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1.
Hum Genet ; 138(10): 1183-1200, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31471722

RESUMO

The glutamate pyruvate transaminase 2 (GPT2) gene produces a nuclear-encoded mitochondrial enzyme that catalyzes the reversible transfer of an amino group from glutamate to pyruvate, generating alanine and alpha-ketoglutarate. Recessive mutations in GPT2 have been recently identified in a new syndrome involving intellectual and developmental disability (IDD), postnatal microcephaly, and spastic paraplegia. We have identified additional families with recessive GPT2 mutations and expanded the phenotype to include small stature. GPT2 loss-of-function mutations were identified in four families, nine patients total, including: a homozygous mutation in one child [c.775T>C (p.C259R)]; compound heterozygous mutations in two siblings [c.812A>C (p.N271T)/c.1432_1433delGT (p.V478Rfs*73)]; a novel homozygous, putative splicing mutation [c.1035C>T (p.G345=)]; and finally, a recurrent mutation, previously identified in a distinct family [c.1210C>T (p.R404*)]. All patients were diagnosed with IDD. A majority of patients had remarkably small stature throughout development, many < 1st percentile for height and weight. Given the potential biological function of GPT2 in cellular growth, this phenotype is strongly suggestive of a newly identified clinical susceptibility. Further, homozygous GPT2 mutations manifested in at least 2 of 176 families with IDD (approximately 1.1%) in a Pakistani cohort, thereby representing a relatively common cause of recessive IDD in this population, with recurrence of the p.R404* mutation in this population. Based on variants in the ExAC database, we estimated that approximately 1 in 248 individuals are carriers of moderately or severely deleterious variants in GPT2.


Assuntos
Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Genes Recessivos , Predisposição Genética para Doença , Mutação , Fenótipo , Transaminases/genética , Adolescente , Alelos , Substituição de Aminoácidos , Deficiências do Desenvolvimento/metabolismo , Ativação Enzimática , Éxons , Feminino , Frequência do Gene , Estudos de Associação Genética , Genética Populacional , Genótipo , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Imagem por Ressonância Magnética , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Moleculares , Linhagem , Conformação Proteica , Sítios de Splice de RNA , Análise de Sequência de DNA , Relação Estrutura-Atividade , Transaminases/química , Transaminases/metabolismo
2.
Stud Health Technol Inform ; 262: 236-239, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31349311

RESUMO

Human genes often, through alternative splicing of pre-messenger RNAs, produce multiple mRNAs and protein isoforms that may have similar or completely different functions. Identification of splice sites is, therefore, crucial to understand the gene structure and variants of mRNA and protein isoforms produced by the primary RNA transcripts. Although many computational methods have been developed to detect the splice sites in humans, this is still substantially a challenging problem and further improvement of the computational model is still foreseeable. Accordingly, we developed DeepDSSR (deep donor splice site recognizer), a novel deep learning based architecture, for predicting human donor splice sites. The proposed method, built upon publicly available and highly imbalanced benchmark dataset, is comparable with the leading deep learning based methods for detecting human donor splice sites. Performance evaluation metrics show that DeepDSSR outperformed the existing deep learning based methods. Future work will improve the predictive capabilities of our model, and we will build a model for the prediction of acceptor splice sites.


Assuntos
Aprendizado Profundo , Sítios de Splice de RNA , Humanos , RNA Mensageiro
3.
Mol Biol (Mosk) ; 53(3): 411-420, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184606

RESUMO

Antithrombin III (AT3) belongs to the superfamily of serine protease inhibitors (serpins) and is a major anticoagulant in physiological conditions. Based on SERPINC1 gene, a minigene coding for human AT3, which is valuable for medicine and biotechnology, was constructed by minimizing the size of lengthy introns and preserving the splicing site-flanking sequences. An analysis of the minigene splicing pattern identified one correct AT3 transcript and two alternatively spliced transcripts, which formed either due to minigene exons 2 and 3 skipping or an aberrant exon insertion via splicing at cryptic splicing sites in intron 1 of the minigene. Site-directed mutagenesis of the cryptic splicing sites successfully optimized the splicing pattern of the AT3 minigene to completely prevent the generation of the alternative transcripts. The presence of the cryptic splicing sites in intron 1 of the minigene was confirmed with Human Splicing Finder v. 3.1 software, thus demonstrating that putative alternative splicing sites are possible to identify in minimized or hybrid introns of minigenes and to eliminate via mutagenesis before experimentally testing the minigene splicing patterns. The approach to the design of minigenes together with the bioinformatical analysis of the nucleotide sequences of minigene introns can be used to construct minigenes in order to generate transgenic animals producing economically valuable proteins in the milk.


