From bedside to bench: how to analyze a splicing mutation.

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
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
(, we are in the fortunate situation that this procedure is becoming a
routine service provided in many hospitals (see 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 [1]. 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 [4]. 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 [12]
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 [17]. A
comprehensive list of specific databases is maintained by the Human Genome Variation
Society (HGVS) and is currently available at [18].
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 [22], NNsplice [23],
AST [24], Spliceport [25], Spliceview [26], HBond [27], Automated Splice Site Analyses
[28], NetGene2 [29], and Human Splicing Finder based on Ensembl release 44 [30] 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 [31], RESCUE-ESE
[22,32], ExonScan [32-34], PESX [35,36] or ESRsearch [37] (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 [8]. 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 [37]. 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, [39] As a result,
the effect of a mutation on pre-mRNA can only be fully elucidated by "wet-lab"
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 [40].
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 [41]. 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 [42]. 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 [39]. 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 [43] (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 [39] 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 [44], exonic [44], allele specific [40] and deep intronic [45], 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
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 [46]. 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 [47]. 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. This will not only represent a clear advantage to the
diagnostic field with important clinical impact for families affected with genetic disease,
but as our knowledge of the complex molecular mechanism of splicing improves, may
also eventually lead to novel therapeutic approaches that take advantage of the recent
advances in RNA chemistry [48].
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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.