Biology subjects

Mohapatra, B.

Publications and source records attributed to Mohapatra, B..

9 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

Implication of a rare variant in OPA1 in Cardiac Pathophysiology: From Cristae Remodelling to Contractile Dysfunction

Abstract Optic Atrophy 1 (OPA1), an important inner mitochondrial membrane GTPase, regulates mitochondrial fusion, maintains cristae structure, calcium buffering, cellular bioenergetics, preserves mtDNA and controls apoptosis. Here we examined the role of OPA1 variants in DCM using whole-exome sequencing (WES) of 5 familial and 10 sporadic DCM cases. A rare de novo OPA1 variant, c.563C>T (p.Pro188Leu), was identified in a DCM patient, which is absent in 100 healthy controls as well as in the 1000 Genomes, IndiGenomes and GenomeAsia 100k databases while it showed very low MAF (0.000069) in GnomAD. Structural modelling predicted the variant to be highly deleterious and revealed marked conformational distortion of the mutant protein (RMSD = 3.5 Angstrom). Molecular docking further demonstrated enhanced accessibility of mutant OPA1 to mitochondrial protease OMA1, suggesting increased OPA1 proteolytic processing and a consequent increase in mitochondrial fragmentation. Functional analysis in stable H9C2 cardiomyoblast cells, demonstrated significantly reduced OPA1 protein expression, extensive mitochondrial fragmentation in mutant-OPA1 expressing cells. The mutant protein caused significant reduction in mitochondrial membrane potential, ATP generation, and oxygen consumption rate (OCR), together with elevated cytosolic Calcium and reactive oxygen species (ROS) levels. qRT-PCR analysis further revealed depletion in mtDNA copy number and increased in expression of intrinsic apoptotic markers Caspase3, 9 and Bax/Bcl-2 ratio. The above findings collectively highlighted the significant impact of the OPA1 mutation on mitochondrial dynamics and cellular health, suggesting a significant correlation with the pathogenesis of DCM. Collectively, these findings suggest that OPA1-mediated mitochondrial dysfunction represents a potential therapeutic avenue for the management of DCM.

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↗

bioRxiv2026

Metagenome-assembled genomes (MAGs) revealed a functionally stratified microbiome in Jeevamrit, enabling co-operative nutrient cycling and rhizospheric growth promotion in the natural farming practices

Jeevamrit, a fermented liquid microbial bioinoculant, is increasingly recognized as a soil and plant growth enhancer in sustainable agricultural practices such as zero-budget natural farming; however, the genetic pool attributed to the functionality by microbial constituents remained poorly resolved. In this study, we reconstructed 16 high-quality metagenome-assembled genomes (MAGs) from Jeevamrit under two critical mixing regimes to elucidate the contributions of key taxa affiliated with Pseudomonadota, Bacillota, and Bacteroidota to nutrient cycling and plant growth promotion. Functional annotation revealed a stratified (upper-middle-lower) metabolic organization with interdependent interactions driving combinatorial functionality. Upper-layer MAGs, including Klebsiella and Pseudaeromonas exhibited organic polymer degradation, glycolytic and oxidative carbon metabolism, respiratory versatility with nutrient acquisition traits such as nitrogen fixation and phosphate/iron solubilization. In middle and lower-layer, Trichococcus, Clostridium, and Veillonella displayed fermentative and reductive metabolisms that facilitate the turnover of partially degraded organic matter and production of organic acids, nitrogen transformations, and metabolic cross-feeding under fluctuating redox conditions. Phylogenetic and taxono-genomic analyses support the designation of eight MAGs as novel species (sp. nov.), for which new names are proposed. A consensus genetic map deciphered traits linked to phytohormone biosynthesis (IAA, cytokinins), quorum-sensing-mediated rhizosphere colonization, and abiotic stress tolerance. Ultimately, this culture-independent metagenome study underpins field-relevant mechanistic insights into an indigenous microbial inoculant, highlighting its potential as a locally adapted solution for sustainable agriculture. ImportanceMicrobial bioinoculants such as Jeevamrit are increasingly used in sustainable agriculture, yet their functional basis remains insufficiently understood due to the limited genome-level resolution of constituent microbiota. This study addresses this gap by applying genome-resolved metagenomics to connect microbial diversity with agriculture-associated ecological functions in a complex fermented local formulation. By integrating metabolic reconstruction with plant-associated functional traits, this study advances understanding of how microbial consortia contribute to nutrient mobilization, rhizosphere competence, and environmental adaptability. This highlights the contribution of yet-to-be-cultivated but metabolically versatile taxa, which are important to the functions of agricultural ecosystems. By uncovering the roles of key microbes and their cooperative metabolic interactions, this work provides a scientific basis for improving Jeevamrit formulations through informed selection or enrichment of functionally important microbes to enhance nutrient delivery and plant growth performance.

