Download Genes and Enzymes in Man

[CANCER RESEARCH 26 Part 1, 2054-2063,September 1966]
Genes and Enzymes in Man
HARRY HARRIS
Galion Laboratory, University College London, Gower Street, London, Englantl
an abnormal enzyme protein with defective catalytic properties,
or in a severe curtailment of the normal rate of synthesis of the
If as is now thought most genes achieve their effects by specific enzyme protein. Homozygotes for such genes show a
directing the synthesis of enzymes, genetic variation will be gross reduction and sometimes an apparently complete absence
largely reflected by enzyme diversity. Such enzyme diversity in of activity of the particular enzyme concerned, and this may
human populations is illustrated by studies on the recently dis
result in quite striking and often serious metabolic and clinical
covered genetically determined polymorphisms involving red consequences. Hétérozygotes
frequently exhibit a partial de
cell acid phosphatase, phosphoglucomutase, adenylate kinase, ficiency of the enzyme, the level of activity in many cases being
and placental alkaline phosphatase. Three of these polymor
only about half that normally present. Such hétérozygotes
are
phisms were discovered in the course of investigating some 10 however usually quite healthy, presumably because the remaining
arbitrarily chosen enzymes in none of which there was any a activity is still adequate for normal metabolic functioning.
priori reason to expect any degree of genetically determined
Most of these genes appear to be relatively uncommon and
variation. The findings thus suggest that genetically determined
have frequencies of between about 0.01 and 0.001 in the popula
polymorphism may be a feature of a substantial number of tion. One may expect, nevertheless, that in aggregate they will
human enzymes. They also indicate that there exists a very high account for an appreciable degree of enzyme variation among
degree of individuality in the enzymic constitution of normal and apparently normal individuals, because many people will be
healthy people.
heterozygous for one or another of them. Thus probably at least
1% of the European population are heterozygous for the gene
Introduction
determing phenylketonuria and have a partial deficiency of the
It is now generally believed that genes exert their effects by enzyme phenylalanine hydroxylase, and this, of course, is only 1
directing the synthesis of enzymes and other proteins. There example out of many. A few cases are also known where a
are, of course, a very large number of different enzymes in the specific enzyme deficiency occurs quite commonly in certain
populations. The most extensively studied case is glucose-6human organism, and many of these probably contain more than
1 structurally distinct polypeptide chain. If current theory is phosphate dehydrogenase deficiency, and here it seems likely
correct one must suppose that the primary structure of each of that its prevalence in certain populations is due to a specific
these different polypeptides is determined by a separate gene selective advantage which the deficiency may confer in situations
locus, and that there are probably also other loci which are where endemic malaria is an important selective agent (16).
Thus until very recently most of our knowledge of genetically
specifically concerned with regulating the rate of synthesis of
determined enzyme variation in human populations was derived
particular polypeptides or groups of polypeptides. Furthermore,
from the study of these enzyme deficiencies, many of which, at
one may anticipate that genetic diversity in a human population
should to a large extent be reflected in enzyme diversity, that is least in homozygotes, are frankly deleterious and in consequence
have generally been regarded as pathologic abnormalities. It is
to say in differences between individuals either in the qualitative
characteristics of the enzymes they synthesize or in differences in indeed important to remember that most of these conditions were
identified in the 1st instance because of some more or less striking
rates of synthesis.
The work I am going to discuss was largely aimed at trying to clinical or metabolic disturbance. They must, therefore, be
regarded as representing a highly selected group of mutants which
get some idea of the extent and character of such genetically
cannot be expected per se to provide us with any clear picture of
determined variation among what may be regarded as normal
people. When my colleagues and I started on this line of work how extensively genetically determined enzyme variation which
just over 3 years ago the information available about this aspect does not result in overt pathologic manifestations may occur in
the general population. Nor can they give us any certain indica
of human variation was very limited. It had of course been tion of what the nature of such "concealed" variation might
recognized for quite a long time that certain rare metabolic dis
be—whether it is, for example, mainly a matter of minor quan
orders, the so-called inborn errors of metabolism, are due to
titative differences in rates of synthesis, attributable to genes at
genetically determined deficiencies of specific enzymes (7). so-called regulator or operator loci, or whether qualitative
Quite a large number of these conditions are now known, and differences involving enzyme structure are an important feature.
judging from the increasing rate at which new examples have
It is not immediately obvious how one can get to grips with
been described in recent years, it seems likely that many more this rather general problem, and my colleagues and I came to
remain to be identified. These conditions have in general been the conclusion that the only practical approach we could adopt
attributed to mutant genes which result either in the synthesis of in the 1st instance was a quite empirical, and perhaps somewhat
Summary
2054
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Genes and Enzymes in Man
simple-minded, one. Our idea was to see whether, if we examined
a series of arbitrarily chosen enzymes in normal individuals in
sufficient detail, we would find genetically determined differences,
and if so whether such differences were common or rare, and
whether they were peculiar to one sort of enzyme rather than
another.
