TBX6 compound inheritance leads to congenital vertebral malformations in humans and mice

Abstract

Congenital vertebral malformations (CVMs) are associated with human TBX6 compound inheritance that combines a rare null allele and a common hypomorphic allele at the TBX6 locus. Our previous in vitro evidence suggested that this compound inheritance resulted in a TBX6 gene dosage of less than haploinsufficiency (i.e. <50%) as a potential mechanism of TBX6-associated CVMs. To further investigate this pathogenetic model, we ascertained and collected 108 Chinese CVM cases and found that 10 (9.3%) of them carried TBX6 null mutations in combination with common hypomorphic variants at the second TBX6 allele. For in vivo functional verification and genetic analysis of TBX6 compound inheritance, we generated both null and hypomorphic mutations in mouse Tbx6 using the CRISPR-Cas9 method. These Tbx6 mutants are not identical to the patient variants at the DNA sequence level, but instead functionally mimic disease-associated TBX6 variants. Intriguingly, as anticipated by the compound inheritance model, a high penetrance of CVM phenotype was only observed in the mice with combined null and hypomorphic alleles of Tbx6. These findings are consistent with our experimental observations in humans and supported the dosage effect of TBX6 in CVM etiology. In conclusion, our findings in the newly collected human CVM subjects and Tbx6 mouse models consistently support the contention that TBX6 compound inheritance causes CVMs, potentially via a gene dosage-dependent mechanism. Furthermore, mouse Tbx6 mutants mimicking human CVM-associated variants will be useful models for further mechanistic investigations of CVM pathogenesis in the cases associated with TBX6.

TBX6 missense variants expand the mutational spectrum in a non‐Mendelian inheritance disease

“…Through identification of different combinations of rare, functional variants and hypomorphic alleles, we have further refined a locus‐dependent dosage model to explain TBX6‐ mediated molecular mechanisms causing CS (Figure ; Liu et al, ; Wu et al, ; N. Yang et al, ). In this model, a dosage of 50% is sufficient for the normal development of the embryo, that is a wild‐type allele in trans with a null/severe hypomorphic allele/mild hypomorphic haplotype or a bi‐allelic mild hypomorphic haplotype would not lead to a CS phenotype in human or mice.…”

“…In contrast, in the setting of bi‐allelic variation involving a rare deleterious homozygous variant(s), severe hypomorphic variant, the greater relative loss of TBX6 function, leads to a more severe phenotype (SCD‐like, multiple vertebral segmentation defects). Complete depletion of TBX6 , that is bi‐allelic null variants (i.e., amorphic alleles), is proposed to cause embryonic lethality as in mouse knockout studies (Chapman & Papaioannou, ; White et al, ) and CRISPR/Cas9 engineered mouse models (Yang et al, ).…”

“…Considering the potential for multigenic modifiers indicated by recent advances (Gonzaga‐Jauregui et al, ), further study based on genome sequencing and enhanced variant detection (e.g., indels and exonic CNV rare variant alleles; noncoding common variant alleles) and more comprehensive genotype–phenotype correlation is necessary. For example, gene dosage modeling in mice may prove informative in future CS studies (Yang et al, ).…”

“…Consistent with our observations in humans, a high penetrance of the CS phenotype was observed only in mice with the allelic combination (bi‐allelic: LoF + hypomorph) modeling compound inheritance in the Tbx6 gene/locus‐edited mice. Moreover, these animals exhibit a strikingly similar phenotype to that observed in humans with TBX6 variants including the type and anatomical location of malformation, that is defects of vertebral column formation involving the lower part of the spine (Yang et al, ).…”

“…The proposed TBX6 compound inheritance model was further confirmed in independent worldwide patient populations (Liu, Wu, Yang, & Takeda; Takeda et al, ). We provided additional evidence for the gene dosage hypothesis and the requirement for bi‐allelic variation at Tbx6/TBX6 by engineering a mouse model to explore compound inheritance and the gene dosage model (Yang et al, ). CS patients affected with compound inheritance at the TBX6 locus present a distinctive pattern of clinical features: simple hemi‐/wedge‐shaped vertebrae in the lower half of the spine, and was thus defined as TBX6 ‐associated congenital scoliosis (TACS; Liu et al, ; Wu et al, ).…”