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Novel and atypical splicing mutation in a compound heterozygous UNC13D defect presenting in Familial Hemophagocytic Lymphohistiocytosis triggered by EBV infection.

Familial Hemophagocytic Lymphohistiocytosis type 3 (FHL3) is a genetic disorder caused by mutations in UNC13D gene, coding the granule primi … ernal allele. This case illustrates an atypical compound heterozygous UNC13D mutation affecting the RNA splicing that generates a typical FHL3 phenotype.

Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family.

Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20−40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin

RNA modifications in cancer.

RNA modifications are emerging as critical cancer regulators that influence tumorigenesis and progression. Key modifications, such as N6-methyladenosine (m6A) and 5-methylcytosine (m5C), are implicated in various cellular processes. These modifications are regulated by proteins that write, erase, and read RNA and modulate RNA stability, splicing, translation, and degradation. Recent studies have highlighted their roles in metabolic reprogramming, signaling pathways, and cell cycle control, which are essential for tumor proliferation and survival. Despite these scientific advances, the precise

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… To date, more than 170 RNA modifications have been identified across all RNA molecule categories. 7 These modifications typically affect RNA splicing, stability, localization, translation, and RNA–RNA and RNA–protein interactions, thereby regulating various biological processes. 8 , 9 , 10 The recent …

Writers, readers, and erasers RNA modifications and drug resistance in cancer.

Drug resistance in cancer cells significantly diminishes treatment efficacy, leading to recurrence and metastasis. A critical factor contributing to this resistance is the epigenetic alteration of gene expression via RNA modifications, such as N6-methyladenosine (m6A), N1-methyladenosine (m1A), 5-methylcytosine (m5C), 7-methylguanosine (m7G), pseudouridine (Ψ), and adenosine-to-inosine (A-to-I) editing. These modifications are pivotal in regulating RNA splicing, translation, transport, degradation, and stability. Governed by "writers," "readers," and "erasers," RNA modifications impact numerou…

Phenotypic variability including Behçet's disease-like manifestations in DADA2 patients due to a homozygous c.973-2A>G splice site mutation.

OBJECTIVES: To describe phenotypic and functional characteristics of patients with the homozygous c.973-2A>G splice site mutation in the adenosine deaminase 2 (ADA2) gene (rs139750129), resulting in deficiency of ADA2 (DADA2). METHODS: We present case synopses of six patients from three unrelated families. Clinical data were analysed and next-generation sequencing (NGS) was performed. We also tested for aberrant RNA splicing and measured ADA2 enzyme activity. RESULTS: One family had common DADA2 symptoms, whereas Behçet's disease-like manifestations were observed in the other two families. W…

PUF60 variants cause a syndrome of ID, short stature, microcephaly, coloboma, craniofacial, cardiac, renal and spinal features.

PUF60 encodes a nucleic acid-binding protein, a component of multimeric complexes regulating RNA splicing and transcription. In 2013, patients with microdeletions of chromosome 8q24.3 including PUF60 were found to have developmental delay, microcephaly, craniofacial, renal and cardiac defects. Very similar phenotypes have been described in six patients with variants in PUF60, suggesting that it underlies the syndrome. We report 12 additional patients with PUF60 variants who were ascertained using exome sequencing: six through the Deciphering Developmental Disorders Study and six through simila…

A Method for Measuring RNA N 6-methyladenosine Modifications in Cells and Tissues.

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. He…

Identification of new alternative splice events in the TCIRG1 gene in different human tissues.

Two transcript variants (TV) of the T cell immune regulator gene 1 (TCIRG1) have already been characterized. TV1 encodes a subunit of the osteoclast vacuolar proton pump and TV2 encodes a T cell inhibitory receptor. Based on the search in dbEST, we validated by RT-PCR six new alternative splice events in TCIRG1 in most of the 28 human tissues studied. In addition, we observed that transcripts using the TV1 transcription start site and two splice forms previously described in a patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies o

Identification and characterization of -3c-g acceptor splice site mutation in human alpha-L-iduronidase associated with mucopolysaccharidosis type IH/S.

DNA screening for mutations in the alpha-L-iduronidase (IDUA) gene was performed in a Chinese mucopolysaccharidosis type IH/S patient. The patient had two different mutations: the maternal allele has L346R (t-g transversion in codon 346) and the paternal allele has 388-3c-g (c-g transversion at position -3 of the 3' splice site of intron 2). In transfected COS-7 cells, L346R showed no appreciable IDUA activity (0.4% of normal activity), although it did not cause an apparent reduction in IDUA mRNA or protein level. The 388-3c-g mutation profoundly affects normal splicing leading to a very unsta

GT to AT transition at a splice donor site causes skipping of the preceding exon in phenylketonuria.

Classical Phenylketonuria (PKU) is an autosomal recessive human genetic disorder caused by a deficiency of hepatic phenylalanine hydroxylase … e of the splice donor site mutation in the human liver is the skipping of the preceding exon during RNA splicing.

Chronic hypoxia induces alternative splicing of transcripts in the goldfish brain.

Several species evolved mechanisms to tolerate periods of severe environmental hypoxia and anoxia. Among them, goldfish are unique as they do not enter a comatose state under such conditions. Taking advantage of the recently published and annotated goldfish genome, we had previously profiled the transcriptomic response of the goldfish brain under normoxic (21 kPa oxygen saturation, N) and hypoxic conditions (2.1 kPa oxygen saturation) after 1 and 4 weeks (1WH, 4WH). Using the RNA-Seq data, we report the occurrence of alternative mRNA splicing (skipped exon, retained intron, alternative 3' or 5…

Identification of a Novel Deep Intronic Mutation in CAPN3 Presenting a Promising Target for Therapeutic Splice Modulation.

Calpainopathy, also known as limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) or LGMD R1 Calpain3-related, is one of the most common genetically characterized forms of limb-girdle muscular dystrophy with a wide range of phenotypic severity. We evaluated a consanguineous family with a clinical phenotype consistent with calpainopathy in whom conventional sequencing did not detect any mutations in the CAPN3 gene. Using whole exome sequencing paired with haplotype analysis, we identified a homozygous deep intronic single base pair deletion in CAPN3 (c.946-29delT). Familial segregation studie

Huntingtin Exists as Multiple Splice Forms in Human Brain.

BACKGROUND: A CAG repeat expansion in HTT has been known to cause Huntington's disease for over 20 years. The genomic sequence of the 67 exon HTT is clear but few reports have detailed alternative splicing or alternative transcripts. Most eukaryotic genes with multiple exons show alternative splicing that increases the diversity of the transcriptome and proteome: it would be surprising if a gene with 67 known exons in its two major transcripts did not present some alternative transcripts. OBJECTIVE: To investigate the presence of alternative transcripts directly in human HTT. METHODS: An ove

AStruct: detection of allele-specific RNA secondary structure in structuromic probing data.

BACKGROUND: Uncovering functional genetic variants from an allele-specific perspective is of paramount importance in advancing our understanding of gene regulation and genetic diseases. Recently, various allele-specific events, such as allele-specific gene expression, allele-specific methylation, and allele-specific binding, have been explored on a genome-wide scale due to the development of high-throughput sequencing methods. RNA secondary structure, which plays a crucial role in multiple RNA-associated processes like RNA modification, translation and splicing, has emerged as an essential foc

Effect of c.1431C > T mutation, a causative mutation of Glanzmann's thrombasthenia, on ITGB3 splicing, gene and protein expression.

BACKGROUND/AIM: Recently, it was reported that the non-synonymous c.1431C > T (p. G477=) mutation of the integrin subunit β3 (ITGB3) gene is the cause of Glanzmann's thrombasthenia (GT). However, the functional consequences of this mutation on the ITGB3 gene and protein expression remain to be elucidated. Therefore, this study was conducted to cover this scientific shortage. METHODS: Peripheral blood samples were collected from Chinese family members (parents and proband and his sister), and DNA was extracted and sequenced using whole-exome and Sanger sequencing. The effect of c.1431C > T mut

In vitro splicing reactions in Drosophila Kc nuclear extracts.

This protocol describes how to generate and analyze products and intermediates in a pre-mRNA splicing reaction. The reaction relies on the use of labeled, capped, synthetic pre-mRNAs, prepared by in vitro transcription, and Drosophila Kc cell culture nuclear extracts. The pre-mRNA substrate is incubated in the nuclear extract under splicing conditions for 1-2 h. The products of the reaction are purified by phenol:chloroform extraction and precipitation with ethanol, and then loaded directly onto a denaturing urea-acrylamide gel. Visualization of the splicing reactions will reveal the pre-mRNA,…

Delpacibart etedesiran improves the molecular pathology of myotonic dystrophy type 1 in the phase 1/2 MARINA study.

Myotonic dystrophy type 1 (DM1) is a rare, autosomal dominant, progressive neuromuscular disease caused by the expansion of CTG repeats in the DM1 protein kinase (DMPK) gene. Expanded CUG repeats in the mutant DMPK transcript bind and sequester splicing factors, especially in the muscleblind-like (MBNL) family, leading to misregulated alternative splicing of mRNA, which is the main driver of DM1 pathology. We designed an antibody-oligonucleotide conjugate (AOC), delpacibart etedesiran (del-desiran), to deliver a DMPK-targeting small interfering RNA to muscle via transferrin receptor 1-mediated

Unveiling the oncogenic function of CCDC12 in regulating RBM47 splicing in breast cancer.

Breast cancer (BC) remains a leading cause of cancer-related mortality, in part due to the lack of effective therapeutic targets. In this study, we report that Coiled-Coil Domain Containing 12 (CCDC12), a relatively uncharacterized member of the CCDC protein family, is upregulated in BC tissues. Functional assays in vitro and in vivo indicate that CCDC12 contributes to malignant phenotypes, including enhanced proliferation and metastatic potential. Transcriptomic analysis is associated with widespread alterations in alternative splicing programs. Among the affected events, RNA Binding Motif Pr

Exon 21 skipping in ARHGAP10: A splicing switch that governs breast cancer metastasis.

Rho GTPase-activating protein 10 (ARHGAP10) is recognized as a tumor suppressor, yet the functional impact of its alternative splicing isoforms on breast cancer metastasis remains unclear. This study aimed to elucidate the role and regulatory mechanism of ARHGAP10 exon 21 skipping in breast cancer progression. Our research results indicate that in metastatic breast cancer cells, the full-length isoform ARHGAP10-L is downregulated, whereas the truncated ARHGAP10-S is upregulated. The RNA-binding protein HNRNPA0 directly binds to intron 21 of ARHGAP10 pre-mRNA, promoting exon-21 skipping and ARH

Identification of biallelic intronic EPM2A mutations in a Lafora disease kindred.

Lafora disease (LD) is a severe autosomal recessive disease, which usually presents as seizure and myoclonus, followed by behavioral changes … tronic mutations. Genetic testing identified two intronic mutations in EPM2A which caused aberrant mRNA splicing. c.301+1 G > A in EPM2A caused aberrant splicing at donor site and resulted in intron retention in transcript NM_005670.4, while c.476+14860 C > A caused aberrant splicing in transcript NM_001368129.2 and NM_001368132.1. Our findings expand the spec …