OBJECTIVE: To explore the genetic variants and phenotypic characteristics of patients with Neurofibromatosis type I (NF1). METHODS: Twenty two NF1 patients who presented at Enze Medical (Center) Group in Taizhou between 2018 and 2024 were selected as the study subjects. Clinical phenotype and family history were collected for the patients. Whole exome sequencing (WES) was carried out for the 22 probands to screen the variants of NF1 gene. Candidate variants were verified by Sanger sequencing of their family members. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No.: K20230902). RESULTS: The 22 probands were diagnosed between the age of 5 months to 47 years old, and have all shown cafe au lait spots on their skin. Seventeen patients exhibited the phenotype at birth, and 11 had various degrees of neurofibromatosis. Among them, probands 1 and 13 underwent surgical resection of the tumor but had recurred, while proband 12 had amputation due to the huge size and serious impact of the neurofibroma and had no recurrence. Five patients had various degrees of scoliosis. In total 22 germline mutations and one somatic mutation were identified among the 22 families, with 5 variants unreported previously, including 1 nonsense mutation c.1603C>T (Q535*), 3 frameshift mutations [c.7268_7269delCA (Thr2423fs), c.2293del (Arg765Alafs*26), and c.5433_5438delinsGC (Phe1812ArgfsTer50)], and 1 deletion involving exons 41-44 of the NF1 gene and adjacent introns. Proband 13 was found to harbor germline mutation c.6796C>T (Gln2266Ter) and somatic mutation c.1019_1020del (Ser340Cysfs Ter12) in the peripheral blood and tumor tissue, respectively. Among the 22 NF1 probands, 6 had received treatment due to severe illness. Proband 1 had tumor resection in the right upper limb, but was found to have malignant lung tumor and died during follow-up. Proband 12 had multiple recurrence of neurofibroma in the left ring finger. Proband 4 underwent spinal correction surgery due to severe scoliosis. Proband 11 had died due to a central nervous system disease. Among the 22 germline mutations, 6 had led to the occurrence of truncated proteins, which may have a more severe impact on the phenotype. CONCLUSION This study investigated the genetic variants and clinical phenotypes of 22 NF1 families and identified 5 novel variants of the NF1 gene, which has expanded the genotypic and phenotypic spectra of the NF1. Preliminary studies have identified an association between truncated mutations, young age, and severe phenotypes, which may provide important clues for prognosis evaluation. For the clinical diagnosis and treatment of NF1, it is necessary to consider the phenotypic characteristics and genetic testing in combination with genetic counseling and long-term follow-up.
Humans ; Neurofibromatosis 1/pathology* ; Male ; Female ; Pedigree ; Adult ; Child ; Child, Preschool ; Middle Aged ; Adolescent ; Infant ; Young Adult ; Neurofibromin 1/genetics* ; Phenotype ; Asian People/genetics* ; Mutation ; Exome Sequencing ; East Asian People

OBJECTIVE: To determine the types of genetic variants in six Chinese pedigrees affected with Marfan syndrome (MFS) and analyze their clinical characteristics and molecular pathogenesis. METHODS: Six MFS pedigrees presented at the Taizhou Enze Medical Center (Group) between 2017 and 2022 were selected as the study subjects. Clinical data of pedigrees were retrospectively analyzed. Peripheral blood samples were collected from the probands and their family members for the extraction of genomic DNA. Whole exome sequencing (WES) was carried out. Candidate variants of the FBN1 gene were verified by Sanger sequencing. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), pathogenicity of the candidate variants was assessed. AlphaFold3 and PyMOL software were used for homology modeling of the FBN1 protein and analysis of its three-dimensional structure and amino acid sequence conservation. This study was approved by the Medical Ethics Committee of Taizhou Enze Medical Center (Group) (Ethics No. 20231002). RESULTS: Cardiovascular system abnormalities were noted in all pedigrees, ocular abnormalities were present in pedigrees 2 and 5, skeletal system abnormalities were presented in pedigrees 1, and 4 to 6. FBN1 gene mutations were identified in all pedigrees, including c.1957_1958dupGT (p.Asp654fs), c.5014T>A (p.Cys1672Ser), c.8135delC (p.Pro2712fs), c.2302G>T (p.Glu768*), c.3473A>G (p.Glu1158Gly) and c.6169C>T (p.Arg2057*), with each involving a different exon. Four variants were rated as pathogenic, one as likely pathogenic, and one as variant of uncertain significance. Among these, c.5014T>A (p.Cys1672Ser), c.1957_1958dupGT (p.Asp654fs), c.8135delC (p.Pro2712fs), and c.2302G>T (p.Glu768*) were unreported previously. Bioinformatic analysis with SIFT and PolyPhen-2 predicted that the c.5014T>A (p.Cys1672Ser) and c.3473A>G (p.Glu1158Gly) variants were deleterious. Protein homologous sequence alignment analysis revealed that the four novel mutation sites are highly conserved across various species. Homology modeling of the FBN1 protein three-dimensional structure indicated that the six variant sites in the amino acid sequence are all close to hydrogen bonds and may alter the secondary and tertiary structures to varying degrees, thereby confirmed the relationship between the variants and MFS. CONCLUSION Four novel variants of the FBN1 gene have been discovered in this study, which has enriched the mutational and phenotypic spectrum of MFS and provided a basis for disease diagnosis and genetic counseling.
