Download Frame Shift Mutation, Exon Skipping, and a Two

Frame Shift Mutation, Exon Skipping, and a Two-Codon Deletion Caused by
Splice Site Mutations Account for Pyruvate Kinase Deficiency
By Hitoshi Kanno, Hisaichi Fujii, David C. Wei, L.C. Chan, Akira Hirono, Ichiro Tsukimoto, and Shiro Miwa
Three novel splice site mutations and two novel missense
mutations were identified by molecular analysis of pyruvate
kinase (PK) deficiency associated with hereditary nonspherocytic hemolytic anemia. A Nepalese PK variant, PK Kowloon, was found to have a homozygous transversion at the
5*-splice site of the seventh intervening sequence (IVS) of
the L-type PK gene (Ivs7["1]gt r tt). Using a reverse transcription polymerase chain reaction (RT-PCR) assay, we
showed that the R-type PK mRNA in the proband’s reticulocytes included the seventh IVS between the seventh and
eighth exon, introducing a stop codon 3 nucleotides downstream of the mutated site. Consequently, the translational
product may lack 44% of the R-PK polypeptide. A transition
at the last nucleotide of exon 9 (1269GCG r GCA) was found
in a Japanese PK variant, PK ‘Kamata.’ The mutation did not
alter the amino acid sequence, but caused skipping of the
ninth exonic sequence in the R-PK transcripts. As a result,
the affected R-type PK lost 51 amino acid residues (373Met423Ala del). A transversion at the splice acceptor site of the
third IVS (Ivs 3[-2]ag r tg) was identified in PK ‘Aomori.’ The
mutation resulted in aberrant splicing at a cryptic splice site
within exon 4, causing deletion of two codons in the aberrant R-PK transcript (95 Gly-96 Pro r del). Both PK ‘Kamata’
and PK ‘Aomori’ had a missense mutation on the other allele,
1044AAG r AAT (348Lys r Asn) and 1075CGC r TGC (359Arg
r Cys), respectively. Although both 348Lys and 359Arg were
located in the sixth loop of A domain (b/a)8 barrel, which
has been shown to contain the substrate and cation binding
sites, the degree of anemia was much more severe in PK
‘Kamata’ than PK ‘Aomori,’ possibly because the 51 amino
acid deletion of PK ‘Kamata’ but the 2 amino-acid deletion
of PK ‘Aomori’ may abolish PK catalytic activity.
q 1997 by The American Society of Hematology.
P
In this report, we describe five novel L-PK gene mutations,
including three splice site mutations, and discuss the aberrant
R-PK transcripts in the variants’ reticulocytes. It is worth
describing these mutations, as they have significantly severe
phenotypes possibly due to quantitative abnormalities of
erythrocyte PK. The mechanisms responsible for the distinct
consequences of these splice site mutations will be discussed.
YRUVATE KINASE (PK) is a key enzyme of anaerobic
glycolysis, which irreversibly catalyzes phosphoenolpyruvate (PEP) to pyruvate. The R (red blood cell)-type
PK (R-PK) is the only isoform that is expressed in mature
erythrocytes,1 and thus functional alterations of R-PK due
to mutations of the L (liver)-type PK (L-PK) gene, which
encodes both R- and L-PK2 would cause hemolysis. PK
deficiency is the second most common erythroenzymopathy
after glucose-6-phosphate dehydrogenase (G6PD) deficiency. This enzymopathy is associated with hereditary nonspherocytic hemolytic anemia, and over 400 cases have been
reported to date.3-5 Biochemical characterization of the variant PK has been standardized,6 and most PK variants have
been shown to have abnormal kinetics and/or decreased enzymatic stability.7 Recent molecular genetic studies of PK
deficiency have demonstrated that most PK variants are
caused by heterogeneous missense mutations occurring
mainly in the catalytically important domain.8,9 Amino acid
changes may alter either hydrophobicity or secondary structure of PK subunits, resulting in decreased affinity for the
substrate, PEP,10 as well as impaired responses to allosteric
effectors such as fructose-1, 6-diphosphate (FDP).11 X-ray
diffraction studies of the cat M1-type isozyme12 showed that
the enzyme is a homotetramer and that each subunit is comprised from four domains, namely N, A, B, and C. Data
concerning the allosteric transition of PK have been obtained
from structural analyses of Escherichia coli PK13 and mutagenesis of Bacillus stearothermophilus PK.14 To elucidate
the genotype-phenotype relationship of PK deficiency, we
analyzed the enzymatic characteristics and gene mutations
in homozygous PK deficiency15 and demonstrated that amino
acid substitutions occurred near either the catalytic center or
effector binding sites. Decreased affinity for PEP and/or FDP
may induce altered allosteric equilibrium, whereas impaired
affinity for adenosine diphosphate (ADP) seemed not to be
responsible for the deficiency because only one mutation
has been reported near the ADP binding site.16 Besides 59
missense mutations including our present data, miscellaneous genetic abnormalities including deletions, insertions,
nonsense and splice site mutations, have been reported.9,17
MATERIALS AND METHODS
Materials. Restriction endonucleases were purchased from Takara Shuzo (Kyoto, Japan) and New England Biolabs (Beverly, MA).
