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Molecular Abnormality of Erythrocyte Pyruvate Kinase Deficiency
in the Amish
By Hitoshi Kanno, Sarnir K. Ballas, Shiro Miwa, Hisaichi Fujii, and Herbert S.Bowman
We describe the cellular and molecular biologic studies of
the erythrocyte pyruvate kinase
(PK) deficiency of
the Amish
deme in Pennsylvania. Nucleotide sequencing of the patient's PK gene showed a point mutation,
CGC to CAC, corresponding to no. 1436 from the translationafinitiatkn site of
the R-type PK (R-PK) mRNA, and it caused a single amino
acid substitution from Arg to His at the 479th amino acid
residue of the R-PK. The substitutedArg residue is located
in the C domainof PK subunit, that is essentialfor both the
intersubunit contact and the allosteric regulation. Because
this enzyme showsthe catalytic activity onlyas a dimer or
tetramer, it is rational that the structural alteration would
result in severePK deficiency. To elucidatethe effect of the
PK deficiency on red blood cell (RBC) membrane, we performed the cellular studies of the patients' RBCs. Ouabaininsensitive K+ efflux was increased
to 142% to 145% of normal controls andnot inhibaed byfurosemide, as previously
observed in HbSC disease RBCs.
0 1994 by The Americen Sochty of Hematology.
P
family (Fig 1).STY,Sr, is a distant consanguineous relativeof SJY
and AB who are believed to be the ancestral pair oftheMifflin
CountyPennsylvaniaAmish
red blood cell (RBC)PK-deficient
gene." Family history showedJY to be one of 10 Old Order Amish
siblings of the STY, Sr, pedigree. Two sisters hadbeenaffected
with the Amish RBC PK-deficient hemolysis, one of whom died. A
subsequent sister born later, AY, likewise was found to have this
RBC disorder.
JY was initially seen at age 1 month, having been icteric at 48
hours of life with deepeningjaundice during the subsequent5 days.
As the icterus gradually faded, he evidenced increasing pallor and
was referred to the Hematology Center, Harrisburg Hospital.He was
transfused with packed RBCs sufficient to maintain the hematocrit
at 26%, and such transfusions were maintained every eighth week.
Thepretransfusionhematologic values were:RBCs,1.56 X lo6/
pL, hemoglobin (Hb), 5.1 g/dL, hematocrit (Hct), 18.5%, mean cell
volume (MCV), 112 fL, reticulocytes, 15.4%. Blood film showed
58 nucleated RBCsIlOO white blood cells (WBCs), macrocytosis,
polychromia, and marked anisocytosis. Total bilirubin was 2.6 mg/
dL, andlactate dehydrogenase (LDH) 1.250U.RBC PK activity was
0.1 U/lO'" RBCs. At 6 months of age, his spleen became palpable5
cm below the left costal margin. Transfusion therapy was continued
until 17 months of age, when splenectomy was performed. Splenic
red pulp preparations showed reticuloendothelial cell, RBCs,and
reticulocyte phagocytosis and increased reticulocytes." Subsequent
examinations have shown normal findings and no transfusion have
beenrequired.Currentexaminationsatage
31 yearshaveshown
YRUVATE KINASE (PK) deficiency is the most common hereditary nonspherocytic hemolytic anemia
caused by a glycolytic enzyme defect. The residual erythrocyte PK activity is not usually related tothe severity of
anemia, whereas the enzymatic characteristics such as a decreased substrate affinity, thermal instability, or impaired
response to the allosteric activator correspond well to the
phenotype.' We have analyzed the R-PK cDNA of six Japanese PK-deficient families, including five true homozygotes
andacompoundheterozygote,andidentifiedfourdistinct
missense mutation^.^^ Another group found point mutations
in the Turkish and Lebanese PK-deficient individuals: and
one mutation found in the Lebanese was identical with the
mutation identified intwo Japanese families? It was noteworthy that these mutations may cause the structural changes near
the potassium-binding sites'; consequently, the variant PKs
showed low affinity with the substrate, phosphoenolpyruvate
(PEP). We have also shown that the variant PK synthesized
in Escherichia coli had a thermal instability? Although the
reticulocyte mRNA is the appropriate source for cloning and
sequencing the R-PK cDNA, the genomic DNA is more convenient for handling. Recently,we have clarified thestructure
of the humanGtype PK (GPK) gene, and it becomes possible
to search the mutation in the genomic DNA analysis.'
