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J Clin Pathol 1999;52:241-244 241


The molecular basis of disorders of red cell


Mary F McMullin

The mature red cell has no nucleus or for example a missense mutation in codon 510
organelles and therefore cannot synthesise pro- (CGA;Arg -e CAA;Gln) is reported in 30% of
tein or lipids. It is totally dependent on glycoly- cases in Northern European and United States
sis to convert glucose into an energy source. populations. Despite patients having the same
Glucose is phosphorylated by hexokinase to genetic abnormality there can be wide variation
glucose-6-phosphate. This is the substrate for in the clinical expression of the disease. Thus
anaerobic glycolysis which proceeds through other genetic or environmental factors influ-
the Embden-Meyerhof pathway to pyruvate ence the phenotype.5 Molecular diagnosis is
with production of ATP (fig 1). Some of the now nearly routine and carrier detection and
glucose-6-phosphate may also be processed by prenatal diagnosis is feasible.
oxidative glycolysis in the pentose phosphate
pathway (fig 2). Around 20 enzymes facilitate
these two major pathways and deficiency of any
of these enzymes may cause haemolytic
anaemia. The incidence of deficiency varies Glucose
from over 400 million cases worldwide in Pentose phosphate
glucose-6-phosphate dehydrogenase (G6PD) Glucose-6-P pathway
deficiency to single case report in some of the (oxidative
other enzymes. I shall discuss the molecular glycolysis)
basis of some of these enzyme deficiencies in Fructose-6-P
this review.
Fructose-1 ,6-P
Deficiency of enzymes involved in the Aldolase|
Embden-Meyerhof pathway
Pyruvate kinase (PK) catalyses the conversion

of phosphoenolpyruvate (PEP) to pyruvate. NAD G3PD
The enzyme exists as four isoenzymes. PR-L is NADH 1,3-BPG 2 3 BPGM
expressed in the liver and PK-R in red cells. AT~~~~~
These two forms are encoded by a single gene,
PK-LR, located at 1 q21 but tissue specific pro- APv3-IG 2,3BPGP
moters generate mRNAs with different 5' end PGM
sequences. PK-M, is the form in muscle and
brain, and PK-M2 is present in fetal and most 2-PG
adult cells including white blood cells and Enolase
platelets. It is encoded by the PK-M gene ADP 2]I-PEP__
located at 15q22 and the isoforms are pro- ATP | _
duced by alternative splicing. During erythroid Pyruvate
differentiation the PK-MK form is progressively LDH
replaced by PK-R.' Lactate
Department of In PK deficiency PK-R is functionally defec-
Haematology, The tive. PK deficiency presents as a non- Figure 1 The Embden-Meyerhof pathway for anaerobic
Queen's University of glycolysis in the erythrocyte. Boxed enzymes cause
Belfast, Institute of spherocytic haemolytic anaemia which can be haemolytic disease when deficient. ADP, adenosine
Clinical Science, Royal anything from mild compensated haemolysis to diphosphate;ATP, adenosine triphosphate; DHAP,
Victoria Hospital, severe anaemia. Approximately 400 patients dihydroxyacetone phosphate; G-3-P,
glyceraldehyde-3-phosphate; G3PD,
Grosvenor Road, with the deficiency have been described and glyceraldehyde-3-phosphate dehydrogenase; GPI, glucose
Belfast BT12 6BA, UK the inheritance is autosomal recessive. Nearly phosphate isomerase; HK, hexokinase; LDH, lactate
M F McMullin 100 gene mutations have been identified.2 6 dehydrogenase; NAD, nicotinamide adenine dinucleotide;
NADH, reduced NAD; PFK, phosphofructokinase; PGK,
Correspondence to: The majority are missense mutations but splic- phosphoglycerate kinase; PGM, phosphoglycerate mutase;
Dr McMullin. ing mutations, insertions, and deletions also PK, pyruvate kinase; TPI, triosephosphate isomerase;
email: occur. The mutations are widely distributed 1,3-BPG, 1,3-bisphosphoglycerate; 2-PG, 2-phosphoglycerate; 2-PEPR 2-phosphoenolpyruvate; 2,3
throughout the gene. Although most mutations BPG, 2,3-biphosphoglycerate; 2,3 BPGM,
Accepted 23 October 1998 occur only once, some appear relatively often; 2,3-biphosphoglycerate mutase; 3-PG, 3-phosphoglycerate.
242 McMullin


