Splice site

拼接部位
  • 文章类型: Journal Article
    改变基因剪接的变异估计占所有致病变异的三分之一,然而,仅仅从DNA测序数据很难预测它们。为了克服这一点,许多小组都在进行基于RNA的分析,资源密集型,特别是诊断实验室。有成千上万的功能验证的变体诱导错误剪接;然而,这些信息没有合并,他们在ClinVar的代表性不足,这对变体解释构成了障碍,并可能导致验证工作的重复。为了解决这个问题,我们开发了SpliceVarDB,一个整合了50,000多个变体的在线数据库,分析了它们对8,000多个人类基因剪接的影响。我们评估了500多个已发布的数据源,并建立了一个可剪接性量表以标准化,协调,并合并一系列实验方案产生的变体验证数据。根据他们的有力支持证据,变异体被归类为“改变剪接”(~25%),“不改变拼接”(~25%),和“低频拼接-改变”(~50%),对应于微弱或不确定的剪接性证据。重要的是,SpliceVarDB中55%的剪接改变变体在规范剪接位点之外(5.6%为深内含子)。这些变体可以支持变体管理诊断途径,并且可以用于提供开发更准确的计算机剪接预测因子所需的高质量数据。这些变体可以通过在线平台访问,SpliceVarDB,具有可视化的附加功能,变异信息,在硅预测中,和验证指标。SpliceVarDB是一个非常大的拼接更改变体集合,可在https://splicevardb.org上获得。
    Variants that alter gene splicing are estimated to comprise up to a third of all disease-causing variants, yet they are hard to predict from DNA sequencing data alone. To overcome this, many groups are incorporating RNA-based analyses, which are resource intensive, particularly for diagnostic laboratories. There are thousands of functionally validated variants that induce mis-splicing; however, this information is not consolidated, and they are under-represented in ClinVar, which presents a barrier to variant interpretation and can result in duplication of validation efforts. To address this issue, we developed SpliceVarDB, an online database consolidating over 50,000 variants assayed for their effects on splicing in over 8,000 human genes. We evaluated over 500 published data sources and established a spliceogenicity scale to standardize, harmonize, and consolidate variant validation data generated by a range of experimental protocols. According to the strength of their supporting evidence, variants were classified as \"splice-altering\" (∼25%), \"not splice-altering\" (∼25%), and \"low-frequency splice-altering\" (∼50%), which correspond to weak or indeterminate evidence of spliceogenicity. Importantly, 55% of the splice-altering variants in SpliceVarDB are outside the canonical splice sites (5.6% are deep intronic). These variants can support the variant curation diagnostic pathway and can be used to provide the high-quality data necessary to develop more accurate in silico splicing predictors. The variants are accessible through an online platform, SpliceVarDB, with additional features for visualization, variant information, in silico predictions, and validation metrics. SpliceVarDB is a very large collection of splice-altering variants and is available at https://splicevardb.org.
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  • 文章类型: Journal Article
    剪接体是一种真核生物多兆道尔顿RNA-蛋白质复合物,可从转录物中去除内含子。剪接体确保通过多个识别事件选择每个外显子-内含子边界。最初,5'剪接位点(5'SS)和分支位点(BS)由U1小核核糖核蛋白(snRNP)和U2snRNP结合,分别,而3'SS主要取决于与分支站点的接近度。大量剪接因子识别剪接位点并通过将snRNA与转录物碱基配对而在snRNP的稳定结合发生之前募集snRNP。这个过程的保真度至关重要,由于剪接因子和U2snRNP成分的突变与许多疾病有关。近年来,在了解如何在酿酒酵母和人类中选择剪接位点方面已经取得了重大进展。这里,我回顾并讨论了当前对剪接体识别剪接位点的理解,重点是人类U2snRNP对分支位点的识别和结合。
    The spliceosome is a eukaryotic multi-megadalton RNA-protein complex that removes introns from transcripts. The spliceosome ensures the selection of each exon-intron boundary through multiple recognition events. Initially, the 5\' splice site (5\' SS) and branch site (BS) are bound by the U1 small nuclear ribonucleoprotein (snRNP) and the U2 snRNP, respectively, while the 3\' SS is mostly determined by proximity to the branch site. A large number of splicing factors recognize the splice sites and recruit the snRNPs before the stable binding of the snRNPs occurs by base pairing the snRNA to the transcript. Fidelity of this process is crucial, as mutations in splicing factors and U2 snRNP components are associated with many diseases. In recent years, major advances have been made in understanding how splice sites are selected in Saccharomyces cerevisiae and humans. Here, I review and discuss the current understanding of the recognition of splice sites by the spliceosome with a focus on recognition and binding of the branch site by the U2 snRNP in humans.
