OBJECTIVE: To summarize the clinical features and spectrum of genetic variants in 12 patients with Loeys-Dietz syndrome (LDS), and to explore the correlation between the type of genetic variants and clinical phenotypes. METHODS: Twelve patients suspected for LDS at Beijing Anzhen Hospital Affiliated to Capital Medical University from January 2015 to January 2022 were selected as the study subjects. Clinical data of the patients were collected. Genomic DNA was extracted from peripheral blood samples and subjected to genetic testing. Pathogenicity of candidate variants was analyzed. RESULTS: The clinical phenotypes of the 12 patients have mainly included cardiovascular, musculoskeletal, craniofacial, skin, ocular and other systemic signs. Four patients (patients 5-1, 5-2, 6, 7) have carried heterozygous missense variants of the TGFBR1 gene, 5 patients (patients 1-1, 1-2, 2, 3, 4) have carried heterozygous variants of the TGFBR2 gene, and 2 patients (patients 8-1, 8-2) had carried heterozygous frameshift variants of the TGFB3 gene. One patient (patient 9) had carried a heterozygous missense variant of the SMAD3 gene. Among these, TGFBR1 c.603T>G (p.1201M) and TGFB3 c.536delA (p.H179FS35) had not been reported previously. CONCLUSION Variants of the TGFBR1, TGFBR2, SMAD3, TGFB2, TGFB3 and SMAD2 genes are mainly associated with LDS. The severity of the disease phenotype caused by the same variant may vary, whilst the clinical phenotype caused by different variant sites may be specific.
Humans ; Loeys-Dietz Syndrome/genetics* ; Receptor, Transforming Growth Factor-beta Type I/genetics* ; Receptor, Transforming Growth Factor-beta Type II/genetics* ; Transforming Growth Factor beta3 ; Face

OBJECTIVE: To detect the expression of cartilage oligomeric matrix protein (COMP) in the synovial chondromatosis of the temporomandibular joint (TMJSC), and to discuss the possible interactions between COMP, transforming growth factor (TGF)-β3, TGF-β1 and bone morphogenetic protein-2 (BMP-2) in the development of this neoplastic disease. METHODS: Patients in Peking University School and Hospital of Stomatology from January 2011 to February 2020 were selected, who had complete medical records, TMJSC was verified histologically after operation. The expressions of COMP, TGF-β3, TGF-β1 and BMP-2 in the TMJSC of the temporomandibular joint were detected by immunohistochemistry and quantitative real-time PCR (RT-PCR) at the protein level and mRNA level respectively, compared with the normal synovial tissue of temporomandibular joint. The histological morphology, protein expression and distribution of TMJSC tissues were observed microscopically, and the positive staining proteins were counted and scored. SPSS 22.0 statistical software was used to analyze the expression differences between the related proteins in TMJSC tissue and the normal synovial tissue of temporomandibular joint and to compare their differences. P < 0.05 indicated that the difference was statistically significant. RESULTS: Immunohistochemical results showed that the positive expression of COMP in TMJSC tissues was mostly found in synovial tissues and chondrocytes adjacent to synovial tissues, and the difference was statistically significant, compared with the normal temporomandibular joint synovial tissues. The positive expression of COMP was significantly different between recurrent TMJSC and non-recurrent ones. The positive expressions of TGF-β3, TGF-β1 and BMP-2 were higher than the normal synovial tissue, and were also mostly found in the synovial cells and adjacent chondrocytes, which was further confirmed by Western blot. According to the RT-PCR results, the expressions of COMP, TGF-β3, TGF-β1 and BMP-2 in TMJSC were higher than those in the normal synovial tissue. CONCLUSION The expression of COMP in TMJSC of temporomandibular joint increased significantly, compared with the normal synovial tissue. There may be interactions between COMP and cytokines related to the proliferation and differentiation, like TGF-β3, TGF-β1 and BMP-2, which may play a potential role in the pathogenesis of TMJSC.
Cartilage Oligomeric Matrix Protein/genetics* ; Chondromatosis, Synovial ; Humans ; Synovial Membrane ; Temporomandibular Joint ; Transforming Growth Factor beta3

Animals ; Cell Line ; Collagen Type I ; biosynthesis ; Hepatic Stellate Cells ; metabolism ; RNA, Messenger ; genetics ; Rats ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Transforming Growth Factor beta3 ; genetics ; metabolism

