Nonsense-mediated RNA Decay Research Articles

UPF1 increases amino acid levels and promotes cell proliferation in lung adenocarcinoma via the eIF2α-ATF4 axis.

Up-frameshift 1 (UPF1), as the most critical factor in nonsense-mediated messenger RNA (mRNA) decay (NMD), regulates tumor-associated molecular pathways in many cancers. However, the role of UPF1 in lung adenocarcinoma (LUAD) amino acid metabolism remains largely unknown. In this study, we found that UPF1 was significantly correlated with a portion of amino acid metabolic pathways in LUAD by integrating bioinformatics and metabolomics. We further confirmed that UPF1 knockdown inhibited activating transcription factor 4 (ATF4) and Ser51 phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), the core proteins in amino acid metabolism reprogramming. In addition, UPF1 promotes cell proliferation by increasing the amino-acid levels of LUAD cells, which depends on the function of ATF4. Clinically, UPF1 mRNA expression is abnormal in LUAD tissues, and higher expression of UPF1 and ATF4 was significantly correlated with poor overall survival (OS) in LUAD patients. Our findings reveal that UPF1 is a potential regulator of tumor-associated amino acid metabolism and may be a therapeutic target for LUAD.

The endogenous HBZ interactome in ATL leukemic cells reveals an unprecedented complexity of host interacting partners involved in RNA splicing.

Adult T-cell leukemia/lymphoma (ATL) is a T-cell lymphoproliferative neoplasm caused by the human T-cell leukemia virus type 1 (HTLV-1). Two viral proteins, Tax-1 and HBZ play important roles in HTLV-1 infectivity and in HTLV-1-associated pathologies by altering key pathways of cell homeostasis. However, the molecular mechanisms through which the two viral proteins, particularly HBZ, induce and/or sustain the oncogenic process are still largely elusive. Previous results suggested that HBZ interaction with nuclear factors may alter cell cycle and cell proliferation. To have a more complete picture of the HBZ interactions, we investigated in detail the endogenous HBZ interactome in leukemic cells by immunoprecipitating the HBZ-interacting complexes of ATL-2 leukemic cells, followed by tandem mass spectrometry analyses. RNA seq analysis was performed to decipher the differential gene expression and splicing modifications related to HTLV-1. Here we compared ATL-2 with MOLT-4, a non HTLV-1 derived leukemic T cell line and further compared with HBZ-induced modifications in an isogenic system composed by Jurkat T cells and stably HBZ transfected Jurkat derivatives. The endogenous HBZ interactome of ATL-2 cells identified 249 interactors covering three main clusters corresponding to protein families mainly involved in mRNA splicing, nonsense-mediated RNA decay (NMD) and JAK-STAT signaling pathway. Here we analyzed in detail the cluster involved in RNA splicing. RNAseq analysis showed that HBZ specifically altered the transcription of many genes, including crucial oncogenes, by affecting different splicing events. Consistently, the two RNA helicases, members of the RNA splicing family, DDX5 and its paralog DDX17, recently shown to be involved in alternative splicing of cellular genes after NF-κB activation by HTLV-1 Tax-1, interacted and partially co-localized with HBZ. For the first time, a complete picture of the endogenous HBZ interactome was elucidated. The wide interaction of HBZ with molecules involved in RNA splicing and the subsequent transcriptome alteration strongly suggests an unprecedented complex role of the viral oncogene in the establishment of the leukemic state.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

RBM47 inhibits hepatocellular carcinoma progression by targeting UPF1 as a DNA/RNA regulator

The mechanisms by which the tumor behaviors of hepatocellular carcinoma (HCC) support growth and metastasis remain largely unknown, and it has become increasingly apparent that molecular dysregulation is of considerable importance for cellular signaling pathways. Recently, RNA-binding motif protein 47 (RBM47) has been suggested to function as a tumor regulator by acting as an RNA binding protein (RBP), but its role in HCC remains ambiguous. Here, in HCC, we identified that RBM47 had an inhibitory influence on tumor behaviors in vitro and accordingly suppressed the growth and metastasis of xenograft tumors in vivo. Additionally, RBM47 was verified to positively regulate Upframeshift 1 (UPF1), which is a crucial protein involved in the nonsense-mediated RNA decay (NMD) process and was previously determined to be an HCC suppressor. Mechanistically, the stability of UPF1 mRNA was demonstrated to be enhanced with its 3’UTR bound by RBM47, which acted as an RNA binding protein. Meanwhile, RBM47 was also proven to promote the transcription of UPF1 as a transcription factor. Taken together, we concluded that RBM47 functioned as a tumor suppressor by upregulating UPF1, acting as a DNA/RNA binding protein at the transcriptional and posttranscriptional levels.

