Pseudoexon Activation Research Articles

CDNA analysis reveals NNT pseudoexon activation in two siblings with familial glucocorticoid deficiency

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Deep intronic mutation and pseudo exon activation as a novel muscular hypertrophy modifier in cattle.

Myostatin is essential for proper regulation of myogenesis, and inactivation of Myostatin results in muscle hypertrophy. Here, we identified an unexpected mutation in the myostatin gene which is almost fixed in Blonde d'Aquitaine cattle. In skeletal muscle, the mutant allele was highly expressed leading to an abnormal transcript consisting of a 41-bp inclusion and premature termination codons and to residual levels of a correctly spliced transcript. This expression pattern, caused by a leaky intronic mutation with regard to spliceosome activity and its apparent stability with regard to surveillance mechanisms, could contribute to the moderate muscle hypertrophy in this cattle breed. This finding is of importance for genetic counseling for meat quantity and quality in livestock production and possibly to manipulate myostatin pre-mRNA in human muscle diseases.

In Vitro Correction of a Pseudoexon-Generating Deep Intronic Mutation in LGMD2A by Antisense Oligonucleotides and Modified Small Nuclear RNAs

Limb-girdle muscular dystrophy type 2A (LGMD2A) is the most frequent autosomal recessive muscular dystrophy. It is caused by mutations in the calpain-3 (CAPN3) gene. The majority of the mutations described to date are located in the coding sequence of the gene. However, it is estimated that 25% of the mutations are present at exon-intron boundaries and modify the pre-mRNA splicing of the CAPN3 transcript. We have previously described the first deep intronic mutation in the CAPN3 gene: c.1782+1072G>C mutation. This mutation causes the pseudoexonization of an intronic sequence of the CAPN3 gene in the mature mRNA. In the present work, we show that the point mutation generates the inclusion of the pseudoexon in the mRNA using a minigene assay. In search of a treatment that restores normal splicing, splicing modulation was induced by RNA-based strategies, which included antisense oligonucleotides and modified small-nuclear RNAs. The best effect was observed with antisense sequences, which induced pseudoexon skipping in both HeLa cells cotransfected with mutant minigene and in fibroblasts from patients. Finally, transfection of antisense sequences and siRNA downregulation of serine/arginine-rich splicing factor 1 (SRSF1) indicate that binding of this factor to splicing enhancer sequences is involved in pseudoexon activation.

Pseudoexon Activation in the HMBS Gene as a Cause of the Nonerythroid Form of Acute Intermittent Porphyria

To the Editor: Acute intermittent porphyria (AIP)1 (MIM 176000) is a low-penetrance, autosomal dominant disorder caused by decreased activity of the third enzyme in the pathway of heme biosynthesis, hydroxymethylbilane synthase (HMBS). Partial deficiency of HMBS can lead to episodic life-threatening neurovisceral attacks of acute porphyria, which are often provoked by drugs, alcohol, or hormonal factors (1). Biochemical analyses of urinary porphobilinogen and porphyrins (fecal porphyrins and plasma porphyrins) can provide the diagnosis of AIP in symptomatic patients, but genetic testing for a defect in the HMBS gene (1) is required to accurately identify asymptomatic affected family members, who can then be counseled on the lifestyle modifications required to limit the risk of attacks. We previously reported on our experience with using direct genomic sequencing and dosage analysis of the HMBS gene for genetic analysis of AIP (2). We found a 98% sensitivity for detecting disease-causing mutations in unrelated symptomatic patients with a biochemical diagnosis of AIP (2), and we speculated that the cause in AIP patients in whom no mutation was found was likely to be mutations in unexplored parts of the gene, possible regulatory variants, or defects at another locus. We investigated a …

Genetic sequencing breakthrough to aid treatment for congenital hyperinsulinism

British Journal of Hospital MedicineVol. 74, No. 2 Clinical NewsGenetic sequencing breakthrough to aid treatment for congenital hyperinsulinismSian EllardSian EllardSearch for more papers by this authorSian EllardPublished Online:27 Sep 2013https://doi.org/10.12968/hmed.2013.74.2.68AboutSectionsView articleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareShare onFacebookTwitterLinked InEmail View article References Flanagan SE, Xie W, Caswell R et al. (2013) Next-generation sequencing reveals deep intronic cryptic ABCC8 and HADH splicing founder mutations causing hyperinsulinism by pseudoexon activation. Am J Hum Genet 92(1): 131–6 Crossref, Medline, Google Scholar FiguresReferencesRelatedDetails 1 February 2013Volume 74Issue 2ISSN (print): 1750-8460ISSN (online): 1759-7390 Metrics History Published online 27 September 2013 Published in print 1 February 2013 Information© MA Healthcare LimitedPDF download

Next-Generation Sequencing Reveals Deep Intronic Cryptic ABCC8 and HADH Splicing Founder Mutations Causing Hyperinsulinism by Pseudoexon Activation

Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ~10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals' fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease.

T.P.28 Pseudo-exon inactivation of the dystrophin gene: Ideal candidates for exon skipping

<h2>Abstract</h2> There are many different types of mutations that can inactivate the dystrophin gene and lead to Duchenne muscular dystrophy (DMD). Genomic deletions of one or more exons deletions account for the most common type of dystrophin gene lesion, and a handful of splice switching antisense oligomers could potentially restore the reading frame of some 40% of all DMD mutations. The removal of one or more exons flanking a frame-shifting genomic deletion could restore the reading frame and allow a Becker MD-like gene transcript be generated from a DMD pre-mRNA. Exon 51 skipping trials are showing great promise as a therapy that may reduce the severity in some DMD patients, but regardless of how efficiently exon skipping can be induced, the resultant dystrophin isoforms will be Becker MD like. There is one type of DMD mutation that could be ideally suited to exon skipping, and although pseudo-exon activation is relatively rare, the application of splice switching to these mutations should result in the generation of a normal, not Becker MD-like, dystrophin gene transcript. A BMD patient was recently diagnosed whose dystrophin defect arose from an as yet uncharacterized mutation in intron 62 that led to the retention of 58 bases of intronic sequence being retained in the mature mRNA. While this pseudo-exon disrupts the reading frame and should have led to DMD, there were low levels of normal dystrophin that reduced disease severity. Antisense oligomers can mask normal exons from the splicing machinery, and we have found that some pseudo-exons are even easier to exclude from the mature mRNA. We describe personalized exon skipping strategies to address two different pseudo-exons, one arising from intron 62 and another from intron 47. In both these cases, normal dystrophin mRNA is induced, and unlike all other splice switching oligomers, compounds targeting these pseudo-exons could be tested in healthy volunteers.

Pseudoexon exclusion by antisense therapy in 6-pyruvoyl-tetrahydropterin synthase deficiency

Antisense oligonucleotide therapy to modulate splicing mutations in inherited diseases is emerging as a treatment option also for metabolic defects. In this article, we report the effect of cellular antisense therapy to suppress pseudoexon activation in primary dermal fibroblasts from patients with mutations in the PTS gene encoding 6-pyruvoyltetrahydropterin synthase (PTPS), which leads to tetrahydrobiopterin and monoamine neurotransmitter deficiency. Pathogenic inclusion of SINE or LINE-derived cryptic exons in different PTPS patients due to the intronic mutations c.84-322A>T, c.163 + 695_163 + 751del57, or c.164-712A>T was demonstrated by transcript analysis in fibroblasts and minigene ex vivo assays. Antisense morpholino oligonucleotides (AMOs) directed to the pseudoexons 3' or 5' splice sites were designed with the aim of preventing the pathological pseudoexon inclusion. At the time of AMO transfection, we investigated patients' cells for correct PTS-mRNA splicing and functional recovery of the PTPS protein. Transcriptional profiling after 24 hr posttransfection revealed a dose- and sequence-specific recovery of normal splicing. Furthermore, PTPS enzyme activity in all three patients' fibroblasts and the pterin profile were close to normal values after antisense treatment. Our results demonstrate proof-of-concept for pseudoexon exclusion therapy using AMO in inherited metabolic disease.

