From bedside to bench: how to analyze a splicing mutation. Diana Baralle, address, etc Diana, can you please provide a table with internet addresses for Important tools from clinicians to investigate alternative splicing? Can you also break up the text in 3-4 subheadings? Key points: Clinical diagnosis can identify mutated candidate genes, which can exhibit altered pre-mRNA splicing patterns The identification of sequence variants involved in splicing helps in understanding splicing regulatory elements as well as disease etiology Currently the effect of mutations can be bioinformatically predicted. These predictions are fairly inaccurate and need to be tested experimentally by analyzing RNA expression or by reporter constructs Introduction One of the principle tasks in clinical genetics is the identification of disease causing mutations in order to be able to improve patient care through accurate diagnosis and prognosis, for their medical or surgical management, prenatal testing, assessment of recurrence risks, and for familial genetic studies as well as advancement of the understanding of a particular genetic condition. Today, with improvements in DNA sequencing protocols and consequent gene sequencing output data, coupled with ever more complete searchable databases, (for example the human gene mutation database (www.hgmd.cf.ac.uk)), we are in the fortunate situation that this procedure is becoming a routine service provided in many hospitals (see www.eddnal.com for a directory of European DNA diagnostic laboratories). Is there such a database for the US? When presented with a new patient, with an estimated more then over 23000 genes in the human genome, molecular genetic analysis needs to be targeted to a specific gene (or small group of genes). This necessitates having a good idea of the possible clinical diagnosis and a prerequisite knowledge of which set of genes could by readily analysed for mutations. Or, in the most favorable cases, which single gene carries a high probability of being mutated in the affected individual. For example, a diagnosis of breast cancer would entail screening of the BRCA1and BRCA2 genes, Neurofibromatosis type 1 of the NF1 gene and long QT syndrome (LQTS) of the 9 genes to date associated with the disorder. Can you include a sentence how these clinical diagnosis is been done? As a result, the screening of such genes will, with high likelihood, yield readily analysed mutations whose connection with the disease has already been verified. Alternatively, checking and if necessary updating and supplementing existing mutation databases can also help identify mutational "hot spots", give clues to phenotype/genotype correlations and thus improve future basic research approaches, diagnostic screening studies and genetic counseling. The introductory paragraph is fairly long, can you break it down and maybe start here with results of genetic variations However, it should be borne in mind, that many genetic screens can also result in nucleotide variations whose affect on gene function has yet to be clarified and understood including those that may simply represent a benign polymorphism and not be pathogenic at all. Preliminary work to try and distinguish which variants are pathogenic and which are disease causing would include; checking for the absence of the variant in a large number of controls, proving that this is a de novo sequence variant, and using bioinformatic techniques to assess the effect of a sequence variant on protein function or splicing function. In many of these cases, subsequent functional studies would have to be performed to confirm pathogenicity. Depending on the type of nucleotide change observed, the potential effect it may have can sometimes be inferred (Fig.1)(see also chapter 9, F Baralle for a discussion of a disease mechanism). For example, if the change was to introduce a stop codon (nonsense mutation) then pathogenicity can be readily inferred. This is also the case with mutations that affect the canonical nucleotides in the either the 5’ or 3’ splices sites (gt and ag dinucleotides respectively). In addition, if the nucleotide change were to result in an amino acid change (missense mutation) or deletion, one could imagine that the functionality of the protein may be affected (there are now several bioinformatics resources that allow predictions to be made on this basis, see chapters in IIF for discussion, as well as table XXX) then these are more likely to be disease causing. Nucleotide changes that are more difficult to assess are those that do not apparently affect any amino acid (same sense sequence variants) and are at times labeled polymorphisms, as well as intronic variations, be they close to or distant from the splices sites. Can you introduce the term and significance of UVs?, most molecular people are not familiar with this Over the last few years, many mutations routinely assumed to be missense, nonsense or even silent have been shown to also cause disease by affecting the pre- mRNA processing of the genes in which they are found. Can you refer here to the fist paper by cooper and mattox? Indeed, genetic analysis of mutations in and around 5’ and 3’ splice sites are responsible for approx 15% of the genetic diseases that are caused by point mutations . Furthermore, for some genes this is much higher