RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants

RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2–9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (<3.4%) of the full-length (FL) transcript. In addition, ESE elements of the alternative exon 3 were mapped by testing four overlapping exonic microdeletions (≥30-bp), revealing an ESE-rich interval (c.202_235del) with critical sequences for exon 3 recognition that might have been affected by germline variants. Next, 26 BRIDGES variants and 16 artificial exon 3 single-nucleotide substitutions were also assayed. Thirty variants impaired splicing with variable amounts (0–65.1%) of the FL transcript, although only c.202G > A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T > C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation.

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Systematic characterization of short intronic splicing-regulatory elements

10.1101/2021.05.17.444464 ◽

2021 ◽

Author(s):

Yuan Gao ◽

Kuan-Ting Lin ◽

Yang Yang ◽

Jialin Bai ◽

Li Wang ◽

...

Keyword(s):

Splice Site ◽

Enrichment Analysis ◽

Regulatory Elements ◽

Hek293 Cells ◽

Splice Site Selection ◽

Aberrant Splicing ◽

Rna Sequences ◽

Splicing Regulatory Elements ◽

Alternatively Spliced

Intronic splicing enhancers and silencers (ISEs and ISSs) are two groups of splicing-regulatory elements (SREs) that play critical roles in determining splice-site selection, particularly for alternatively spliced introns or exons. SREs are often short motifs; their mutation or dysregulation of their cognate proteins frequently causes aberrant splicing and results in disease. To date, however, knowledge about SRE sequences and how they regulate splicing remains limited. Here, using an SMN2 minigene, we generated a complete pentamer-sequence library that comprises all possible combinations of 5 nucleotides in intron 7, at a fixed site downstream of the 5′ splice site. We systematically analyzed the effects of all 1023 mutant pentamers on exon 7 splicing, in comparison to the wild-type minigene, in HEK293 cells. Our data show that the majority of pentamers significantly affect exon 7 splicing: 584 of them are stimulatory and 230 are inhibitory. To identify actual SREs, we utilized a motif set enrichment analysis (MSEA), from which we identified groups of stimulatory and inhibitory SRE motifs. We experimentally validated several strong SREs in SMN1/2 and MAPT minigene settings. Our results provide a valuable resource for understanding how short RNA sequences regulate splicing. Many novel SREs can be explored further to elucidate their mechanism of action.

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Distribution of Fitness and Virulence Effects Caused by Single-Nucleotide Substitutions in Tobacco Etch Virus

Journal of Virology ◽

10.1128/jvi.00524-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 12979-12984 ◽

Cited By ~ 136

Author(s):

Purificación Carrasco ◽

Francisca de la Iglesia ◽

Santiago F. Elena

Keyword(s):

Single Case ◽

Site Directed Mutagenesis ◽

Relative Fitness ◽

In Planta ◽

Nucleotide Substitutions ◽

Phenotypic Effect ◽

Single Nucleotide ◽

Mutant Genotype

ABSTRACT Little is known about the fitness and virulence consequences of single-nucleotide substitutions in RNA viral genomes, and most information comes from the analysis of nonrandom sets of mutations with strong phenotypic effect or which have been assessed in vitro, with their relevance in vivo being unclear. Here we used site-directed mutagenesis to create a collection of 66 clones of Tobacco etch potyvirus, each carrying a different, randomly chosen, single-nucleotide substitution. Competition experiments between each mutant and the ancestral nonmutated clone were performed in planta to quantitatively assess the relative fitness of each mutant genotype. Among all mutations, 40.9% were lethal, and among the viable ones, 36.4% were significantly deleterious and 22.7% neutral. Not a single case of beneficial effects was observed within the level of resolution of our measures. On average, the fitness of a genotype carrying a deleterious but viable mutation was 49% smaller than that for its unmutated progenitor. Deleterious mutational effects conformed to a beta probability distribution. The virulence of a subset of viable mutants was assessed as the reduction in the number of viable seeds produced by infected plants. Mutational effects on virulence ranged between 17% reductions and 24.4% increases. Interestingly, the only mutations showing a significant effect on virulence were hypervirulent. Competitive fitness and virulence were uncorrelated traits.