Taq DNA polymerase (AmpliTaq) was obtained from Perkin Elmer
Cetus (Norwalk, CT). Expand reverse transcriptase was purchased
from Boehringer (Mannheim, Germany). For the long polymerase
chain reaction (PCR) experiment, a commercial kit (LAPCR kit
version 2) from Takara Shuzo was used. Oligonucleotides were synthesized with a DNA synthesizer (Applied Biosystems, Model 380A,
Foster City, CA). DNA sequence data were obtained using an automated sequencer (Applied Biosystems, Model 373A). The computer
software DNASIS (Hitachi Software Engineering, Yokohama, Japan), was used to analyze DNA and amino acid profiles.
From the Okinaka Memorial Institute for Medical Research,
Tokyo; Department of Blood Transfusion Medicine, Tokyo Women’s
Medical College, Tokyo; the Department of Pediatrics, Toho University School of Medicine, Tokyo, Japan; and the Department of Pathology, University of Hong Kong, Hong Kong.
Submitted October 25, 1996; accepted January 22, 1997.
Supported, in part, by a Scientific Research Grant from the Ministry of Education, Science, Sports, and Culture, by the Mitsui Life
Social Welfare Foundation, Tokyo, and by a Research Grant for
Specific Diseases from the Ministry of Health and Welfare, Tokyo,
Japan.
Address reprint requests to Hitoshi Kanno, MD, PhD, Okinaka
Memorial Institute for Medical Research, 2-2-2 Toranomon, Minatoku, Tokyo 105, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/8911-0023$3.00/0
Blood, Vol 89, No 11 (June 1), 1997: pp 4213-4218
AID
Blood 0022
/
5H36$$$421
4213
05-01-97 18:09:20
blda
WBS: Blood
4214
KANNO ET AL
Clinical features of PK-deficient subjects. Laboratory data of
nonidentical twin Nepalese girls with PK deficiency (PK Kowloon)
have been reported elsewhere in detail.18 Intrauterine growth retardation of the twins was noted, and they received exchange blood transfusion during the neonatal period. Hemoglobin levels in the twins
at 3 months of age were 1.8 and 2.8 g/100 mL. One of the twins
had a recent splenectomy. Histological assessment shows marked
hemosiderosis with iron deposition in the hepatocytes. There are no
other abnormalities or evidence of cirrhosis.
A Japanese girl (MT, PK ‘Kamata’) received exchange blood
transfusion due to severe neonatal jaundice (total bilirubin, 36 mg/
100 mL), and was diagnosed as having congenital chronic hemolytic
anemia of unknown ethiology. As she required red blood cell (RBC)
transfusion once a month, splenectomy was performed at 8 years of
age, and since then anemia has been slightly improved with hemoglobin levels of 4-6 to 7-9 g/100 mL. The hematological data at 8
years of age were as follows: RBC count 2.66 1 106/mL, hemoglobin
8.7 g/100 mL, hematocrit 26.1%, and reticulocytes 30%. Serum
indirect bilirubin was 6.4 mg/100 mL.
In 1982, an 11-year-old Japanese girl (TF, PK ‘Aomori’) was
diagnosed as having congenital hemolytic anemia. The hematological data at 21 years of age were as follows: RBC count 3.36 1 106/
mL, hemoglobin 11.1 g/100 mL, hematocrit 32.6%, and reticulocytes
18.8%. Serum indirect bilirubin was 4.8 mg/100 mL.
Enzymatic and biochemical analyses of PK variants. Blood samples were obtained under informed consent from the probands. Because the Nepalese twins had recently been splenectomized, we
obtained liver specimens for isozyme and RNA analyses with their
parents’ informed consent. RBC enzyme activities and glycolytic
intermediates were measured by protocols standardized by the International Committee for Standardization in Hematology (ICSH)19 or
as described by Minakami et al.20
We separated the reticulocyte-enriched fraction from normochromic RBC by density gradient centrifugation. Heparinized whole
blood was passed through a cellulose column to remove white blood
cells (WBC) and platelets and then purified RBC were mixed with
19 volumes of density gradient mixture, 35% percoll, 20% urographin, as described.21 Ten-milliliter samples of the suspensions
were centrifuged at 35,000g and 47C for 20 minutes.
RBC PK was partially purified by precipitation with 280 g/L
ammonium sulfate, in which both R- and M2-type PK were precipitated. Protein extracts prepared from human liver were homogenized
in PK sample buffer containing 10 mmol/L Tris/HCl, pH7.5, 100
mmol/L KCl, 2 mmol/L 2-mercaptoethanol, 10 mmol/L e-aminocaproic acid, and 10 mmol/L EDTA. Polyclonal antibody against the
rat L-PK was kindly provided by Prof T. Noguchi, Fukui Medical
School (Fukui, Japan) and used to characterize the isozyme expression of the variant RBC and liver. Aliquots of 100 mg of protein
extracts prepared from RBCs and liver were separated on a 10%
sodium dodecyl sulfate (SDS)-polyacrylamide gel. Proteins were
transferred onto a nylon membrane (Hybond N, Amersham, UK) in
buffer containing 25 mmol/L Tris, 192 mmol/L glycine, pH 8.3, at
30 V, 47C, overnight in a mini trans-blot electrophoretic transfer cell
(BioRad, Hercules, CA). The membrane was incubated with antirat L-PK antibody, followed by chemiluminescence detection using
a Western-light Protein Detection Kit (Tropix, Bedford, MA).
Amplification and sequencing of flanking regions of the human LPK gene. High molecular weight genomic DNA was purified by
the standard protocol.22 Each exon of human L-PK gene was amplified by PCR using intronic primers and sequenced as described.15
Structural analysis of R-PK transcripts and genomic sequences.
Reticulocyte RNA was purified by the guanidinium isothiocyanate/
cesium chloride method.23 Aliquots of 2 mg of total reticulocyte
RNA were reverse transcribed in 20-mL reaction mixtures containing
11 Expand reverse transcriptase buffer, 10 mmol/L dithiothreitol, 1
AID
Blood 0022
/
5H36$$$421
Table 1. PCR Primers Used in This Study
To create appropriate restriction sites, the underlined nucleotide
were replaced.
R-1
5*-CTTAAAGGTGGGGCTTTGGA-3*
R-2
5*-CCCAGGCCTACACTGAAAGC-3*
R-3
5*-TGTGGGCTGGAGAACGTAGACT-3*
R-4
5*-ATCTCCCTAGTGGTCCAGAA-3*
R-5
5*-CACCATGATGCCGTCGCTCA-3*
R-6
5*-CCAGGAAAACCTTCTCTGCT-3*
R-7
5*-AGGACGTCCGAGACCTGCGCTT-3*
R-8
5*-TAGCTCCTCAAACAGCTGC-3*
R-9
5*-GCGGTGAAGATGCAGCCTGC-3*
R-10
5*-GGCGTTCTGAGAAATGGTAATGG-3*
R-11
5*-CTCGTGGGAGCCGTGGGAGAAG-3*
R-12
5*-AGGTGAGCGACGGCATCATGGT-3*
R-13
5*-CATCTTCTGAGCCAGGATCA-3*
R-14
5*-CTGTGTGGCACAGACAACAGGCTT-3*
EX-7
5*-CCTTTGTGCGGAAAGCCAGC-3*
IVS-7
5*-AGGTGATGGGGAATAGCGAC-3*
mmol/L deoxyribonucleoside triphosphates (dNTPs), 20 U of ribonuclease inhibitor, 50 pmol of R-1 primer, and 50 U of Expand
reverse transcriptase. Aliquots of 1/10 of the cDNA mixture were
then subjected to PCR amplification in 50-mL mixtures of 11
LAPCR buffer (included in the kit), 0.4 mmol/L dNTP with 10 pmol
each of primers R-2/R-3 and 2.5 U of LATaq DNA polymerase.
The reaction mixtures were subjected to 40 cycles of amplification
consisting of 947C for 20 seconds, 607C for 30 seconds, and 727C
for 210 seconds in GeneAmp PCR System 9600 (Perkin Elmer
Cetus). The amplified cDNA spanned 1,819 bp covering the entire
coding sequence and parts of the 5*- and 3*-untranslated regions.
The amplified product was subsequently subcloned into the pCR II
vector using a TA cloning kit (InVitrogen, San Diego, CA), and
sequenced using PK gene-specific primers (Table 1) and a Dye
termination cycle sequencing kit (Perkin Elmer).
To verify the nucleotide changes in the genomic DNA, aliquots
of 0.5 mg of genomic DNA were amplified by PCR in 50-mL mixtures of 10 mmol/L Tris/HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/
L MgCl2 , 0.001% gelatin, 0.2 mmol/L dNTP with 25 pmol of each
primer listed in Table 2, and 1.25 U of AmpliTaq DNA polymerase.
