## 1 Introduction  
Adolescent idiopathic scoliosis (AIS) is a structural lateral spinal curvature \(\geq\)10 deg that affects 2-3% of healthy children [1], with females at the greatest risk for severe progression [2, 3, 4]. In individuals with severe progressive curvatures, life-long problems of cosmetic deformity, respiratory compromise, back pain, and degenerative disease often arise and in many cases require surgical intervention, placing a significant economic burden upon our healthcare system [5]. AIS is known to be highly heritable, however, our knowledge of its etiology is severely limited, both in terms of the individuals at risk for curve initiation and those likely to experience curve progression. Understanding key risk variants or genetic pathways leading to AIS holds the potential to improve patient care by way of targeted clinical treatments or prognostics for detecting AIS risk or risk of curvature progression.  
Multiple studies support the genetic foundation of AIS, with sibling recurrence risks reported to be near 18%, and heritability estimates of approximately 87.5% [6; 7; 8]. To understand the genetics of AIS, traditional approaches including genome wide association studies (GWAS), exome sequencing, and familial linkage studies have been applied to multiple populations. GWAS of unrelated individuals with AIS have identified potential common risk alleles, notably those in or near _LBX1_[9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23] and _GPR126/ADGRG6_[24; 25; 26; 23; 27], which are the most well-replicated genetic findings to date across populations. Next-generation sequencing studies (i.e., whole exome sequencing) both within families and unrelated individuals with AIS have identified _rare_ variants in extracellular matrix genes that may contribute to the AIS phenotype [28; 29; 30]. Recent genetic and functional studies have led to varied hypotheses of AIS etiology, including dysfunction within neuroinflammatory pathways [31; 32], the cartilage matrisome [33], cilia, the cytoskeleton [34; 35; 36; 37; 38], or the vestibular system [39; 40; 41; 42; 43]. However, the inability thus far to relate specific genetic variants to the biology of AIS and the wide variation in which AIS presents are indicative of the complex heterogeneity of this disorder.  
Disease susceptibility variants for complex diseases may collectively be common in the general population, but specific variants may be rare within families or affected individuals. Family studies present unique advantages over case-control studies, as they may reveal rare disease-associated variants enriched within the family that are amenable to targeted sequencing. Sequencing studies of multiple large families can reveal multiple variants within a gene or molecular pathway that contribute to the disease phenotype [44; 45].  
Within our laboratory, a pilot exome sequencing study of five AIS families identified an enrichment of damaging uncommon variants in cilia, extracellular matrix, and cytoskeletal genes, although no specific variants or genes were found to be present across all families [46].  
This led us to propose the hypothesis that the development of AIS may be due to damaging variants within a specific set of pathways or molecular classes, rather than being driven by just a few select 'AIS genes'. In this study, we expand upon our previous work and present exome sequencing of affected individuals from 23 AIS families, interpreted using gene enrichment analyses to identify overrepresented functional categories. We then investigate specific genes containing variants in multiple families via genotyping confirmation in additional affected and unaffected members of the family to assess how closely these variants track with the AIS phenotype.