Assuntos
Processamento Alternativo/genética , Antitrombina III/genética , Sítios de Splice de RNA/genética , Éxons/genética , Humanos , Íntrons/genética
4.
Mol Biol (Mosk) ; 53(3): 524-528, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184618

RESUMO

Proteins with homo-repeats of more than 4 amino acid residues in length were examined to understand whether some splicing sites in pre-mRNA may be attributed to homo-repeats in human proteins. The human proteome was found to contain a total of 404 proteins with homo-repeats that account for at least one splicing site in pre-mRNA. Pre-mRNA splicing sites were more often found in the C-terminal part (67%) than in the middle orN-terminal part of a homo-repeat. Ten homo-repeats were identified to have two splicing sites per repeat. The repeats were lysine homo-repeats in all but one case.


Assuntos
Proteínas/análise , Proteínas/química , Precursores de RNA/genética , Sítios de Splice de RNA/genética , Sequências Repetitivas de Aminoácidos/genética , Humanos , Lisina/genética , Lisina/metabolismo , Proteínas/genética , Proteoma/análise , Proteoma/química , Proteoma/genética , Processamento de RNA/genética
5.
Biochim Biophys Acta Gene Regul Mech ; 1862(6): 634-642, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31042550

RESUMO

Removal of introns by pre-mRNA splicing is fundamental to gene function in eukaryotes. However, understanding the mechanism by which exon-intron boundaries are defined remains a challenging endeavor. Published reports support that the recruitment of U1 snRNP at the 5'ss marked by GU dinucleotides defines the 5'ss as well as facilitates 3'ss recognition through cross-exon interactions. However, exceptions to this rule exist as U1 snRNP recruited away from the 5'ss retains the capability to define the splice site, where the cleavage takes place. Independent reports employing exon 7 of Survival Motor Neuron (SMN) genes suggest a long-distance effect of U1 snRNP on splice site selection upon U1 snRNP recruitment at target sequences with or without GU dinucleotides. These findings underscore that sequences distinct from the 5'ss may also impact exon definition if U1 snRNP is recruited to them through partial complementarity with the U1 snRNA. In this review we discuss the expanded role of U1 snRNP in splice-site selection due to U1 ability to be recruited at more sites than predicted solely based on GU dinucleotides.


Assuntos
Sítios de Splice de RNA , Processamento de RNA/fisiologia , Ribonucleoproteína Nuclear Pequena U1/fisiologia , Processamento Alternativo , Éxons/genética , Humanos , Íntrons/genética , Mutação , Processamento de RNA/genética , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/genética , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor
6.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035721

RESUMO

Protein kinase Mζ is considered important for memory formation and maintenance in different species, including invertebrates. PKMζ participates in multiple molecular pathways in neurons, regulating translation initiation rate, AMPA receptors turnover, synaptic scaffolding assembly, and other processes. Here, for the first time, we established the sequence of mRNA encoding PKMζ homolog in land snail Helix lucorum. We annotated important features of this mRNA: domains, putative capping sites, translation starts, and splicing sites. We discovered that this mRNA has at least two isoforms, and one of them lacks sequence encoding C1 domain. C1 deletion may be unique for snail because it has not been previously found in other species. We performed behavioral experiments with snails, measured expression levels of identified isoforms, and confirmed that their expression correlates with one type of learning.