Adolescent ; Adult ; Child ; Female ; Humans ; Male ; Middle Aged ; Young Adult ; China ; East Asian People/genetics* ; Exome Sequencing ; Fibrillin-1/genetics* ; Marfan Syndrome/genetics* ; Mutation ; Pedigree ; Retrospective Studies ; Adipokines

Objective:To determine the types of genetic variants in six Chinese pedigrees affected with Marfan syndrome (MFS) and analyze their clinical characteristics and molecular pathogenesis.Methods:Six MFS pedigrees presented at the Taizhou Enze Medical Center (Group) between 2017 and 2022 were selected as the study subjects. Clinical data of pedigrees were retrospectively analyzed. Peripheral blood samples were collected from the probands and their family members for the extraction of genomic DNA. Whole exome sequencing (WES) was carried out. Candidate variants of the FBN1 gene were verified by Sanger sequencing. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), pathogenicity of the candidate variants was assessed. AlphaFold3 and PyMOL software were used for homology modeling of the FBN1 protein and analysis of its three-dimensional structure and amino acid sequence conservation. This study was approved by the Medical Ethics Committee of Taizhou Enze Medical Center (Group) (Ethics No. 20231002). Results:Cardiovascular system abnormalities were noted in all pedigrees, ocular abnormalities were present in pedigrees 2 and 5, skeletal system abnormalities were presented in pedigrees 1, and 4 to 6. FBN1 gene mutations were identified in all pedigrees, including c. 1957_1958dupGT (p.Asp654fs), c. 5014T>A (p.Cys1672Ser), c. 8135delC (p.Pro2712fs), c. 2302G>T (p.Glu768*), c. 3473A>G (p.Glu1158Gly) and c. 6169C>T (p.Arg2057*), with each involving a different exon. Four variants were rated as pathogenic, one as likely pathogenic, and one as variant of uncertain significance. Among these, c. 5014T>A (p.Cys1672Ser), c. 1957_1958dupGT (p.Asp654fs), c. 8135delC (p.Pro2712fs), and c. 2302G>T (p.Glu768*) were unreported previously. Bioinformatic analysis with SIFT and PolyPhen-2 predicted that the c. 5014T>A (p.Cys1672Ser) and c. 3473A>G (p.Glu1158Gly) variants were deleterious. Protein homologous sequence alignment analysis revealed that the four novel mutation sites are highly conserved across various species. Homology modeling of the FBN1 protein three-dimensional structure indicated that the six variant sites in the amino acid sequence are all close to hydrogen bonds and may alter the secondary and tertiary structures to varying degrees, thereby confirmed the relationship between the variants and MFS. Conclusion:Four novel variants of the FBN1 gene have been discovered in this study, which has enriched the mutational and phenotypic spectrum of MFS and provided a basis for disease diagnosis and genetic counseling.

