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Abstract Introduction: The aim of this study was to extend our knowledge of the genetic background of Argentinean pediatric patients with developmental and epileptic encephalopathy (DEE) applying a next generation sequencing (NGS) panel. Methods: Thirty one patients with DEE were studied, including these phenotypes: Dravet syndrome (n:7), Dravet like syndrome (n:3), West syndrome (WS) (n:6), WS that evolved to Lennox-Gastaut syndrome (LGS) (n:4), epilepsy of infancy with migrating focal seizures (n:2), continuous spikes and waves during slow sleep evolving to LGS (n:1), LGS (n:1), myoclonic status in non-progressive encephalopathy (n:1), myoclonic atonic epilepsy (n:1), epileptic encephalopathy with multifocal spikes (n:1) and unclassified epileptic encephalopathy (n:4). Fifty-two genes frequently associated with DEE were studied by NGS in genomic DNA from peripheral blood. Results: Relevant variants were detected in 12 cases; 6 novel pathogenic or likely pathogenic variants, 6 previously reported as pathogenic and 1 variant of unknown sig nificance. Single-nucleotide heterozygous variants were identified in the SCN1A (5), GABRG2 (1), STXBP1 (2) genes, a mosaic variant in SCN2A (1) and a homozygous variant in SCN1B (1). Additionally, a heterozygous deletion involving the SCN1A, SCN2A and SCN3A genes (1), and the most frequent triplet repeat expansion in the ARX gene (1) were detected. Discussion: Genetic diagnosis was made in 39% of patients. We emphasize the importance of considering mosaic variants, copy number variants and hereditary forms when designing and interpreting molecular studies, to optimize diagnosis and management of patients. Approximately 42% of the de tected variants were novel, expanding the knowledge of the molecular basis of DEEs in Latin-American patients.
Resumen Introducción: El objetivo del estudio fue ampliar el conocimiento de las bases moleculares de las encefalopatías epilépticas y del desarrollo (EED) en pacientes pediátricos argentinos aplicando un panel de secuenciación de nueva generación (NGS). Métodos: Se analizaron 31 pacientes con los fenotipos clínicos de síndrome de Dra vet (n:7), síndrome símil Dravet (n:3), síndrome de West (SW) (n:6), SW que evoluciona a síndrome de Lennox Gastaut (SLG)(N:4), epilepsia de la infancia con crisis focales migratorias (n:2), actividad de punta onda continua durante el sueño que evolucionan a SLG (n:1), SLG (n:1), encefalopatía no progresiva con estatus mioclónico (n:1), epilepsia mioclónica atónica (n:1), encefalopatía epiléptica con espigas multifocales (n:1) y encefalopatía epiléptica indeterminada (n:4). Se estudiaron los 52 genes más frecuentemente asociados a EED a través de NGS, en ADN extraído de sangre periférica. Resultados: Se identificaron variantes relevantes en 12 casos, de las cuales 5 fueron nuevas y 6 previamente reportadas como patogénicas o posiblemente patogénicas, mien tras que una variante fue clasificada como de significado incierto. Variantes heterocigotas, de nucleótido único, se identificaron en los genes SCN1A (5), GABRG2 (1), STXBP1 (2), una variante en mosaico en SCN2A (1) y otra homocigota en SCN1B (1). Además, se detectó una deleción que involucra a los genes SCN1A, SCN2A y SCN3A (1) y la expansión de repeticiones de tripletes más frecuente en el gen ARX (1). Discusión: Se alcanzó el diagnóstico molecular en el 39% de los pacientes. Remarcamos la importancia de considerar variantes en mosaico, variantes en el número de copias y formas heredadas al momento de diseñar e interpretar los estudios moleculares, de tal forma de optimizar el diagnóstico y seguimiento de los pacientes con EED. Cabe destacar, que el 42% de las variantes detectadas fueron nuevas, ampliando nuestro conocimiento sobre las bases mole culares de las EED en población latino americana.
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Modeling a protein functional network in concerned species is an efficient approach for identifying novel genesin certain biological pathways. Tea plant (Camellia sinensis) is an important commercial crop abundant innumerous characteristic secondary metabolites (e.g., polyphenols, alkaloids, alkaloids) that confer tea qualityand health benefits. Decoding novel genes responsible for tea characteristic components is an important basisfor applied genetic improvement and metabolic engineering. Herein, a high-quality protein functional networkfor tea plant (TeaPoN) was predicted using cross-species protein functional associations transferring andintegration combined with a stringent biological network criterion control. TeaPoN contained 31,273 nonredundant functional interactions among 6,634 tea proteins (or genes), with general network topologicalproperties such as scale-free and small-world. We revealed the modular organization of genes related to themajor three tea characteristic components (theanine, caffeine, catechin) in TeaPoN, which served as strongevidence for the utility of TeaPoN in novel gene mining. Importantly, several case studies regarding geneidentification for tea characteristic components were presented. To aid in the use of TeaPoN, a concise webinterface for data deposit and novel gene screening was developed (http://teapon.wchoda.com). We believe thatTeaPoN will serve as a useful platform for functional genomics studies associated with characteristic secondarymetabolites in tea plant.
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Objective@#To analyze variants of PRRT2 gene in two children with paroxysmal kinesigenic dyskinesia.@*Methods@#Genomic DNA of the two children and their parents was extracted from peripheral venous blood samples. All exons and their flanking regions of the PRRT2 gene were subjected to PCR and Sanger sequencing.@*Results@#The two children were found to respectively harbor a c. 282dupA and a c. 715_716dupCC variant in exon 2 of the PRRT2 gene, which were both inherited from their mothers. Pooling together their frequencies in general population, genetic models, related literature and impact on protein function, the two novel variants were both predicted to be pathogenic.@*Conclusion@#The c. 282dupA and c. 715_716dupCC variants probably underlie the disease in the two children.
ABSTRACT
The brown planthopper (BPH) Nilaparvata lugens (Stål) (Homoptera: Delphacidae) is considered a threat to rice (Oryza sativa ssp.) crop in many parts of the world including India. Among the BPH-resistance (R) genes so far reported in rice, most of them are ineffective against BPH biotype 4 predominant in the Indian sub-continent. In this study, we show the introgression line RPBio4918-230S was identified as BPH resistant after five years of rigorous screening at seedling stage and two years at tillering and reproductive stages. The inheritance of resistance indicated that two recessive genes are involved at seedling and reproductive stages. The allelic relation with known genes using linked reported markers suggested that the genes present in RPBio4918-230S are different. We report here the genetics of the two newly introgressed BPH resistance genes from O. nivara in the background of Swarna which are effective at all the important growth stages. The genes have been tentatively named as bph39(t) and bph40(t). The honeydew area (feeding rate) and days to wilt parameters observed at 30 days after sowing in BC1F3 indicated that newly introgressed genes have both antibiosis and tolerance mechanisms for resistance. The BPH resistance genes identified in this study would facilitate the breeding of broad spectrum and durable resistance in rice against BPH biotype 4.
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Rv2742 is a novel gene identified from Mycobacterium tuberculosis H37Rv by the proteogenomics strategy. The aim of this study was to establish a system of soluble expression and purification of the missing protein Rv2742 in M. tuberculosis H37Rv, to provide reference for further research on the biological function of Rv2742. The soluble protein was not successfully induced by prokaryotic expression vectors pGEX-4T-2-Rv2742, pET-32a-Rv2742, pET-28a-Rv2742 and pMAL-c2X-Rv2742. After the codon of novel gene Rv2742 was optimized according to E. coli codon usage frequency, only the recombinant strain containing plasmid pMAL-c2X-Rv2742 could produce soluble products of Rv2742 encoding gene. In addition, the expression effects of the desired fusion protein were also analyzed under different conditions including hosts, culture temperatures and IPTG concentrations. The optimum expression conditions were as follows: Rosetta (DE3) host, 16 °C culture temperature and 0.5 mmol/L IPTG. After being purified by affinity chromatography with amylose resin, the fusion protein sequence was confirmed by LC-MS/MS. These results indicated that the novel gene Rv2742 product could be successfully induced and expressed in a soluble form by the expression system pMAL-c2X with MBP tag. Our findings provide reference for studies on potential interaction and immunogenicity.
Subject(s)
Chromatography, Liquid , Cloning, Molecular , Escherichia coli , Mycobacterium tuberculosis , Genetics , Recombinant Fusion Proteins , Tandem Mass SpectrometryABSTRACT
PURPOSE: Although breast cancer the most common cancer for women remains a significant health problem, it has not been systematically studied until now. In an attempt to identify novel genes implicated in breast cancer development, we performed a suppression subtraction hybridization (SSH) with human breast cancer tissues, as well as with cloned genes, that are expressed more than in normal tissue. METHODS: After the identification of a novel gene, RT-PCR was performed to determine its mRNA expression in human breast cancers. In order to learn more about the expression profile of this gene, PCR was performed using various commercially available normal or carcinoma cell lines. The novel gene was found to be strongly expressed in breast cancer tissues and other carcinoma cell lines. To determine whether this novel gene was associated with cell cycle regulation, normal WI-38 fibroblast cells were stimulated with media containing 0.1% FBS for 48hours. RESULT: From the experimental results of the SSH, a novel clone, "Clone 135" which was strongly expressed in tumor compared to matched normal tissue, has been found. The novel clone was identified as being expressed in several tumor tissues and carcinoma cell lines. The time-course expression of this novel gene in the WI-38 (8PDL) normal lung cell line indicated a significant increase for G1-phase arrest. CONCLUSION: We have used a suppression subtractive hybridization (SSH) to generate a profile of genes overexpressed in human breast cancer. We have screened novel genes, of which "Clone 135" scored as a candidate oncogene that was overexpressed in tumor compared to matched normal tissue.
