The endothelial glycocalyx（EG）is a villous polyglycoprotein complex that covers the apical membrane of endothelial cells，situated between the blood vessel wall and the bloodstream.Due to its unique physiological position，the EG plays a crucial role in vascular permeability，mechanical conduction，coagulation，inflammation，and other processes.EG injury can cause endothelial barrier dysfunction，increase vascular permeability，and promote inflammation and thrombosis.Recent studies have revealed that the involvement of EG in inflammatory responses may contribute significantly to the pathogenesis of certain rheumatic and immune diseases as well as multi-system inflammatory syndrome，Kawasaki disease，rheumatoid arthritis，systemic lupus erythematosus and antiphospholipid syndrome，etc.This review paper aims to provide an overview of the structure and function of EG while highlighting its research progress in diagnosing and treating rheumatic and immune diseases.

OBJECTIVE: To explore the genetic basis for 7 families with gonadal mosaicism for Duchenne muscular dystrophy (DMD). METHODS: For the 7 families presented at the CITIC Xiangya Reproductive and Genetic Hospital from September 2014 to March 2022, clinical data were collected. Preimplantation genetic testing for monogenic disorders (PGT-M) was carried out for the mother of the proband from family 6. Peripheral venous blood samples of the probands, their mothers and other patients from the families, amniotic fluid samples from families 1 ~ 4 and biopsied cells of embryos cultured in vitro from family 6 were collected for the extraction of genomic DNA. Multiplex ligation-dependent probe amplification (MLPA) was carried out for the DMD gene, and short tandem repeat (STR)/single nucleotide polymorphism (SNP)-based haplotypes were constructed for the probands, other patients, fetuses and embryos. RESULTS: The results of MLPA showed that the probands and the fetuses/probands' brothers in families 1 ~ 4, 5, 7 had carried the same DMD gene variants, whilst the probands' mothers were all normal. The proband in family 6 carried the same DMD gene variant with only 1 embryo (9 in total) cultured in vitro, and the DMD gene of the proband's mother and the fetus obtained through the PGT-M were normal. STR-based haplotype analysis showed that the probands and the fetuses/probands' brothers in families 1 ~ 3 and 5 have inherited the same maternal X chromosome. SNP-based haplotype analysis showed that the proband from family 6 has inherited the same maternal X chromosome with only 1 embryo (9 in total) cultured in vitro. The fetuses in families 1 and 6 (via PGT-M) were both confirmed to be healthy by follow up, whilst the mothers from families 2 and 3 had chosen induced labor. CONCLUSION Haplotype analysis based on STR/SNP is an effective method for judging gonad mosaicism. Gonad mosaicisms should be suspected for women who have given births to children with DMD gene variants but with a normal peripheral blood genotype. Prenatal diagnosis and reproductive intervention may be adapted to reduce the births of further affected children in such families.
Male ; Pregnancy ; Child ; Humans ; Female ; Muscular Dystrophy, Duchenne/diagnosis* ; Dystrophin/genetics* ; Mosaicism ; Exons ; Prenatal Diagnosis/methods* ; Nucleotides

OBJECTIVE: To summarize the genetic characteristics of 671 Chinese pedigrees affected with Duchenne/Becker muscular dystrophy (DMD/BMD). METHODS: Clinical data of the pedigrees were collected. Multiplex PCR, multiple ligation dependent probe amplification (MLPA), next generation sequencing (NGS), Sanger sequencing and long read sequencing were used to detect the variant of DMD gene in the probands and their mothers, and prenatal diagnosis was provided for high risk pregnant women. RESULTS: Among 178 pedigrees analyzed by multiplex PCR, 44 variants of the DMD gene were detected, with the genetic diagnosis attained in 110 pedigrees. Among 493 pedigrees analyzed by MLPA in combination with NGS or Sanger sequencing, 294 pathogenic/possible pathogenic variants were identified, among which 45 were unreported previously, and the genetic diagnosis attained in 484 pedigrees. Structural variants of the DMD gene were identified in two pedigrees by long-read sequencing. Among 444 probands, 341 have inherited the DMD gene variant from their mothers (76.8%). Among 390 women with a high-risk, 339 have opted to have natural pregnancy and 51 chose preimplantation genetic testing for monogenetic disease (PGT-M). The detection rate of neonatal patients and carriers following natural pregnancy was significantly higher than that for PGT-M. CONCLUSION Combined application of MLPA, NGS, Sanger sequencing and long-read sequencing is an effective strategy to detect DMD/BMD. PGT-M can effectively reduce the risk of fetuses. Above finding has expanded the spectrum of DMD gene variants and provided a basis for reproductive intervention for pregnancies with a high risk for DMD/BMD.
China ; Dystrophin/genetics* ; Exons ; Female ; Genetic Testing ; Humans ; Infant, Newborn ; Multiplex Polymerase Chain Reaction ; Muscular Dystrophy, Duchenne/genetics* ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis

OBJECTIVE: To analyze the (CGG)n repeats of FMR1 gene among patients with unexplained mental retardation. METHODS: For 201 patients with unexplained mental retardation, the (CGG)n repeats of the FMR1 gene were analyzed by PCR and FragilEase RESULTS: For the 201 patients with unexplained mental retardation, 15 were identified with full mutations of the FMR1 gene. The prevalence of fragile X syndrome (FXS) in patients with unexplained mental retardation was determined as 7.5% (15/201). Prenatal diagnosis was provided for 6 pregnant women with pre- or full mutations. Analysis revealed that women with mental retardation and full FMR1 mutations exhibited a skewed XCI pattern with primary expression of the X chromosome carrying the mutant allele. CONCLUSION FXS has a high incidence among patients with unexplained mental retardation. Analysis of FMR1 gene (CGG)n repeats in patients with unexplained mental retardation can facilitate genetic counseling and prenatal diagnosis for their families. FMR1 gene (CGG)n repeats screening should be recommended for patients with unexplained mental retardation.
Female ; Fragile X Mental Retardation Protein/genetics* ; Fragile X Syndrome/genetics* ; Humans ; Intellectual Disability/genetics* ; Mutation ; Pregnancy ; Prenatal Diagnosis

OBJECTIVETo screen for FOXL2 gene mutations in 6 patients with blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES), and explore their genotype-phenotype correlation.

METHODSPeripheral venous blood samples were collected from the patients for the extraction of genomic DNA. PCR and Sanger sequencing were employed to analyze the coding region and flanking sequences of the FOXL2 gene. Pathogenicity of the identified mutations was verified through literature review and bioinformatic analysis.

RESULTSA heterozygous c.672_701dup30 mutation was found in the probands from the two familial cases, while three heterozygous mutations (two were novel), namely c.462_468del (p.Pro156Argfs*113), c.251T to A (p.Ile84Asn) and c.988_989insG (p.Ala330Glyfs*204) were detected in the three sporadic cases. Literature review and bioinformatic analysis indicated that all these mutations are pathogenic.

CONCLUSIONIdentification of causative mutations in the BPES patients has provided a basis for genetic counseling and reproductive guidance. The novel mutations have enriched the mutation spectrum of the FOXL2 gene.

Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Blepharophimosis ; diagnosis ; genetics ; China ; Female ; Forkhead Box Protein L2 ; Forkhead Transcription Factors ; genetics ; Genetic Association Studies ; Humans ; Male ; Molecular Sequence Data ; Pedigree ; Skin Abnormalities ; diagnosis ; genetics ; Urogenital Abnormalities ; diagnosis ; genetics ; Young Adult

Objective To explore mutation diagnosis and discuss the pathogenic and clinical characteristics of neurofibromatosis type 1 (NF1).Methods DNA sequencing combined with denaturing highperformance liquid chromatography (DHPLC) method was used to diagnose patients and parents.Results A new nonsense mutations c.503C ＞ G(p.S168 *) was identified.Conclusions NF1 is a rare autosomal dominant genetic disease.Most of them are caused by new mutations.Genetic diagnosis of sporadic cases is very important for treatment and the future generations.

Objective To explore the effects of Na+ H-exchanger 1(NHE1) knockdown on ATP binding cassette transporter A1 (ABCA1) protein expression levels and cholesterol efflux in the hypoxic RAW264.7 cells.Methods The RAW264.7 cells were infected with lentiviral vectors expressing shRNA specific for NHE1(siNHE1) or scramble RNA (siNC).The expression of NHE1 at mRNA or protein level was detected by qRT-PCR and Western blotting respectively in the infected cells after 24 h in a hypoxia condition.In the meantime,the methods of SNARF-1,Fluo-4 NW andSuc-LLVY-aminoluciferin were employed to determine NHE1 activity,intracellular Ca2+ ([Ca2+]i) and calpain activity,respectively.Furthermore,ABCA1 protein levels were detected by Western blotting in the 24 h hypoxic cells.In parallel,the intracellular cholesterol content and cholesterol efflux were analyzed by the methods of combined enzymatic HLPC and 3 H-cholesterol.Results The hypoxia condition versus the normoxia condition up-regulated NHE1 mRNA and protein expression level and activity by 2.48 folds,1.28 folds and 61.96％ (all P＜0.05),and increased[Ca2+]i and calpain activity by 4.51 folds and 2.41 folds(all P＜0.05).Whereas the NHE1 mRNA and protein expression and activity at the presence of hypoxia were inhibited by siNHE1 with the inhibition ratio of 84.95％,60.75％ and 66.44％,respectively (all P＜0.05)and[Ca2+]i and calpain activity were reduced by 59.23％ and 54.66％ (P＜0.05).Furthermore,the ABCA1 protein level was 61.67％ lower in the hypoxic cells than in the normoxic cells (P＜0.05),and siNHE1 was increased by 56.52％ after treatment of Hypoxia.Hypoxia elevated intracellular total cholesterol and cholesterol ester by 74.57 ％ and 101.81％ (all P＜0.05).Treatment with siNHE1 in the hypoxia condition can reduce total cholesterol and cholesterol ester by 34.24 ％ 及 49.66 ％ (all P＜0.05).Hypoxia reduced the cholesterol efflux by 34.79％(P＜0.05),which were partially reversed by siNHE1.Conclusions NHE1 might play an important role in hypoxia-induced ABCA1 protein attenuation and reverse cholesterol transport dysfunction through[Ca2+]i/calpain pathway.