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  • 文章类型: Journal Article
    背景:对Euglenida中核内含子的研究不足。本研究旨在通过识别Euglenagracilis中的大量内含子来研究Euglenida中的核内含子(E.gracilis),包括顺式剪接的常规和非常规内含子,以及交叉剪接的外突。我们还检查了这些内含子的序列特征。
    结果:共鉴定出28,337个内含子和11,921个外端。常规和非常规内含子具有不同的剪接位点特征;前者是典型的GT/C-AG剪接位点,而后者能够与其末端序列形成结构化基序。我们观察到短内含子对规范的GT-AG内含子具有偏好。值得注意的是,普通E.gracilis中的常规内含子和反离子表现出明显的富含胞苷的聚嘧啶束,与在其他生物体中观察到的富含胸苷的区域相反。此外,E.gracilis中的SL-RNA,以及其他反式剪接物种,可以与相应的U6形成一个最近发现的主题,称为扩展的U6/5\'ss双工。我们还描述了一种新型的可变剪接模式。确定了该原生生物中内含子的串联重复序列,它们的含量与人类相当。
    结论:我们的发现突出了E.gracilis内含子的独特特征,并提供了对这些内含子剪接机制的见解,以及Euglenida的基因组学和进化。
    BACKGROUND: Nuclear introns in Euglenida have been understudied. This study aimed to investigate nuclear introns in Euglenida by identifying a large number of introns in Euglena gracilis (E. gracilis), including cis-spliced conventional and nonconventional introns, as well as trans-spliced outrons. We also examined the sequence characteristics of these introns.
    RESULTS: A total of 28,337 introns and 11,921 outrons were identified. Conventional and nonconventional introns have distinct splice site features; the former harbour canonical GT/C-AG splice sites, whereas the latter are capable of forming structured motifs with their terminal sequences. We observed that short introns had a preference for canonical GT-AG introns. Notably, conventional introns and outrons in E. gracilis exhibited a distinct cytidine-rich polypyrimidine tract, in contrast to the thymidine-rich tracts observed in other organisms. Furthermore, the SL-RNAs in E. gracilis, as well as in other trans-splicing species, can form a recently discovered motif called the extended U6/5\' ss duplex with the respective U6s. We also describe a novel type of alternative splicing pattern in E. gracilis. The tandem repeat sequences of introns in this protist were determined, and their contents were comparable to those in humans.
    CONCLUSIONS: Our findings highlight the unique features of E. gracilis introns and provide insights into the splicing mechanism of these introns, as well as the genomics and evolution of Euglenida.
