Generation and characterization of iPSC lines from Friedreich's ataxia patient (FRDA) with GAA.TTC repeat expansion in the Frataxin (FXN) gene's first intron (IGIBi016-A) and a non-FRDA healthy control individual (IGIBi017-A).

Friedreich's ataxia is a spinocerebellar degenerative disease caused by microsatellite (GAA.TTC)n repeat expansion in the first intron of FXN gene. Here, we developed iPSC lines from an FRDA patient (IGIBi016-A) and non-FRDA healthy control (IGIBi017-A). Both iPSC lines displayed typical iPSC morphology, expression of pluripotency markers, regular karyotypes (46, XY; 46, XX), capacity to grow into three germ layers, and FRDA hallmark -GAA repeat expansion and decreased FXN mRNA. Through these iPSC lines, FRDA phenotypes may be replicated in the in vitro assays, by creating neuron subtypes, cardiomyocytes and 3D organoids, for molecular and cellular biomarkers and therapeutic applications.

Generation of an Induced pluripotent stem cell (iPSC) line (IGIBi011-A) from a Spinocerebellar ataxia type 12 gait dominant patient.

The PPP2R2B gene, expressed highly in the brain, harbours trinucleotide CAG repeats in the 5'UTR region, in the range of 7-42 repeats. Individuals carrying CAG repeats greater than 43 have been associated to manifest a neurodegenerative disease condition termed as Spinocerebellar Ataxia type 12 (SCA12). An iPSC line from an adult male diagnosed with SCA12 presenting symptoms of gait (Gait Dominance) was generated. It showed pluripotency and trilineage markers without any chromosomal abnormality. This line can be utilized as an essential resource in enhancing our understanding of the molecular pathogenic mechanisms underlying SCA12 by facilitating generation of various neuronal cell types.

Generation of two human induced pluripotent stem cell lines, IGIBi012-A and IGIBi013-A from Friedreich's ataxia (FRDA) patients with homozygous GAA repeat expansion in FXN gene.

Friedreich's ataxia is a neurodegenerative disorder caused by the hyper expansion of (GAA-TTC)n triplet repeats in the first intron of the FXN gene. Here, we generated iPSC lines from two individuals with FRDA, both of whom have homozygous GAA repeat expansion in the first intron of FXN gene. Both iPSC lines demonstrated characteristics of pluripotency, including expression of pluripotency markers, stable karyotypes and ability to develop into all three germ layers, and presence of GAA repeat expansion with reduced FXN mRNA expression. These iPSC lines will serve as invaluable tools for investigating the pathophysiology and phenotypes of FRDA.

Generation and characterization of two human iPSC lines, IGIBi014-A and IGIBi015-A, from Friedreich's ataxia (FRDA) patients with pathogenic (GAA/TTC)n repeat expansion in first intron of the Frataxin (FXN) gene.

Friedreich's ataxia (FRDA) is a rare neurodegenerativedisorder caused by over expansion of GAA repeats in thefirstintron ofFXN gene. Here, we generated two iPSC lines from FRDA patients with biallelic expansion of GAA repeats in the first intron ofFXNgene.IGIBi014-A and IGIBi015-Aboth iPSC lines demonstrated characteristics of pluripotency, normal karyotypes (46, XY),the capacity to differentiate into all three germ layers, and the ability to sustain the GAA repeat expansion with decreased FXN mRNA expression. These cell lines will be utilized to comprehend the pathophysiology of the illness and the FRDA's predictive phenotypes.

Sequencing through hyperexpanded Friedreich's ataxia-GAA repeats by nanopore technology: implications in genotype-phenotype correlation.

Friedreich's ataxia, an autosomal recessive disorder, is caused by tandem GAA nucleotide repeat expansions in intron 1 of the frataxin gene. The GAA repeats over 66 in number are considered as pathogenic, and commonly occurring pathogenic repeats are within a range of 600-1200. Clinically, the spectrum of features is confined mainly to neurological tissues; however, cardiomyopathy and diabetes mellitus have been reported in 60 and 30% of the subjects, respectively. The accurate detection of GAA repeat count is of utmost importance for clinical genetic correlation, and no study so far has attempted an approach that is of high-throughput nature and defines the exact sequence of GAA repeats. Largely, the method for detection of GAA repeats so far is either through the conventional polymerase chain reaction-based screening or Southern blot, which remains the gold standard method. We utilized an approach of long-range targeted amplification of FXN-GAA repeats using Oxford Nanopore Technologies MinION platform for accurate estimation of repeat length. We were able to achieve successful amplification of GAA repeats ranging from ∼120 to 1100 at ∼2600× mean coverage. The total throughput achievable through our protocol can allow for screening of up to 96 samples per flow cell in less than 24 h. The proposed method is clinically scalable and deployable for day-to-day diagnostics. In this paper, we demonstrate to resolve the genotype-phenotype correlation of Friedreich's ataxia patients with better accuracy.

Generation of two induced pluripotent stem cell (iPSC) lines from patients with Duchenne muscular dystrophy (IGIBi006-A and IGIBi008-A) carrying exonic deletions in the dystrophin gene.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with defect in dystrophin gene that shows features of degeneration of muscle tissue at an early age. Here, we describe iPSC lines generated from LCL of two patients of Indian origin carrying 46-48 and 49-50 exons deletions in DMD. The resulting iPSC lines IGIBi006-A and IGIBi008-A showed all the characteristic features of pluripotency, differentiated into cells of three germ layers in vitro and have no major genetic alterations due to reprogramming process. These lines can serve as a useful cell model for studying disease pathogenesis and will aid in precision therapy.

Lab resource: Single cell line generation and characterization of a human-derived induced pluripotent stem cell line (IGIBi005-A) from a patient with spastic paraplegia/ataxia/ALS phenotype due to the mutation of the gene Kinesin Family Member 5A (KIF5A).

Human Kinesin Family Member 5A (KIF5A) gene mutations have been identified as a putative genetic cause of amyotrophic lateral sclerosis (ALS). Disease modelling using human-induced pluripotent stem cells (HiPSCs) is the next-generation approach to studying numerous human diseases. For the current investigation, we report the generation of patient-specific KIF5A iPSC lines with a mutation at the splice site mutation (c.3020 + 3 A > T) in the intronic region. The resulting line displayed markers for pluripotency, a healthy karyotype, the ability to differentiate into three germ layers in vitro, vector clearance, the KIF5A mutation, STR-based genomic identity, and contamination-free culture.

Biallelic cGMP-dependent type II protein kinase gene (PRKG2) variants cause a novel acromesomelic dysplasia.

BACKGROUND: C-type natriuretic peptide (CNP), its endogenous receptor, natriuretic peptide receptor-B (NPR-B), as well as its downstream mediator, cyclic guanosine monophosphate (cGMP) dependent protein kinase II (cGKII), have been shown to play a pivotal role in chondrogenic differentiation and endochondral bone growth. In humans, biallelic variants in NPR2, encoding NPR-B, cause acromesomelic dysplasia, type Maroteaux, while heterozygous variants in NPR2 (natriuretic peptide receptor 2) and NPPC (natriuretic peptide precursor C), encoding CNP, cause milder phenotypes. In contrast, no variants in cGKII, encoded by the protein kinase cGMP-dependent type II gene (PRKG2), have been reported in humans to date, although its role in longitudinal growth has been clearly demonstrated in several animal models. METHODS: Exome sequencing was performed in two girls with severe short stature due to acromesomelic limb shortening, brachydactyly, mild to moderate platyspondyly and progressively increasing metaphyseal alterations of the long bones. Functional characterisation was undertaken for the identified variants. RESULTS: Two homozygous PRKG2 variants, a nonsense and a frameshift, were identified. The mutant transcripts are exposed to nonsense-mediated decay and the truncated mutant cGKII proteins, partially or completely lacking the kinase domain, alter the downstream mitogen activation protein kinase signalling pathway by failing to phosphorylate c-Raf 1 at Ser43 and subsequently reduce ERK1/2 activation in response to fibroblast growth factor 2. They also downregulate COL10A1 and upregulate COL2A1 expression through SOX9. CONCLUSION: In conclusion, we have clinically and molecularly characterised a new acromesomelic dysplasia, acromesomelic dysplasia, PRKG2 type (AMDP).

Generation of induced pluripotent stem cell line (IGIBi007-A) from a patient with a novel acromesomelic dysplasia, PRKG2 type (AMDP).

Biallelic PRKG2 (Protein Kinase, cGMP dependent Type-2) mutations cause a novel acromesomelic dysplasia PRKG2 type. We report generation of induced pluripotent stem cell line from lymphoblastoid cell lines of the patient carrying the reported frameshift mutation (p.Asn164Lysfs*2). The derived iPSC line exhibits all the features of pluripotency, free of major genetic alterations due to reprogramming process and has the capability to differentiate into three germ layers. This iPSC cell line may provide an opportunity to investigate the effect of PRKG2 mutations upon FGF (fibroblast-growth-factor) induced MAPK signalling involved in chondrocyte proliferation in-vitro and may aid in possible therapeutic screening of novel biomolecules.

Investigations of Huntington's Disease and Huntington's Disease-Like Syndromes in Indian Choreatic Patients.

BACKGROUND: The diagnostic workup for choreiform movement disorders including Huntington's disease (HD) and those mimicking HD like phenotype is complex. OBJECTIVE: The aim of the present study was to genetically define HD and HD-like presentations in an Indian cohort. We also describe HTT-CAG expansion manifesting as neuroferritinopathy-like disorder in four families from Punjab in India. MATERIALS AND METHODS: 159 patients clinically diagnosed as HD and HD-like presentations from various tertiary neurology clinics were referred to our centre (CSIR-IGIB) for genetic investigations. As a first tier test, CAG-TNR for HTT was performed and subsequently HD-negative samples were screened for JPH3 (HDL2), TBP (SCA17), ATN1 (DRPLA), PPP2R2B (SCA12) and GGGGCC expansion in C9orf72 gene. Four families presenting as neuroferritinopathy-like disorder were also investigated for HTT-CAG expansion. RESULTS: 94 of 159 (59%) patients were found to have expanded HTT-CAG repeats. Pathogenic repeat expansion in JPH3, TBP, ATN1 and C9orf72 were not found in HD negative cases. Two patients were positive for SCA12-CAG expansion in pathogenic length, whereas 5 cases harboured TBP-CAG repeats falling in reduced penetrance range of 41- 48 repeats for SCA17. Four unrelated families, presented with atypical chorea and brain MRI findings suggestive of basal ganglia abnormalities mimicking neuroferritinopathy were found to harbour HTT-CAG expansion. CONCLUSION: We present SCA12 as a new reported phenocopy of HD which should be considered for diagnostic workout along with SCA17 for HD-like syndromes. This study also illustrates the necessity, to consider evolving HD like phenotype, as a clinical diagnosis for cases with initial manifestations depicting neuroferritinopathy.

Encephalopathy due to defective mitochondrial and peroxisomal fission 2 caused by a novel MFF gene mutation in a young child.

Encephalopathy due to defective mitochondrial and peroxisomal fission 2 caused by mitochondrial fission factor (MFF) gene mutation is a rare neurogenetic disorder. Pathogenic MFF mutations have been described in three reports in literature so far. We report a young child of Indian descent who presented to us with global developmental followed by regression of acquired milestones, spasticity, visual and auditory impairment, and was found to harbor a novel pathogenic homozygous MFF truncating variant c.433C>T; p.Arg145Ter. Cellular imaging of patient lymphoblastoid cell line had shown abnormal shapes of mitochondria due to fission defects. The patient has been started on mitochondrial cocktail with some improvement.

Synthetic transcription elongation factors license transcription across repressive chromatin.

The release of paused RNA polymerase II into productive elongation is highly regulated, especially at genes that affect human development and disease. To exert control over this rate-limiting step, we designed sequence-specific synthetic transcription elongation factors (Syn-TEFs). These molecules are composed of programmable DNA-binding ligands flexibly tethered to a small molecule that engages the transcription elongation machinery. By limiting activity to targeted loci, Syn-TEFs convert constituent modules from broad-spectrum inhibitors of transcription into gene-specific stimulators. Here we present Syn-TEF1, a molecule that actively enables transcription across repressive GAA repeats that silence frataxin expression in Friedreich's ataxia, a terminal neurodegenerative disease with no effective therapy. The modular design of Syn-TEF1 defines a general framework for developing a class of molecules that license transcription elongation at targeted genomic loci.

Exploring the Cytotoxic Potential of Triterpenoids-enriched Fraction of Bacopa monnieri by Implementing In vitro, In vivo, and In silico Approaches.

BACKGROUND: Bacopa monnieri (BM) is a herbaceous plant traditionally used from time immemorial in Ayurvedic and folklore medicines. We hypothesized that the extract of the whole plant might contain numerous molecules with having antitumor activities that could be very effective in killing of human cancer cells. OBJECTIVES: This work investigated anticancer activity of bioactive fraction of BM. MATERIALS AND METHODS: The hydroalcoholic extract of BM was fractionated with different solvent, namely, hexane, dichloromethane (DCM), acetone, methanol, and water. The in vitro anticancer activity was performed against various Human Cancer Cell lines, namely, Colon (HT29, Colo320, and Caco2), Lung (A549), Cervix (HeLa, SiHa), and Breast (MCF-7, MDAMB-231). Further, DCM fraction was evaluated in vivo for anticancer activity against Ehrlich ascites carcinoma (EAC) tumor-bearing mice since it showed the best cytotoxicity at 72 h (IC50 41.0-60.0 µg/mL). The metabolic fingerprinting of these extract were carried out using high-performance thin-layer chromatography along with quantification of bacoside A, bacoside B, cucurbitacin B, cucurbitacin E, and bittulinic acid. RESULTS: Oral administration of DCM fraction at a dose of 40 mg/kg rendered prominent reduction of tumor regression parameters such as tumor weight, packed cell volume, tumor volume and viable tumor cell count as compared to the untreated mice of the EAC control group. The anticancer activity of DCM fraction may be due to the presence of large amount of bacoside A, B and cucurbitacins. The molecular docking studies of major metabolites with targeted proteins predicted the anticancer activity of DCM fraction which was in support of in vivo activity. CONCLUSION: The in vitro, in vivo, analytical and in silico studies on DCM fraction of Bacopa monieri has proved its great potential for development of anticancer phytopharmaceuticals. SUMMARY: A new HPTLC method has been developed and validated for the qualitative and quantitative analysis of bacoside A, B, cucurbitacin B, D, E and bittulinic acid in Bacopa monnieri extract. Enrichment of active anticancer metabolites was done by polarity based fractionations of hydroalcoholic extract of Bacopa. DCM fraction of a hydroalcoholic extract of Bacopa showed anticancer potential against human cancer cell line (IC50 41.0-60.0 µg/mL) and in EAC treated mice (at a dose of 40 mg/kg body weight). The anticancer activity of Bacopa may be due to the presence of bacosides and cucurbitacin and it was confirmed by in silico screening. Abbreviations used: DBM: DCM fraction of Bacopa monnieri; DCM: Dichloromethane; EAC: Ehrlich ascites carcinoma; HCT: Hematocrit; HGB: Hemoglobin; HPTLC: High performance thin layer chromatography; ICH: International council for Harmonisation; LOD: Limit of detection; LOQ: Limit of quantification; LYM: Lymphocytes; MCH: Mean corpuscular hemoglobin; MCHC: Mean corpuscular haemoglobin concentration (MCHC); MCV: Mean corpuscular volume; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PLT: Platelet; RBC: Red blood cell; RDW: Red blood cell distribution width; RSD: Relative standard deviation; WBC: White blood cells.

