From the Laboratoire Centre de Recherche
Thérapeutique en Ophtalmologie, Faculté de Médecine
Necker, 156 rue de Vaugirard 75015 Paris, France,
§ Laboratoire de Biochimie, Hôpital Kremlin
Bicêtre, AP-HP Hôpital de Bicêtre, France,
Service de Neuropédiatrie, Hôpital Necker
Enfants-Malades, 149 rue de Sèvres, 75015 Paris, France, and
** Department of Biological Sciences, University of
Pittsburgh, Pittsburgh, Pennsylvania 15260
Received for publication, October 30, 2002, and in revised form, December 30, 2002
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ABSTRACT |
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An intronic point mutation was identified in the
E1 Accurate pre-mRNA splicing is vital for the conversion of
nascent transcripts into mRNA templates for protein synthesis. In humans, this involves the removal of 7-8 introns on average from each
nascent transcript (1). Accurate splicing depends upon the recognition
of the 5'-splice site, branch site, and polypyrimidine tract/3'-splice
site at the boundaries of each intron. However, the splice sites, which
are short and highly degenerate, do not contain sufficient sequence
information for their accurate recognition (2). Consequently, the
sequences outside of but in the vicinity of the splice sites play
highly important roles in modulating the strengths of the natural
splice sites. Exonic and intronic splicing enhancers
(ESEs1 and ISEs,
respectively) exert positive roles, whereas exonic and intronic
splicing silencers exert negative roles. In constitutive splicing, these sequence elements favor not only the recognition of the
authentic splice sites but disfavor the recognition of cryptic splice
sites. Splicing control elements are also highly important in
specifying alternative splicing events. Alternative splicing generates
remarkable proteomic diversity in multicellular organisms and can give
rise to tissue-specific or developmentally controlled production of
protein isoforms from a single gene.
The recognition of splicing control elements is normally accomplished
by two types of protein families, the heterogeneous nuclear
ribonucleoprotein, which are frequently involved in negative control,
or by the SR splicing factors, which are involved in splicing
enhancement through the use of ESEs. SR splicing factors are
RNA-binding proteins containing a carboxyl terminal effector domain
that is rich in serine arginine dipeptides. SR splicing factors are
thought to promote splice site recognition by engaging in
protein-protein interactions with subunits of the essential splicing
factors, U2AF and U1 small nuclear ribonucleoprotein.
Pyruvate dehydrogenase (PDH) defects are major causes of lactic
acidosis and Leigh's encephalomyelopathies in infancy and childhood
(3, 4). The PDH complex is responsible for the conversion of pyruvate
to acetyl-CoA. The majority of the molecular defects has been localized
in the coding regions of E1 Case Report--
The patient, a boy, was born at term to
unrelated parents, but his mother's sister died suddenly at the age of
2-3 months. The patient was first examined at 3 months for hypotonia.
At 4 months of age, his general state was impaired. At 9.5 months, seizures appeared. At this time, lactates were 2.8-3.9 and pyruvates were 0.33 and 0.41 mmol/liter before and after meals, respectively, and
the lactate in cerebrospinal fluid was 3.2 mmol/liter. The MRI was
normal. At 16 months of age, he suffered from ataxia and developmental
delay and he was treated by a ketogenic diet (80% lipids) and thiamine
(500 mg/day), which improved the neurological and biological
features but maintained the mental retardation.
Biochemical Studies--
Pyruvate dehydrogenase complex activity
was determined by measuring the release of
14CO2 from 0.2 mM
[1-14C]pyruvic acid in fibroblast homogenates and
lymphocytes (16).
For Western blot analysis, 7 µg of protein from total fibroblasts was
separated on a 10% SDS-polyacrylamide gel and transferred onto a
nitrocellulose membrane. The blots were probed with anti-PDH complex
holoenzyme antibodies. All of the PDH subunits were visualized by use of the ECL detection system (Amersham Biosciences).
