ObjectiveBased on the TCM theory of "Yang transforms materials to Qi while Yin constitutes material form"， this paper explored the effects of Zuogui Wan and Yougui Wan on the molecular mechanism of mitochondrial biogenesis during the adipogenic differentiation process of rat bone marrow mesenchymal stem cells （BMSCs） by mediating peroxisome proliferator-activated receptor γ coactivator-1α （PGC-1α） and peroxisome proliferators-activated receptor γ （PPARγ）， providing theoretical support for the prevention and treatment of postmenopausal osteoporosis （PMOP） using Zuogui Wan and Yougui Wan. MethodsBMSCs were divided into a blank group， Zuogui Wan （ZGW） group， Yougui Wan （YGW） group， and Progynova group. Cell identification was performed using flow cytometry. The growth curves of BMSCs were plotted using the methylthiazolyldiphenyl-tetrazolium bromide （MTT） method， and the effects of Zuogui Wan and Yougui Wan on the proliferation of BMSCs were detected. The Oil red O staining method was used to detect lipid droplet formation. The Western blot method was used to detect the expression of adipogenesis-related factors PPARγ， CCAAT/enharcer-binding protein （C/EBP）α， C/EBPβ， lipoprotein lipase （LPL） protein， brown adipose tissue-related （BAT） proteins PGC-1α， uncoupcing protein 1 （UCP1）， PR domdin-containing protein 16 （PRDM16）， mitochondrial biogenesis-related PGC-1α， nuclear respiratory factor 1 （Nrf1）， nuclear factor E₂-related factor 2 （Nrf2）， and mitochondrial transcription factor A （TFAM）. The expression of adipogenesis-related factors PPARγ， C/EBPα， C/EBPβ， LPL genes， and the copy number of cytochrome B （CytoB mtDNA） gene was detected using real-time polymerase chain reaction （Real-time PCR）. Mitochondrial ultrastructure was detected using transmission electron microscopy. ResultsCompared with that in the blank group， the proliferation ability of BMSCs in each treatment group increased continuously as the intervention progressed， and lipid droplets significantly decreased after the drug intervention. The mRNA and protein expression levels of adipogenesis-related factors PPARγ， C/EBPα， C/EBPβ， and LPL were significantly downregulated （P<0.01）， while those of the BAT-related factors PGC-1α， UCP1， PRDM16 were significantly upregulated （P<0.01）. The number of mitochondria increased， accompanied by reduced swelling. The double membrane and cristae structure were clear， and the internal cristae rupture was reduced. The copy number of CytoB mtDNA in each treatment group was significantly increased （P<0.01）. The protein expression levels of mitochondrial biogenesis-related PGC-1α， Nrf1， Nrf2， and TFAM in each treatment group were significantly increased （P<0.01）. ConclusionBoth Zuogui Wan and Yougui Wan can prevent and treat PMOP by intervening in mitochondrial biogenesis in BMSCs through PGC-1α/PPARγ.

Objective To identify the clinical features and pathogenic variants in two unrelated families with gna-thodiaphyseal dysplasia(GDD),a rare genetic bone disorder.Methods Facial and limb deformities and skeletal morphology were observed in the probands and their family members.Peripheral blood samples(3-4 mL)were col-lected from the probands and their parents.Genomic DNA was extracted by standard phenol-chloroform method.Whole exome sequencing(WES)was performed to screen for candidate pathogenic gene variants of the probands.PCR-Sanger sequencing was used to validate the candidate pathogenic variants in the probands and their family members.The pathogenic variants responsible for GDD in the target families were determined through co-segregation of the pathogenic variants in the affected families,evolutionary conservation at the mutation sites,population allele frequency analysis and bioinformatics analysis.Results Heterozygous missense variants in the ANO5 gene were identified in both GDD probands.In family 1,the pathogenic variant was c.1 066T＞G located in the exon 11 of the ANO5 gene,while in family 2,the pathogenic variant was c.1 553G＞A located in the exon 15 of the ANO5.These two variants resulted in the substitutions of amino acid cysteine with glycine at position 356(p.Cys356Gly)and amino acid glycine with glutamic acid at position 518(p.Gly518Glu)in the ANO5 protein,respectively.Conclusions This study first identified the pathogenic variant c.1 066T＞G(p.Cys356Gly)in Chinese population,provided important evidence for prediction of disease prognosis and development of potential prenatal genetic diagnosis.

