Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2959-2962
The Relationship of the 
5, 8, and 24 Variant Alleles in
African Americans to Triosephosphate Isomerase (TPI) Enzyme
Activity and to TPI Deficiency
By
Arthur Schneider,
Linda Forman,
Beryl Westwood,
Catherine Yim,
James Lin,
Satinder Singh, and
Ernest Beutler
From the Department of Pathology, Finch University of Health
Sciences/The Chicago Medical School, North Chicago, IL; the Department
of Molecular and Experimental Medicine, The Scripps Research Institute,
La Jolla, CA; and the Laboratory Service, Veterans Affairs Medical
Center, North Chicago, IL.
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ABSTRACT |
In 424 African-American and 75 white subjects, we found that the
5 (TPI 592 A
G), 8 (TPI 589 GA), and 24
(TPI 573 TG) variants in the triosephosphate isomerase (TPI)
gene occurred frequently (41.0%) in the African-American subjects but
did not occur in the whites. These data suggest that this set of
polymorphisms may turn out to be one of the higher-incidence molecular
markers of African lineage, a surprising finding because others had
reported that these nucleotide substitutions were restricted to a small subset of African Americans who had been characterized as
TPI-deficiency heterozygotes. Additionally, we investigated the
relationship of these variants to TPI-enzyme activity. Although the
variant substitutions (occurring in three haplotypes: 5 alone, 5
8, and 5 8 24) were associated with moderate reduction in
enzyme activity, severe-deficiency heterozygotes could not be
identified with certainty, and none of the haplotypes were restricted
to subjects with marked reduction of enzyme activity. Three subjects were homozygous for the 5 8 haplotype, a finding inconsistent with the putative role of this haplotype as the cause of a null variant
incompatible with life in homozygotes. Despite these findings, the
possibility remains that the 5 8 or 5 8 24 haplotypes may in some instances contribute to compound heterozygosity and clinical TPI deficiency.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
TRIOSEPHOSPHATE ISOMERASE (TPI)
deficiency is a rare autosomal recessive disorder characterized by
hereditary nonspherocytic hemolytic anemia, severe progressive
crippling neuromuscular disability, increased propensity to infection,
and death in early childhood in most cases.
In the more than 30 years since its original description,1
fewer than 40 homozygous or compound heterozygous clinically affected
subjects have been described. Only 21 families have been characterized
at the molecular level, and these characterized defects have been the
subject of recent reviews.2,3 The distribution of the
genetically characterized families has been worldwide, with cases
reported from the US, UK, France, Germany, Greece, Turkey, Hungary, and
Australia. None of these families have been of African origin.
Despite the rarity of homozygous clinically significant enzyme
deficiency, clinically unaffected heterozygotes have been reported in
rather high incidence in several population surveys.4-6 Of particular note was the report several years ago of a survey based on
automated red cell enzyme activity estimation that described an
extremely high incidence of putative heterozygotes in African Americans
(7 in a study group of 146).4 The absence of clinically affected homozygotes in this population was unexplained, but it was
suggested that it was likely that the defect would prove to be a null
allele, incompatible with life in the homozygous state. This suggestion
was consistent with a subsequent observation in mice of a series of
induced mutations with nucleotide substitutions consistent with null
alleles that were found to be lethal in homozygotes at an early
postimplantation stage of embryonic development.7,8
More recently, Watanabe et al9 studied the same group of 7 African-American subjects reported earlier by Mohrenweiser and Fielek.4 All were reported to have base substitutions in
each of two sites, 5 and 8 base pairs upstream of the
transcription start site. Three of these subjects had yet another base
substitution at an additional site, 24 base pairs upstream of the
start of transcription. The substitutions were AG at position
5, GA at position 8, and TG at position
24.
These were provocative data. However, a number of questions remained
unanswered. We therefore set out to more fully assess the significance
of these abnormalities.
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MATERIALS AND METHODS |
Sample Collection, Labeling, and Requirements for Waiver of
Informed Consent
Discarded blood samples were obtained from the clinical hematology
laboratory of the Veterans Affairs Medical Center (North Chicago, IL)
and were refrigerated until analysis. The specimens, collected in EDTA, were originally obtained for performance of routine
hematology testing ordered for patient care purposes. The phlebotomists
coded the specimens as African American or white, determined by visual
assessment at the time of blood collection. A total of 424 African-American subjects and 75 white subjects were available for
study.