Because we would need to examine the selected enzymes in
quite a large number of different people, and because we wished
to carry out family studies on any enzyme differences that
turned up, we were in the 1st instance largely forced to confine
our attention to enzymes present in blood. We had, of course,
also to make some decision about the kind of technics we would
utilize in looking for such differences. A wide variety of methods
suitable for examining the many different properties of enzyme
proteins are available, and it would have been impractical to
attempt to utilize more than a few of these. In practice we have
mainly relied initially on the technic of starch gel electrophoresis.
This is known to be capable, if one gets the conditions right, of
detecting quite subtle differences in molecular charge and
molecular size. It is however not designed to pick up other sorts
of molecular difference, and it is also not very sensitive in the
detection of small quantitative differences. Thus we could expect
to detect at best only a proportion of all possible forms of enzyme
variation.
Despite this limitation, we have found during the course of
examining some 10 arbitrarily chosen enzymes in varying degrees
of detail, 3 quite striking examples of genetically determined
polymorphism, as well as a number of relatively rare variants.
Red Cell Acid Phosphatase
The 1st enzyme we selected for study on this arbitrary basis
was red cell acid phosphatase (12). This enzyme is known to
differ both in its pattern of substrate specificity and in its in
hibition characteristics from the acid phosphatases present in
other tissues, and it is thought to occur only in the erythrocyte.
Its precise function, however, is not known.
A fairly simple method was developed for detecting the enzyme
after electrophoresis in starch gel. The surface of the gel is
incubated in a reaction mixture containing phenolphthalein
diphosphate at pH 6.0, so that at any site of acid phosphatase
activity, free phenolphthalein is liberated. This can then be
detected by making the surface of the gel alkaline, so that the
sites of enzyme activity appear as bright red zones.
Qtì
/,
e
BA
Phenotype
Genotype
P°P°
B
CA
CB
pepo
CHART1. Diagram of isoenzyme components seen in the various
red cell acid phosphatase phenotypes after electrophoresis at pH
6.0.
TABLE 1
SEGREGATION
OF RED CELL ACIDPHOSPHATASE
PHENOTYPESIN
216 FAMILIES
PARENTSAxOFMATINGS42511455150782491611216CHILDRENA831324672BA25202525235711
AAxBAAxBAxCAAxCBBA
BABAxBBA
x
CABA
x
CBBxBBxCABxCBCAxCACAxCBCBx
x
CBTotalsNo.
TABLE 2
MEANS AND STANDARDDEVIATIONS OF RED CELL ACID
PHOSPHATASEACTIVITY IN INDIVIDUALSOF KNOWN
PHENOTYPES
The Activity is expressed in arbitrary units based on the quan
tity of p-nitrophenol
liberated from p-nitrophenol
phosphate
under standard conditions (20).
ofindividuals33124811126Mean
activity122.4153.9188.3183.8212.3Standard
PhenotypeABABCACBNo.
deviation16.817.319.519.823.1
When hemolysates from a series of normal individuals were
examined using this procedure, it was found that every sample
showed more than 1 zone of enzyme activity. We were evidently
dealing with what is now generally called a set of isoenzymes.
Furthermore there were clear-cut person to person differences in
the number, the mobilities, and the relative activities of these
isoenzyme components (Chart 1). Five distinct phenotypes were
soon identified. They are now referred to as A, BA, B, CA, and
CB, and in the British population occur with frequencies of
about 0.13, 0.43, 0.36, 0.03, and 0.5, respectively. The initial
family studies showed that these phenotypes are genetically
determined and led to the hypothesis that 3 allelic genes (Pa,
Pb, and Pc) at an autosomal locus are involved (phenotypes A
and B being produced by the homozygous genotypes Papa and
PbPb, respectively, and phenotypes BA, CA, and CB, by the
heterozygous genotypes P"Pb, P"PC, and PbPc). The hypothesis
predicted the occurrence of a 6th phenotype corresponding to
the genotype PCPC.Gene frequency considerations indicated that
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Harry Harris
aLJ
et
100
140
180
220
RED CELL ACID PHOSPHATASE ACTIVITY
CHART 2. Distribution of red cell acid phosphatase activities in the general population (top line) and in the separate phenotypes.
The curves are constructed from the values given in Table 2 and from the relative frequencies of the phenotypes observed in a randomly
selected population.