The reaction mixtures were subjected to 30 cycles of amplification
consisting of 947C for 20 seconds, 607C for 20 seconds, and 727C
for 20 seconds in a GeneAmp PCR System 9600. The amplified
DNA was digested with the restriction endonucleases listed in Table
2 to verify the nucleotide changes found in the cDNA.
Southern blot hybridization of the RT-PCR products. Aliquots
of reticulocyte cDNA were amplified in the same reaction solution
used for genomic DNA amplification described above. Primers R-4
and R-5 flanking exon 7 were used. The PCR products were separated on a 1.5% agarose gel and transferred onto a nylon membrane.
Hybridization was performed in 51 SSC (11 SSC is 0.15 mol/L
NaCl, 0.015 mol/L sodium citrate, pH 7.0), 1% blocking reagent
(supplied with DIG Nucleic Acid Detection Kit, Boehringer), 0.1%
N-lauroylsarkosine, 0.02% SDS at 607C. The final washing conditions were 657C in 0.11 SSC-0.1% SDS. Oligonucleotide probes,
EX-7 and IVS-7 were labeled at their 3* ends with digoxigenin (DIG
oligonucleotide 3*-end labeling kit) and used as probes. Positive
bands were detected by chemiluminescence (DIG nucleic acid detection kit; Boehringer Mannheim Biochemica, Mannheim, Germany).
RESULTS
Homozygous 5*-splice site mutation of IVS 7 identified in
PK Kowloon. RBC enzyme analysis of the PK Kowloon
05-01-97 18:09:20
blda
WBS: Blood
SPLICING MUTATIONS IN PK DEFICIENCY
4215
Table 2. Strategy for Detection of L-PK Gene Mutations
Ivs7[/1]gtrtt
1269GCGrGCA
Ivs 3[02]agrtg
1044AAGrAAT
1075CGCrTGC
Primers
PCR Products
(bp)
Restriction
Enzyme
R-6/R-7
R-8/R-9
R-10/R-11
R-12/R-13
R-12/R-14
351
170
179
83
134
Hph I
Pst I
Bfa I
Hph I
Hha I
family showed almost normal PK activity in both twins (twin
1, 13.5; twin 2, 11.1 IU/g Hb, normal range, 12.0 to 15.6),
whereas PK activity of the mother was decreased (9.03 IU/
g Hb). It seemed most likely that there was contamination
by normal RBC in the probands’ blood, because the twins
remained transfusion-dependent. To exclude PK activity derived from normal RBCs, we separated reticulocytes and
young RBCs by density gradient centrifugation using percoll/urographin21 and measured PK activity. PK activity of
the fractionated RBCs of twin 1 was decreased to below the
normal range, to about 75% of the normal mean value, 10.4
IU/g Hb.
Immunoblot analysis showed a decrease in amount of the
R-PK subunit in the reticulocyte fraction (Fig 1, lane 2), but
not in unfractionated samples (Fig 1, lane 3). The L-PK
subunit was not detected in the liver extract (lane 5).
Genomic DNA sequencing showed that the variant had a
transversion at /1 position of the 5*-splice site of intron 7
(Ivs 7[/1] gt r tt). As the mutation destroyed an Hph I
restriction site, the genomic DNAs of the family members,
as well as a normal control, were amplified by PCR using
primers R-6/R-7, and digested with Hph I. Both twins were
homozygous for this mutation, while the parents were heterozygotes (data not shown). To elucidate the effect of the
mutation on RNA splicing, we amplified the 1.7-kb R-PK
cDNA of PK Kowloon. Agarose gel electrophoresis demonstrated two RT-PCR products of different sizes. One of the
Fig 1. Immunoblot analysis of erythrocytes and liver extracts with
anti-L–PK. Labeled bands, which migrated at positions corresponding to about 62 (arrow designated as R) and 60 (arrow designated
as L) kD, were R-and L-PK, respectively. Lane 1, normal erythrocytes;
lane 2, low density fraction of erythrocytes of PK Kowloon; lane 3,
unfractionated erythrocytes of PK Kowloon; lane 4, normal liver; lane
5, liver of PK Kowloon.