PURPOSE: To observe the changes of gait behavior and the expression of wound healing factors of transforming growth factor-β1 (TGF-β1), TGF-β3 and cAMP response element binding protein-1 (CREB-1) during the healing of Achilles tendon in a rat model, and to investigate whether gait analysis can be used to evaluate the tendon healing. METHODS: Achilles tendon of 40 healthy male Sprague-Dawley rats were transected and sutured to establish the Achilles tendon injury (ATI) model. They were randomly divided into 4 groups based on the observational time point at 1, 2, 4 and 6 weeks after injury (n = 10 for each group). Before modeling, 9 rats were randomly selected for CatWalk gait analysis, which contained step cycle, single stance time and average speed. Data were recorded as the normal controls. After then, ATI models were established in the left hind limbs of the all 40 rats (ATI group), while the right hind limbs were only cut and sutured without injury of the Achilles tendon (sham operation group). At 1, 2, 4 and 6 weeks after injury, the gait behavior of the corresponding group of rats (n = 9) as observed and recorded by CatWalk platform. After then, the rats were sacrificed and Achilles tendon of both limbs was harvested. The tendon healing was observed by gross anatomy and histological examination, and the protein and mRNA expression of TGF-β1, TGF-β3, CREB-1 were observed by immunohistochemistry and qPCR. The results of tendon gross grading were analyzed by Wilcoxon rank sum test, and other data were analyzed by one-way analysis of variance among multiple groups. RESULTS: Compared with normal controls, all gait indexes (step cycle, single stance time and average speed) were greatly affected following ATI, which however improved with time. The step cycle was significantly lower at 1, 2 and 4 weeks after ATI (compared with normal controls, all p < 0.05), but almost returned to the normal level at 6 weeks ((0.694 ± 0.102) vs. (0.503 ± 0.094) s, p > 0.05). The single stance time of the ATI group was significantly shorter at 1 and 2 weeks after operation ((0.078 ± 0.010) s at 1 week, (0.078 ± 0.020) s at 2 weeks, all p < 0.001) and revealed no significant difference at 4 weeks (p = 0.120). The average speed of ATI group at 1, 2, 4, 6 weeks was significantly lower than that in the normal control group (all p < 0.001). Gross observation showed that the grade of local scar adhesion in ATI group increased significantly at 2, 4 and 6 weeks, compared with the sham operation group (all p < 0.001). Extensive adhesion was formed at 6 weeks after ATI. The results of HE staining showed that the number of fibroblast increased gradually and arranged more orderly in ATI group at 1, 2 and 4 weeks (all p < 0.001), and decreased at 6 weeks, but it was still significantly higher than that of the sham operation group (p < 0.001). Immunohistochemistry showed that the positive expression of TGF-β1, TGF-β3, CREB-1 in ATI group was higher than that in the sham operation group at 4 time points (all p < 0.05), which reached the peak at 2 weeks after operation and decreased at 4 weeks (p = 0.002, p < 0.001, p = 0.041, respectively). The results of qPCR suggested that the mRNA expression of TGF-β1, TGF-β3, CREB-1 in ATI group was higher than that in the sham operation group at all-time points (all p < 0.05), which reached the peak at 2 weeks after operation, decreased at 4 weeks, and significantly decreased at 6 weeks (all p < 0.001). CONCLUSION Gait behavior indexes are associated with Achilles tendon healing. The study gives an insight of TGF-β1, TGF-β3, CREB-1 changes in the coursing of Achilles tendon healing and these cytokines may be able to be used to regulate the Achilles tendon healing.
Achilles Tendon ; Animals ; CREB-Binding Protein ; Gait Analysis ; Male ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1/genetics* ; Transforming Growth Factor beta3 ; Wound Healing

OBJECTIVETo construct and identify the stable expression system of NIH3T3 fibroblast with eukaryotic expression vector of human transforming growth factor beta3 (pcDNA3.1 (-)/TGFbeta3). So as to investigate the proliferation of NIH3T3 fibroblasts transfected with hTGFbeta3 gene stably.

