease of PC:A and PC:Ag in this pedigree.

Clinical characteristics and genetic analysis of a combined inherited antithrombin and factor Ⅶ deficiency pedigree / 中华检验医学杂志

Objective:To study the clinical characteristics and gene mutations in a family with combined inherited antithrombin (AT) and factor Ⅶ (FⅦ) deficiency, and explore the relationship between AT gene, F7 gene mutations and diseases. Methods:Pedigree investigation. Blood samplesand clinical dataswere collected fromthe proband and her family members (a total of 16 people in 3 generations) who admitted to the First Affiliated Hospital of Wenzhou Medical University in November 2018. The prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), antithrombin activity (AT: A), antithrombin antigen (AT: Ag), protein C activity (PC: A), protein S activity (PS: A), FⅦ activity (FⅦ: C), FⅦ antigen (FⅦ: Ag) and other indicators were detectedto confirm the diagnosis. DNA direct sequencing analysis of all exons, flanking sequences, 5′ and 3′ untranslated regions of AT genes and F7 genes, and the mutation sites were confirmed by clone sequencingor reverse sequencing. Results:The AT: A and AT: Ag of the proband were 46% and 135 mg/L, respectively (reference range: 250-360 mg/L), some of her family members′ s (father, aunt, two cousins, younger brother and nephew) AT: A and AT: Ag were reduced to 50% of normal range. Her father (Ⅰ 2), aunt (Ⅰ 4), elder brother (Ⅱ 7), younger brother (Ⅱ 8), and nephew (Ⅲ 3)′s FⅦ: C were 45%, 50%, 48%, 47% and 48%, respectively; and their FⅦ:Ag was within the normal range. Genetic analysis revealed that the proband(Ⅱ 6) and some of her family members (father, aunt, two cousins, younger brother and nephew) took rs3138521 polymorphism in the 5′ untranslated region of AT gene. Her father (Ⅰ 2), aunt (Ⅰ 4), elder brother (Ⅱ 7), younger brother (Ⅱ 8), nephew (Ⅲ 3) took c.1091G>A heterozygous missense mutationin exon 8 of F7 gene, resulting in p.Arg304Gln. Conclusion:The rs3138521 in AT gene and c.1091G>A in F7 gene, which may be the molecular mechanism leading to combined inherited AT and FⅦ deficiency in this family.

Analysis of the reasons of contrast agent venues intravasation during transvaginal four-dimensional hysterosalpingo-contrast sonography / 中华超声影像学杂志

Objective:To explore the influencing factors of uterine myometrial and parauterine venous plexus reflux during transvaginal four-dimensional contrast-enhanced hysterosalpingography, which will help reduce and avoid the occurrence of reflux and improve the accuracy of diagnosis.Methods:A total of 306 infertile patients who underwent transvaginal four-dimensional contrast-enhanced hysterosalpingograph from June 2016 to June 2017 in the Third Affiliated Hospital of Guangzhou Medical University were retrospectively enrolled. The ultrasonographic characteristics were reviewed and the correlations between reflux and endometrial thickness, days after menstruation, intrauterine operation history and patency of fallopian tube were analyzed.Results:The incidence of countercurrent during four-dimensional contrast-enhanced hysterosalpingography was 14.71%. The chi-square test showed that the endometrial thickness and the days after menstruation had statistically significant effects on the incidence of reflux ( P=0.031 and <0.001, respectively). There were no statistically significant effects of intrauterine operation history and patency of the fallopian tube on the incidence of reflux ( P=0.610, 0.137). Logistic regression analysis showed that the incidence of reflux was associated with endometrial thickness ( B=-1.171, P<0.001) and the days after menstruation ( B=0.439, P=0.015). Conclusions:Transvaginal ultrasound measurement of endometrial thickness before angiography and selection of appropriate examination time according to the menstrual cycle can effectively reduce the incidence of reflux, and adverse reactions, and improve the accuracy of four-dimensional contrast-enhanced hysterosalpingography.