AID
Blood 0022
/
5H36$$$421
Normal Allele
178
115
95
60
94
/
/
/
/
/
88 / 58 / 27
55
84
14 / 9
40
Mutant Allele
205 / 88 / 58
95 / 55 / 20
179
69 / 14
134
RT-PCR products had the normal R-PK cDNA sequence,
while the other cDNA with a slightly larger molecular weight
showed an insertion of 96 nucleotides, which was identical
to the seventh IVS between exons 7 and 8. Southern blot
hybridization of the RT-PCR products flanking exon 7 of
the L-PK gene showed an additional larger cDNA corresponding to IVS 7 in PK Kowloon, as well as a 336-bp
cDNA corresponding to the normally spliced transcript (Fig
2, lane 1). The oligonucleotide complementary to the IVS 7
(Fig 2, lanes 3 and 4) hybridized with the 432-bp cDNA in
PK Kowloon (lane 3), but not in the control (lane 4). These
observation led us to conclude that the 5*-splice site mutation
caused aberrant splicing of the primary transcript of the LPK gene, resulting in insertion of the IVS 7 in the mature
transcript. Although both the R-PK subunits and transcripts
were detected in reticulocytes of the patient, it is likely that
most of the molecules were derived from contaminating normal RBC and reticulocytes due to recurrent blood transfusion, because L-PK expression was hardly observed in the
patient’s liver as shown by immunoblotting (Fig 1). Translation of the aberrant mRNA terminates prematurely in the
IVS 7 (AGttga). The expected length of the truncated RPK subunit was 322 amino acid residues, meaning that the
Fig 2. Southern blot hybridization of RT-PCR products. Oligo-dT
primed reticulocyte RNA was used for PCR with primers R-4 and R5 flanking exon 7 of the R-PK cDNA. Normal R-PK cDNA is 336 bp
long, which includes 25, 271, and 40 bp cDNA sequences of exon 6,
7, and 8. If the 96 bases of the seventh intervening sequence are
retained, the aberrant cDNA becomes 432 bp long. Oligonucleotide
EX-7 hybridized with exon 7 (lane 1, PK Kowloon, lane 2; control), and
IVS-7 hybridized with intron 7 (lane 3, PK Kowloon, lane 4; control).
05-01-97 18:09:20
blda
WBS: Blood
4216
KANNO ET AL
polypeptide lost 44% of the R-PK. We were unable to detect
the short R-PK subunit immunologically.
1269A mutation identified in PK ‘Kamata’ is responsible
for the skipping of exon 9 sequence. RBC enzyme analysis
showed that PK activity of the proband was decreased to
44% of the normal mean value (6.0 IU/g Hb). The PK activity of the mother was also below the normal range (11.6
IU/g Hb). Glycolytic intermediates upstream of PK were
accumulated in the proband: 2,3-diphosphoglycerate (2,3DPG), 12320 (normal range, 3,438 to 5,218); 3-phosphoglycerate (3PG), 185.9 (25.6 to 55.2); 2-phosphoglycerate
(2PG), 41.9 (3.0 to 11.0); PEP 152.8 (8.9 to 19.7)(nmol/mL
RBC).
The reticulocyte R-PK cDNA of PK ‘Kamata’ amplified
with R-2/R-3 primers showed double bands of about 1.7 and
1.6 kb. Sequencing of a short cDNA indicated that the RPK cDNA lacked exon 9. The skipping of exon 9 caused a
deletion of 51 amino acids (373Met-423Ala del). These
amino acid residues included residues essential for the function of the enzyme, and thus the variant PK may have no
activity. To explore the molecular abnormality accounting
for the exon skipping, we analyzed cDNA and genomic DNA
sequences of PK ‘Kamata’ and found that the proband was
a compound heterozygote of 1269GCG r GCA (423Ala r
Ala)/1044AAG r AAT (348Lys r Asn, K348N). These
mutations were verified by PCR-restriction fragment length
polymorphism (RFLP) analysis using the primers and restriction endonucleases listed in Table 2. The 1269 G r A mutation does not alter the amino acid sequence, but is responsible for aberrant splicing at the 5*-splice site of IVS 9. 348
Lys is evolutionarily conserved from chicken through humans and is located in the sixth alpha helix of the A domain
(Aa6) of the PK subunit.12 Recently Mattevi et al13 elucidated the three-dimensional structure of the allosteric type
PK of E coli and demonstrated that 292Arg on the Aa6,
which corresponded to 337Arg of the human R-PK, participated in substrate binding in the allosterically active state.
K348N may cause decreased affinity for PEP due to conformational changes of the active site.
Novel splice acceptor site mutation identified in PK ‘Aomori.’ RBC enzyme assay of the proband showed that PK
activity was decreased to 49% of the normal mean value
(6.7 IU/g Hb). Glycolytic intermediates upstream of PK were
accumulated: 2,3-DPG, 7560; 3PG, 121.5; 2PG, 14.4; PEP
44.2 (nmol/mL RBC). Sequencing of the R-PK cDNA and
L-PK gene showed that the variant was a compound heterozygote of two novel point mutations; Ivs 3 [-2]ag r tg and
1075 CGC r TGC (359 Arg r Cys, R359C). The splice
acceptor site mutation induced cryptic splice site utilization
in the 5*-end sequence of exon 4 (ag r tgGGCCAG). Consequently, the R-PK transcript lacked the first six nucleotides
of exon 4, resulting in deletion of two amino acids in the
R-PK (95 Gly-96 Pro r del). R359C may cause decreased
affinty for PEP, because the substituted 359Arg was located
in the same alpha helix (Aa6)12 as 348 Lys.