In 1963, five PK-deficient kindred found in the Mifflin
County Amish deme in Pennsylvania were reported? Previous studies showed that all the Amish PK variants descended
from a common ancestor, and consanguineous marriage was
often noted in the family." Therefore, the PK-deficient subjects in the population are homozygous with an identical
mutation. The clinical manifestation of the Amish PK variant
was reported to be severe, and sometimes death occurred
in the neonatal or infantile period unless splenectomy was
performed.' To clarify the molecular abnormality of the
Amish PK variant might be useful not only for the understanding of the relationship between the structure and the
function of PK but also for the screening of the variant gene
in the population. In this report, we present the molecular
abnormality identified in an Amish family, and discuss the
structure-function relationship of PK and the cellular characteristics of PK-deficient erythrocytes.
MATERIALS AND METHODS
Clinical features of the Amish PK-deficient subjects. AY and JY
are siblings, being brother and sister and members of the STY,Sr,
Blood, Vol 83, No 8 (April 15). 1994: pp 2311-2316
From the Okinaka Memorial Institute for Medical Research; the
Department of Blood Transfusion Medicine, the Tokyo Women's
Medical College, Tokyo, Japan; the Cardeza Foundationfor Hematologic Research, Jefferson Medical College, Philadelphia; and the
Harrisburg Hospital, Harrisburg, PA.
Submitted June 18, 1993; accepted December 8,1993.
Supported in part by a Scientific Research Grant from the Ministry
of Education, Science, and Culture, and by a Research Grant for
Specific Diseases from the Ministry of Health and Welfare, Japan,
andin part by the National Institutes of Health Comprehensive
Sickle Cell Grant No. HL 38632.
Address reprint requests to Hitoshi Kanno, MD, Okinaka Memorial Institute for Medical Research, 2-2-2 Toranomon, Minato-h,
Tokyo 105, Japan.
The publication costs of this article weredefrayed 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.
8 1994 by The American Society of Hematology.
O006-497I/94/8308-00I9$3.00/0
2311
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2312
1.22
KANNO ET AL
2.46
DIED
2.64
1.04
0.17
3.3
AY
0.1
3.75
1.35
0.46
JY
Fig 1. The pedigree of the Amish PK-deficient family. Number
indicates erythrocyte PK activity, units per 10'' RBCs.
RBCs 2.55 X 106/pL, Hb 10 g/dL, Hct 29.6%, MCV 116 fL, and
reticulocytes 26%. In the blood film, macrocytes, polychromasia,
and echinocyte were noted. LDH was 278 U.
Materials. Blood samples from the probands were obtained under informed consent. High molecular weight genomic DNAs were
purified by the standard protocol." Restriction endonucleases and
modifying enzymes 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). DNA was sequenced using a DNA sequencing system
(Model 373A; Applied Biosystems, Foster City, CA). The computer
software, DNASIS (Hitachi Software Engineering, Yokohama, Japan), was used toanalyze the hydrophobicity properties of the variant
protein based on the theory of Hopp and Woods."
Hematologic data and cellular studies about PK-deficientRBCs.
Routine hematologic methods were used to determine the Hb level,
Hct, RBC indices, and reticulocyte count. Whole RBC deformability
and RBC sodium (Na+) and potassium (K') concentrations were
determined as previously de~cribed.'~
The ouabain-insensitive component of K+-efflux was measured as described previo~sly.'~
Amplification and sequencing of the humanL-PK gene. Oligonucleotides used for the amplification of the exons of human L-PK
gene are listed inthe Table 1. L1 (S-CCCGGGAGGCAGAGGCCGCAGT-3') and L2 (5'-CAAAAGCTCCATCTGGACATT-3')
were used for amplifying the 3' end sequences of exon 10. Polymerase chain reaction (PCR) was performed in a total volume of 100
pL containing 1 pg of the template DNA, deoxynucleotides at 200
mmol/L each, a pair of primers, 10 mmoVL Tris/HC1 @H 8.3), 50
mmol/L KCl, 1.5 to 3 mmol/L MgCI2, 0.001% gelatin, and 5 U of
Taq DNA polymerase. The amplified DNA fragments were subcloned into pBluescript (Stratagene, La Jolla, CA) by the restriction
sites within the DNA or primers. Exons 1, 3 through 12, and the
adjacent intron sequences were sequenced by the dideoxy chain
termination method with fluorescent primers.