Evaluating the activity of nonsense-mediated RNA decay via Nanopore direct RNA sequencing

Nonsense-mediated mRNA decay (NMD) and its regulation play an important role in eliminating faulty transcripts and controlling gene expression. However, measuring NMD activity and characterizing its targets remain challenging. In this study, we set out to establish Nanopore direct RNA sequencing in combination with quantitative real-time PCR (qPCR) as a method for analyzing NMD activity and its targets in cultured cell lines and clinical tissue samples. Nanopore RNA sequencing could detect more isoforms than short-read sequencing, especially in identifying novel isoforms and predicting isoforms annotated with premature termination codon (PTC). Changes in transcriptional isoforms of five genes (PRS, RPL12, SRSF2, PPIA, and TMEM208) could faithfully reflect NMD activity in the three cell lines and prostate cancer (PCA) samples. NMD activity in PCA samples varied, but some patients showed an increased trend. Together, Nanopore sequencing was superior in identifying NMD targets and evaluating NMD activity compared with short-read sequencing, and the NMD markers we screened may be used for measuring NMD activity in clinical patients.

A Novel Role of SMG1 in Cholesterol Homeostasis That Depends Partially on p53 Alternative Splicing.

Simple Summaryp53 isoforms have been reported in various tumor types. Both p53β and p53γ were recently reported to retain functionalities of full-length p53α. A role for p53 and p53 loss in cholesterol metabolism has also emerged. We show that SMG1, a phosphatidylinositol 3-kinase-related kinase, when inhibited in p53 wild-type MCF7 and HepG2 cells, significantly alters the expression of cholesterol pathway genes, with a net increase in intracellular cholesterol and an increased sensitivity to Fatostatin in MCF7. We confirm a prior report that SMG1 inhibition in MCF7 cells promotes expression of p53β and show the first evidence for increases in p53γ. Further, induced p53β expression, confirmed with antibody, explained the loss of SMG1 upregulation of the ABCA1 cholesterol exporter where p53γ had no effect on ABCA1. Additionally, upregulation of ABCA1 upon SMG1 knockdown was independent of upregulation of nonsense-mediated decay target RASSF1C, previously suggested to regulate ABCA1 via a “RASSF1C-miR33a-ABCA1” axis.SMG1, a phosphatidylinositol 3-kinase-related kinase (PIKK), essential in nonsense-mediated RNA decay (NMD), also regulates p53, including the alternative splicing of p53 isoforms reported to retain p53 functions. We confirm that SMG1 inhibition in MCF7 tumor cells induces p53β and show p53γ increase. Inhibiting SMG1, but not UPF1 (a core factor in NMD), upregulated several cholesterol pathway genes. SMG1 knockdown significantly increased ABCA1, a cholesterol efflux pump shown to be positively regulated by full-length p53 (p53α). An investigation of RASSF1C, an NMD target, increased following SMG1 inhibition and reported to inhibit miR-33a-5p, a canonical ABCA1-inhibiting miRNA, did not explain the ABCA1 results. ABCA1 upregulation following SMG1 knockdown was inhibited by p53β siRNA with greatest inhibition when p53α and p53β were jointly suppressed, while p53γ siRNA had no effect. In contrast, increased expression of MVD, a cholesterol synthesis gene upregulated in p53 deficient backgrounds, was sensitive to combined targeting of p53α and p53γ. Phenotypically, we observed increased intracellular cholesterol and enhanced sensitivity of MCF7 to growth inhibitory effects of cholesterol-lowering Fatostatin following SMG1 inhibition. Our results suggest deregulation of cholesterol pathway genes following SMG1 knockdown may involve alternative p53 programming, possibly resulting from differential effects of p53 isoforms on cholesterol gene expression.

Peripheral Ion Channel Gene Screening in Painful- and Painless-Diabetic Neuropathy.