Assuntos
Aprendizagem , Proteína Quinase C/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Isoenzimas , Modelos Biológicos , Família Multigênica , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/química , Proteína Quinase C/genética , Sítios de Splice de RNA , Relação Estrutura-Atividade , Transcrição Genética
7.
Nat Genet ; 51(6): 1011-1023, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110352

RESUMO

It is not clear how spontaneous DNA double-strand breaks (DSBs) form and are processed in normal cells, and whether they predispose to cancer-associated translocations. We show that DSBs in normal mammary cells form upon release of paused RNA polymerase II (Pol II) at promoters, 5' splice sites and active enhancers, and are processed by end-joining in the absence of a canonical DNA-damage response. Logistic and causal-association models showed that Pol II pausing at long genes is the main predictor and determinant of DSBs. Damaged introns with paused Pol II-pS5, TOP2B and XRCC4 are enriched in translocation breakpoints, and map at topologically associating domain boundary-flanking regions showing high interaction frequencies with distal loci. Thus, in unperturbed growth conditions, release of paused Pol II at specific loci and chromatin territories favors DSB formation, leading to chromosomal translocations.


Assuntos
Quebras de DNA de Cadeia Dupla , Loci Gênicos , Neoplasias/genética , Neoplasias/metabolismo , RNA Polimerase II/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Reparo do DNA , Elementos Facilitadores Genéticos , Etoposídeo/farmacologia , Citometria de Fluxo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica/métodos , Íntrons , Neoplasias/patologia , Regiões Promotoras Genéticas , Sítios de Splice de RNA , Inibidores da Topoisomerase/farmacologia , Sítio de Iniciação de Transcrição
8.
Methods Mol Biol ; 1962: 97-120, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31020556

RESUMO

EuGene is an integrative gene finder applicable to both prokaryotic and eukaryotic genomes. EuGene annotated its first genome in 1999. Starting from genomic DNA sequences representing a complete genome, EuGene is able to predict the major transcript units in the genome from a variety of sources of information: statistical information, similarities with known transcripts and proteins, but also any GFF3 structured information supporting the presence or absence of specific types of elements. EuGene has been used to find genes in the plants Arabidopsis thaliana, Medicago truncatula, and Theobroma cacao; tomato, sunflower, and Rosa genomes; and in the nematode Meloidogyne incognita genome, among many others. The large fraction of plant in this list probably influenced EuGene development, especially in its capacities to withstand a genome with a large number of repeated regions and transposable elements.Depending on the sources of information used for prediction, EuGene can be considered as purely ab initio, purely similarity based, or hybrid. With the general availability of NGS-transcribed sequence data in genome projects, EuGene adopts a default hybrid behavior that strongly relies on similarity information. Initially targeted at eukaryotic genomes, EuGene has also been extended to offer integrative gene prediction for bacteria, allowing for richer and robust predictions than either purely statistical or homology-based prokaryotic gene finders.This text has been written as a practical guide that will give you the capacity to train and execute EuGene on your favorite eukaryotic genome. As the prokaryotic case is simpler and has already been described, only the main differences with the eukaryotic version were reported.


Assuntos
Biologia Computacional/métodos , Células Eucarióticas , Células Procarióticas , Software , Arabidopsis/genética , Bases de Dados Genéticas , Internet , Aprendizado de Máquina , Modelos Estatísticos , Anotação de Sequência Molecular , Plantas/genética , Proteoma/genética , Sítios de Splice de RNA , RNA não Traduzido , Transcriptoma , Navegador
9.
Methods Mol Biol ; 1962: 179-191, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31020560

RESUMO

Alignment-based gene identification methods utilize sequence conservation between orthologous protein-coding genes to annotate genes in newly sequenced genomes. CESAR is an approach that makes use of existing genome alignments to transfer genes from one genome to other aligned genomes, and thus generates comparative gene annotations. To accurately detect conserved exons that exhibit an intact reading frame and consensus splice sites, CESAR produces a new alignment between orthologous exons, taking information about the exon's reading frame and splice site positions into account. Furthermore, CESAR is able to detect most evolutionary splice site shifts, which helps to annotate exon boundaries at high precision. Here, we describe how to apply CESAR to generate comparative gene annotations for one or many species, and discuss the strengths and limitations of this approach. CESAR is available at https://github.com/hillerlab/CESAR2.0 .