Pseudoexon Activation Caused by Intronic Double‐deletion Events in the DMD Gene

Gross genomic rearrangements (GGRs) including copy number variations are increasingly being recognized as an important source of the “missing heritability” for both monogenic and complex diseases. The functional consequences of GGRs involving coding sequences or splice sites are often self-evident. By contrast, the functional consequences of GGRs involving deep intronic sequences often need to be sought by laborious experimental testing. In this issue, Khelifi et al. (Hum Mutat 32:467–475, 2011) characterized two unusual and complex intronic GGRs in the DMD gene at the nucleotide level through array-CGH and direct genomic sequencing. Both events comprise two deletions within single introns, the intervening sequence in the first event being also inverted. Each event was shown to give rise to pseudoexon activation by RT-PCR analysis using total RNA isolated from frozen muscle biopsy samples from the patients, suggesting pathogenic relevance. The authors performed a series of deletion experiments using a splicing reporter minigene assay, providing compelling evidence that an upstream intronic 592-bp sequence plays a crucial role in repressing pseudoexon activation in the wild-type allele. However, a combination of in silico analysis and experimental validation failed to identify any splicing regulatory elements that could have accounted for the inclusion/exclusion of the 166-bp pseudoexon. In addition, MFold prediction analysis of possible RNA secondary structure involvement in splicing regulation was inconclusive. Other factors such as nucleosome positioning and specific histone modifications may well turn out to play a role in regulating exon recognition. In reporting these two cases of DMD pseudoexon activation resulting from pure intronic double-deletion events, Khelifi et al. remind us that we still have some way to go before we are able to decode all the regulatory elements lurking in our own genome.

Pure intronic rearrangements leading to aberrant pseudoexon inclusion in dystrophinopathy: a new class of mutations?

We report on two unprecedented cases of pseudoexon (PE) activation in the DMD gene resulting from pure intronic double-deletion events that possibly involve microhomology-mediated mechanisms. Array comparative genomic hybridization analysis and direct genomic sequencing allowed us to elucidate the causes of the pathological PE inclusion detected in the RNA of the patients. In the first case (Duchenne phenotype), we showed that the inserted 387-bp PE was originated from an inverted ∼57 kb genomic region of intron 44 flanked by two deleted ∼52 kb and ∼1 kb segments. In the second case (Becker phenotype), we identified in intron 56 two small deletions of 592 bp (del 1) and 29 bp (del 2) directly flanking a 166-bp PE located in very close proximity (134 bp) to exon 57. The key role of del 1 in PE activation was established by using splicing reporter minigenes. However, the analysis of mutant constructs failed to identify cis elements that regulate the inclusion of the PE and suggested that other splicing regulatory factors may be involved such as RNA structure. Our study introduces a new class of mutations in the DMD gene and emphasizes the potential role of underdetected intronic rearrangements in human diseases.

Mutation deep within an intron of MSH2 causes Lynch syndrome

Lynch syndrome, a heritable form of cancer predisposition, is caused by germline mutations within genes of the DNA mismatch repair family, and can be rapidly identified in young onset cancer patients through the detection of loss of expression of at least one of these genes in tumour samples. To date, such causative mutations have only been identified within exonic and splice site regions. Though this approach has been successful in the majority of families, a considerable number remain in which no mutation has been found. To address this situation, we used an alternative mutation discovery procedure which involved haplotype analysis of the locus containing the gene lost in the tumour and delineation of segregating haplotypes, followed by an investigation of splicing aberrations to uncover cryptic splice sites which lay outside the genomic regions routinely examined for mutations. In this report, we show that an intronic mutation 478bp upstream of exon 2 in the MSH2 gene causes Lynch syndrome through creation of a novel splice donor site with subsequent pseudoexon activation, thus highlighting the need for more extensive sequencing approaches in families where routine procedures fail to find a mutation.

