TBX6 null variants and a common hypomorphic allele in congenital scoliosis

Abstract

Background: Congenital scoliosis is a common type of vertebral malformation. Genetic susceptibility has been implicated in congenital scoliosis.

Methods: We evaluated 161 Han Chinese persons with sporadic congenital scoliosis, 166 Han Chinese controls, and 2 pedigrees, family members of which had a 16p11.2 deletion, using comparative genomic hybridization, quantitative polymerase-chain-reaction analysis, and DNA sequencing. We carried out tests of replication using an additional series of 76 Han Chinese persons with congenital scoliosis and a multicenter series of 42 persons with 16p11.2 deletions.

Results: We identified a total of 17 heterozygous TBX6 null mutations in the 161 persons with sporadic congenital scoliosis (11%); we did not observe any null mutations in TBX6 in 166 controls (P<3.8×10(-6)). These null alleles include copy-number variants (12 instances of a 16p11.2 deletion affecting TBX6) and single-nucleotide variants (1 nonsense and 4 frame-shift mutations). However, the discordant intrafamilial phenotypes of 16p11.2 deletion carriers suggest that heterozygous TBX6 null mutation is insufficient to cause congenital scoliosis. We went on to identify a common TBX6 haplotype as the second risk allele in all 17 carriers of TBX6 null mutations (P<1.1×10(-6)). Replication studies involving additional persons with congenital scoliosis who carried a deletion affecting TBX6 confirmed this compound inheritance model. In vitro functional assays suggested that the risk haplotype is a hypomorphic allele. Hemivertebrae are characteristic of TBX6-associated congenital scoliosis.

Conclusions: Compound inheritance of a rare null mutation and a hypomorphic allele of TBX6 accounted for up to 11% of congenital scoliosis cases in the series that we analyzed. (Funded by the National Basic Research Program of China and others.).

Figure 1. Effect of the 16p11.2 Deletion on TBX6

Panel A shows the region affected by the proximal 16p11.2 deletion based on the human genome assembly hg19. A pair of long direct genomic repeats (shown in orange) can mediate 0.6-Mb recurrent deletions. The genes affected by this deletion, including TBX6 (circled), are shown. Panel B shows three common single-nucleotide polymorphisms (SNPs) in TBX6. Two SNPs, rs3809624 and rs3809627, are located in the 5' noncoding region, whereas rs2289292 is a synonymous SNP in the last exon. Panels C and D show two pedigrees with 16p11.2 deletions (SE1 and SE2). Squares denote male pedigree members, circles female pedigree members, solid symbols members with congenital scoliosis, and open symbols unaffected members; the probands are indicated by black arrows. The red bracket represents the deletion allele, and the nonreference alleles of rs2289292, rs3809624, and rs3809627 on the nondeletion chromosomes are shown in blue. The slash denotes a deceased family member. NA denotes not available.

Figure 2. Spinal Radiographs in Patients with TBX6-Associated Congenital Scoliosis

The vertebrae with malformations are indicated by arrows. The patient number is shown above each radiograph.

Figure 3. Results of In Vitro Functional Assays of Common TBX6 Variants and the Mechanistic Model of TBX6-Associated Congenital Scoliosis

Panel A shows the results of luciferase reporter assays. Two cell types (P19CL6 cells and human induced pluripotent stem cells [hiPSCs]) with high levels of expression of TBX6 during induced differentiation were used. The nonreference alleles of rs3809624 and rs3809627 are shown in blue, and the risk haplotype is underlined. At least three independent experiments of normalized luciferase activity were quantified, with the graph showing mean values and their standard errors (I bars). Panel B shows a simplified genetic model of TBX6-associated congenital scoliosis. TBX6 expression is critical for normal vertebral formation (the reference allele of TBX6 is indicated by a black line). Heterozygous hypomorphic variants (shown in blue) cause only a moderate reduction in TBX6 expression. Even homozygous hypomorphic variants do not reduce TBX6 expression dramatically. Heterozygous TBX6 null mutations (indicated by red brackets) may reduce gene expression by one half. Independently, these mutations hardly reach the gene-dosage threshold for congenital scoliosis. In combination, however, a null allele and a hypomorphic allele of TBX6 cause further reductions in gene expression that may confer a high risk of congenital scoliosis.