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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3585-3588
RED CELLS
Estimating the prevalence of pyruvate kinase deficiency from the
gene frequency in the general white population
Ernest Beutler and
Terri Gelbart
From the Department of Molecular and Experimental Medicine, The
Scripps Research Institute, La Jolla, CA.
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Abstract |
Pyruvate kinase (PK) deficiency is the most common cause of
hereditary nonspherocytic hemolytic anemia. The prevalence of this deficiency is unknown, though some estimates have been made based on the frequency of low red cell PK activity in the
population. An additional 20 patients with hereditary
nonspherocytic hemolytic anemia caused by PK deficiency have been
genotyped. One previously unreported mutation 1153C T (R385W) was
encountered. The relative frequency of PK mutations in patients with
hemolytic anemia caused by PK deficiency was calculated from the
18 white patients reported here and from 102 patients previously
reported in the literature. DNA samples from 3785 subjects from
different ethnic groups have been screened for the 4 more
frequently encountered mutations c.1456 CT(1456T), c.1468
CT(1468T), c.1484 CT(1484T), and c.1529 G6A (1529A)by
allele-specific oligonucleotide hybridization. Among white patients the
frequency of the 1456T mutation was 3.50 × 10 3; that
of the 1529A mutation was 2.03 × 103. Among African
Americans the frequency of the 1456T mutation was
3.90 × 103 The only mutation found in the limited
number of Asians tested was 1468T at a frequency of
7.94 × 103. Based on the gene frequency of the 1529A
mutation in the white population and on its relative abundance in
patients with hemolytic anemia caused by PK deficiency, the prevalence
of PK deficiency is estimated at 51 cases per million white
population. This number would be increased by inbreeding and
decreased by failure of patients with PK deficiency to survive.
(Blood. 2000;95:3585-3588)
© 2000 by The American Society of Hematology.
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Introduction |
Pyruvate kinase (PK) deficiency is probably the most
common cause of hereditary nonspherocytic hemolytic
anemia.1 The true prevalence of this disorder is unknown
because diagnoses are made not only in specialized centers throughout
the world but also in commercial and hospital laboratories.
The red cells of heterozygotes for PK deficiency generally have
approximately half the normal level of enzyme activity. Based on the
lowered erythrocyte enzyme activity of heterozygotes, a few efforts
have been made to obtain some information regarding the prevalence of
this disorder. Assays of red cell PK in 214 normal subjects led Blume
et al2 to conclude that approximately 1.4% of the German
subjects were heterozygous for the deficiency. Mohrenweiser3 reported that among 697 newborns, 84% of
whom were white, 1 (0.15%) manifested red cell PK activity that was more than 3 standard deviations below the mean. In a subsequent study,
however, he suggested that the frequency of heterozygous subjects was
18 of 1736 white newborns.4 However, because
red cell PK activity varies widely from person to person, there is bound to be overlap between normal and heterozygous levels.
Detection of the existence of mutations based on the DNA analysis is
more robust than is the estimation of enzyme levels. Although it is not
feasible to perform complete sequence analysis on thousands of persons,
it is possible to determine the frequency of those mutations that are
the most common causes of PK deficiency. Based on the known frequency
of such mutations in patients with the disease, it is possible to
extrapolate from the general population to obtain some estimate of the
number of persons in the population who may have the PK-deficient genotype.
We now report the genotypes of an additional 20 patients with PK
deficiency. Based on the accumulated information of the occurrence of
mutations in patients with PK deficiency and a survey of more than 3500 normal subjects, we estimate the prevalence of PK deficiency in the
white population at large.
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Patients and methods |
Patients with PK deficiency had documented hemolytic anemia and
deficiency of erythrocyte PK activity when assayed by standard methods
as modified from previously published methods.5 When the
more common mutations were not detected by restriction analysis or
direct sequencing, the entire coding region was sequenced as described
previously.6
DNA samples from 3785 anonymous persons, identified only by ethnic
origin, were examined for 4 PK mutations: c.1456 CT(1456T), c.1468 CT(1468T), c.1484 CT(1484T), and c.1529 GA (1529A). These samples were derived from several different sources.
Approximately 3500 samples were obtained from patients attending a
health-screening clinic. Ethnic origin was based on
self-identification. Approximately 300 additional samples from African
Americans, identified as such by the phlebotomist, were from discarded
diagnostic samples. The segment of DNA containing these mutations was
amplified by means of the polymerase chain reaction (PCR) with the
following 2 primers: sense 5'-CTCGTTCACCACTTTCTTGC-3' and
antisense 5'-GAGGCAAGGCCCTTTGAGTG-3'. The PCR mixture
contained 34 mmol/LTris-HCl, pH 8.8, 8.3 mmol/L ammonium sulfate,
1.5 mmol/L MgCl2, 85 µg/mL bovine serum albumin, 0.2 mmol/L each dATP, dCTP, dGTP, and dTTP, 120 ng of each oligonucleotide primer listed above, 200 ng genomic DNA, and 1 U Taq polymerase. After
a 4-minute denaturation at 98°C, 30 cycles of PCR at 94°C for
30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds
were performed.