for example in NF1 and ATM, it has been shown that mutations that cause splicing alterations occur in approximately 50% of the affected patients [2,3]. Of these mutations, 24% would have been mis-assessed as frameshift, missense or nonsense mutations if the analysis had been limited to genomic sequences. As a result of these studies and reappraisals, it has been recently proposed that up to 60% of mutations that cause disease may do so through disruption of pre-mRNA splicing . Mutation analyses exclusively performed at the genomic DNA level, are often not sufficient to correctly identify and characterize these mutations. For this reason, analysis of mRNA splicing patterns would be desirable for proper and more complete genetic diagnosis. When possible this could be done by in vivo analysis of patient samples directly and/or by employing reliable minigene splicing assays in vitro or in cell culture analysis. As outlined in chapters IID To further accentuate the importance of testing to see if a nucleotide change affects the pre-mRNA splicing process, we give an example of a borderline diagnosis, a common clinical scenario. Is borderline diagnosis as standart term? Can you explain maybe this could be moved as an example in the introduction A patient with an ECG reading of QTc exceeding 500ms poses a negligible cardiac diagnostic challenge, whereas diagnostic certainty considerably decreases in asymptomatic persons with QTc values termed intermediate can you explain/elaborate on ECG, Qtc and LQT. In fact, although these values impart a much lower risk factor it does not exclude a patient from harboring a potentially lethal LQT mutation. In these cases, correct diagnosis is of paramount importance, as identification of one such mutation would allow the appropriate life saving medication to be administered to the patient as well as screening of all at risk family members. Indeed, a scenario of this type led to the identification of the first splicing mutation in LQT and subsequently the discovery of many more of these types of mutation in the field [5-7]. The reason that so many disease-causing mutations are now being shown to result in pathology due to aberrant splicing of the gene in which they are found, is that the removal of introns from pre/messenger RNA by splicing is a very complex step in eukaryotic gene expression which necessitates a more widespread use than previously thought of cis and trans-acting elements in order to identify the exon (see chapter 4 Hertel and 5 Luhrmann for discussion). As a result, the widespread occurrence of this class of mutation was previously underestimated. Firstly, one has to consider the conserved albeit degenerate ‘core’ cis-acting sequences that include 5' and 3' splice sites, branch-point sequence and polypyrimidine tract. In addition to these essential sequence elements, the overall fidelity of splicing is enhanced by highly degenerate as well as context specific enhancer and silencer elements that may be variably present in any particular system: exon splicing enhancers (ESEs); exon splicing silencers (ESS); intron splicing enhancers (ISEs) and intron splicing silencers (ISS). It is the mutations in such enhancer or silencer sequences, as well as mutations in the trans-acting factors that bind these sequences, that often lead to the harder to spot significant defects in splicing patterns and alterations in protein expression [8-11]. The number of mutations occurring at the pre-mRNA splicing level have risen to an extent where databases partially or totally dedicated to collecting mRNA splicing defects now exist. Probably the most publicized example is the Human Gene Mutation Database (HGMD) that acts as a general repository of pathological gene mutations  but other databases are also being established along these lines such as the Alternative Splicing Mutation Database (ASMD) [13,14] (can you include a table of databases, bioinformaitc tools useful for clinicians?, including their web addresses). In addition, for particular aberrant splicing events such as cryptic splice site activation, researchers and diagnosticians can also be referred to the recently established DBASS3 and DBSSS5 databases [15,16]. Finally, there is also a growing list of locus-specific databases that are exclusively focused on particular genes of interest such as CFTR or HPRT . A comprehensive list of specific databases is maintained by the Human Genome Variation Society (HGVS) and is currently available at www.hgvs.org/dblist/dblist.html . Although none of these databases contain predictive information with regards to newly discovered mutations they have the potential to save a lot of work by acting as an easy reference source for clinicians. Mutation testing procedures. Identification of the cis and trans-regulatory elements that control the splicing of a given gene is essential for interpreting how the changes in splicing may lead either to disease or conversely, to an amelioration of the effects of certain genetic lesions. In recent years, efforts have been made to characterize cis-acting splicing regulatory elements such as; 5'ss, 3'ss, and branch-point using position weight matrices that are calculated from collections of splice sites [19-21], or ESE, ESS, ISE and ISS sequences using in vitro and in vivo selection methods. An important resource for