Genetic Variants of TBX5 in Holt-Oram Syndrome Patients

Objectives: The Holt-Oram syndrome (HOS) is a rare autosomal dominant disease defined by upper limb anomalies and congenital heart defects. In more than 70% of HOS cases mutations of the TBX5 gene are responsible for the development of the specific phenotype with variable severity. We screened five HOS patients to identify novel mutations within the TBX5 gene and/or the corresponding regulatory elements.

Methods: Sequencing of all TBX5 coding exons, the TBX5 promoter and enhancer was done by Sanger Sequencing. Three-dimensional analysis of the protein structure was used to predict the relevance of selected mutations for their protein activity. Luciferase assays with selected mutated TBX5 sequences were used to investigate the functional activity versus wild-type TBX5. Genetic variants in regulatory elements were predicted for their relevance using general databases.

Results: We identified three patients with a TBX5 mutation in the TBX5 coding sequence. These patients carry additional mutations within corresponding regulatory elements. Two patients show only mutations within the promotor and/or enhancer elements with a wild type TBX5 coding sequence.

One out of the three identified TBX5 mutations is a so far unknown mutation at position 920 (C→A) within exon four. This mutation causes an amino acid change at position 85 (proline → threonine). Alignment of the sequence verified this position as highly conserved across the T genes and across several species. Furthermore the mutation is located in the DNA-binding site of the transcription factor. The Pro85Thr mutation changes the torsion angle from 180 to 0 degree thereby provoking major conformational and functional alterations of the protein. The transcriptional activity of the mutated TBX5 is dramatically reduced compared with the wild type TBX5 sequence on the ANF promoter in luciferase assays.

Conclusion: We identified a novel TBX5 mutation in a highly conserved area of the TBX5 gene, which is disease causing for HOS. Three-dimensional analysis predicted and functional investigation of the mutated TBX5 gene confirmed a severe reduction of the TBX5 function. The combined emergence of mutations in the TBX5 coding sequences and/or in different regulatory elements of the TBX5 gene could be responsible for the variable severity of the phenotype.