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PubMed1995

Alternative splicing of natriuretic peptide A and B receptor transcripts in the rat brain.

1. In the present study we searched for variants of alternative splicing of guanylyl cyclase A and B mRNA in rats in vivo. 2. Guanylyl cyclase A2 and guanylyl cyclase B2 isoforms of guanylyl cyclase produced by alternative splicing leading to the deletion of exon 9 of both transcripts were quantified in several rat organs. 3. Only one alternative splicing was found in the regulatory domain, encoded by exons 8-15. 4. Quantification of the guanylyl cyclase B2 isoform in different rat organs and in cultured aortic smooth muscle cells showed that this alternative splicing was tissue-specific and occurred predominantly in the central nervous system where the alternatively spliced variant represented more than 50% of the guanylyl cyclase B mRNA. 5. The same alternative splicing existed for guanylyl cyclase A mRNA but at very low levels in the organs studied. 6. Alternative splicing of guanylyl cyclase B exon 9 in the brain may play an important role in signal transduction, since the expressed protein possesses a constitutionally active guanylyl cyclase acting independently of C-type natriuretic peptide regulation.

Alternative Splicing↗
PubMed2005

Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene.

BACKGROUND: haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3' UTR regions. OBJECTIVES, PATIENTS AND METHODS: In the present study we performed a detailed RNA analysis of three groups of patients. The first included control patients with known splicing defects, the second included two patients with already identified nucleotide changes close to splicing sites, that could potentially alter the normal splicing process, and a third group of 11 unrelated patients whose genomic DNA have already been screened for mutations by DHPLC and direct sequencing with no mutation being identified. RESULTS: Both candidate splice site mutations were shown to result in either skipping or alternative splicing of at least one exon, therefore these DNA changes must be considered as causal for the patients' HA phenotype. In contrast, no abnormalities on the RNA level were observed in any of 11 unrelated patients without mutations in the FVIII gene. CONCLUSIONS: These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein.

DNA Mutational Analysis↗
PubMed2026

Interleukin-23 Receptor and Interleukin-17 Receptor A: Splice Variants, Isoforms and Their Relationship With Periodontitis-A Systematic Review and Bioinformatic Analysis.

This systematic review aimed to: (1) identify the splicing variants of IL23R and IL17RA reported in the literature; (2) perform a multiple alignment analysis to describe the isoforms of IL-23R and IL-17RA; and (3) compare the expression levels of IL-23R, IL-17RA, and their soluble isoforms (sIL-23R and sIL-17RA) in patients with periodontitis and periodontally healthy individuals. The study protocol followed PRISMA guidelines and was registered in PROSPERO (CRD420251267367). Six databases (PubMed, ScienceDirect, Scopus, Web of Science, EBSCO, and Google Scholar) were searched without restrictions on year or language. The descriptors used were: 'Interleukin-23 Receptor,' 'IL-23R,' 'Interleukin-17 Receptor A' 'IL-17RA,' 'Alternative Splicing,' 'Splice Variants,' 'Isoforms,' and 'Periodontitis.' The bioinformatics analysis was performed using CLUSTALW (V.1.83), InterPro and DeepTMHMM. Risk of bias was assessed with the QUIN and JBI tools for cross-sectional studies. Of 104 articles, four in vitro studies and eight cross-sectional studies were included. Qualitative analysis revealed that to date there are 32 splicing variants of the IL23R gene, while only one splicing variant has been reported for IL17RA. CLUSTALW, InterPro and DeepTMHMM analysis showed that these splicing variants result in 23 isoforms which can be soluble forms, complete intracellular peptides, truncated extracellular or intracellular peptides, or complete structures with truncated extracellular and/or intracellular domains. All studies had a low risk of bias. IL-23R and IL-17RA exhibit structural diversity resulting from alternative splicing, with IL-23R demonstrating significantly greater isoform complexity. However, the biological significance of these isoforms in periodontitis remains unclear and requires further investigation.

Humans↗
PubMed2006

SRp54 (SFRS11), a regulator for tau exon 10 alternative splicing identified by an expression cloning strategy.