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Potential Spectrum of Missense and Nonsense Factor (F)VIII Gene (F8) Alleles Based on Single Nucleotide Substitutions - How Many Remain To Be Identified?.

Blood ◽

10.1182/blood.v108.11.4039.4039 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4039-4039

Author(s):

Kevin R. Viel ◽

Manana Khachidze ◽

Laura Almasy ◽

Arthur R. Thompson ◽

Tom E. Howard

Keyword(s):

Replacement Therapy ◽

Hemophilia A ◽

Protein Domain ◽

Reference Sequence ◽

Nucleotide Position ◽

Loss Of Function ◽

Coding Region ◽

Nucleotide Substitutions ◽

Single Nucleotide ◽

The Impact

Abstract Regardless of advances in prenatal diagnosis, carrier detection and gene therapy for hemophilia-A, new patients with bleeding diatheses due to inadequate plasma FVIII activity (FVIII:C) levels will still require specialized management at treatment centers. In the ‘post-genome era’ the possibility exists for personalized medicine, in which an individual’s genetic information will be used to tailor prophylactic and/or treatment regimens that will optimize patient outcomes. As listed in the HAMSTeRS database, ~1,000 distinct loss-of-function F8 variants, representing all mutation types including inversions, insertion/deletions and single nucleotide substitutions (SNSs), have been associated with deficiencies of FVIII. To estimate how soon a complete catalogue of every possible mutation affecting FVIII:C levels may become available, we first determined the theoretical number of potential missense and nonsense F8 alleles, whether loss-of-function or not, based on each possible SNS in the coding region as compared to the reference sequence. While the impact of a missense change on FVIII:C, if any, is not always obvious, in contrast to premature-termination codons (PTCs), which are almost always deleterious, findings from a recent resequencing study raises the possibility that non-hemophilic structural differences between a patient’s endogenous FVIII protein and the infused “wildtype” molecule may increase risk of alloimmunization during replacement therapy. Wildtype FVIII contains 2,351 amino acid (aa) residues: 2,332 in the mature protein and 19 in the signal peptide (SP). Appropriate SNSs within the codons for 793 of these residues would create a PTC (UAA, UAG, UGA). Since three distinct base substitutions are possible at each of the three codon positions, 996 nonsense alleles could theoretically arise naturally. As only 123 distinct nonsense mutations are listed in HAMSTeRS, <15% of the theoretical number, many more likely await discovery. Since suitable SNSs within codons for every residue allow for as many as 15,631 naturally-occurring missense variations, of which 462 are in HAMSTeRS, only 2% of all possible alleles, even more mutations of this type likely remain to be identified. The Table presents the number of possible nonsense and missense F8 alleles and the FVIII protein domain/region affected. Although substantial time and diligent surveillance will be required to document the complete allelic architecture of hemophilia-A, since SNS-mutations can occur at every F8 nucleotide, not just those already identified, doing so could potentially have far reaching implications with respect to personalizing both the current strategy of replacement therapy, based on intravenous infusions and future gene-based methods. Table. Potential nonsense and missense F8 alleles based on the FVIII domain/region and position in codon Nucleotide Position in Codon Domain/Region Amino Acids 1st Codon 2nd Codon 3rd Codon Nonsense (Missense) Nonsense (Missense) Nonsense (Missense) SP 0001–0019 3 (51) 1 (56) 3 (23) A1 0001–0336 61 (911) 27 (981) 38 (317) a1 0337–0372 6 (101) 0 (108) 2 (50) A2 0373–0719 70 (935) 41 (1000) 55 (353) a2 0720–0740 5 (55) 3 (60) 4 (26) B 0741–1648 221 (2400) 139 (2585) 35 (1032) a3 1649–1689 12 (111) 4 (119) 4 (53) A3 1690–2019 71 (887) 15 (975) 45 (364) C1 2020–2172 30 (417) 17 (442) 20 (154) C2 2173–2332 35 (432) 14 (466) 15 (167)

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Practical approach to the genetic diagnosis of unsolved dystrophinopathies: a stepwise strategy in the genomic era

Journal of Medical Genetics ◽

10.1136/jmedgenet-2020-107113 ◽

2020 ◽

pp. jmedgenet-2020-107113 ◽

Cited By ~ 1

Author(s):

Zhiying Xie ◽

Chengyue Sun ◽

Yilin Liu ◽

Meng Yu ◽

Yiming Zheng ◽

...

Keyword(s):

Genetic Testing ◽

Comprehensive Evaluation ◽

Genetic Diagnosis ◽

Regulatory Elements ◽

Diagnostic Value ◽

Molecular Defect ◽

Aberrant Splicing ◽

Splicing Regulatory Elements ◽

Long Read ◽

Complex Structural

ObjectiveTo investigate the diagnostic value of implementing a stepwise genetic testing strategy (SGTS) in genetically unsolved cases with dystrophinopathies.MethodsAfter routine genetic testing in 872 male patients with highly suspected dystrophinopathies, we identified 715 patients with a pathogenic DMD variant. Of the 157 patients who had no pathogenic DMD variants and underwent a muscle biopsy, 142 patients were confirmed to have other myopathies, and 15 suspected dystrophinopathies remained genetically undiagnosed. These 15 patients underwent a more comprehensive evaluation as part of the SGTS pipeline, which included the stepwise analysis of dystrophin mRNA, short-read whole-gene DMD sequencing, long-read whole-gene DMD sequencing and in silico bioinformatic analyses.ResultsSGTS successfully yielded a molecular diagnosis of dystrophinopathy in 11 of the 15 genetically unsolved cases. We identified 8 intronic and 2 complex structural variants (SVs) leading to aberrant splicing in 10 of 11 patients, of which 9 variants were novel. In one case, a molecular defect was detected on mRNA and protein level only. Aberrant splicing mechanisms included 6 pseudoexon inclusions and 4 alterations of splice sites and splicing regulatory elements. We showed for the first time the exonisation of a MER48 element as a novel pathogenic mechanism in dystrophinopathies.ConclusionOur study highlights the high diagnostic utility of implementing a SGTS pipeline in dystrophinopathies with intronic variants and complex SVs.

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The effect of disease-associated HRPT2 mutations on splicing

Journal of Endocrinology ◽

10.1677/joe-09-0038 ◽

2009 ◽

Vol 201 (3) ◽

pp. 387-396 ◽

Cited By ~ 8

Author(s):

Michael A Hahn ◽

Julie McDonnell ◽

Deborah J Marsh

Keyword(s):

Splice Site ◽

Hot Spot ◽

Splice Sites ◽

Loss Of Function ◽

Splice Enhancer ◽

Aberrant Splicing ◽

Acceptor Splice Site ◽

Web Based ◽

Exonic Splice

Mutations in the tumour suppressor HRPT2 occur in patients with parathyroid carcinoma, kidney tumours and Hyperparathyroidism–Jaw Tumour syndrome. Disruption of exonic splicing through mutation of donor/acceptor splice sites or exonic splice enhancer (ESE) sites leads to loss of function of a number of major tumour suppressors including BRCA1, APC and MLH1. Given that the effect of HRPT2 mutations on splicing has not been widely studied, we used an in vitro splicing assay to determine whether 17 HRPT2 mutations located in hot-spot and other exons predicted to disrupt ESE consensus sites led to aberrant splicing. Using two independent web-based prediction programs, the majority of these mutations were predicted to disrupt ESE consensus sites; however, aberrant splicing of HRPT2 transcripts was not observed. Canonical donor or acceptor splice site mutations were also investigated using this splicing assay and transcripts assessed from tumour tissue. Splice site mutations were shown to lead to either exon skipping or retention of intronic sequences through the use of cryptic splice sites comprised of non-classical splicing signals. Aberrant splicing caused by disruption of ESE sites does not appear to have a major role in HRPT2-associated disease; however, premature truncation of parafibromin as the result of canonical donor or acceptor splice site mutations is associated with pathogenicity. Functional splicing assays must be undertaken in order to confirm web-based software predictions of the modification of putative ESE sites by disease-associated mutations.