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  • 文章类型: Journal Article
    背景:基因编码序列中的单核苷酸变体(SNV)可以显着影响前mRNA剪接,对致病机制和精准医学有着深远的影响。在这项研究中,我们的目标是利用完善的全长基因剪接测定(FLGSA)与SpleeAI结合,前瞻性地解释四外显子SPINK1基因内所有潜在编码SNV的剪接效应,与慢性胰腺炎相关的基因。
    结果:我们的研究开始于对先前使用FLGSA评估的27个SPINK1编码SNV的回顾性分析,对35种新的FLGSA测试的SPINK1编码SNV进行了前瞻性分析,然后是数据外推,并以进一步的验证结束。总的来说,我们分析了67个SPINK1编码SNV,占720种可能的编码SNV的9.3%。在这67个FLGSA分析的SNV中,12个被发现影响拼接。通过对FLGSA结果和SpliceAI预测的详细比较,我们推断,SPINK1基因中剩余的653个未测编码SNV不太可能显著影响剪接.在12个改变剪接的事件中,九个产生正常剪接和异常剪接的转录本,而其余三个只产生异常剪接的转录本。这些影响剪接的SNV仅在外显子1和2中发现,特别是在这些外显子的第一个和/或最后一个编码核苷酸处。在12个改变剪接的事件中,11个是错义变体(506个潜在错义变体的2.17%),一个是同义的(164个潜在同义变异中的0.61%).值得注意的是,将SpliceAI截止值调整为0.30而不是常规的0.20将提高特异性而不降低灵敏度.
    结论:通过将FLGSA与SpleeAI集成,我们已经确定,SPINK1中所有可能编码SNV的不到2%(1.67%)显著影响剪接结果.我们的发现强调了在研究基因的更广泛的基因组序列背景下进行剪接分析的关键重要性,并强调了与中间SpleeAI评分(0.20至0.80)相关的固有不确定性。这项研究通过成为第一个前瞻性地解释疾病相关基因中所有潜在编码SNV的高准确度,代表了在外显子组和基因组测序时代从回顾性变异分析转向前瞻性变异分析的有意义的尝试。
    BACKGROUND: Single-nucleotide variants (SNVs) within gene coding sequences can significantly impact pre-mRNA splicing, bearing profound implications for pathogenic mechanisms and precision medicine. In this study, we aim to harness the well-established full-length gene splicing assay (FLGSA) in conjunction with SpliceAI to prospectively interpret the splicing effects of all potential coding SNVs within the four-exon SPINK1 gene, a gene associated with chronic pancreatitis.
    RESULTS: Our study began with a retrospective analysis of 27 SPINK1 coding SNVs previously assessed using FLGSA, proceeded with a prospective analysis of 35 new FLGSA-tested SPINK1 coding SNVs, followed by data extrapolation, and ended with further validation. In total, we analyzed 67 SPINK1 coding SNVs, which account for 9.3% of the 720 possible coding SNVs. Among these 67 FLGSA-analyzed SNVs, 12 were found to impact splicing. Through detailed comparison of FLGSA results and SpliceAI predictions, we inferred that the remaining 653 untested coding SNVs in the SPINK1 gene are unlikely to significantly affect splicing. Of the 12 splice-altering events, nine produced both normally spliced and aberrantly spliced transcripts, while the remaining three only generated aberrantly spliced transcripts. These splice-impacting SNVs were found solely in exons 1 and 2, notably at the first and/or last coding nucleotides of these exons. Among the 12 splice-altering events, 11 were missense variants (2.17% of 506 potential missense variants), and one was synonymous (0.61% of 164 potential synonymous variants). Notably, adjusting the SpliceAI cut-off to 0.30 instead of the conventional 0.20 would improve specificity without reducing sensitivity.
    CONCLUSIONS: By integrating FLGSA with SpliceAI, we have determined that less than 2% (1.67%) of all possible coding SNVs in SPINK1 significantly influence splicing outcomes. Our findings emphasize the critical importance of conducting splicing analysis within the broader genomic sequence context of the study gene and highlight the inherent uncertainties associated with intermediate SpliceAI scores (0.20 to 0.80). This study contributes to the field by being the first to prospectively interpret all potential coding SNVs in a disease-associated gene with a high degree of accuracy, representing a meaningful attempt at shifting from retrospective to prospective variant analysis in the era of exome and genome sequencing.
