First Genotype-Phenotype Study in TBX4 Syndrome: Gain-of-Function Mutations Causative for Lung Disease

Abstract

Rationale: Despite the increased recognition of TBX4 (T-BOX transcription factor 4)-associated pulmonary arterial hypertension (PAH), genotype-phenotype associations are lacking and may provide important insights. Objectives: To compile and functionally characterize all TBX4 variants reported to date and undertake a comprehensive genotype-phenotype analysis. Methods: We assembled a multicenter cohort of 137 patients harboring monoallelic TBX4 variants and assessed the pathogenicity of missense variation (n = 42) using a novel luciferase reporter assay containing T-BOX binding motifs. We sought genotype-phenotype correlations and undertook a comparative analysis with patients with PAH with BMPR2 (Bone Morphogenetic Protein Receptor type 2) causal variants (n = 162) or no identified variants in PAH-associated genes (n = 741) genotyped via the National Institute for Health Research BioResource-Rare Diseases. Measurements and Main Results: Functional assessment of TBX4 missense variants led to the novel finding of gain-of-function effects associated with older age at diagnosis of lung disease compared with loss-of-function effects (P = 0.038). Variants located in the T-BOX and nuclear localization domains were associated with earlier presentation (P = 0.005) and increased incidence of interstitial lung disease (P = 0.003). Event-free survival (death or transplantation) was shorter in the T-BOX group (P = 0.022), although age had a significant effect in the hazard model (P = 0.0461). Carriers of TBX4 variants were diagnosed at a younger age (P < 0.001) and had worse baseline lung function (FEV₁, FVC) (P = 0.009) than the BMPR2 and no identified causal variant groups. Conclusions: We demonstrated that TBX4 syndrome is not strictly the result of haploinsufficiency but can also be caused by gain of function. The pleiotropic effects of TBX4 in lung disease may be in part explained by the differential effect of pathogenic mutations located in critical protein domains.

Keywords: TBX4; gain-of-function; interstitial lung disease; lung developmental disease; pulmonary arterial hypertension.

Figure 1.

Lolliplot depicting TBX4 (T-BOX transcription factor 4) mutation spectrum. Recurrently mutated positions are represented by a proportionally sized lollipop. Critical protein domains are highlighted, inclusive of the DNA binding T-BOX, nuclear localization segments (NLS1 and NLS2), and transactivation domains. Variants are grouped by primary associated phenotype and color-coded, taking into account the functional assessment of missense and inframe insertion/deletions (indels). GoF = gain-of-function; LoF = loss-of-function; PAH = pulmonary arterial hypertension.

Figure 2.

Functionally assessed TBX4 (T-BOX transcription factor 4) variants by luciferase assay inducing gain or loss of function. (A) Schematic representation of the in vitro dual-luciferase reporter assay. We cotransfected three different plasmids: Firefly luciferase with x3 T-BOX motifs as the promoter, Renilla luciferase, and TBX4 overexpression plasmid (wild type/mutated). (B) Variants inducing gain or loss of function grouped by primary phenotype; lung disease (perinatal-, childhood-, and adult-onset) or small patella syndrome (SPS). The y-axis represents the relative luciferase units; the dashed line marks the level of the wild type. Data are shown as box plots representing median ± quartiles. Dots represent biological replicates with corresponding batches in different shapes. CMV = Cytomegalovirus promoter; Flag = Flag tag; Myc = partial cMyc tag.

Figure 3.

Structural analysis of TBX4 (T-BOX transcription factor 4) sequence variants. The crystal structure of TBX5 (T-BOX transcription factor 5) bound to DNA, Protein Database code 2X6V, was used for structural analysis. They share 52.6% sequence together with the full-length proteins and 93.9% in the T-BOX domain. (A) Sequence alignment of TBX4 and TBX5 in the T-BOX region (highlighted in gray) containing the DNA-binding motif as well as the nuclear localization segment 1 (NLS1, in cyan) and the nuclear export segment (NES, in yellow) (29, 30). TBX4 missense variants are indicated in bold/blue, with indels highlighted in magenta. Residues visible in the TBX5 structure are shown in light blue letters. (B) Mutations plotted on the TBX5 crystal structure as spheres. Cyan, yellow, and magenta spheres correspond to the NLS1, NES regions, and indels as indicated in A. When annotating loss-of-function variants on the TBX4 sequence, they are highly enriched in the T-BOX, particularly the NLS1 and NES. (C) Some mutations of the noninterface residues, such as TBX4 p.Glu86 and p.Tyr127 (corresponding residues p.Glu73 and p.Tyr114 in TBX5, respectively), make essential interactions to stabilize the secondary structural elements required for T-BOX binding to DNA. Clustal Omega was used for sequence alignment. Figures were generated using PyMOL Molecular Graphics System.

Figure 4.

Age at diagnosis of lung disease by TBX4 (T-BOX transcription factor 4) genotype. (A) Variants in the T-BOX and second nuclear localization segment (NLS2) versus other domains. (B) Likely pathogenic/pathogenic gain-of-function (GoF) versus loss-of-function (LoF) missense variants.

Figure 5.

Time to death or lung transplantation (years) by TBX4 (T-BOX transcription factor 4) protein domain genotype. Event-free survival was shorter in the T-BOX domain variant group, although age had a significant effect in the hazard model. CI = confidence interval; *Variables with P < 0.05.