The amplified DNA was spotted in duplicate on Nytran SuPerCharge
membranes (Schleicher and Schuell, Keene, NH) and probed with the
wild-type and mutant probes for each mutation studied (Table
1). All filters contained controls of
normal, heterozygous, and homozygous samples. All positive results were
confirmed by restriction analysis or direct sequencing.
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Results |
Patient studies
The mutations found in the 20 patients with PK
deficiency are summarized in Table 2. In
one patient, only 1 of the 2 mutations was identified. It is possible
that the missing mutation, denoted "?," was not found because it
was not in the coding region of the gene or because of technical
reasons. One mutation that has not been reported previously is 1153T.
The deduced amino acid change for this mutation is arginine to
tryptophan at amino acid 385 (R385W). This new mutation was found with
the 1456T mutation in a 1-year-old boy. He was born at full term and
had a bilirubin level of 14 mg/dL. His reticulocyte count was 28%, and
his hematocrit level was 32%. He did well after exchange transfusion.
At 3 months of age he was found to have a hemoglobin level of 5 g/dL and a reticulocyte count of 9.9%. He is transfused
every 3 to 4 weeks. His parents are of Irish and mixed European
ancestry.
Population studies
Heterozygotes for each of the 4 mutations for
which the population was screened were detected in the
population survey except for the 1484T mutation, but there were no
homozygotes. The results are shown in Table
3. The gene frequency for the 1529A
mutation was 0.00203 ± 0.0006 (mean ± 1 SE) in the white
population; that of the 1456T mutation was 0.00350 ± 0.0008 in
the white population and 0.0039 ± 0.0031 in the African American
population. The 1468T mutation was found only among Asians. In the
small group of 126 persons examined, it was encountered twice, giving a
gene frequency of 0.00794 ± 0.00559. The 1484T mutation was not
found in any of the subjects studied.
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Discussion |
Estimating the prevalence of uncommon autosomal recessive
diseases is a difficult challenge. For example, numerous attempts have
been made to estimate the prevalence of Gaucher disease in the Jewish
population. One approach has been to attempt to identify all cases in a
target population by surveying physicians, hospitals, or
both.7 This method depends on accurate and complete
ascertainment of cases and thus tends to underestimate the true
incidence of the disease. A second means of estimating prevalence in
the population is to attempt to identify heterozygotes by
measuring the gene product, which tends to be half
the normal level in heterozygotes 8,9 However, there is
always overlap between heterozygous and normal values and sometimes
between those of heterozygous and homozygous subjects. Correcting for
this overlap introduces a large error and, indeed, is often ignored,
giving inaccurate estimates. The actual documentation of mutations at
the DNA level is the most accurate way of identifying heterozygotes,
but, unless complete sequence analysis is carried out, only the
mutations that are selected for detection will be identified, and
correction must be made for patients with other mutations.
In the case of PK deficiency, the only method that has been
used to estimate population prevalence is that of detecting
heterozygotes on the basis of red cell enzyme activity.2-4
Three of the most prevalent mutations in patients with PK deficiency
are 1529A, 1456T, and 1468T; 1529A is most common in northern and
central Europe,6,10 1456T is most common in southern
Europe,11 and 1468T is most common in Asia. Each of these
mutations is found in the context of its own haplotype, arguing that
each has a unique origin. Each is in the C domain of the enzyme,
involved in the intersubunit contact of the homotetramer catalytic
unit.12
If the gene frequencies of the mutations that cause a disease
are known, its prevalence may be estimated by applying the
Hardy-Weinberg equilibrium. When this is done, the assumption is made
that the penetrance of the clinical disorder is 100%. How well the
clinical expression approaches this ideal can be estimated by comparing the relative frequency of the mutations that are encountered in the
general population with the frequency in the patient population. We
have previously studied the ratio of various Gaucher disease mutations
in the Jewish population with Gaucher disease on the one hand and in
the general population on the other. Here we found marked
overrepresentation of the 1226G mutation in the general population compared with the patient population.13 This
implied that many of the persons who carry this particular mutation
never came to medical attention as patients with Gaucher disease, and this is, indeed, the case.