The deep intronic c.903+469T&gt;C mutation in the MTRR gene creates an SF2/ASF binding exonic splicing enhancer, which leads to pseudoexon activation and causes the cblE type of homocystinuria

Deep intronic mutations are often ignored as possible causes of human diseases. A deep intronic mutation in the MTRR gene, c.903+469T>C, is the most frequent mutation causing the cblE type of homocystinuria. It is well known to be associated with pre-mRNA missplicing, resulting in pseudoexon inclusion; however, the pathological mechanism remains unknown. We used minigenes to demonstrate that this mutation is the direct cause of MTRR pseudoexon inclusion, and that the pseudoexon is normally not recognized due to a suboptimal 5′ splice site. Within the pseudoexon we identified an exonic splicing enhancer (ESE), which is activated by the mutation. Cotransfection and siRNA experiments showed that pseudoexon inclusion depends on the cellular amounts of SF2/ASF and in vitro RNA-binding assays showed dramatically increased SF2/ASF binding to the mutant MTRR ESE. The mutant MTRR ESE sequence is identical to an ESE of the alternatively spliced MST1R proto-oncogene, which suggests that this ESE could be frequently involved in splicing regulation. Our study conclusively demonstrates that an intronic single nucleotide change is sufficient to cause pseudoexon activation via creation of a functional ESE, which binds a specific splicing factor. We suggest that this mechanism may cause genetic disease much more frequently than previously reported. Hum Mutat 31:437–444, 2010. © 2010 Wiley-Liss, Inc.

Alternative splicing: role of pseudoexons in human disease and potential therapeutic strategies

What makes a nucleotide sequence an exon (or an intron) is a question that still lacks a satisfactory answer. Indeed, most eukaryotic genes are full of sequences that look like perfect exons, but which are nonetheless ignored by the splicing machinery (hence the name 'pseudoexons'). The existence of these pseudoexons has been known since the earliest days of splicing research, but until recently the tendency has been to view them as an interesting, but rather rare, curiosity. In recent years, however, the importance of pseudoexons in regulating splicing processes has been steadily revalued. Even more importantly, clinically oriented screening studies that search for splicing mutations are beginning to uncover a situation where aberrant pseudoexon inclusion as a cause of human disease is more frequent than previously thought. Here we aim to provide a review of the mechanisms that lead to pseudoexon activation in human genes and how the various cis- and trans-acting cellular factors regulate their inclusion. Moreover, we list the potential therapeutic approaches that are being tested with the aim of inhibiting their inclusion in the final mRNA molecules.

Pseudoexon activation in the PKHD1 gene: a French founder intronic mutation IVS46+653A&gt;G causing severe autosomal recessive polycystic kidney disease

Pseudoexon activation in the <i>PKHD1</i> gene: a French founder intronic mutation IVS46+653A&gt;G causing severe autosomal recessive polycystic kidney disease

Disease-causing mutations improving the branch site and polypyrimidine tract: Pseudoexon activation of LINE-2 and antisenseAlulacking the poly(T)-tail

Cryptic exons or pseudoexons are typically activated by point mutations that create GT or AG dinucleotides of new 5' or 3' splice sites in introns, often in repetitive elements. Here we describe two cases of tetrahydrobiopterin deficiency caused by mutations improving the branch point sequence and polypyrimidine tracts of repeat-containing pseudoexons in the PTS gene. In the first case, we demonstrate a novel pathway of antisense Alu exonization, resulting from an intronic deletion that removed the poly(T)-tail of antisense AluSq. The deletion brought a favorable branch point sequence within proximity of the pseudoexon 3' splice site and removed an upstream AG dinucleotide required for the 3' splice site repression on normal alleles. New Alu exons can thus arise in the absence of poly(T)-tails that facilitated inclusion of most transposed elements in mRNAs by serving as polypyrimidine tracts, highlighting extraordinary flexibility of Alu repeats in shaping intron-exon structure. In the other case, a PTS pseudoexon was activated by an A>T substitution 9 nt upstream of its 3' splice site in a LINE-2 sequence, providing the first example of a disease-causing exonization of the most ancient interspersed repeat. These observations expand the spectrum of mutational mechanisms that introduce repetitive sequences in mature transcripts and illustrate the importance of intronic mutations in alternative splicing and phenotypic variability of hereditary disorders.

