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.

In situ bioimaging is a powerful tool for directly observing the localization, expression, and interactions of nucleic acids or protein targets within cells, providing essential insights into cell function and disease mechanisms. In recent years, the CRISPR/Cas9 system, a revolutionary gene-editing tool, has been applied to develop efficient in situ imaging techniques. This paper reviews recent CRISPR/Cas9-based imaging methods utilizing Cas9 protein, engineered single-guide RNA (sgRNA), and coupled fluorescent tags, and compares their application in living and fixed cells. It focuses on the specificity, signal amplification efficiency, and multi-modal imaging capabilities of these methods, with further discussion based on current research, aiming to offer a comprehensive overview of CRISPR/Cas9-based in situ bioimaging techniques, with some valuable reference and guidance for research in related fields.

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.

ObjectiveTo explore the possible mechanism of the Yiqi Jiedu formula （YQ） in treating ischemic stroke （IS） from the perspective of the microbial-gut-brain axis （MGBA）. MethodRats were randomly divided into five groups， with six in each group， including sham surgery group， model group， and low， medium， and high dose YQ groups （1， 5， and 25 mg·kg^-1）. Except for the sham surgery group， all other groups were established with a middle cerebral artery occlusion （MCAO） model using the thread occlusion method. The success of modeling was determined through neurobehavioral scoring， and the protective effect of YQ on IS was evaluated. Then， the changes in gut microbiota before and after MCAO modeling and YQ administration were compared using 16S rDNA sequencing technology， and the possible biological pathways related to the effect of this formula were analyzed. The expression of inflammatory factors such as interleukin-6 （IL-6）， interleukin-17A （IL-17A）， and interleukin-10 （IL-10） in serum was detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the expression of tight junction proteins ZO-1 and Occludin in brain and intestinal tissue， and hematoxylin-eosin staining （HE） was used to observe pathological changes in the cerebral cortex and colon， so as to validate the possible mechanism of action. ResultYQ significantly improved the neurobehavioral score of MCAO rats （P<0.01） and played a good regulatory role in intestinal microbial disorders caused by enriched pathogens and opportunistic pathogens during the acute phase. Among them， significantly changed microorganisms include Morgentia， Escherichia Shigella， Adlercreutzia， and Androbacter. Bioinformatics analysis found that these bacteria may be related to the regulation of inflammation in the brain. Compared with the blank group， the detection of inflammatory factors in the serum of IS model rats showed an increase in inflammatory factors IL-6 and IL-17A （P<0.01） and a decrease in the content of anti-inflammatory factor IL-10 （P<0.01）. Compared with the model group， the content of inflammatory factors IL-6 and IL-17A in the serum of the treatment group decreased （P<0.05）， and that of anti-inflammatory factor IL-10 increased （P<0.01）. The expression results of barrier proteins ZO-1 and Occludin in brain and intestinal tissue showed that the expression levels of both decreased in IS model rats （P<0.05）， while the expression levels of both increased in the treatment group （P<0.05）. ConclusionAcute cerebral ischemia can lead to an imbalance of intestinal microbiota and damage to the intestinal barrier， and it can increase intestinal permeability. YQ can regulate intestinal microbiota imbalance caused by ischemia， inhibit systemic inflammatory response， and improve the disruption of the gut-blood brain barrier， preventing secondary cascade damage to brain tissue caused by inflammation. The MGBA may be an important mechanism against the IS.

Abstract Biomarkers could improve the understanding of the causes of obesity and its association with chronic diseases for people. The purpose of the review is to summarize recent advances in transcriptomic, proteomic, and metabolomic phenotypic biomarkers of obesity in order to deepen the understanding of the etiology of obesity and its metabolic consequences. In the precise prevention and control of childhood obesity, different groups of biomarkers can improve the accuracy of the word "obesity" and help early detection of specific biomarkers with risk characteristics, so as to realize the transformation of childhood obesity from a one size fits all prevention and control strategy to a personalized prevention and control plan during the development of obesity.