Assuntos
Éxons , Anotação de Sequência Molecular/métodos , Sítios de Splice de RNA , Análise de Sequência de DNA/métodos , Software , Animais , Sequência de Bases , Sequência Conservada , Apresentação de Dados , Evolução Molecular , Genoma , Genômica/métodos , Humanos , Camundongos , Fases de Leitura
10.
Gene ; 705: 113-126, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31009682

RESUMO

Identification of splice sites is imperative for prediction of gene structure. Machine learning-based approaches (MLAs) have been reported to be more successful than the rule-based methods for identification of splice sites. However, the strings of alphabets should be transformed into numeric features through sequence encoding before using them as input in MLAs. In this study, we evaluated the performances of 8 different sequence encoding schemes i.e., Bayes kernel, density and sparse (DS), distribution of tri-nucleotide and 1st order Markov model (DM), frequency difference distance measure (FDDM), paired-nucleotide frequency difference between true and false sites (FDTF), 1st order Markov model (MM1), combination of both 1st and 2nd order Markov model (MM1 + MM2) and 2nd order Markov model (MM2) in respect of predicting donor and acceptor splice sites using 5 supervised learning methods (ANN, Bagging, Boosting, RF and SVM). The encoding schemes and machine learning methods were first evaluated in 4 species i.e., A. thaliana, C. elegans, D. melanogaster and H. sapiens, and then performances were validated with another four species i.e., Ciona intestinalis, Dictyostelium discoideum, Phaeodactylum tricornutum and Trypanosoma brucei. In terms of ROC (receiver-operating-characteristics) and PR (precision-recall) curves, FDTF encoding approach achieved higher accuracy followed by either MM2 or FDDM. Further, SVM was found to achieve higher accuracy (in terms of ROC and PR curves) followed by RF across encoding schemes and species. In terms of prediction accuracy across species, the SVM-FDTF combination was optimum than other combinations of classifiers and encoding schemes. Further, splice site prediction accuracies were observed higher for the species with low intron density. To our limited knowledge, this is the first attempt as far as comprehensive evaluation of sequence encoding schemes for prediction of splice sites is concerned. We have also developed an R-package EncDNA (https://cran.r-project.org/web/packages/EncDNA/index.html) for encoding of splice site motifs with different encoding schemes, which is expected to supplement the existing nucleotide sequence encoding approaches. This study is believed to be useful for the computational biologists for predicting different functional elements on the genomic DNA.


Assuntos
Biologia Computacional/métodos , Sítios de Splice de RNA , RNA Mensageiro/metabolismo , Algoritmos , Animais , Arabidopsis , Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Aprendizado de Máquina , Processamento de RNA , Curva ROC , Trypanosoma brucei brucei/genética
11.
Nat Commun ; 10(1): 1777, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30992453

RESUMO

Nerve conduction (NC) studies generate measures of peripheral nerve function that can reveal underlying pathology due to axonal loss, demyelination or both. We perform a genome-wide association study of sural NC amplitude and velocity in 7045 Icelanders and find a low-frequency splice-donor variant in PRPH (c.996+1G>A; MAF = 1.32%) associating with decreased NC amplitude but not velocity. PRPH encodes peripherin, an intermediate filament (IF) protein involved in cytoskeletal development and maintenance of neurons. Through RNA and protein studies, we show that the variant leads to loss-of-function (LoF), as when over-expressed in a cell line devoid of other IFs, it does not allow formation of the normal filamentous structure of peripherin, yielding instead punctate protein inclusions. Recall of carriers for neurological assessment confirms that from an early age, homozygotes have significantly lower sural NC amplitude than non-carriers and are at risk of a mild, early-onset, sensory-negative, axonal polyneuropathy.


Assuntos
Condução Nervosa/genética , Periferinas/genética , Polineuropatias/genética , Sítios de Splice de RNA/genética , Nervo Sural/fisiopatologia , Adulto , Idade de Início , Idoso , Axônios/patologia , Estudos de Casos e Controles , Linhagem Celular , Feminino , Seguimentos , Estudo de Associação Genômica Ampla , Homozigoto , Humanos , Islândia/epidemiologia , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Polineuropatias/epidemiologia , Polineuropatias/fisiopatologia , Prevalência , Processamento de RNA/fisiologia
12.
Science ; 364(6438): 362-367, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30975767

RESUMO

The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5' splice site (5'SS) and branch point sequence, associates with the U4/U6.U5 tri-snRNP to form the fully assembled precatalytic pre-B spliceosome. Here, we report cryo-electron microscopy structures of the human pre-B complex captured before U1 snRNP dissociation at 3.3-angstrom core resolution and the human tri-snRNP at 2.9-angstrom resolution. U1 snRNP inserts the 5'SS-U1 snRNA helix between the two RecA domains of the Prp28 DEAD-box helicase. Adenosine 5'-triphosphate-dependent closure of the Prp28 RecA domains releases the 5'SS to pair with the nearby U6 ACAGAGA-box sequence presented as a mobile loop. The structures suggest that formation of the 5'SS-ACAGAGA helix triggers remodeling of an intricate protein-RNA network to induce Brr2 helicase relocation to its loading sequence in U4 snRNA, enabling Brr2 to unwind the U4/U6 snRNA duplex to allow U6 snRNA to form the catalytic center of the spliceosome.


Assuntos
Sítios de Splice de RNA , Processamento de RNA , Spliceossomos/metabolismo , Microscopia Crioeletrônica , Humanos , Conformação Proteica , Dobramento de RNA , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/química , Spliceossomos/ultraestrutura
13.
Biol Direct ; 14(1): 6, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975175

RESUMO

BACKGROUND: Splice sites prediction has been a long-standing problem in bioinformatics. Although many computational approaches developed for splice site prediction have achieved satisfactory accuracy, further improvement in predictive accuracy is significant, for it is contributing to predict gene structure more accurately. Determining a proper window size before prediction is necessary. Overly long window size may introduce some irrelevant features, which would reduce predictive accuracy, while the use of short window size with maximum information may performs better in terms of predictive accuracy and time cost. Furthermore, the number of false splice sites following the GT-AG rule far exceeds that of true splice sites, accurate and rapid prediction of splice sites using imbalanced large samples has always been a challenge. Therefore, based on the short window size and imbalanced large samples, we developed a new computational method named chi-square decision table (χ2-DT) for donor splice site prediction. RESULTS: Using a short window size of 11 bp, χ2-DT extracts the improved positional features and compositional features based on chi-square test, then introduces features one by one based on information gain, and constructs a balanced decision table aimed at implementing imbalanced pattern classification. With a 2000:271,132 (true sites:false sites) training set, χ2-DT achieves the highest independent test accuracy (93.34%) when compared with three classifiers (random forest, artificial neural network, and relaxed variable kernel density estimator) and takes a short computation time (89 s). χ2-DT also exhibits good independent test accuracy (92.40%), when validated with BG-570 mutated sequences with frameshift errors (nucleotide insertions and deletions). Moreover, χ2-DT is compared with the long-window size-based methods and the short-window size-based methods, and is found to perform better than all of them in terms of predictive accuracy. CONCLUSIONS: Based on short window size and imbalanced large samples, the proposed method not only achieves higher predictive accuracy than some existing methods, but also has high computational speed and good robustness against nucleotide insertions and deletions. REVIEWERS: This article was reviewed by Ryan McGinty, Ph.D. and Dirk Walther.


