Browsing by Author "Subodh Kumar Singh"

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A novel GLI3c.750delC truncation mutation in a multiplex Greig cephalopolysyndactyly syndrome family with an unusual phenotypic combination in a patient
(Elsevier B.V., 2014) Rashmi Patel; Fanish Mani Tripathi; Subodh Kumar Singh; Anjali Rani; Visweswar Bhattacharya; Akhtar Ali
Greig cephalopolysyndactyly (GCPS) syndrome is an autosomal dominant disorder with high penetrance in majority of cases, characterized by a triad of polysyndactyly, macrocephaly and hypertelorism. GCPS is known to be caused by mutations in the transcription factor GLI3 gene (7p13) which results in functional haploinsufficiency of this gene. The present study reports a large multiplex family having 12 members affected with GCPS in 3 generations and several unaffected members showing autosomal dominant pattern of inheritance with complete penetrance. Interestingly an affected member of the family had unusual features including thumb which is although biphalangeal (confirmed with X-ray) but morphologically looks like finger and a unilateral tiny bony outgrown (externally indistinguishable) on the distal phalanx of the first toe of the left foot. This member also presented with mild ichthyosis. Although it is also possible that one or more of these features are coincidentally present in this member and might not be part of GCPS. Resequencing of the GLI3 gene detected a novel frame-shift mutation c.750delC in heterozygous state transmitting in the family and co-segregating with the disorder suggesting it to be the causal for the GCPS phenotype in the family. In silico analysis suggests that this mutation creates a truncated GLI3 protein resulting in its haploinsufficiency leading to GCPS syndrome. Furthermore, genotype-phenotype correlation is supported by the mutation as it lies in the amino terminal domain of the protein. © 2014.
A novel non-coding RNA within an intron of CDH2 and association of its SNP with non-syndromic cleft lip and palate
(Elsevier B.V., 2018) Priyanka Kumari; Subodh Kumar Singh; Rajiva Raman
Background: Genome-wide linkage analysis and whole genome sequencing in a Van der Woude syndrome (VWS) family revealed that the SNP, rs539075, within intron 2 of the cadherin 2 gene (CDH2) co-segregated with the disease phenotype. Results: A study with nonsyndromic cleft lip with or without cleft palate (NSCL ± P) cases (N = 292) and controls (N = 287) established association of this SNP with NSCL ± P as a risk factor. RT-PCR based expression analysis of the SNP-harbouring region of intron 2 of CDH2 in the clefted lip and/or palate tissues of 16 patients revealed that the mutant allele expressed in all those individuals having it (hetero-/homozygous), whereas the wild type allele expressed in <50% of the samples in which it was present. The intronic transcript was also present in the prospective lip and palate region of 13.5 dpc mouse embryo, detected by RNA in situ hybridization and RT-PCR. Conclusions: These results including the in silico, characterization of the ~200 nt-intronic transcript showed that conformationally it fits best with noncoding small RNA, possibly a precursor of miRNA. Its function in the orofacial organogenesis remains to be elucidated which will enable us to define the role of this mutant ncRNA in the clefting of lip and palate. © 2018 Elsevier B.V.
Coding region of IRF6 gene may not be causal for Van der Woude syndrome in cases from India
(2009) Akhtar Ali; Subodh Kumar Singh; Rajiva Raman
Objective: Evaluation of the IRF6 gene in Van der Woude syndrome cases from an Indian population. Subjects: Nine affected and four unaffected individuals from seven families with Van der Woude syndrome as well as five normal controls (with no history of Van der Woude or any other congenital malformation and belonging to the same geographical area as the families with Van der Woude syndrome). Method: Direct sequencing of all coding regions and exon-intron boundaries of the IRF6 gene. Results: Five novel variants: IVS1+3900 A>G, 191 T>C, IVS4+775 C>T, IVS8+218 C>T, 1511 T>A (Ser 416 Arg) and two known variants: IVS6+27 C>G, 1083 G>A (V274I) were detected.Except for one, all were in noncoding regions either in 3′UTR or in introns. There was only one mutation in the coding region, detected in a normal control. Conclusion: The present report indicates that point mutations in the coding region of the IRF6 gene may not be a major cause of Van der Woude syndrome in Indian populations.
Extensive facial damage caused by a blast injury arising from a 6 volt lead accumulator
(Churchill Livingstone, 1999) Subodh Kumar Singh; P. Jain; J.K. Sinha
Low-voltage electrical injuries are relatively uncommon. Injury caused by flow of heavy current due to short-circuiting a low-voltage battery has not been described in the English literature. A 9-year-old boy connected two thin household electrical wires to the two terminals of a 6 volt (lead accumulator) battery and pressed the other two ends between his teeth. This resulted in a blast causing a compound comminuted fracture of the mandible and extensive tissue damage in the oral cavity. The low internal resistance of a lead accumulator (~ 0.03 ohms) permits the flow of a heavy current (~ 200 amps) when short-circuited. This instantaneously vaporises a minuscule portion of wire at ~ 2000 K resulting in a sudden rise of intraoral pressure to 30 kg cm-2 leading to tissue damage.
Genetic heterogeneity in Van der Woude syndrome: identification of NOL4 and IRF6 haplotype from the noncoding region as candidates in two families
(Springer India, 2018) Priyanka Kumari; Akhtar Ali; Subodh Kumar Singh; Amit Chaurasia; Rajiva Raman
Van der Woude syndrome (VWS) shows an autosomal dominant pattern of inheritance with two known candidate genes, IRF6 and GRHL3. In this study, by employing genome-wide linkage analyses on two VWS affected families, we report the cosegregation of an intronic rare variant in NOL4 in one family, and a haplotype consisting of three variants in the noncoding region of IRF6 (introns 1, 8 and 3′UTR) in the other family. Using mouse, as well as human embryos as a model, we demonstrate the expression of NOL4 in the lip and palate primordia during their development. Luciferase, as well as miRNA-transfection assays show decline in the expression of mutant NOL4 construct due to the creation of a binding site for hsa-miR-4796-5p. In family 2, the noncoding region IRF6 haplotype turns out to be the candidate possibly by diminishing its IRF6 expression to half of its normal activity. Thus, here we report a new candidate gene (NOL4) and a haplotype of IRF6 for VWS, and highlight the genetic heterogeneity of this disorder in the Indian population. © 2018, Indian Academy of Sciences.
GLI3 mutations in syndromic and non-syndromic polydactyly in two Indian families
(Blackwell Publishing, 2016) Rashmi Patel; Chandra Bhan Singh; Visweswar Bhattacharya; Subodh Kumar Singh; Akhtar Ali
The GLI3 protein is a zinc finger transcription factor, expressed early in development. The GLI3 gene exhibits allelic heterogeneity as mutations in this gene are associated with several developmental syndromic and non-syndromic polydactyly. The present study reports two cases: first, a familial case of Greig Cephalopolysyndactyly Syndrome (GCPS); the second is a sporadic case with both postaxial polydactyly (PAP) type A and B. Resequencing of GLI3 gene reveals a previously reported nonsense truncation mutation g.42007251G>A (p.R792X; rs121917714) in the GCPS family and a novel single nucleotide insertion g.42004239_42004240insA (p.E1478X) in the sporadic case of postaxial polydactyly (PAP). Both nonsense truncation mutations; p.R792X (GCPS) and p.E1478X (PAP) introduce a premature stop codon leading to loss of C-terminal domains. © 2016 Japanese Teratology Society.
MTHFR 677TT alone and IRF6 820GG together with MTHFR 677CT, but not MTHFR A1298C, are risks for nonsyndromic cleft lip with or without cleft palate in an Indian population.
(2009) Akhtar Ali; Subodh Kumar Singh; Rajiva Raman
AIM: To determine the association of three SNPs, IRF6 G820A, MTHFR C677T, and MTHFR A1298C, with nonsyndromic cleft lip with or without cleft palate (NSCL/P) in an Indian population. METHOD: A total of 323 NSCL/P patients, 116 of their mothers, 108 of their fathers, and 214 normal controls have been examined for the above three SNPs. RESULT: Frequency of IRF6 GG was 65% in controls, 78% in cases, 84% in case-fathers, and 80% in case-mothers. MTHFR 677T homozygosity was lower than 1% in controls and unaffected parents, while in the group of probands it was much higher (3.4%; OR 4.30). The frequency of CT genotype was also high in the cases and case-mothers (OR 1.89 and 2.2, respectively). MTHFR A1298C did not reveal a statistically significant deviation in allele and genotype frequencies. CONCLUSION: While MTHFR 677T homozygotes show a significant association with NSCL/P, heterozygotes 677CT are minor risk factors. MTHFR A1298C does not show a risk in any combination of alleles. IRF6 820GG too forms a minor risk. However, combined genotypes IRF6 GG/MTHFR 677CT together form greater risk for NSCL/P.