DISCUSSION
Approximately 15% of point mutations accounting for
human genetic diseases cause aberrant mRNA splicing.24 In
AID
Blood 0022
/
5H36$$$421
RBC enzymopathies, splicing defects are known to be the
common abnormalities in phosphofructokinase (PFK) deficiency.25 Aberrant splicing may reduce or diminish mRNA
levels, as it can cause exon skipping, nucleotide insertion/
deletion or premature translational termination. In PK deficiency, seven splice site mutations have been identified to
date including those in the present cases.11,15,26,27,40 However,
none have been analyzed at the cDNA level. In this study,
we described three splicing defects of the human L-PK gene
associated with PK deficiency, and by analyzing reticulocyte
PK mRNA structure, we confirmed that each mutation resulted in a distinct molecular abnormality.
A Nepalese PK variant, PK Kowloon, is one of the most
severe PK deficiencies so far reported. The probands were
true homozygotes of the 5* splice site mutation at IVS 7.
Splice donor site mutations at /1 position have been reported to cause either skipping of preceded exons or utilization of cryptic splice sites.28,29 In PK Kowloon, however,
only the transcripts that retained seventh IVS were detected.
We searched for potential splice sites using statistical scoring
according to the theory of Shapiro and Senepathy.30 Using
the nucleotide sequence data around 5*-splice sites of 97
human genes,31 the 5*-splice site of the IVS 7 scored as 90.6,
and the adjacent 5* and 3* GT sequences counted as 57.0 or
45.0, respectively. Even with gt r tt mutation, the score was
73.4, which may still be sufficient for efficient splicing at
the mutated site and, therefore, cryptic sites were not used.
The skipping of exon 7 would not occur, possibly because
the length of exon 7, 271 bp, is much longer than that of
IVS 7, 96 bp. Thus, the splice donor site mutation may cause
retention of IVS 7.
Two PK isozymes, the L- and M2-type, expressed in normal liver,32 and those isozymes were encoded by distinct
structural genes.33 Although PK Kowloon had virtually no
L-PK in liver, the M2-PK activity had been detected by
polyacrylamide gel electrophoresis (data not shown). Biochemical data of the probands showed the increments of
serum transaminases, alkaline phosphatase, as well as hyperbilirubinemia. We believed that these hepatic abnormalities
were likely due to hemosiderosis, but not to the PK deficiency in the liver.
Several examples have been reported of nucleotide substitutions occurring at the last base of exons being a cause
of aberrant splicing.34,35 Ohshima and Gotoh31 have shown
statistically that 80% of the last nucleotides of 97 donor sites
of human genes were guanine. Although both G r A and G r
C27 at 1,269 of the L-PK gene caused small decreases in the
splicing score, the skipping of exon 9 may have occurred
due to the relatively short (94 bp) intronic length.
Mutations of the invariant ag (02) at 3*-splice sites have
been reported to be responsible for usage of cryptic sites36
or exon skipping.37 The splice acceptor site mutation at IVS
3 of PK ‘Aomori’ caused cryptic utilization of the exonic
sequence within exon 4, resulting in a two-amino acid deletion near the amino-terminal region of the affected PK subunit. Because amino acid substitutions have not been identified in this region, with only one exception,16 we speculate
that the variant PK with the two amino-acid deletion might
show catalytic activity; a similar situation was reported in a
05-01-97 18:09:20
blda
WBS: Blood
SPLICING MUTATIONS IN PK DEFICIENCY
4217
molecular study of G6PD deficiency. An 8-amino acid deletion was identified in a Japanese G6PD variant, G6PD
Nara.38 The variant enzyme had residual activity even with
the deletion, and Rawland39 recently showed that the region
where the amino acid deletion occurred was not essential
for G6PD enzymatic activity by analyzing the tertiary structure of the Leuconostoc homolog. This speculation was supported by the observation that accumulation of glycolytic
intermediates in erythrocytes, as well as the degree of anemia
of PK ‘Aomori,’ seemed to be much milder than those in
PK ‘Kamata,’ although the missense mutations at the other
alleles were supposed to have similar effects on the catalytic
activity.
The present study has unveiled the molecular lesions of
severe PK deficiency with marked structural alterations of
the affected PK subunit. The consequences of the mutations
were demonstrated by analyzing reticulocyte mRNA structure. Reticulocyte RNA seems a useful source of experimental material for analyzing molecular defects of severe hemolytic anemia due to PK deficiency.