61 P
PGM
G1.6diP
G6P-
NADP
FORMAZAN
G6PD
6PG
MTT
CHART 3. Sequence of reactions in the detection of phosphoglucomutase (PGM) after starch gel electrophoresis.
The under
lined components are contained in the reaction mixture. GIP,
glucose-1-phosphate;
Gl,6 di P, glucose-l,6-diphosphate;
G6P,
glucose-6-phosphate;
6PG, 6-phosphogluconate;
G6PD, glucose-6phosphate dehydrogenase;
NADP, nicotinamide adenine dinucleotide phosphate; NADPH2, reduced form of NADP; PMS,
phenazine methosulphate;
MTT, monotetrazolium
salt.
this would be fairly uncommon (about 1 in 625) of the general
population, and indeed a few examples of what is probably this
phenotype have now been observed (15).
With the 5 common phenotypes 15 different mating types are
possible and the segregation pattern in most of these has now
been studied (Table 1). This quite extensive family data has
proved to be fully consistent with the hypothesis, and the findings
have also been confirmed by several other groups working with
different populations. There seems little doubt, therefore, that
these acid phosphatase variations reflect a polymorphism in
volving at least 3 alÃ-eles,and a variety of studies on the properties
of the isoenzymes in the different phenotypes makes it appear
reasonably certain that these alÃ-elesdetermine the synthesis of
structurally different forms of the enzyme.
A particularly interesting feature of the polymorphism is that
the qualitative differences between the phenotypes are reflected
quantitatively by differences in the levels of enzyme activity
(20). Levels of total acid phosphatase activity were determined
by a standard method in a series of hemolysates from individuals
of the different phenotypes. Although there was considerable
variation in activity between individuals of any 1 phenotype,
nevertheless quite marked differences between the mean values
for different phenotypes could be demonstrated (Table 2). Using
these values one may examine the question as to whether the
quantitative effects of the 3 postulated alÃ-elesare additive in a
simple way or not. If they are additive, one could expect the
following relationships to be true:
a)
b)
+
= BÄ
CÄ- JA = ÜB- }B
where A, BA, B, etc., are the mean values for the various pheno
types. It will be seen that the results support the idea of simple
additivity rather _well (JA_+ JB = 155.35, BÄ = 153.9, CÄ1A = 122.6, and C13 - |B = 118.15).
It is of interest to note that if one determines red cell acid
phosphatase activities in a series of randomly selected individuals
one obtains a continuous unimodal distribution which, in fact,
is not dissimilar in form to the distributions usually obtained
when other enzymes in man are examined quantitatively in
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VOL. 26
Genes and Enzymes in Man
g
f
•
C
b
Origin
Type
1
2-1
1
2-1
CHART4. Photograph and diagram of phosphoglucomutase isoenzyme patterns obtained by starch gel electrophoresis at pH 7.4.
PEP, phosphoenol pyruvate; LDH, Lactic dehydrogenase.
randomly selected populations. In particular, the variance when
related to the mean is of the same order of magnitude as is found
with many other enzymes. In the present case, however, it is
clear that the over-all distribution represents a summation of a
series of separate but overlapping distributions corresponding to
each of the qualitatively different phenotypes (Chart 2). Further
more, the genetic component of the variance of the over-all
distribution can be largely, if not entirely, attributed simply to
the effects of the 3 alÃ-eles.It is not unreasonable to suppose that
the genetic component of other examples of continuous variation
in enzyme levels may have a similar underlying basis.
Phosphoglucomutase
Phosphoglucomutase provided the 2nd example of common
enzyme polymorphism to be found in the course of this work (21).
This enzyme catalyzes the reversible transfer of phosphate
between glucose-1-phosphate and glueose-6-phosphate, and it
has an important role in carbohydrate metabolism. Its detection
following starch gel electrophoresis was accomplished via the
sequence of reactions shown in Chart 3. The underlined com
ponents are included in the reaction mixture, and the sites of
Phosphoglucomutase activity are located by the deposition of a
blue colored formazan formed by the reduction of the tetrazolium
salt.
When hemolysates from different individuals are subjected to
SEPTEMBER 1966
starch gel electrophoresis and this reaction system is applied,
one obtains the rather complex isoenzyme patterns shown in
Chart 4. At least 7 different zones of activity (a-g) may be
detected, and 3 quite distinct types of pattern can be identified in
different individuals. These are referred to as PGM 1, PGM
2-1, and PGM 2. The phenotypes differ in the occurrence of
components a, b, c and d, a and c being present in PGM 1 and
PGM 2-1, but not in PGM 2, while b and d are present in PGM
2-1 and PGM 2 but not in PGM 1. Components e, f, and g are
present in all the 3 phenotypes.
In the British population the incidence of the 3 phenotypes is
approximately as follows: PGM 1, 0.58; PGM 2-1, 0.36; and
PGM 2, 0.06. Studies on the segregation of these phenotypes in
more than 150 different families involving all the possible mating
types make it clear that 2 autosomal alÃ-eles(PGMi1 and PGM?)
determine these differences. Phenotypes PGM 1 and PGM 2
represent the homozygotes PGM^PGMi1 and PGM ¿POMf, and
phenotypes PGM 2-1 the hétérozygote
PGMfPGMf.
This suggests that the isoenzyme components a and c deter
mined by PGMi1 may be molecular alternatives of components
b and d determined by PGMf. Possibly these isoenzymes contain
a common polypeptide chain, and the difference between the 2
homozygous phenotypes depends on a small structural difference
in this, which involves perhaps a single amino acid substitution.
If this is so, then one would presume that this polypeptide chain
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Hairy Harris
ADENYLATE KINASE. *ATP+AMP
2 ADP,
AK
ATP »AMP
ADP
AMP »ATP
AK
ADP
PEP
Gluœse
Pyruvate
G6P
Lattate
6PG
NAD
NADPH2
Formazan
a
b
CHART5. Two different reaction sequences (a and 6) utilized in the detection of adenylate
sis. The components underlined are contained in the reaction mixtures (5).
is not present in the isoenzyme components e, f, and g, as they
appear to be uninfluenced by this gene substitution, and their
structures are presumably therefore determined by other loci.
Support for this idea has now been obtained (10) by the discovery
of an uncommon variant involving components e, f, and g, but
not affecting a, b, c, or d. This variant was found to segregate
independently from phenotypes 1, 2-1, and 2, and the family
studies indicated that it was determined at a separate and not
closely linked locus (designated PGMÃ-). A number of other
2058
kinase (AK) after starch gel electrophore-
relatively uncommon phosphoglucomutase phenotypes have
also been found during the course of these population and
family studies. They appear to be due to different rare genes, 3
of which are probably alÃ-elesat the PGMt locus, while the other
is probably a rare alÃ-eleat the PG.l/2 locus.
It is also possible that other structurally distinct polypeptide
chains may be contained in these isoenzyme components. These
might for instance account for the mobility differences between
isoenzymes a and c, or between e, f, and g, and would if present
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VOL. 26
Genes and Enzymes in Man
pH 8-5
pH 7-0
[ÎÕÕÕÕTÕÕ1
Hb
Insert
G
nimm umiliili
AK
AK
AK
AK
2-1
2-1
1
1
CHART 6. Diagram of zones of adenylate kinase activity ob
served in hemolysates from individuals of phenotype AK l and
AK 2-1, after starch gel electrophoresis at pH 8.5 and at pH 7.O.
The hemoglobin zone (Hb) is also shown.
imply the existence of further loci involved in the determination
of this enzyme. No doubt structural studies on the isolated
isoenzyme components will enable these questions to be resolved.
Unlike red cell acid phosphatase, phosphoglucomutase occurs
in many different tissues, and it was therefore of some importance
to see whether the isoenzyme components and the polymorphism
found in erythrocytes also occurred elsewhere. It has in fact been
possible to demonstrate that this is the case. The tissues studied
included liver, kidney, muscle, brain, skin, and placenta. The
phosphoglucomutase isoenzymes have also been demonstrated in
tissue culture cells grown in vitro. The tissue cultures were
started from small skin biopsies from different individuals and
were kept going for up to 10 passages for more than 3 months.
The cells were harvested at different times and the phosphogluco
mutase examined. In each case the PGM phenotype found was
the same as that originally observed in the red cells of the donor
whose skin was used to start the culture.
Adenylate
and also skeletal muscle includes several distinct isoenzyme
components, and 2 relatively common phenotypes have been
identified (Chart 6). One of these (AK 1) is observed in about
90% of the English population, while the other (AK 2-1) is
present in about 10%- Family studies make it clear that the
difference between the phenotypes is genetically determined and
in 49 families in which 1 parent was AK 1 and the other AK
2-1, it was found that among the 112 children, 57 were AK l and
55 AK 2-1. In contrast to this, in 62 families where both parents
were AK 1, all the 141 children were also AK 1. These results
and also the segregation pattern in several quite extensive
pedigrees indicate that a pair of autosomal allelic genes (AK1
and AK?) are involved, AK l individuals being homozygotes
(AK1AK1) and AK 2-1 individuals hétérozygotes
(AK1AK?).