this type of research that will assist our diagnostic capabilities, is the study of disease associated mutations or variants that are known disrupt pre-mRNA splicing. These approaches have provided the scientific community with several bioinformatics methodologies with which to assess splice sites such as MaxEntScan , NNsplice , AST , Spliceport , Spliceview , HBond , Automated Splice Site Analyses , NetGene2 , and Human Splicing Finder based on Ensembl release 44  as well as a list of positively and negatively acting elements involved in splicing. These are available in web-accessible servers or programs such as ESEfinder , RESCUE-ESE [22,32], ExonScan [32-34], PESX [35,36] or ESRsearch  (bioinformatics approaches to alternative splicing are discussed in section II.F of the book). In all these cases, a key question is the degree of reliance that one can place on each of these approaches with regards to the routine identification of possible splicing mutations and whether these can be used clincially. Due to the larger dataset available and greater conservation, the prediction programs that deal with the 5' and 3' splice sites strength currently fair better than those that deal with the more degenerate splicing enhancer and silencer elements. I think it should be clearly state that none of the prediction methods are really accurate (with the exceptions of mutations in the GT-Ag and need experimental backup), which is one reason for the book) However, it should be noted that a high number of false positive and false negative hits are generated with the available prediction programs and raises the issue of practical applicability of these predictions to medical genetics . In fact, it has also been shown that many computationally predicted candidates turn out to be inactive when tested experimentally in both homologous and heterologous extent . It is also true that many more as yet unidentified motifs will also have splicing regulatory activity [37,38]. The reason for these discrepancies resides in the great role played by "genetic context, can this be defined in the glossary?" in the pre-mRNA splicing process,  As a result, the effect of a mutation on pre-mRNA can only be fully elucidated by "wet-lab" experiments. The simplest and fastest method of testing whether a suspected disease causing mutation affects splicing of the gene in which it finds itself in or not, comes from RNA analysis of the affected tissue through a reverse transcriptase reaction followed by PCR using primers that amplify, preferably from exons as far away from the mutation location as possible (described in chapter 18, smith, beggs). Though apparently straightforward, this approach carries problems. Firstly, the patient or the appropriate tissue may not always be available. The majority of samples for clinical diagnostics are nearly always leukocytes from which, usually only the DNA is extracted. Extracted RNA is a relatively simple procedure, however, it is important to remember that the gene of interest may not be expressed in this tissue. Moreover, in the case of alternatively spliced exons, leukocytes may only provide a limited set of the possible splicing outcomes, representing a serious limitation if the eventual cis-acting mutations have cell specific effects. What about shipping the samples between clinicians and labs, paraffin sections? Another point to keep in mind when performing these types of experiments is the potential presence of allele specific polymorphisms. Minimal alterations in alternatively spliced products can result in disuse, and making sure that we look at any eventual effect in the mRNA splicing of the specific allele is of extreme importance . Lastly, the mutation may favor an alternative splicing event that introduces a premature termination codon (PTC). Indeed one third of alternative splicing events are thought to be of this type . In these cases, a regulatory mechanism known as nonsense mediated decay, in which the quality of the mRNA is assessed and if found to carry PTC selectively degrades these transcripts, is now known to exist in eukaryotic cells . This process will effectively screen any deleterious effect on pre-mRNA splicing of the mutation both at the molecular biology level. Methods to circumvent this problem such as stable cell culture of the patient cell lines together with blocking of the NMD pathway with antibiotics exist but are time consuming. Although direct analysis is an obvious first approach, medical screening of mutations needs a fast, user friendly, experimentally controlled and easily repetitive methodology. Two principle methods, in vitro splicing assays and mingene splicing assays have being used over the years (chapter 26 mayeda, 31 stamm, 32 tazi). Briefly, in vitro splicing uses bacterial polymerases to radioactively transcribe DNA sequences. The RNA is subsequently incubated with nuclear extract in which the splicing reaction occurs. The products of the splicing reaction are then visualized on polyacrylamide denaturing gels. This approach has the drawback that it is normally performed with relatively short pieces of DNA. For this reason it is difficult to take into account all the cis-acting elements and that the sum of these determine the amount of inclusion of that exon in the final transcript . Also not all in vitro construcs splice