RESUMEN

Gitelman syndrome (GS) is the most prevalent genetic tubulopathy characterized by several electrolyte abnormalities, including hypokalemia, hypomagnesemia, hypocalciuria, metabolic alkalosis, and hyperreninemic hyperaldosteronism. These features are caused by a bi-allelic mutation in the SLC12A3 gene. In this report, we present a case of GS in an asymptomatic woman who incidentally exhibited hypokalemia during an antenatal check-up. Her biochemical profile was consistent with GS. Genetic analysis revealed two heterozygous variants in trans, namely, NM_001126108.2:c.625C>T; p.(Arg209Trp) and c.965C>T; p.(Ala322Val). The c.625C>T; p.(Arg209Trp) variant has previously been experimentally confirmed as a loss-of-function (LOF) variant. However, the functional impact of the c.965C>T variant, located at the 5 prime end of exon 8, has not been fully elucidated. Through the utilization of both complementary DNA (cDNA) and minigene analysis, we confirmed that the c.965C>T variant can generate two distinct cDNA transcripts. The first transcript carries a missense mutation, p.(Ala322Val) in the full SLC12A3 transcript, while the second transcript consists of an in-frame deletion of both exons 7 and 8 in the SLC12A3 transcript, in which may result in the loss of transmembrane regions 5 - 6 involved in chloride transport. Our findings provide insights into the intricate mechanisms of splicing, highlighting how a variant in one exon can remotely influence the transcription of an upstream exon, as observed with the variant in exon 8 impacting the transcription of exon 7.

Asunto(s)

Síndrome de Gitelman , Mutación Missense , Miembro 3 de la Familia de Transportadores de Soluto 12 , Síndrome de Gitelman/genética , Humanos , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Femenino , Empalme del ARN/genética , Adulto

RESUMEN

BACKGROUND: Alternative splicing of pre-mRNA is a fundamental regulatory process in multicellular eukaryotes, significantly contributing to the diversification of the human proteome. RNA-binding fox-1 homologue 2 (RBFOX2), a member of the evolutionarily conserved RBFOX family, has emerged as a critical splicing regulator, playing a pivotal role in the alternative splicing of pre-mRNA. This review provides a comprehensive analysis of RBFOX2, elucidating its splicing activity through direct and indirect binding mechanisms. RBFOX2 exerts substantial influence over the alternative splicing of numerous transcripts, thereby shaping essential cellular processes such as differentiation and development. MAIN BODY OF THE ABSTRACT: Dysregulation of RBFOX2-mediated alternative splicing has been closely linked to a spectrum of cardiovascular diseases and malignant tumours, underscoring its potential as a therapeutic target. Despite significant progress, current research faces notable challenges. The complete structural characterisation of RBFOX2 remains elusive, limiting in-depth exploration beyond its RNA-recognition motif. Furthermore, the scarcity of studies focusing on RBFOX2-targeting drugs poses a hindrance to translating research findings into clinical applications. CONCLUSION: This review critically assesses the existing body of knowledge on RBFOX2, highlighting research gaps and limitations. By delineating these areas, this analysis not only serves as a foundational reference for future studies but also provides strategic insights for bridging these gaps. Addressing these challenges will be instrumental in unlocking the full therapeutic potential of RBFOX2, paving the way for innovative and effective treatments in various diseases.

Asunto(s)

Neoplasias , Factores de Empalme de ARN , Humanos , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Progresión de la Enfermedad , Empalme Alternativo/genética , Empalme del ARN/genética

