RESUMO
Objective: To analyze the distribution characteristics of UGT1A1 mutant genes (including enhancers, promoters, and exons 1-5) and further explore the correlation between UGT1A1 genotype and clinical phenotypes in patients with inherited hyperunconjugated bilirubinemia. Methods: Patients diagnosed with hereditary hyperunconjugated bilirubinemia at Nanjing Second Hospital from June 2015 to December 2022 were retrospectively analyzed. The UGT1A1 gene was examined using Sanger sequencing in all patients. Complete blood count, liver function, and abdominal imaging examinations were performed. Comparison of categorical variable data using χ(2) testor Fisher percision tests. Comparison of continaous veriable data with normal distribution using t-test. Results: 112 cases (male:female ratio 81:31, aged 9-70 years) had inherited hyperunconjugated bilirubinemia, with a total of 14 mutation sites identified, of which seven were confirmed mutations, and the frequency ranged from high to low: (TA)n accounted for 50%, c.211G>A (p.G71R) accounted for 49.10%, 1456T>G (p.Y486D) accounted for 16.96%, c.686C>A (p.R229W) accounted for 12.5%, 1091C>T (p.P364L) accounted for 8.04%, and c- 3279T>G accounted for 0.982%. Simultaneously, all patients had one to four mutations, of which only one mutation was the most common (55.36%), followed by two mutations (37.5%), and rare three and four mutations (5.36% and 1.78%). There was no statistical significance in total bilirubin (TBil) levels among the four groups (F=0.652, P=0.583). One mutation was most common in (TA)n and c.211G>A (p.G71R), among which TA6/TA7 (n=10) and TA7/TA7 (n=14) mutations were statistically significant in TBil (t=2.143, P=0.043). The c.211G>A (p.G71R) heterozygous (n=9) and isolated (n=15) mutation had no statistical significance in TBil (t=0.382, P=0.706). The GS group accounted for 75%, the intermediate group accounted for 16.9%, and the CNS-â ¡ group accounted for 8%. TBil was statistically significant among the three groups (F=270.992, P<0.001). There was no statistically significant difference (χ(2)=3.317, P=0.19) between mutation 1 (44 cases, 14 cases, and 4 cases, respectively) and mutations ≥ 2 (40 cases, 5 cases, and 5 cases, respectively) in the GS group, intermediate group, and CNS-II group. Conclusion: The number of UGT1A1 gene mutation sites may have no synergistic effect on TBil levels in patients with inherited hyperunconjugated bilirubinemia. TA7/TA7 mutations are not uncommon, and TBil levels are relatively high.
Assuntos
Genótipo , Glucuronosiltransferase , Mutação , Fenótipo , Humanos , Glucuronosiltransferase/genética , Estudos Retrospectivos , Hiperbilirrubinemia Hereditária/genética , Bilirrubina/sangue , Masculino , Feminino , Éxons , AdultoRESUMO
HLA-DQA1*02:32 differs from DQA1*02:01:01 by one nucleotide substitution in codon 210 in exon 4.
Assuntos
Alelos , Éxons , Cadeias alfa de HLA-DQ , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade , Humanos , Cadeias alfa de HLA-DQ/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade/métodos , Sequência de Bases , Códon , Análise de Sequência de DNA/métodosRESUMO
The novel HLA-C*15:274 allele, first described in a potential bone marrow donor from Brazil.
