Lagnajeet Pradhan

A Mouse Model of Human Congenital Heart Disease High Incidence of Diverse Cardiac Anomalies and Ventricular Noncompaction Produced by Heterozygous Nkx2-5 Homeodomain Missense Mutation

Background—Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5, in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results—We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5+/R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5+/R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5+/+ or Nkx2-5+/− mice. Conclusions—The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain (R52G) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.Background— Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5 , in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results— We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5 +/ R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5 +/ R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5 +/+ or Nkx2-5 +/− mice. Conclusions— The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain ( R52G ) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.

NuProPlot: nucleic acid and protein interaction analysis and plotting program

Growing numbers of protein and nucleic acid complex structures are being determined and deposited in the Protein Data Bank and the Nucleic Acid Database. With the increasing complexity of these structures, it is challenging to analyse and visualize the three-dimensional interactions. The currently available programs for such analysis and visualization are limited in their applications. They can only analyse a subset of protein-nucleic acid complexes and require multiple iterations before obtaining plots that are suitable for presentation. An interactive web-based program, NuProPlot (http://www.nuproplot.com), has been developed which can automatically identify hydrogen, electrostatic and van der Waals interactions between proteins and nucleic acids and generate a plot showing all of the interactions. Protein-DNA and protein-RNA interactions can be visualized in simple two-dimensional schematics. Interactive schematic drawing options allow selection of the plotted area and repositioning of the individual interactions for better legibility. NuProPlot is a fully automated and user-friendly program providing various custom options. NuProPlot represents a greatly improved option for analysis and presentation of protein-nucleic acid interactions.

Crystallization and preliminary X-ray analysis of the cardiac transcription factor complex of NKX2.5 and TBX5 with DNA

Heart development depends on timely expression of genes regulated by combinatorial interactions of master cardiac transcription factors. To elucidate the molecular basis of their interactions, a ternary complex of cardiac transcription factors, NKX2.5 and TBX5, and their target DNA was studied using X-ray crystallography. Here, the purification, crystallization and preliminary X-ray crystallographic analyses of the NKX2.5 homeodomain and TBX5 DNA-binding domain complex with a DNA element from the -252 promoter region of the atrial natriuretic factor are reported. The crystal diffracted to 2.88 Å resolution and belonged to space group P21, with unit-cell parameters a = 69.30, b = 77.78, c = 77.60 Å, β = 108.31°. Two sets of ternary complexes are present in an asymmetric unit with a solvent content of 54%.

A Forkhead Box Protein C2 Inhibitor: Targeting Epithelial-Mesenchymal Transition and Cancer Metastasis

The epithelial–mesenchymal transition (EMT) has been suggested as a new target for therapeutic intervention of metastatic cancer. Forkhead box protein C2 (FOXC2) is known to be necessary for initiating and maintaining EMT, and therefore bestows on cancer cells metastatic and cancer stem cell (CSC)‐like phenotypes, allowing cells to acquire higher motility, invasiveness, self‐renewal, and therapy resistance. Here, we describe the first inhibitor of FOXC2, MC‐1‐F2. MC‐1‐F2 was able to induce cadherin switching and reverse EMT through the degradation of FOXC2 and blocking of its nuclear localization. In addition, MC‐1‐F2 was very effective in inhibiting cancer cell migration and invasion. As the first small‐molecule inhibitor of FOXC2 and the first compound targeting EMT‐associated transcription factor, MC‐1‐F2 will pave the way for a new anticancer therapeutic agent targeting metastatic cancer and help to elucidate the network of EMT signaling pathways.

A Mouse Model of Human Congenital Heart DiseaseClinical Perspective

Background—Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5, in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results—We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5+/R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5+/R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5+/+ or Nkx2-5+/− mice. Conclusions—The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain (R52G) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.Background— Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5 , in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results— We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5 +/ R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5 +/ R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5 +/+ or Nkx2-5 +/− mice. Conclusions— The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain ( R52G ) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.

A Mouse Model of Human Congenital Heart DiseaseClinical Perspective: High Incidence of Diverse Cardiac Anomalies and Ventricular Noncompaction Produced by Heterozygous Nkx2-5 Homeodomain Missense Mutation

Background—Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5, in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results—We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5+/R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5+/R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5+/+ or Nkx2-5+/− mice. Conclusions—The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain (R52G) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.Background— Heterozygous human mutations of NKX2-5 are highly penetrant and associated with varied congenital heart defects. The heterozygous knockout of murine Nkx2-5 , in contrast, manifests less profound cardiac malformations, with low disease penetrance. We sought to study this apparent discrepancy between human and mouse genetics. Because missense mutations in the NKX2-5 homeodomain (DNA-binding domain) are the most frequently reported type of human mutation, we replicated this genetic defect in a murine knockin model. Methods and Results— We generated a murine model in a 129/Sv genetic background by knocking-in an Nkx2-5 homeodomain missense mutation previously identified in humans. The mutation was located at homeodomain position 52Arg→Gly (R52G). All the heterozygous neonatal Nkx2-5 +/ R52G mice demonstrated a prominent trabecular layer in the ventricular wall, so called noncompaction, along with diverse cardiac anomalies, including atrioventricular septal defects, Ebstein malformation of the tricuspid valve, and perimembranous and muscular ventricular septal defects. In addition, P10 Nkx2-5 +/ R52G mice demonstrated atrial sepal anomalies, with significant increase in the size of the interatrial communication and fossa ovalis, and decrease in the length of the flap valve compared with control Nkx2-5 +/+ or Nkx2-5 +/− mice. Conclusions— The results of our study demonstrate that heterozygous missense mutation in the murine Nkx2-5 homeodomain ( R52G ) is highly penetrant and result in pleiotropic cardiac effects. Thus, in contrast to heterozygous Nkx2-5 knockout mice, the effects of the heterozygous knockin mimic findings in humans with heterozygous missense mutation in NKX2-5 homeodomain.