RESUMEN

Male infertility due to asthenozoospermia is quite frequent, but its etiology is poorly understood. We recruited two infertile brothers, born to first-cousin parents from Pakistan, displaying idiopathic asthenozoospermia with mild stuttering disorder but no ciliary-related symptoms. Whole-exome sequencing identified a splicing variant (c.916+1G>A) in ARMC3, recessively co-segregating with asthenozoospermia in the family. The ARMC3 protein is evolutionarily highly conserved and is mostly expressed in the brain and testicular tissue of human. The ARMC3 splicing mutation leads to the exclusion of exon 8, resulting in a predicted truncated protein (p.Glu245_Asp305delfs*16). Quantitative real-time PCR revealed a significant decrease at mRNA level for ARMC3 and Western blot analysis did not detect ARMC3 protein in the patient's sperm. Individuals homozygous for the ARMC3 splicing variant displayed reduced sperm motility with frequent morphological abnormalities of sperm flagella. Transmission electron microscopy of the affected individual IV: 2 revealed vacuolation in sperm mitochondria at the midpiece and disrupted flagellar ultrastructure in the principal and end piece. Altogether, our results indicate that this novel homozygous ARMC3 splicing mutation destabilizes sperm flagella and leads to asthenozoospermia in our patients, providing a novel marker for genetic counseling and diagnosis of male infertility.

Asunto(s)

Astenozoospermia , Consanguinidad , Homocigoto , Linaje , Empalme del ARN , Cola del Espermatozoide , Adulto , Humanos , Masculino , Astenozoospermia/genética , Astenozoospermia/patología , Secuenciación del Exoma , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Mutación , Empalme del ARN/genética , Motilidad Espermática/genética , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Cola del Espermatozoide/metabolismo , Espermatozoides/ultraestructura , Espermatozoides/patología

RESUMEN

Advances in next-generation sequencing technologies have led to elucidation of sensorineural hearing loss genetics and associated clinical impacts. However, studies on the functional pathogenicity of variants of uncertain significance (VUS), despite their close association with clinical phenotypes, are lacking. Here we identified compound heterozygous variants in ESRRB transcription factor gene linked to DFNB35, specifically a novel splicing variant (NM_004452.4(ESRRB): c.397 + 2T>G) in trans with a missense variant (NM_004452.4(ESRRB): c.1144C>T p.(Arg382Cys)) whose pathogenicity remains unclear. The splicing variant (c.397 + 2T>G) caused exon 4 skipping, leading to premature stop codon formation and nonsense-mediated decay. The p.(Arg382Cys) variant was classified as a VUS due to its particularly higher allele frequency among East Asian population despite disease-causing in-silico predictions. However, functional assays showed that p.(Arg382Cys) variant disrupted key intramolecular interactions, leading to protein instability. This variant also reduced transcriptional activity and altered expression of downstream target genes essential for inner ear function, suggesting genetic contribution to disease phenotype. This study expanded the phenotypic and genotypic spectrum of ESRRB in DFNB35 and revealed molecular mechanisms underlying ESRRB-associated DFNB35. These findings suggest that variants with high allele frequencies can also possess functional pathogenicity, providing a breakthrough for cases where VUS, previously unexplored, could be reinterpreted by elucidating their functional roles and disease-causing characteristics.

Asunto(s)

Pérdida Auditiva Sensorineural , Receptores de Estrógenos , Femenino , Humanos , Masculino , Codón sin Sentido/genética , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Pérdida Auditiva/genética , Pérdida Auditiva Sensorineural/genética , Mutación Missense , Linaje , Empalme del ARN/genética , Receptores de Estrógenos/genética

RESUMEN

The contribution of splicing variants to molecular diagnostics of inherited diseases is reported to be less than 10%. This figure is likely an underestimation due to several factors including difficulty in predicting the effect of such variants, the need for functional assays, and the inability to detect them (depending on their locations and the sequencing technology used). The aim of this study was to assess the utility of Nanopore sequencing in characterizing and quantifying aberrant splicing events. For this purpose, we selected 19 candidate splicing variants that were identified in patients affected by inherited retinal dystrophies. Several in silico tools were deployed to predict the nature and estimate the magnitude of variant-induced aberrant splicing events. Minigene assay or whole blood-derived cDNA was used to functionally characterize the variants. PCR amplification of minigene-specific cDNA or the target gene in blood cDNA, combined with Nanopore sequencing, was used to identify the resulting transcripts. Thirteen out of nineteen variants caused aberrant splicing events, including cryptic splice site activation, exon skipping, pseudoexon inclusion, or a combination of these. Nanopore sequencing allowed for the identification of full-length transcripts and their precise quantification, which were often in accord with in silico predictions. The method detected reliably low-abundant transcripts, which would not be detected by conventional strategies, such as RT-PCR followed by Sanger sequencing.