Having said this, due to ease of manipulation through various biochemical approaches in vitro splicing is still very much used especially in the study of the molecular mechanism involved in the recognition of an exon  (chapter 26, mayeda). For these reasons, the most common technique in use today for the analysis of the effect of a mutation on pre-mRNA splicing is the minigene splicing assay. Whatever type of minigene system is used, the basic methodology remains the same and the basic principle is shown in figure 2 ( I don’t think this figure is included in the ppt file, you included mainly a map of a vector) The genomic region of interest is amplified from normal and affected individuals and cloned into a plasmid between a ubiquitous transcriptional promoter and a gene segment for poly A 3’ end formation. To avoid eventual NMD effects the DNA fragment can be inserted in the correct reading frame phase, (if not already the case), by the addition or subtraction of the appropriate number of nucleotides as well as the addition of a Met initiation codon for the start of translation through PCR mutagenesis. The minigene plasmid is then transiently transfected in an appropriate cell line where it is transcribed by the cellular RNA polymerase II and the resulting pre-mRNA processed to obtain a mature mRNA. The mRNA splicing pattern is analyzed mainly by RT-PCR with primers specifically designed to amplify processed transcripts derived from the minigene to distinguish them from endogenous transcripts. Finally, the spliced products are visualised on an agarose gel. The size of the genomic region amplified dictates the type of minigene utilised. Due to the fact that exon definition is often the sum of complex antagonistic and/or synergistic interactions mediated by different splicing elements that can occur across both introns and exons  it is preferable that as much homologous not clear why homologous, patient DNA? Flanking DNA? genomic sequence as possible is used. If amplification of a 3 exon two intron segment is possible (with the affected exonic of intronic sequence located centrally) then this can be cloned directly between the promoter and the poly A already present in a plasmid such as pCDNA3 (invitrogen). Often however, for practical reasons this is not feasible as the length of the amplified fragment will be too large. In these cases, the introns can be deleted internally or a hybrid minigene may be utilised. This is a plasmid that, as before, contains a ubiquitous transcriptional promoter and a gene segment for poly A 3’ end formation but carries at least two exons separated by an intron that contains a cloning site for your amplified fragment. One such example is the PTB minigene ( can you add this minigene into the reagent collection) that has been used successfully in identifying a diverse array of mutations from, splice site , exonic , allele specific  and deep intronic , that have been shown to affect pre-mRNA splicing. In addition, availability of such research tools has greatly aided in the characterization of the molecular mechanisms behind these aberrant splicing events. The PTB minigene is a hybrid construct containing exons from -globin and fibronectin, under the control of the -globin promoter. The intronic region between the two fibronectin exons contains a unique NdeI restriction site into which the genomic region of interest can be cloned. In the case of exonic or intronic mutations close to the splice sites this would consist of the exon together with an appropriate amount of flanking intronic sequence. In the case of deeper intronic mutations the two exons flanking the intron carrying the mutation and the entire intron itself may be inserted into the minigene at the NdeI site. Aside from PTB, a variation on the minigene theme is exemplified by that utilised in the identification of ESEs by in vivo selection . This hybrid minigene (SXN13) ( can you add this minigene into the reagent collection) consists of a 34 nucleotide alternative exon flanked by duplicated intron 1 from human -globin such that the first and third exons are globin exons 1 and 2. This alternative exon, which is only partially recognized by the splicing machinery in normal conditions contains a small cassette into which oligos of 13 nucleotides mimicking the suspected wild type or mutated ESE or ESS elements may be cloned. The effects of this insertion that in theory should cause increased or decreased inclusion respectively can then be analysed. Can you show these minigenes in linear form, i.e. not as plasmid circles One of the main drawbacks to date in this type of analysis being applied in clinical diagnosis is that this type of methodology requires a certain degree of molecular biology skill. The latest generation of minigene splicing assays (pSpliceExpress), however, goes some way to making this a feasible option . This method, described in chapter 31 stamm, makes this system simpler and more amenable for high throughput analysis as it uses a recombination method where the need for appropriate restriction sites is removed, and the procedure highly streamlined. Further characterization of the molecular mechanism involved may use minigene splicing assays in combination with protein over-expression, RNA interference knock down methods and targeted oligonucleotide treatment and