If this is so the gene frequency of AK2 in the English population
should be about 0.05 and one would expect the homozygote
AKïAKïto occur with a frequency of about 1 in 400. Several
individuals who are thought to have this uncommon genotype
have indeed been identified. In each case the adenylate kinase
electrophoretic pattern was quite distinctive, most though not
all of the activity being present in a zone with the same mobility
as the most cathodic component in AK 2-1. This work is still in
its early stages, and it remains to be seen how the various
isoenzyme components are related to one another and whether
all or only some of them are involved in the polymorphism.
Placen t al Alkaline
Phosphatase
The various enzymes studied in this screening program were
selected because, among other reasons, they were known to occur
Type
SI
©
pH 8-6
Kinase
The 3rd polymorphism discovered in this screening program
of arbitrarii}^ selected enzymes involves the enzyme adenylate
kinase (5). It catalyzes the reversible reaction1
INSERT
2ADP^±ATP +AMP
and 2 procedures for detecting the enzyme activity after starch
gel electrophoresis have been developed. One of these uses ADP
as substrate, and the other an equimolar mixture of ATP and
AMP. Although each of these procedures require complex and
different multienzyme reaction mixtures (see Chart 5), they both
reveal the same pattern of zones of activity.
It has been found that the enzyme as it occurs in erythrocytes
1The abbreviations
used are: ADP, adenosine diphosphate;
ATP, adenosine triphosphate;
AMP, adenosine monophosphate.
SEPTEMBER
pH6.O
0
INSERT
CHART 7. Placental alkaline phosphatase
electrophoresis at pH 8.6 and pH 6.O.
patterns
obtained
1966
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by
2059
Harry Harris
in the erythrocyte, and this is obviously convenient if one wishes
to carry out population and family investigations. However, the
erythrocyte is a rather specialized cell type, and even though it
is often possible to demonstrate that many red cell enzymes are
present in essentially the same form in other tissues, it might be
considered that a survey restricted to 1 cell type could present a
somewhat biased picture of human enzyme variation in general.
Analogous studies on enzymes localized to other tissues are for
obvious reasons very much more difficult to pursue and so far
have not been carried out in any systematic way. However, it
has been possible to investigate in some detail 1 particular
example of a polymorphism involving what can be regarded as
an organ-specific enzyme, and the results illustrate something of
the possibilities and problems which such enzymes may present.
The enzyme is an alkaline phosphatase present in quite large
amounts in the human placenta. It appears to be peculiar to this
organ and to be different from the alkaline phosphatases present
in other tissues such as liver, kidney, and bone.
Following earlier work by Boyer (2) it has now been possible
to demonstrate that placentae may be classified into at least 6
distinct phenotypes according to the elect rophoretic behavior of
the alkaline phosphatases they contain (19). The electrophoretic
patterns are illustrated in Chart 7, and one may note that com
plete discrimination of the 6 phenotypes requires electrophoresis
at 2 different pHs. The 6 phenotypes are referred to as S, F, I,
SF, SI, and FI. In types S, F, and I most of the alkaline phos
phatase activity is present in a single rapidly moving component,
which, however, has a different mobility in each type. In the I
phenotype, the characteristic component has a mobility very
close to that of the F phenotype at pH 8.6, but is indistinguishable
TABLE 3
OBSERVEDANDEXPECTEDNUMBERSOF 6 PLACENTAL
ALKALINE
PHOSPHATASE
TYPESIN A POPULATIONSAMPLEASSUMINGA
HARDY-WEINBEHG
EQUILIBRIUM
Placenta! alkaline
in
in
incidence"r'2prP22gr2pq5s(p+9+r)20.4100.3460.0730.1150.0490.0081.001Expecte
phosphatase
typeSSFFSIFIITotalsExpected
populationsample135.9114.724.238.216.12.7
populationsample141111283
1p = 0.27, g = 0.09, and r = 0.64 (19).