OBJECTIVE: To explore the genetic basis for three Chinese patients with McCune-Albright syndrome (MAS). METHODS: Three children who had respectively presented at Shandong Provincial Hospital in April 2019 and Peking Union Medical College Hospital in August 2020 and May 2021 were selected as the research subjects. Peripheral blood samples of the probands and their family members were taken for the extraction of genomic DNA. Potential variants were screened by whole exome sequencing (WES), and candidate variants were validated by Sanger sequencing of the patients and their family members. RESULTS: The proband from family 1 was found to harbor a heterozygous c.601C>T (p.R201C) missense variant in exon 8 of the GNAS gene, whilst the probands from families 2 and 3 were both found to harbor a heterozygous c.602G>A (p.R201H) missense variant in exon 8 of the GNAS gene. Both variants were known to be pathogenic, and all probands were found to be mosaics for the corresponding variants but with various degrees. CONSLUSION WES can effectively diagnose MAS and other somatic genetic disorders. In this study, the combined WES and Sanger sequencing have verified the degree of mosaicisms of pathogenic variants in the three MAS patients, albeit no apparent correlation was found between the degree of mosaicisms and the phenotype of patients. Above finding has provided a basis for genetic counseling and prenatal diagnosis for the affected families.
Humans ; Mutation ; Fibrous Dysplasia, Polyostotic/genetics* ; East Asian People ; Exons ; Phenotype ; Pedigree

Objective To evaluate the safety and efficacy of telescopic intramedullary rod for treatment of femur fracture or deformity correction in children with osteogenesis imperfecta,and to analysis the result of prevention recurrent fracture as well as the complication.Methods Data of patients who were treated by telescopic intramedullary rod for recurrent femur fracture or curved femoral deformity from March 2015 to December 2015 were prospectively analyzed.There were 39 boys and 26 girls.The average age of the patients was 9 years 2 months,ranging from 3 years 5 months to 13 years 4 month.All the patients had suffered from recurrent femur fractures leading to femoral deformity.The mean angulation angle was 58° (range,30°-95°).Among 69 sides,there were 21 sides of new fracture and 48 sides of deformity.Sixty-one patients were operated at one side and the other 4 patients were treated bilaterally.According to the modified Sillence classification system,there were 5 cases of type Ⅰ,17 type Ⅲ,34 type Ⅴ,3 type Ⅴ,2 type Ⅵ and 4 type ⅩⅤ.Results All the 65 patients were followed up for a mean period of 32 months (range,15-43).The average healing time of the osteotomy site or fracture site of the femur was 8 weeks (range,7-12).The patient was encouraged to begin weight bearing and walking when the Ⅹ-ray film showed healing of the osteotomy or fracture site.By the latest follow up,80％ of the patients could stand and walk independently,The incidence of femur fracture decreased significantly to the level of 0.5±0.2/year,compared to 2.7±1.8/year before operation.All the parents of the children were satisfied with the result of deformity correction.The children's self care and motion ability improved obviously after operation.During follow up,6 patients suffered from recurrent fracture of the femur by various degree,1 of them was treated by open reduction and telescopic rodding surgery,while the other 5 patients were treated conservatively because the fracture displaced or angulated minimally and 4 patients healed uneventfully while 1 patient need plate fixation to augment the axial stability.In 3 patients (1 type Ⅳ,2 type Ⅲ) the intubator failed to elongate with the growth of the distal femoral epiphysis,and in 2 patients the obturator migrated proximally which needed to be re-fixed.Low toxic infections occurred in 2 patients (type Ⅵ) which were treated successfully by removal of the rod and antibiotics.Conclusion The telescopic intramedullary rod can maintain the correction of the femur deformity and improve the quality the bone,thus prevent the recurrent fracture of the femur in children with osteogenesis imperfecta effectively.

Objective To study the method and therapeutic effect of Peter-Williams intramedullary nail in children with tibia fracture due to osteogenesis imperfecta.Methods A total of 30 children of tibia fracture due to osteogenesis imperfecta,including 17 males and 13 females in Department of the Third Orthopaedics and Osteogenesis Imperfecta Center,Tianjin Wuqing District People's Hospital from June 2015 to January 2017,were operated on the shaft of tibia with Peter-Williams intramedullary nail.Twelve cases were left tibia fracture,13 cases were right tibia fracture,and 5 cases were bilateral tibia fracture.Twenty of 35 tibias were accompanied with no tibial deformity,and 15 of 35 tibias were accompanied with different degrees of anterior arch bowing deformity.The movement and life self-care ability were evaluated according to Functional independence measure (WeeFIM) evaluation form.Results All of the children were followed up for an average of 18 months(ranging from 10 to 26 months).The average operating time was 1.2 hours(0.75-2.50 hours).The average bleeding was 60 mL(30-100 mL).The average bone healing time was 10 weeks (8-16 weeks).Patients began to exercise on bed after removing plaster,and to walk after X-ray showing bone healing.Parents of all children were satisfied with surgical operation,and the WeeFIM score was improved by (60.76 ± 15.57) scores (64-85 scores) preoperatively to (82.00 ± 7.50) scores (75-91 scores) postoperatively.There was a statistically significant difference in scores before and after the treatment(t =2.765,P =0.003).There was no infection,osteomyelitis,nerve damage,or vascular damage.Conclusions Therapy of Peter-Williams intramedullary nail in children with tibia fracture due to osteogenesis imperfect is advantaged.It causes less blood,less damage,with no intruding joint,correcting deformity and improving lower limb functions and life quality.

OBJECTIVETo explore genetic mutations and clinical features of osteogenesis imperfecta type V.

METHODSClinical record of five patients (including one familial case) with osteogenesis imperfecta type V were retrospectively analyzed. Peripheral blood samples of the patients, one family member, as well as healthy controls were collected. Mutation of IFITM5 gene was identified by PCR amplification and Sanger sequencing.

RESULTSA heterozygous mutation (c.-14C>T) in the 5-UTR of the IFITM5 gene was identified in all of the patients and one mother. The clinical findings included frequent fractures and spine and/or extremities deformities, absence of dentinogenesis imperfecta, absence of hearing impairment, and blue sclera in 1 case. Radiographic findings revealed calcification of the interosseous membrane between the radius-ulna in all cases. Hyperplastic callus formation was found in 3 cases. Four had radial-head dislocation.

CONCLUSIONA single heterozygous mutation c.-14C>T was found in the 5-UTR of the IFITM5 gene in 5 patients with osteogensis imperfecta type V. The patients showed specific radiological features including calcification of interosseous membrane, hyperplastic callus formation, and radial-head dislocation.

Adolescent ; Adult ; Child ; Child, Preschool ; Female ; Humans ; Male ; Mutation ; Osteogenesis Imperfecta ; diagnostic imaging ; genetics ; Young Adult

Objective To evaluate the efficacy of prevention of recurrent femur fracture in children with osteogenesis imperfecta.Methods Data of 35 patients (19 boys,16 girls) who were treated for femur deformity due to osteogenesis imperfecta at our hospital from Jun 2009 to Jun 2013 were retrospectively analyzed.The average age of the patients was 9 years 3 months,ranging from 3 years 6 months to 13 years 1 month.All the patients had suffered from recurrent femur fractures before which led to femoral deformity.The mean angulation angle was 55° (range,10°-90°).10 patients were operated at one side;the other 25 patients were treated bilaterally.None of them received operation before for fractures.The average incidence of femur fracture before operation was 2.4/year (range,2-4).According to the modified Sillence classification system,9 were type Ⅲ OI,14 type Ⅳ,and 12 type I.The curved deformity of the femurs were corrected by osteotomy and stabilized by telescopic intramedullary rods in 35 patients.Results All the 35 patients were followed up for a mean period of 62 months (range,36-72).The average healing time of the osteotomy site of the femur was 8.5 weeks (range,7-12).The patient was encouraged to begin weightbearing and walking when the Xray film showed healing of the osteotomy site.By the latest follow up,80％ of the patients could stand and walk independently.The incidence of femur fracture decreased significantly to the level of 0.3±0.1/year,compared with the level of 2.4± 1.3/year before operation.All the parents of the children were satisfied with the result of deformity correction.At the latest follow up,the Barthel index score increased from 71.82 (range,51-92) before operation to 92.32 (range,82-100) postoperatively,while the WeeFIM score increased from 53.32 (range,42-72) before operation to 78 (range,70-86) postoperatively.The difference of Barthel index score and WeeFIM score between that before and post operation was significant.The children's selfcare and motion ability improved obviously after operation.During follow up,22 patients suffered from recurrent fracture of the femur by various degree,7 of them were treated by open reduction and telescopic rodding surgery,while the other 15 patients were treated conservatively because the fracture displaced or angulated minimally and the fracture healed uneventfully.Conclusion The telescopic intramedullary rod can maintain the correction of the femur deformity and improve the quality of the bone,thus prevent the recurrent fracture of the femur in children with osteogenesis imperfecta effectively.

OBJECTIVETo identify deletion of large fragment in COL1A1/2 genes among patients with osteogenesis imperfecta (OI).

METHODSGenomic DNA was extracted from peripheral blood samples by a standard SDS-proteinase K-phenol/chloroform method. Multiplex ligation-dependent probe amplification (MLPA) was performed to detect gross deletions of the COL1A1/2 genes among 46 patients affected with OI, in whom no mutation was detected in the sequences of the COL1A1/2 genes.