OBJECTIVETo study the prediction performance evaluation with five kinds of bioinformatics software (SIFT, PolyPhen2, MutationTaster, Provean, MutationAssessor).

METHODSFrom own database for genetic mutations collected over the past five years, Chinese literature database, Human Gene Mutation Database, and dbSNP, 121 missense mutations confirmed by functional studies, and 121 missense mutations suspected to be pathogenic by pedigree analysis were used as positive gold standard, while 242 missense mutations with minor allele frequency (MAF)>5% in dominant hereditary diseases were used as negative gold standard. The selected mutations were predicted with the five software. Based on the results, the performance of the five software was evaluated for their sensitivity, specificity, positive predict value, false positive rate, negative predict value, false negative rate, false discovery rate, accuracy, and receiver operating characteristic curve (ROC).

RESULTSIn terms of sensitivity, negative predictive value and false negative rate, the rank was MutationTaster, PolyPhen2, Provean, SIFT, and MutationAssessor. For specificity and false positive rate, the rank was MutationTaster, Provean, MutationAssessor, SIFT, and PolyPhen2. For positive predict value and false discovery rate, the rank was MutationTaster, Provean, MutationAssessor, PolyPhen2, and SIFT. For area under the ROC curve (AUC) and accuracy, the rank was MutationTaster, Provean, PolyPhen2, MutationAssessor, and SIFT.

CONCLUSIONThe prediction performance of software may be different when using different parameters. Among the five software, MutationTaster has the best prediction performance.

Computational Biology ; methods ; DNA Mutational Analysis ; methods ; Gene Frequency ; Humans ; Mutation, Missense ; genetics ; Polymorphism, Single Nucleotide ; genetics ; Reproducibility of Results ; Software

OBJECTIVETo detect SCN4A gene mutation in a pedigree with paramyotonia congenita in order to facilitate genetic counseling and assisted reproduction.

METHODSClinical data of the family was collected. DNA was extracted from peripheral blood samples. Potential mutation of the SCN4A gene was screened using PCR-Sanger sequencing. Potential mutation was detected in 3 affected relatives, 4 unaffected relatives and 100 unrelated healthy controls. Bioinformatics software was used to predict the effect of mutation on the protein function and conservation of the sequence at the mutation site across various species.

RESULTSA novel missense mutation c.4427T>C (p.Met1476Thr) was detected in the exon 24 of the SCN4A gene in the proband and other 3 affected relatives, but not in 4 unaffected relatives and 100 unrelated controls. Bioinformatic analysis indicated that the codon is highly conserved across various species, and that the mutation has caused damage to the structure and function of SCN4A protein.

CONCLUSIONThe c.4427 T>C (p.Met1476Thr) mutation of the SCN4A gene may contribute to the paramyotonia congenita. Detection of SCN4A gene mutation is an effective method for the diagnosis of paramyotonic congenita.

Adult ; Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Exons ; Female ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation, Missense ; Myotonic Disorders ; genetics ; NAV1.4 Voltage-Gated Sodium Channel ; genetics ; Pedigree ; Point Mutation ; Sequence Alignment

OBJECTIVETo discuss the genetic diagnosis of congenital adrenal hyperplasia (CAH) and investigate the resource of gene mutations in CAH.

METHODEnzymatic methods with restriction endonucleases that specifically recognized the mutation sites were used to detect the gene mutations in patients with CAH and their relatives. Polymerase chain reaction and direct sequencing were used to identify the mutations in 21-hydroxylase gene, and short tandem repeat (STR) typing was used to determine the sources of the mutations.

RESULTSOne CAH patient had two known mutations in 21-hydroxylase gene, namely the I2g and I172N mutations. The former mutation was inherited from the biological mother and the latter was not inherited.

CONCLUSIONThe 9 common mutations of CAH are also the hotspots for new mutations.

Adrenal Hyperplasia, Congenital ; diagnosis ; genetics ; Gene Deletion ; Genotype ; Humans ; Mutation ; Point Mutation ; Polymerase Chain Reaction ; Steroid 21-Hydroxylase ; genetics