NOTE ADDED IN PROOF
40
Bianchi et al reported a splice site mutation recently.
ACKNOWLEDGMENT
We thank Y. Okamura, A. Sakuma, M. Watanabe, and J. Oka for
their technical assistance. We are also indebted to Dr J.C. Wilson
for managing the patient and providing the blood sample.
REFERENCES
1. Imamura K, Tanaka T, Nishina T, Nakashima K, Miwa S:
Studies on pyruvate kinase (PK) deficiency. II. Electrophoretic, kinetic, and immunological studies on pyruvate kinase of erythrocytes
and other tissues. J Biochem (Tokyo) 74:1165, 1973
2. Noguchi T, Yamada K, Inoue H, Matsuda T, Tanaka T: The
L- and R-type isozymes of rat pyruvate kinase are produced from a
single gene by use of different promoters. J Biol Chem 262:14366,
1987
3. Valentine WN, Tanaka KR, Paglia DE: Pyruvate kinase and
other enzyme deficiency disorders of the erythrocyte, in Scriver CR,
Beaudet AL, Sly WS, Valle D (eds): The Metabolic Basis of Inherited Disease (ed 6). New York, NY, McGraw-Hill, 1989, p 2341
4. Tanaka KR, Paglia DE: Pyruvate kinase and other enzymopathies of the erythrocyte, in Scriver CR, Beaudet AL, Sly WS, Valle
D (eds): The Metabolic and Molecular Bases of Inherited Disease
(ed 7). New York, NY, McGraw-Hill, 1995, p 3485
5. Miwa S, Kanno H, Fujii H: Pyruvate kinase deficiency. Historical perspectives and molecular genetics. Am J Hematol 42:31, 1993
6. International Comittee for Standardization of Haematology:
Recommended methods for the characterization of red cell pyruvate
kinase variants. Br J Haematol 43:275, 1979
7. Miwa S, Fujii H, Takegawa S, Nakatsuji T, Yamato K, Ishida
Y, Ninomiya N: Seven pyruvate kinase variants characterized by
the ISCH recommended methods. Br J Haematol 45:575, 1980
8. Beutler E, Baronciani L: Mutations in pyruvate kinase. Hum
Mutat 7:1, 1996
9. Miwa S, Fujii H: Molecular basis of erythroenzymopathies
associated with hereditary hemolytic anemia: Tabulation of mutant
enzymes. Am J Hematol 51:122, 1996
10. Kanno H, Fujii H, Miwa S: Low substrate affinity of pyruvate
kinase variant (PK Sapporo) due to a single amino acid substitution
AID
Blood 0022
/
5H36$$$421
(426Arg r Gln) associated with hereditary hemolytic anemia. Blood
81:2439, 1993
11. Kanno H, Ballas SK, Miwa S, Fujii H, Bowman HS: Molecular abnormality of erythrocyte pyruvate kinase deficiency in the Amish. Blood 83:2311, 1994
12. Muirhead H, Clayden DA, Barford D, Lorimer CG, Fothergill-Gilmore LA, Schlitz E, Schmitt W: The structure of cat muscle
pyruvate kinase. EMBO J 5:475, 1986
13. Mattevi A, Valentini G, Rizzi M, Speranza ML, Bolognesi
M, Coda A: Crystal structure of Escherichia coli pyruvate kinase
type I: Molecular basis of the allosteric transition. Structure 3:729,
1995
14. Walker D, Chia WN, Muirhead H: Key residues in the allosteric transition of Bacillus stearothermophilus pyruvate kinase identified by site-directed mutagenesis. J Mol Biol 228:265, 1992
15. Kanno H, Wei DC, Chan LC, Mizoguchi H, Ando M, Nakahata T, Narisawa K, Fujii H, Miwa S: Hereditary hemolytic anemia
caused by diverse point mutations of pyruvate kinase gene found in
Japan and Hong Kong. Blood 84:3505, 1994
16. Uenaka R, Nakajima H, Noguchi T, Imamura K, Hamaguchi
T, Tomita K, Yamada K, Kuwajima M, Kono N, Tanaka T, Matsuzawa Y: Compound heterozygous mutations affecting both hepatic
and erythrocyte isozymes of pyruvate kinase. Biochem Biophys Res
Commun 208:991, 1995
17. Baronciani L, Bianchi P, Zanella A: Hematologically important mutations: Red cell pyruvate kinase. Blood Cells Mol Dis
22:85, 1996
18. Wei DC, Chan LC, Li A: Homozygous pyruvate kinase deficiency in Hong Kong ethnic minorities. J Paediatr Child Health
28:334, 1992
19. Beutler E, Blume KG, Kaplan JC, Löhr GW, Ramot B, Valentine WN: International Committee for Standardization in Haematology: Recommended methods for red cell enzyme analysis. Br J
