Identification Of A Variant In SM0C2 Gene In The Pathogenesis Of Multiple Epiphyseal Dysplasia

BackgroundMultiple epiphyseal dysplasia(MED,MIM 132400)is a kind of skeletal dysplasia and chondrodysplasia.Eight different genes,including COMP,COL9A1,COL9A2,COL9A3,MATN3,DTDST,CANT1 and COL2A1 gene,have been identified as MED pathogenic genes so far,which inherited in either autosomal dominant or autosomal recessive pattern.MED is characterized by mild to moderate shortness of stature and early-onset arthritis,especially in hip and knee joints.Radiographic findings include delayed and irregular ossification of the epiphyses in multiple joints.The epiphyseal plate,also known as growth plate,is a hyaline cartilage plate that sits between the diaphysis and the epiphysis at each end of a long bone.The proliferation and differentiation of chondrocytes in the growth plates drives the longitudinal growth of long bones.The abnormalities in proliferation,differentiation,apoptosis and ossification lead to delay in development of long bones and cause MED.Except chondrocytes,there is a large number of cartilage extracellular matrix which consists of collagen fibers and extrafibrillar cartilage matrix in the growth plates.The mutual interaction between fibrillar and extrafibrillar matrix is mediated by the molecular network,which is composed of collagen and non-collagen molecules,such as collagen IX,matrilins and COMP.The unbalance of the molecular network plays important role in the pathogenic mechanism of skeletal dysplasia and chondrodysplasia,such as MED.Bone morphogenetic protein(BMP)is a secreted protein with various biological functions and the only growth factor to induce mesenchymal cells to differentiate into bone tissues.BMP signal pathway is composed of BMP ligands,receptors and SMADs.It is involved in cell proliferation,differentiation and apoptosis and plays important role in the development of skeletal system.Therefore,the abnormalities in BMP signaling pathway led to various skeletal diseases.SMOC2(SPARC-related modular calcium-binding protein 2)gene,the full length of which is 232Kb,locates at 6q27 and consists of 13 exons.The protein encoded by SMOC2 gene is a secretory protein,which was originally identified in the cartilage extracellular matrix.The extracellular calcium-binding domain(EC domain)in SMOC2 and other SMOC proteins has many important biological functions.SMOC proteins bind to collagen,heparan sulfate proteoglycan(HSPG)and integrin through the EC domain.Previous studies showed that SMOC could mediate BMP signaling pathway by interacting with HSPG.In addition,SMOC2 also takes part in embryogenesis,cell cycle,cell adhesion,fibrosis,calcification,angiogenesis,tumor progression and so on.Objectives1.To identify the pathogenic gene of a pedigree with MED;2.To verify the pathogenicity of the variant in SMOC2 gene using knock-in mouse model;3.To investigate the molecular mechanism of epiphyseal dysplasia caused by the variant in SMOC2 gene.Methods1.Genomic DNA extracted from the family members with MED was used to perform STR linkage analysis and whole-exome sequencing to find out the pathogenic gene.2.Mutant Smoc2(mSmoc2)knock-in mouse model was constructed and the body lengths and long bone lengths of all studied mice were measured by X-ray and vernier caliper.Micro-CT was used to detect the morphology,bone mineral density and bone volume of knee joints.Transmission electron microscopy(TEM)was used to observe the abnormal ultrastructure in chondrocytes.After the skeleton tissue paraffin sections being prepared,HE staining,safranine and Fast Green double dyeing,IHC,IF or TUNEL were performed to analyze the morphology of growth plates,the abilities of proliferation,differentiation and apoptosis of chondrocytes and the expression levels of the biomarkers.3.HEK293 cells with stable expression of wild-type or mutant-type SMOC2 gene were established by recombinant lentiviruses.BMP signaling pathway and MAPK signaling pathway were detected by WB and the mutual interactions between matrix proteins and wild-type or mutant SMOC2 were detected by Co-IP,SPBA and DPLA.Results1.Identification of SMOC2 gene as the pathogenic gene in a pedigree with MEDA pedigree with MED was found in Linyi,Shandong province.The male/female patient ratio in this pedigree was close to 1,which suggested an autosomal dominant inheritance pattern.All the patients had a history of pain in joints and a shorter stature than average.The epiphyses of some patients proceeded heterogeneous ossification and osteoporosis.We found no linkage between the known MED pathogenic genes and the MED phenotype in this pedigree by STR linkage analysis.To find the pathogenic gene,we performed whole-exome sequencing on IV9 and found 9 variants.Only NM 001166412.2:c.1076T>G,p.Leu359Arg mutation in exon 11 of SMOC2 gene was confirmed complete cosegregation with the MED phenotype in this pedigree and conservation in evolution.Therefore,we initially identified SMOC2 gene as the pathogenic gene in this pedigree.2.Verification of the pathogenicity of the variant in SMOC2 gene by mSmoc2 knock-in mouse model.The mSmoc2 knock-in mouse model was