## 5 Conclusions  
This work presents a novel set of candidate variants found in affected individuals from 23 AIS families. In agreement with our previous WES study of five AIS families, we observed an overrepresentation of variants in cytoskeletal and ECM functional categories, with few specific genes shared across families. Overall, this work paints a picture of AIS genetic etiology as highly polygenic and specific to individual family lineages. An analytical approach that integrates data from family-based sequencing with genetic association studies, an understanding of study population variation, population stratification and genetic heterogeneity, and advances in clinical phenotyping will enhance our ability to define the genetic complexity of this disorder. **Supplementary Materials:** The following are available online at [[https://www.mdpi.com/article/10.3390/genes12060922/s1](https://www.mdpi.com/article/10.3390/genes12060922/s1)]([https://www.mdpi.com/article/10.3390/genes12060922/s1](https://www.mdpi.com/article/10.3390/genes12060922/s1)): File S1: Family Pedigrees. Degree of spinal curvature is indicated with a D (degrees). Individuals marked \"confirmed positive\" were confirmed as affected with AIS but were unable to provide us with an X-ray for measurement. Exome sequenced individuals are indicated. File S2: Table S1: Clinical information from all exome sequenced individuals; Table S2: Enriched cytobands from MAF < 0.05 gene list; Table S3: Genotyping of genes in multiple families and in functional categories of interest; Table S4: Full Gene Ontology (GO) Term enrichment results(\(p\) < 0.01) for all functional categories using DAVID; File S3: Detail of all filtered variants, sorted by family. All filtered variants had a minor allele frequency (MAF) < 0.05, were present in all sequenced members of the family, and were predicted to be damaging to the resulting protein by at least one algorithm. Details of the exome sequencing strategy and bioinformatic filtering process are provided in the Methods. Conceptualization: N.H.M., K.L.J., M.R.G.T., E.E.B. and E.A.T. ; Methodology: C.I.W., M.T.C., A.M.M., G.D.T., M.R.B., E.E.B. and R.B. ; Software: G.D.T. and K.L.J. ; Validation: C.I.W. ; Formal analysis: K.L.J., G.D.T. and E.A.T. ; Investigation: N.H.M. ; Resources: N.H.M. ; Data curation: N.H.M. and E.A.T. ; Writing--original draft preparation: E.A.T. ; Writing--review and editing: N.H.M. and A.M.M. ; Visualization: E.A.T. ; Supervision: N.H.M., E.E.B. and E.A.T. ; Project administration: N.H.M. ; Funding acquisition: N.H.M. and E.E.B. All authors have read and agreed to the published version of the manuscript. Funding Funding for NHM's laboratory and this study was provided by NIH/NIAMS R01 AR068292. Additional funds were provided by Children's Hospital Colorado. Institutional Review Board Statement: Written informed consent was obtained from study subjects who were enrolled in accordance with protocols approved by the Johns Hopkins School of Medicine Institutional Review Board and the University of Colorado Anschutz Medical Campus Institutional Review Board (Colorado Multiple Institutional Review Board, Studies #06-1161 and #07-0417). All procedures involving human participants were performed in accordance with the ethical standards of these institutional review boards, the 1964 Declaration of Helsinki and its later amendments, or comparable ethical standards. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The authors affirm that all data necessary for confirming the conclusions of this article are represented fully within the article and its supplementary files, including the complete lists of filtered variants for each family. Patient data was collected and managed using REDCap (Research Electronic Data Capture) tools hosted at the University of Colorado Anschutz Medical Campus.