TABLE 4
PLACENTALALKALINE PHOSPHATASEPHENOTYPESIN 190 DIZYGOTICTWIN PAIRS (19)
twinsLike
Dizygotic
pairsS
SSF
SFF
FSI
SII
IFI
FIUnlike
pairsS
SFS
FS
SIS
IS
FISF
FSF
SISF
ISF
FIF
SIF
IF
FISI
ISI
FII
FIExpected
incidence assuming 3 alÃ-eles
r0.25r2(l
with frequencies p, g, and
inci
dence where p =
0.27, q - 0.09,
Xumbers5337611141124121301471320112078190.04
0.640.27540.19490.02940.05310.00240.01720.18140.01500.06040.00160.01000.05920.03540.0
r =
r)20.5pr[pr
+
r)]0.25p2(l
+ (1 + p)(l +
P)20.5qr{qr
+
r)]0.25<?'(1
+ (1 + ?)(1 +
?)'0.5pq[pq+
+
?)]pr»(l
(1 +p)(l
+
r)0.5p +
V?r2(l
r)O.SgV2pqr*p'r(l
+
p)pqr(l+
2r)pc2rpqr(\
+
2p)PV0.5pVP29d
+
P)?V(1+
q)pqr(l+
2c)P92U
+
?)TotaisExpected
+
190
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Genes and Enzymes in Man
from that of the S phenotype at pH 6.0. In phenotypes SF, SI,
and FI, 3 such components are found; 2 of them in each case
have mobilities similar to the component present in S, F, or I,
while the 3rd has an intermediate mobility. There are reasons to
think that, this 3rd component in these 3 phenotypes may repre
sent a "hybrid" enzyme containing polypeptide chains char
acteristic of the 2 other components present. In each of the 6
phenotypes at least 1 other component which migrates only
very slowly may be seen. These slow components do in fact
exhibit slight differences in mobility in the different phenotypes,
and these can be shown to correlate with the more striking
differences observed in the major and more rapidly moving
•components.
On the basis of the phenotypic patterns and their relative
frequencies, it is possible to construct a simple genetic hypothesis
which will account for these variations. This suggests that 3
autosomal allelic genes are concerned, phenotypes F, I, and S
representing the 3 homozygous genotypes, and phenotypes SF,
SI, and FI the corresponding hétérozygotes.
From the observed
incidence of the 6 phenotypes in the British population, one may
readily obtain values for the frequencies of the 3 postulated
genes. They are 0.27, 0.09, and 0.64, and using these values one
finds a very good agreement between the observed incidence of
the 6 phenotypes and those expected assuming a Hardy-Weinberg equilibrium (Table 3).
It is of course obvious that family studies of the ordinary kind
are impractical in the case of a characteristic peculiar to the
placenta. However, it occurred to us that we might be able to
test the hypothesis by studying a series of pairs of placentae
from dizygotic twins, because such twin pairs can be regarded
as pairs of sibs. We were fortunate in being able to obtain such
material from an extensive investigation of twin births which is
being carried out in the Birmingham area under the general
direction of Dr. John Edwards. So far we have been able to
examine the alkaline phosphatase types in 380 placentae from
190 dizygotic twin pairs. The findings are summarized in Table
4. In 112 pairs the alkaline phosphatase phenotypes were the
same, and in 78 pairs they were different. This result excludes
the possibility that these placental phenotypes are determined
by the maternal genotype, because if this were so none of the
pairs should have shown any differences.
If the phenotypes depend on the fetal genotype then one may
test the hypothesis by calculating the expected incidence of the
different sorts of sib pair using the gene frequencies previously
obtained. This is shown in Table 4, and it will be seen that there
is quite a good agreement between the numbers of the different
sorts of twin pair observed and those expected according to the
hypothesis.
Thus the evidence strongly suggests that these placental
alkaline phosphatase phenotypes are determined by the fetal
genotype and that at least 3 autosomal alÃ-elesare concerned.
The biologic significance of the polymorphism is still quite
obscure, but it might well be of importance in problems con
cerned with maternal-fetal interaction. It may also perhaps be
worth considering other enzyme polymorphisms from this point
of view. The phosphoglucomutase polymorphism, for example,
can also be readily demonstrated in the placenta, and here again
it has been shown that the placental phenotype is determined
by the fetal and not the maternal genotype.
Discussion
In the past it has generally been assumed, at least implicitly,
that the pattern of enzyme synthesis within the species is more
or less standard and uniform. Deviations from this so-called
normal state, such as the enzyme deficiencies characteristic of
the inborn errors of metabolism, could be attributed to deleterious
mutations which were usually kept at a fairly low frequency by
the process of natural selection.
Although the present work is still in its early stages, a rather
different and perhaps in some ways unexpected picture is be
ginning to emerge. In the course of examining some 10 arbitrarily
chosen enzymes, in none of which we had any particular reason
to expect any degree of variation, and not all of which have been
examined in great detail or by perhaps the most suitable methods,
we have come across 3 quite striking examples of enzyme poly
morphism. Although one can hardly draw firm numerical con
clusions from such a small series, it seems likely, unless we have
been excessively lucky in our choice of enzymes, that poly
morphism to a similar degree may be a fairly common phenom
enon among the very large number of enzymes that occur in
the human organism.
This implies a considerable degree of individual diversity, and
some idea of how extensive this may be can be obtained by con
sidering together the various enzymes which are now known to
exhibit some degree of polymorphism in the English population.
