Abstract
Congenital tooth agenesis (CTA) is one of the most common craniofacial anomalies. Its frequency varies among different population depending upon the genetic heterogeneity. CTA could be of familial or sporadic and syndromic or non-syndromic. Five major genes are found to be associated with non-syndromic CTA, namely PAX9, MSX1, EDA1, AXIN2, and WNT10A. Very few studies have been carried out so far on CTA on this Indian population making this study unique and important. This study was initiated to identify potential pathogenic variant associated with congenital tooth agenesis in an India family with molar tooth agenesis. CTA was investigated and a novel c.336C > G variation was identified in the exon 3 of PAX9, leading to substitution of evolutionary conserved Cys with Trp at 112th amino acid position located at the functionally significant DNA-binding paired domain region. Functional analysis revealed that p.Cys112Trp mutation did not prevent the nuclear localization although mutant protein had higher cytoplasmic retention. EMSA using e5 probe revealed that mutant protein was unable to bind with the paired-domain-binding site. Subsequently, GST pull-down assay revealed lower binding activity of the mutant protein with its known interactor MSX1. These in vitro results were consistent with the computational results. The in vitro and computational observations altogether suggest that c.336C > G (p.Cys112Trp) variation leads to loss of function of PAX9 leading to CTA in this family.
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Acknowledgements
We are thankful to all the members of the DEN14 family and all control individuals who provided their valuable time to participate in this study. Prof. S. Ganesh, Department of Biological Science and Bioengineering, IIT Kanpur, India, for his kind gift of pCDNA3.1 ( +) Myc-His(A) vector. We are grateful to Department of Biotechnology, Govt. of India for providing funds for this study. TS acknowledges the receipt of Junior and Senior Research Fellowship from University Grants Commission, Govt. of India (http://www.ugc.ac.in/).
Funding
This study was supported by The Department of Biotechnology, Govt. of India (Grant Number: BT/PR12638/MED/12/467/2009 dated 18/03/2010; http://dbtindia.nic.in/index.asp).
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P.D. and T.S. conceived and planned the experiments. S.K. identified and performed clinical investigations. A.G. planned the experiment for GST pull-down. P.D. supervised and arranged funding. P.R. did bioinformatics analysis. T.S. performed all the experiments. P.D. and T.S. wrote the manuscript. T.S., S.K., A.G., and P.D. critically reviewed the findings and the manuscript.
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Communicated by Shuhua Xu.
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Sarkar, T., Ranjan, P., Kanathur, S. et al. An in vitro and computational validation of a novel loss-of-functional mutation in PAX9 associated with non-syndromic tooth agenesis. Mol Genet Genomics 298, 183–199 (2023). https://doi.org/10.1007/s00438-022-01970-7
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DOI: https://doi.org/10.1007/s00438-022-01970-7