Asunto(s)

Secuenciación de Nucleótidos de Alto Rendimiento , Secuenciación de Nanoporos , Distrofias Retinianas , Humanos , Distrofias Retinianas/genética , Distrofias Retinianas/diagnóstico , Secuenciación de Nanoporos/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Empalme Alternativo/genética , Empalme del ARN/genética , Exones/genética

RESUMEN

Vascular Ehlers-Danlos syndrome or Ehlers-Danlos syndrome type IV (vEDS) is a connective tissue disorder characterised by skin hyperextensibility, joint hypermobility and fatal vascular rupture caused by COL3A1 mutations that affect collagen III expression, homo-trimer assembly and secretion. Along with collagens I, II, V and XI, collagen III plays an important role in the extracellular matrix, particularly in the inner organs. To date, only symptomatic treatment for vEDS patients is available. Fibroblasts derived from vEDS patients carrying dominant negative and/or haploinsufficiency mutations in COL3A1 deposit reduced collagen III in the extracellular matrix. This study explored the potential of an antisense oligonucleotide (ASO)-mediated splice modulating strategy to bypass disease-causing COL3A1 mutations reported in the in-frame exons 10 and 15. Antisense oligonucleotides designed to redirect COL3A1 pre-mRNA processing and excise exons 10 or 15 were transfected into dermal fibroblasts derived from vEDS patients and a healthy control subject. Efficient exon 10 or 15 excision from the mature COL3A1 mRNA was achieved and intracellular collagen III expression was increased after treatment with ASOs; however, collagen III deposition into the extracellular matrix was reduced in patient cells. The region encoded by exon 10 includes a glycosylation site, and exon 15 encodes hydroxyproline and hydroxylysine-containing triplet repeats, predicted to be crucial for collagen III assembly. These results emphasize the importance of post-translational modification for collagen III homo-trimer assembly. In conclusion, while efficient skipping of target COL3A1 exons was achieved, the induced collagen III isoforms generated showed defects in extracellular matrix formation. While therapeutic ASO-mediated exon skipping is not indicated for the patients in this study, the observations are restricted to exons 10 and 15 and may not be applicable to other collagen III in-frame exons.

Asunto(s)

Colágeno Tipo III , Síndrome de Ehlers-Danlos , Exones , Fibroblastos , Mutación , Oligonucleótidos Antisentido , Humanos , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/terapia , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Exones/genética , Fibroblastos/metabolismo , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Células Cultivadas , Empalme del ARN/genética , Masculino , Matriz Extracelular/metabolismo , Síndrome de Ehlers-Danlos Tipo IV

RESUMEN

Acute liver failure (ALF) is characterized by the rapidly progressive deterioration of hepatic function, which, without effective medical intervention, results in high mortality and morbidity. Here, using proteomic and transcriptomic analyses in murine ALF models, we found that the expression of multiple splicing factors was downregulated in ALF. Notably, we found that KH-type splicing regulatory protein (KHSRP) has a protective effect in ALF. Knockdown of KHSRP resulted in dramatic splicing defects, such as intron retention, and led to the exacerbation of liver injury in ALF. Moreover, we demonstrated that KHSRP directly interacts with splicing factor 3b subunit 1 (SF3B1) and enhances the binding of SF3B1 to the intronic branch sites, thereby promoting pre-mRNA splicing. Using splicing inhibitors, we found that Khsrp protects against ALF by regulating pre-mRNA splicing in vivo. Overall, our findings demonstrate that KHSRP is an important splicing activator and promotes the expression of genes associated with ALF progression by interacting with SF3B1; thus, KHSRP could be a possible target for therapeutic intervention in ALF.