Neuropathic pain is common in diabetic peripheral neuropathy (DN), probably caused by pathogenic ion channel gene variants. Therefore, we performed molecular inversion probes-next generation sequencing of 5 transient receptor potential cation channels, 8 potassium channels and 2 calcium-activated chloride channel genes in 222 painful- and 304 painless-DN patients. Twelve painful-DN (5.4%) patients showed potentially pathogenic variants (five nonsense/frameshift, seven missense, one out-of-frame deletion) in ANO3 (n = 3), HCN1 (n = 1), KCNK18 (n = 2), TRPA1 (n = 3), TRPM8 (n = 3) and TRPV4 (n = 1) and fourteen painless-DN patients (4.6%—three nonsense/frameshift, nine missense, one out-of-frame deletion) in ANO1 (n = 1), KCNK18 (n = 3), KCNQ3 (n = 1), TRPA1 (n = 2), TRPM8 (n = 1), TRPV1 (n = 3) and TRPV4 (n = 3). Missense variants were present in both conditions, presumably with loss- or gain-of-functions. KCNK18 nonsense/frameshift variants were found in painless/painful-DN, making a causal role in pain less likely. Surprisingly, premature stop-codons with likely nonsense-mediated RNA-decay were more frequent in painful-DN. Although limited in number, painful-DN patients with ion channel gene variants reported higher maximal pain during the night and day. Moreover, painful-DN patients with TRP variants had abnormal thermal thresholds and more severe pain during the night and day. Our results suggest a role of ion channel gene variants in neuropathic pain, but functional validation is required.

Transcriptional and post-transcriptional regulation of young genes in plants.

BackgroundNew genes continuously emerge from non-coding DNA or by diverging from existing genes, but most of them are rapidly lost and only a few become fixed within the population. We hypothesized that young genes are subject to transcriptional and post-transcriptional regulation to limit their expression and minimize their exposure to purifying selection.ResultsWe performed a protein-based homology search across the tree of life to determine the evolutionary age of protein-coding genes present in the rice genome. We found that young genes in rice have relatively low expression levels, which can be attributed to distal enhancers, and closed chromatin conformation at their transcription start sites (TSS). The chromatin in TSS regions can be re-modeled in response to abiotic stress, indicating conditional expression of young genes. Furthermore, transcripts of young genes in Arabidopsis tend to be targeted by nonsense-mediated RNA decay, presenting another layer of regulation limiting their expression.ConclusionsThese data suggest that transcriptional and post-transcriptional mechanisms contribute to the conditional expression of young genes, which may alleviate purging selection while providing an opportunity for phenotypic exposure and functionalization.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12915-022-01339-7.

MEX3A promotes the malignant progression of ovarian cancer by regulating intron retention in TIMELESS

The latest research shows that RNA-binding proteins (RBPs) could serve as novel potential targets for cancer therapy. We used bioinformatics analysis to screen and identify the key RBPs in ovarian cancer, from which we found that Mex-3 RNA Binding Family Member A (MEX3A) was intimately associated with the clinical prognosis of ovarian cancer. Nevertheless, little is known about its biological roles in ovarian cancer. In this case, we observed that MEX3A was highly overexpressed in fresh-frozen ovarian cancer tissues. MEX3A knockdown suppressed the development and invasion of ovarian cancer cells, while MEX3A overexpression promoted the proliferation and invasion of ovarian cancer cells. Mechanistically, TIMELESS was the critical downstream target gene of MEX3A, as demonstrated through alternative splicing event analysis based on RNA-seq. MEX3A knockdown resulted in retention of intron twenty-three of TIMELESS mRNA and decreased TIMELESS mRNA owing to stimulation of nonsense-mediated RNA decay (NMD). Additionally, we found that TIMELESS overexpression with MEX3A knockdown partially restored the proliferation ability of ovarian cancer cells. The results of this paper demonstrated that the MEX3A/TIMELESS signaling pathway was a key regulator of ovarian cancer, and MEX3A was a novel possible treatment target for ovarian cancer patients.

Nonsense-mediated RNA decay: an emerging modulator of malignancy.