Assuntos
Biologia Computacional/métodos , Sítios de Splice de RNA , Distribuição de Qui-Quadrado
14.
Int J Hematol ; 109(5): 603-611, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30850927

RESUMO

X-Linked severe combined immunodeficiency (X-SCID) is a severe form of primary immunodeficiency characterized by absence of T cells and NK cells. X-SCID is caused by a loss-of-function mutation in the IL2RG gene that encodes common gamma chain (γc), which plays an essential role in lymphocyte development. We report the first case of hypomorphic X-SCID caused by a synonymous mutation in the IL2RG gene leading to a splice anomaly, in a family including two patients with diffuse cutaneous warts, recurrent molluscum contagiosum, and mild respiratory infections. The mutation caused aberrant splicing of IL2RG mRNA, subsequently resulted in reduced γc expression. The leaky production of normally spliced IL2RG mRNA produced undamaged protein; thus, T cells and NK cells were generated in the patients. Functional assays of the patients' T cells and NK cells revealed diminished cytokine response in the T cells and absent cytokine response in the NK cells. In addition, the TCR repertoire in these patients was limited. These data suggest that a fine balance between aberrant splicing and leaky production of normally spliced IL2RG mRNA resulted in late-onset combined immunodeficiency in these patients.


Assuntos
Subunidade gama Comum de Receptores de Interleucina , Mutação , Sítios de Splice de RNA , Processamento de RNA , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Adolescente , Feminino , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Masculino , Processamento de RNA/genética , Processamento de RNA/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/patologia
15.
Mol Vis ; 25: 1-11, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820140

RESUMO

Purpose: To identify the mutation for Volkmann cataract (CTRCT8) at 1p36.33. Methods: The genes in the candidate region 1p36.33 were Sanger and parallel deep sequenced, and informative single nucleotide polymorphisms (SNPs) were identified for linkage analysis. Expression analysis with reverse transcription polymerase chain reaction (RT-PCR) of the candidate gene was performed using RNA from different human tissues. Quantitative transcription polymerase chain reaction (qRT-PCR) analysis of the GNB1 gene was performed in affected and healthy individuals. Bioinformatic analysis of the linkage regions including the candidate gene was performed. Results: Linkage analysis of the 1p36.33 CCV locus applying new marker systems obtained with Sanger and deep sequencing reduced the candidate locus from 2.1 Mb to 0.389 Mb flanked by the markers STS-22AC and rs549772338 and resulted in an logarithm of the odds (LOD) score of Z = 21.67. The identified mutation, rs763295804, affects the donor splice site in the long non-coding RNA gene RP1-140A9.1 (ENSG00000231050). The gene including splice-site junctions is conserved in primates but not in other mammalian genomes, and two alternative transcripts were shown with RT-PCR. One of these transcripts represented a lens cell-specific transcript. Meta-analysis of the Cross-Linking-Immuno-Precipitation sequencing (CLIP-Seq) data suggested the RNA binding protein (RBP) eIF4AIII is an active counterpart for RP1-140A9.1, and several miRNA and transcription factors binding sites were predicted in the proximity of the mutation. ENCODE DNase I hypersensitivity and histone methylation and acetylation data suggest the genomic region may have regulatory functions. Conclusions: The mutation in RP1-140A9.1 suggests the long non-coding RNA as the candidate cataract gene associated with the autosomal dominant inherited congenital cataract from CCV. The mutation has the potential to destroy exon/intron splicing of both transcripts of RP1-140A9.1. Sanger and massive deep resequencing of the linkage region failed to identify alternative candidates suggesting the mutation in RP1-140A9.1 is causative for the CCV phenotype.


Assuntos
Catarata/congênito , Cromossomos Humanos Par 1/química , Mutação , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Acetilação , Adulto , Sequência de Bases , Sítios de Ligação , Catarata/diagnóstico , Catarata/genética , Catarata/patologia , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Éxons , Família , Feminino , Genes Dominantes , Loci Gênicos , Marcadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/genética , Histonas/metabolismo , Humanos , Íntrons , Masculino , Metilação , Pessoa de Meia-Idade , Linhagem , Sítios de Splice de RNA , Processamento de RNA , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo
16.
Mol Vis ; 25: 155-164, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820151