Analytic Methods
Enzyme activity.
TPI activity was assayed, as previously described,10 on
lysates of washed red cells separated from leukocytes, platelets, and
plasma. The method is a kinetic assay based on the conversion of
glyceraldehyde-3-phosphate to dihydroxyacetone phosphate, linked to the
subsequent conversion of dihydroxyacetone phosphate to 
-glycerophosphate in the presence of excess -glycerophosphate dehydrogenase and reduced nicotinamide-adenine dinucleotide
(NADH). Hemoglobin was assayed by the ferricyanide-cyanide
method at 540 nm.10 Enzyme activity was expressed in
international units (micromoles of substrate utilized per minute) per
gram of hemoglobin.
Polymerase chain reaction (PCR) amplification.
The 50 µL volume for PCR contained 670 mmol/L Tris pH 8.8, 166 mmol/L
(NH4)2SO4, 67 mmol/L
MgCl2, 1.7 mg/mL bovine serum albumin, 2.5 µL dimethyl
sulfoxide (DMSO), 0.025 µmoles of each dNTP, 1.25 units
AmpliTaq DNA Polymerase (PE Applied Biosystems, Foster
City, CA), 150 ng forward primer 5
-GGCCATGGCGGAGGACGGCG (corresponding with genomic positions 520 to 539 in GenBank TPI sequence HSTPI1G, revised March 24, 1997), 150 ng reverse primer 5-GCCAGACCCCTCCTCGGCGA (corresponding with genomic positions 777 to 758), and 100 to 150 ng
genomic DNA. PCR was conducted for 30 cycles as follows: denature 30 seconds at 92°C, anneal 30 seconds at 50°C, extend 40 seconds
at 72°C. Cycling was preceded by denaturation at 92°C for 3 minutes and followed by extension at 72°C for 7 minutes.
Restriction digests.
After PCR, a 10 µL aliquot was removed and subjected to restriction
digestion in a system consisting of, in addition to the sample, 5 µL
of buffer, 1.5 µL of the appropriate restriction endonuclease
(diluted to contain 1.5 × 10
3 units), and water
sufficient to bring the volume to 50 µL. The digest was incubated for
3 hours at 55°C, precipitated in ethanol, resolved by
electrophoresis on a 10% acrylamide gel, and visualized by ethidium
bromide staining. For the 5, 8, and 24 variants, the
restriction endonucleases were TseI, MscI, and
SfcI, respectively. The enzymes and the appropriate buffers for
each enzyme were purchased from New England BioLabs (Beverly, MA).
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RESULTS |
The haplotype frequencies are indicated in
Table 1. The relationship of all genotypes
to enzyme activity is shown in
Table 2, and the distribution of values for the more common genotypes is
depicted in Fig 1. The mean TPI activity was somewhat
reduced in African Americans as compared with whites. However, in the African-American group lacking variant alleles, mean TPI activity was
essentially the same as in whites. In the genotype groups, 5/normal,
(5 8)/normal, and (5 8 24)/normal, there appeared to be
a successive reduction in enzyme activity. Of special interest was the
finding of three (5 8)/(5 8) homozygotes. The enzyme activities in these three subjects were all somewhat reduced. Severe-deficiency heterozygotes could not be identified with certainty. To explore the possibility that such heterozygotes were undetectable because of overlap with the lower ranges of normal, we compared the
genotypes of subjects with the lowest and highest activity values
(Table 3). The lowest activity was found in a (5
8)/(5 8) homozygote; most of the subjects with lowest
activities displayed the (5 8)/normal genotype, and the
5/normal genotype also occurred. Most of the subjects in the
high-activity group did not have any of the promoter variants.
Comparison of the lowest and highest activity groups by quartiles
(Table 4) showed a considerable excess of 5 8
haplotypes in the lowest quartile.
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| Fig 1.
