Biology subjects

Maddhesiya, J.

Publications and source records attributed to Maddhesiya, J..

5 recordsLinked to original sources

bioRxiv2026

Unravelling the role of IRX4 variants in non-syndromic and Down syndrome associated congenital heart disease

IRX4 is a TALE- homeodomain transcription factor which is essential for cardiac development. In murine models, Irx4 deficiency leads to impaired ventricular function and results in cardiomyopathy. To elucidate the role of IRX4 in human congenital heart disease (CHD), Sanger sequencing of the IRX4 gene was performed in 205 individuals with non-syndromic CHD, 24 Down syndrome (DS) cases with CHD, 27 DS cases without CHD, and 150 healthy control individuals. Two novel (p.Ser24Asn and p.Thr217Iso) and one reported variant (rs2232376) were identified in non-syndromic CHD. Concurrently, rs2232376 was also detected in DS with CHD. The first novel (p.Ser24Asn) and reported (rs2232376) variants lie in the N-terminal region while the second novel (Thr217Iso) variant lies within the TALE homeodomain. In silico structural modelling suggested that both the novel variants (p.Ser24Asn and Thr217Iso) induce conformational changes in the IRX4 protein, potentially altering its DNA-binding affinity. A significant reduced expression of IRX4 muteins was noted in Western blotting by both variants (p.Ser24Asn and Thr217Iso). Furthermore, luciferase reporter assays demonstrated decline in the activity of Nanog promoter and HEY2 enhancer in response to both the variants which was further corroborated by decrease mRNA expression in qRT-PCR. Additional downstream targets, including Nfyc, Nppa, and Bmp10, also exhibited anomalous expression due to both the variants (p.Ser24Asn and Thr217Iso). Altogether, the aberrant expression of muteins as well as downstream target genes along with compromised activities of promoters substantiate the pathogenic potential of the identified IRX4 variants and underscore the critical role of IRX4 in regulating multiple stages of cardiogenesis.

genetics↗

bioRxiv2026

Ethanol-induced activation of BMP signaling and reprogramming of cardiomyocytes' transcriptome

Congenital heart disease (CHD) comprises a diverse group of structural heart defects present at birth due to complex interactions between genetic and environmental factors. Prenatal alcohol exposure (PAE) is a known environmental factor that disrupts fetal cardiogenesis and increases the risk of CHD. However, the molecular mechanisms behind ethanol (EtOH)-induced CHD remain obscure. This study investigated the effects of EtOH on bone morphogenetic protein (BMP) signaling and transcriptomic reprograming in HL-1 cardiomyocytes. HL-1 cells were treated with varying concentrations of EtOH (25, 50, and 100 mM) for 24 h. 100 mM of EtOH exposure significantly enhanced SMAD1/5 phosphorylation and upregulated BMP-responsive genes, namely Id1, Gata4, Mef2c, and Nkx2.5. Increased histone acetyltransferase activity further validated activation of BMP signaling through histone hyperacetylation. These effects were reversed by the BMP pathway inhibitor LDN-193189, confirming pathway-specific activation. Further, transcriptome analysis following 100 mM EtOH treatment identified 3,876 differentially expressed genes. KEGG enrichment analysis revealed significant dysregulation of cardiogenic pathways, including TGF-{beta}, Hedgehog, PI3K-Akt, Notch, FoxO, and calcium signaling pathways, along with extracellular matrix-receptor interaction and focal adhesion pathways. Gene Ontology analysis highlighted disturbances in heart development, cellular differentiation, apoptosis, extracellular matrix (ECM) organization, and chromatin regulation. Network analysis identified key hub genes, viz. Kras, Fn1, Col1a1, Prkaca, Fbn1, Col6a1, Col6a2, Ccnd1, Col1a2 and Myc which are upregulated and Hsp90aa1, Mdm2, Jun, Hras, Il6, Hsp90ab1, Pdgfra, Cdkn1a, Pparg, Fos and Hspa8 are downregulated which were subsequently validated by qRT-PCR. Collectively, these findings provide novel insights into the molecular basis of EtOH-induced CHD and identify potential biomolecule candidates for future therapeutic investigation.

cell biology↗

bioRxiv2025

Deciphering the functional association of novel variants of BMP7 in isolated congenital heart disease by integrating in vitro and in silico approaches