The tau gene encodes a microtubule-associated protein that is critical for neuronal survival and function. Splicing defects in the human tau gene lead to frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), an autosomal dominant neurodegenerative disorder. Genetic mutations associated with FTDP-17 often affect tau exon 10 alternative splicing. To investigate mechanisms regulating tau exon 10 alternative splicing, we have developed a green fluorescent protein reporter for tau exon 10 skipping and an expression cloning strategy to identify splicing regulators. A role for SRp54 (also named SFRS11) as a tau exon 10 splicing repressor has been uncovered using this strategy. The overexpression of SRp54 suppresses tau exon 10 inclusion. RNA interference-mediated knock-down of SRp54 increases exon 10 inclusion. SRp54 interacts with a purine-rich element in exon 10 and antagonizes Tra2beta, an SR-domain-containing protein that enhances exon 10 inclusion. Deletion of this exonic element eliminates the activity of SRp54 in suppressing exon 10 inclusion. Our data support a role of SRp54 in regulating tau exon 10 splicing. These experiments also establish a generally useful approach for identifying trans-acting regulators of alternative splicing by expression cloning.

Alternative Splicing↗
PubMed1995

Genetic enhancement of RNA-processing defects by a dominant mutation in B52, the Drosophila gene for an SR protein splicing factor.

SR proteins are essential for pre-mRNA splicing in vitro, act early in the splicing pathway, and can influence alternative splice site choice. Here we describe the isolation of both dominant and loss-of-function alleles of B52, the gene for a Drosophila SR protein. The allele B52ED was identified as a dominant second-site enhancer of white-apricot (wa), a retrotransposon insertion in the second intron of the eye pigmentation gene white with a complex RNA-processing defect. B52ED also exaggerates the mutant phenotype of a distinct white allele carrying a 5' splice site mutation (wDR18), and alters the pattern of sex-specific splicing at doublesex under sensitized conditions, so that the male-specific splice is favored. In addition to being a dominant enhancer of these RNA-processing defects, B52ED is a recessive lethal allele that fails to complement other lethal alleles of B52. Comparison of B52ED with the B52+ allele from which it was derived revealed a single change in a conserved amino acid in the beta 4 strand of the first RNA-binding domain of B52, which suggests that altered RNA binding is responsible for the dominant phenotype. Reversion of the B52ED dominant allele with X rays led to the isolation of a B52 null allele. Together, these results indicate a critical role for the SR protein B52 in pre-mRNA splicing in vivo.

Alleles↗
PubMed1996

Functional properties of p54, a novel SR protein active in constitutive and alternative splicing.

The p54 protein was previously identified by its reactivity with an autoantiserum. We report here that p54 is a new member of the SR family of splicing factors, as judged from its structural, antigenic, and functional characteristics. Consistent with its identification as an SR protein, p54 can function as a constitutive splicing factor in complementing splicing-deficient HeLa cell S100 extract. However, p54 also shows properties distinct from those of other SR family members, p54 can directly interact with the 65-kDa subunit of U2 auxiliary factor (U2AF65), a protein associated with the 3' splice site. In addition, p54 interacts with other SR proteins but does not interact with the U1 small nuclear ribonucleoprotein U1-70K or the 35-kDa subunit of U2 auxiliary factor (U2AF35). This protein-protein interaction profile is different from those of prototypical SR proteins SC35 and ASF/SF2, both of which interact with U1-70K and U2AF35 but not with U2AF65. p54 promotes the use of the distal 5' splice site in E1A pre-mRNA alternative splicing, while the same site is suppressed by ASF/SF2 and SC35. These findings and the differential tissue distribution of p54 suggest that this novel SR protein may participate in regulation of alternative splicing in a tissue- and substrate-dependent manner.

Alternative Splicing↗
PubMed1998

An intronic sequence element mediates both activation and repression of rat fibroblast growth factor receptor 2 pre-mRNA splicing.

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) is an example of highly regulated alternative splicing in which exons IIIb and IIIc are utilized in a mutually exclusive manner in different cell types. The importance of this splicing choice is highlighted by studies which indicate that deregulation of the FGF-R2 splicing is associated with progression of prostate cancer. Loss of expression of a IIIb exon-containing isoform of FGF-R2 [FGF-R2 (IIIb)] accompanies the transition of a well-differentiated, androgen-dependent rat prostate cancer cell line, DT3, to the more aggressive, androgen-independent AT3 cell line. We have used transfection of rat FGF-R2 minigenes into DT3 and AT3 cancer cell lines to study the mechanisms that control alternative splicing of rat FGF-R2. Our results support a model in which an important cis-acting element located in the intron between these alternative exons mediates activation of splicing using the upstream IIIb exon and repression of the downstream IIIc exon in DT3 cells. This element consists of 57 nucleotides (nt) beginning 917 nt downstream of the IIIb exon. Analysis of mutants further demonstrates that an 18-nt "core sequence" within this element is most crucial for its function. Based on our observations, we have termed this sequence element ISAR (for intronic splicing activator and repressor), and we suggest that factors which bind this sequence are required for maintenance of expression of the FGF-R2 (IIIb) isoform.