Asunto(s)

Fallo Hepático Agudo , Precursores del ARN , Factores de Empalme de ARN , Empalme del ARN , Proteínas de Unión al ARN , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/genética , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Empalme del ARN/genética , Fallo Hepático Agudo/metabolismo , Fallo Hepático Agudo/genética , Precursores del ARN/metabolismo , Precursores del ARN/genética , Ratones , Humanos , Ratones Endogámicos C57BL , Masculino , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Modelos Animales de Enfermedad , Unión Proteica , Transactivadores

RESUMEN

Biallelic variants in USH2A are associated with retinitis pigmentosa (RP) and Type 2 Usher Syndrome (USH2), leading to impaired vision and, additionally, hearing loss in the latter. Although the introduction of next-generation sequencing into clinical diagnostics has led to a significant uplift in molecular diagnostic rates, many patients remain molecularly unsolved. It is thought that non-coding variants or variants of uncertain significance contribute significantly to this diagnostic gap. This study aims to demonstrate the clinical utility of the reverse transcription-polymerase chain reaction (RT-PCR)-Oxford Nanopore Technology (ONT) sequencing of USH2A mRNA transcripts from nasal epithelial cells to determine the splice-altering effect of candidate variants. Five affected individuals with USH2 or non-syndromic RP who had undergone whole genome sequencing were recruited for further investigation. All individuals had uncertain genotypes in USH2A, including deep intronic rare variants, c.8682-654C>G, c.9055+389G>A, and c.9959-2971C>T; a synonymous variant of uncertain significance, c.2139C>T; p.(Gly713=); and a predicted loss of function duplication spanning an intron/exon boundary, c.3812-3_3837dup p.(Met1280Ter). In silico assessment using SpliceAI provided splice-altering predictions for all candidate variants which were investigated using ONT sequencing. All predictions were found to be accurate; however, in the case of c.3812-3_3837dup, the outcome was a complex cryptic splicing pattern with predominant in-frame exon 18 skipping and a low level of exon 18 inclusion leading to the predicted stop gain. This study detected and functionally characterised simple and complex mis-splicing patterns in USH2A arising from previously unknown deep intronic variants and previously reported variants of uncertain significance, confirming the pathogenicity of the variants.

Asunto(s)

Proteínas de la Matriz Extracelular , Empalme del ARN , Síndromes de Usher , Humanos , Proteínas de la Matriz Extracelular/genética , Síndromes de Usher/genética , Femenino , Masculino , Empalme del ARN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Exones/genética , Mutación/genética , Retinitis Pigmentosa/genética , Adulto , ARN Mensajero/genética , ARN Mensajero/metabolismo , Intrones/genética , Persona de Mediana Edad

RESUMEN

BACKGROUND: Global developmental delay with speech and behavioral abnormalities (OMIM: 619243) is an autosomal dominant disease caused by variants in TNRC6B gene. METHOD: We reviewed and summarized clinical manifestations and genotypes in patients previously reported with TNRC6B gene variants. We used several prediction tools to predict pathogenicity and performed minigene assays to verify the function of the synonymous variant affecting RNA splicing. RESULT: The patient presented with convulsive seizures and developmental delay. WES combined with functional studies diagnosed a child with a synonymous variant in TNRC6B gene. Through minigene assay and Sanger sequencing, we demonstrated that c.3141G > A variant induced exon 7 skipping and the synonymous variant was pathogenic. CONCLUSION: Synonymous variants do not change the amino acids encoded by the codon, so we usually consider synonymous variants to be benign and ignore their pathogenicity. Minigene assay is a valuable tool to identify the effect of variation on RNA splicing and identify synonymous variants' benign or pathogenic. We showed that the synonymous variant was pathogenic by minigene assay. WES combined with minigene assay establishes a robust basis for genetic counseling and diagnosing diseases.

Asunto(s)

Empalme del ARN , Humanos , Empalme del ARN/genética , Discapacidades del Desarrollo/genética , Exones/genética , Masculino , Mutación Silenciosa , Secuenciación del Exoma/métodos , Femenino , Genotipo , Niño , Preescolar

Asunto(s)

Discapacidades del Desarrollo , ARN , Enfermedades Raras , Humanos , Discapacidades del Desarrollo/genética , Enfermedades Raras/genética , ARN/genética , Empalme del ARN/genética , Empalmosomas/genética

