Exome Sequencing Analysis Identifies Frequent Oligogenic Involvement And FLNB Variants In Adolescent Idiopathic Scoliosis

BackgroundAdolescent idiopathic scoliosis(AIS)refers to a three-dimensional deformity of the spine with lateral curvature greater than 10 ° and vertebral body rotation that begins at adolescence.The etiology of AIS is still unexplained.Epidemiological studies showed that the incidence of AIS is about 2% to 3%,and it is one of the most common spinal deformities.In the diagnosis of this disease,it needs to be diagnosed only after excluding other potential diseases such as congenital scoliosis and connective tissue diseases.It lacks specific molecular markers for early screening.In terms of treatment,patients need to be followed closely after diagnosis to see if the angle progresses.For progressive scoliosis,follow-up can assess the speed of progress of the curve and determine whether brace treatment or invasive surgery is required.For patients with non-progressive scoliosis,spinal radiography still needs to be performed during follow-up,thus patients will suffer a lot of radiation.There is currently no effective means or molecular markers to predict whether the curves will progress.Therefore,in-depth research on the mechanism of AIS pathogenesis and curves progression will help better serve clinical treatment and reduce patient pain and costs.Studies based on pedigrees and twins suggest that genetic factors play a non-negligible role in the pathogenesis of AIS.With the development of next-generation sequencing(NGS),genome wide association studies(GWAS)and whole-exome sequencing(WES)have been performed in different ethnic AIS populations.Several single nucleotide polymorphisms(SNP)or rare mutations related to the onset of AIS or the severity of the disease were also discovered.So far,susceptibility sites or common mutations in more than 20 genes have been identified as being associated with AIS.Nonetheless,there is no single gene that can explain the pathophysiology of AIS.And some problems occurred in genetic research have not been reasonably explained.Firstly,the majority of AIS patients are sporadic cases.Secondly,some candidate genes are incomplete in families with autosomal dominant inheritance.It is suggested that the genetic background of AIS needs to be explained from a new genetic perspective.Oligogenic inheritance refers to the occurrence of a disease or trait caused by the simultaneous change of several genes.One study has suggested a greater genetic load is required for males to be affected.And males would also be more likely to transmit the disease to their children and to have siblings with AIS.Another study declaimed that 15.8% of the first-degree relatives of individuals with scoliosis were affected compared with 2.4% of second-degree relatives and 1.4% of third-degree relatives.These data also support the oligogenic inheritance of AIS,since individuals who are more closely related are expected to inherit genes that are more similar.A recent exon sequencing analysis study revealed that some patients with AIS carry some mutations in extracellular matrix-related genes,and the number of mutations carried is related to some patients’ phenotype.Therefore,we hypothesized that oligogenic inheritance may be a genetic pattern in some patients with AIS.ObjectiveTo identify novel susceptible genes in Chinese Han patients with AIS and to study the nature of oligogenic inheritance in AIS.By studying the interactions between AIS-related genes,it might help to understand the mechanism of AIS.By studying the correlation between gene mutation load and clinical features such as curve progression,it could help provide clues and basis for early diagnosis and clinical intervention selection of AIS.Materials and methods1.Blood samples were collected from 40 Chinese AIS trios and exome sequencing was performed.Genes implicated in human idiopathic scoliosis,and genes whose inactivation or mutation result in an idiopathic scoliosis phenotype in mice or zebrafish were included and considered AIS-related genes.The rare-damaging variants of the above genes are screened in the exon data to study the proportion of oligogenic inheritance patterns in AIS trios;2.A total of 183 patients with sporadic AIS and 153 controls were included,and all exons were sequenced to analyze whether the frequency of oligogenic inheritance patterns of AIS-related genes was different between the two groups;3.Exon data of the Chinese Han population(222 cases)was extracted from the 1000 Genomes Project Database to comprehensively analyze the frequency of rare-damaging mutations and oligogenic genetic patterns of AIS-related genes in the Chinese population;4.Univariate and multivariate Cox regression analyses were performed to identify the prognostic values of sex,age of curve onset,curve magnitude at presentation,Risser sign,body mass index,and the number of rare damaging variants to investigate whether oligogenic inheritance can be used to predict