can be used to determine the role played by trans-acting factors in the regulation of constitutive and alternative splicing (see chapters 32, 33 and 42 aartsma rus). In additions, reporter genes can be modified to identify possibly regulatory substances (chapter 41 stoilov, and RNA- containing trans-acting factors chapter 40 schumperli,) can be generated. If successful, the elucidation of the mechanisms regulating pre-mRNA processing will eventually allow development of additional drugs targets and novel diagnostic and therapeutic approaches. Some of these novel approaches (ie. small molecule modification of trans-acting factor activities or use of antisense oligonucleotides carrying functional tails) are described in Chapters Z and W. Concluding remarks Classical routine strategies of mutation analysis, whereby the more common types and locations of mutations are sought first, have historically been extremely fruitful. However, in many cases researchers are still unable to establish the disease causing mutation. A number of these will be because the mutation is located in an atypical region, for example in an intronic region a non mature mRNA coding region (intron), in the promoter region, in a distant regulator gene, and even through missclassification of sequence variations as benign variants. In order to improve our diagnostic capabilities, it is essential to introduce corrections to our future mutational analyses. It is now clear that defects in pre-mRNA processing are one of the major causes of human diseases and that they are often missed in routine classical analyses., An essential step to improve today's clinical diagnostic testing would be to routinely employ some form of splicing assay when testing for disease causing mutations. In the past, this has been certainly hampered by the need for considerable expertise in molecular biology. However the advances in the molecular techniques outlined above make it now feasible to integrate these types of analyses in routine mutation screening. 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Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9. Hum Mol Genet 8, 2339-2349. Lewis, B.P., Green, R.E. and Brenner, S.E. (2003). Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc Natl Acad Sci U S A 100, 189-92. Maquat, L.E. (2004). Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics. Nat Rev Mol Cell Biol 5, 89-99. Matlin, A.J. and Moore, M.J. (2007). Spliceosome assembly and composition. Adv Exp Med Biol 623, 14-35. Baralle, M., Baralle, D., De Conti, L., Mattocks, C., Whittaker, J., Knezevich, A., Ffrench-Constant, C. and Baralle, F.E. (2003). Identification of a mutation that perturbs NF1, a gene splicing using genomic DNA samples and a minigene assay. J Med Genet 40, 220-2. Pagani, F., Buratti, E., Stuani, C., Bendix, R., Dork, T. and Baralle, F.E. (2002). A new type of mutation causes a splicing defect in ATM. Nat Genet 30, 426-9. Singh, G. and Cooper, T.A. (2006). Minigene reporter for identification and analysis of cis elements and trans factors affecting pre-mRNA splicing. Biotechniques 41, 177-81. Kishore, S., Khanna, A. and Stamm, S. (2008). Rapid generation of splicing reporters with pSpliceExpress. Gene 427, 104-10. Bonetta, L. (2009). RNA-based therapeutics: ready for delivery? Cell 136, 581-4. Figure 1 Representation of reasoning to follow upon identification of candidate sequence variants. Figure 2 Schematic representation of minigene splicing assays: A) The most basic minigene is composed of a plasmid containing a promoter and a poly A signal with a multiple cloning site (MCS) between the two. In the MCS the region of the gene in which the suspected mutation is found is inserted. A minimum of three exons/two introns need to be inserted in the MCS with the exon whose pre-mRNA processing is thought to be affected by the mutation. be it intronic or exonic, being the central exon. B) PTB hybrid minigene composed of a alpha-globin gene promoter and SV40 enhancer sequences (indicated by the arrow at the start of the gene) to allow polymerase II transcription in the transfected cell lines. This is followed by a series of exonic and intronic sequences (indicated by boxes and lines, respectively) that derive from alpha-globin (black boxes) and fibronectin exons (grey boxes), while at the 3’ end a functional polyadenylation site, derived from the alpha-globin gene, is present. The genomic DNA region of interest that contains a putative splicing mutation is introduced into the minigene in a unique restriction site (NdeI). In the case of deep intronic mutations, hybrid minigenes are created in which the two exons flanking the intron carrying the mutation and the intron itself (or a shortened version of it) are inserted into the minigene at the NdeI site. (C) Schematic representation of the hybrid minigene SXN13. This minigene consists of a 34 nucleotide alternative exon flanked by duplicated intron 1 from human alpha-globin such that the first and third exons are globin exons 1 and 2. In the absence of a splicing enhancer this element is predominately skipped due to its small size and a non-canonical 5’ splice site. Regions of exonic DNA suspected of having enhancer activity can be cloned into the alternative exon and tested for their effect on splicing.
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