The source of recombinant collagen is clean, and it has the advantages of flexible sequence design, high yield and high purity, so it has a wide application prospect as biomaterials in tissue engineering and other fields. However, how to promote the cross-linking of recombinant collagen molecules and make them form a more stable spatial structure is the difficulty to be overcome in the design of recombinant collagen nanomaterials. Unnatural amino acid O-(2-bromoethyl)-tyrosine was incorporated into collagen by two-plasmid expression system. The results showed that high-purity collagen incorporated with unnatural amino acid could be obtained by induction with final concentration of 0.5 mmol/L IPTG and 0.06% arabinose at 25 °C for 24 hours. The intermolecular cross-linking through thioether bond was formed between collagen molecule incorporated with unnatural amino acid and collagen molecule with cysteine mutation in pH 9.0 NH4HCO3 buffer, which formed aggregates with the largest molecular size up to 1 micrometre. The results pave the way for the design of recombinant collagen biomaterials.
Amino Acids ; Biocompatible Materials ; Collagen/genetics* ; Sulfides

Humans ; Nephritis, Hereditary/genetics* ; Astigmatism ; Mutation ; Collagen Type IV/genetics*

OBJECTIVEThe aim of this study was to investigate the expression of collagen in the process of masseter muscle reattachment to the cortical and cancellous bones of mandible.

METHODSA total of nine adult goats were used in the study. One was the control. The other eight were treated with bilateral detachment of the masseter muscles. The biopsies of bone and muscle were taken at 2, 4, 8 and 12 weeks after the operation. The characteristics of the healing muscle-bone interfaces were examined using immunohistochemical techniques.

RESULTSImmunohistochemical analysis illustrated that the locations of collagen type I, II and III were different during the healing process, but similar in the cortical and cancellous bones.

CONCLUSIONThis study demonstrates that the distribution of the three types of collagens at the muscle-bone interfaces is associated with time, but not related with their locations.

Animals ; Collagen ; biosynthesis ; genetics ; Collagen Type I ; biosynthesis ; genetics ; Collagen Type II ; biosynthesis ; genetics ; Collagen Type III ; biosynthesis ; genetics ; Female ; Goats ; Male ; Mandible ; metabolism ; pathology ; surgery ; Masseter Muscle ; metabolism ; pathology ; surgery ; Wound Healing ; physiology

Collagen Type IV/genetics* ; Genetic Testing ; Humans ; Nephritis, Hereditary/therapy*

OBJECTIVE: To explore the genetic basis of two fetuses with an osteogenesis imperfecta (OI) phenotype. METHODS: Two fetuses diagnosed at the Affiliated Hospital of Weifang Medical College respectively on June 11, 2021 and October 16, 2021 were selected as the study subjects. Clinical data of the fetuses were collected. Amniotic fluid samples of the fetuses and peripheral blood samples of their pedigree members were collected for the extraction of genomic DNA. Whole exome sequencing (WES) and Sanger sequencing were carried out to identify the candidate variants. Minigene splicing reporter analysis was used to validate the variant which may affect the pre-mRNA splicing. RESULTS: For fetus 1, ultrasonography at 17+6 weeks of gestation had revealed shortening of bilateral humerus and femurs by more than two weeks, in addition with multiple fractures and angular deformities of long bones. WES revealed that fetus 1 had harbored a heterozygous c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in exon 49 of the COL1A1 gene (NM_000088.4). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), it was classified as a pathogenic variant (PVS1+PS2+PM2_Supporting) for disrupting the downstream open reading frame resulting in premature translational termination, being de novo in origin, and lacking records in the population and disease databases.For fetus 2, ultrasonography at 23 weeks of gestation also revealed shortening of bilateral humerus and femurs by one and four weeks, respectively, in addition with bending of bilateral femurs, tibias and fibulas. Fetus 2 had harbored a heterozygous c.1557+3A>G variant in intron 26 of the COL1A2 gene (NM_000089.4). Minigene experiment showed that it has induced skipping of exon 26 from the COL1A2 mRNA transcript, resulting in an in-frame deletion (c.1504_1557del) of the COL1A2 mRNA transcript. The variant was inherited from its father and had been previously reported in a family with OI type 4. It was therefore classified as a pathogenic variant (PS3+PM1+PM2_Supporting+PP3+PP5). CONCLUSION The c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in the COL1A1 gene and c.1557+3A>G variant in the COL1A2 gene probably underlay the disease in the two fetuses. Above findings not only have enriched the mutational spectrum of OI, but also shed light on the correlation between its genotype and phenotype and provided a basis for genetic counseling and prenatal diagnosis for the affected pedigrees.
Pregnancy ; Female ; Humans ; Osteogenesis Imperfecta/genetics* ; Collagen Type I, alpha 1 Chain ; Collagen Type I/genetics* ; Mutation ; Fetus

