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Identical Point Mutations of the R-type Pyruvate Kinase (PK) cDNA Found in
Unrelated PK Variants Associated With Hereditary Hemolytic Anemia
By Hitoshi Kanno, Hisaichi Fujii, Akira Hirono, Mitsuhiro Omine, and Shiro Miwa
We cloned and sequenced the cDNAs for R-type pyruvate
kinase (R-PK) from three homozygous PK patients. A point
mutation (““ACG to AIG) identified in the R-PK cDNA of PK
Nagasaki was identical with that previously identified in an
unrelated family, PK Tokyo. The mutation has also been
identified in the PK L gene of a Lebanese family, PK Beirut, by
Neubauer et al (Blood 77:1871,1991). These results suggest
either that the point mutation is very old or that the same
mutation occurs sporadically in the same hot spot in unrelated families. A point mutation, ‘ V A G to AAG, was detected in both PK Fukushima and PK Maebashi3his mutation
causes a single amino acid substitution, from Gin'" to Lys, in
R-PK. Consequently, the hydrophobicity properties near the
mutated site are drastically changed. A silent mutation
(”%AGG to CGG) was also identified in those variants. The
screening of these three point mutations showed only the
silent mutation in a normal individualas well as in individuals
with PK variants. These results indicate that the multiplicity
of the mutant PK allele is smaller than expected, and that the
silent mutation is a polymorphic change commonly distributed in the Japanese population.
o 1992by The American Society of Hematology.
P
CAAGGGTAGGCTGGGCCAGAG-3’)were used for amplifying
the exon 12 region. 1261C (5’-GTGAAGATGCAGCATGC-3’)
and 1261A (5’-GTGAAGATGAAGCATGC-3’)
were allelespecific oligonucleotides (ASOs) which detected a single nucleotide change at no. 1261of R-PK cDNA.
cDNA synthesis, ampli@ation, and sequencing of R-type PK
mRNA. Purification of reticulocyte RNA from patients, PK
Nagasaki, PK Fukushima, and PK Maebashi, cDNA synthesis, and
amplification of the R-PK cDNA were performed as described
previously! Briefly, the R-PK cDNA were amplified with four sets
of primers, and subcloned into pBluescript (Stratagene, La Jolla,
CA). Whole coding and 55 bp 3’ noncoding sequences were
sequenced by the dideoxy method.
A S 0 hybridization for detecting point mutations in genomic DNA
from other PK variants. To detect the mutations identified in PK
Nagasaki, PK Fukushima, and PK Maebashi in the PK L gene of
other PK variants, we used A S 0 hybridization technique. Because
both ’”‘ACG to ATG and Iz1CAG to &4G mutations are located
in exon 9 of human L-PK gene (our unpublished results, January
1990), we amplified a 330-bp genomic DNA fragment that flanked
whole exon 9 sequences using primers L10 and L12 under conditions previously described.’ To detect the ‘”‘C to T mutation, either
ASOWT or ASOPK primers’ were used, and 1261C and 1261A
were used for lZ6’Cto A mutation. Hybridization was performed in
5X SSPE (1X SSPE is 0.18 mol/L NaCl, 10 mmol/L phosphate pH
7.4, 1 mmol/L EDTA), 5X Denhardt’s solution, 0.1% sodium
dodecyl sulfate (SDS), 100 p,g/mL denatured salmon sperm DNA,
at 57°C for ASOWT, 54°C for ASOPK, 55°C for 1261C, and 52°C
for 1261A. The final washing conditions were 60°C for ASOWT,
57°C for ASOPK, 58°C for 1261C, and 56°C for 1261A in 6X SSC
YRUVATE KINASE (PK) is a key glycolytic enzyme
and has four isozymes in mammals.’.‘ The type-R
(R-PK) is specifically expressed in erythrocytes, and in
other cells of hematopoietic lineage, type-M2 (M,-PK) is
expressed. Congenital nonspherocytic hemolytic anemia
(CNSHA) due to a deficiency of PK activity was first
reported in 1961,’ and it is now recognized as the most
common cause among glycolytic enzyme defects.4~~
Over 300
cases of PK deficiency have been reported, and most PK
variants are considered to be a result of a structural change
in the PK L gene, which encodes both type L (L-PK) and
R.6 Recently, we cloned the cDNA for human R-PK, and
identified a single amino acid substitution (Th1384to Met) in
the R-PK of the homozygous patients, PK T ~ k y o .This
~,~
substitution caused PK instability, directly demonstrated by
expression of the recombinant PK with the point mutation:
More recently, Neubauer et al’ reported point mutations
found in the human L-PK gene of Turkish and Lebanese
PK-deficient individuals. In this report, we describe two
point mutations found in unrelated PK-deficient families
associated with CNSHA and a polymorphic nucleotide
change, which is commonly distributed in the Japanese
population.
