Correlation Analysis And Pathogenicity Study Of Genes Related To Lumbar Intervertebral Disc Degeneration And Congenital Scoliosis

Background:Lumbar intervertebral disc degeneration is a common lumbar spine disease,which can gradually increase with age.Previous studies have suggested that lumbar disc degeneration is a clinical disease caused by multiple factors.At present,studies have suggested that the genetic polymorphisms of genes related to lumbar intervertebral disc degeneration are widely involved in the pathogenesis of lumbar intervertebral disc degeneration.With the completion of the Human Genome Project(HGP),the Human Genome Haplotype Project(HapMap),genome-wide association research methods are widely used in genetic studies of complex diseases.Using the existing SNP database and the application of genome-wide association studies(GWAS),many genes related to lumbar intervertebral disc degeneration and susceptibility to disease sites were screened out.The gene types can be divided into four categories:1.Genes related to lumbar intervertebral disc structure;2.Genes related to lumbar intervertebral disc metabolism function;3.Genes related to lumbar intervertebral disc inflammation and apoptosis;4.Pain associated with lumbar intervertebral discs genes.The screening of these genes and pathogenic sites will help improve the accuracy of the molecular diagnosis of the disease in the future,and also lay the foundation for the future treatment at the molecular levels.At present,studies had suggested that the degeneration of lumbar intervertebral discs is related to metabolism,including the energy metabolism of lumbar intervertebral disc nucleus pulposus and leptin regulation.FTO gene is a popular research gene in the fields of obesity,type Ⅱ diabetes,cancer,etc.It is widely involved in energy metabolism activities of various tissues of the body,and FTO gene is also closely related to leptin metabolism.FTO gene may interfere with the normal metabolism of the lumbar intervertebral disc through the regulation of leptin,thus promoting the degeneration of lumbar intervertebral disc tissue.Two previous studies have reported the association of FTO gene with lumbar intervertebral disc degeneration.However,due to the limitation of the study sample size,the final verification efficiency is low.In this study,in order to improve the reliability of genetic association analysis research,we included a total of 999 Chinese Han population.This is so far the largest cohort to study the risk of FTO gene and lumbar intervertebral disc degeneration.Based on the cohort,we explored the association between polymorphisms in the FTO gene and lumbar intervertebral disc degeneration.Objects:In the present study,a large sample cohort was used to investigate association between 19 polymorphic sites of FTO gene and the risk of lumbar intervertebral disc degeneration in Chinese Han population.The study can provide candidate variants for clinical molecular diagnosis.Methods:1.Participants:The case group was enrolled from patients with lumbar and leg pain who were treated in the Orthopaedics Department of Peking Union Medical College Hospital between October 2012 and September 2017.The control group was enrolled from a normal population who had undergone a physical examination at the Peking Union Medical College Hospital Health Examination Center during the same period and who voluntarily participated in the project.According to the strict enrollment process,the cases and control populations meet the requirements of the study;2.According to the gene candidate site data provided by the International Homo sapiens Homo sapiens Project(http://www.hapmap.org)and the NCBI SNP Database(http://www.ncbi.nlm.nih.gov/SNP),applications Haploview software preferentially selected SNPs with a minimum allele frequency of 5%or more in the FTO gene.A total of 19 sites were selected;3.Whole blood DNA was extracted,and the polymorphic sites were detected using the Sequenom MassARRAY SNP platform;4.Hardy-Weinberg(HW)balance test and linkage disequilibrium(LD)analysis based on case and control group;5.Data analysis were performed by these approaches,including:allele and genotype correlation analysis,haplotype correlation analysis,and SNP-SNP interaction analysis.Results:1.According to strict enrollment processes,the case group included 502 people and the control group included 497 people.After screening preoperative MRI data,502 people in the case group had different degrees of lumbar intervertebral disc degeneration symptoms.There were 2309 segments,which suffer from lumbar intervertebral disc degeneration;2.The results of Hardy-Weinberg(HW)balance between the case group and the control group show that 17 SNPs are consistent with the HW balance test;3.In the 17 SNPs that meet the Hardy-Weinberg equilibrium,the rs1121980 polymorphism site C→T within the 95%confidence interval(1.024-1.666)demonstrated the P value was less than 0.05(0.0309).The T allele is the risk allele of the rs1121980;4.Genotype association analysis showed that AG genotype of rs2689247 and AC genotype of rs16952955 were relatively low risk factors for the development of lumbar intervertebral disc degeneration;5.Haplotype analysis demonstrated that there are two linked haplotype blocks in 17 FTO polymorphisms.6.In the analysis of the interaction between SNP and SNP,there are five polymorphic linkage sites associated with lumbar intervertebral disc degeneration.Conclusions:1.In Chinese Han population,the genetic polymorphism variation of FTO gene is associated with the risk of lumbar intervertebral disc degeneration.The T allele of rs1121980 is a risk allele of lumbar intervertebral disc degeneration,which can be used as a biomarker for the screening and prognosis of lumbar intervertebral disc degeneration diseases;2.Patients with the AG genotype of rs2689247 and the AC genotype of rs16952955 had a relatively lower risk of morbidity than the control group,suggesting that the combination of these two genotypes may serve as an indicator of disease;3.This study also found that two haplotypes and five SNP-SNP combinations were associated with lumbar intervertebral disc degeneration in cohort association analysis and could be used as candidate polymorphic loci for genetic screening and functional experiments.Biological functions need to be studied in the future.Background:Congenital Scoliosis is a congenital skeletal malformation in which abnormal vertebral development can lead to lateral curvature of the spine ≥10°.Congenital scoliosis has the characteristics of rapid progress,severe