Assuntos
Alelos , Éxons , Antígenos HLA-C , Teste de Histocompatibilidade , Humanos , Antígenos HLA-C/genética , Teste de Histocompatibilidade/métodos , Análise de Sequência de DNA/métodos , Doadores de Tecidos , Brasil , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequência de BasesRESUMO
Spinal muscular atrophy (SMA) genes, SMN1 and SMN2 (hereinafter referred to as SMN1/2), produce multiple circular RNAs (circRNAs), including C2A-2B-3-4 that encompasses early exons 2A, 2B, 3 and 4. C2A-2B-3-4 is a universally and abundantly expressed circRNA of SMN1/2. Here we report the transcriptome- and proteome-wide effects of overexpression of C2A-2B-3-4 in inducible HEK293 cells. Our RNA-Seq analysis revealed altered expression of ~ 15% genes (4172 genes) by C2A-2B-3-4. About half of the affected genes by C2A-2B-3-4 remained unaffected by L2A-2B-3-4, a linear transcript encompassing exons 2A, 2B, 3 and 4 of SMN1/2. These findings underscore the unique role of the structural context of C2A-2B-3-4 in gene regulation. A surprisingly high number of upregulated genes by C2A-2B-3-4 were located on chromosomes 4 and 7, whereas many of the downregulated genes were located on chromosomes 10 and X. Supporting a cross-regulation of SMN1/2 transcripts, C2A-2B-3-4 and L2A-2B-3-4 upregulated and downregulated SMN1/2 mRNAs, respectively. Proteome analysis revealed 61 upregulated and 57 downregulated proteins by C2A-2B-3-4 with very limited overlap with those affected by L2A-2B-3-4. Independent validations confirmed the effect of C2A-2B-3-4 on expression of genes associated with chromatin remodeling, transcription, spliceosome function, ribosome biogenesis, lipid metabolism, cytoskeletal formation, cell proliferation and neuromuscular junction formation. Our findings reveal a broad role of C2A-2B-3-4, and expands our understanding of functions of SMN1/2 genes.
Assuntos
Éxons , Atrofia Muscular Espinal , Proteoma , RNA Circular , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio Motor , Transcriptoma , Humanos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Proteoma/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Células HEK293 , Éxons/genética , Regulação da Expressão GênicaRESUMO
The novel HLA-C*08:275 allele, first described in a potential bone marrow donor from Brazil.
Assuntos
Alelos , Éxons , Antígenos HLA-C , Humanos , Antígenos HLA-C/genética , Brasil , Teste de Histocompatibilidade , Doadores de Tecidos , Sequência de Bases , Análise de Sequência de DNA/métodos , Alinhamento de Sequência , CódonRESUMO
Nucleotide substitution in codon 238 of HLA-C*12:02:02:01 results in a novel allele, HLA-C*12:02:53.
Assuntos
Alelos , Sequência de Bases , Códon , Éxons , Antígenos HLA-C , Teste de Histocompatibilidade , Humanos , Antígenos HLA-C/genética , Taiwan , Análise de Sequência de DNA , Povo Asiático/genética , Alinhamento de Sequência , Polimorfismo de Nucleotídeo Único , Substituição de AminoácidosRESUMO
Five novel HLA-C alleles detected by next-generation sequencing: HLA-C*02:02:73, -C*03:04:106, -C*06:382, -C*07:1114Q and -C*12:408.
Assuntos
Alelos , Antígenos HLA-C , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade , Humanos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Antígenos HLA-C/genética , Teste de Histocompatibilidade/métodos , Éxons , Análise de Sequência de DNA/métodosRESUMO
The novel HLA-B*35:593 allele, first described in a potential bone marrow donor from Brazil.
Assuntos
Alelos , Éxons , Humanos , Teste de Histocompatibilidade/métodos , Análise de Sequência de DNA/métodos , Doadores de Tecidos , Brasil , Antígeno HLA-B35/genética , Antígenos HLA-B/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequência de BasesRESUMO
Pre-mRNA splicing is a fundamental step in gene expression, conserved across eukaryotes, in which the spliceosome recognizes motifs at the 3' and 5' splice sites (SSs), excises introns, and ligates exons. SS recognition and pairing is often influenced by protein splicing factors (SFs) that bind to splicing regulatory elements (SREs). Here, we describe SMsplice, a fully interpretable model of pre-mRNA splicing that combines models of core SS motifs, SREs, and exonic and intronic length preferences. We learn models that predict SS locations with 83 to 86% accuracy in fish, insects, and plants and about 70% in mammals. Learned SRE motifs include both known SF binding motifs and unfamiliar motifs, and both motif classes are supported by genetic analyses. Our comparisons across species highlight similarities between non-mammals, increased reliance on intronic SREs in plant splicing, and a greater reliance on SREs in mammalian splicing.
