A TBX5 NONSENSE MUTATION IN SIBLINGS WITH DIVERGENT PHENOTYPES ASSOCIATED WITH ISOLATED SEPTAL DEFECTS

  Objective: Heart septal defects (HSD) account for 50% of the congenital heart malformations and are characterized by the hole in the wall of tissue which separates the heart chambers. The known causes of the SD are multifactorial and complex inheritance.Methods: Isolated 15 subjects with ostium secundum atrial SD (OS-ASD) and one subject with perimembranous ventricular SD (VSD) among 125 clinically diagnosed SD were included in the study. Sanger sequencing was performed for all the exons of TBX5 genes using genomic DNA of these patients.Results: Sequence variation c.444 G>A substitution, leads to the alteration of tryptophan residue into premature stop codon at codon 148. We observed a divergent phenotype within a family of four, where one sibling and the mother had OS-ASD, another sibling had phenotype of perimembranous VSD, and the father had normal genotype.Conclusion: We believe that this novel sequence variant in TBX5 gene is one of the factors in the SD and may hold a key determining the role of TBX5 gene in the heart development

EVALUATION OF THE PROTECTIVE EFFECT OF GALLIC ACID AGAINST ARSENIC-INDUCED GENOTOXICITY IN HEPG2 CELL LINE

Objective: Arsenic has cytotoxic as well as mutagenic effect in human health due to its indirect effect on oxidative stress on the cells. We aimed to find out the effect of gallic acid (GA), a well-known natural antioxidant in ameliorating in heavy metal toxicity. Methods: MTT assay was performed to determine the cytotoxicity of sodium arsenite (NaAsO2) on HepG2 cells with the cytoprotectant GA at varying concentrations for exposure durations of 6 h, 12 h, and 24 h. Similarly, the alkaline version of the comet assay was performed to investigate the genotoxicity and assessment of oxidative stress of the cells using flow cytometry. Results: Cells treated with NaAsO2 at various doses spanning a broad range of concentrations (5–500 μM) showed a dose- and time-dependent decrease in cellular viability as observed. However, the effect of the proposed protectant, GA showed an increase in cellular viability in a concentration-dependent manner. Conclusion: We assessed the cytotoxicity and genotoxicity induced by NaAsO2 to provide insight into the role of GA on arsenic-induced toxicity in liver cells and to shed light on its possible ameliorative effect at low concentrations in a time-dependent manner

SUBMICROSCOPIC CHROMOSOMAL VARIATIONS IN CHILDREN WITH IDIOPATHIC INTELLECTUAL AND DEVELOPMENTAL DISABILITIES

Objective: Intellectual disability is the most common developmental disorder that originates before the age of 18 years and is characterized by limitation in intellectual functioning and adaptive behaviour. The fact that >30 to 50% of all causes are still unknown in etiology is increasing the burden of the clinical evaluators and managers handling these children. The purpose of this study was to have an optimal genetic diagnostic evaluation to assist paediatricians in providing medical advice for children with intellectual disabilities and global developmental delays. Methods: The study was initiated with 385 cases; however, only 201 cases had no cytogenetic abnormality and negative for PCR test for FXS. However, these subjects showed characteristic signs of facial dysmorphisms, developmental delay, mild to severe intellectual disability, which were unique and unspecific with lack of major hallmarks for any particular syndrome/phenotype, considered as “idiopathic” and tested for MLPA analysis and subsequently confirmed by FISH and RT-qPCR. Results: A total of 23 (11.44%) cases were found to have submicroscopic chromosomal variations [microdeletions (18 cases), microduplications (5 cases)]. We categorized the aberrations detected in these cases as novel and as variants of uncertain significance. All these cases showed clear evidence for segregation of the variation and were provided with the required genetic counselling. Conclusion: MLPA method gives a better yield in combination with karyotype analysis. The detection rate as per current analysis suggests that the use of MLPA could be a robust, high throughput yet cost-effective technique for use in a diagnostic set up

Cytogenetic evaluation of patients with clinical spectrum of Turner syndrome

Aim: The objective of this study was to correlate the genotype, of female patients, withshort stature and primary amenorrhea. Materials and Methods: One hundred and forty-six subjects were recruited during 2005-2012. Microscopic and automated karyotyping analyses were done by using chromosomes isolated from the lymphocytes using Giemsa banding (GTG) to identify chromosome abnormalities. Results: A total of 146 clinically suspected Turner syndrome (TS) subjects were recruited for the study, of which, 61 patients were identified to have chromosome abnormalities. The chromosomal abnormalities detected were as follows: Monosomy X (n = 19, 13.01%), triple X syndrome (n = 4, 2.7%), mosaic TS (n = 12, 8.21%), XY gonadal dysgenesis (n = 13, 8.9%), and structural abnormalities including X chromosome (n = 15, 10.27%) and one patient each with autosomal changes involving 9qh inversion and translocation of chromosomes 12 and 14. Conclusion: Karyotype abnormalities accounting for 46% in this study emphasize the need for karyotype testing in cases of short stature with primary amenorrhea

ANALYSIS OF GENE COPY NUMBER VARIATIONS IN PATIENTS WITH CARDIAC SEPTAL DEFECTS USING MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION: CNVs analysis in CSDs

Objective: Cardiac septal defects (CSDs), the most common human congenital heart malformations are complex and heterogeneous. Progress in molecular biology has helped to identify many genes responsible for cardiac morphogenesis. However, etiologic factors in familial as well as isolated syndromes are being identified; the root genetic cause still needs to be resolved and its mechanism is yet to be revealed. The objective of this study is to identify DNA copy number variations (CNVs) and their possible association with septal defects. Methods: Multiplex ligation-dependent probe amplification (MLPA) was used to detect DNA copy number in non-syndromic CSDs using the P311-A1 Kit consisting of probes for the key genes, namely, NKX2-5 (NK2 transcription factor related, locus 5), GATA4 (GATA binding protein 4), TBX5 (T-box transcription factor), bone morphogenetic protein 4, and CRELD1 (cysteine rich with EGF-like domains 1). Results: We studied 124 clinically diagnosed CSD subjects, of which 111 (89.5%) had atrial septal defects and 13 (10.5%) had ventricular septal defects. MLPA assay was carried out in all these patients after a thorough clinical and cytogenetic screening. CNVs were identified in 16 (12.9%) cases, of which heterozygous deletions and heterozygous duplications were detected (8 patients each) with apparent phenotypes. Conclusion: MLPA could be a useful assay for the detection of CNVs and to be adopted as the first line of screening in patients with congenital heart diseases

Multiplex ligation-dependant probe amplification study of children with idiopathic mental retardation in South India

Background: Mental retardation (MR) is a heterogeneous dysfunction of the central nervous system exhibiting complex phenotypes and has an estimated prevalence of 1-3% in the general population. However, in about 50% of the children diagnosed with any form of intellectual disability or developmental delay the cause goes undetected contributing to idiopathic intellectual disability. Materials and Methods: A total of 122 children with developmental delay/MR were studied to identify the microscopic and submicroscopic chromosome rearrangements by using the conventional cytogenetics and multiplex ligation dependent probe amplification (MLPA) analysis using SALSA MLPA kits from Microbiology Research Centre Holland [MRC] Holland. Results: All the recruited children were selected for this study, after thorough clinical assessment and metaphases prepared were analyzed by using automated karyotyping system. None was found to have chromosomal abnormality; MLPA analysis was carried out in all subjects and identified in 11 (9%) patients. Conclusion: Karyotype analysis in combination with MLPA assays for submicroscopic micro-deletions may be recommended for children with idiopathic MR