METHODSThe stable transfection of NIH3T3 fibroblasts with recombinant plasmid expressing hTGFbeta3 was established by using LipofectamineTM2000 and G418 selection. The mRNA and protein expression of TGFbeta3 were detected by the RT-PCR and Western blot method, respectively. Microscope and MTT were adopted to examine the proliferation of the stable expression system of fibroblasts with hTGFbeta3.

RESULTSAfter G418 selection, RT-PCR and Western blot analysis, 7 out of 10 cell lines transfected with pcDNA3.1 (-)/TGFbeta3 expressed with very high level of TGFbeta3, as compared with vector control transfectants that showed no expression, and compared with the other cell lines that expressed relatively low level. The stable transfection of NIH3T3 fibroblasts growth slowed down significantly (P < 0.05).

CONCLUSIONThe stable expression system of NIH3T3 fibroblast with hTGFbeta3 were constructed successfully. The TGFbeta3 gene could inhibit the proliferation of NIH3T3 fibroblasts in vitro.

Animals ; Cell Proliferation ; Fibroblasts ; metabolism ; Humans ; Mice ; NIH 3T3 Cells ; Plasmids ; Transfection ; Transforming Growth Factor beta3 ; genetics ; metabolism

OBJECTIVETo construct adeno-associated virus (AAV) expression system for transforming growth factor beta3 (TGFbeta3 ) and detect its biological effect on proteoglycan synthesis of the earlier and later dedifferentiated rabbit lumbar disc nucleus pulpous (NP) cells, which was compared with that of adenovirus (AV) expression system for TGFbeta1.

METHODSTGFbeta3 gene was obtained using PCR. Its upstream contained restriction enzyme site Kpn I, and its downstream contained restriction enzyme site Sal I. Using the restriction enzyme sites of PCR product of TGFbeta3 and the corresponding multiple cloning site (MCS) in plasmid AAV, TGFbeta3 was subcloned into AAV. The recombinant plasmid AAV-TGFbeta3 was transfected into H293 cells with Lipofectamine 2000, and the expression of TGFbeta3 gene was detected using immunofluorescent analysis. After AAV-TGFbeta3 virus particle with infectious activity was packaged, TGFbeta3 expression in NP cells was detected by immunoblotting, and its biological effect on proteoglycan synthesis was detected by antonopulos method and compared with that of AV-TGFbeta1 in the earlier and later dedifferentiated NP cells.

RESULTSFor the earlier dedifferentiated NP cells, AAV-TGFbeta3 slowly and stably enhanced proteoglycan synthesis, but AV-TGFbeta1 rapidly and transiently enhanced its synthesis. For the later dedifferentiated NP cells, AAV-TGFbeta3 stably enhanced proteoglycan synthesis, but AV-TGFbeta1 inhibited its synthesis.

CONCLUSIONAAV expression system can mediate TGFbeta3 gene to be expressed stably, and AAV-TGFbeta3 can enhance proteoglycan synthesis of the earlier and later dedifferentiated NP cells.

Animals ; Cell Line ; DNA, Recombinant ; genetics ; Dependovirus ; genetics ; metabolism ; Female ; Humans ; Intervertebral Disc ; cytology ; Placenta ; cytology ; Plasmids ; genetics ; Polymerase Chain Reaction ; Pregnancy ; Proteoglycans ; biosynthesis ; Rabbits ; Transforming Growth Factor beta1 ; genetics ; Transforming Growth Factor beta3 ; genetics ; Viral Proteins ; biosynthesis

OBJECTIVETo investigate the effects of interactions among environmental factors, bone morphogenetic protein-4 (BMP4) and transforming growth factor beta-3 (TGF-β(3)) polymorphisms on nonsyndromic cleft lip and cleft palate (NSCLP).

METHODSThe data of environmental exposures were collected with questionnaires. Genotypes were determined with techniques of polymerase chain reaction-restriction fragment length polymorphism. Interactions between genes, environmental factors and NSCLP were analyzed using multifactor dimensionality reduction (MDR) method. The interactions were validated by logistic regression analysis.

RESULTSThere was no correlation between three single nucleotide polymorphism (SNP) associated with NSCLP. The developmental accident of NSCLP had higher risk in the interaction between BMP4 T538C, maternal passive smoking and infection in first trimester pregnancy, as well as in the interaction of six factors between TGF-β(3) G15572-, maternal passive smoking, infections, multivitamin supplement in the first trimester pregnancy, paternal smoking and high risk drinking before realizing pregnancy than in other interactions of environmental factors. The results could be validated by logistic regression analysis.