OBJECTIVE: To explore the genetic basis for a pedigree affected with Alport syndrome. METHODS: Next generation sequencing and Sanger sequencing was applied to detect potential variants of the COL4A3, COL4A4 and COL4A5 genes among members from the pedigree and 100 unrelated healthy controls. RESULTS: The proband and his twin brother were found to carry two novel variants, namely c.4953G>A and c.4623C>A, of the COL4A4 gene, which were respectively inherited from her father and mother. The same variants were not detected among the 100 healthy controls and medical literature. Based on the guidelines of the American College of Medical Genetics and Genomics, both the c.4953G>A and c.4623C>A variants were predicted to be pathogenic (PVS1+PM2_supporting+PP1). CONCLUSION The c.4953G>A and c.4623C>A variants of the COLA4A gene probably underlay the Alport syndrome in this pedigree. Above finding has enriched the spectrum of COLA4A gene variants.
Autoantigens/genetics* ; Child ; Collagen Type IV/genetics* ; Female ; Humans ; Male ; Mutation ; Nephritis, Hereditary/genetics* ; Pedigree

OBJECTIVETo characterize the role of Smads proteins in alpha 2 (I) collagen (COL1A2) gene expression induced by bone morphogenetic protein-2 (BMP-2) in odontoblast cell line MDPC-23.

METHODSEndogenous Smad protein expression was determined by immunocytochemistry. Smads function and their role in COL1A2 gene expression were investigated in cotransfection experiments using promoter-luciferase reporter gene construct.

RESULTSMDPC-23 cells expressed Smad1, Smad5 and Smad6. BMP-2 promoted the activation of COL1A2 promoter reporter construct. Transient overexpression of Smad1 or Smad5 was enhanced, while overexpression of Smad6 inhibited BMP-2-induced COL1A2 promoter activity. BMP-2 inducibility could be blocked by overexpression of Smad1 or Smad5 dominant negative mutant.

CONCLUSIONSSmad signaling is functioning and appears to be involved in BMP-2-induced COL1A2 collagen transcription in MDPC-23. Smad signaling may play an important role in odontoblast differentiation and dentin extracellular matrix formation mediated by BMP-2.

Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; genetics ; Cell Line ; Collagen ; genetics ; Collagen Type I ; Mice ; Odontoblasts ; cytology ; metabolism ; Smad Proteins ; physiology ; Transforming Growth Factor beta ; genetics

OBJECTIVETo study a family affected with osteogenesis imperfecta for potential mutations in COL1A1 gene.

METHODSClinical data of an affected family was collected. Potential mutation of the COL1A1 gene was screened using polymerase chain reaction and direct sequencing. Suspected mutation was detected in 20 unaffected relatives and 200 unrelated healthy controls.

RESULTSAnalysis of RNA splicing has revealed a c.3208G/A mutation, which created a new splice sites and led to a frameshift mutation. The same mutation was not detected in the unaffected relatives or the 200 healthy controls.

CONCLUSIONMutations of the COL1A1 gene are one of the major causes of osteogenesis imperfecta in Chinese population. Our finding has enriched the mutation spectrum of type I collagen genes.

Adult ; Child, Preschool ; Collagen Type I ; genetics ; Female ; Humans ; Male ; Mutation ; Osteogenesis Imperfecta ; genetics ; RNA Splicing

Collagen Type IV ; genetics ; Female ; Fetal Development ; Humans ; Nephritis, Hereditary ; diagnosis ; genetics ; Pregnancy ; Prenatal Diagnosis ; methods

Hereditary angiopathy with nephropathy, aneurysms and muscle cramps (HANAC) syndrome is an autosomal dominant genetic disease caused by COL4A1 gene mutation, with major clinical manifestations of white matter lesion, aneurysm, retinal artery tortuosity, polycystic kidney, microscopic hematuria and muscle cramps. This article reports the clinical features and genotype of one toddler with HANAC syndrome caused by COL4A1 gene mutation. The boy, aged 1 year and 8 months, had an insidious onset, with the clinical manifestations of pyrexia and convulsion, white matter lesions in the periventricular region and the centrum semiovale on both sides, softening lesions beside the left basal ganglia, retinal arteriosclerosis, microscopic hematuria and muscle cramps. Whole exome sequencing revealed a pathogenic de novo heterozygous mutation in the COL4A1 gene, (NM_001845) c.4150+1(IVS46)G>T, and therefore, the boy was diagnosed with HANAC syndrome. COL4A1 gene mutation detection should be performed for children with unexplained white matter lesion, stroke, hematuria, polycystic kidney, cataract and retinal artery tortuosity or families with related history.
Aneurysm ; Collagen Type IV ; genetics ; Genotype ; Humans ; Infant ; Male ; Muscle Cramp ; genetics ; Mutation ; Syndrome