A similar picture emerges in the case of PK deficiency. Table
4 summarizes the frequency of the 1529A
mutation among patients with hemolytic anemia who were of European,
non-Gypsy ancestry. (Gypsies have been excluded because they have a
unique PK deletion12). Among this population, the 1529A
mutation is the most common, accounting for 28.3% of all the
deficiency-producing alleles; its frequency in the general white
population is 0.00203. The 1456T mutation, on the other hand,
represents only 15.4% of the disease-producing alleles in the patient
population, but its frequency in the general white population, at
0.00350, is appreciably higher than that of the 1529A. It is of
interest, in this respect, that the 1456T mutation is only rarely found
in the homozygous state in patients with hemolytic anemia and that when
the homozygous state has been documented, the anemia is very mild or
does not exist at all.14 Further attesting to the
relatively mild nature of this mutation, there is a tendency for it to
be found together with null (nonsense) mutations rather than missense
mutations.
However, within these limitations, population data can be used
to estimate the prevalence of PK deficiency in the white population. The approach that we have adopted is to select an "index
mutation." The frequency of this mutation in the patient population
is taken to be pi, and its frequency in the general
population under investigation is taken to be gi. The index
mutation should have penetrance that approaches 100%, and it should be
one of the more prevalent mutations in the patient population. In the
case of PK deficiency, the 1529A mutation has been selected as the
index mutation. It is the most common mutation among northern Europeans
with PK deficiency, and its penetrance seems to be high because most
homozygotes for this mutation have severe hemolytic anemia. Moreover,
we know of no instances of siblings found in family studies to be
homozygous for this mutation who did not also have hemolytic
anemia. If the gene frequency of the index mutation,1529A
mutation, in the general population is gi and the frequency
of all other PK mutations is go, then the frequency of all
homozygotes and compound heterozygotes for PK deficiency
mutations is gi2 + 2 gigo + go2, where gi2 is the population frequency of
homozygotes for the 1529A mutation, 2 gigo is
that of compound heterozygotes of 1529A mutations and other
mutations, and go2 is that of homozygotes and
compound heterozygotes of all other mutations. In this study we
found the value of gi to be 0.00203, but how do
we obtain the value of the other mutations, go? If we
assume that the representation of all mutations in the patient
population is the same as in the general population, then the ratio of
the index mutation to all other mutations in the general population
will be
gi/go = pi/po.
Therefore, go = po×gi/pi
where pi is the frequency of the index mutation in the
patient population and po that of all other PK mutations in
the patient population. The fractional frequency of the occurrence of
the 1529A mutation in the patient population, pi, was found to be 0.283 (Table 4). The value of po, representing all
other mutations in the patient population, is then
1  0.283 or 0.717, and the value of go, the
frequency of mutations other than 1529A in the general population, is
then calculated to be 0.00514. Using the values of 0.00203 for
gi and 0.00514 for go, the value of the
expression gi2 + 2 gigo + go2 is 4.12 × 106 + 20.9 × 106 + 26.4 × 106, or 51 per million white population, approximately 10 000 patients in the
United States. The standard error of the estimate of 51 per million can
be assessed with the delta method,18 which uses a
multivariate Taylor series to incorporate the sampling variability in
both gi and pi into an overall estimate
of the variance of gi2/pi2 because
gi2 + 2 gigo + go2 reduces to
this ratio. As expected from the relatively small numbers, this
method provides a the standard error is 32.5 per million. This estimate
would be increased by inbreeding, as in the Pennsylvania Amish
community and in Gypsies, and would be decreased by failure of
PK-deficient patients to survive. The number of patients actually
identified seems to be smaller. In the past 25 years we have diagnosed
PK deficiency in 201 patients. Because we do not know what percentage of all diagnoses in the United States were made in our laboratory, it
is not possible to calculate the number of patients actually diagnosed
in the United States. However, because there is a limited number of
laboratories that carry out these assays, it appears that the actual
cases of PK deficiency diagnosed falls far short of the 10 000 case estimate.
As we have pointed out above, the assumption that the
distribution of mutations in the patient population is the same as in the general population is not entirely accurate; mild mutations such as
1456T are underrepresented among patients. Nevertheless, the approach
we have used has the effect of correcting for those mutations that are
underrepresented in the patient population because the estimate of the
frequency in the general population depends on representation in the
patient population. It thus has the effect of providing a fairly
accurate picture of the patients in whom the disease actually develops.
| |
Acknowledgment |
The authors thank Dr James Koziol for his help in calculating the
estimate of error.
| |
Footnotes |
Submitted November 29, 1999; accepted January 21, 2000.
Supported by National Institutes of Health grants HL25552 and RR00833
and the Stein Endowment Fund.
Reprints: Ernest Beutler, Department of Molecular and
Experimental Medicine, The Scripps Research Institute, 10550 North
Torrey Pines Rd, La Jolla, CA 92037.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction