RESUMO
Oprm1, the gene encoding the µ-opioid receptor, has multiple reported transcripts, with a variable 3' region and many alternative sequences encoding the C-terminus of the protein. The functional implications of this variability remain mostly unexplored, though a recurring notion is that it could be exploited by developing selective ligands with improved clinical profiles. Here, we comprehensively examined Oprm1 transcriptional variants in the murine central nervous system, using long-read RNAseq as well as spatial and single-cell transcriptomics. The results were validated with RNAscope in situ hybridization. We found a mismatch between transcripts annotated in the mouse genome (GRCm38/mm10) and the RNA-seq results. Sequencing data indicated that the primary Oprm1 transcript has a 3' terminus located on chr10:6,860,027, which is â¼ 9.5 kilobases downstream of the longest annotated exon 4 end. Long-read sequencing confirmed that the final Oprm1 exon included a 10.2 kilobase long 3' untranslated region, and the presence of the long variant was unambiguously confirmed using RNAscope in situ hybridization in the thalamus, striatum, cortex and spinal cord. Conversely, expression of the Oprm1 reference transcript or alternative transcripts of the Oprm1 gene was absent or close to the detection limit. Thus, the primary transcript of the Oprm1 mouse gene is a variant with a long 3' untranslated region, which is homologous to the human OPRM1 primary transcript and encodes the same conserved C-terminal amino acid sequence.
Assuntos
Prosencéfalo , Receptores Opioides mu , Medula Espinal , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Animais , Camundongos , Medula Espinal/metabolismo , Prosencéfalo/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Éxons , Regiões 3' não TraduzidasRESUMO
BACKGROUND: Cutis laxa constitutes a diverse group of connective tissue diseases, both inherited and acquired, characterized by loose skin and varying systemic involvement, including pulmonary lesions. While cutis laxa has been linked to conditions like emphysema, asthma, and bronchiectasis, the specific pathological and radiological characteristics underlying pulmonary complications related to cutis laxa remain unclear. CASE PRESENTATION: A 36-year-old woman, diagnosed with cutis laxa at birth, presented to our outpatient clinic with severe obstructive ventilatory impairment, evident in pulmonary function tests (expiratory volume in one second (FEV1)/forced vital capacity (FVC): 34.85%; %residual volume [RV]: 186.5%; %total lung capacity [TLC]: 129.2%). Pulmonary function tests also indicated small airway disease (%FEF50%, 7.9%; %FEF75%, 5.7%; and %FEF25-75%, 6.8%). Computed tomography (CT) revealed the lack of normal increase in lung attenuation on expiratory CT scan, with no discernible emphysematous changes. Exome sequencing was performed to confirm the association between the pulmonary lesions and cutis laxa, revealing a frameshift variant in exon 30 of the elastin gene (ELN). Further analysis employing a parametric response map revealed a longitudinal increase in the percentage of functional small airway disease (fSAD) from 37.84% to 46.61% over the 8-year follow-up, despite the absence of overt changes in CT findings, specifically the lack of normal increase in lung attenuation on expiratory CT scan. Over the same follow-up interval, there was a modest reduction of 25.6 mL/year in FEV1 coupled with a significant increase in %RV. Pulmonary function test metrics, reflective of small airway disease, exhibited a continual decline; specifically, %FEF50%, %FEF75%, and %FEF25-75% diminished from 7.9% to 7.0%, 5.7% to 4.6%, and 6.8% to 5.4%, respectively. CONCLUSIONS: This case highlighted an instance of autosomal dominant cutis laxa arising from a frameshift variant in exon 30 of ELN, accompanied by small airway disease. Comprehensive investigation, utilizing quantitative CT analysis, revealed a longitudinal increase in fSAD percentage with a mild reduction in FEV1. These findings indicate that elastin deficiency may not only diminish elastic fibers in the skin but also be implicated in small airway disease by impacting components of the extracellular matrix in the lungs.
Assuntos
Cútis Laxa , Elastina , Mutação da Fase de Leitura , Tomografia Computadorizada por Raios X , Humanos , Cútis Laxa/genética , Feminino , Adulto , Elastina/genética , Japão , Éxons/genética , Seguimentos , Testes de Função Respiratória , Pulmão/diagnóstico por imagem , Pulmão/fisiopatologia , Pulmão/patologia , População do Leste AsiáticoRESUMO
Congenital bilateral absence of vas deferens (CBAVD) is a urological syndrome of Wolffian ducts and is responsible for male infertility and obstructive azoospermia. This study is designed to explore the integrity of exon 10 of CFTR and its role in male infertility in a cohort of CBVAD patients in Pakistan. Genomic DNA was extracted from 17 male patients with CBAVD having clinical symptoms, and 10 healthy controls via phenol-chloroform method. Exon 10 of the CFTR gene was amplified, using PCR with specific primers and DNA screening was done by Sanger sequencing. Sequencing results were analyzed using freeware Serial Cloner, SnapGene, BioEdit and FinchTV. Furthermore, bioinformatics tools were used to analyze the mutations and their impact on the protein function and stability. We have identified 4 mutations on exon 10 of CFTR in 6 out of 17 patients. Two of the mutations were missense variants V456A, K464E, and the other two were silent mutations G437G, S431S. The identified variant V456A was present in 4 of the studied patients. Whereas, the presence of K464E in our patients further weighs on the crucial importance for its strategic location to influence the gene function at post-transcriptional and protein level. Furthermore, Polyphen-2 and SIFT analyze the mutations as harmful and deleterious. The recurrence of V456A and tactically conserved locality of K464E are evidence of their potential role in CBAVD patients and in male infertility. The data can contribute in developing genetic testing and treatment of CBAVD.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Éxons , Infertilidade Masculina , Mutação , Ducto Deferente , Humanos , Masculino , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Ducto Deferente/anormalidades , Paquistão , Infertilidade Masculina/genética , Mutação de Sentido Incorreto , Adulto , Transtorno 46,XY do Desenvolvimento Sexual/genética , Transtorno 46,XY do Desenvolvimento Sexual/diagnóstico , Estudos de Coortes , Estudos de Casos e Controles , Doenças Urogenitais MasculinasRESUMO
Previously, we showed that the germ cell-specific nuclear protein RBMXL2 represses cryptic splicing patterns during meiosis and is required for male fertility (Ehrmann et al., 2019). Here, we show that in somatic cells the similar yet ubiquitously expressed RBMX protein has similar functions. RBMX regulates a distinct class of exons that exceed the median human exon size. RBMX protein-RNA interactions are enriched within ultra-long exons, particularly within genes involved in genome stability, and repress the selection of cryptic splice sites that would compromise gene function. The RBMX gene is silenced during male meiosis due to sex chromosome inactivation. To test whether RBMXL2 might replace the function of RBMX during meiosis we induced expression of RBMXL2 and the more distantly related RBMY protein in somatic cells, finding each could rescue aberrant patterns of RNA processing caused by RBMX depletion. The C-terminal disordered domain of RBMXL2 is sufficient to rescue proper splicing control after RBMX depletion. Our data indicate that RBMX and RBMXL2 have parallel roles in somatic tissues and the germline that must have been conserved for at least 200 million years of mammalian evolution. We propose RBMX family proteins are particularly important for the splicing inclusion of some ultra-long exons with increased intrinsic susceptibility to cryptic splice site selection.
Assuntos
Éxons , Sítios de Splice de RNA , Splicing de RNA , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Humanos , Éxons/genética , Sítios de Splice de RNA/genética , Masculino , Meiose/genética , Animais , Ribonucleoproteínas Nucleares HeterogêneasRESUMO
Antibodies that gain specificity by a large insert encoding for an extra domain were described for the first time in 2016. In malaria-exposed individuals, an exon deriving from the leukocyte-associated immunoglobulin-like 1 (LAIR1) gene integrated via a copy-and-paste insertion into the immunoglobulin heavy chain encoding region. A few years later, a second example was identified, namely a dual exon integration from the leukocyte immunoglobulin-like receptor B1 (LILRB1) gene that is located in close proximity to LAIR1. A dedicated high-throughput characterization of chimeric immunoglobulin heavy chain transcripts unraveled, that insertions from distant genomic regions (including mitochondrial DNA) can contribute to human antibody diversity. This review describes the modalities of insert-containing antibodies. The role of known DNA mobility aspects, such as genomic translocation, gene conversion, and DNA fragility, is discussed in the context of insert-antibody generation. Finally, the review covers why insert antibodies were omitted from the past repertoire analyses and how insert antibodies can contribute to protective immunity or an autoreactive response.
Assuntos
Éxons , Recombinação V(D)J , Humanos , Recombinação V(D)J/genética , Éxons/genética , Animais , Anticorpos/imunologia , Anticorpos/genética , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores Imunológicos/imunologia , Diversidade de Anticorpos/genéticaRESUMO
It is unclear how patterns of regional genetic differentiation in the UK and Ireland might impact the protein-coding fraction of the genome. We exploit UK Biobank (UKB) and Viking Genes whole exome sequencing data to study regional genetic differentiation across the UK and Ireland in protein coding genes, encompassing 44,696 unrelated individuals from 20 regions of origin. We demonstrate substantial exonic differentiation among Shetlanders, Orcadians, individuals with full or partial Ashkenazi Jewish ancestry and in several mainland regions (particularly north and south Wales, southeast Scotland and Ireland). With stringent filtering criteria, we find 67 regionally enriched (≥5-fold) variants likely to have adverse biomedical consequences in homozygous individuals. Here, we show that regional genetic variation across the UK and Ireland should be considered in the design of genetic studies and may inform effective genetic screening and counselling.
Assuntos
Éxons , Variação Genética , Humanos , Irlanda , Reino Unido , Éxons/genética , Sequenciamento do Exoma , Genética Populacional , Judeus/genética , Genoma Humano , Polimorfismo de Nucleotídeo ÚnicoRESUMO
A boy with nonambulatory Duchenne muscular dystrophy (DMD) tested positive for exon 63 duplication and exhibited intellectual disability, overweight and dyslipidaemia. The patient underwent a comprehensive multidisciplinary approach involving pharmacological and non-pharmacological interventions. Despite challenges, such as socioeconomic constraints and limited access to advanced therapies, the patient received tailored care. The management included prednisone medication, dietary modifications and psychological support. The patient's journey highlighted the complex interplay of medical and psychosocial factors affecting DMD patients in resource-limited settings. Regular monitoring and the involvement of the patient's family in a peer group were arranged to improve overall quality of life. The case underscores the need for accessible and holistic care for DMD patients, addressing both medical and psychosocial challenges.
Assuntos
Éxons , Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Masculino , Criança , Qualidade de Vida , Prednisona/uso terapêutico , Deficiência Intelectual/genéticaRESUMO
One nucleotide substitution in codon 136 of HLA-B*40:02:01:01 results in a novel allele, HLA-B*40:78.
Assuntos
Alelos , Sequência de Bases , Códon , Éxons , Antígeno HLA-B40 , Teste de Histocompatibilidade , Humanos , Taiwan , Teste de Histocompatibilidade/métodos , Antígeno HLA-B40/genética , Análise de Sequência de DNA/métodos , Povo Asiático/genética , Alinhamento de Sequência , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Alternative splicing (AS) is a crucial mechanism contributing to proteomic diversity, which is highly regulated in tissue- and development-specific patterns. Retinal tissue exhibits one of the highest levels of AS. In particular, photoreceptors have a distinctive AS pattern involving the inclusion of microexons not found in other cell types. PROM1 whose encoded protein Prominin-1 is located in photoreceptor outer segments (OSs), undergoes exon 4 inclusion from the 12th post-conception week of human development through adulthood. Exon 4 skipping in PROM1 is associated with late-onset mild maculopathy, however its role in photoreceptor maturation and function is unknown. In this study retinal organoids, a valuable model system, were employed in combination with phosphorodiamidate morpholino oligos (PMOs) to assess the role of exon 4 AS in the development of human retina. Retinal organoids were treated with the PMOs for four weeks after which RT-PCR, western blotting and immunofluorescence analysis were performed to assess exon 4 exclusion and its impact on photoreceptors. The transcriptome of treated ROs was studied by bulk RNA-Seq. Our data demonstrate that 55% skipping of PROM1 exon 4 resulted in decreased Prominin-1 expression by 40%, abnormal accumulation of cones in the basal side of the retinal organoids as well as detectable cone photoreceptor cilium defects. Transcriptomic and western blot analyses revealed decreased expression of cone, inner segment and connecting cilium basal body markers, increased expression of genes associated with stress response and the ubiquitin-proteasome system, and downregulation of autophagy. Importantly, the use of retinal organoids provides a valuable platform to study AS and unravel disease mechanisms in a more physiologically relevant context, opening avenues for further research and potential therapeutic interventions. Together our data indicate that cones may be more sensitive to PROM1 exon 4 skipping and/or reduced Prominin-1 expression, corroborating the pathogenesis of late-onset mild maculopathy.
Assuntos
Antígeno AC133 , Processamento Alternativo , Humanos , Processamento Alternativo/genética , Antígeno AC133/metabolismo , Antígeno AC133/genética , Organoides/metabolismo , Éxons/genética , Retina/metabolismo , Retina/crescimento & desenvolvimento , Células Fotorreceptoras de Vertebrados/metabolismoRESUMO
BACKGROUND: Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. METHODS: We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status. RESULTS: Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. CONCLUSIONS: Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.
Assuntos
Sistemas CRISPR-Cas , Distrofina , Éxons , Edição de Genes , Terapia Genética , Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofina/genética , Edição de Genes/métodos , Terapia Genética/métodos , Linhagem Celular , Deleção de Sequência , Mioblastos/metabolismoRESUMO
HLA-C*01:262 differs from HLA-C*01:02:01:01 by one nucleotide substitution in codon 150 in exon 3.
Assuntos
Alelos , Sequência de Bases , Códon , Éxons , Antígenos HLA-C , Teste de Histocompatibilidade , Análise de Sequência de DNA , Humanos , Antígenos HLA-C/genética , Teste de Histocompatibilidade/métodos , Análise de Sequência de DNA/métodos , Alinhamento de Sequência , Polimorfismo de Nucleotídeo ÚnicoRESUMO
HLA-DQA1*01:02:24 differs from HLA-DQA1*01:02:01:03 by one nucleotide substitution in codon 167 in exon 3.
Assuntos
Alelos , Sequência de Bases , Éxons , Cadeias alfa de HLA-DQ , Teste de Histocompatibilidade , Análise de Sequência de DNA , Humanos , Cadeias alfa de HLA-DQ/genética , Análise de Sequência de DNA/métodos , Códon , Alinhamento de SequênciaRESUMO
HLA-B*49:01:01:22 differs from HLA-B*49:01:01:01 by a single nucleotide C->G change in intron 5 at gDNA 2451.
Assuntos
Alelos , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade , Íntrons , Transplante de Rim , Doadores de Tecidos , Humanos , Teste de Histocompatibilidade/métodos , Índia , Éxons , Sequência de Bases , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , Antígeno HLA-B39/genéticaRESUMO
The novel HLA-B*18:37:03 allele, first described in a potential bone marrow donor from Brazil.
Assuntos
Alelos , Éxons , Humanos , Sequência de Bases , Transplante de Medula Óssea , Brasil , Teste de Histocompatibilidade , Antígeno HLA-B18/genética , Antígeno HLA-B18/imunologia , Alinhamento de Sequência , Análise de Sequência de DNA , Doadores de TecidosRESUMO
HLA-DQB1*05:305 shows one single nucleotide substitution at position 664 compared with HLA-DQB1*05:03:01:01.
Assuntos
Alelos , Éxons , Cadeias beta de HLA-DQ , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade/métodos , Cadeias beta de HLA-DQ/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodosRESUMO
HLA-DRB3*02:202 differs from DRB3*02:112 by one nucleotide substitution in codon 51 in exon 2.
Assuntos
Alelos , Sequência de Bases , Éxons , Cadeias HLA-DRB3 , Teste de Histocompatibilidade , Análise de Sequência de DNA , Humanos , Códon , Teste de Histocompatibilidade/métodos , Cadeias HLA-DRB3/genética , Alinhamento de Sequência , Análise de Sequência de DNA/métodosRESUMO
HLA-A*26:247 differs from HLA-A*26:01:01:01 by one nucleotide substitution in codon 245 in exon 4.