Nonsense-mediated RNA decay (NMD) is a highly conserved RNA turnover pathway that selectively degrades RNAs harbouring truncating mutations that prematurely terminate translation, including nonsense, frameshift and some splice-site mutations. Recent studies show that NMD shapes the mutational landscape of tumours by selecting for mutations that tend to downregulate the expression of tumour suppressor genes but not oncogenes. This suggests that NMD can benefit tumours, a notion further supported by the finding that mRNAs encoding immunogenic neoantigen peptides are typically targeted for decay by NMD. Together, this raises the possibility that NMD-inhibitory therapy could be of therapeutic benefit against many tumour types, including those with a high load of neoantigen-generating mutations. Complicating this scenario is the evidence that NMD can also be detrimental for many tumour types, and consequently tumours often have perturbed NMD. NMD may suppress tumour generation and progression by degrading subsets of specific normal mRNAs, including those encoding stress-response proteins, signalling factors and other proteins beneficial for tumours, as well as pro-tumour non-coding RNAs. Together, these findings suggest that NMD-modulatory therapy has the potential to provide widespread therapeutic benefit against diverse tumour types. However, whether NMD should be stimulated or repressed requires careful analysis of the tumour to be treated.

Retroviral RNA Processing.

This review is an accompaniment to a Special Issue on “Retroviral RNA Processing”. It discusses post-transcriptional regulation of retroviruses, ranging from the ancient foamy viruses to more modern viruses, such as HIV-1, HTLV-1, Rous sarcoma virus, murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus. This review is not comprehensive. However, it tries to address some of the major questions in the field with examples of how different retroviruses express their genes. It is amazing that a single primary RNA transcript can have so many possible fates: genomic RNA, unspliced mRNA, and up to 50 different alternatively spliced mRNAs. This review will discuss the sorting of RNAs for packaging or translation, RNA nuclear export mechanisms, splicing, translation, RNA modifications, and avoidance of nonsense-mediated RNA decay.

No-nonsense: insights into the functional interplay of nonsense-mediated mRNA decay factors.

Nonsense-mediated messenger RNA decay (NMD) represents one of the main surveillance pathways used by eukaryotic cells to control the quality and abundance of mRNAs and to degrade viral RNA. NMD recognises mRNAs with a premature termination codon (PTC) and targets them to decay. Markers for a mRNA with a PTC, and thus NMD, are a long a 3′-untranslated region and the presence of an exon-junction complex (EJC) downstream of the stop codon. Here, we review our structural understanding of mammalian NMD factors and their functional interplay leading to a branched network of different interconnected but specialised mRNA decay pathways. We discuss recent insights into the potential impact of EJC composition on NMD pathway choice. We highlight the coexistence and function of different isoforms of up-frameshift protein 1 (UPF1) with an emphasis of their role at the endoplasmic reticulum and during stress, and the role of the paralogs UPF3B and UPF3A, underscoring that gene regulation by mammalian NMD is tightly controlled and context-dependent being conditional on developmental stage, tissue and cell types.

Landscape and regulation of alternative splicing and alternative polyadenylation in a plant pathogenic fungus.

Alternative splicing (AS) and alternative polyadenylation (APA) contribute significantly to the regulation of gene expression in higher eukaryotes. Their biological impact in filamentous fungi, however, is largely unknown. Here we combine PacBio Isoform-Sequencing and strand-specific RNA-sequencing of multiple tissues and mutant characterization to reveal the landscape and regulation of AS and APA in Fusarium graminearum. We generated a transcript annotation comprising 51 617 isoforms from 17 189 genes. In total, 4997 and 11 133 genes are alternatively spliced and polyadenylated, respectively. Majority of the AS events alter coding sequences. Unexpectedly, the AS transcripts containing premature-termination codons are not sensitive to nonsense-mediated messenger RNA decay. Unlike in yeasts and animals, distal APA sites have strong signals, but proximal APA isoforms are highly expressed in F. graminearum. The 3'-end processing factors FgRNA15, FgHRP1, and FgFIP1 play roles in promoting proximal APA site usage and intron splicing. A genome-wide increase in intron inclusion and distal APA site usage and downregulation of the spliceosomal and 3'-end processing factors were observed in older and quiescent tissues, indicating intron inclusion and 3'-untranslated region lengthening as novel mechanisms in regulating aging and dormancy in fungi. This study provides new insights into the complexity and regulation of AS and APA in filamentous fungi.

SF3B4 Frameshift Variants Represented a More Severe Clinical Manifestation in Nager Syndrome.