Relevant data on 7 such enzymes are given in Table 5. For the
present purpose only those variations where 2 or more allelic
genes have been found to have frequencies greater than 0.01 have
been included. In the case of 1 enzyme, serum cholinesterase,
variation at 2 different loci fall into this category, so that 8 loci
are represented in all. Of these, at least 6 can be regarded as
"structural" loci since the variation produced appears to involve
qualitative differences. In the other 2 cases (serum cholinesterase
E2 and acetyl transferase) only quantitative differences in enzyme
level have so far been identified, but it is possible that these may
also reflect structural differences in the enzyme protein present.
Each of these variations occurs independently of the others so
TABLE 5
ENZYMEPOLYMORPHISM
IN THE ENGLISHPOPULATION
EnzymeRed
of
ofalÃ-eles
2 randomly
with
of
selected
indi
frequency
commonest viduals being erence(11)(21)(19)(18)(5)
greater
phenotype0.430.580.410.500.900.960.900.960.037Probabil
of the same
than0.0132322222Frequency
phenotype0.340.470.310.500.820.920.820.
phosphatasePhosphoglucomutasePlacental
cell acid
alkaline phos
phataseAcetyl
transferaseAdenylate
kinaseSerum
cholinesteraseLocus
EtLocus
EÃŽG-PhosphogluconatedehydrogenaseCombinedNo.
SEPTEMBER 1966
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2061
Harry Harris
that quite a large number of different phenotypic combinations
may be found in the general population. Indeed, the commonest
of these will occur in less than 4% of people, and the probability
that 2 randomly selected individuals would be found to have the
same combination of phenotypes is less than 1 in 70. Thus just
taking into account this very limited series of examples, quite a
high degree of individual differentiation in enzymic make-up is
demonstrable, and it is of interest that most of this is probably
attributable to variation in enzyme structure. Since these various
enzyme combinations are all to be found among individuals whc
can be regarded as normal and healthy, it is apparent that there
must be many different versions of so-called normality. Indeed
one may plausibly imagine that in the last analysis every in
dividual may be found to have a unique enzymic constitution.
Besides the relatively common variations listed in Table 5, a
number of rather less frequent variants of some of these enzymes
have also been discovered during the course of population and
family studies. For example several rare phosphoglucomutase
phenotypes are known (10), and a number of relatively un
common placenta! alkaline phosphatase phenotypes have also
been identified (19). In each case these unusual phenotypes
appear to be determined by rare genes in heterozygous combina
tion with one or another of their common alÃ-eles.Uncommon
variants of serum cholinesterase (9) and of 6-phosphogluconate
dehydrogenase (6) have also been found. One may also note that
extensive surveys have also been carried out on several enzymes
which, at least with the methods used, do not show common
polymorphism, and in a number of cases rare variants have also
come to light here. These include variants of lactate dehydro
genase (3, 4,14,17, 24), red cell esterase (23), carbonic anhydrase
(22), and catalase (1).
Since the screening programs required to uncover such infre
quent variations are necessarily very laborious and often techni
cally difficult, it is not surprising that as yet only a few enzymes
have been looked at in this way. However, one can be reasonably
certain, even from the data available at the moment, that a large
number of such rare variants must exist. The genes which
determine them appear to have frequencies of between about
0.01 and 0.001 in the general population. They are in this respect
similar to the genes which determine many of the classical inborn
errors of metabolism, though they have been discovered in a
quite different way, and they are not necessarily associated with
a reduction in enzyme activity.
Each of the different enzyme polymorphisms and rare variants
which have been mentioned poses a variety of intriguing problems
both in biochemistry and genetics. One would like to know, for
example, what is the precise nature of the structural differences
between the variant forms of a given enzyme and whether these
are reflected in kinetic differences and in differences in functional
activity. It is notable that several of these enzymes apparently
occur in multiple molecular forms even in homozygous indivi
duals. The recognition of these so-called isoenzyme systems is a
fairly new development in enzymology, and the further in
vestigation of these particular examples and of their variant
forms may well help to throw light on the general biologic
significance of this phenomenon.
One would also like to know why these different enzyme
phenotypes occur with the particular frequencies that we observe;
why some occur quite commonly while others are relatively rare;
2062
and why, as is the case, for example, with red cell acid phospha
tase and placental alkaline phosphatase, the gene frequencies
may vary quite widely from one population to another. Pre
sumably selective differences are important here, but at present
we have virtually no idea what these might be. However, one
may reasonably hope that if the metabolic and functional
differences which presumably derive from the various enzyme
differences can be elucidated, this may provide us with some
indication of what selective factors may be important.
References
1. Baur, E. W. Catalase Abnormality in a Caucasian Family in
the United States. Science, HO: 816-17, 1963.