RESUMEN

BACKGROUND: The RNA N6-methyladenosine (m6A) modification has become an essential hotspot in epigenetic modulation. Serine-arginine protein kinase 1 (SRPK1) is associated with the pathogenesis of various cancers. However, the m6A modification of SRPK1 and its association with the mechanism of in lung adenocarcinoma (LUAD) remains unclear. METHODS: Western blotting and polymerase chain reaction (PCR) analyses were carried out to identify gene and protein expression. m6A epitranscriptomic microarray was utilized to the assess m6A profile. Loss and gain-of-function assays were carried out elucidate the impact of METTL3 and SRPK1 on LUAD glycolysis and tumorigenesis. RNA immunoprecipitation (RIP), m6A RNA immunoprecipitation (MeRIP), and RNA stability tests were employed to elucidate the SRPK1's METTL3-mediated m6A modification mechanism in LUAD. Metabolic quantification and co-immunoprecipitation assays were applied to investigate the molecular mechanism by which SRPK1 mediates LUAD metabolism. RESULTS: The epitranscriptomic microarray assay revealed that SRPK1 could be hypermethylated and upregulated in LUAD. The main transmethylase METTL3 was upregulated and induced the aberrant high m6A levels of SRPK1. Mechanistically, SRPK1's m6A sites were directly methylated by METTL3, which also stabilized SRPK1 in an IGF2BP2-dependent manner. Methylated SRPK1 subsequently promoted LUAD progression through enhancing glycolysis. Further metabolic quantification, co-immunoprecipitation and western blot assays revealed that SRPK1 interacts with hnRNPA1, an important modulator of PKM splicing, and thus facilitates glycolysis by upregulating PKM2 in LUAD. Nevertheless, METTL3 inhibitor STM2457 can reverse the above effects in vitro and in vivo by suppressing SRPK1 and glycolysis in LUAD. CONCLUSION: It was revealed that in LUAD, aberrantly expressed METTL3 upregulated SRPK1 levels via an m6A-IGF2BP2-dependent mechanism. METTL3-induced SRPK1 fostered LUAD cell proliferation by enhancing glycolysis, and the small-molecule inhibitor STM2457 of METTL3 could be an alternative novel therapeutic strategy for individuals with LUAD.

Asunto(s)

Adenocarcinoma del Pulmón , Adenosina , Glucólisis , Neoplasias Pulmonares , Metiltransferasas , Proteínas Serina-Treonina Quinasas , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Glucólisis/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Animales , Regulación Neoplásica de la Expresión Génica , Ratones , Línea Celular Tumoral , Ratones Desnudos , Empalme del ARN/genética , Proteínas de Unión a Hormona Tiroide , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Proliferación Celular/genética

RESUMEN

Juvenile polyposis syndrome (JPS) is an inherited autosomal dominant condition that predisposes to the development of juvenile polyps throughout the gastrointestinal (GI) tract, and it poses an increased risk of GI malignancy. Germline causative variants were identified in the SMAD4 gene in a subset (20%) of JPS cases. Most SMAD4 germline genetic variants published to date are missense, nonsense, and frameshift mutations. SMAD4 germline alterations predicted to result in aberrant splicing have rarely been reported. Here, we report two unrelated Italian families harboring two different SMAD4 intronic variants, c.424+5G>A and c.425-9A>G, which are clinically associated with colorectal cancer and/or juvenile GI polyps. In silico prediction analysis, in vitro minigene assays, and RT-PCR showed that the identified variants lead to aberrant SMAD4 splicing via the exonization of intronic nucleotides, resulting in a premature stop codon. This is expected to cause the production of a truncated protein. This study expands the landscape of SMAD4 germline genetic variants associated with GI polyposis and/or cancer. Moreover, it emphasizes the importance of the functional characterization of SMAD4 splicing variants through RNA analysis, which can provide new insights into genetic disease variant interpretation, enabling tailored genetic counseling, management, and surveillance of patients with GI polyposis and/or cancer.

