## 4 Discussion  
In this study, we report an enrichment of predicted damaging variants in cytoskeletal and extracellular matrix (ECM) functional categories within adolescent idiopathic scoliosis (AIS) families. Additionally, this cohort showed minimal overlap in specific genes across families. Our results support and add to the growing evidence that AIS is a highly polygenic disorder [7; 8; 28; 79; 80] in which multiple variants of variable effect size, potentially in combination with epigenetic and environmental phenomenon [81], contribute to the disease phenotype. The ECM is a dynamic molecular network of proteoglycans, glycoproteins, minerals, and related proteins that plays a critical role within musculoskeletal tissues [82]. The ECM provides critical structural networks and scaffolding to tissues, and contributes to cellular signaling, growth, and repair [83]. Studies of genes encoding the \"matrisome\", the proteins composing and relating to the ECM, have identified dozens of causal ECM mutations for connective tissue and musculoskeletal disorders [83]. A polygenic burden of rare variants in musculoskeletal collagen genes was linked to AIS risk in a large case-control cohort [28], and our group previously showed mild variant enrichments in our pilot exome sequencing study of five AIS families [46]. Candidate ECM genes including _HSPG2_[29] and _FBN2_[30] have also been linked to AIS in specific cohorts. Several collagen genes (_COL8A2_, _COL4A3_, _COL6A5_, _COL7A1_, _COL7A1_, _COL21A1_, _COL9A2_, _COL9A3_, _COL4A6_) and perlecan (_HSPG2_) appeared in our families (Supplementary Files S3). Candidate studies of ECM genes in AIS [84; 85; 63; 86] were launched, in part, because scoliosis is a common phenotype of monogenic connective tissue disorders including Marfan and Ehlers-Danlos syndromes (EDS) [72; 74]. _TNXB_ (tenascin XB) variants, which have been associated with classic EDS, were found in two families in our current cohort as well as one family in our previous study [46]. The _TNXB_ variant within Family G (TNXB:NM_019105:exon24:c.C8192G:p.P2731R) appeared in the Leiden Open Variation Database for EDS but was predicted as \"benign\" ([[https://databases.lovd.nl/shared/variants/0000313458#00021614](https://databases.lovd.nl/shared/variants/0000313458#00021614)]([https://databases.lovd.nl/shared/variants/0000313458#00021614](https://databases.lovd.nl/shared/variants/0000313458#00021614)), accessed on 4 May 2021). Functional data will be required to determine the pathogenicity of these variants. In a recent review, [PERSON] et al. proposed that genetic variants affecting the cartilage matrisome, specifically of the intravertebral disc, may be involved in a subset of AIS cases [33]. Our results provide support to polygenic variants in ECM genes as a contributing factor to AIS in some family lineages. The cytoskeleton comprises actin, microtubule, and intermediate filament networks and is responsible for a multitude of functions including molecular transport, cellular stability, molecular signaling, cell migration, and cell division [87]. The cytoskeleton and ECM are intricately connected within musculoskeletal tissues and play important roles in mechanotransduction, tissue stability and response to biomechanical loading [88; 89]. Every family within our dataset had at least one cytoskeletal genetic variant, and 20/23 families showed an enriched cytoskeletal GO term (Supplementary Files S2). Variants in the centroid protein _POC5_ have been associated with scoliosis through human and animal studies [90; 91; 92]. The cytoskeletal kinesin _kif6_ was shown to be necessary for proper spine development in zebrafish [34], and mutations in _kif6_ also appeared in a zebrafish ENU genetic screen for scoliosis [93]. Defects in cilia, microtubule-based projections critical for cell signaling and fluid flow, have been linked to scoliosis in animal models [31; 34; 35; 37; 94; 95]. A variant in the ciliary kinesin _KIF7_ was found within one AIS family and specific _KIF7_ mutations produced scoliosis in zebrafish [38].