RESUMO

Purpose: To identify the genetic basis for retinitis pigmentosa (RP) in a cohort of Jewish patients from Caucasia. Methods: Patients underwent a detailed ophthalmic evaluation, including funduscopic examination, visual field testing, optical coherence tomography (OCT), and electrophysiological tests, electroretinography (ERG) and visual evoked potentials (VEP). Genetic analysis was performed with a combination of whole exome sequencing (WES) and Sanger sequencing. Bioinformatic analysis of the WES results was performed via a customized pipeline. Pathogenicity of the identified intronic variant was evaluated in silico using the web tool Human Splicing Finder, and in vitro, using a minigene-based splicing assay. Linkage disequilibrium (LD) analysis was used to demonstrate a founder effect, and the decay of LD over generations around the mutation in Caucasus Jewish chromosomes was modeled to estimate the age of the most recent common ancestor. Results: In eight patients with RP from six unrelated families, all of Caucasus Jewish ancestry, we identified a novel homozygous intronic variant, located at position -9 of PDE6B intron 15. The c.1921-9C>G variant was predicted to generate a novel acceptor splice site, nine bases upstream of the original splice site of intron 15. In vitro splicing assay demonstrated that this novel acceptor splice site is used instead of the wild-type site, leading to an 8-bp insertion into exon 16, which is predicted to cause a frameshift. The presence of a common ancestral haplotype in mutation-bearing chromosomes was compatible with a founder effect. Conclusions: The PDE6B c.1921-9C>G intronic mutation is a founder mutation that accounts for at least 40% (6/15 families) of autosomal recessive RP among Caucasus Jews. This result is highly important for molecular diagnosis, carrier screening, and genetic counseling in this population.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Mutação da Fase de Leitura , Judeus , Sítios de Splice de RNA , Retinite Pigmentosa/genética , Adulto , Idoso , Biologia Computacional , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/deficiência , Eletrorretinografia , Potenciais Evocados Visuais/fisiologia , Éxons , Feminino , Efeito Fundador , Expressão Gênica , Genes Recessivos , Homozigoto , Humanos , Íntrons , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Retina/diagnóstico por imagem , Retina/metabolismo , Retina/patologia , Retinite Pigmentosa/diagnóstico por imagem , Retinite Pigmentosa/etnologia , Retinite Pigmentosa/patologia , Sibéria/etnologia , Tomografia de Coerência Óptica , Sequenciamento Completo do Exoma
17.
Mol Genet Genomic Med ; 7(5): e608, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30916489

RESUMO

BACKGROUND: Autosomal recessive congenital ichthyoses (ARCI) have been associated with different phenotypes including: harlequin ichthyosis (HI), congenital ichthyosiform erythroderma (CIE), and lamellar ichthyosis (LI). While pathogenic variants in all ARCI genes are associated with LI and CIE phenotypes, the unique gene associated with HI is ABCA12. In HI, the most severe ARCI form, pathogenic variants in both ABCA12 gene alleles usually have a severe impact on protein function. The presence of at least one non-truncating variant frequently causes a less severe congenital ichthyosis phenotype (LI and CIE). METHODS: We report the case of a 4-year-old Ecuadorian boy with a severe skin disease. Genetic diagnosis was performed by NGS. In silico predictions were performed using Alamut software v2.11. A review of the literature was carried out to identify all patients carrying ABCA12 splice-site and missense variants, and to explore their genotype-phenotype correlations. RESULTS: Genetic testing revealed a nonsense substitution, p.(Arg2204*), and a new missense variant, p.(Val1927Leu), in the ABCA12 gene. After performing in silico analysis and a comprehensive review of the literature, we conclude that p.(Val1927Leu) affects a well conserved residue which could either disturb the protein function or alter the splicing process, both alternatives could explain the severe phenotype of our patient. CONCLUSION: This case expands the spectrum of ABCA12 reported disease-causing variants which is important to unravel genotype-phenotype correlations and highlights the importance of missense variants in the development of HI.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Ictiose Lamelar/genética , Mutação com Perda de Função , Fenótipo , Pré-Escolar , Códon sem Sentido , Humanos , Ictiose Lamelar/patologia , Masculino , Mutação de Sentido Incorreto , Sítios de Splice de RNA
18.
Mil Med ; 184(Suppl 1): 16-20, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30901429

RESUMO

METHODS: Ten patients having split-thickness skin grafting for burn injury were treated with the fish skin xenografts. RESULTS: There were no adverse reactions noted on the use of the fish skin grafts. No patient had any reaction to the fish skin and there was a zero incidence of infection. The handling of the fish skin was excellent, a robust and pliable xenograft that was easy to apply.The quality of donor site healing was judged to be good in all cases. Both the analgesic effect noted and the relatively short average times until 100% re-epithelialization are promising. We also illustrate two cases where the dressing was used to treat superficial burns.


