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

Abstract

Congenital scoliosis (CS) is a birth defect with variable clinical and anatomical manifestations due to spinal malformation. The genetic etiology underlying about 10% of CS cases in the Chinese population is compound inheritance by which the gene dosage is reduced below that of haploinsufficiency. In this genetic model, the trait manifests as a result of the combined effect of a rare variant and common pathogenic variant allele at a locus. From exome sequencing (ES) data of 523 patients in Asia and two patients in Texas, we identified six TBX6 gene-disruptive variants from 11 unrelated CS patients via ES and in vitro functional testing. The in trans mild hypomorphic allele was identified in 10 of the 11 subjects; as anticipated these 10 shared a similar spinal deformity of hemivertebrae. The remaining case has a homozygous variant in TBX6 (c.418C>T) and presents a more severe spinal deformity phenotype. We found decreased transcriptional activity and abnormal cellular localization as the molecular mechanisms for TBX6 missense loss-of-function alleles. Expanding the mutational spectrum of TBX6 pathogenic alleles enabled an increased molecular diagnostic detection rate, provided further evidence for the gene dosage-dependent genetic model underlying CS, and refined clinical classification.

Keywords: TBX6 gene; compound inheritance model; congenital scoliosis (CS); gene dosage; genotype-phenotype correlation.

Figure 1:. The schematic view of six TBX6 gene-disruptive variants.

(A) Schematic view of exons 1-9 and locations of variants (orange stars). (B) Overview of TBX6 protein with the DNA binding region (T-box domain, orange bar). The TBX6 variants are shown above the protein with black balls and counting. Four missense variants (p.R119H, p.L140F, p.D142Y, p.P145L) and the in-frame insertion variant (p.W158_E159insG) are enriched in the T-box region domain, one variant (p.R378) was not located in the T-box region domain. (C) Space distribution of the 5 gene-disruptive variants in the T-box domain in TBX6 protein. The model of T-box domain from TBX6 (89-272 amino acids) in complex with DNA was built by Swiss-Model (https://www.swissmodel.expasy.org/) using the crystal structure of TBX3 (PDB ID code: 1H6F) as model and presented by PyMOL. Two of the identified variants (p.R119H and p.W158_E159insG, red) locate close to the DNA-binding region.

Figure 2:. Relatively transcription activity of mutated TBX6.

A previously reported nonsense variant c.699G>A (p.Trp233Ter) was used as a positive control, whose transcriptional activities (TA) were shown to be significantly decreased compared to the wild-type control. *** P < 0.001; ** P < 0.01. The decreased transcriptional activity was observed in c.356G>A, c.424G>T, c.434C>T, c.473_475dupGGG TBX6 plasmid transfection from DISCO cohort, as well as the c.1133G>A from US-P1 (black bars). These variants were all found in trans with the T-C-A haplotype in patients with hemi-/wedge-shaped vertebrae. The remaining variants in our cohort were identified statistically nonsignificant less transcriptional activity (gray bars). The variant (c.418C>T) from US-P2 also showed statistically significant decreased transcriptional activity (black bar), which was found in homozygous state in a patient clinically diagnosed as SCD (multiple levels of vertebral malformations).

Figure 3:. Intracellular distributions of mutant TBX6 protein.

The cell nucleus could be visualized by DAPI (blue). The wild-type TBX6 protein (Myc-tagged, green) located inside the cell nucleus, while the mutated TBX6 (FLAG-tagged, red) (c.356G>A, c.418C>T and c.473_475dupGGG) showed obviously mis-localized at the extra-nuclear area (white arrows).

Figure 4:. Spinal radiographs of TACS patients.

Ten patients (XH026, XH070, XH071, XH171, XH246, XH261, XH329, XH421, XH635, US-P1) present as TACS (simple hemivertebrae/butterfly vertebrae involving the lower part of the spine, simple rib anomaly, and fewer vertebrae and intraspinal defects), the similar phenotype as the 16p11.2 del patient (XH522) we previously reported (Liu et al.). The vertebral malformations were indicated in the white arrows. The remaining one patient (US-P2) present a much more severe phenotype compared with TACS (a more severe one, extensive segmentation defects with multiple abnormalities of the ribs manifesting as CS clinically, SCD-like). The extensive vertebral malformations were indicated by the white bracket.

Figure 5:. Gene dosage model of TBX6-Associated Congenital Scoliosis (TACS).

In our putative model, the phenotypes in TACS patients vary along with the dosage alterations of TBX6 expression. Slight loss of TBX6 expression caused by heterozygous hypomorphic haplotype or bi-allelic hypomorphic haplotype could be regarded as tolerable mutational dosage (green part) and would not lead to any spinal phenotype. One heterozygous severe hypomorphic (gene-disruptive) or null allele would still be tolerable (yellow part). Both of severe hypomorphic or null allele in trans with a mild hypomorphic haplotype could reduce the TBX6 expression level to threshold of a high CS penetrance (upper half part of the red part), also known as compound inheritance model, in which TACS is the most common phenotype. Bi-allelic series consist of two severe hypomorphic alleles or a null allele in trans with a severe hypomorphic allele, which would lead to an extreme loss of expression of TBX6 (lower half of the red part) contributing to a much more severe phenotype. Bi-allelic null variants would be embryonic lethal.