Alternative Splicing↗
PubMed1999

SL1 trans splicing and 3'-end formation in a novel class of Caenorhabditis elegans operon.

Many Caenorhabditis elegans genes exist in operons in which polycistronic precursors are processed by cleavage at the 3' ends of upstream genes and trans splicing 100 to 400 nucleotides away, at the 5' ends of downstream genes, to generate monocistronic messages. Of the two spliced leaders, SL1 is trans spliced to the 5' ends of upstream genes, whereas SL2 is reserved for downstream genes in operons. However, there are isolated examples of what appears to be a different sort of operon, in which trans splicing is exclusively to SL1 and there is no intercistronic region; the polyadenylation signal is only a few base pairs upstream of the trans-splice site. We have analyzed the processing of an operon of this type by inserting the central part of mes-6/cks-1 into an SL2-type operon. In this novel context, cks-1 is trans spliced only to SL1, and mes-6 3'-end formation occurs normally, demonstrating that this unique mode of processing is indeed intrinsic to this kind of operon, which we herein designate "SL1-type." An exceptionally long polypyrimidine tract found in the 3' untranslated regions of the three known SL1-type operons is shown to be required for the accumulation of both upstream and downstream mRNAs. Mutations of the trans-splice and poly(A) signals indicate that the two processes are independent and in competition, presumably due to their close proximity, raising the possibility that production of upstream and downstream mRNAs is mutually exclusive.

Animals↗
PubMed2006

Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is associated with mutations in the PITX2 gene that encodes a homeobox transcription factor. Several intronic PITX2 mutations have been reported in Axenfeld-Rieger patients but their effects on gene expression have not been tested. METHODS: We present two new families with recurrent PITX2 intronic mutations and use PITX2c minigenes and transfected cells to address the hypothesis that intronic mutations effect RNA splicing. Three PITX2 mutations have been analyzed: a G>T mutation within the AG 3' splice site (ss) junction associated with exon 4 (IVS4-1G>T), a G>C mutation at position +5 of the 5' (ss) of exon 4 (IVS4+5G>C), and a previously reported A>G substitution at position -11 of 3'ss of exon 5 (IVS5-11A>G). RESULTS: Mutation IVS4+5G>C showed 71% retention of the intron between exons 4 and 5, and poorly expressed protein. Wild-type protein levels were proportionally expressed from correctly spliced mRNA. The G>T mutation within the exon 4 AG 3'ss junction shifted splicing exclusively to a new AG and resulted in a severely truncated, poorly expressed protein. Finally, the A>G substitution at position -11 of the 3'ss of exon 5 shifted splicing exclusively to a newly created upstream AG and resulted in generation of a protein with a truncated homeodomain. CONCLUSION: This is the first direct evidence to support aberrant RNA splicing as the mechanism underlying the disorder in some patients and suggests that the magnitude of the splicing defect may contribute to the variability of ARS phenotypes, in support of a gene dosage model of Axenfeld-Rieger syndrome.

Abnormalities, Multiple↗
PubMed2005

Dual effect of the SR proteins ASF/SF2, SC35 and 9G8 on HIV-1 RNA splicing and virion production.

In HIV-1 infected cells transcription of the integrated provirus generates the single full length 9 kb viral RNA, a major fraction of which is spliced to produce the single-spliced 4 kb RNAs and the multiple-spliced 2 kb RNAs. These spliced RNAs are the messengers for the Env glycoproteins and the viral regulatory factors. The cellular SR and hnRNP proteins were shown in vitro to control alternative splicing by binding cis-regulatory elements on the viral RNA. To better understand in vivo the role of the SR proteins on HIV-1 genomic RNA splicing and virion production, we used a human cell line expressing high levels of complete HIV-1 and either one of the ASF/SF2, SC35, and 9G8 SR proteins. Results show that over-expressing SR proteins caused a large reduction of genomic RNA and that each SR protein modified the viral 9 kb RNA splicing pattern in a specific mode. In fact, ASF/SF2 increased the level of Vpr RNA while SC35 and 9G8 caused a large increase in Tat RNA. As expected, overexpressing SR proteins caused a strong reduction of total Gag made. However, we observed by immuno-confocal microscopy an accumulation of Gag at the plasma membrane and in intracellular compartments while there is a dramatic reduction of Env protein made in most cells. Due to the negative impact of the SR proteins on the levels of genomic RNA and HIV-1 structural proteins much less virions were produced which retained part of their infectivity. In conclusion, SR proteins can down-regulate the late steps of HIV-1 replication.