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  • 文章类型: Journal Article
    人UlsnRNA由多基因家族编码,所述多基因家族由转录的变体和缺陷的假基因组成。已经证明许多变体U1(vU1)snRNA不仅可以转录,而且可以通过添加三甲基化鸟苷帽进行处理,打包到snRNPs中,组装成剪接体,然而,它们促进前mRNA剪接的能力,到目前为止,没有经过测试。最近对人类snRNA基因的系统分析鉴定了178个U1snRNA基因,这些基因在基因组中以串联阵列或多个染色体上的单个基因存在。其中,发现15在人体组织和细胞系中表达,尽管其内源性基因座的水平明显较低,小于规范U1snRNA的0.001%。在这项研究中,我们发现,将变体置于RNA1-1基因调控元件的背景下,可将许多变体的表达提高到与标准U1snRNA相当的水平.应用先前建立的基于HeLa细胞的小基因报告基因测定来检查vU1snRNA支持pre-mRNA剪接的能力表明,即使外源表达的变体snRNA在细胞核中富集,只有少数对剪接有可测量的影响。
    The human U1 snRNA is encoded by a multigene family consisting of transcribed variants and defective pseudogenes. Many variant U1 (vU1) snRNAs have been demonstrated to not only be transcribed but also processed by the addition of a trimethylated guanosine cap, packaged into snRNPs, and assembled into spliceosomes; however, their capacity to facilitate pre-mRNA splicing has, so far, not been tested. A recent systematic analysis of the human snRNA genes identified 178 U1 snRNA genes that are present in the genome as either tandem arrays or single genes on multiple chromosomes. Of these, 15 were found to be expressed in human tissues and cell lines, although at significantly low levels from their endogenous loci, <0.001% of the canonical U1 snRNA. In this study, we found that placing the variants in the context of the regulatory elements of the RNU1-1 gene improves the expression of many variants to levels comparable to the canonical U1 snRNA. Application of a previously established HeLa cell-based minigene reporter assay to examine the capacity of the vU1 snRNAs to support pre-mRNA splicing revealed that even though the exogenously expressed variant snRNAs were enriched in the nucleus, only a few had a measurable effect on splicing.
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  • 文章类型: Journal Article
    先天性蛛网膜挛缩症(CCA)是一种罕见的结缔组织疾病,以蛛网膜畸形为特征,多发关节挛缩,进行性脊柱侧后凸,胸部畸形和异常皱褶的耳朵。FBN2是目前已知与CCA相关的唯一基因。在这项研究中,我们报道了一个产前骨骼病例,心脏和脊髓畸形.他父亲的四肢拉长,近端指间关节挛缩,高度近视和脊柱侧弯。我们对胎儿亲本三重奏和杂合变体进行了全外显子组测序(WES)(hg19chr5:127,673,685,c.35984A>G,NM_001999.4)在FBN2基因的内含子27中成功鉴定,继承自父亲。进行逆转录聚合酶链反应(RT-PCR)以评估该变体的潜在剪接效果,这证实了该变体通过破坏剪接供体位点而导致外显子27(126bp)的缺失,并破坏了第17个钙结合表皮生长因子样(cbEGF)结构域。我们的研究不仅发现了发病个体的病因,而且扩大了FBN2基因的突变谱,而且还为这个家庭提供遗传咨询和生育指导。
    Congenital contractural arachnodactyly (CCA) is a rare connective tissue disorder characterized by arachnodactyly, multiple joint contractures, progressive kyphoscoliosis, pectus deformity and abnormal crumpled ears. FBN2 is the only gene currently known to be associated with CCA. In this study, we report on a prenatal case presented with skeletal, cardiac and spinal malformations. And his father had elongated limbs, contractures of the proximal interphalangeal joints, high myopia and scoliosis. We conducted whole exome sequencing (WES) on the fetus-parental trio and a heterozygous variant (hg19 chr5:127,673,685, c.3598 + 4A > G, NM_001999.4) in intron 27 of the FBN2 gene was successfully identified, inherited from the father. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate the potential splicing effect of this variant, which confirmed that the variant caused a deletion of exon 27 (126 bp) by disrupting the splice-donor site and destroyed the 17th calcium-binding epidermal growth factor-like (cbEGF) domain. Our research not only finds the etiology of the disease in affected individuals and expands the mutation spectrum of FBN2 gene, but also provides genetic counseling and fertility guidance for this family.