RESULTSHeterozygous deletions of the entire COL1A1 gene and exon 20 of the COL1A2 gene were detected in probands A and B, respectively, and no gross deletion was found in the remaining 44 samples. The MLPA result of proband A was confirmed by fluorescence quantitative PCR (Q-PCR) in his family. A further conjunction point analysis through gap-PCR and DNA sequencing revealed deletion of exons 17 to 23 in the COL1A2 gene, and a 637 bp-insertion from chromosome 5 in the proband B.

CONCLUSIONTwo gross deletions have been found in the genes coding for collagen type I in the Chinese OI population, and the deletion of exons 17 to 23 in the COL1A2 gene is a novel mutation. This work not only has expanded the mutation spectrum of the COL1A1/2 gene, but also provided a support for prenatal genetic diagnosis for the families.

Adolescent ; Adult ; Child ; Child, Preschool ; Collagen Type I ; genetics ; Female ; Gene Deletion ; Humans ; Male ; Middle Aged ; Multiplex Polymerase Chain Reaction ; Osteogenesis Imperfecta ; genetics

OBJECTIVETo detect potential mutations of COL1A1 and COL1A2 genes with polymerase chain reaction-high-resolution melting analysis(PCR-HRMA) in a proband diagnosed with osteogenesis imperfecta (OI).

METHODSPeripheral blood samples were collected from the proband and members of his family as well as healthy controls. The mutations were detected by PCR-HRMA and confirmed by direct sequencing. Potential effects of the mutations were predicted using softwares including PolyPhen, SIFT and Align GVGD.

RESULTSThe PCR-HRMA has indicated mutations in exon 45 of the COL1A1 gene in the proband as well as his parents, which were presented as the difference in the melting curves between the patients and the control samples. Sequencing analysis confirmed that the proband has carried two heterozygous mutations (c.3235G>A, p.Gly1079Ser and c.3247G>A, p.Ala1083Thr) in exon 45 of the COL1A1 gene. Among them, c.3235G>A was predicted to have impeded alpha helix structure domain, which was inherited from the father who also had OI. c.3247G>A was inherited from mother who had a normal phenotype. All three softwares predicted that the c.3235G>A mutation can interfere with the function of the protein, while the c.3247G>A may have a benign effect by PolyPhen analysis.

CONCLUSIONThe study identified two mutations (c.3235G>A and c.3247G>A) occurred simultaneously in COL1A1 gene in a case. The case is the first reported in human collagen mutation database. As identified,mutation of c.3235G>A may be the major cause of the disease in the proband.

Adolescent ; Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Case-Control Studies ; Child ; Child, Preschool ; China ; Collagen Type I ; genetics ; Exons ; Female ; Humans ; Male ; Molecular Sequence Data ; Mutation ; Osteogenesis Imperfecta ; genetics ; Pedigree ; Point Mutation

Objective To evaluate specific clinical and imaging features of osteogenesis imperfecta V and to improve diagnostic accuracy of this disease. Methods Data of 15 patients with osteogenesis imperfecta type V were retrospectively analyzed for their clinical and imaging features. There were 10 males and 5 females, aged from 1 year and 30 years old (median age,12.5 years ). All 15 patients had plain X-ray, and 4 of 15 had CT. All data were analyzed by 3 experienced deputy chief doctors in OI according to OI V standard. Results X-ray:calcification of the interosseous membrane between radius-ulna was detected in all patients and calcification of the interosseous membrane between tibia-fibula was detected in 2 of 15 patients. Dislocation of the radial head was seen in 13 of 15 patients,bilateral in 9 and utilateral in 4.All patients showed restriction in the pronation and supination of the forearm and restricton in the flexion and extention of the elbow joint. Patients with dislocation of raidal head were associated with large coronoid process and olecranon of the ulna. Hyperplastic callus of the extremities were detected in 7 of 15 patients (7 at femur , 3 at humerus, 1 at tibia.In early stage, hyperplastic callus showed thin cortice, and clear boundares with the diaphysis showing and low density, irregular, mesh-like lamellation inside. In the later stage, there were dense calcification inside hyperplastic callus, and no difference in density with the diaphysis. Diaphysis surrounded by hyperplastic callus had clear boundaries with the hyperplastic callus. No cortical destruction was detected. CT:there were sparse needle-dot calcification inside hyperplastic callus, with the patterns of granular, ring-and-arch,irregular streaky mineralization. The cross section of proximal femoral shaft showed irregular shape , flat square shape and tiny medullary cavity, with no calcification on the edge of hyperplastic callus. CT value:-91 HU inside hyperplastic callus; 283 HU in femoral shaft. Conclusions Interosseous membrane between radius-ulna or tibia-fibula, hyperplastic callus ,dislocation of the radial head are specific features in osteogenesis imperfecta V. X-ray can make a definitive diagnosis of osteogenesis imperfecta V. CT scan is helpful in the differential diagnosis of osteogenesis imperfecta V from osteosarcoma.