Cell Line↗
PubMed2004

Variation in alternative splicing across human tissues.

BACKGROUND: Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues. RESULTS: Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues. CONCLUSIONS: This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells.

Alternative Splicing↗
PubMed2006

SRp30a (ASF/SF2) regulates the alternative splicing of caspase-9 pre-mRNA and is required for ceramide-responsiveness.

Two splice variants are derived from the caspase-9 gene, proapoptotic caspase-9a and antiapoptotic caspase-9b, by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. Previous studies from our laboratory have shown that the alternative splicing of caspase-9 and the phosphorylation status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and ceramide via the action of protein phosphatase-1. In this study, a link between ceramide, SR proteins, and the alternative splicing of caspase-9 was established. The downregulation of SRp30a in A549 cells by RNA interference technology resulted in an increase in the caspase-9b splice variant, with a concomitant decrease in the caspase-9a splice variant, thereby significantly decreasing the caspase-9a/9b ratio from 1.67 +/- 0.11 to 0.56 +/- 0.08 (P < 0.005). The specific downregulation of SRp30a also inhibited the ability of exogenous ceramide treatment to induce the inclusion of the exon 3, 4, 5, and 6 cassette. Therefore, we have identified SRp30a as an RNA trans-acting factor that functions as a major regulator of caspase-9 pre-mRNA processing and is required for ceramide to mediate the alternative splicing of caspase-9.

Alternative Splicing↗
PubMed2000

Breeding stock-specific variation in peptidylglycine alpha-amidating monooxygenase messenger ribonucleic acid splicing in rat pituitary.

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, we made the unexpected observation that Charles River Sprague Dawley rats expressed forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats did not. Forms of PAM containing exon A were expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differed between rat breeders, and full-length PAM-1 was not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, did not vary with rat breeder. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance.

Alternative Splicing↗
PubMed2004

Stop codon-mediated suppression of splicing is a novel nuclear scanning mechanism not affected by elements of protein synthesis and NMD.

The pre-mRNA splicing machine must frequently discriminate between normal and many potential 5'splice sites that match the consensus sequence but remain latent. Suppression of splicing (SOS) at such latent 5'splice sites is required for the maintenance of an open reading frame, and to ensure that only RNAs that encode for functional proteins will be formed. In this study we show that SOS is a novel mechanism distinct from the known RNA surveillance mechanisms. First, SOS is distinct from nonsense-mediated mRNA decay (NMD) because it is not dependent on translation and is not affected by RNAi-mediated down-regulation of hUpf1 and hUpf2--two key components of the NMD pathway. Second, SOS is distinct from nonsense-associated alternative splicing (NAS), because a mutant of hUpf1, which was shown to abrogate NAS, does not activate latent splicing. Elucidating the mechanism of SOS is pertinent to human disease in view of the large number of human genes that harbor latent splice sites.

5' Untranslated Regions↗
PubMed2006

Inference of splicing regulatory activities by sequence neighborhood analysis.

Sequence-specific recognition of nucleic-acid motifs is critical to many cellular processes. We have developed a new and general method called Neighborhood Inference (NI) that predicts sequences with activity in regulating a biochemical process based on the local density of known sites in sequence space. Applied to the problem of RNA splicing regulation, NI was used to predict hundreds of new exonic splicing enhancer (ESE) and silencer (ESS) hexanucleotides from known human ESEs and ESSs. These predictions were supported by cross-validation analysis, by analysis of published splicing regulatory activity data, by sequence-conservation analysis, and by measurement of the splicing regulatory activity of 24 novel predicted ESEs, ESSs, and neutral sequences using an in vivo splicing reporter assay. These results demonstrate the ability of NI to accurately predict splicing regulatory activity and show that the scope of exonic splicing regulatory elements is substantially larger than previously anticipated. Analysis of orthologous exons in four mammals showed that the NI score of ESEs, a measure of function, is much more highly conserved above background than ESE primary sequence. This observation indicates a high degree of selection for ESE activity in mammalian exons, with surprisingly frequent interchangeability between ESE sequences.

