Browsing by Author "Manohar Lal Yadav"

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A gain-of-function mutation in CITED2 is associated with congenital heart disease
(Elsevier B.V., 2021) Manohar Lal Yadav; Dharmendra Jain; Neelabh; Damyanti Agrawal; Ashok Kumar; Bhagyalaxmi Mohapatra
CITED2 is a transcription co-activator that interacts with TFAP2 and CBP/ P300 transcription factors to regulate the proliferation and differentiation of the cardiac progenitor cells. It acts upstream to NODAL-PITX2 pathways and regulates the left-right asymmetry. Both human genetic and model organism studies have shown that altered expression of CITED2 causes various forms of congenital heart disease. Therefore, we sought to screen the coding region of CITED2 to identify rare genetic variants and assess their impact on the structure and function of the protein. Here, we have screened 271 non-syndromic, sporadic CHD cases by Sanger's sequencing method and detected a non-synonymous variant (c.301C>T, p.P101S) and two synonymous variants (c.21C>A, p.A7A; c.627C>G, p.P209P). The non-synonymous variant c.301C>T (rs201639244) is a rare variant with a minor allele frequency of 0.00011 in the gnomAD browser and 0.0018 in the present study. in vitro analysis has demonstrated that p.P101S mutation upregulates the expression of downstream target genes Gata4, Mef2c, Nfatc1&2, Nodal, Pitx2, and Tbx5 in P19 cells. Luciferase reporter assay also demonstrates enhanced activation of downstream target promoters. Further, in silico analyses implicate that increased activity of mutant CITED2 is possibly due to phosphorylation of Serine residue by proline-directed kinases. Homology modeling and alignment analysis have also depicted differences in hydrogen bonding and tertiary structures of wild-type versus mutant protein. The impact of synonymous variations on the mRNA structure of CITED2has been analyzed by Mfold and relative codon bias calculations. Mfold results have revealed that both the synonymous variants can alter the mRNA structure and stability. Relative codon usage analysis has suggested that the rate of translation is attenuated due to these variations. Altogether, our results from genetic screening as well as in vitro and in silico studies support a possible role of nonsynonymous and synonymous mutations in CITED2contributing to pathogenesis of CHD. © 2021
Computational exploration of TITIN variations: insights from whole exome sequencing and molecular dynamics simulation study
(Taylor and Francis Ltd., 2025) Amrita Mukhopadhyay; Bharti Devi; Anurag T.K. Baidya; Manohar Lal Yadav; Rajnish Kumar; Bhagyalaxmi Mohapatra
Titin (TTN), the largest known human protein (∼4 MDa), is considered as a key component for sarcomere integrity and function. Mutations in the TTN gene play a pivotal role in the genetic underpinnings of Dilated Cardiomyopathy (DCM). In the present study, we have conducted whole exome sequencing (WES) on 15 patients (5 familial and 10 sporadic) diagnosed with idiopathic DCM and identified 88 exonic variants. Here, we also report for the first time four novel variants comprising two frame-shifts, one missense, and one stop-codon variant. These variants are 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 have performed several bioinformatics analyses including pathogenicity prediction, protein stability, and protein-protein docking followed by molecular dynamics (MD) simulations on both wild-type and mutant TTN fragments with their corresponding interacting partners. We reveal that variations in the A-band domain significantly alter the protein’s 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. © 2025 Informa UK Limited, trading as Taylor & Francis Group.
Identification and characterization of genetic variants of TGFB1 in patients with congenital heart disease
(Elsevier B.V., 2022) Manohar Lal Yadav; Ashutosh Narayan Bhasker; Ashok Kumar; Bhagyalaxmi Mohapatra
Background: Congenital heart diseases (CHDs) are believed to be caused by abnormal gene functioning during embryonic heart development. Transforming growth factor-beta1 (TGFB1) is known to express in the early embryonic heart and regulates heart development. Methods: In this study, the coding region of TGFB1 was screened for 238 CHD patients by Sanger sequencing. Case-control association study was performed to identify the risk allele for CHD. In silico and in vitro approaches were used to elucidate the role of rare missense variant of TGFB1 using P19 cell line. Results: We identified a rare missense variant (c.29C > G; p.P10R) in the signal peptide of the TGFB1 in two cases (MAF = 0.0042017), which was absent in 200 healthy controls. Although this variation is reported in the gnomAD (rs1800470, MAF =0.0002386) and the ExAC database (MAF = 0.00064), it is not reported in INDEX-db and GenomeAsia 100K databases. We also found three polymorphisms, namely c.29C > T; p.P10L, c.74G > C; p.R25P and c.788C > T; p.T263I. Case-control studies revealed that c.29C > T (rs1800470) variation is a risk factor, significantly associated with the CHD phenotype (OR = 1.4361, P = 0.0083). However, c.74G > C (rs1800471) and c.788C > T (rs1800472) alleles are not associated with the disease. Additionally, two rare synonymous variations, i.e. c.348C > T; p.T116T (MAF = 0.0042017) and c.501C > T; p.H167H (MAF = 0.00210084) were also identified in two and one cases, respectively. These were absent in the 200 controls. In silico analysis showed that missense variation p.P10R enhances the formation of the α-helix in the signal peptide, which possibly increases the TGFB1 secretion. The luciferase-reporter assay demonstrated significantly increased activity of p(SBE)4 (P = 0.016) and p(CAGA)12 (P = 0.0004) promoters in response to p.P10R mutant versus wild-type TGFB1. Conclusion: The p.P10R variant of TGFB1 implicated a gain-of-function activity which is potentially deleterious, while the c.29C > T variation is a risk factor associated with the CHD. © 2021 Elsevier B.V.
Implication of rare genetic variants of NODAL and ACVR1B in congenital heart disease patients from Indian population
(Elsevier Inc., 2021) Manohar Lal Yadav; Prashant Ranjan; Parimal Das; Dharmendra Jain; Ashok Kumar; Bhagyalaxmi Mohapatra
NODAL signaling plays an essential role in vertebrate embryonic patterning and heart development. Accumulating evidences suggest that genetic mutations in TGF-β/NODAL signaling pathway can cause congenital heart disease in humans. To investigate the implication of NODAL signaling in isolated cardiovascular malformation, we have screened 300 non-syndromic CHD cases and 200 controls for NODAL and ACVR1B by Sanger sequencing and identified two rare missense (c.152C > T; p.P51L and c.981 T > A; p.D327E) variants in NODAL and a novel missense variant c.1035G > A; p.M345I in ACVR1B. All these variants are absent in 200 controls. Three-dimensional protein-modelling demonstrates that both p.P51L and p.D327E variations of NODAL and p.M345I mutation of ACVR1B, affect the tertiary structure of respective proteins. Variants of NODAL (p.P51L and p.D327E) and ACVR1B (p.M345I), significantly reduce the transactivation of AR3-Luc, (CAGA)12-Luc and (SBE)4-Luc promoters. Moreover, qRT-PCR results have also deciphered a reduction in the expression of cardiac-enriched transcription factors namely Gata4, Nkx2-5, and Tbx5 in both the mutants of NODAL. Decreased expression of, Gata4, Nkx2-5, Tbx5, and lefty is observed in p.M345I mutant of ACVR1B as well. Additionally, reduced phosphorylation of SMAD2/3 in response to these variants, suggests impaired NODAL signaling and possibly responsible for defective cell fate decision and differentiation of cardiomyocytes leading to CHD phenotype. © 2021 Elsevier Inc.