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  • 文章类型: Journal Article
    HLA-DRB1*11:01:01:12N与HLA-DRB1*11:01:01:03的不同之处在于c.6521G>C的内含子3中的一个核苷酸取代,hg19.
    HLA-DRB1*11:01:01:12N differs from HLA-DRB1*11:01:01:03 by one nucleotide substitution in intron 3 at position c.652+1G>C, hg19.
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  • 文章类型: Journal Article
    杜氏肌营养不良是一种罕见且致命的遗传性疾病,由于DMD基因突变而导致进行性肌肉萎缩。我们使用CRISPR-Cas9Prime编辑技术开发了不同的策略来纠正携带外显子52或外显子45至52缺失的DMD基因中的移码突变。使用优化的epegRNA,我们能够在多达32%的HEK293T细胞和28%的患者成肌细胞中诱导外显子53剪接供体位点的GT核苷酸的特异性取代。我们还实现了高达44%和29%的外显子53的GT剪接位点的G核苷酸缺失,以及在HEK293T细胞和人成肌细胞的外显子51的GT剪接供体位点之间插入17%和5.5%的GGG,分别。外显子51和外显子53的剪接供体位点的修饰引起它们的跳跃,并分别允许外显子50连接到外显子53和允许外显子44连接到外显子54。如通过蛋白质印迹所证明的,这些校正恢复了肌营养不良蛋白的表达。因此,Prime编辑用于诱导特定的替换,外显子51和53的剪接供体位点中的插入和缺失,以分别校正携带外显子52和外显子45至52的缺失的DMD基因中的移码突变。
    Duchenne muscular dystrophy is a rare and lethal hereditary disease responsible for progressive muscle wasting due to mutations in the DMD gene. We used the CRISPR-Cas9 Prime editing technology to develop different strategies to correct frameshift mutations in DMD gene carrying the deletion of exon 52 or exons 45 to 52. With optimized epegRNAs, we were able to induce the specific substitution of the GT nucleotides of the splice donor site of exon 53 in up to 32% of HEK293T cells and 28% of patient myoblasts. We also achieved up to 44% and 29% deletion of the G nucleotide of the GT splice site of exon 53, as well as inserted 17% and 5.5% GGG between the GT splice donor site of exon 51 in HEK293T cells and human myoblasts, respectively. The modification of the splice donor site for exon 51 and exon 53 provoke their skipping and allowed exon 50 to connect to exon 53 and allowed exon 44 to connect to exon 54, respectively. These corrections restored the expression of dystrophin as demonstrated by western blot. Thus, Prime editing was used to induce specific substitutions, insertions and deletions in the splice donor sites for exons 51 and 53 to correct the frameshift mutations in DMD gene carrying deletions of exon 52 and exons 45 to 52, respectively.
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  • 文章类型: Journal Article
    预测编码和非编码变体对剪接的影响是具有挑战性的,特别是在非规范剪接位点,导致患者漏诊。现有的拼接预测工具是互补的,但是知道哪个用于每个拼接上下文仍然是困难的。这里,我们描述介绍,它使用机器学习来整合来自几个拼接检测工具的预测,附加拼接规则,和基因结构特征,以全面评估变体影响剪接的可能性。通过对21,000个剪接改变变体的广泛基准测试,Introme优于所有工具(auPRC:0.98)用于检测临床上有意义的剪接变体。Introme可在https://github.com/CCICB/introme获得。
    Predicting the impact of coding and noncoding variants on splicing is challenging, particularly in non-canonical splice sites, leading to missed diagnoses in patients. Existing splice prediction tools are complementary but knowing which to use for each splicing context remains difficult. Here, we describe Introme, which uses machine learning to integrate predictions from several splice detection tools, additional splicing rules, and gene architecture features to comprehensively evaluate the likelihood of a variant impacting splicing. Through extensive benchmarking across 21,000 splice-altering variants, Introme outperformed all tools (auPRC: 0.98) for the detection of clinically significant splice variants. Introme is available at https://github.com/CCICB/introme .