Assuntos
Éxons , Íntrons , Precursores de RNA , Sítios de Splice de RNA , Splicing de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Animais , Íntrons/genética , Éxons/genética , Genes de Plantas , Modelos Genéticos , Spliceossomos/metabolismo , Spliceossomos/genética , Plantas/genética , Humanos , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismoRESUMO
Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is hampered by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically inactive CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identifies not only known SREs but also a previously unknown distal intronic SRE, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.
Assuntos
Sistemas CRISPR-Cas , Éxons , Íntrons , Splicing de RNA , RNA Guia de Sistemas CRISPR-Cas , Proteína 2 de Sobrevivência do Neurônio Motor , Humanos , Splicing de RNA/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , RNA Guia de Sistemas CRISPR-Cas/genética , Íntrons/genética , Éxons/genética , Células HEK293 , Oligonucleotídeos Antissenso/genética , Atrofia Muscular Espinal/genética , Sequências Reguladoras de Ácido Nucleico/genética , Precursores de RNA/genética , Precursores de RNA/metabolismoRESUMO
Expression quantitative trait loci (eQTL) studies typically consider exon expression of genes and discard intronic RNA sequencing reads despite their information on RNA metabolism. Here, we quantify genetic effects on exon and intron levels of genes and their ratio in lymphoblastoid cell lines, revealing thousands of cis-QTLs of each type. While genetic effects are often shared between cis-QTL types, 7814 (47%) are not detected as top cis-QTLs at exon levels. We show that exon levels preferentially capture genetic effects on transcriptional regulation, while exon-intron-ratios better detect those on co- and post-transcriptional processes. Considering all cis-QTL types substantially increases (by 71%) the number of colocalizing variants identified by genome-wide association studies (GWAS). It further allows dissecting the potential gene regulatory processes underlying GWAS associations, suggesting comparable contributions by transcriptional (50%) and co- and post-transcriptional regulation (46%) to complex traits. Overall, integrating intronic RNA sequencing reads in eQTL studies expands our understanding of genetic effects on gene regulatory processes.
Assuntos
Éxons , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Íntrons , Locos de Características Quantitativas , Humanos , Íntrons/genética , Éxons/genética , Transcrição Gênica , Linhagem Celular , Análise de Sequência de RNA/métodos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive chorioretinal degenerative disease without approved therapeutic drugs. It is caused by mutations in CYP4V2 gene, and about 80% of BCD patients carry mutations in exon 7 to 11. Here, we apply CRISPR/Cas9 mediated homology-independent targeted integration (HITI)-based gene editing therapy in HEK293T cells, BCD patient derived iPSCs, and humanized Cyp4v3 mouse model (h-Cyp4v3mut/mut) using two rAAV2/8 vectors via sub-retinal administration. We find that sgRNA-guided Cas9 generates double-strand cleavage on intron 6 of the CYP4V2 gene, and the HITI donor inserts the carried sequence, part of intron 6, exon 7-11, and a stop codon into the DNA break, achieving precise integration, effective transcription and translation both in vitro and in vivo. HITI-based editing restores the viability of iPSC-RPE cells from BCD patient, improves the morphology, number and metabolism of RPE and photoreceptors in h-Cyp4v3mut/mut mice. These results suggest that HITI-based editing could be a promising therapeutic strategy for those BCD patients carrying mutations in exon 7 to 11, and one injection will achieve lifelong effectiveness.
Assuntos
Sistemas CRISPR-Cas , Distrofias Hereditárias da Córnea , Família 4 do Citocromo P450 , Edição de Genes , Terapia Genética , Células-Tronco Pluripotentes Induzidas , Doenças Retinianas , Humanos , Edição de Genes/métodos , Animais , Células HEK293 , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/terapia , Distrofias Hereditárias da Córnea/patologia , Distrofias Hereditárias da Córnea/metabolismo , Camundongos , Células-Tronco Pluripotentes Induzidas/metabolismo , Terapia Genética/métodos , Família 4 do Citocromo P450/genética , Família 4 do Citocromo P450/metabolismo , Modelos Animais de Doenças , Mutação , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Vetores Genéticos/genética , Íntrons/genética , Éxons/genéticaRESUMO
Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development.