Bone morphogenetic protein7 (BMP7), an important member of the TGF{beta} superfamily, is known to be vital for embryonic growth and development. To decipher the role of BMP7 in congenital cardiac malformations, genetic screening of 285 CHD cases along with 400 healthy controls individuals was performed by Sanger sequencing method. Five missense variants were identified in 5 unrelated CHD probands with distinct phenotypes. Three novel missense (p.D85V, p.R175W, and p.A283T) variants in the pro-peptide region and two other variants (p.M315I and p.N321S) in the mature domain were documented. In vitro functional analysis revealed WT as well as all five mutant BMP7 proteins localized within ER compartment, which was confirmed by ER-Tracker and SERCA2 staining. Western blot analysis demonstrated enhanced phosphorylation of SMAD1/5 associated with these variants. Furthermore, transactivation assays showed increased activity of BMP-responsive promoters-Id1-luc, Id3-luc, Tlx2-luc, and p(SBE)4-luc with a synergistic effect observed upon AKL2 co-expression. Additionally, overexpression of downstream targets, namely Bmp2, Bmp7, Nkx2.5, Gata4, Irx4, Smad1, Smad4, and Smad5, alongside downregulation of left-right patterning genes (Nodal and Pitx2) and the BMP antagonist Chordin, was observed. Likewise, EdU staining revealed increased cellular proliferation associated with all these variants. Moreover, modeling of secondary and tertiary structures also suggested that these variants might induce conformational changes in BMP7, potentially strengthening its binding affinity with the receptor and thereby amplifying SMAD signaling. Collectively, in vitro and in silico analyses suggest that these BMP7 variants exhibit gain-of-function (GoF) activity, disrupting normal BMP signaling pathways and thereby contributing to the development of CHD.

molecular biology↗

bioRxiv2025

Implication of novel variants of BMP2 in isolated congenital heart disease: Functional characterization by in silico and invitro approaches

Bone morphogenic protein2 (BMP2), a member of TGF-{beta} super-family, known to play a wide range of roles during embryonic development, particularly in the formation of bone/skeleton, differentiation of neurons, skeletal muscle, and development of cardiac valve septa and outflow tract. BMP2 haploinsufficiency is reported to cause multiple congenital malformations including cardiac defects mainly endocardial cushion formation and chamber specification. To investigate the functional relevance of BMP2 variations in isolated CHD cases, we performed genetic screening of BMP2 in 285 CHD probands along with 400 healthy controls by Sangers method. Five non-synonymous variants namely, an already known variant p.Ser37Ala in N-terminal region, one nonsense variant, p.Lys241X in pro-peptide region and three missense variants p.His321Leu, p.Glu328Lys and p.Ser351Cys in mature domain, were identified in 8 unrelated CHD cases. In vitro functional analysis by western blotting depicted an increase in phosphorylation of SMAD1/5 due to all five variants. Furthermore, overexpression of cardiac-specific downstream target genes namely Smad1, Smad4, Smad5, Nkx2.5, Gata4 and Irx4 of the BMP pathway was observed in response to all the variants. Luciferase assay also validated the enhanced expression of multiple downstream promoters Id1-luc, Id3-luc, Tlx2-luc, and p(SBE)4-luc. Additionally, computational analysis of RNA structural features and protein secondary and tertiary structural changes also highlighted the increased activity of mutants, possibly due to enhanced interactions of mutant proteins with their binding partners owing to more stable structures. Overall, this is the first study which characterized the functional association of BMP2 variants with the pathogenesis of CHD by in vitro and in silico methods.

genetics↗

bioRxiv2025

Functionally significant, novel variants of BMP4 are associated with isolated congenital heart disease

AbstractBone morphogenetic proteins (BMPs) are multipotent cytokines of TGF{beta} super family, involved in wide range of biological processes including embryonic development, tissue differentiation, cell proliferation, migration and organogenesis. BMP signaling also plays a pivotal role during different phases of cardiogenesis. Although genetic variations in several components of BMP signaling have been linked to congenital heart disease (CHD), many of these findings lack thorough functional validation. To assess the role of BMPs in CHD, Sanger sequencing of BMP4 gene was conducted in 285 CHD cases along with 400 healthy controls. Four missense novel pathogenic variants were detected in four unrelated CHD probands with heterogeneous phenotypes. All the four variants (p.R113G, p.E151V, p.T197I and p.R226W) were located in BMP4 pro-peptide domain. Western blotting analysis revealed a significant increase in the phosphorylation of SMAD1/5 caused by all the four variants. Further, all the four variants enhance the transactivation of BMP-responsive promoters, Id1-luc and Id3-luc in luciferase reporter assay. Moreover, qRT-PCR analysis validating the enhanced endogenous expression of downstream targets namely Smad1, Smad5, Id1, Id3, and Irx4 which further confirm the augmented activity due to all the four variants. Besides, our computational modeling of RNA structures and its features, modifications in secondary and tertiary structures and various physiochemical properties are speculated to enhanced the binding of BMP4 muteins with its respective partner and thereby boosting the SMAD-dependent BMP signaling. Altogether, both in vitro and in silico observations unveiling the gain-of-function activity of mutants which potentially perturbing the normal BMP signaling/ dynamics, consequently inducing CHD.

genetics↗