MATERIALS AND METHODS
Patients. Case reports, laboratory data, and the enzymatic
properties of PK Nagasaki, PK Fukushima, and PK Maebashi have
been reported
Because of the consanguineous marriage of the parents, these patients are considered to be homozygous for the mutant PK alleles. Clinical histories and kinetic
studies of compound heterozygotes of the PK variant, PK Osaka,
From the Okinaka Memorial Institute for Medical Research, Tokyo;
PK Beppu, PK Niigata, and PK Nichinan have been des~ribed.l~-’~ the Department of Blood Transfusion Medicine, Tokyo Women’s
Materials. Restriction endonucleases and modifying enzymes
Medical College, and the Department of Medicine, Showa University
were purchased from Takara Shuzo (Japan) and New England
Fujigaoka Hospital, Tokyo, Japan.
Biolabs (Beverly, MA). Taq DNA polymerase (AmpliTaq) was
Submitted August 30, 1991;accepted October 23, 1991.
obtained from Cetus (Norwalk, CT). Modifying T7 DNA polySupported in part by a Scientific Research Grant from the Ministry
merase (Sequenase; United Biochemicals, Cleveland, OH) was
of Education, Science, and Culture, and by a Research Grant for
used for DNA sequencing. a ”P dCTP and y 32PATP were purSpecGc Diseases from the Ministry of Health and Welfare, Japan.
chased from Amersham (UK). The computer software, DNASIS
Address reprint requests to Hitoshi Kanno, MD, Okinaka Memorial
(Hitachi Software Engineering, Japan), was used for analysis of the
Institute for Medical Research, 2-2-2 Toranomon, Minato-ku, Tokyo
hydrophobicity properties of the variant protein based on the
105, Japan.
theory of Hopp and Woods.I6
The publication costs of this article were defrayed in part by page
Oligonucleotides. Oligonucleotides used for cDNA cloning have
charge payment. This article must therefore be hereby marked
been described previously.’ L10 (5’-GTCCTACAACTTTGA“advertisement” in accordance with 18 U.S.C.section I734 solely to
CATCC-3’) and L12 (5-TAGCTCCTCAAACAGCTGC-3‘)were
indicate this fact.
used for amplifying the exon 9-10 region of the PK L gene. L21
0 1992 by The American Society of Hematology.
(5’-GAAAGCrCCGTGGCCCTCCG-3’) and L22 (5’-GTA0006-49711921 7905-OO26$3.00/0
Blood, Vol79, No 5 (March 1). 1992: pp 1347-1350
1347
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KANNO ET AL
1348
(1XSSC is 0.15 mol/L NaCI, 0.015 mol/L sodium citrate, pH 7.0).
0.1% SDS.
Detection of the "2 to C silent mutation in the Japanese
population. To test whether the silent mutation, '""A to C, is
present in other PK variants and normal subjects, we amplified 0.5
+g of the genomic DNA from a normal subject and PK variants by
polymerase chain reaction (PCR) with primers L21 and L22 under
the conditions previously described." One fifth of the products were
digested with BspHl (NEB) according to the manufacturer's
recommended conditions and electrophoresed in a composite gel
of 3% NuSieve agarose (FMC Bioproducts, Rockland, ME) and
1% HGT agarose (FMC Bioproducts).
RESULTS
-2
Identijication of the same mutation as PK Tokyo in the
R-PK cDNA of PK Nagasaki. Sequencingof the R-type PK
cDNA from PK Nagasaki showed that the variant had the
same point mutation as PK Tokyo, ""ASG to ATG. This
mutation causes a single amino acid substitution at Thru" to
Met, which causes thermal instability of the PK.8 This point
mutation was also identical with that in the PK L gene of a
Lebanese PK-deficient patient, PK Beirut, reported by
Neubauer et a1.9
Single amino acid substitution, Gln"' to Lys, caused by a
point mutation, l2''C to A, identijied in the R-type PK cDNA in
both PK Fukushima and PK Maebashi. Sequencing of the
R-PK cDNA from PK Fukushima demonstrated a point
mutation, '&'CAG to AAG (Fig l), which caused a single
amino acid s u b s t i t ~ t i o ~ G 1to
n ~Lys.
~ ' Although the site was
not located in the active site, which has been proposed from
the tertiary structure deduced from cat muscle PK,I7 this
substitution causes a diverse change of the net charge in the
eighth a helix of the A domain of PK.I7The hydrophobicity
properties of the R-type PK near the mutated site are
drastically changed by this substitution (Fig 2). The R-PK
cDNA of PK Maebashi also had the same point mutation.
3'
3'
Control
G A T C
G A T C
G
Ala
c
G
T
G
T
His A
C
421
Glnl A
A
C
4
LYS
(0
G
Met T
G
T
A
G
LYs A
G
A
Val T
G
5'
21[
A
\i
5'
Fig 1. Identificationof a point mutation in R-type PK cDNA of PK
Fukushima. The arrow indicates poskions of the nucleotide change in
the sequence ladder of normal or PK FukushimacDNA.
410
420
430
No.of R-type PK aminoadd residue
Fig 2. Hydrophobicity profiles near the mutated ske in the R-type
PK of PK Fukushima. The solid line indicates the hydrophobic properties of normal R-PK from no. 410 to no. 430 amino acid residue.' The
arrow shows the position of no. 421 amino acid residue that is
substituted from Gln to Lys. The dotted line indicates the altered
hydrophobic propertiesof PK Fukushima.
A silent point mutation, "%CC to CCC, was detected in
the R-type PK cDNA of PK Fukushima and PK Maebashi. A
point mutation, "'AGG to CGG, was also detected in the
R-type PKcDNA ofboth PK-Fukushima and PK Maebashi.
This mutation does not change an amino acid residue (Ser).
We evaluated the existence of this nucleotide change in the
genomic DNA from normal subjects and PK variants by
amplifying exon 12 of the PK L gene, and by BspHI
digestion of the products. Because the point mutation
altered the recognition sequence of this restriction endonuclease, from TCGCGA to TCGCGC, the mutation was
detected by the disappearance of the BspHI cutting site in
the PCR products. Figure 3 shows the agarose electrophoresis of the BspHI digest of PCR samples. This mutation was
found in the PK L gene of both PK-deficient patients and a
normal individual, and 8 PK alleles of 18 have this mutation. Both PK Tokyo and PK Nagasaki, the variants with
""C to T mutation, are homozygous for adenine at no.
1705, and in contrast, both PK Fukushima and PK Maebashi, those with Iz6'Cto A mutation, are homozygous for
cytosine at the polymorphic base. Among four other compound heterozygous PK variants, PK Nichinan and PK
Beppu are homozygous for ImA, and the others, PK Niigata
and PK Osaka, are heterozygous for the nucleotide at 1705.
A S 0 hybridizationfor screening the point mutations in other
Japanese PK families. Because both lls'C to T and IZ6'Cto
A mutation were identified in unrelated families, we
screened these mutations in other PKvariants, who showed
slow-electrophoreticmobility or thermal instability in their
enzymatic properties.'.'.'' All four PK variants examined are
considered to be compound heterozygotes. AS0 hybridization demonstrated that PK Nagasaki was homozygous for
the point mutation as well as PK Tokyo (Fig 4), and that
both PK Fukushima and PK Maebashi were homozygous
for the 1261C
to A mutation. Among other PK variants
tested, none had these point mutations.