Objective:To evaluate the effect of robot-assisted laparoscopic technology via abdominal approach for patients with primary retroperitoneal tumors.Methods:A retrospective cohort analysis was conducted for the clinical data of 71 patients who underwent robot-assisted laparoscopic resection of primary retroperitoneal tumor via abdominal approach at the Department of Urology of Sir Run Run Shaw Hospital,Zhejiang University School of Medicine from January 2015 to December 2023. There were 35 male and 36 female patients. The age ( M(IQR)) was 56(21) years (range: 21 to 83 years). The median tumor diameter was 46 (31) mm (range: 15 to 134 mm). Postoperative pathology revealed 58 benign and 13 malignant cases. Patients were divided into non-adherent group ( n=47) and adherent group ( n=24) based on whether the tumor was adhered to major organs or vessels. Perioperative and postoperative situation were compared between the two groups. Data comparisons were conducted using independent samples t-test for normally distributed continuous variables, Mann-Whitney U tests for non-normally distributed data, χ2 test or Fisher′s exact test for categorical variables. Kaplan-Meier survival analysis was employed to estimate 3-year recurrence or metastasis rate and 3-year mortality rate. Results:Operative time was 120(60) minutes (range: 45 to 440 minutes), intraoperative blood loss was 50 (80) ml (range: 10 to 2 000 ml). The median change of intraoperative mean arterial pressure was 40 (19) mmHg(1 mmHg=0.133 kPa)(range: 10 to 112 mmHg). Intraoperative blood transfusion was required in 7 cases, whereas 64 cases did not necessitate transfusion. The change in hemoglobin levels before and after surgery was (17.9±13.6) g/L (range:-16 to 53 g/L), and the median change in serum creatinine levels was 2.0 (14.5) μmol/L (range:-71.0 to 100.4 μmol/L). Postoperative fasting duration was 2.0 (1.5) days (range: 1 to 6 days), and the median hospital stay was 10.0 (7.5) days (range: 4 to 24 days). No perioperative mortality occurred in any of the patients. The non-adherent group had shorter operation time, less estimated blood loss, lower blood transfusion rate, smaller delta value of hemoglobin before and after surgery, larger delta value of creatinine before and after surgery, fewer postoperative complications, shorter postoperative fasting time, and shorter length of hospital stay than the adherent group(all P<0.05), while there was no significant difference in mean arterial pressure fluctuation between the two groups ( P>0.05). Follow-up data were available for 69 patients, with a median follow-up duration of 39 (43) months (range: 4 to 88 months). Among these patients, 40 completed the 3-year follow-up. The 3-year recurrence or metastasis rate was 10.14%, and the 3-year mortality rate was 2.90%. Conclusions:Robot-assisted laparoscopic technology via abdominal approach for resection of primary retroperitoneal tumors is safe and feasible. It can also achieve secure surgical outcome for primary retroperitoneal tumors adherent to surrounding organs or vessels, albeit with increased surgical complexity and slower postoperative recovery compared to non-adherent cases.

To investigate the impact of miR-142-5p on intestinal epithelial cells,an expression vector for overexpressing miR-142-5p lentivirus was constructed and administered to mice to observe pathological changes in their colon tissue.The miR-142-5p gene was integrated into the green fluo-rescent lentiviral vector plenti-CMV/TO eGFP-Puro(plenti),and the resulting recombinant vec-tor was named plenti miR-142-5p following confirmation through sequencing.293T cells were transfected with the recombinant plasmid and packaging-assisted plasmid,yielding a packaged miR-142-5p overexpressing lentiviral vector(LV-miR-142-5p).The recombinant lentivirus suspen-sion was collected and concentrated via ultrafast centrifugal precipitation.Virus titer was deter-mined using real-time PCR.The virus suspension was then injected into the tail vein of 6-8-week-old BALB/c mice,after which their colonic tissues were dissected for HE staining and microscopic observation.The results showed that miR-142-5p homologous recombination into plenti vector and sequencing results were consistent with the expected sequence.plenti-miR-142-5p was co-transfect-ed with the packaging helper plasmid in 293T cells,and the successful transfection was confirmed based on the green fluorescence expression,and the real-time PCR results showed that the lentiviral titer was 1.23 × 109 IU/mL.Colon tissue slices from infected mice exhibited compromised colon integrity and significant inflammatory response.It was proved that LV-miR-142-5p was suc-cessfully constructed,and miR-142-5p caused damage to intestinal epithelial cells of mice.