Assuntos
Processamento Alternativo , Diferenciação Celular , Cromatina , Ribonucleoproteínas Nucleares Heterogêneas , Neurônios , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Fatores de Transcrição , Processamento Alternativo/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Animais , Diferenciação Celular/genética , Cromatina/metabolismo , Camundongos , Neurônios/metabolismo , Neurônios/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Transcrição Gênica , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Éxons/genética , Humanos , Autorrenovação Celular/genéticaRESUMO
Compared with HLA-DRB1*12:02, the alleles HLA-DRB1*12:92 and HLA-DRB1*12:101 each show one nucleotide substitution respectively.
Assuntos
Alelos , Cadeias HLA-DRB1 , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade , Humanos , Cadeias HLA-DRB1/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade/métodos , Éxons , Polimorfismo de Nucleotídeo Único , Sequência de Bases , Análise de Sequência de DNA/métodosRESUMO
HLA-DQB1*06:466 differs from HLA-DQB1*06:01:01:01 by a single nucleotide substitution at position 571GâA.
Assuntos
Alelos , Povo Asiático , Doadores de Sangue , Éxons , Sangue Fetal , Cadeias beta de HLA-DQ , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cadeias beta de HLA-DQ/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Povo Asiático/genética , Teste de Histocompatibilidade/métodos , Polimorfismo de Nucleotídeo Único , Sequência de Bases , Análise de Sequência de DNA/métodos , População do Leste AsiáticoRESUMO
HLA-C*03:620 differs from the HLA-C*03:04:01:02 allele by one nucleotide substitution in the exon 3.
Assuntos
Alelos , Povo Asiático , Sequência de Bases , Éxons , Antígenos HLA-C , Teste de Histocompatibilidade , Humanos , Antígenos HLA-C/genética , Povo Asiático/genética , Análise de Sequência de DNA/métodos , Códon , Alinhamento de Sequência , Polimorfismo de Nucleotídeo Único , População do Leste AsiáticoRESUMO
HLA-DPB1*05:01:20 differs from HLA-DPB1*05:01:01:01 by one nucleotide in exon 3.
Assuntos
Alelos , Povo Asiático , Sequência de Bases , Éxons , Cadeias beta de HLA-DP , Teste de Histocompatibilidade , Análise de Sequência de DNA , Humanos , Cadeias beta de HLA-DP/genética , Povo Asiático/genética , Análise de Sequência de DNA/métodos , Doadores de Tecidos , Alinhamento de Sequência , Códon , População do Leste AsiáticoRESUMO
HLA-B*15:659 differs from HLA-B*15:02:01:01 by one nucleotide in exon 2.
Assuntos
Alelos , Povo Asiático , Sequência de Bases , Éxons , Teste de Histocompatibilidade , Análise de Sequência de DNA , Humanos , Povo Asiático/genética , Análise de Sequência de DNA/métodos , Antígeno HLA-B15/genética , Antígeno HLA-B15/imunologia , Alinhamento de Sequência , Códon , Doadores de Tecidos , População do Leste AsiáticoRESUMO
Genomic full-length sequence of HLA-B*37:46 was identified by a group-specific sequencing approach in a Chinese individual.
Assuntos
Alelos , Povo Asiático , Antígenos HLA-B , Teste de Histocompatibilidade , Análise de Sequência de DNA , Humanos , Antígenos HLA-B/genética , Análise de Sequência de DNA/métodos , Teste de Histocompatibilidade/métodos , Povo Asiático/genética , Éxons , Sequência de BasesRESUMO
HLA-B*15:02:15 differs from HLA-B*15:02:01:01 by one nucleotide in exon 2.