Assuntos
Alelos , Sequência de Bases , Éxons , Antígenos HLA-A , Teste de Histocompatibilidade , Análise de Sequência de DNA , Humanos , Códon , Teste de Histocompatibilidade/métodos , Antígenos HLA-A/genética , Alinhamento de Sequência , Análise de Sequência de DNA/métodosRESUMO
T cell receptor (TCR) sensitivity to peptide-major histocompatibility complex (MHC) dictates T cell fate. Canonical models of TCR sensitivity cannot be fully explained by transcriptional regulation. In this work, we identify a posttranscriptional regulatory mechanism of TCR sensitivity that guides alternative splicing of TCR signaling transcripts through an evolutionarily ultraconserved poison exon (PE) in the RNA-binding protein (RBP) TRA2ß in mouse and human. TRA2ß-PE splicing, seen during cancer and infection, was required for TCR-induced effector T cell expansion and function. Tra2ß-PE skipping enhanced T cell response to antigen by increasing TCR sensitivity. As antigen levels decreased, Tra2ß-PE reinclusion allowed T cell survival. Finally, we found that TRA2ß-PE was first included in the genome of jawed vertebrates that were capable of TCR gene rearrangements. We propose that TRA2ß-PE splicing acts as a gatekeeper of TCR sensitivity to shape T cell fate.
Assuntos
Processamento Alternativo , Éxons , Receptores de Antígenos de Linfócitos T , Fatores de Processamento de Serina-Arginina , Animais , Humanos , Camundongos , Sobrevivência Celular , Sequência Conservada , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
PURPOSE: Genetic hypomorphic defects in X chromosomal IKBKG coding for the NF-κB essential modulator (NEMO) lead to ectodermal dysplasia and immunodeficiency in males and the skin disorder incontinentia pigmenti (IP) in females, respectively. NF-κB essential modulator (NEMO) Δ-exon 5-autoinflammatory syndrome (NEMO-NDAS) is a systemic autoinflammatory disease caused by alternative splicing and increased proportion of NEMO-Δex5. We investigated a female carrier presenting with IP and NEMO-NDAS due to non-skewed X-inactivation. METHODS: IKBKG transcripts were quantified in peripheral blood mononuclear cells isolated from the patient, her mother, and healthy controls using RT-PCR and nanopore sequencing. Corresponding proteins were analyzed by western blotting and flow cytometry. Besides toll-like receptor (TLR) and tumor necrosis factor (TNF) signaling, the interferon signature, cytokine production and X-inactivation status were investigated. RESULTS: IP and autoinflammation with recurrent fever, oral ulcers, hepatitis, and neutropenia, but no immunodeficiency was observed in a female patient. Besides moderately reduced NEMO signaling function, type I interferonopathy, and elevated IL-18 and CXCL10 were found. She and her mother both carried the heterozygous variant c.613 C > T p.(Gln205*) in exon 5 of IKBKG previously reported in NEMO-deficient patients. However, X-inactivation was skewed in the mother, but not in the patient. Alternative splicing led to increased ratios of NEMO-Dex5 over full-length protein in peripheral blood cell subsets causing autoinflammation. Clinical symptoms partially resolved under treatment with TNF inhibitors. CONCLUSION: Non-skewed X-inactivation can lead to NEMO-NDAS in females with IP carrying hypomorphic IKBKG variants due to alternative splicing and increased proportions of NEMO-∆ex5.
Assuntos
Éxons , Quinase I-kappa B , Incontinência Pigmentar , Inativação do Cromossomo X , Humanos , Feminino , Incontinência Pigmentar/genética , Incontinência Pigmentar/diagnóstico , Quinase I-kappa B/genética , Éxons/genética , Doenças Hereditárias Autoinflamatórias/genética , Doenças Hereditárias Autoinflamatórias/diagnóstico , Mutação/genética , Citocinas/metabolismo , Adulto , Processamento Alternativo , Transdução de SinaisRESUMO
HLA-B*51:411 differs from HLA-B*51:01:01:01 by one nucleotide substitution in codon 235 in exon 4.