To investigate the impact of miR-142-5p on intestinal epithelial cells,an expression vector for overexpressing miR-142-5p lentivirus was constructed and administered to mice to observe pathological changes in their colon tissue.The miR-142-5p gene was integrated into the green fluo-rescent lentiviral vector plenti-CMV/TO eGFP-Puro(plenti),and the resulting recombinant vec-tor was named plenti miR-142-5p following confirmation through sequencing.293T cells were transfected with the recombinant plasmid and packaging-assisted plasmid,yielding a packaged miR-142-5p overexpressing lentiviral vector(LV-miR-142-5p).The recombinant lentivirus suspen-sion was collected and concentrated via ultrafast centrifugal precipitation.Virus titer was deter-mined using real-time PCR.The virus suspension was then injected into the tail vein of 6-8-week-old BALB/c mice,after which their colonic tissues were dissected for HE staining and microscopic observation.The results showed that miR-142-5p homologous recombination into plenti vector and sequencing results were consistent with the expected sequence.plenti-miR-142-5p was co-transfect-ed with the packaging helper plasmid in 293T cells,and the successful transfection was confirmed based on the green fluorescence expression,and the real-time PCR results showed that the lentiviral titer was 1.23 × 109 IU/mL.Colon tissue slices from infected mice exhibited compromised colon integrity and significant inflammatory response.It was proved that LV-miR-142-5p was suc-cessfully constructed,and miR-142-5p caused damage to intestinal epithelial cells of mice.

Objective:To determine the types of genetic variants in six Chinese pedigrees affected with Marfan syndrome (MFS) and analyze their clinical characteristics and molecular pathogenesis.Methods:Six MFS pedigrees presented at the Taizhou Enze Medical Center (Group) between 2017 and 2022 were selected as the study subjects. Clinical data of pedigrees were retrospectively analyzed. Peripheral blood samples were collected from the probands and their family members for the extraction of genomic DNA. Whole exome sequencing (WES) was carried out. Candidate variants of the FBN1 gene were verified by Sanger sequencing. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), pathogenicity of the candidate variants was assessed. AlphaFold3 and PyMOL software were used for homology modeling of the FBN1 protein and analysis of its three-dimensional structure and amino acid sequence conservation. This study was approved by the Medical Ethics Committee of Taizhou Enze Medical Center (Group) (Ethics No. 20231002). Results:Cardiovascular system abnormalities were noted in all pedigrees, ocular abnormalities were present in pedigrees 2 and 5, skeletal system abnormalities were presented in pedigrees 1, and 4 to 6. FBN1 gene mutations were identified in all pedigrees, including c. 1957_1958dupGT (p.Asp654fs), c. 5014T>A (p.Cys1672Ser), c. 8135delC (p.Pro2712fs), c. 2302G>T (p.Glu768*), c. 3473A>G (p.Glu1158Gly) and c. 6169C>T (p.Arg2057*), with each involving a different exon. Four variants were rated as pathogenic, one as likely pathogenic, and one as variant of uncertain significance. Among these, c. 5014T>A (p.Cys1672Ser), c. 1957_1958dupGT (p.Asp654fs), c. 8135delC (p.Pro2712fs), and c. 2302G>T (p.Glu768*) were unreported previously. Bioinformatic analysis with SIFT and PolyPhen-2 predicted that the c. 5014T>A (p.Cys1672Ser) and c. 3473A>G (p.Glu1158Gly) variants were deleterious. Protein homologous sequence alignment analysis revealed that the four novel mutation sites are highly conserved across various species. Homology modeling of the FBN1 protein three-dimensional structure indicated that the six variant sites in the amino acid sequence are all close to hydrogen bonds and may alter the secondary and tertiary structures to varying degrees, thereby confirmed the relationship between the variants and MFS. Conclusion:Four novel variants of the FBN1 gene have been discovered in this study, which has enriched the mutational and phenotypic spectrum of MFS and provided a basis for disease diagnosis and genetic counseling.

