Genetic Etiology Study Of Congenital Pseudarthrosis

Background:Congenital pseudarthrosis(CP)is characterized by deossification of the long bone progressed to bowing,pathological fractures,or pseudarthrosis in early life with intractable non-healing.The onset of tibia(congenital pseudarthrosis of tibia,CPT)is the most common,sporadically seem in fibula,ulna,radius and clavicle.Currently the pathogenesis of CP is still not well understood,and it is still a puzzle and intractable disease in pediatric orthopedics.Surgery is the main treatment method but it requires multiple operations and the long-term outcome is not satisfied.55.4%-84%of CP meet the clinical diagnostic criteria of neurofibromatosis 1(NF1,OMIM:162200),which is an autosomal dominant disorder caused by the haploinsufficiency of the NF1gene.These patients named as NF1-CP.Previous studies have mostly focused on NF1-CP,but rarely on non NF1-CP.Therefore,further exploration of the genetic basis of CP will provide a new basis for understanding the etiology and pathogenesis of CP.Methods:CP patients admitted to Hunan children’s Hospital with peripheral blood or periosteum samples were collected.According to the NF1 clinical diagnostic criteria,the patients were divided into two groups:NF1-CP and non NF1-CP.131 sporadic patients and five patients with family history(three from three monozygotic twins with discordant CP,and two from one family)obtained ethical and informed consent were included for genetic testing.First,55 NF1-CP and 20 non NF1-CP patients enrolled in 2017 were analyzed genotype-phenotype characteristics.Probands’peripheral blood was performed whole exome sequencing(WES),supplemented by trio’s Sanger sequencing and the multiplex ligation-dependent probe amplification(MLPA)searching for NF1 exon deletions/duplications.Whole genome sequencing(WGS)using peripheral blood was used to identify the germline pathogenic single nucleotide variants(SNVs)and structural variations(SVs)in the two monozygotic twins with NF1,especially in the individuals uniquely with CP.Second,somatic variants in periosteum were further screened.The periosteum of 43 non NF1-CP and six random NF1-CP were applied WES.The identified variants were verified by MLPA,ultra high amplicon sequencing(UHAS)and Sanger sequencing in family members.the genotypic spectrum of the NF1 gene and the phenotypic spectrum of CP were summarized basing on the germline and somatic variants.Third,multi-omics analysis was performed to identify other disease-causing genes for further investigate the genetic basis.Eighteen collected periosteal samples(6 NF1~-,10 NF1~+,and 2 controls)were performed full length transcriptome sequencing via Nanopore.By integrating the rare variants from WES,the published m RNA differentially expressed genes from periosteal osteoprogenitor cells and differentially expressed proteins from the periosteal mass spectrometry,multi-omics analysis was applied to explore the new disease related genes and pathways.q PCR,Western Blot(WB)and immunohistochemistry were performed to verify the expression of the prioritized genes.Results:First,genotype-phenotype analysis of 75 patients found that80%of the NF1-CP and 0%of the non NF1-CP were detected NF1pathogenic germline variants.In the two identical twins with NF1 but discordant CP,the pathogenic germline variants were identified in the NF1 gene but not in other genes from the CP affected individuals specifically.Nearly 50%of the germline pathogenic variants in the NF1gene were inherited,and the consistency of CP phenotype is less than 5%among the parent-child pairs or enzygotic siblings with the same NF1variant.In addition,one NF1-CP was found a pathogenic variant in RASA1 but not in NF1;non NF1-CP was found one pathogenic variant in ALPL related to hypophosphatesteremia and one in fibrinogen FBN1 each.The phenotypic analysis revealed that the onset of CP was almost within one year old.Significantly higher proportion of non NF1-CP were found cystic lesion and mucoid denaturation than NF1-CP.NF1-CP with NF1pathogenic variants presented“sucked candy”narrowing of the bone in morphology.Second,9/43(21%)of non NF1-CP patients had NF1 somatic variants in the periosteum,including one non NF1-CP from the monozygotic twin without NF1.In addition,5/6 NF1-CP patients were found somatic biallelic inactivation in periostea.Combined with the previously reported cases,approximately 67%of the NF1-CP had NF1somatic biallelic inactivation(also known as"second hit")in the pseudarthrosis tissue.Additionally,12%of the non NF1-CP without NF1variants were found variants in MET,NRAS and SOS1 from tumor-related RAS signaling pathway,as well as variants in ALPL and COL5A1.Third,of the 68 samples without NF1 mutations found,135 rare and damaging variants involved genes with 84%were found in only one sample,and 80%of the samples had multiple rare and damaging variants.Combining the differentially expressed genes in transcriptome and proteome,the related genes were mainly enriched in biological processes related to bone development,and in signaling pathways of ECM-receptor interaction,PI3K-Akt or RAS/MAPK.Additionally,about 1%of the THBS4 transcript were found 3’UTR extension(891 bp)in enhancer/promoter region.THBS4 was significantly higher expressed in CP(particularly NF1-CP)than the controls in the bulk m RNA and protein from periostea,as well as the m RNA derived from the CP periosteal progenitor cells.The expression level was significantly positively correlated with the number of alternative polyadenylation sites(APA).WGS of periosteum did not reveal any heterozygous or mosaic variants of THBS4(variant allele frequency>10%)surrounding 10 kb range.Immunohistochemistry showed that the coding thrombospondin 4 was mainly localized in the collagen fibers,vascular endothelium,and chondrocytes.Conclusion:(1)This study found that NF1 gene was the main disease-causing gene of CP,including germline variants and somatic variants.The overall positive rate of the NF1 variation was 50%in the sequenced 136 CP patients.80%of the NF1-CP were detected NF1germline variants,and 67%of the NF1-CP were detected NF1 somatic‘Second hit’.Approximately 21%of the non NF1-CP were found NF1somatic variation in the periosteum.Parent-child pairs and enzygotic siblings carrying the same variants of the NF1 gene exhibited less than5%consistency with the CP phenotype.(2)12%of the 68 CP without NF1 variants identified were found pathogenic variants in RAS signaling pathway,phosphatase or fiber related MET,NRAS,SOS1,RASA1 and ALPL genes that may be associated with CP.The results provided a basis for precisely diagnosis and individualized intervention in clinic.(3)This study first found that THBS4 was significantly over expressed in the CP periosteum and its derived osteoprogenitor cells.There were 1%of THBS4 transcripts prolonged in the enhancer/promotor region near 3’UTR.This suggests that THBS4 might be related to CP pathogenesis.