Molecular and Genetic Analysis--
The DNA of the
patient was prepared from lymphocytes and fibroblasts. The DNA
of the mother was purified from blood using proteinase K/sodium dodecyl
sulfate extraction method. The individual exons and the exon-intron
boundaries were amplified (primers available on request). The sequence
of the short intron 7 (181 bp) was amplified in a single PCR fragment
with exons 7-8 with forward primer 5'-GGC AGA GCA GCA GCT GTT AG-3'
and reverse primer 5'-CAG CTT CAG CAG GCA CAT GG-3'.
Total RNA was extracted from cultured fibroblasts and from fresh
lymphocytes using High Pure RNA Isolation Kit (Roche Molecular Biochemicals). cDNA was prepared from 2 µg of RNA using
Omniscript reverse transcriptase kit (Qiagen) and oligo(dT) primer and
amplified in two overlapping PCR fragments (Fig. 2A), GH
(from bases
All of the PCR products were purified with Concert Rapid PCR
purification system (Invitrogen) and sequenced with the Big Dye terminator kit on a 3100 ABI Prism sequencer (Applied Biosystems).
Cloning of the Full-length E1 Detection of an Aberrant Splicing in Vitro--
The PCR fragment
containing exon 7, intron 7, and exon 8 was performed from the genomic
DNA of the patient and a control using the following primers: sense,
5'-GCT-GTT-AGA-GAT-GAT-GAA-GCC-TCG-AGA-AAG-AAG-3', and
antisense, 5'-GGG-AGG-CAG-TCT-TTG-AAC-TTC-TAG-AGA-TAA-ATG-3'. These primers introduced sites for XhoI and XbaI
restriction enzymes in the 5' and 3' ends of the PCR product,
respectively, that were used for the insertion of the PCR fragment into
the pET01 Exon Trap vector (Mobitec). After digestion with
XhoI and XbaI, this fragment was 494-bp long: 45 bp of intron 6, exon 7 (156 bp), intron 7 (181 bp), and exon 8 (72 bp)
and 40 bp of intron 8.
Three independent transfections were carried out in COS-7 cells using
LipofectAMINE reagent (Invitrogen) according to the manufacturer's
instructions. Total RNA was obtained with RNA-PLUSTM
extraction solution (Quantum-Bioprobe). 2 µg of RNA was used for
RT-PCR with the First Strand cDNA Synthesis kit (Amersham Biosciences) using the specific primer (cDNA primer number 01) from
the Exon Trap kit. The PCR was performed with the 5'-PCR primer 02 and
3'-primer 03 also from the kit. With this construction and PCR primers,
the expected size of the normal product is 477 bp. The PCR fragment was
visualized on a 1.5% agarose gel.
SR Protein ESE Motif Analyses (18, 19) in Normal and Mutated
Intron 7--
The nucleotide sequences of normal and mutated intron 7 were searched with the ESEfinder program using the default settings (exon.cshl.org/ESE/).
The patient was diagnosed as having a defect in PDH activity
because of neonatal hypotonia, seizures, and mental retardation associated with hyperlactacidemia and a normal lactate/pyruvate ratio. The assays of enzymatic PDH activity confirmed a partial pyruvate dehydrogenase defect in cultured fibroblasts (0.50 nmol/mg protein/min; normal control range of 0.64-1.01) and in isolated lymphocytes. Respiratory chain complex activities were normal in muscle biopsy.