deformity,and many complications.It can cause severe paralysis in patients.It is one of the major diseases causing disability in adolescents,and brings enormous burdens to families and society.Congenital scoliosis can be clinically divided into three types:Type I is a partial or complete disorder of one or more vertebrae,Type II is a partial or complete bad segmentation of two or more vertebrae,and Type III is mixed at the same time.Congenital scoliosis spinal deformity can exist alone,but also with other systolic malformations,such as heart,kidney,reproductive system and other deformities coexist.Congenital scoliosis is a congenital disorder caused by disruption of somite development during embryonic development.Embryonic somitogenesis is mediated and regulated by multiple signaling pathways and genes,including NOTCH,WNT/H-catenin,fibroblast growth factor pathway(FGF),and TGF-β signaling pathways.TGF-β and its multiple downstream signaling networks(such as BMP,Nodal,GDF family,etc.)play an important role in somitogenesis.GDF3 is a ligand of TGF-β supramolecular family,also is an important component of Nodal signaling pathway downstream of TGF-β.GDF3 is also an inhibitor of BMP signaling pathway and can inhibit cartilage proliferation and differentiation,thereby affecting the process of osteogenesis and bone development.GDF3 gene mutations are associated with scoliosis,Klippel-Feil syndrome,and ocular malformation.Patients with mutations in the GDF3 gene have phenotypes of spinal deformity:Klippel-Feil syndrome,scoliosis of thoracolumbar spine,loss of ribs,etc.Therefore,GDF3 gene can be an important candidate gene for congenital scoliosis.At present,the genetic screening methods for congenital genetic diseases include genomic DNA sequencing,transcriptome sequencing,and proteomics,metabolomics,etc.Recently,whole exome sequencing or high-genome sequencing is used for detecting mutations form genomic scale.The whole exome sequencing has the advantages of low cost,relatively small amount of data,and easy exploration of certain pathogenic locus from the perspective of gene expression.Genes with different pathogenic locus may cause different disease phenotypes and gene function changes.The genetic pathogenic sites can be broadly classified into missense mutations,nonsense mutations,deletion mutations,insertion mutations,frameshift mutations and copy number variations.The comprehensive evaluation of these mutation sites,including the verification of genetics and molecular biology,can provide a powerful theoretical basis for further study of gene function.Objects:This study was based on the sequencing of whole exomes in a large sample of patients with congenital scoliosis.The mutantion of GDF3 gene were screened.Then we used bioinformatic approaches and performed downstream molecular biology assays to interpret pathogenicity of these mutations.Methods:1.According to the inclusion and exclusion criteria,a total of 464 patients with sporadic congenital scoliosis were enrolled in our study;2.Whole blood DNA was extracted and then whole exome sequencing was performed;3.The verification of whole exome sequencing data was used by PCR Sanger sequencing;4.We screened of GDF3 missense mutations from the patient cohort,then performing multiple alignments and comparing the data with HGMD,Clinvar databases;5.Assessment of pathogenicity and conservation of GDF3 gene variation sites:Candidate mutaions were evaluated using SIFT,PolyPhen-2,and MutationTaster.Homologous sequence comparison were performed using the ClustalW multiple sequence alignment software;6.Analysis of GDF3 gene structure:Three-dimensional modeling of GDF3 protein was performed using Swiss-model server;DUET software was used to analyze local conformational changes and energy changes;UCSF Chimera protein was used to analyze hydrogen bond formation conformation;Ligplot software was used to predict molecular hydrogen bonds,hydrophobic bonds,etc;7.Dual luciferase reporter gene assays were used to demonstrate the effect of mutation sites on GDF3 transcriptional activity;8.Western blot assays were used to demonstrate the effect of the mutation site on the function of the GDF3 protein.Results:1.Through whole exome sequencing,13 congenital scoliosis patients were found to carry five GDF3 gene mutations;2.Sanger sequencing confirmed that the mutation carried by GDF3 gene in 13 CS patients were consistent with the results of whole exon sequencing;3.According to database comparisons,it was initially determined that the new mutations were c.250 C>T and c.251 G>T.The c.644 A>G was a new mutation of which the pathogenicity was unknown.And c.635 C>T,c.751 G>A were mutation that were related to clinical phenotype but unknown pathogenicity;4.Multiple homologous sequence alignments of five GDF3 gene mutation sites were performed using ClustalW software.The R84 locus was calculated as a highly conserved amino acid site;the rest of the loci were conserved in at least five species;5.The results of dual luciferase reporter assyas showed that mutations in the c.251 G>T,c.635 C>T,and c.751 G>A could interfere with the transcriptional activation of the GDF3 gene itself,whereas c.250 C>T and c.644 A>G mutation had no significant effect on the transcriptional activity of GDF3 gene;6.Western Blot results showed that R84L,S212L,N215S,and A251T could significantly decrease the expression of GDF3 protein in mature cells compared with the normal group.In the supernatant,S212L,N215S and A251T mutations had a greater effect on the maturation of GDF3 protein.The R84C did not affect the maturation of GDF3 protein either in the supernatant or in the cells;7.DUET software predicts that the R84L site can increase thermodynamic changes in the peptide chain;UCSF Chimera software found that after the N215S mutation,the conformation of the local peptide chain changed;Ligplot software found that the 212th serine was mutated to leucine and then to form a third hydrophobic bond with threonine at position 136.Conclusions:1.In this study,subjects were sequenced through whole exome sequencing.After data analysis and clinical diagnosis procedures,a total of 13 patients with 5 rare mutations in GDF3 were enrolled in the next assays;2.Through functional verification,c.635C>T,c.251G>T,c.751G>A can affect the transcription activity of downstream SOX9 and the maturation of GDF3 protein in the molecule level;3.c.644A>G only affected the maturation of GDF3 protein and had no significant effect on transcriptional regulation.The c.250 C>T site had no significant effect on the biological function of GDF3.