Decreased expression of MGMT in correlation with aberrant DNA methylation in esophageal cancer patients from North India.

O-6-methylguanine-DNA methyltransferase, DNA repair gene, has been found to be involved with the pathogenesis of the esophageal cancer. DNA hypermethylation and other factors have been suggested to downregulate O-6-methylguanine-DNA methyltransferase. In this communication, the methylation status of O-6-methylguanine-DNA methyltransferase gene and the corresponding O-6-methylguanine-DNA methyltransferase protein expression in esophageal cancer from North India has been studied. In all, 80 samples of tumor tissue along with adjacent normal tissue as controls were analyzed for messenger RNA level of O-6-methylguanine-DNA methyltransferase gene, protein expression, and subcellular localization. The messenger RNA expression was studied using real-time quantitative polymerase chain reaction, protein expression, and its subcellular localization by Western blotting and immunohistochemistry. DNA methylation was assessed through methylation-specific polymerase chain reaction. Clinicopathological parameters were recorded and correlated with the O-6-methylguanine-DNA methyltransferase expression. O-6-methylguanine-DNA methyltransferase messenger RNA expression was found to be downregulated in 65% cases (52/80). The expression of O-6-methylguanine-DNA methyltransferase at the protein level was also found to be absent in 65% (52/80) cases. In all, 52 cases had low or no expression of the protein, whereas out of those 28 remaining cases, 11.25% (09/80) cases had high O-6-methylguanine-DNA methyltransferase protein expression. The absence of O-6-methylguanine-DNA methyltransferase protein coincided with the methylated cases in 84% (38/45), whereas in 07 cases, out of the 45 methylated, O-6-methylguanine-DNA methyltransferase protein was present. The aggressive esophageal cancer patients having methylated O-6-methylguanine-DNA methyltransferase had more than 50% cases with no/mild expression of the O-6-methylguanine-DNA methyltransferase protein ( p > 0.001). Loss of O-6-methylguanine-DNA methyltransferase protein was very frequent in the incidence of esophageal cancer from North Indian patients, and methylation of the promoter region of O-6-methylguanine-DNA methyltransferase was significantly associated in its downregulation.

Evaluation of BODE index and its relationship with systemic inflammation mediated by proinflammatory biomarkers in patients with COPD.

INTRODUCTION: BODE index, a multidimensional grading system which is based on Body mass index, airway Obstruction, Dyspnea scale, and Exercise capacity, has been increasingly used for the evaluation of chronic obstructive pulmonary disease (COPD). Many of the systemic manifestations of COPD are shown to be mediated by elevated levels of proinflammatory biomarkers. OBJECTIVE: We aimed to investigate the relationship between the BODE index, its components, disease severity, and proinflammatory biomarkers like C-reactive protein (CRP), tumor necrosis factor (TNF)-α, and interleukin (IL)-6. MATERIALS AND METHODS: A cross-sectional study which included 290 clinically stable COPD patients and 80 smoker controls was conducted. Medical history, body mass index, pulmonary function tests, 6-minute walking test, and modified scale of Medical Research Council dyspnea scale were evaluated. BODE scores were determined. Systemic inflammation was evaluated with the measurement of CRP, TNF-α, and IL-6 in the serum samples of all studied subjects. The correlation between inflammatory biomarkers and BODE index was assessed in COPD patients. RESULTS: We found a significant relationship between COPD stages and BODE index. Our analysis showed significant association between systemic biomarkers and components of the BODE index. Both TNF-α and CRP levels exhibited weak but significant correlation with BODE index. Serum IL-6 concentrations exhibited significant correlation with 6-minute walking test, modified scale of Medical Research Council, and BODE index (r=0.201, P=0.004; r=0.068, P=0.001; and r=0.530, P=0.001, respectively). Also, an inverse and significant correlation was observed between BODE index and FEV1 (r=0.567, P=0.001). IL-6 exhibited a highly significant and inverse correlation with FEV1 (r=-0.580, P=0.001). CONCLUSION: BODE index should be considered for evaluating patients with COPD. Also, IL-6 seems to be a potential biomarker that may enable determination of the severity and prediction of the course of the disease.