Objective:To evaluate the effect of robot-assisted laparoscopic technology via abdominal approach for patients with primary retroperitoneal tumors.Methods:A retrospective cohort analysis was conducted for the clinical data of 71 patients who underwent robot-assisted laparoscopic resection of primary retroperitoneal tumor via abdominal approach at the Department of Urology of Sir Run Run Shaw Hospital,Zhejiang University School of Medicine from January 2015 to December 2023. There were 35 male and 36 female patients. The age ( M(IQR)) was 56(21) years (range: 21 to 83 years). The median tumor diameter was 46 (31) mm (range: 15 to 134 mm). Postoperative pathology revealed 58 benign and 13 malignant cases. Patients were divided into non-adherent group ( n=47) and adherent group ( n=24) based on whether the tumor was adhered to major organs or vessels. Perioperative and postoperative situation were compared between the two groups. Data comparisons were conducted using independent samples t-test for normally distributed continuous variables, Mann-Whitney U tests for non-normally distributed data, χ2 test or Fisher′s exact test for categorical variables. Kaplan-Meier survival analysis was employed to estimate 3-year recurrence or metastasis rate and 3-year mortality rate. Results:Operative time was 120(60) minutes (range: 45 to 440 minutes), intraoperative blood loss was 50 (80) ml (range: 10 to 2 000 ml). The median change of intraoperative mean arterial pressure was 40 (19) mmHg(1 mmHg=0.133 kPa)(range: 10 to 112 mmHg). Intraoperative blood transfusion was required in 7 cases, whereas 64 cases did not necessitate transfusion. The change in hemoglobin levels before and after surgery was (17.9±13.6) g/L (range:-16 to 53 g/L), and the median change in serum creatinine levels was 2.0 (14.5) μmol/L (range:-71.0 to 100.4 μmol/L). Postoperative fasting duration was 2.0 (1.5) days (range: 1 to 6 days), and the median hospital stay was 10.0 (7.5) days (range: 4 to 24 days). No perioperative mortality occurred in any of the patients. The non-adherent group had shorter operation time, less estimated blood loss, lower blood transfusion rate, smaller delta value of hemoglobin before and after surgery, larger delta value of creatinine before and after surgery, fewer postoperative complications, shorter postoperative fasting time, and shorter length of hospital stay than the adherent group(all P<0.05), while there was no significant difference in mean arterial pressure fluctuation between the two groups ( P>0.05). Follow-up data were available for 69 patients, with a median follow-up duration of 39 (43) months (range: 4 to 88 months). Among these patients, 40 completed the 3-year follow-up. The 3-year recurrence or metastasis rate was 10.14%, and the 3-year mortality rate was 2.90%. Conclusions:Robot-assisted laparoscopic technology via abdominal approach for resection of primary retroperitoneal tumors is safe and feasible. It can also achieve secure surgical outcome for primary retroperitoneal tumors adherent to surrounding organs or vessels, albeit with increased surgical complexity and slower postoperative recovery compared to non-adherent cases.

BACKGROUND:The most prominent transcription factor activated by tumor stem cells in osteosarcoma is EZH2,and silencing of EZH2 has been reported to inhibit osteosarcoma cell growth.Studies have confirmed that bovine serum albumin-chitosan nanoparticles are a drug delivery vector with excellent biocompatibility and biodegradability,and the albumin carrier can provide tumor-targeted drug delivery function. OBJECTIVE:To investigate the effect and mechanism of bovine serum albumin-chitosan nanoparticles loaded with EPZ6438(EZH2 inhibitor)for the treatment of osteosarcoma. METHODS:(1)Bovine serum albumin-chitosan nanoparticles loaded with and without EPZ6438 were prepared.The drug encapsulation rate and drug release rate of serum albumin-chitosan nanoparticles loaded with EPZ6438 were detected.(2)MG-63 cells were divided into four groups and added with PBS(control group),serum albumin-chitosan nanoparticle extract solution(blank nanoparticle group),EPZ6438 solution(free drug group),and serum albumin-chitosan nanoparticle extract loaded with EPZ6438(drug-loaded nanoparticle group),respectively.After 3 days of culture,cell apoptosis was detected by flow cytometry and the expression of caspase-3 mRNA was detected by RT-PCR.