Phosphofructokinase (PFK) catalyses the con-
version of fructose-6-phosphate to fructose 1-6
phosphate. Over 35 cases of PFK deficiency
G peroxidase have been reported. There are different genes
for the different PFK proteins in muscle
(PFK-M), liver (PFK-L), and platelets (PFK-
GSH GSSG P). It is a multisystem disease, the expression of
which varies owing to the variable expression in
the tissues. Manifestations include haemolytic
GSSG reductase anaemia and myopathy. The commonest form
is Tarui's disease or glycogen storage disease
type VII. At least 11 mutations of the PFK-M
Glucose gene, which is located at 1 cen-q32, have been
NADP NADPH described-splicing defects, nucleotide dele-
tions, and point mutations.'
Triosephosphate isomerase (TPI) catalyses the
Embden- phosphate 6PGD
interconversion of dihydroxyacetone phos-
Meyerhof pathway phate and glyceraldehyde-3-phosphate. Over
pathway (oxidative) 35 cases of TPI deficiency have been reported.
(glycolytic) Pentose It presents as a multisystem disorder with pro-
phosphate gressive neurological dysfunction, cardiomy-
opathy, and increased susceptibility to infec-
tion. Affected individuals die in early
childhood. The inheritance is autosomal reces-
sive but it is interesting, given the rarity of the
disorder, that 5% of African Americans are
heterozygotes. The gene is encoded at l2p13.
Figure 2 The pentose phosphate pathway for red cell Although mutations have been described in
oxidative glycolysis. G6IP glucose-6-phosphate; G6PD,
glucose 6-phosphate dehydrogenase; GSH, glutathione; single families, the majority of cases are due to
GSSG, oxidised glutathione; H202, hydrogen peroxide; a missense mutation, GAG;Glu -e GAC;Asp,
NADP1 nicotinamide adenine dinucleotide phosphate; 6PG, in codon 104, in contrast to the genetic
6-phosphogluconolactone; 6PGD, 6-phosphogluconolactone heterogeneity of other disorders."' There is evi-
dence for a single origin for this mutation in a
single ancestor."
Hexokinase catalyses the first step in the PHOSPHOGLYCERATE KINASE DEFICIENCY
Embden-Meyerhof pathway, the conversion of Phosphoglycerate kinase (PGK) catalyses the
glucose to glucose-6-phosphate. The enzyme is interconversion of 1,3 diphosphoglycerate and
located at lOpl 1.2. Approximately 20 cases of 3-phosphoglycerate. Approximately 20 cases of
hexokinase deficiency have been described and PGK deficiency have been described. Inherit-
the patients present with non-spherocytic ance is X linked, the gene being located at
haemolytic anaemia. It is an autosomal reces- Xql3. Affected males usually present with
sive disorder. The molecular defect has been haemolytic anaemia and neurological abnor-
described in at least one patient in whom there malities, including mental retardation and
was a point mutation in one allele of the myopathy. Females have mild haemolytic anae-
hexokinase gene and a deletion in the other mia owing to the lyonisation of the X chromo-
allele producing clinical disease.7 some. At least nine mutations have been
described, each resulting in a unique point
Glucose phosphate isomerase (GPI) catalyses OTHER ENZYME DEFICIENCIES
the conversion of glucose-6-phosphate to Deficiencies of the enzymes aldolase, enolase,
fructose-6-phosphate. The GPI gene is located 2,3-bisphosphoglycerate mutase (2,3-BPGM),
at l9cen-q13. Over 45 cases of GPI deficiency and lactate dehydrogenase (LDH) have all
have been described, generally presenting with been reported in isolated cases, and haemolysis
compensated haemolytic anaemia but in one has been described as part of the deficiency.
case associated with hydrops fetalis and in two Molecular abnormalities have now been de-
cases associated with mental retardation. The scribed in all of these deficiencies.'
inheritance is autosomal recessive and cases are
compound heterozygotes or homozygotes. Ap- Abnormalities of erythrocyte nucleotide
proximately 20 mutations have been described metabolism
which are distributed throughout the gene.8 9 Nucleotide metabolic pathways are essential
These are missense mutations, deletions, and for maintenance of the energy pool of red cells.
nonsense mutations. GPI is identical to neu- Pyrimidine 5' nucleotidase (P5'N), adenosine
roleptin, a neurotrophic factor for spinal and deaminase, and adenylate kinase are vital to
sensory neurones, and may have a function nucleotide cycling, and abnormalities of these
outside carbohydrate metabolism. enzymes are associated with decreased red cell
The molecular basis of disorders of red cell enzymes 243