2. Boyer, S. H. Alkaline Phosphatase in Human Sera and Pla
centae. Ibid., 134: 1002-04, 1961.
3. Boyer, S. H., Fainer, D. C., and Watson-Williams,
E. J.
Lactic Dehydrogenase Variant from Human Blood: Evidence
for Molecular Subunits. Ibid., 141: 642-43, 1963.
4. Davidson, R. G., Fildes, R. A., Glen-Bott, A. M., Harris, H.,
Robson, E, B., and Cleghorn, T. E. Genetical Studies on a
Variant of Human Lactate Dehydrogenase (Subunit A). Ann.
Human Genet. London, 29: 5-17, 1965.
5. Fildes, R. A., and Harris, H. Genetically Determined Varia
tion of Adenylate Kinase in Man. Nature, 209: 261-63, 1966.
6. Fildes, R. A., and Parr, C. W. Various Forms of Human
Erythrocyte Phosphogluconate
Dehydrogenase, p. 229. Sixth
Intern. Congr. Biochem., New York, 1964.
7. Harris, H. The "Inborn Errors" Today. In: Garrod's Inborn
Errors of Metabolism, pp. 120-97. London: Oxford University
Press, 1963.
8. Harris, H., Hopkinson, D. A., Robson, E. B., and Whittaker,
M. Genetical Sgudies on a New Variant of Serum Cholinester
ase Detected by Electrophoresis. Ann. Human Genet. London,
Õ6:359-82, 1963.
9. Harris, H., and Whittaker,
M. Differential
Inhibition
of
Human Serum Cholinesterase with Fluoride: Recognition of
Two New Phenotypes. Nature, 191: 496-98, 1961.
10. Hopkinson, D. A., and Harris, II. Evidence for a Second
'Structural' Locus Determining Human Phosphoglucomutase.
Ibid., 208: 410-12, 1965.
11. Hopkinson, D. A., Spencer, N., and Harris, H. Red Cell Acid
Phosphatase Variants: a New Human Polymorphism. Ibid.,
199: 969-71, 1963.
12.
. Genetical Studies on Human Red Cell Acid Phos
phatase. Am. J. Human Genet., 16: 141-54, 1964.
13. Kalow, W., and Staron, N. On the Distribution
and Inheri
tance of Atypical Forms of Human Serum Cholinesterase as
Indicated by Dibucaine Numbers. Can. J. Biochem. Plrysiol.,
35: 1305-20, 1957.
14. Kraus, A. P., and Neely, C. L. Human Erythrocyte Lactate
Dehydrogenase:
Four Genetically
Determined
Variants.
Science, 145: 595-97,1964.
15. Lai, L., Nevo, S., and Steinberg, A. G. Acid Phosphatase of
Human Red Cells: Predicted Phenotype Conforms to a Gen
etic Hypothesis. Ibid., 145:1187-88, 1964.
16. Motulsky, A. Hereditary Red Cell Traits and Maleria. Am. J.
Trop. Med. Hyg., IS: 147-55, 1964.
17. Nance, W. E., Claflin, A., and Smithies, C. Lactate Dehy
drogenase: Genetic Control in Man. Science, 142: 1075-77,
1963.
18. Price Evans, D. A., and White, T. A. Human Acetylation
Polymorphism. J. Lab. Clin. Med., 63: 394-403, 1964.
CANCER
RESEARCH
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Genes and Enzymes in Man
19. Robson, E. B., and Harris, H. Genetics of the Alkaline Phosphatase Polymorphism of the Human Placenta. Nature, 207:
1257-59, 1965.
20. Spencer, N., Hopkinson, D. A., and Harris, H. Quantitative
Differences and Gene Dosage in the Human Red Cell Acid
Phosphatase Polymorphism. Ibid., 201: 299-300, 1964.
21.
. Phosphoglucomutase
Polymorphism
in Man. Ibid.,
204: 742-45, 1964.
22. Tashian, R. E., Plato, C. C., and Shows, T. B., Jr. Inherited
Variant of Erythrocyte Carbonic Anhydrase in Micronesians
from Guam and Saipan. Science, 140: 53-54, 1963.
23. Tashian, R. E., and Shaw, M. W. Inheritance of an Erythrocyte Acetylesterase Variant in Man. Am. J. Human Genet.,
14: 295-300, 1962.
24. Vessel, E. S. Polymorphism of Human Lactate Dehydrogenase
Isoenzymes. Science, ¡48:1103-05, 1965.
SEPTEMBER 1966
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2063
Genes and Enzymes in Man
Harry Harris
Cancer Res 1966;26:2054-2063.
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