Identification of compound heterozygous mutations of F11 gene in a pedigree affected with heriditary coagulation factor XI deficiency / 中华医学遗传学杂志

OBJECTIVE@#To identify potential mutations of F11 gene in a pedigree affected with hereditary coagulation factor XI (FXI) deficiency and explore its molecular pathogenesis.@*METHODS@#Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), coagulation factor VIII activity (FVIIIC), coagulation factor IX activity (FIXC), coagulation factor XI activity (FXIC), coagulation factor XII activity (FXIIC) and lupus anticoagulation (LA) of the proband and eight family members were determined. FXI antigen (FXIAg) was determined by enzyme-linked immunosorbent assay (ELISA). For the proband, potential mutations in the exons, flanking introns and 5'-, 3'-untranslated regions of the F11 gene were screened by direct DNA sequencing. The results were confirmed by reverse sequencing. Suspected mutations were detected in other family members. ClustalX-2.1-win and four online bioinformatic tools (PolyPhen-2, PROVEAN, SIFT, and Mutation Taster) were used to study the conservation and possible impact of the mutations. The structure of the mutational sites was processed with Swiss-PdbViewer.@*RESULTS@#The propositus had prolonged APTT (69.6 s), whose FXIC and FXIAg were reduced to 6.0% and 10.7%, respectively. Her mother, elder sister, one younger sister, little brother, daughter and son showed slightly prolonged APTT and moderate FXIC and FXIAg levels. Gene sequencing revealed that the propositus carried a heterozygous nonsense mutation c.738G>A (p.Trp228stop) in exon 7 and a heterozygous mutation c.1556G>C (p.Trp501Ser) in exon 13. Her mother, elder sister and daughter were heterozygous for the p.Trp228stop mutation, while one younger sister and little brother and son were heterozygous for p.Trp501Ser. Her husband and the youngest sister were of the wild type. Phylogenetic analysis suggested that Trp501 was highly conserved among all homologous species. The p.Trp501Ser was predicted to be "probably damaging","deleterious", "affect protein function" and "disease causing" corresponding to PolyPhen-2, PROVEAN, SIFT and Mutation Taster. Model analysis demonstrated that the non-polar Trp501 has two benzene rings, forming a hydrogen bond with Gln512 in the wild type. Once substituted by Ser501, the side chain may form another hydrogen bond with the benzene of His396. This may affect the normal space conformation and stability of FXI protein.@*CONCLUSION@#The compound heterozygous mutations of the F11 gene probably accounted for the low FXI concentration in this pedigree.

Construction and fluorescence intensity of lipid ultrasound microbubbles with monoclonal antibody targeted to leukaemia inhibitory factor receptor / 中华超声影像学杂志

Objective To investigate fluorescence intensity of lipid ultrasound microbubbles constructed in vitro and targeted to leukaemia inhibitory factor receptor (LIFR) with a monoclonal antibody.MethodsThe LIFR-targeted ultrasound mierobubbles (MB-BSB-LIFR-AB) were constructed using a technology of biotin-avidin bridge.FITC labeled Avidin was incubated with lipid ultrasound microbubbles (MB) and biotinylated lipid microbubbles (MB-B).Two dilutions (1:4 and 1:16) of DTAF second antibody were incubated with four types of ultrasound microbubbles,including MB,MB-B,biotinavidin-MB (MB-BS),MB-BSB-LIFR-AB.The fluorescence intensity of microhubbles were graded as 0,1,2to 3.ResultsAfter incubating with FITC-avidin,MB-B displayed bright green fluorescence ( grade 3),but MB had no fluorescence ( grade 0).After incubating with two dilutions of DTAF second antibody (1:4 and 1:16),MB-BSB-LIFR-AB displayed brightest green fluorescence (grade 3) in both concentration,while MB-BS and MB-B only displayed dim green fluorescence (grade 1 ) at the dilution of 1:4,with MB displaying no fluorescence at either dilution (grade 0).Conclusions LIFR monoclonal antibody can be effectively conjugated to MB-B with biotin-avidin bridge.Fluorescence detection is a simple method for investigating the conjugation reliability of targeted lipid ultrasound microbubbles.