Novel GLI3 pathogenic variants in complex pre- and postaxial polysyndactyly and Greig cephalopolysyndactyly syndrome
(John Wiley and Sons Inc, 2021) Rashmi Patel; Subodh Kumar Singh; Visweswar Bhattacharya; Akhtar Ali
Polydactyly is a limb malformation and can occur as nonsyndromic polydactyly, syndromic polydactyly, or along with other limb defects. A few genes have been identified that cause various forms of syndromic and nonsyndromic polydactyly, of which GLI3 has been extensively explored. In the present study, GLI3 gene was screened by direct resequencing in 15 polydactyly cases with or without other anomalies. GLI3 screening revealed two novel pathogenic variants, NM_000168.6:c.3414delC [p.(H1138Qfs*68)] and NM_000168.6:c.1862C>T [p.(P621L)], found in two unrelated cases of familial complex pre- and postaxial polysyndactyly and sporadic Greig cephalopolysyndactyly syndrome (GCPS), respectively. The first pathogenic GLI3 variant, NM_000168.6:c.3414delC, causes premature protein truncation at the C-terminal domain of GLI3. Alternatively, the second pathogenic variant, NM_000168.6:c.1862C>T, lies in the DNA binding domain of GLI3 protein and may affect its hydrophobic interaction with DNA. Both pathogenic GLI3 variants had reduced transcriptional activity in HEK293 cells that likely had led to haploinsufficiency and, consequently, the clinical phenotypes. Overall, the present study reports a novel familial case of complex pre- and postaxial polysyndactyly and the underlying novel pathogenic GLI3 variant expanding the clinical criteria for GLI3 mutational spectrum to complex pre- and postaxial polysyndactyly. Furthermore, this study also reports a novel GLI3 pathogenic variant linked to GCPS, highlighting the known genotype–phenotype correlation. © 2020 Wiley Periodicals LLC
Novel HOXD13 variants in syndactyly type 1b and type 1c, and a new spectrum of TP63-related disorders
(Springer Nature, 2022) Rashmi Patel; Subodh Kumar Singh; Visweswar Bhattacharya; Akhtar Ali
Syndactyly is the most common limb defect depicting the bony and/or cutaneous fusion of digits. Syndactyly can be of various types depending on the digits involved in the fusion. To date, eight syndactyly-associated genes have been reported, of which HOXD13 and GJA1 have been explored in a few syndactyly but most of them have unknown underlying genetics. In the present study HOXD13, GJA1 and TP63 genes have been screened by resequencing in 24 unrelated sporadic cases with various syndactyly. The screening revealed two pathogenic HOXD13 variants, NM_000523:c.500 A > G [p.(Y167C)], and NM_000523:c.961 A > C [p.(T321P)] in syndactyly type 1b and type 1c, respectively. This is the first report to identify HOXD13 pathogenic variant in syndactyly type 1b and third report in syndactyly type 1c pathogenesis. Furthermore, this study also reports a TP63 pathogenic variant, NM_003722:c.953 G > A [p.(R318H)] in Ectrodactyly and Cleft lip and palate (ECLP). In conclusion, the current study expands the clinical spectrum of HOXD13 and TP63-related disorders. © 2021, The Author(s), under exclusive licence to The Japan Society of Human Genetics.
TGFβ3, MSX1, and MMP3 as Candidates for NSCL±P in an Indian Population
(SAGE Publications Ltd, 2019) Priyanka Kumari; Subodh Kumar Singh; Rajiva Raman
Objective: To evaluate the association of transforming growth factor β3 (TGFβ3), muscle segment homeobox 1 (MSX1), Metalloproteinases 3 (MMP3), and MMP9 genes as candidates for nonsyndromic cleft lip and/or palate in an Indian population. Design: Case–control association study, mutational screening, and functional evaluation of obtained mutations. Setting: Mutational screening of the developmental genes, TGFβ3 and MSX1, along with functional evaluation and association of promoter region SNPs—one each in MMP3 and MMP9. Patients, Participants: Two hundred forty five NSCL±P cases from G. S. Memorial Plastic Surgery Hospital and Trauma Center, Varanasi and 201 healthy controls without a family history of congenital malformations from nearby schools, primary health centers, and the university hospital. Main Outcome Measure(s): Sequencing, SSCP, and PCR-RFLP were used for candidate gene screening. MatInspector and electrophoretic mobility shift assay (EMSA) were used to check the differential transcription factor binding of the variants at promoter region. Luciferase assay was used to test the transcriptional potential of the variant, and evaluation of the alternative splice site was carried out using exon-trapping experiment. Results: Metalloproteinases3 −1171 5A/6A was associated with NSCL±P, whereas MMP9 −1562 C/T did not show association. A rare variant in the promoter region of TGFβ3 (rs117462711) creates a differential binding site, confirmed by EMSA. Luciferase assay showed 3.7-fold increased expression level in mutant construct. A synonymous change in MSX1 (rs34165410) showed association with NSCL±P, which may create an alternative splice site or lead to low codon usage. Exon-trapping experiment failed to confirm alternative splicing, indicating low codon usage frequency of the mutant affecting the gene function. Conclusions: TGFβ3, MSX1, and MMP3 are candidates for NSCL±P. © 2018, American Cleft Palate-Craniofacial Association.