RESULTS
Cellular studies of the PK-deficient RBCs. Hematologic
characteristics of the subjects studied and the rheologic prop-
erties of PK-deficient RBCs are listed in Table 2. Because
both JY and AY had been splenectomized, the anemia was
not severe on the examination. The MCV in PK deficiency
was increased, and it seemed to be reflectedon the high
reticulocyte counts, which were usually observed in PK deficiency after splenectomy. It should be noted that the PKdeficient RBCs showed increased maximum deformability
as well as increased deformability at isotonic conditions.
To elucidate further the hydration status and surface areato-volume ratio of PK-deficient RBCs,we measured the density distribution and cation content of these cells (Fig 2,
Table 3). There is a significant shift to lower density in PKdeficient RBC populations as seen in Fig 2. Table 3 shows
that the sodium content of PK-deficient cells is similar to
that of controls, whereas the potassium and total cation contents are significantly increased to 117% to 121% of normal
controls.
To investigate the nature of the abnormal cation regulation
in PK-deficient RBCs further, we determined the ouabaininsensitive K' efflux in the presence or absence of 1.0 mmol/
L furosemide, an inhibitor of Cl- transport and chloridedependent K+ flux. Table 4 shows that PK-deficient cells
had a markedly increased K+ efflux. The first-order rate
constant for K' efflux of the PK-deficient cells was 0.034
to 0.035 h-', compared with 0.024 for control cells (142%
to 145% increase). The addition of furosemide reduced the
rate constant for K+ efflux of normal cells, but did not decrease that of the PK-deficient cells.
Identification of a point mutation in the L-PK gene of the
Amish PK variant. Sequencing of the L-PK gene from the
Amish PK variant showed that the variant had a single nucleotide change at the 3'-end nucleotide of exon 10 of human
L-PK gene, corresponding tono. 1436 of humanR-PK
cDNA, 1436 CGC -+ CAC. This missense mutation caused
a single aminoacid substitution; 479 Arg His (R479H),
which subsequently decreased the hydrophobicity properties
near the mutated site (Fig 3). The predicted secondary structure by the theory of Chou and Fasman showed that the core
of a-helix might be formed by this amino acid change (data
not shown). This Arg residue is well conserved during evolution, including chicken M1-, cat M1-,ratR-, and human
M2-type PK (Fig 4).16-19
Demonstration of the point mutation by the Hpa II digestion of the genomic PCR products. To confirm this nucleotide change in genomic DNAs of the probands, the 3' end
-+
Table 1. Oligonucleotidesfor PCR of Genomic PK
Exon
I
111
IV-v
VI
VII-VIII
IX-x
XI
XI I
Sense Primer
5'-CTAAAGClTCTGTGGGGACAGGGTGGC-3'
5'-GTGACATGCAGTCCCTGAGCICCC-3'
5'-GGCGlTCTGAGAAFsGTAATGG-3'
5"GACTCCGGAGCTCAGAACTCA-3'
5"CTGACCGCAGCTGGCTCl"rCATG-3'
5"GGTGTCAGAGAGGTAsClTGGGC-3'
5'-LAAlTCACCACl"rClTGC-3'
5'-CCACAGCTGTCCAATGAllTG-3'
Antisense Primer
5"GAGGCTCTGAAGAACGTACG-3'
5"ATACCAATAGGCCTGTGTGGCT-3'
5"TCCAClTCCGACTCTGGACC-3'
5"CAGCGCACGGATGTGATCAGG-3'
5"CCCTGCAGTGTGGGTAlTCACCCA-3'
5"CAAAGGATCCATCTGGACAlTCC-3'
5'-CTCCGGATsCAAATGGTAGGAG-3'
5"AAGGCATCTTAGGGCCTGChGAGC-3'
Product (bp)
374
260
559
Sac
272
626
531
281
450
Restriction Sites
HindlllISty l
Sac IIPst l
€CO RIIApa I
IIBcl l
Pvu IIIPst I
Kpn IIBamHI
€coRIIBamHI
Pvu IIIfSt I
The primers used in sequencing the entire coding region for the R-type PK are shown. To create appropriate restriction sites, the underlined
nucleotides are added or substituted to the primers.
