Studies On The Molecular Pathogensis And Preimplantation Genetic Test Of Pseudoachondroplasia

Objective:Pseudoachondroplasia(PSACH)is known as an autosomal dominant disorder associated with mutations in the gene of cartilage oligomeric matrix protein(COMP)and characterized by disproportionate short stature,abnormal joints,and early onset osteoarthritis.Patients often have no abnormalities of height and weight at birth,and growth retardation begins to be observed around 18–24 months.The adult height of individuals with PSACH usually ranges from 82–130 cm.COMP is an extracellular matrix glycoprotein and a member of the pentameric subgroup of the thrombospondin gene family.COMP is a pentameric protein consisting with 5 subunits,each with an N-terminal domain,four epidermal growth factor regions,seven T3 repeat regions,and a C-terminal globular region(CTD).The T3 repeat region and the CTD region are rich in calcium-binding sites,which play a dominant role in protein folding.Of the more than200 mutations causing PSACH,85% are located in the T3 repeat region and 15% are located in the CTD region.Electron microscopy studies have found that a large amount of mutant COMP and other extracellular matrix proteins accumulate in the rough endoplasmic reticulum(ER)of chondrocytes in patients with T3 mutations,which leads to ER stress resulting in premature chondrocyte cell death and impaired endochondral bone formation.Although the mechanism of PSACH with T3 mutations is well defined,it's still unclear in PSACH caused by CTD mutations.In this study,we designed,optimized and implemented a PGT-M program for PSACH based on NGS and SNP linkage analysis to avoid the birth of children with high genetic risks.At the same time,PSACH specific menstrual blood derived mesenchymal stem cell(MenSCs)and induced pluripotent stem cell(iPS)cell line were established to overcome the shortage of human samples and the limitation of model animals.In vitro and computer simulation methods were used to explore the effect of c.1675G>A(p.E559K)mutation on the structure and function of COMP and the pathogenesis of PSACH,which will lay a foundation for the treatment of PSACH.Method:Clinical information and samples of PSACH families were collected and the genetic mutation site carried by the families was indentified.The pathogenicity of the mutation site was evulated using database and bioinformatics prediction softwares.And the mutation was classified according to the guidline published by American Society ofMedical Genetics and Genomics(ACMG).Controlled ovarian hyperstimulation was carried out on the 2nd-3rd day of menstruation.ICSI was performed after oocytes retrieval.The fertilized oocytes were cultured to the blastocyst stage and the trophoblast cells were biopsied.The preimplantation genetic test was carried out using NGS and SNP linkage analysis protocol.The menstrual blood was collected from PSACH patient and normal people on the 2nd-3rd day of menstruation.The MenSCs were isolated and characterized.PSACH-specific i PS cell line was obtained by reprogramming of MenSCs.The pluripotency of i PS cells was identified by immunofluorescence detection,relative expression of endogenous pluripotency genes and exogenous reprogramming genes and embryoid differentiation experiment.The mutation site was verified by COMP gene sequencing.The stem cells from normal control group and PSACH group were differentiated to chondrocytes in vitro.The cells were collected on day 0,7 and 14.The transcription levels of COMP,Sox9,COL2A1,COL1A2,COL1A1 and ACAN were detected by real-time PCR.The apoptosis of chondrocytes was detected by TUNEL assay.At the same time,the ultrastructures of chondrocytes were observed by electron microscopy and the expressions of ER stress-specific markers were detected by real-time PCR.The structure model of wild-type comp protein was obtained from the protein structure database.The point mutation was used to establish the three-dimensional model of c.1675G>A(p.E559K)mutant protein,and the rationality of the structure model was evaluated.The molecular dynamics simulation was carried out by GROMACS.RMSD,RMSF,RG,SASA and secondary structure were calculated and analyzed.The results were plotted by Xmtrace and R software.The study was approved by the Ethics Committee of Shengjing Hospital of China Medical University and Written informed consent was provided by all subjects.Result : The c.1675G> A(p.E559K)heterozygous mutation was identified as the pathogenic mutation site of this family by genetic experts.4 blastocysts were obtained after ICSI.The results of preimplantation genetic test showed that one embryo was recommended for transplantation.Thawed frozen blastocyst was transferred.Amniocentesis was carried out in the middle of pregnancy without abnormalities.The patient has delivered a healthy baby boy in June 2019 and the baby has been growing well.The isolated cells from menstrual blood samples could differentiate to osteoblasts,chondrocytes and adipocytes.CD44?CD73 and CD105 were positive.CD34?CD38 and CD45 were negative.A PSACH-specific iPS cell line expressing the surface markers of OCT4,SOX2 and Nanog was obtained by reprogramming of MenSCs with lentivirus.Real-time PCR indicated that the cells did not express exogenous reprogramming factors,but endogenous pluripotent stem cell markers.The embryoid differentiation experiment in vitro confirmed that the cell line could differentiate into three germ layers.DNA sequencing confirmed that the cell line carried the c.1675G>A(p.E559K)heterozygous mutation on COMP gene,which can be used for further experimental studies.The stem cells from patients and healthy people were differentiated to chondrocytes in vitro.Both cell lines formed white chondrogenic pellets,but pellets from the patient MenSCs were smaller and irregular in morphology,with a rough surface.MenSCs derived from the PSACH patient exhibited a reduced chondrocyte formation ratio and abnormal expressions of chondrocytes specific markers suggesting limited chondrocytes formation.TUNEL assay at day 21 indicated increased apoptosis of PSACH-MenSCs-derived chondrocytes compared with wild-type.Ultrastructure analysis showed that the rough ER in PSACH derived chondrocytes was slightly distended.Real-time PCR expression analysis of ER stress marker genes showed that increased expression levels of ATF6,caspase-3,CHOP and decreased bcl-2 expression in PSACH derived chondrocytes on day 21 indicating ER stress was happening.The 200 ns simulations were performed using GROMACS and the molecular dynamics simulation trajectories were analyzed and compared.Both the wild-type and c.1675G>A(p.E559K)systems reached a stable conformation in approximately 125 ns.A larger RMSD was observed in the E559 k system,suggesting higher conformational fluctuations in the c.1675G>A(p.E559K)system.The region between residue 375 and 425 on T3 repeat region of the E559 k system fluctuated more as compared with the same region of the wild-type system.c.1675G>A(p.E559K)system exhibited a higher average Rg and SASA value.The observations suggested that the stability of the mutant structure was decreased and the interactions with calcium and other molecules in mutant structure were affected resulting in misfolding of the protein,which might lead to ER stress and finally affect the survival of chondrocytes.Conclusion: For patients with pseudoachondroplasia,preimplantation genetic testing caneffectively avoid the birth of children with high genetic risks.A PSACH disease-specific MenSCs and i PS cell line were obtained for future study.The stem cell derived chondrocytes recapitulated the partial of pathological features of PSACH.Molecular dynamics simulation outcomes suggested that the interactions with calcium and other molecules in mutant structure were affected resulting in misfolding of the protein,which might lead to ER stress and finally affect the survival of chondrocytes.