Nager syndrome (NS) is a rare disease marked with craniofacial and preaxial limb anomalies. In this report, we summarized the current evidence to determine a possible genotype-phenotype association among NS individuals. Twenty-four articles comprising of 84 NS (including 9 patients with a severe form of NS [Rodriguez syndrome]) patients were examined, of which 76% were caused by variants in SF3B4 (OMIM *605593, Splicing Factor 3B, Subunit 4). Within the SF3B4 gene, variants located in exon 3 commonly occurred (20%) from a total identified variant, while hotspot location was identified in exon 1 (12%), and primarily occurred as frameshift variants (64%). Thirty-five distinct pathogenic variants within SF3B4 gene were identified with two common sites, c.1A > G and c.1060dupC in exons 1 and 5, respectively. Although no significant genotype-phenotype association was found, it is notable that patients with frameshift SF3B4 variants and predicted to lead to nonsense-mediated RNA decay (NMD) of the transcripts tended to have a more severe clinical manifestation. Additionally, patients harboring variants in exons 2 and 3 displayed a higher proportion of cardiac malformations. Taken together, this article summarizes the pathogenic variants observed in SF3B4 and provides a possible genotype-phenotype relationship in this disease.

Chronic Enteropathy Associated with Solute Carrier Organic Anion Transporter Family, Member 2A1 (SLCO2A1) with Positive Immunohistochemistry for SLCO2A1 Protein.

Chronic enteropathy associated with solute carrier organic anion transporter family, member 2A1 (SLCO2A1) (CEAS) is a rare autosomal recessive hereditary disease characterized by chronic persistent anemia and hypoproteinemia. Its diagnosis typically requires a genetic analysis. The efficacy of immunohistochemical staining with SLCO2A1 polyclonal antibody as a pre-diagnostic tool for CEAS has been previously reported. We herein report a patient with CEAS in whom immunohistochemical staining confirmed SLCO2A1 protein expression. The immunopositive results may have been due to nonsense-mediated RNA decay. As immunohistochemical staining of SLCO2A1 protein may show immunopositive results, a genetic analysis should also be performed when CEAS is strongly clinically suspected.

The edited UPF1 is correlated with elevated asparagine synthetase in pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinomas (PDACs) is a malignant disorder and is the most common pancreatic cancer type. The malignant cells depend on the uptake of asparagine (Asn) for growth. The synthesis of Asn occurs through the enzyme asparagine synthetase (ASNS). Interestingly, ASNS is known as is direct target of nonsense-mediated RNA decay (NMD). We have previously reported that NMD major factor UPF1 mutations in the pancreatic tumors. However, the relationship between NMD and the level of ASNS is unknown. We constructed point mutations by site-specific mutagenesis. To evaluate NMD magnitude, we assessed the expression ratio of an exogenously expressed wild-type and mutated β-globin mRNA with N39 allele, and five known NMD targets. Then, reverse transcription-polymerase chain reaction (RT-PCR), RT-qPCR and western bolt to determine RNA or protein levels, after knockdown of endogenous UPF1 by small RNA interference in the cells. An RNA editing event (c.3101 A > G) at UPF1 transcripts resulting in an Asparagine (p.1034) changed to a Serine is found in one primary PDAC patient. The edited UPF1 increases the ability of degrading of NMD provoking transcripts, such as β-globin mRNA with N39 allele and 5 out of 5 known endogenous NMD substrate mRNAs, including ASNS. In addition, ASNS mRNA is subjected to NMD degradation by virtue of its possessing uORFs at the 5'UTR. A reduction of endogenous ASNS RNA and the increased protein expression level is found either in the PDAC patient or in the cells with edited UPF1 at c.3101 A > G relative to the controls. This edited UPF1 found in the PDAC results in hyperactivated NMD, which is tightly correlation to elevated expression level of ASNS. The targeting of knockdown of ASNS may improve the antitumor potency in PDACs.

Genotype-Phenotype Comparison in POGZ-Related Neurodevelopmental Disorders by Using Clinical Scoring.