CONCLUSIONSThe NSCLP is induced by the interactions between genes and environmental risk factors.

Bone Morphogenetic Protein 4 ; genetics ; Brain ; abnormalities ; Cleft Lip ; genetics ; Cleft Palate ; genetics ; Female ; Genotype ; Humans ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Pregnancy ; Risk Factors ; Smoking ; Transforming Growth Factor beta ; Transforming Growth Factor beta3 ; genetics

A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells (ADSCs). The recombinant pIRES-EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFP. LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups: experimental group (MMP group), negative control group (no MMP) and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen (ColII) was detected by using Alcian blue staining and immunohistochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and sequencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differentiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.
Adipose Tissue ; metabolism ; Animals ; Chondrogenesis ; genetics ; Female ; Male ; Rabbits ; Recombinant Fusion Proteins ; genetics ; metabolism ; Stem Cells ; metabolism ; Transforming Growth Factor beta3 ; genetics ; metabolism

The present study was aimed to explore the relationship of transforming growth factor (TGF) beta3 gene SfaNI polymorphism (rs3917201 locus) and non-syndromic cleft lip with or without cleft palate (NSCL/P) in people of the Uygur's Nationality and Han's in Xinjiang, China. TGFbeta3 gene fragment including SfaNI was amplified and purified as the template of the primer extension reaction thenafter. The single base extension reaction was carried out u sing SNP specific extension primer. The products were purified and analyzed by MALDI-TOF. The test showed that there were not significantly different frequencies of AA, AG, GG genotypes and alleles between the whole NSCL/P group and the whole control group (P > 0.05). Within the Uygurs or Hans, the frequencies of genotypes between the whole NSCL/P group and the whole control group were not significantly different (P > 0.05). The distributions of the A, G alleles between the NSCL/P group and the control group were not significantly different within the Uygurs (P > 0.05), but significant different within the Hans (P < 0.05). In all the NSCL/P patients, frequencies of genotypes and alleles were not significantly different between Uygurs and Hans (P > 0.05), and not significantly different (P > 0.05) either between Uygurs and Hans in all the healthy persons. The results proved that TGFbeta3 gene SfaNI polymorphism may not be related to NSCL/P in Xinjiang Uygur people, while the occurrence of NSCL/P in Han population may be related to frequency of the A and G allele of SfaNI polymorphism.
Alleles ; China ; Cleft Lip ; ethnology ; genetics ; Cleft Palate ; ethnology ; genetics ; Ethnic Groups ; Gene Frequency ; Genotype ; Humans ; Polymorphism, Single Nucleotide ; Transforming Growth Factor beta3 ; genetics

OBJECTIVETo investigate possible association between the single nucleotide polymorphisms (SNPs) of transforming growth factor beta3 (TGF-beta3) gene and essential hypertension (EH) in Chinese.

METHODSThe promoter region, exons, as well as part of the introns of TGF-beta3 gene were sequenced by a fluorescent labeling automatic sequencing method to detect and characterize the SNPs in 24 DNA samples from a Chinese population. Then we conducted a case-control study using 396 patients with hypertension (case) and 214 nomortensive subjects (control). The three SNPs including Thr63Asn, SS5608219 and SS5608220 were genotyped by PCR-RFLP or real-time allele-specific PCR in subjects studied.

RESULTSSeven SNPs in the exons, introns and 3'untranslated region (3'UTR) of TGF-beta3 gene were identified. Among them, 2 SNPs were found to be novel genetic variants and one of the two located in the exon 1 and produced substitution of amino acid. However, no differences were found between hypertensives and nomortensives in genotype distribution and allele frequency of SS5608219, Thr63Asn or SS5608220 polymorphisms.

CONCLUSIONSTwo novel SNPs of TGF-beta3 gene were identified in Chinese. One of them produces a threonine to asparagines substitution in codon 63 (Thr63Asn). But no association was found between TGF-beta3 gene polymorphisms and EH in Chinese.

Aged ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Exons ; Female ; Genotype ; Humans ; Hypertension ; genetics ; Middle Aged ; Polymorphism, Single Nucleotide ; Transforming Growth Factor beta3 ; genetics