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PYRUVATE KINASE DEFICIENCY IN THE AMISH
2313
Table 2. Hematologic and Rheologic Data on the Patients Studied
Max Dl
Age/
Sex
Hb
(g/dL)
Het
MCV
Reticulocytes
(%)
(fL)
(X)
29F
31/M
11.0
9.3
37.3
27.3
116.1
115.8
18.5
25.4
Patient
AY
JY
% of Normal
111
120
112
120
Maximum deformability index (Max Dl) was attained by osmotic
gradient ektacytometry. Dl 290 is the deformability index of RBCs
suspended in a buffer the osmolarity of which is 290 mOsmkg.
region of exon 10 was amplified by PCR and examined the
Hpa I1 cleavage site, that should be abolished by the nucleotide change. A 201-bp fragment flanking the 3' end of exon
10 was amplified by PCR. When the Hpa I1 site is intact,
theband becomes four fragments, 132, 39, 28,and 2 bp,
r
a
w
I
.
-!P=+
,
Table 3. Cation Content of PK-Deficient RBCs
Dl 290
?
i
Patient
Control
P
Variable
AY
JY
n=3
Value
Na', mEq/L RBCs
K', mEq/L RBCs
Na* + K-, mEq/L RBCs
13.6
99.5
113.1
11.9
102.0
113.9
11.9 z 3.30
84.6 2 2.04
96.4 2 1.39
>.05
c.025
c.005
after Hpa I1 digestion. As shown in Fig 5, one of the Hpa
I1 sites was abolished in the PCR products of the probands,
meaning the homozygous mutation in the probands' L-PK
gene.
DISCUSSION
Here we report a molecular abnormality in the Amish PK
deficiency. The probands had suffered from severe hemolytic
anemia, and required transfusion every 8 weeks. After splenectomy the anemia was compensated, with a Hb level of
about 9 to 10 g/dL. Cellular studies of the PK-deficient RBCs
showed increased K' content and a markedly expanded ouabain-insensitive K' efflux. The pathophysiology of PK-deficient erythrocytes has been
Keitt'" has shown
that PK-deficient erythrocytes were almost entirely dependent on mitochondrial oxidative phosphorylation rather than
on glycolysis to maintain adenosine triphosphate (ATP) levels. A rapid decrease of ATP levels because of decreased
oxidative phosphorylation during maturation of erythrocytes
allows massive losses of potassium and water, resulting in
the formation of spiculated erythrocytes, ie, echinocytes.
These cells usually have decreased deformability, and are
easily sequestered by the reticuloendothelial system." Splenectomy may prevent reticulocytes and young erythrocytes
from being trapped by the reticuloendothelial system, resulting in improvement of the anemia in most cases. The
hematologic data of the Amish PK deficiency showed postsplenectomy reticulocytosis, which was usually observed in
PK deficiency, and the large, lower density cells with increased deformability may reflect mainly the increased number of reticulocytes. Despite the increased K' content, these
cells are prone to lose K' andwaterrapidly
because of
impaired ATP availability, and become dehydrated and en-
Table 4. Effect of Furosemide on Ouabain-Insensitive K' Efflux of
Normal and PK-Deficient RBCs
- 3 In K- ConcentrationlmL RECs
~~
PK-Deficient RECs
Normal
-.
C
AY
JY
Fig 2. The actual density profile of PK-defificientRBC (Ay and JY)
and a normal control (C).
Additive
RECs
AY
JY
None
1.0 mmol/L furosemide
0.024
0.014
0.034
0.037
0.035
0.037
Cells were incubated for 2 hours at 37°C in 10 mmol/L HEPES (pH
7.5). 5 mmol/L glucose, 0.2 mmol/L ouabain, and NaCl to bring the
final osmolarity to 290 mOsm/kg, in the presence or absence of the
indicated additive. Values shown are the negative In of the calculated
intracellular K+ concentration as a function of time.
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KANNO ET AL
2314
5 1
I
1
0
-1
-2
-3
-4
-5
Arg-His
-q
p"----d
.....................................................
l'
471
.................................................................
I
48 1
ity were unfavorable characteristics of this variant, which
may be responsible for the phenotype.
There are four mammalian PK isozymes23:type-M1, M2,
L, and R. The M-type PK (M-PK) gene encodes both M1
andM2 isozyme^.^^^^^ In contrast, L and R isozymes are
encoded by the L-PK gene.8." Both the M- and L-PK gene
of rats and humans have 12 exons.8,18,25,26
Alternative splicing
produces two distinct mRNAs, which contain either exon 9
(Ml) or 10 (M2) of the M-PK gene. Exons 1 and 2 of the
L-PK gene are specifically transcribed to the R- and L-PK
mRNA by the tissue-specific promoter. It is of interest that
among four isoenzymes only M1-type is not allosterically
regulated." Because M1 and M2 isozymes differ only in the
exon 9-10 region, exon 10 should encode the amino acid
residues responsible for the allosteric property. Comparison
of exon 10 between the M- and L-PK gene disclosed that
there was an extensive homology, indicating that the exon
10-encoding region of L-PK gene also contained the allosteric structure (Fig 4). It seemed to be rational that the variant
showed lower response to the allosteric effector, because the
missense mutation identifiedin exon 10 would cause the
drastic change of hydrophobicity and secondary structure.