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1349
POINT MUTATIONS IN PYRUVATE KINASE DEFICIENCY
M
Fig 3. Ethidium bromide staining of the agarose gel in which
the BspHI-digested PCR products of exon 12 from the normal
subject and the PK variants were
electrophoresed. The PCR products are resistant t o the BspHl
digest, when the "A t o C polymorphism is present. IftheBspHl
site remains, the 144-bp products are digested into two fragments, 84 and 60 bp. M, molecular standard, pBR322 digested
Mspl; N, normal control; 1, PK
Fukushima; 2, PK Maebashi; 3,
PK Tokyo; 4, PK Nagasaki; 5, PK
Niigata; 6, PK Nichinan; 7, PK
Osaka; 8, PK Beppu.
N
1
2
5
6
7
8
f- 144
*.mi-
DISCUSSION
1
4
bP
We report here two point mutations in three homozygous
PK variants, and a polymorphic nucleotide change found in
the Japanese population. Thc PKvariants that wcre considered unique have proven to be identical from our present
results; the point mutation found in PK Nagasaki is idcntical with those previously detected in another Japanese
family with PK variant, PK Tokyo'." and a Lcbancsc PK
variant, PK Beirut, reported by Neubauer et al." When the
biochemical characters of these three variants wcre compared,U." the parameters such as thermal stability, electrophoretic mobility, optimum pH, ATP inhibition, and FDP
(Fructose-l,6-diphosphate)activation were almost idcntical; however, thcre were some discrepancies in nucleotide
specificity between the three variants and in the Michaelis
constant for ADP and phosphoenolpyruvate between our
N
3
2
3
4
5
data" and that of Neubauer et al." These discrepancies may
be caused by the mutant enzymes being unstable during
biochemical characterization. Similar discrepancies have
been described in the biochemical and genetic analyses of
the glucose-6-phosphate dchydrogenase deficiency, one of
the major genetic defects which causes CNSHA.''
The fact that thc mutation has been found in three
unrelated families of different races means either that this
mutation occurred long before racial divergence between
Lebanesc and Japanese, or that it occurred sporadically in
the same hot spot in the human L-PK gene in unrelated
families. We are inclined to favor the former explanation,
bccause PK Tokyo and PK Nagasaki seem to be derived
from a common evolutional origin judging from the fact
that both of them are homozygous for the adenine at no.
1705 of the R-PK cDNA, which are polymorphic nucle-
6
7
a
A
B
C
D
e
Fig 4. Dot-blot hybridization of
PCR-amplifiedgenomic DNAflanking
the exon 9-10 region of the PK L gene
from normal subject and the PK variants. N, normal control; 1, PK Fukushima:. 2.. PK Maebashi;. 3.. PK Tokvo:
. 4. PK Nagasaki; 5, PK Niigata; 6, PK
Nichinan; 7, PK Osaka; 8, PK Beppu.
AS0 probes used in the hybridization were: A, ASO-WT; B, ASO-PK;
C, 1261C; D, 1261A.
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1350
KANNO ET AL
otides in the Japanese population. A similar speculation
can be applied to individuals with PK Fukushima and PK
Maebashi, who are homozygous for cytosine at no. 1705.
These results indicate that these mutations occurred in a
common ancestor. Further information about DNA polymorphisms of the PK gene will provide insights into the
origin of these PKvariants.
The point mutation found in PK Fukushima causes a
single amino acid substitution, Gln4” to Lys. The hydrophobicity properties are drastically changed by this substitution
(Fig 2). As a result, stability of the patient’s PK is severely
impaired. It should be noted that both the mutation
identified in PK Tokyo, ‘‘”C to T, and this mutation reside
in exon 9 of the human PK L gene. This observation
suggests that the exon 9-encoded region may be functionally important for PK activity. The relationship between
structure and function of PK will be further clarified by
structural studies on PKvariants.
ACKNOWLEDGMENT
We are indebted to Drs M. Sanada, H. Uno, T. Hosokawa, M.
Mizutori, M. Karasawa, and G. Tsujino for providing the blood
samples of PK-deficient patients. We thank K. Takizawa, Y.
Okamura, and A. Kawaguchi for their technical assistance.
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1992 79: 1347-1350
Identical point mutations of the R-type pyruvate kinase (PK) cDNA
found in unrelated PK variants associated with hereditary hemolytic
anemia
H Kanno, H Fujii, A Hirono, M Omine and S Miwa
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