Immunochemical analysis using an antibody to the whole complex revealed
that there was a slight decrease of both immunoreactive E1 PDH gene from a boy with delayed development
and lactic acidosis, an X-linked disorder associated with a partial
defect in pyruvate dehydrogenase (PDH) activity. Protein analysis
demonstrated a corresponding decrease in immunoreactivity of the
and
subunits of the PDH complex. In addition to the normal spliced
mRNA product of the E1
PDH gene, patient samples
contained significant levels of an aberrantly spliced mRNA with the
first 45 nucleotides of intron 7 inserted in-frame between exons 7 and
8. The genomic DNA analysis found no mutation in the coding regions but
revealed a hemizygous intronic G to A substitution 26 nucleotides
downstream from the normal exon 7 5'-splice site. Splicing experiments
in COS-7 cells demonstrated that this point mutation at intron 7 position 26 is responsible for the aberrant splicing phenotype, which
involves a switch from the use of the normal 5'-splice site (intron 7 position 1) to the cryptic 5'-splice site downstream of the mutation
(intron 7 position 45). The intronic mutation is unusual in that it
generates a consensus binding motif for the splicing factor, SC35,
which normally binds to exonic enhancer elements resulting in increased
exon inclusion. Thus, the aberrant splicing phenotype is most likely
explained by the generation of a de novo splicing enhancer
motif, which activates the downstream cryptic 5'-splice site. The
mutation documented here is a novel case of intron retention
responsible for a human genetic disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PDH gene at chromosome Xp22.1
(gene symbol PDHA1, MIM catalog 312170) (5). Also,
some rare cases are because of mutations in another gene, the
Hs-PDX1 coding for the E3-binding protein of the PDH
complex, and most of them are splicing site mutations (6). Recently, two exonic mutations leading to aberrant splicing in the E1
PDH gene and the excision of exon 6 have been described previously (7). To date, there is no evidence for developmentally regulated alternative splicing events or tissue-specific control of the expression of this gene. This paper describes a new PDH deficiency explained by a novel intronic splicing mutation of the E1
PDH gene in a patient with an encephalopathy and lactic acidosis. A large number of inherited human diseases involve splicing errors caused by mutations within the natural splice sites or within exonic
enhancer sequences (8-10). In addition, mutations within introns that
generate cryptic splice sites have been shown to result in the aberrant
inclusion of intron sequences (11-15). Here we show a novel case in
human pathology of a mutation responsible for the generation of a
de novo splicing control element, most probably an enhancer
that favors the recognition of a cryptic 5'-splice site downstream of
the mutation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
46 to 616) and CD (from bases 528 to 1325). Because of an
abnormal CD PCR product, we performed a RT-PCR with primers PDS30-32,
which amplify only the exons 6-9 of the cDNA (281 bp).
cDNA--
Total cDNA
from the fibroblasts of the patient (as described in molecular
analysis) was submitted to a PCR with primers G and D to obtain the
full-length PDH E1
cDNA. The RT-PCR product was cloned into the
pGEM-T Easy vector (Promega). The presence of the insert was checked
after digestion with EcoRI. The positive clones were
analyzed by PCR with primers PDH30-32 (17) to differentiate the normal
from the mutated clones and sequenced as above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and E1
subunits with no evidence for any form with altered size (Fig.
1).
View larger version (58K):
[in a new window]
Fig. 1.
Western blotting of total fibroblasts with
the anti-PDH holoenzyme. Proteins (7 µg) from a control and from
the patient were separated on a 10% SDS-polyacrylamide gel under
reducing conditions, transferred to a nitrocellulose membrane, and
probed with anti-PDH holoenzyme.
Direct DNA sequencing was performed for all 11 exons including their
exon-intron boundaries of the E1 PDH gene with the result that no mutation was found.
RT-PCR was performed by using RNA obtained from fibroblasts and
lymphocytes (patient and control) for sequencing analysis. The product
GH (5' end) was normal (Fig.
2A); however, the analysis of
fragment CD (3' end) displayed a superposition of two sequences in both
directions between exons 7 and 8. This prompted us to perform a new
RT-PCR with primers PDH30-32, which amplified from a part of exon 6 until the middle of exon 9 (281 bp). These results showed the same
abnormal RT-PCR pattern in both tissues, an elongated band together
with the normal band in smaller amounts (Fig. 2B, lanes 1 and 2). The full-length PDH E1
cDNA was cloned from the patient sample for sequencing in pGEM-T
Easy. The presence of the insert was checked with EcoRI
digestion (data not shown). We distinguished the plasmids containing
the normal from the mutated cDNA by PCR with primers PDH30-32
(Fig. 2B, lanes 3-5). The mutated sequence was
found to have an insertion of 45 bases between exons 7 and 8. The
insertion corresponded to the genomic region of the intron 7, giving a
longer mRNA coding region for 15 new amino acids. This is an
in-frame insertion that generates a protein of 405 rather than 390 residues.