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Splicing Regulatory Elements within tat Exon 2 of Human Immunodeficiency Virus Type 1 (HIV-1) Are Characteristic of Group M but Not Group O HIV-1 Strains

Journal of Virology ◽

10.1128/jvi.73.12.9764-9772.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 9764-9772 ◽

Cited By ~ 15

Author(s):

Patricia S. Bilodeau ◽

Jeffrey K. Domsic ◽

C. Martin Stoltzfus

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Splice Site ◽

Regulatory Elements ◽

Splice Sites ◽

Exon 2 ◽

Immunodeficiency Virus ◽

Splicing Regulatory Elements ◽

Hiv 1

ABSTRACT In the NL4-3 strain of human immunodeficiency virus type 1 (HIV-1), regulatory elements responsible for the relative efficiencies of alternative splicing at the tat, rev, and theenv/nef 3′ splice sites (A3 through A5) are contained within the region of tat exon 2 and its flanking sequences. Two elements affecting splicing of tat, rev, and env/nef mRNAs have been localized to this region. First, an exon splicing silencer (ESS2) in NL4-3, located approximately 70 nucleotides downstream from the 3′ splice site used to generatetat mRNA, acts specifically to inhibit splicing at this splice site. Second, the A4b 3′ splice site, which is the most downstream of the three rev 3′ splice sites, also serves as an element inhibiting splicing at the env/nef 3′ splice site A5. These elements are conserved in some but not all HIV-1 strains, and the effects of these sequence changes on splicing have been investigated in cell transfection and in vitro splicing assays. SF2, another clade B virus and member of the major (group M) viruses, has several sequence changes within ESS2 and uses a differentrev 3′ splice site. However, splicing is inhibited by the two elements similarly to NL4-3. As with the NL4-3 strain, the SF2 A4b AG dinucleotide overlaps an A5 branchpoint, and thus the inhibitory effect may result from competition of the same site for two different splicing factors. The sequence changes in ANT70C, a member of the highly divergent outlier (group O) viruses, are more extensive, and ESS2 activity in tat exon 2 is not present. Group O viruses also lack the rev 3′ splice site A4b, which is conserved in all group M viruses. Mutagenesis of the most downstream rev3′ splice site of ANT70C does not increase splicing at A5, and all of the branchpoints are upstream of the two rev 3′ splice sites. Thus, splicing regulatory elements in tat exon 2 which are characteristic of most group M HIV-1 strains are not present in group O HIV-1 strains.

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Characterization of the Jembrana Disease Virustat Gene and the cis- andtrans-Regulatory Elements in Its Long Terminal Repeats

Journal of Virology ◽

10.1128/jvi.73.1.658-666.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 658-666 ◽

Cited By ~ 18

Author(s):

Hexin Chen ◽

Graham Wilcox ◽

Gde Kertayadnya ◽

Charles Wood

Keyword(s):

Regulatory Elements ◽

Site Directed Mutagenesis ◽

Bovine Immunodeficiency Virus ◽

Loop Structure ◽

Nucleotide Substitutions ◽

Stem Loop ◽

Loop Region ◽

Stem Loop Structure ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Jembrana disease virus (JDV) is a newly identified bovine lentivirus that is closely related to the bovine immunodeficiency virus (BIV). JDV contains a tat gene, encoded by two exons, which has potent transactivation activity. Cotransfection of the JDVtat expression plasmid with the JDV promoter chloramphenicol acetyltransferase (CAT) construct pJDV-U3R resulted in a substantial increase in the level of CAT mRNA transcribed from the JDV long terminal repeat (LTR) and a dramatic increase in the CAT protein level. Deletion analysis of the LTR sequences showed that sequences spanning nucleotides −68 to +53, including the TATA box and the predicted first stem-loop structure of the predicted Tat response element (TAR), were required for efficient transactivation. The results, derived from site-directed mutagenesis experiments, suggested that the base pairing in the stem of the first stem-loop structure in the TAR region was important for JDV Tat-mediated transactivation; in contrast, nucleotide substitutions in the loop region of JDV TAR had less effect. For the JDV LTR, upstream sequences, from nucleotide −196 and beyond, as well as the predicted secondary structures in the R region, may have a negative effect on basal JDV promoter activity. Deletion of these regions resulted in a four- to fivefold increase in basal expression. The JDV Tat is also a potent transactivator of other animal and primate lentivirus promoters. It transactivated BIV and human immunodeficiency virus type 1 (HIV-1) LTRs to levels similar to those with their homologous Tat proteins. In contrast, HIV-1 Tat has minimal effects on JDV LTR expression, whereas BIV Tat moderately transactivated the JDV LTR. Our study suggests that JDV may use a mechanism of transactivation similar but not identical to those of other animal and primate lentiviruses.