Asunto(s)

Poliposis Intestinal , Síndromes Neoplásicos Hereditarios , Empalme del ARN , Proteína Smad4 , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación de Línea Germinal , Poliposis Intestinal/genética , Poliposis Intestinal/congénito , Intrones/genética , Síndromes Neoplásicos Hereditarios/genética , Linaje , Empalme del ARN/genética , Proteína Smad4/genética

Asunto(s)

Homocigoto , Humanos , Masculino , Apraxias/genética , Femenino , Neuropatía Hereditaria Motora y Sensorial/genética , Neuropatía Hereditaria Motora y Sensorial/patología , Mutación , Linaje , Empalme del ARN/genética

RESUMEN

BACKGROUND: Primary ciliary dyskinesia (PCD) is an autosomal recessive hereditary disease characterized by recurrent respiratory infections. In clinical manifestations, DNAH5 (NM_001361.3) is one of the recessive pathogenic genes. Primary familial brain calcification (PFBC) is a neurodegenerative disease characterized by bilateral calcification in the basal ganglia and other brain regions. PFBC can be inherited in an autosomal dominant or recessive manner. A family with PCD caused by a DNAH5 compound heterozygous variant and PFBC caused by a MYORG homozygous variant was analyzed. METHODS: In this study, we recruited three generations of Han families with primary ciliary dyskinesia combined with primary familial brain calcification. Their clinical phenotype data were collected, next-generation sequencing was performed to screen suspected pathogenic mutations in the proband and segregation analysis of families was carried out by Sanger sequencing. The mutant and wild-type plasmids were constructed and transfected into HEK293T cells instantaneously, and splicing patterns were detected by Minigene splicing assay. The structure and function of mutations were analyzed by bioinformatics analysis. RESULTS: The clinical phenotypes of the proband (II10) and his sister (II8) were bronchiectasis, recurrent pulmonary infection, multiple symmetric calcifications of bilateral globus pallidus and cerebellar dentate nucleus, paranasal sinusitis in the whole group, and electron microscopy of bronchial mucosa showed that the ciliary axoneme was defective. There was also total visceral inversion in II10 but not in II8. A novel splice variant C.13,338 + 5G > C and a frameshift variant C.4314delT (p. Asn1438lysfs *10) were found in the DNAH5 gene in proband (II10) and II8. c.347_348dupCTGGCCTTCCGC homozygous insertion variation was found in the MYORG of the proband. The two pathogenic genes were co-segregated in the family. Minigene showed that DNAH5 c.13,338 + 5G > C has two abnormal splicing modes: One is that part of the intron bases where the mutation site located is translated, resulting in early translation termination of DNAH5; The other is the mutation resulting in the deletion of exon76. CONCLUSIONS: The newly identified DNAH5 splicing mutation c.13,338 + 5G > C is involved in the pathogenesis of PCD in the family, and forms a compound heterozygote with the pathogenic variant DNAH5 c.4314delT lead to the pathogenesis of PCD.

Asunto(s)

Calcinosis , Mutación , Linaje , Humanos , Masculino , Calcinosis/genética , Calcinosis/patología , Femenino , Dineínas Axonemales/genética , Adulto , Trastornos de la Motilidad Ciliar/genética , Encefalopatías/genética , Fenotipo , Células HEK293 , China , Empalme del ARN/genética , Persona de Mediana Edad , Glicósido Hidrolasas

RESUMEN

Genetic variation that influences gene expression and splicing is a key source of phenotypic diversity1-5. Although invaluable, studies investigating these links in humans have been strongly biased towards participants of European ancestries, which constrains generalizability and hinders evolutionary research. Here to address these limitations, we developed MAGE, an open-access RNA sequencing dataset of lymphoblastoid cell lines from 731 individuals from the 1000 Genomes Project6, spread across 5 continental groups and 26 populations. Most variation in gene expression (92%) and splicing (95%) was distributed within versus between populations, which mirrored the variation in DNA sequence. We mapped associations between genetic variants and expression and splicing of nearby genes (cis-expression quantitative trait loci (eQTLs) and cis-splicing QTLs (sQTLs), respectively). We identified more than 15,000 putatively causal eQTLs and more than 16,000 putatively causal sQTLs that are enriched for relevant epigenomic signatures. These include 1,310 eQTLs and 1,657 sQTLs that are largely private to underrepresented populations. Our data further indicate that the magnitude and direction of causal eQTL effects are highly consistent across populations. Moreover, the apparent 'population-specific' effects observed in previous studies were largely driven by low resolution or additional independent eQTLs of the same genes that were not detected. Together, our study expands our understanding of human gene expression diversity and provides an inclusive resource for studying the evolution and function of human genomes.

Asunto(s)

Regulación de la Expresión Génica , Variación Genética , Genoma Humano , Internacionalidad , Sitios de Carácter Cuantitativo , Empalme del ARN , Grupos Raciales , Femenino , Humanos , Masculino , Artefactos , Sesgo , Línea Celular , Estudios de Cohortes , Conjuntos de Datos como Asunto , Epigenómica , Evolución Molecular , Regulación de la Expresión Génica/genética , Genética de Población , Genoma Humano/genética , Linfocitos/citología , Linfocitos/metabolismo , Sitios de Carácter Cuantitativo/genética , Grupos Raciales/genética , Empalme del ARN/genética , Análisis de Secuencia de ARN

RESUMEN

Disrupted alternative splicing plays a determinative role in neurological diseases, either as a direct cause or as a driver in disease susceptibility. Transcriptomic profiling of aged human postmortem brain samples has uncovered hundreds of aberrant mRNA splicing events in Alzheimer's disease (AD) brains, associating dysregulated RNA splicing with disease. We previously identified a complex array of alternative splicing combinations across apolipoprotein E receptor 2 (APOER2), a transmembrane receptor that interacts with both the neuroprotective ligand Reelin and the AD-associated risk factor, APOE. Many of the human APOER2 isoforms, predominantly featuring cassette splicing events within functionally important domains, are critical for the receptor's function and ligand interaction. However, a comprehensive repertoire and the functional implications of APOER2 isoforms under both physiological and AD conditions are not fully understood. Here, we present an in-depth analysis of the splicing landscape of human APOER2 isoforms in normal and AD states. Using single-molecule, long-read sequencing, we profiled the entire APOER2 transcript from the parietal cortex and hippocampus of Braak stage IV AD brain tissues along with age-matched controls and investigated several functional properties of APOER2 isoforms. Our findings reveal diverse patterns of cassette exon skipping for APOER2 isoforms, with some showing region-specific expression and others unique to AD-affected brains. Notably, exon 15 of APOER2, which encodes the glycosylation domain, showed less inclusion in AD compared to control in the parietal cortex of females with an APOE ɛ3/ɛ3 genotype. Also, some of these APOER2 isoforms demonstrated changes in cell surface expression, APOE-mediated receptor processing, and synaptic number. These variations are likely critical in inducing synaptic alterations and may contribute to the neuronal dysfunction underlying AD pathogenesis.

Asunto(s)

Empalme Alternativo , Enfermedad de Alzheimer , Proteínas Relacionadas con Receptor de LDL , Proteína Reelina , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Empalme Alternativo/genética , Isoformas de Proteínas/genética , Análisis de Secuencia de ARN , Femenino , Anciano , Encéfalo/metabolismo , Encéfalo/patología , Apolipoproteínas E/genética , Masculino , Hipocampo/metabolismo , Hipocampo/patología , Anciano de 80 o más Años , Empalme del ARN/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo

RESUMEN

HP1α/CBX5 is an epigenetic regulator with a suspected role in multiple sclerosis (MS). Here, using high-depth RNA sequencing on monocytes, we identified a subset of MS patients with reduced CBX5 expression, correlating with progressive stages of the disease and extensive transcriptomic alterations. Examination of rare non-coding RNA species in these patients revealed impaired maturation/degradation of U snRNAs and enhancer RNAs, indicative of reduced activity of the Integrator, a complex with suspected links to increased MS risk. At protein-coding genes, compromised Integrator activity manifested in reduced pre-mRNA splicing efficiency and altered expression of genes regulated by RNA polymerase II pause-release. Inactivation of Cbx5 in the mouse mirrored most of these transcriptional defects and resulted in hypersensitivity to experimental autoimmune encephalomyelitis. Collectively, our observations suggested a major contribution of the Integrator complex in safeguarding against transcriptional anomalies characteristic of MS, with HP1α/CBX5 emerging as an unexpected regulator of this complex's activity. These findings bring novel insights into the transcriptional aspects of MS and provide potential new criteria for patient stratification.

Asunto(s)

Homólogo de la Proteína Chromobox 5 , Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Transcriptoma , Humanos , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Animales , Ratones , Transcriptoma/genética , Femenino , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Masculino , Adulto , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Persona de Mediana Edad , Empalme del ARN/genética , Regulación de la Expresión Génica , Monocitos/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Ratones Endogámicos C57BL , Perfilación de la Expresión Génica/métodos

RESUMEN

Pseudouridine (Ψ), the most abundant RNA modification, plays a role in pre-mRNA splicing, RNA stability, protein translation efficiency, and cellular responses to environmental stress. Dysregulation of pseudouridylation is linked to human diseases. This review explores recent insights into the role of RNA pseudouridylation alterations in human disorders and the therapeutic potential of Ψ. We discuss the impact of the reduction of Ψ levels in ribosomal, messenger, and transfer RNA in RNA processing, protein translation, and consequently its role in neurodevelopmental diseases and cancer. Furthermore, we review the success of N1-methyl-Ψ messenger RNA vaccines against COVID-19 and the development of RNA-guided pseudouridylation enzymes for treating genetic diseases caused by premature stop codons.

Asunto(s)

COVID-19 , Seudouridina , Humanos , Seudouridina/metabolismo , Seudouridina/genética , COVID-19/genética , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Neoplasias/patología , SARS-CoV-2/genética , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/terapia , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/patología , Vacunas contra la COVID-19 , Biosíntesis de Proteínas , Empalme del ARN/genética , ARN de Transferencia/genética , ARN de Transferencia/metabolismo

RESUMEN

N6-methyladenosine (m6A) is the most abundant modification to mRNAs. Loss-of-function studies of main m6A regulators have indicated the role of m6A in pre-mRNA splicing. Recent studies have reported the role of splicing in preventing m6A deposition. Understanding the interplay between m6A and mRNA splicing holds the potential to clarify the significance of these fundamental molecular mechanisms in cell development and function, thereby shedding light on their involvement in the pathogenesis of myriad diseases.

Asunto(s)

Adenosina , Metilación de ARN , Empalme del ARN , ARN Mensajero , Animales , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

Recent studies have highlighted the essential role of RNA splicing, a key mechanism of alternative RNA processing, in establishing connections between genetic variations and disease. Genetic loci influencing RNA splicing variations show considerable influence on complex traits, possibly surpassing those affecting total gene expression. Dysregulated RNA splicing has emerged as a major potential contributor to neurological and psychiatric disorders, likely due to the exceptionally high prevalence of alternatively spliced genes in the human brain. Nevertheless, establishing direct associations between genetically altered splicing and complex traits has remained an enduring challenge. We introduce Spliced-Transcriptome-Wide Associations (SpliTWAS) to integrate alternative splicing information with genome-wide association studies to pinpoint genes linked to traits through exon splicing events. We applied SpliTWAS to two schizophrenia (SCZ) RNA-sequencing datasets, BrainGVEX and CommonMind, revealing 137 and 88 trait-associated exons (in 84 and 67 genes), respectively. Enriched biological functions in the associated gene sets converged on neuronal function and development, immune cell activation, and cellular transport, which are highly relevant to SCZ. SpliTWAS variants impacted RNA-binding protein binding sites, revealing potential disruption of RNA-protein interactions affecting splicing. We extended the probabilistic fine-mapping method FOCUS to the exon level, identifying 36 genes and 48 exons as putatively causal for SCZ. We highlight VPS45 and APOPT1, where splicing of specific exons was associated with disease risk, eluding detection by conventional gene expression analysis. Collectively, this study supports the substantial role of alternative splicing in shaping the genetic basis of SCZ, providing a valuable approach for future investigations in this area.

Asunto(s)

Empalme Alternativo , Exones , Estudio de Asociación del Genoma Completo , Esquizofrenia , Transcriptoma , Humanos , Esquizofrenia/genética , Empalme Alternativo/genética , Exones/genética , Predisposición Genética a la Enfermedad , Empalme del ARN/genética , Polimorfismo de Nucleótido Simple