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Direct sequencing of the genomic DNA of the patient revealed that
intron 7 of the E1 PDH gene contained a hemizygous G to A
substitution at +26 (759 + 26G
A) (Fig.
3A). Furthermore, the mutation
was positioned within the 45-nucleotide insertion found in the aberrant
mRNA (Fig. 4). Direct sequencing of
the genomic DNA of the mother showed a heterozygous pattern (Fig.
3B). On screening for this mutation by PCR in genomic DNA
samples from 30 controls, all showed the wild type sequence.
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To clarify whether this intronic mutation was responsible for the
splicing error, we conducted an exon trapping experiment. COS-7 cells
were transiently transfected with a splicing reporter plasmid (pET)
containing the genomic sequence from the end of intron 6 to the
beginning of intron 8 with either the normal or the +26G>A intron 7. After harvesting RNA from the cells, RT-PCR was used to identify the
spliced mRNAs with primers 02 and 03 from the Exon Trap kit. These
results show that only the normal spliced product of 477 bp is produced
in the control sample, which contains the normal DNA sequence (Fig.
5, lane 1). However, in the
cells transfected with the mutant splicing reporter, both the normal
and abnormal spliced products (477 and 522 bp) were produced (Fig. 5,
lane 2). Moreover, the abnormally spliced product was
predominant.
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We next performed a search of intron 7 sequences (normal
and mutated) of the E1 PDH gene to identify binding
motifs of known splicing enhancer proteins. Binding motifs that predict
functional ESEs for four of the most well characterized SR proteins,
SF2/ASF, SC35, SRp40, and SRp55, were found in the 5' portion of
intron 7 for the control and patient
sequence (see Table I). Remarkably, this analysis identified one
new high affinity SC35 binding motif in the patient but not the control
sequence that overlaps with the point mutation at +26. This sequence is
shown in boldface in Table I, and the mutated nucleotide is underlined.
Note that the new SC35 binding motif also overlaps with an SF2/ASF
motif (also highlighted in boldface in the Table I). Thus, one or both of these newly generated splicing enhancer motifs in the patient DNA
may be responsible for the aberrant splicing phenotype because of its
close proximity to the cryptic 5' splice site at nucleotide 46 of
intron 7.
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DISCUSSION |
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PDH deficiency appears to be a frequent inborn error of energetic
metabolism in children with neurological disorders. The beginning of
the disorder appears during fetal development of the brain, and the
clinical symptoms vary considerably and range from early neonatal
lactic acidosis and with mental retardation or intermittent ataxia.
Many different mutations have been described in the coding regions of
the E1 PDH gene. Only one publication (5) with a new case
of the PDH defect described two exonic mutations leading to aberrant
splicing in the E1
PDH gene and excision of exon 6. In
many patients with typical PDH deficiencies as with our patient
described here, the E1
PDH gene is found normal without
any mutation in the exons or the exon-intron boundaries. In a few cases
of PDH deficiency, mutations have been identified in another gene, the
Hs-PDX1 gene, which encodes the E3-binding protein of the PDH complex (6). In all of these cases, the E3-binding
protein (also called protein X) was absent as determined by
immunoblotting. In this study, no elongated forms of the E1
subunit
were detected with the exception of a slight decrease of E1
and
E1
immunoreactivity compared with control samples. The abnormal
protein is probably unstable, and the amount of E1
protein
corresponds to the translation of the normal E1
cDNA detected by
RT-PCR. The gene was abnormally spliced in two tissues, fibroblasts and
lymphocytes. Together with the normal product, one aberrant product
appeared corresponding to a mRNA with partial intron 7 retention.
The unusual feature of this mutation is that the G to A change at
position 26 does not alter an authentic splice site nor does it
generate a cryptic splice site. Rather, the mutation activates a
cryptic 5'-splice site, which lies 20 nucleotides downstream of the
mutated site within the same intron. The score for the newly generated
SC35 binding motif (3.62) compares favorably with the highest possible
score for a SC35 binding motif so far reported in the literature
(3.95). The score is a measure of the match of each nucleotide position
to the consensus SC35 sequence motif.