Binding Sites↗
PubMed2004

Expression of splicing factors in human ovarian cancer.

Alternative splicing represents an important nuclear mechanism in the post-transcriptional regulation of gene expression, which is frequently altered during tumorigenesis. Previously, we have described marked changes in alternative splicing of the CD44 gene in ovarian and breast cancer. In the latter one we described also a specific induction of splicing factors during tumor development. Now we have focussed our studies on the expression profiles of splicing factors, including classical SR proteins, Tra2 and YB-1 in physiological and malignant ovarian tissues by RT-PCR and Western blot analysis. We detected changed expression pattern with higher levels of phosphorylated 30 kDa SR proteins as well as relatively high concentrations of hyperphosphorylated Tra2 protein isoforms in ovarian cancer. RT-PCR analysis revealed a marked induction of SC35 and ASF/SF2 as well as mRNA levels in malignant ovarian tissue. These results suggest gene-specific alterations of expression rather than a general induction of the splicing machinery. Together with previously performed functional studies of CD44 splicing these findings implicate that altered expression profiles of SR proteins, Tra2beta and YB-1 might be responsible for the known changes of alternative CD44 splicing in ovarian cancer.

Antigens, CD↗
PubMed1998

Defective RNA splicing resulting from a mutation in the cyclic guanosine monophosphate-phosphodiesterase beta-subunit gene.

PURPOSE: The authors have previously reported a 3' splice site mutation in intron 2 of the rod cyclic guanosine monophosphate-phosphodiesterase (cGMP) beta-subunit (beta-PDE) gene in a patient with compound heterozygous autosomal recessive retinitis pigmentosa. The purpose of this study is to determine whether this mutation interferes with RNA splicing, what products it generates, and whether the resultant mRNA is able to support the synthesis of the native protein. METHODS: Two expression constructs were prepared by subcloning genomic DNA fragments (one from the control subject DNA and the other from the patient's DNA) to the pCIS2 expression vector. Recombinant plasmid DNA was introduced into 293 human embryonic kidney cells using the calcium phosphate-mediated transfection procedure. Northern blot hybridization, reverse transcription-polymerase chain reaction, and sequencing were used for RNA analysis. RESULTS: Four major products were present in the RNA pool isolated from cells transfected with the expression construct containing the splice site mutation. One of the transcripts resulted from the activation of a cryptic 3' splice site located in exon 3, 12 nucleotides downstream of the mutated site. The second fragment was longer than the correctly spliced mRNA by approximately 1 kb and contained unspliced intron 2. Two other high molecular weight products corresponded to intermediate lariats. CONCLUSIONS: An acceptor splice site mutation in intron 2 of the beta-PDE gene leads to the accumulation of pre-mRNA and intermediate lariats and a 12-nucleotide shorter beta-PDE transcript produced by the use of a cryptic splice site located in exon 3. In the normal beta-PDE mRNA, these 12 nucleotides code for ValPheLeuLys. These amino acids are highly conserved in the putative noncatalytic cGMP-binding domain I of beta-PDE from several species and, probably, are important for the correct folding and function of the protein.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2025

Tandem splice acceptor sites: Profiling their relevance to human disease.

PURPOSE: Interpretation of variation, particularly the creation or disruption of tandem splice acceptor sites (NAGNnAG variants), challenges genomic medicine practice. METHODS: We analyzed the creation and disruption of dinucleotide AG sites within &#xb1;30 bases of natural splice-acceptor sites in the GRCh37 human reference genome. These results were compared with variant data from the ClinVar and gnomAD databases, as well as with data from 779 National Institutes of Health Undiagnosed Diseases Program study participants. Using RNA sequencing, we assessed the splicing at NAGNnAG variants for 107 of the Undiagnosed Diseases Program participants and compared the empirical data with SpliceAI predictions. RESULTS: Creation or disruption of NAGNnAG sites within 30 bases of the natural splice acceptor are enriched in ClinVar compared with gnomAD; however, such variants in the 2 databases are rarely differentiated by SpliceAI scores. Empirical evaluation via RNA sequencing analysis supported novel acceptor site usage from -21 to +30; splice-altering variants did not predominate in a specific region or have SpliceAI scores invariantly, suggesting increased spliceogenicity. CONCLUSION: NAGNnAG variants within 30 bp of the natural splice acceptor have a high probability of clinical relevance and are poorly contextualized for clinical utility. Their interpretation benefits from empirical evaluation via RNA analysis.

Humans↗