## 3 Results  
To identify rare and low frequency variants associated with familial AIS, we performed whole exome sequencing on 23 multigenerational IS families (3-5 individuals per family, 86 individuals in total). Pedigrees for all families are provided in Supplementary Files S1, and clinical information for sequenced individuals is provided in Supplementary Files S2. Whole exome sequencing was performed using DNA extracted from whole blood, as described in the Methods. Illumina HiSeq reads were mapped to the human reference genome (hg39), and a minimum of 50X average coverage was obtained for each sample. We then filtered the list of variants, requiring that each retained variant was present in all sequenced members of the family, was predicted to be damaging, and had an ExAC minor allele frequency (MAF) of <0.05. This MAF filter thus included both low-frequency (MAF 1-5%) and rare variants (MAF < 1%). These filters resulted in a total of 1448 variants in 1160 genes across the 23 families, with 11 to 128 variants identified in each family (median = 51 variants). Figure 2 provides a summary of variant information identified across all families. Nonsynonymous single nucleotide polymorphisms (SNPs) constituted the majority of filtered variants (88%, \(n\) = 1281), followed by non-frameshift deletions (4%, \(n\) = 52), non-frameshift insertions (2%, \(n\) = 34), frameshift deletions (2%, \(n\) = 25), stop gains (2%, \(n\) = 22), frameshift insertions (2%, \(n\) = 22), and splice sites (1%, \(n\) = 12). About half (48%) of variants had a minor allele frequency (MAF) of 0.01-0.05, 22% had an MAF 0.001-0.01, 18% had an MAF < 0.001, and 12% were predicted to be novel. Chromosomes with the largest number of variants were chromosome 1 (\(n\) = 150) and chromosome 19 (\(n\) = 119). Full details of each variant, sorted by family, are provided in Supplementary Files S3. We next searched for genes containing variants in multiple families, with the hypothesis that shared genes would be more likely to contribute to the IS phenotype. Most variant-containing genes (\(n\) = 1028 of 1160 total, 88.6%) were specific to only one family. 132 genes were shared by at least 2 families, 38 were shared between 3+ families, and 10 were shared by 4+ families. No genes were shared among all families. Table 1 provides a list of genes found within multiple families. Of this list, four genes had a previously observed association with either idiopathic, degenerative, or infantile scoliosis (_DLL3_[58; 59], _AHNAK_[60], _TTN_[61], _ANKRD11_[62]). Although few genes were shared across families, we hypothesized that AIS families may share an enrichment of variants in specific functional categories. To identify categories of genes which were overrepresented with damaging variants within the AIS families, we performed gene set overrepresentation analysis (GOA) of our resulting filtered gene lists. We entered the MAF < 0.05 gene lists from individual families and combined data from all families into DAVID to identify overrepresented gene ontologies (GO Terms) (Table 2). The most overrepresented GO Cell Component categories across all families using DAVID were \"microtubule\" (\(p\) = 1.62 \(\times\) 10\({}^{4}\), 1.98 fold enrichment), \"slit diaphragm\" (\(p\) = 8.88 \(\times\) 10\({}^{4}\), 10.66 fold enrichment), and \"intermediate filament\" (\(p\) = 8.88 \(\times\) 10\({}^{4}\), 2.57 fold enrichment). Categories within extracellular matrix (ECM) GO terms, including \"proteinaceous extracellular matrix\" and \"collagen trimer\" also showed mild enrichments (Table 2) and the top enriched KEGG term was \"ECM receptor activation\" (Figure 2E). A full list of all GO functional categories is provided in Supplementary Files S2. We also used GOA to analyze an overrepresentation of cytogenic bands, as specific chromosomal regions have been linked with IS development by linkage analyses [63; 64; 65; 66; 67; 68; 69; 70; 71]. The most overrepresented band was 16p13.3 (\(p\) < 0.001), as described in Supplementary Files S2.