It has been estimated that 15% of inherited human diseases involve
splicing errors because of mutations in splice sites or in splicing
control sequences (20). Most splicing mutations are distributed within
the coding exons or in the adjacent 5'- and 3'-splice sites, which
frequently lead to exon skipping. In human disease genes,
there are numerous mutations in ESE control sequences that have been
documented to cause aberrant exon skipping (10). Although ISEs have
also been shown to promote splice site recognition, these sequences do
not normally bind to SR splicing factors (21). Using ESE motif
prediction tools (18, 19), we found that this mutation in the patient
intron generates a new SC35 binding motif in the transcript, which is
slightly upstream from the cryptic site (Table I). An ASF/SF2 sequence
overlaps the SC35 binding motif. Whether SC35 and ASF/SF2 bind
simultaneously or competitively to the patient intron sequence is not
known. Moreover, when an "exonic enhancer" type of sequence
is introduced in an intronic sequence near a splice site, it normally
inhibits splicing (22). Thus, the new SC35 binding site could inhibit the wild type 5'-splicing site or enhance the cryptic 5'-splice site or
both. The SR factors, SC35 and ASF/SF2, have already been described as
having antagonistic effects on intronic enhancer-dependent splicing of the -tropomyosin alternative exon 6A (23).
The intronic mutation identified here is located within a small intron,
which may play a role in the partial intron 7 retention mechanism.
Although on average most vertebrate introns are much larger than their
flanking exons, small introns are not unusual. An examination of
splicing phenotypes of other human disease genes indicates that intron
inclusion can be observed in genes containing a splice site mutation
within a small human intron. In our case, the mutation (759 +26GA)
was found in a quadruplet of Gs in intron 7 and created an intronic
retention. Intron 7 is small (181 bp) and G-rich. Furthermore, the
mutation is found in an evolutionarily conserved region in many animal
species and yeast. Multiple G triplets and quadruplets are commonly
found in intronic sequences near 5'-splice sites, and in some contexts,
these sequences function as intronic enhancers (24, 25). Moreover, a
recent computational analysis revealed that G triplets are found in
most of the highest scoring pentamer sequences in small human introns
(2). We consider that GGGG from positions +24-27 of the intron 7 of
the E1
PDH gene may function normally as an enhancer of
the authentic 5'-splice site, and the G
A mutation in the patient
intron may consequently weaken the authentic site. However, this is not
a complete loss of recognition, because a small proportion of the
patient mRNAs uses the authentic 5'-splice site.
The protein levels of the SR splicing factors vary naturally over a very wide range, and our results support the notion already put forward by other groups (26) that changes in the ratio of these proteins can affect the spliceosome assembly and alternative splicing regulation of a variety of pre-mRNAs in vivo. Further studies will be necessary to confirm our hypothesis by performing in vitro splicing experiments (27).
In conclusion, this study demonstrates a causal relationship between an
inherited intronic mutation in the E1 PDH gene and the
activation of a downstream cryptic 5'-splice site with devastating consequences for human brain development.
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ACKNOWLEDGEMENTS |
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We are grateful to Douglas Black for helpful discussion. We thank Garry Brown for providing protocols for experimental procedures. We thank Association Retina France and Université René-Descartes.
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FOOTNOTES |
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* This work is supported in part by Contract QLG2-CT-1999-00660 from the European Union.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a grant from "Federation des Maladies Orphelines (AFRG)."
To whom correspondence should be addressed. Tel.:
0033-1-45-67-55-80; Fax: 0033-1-40-61-54-74; E-mail:
marsac@necker.fr.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M211106200
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ABBREVIATIONS |
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The abbreviations used are: ESE, exonic splicing enhancer; ISE, intronic splicing enhancer; PDH, pyruvate dehydrogenase; SR, specific serine/arginine-rich proteins; RT, reverse transcriptase.
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