Haematol 35:331, 1977
20. Minakami S, Suzuki C, Saito T, Yoshikawa H: Studies on
erythrocyte glycolysis. I. Determinations of the glycolytic intermediates in human erythrocytes. J Biochem (Tokyo) 58:543, 1965
21. Rijksen G, Veerman AJP, Schipper-Kester GPM, Staal GEJ:
Diagnosis of pyruvate kinase deficiency in a transfusion-dependent
patient with severe hemolytic anemia. Am J Hematol 35:187, 1990
22. Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: A
Laboratory Manual (ed 2). Cold Spring Harbor, NY, Cold Spring
Harbor Laboratory Press, 1989
23. Chirgwin JJ, Przbyla AE, MacDonald RJ, Rutter WJ: Isolation
of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294, 1979
24. Krawczak M, Reiss J, Cooper DN: The mutational spectrum
of single base-pair substitutions in mRNA splice junctions of human
genes: Causes and consequences. Hum Genet 90:41, 1992
25. Raben N, Sherman JB: Mutations in muscle phosphofructokinase gene. Hum Mutat 6:1, 1995
26. Lenzner C, Nürnberg P, Thiele B-J, Reis A, Brabec V, Sakalova A, Jacobasch G: Mutations in the pyruvate kinase L gene in
patients with hemolytic anemia. Blood 83:2817, 1994
27. Bianchi P, Terragna C, Zappa M, Alfinito F, Zanella A: Molecular characterization of L-PK gene in pyruvate kinase (PK) deficient Italian patients. Blood 84:14a, 1994 (abstr, suppl 1)
28. Sakuraba H, Eng CM, Desnick RJ, Bishop DF: Invariant exon
skipping in the human alpha-galactosidase A pre-mRNA: ag / 1 to
t substitution in a 5*-splice site causing Fabry disease. Genomics
12:643, 1992
29. Formstone CJ, Wacey AI, Berg LP, Rahman S, Bevan D,
Rowley M, Voke J, Bernardi F, Legnani C, Simioni P: Detection
and characterization of seven novel protein S (PROS) gene lesions:
05-01-97 18:09:20
blda
WBS: Blood
4218
KANNO ET AL
Evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy. Blood 86:2632, 1995
30. Shapiro MB, Senepathy P: RNA splice junctions of different
classes of eukaryotes: Sequence statics and functional implications
in gene expression. Nucleic Acids Res 15:7155, 1987
31. Ohshima Y, Gotoh Y: Signals for the selection of a splice
site in pre-mRNA: Computer analysis of splice junction sequences
and like sequences. J Mol Biol 195:247, 1987
32. Imamura K, Tanaka T: Multimolecular forms of pyruvate
kinase from rat and other mammalian tissues. J Biochem (Tokyo)
71:1043, 1972
33. Noguchi T, Inoue H, Tanaka T: The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by
alternative RNA splicing. J Biol Chem 261:13807, 1986
34. Akli S, Chelly J, Mezard C, Gandy S, Kahn A, Poenaru L:
A ‘‘G’’ to ‘‘A’’ mutation at position -1 of 5* splice site in a late
infantile form of Tay-Sachs disease. J Biol Chem 265:7324, 1990
35. Lind B, van SW, Schwartz M, Thorsen S: Splice site mutation
in the human protein C gene associated with venous thrombosis:
AID
Blood 0022
/
5H36$$$421
Demonstration of exon skipping by ectopic transcript analysis. Blood
82:2423, 1993
36. Carstens RP, Fenton WA, Rosenberg LR: Identification of
RNA splicing errors resulting in human ornithine transcarbamylase
deficiency. Am J Hum Genet 48:1105, 1991
37. David D, Tavares A, Lavinha J: Characterization of a splicing
mutation in the factor VIII gene at the RNA level. Hum Genet
95:109, 1995
38. Hirono A, Fujii H, Shima M, Miwa S: G6PD Nara: A new
class I glucose-6-phosphate dehydrogenase variant with an eight
amino acid deletion. Blood 82:3250, 1993
39. Rawland P: The 3-dimensional structure of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides refined at
2.0Å. Structure 2:1073, 1994
40. Bianchi P, Baronciani L, Bredi E, Zappa M, Vercellati C,
Alfinito F, Barraco F, Zanella A: Molecular study of erythrocyte
pyruvate kinase deficiency in Italy. Blood 88:4b, 1996 (abstr,
suppl 1)
05-01-97 18:09:20
blda
WBS: Blood