TPI activity compared by race and genotype. When TPI
activity of African Americans (AA) with no variant substitutions is
compared with that of AA with the (5 8)/normal genotype
(distributions shown in darker lines for emphasis), a clear-cut
reduction in enzyme activity is apparent in the variant group. However,
it is also noteworthy that decreased enzyme activity is not restricted
to the range for previously established heterozygotes, which
approximates one half of normal mean activity (AA normal/normal: mean
= 2002.7 IU/g hgb, standard deviation (SD) = 454.4; AA [5
8]/normal: mean = 1700.2, SD = 393.9). These differences are
highly significant (P = 8.6 × 108,
t-test). TPI activity in the total group of AA is less than in
whites (AA all genotypes: mean = 1912.9 IU/g hgb, SD = 442.6; white
normal/normal: mean = 2041.9, SD = 366.3). This difference is
significant (P = .008, t-test). However, when TPI
enzyme activity of AA with no variant substitutions is compared with
the white group, the differences between the two groups diminish and
are probably of no significance (AA normal/normal: mean = 2002.7 IU/g hgb, SD = 454.4; white normal/normal: mean = 2041.9, SD
= 366.3; P = .444, t-test). When enzyme activity of
AA with no variant substitutions is compared with AA with the
5/normal genotype, the variant genotype is associated with a modest
reduction in enzyme activity (AA normal/normal: mean = 2002.7 IU/g
hgb, SD = 454.4; AA 5/normal: mean = 1867.7, SD = 376.2).
These differences are significant (P = .008, t-test).
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Table 3.
Genotypes in African-American Subjects With Lowest
and Highest Triosephosphate Isomerase (TPI) Activities
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Table 4.
Frequency of Variant Haplotypes in Lowest and
Highest Quartiles of Triosephosphate Isomerase Activity in
African-American Subjects
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DISCUSSION |
The data are surprising in that they are strikingly dissimilar to those
reported by others.4,9 Rather than occurring only in a
small subset of African-American subjects, the 5, 8, and 24
nucleotide substitutions are evidently high-incidence polymorphic
markers of African Americans, and probably of African lineage in
general. Indeed, this set of polymorphisms may well turn out to be one
of the higher incidence molecular markers of African lineage.
Also, in contrast to the findings of Mohrenweiser and
Fielek,4 who had reported a 4.79% incidence of
severe-deficiency heterozygotes in their group of African-American
subjects, we were unable to identify with certainty a distinct
population of such heterozygotes. Whether this discrepancy indicates
the absence of heterozygotes or, alternatively, undetectability because
of overlap with lower-level normal values is unclear. It may well be
that so-called deficiency heterozygosity represents nothing more than
the lower end of the distribution of the enzyme activity curve in the
African-American group. Also, because most (but not all) of the
subjects with lower activity values carry the 5 8 haplotype, it
might well be that the invariable association of this haplotype with
low-enzyme activity in the small group reported by Watanabe et
al9 could have occurred entirely by chance. Even though the
true incidence of severe-deficiency heterozygosity in the
African-American population remains unknown, there is no question that
this set of nucleotide substitutions in the TPI promoter is associated
with modest reduction of enzyme activity. In fact, the somewhat lower
TPI activity characteristic of the African-American population appears
to be closely linked to these substitutions.
The clinical importance of these promoter polymorphisms remains
unclear. That these variant substitutions can contribute to severe TPI
deficiency is suggested by our observation of an obligate heterozygote
(father of a clinically affected TPI deficiency patient), in whom the
only abnormality shown thus far is the 5 8 24 variant haplotype. Whether it is the promoter polymorphisms themselves that
result in lower enzyme activity or whether there are functionally important variant sequences elsewhere in the gene (possibly far upstream) in linkage disequilibrium with the promoter polymorphisms is
not yet established. Studies now in progress with reporter constructs
should clarify this question. Additionally, the finding of three 5
8 homozygotes in our study group precludes the validity of the
suggestion that the 5 8 haplotype is a null variant incompatible with life in homozygotes.9 Finally, the association of a
set of high-incidence polymorphisms with moderately reduced enzyme activity is supportive of our previous suggestion that modest reduction
of TPI activity may provide a selective advantage for survival.11
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FOOTNOTES |
Submitted April 14, 1998;
accepted June 16, 1998.
Supported in part by Grants No. HL25552-19 and RR00833 from the
National Institutes of Health, Bethesda, MD.
Address correspondence to Arthur Schneider, MD, Department of
Pathology, Finch University of Health Sciences/The Chicago Medical School, 3333 Green Bay Rd, North Chicago, IL 60064;
e-mail:schneidr{at}finchcms.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We gratefully acknowledge the technical staff of the Laboratory
Hematology Unit of the North Chicago Veterans Affairs Medical Center,
who collected and appropriately designated the blood samples used in
this study.
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Abstract
Introduction
Abstract