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A rapid, super-selective method for detection of single nucleotide variants in C. elegans

10.1101/2020.04.01.020818 ◽

2020 ◽

Author(s):

Denis Touroutine ◽

Jessica E. Tanis

Keyword(s):

Low Cost ◽

Primer Design ◽

Site Directed Mutagenesis ◽

Amplification Efficiency ◽

Single Nucleotide Variants ◽

Nucleotide Substitutions ◽

Single Nucleotide ◽

Colony Pcr ◽

Sequence Composition ◽

C Elegans

ABSTRACTWith the widespread use of single nucleotide variants generated through mutagenesis screens, the million mutation project, and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end point PCR in Caenorhabditis elegans. Each SS primer consists of a 5’ “anchor” that hybridizes to the template, followed by a non-complementary “bridge,” and a “foot” corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3’ nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length, stability, and sequence composition of each SS primer segment affected selectivity and efficiency in order to develop simple rules for primer design that allow for distinction between any mismatches in various genetic contexts over a broad range of annealing temperatures. Manipulating bridge length affects amplification efficiency, while modifying the foot sequence can increase discriminatory power. Flexibility in the positioning of the anchor enables SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. In summary, we have demonstrated flexibility in design of SS primers and their utility for genotyping in C. elegans. Since SS primers reliably detect single nucleotide variants, we propose that this method could have broad application for SNP mapping, screening of CRISPR mutants, and colony PCR to identify successful site-directed mutagenesis constructs.

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Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis studies

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2010.0063 ◽

2010 ◽

Vol 365 (1548) ◽

pp. 1975-1982 ◽

Cited By ~ 110

Author(s):

Rafael Sanjuán

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Nucleotide Substitutions ◽

Single Nucleotide ◽

Single Stranded Dna ◽

Dna Viruses ◽

Fitness Effects ◽

Lethal Mutations ◽

Mean Fitness ◽

The Mean

The fitness effects of mutations are central to evolution, yet have begun to be characterized in detail only recently. Site-directed mutagenesis is a powerful tool for achieving this goal, which is particularly suited for viruses because of their small genomes. Here, I discuss the evolutionary relevance of mutational fitness effects and critically review previous site-directed mutagenesis studies. The effects of single-nucleotide substitutions are standardized and compared for five RNA or single-stranded DNA viruses infecting bacteria, plants or animals. All viruses examined show very low tolerance to mutation when compared with cellular organisms. Moreover, for non-lethal mutations, the mean fitness reduction caused by single mutations is remarkably constant (0.10–0.13), whereas the fraction of lethals varies only modestly (0.20–0.41). Other summary statistics are provided. These generalizations about the distribution of mutational fitness effects can help us to better understand the evolution of RNA and single-stranded DNA viruses.

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Role for Fox-1/Fox-2 in Mediating the Neuronal Pathway of Calcitonin/Calcitonin Gene-Related Peptide Alternative RNA Processing

Molecular and Cellular Biology ◽

10.1128/mcb.01015-06 ◽

2006 ◽

Vol 27 (3) ◽

pp. 830-841 ◽

Cited By ~ 40

Author(s):

Hua-Lin Zhou ◽

Andrew P. Baraniak ◽

Hua Lou

Keyword(s):

Splice Site ◽

Critical Role ◽

Regulatory Elements ◽

Calcitonin Gene Related Peptide ◽

Related Peptide ◽

Alternative Processing ◽

Neuronal Pathway ◽

Calcitonin Gene ◽

Splicing Regulatory Elements ◽

Exon 4

ABSTRACT Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3′ splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF65 binding to the 3′ splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.

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