The genetic and molecular basis of congenital scoliosis

Abstract

Congenital Scoliosis (CS) is a birth defect of the spinal curvature, characterized by congenital vertebral malformations (CVMs) that affects 0.5-1 of 1,000 live births. Although heritable or de novo mutations have been implicated in the development of CS, the complex genetic etiology of this disorder remains largely elusive. In our study, we recruited a CS cohort in Hong Kong and performed whole-exome sequencing for 67 patients and their close family members to investigate the underlying genetic etiology of CS. Copy number variants (CNVs) were first identified using both candidate gene and family-based approaches. We identified 12 CNVs in four CVM-associated genes (TBX6, NOTCH2, DSCAM, and SNTG1) and verified the compound heterozygous model of TBX6 by CNV analysis and haplotype sequencing. Through inheritance pattern analysis in the patients’ families, we determined eight recessive and 64 de novo CNVs in 15 novel genes, including DHX40, NBPF20, RASA2, and MYSM1. The genetic screens for single nucleotide variants (SNVs) in CS patients were performed through both candidate gene approach and family studies. For the candidate gene approach, we identified a rare de novo p.R16L missense mutation in TBXT, an essential transcription factor controlling somitogenesis, in one CS patient and validated its pathogenicity by electrophoretic mobility shift and luciferase assays. Besides TBXT, the canonical Wnt/b-catenin signaling is also essential for mesoderm development. However, the function of non-canonical Wnt/planar cell polarity (Wnt/PCP) signaling in somitogenesis remains undetermined. Here, we discovered that the deletion of two core PCP components, Vangl1 and Vangl2, leads to defective somitogenesis and spinal malformations in mice. Through a multi-center collaboration, we identified nine VANGL1 and eight VANGL2 rare missense variants in a multiethnic cohort. Several mutations cause loss-of-function and dominant-negative effects, as shown by reduced protein expression and compromised membrane presentation of Vangl proteins. In the zebrafish model, mutant VANGL mRNA failed to restore the convergent extension defects caused by the knockdown of endogenous Vangl2. Furthermore, we generated a p.R258H Vangl1 knock-in mouse model to test the in vivo functionality of the human p.R256H VANGL1 mutation. We found that the Vangl1R258H mice showed vertebral malformations in a Vangl2 dose-dependent manner, resembling human CVMs. What’s more, our family-based filtration of SNVs also identified additional variants in other PCP genes. Overall, our findings determined new CNVs associated with CS, verified the previously reported TBX6 compound heterozygous model, and identified rare missense SNVs in TBXT and VANGL1/2 that may contribute to the development of CS.