Assuntos
Queimaduras/cirurgia , Produtos Pesqueiros/normas , Sítios de Splice de RNA , Transplante de Pele/instrumentação , Transplante de Pele/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Bandagens/normas , Feminino , Peixes , Humanos , Masculino , Pessoa de Meia-Idade , Cicatrização
19.
Science ; 363(6428): 710-714, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30705154

RESUMO

During exon ligation, the Saccharomyces cerevisiae spliceosome recognizes the 3'-splice site (3'SS) of precursor messenger RNA (pre-mRNA) through non-Watson-Crick pairing with the 5'SS and the branch adenosine, in a conformation stabilized by Prp18 and Prp8. Here we present the 3.3-angstrom cryo-electron microscopy structure of a human postcatalytic spliceosome just after exon ligation. The 3'SS docks at the active site through conserved RNA interactions in the absence of Prp18. Unexpectedly, the metazoan-specific FAM32A directly bridges the 5'-exon and intron 3'SS of pre-mRNA and promotes exon ligation, as shown by functional assays. CACTIN, SDE2, and NKAP-factors implicated in alternative splicing-further stabilize the catalytic conformation of the spliceosome during exon ligation. Together these four proteins act as exon ligation factors. Our study reveals how the human spliceosome has co-opted additional proteins to modulate a conserved RNA-based mechanism for 3'SS selection and to potentially fine-tune alternative splicing at the exon ligation stage.


Assuntos
Processamento Alternativo , Proteínas de Transporte/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/metabolismo , Éxons , Proteínas Nucleares/metabolismo , Spliceossomos/química , Biocatálise , Microscopia Crioeletrônica , Células HeLa , Humanos , Conformação Proteica , Precursores de RNA/genética , Sítios de Splice de RNA
20.
Eur J Med Genet ; 62(7): 103631, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30790670

RESUMO

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is one of the most common causes of sudden cardiac death (SCD) during childhood and in adolescence. Trans-2, 3-enoyl-CoA reductase-like (Tecrl) gene mutations (Arg196Gln and c.331+1G > A splice site mutation) were first reported in CPVT. Tecrl homozygous c.331+1G > A splice site mutation in iPSCs revealed a definite correlation between Tecrl and Ca2+ transport in cardiomyocytes. However, no other researchers have confirmed Tecrl mutations in CPVT with literature review. In this study, a case of compound heterozygosity in the Tecrl gene (Arg196Gln and c.918+3T > G splice site mutation) was first identified in a 13-year-old boy with CPVT by whole-exome sequencing (WES) and confirmed by Sanger sequence. Support vector machine and neural network analysis predicted that Arg196Gln mutation could decrease the stability of Tecrl structure, the confidence scores were -0.8929 and -0.9930. A STRUM server also confirmed that Arg196Gln mutation may decrease the binding capacity of the substrate and cause an amino acid substitution immediately upstream of the 3-oxo-5-alpha steroid 4-dehydrogenase domain. According to the "human splicing finder" indication and Alamut Visual Splicing Prediction, the c.918 + 3T > G mutation could influence Tecrl variable splicing. Thus, we confirmed that Tecrl as a new gene which is associated with CPVT.


Assuntos
Oxirredutases/genética , Taquicardia Ventricular/genética , Adolescente , Sítios de Ligação , Cálcio/metabolismo , Heterozigoto , Humanos , Masculino , Mutação , Miócitos Cardíacos/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Linhagem , Ligação Proteica , Estabilidade Proteica , Sítios de Splice de RNA , Taquicardia Ventricular/patologia
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