POGZ-related disorders (also known as White-Sutton syndrome) encompass a wide range of neurocognitive abnormalities and other accompanying anomalies. Disease severity varies widely among POGZ patients and studies investigating genotype-phenotype association are scarce. Therefore, our aim was to collect data on previously unreported POGZ patients and perform a large-scale phenotype-genotype comparison from published data. Overall, 117 POGZ patients’ genotype and phenotype data were included in the analysis, including 12 novel patients. A severity scoring system was developed for the comparison. Mild and severe phenotypes were compared with the types and location of the variants and the predicted presence or absence of nonsense-mediated RNA decay (NMD). Missense variants were more often associated with mild phenotypes (p = 0.0421) and truncating variants predicted to escape NMD presented with more severe phenotypes (p < 0.0001). Within this group, variants in the prolin-rich region of the POGZ protein were associated with the most severe phenotypes (p = 0.0004). Our study suggests that gain-of-function or dominant negative effect through escaping NMD and the location of the variants in the prolin-rich domain of the protein may play an important role in the severity of manifestations of POGZ–associated neurodevelopmental disorders.

Exon-skipping antisense oligonucleotides for cystic fibrosis therapy.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity but is expressed at a very low level due to nonsense-mediated messenger RNA (mRNA) decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable, and read-through drugs are inefficient partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktailof two splice-site-targeting ASOs induced the expression of CFTR mRNA without the premature-termination-codon-containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.

De novo STXBP1 Mutations in Two Patients With Developmental Delay With or Without Epileptic Seizures

Objectives: Mutations in the STXBP1 gene have been associated with epileptic encephalopathy. Previous studies from in vitro neuroblastoma 2A cells showed that haploinsufficiency of STXBP1 is the mechanism for epileptic encephalopathy. In this ex vivo study, STXPB1 DNA mutations and RNA expression were assessed from two patients to help understand the impact of STXBP1 mutations on the disease etiology and mechanism.Methods: Microarray analysis and DNA sequencing were performed on two children with development delay, one with and one without infantile spasms. Different pathogenic mutations of STXBP1 were identified in the patients and RNA expression of STXPB1 was then performed by RT-Q-PCR on RNA extracted from blood samples of each patient.Results: Pathogenic deletion [of exons 13–20 and 3′ downstream of STXBP1] and nonsense mutation [c.1663G>T (p.Glu555X) in exon 18 of STXBP1] were detected from the two patients, respectively. RNA analysis showed that 1) the deletion mediated RNA decay, and that 2) no RNA decay was identified for the nonsense mutation at codon 555 which predicts a truncated STXBP1 protein.Significance: Our RNA expression analyses from the patient blood samples are the first ex vivo studies to support that both haploinsufficiency and truncation of STXBP1 protein (either dominant negative or haploinsufficiency) are causative mechanisms for epileptic encephalopathies, intellectual disability and developmental delay. The RNA assay also suggests that escape from nonsense-mediated RNA decay is possible when the nonsense mutation resides <50 nucleotides upstream of the last coding exon-exon junction even in the presence of additional non-coding exons that are 3′ downstream of the last coding exon.

Abstract 12946: Identification of a LMNA Synonymous Variant Associated With Severe Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is one of the most common cardiac phenotypes caused by mutations of lamin A/C (LMNA) gene in humans. Almost 50% of LMNA variants reported in ClinVar as pathogenic or likely pathogenic are due to frameshift, nonsense, or splicing variants, predictive of haploinsufficiency for LMNA protein. In contrast, synonymous variants that do not immediately suggest a haploinsufficiency mechanism are much harder to interpret. We evaluated a cohort of 57 patients who underwent heart transplant for dilated cardiomyopathy for LMNA variants and identified a novel synonymous variant c.936G&gt;A in the last nucleotide of exon 5 of LMNA in a DCM family. Clinically, LMNA c.936G&gt;A carriers presented with severe DCM, conduction disease and high creatine-kinase levels. Sanger sequencing showed the presence of LMNA c.936G&gt;A in the genomic DNA but not in the cDNA derived from one family member’s heart tissue. RT-PCR using RNA from the patient’s heart tissue was performed with primers that amplified LMNA exons 4-6. DNA fragments amplified by RT-PCR were then cloned into pcDNAII vector and 40 clones were picked and sequenced. An aberrantly spliced mRNA containing the variant and a premature stop codon was identified in 1 of the 40 clones. Real-time quantitative PCR showed significantly lower LMNA mRNA levels in the patient’s heart compared to the controls. Our findings suggest that the c.936G&gt;A LMNA variant resulted in aberrant RNA splicing, which possibly triggers nonsense-mediated RNA decay, leading to reduced mRNA and likely protein expression. These results expand the understanding on the association between synonymous LMNA variants and the molecular pathogenesis of LMNA-DCM.