Subject(s)
Female , Humans , Breast Neoplasms , Breast , Cell Cycle , Cell Line , Clone Cells , Fibroblasts , Lung , Oncogenes , Polymerase Chain Reaction , RNA, MessengerABSTRACT
PURPOSE: Although breast cancer the most common cancer for women remains a significant health problem, it has not been systematically studied until now. In an attempt to identify novel genes implicated in breast cancer development, we performed a suppression subtraction hybridization (SSH) with human breast cancer tissues, as well as with cloned genes, that are expressed more than in normal tissue. METHODS: After the identification of a novel gene, RT-PCR was performed to determine its mRNA expression in human breast cancers. In order to learn more about the expression profile of this gene, PCR was performed using various commercially available normal or carcinoma cell lines. The novel gene was found to be strongly expressed in breast cancer tissues and other carcinoma cell lines. To determine whether this novel gene was associated with cell cycle regulation, normal WI-38 fibroblast cells were stimulated with media containing 0.1% FBS for 48hours. RESULT: From the experimental results of the SSH, a novel clone, "Clone 135" which was strongly expressed in tumor compared to matched normal tissue, has been found. The novel clone was identified as being expressed in several tumor tissues and carcinoma cell lines. The time-course expression of this novel gene in the WI-38 (8PDL) normal lung cell line indicated a significant increase for G1-phase arrest. CONCLUSION: We have used a suppression subtractive hybridization (SSH) to generate a profile of genes overexpressed in human breast cancer. We have screened novel genes, of which "Clone 135" scored as a candidate oncogene that was overexpressed in tumor compared to matched normal tissue.
Subject(s)
Female , Humans , Breast Neoplasms , Breast , Cell Cycle , Cell Line , Clone Cells , Fibroblasts , Lung , Oncogenes , Polymerase Chain Reaction , RNA, MessengerABSTRACT
In rat that is helpless at birth, the cerebellum is in a corresponding state of immaturity, and its histogenesis and morphogenesis mainly occur after birth. The times and sites of origin of the four types of cerebellar local-circuit neurons, as well as their migration routes to specific positions in the cortex, their distinctive patterns of differentiation and growth, and their synaptogenesis, have been well studied. The stage-specific genes in the postnatal rat cerebellum may be related with these kind of neural development in the cerebellum. To clone the genes related with neural development in the postnatal cerebellum, developmentally differentially expressed genes were screened from postnatal rat cerebellum with ordered differential display (ODD) and the developmental expression pattern in the postnatal rat cerebella was investigated with in situ hybridization histochemistry. One novel postnatal stage-specific gene (PKrCb1) was cloned by ODD with 7 cDNA pools (P0, P3, P7, P12, P18, P25, adult rat cerebella). To investigate the developmental expression pattern of this novel gene on the cell level, in situ hybridization histochemistry was performed in the developing and adult rat brain sections. The developmental expression pattern of PKrCb1 in the cerebellum was well matched with spatiotemporal migration pattern of granule cells and it may be suspected that PKrCb1 is related with migration of granule cells from external granular layer to internal granular layer. From the results, it is suggested that the methods used in this experiment will be the powerful methods for the cloning and primary function study of the genes related with cerebellar development.
Subject(s)
Adult , Animals , Humans , Rats , Brain , Cerebellum , Clone Cells , Cloning, Organism , DNA, Complementary , In Situ Hybridization , Morphogenesis , Neurons , ParturitionABSTRACT
A marine bacterium MWYL1,originally isolated from the roots of Spartina anglica growing in a salt marsh near seaside,was identified as a member of the genus of Marinomonas via morphology characterization、physiological test and 16S rDNA sequencing and Blast analysis.The strain was short,rod,gram-negative,grew aerobically and optimally at 28℃.The analysis of 16S rDNA sequence suggests that the sequence similarity values are 97% and 95% with Marinomonas pontica and Marinomonas dokdonensis,respectively.One fosmid clone producing melanin was directly isolated by plating from the genomic library of Marinomonas MWYL1.The novel functional gene cluster involved in melanin biosynthesis was screened after subcloning and sequencing of the 14kb insert in pUC18,further more,the putative functional genes was preminary analyzed using bioinformatics.
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Objective: To clone novel gene fragments differentially expressed from diabetic rats supplemented with selenium and detect their expression distribution in various tissues. Method: cDNA fragments from former research project were cloned, sequenced and BLASTn analysed. The RT-PCR of the five novel genes were made using the primers designed according to the sequence of cDNA to observe the expression changes in liver of various groups and their expression distributions in various tissues. Results: Se-2, Se-6, Se-10, Se-14 and Se-18 cDNA were shown to be the novel gene fragments for no matched gene with them in GenBank. The expression levels of four cDNAs, including Se-2, Se-10, Se-14, Se-18, in DM group and DM+Se group were obviously lower than those in NC group. The expression level of DM+Se group was higher than those in DM group (P