Pseudo‐exon activation caused by a deep‐intronic mutation in the fibrinogen γ‐chain gene as a novel mechanism for congenital afibrinogenaemia

Congenital afibrinogenaemia, characterized by severe fibrinogen deficiency, is caused by mutations within FGA, FGB or FGG. Conventional sequencing of coding regions and splice signals of these three genes did not reveal any mutation in an afibrinogenaemic proband. After confirming disease co-segregation with the fibrinogen cluster, full intron sequencing was tackled leading to the identification of a novel transvertion within FGG intron 6 (IVS6-320A-->T). Its effect on mRNA processing was evaluated in-vitro: the in-frame inclusion of a 75-bp pseudo-exon carrying a premature stop was found, representing the first report of pseudo-exon activation as a mechanism leading to afibrinogenaemia.

RNA structure is a key regulatory element in pathological ATM and CFTR pseudoexon inclusion events

Genomic variations deep in the intronic regions of pre-mRNA molecules are increasingly reported to affect splicing events. However, there is no general explanation why apparently similar variations may have either no effect on splicing or cause significant splicing alterations. In this work we have examined the structural architecture of pseudoexons previously described in ATM and CFTR patients. The ATM case derives from the deletion of a repressor element and is characterized by an aberrant 5′ss selection despite the presence of better alternatives. The CFTR pseudoexon instead derives from the creation of a new 5′ss that is used while a nearby pre-existing donor-like sequence is never selected. Our results indicate that RNA structure is a major splicing regulatory factor in both cases. Furthermore, manipulation of the original RNA structures can lead to pseudoexon inclusion following the exposure of unused 5′ss already present in their wild-type intronic sequences and prevented to be recognized because of their location in RNA stem structures. Our data show that intrinsic structural features of introns must be taken into account to understand the mechanism of pseudoexon activation in genetic diseases. Our observations may help to improve diagnostics prediction programmes and eventual therapeutic targeting.

Pseudoexon activation in the DMD gene as a novel mechanism for Becker muscular dystrophy.

We report the characterization of two deep intronic mutations in the Duchenne muscular dystrophy (DMD) gene of two unrelated Becker muscular dystrophy (BMD) patients, causing the aberrant inclusion of a pseudoexon in the mature transcripts. These two mutations were identified by the use of RT-PCR on transcripts isolated from muscle. The first abnormally large transcript resulting from a 58-bp insertion between exon 62 and exon 63 was identified in a BMD patient with mental retardation. The origin of this transcript was a mutation in intron 62 (IVS62-285A>G), which resulted in the occurrence of a high quality donor splice site. The IVS25+2036A>G in intron 25 was identified in a subclinical BMD patient with high CK levels. The mutation reinforces the strength of a pre-existing acceptor splice site, resulting in activation of an intronic pseudoexon of 95 bp. By using DHPLC, the patient's mother was found to be a somatic mosaic. The insertion of these newly recognized extra exons leads to premature termination codons, but we could observe that some degree of normal splicing was taking place in both patients. The detection of these residual full length transcripts is consistent with the clinical presentation and dystrophin analyses. This is the first report of pseudoexon activation as a mechanism for Becker muscular dystrophy, and this reveals further the diversity of genetic abnormalities causing BMD.

Pseudoexon Activation as a Novel Mechanism for Disease Resulting in Atypical Growth-Hormone Insensitivity

Inherited growth-hormone insensitivity (GHI) is a heterogeneous disorder that is often caused by mutations in the coding exons or flanking intronic sequences of the growth-hormone receptor gene (GHR). Here we describe a novel point mutation, in four children with GHI, that leads to activation of an intronic pseudoexon resulting in inclusion of an additional 108 nt between exons 6 and 7 in the majority of GHR transcripts. This mutation lies within the pseudoexon (A(-1)-->G(-1) at the 5' pseudoexon splice site) and, under in vitro splicing conditions, results in inclusion of the mutant pseudoexon, whereas the wild-type pseudoexon is skipped. The presence of the pseudoexon results in inclusion of an additional 36-amino acid sequence in a region of the receptor known to be involved in homo-dimerization, which is essential for signal transduction.