The amino acid substitution (R479H) resides in the third ahelix of the C domain of PK subunit, deduced from the
tertiary structure of cat muscle PK." The C domain is considered to be important for the intersubunit contact as well as
the allosteric regulation. Because PK has kinetic activity
only as either dimer or tetramer, the structural change may
interfere with tetramer formation, resulting in a drastic loss
of activity.
The identification of the molecular lesion of erythrocyte
County Pennsylvania Amish
PK deficiency in the Mienables us to detect the mutation by a simple procedure,
PCR and restriction endonuclease digestion. Thus, it can be
applied to genetic counseling or the prenatal diagnosis of
the Amish PK-deficient hemolytic anemia, which has been
known as a most severe form of PK deficiency. As only the
Geauga County Ohio Amish are related to the MiWin County
Pennsylvania Amish deme by an ancestral genetic member
B
)
:
'no other demes of Amish within the United States
(ie, A
are believed to exist sharing this missense mutation or the
erythrocyte PK deficiency hemolytic anemia.
I
490
Fig 3. Hydrophobicity profiles neer the mutated site. The dolted
line indicatesthe hydrophobicity propertiesof normel R-PK from no.
471 to no. 490 amino acid noidues, and the solid line s h o w the
altered hydrophobicprop.rtkr of the Amish PK variant. The arrows
show the position of the 479th emino acid residue, whkh is substituted from Arg to His, and the shift of the hydrophobicity profile
caused bythe amino acid substitution.
trapped in the splenic environment. This hypothesis is supported by the finding of entrapped reticulocytes in the splenic
red pulp of the patient's spleen." It should be noted that the
potassium efflux of the Amish PK-deficient erythrocytes was
increased up to 142% to 145% of normal controls. Activation
of Na+-independent K+/Cl- cotransport seemed unlikely in
PK deficiency, because the addition of 1.0 mmol/L furosemide (an inhibitor of KC1 cotransport) to the medium did
not reduce the rate constant of K+ efflux. The K+ efflux may
be increased by a mechanism similar tothat observed in
Hbsc RBcs.ls
The reported enzymatic characteristics" of the identical
variant PK were a low Michaelis constant (Km) for PEP,
low Km for adenosine diphosphate, narrow nucleotide specificity, less ATP inhibition than normal, more fructose-l, 6diphosphate (FDP) required for 50% activation, thermal instability, normal optimum pH, and normal migration in polyacrylamide gel electrophoresis. It should be noted that lower
response to the allosteric effector, FDP, and thermal instabil-
Fig4.
The comparison of the aminoacid sequence of the C domain of PK, encoded by exon 9
(Ml)or 10 ( M 2 ,R). Open boxes surrounded by solid
lines indicate amino acid residues, which are conserved in all species examined. Boxes with dotted
lines showthe amino acids, which are only found
in
the allosterkdy r e g M isozymes, M2 and R (L).
The arrow polnb the Arg residue, which is substituted to His in the Amish PK variant. The numbers
indicate the amino acid comrponding to the Arg
residues.
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PYRUVATEKINASEDEFICIENCY
M
1
2
IN THE AMISH
2315
3
-
bp
171
132
39
28
Fig 5. Hpa II digestion of the PCR productsflanking the 3' end of
exon 10 from the probands and a normal control. Becausethere are
three Hpa II recognitionsites in the amplified DNA, the PCR product
of normal control is digested into four fragments: 132, 39, 28, and 2
bp. When the 1436 G to A mutation is present, the 201-bp products
are digested into three fragments:171,28,
and 2bp. M, pBR322
digested with Msp I; 1, normal control; 2, the AmishPK-deficient
subject JV; 3, the Amish PK-deficient subject AV.
ACKNOWLEDGMENT
WC are indebted to Y. Okamura, A. Kawaguchi, and M. Watanabe
for their technical assistance.
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1994 83: 2311-2316
Molecular abnormality of erythrocyte pyruvate kinase deficiency in
the Amish
H Kanno, SK Ballas, S Miwa, H Fujii and HS Bowman
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