Download From bedside to bench: how to analyze a splicing

From bedside to bench: how to analyze a splicing mutation.
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).
When presented with a new patient, with 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.
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 counselling.
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). 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) 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.
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 premRNA processing of the genes in which they are found. 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.
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. 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. 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. 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 transacting 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]. 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 www.hgvs.org/dblist/dblist.html [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]. 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.
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" 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" 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. 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.
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 X and Y respectively).
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]. 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 Z).
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. 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 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
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 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 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 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 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)
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.
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 X, 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 and RNA interference knock
down methods and can be used to determine the role played by trans-acting factors in the
regulation of constitutive and alternative splicing (see chapters X and X). 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 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.