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  • 文章类型: Case Reports
    原发性纤毛运动障碍(PCD)是一种罕见的遗传性孤儿疾病,导致各种表型,包括不孕症。科学文献中报道了大约50种基因变异导致PCD,其中,最近报道了动力蛋白轴突组装因子4(DNAAF4)。DNAAF4与多单位动力蛋白蛋白的预组装有关,该蛋白对运动纤毛和鞭毛的正常功能至关重要。在目前的研究中,招募了一个中国家庭的病人,被诊断患有PCD和弱精子症。受影响的个体是来自非近亲家庭的32岁男性。他的脊柱结构异常,脊髓弯曲的角度被诊断为脊柱侧弯。医疗报告,实验室结果,和影像学数据进行了调查。全外显子组测序,桑格测序,免疫荧光分析,苏木精-伊红染色,和硅基功能分析,包括蛋白质建模和对接研究,被使用。结果鉴定了DNAAF4疾病相关变体并证实了它们的致病性。通过全外显子组测序的遗传分析鉴定了受影响个体中的两种致病性双等位基因变体。鉴定的变体是半合子剪接位点c.784-1G>A和DNAAF4基因座处的杂合20.1Kb缺失,导致截短和无功能的DNAAF4蛋白。免疫荧光分析表明,精子鞭毛中不存在内动力蛋白臂,精子形态分析显示,小精子有扭曲和弯曲的鞭毛或缺乏鞭毛。目前的研究发现了导致PCD和弱精子症的新型双等位基因变异,扩大PCD中DNAAF4致病变异的范围,并与弱精子症的病因相关。这些发现将提高我们对PCD病因的认识。
    Primary ciliary dyskinesia (PCD) is a rare hereditary orphan condition that results in variable phenotypes, including infertility. About 50 gene variants are reported in the scientific literature to cause PCD, and among them, dynein axonemal assembly factor 4 ( DNAAF4 ) has been recently reported. DNAAF4 has been implicated in the preassembly of a multiunit dynein protein essential for the normal function of locomotory cilia as well as flagella. In the current study, a single patient belonging to a Chinese family was recruited, having been diagnosed with PCD and asthenoteratozoospermia. The affected individual was a 32-year-old male from a nonconsanguineous family. He also had abnormal spine structure and spinal cord bends at angles diagnosed with scoliosis. Medical reports, laboratory results, and imaging data were investigated. Whole-exome sequencing, Sanger sequencing, immunofluorescence analysis, hematoxylin-eosin staining, and in silico functional analysis, including protein modeling and docking studies, were used. The results identified DNAAF4 disease-related variants and confirmed their pathogenicity. Genetic analysis through whole-exome sequencing identified two pathogenic biallelic variants in the affected individual. The identified variants were a hemizygous splice site c.784-1G>A and heterozygous 20.1 Kb deletion at the DNAAF4 locus, resulting in a truncated and functionless DNAAF4 protein. Immunofluorescence analysis indicated that the inner dynein arm was not present in the sperm flagellum, and sperm morphological analysis revealed small sperm with twisted and curved flagella or lacking flagella. The current study found novel biallelic variants causing PCD and asthenoteratozoospermia, extending the range of DNAAF4 pathogenic variants in PCD and associated with the etiology of asthenoteratozoospermia. These findings will improve our understanding of the etiology of PCD.
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