microbiology↗

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

Identification and functional characterization of LDB3 gene variants in DCM patients from Indian Population

The LIM domain-binding protein 3 (LDB3), also known as Z-band alternatively spliced protein (ZASP), is a key component of the Z-disc, essential for maintaining sarcomeric integrity and transmitting contractile force in cardiomyocytes. Genetic variants in LDB3 have been implicated in cardiomyopathies, including dilated cardiomyopathy (DCM). In this study, we have screened 100 idiopathic DCM cases along with controls by Sanger sequencing and identified nine non-synonymous (p.S184I, p.D193N, p.K204R, p.R229C, p.P295T, p.Q402P, p.F465I, p.F496L, p.P606S), seven synonymous (p.T91=, p.A152=, p.D168=, p.S182=, p.A279=, p.S347=, p.A358=), and seven intronic (c.322-50G>C, c.548+53A>C, c.718+47G>C, c.718+81G>A, c.755+11G>A, c.755+86G>A, c.*30C>G) variants, among which six (p.S184I, p.D193N, p.P295T, p.F465I, p.F496L, p.P606S) missense variants were novel. In-vitro studies demonstrated that p.K204R increased LDB3 expression whereas p.S184I, p.F465I, p.F496L, p.P606S reduced LDB3 expression. Immunostainning revealed cytoplasmic aggregation of the p.K204R-LDB3 protein. The variant p.R229C also over-expressed the mutant protein. In case of variant, p.P295T, Z-disc was severely disrupted indicating impaired Z-disc integrity. Furthermore, synonymous variants showed altered mRNA folding, stability, and codon usage bias, potentially affecting translation efficiency. In-silico analyses predicted p.F465I, p.F496L, p.P606S, and p.S184I variants to be deleterious, significantly altering protein structure. Structural modeling using AlphaFold2 showed high RMSD values, suggesting conformational destabilization. Collectively, these findings highlight LDB3 as a hypermutable candidate gene for DCM in our cohort that may contribute to DCM pathogenesis by perturbing protein structure, expression, and cellular localization, underscoring the critical role of LDB3 in cardiac muscle function and disease.

genetics↗

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↗

bioRxiv2024

Implication of TITIN Variations in Dilated Cardiomyopathy: Integrating Whole Exome Sequencing With Molecular Dynamics Simulation Study

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure, characterized by ventricular dilation and impaired systolic function. Variations in the TITIN (TTN) gene, which encodes the giant muscle protein TTN, play a pivotal role in the genetic underpinnings of DCM. We conducted WES on 15 patients (5 familial and 10 sporadic) diagnosed with idiopathic DCM and identified 88 exonic variants including four novel variants. These variants were predominantly located in the A-band region (39 variants) of TTN, a critical region for its mechanical stability and interaction with other sarcomeric proteins, followed by the I-band domain (33 variants), Z-disc domain (7 variants) and M-band region (9 variants). To discern the functional repercussions of these variations, we performed several bioinformatics analyses including pathogenicity prediction, protein stability, and protein-protein docking followed by MD simulations on both wild-type and mutant TTN fragments with their corresponding interacting partners (TCAP, MYH7, LMNA). We revealed that variations in the A-band domain significantly alter the proteins structural dynamics, leading to decreased mechanical stability and altered protein-protein interactions. These changes are likely to disrupt sarcomere function, thereby elucidating their role in the pathogenesis of DCM. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/622829v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@15d3eb1org.highwire.dtl.DTLVardef@bf096dorg.highwire.dtl.DTLVardef@4141dorg.highwire.dtl.DTLVardef@1c4948b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