Objective:To explore the changes of T follicular regulatory (T FR) cells/T follicular helper (T FH) cells and their related cytokines in peripheral blood of children with dust mite allergic asthma, and their clinical significance. Methods:A total of 25 children with acute dust mite allergic asthma (the asthma group) in Affiliated Hospital of Jiangnan University from January to December 2021 and 16 age- and sex-matched healthy volunteers (the healthy control group) at the same time were enrolled in the retrospective study.The percentages of peripheral T FR cells and T FH cells of the 2 groups were measured by flow cytometry.The plasma levels of cytokines[interleukin (IL)-10, IL-21] of the 2 groups were assessed by the flow cytometric microsphere-based array technology.The specific IgE (sIgE) levels of dust mites in 2 groups were detected by fluorescence enzyme immunoassay.The percentage of eosinophils in peripheral blood detected by blood cell analyzer.Data between groups were compared by t-test, and the correlation among indicators was analyzed by Spearman rank correlation analysis. Results:The asthma group had evident T FR cells/T FH cells immune imbalance.Compared with the healthy control group, the asthma group had a significantly lower T FR cells level[(0.11±0.03)% vs.(0.13±0.03)%], a significantly higher T FH cells level[(5.07±1.75)% vs.(3.80 ± 1.60)%], and a significantly lower ratio of T FR cells /T FH cells(0.02±0.01 vs.0.05±0.03) ( t=2.29, 2.30, 3.71; all P<0.05). Compared with the healthy control group, the asthma group had a significantly higher IL-21 level[(547.85±195.13) ng/L vs.(404.94±110.41) ng/L], and a significantly lower IL-10 level[(10.18±3.49) ng/L vs.(14.79±5.65) ng/L] ( t=2.60, 3.15; all P<0.05). The ratio of T FR cells/T FH cells in asthma group was negatively correlated with sIgE ( r=-0.444 2, P=0.026 1), but not related to the eosinophil percentage ( r=-0.135 2, P=0.519 3). Conclusions:Children with dust mite allergic asthma suffer from T FR cells/T FH cells subset imbalance.The imbalanced T FR cells, T FH cells and their related cytokines IL-10 and IL-21 may play a role in regulating the production of asthma sIgE.

Apolipoprotein A-I (ApoA-I), the main protein component of high-density lipoprotein (HDL), plays a pivotal role in reverse cholesterol transport (RCT). Previous studies indicated a reduction of serum ApoA-I levels in various types of cancer, suggesting ApoA-I as a potential cancer biomarker. Herein, ectopically overexpressed ApoA-I in MDA-MB-231 breast cancer cells was observed to have antitumor effects, inhibiting cell proliferation and migration. Subsequent studies on the mechanism of expression regulation revealed that estradiol (E2)/estrogen receptor α (ERα) signaling activates

Objective:To compare the prevalence of non-alcoholic fatty liver disease (NAFLD) in women with or without polycystic ovary syndrome (PCOS), and to evaluate association between PCOS and NAFLD.Methods:A cross-sectional study was performed including 122 PCOS patients (PCOS group) and 107 age, and body mass index (BMI)-matched women (control group). Anthropometric parameters, liver enzyme, lipid profile, glucose and insulin levels, sex hormones and hepatic ultrasonography were measured in all subjects. The clinical features, laboratory parameters and prevalence of NAFLD were compared between PCOS group and control group. The related factors were evaluated between PCOS and NAFLD, finally the role of insulin resistance (IR) and hyperandrogenism (HA) was analysed.Results:Women with PCOS had a significantly higher prevalence of NAFLD than those without PCOS (62.6% vs. 76.2%, P=0.025). Logistic regression found that HOMA-IR and FAI were associated with NAFLD in PCOS women ( OR=1.686, 95% CI=1.279-2.223; OR=1.167, 95% CI=1.039-1.311), however, there was no significant correlation between FAI and NAFLD after adjustment for HOMA-IR ( P>0.05). Conclusion:NAFLD is more prevalent in women with PCOS than in those without. Insulin resistance and HA drive risk of NAFLD in young female with PCOS. IR may be an independent risk factor for NAFLD, and the association between HA and NAFLD is not independent but is mediated by IR.