survival. P5'N is the most common of these The situation with the sporadic variety is dif-
disorders but the precise molecular defect has ferent. The same mutation appears to have
not been clarified because the gene has not arisen independently in cases in different
been identified. areas.'7 This suggests that mutations capable of
Adenosine deaminase deficiency has been causing chronic haemolysis are restricted to a
detected in red cells as part of autosomal small number of changes that can produce an
recessive severe combined immunodeficiency. enzyme with low red cell activity but consider-
Adenylate kinase deficiency has also rarely able activity in other tissues.
been described as a cause of haemolytic anae- Three dimensional models of human G6PD
mia and in some cases mental retardation."3 have allowed some insight into how mutations
The molecular defect is not yet clear. can cause clinical disease.'6 In the sporadic
form many of the mutations are situated at the
Deficiency of enzymes involved in the dimer interface and affect NADP binding.
pentose phosphate pathway Binding NADP causes increased stability of the
GLUCOSE-6-PHOSPHATE DEHYDROGENASE dimeric form, and mutations affecting dimer
By far the most common abnormality occur- formation or stability have a dramatic effect on
ring in the pentose phosphate pathway is a NADP binding.
defect in glucose-6-phosphate dehydrogenase The polymorphic mutations are not associ-
(G6PD). This enzyme oxidizes glucose-6- ated with any one area of the tertiary structure.
phosphate (G6P) to 6-phosphogluconolactone However, G6PDA- mutations result in loss of
(6PG) with concomitant reduction of nicotina- folding of the protein and this form may have a
mide adenine dinucleotide phosphate (NADP) shorter half life and account for the disease
to the reduced form, NADPH. (fig 2) In the phenotype. Further research in this area may
red cell this is the only source of NADPH elucidate the relation between mutations and
which maintains glutathione (GSH) in the the presenting disease.
reduced state. GSH in turn reduces peroxides
and other reactive oxygen species. G6PD is OTHER ENZYME DEFICIENCIES
therefore essential to protect the red cell from Two enzymes are required for glutathione
oxidative damage. (GSH) synthesis, y-glutamyl synthetase and
The G6PD gene is located on the long arm GSH synthetase, and reduced activity of these
of the X chromosome (Xq28). It consists of 13 enzymes has been associated with mild haemo-
exons which encode a 515 amino acid protein lysis with and without other more generalised
with a molecular weight of 59 kDa. abnormalities in isolated cases.8 19 The mo-
The active form of the enzyme exists as a lecular defect in individual cases has not been
dimer or tetramer and tightly binds NADP. isolated.
G6PD deficiency affects over 400 million peo- Glutathione reductase and glutathione per-
ple worldwide. In most cases it exists as a bal- oxidase levels have been reported as decreased
anced polymorphism, affected individuals hav- in various nutritional deficiencies and in
ing the advantage of resistance to malaria. isolated case reports. No definite link to
These people are asymptomatic unless exposed haemolysis has been established. Likewise
to an agent which precipitates an episode of 6-phosphogluconate dehydrogenase (6PGD)
acute haemolysis.'4 A small subset of cases, deficiency has been well documented but has
usually with more severe chronic haemolysis, no significance for red cell survival.
occurs sporadically worldwide.
The vast majority of abnormalities in the Conclusion
G6PD gene are point mutations causing single It is evident that functionally deficient enzymes
amino acid substitutions. This contrasts with can cause characteristic disorders of the red
many other genetic disorders and implies that cell, although many such enzymes have a more
only disorders with some residual G6PD activ- widespread occurrence in the body. Molecular
ity exist. Five small deletions have been analysis of many of these defects has now
described in which the number of bases deleted become semi-routine and opens avenues for
is a multiple of 3, leading to the deletion of a prenatal diagnosis for the first time and the
few amino acids and residual enzymic activity. hope of cure with gene therapy.
The one stop codon mutation reported which
results in a truncated protein of 427 amino 1 Arya, R, Layton DM, Bellingham AJ. Hereditary red cell
acids exists only in the heterozygous state, as enzymopathies. Blood Reviews 1995;9:165-75.
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thies associated with hereditary anemia: tabulation of
found to have residual enzymic activity.'5 16 It mutant enzymes. Am J Hematol 1996;51:122-32.
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the molecular defects in pyruvate kinase deficient patients
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In some populations the gene frequency of Mol Dis 1995;21:49-55.
4 Kanno H, Fujii H, Wei DC, et al. Frame shift mutation,
G6PD deficiency is very high, but very few exon skipping, and a two-codon deletion caused by splice
mutations may exist in individual populations. site mutations account for pyruvate kinase deficiency. Blood
Thus some of these mutations may have a sin- 5 Lenzner C, Nurnberg P, Jacobasch G, et al. Molecular
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tral Europe with hereditary hemolytic anemia. Blood 1997;
deficiency was initially characterised biochemi- 89:1793-9.
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terization of PK-LR gene in pyruvate kinase-deficient Ital-
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produce nonspherocytic hemolytic anemia. Blood Cells Mol
the same. Dis 1995;21:2-8.
244 McMullin

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