Author's response to reviews Title: Functional analysis of the novel TBX5 c.1333delc mutation resulting in an extended TBX5 protein Authors: Johann Bohm (Johann.Boehm@titus.u-strasbg.fr) Wolfram Heinritz (wolfram.heinritz@medizin.uni-leipzig.de) Mihailo Vujic (mihailo.vujic@vgregion.se) Britt-Marie Ekman-Joelsson (britt-mari.ekman-joelsson@vgregion.se) Juergen Kohlhase (jkohlhase@humangenetik-freiburg.de) Ursula Froster (reichsi@medizin.uni-leipzig.de) Version: 2 Date: 21 April 2008 Author's response to reviews: see over
BMC Medical Genetics Illkirch, 21st of April, 2008 MS: 2226828441829521 Functional analysis of the novel TBX5 c.1333delc mutation resulting in an extended TBX5 protein Dear Madam, Dear Sir, thank your for considering our manuscript for publication in BMC Medical Genetics. We have carefully read the reviewers reports and we would like to respond to the concerns. Reviewer Malcolm Logan 1) p9 In transfection experiments the tagged proteins are visible as dot-like structures in the nucleus. While the co-localization with SALL4 looks convincing, is it true to say this shows the distribution corresponds to its location of biological function. We are looking at overexpression here in a (kidney-derived) tissue culture cell. This cell line does not represent an endogenous environment. It has not been shown how appropriate or otherwise this cell line is to analyze Tbx5 localisation/activity. I think statements regarding positional determination could be removed. We agree with Mr. Logan. The cell culture model used for our experiments does not represent an in vivo study. It is therefore speculative to talk about positional determination biological role of overexpressed proteins in COS7 cells. However, Fan et al. (2003) and others have used this procedure to analyze the impact of TBX5 mutations. Nevertheless we have replaced positional determination by intranuclear distribution 2) p12 Altered protein configuration or stearic hindrance effects on NLS are not directly tested. We agree with Mister Logan. Indeed Fan et al. (2003b) compared the predicted structure of TBX5 mutants to wild-type TBX5 by creating a model of the DNA-binding domain of TBX5 based on the crystal structure of TBX3. However, Fan and colleagues tested missense mutations within the DNA-binding domain. The mutation described in our manuscript is completely different. It would be highly speculative to predict the influence of the additional amino acids on the structure of the protein. By testing the nuclear localization we can assume that the gross protein structure is conserved and that the NLS is apparently not masked. However, we modified the sentence to clarify this point:
It is conceivable that an altered protein configuration or steric hindrance may silence the activity of the NLS motifs. We therefore wanted to analyze the intracellular distribution of the mutated TBX5 protein 3) p10 the mutation..abrogates activation of the ANF promoter p11. was severely affected as compared to wild-type. The mutant form of the protein is completely dead in the luciferase assay. We agree with Mister Logan. This sentence has been corrected in the manuscript to was not functional as compared to the wild type protein 4) Minor points It could be helpful to have a diagram to show the site of and how this missense mutation is predicted to lead to the addition of additional residues. We have added a diagram as supplementary figure p9 The functional domain of TBX5 is a T-Box motif. Surely this refers to just one of the functional domains of the protein. We agree with Mister Logan. This sentence has been corrected to An important functional domain of TBX5 is the T-Box motif p12 prolonged protein. change to elongated We have replaced prolonged by elongated. p12 For specific organs like heart and upper limbs, the nuclear localization of TBX5 during development is essential. Since Tbx5 is a transcription factor its nuclear localization will be essential wherever the protein is required to act as a transcription factor. We agree with Mister Logan. This sentence has been replaced by For the development of specific organs like heart and upper limbs, TBX5 is an essential transcription factor. P11.It is not clear what p.h445fsx136 refers to p = protein, H445 = histidine 445, fs = frameshift, X136 = additional 136 amino acids. We have put the abbreviation in brackets. It might now be clear that is refers to c1333delc. Reviewer Benoit Bruneau 1) while the mutant TBX5 plasmid does not activate ANF under the single condition tested, a range of amounts of transfected plasmid should be attempted to indicate if this is simply a dosage effect.
We agree with Mister Bruneau. We have therefore tested the activation of the ANF promoter by rising amounts of the TBX5wt and TBXmut constructs. We do not observe any change in luciferase activity. A dosage effect can therefore be ruled out. We have added this observation in the manuscript: An increased amount of transfected TBX5mut plasmid did not induce luciferase activity, ruling out a dosage effect. 2) transactivation with partners of Tbx5, including Nkx2-5, Gata4, and Sall4 (and combinations of these) should be performed to determine if this mutation affects thee interactions. And 3) Co-immunoprecipitations between TBX5 and the above-mentioned factors should be done to determine if the elongated protein can still associate with these partner proteins. We partially agree with Mister Bruneau. Indeed, it would be interesting to analyze the detailed characteristics of the elongated TBX5 protein including co-immunoprecipitations and transactivation of the ANF promoter with known interaction partners. It would also be interesting to test the activation of the known target gene FGF10. We have carried out the transactivation experiments with SALL4. We do not see any difference between wild type and mutated TBX5, suggesting that the interaction of SALL4 and TBX5 is not affected by the mutation. We did not include these results in the manuscript as other transactivation and interaction experiments (as proposed by Mister Bruneau) would be necessary. However, these experiments are very time-consuming and would go beyond the scope of this project. We have identified an unusual mutation in a patient with classical Holt-Oram syndrome. As the frameshift mutation presumably encompasses NMD, we assume that the protein is present and we speculate that it is somehow misfolded. Therefore the experiments we carried out are straight forward and sufficient with respect to our aim: we clearly show a loss of function of the elongated protein. 4) Minor points Reference #1: the word "corrected" is not part of the title, but indicates that the title was corrected from a previously incorrect title; please remove this word. The reference has been corrected. Reference to the T-box sites in the ANF promoter should include Bruneau et al 2001, in which the functionality of three such sites was demonstrated. We have included this reference.
Reviewer Philippe Debeer An extra figure with an XRay and/or clinical pictures would be interesting for readers who are not familiar with the syndrome. We have added an X-Ray and a clinical picture of the patient (fig 1). Additional comments 1) The experiments proposed by the reviewers were performed in cooperation with the PhD student Alexander Craig at the Institute of Human Genetics in Freiburg, Germany. Please note the modified authors list. 2) All experiments have been carried out in the cell culture model. Therefore we do not have a specific approval of an ethics committee. However, all kind of cell culture experiments with the cells mentioned in the manuscript have generally been approved by the ethics committee of the Freiburg University. 3) We have obtained informed consent including publication of the case report. For any questions please contact Britt-Marie Ekman-Joelsson or Wolfram Heinritz. 4) We have modified the manuscript style in accordance to the example shown on http://www.biomedcentral.com/info/ifora/medicine_journals We are highly interested in publishing our manuscript in BMC Medical Genetics and we would be pleased to hear that our comments and corrections meet the journal s and the reviewers expectations. If you have any further queries please do not hesitate to contact us. Best regards, Johann Böhm