A novel de novo TBX5 mutation in a patient with Holt-Oram syndrome leading to a dramatically reduced biological function

Abstract

Background: The Holt-Oram syndrome (HOS) is an autosomal dominant disorder affecting 1/100.000 live births. It is defined by upper limb anomalies and congenital heart defects with variable severity. We describe a dramatic phenotype of a male, 15-month-old patient being investigated for strict diagnostic criteria of HOS.

Methods and results: Genetic analysis revealed a so far unpublished TBX5 mutation, which occurs de novo in the patient with healthy parents. TBX5 belongs to the large family of T-box transcription factors playing major roles in morphogenesis and cell-type specification. The mutation located in the DNA-binding domain at position 920 (C→A) leads to an amino acid change at position 85 (proline → threonine). Three-dimensional analysis of the protein structure predicted a cis to trans change in the respective peptide bond, thereby probably provoking major conformational and functional alterations of the protein. The p.Pro85Thr mutation showed a dramatically reduced activation (97%) of the NPPA promoter in luciferase assays and failed to induce NPPA expression in HEK 293 cells compared to wild-type TBX5 protein. The mutation did not interfere with the nuclear localization of the protein.

Conclusion: These results suggest that the dramatic functional alteration of the p.Pro85Thr mutation leads to the distinctive phenotype of the patient.

Keywords: Congenital heart disease; Holt–Oram syndrome; TBX5; de novo mutation; heart‐hand syndrome; loss‐of function; transcription factor.

Figure 1

Upper limb anomalies and cardiac defects of the patient. (A) Photographs of the body and upper limbs showing the deformation of the upper arms and thumbs on both hands. (B) X‐rays of both upper limbs. (C) X‐ray of the body. (D) Echocardiogram showing the two VSDs (marked by *).

Figure 2

Identification of the p.Pro85Thr mutation in the TBX5 gene. Chromatograms of exon 4 showing the sequences of the unaffected parents and the c.920_C>A mutation of the HOS patient. For the parents, the forward sequences are shown.

Figure 3

Location and conservation of the p.Pro85Thr mutation. (A) Schematic view of the interaction between TBX5 and DNA. The location of p.Pro85 and the cis‐peptide bond is indicated. (B) Vicinity of Phe84‐Pro85 and the TBX5 C‐terminus involved in DNA binding. (C) Alignment of human TBX5 protein sequence with TBX5 proteins of multiple species. (D) Alignment of human TBX5 protein with other members of the T‐box gene family. Numbers refer to amino acid positions of the human TBX5 sequence.

Figure 4

Functional analysis of the p.Pro85Thr mutant. (A) Activation of NPPA promoter‐driven luciferase activity in HEK 293 cells. Results are presented as the mean ± SE of four independent experiments. *P < 0.05, ***P < 0.001. (B) Induction of NPPA gene expression (NM_006172.3) in HEK 293 cells after transfection with wild‐type or mutant TBX5 sequences. 1: untransfected, 2: wild‐type TBX5, 3: p.Pro85Thr TBX5, 4: aq.bidest. (C) qRT‐PCR analysis of TBX5 (NM_000192.3) and NPPA expression in HEK293 cells. Fold gene expression was determined after normalization to an internal control (β‐ACTIN, NM_001101.3).

Figure 5

Nuclear localization of wild‐type (A) and mutant (B) TBX5 protein. Distribution of wild‐type and mutant TBX5 was analyzed by immunohistochemical staining using an anti‐flag antibody. Merged images show a combined staining of nuclei (blue), cytoplasm (red) and TBX5 (green). White arrows indicate cytoplasmic areas around the nuclei of TBX5 expressing cells. Scale bars represent 20 μ m.