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In this study, we analyze exome\/genome sequencing data from 873 probands with congenital vertebral malformation and 3794 control individuals. Clinical interpretation identifies Mendelian etiologies in 12.0% of the probands and reveals a muscle-related disease mechanism. Gene-based burden test of ultra-rare variants identifies risk genes with large effect sizes (<jats:italic>ITPR2<\/jats:italic>, <jats:italic>TBX6<\/jats:italic>, <jats:italic>TPO<\/jats:italic>, <jats:italic>H6PD<\/jats:italic>, and <jats:italic>SEC24B<\/jats:italic>). To further investigate the biological relevance of the genetic association signals, we perform single-nucleus RNAseq on human embryonic spines. The burden test signals are enriched in the notochord at early developmental stages and myoblast\/myocytes at late stages, highlighting their critical roles in the developing spine. 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