Human TBX1 missense mutations cause gain of function resulting in the same phenotype as 22q11.2 deletions

Abstract

Deletion 22q11.2 syndrome is the most frequent known microdeletion syndrome and is associated with a highly variable phenotype, including DiGeorge and Shprintzen (velocardiofacial) syndromes. Although haploinsufficiency of the T-box transcription factor gene TBX1 is thought to cause the phenotype, to date, only four different point mutations in TBX1 have been reported in association with six of the major features of 22q11.2 deletion syndrome. Although, for the two truncating mutations, loss of function was previously shown, the pathomechanism of the missense mutations remains unknown. We report a novel heterozygous missense mutation, H194Q, in a familial case of Shprintzen syndrome and show that this and the two previously reported missense mutations result in gain of function, possibly through stabilization of the protein dimer DNA complex. We therefore conclude that TBX1 gain-of-function mutations can result in the same phenotypic spectrum as haploinsufficiency caused by loss-of-function mutations or deletions.

Figure 1.

Identification of a mutation in the TBX1 gene in a family with the 22q11 deletion phenotype. A, Pedigree and electropherograms of the mutated genomic sequence of exon 5 of the TBX1 gene from the affected family members and the unaffected mother. The position of the mutated nucleotide is indicated by a black arrow; the identified mutation c.582C→G leads to the amino acid substitution H194Q. B, Alignment of a part of the T-box domain of TBX1 with homologues from various species (UCSC Genome Bioinformatics) and other members of the T-box family. The conserved amino acids are shown with a gray background, and similar amino acids with a light gray background. Residue 194 is indicated with a black box. C, Scheme of TBX1C indicating the T-box (pink), the novel H194Q mutation (red arrowhead), and the mutations published by Yagi et al. and Paylor et al.

Figure 2.

Transcriptional activation of a luciferase reporter constructs by wild-type (WT) and mutant TBX1C. A–D, JEG-3 cells were transiently transfected with reporter constructs containing a promoter without the T-Box binding sites (tkGL2 [white bars]) or with T-Box binding sites (2xTtkGL2, bars with different shades of gray). Cotransfection was performed with either a CMV control vector, the TBX1C wild-type construct, or the mutant constructs. Results are normalized for transfection efficiency to a cotransfected renilla luciferase vector and expressed as average values ± SEM of three independent transfections. The results were confirmed by repeating the triple transfections and measurements for two to three times respectively. A and B, Representative examples of results of single experiments with three independent transfections for each combination. In comparison to the CMV vector TBX1C shows a 30–40-fold increased activation on a reporter containing T-Box binding elements (2xTtkGL2 [gray bars]), whereas there is virtually no activation of the reporter construct without T-Box binding sites (tkGL2 [white bars]). The truncating mutation 1223delC shows a clear lack of activation, whereas the missense mutations indicate an increase in activation of the luciferase reporter construct. Due to the small number of measurements, none of these results from a single experiment reaches statistical significance with the Mann-Whitney-Wilcoxon test. Similar results were obtained with two further experiments with three independent transfections each (data not shown). C, To compare results from independent experiments, the activity of mutant TBX1C was normalized to that of wild-type TBX1C, and P values were obtained using the Mann-Whitney-Wilcoxon test for unpaired samples of totally nine independent transfections in three independent experiments (six transfections in two experiments for 1223delC). D, To simulate heterozygosity of the mutations, cells were simultaneously transfected with equal amounts of wild-type and mutant TBX1C construct DNA. No obvious difference was observed in comparison with cells transfected with mutant TBX1C only.

Figure 3.

Expression of wild-type (WT) and mutant TBX1C in JEG3 cells. JEG3 cells were transiently transfected with CMV, wild-type and mutant TBX1C and TBX1C expression measured with a Taqman assay. No increased expression was observed for the mutants in comparison to the wild type. Results are normalized for transfection efficiency against the cotransfected renilla vector and expressed as average value ± SEM of quadruplicate measurements of two independent transfections, respectively. By use of the Mann-Whitney-Wilcoxon test for unpaired samples, P values for comparison with the wild-type levels are 1 each for F148Y and H194Q, 0.24 for G310S, and 0.44 for 1223delC.

Figure 4.

A, Three-dimensional model of human Tbx1 in complex with DNA (orange). The subunits of the dimeric protein are shown in backbone presentation (blue and cyan) and the α-helices and β-sheets are depicted schematically. The three loops predicted to form the dimer interface are shown (magenta), and residues D155 and K202 that form salt bridges (black dotted lines) across the dimer interface are shown in stick presentation. A prime distinguishes residues belonging to different subunits. Residues F148 and H194, for which mutations were observed, are shown in stick presentation, and sequence positions that were predicted to form novel interactions with these residues in the mutant proteins are shown as balls. The spatial vicinity (black boxes) of these residues is shown as an enlargement for the wild-type and mutant proteins in panels B–E. B–E, Detailed analysis of the structural effects of F148Y and H194Q mutants. Models of the wild-type and mutant proteins are shown in the left and right panels, respectively. Structurally important residues are shown in stick presentation and are colored according to the atom types. Green dotted lines indicate important hydrogen bonds, which are apparently affected by the mutations, and the site of the hydrogen bond is marked (green arrow). B and C, Effect of the F148Y mutation. The presence of a tyrosine at position 148 allows the formation of a novel side-chain hydrogen bond to the carbonyl oxygen of M207 (E), which cannot be formed by the phenylalanine in the wild type (D). D and E, Effect of the H194Q mutation. The presence of a glutamine at position 194 allows the formation of a novel side-chain hydrogen bond to the carbonyl oxygen of G227 (E), which cannot be formed by the histidine in the wild type (D).