(3)Twelve nude mice were selected and the subcutaneous tumor-bearing mouse model was established by injecting MG-63 cell suspension under the armpit.After successful modeling,the mice were randomly divided into four groups for intervention.Normal saline(control group),serum albumin-chitosan nanoparticle solution(blank nanoparticle group),EPZ6438 solution(free drug group)and serum albumin-chitosan nanoparticle solution loaded with EPZ6438(drug-loaded nanoparticle group)were injected into tumor tissues,with three animals in each group.After 7 days of injection,the tumor volume and frozen sections of tumor tissue were observed by TUNEL staining. RESULTS AND CONCLUSION:(1)The drug encapsulation rate of the nanoparticles was about 8.8%,and the nanoparticles had a good drug release effect in pure water.The drug release amount was(34.72±1.93)μg at 24 hours,(48.58±1.10)μg at 72 hours,(49.18±1.24)μg at 120 hours,and(50.25±1.13)μg at 168 hours.The drug release reached the plateau at 120 hours,and the release rate was about 97.9%.(2)After 3 days of cell culture with MG-63,the apoptotic rate in the control group and blank nanoparticle group was lower than that in the free drug group and drug-loaded nanoparticle group(P<0.001),and the expression of caspase 3 mRNA was lower than that in the free drug group and drug-loaded nanoparticle group(P<0.000 1).(3)After 7 days of injection,the tumor volume of nude mice in the drug-loaded nanoparticle group was smaller than that in the other three groups(P<0.05),and the percentage of TUNEL-positive cells in tumor tissue was higher than that in the other three groups(P<0.000 1).(4)The results verify that serum albumin-chitosan nanoparticles loaded with EPZ6438 can inhibit the growth of osteosarcoma by inducing apoptosis of tumor cells.

Upon penetration into the host organism,the decapsulated larvae(intestinal infective stage 1 larvae)of Trichinella spiralis(T.spiralis)proceed to invade the host's small intestinal epithelial cells to continue their development,which is a critical step for T.spiralis infection and pathogenesis.Based on our prior research,the Ts-DNase Ⅱ-7 protein,which is present in the adult stage of T.spiralis,has a role in promoting the parasite's invasion of the intestinal epithelium.This is achieved by disrupting the integrity of the intestinal barrier,consequently promoting the parasitization of these cells in the host organism.Ts-DNase Ⅱ-7 can induce differential expression of various miRNAs,among which miR-142-5p has been reported to be involved in disrupting the intestinal barrier.Therefore,in this study,we investigated the mechanism by which Ts-DNase Ⅱ-7-induced miR-142-5p regulates intestinal epithelial cell barrier function by affecting target genes.The experiment was divided into Ts-DNase Ⅱ-7-treated group,miR-142-5p overexpression group(OE),miR-142-5p inhibition group(Inhibition),negative control group(NC)and control group(Control),lentiviral vectors for overexpression and suppression of miR-142-5p expression were used to infect human colorectal adenocarcinoma cells Caco-2 at an MOI of 80,and puromycin(8 mg/L)was used to screen cells to obtain cell lines that stably suppressed the expression of miR-142-5p and cell lines that miR-142-5p was overexpressed,cell transfection efficiency was assessed by fluorescence microscopy,and the relative expression of miR-142-5p was detected by RT-qPCR in each group of cells.The target gene Claudin-1(CLDN1)was predicted and validated using miR target prediction database,RT-qPCR,Western blot and dual luciferase assay.HE staining,Western blot and quantitative analysis of monolayer transmembrane electrical resistance(TEER)and FITC permeability were then applied to elucidate the mechanism of action of miR-142-5p.Caco-2 cells were lentivirally infected and successful transfection was verified by fluorescence microscopy and RT-qPCR.Dual luciferase and Western blot results showed that CLDN1 was a direct target gene of miR-142-5p(P＜0.001).Compared to the Ts-DNaseⅡ-7 alone treatment group and the OE+Ts-DNase Ⅱ-7 group,the miR-142-5p inhibited expression group and alleviated Ts-DNase Ⅱ-7-induced down-regulation of CLDN1 mRNA and protein levels(P＜0.01 and P＜0.05,respectively)and re-versed the decrease in TEER and elevated permeability(P＜0.01).Ts-DNase Ⅱ-7 up-regulates miR-142-5p expression,causing reduced CLDN1 expression and impaired intestinal barrier function in Caco-2 monolayer cells,which in turn promotes T.spiralis invasion of the intestinal epithelium to achieve further development.