the curve progression of AIS;5.Using the above-mentioned exome sequencing data to perform a gene-based burden test to study susceptible genes that play an important role in the pathogenesis of AIS patients,and further verify the role of candidate genes found in genetically engineered animal models in pathogenesis of AIS;6.To study whether the frequency of genes in the interactome of AIS-related genes is also significantly different in patients and controls,to further study the genetic characteristics of oligogenic inheritance in AIS;7.Immunofluorescence experiments were performed to study the effect of AIS-related FLNB gene mutation on its protein localization,interaction with Flna protein,and the formation of intracellular actin tension filaments;8.In a twin family,In Silico mutation modeling was used to evaluate the effect of gene mutations on Flnb protein structure,and co-immunoprecipitation experiments were performed to study the effect of gene mutations on the interaction of FLNB and OFD1 proteins;9.In trios 22 and 27,In Silico mutation modeling was used to evaluate the effect of gene mutations on Flnb protein structure,and co-immunoprecipitation experiments were performed to study the effect of gene mutations on the interaction of FLNB and TTC26 proteins.Results1.In the trios(n=40),42.5%(17/40)of patients carried mutations in AIS-related genes.In particular,we identified variants in more than one AIS-associated gene in 8 trios,that is,20%(8/40)of the trios fit the oligogenic pattern.We also highlighted 5 individuals harbouring rare damaging variants in FLNB;2.when combined the data of AIS patients from trios and sporadic cases,we found that 33.93% of patients carried rare damaging variants(i.e.,oligogenic inheritance)of two or more AIS-related genes,which was significantly higher than the control population(37/186 versus 3/150,odds ratio [OR],9.946;95% confidence interval [CI],3.008-32.893;p = 2.00E-06);3.The frequency of oligogenic inheritance was also significantly higher in AIS patients than in the control from the 1000 Human Genome Project(37/186 versus 8/214,OR,5.321;95% CI,2.417-11.714;p = 6.00E-06);4.Univariate survival analysis revealed that curve magnitude at first presentation(>23 vs.≤23,p=2.00E-03,hazard ratio [HR] =2.137,95% CI=1.324-3.448)and the number of rare damaging variants((>=2 vs.0 or 1,p=7.69E-11,HR=5.098,95% CI=3.121-8.328)were of prognostic significance.In the multivariate analysis,both curve magnitude at presentation(>23 vs.≤23,p=2.50E-02,HR=1.781,95% CI=1.074-2.955)and the number of rare damaging variants(>=2 vs.0 or 1,p=3.29E-07,HR=4.304,95% CI=2.458-7.537)proved to be independent prognostic factors associated with curve progression;5.Rare damaging variants in FLNB(p=6.30E-05),TTC26(p=3.07E-04),PTK7(p=5.37E-03),CNTNAP2(p=7.12E-03),FBN1(p=7.12E-03),and TTLL3(p=2.33E-02)were overrepresented in AIS patients in gene-based burden test;6.We identified rare damaging FLNB variants in 25/223(11.21%)of AIS patients,16/25(64.00%)of whom exhibited an additional variant of an AIS-associated gene(e.g.,TTC26,PTK7,TTLL3,or OFD1);7.FLNA,encoding an interaction partner of FLNB,was also significantly enriched with rare damaging variants in AIS patients(p=2.58E-03,OR=1.781,95% CI=1.074-2.955);8.Some FLNB variants(including p.M1803 L,p.S2503 G,and p.T2166M)resulted in cytoplasmic focal accumulation and some other FLNB variants(including p.R566 L,p.A2282 T,p.S2503 G,p.R199 Q,and p.R2003H)altered actin dynamics;9.In the twin family,the H2003 side chain of the mutant(R2003H)FLNB protein can form a new strong hydrogen bond interaction with E2078.Co-immunoprecipitation experiments suggest that when two genes are mutated at the same time(FLNB,p.R2003 H,OFD1,p.Y437F),the interaction between the two proteins was significantly reduced;10.In the trio 22 and 27,in the structure of mutant(A2282T)FLNB protein,intermolecular hydrogen bonds can be formed between the side chains of T2282,while in the structure of mutant(R566L)FLNB protein,L566 does not form any hydrogen bond surrounding amino acids.Co-immunoprecipitation experiments suggest that when two genes are mutated at the same time(FLNB,p.R566 L,TTC26,p.R297C;FLNB,p.A2282 T,TTC26,p.R50C),the interaction between the two proteins was enhanced;ConclusionOur study demonstrated that about one-third of AIS patients have an oligogenic inheritance pattern.More importantly,the number of rare damaging variants was recognized as an independent prognostic factor for curve progression,indicating that a higher genetic load might lead to a severe phenotype.In addition,FLNB was found to be a new susceptibility gene in AIS.It is worth noting that about two-thirds of patients carrying mutations of FLNB gene also carry mutations in other AIS-related genes.In family-based mechanism studies,the simultaneous mutation of two AIS-related genes can affect the interaction of the two proteins,suggesting that the interaction of FLNB with other genes plays an important role in the pathogenesis of AIS.