Nanocurcumin: a novel antifilarial agent with DNA topoisomerase II inhibitory activity.

OBJECTIVE: The aim of this study is to evaluate the antifilarial, antiwolbachial and DNA topoisomerase II inhibitory activity of nanocurcumin (nano-CUR). METHODS: Nano-CUR formulations (F1-F6) were prepared using free radical polymerization and were characterized by particle size, morphology, encapsulation efficiency and in vitro release kinetics. Antifilarial potential was evaluated in vivo against Brugian filariasis in an experimental rodent model, Mastomys coucha, by selecting the formulation that maximized parasite elimination characteristics. Wolbachial status was determined by PCR and a relaxation assay was used to estimate DNA topoisomerase II inhibitory activity. RESULTS: Nano-CUR (F3) having a 60 nm diameter and 89.78% entrapment efficiency showed the most favorable characteristics for the elimination of filarial parasites. In vivo pharmacokinetic and organ distribution studies demonstrate significantly greater C(max) (86.6 ± 2.56 ng ml(-1)), AUC0-∞ (796 ± 89.8 ng d ml(-1)), MRT (19.5 ± 7.82 days) and bioavailability of CUR (70.02%) in the organs from which the adult parasites were recovered. The optimized nano-CUR (F3) (5 × 5 mg/kg, orally) significantly augmented the microfilariciadal and adulticidal action of CUR over free CUR (5 × 50 mg/kg, orally) or Diethylcarbamizine (50 mg/kg, orally) against the Brugia malayi Mastomys coucha rodent model. The PCR results showed complete elimination of wolbachia from the recovered female parasites. Interestingly, nano-CUR was also found to be a novel inhibitor of filarial worm DNA topoisomerase II, Setaria Cervi in vitro. CONCLUSION: This study recognizes the beforehand antimicrofilarial, antimacrofilarial, anti-wolbachial activity of nano-CUR (F3) over free forms and additionally its strong inhibitory action against the major target filarial parasite enzyme DNA topoisomerase II in vitro.

Mutation and protein expression analysis of CYP1A1 gene-a study on female breast cancer cases from India.

Increased risk may be associated with exposure to genotoxic agents during breast development because the undifferentiated ductal elements of breast are more susceptible to the action of genotoxic early in life and thus an impairment in Cytochrome P 4501A1 (CYP1A1) may contribute to the development of breast cancer. Therefore, we carried out the population-based study in a total of 105 Indian female breast cancer cases with equal normal adjacent controls. A total of 20 samples (20/105, 19.04 %) showed final mutations in the exon 7 of the CYP1A1 gene where 5 cases harbored frame shift mutation (deletion of G nucleotide), and the remaining were missense mutation observed in 15 cases of breast cancer with significant association to histological grade (chi square -7.20, p = 0.02), tumor stage (chi square -6.36, p = 0.01), menopausal stage (chi square -9.76, p = 0.001), and ER status (chi square -4.22, p = 0.03). We further did protein expression analysis of CYP1A1 through immunohistochemistry where 66 cases showed down or no expression (+) (66/105, 62.85 %), 28 cases with moderate expression (++) (28/105, 26.66 %), and 11 cases with high expression (+++) (11/105, 10.47 %). Highly significant associations were observed between protein expression and clinico-pathological variables like Her 2 category (chi square = 31.73, p < 0.0001) and tumor stage (chi square = 10.27, p = 0.005). Importantly, mutation(s) of the type like deletion of A nucleotide and missense mutation (Gly > Val) exclusively showed low (+) or no expression for the CYP1A1 protein when studied in relation to each other. In summary, CYP1A1 may be associated with breast cancer and its down regulation may serve as an important tool in the field of biomarker study.