generated by ES cell gene targeting.First,we measured the lengths and weights of the littermates at P30 and P63.The average body lengths and the average weights of mSmoc2 knock-in mice were obviously reduced as compared with wild-type mice at both P30 and P63 group.Then,the lengths of tibia and femur were measured at P30 and P63.The average lengths of femur and tibia from mSmoc2 knock-in mice were noticeably reduced as compared with wild-type mice.To address the delay on the long bone development in mSmoc2 knock-in mice,we performed pathological analysis on tibial growth plates of all studied mice at P21 and P63.The growth plates of mice at P21 were analyzed considering the growth plates of mice at P30 began to narrow.The average ratio of the proliferative zones to the growth plates was reduced and that of hypertrophic zones to the growth plates was expanded in mSmoc2 knock-in mice in both two groups.In the tibial growth plates of wild-type mice at P21,the chondrocytes in the proliferative zones were closely aligned,well organized and arranged along the long axis of the tibia.The chondrocytes in the proliferative zones of the tibial growth plates of mSmoc2 knock-in mice were disorganized and failed to arranged into a column.Some hypocellular areas could be observed in the proliferative zones and hyaline cartilage of proximal tibia.These changes are much more obviously in P63 mice.To determine the mechanism of the changes in mSmoc2 knock-in mice tibial growth plates,we determined the relative levels of proliferation,hypertrophy and apoptosis in growth plates and primary chondrocytes.We counted the number of PCNA-positive or EdU-positive cells as compared with hematoxylin-stained or DAPI-stained cells in proliferative and hypertrophic zones of tibial growth plates or primary chondrocytes from all studied mice.We found the ratios of PCNA-positive or EdU-positive cells in mSmoc2 knock-in mice are reduced as compared with wild-type mice.Ihh,Runx2 and Collagen X are markers of the prehypertrophic and hypertrophic zone.To test chondrocyte differentiation in the growth plates,we detected the expression of Ihh,Runx2 and Collagen X by IHC and real-time PCR.The results of IHC showed that the expression area of Ihh was wider in mSmoc2 knock-in mice than wild-type mice.The expression of Collagen X and Runx2 was higher in mSmoc2 knock-in mice than wild-type mice.The results of real-time PCR showed the up-regulation of the relative mRNA expression of Collagen X and Ihh in primary chondrocytes of mSmoc2 knock-in mice.The relative apoptosis was calculated by comparing the number of apoptotic chondrocytes to number of DAPI-stained chondrocytes in the hypertrophic zones.As expected,mSmoc2 knock-in mice showed less apoptotic chondrocytes in the hypertrophic zone as compared with wild-type mice.In order to find whether mSmoc2 knock-in mice got arthritis,we tested one-year-old littermates and nine-month-old littermates who experienced treadmill experiments for 3 months by micro-CT.The results showed that the values of BMD and BV/TV of heterozygous mSmoc2 knock-in mice were slight lower than those of wild-type mice.Safranine and fast green double dyeing showed that the erosions on the surface of knee joints in heterozygous mSmoc2 knock-in mice were not more than wild-type mice.3.Study on the pathogenic mechanism of epiphyseal dysplasia caused by the variant in SMOC2 gene.We tested the BMP signal pathway in vivo and in vitro because BMP played important role in skeleton development and disordered BMP signaling could lead to severe skeletal diseases.The phosphorylation level of SMAD1/5/9 was lower in wild-type or mutant SMOC2 gene stable expression HEK293 cells than that in control group.In vivo,the phosphorylation level of Smad1/5/9 in chondrocytes of mSmoc2 knock-in mice was lower than that of wild-type mice.That meant overexpressed wild-type or mutant SMOC2 gene could inhibit BMP signaling pathway.To elucidate the exact mechanism of overexpressed wild-type or mutant SMOC2 gene inhibiting BMP signaling pathway,we tested MAPK signaling pathway and found no differences.Following that,we found the phosphorylation level of SMAD1/5/9 could be rescued by activated BMP receptors.Then,Co-IP and SPBA showed wild-type and mutant SMOC2 protein could bind to BMPRIB.However,the ability of mutant SMOC2 bind to HSPG or COL9A1 was declined.Therefore,overexpressed SMOC2 could inhibited BMP signaling by excessively binding to BMPRIB and inhibit the activating of BMP receptors.The ability of mutant SMOC2 binding to matrix molecular declined and much mutant SMOC2 became dissociative.Therefore,mutant SMOC2 bound to BMPRIB excessively and inhibited the activating of BMP receptors.SignificanceIn this study,we identified SMOC2 gene as a novel MED pathogenic gene.The variant in EC dominant of SMOC2 gene could inhibit BMP signaling pathway and led to disorganized chondrocytes with low density,reduced proliferative zone and expanded hypertrophic zone in growth plates and dysregulated differentiation and apoptosis.The results above revealed the pathogenic mechanism of MED and provide new insights in genetic diagnosis and treatment of MED.