Leader
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 DEFICIENCY
Pyruvate kinase (PK) catalyses the conversion
TP.
DHAP .-
-
I-I
I
X|
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,
M.McMullinmqub.ac.uk 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
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.
has the one splice site mutation which has been 2 Miwa S, Hisaichi F. Molecular basis of erythroenzymopa-
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.
seems probable that total absence of G6PD 3 Baronciani L, Magalhaes IQ, Mahoney DH, et al. Study of
the molecular defects in pyruvate kinase deficient patients
activity would be incompatible with life. affected by nonspherocytic hemolytic anemia. Blood Cells
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
1997;89:4213-18.
Thus some of these mutations may have a sin- 5 Lenzner C, Nurnberg P, Jacobasch G, et al. Molecular
gle origin. Despite the fact that G6PD analysis of 29 pyruvate kinase-deficient patients from cen-
tral Europe with hereditary hemolytic anemia. Blood 1997;
deficiency was initially characterised biochemi- 89:1793-9.
cally and variants from different areas were 6 Zanella A, Bianchi P, Baronciano L, et al. Molecular charac-
terization of PK-LR gene in pyruvate kinase-deficient Ital-
given individual names, molecular analysis has ian patients. Blood 1997;89:3847-52.
shown that many of the so called variants are 7 Bianchi M, Magnani M. Hexokinase mutations that
produce nonspherocytic hemolytic anemia. Blood Cells Mol
the same. Dis 1995;21:2-8.
244 McMullin
8 Kanno H, Fujii H, Hirono, et al. Molecular analysis of glu- 13 Toren A, Brok-Simoni F, Ben-Bassat I, et al. Congenital
cose phosphate isomerase deficiency associated with haemolytic anaemia associated with adenylate kinase
hereditary hemolytic anemia. Blood 1996;88:2321-5. deficiency. BrJ Haematol 1994;87:376-80.
9 Baronciani L, Zanella A, Bianchi P, et al. Study of the 14 Beutler E. G6PD: population genetics and clinical manifes-
molecular defects in glucose phosphate isomerase-deficient tations. Blood Rev 1996;10:45-52.
patients affected by chronic hemolytic anemia. Blood 1996; 15 Chang J-G, Liu T-C. Glucose-6-phosphate dehydrogenase
88:2306-10. deficiency. Crit Rev Oncol Hematol 1995;20: 1 -7.
10 Schneider A, Westwood B, Yim C, et al. Triosephosphate 16 Mason PJ. New insights into G6PD deficiency. BrJ Haema-
isomerase deficiency: repetitive occurrence of point muta-
tion in amino acid 104 in multiple apparently unrelated tol 1996;94:585-91.
families. Am Hematol 1995;50:263-8. 17 Mason PJ, Sonati MF, MacDonald D, et al. New glucose-6-
11 Arya R, Lalloz MRA, Bellingham, et al. Evidence for phosphate dehydrogenase mutations associated with
founder effect of the Glul04Asp substitution and identifi- chronic anemia. Blood 1995;85:1377-80.
cation of new mutations in triosephosphate isomerase defi- 18 Lee GR, Bithell TC, Foerster J, et al. Wintrobe's clinical
ciency. Hum Mutat 1997:10:290-4. hematology, 9th ed. Philadelphia: Lea and Febiger, 1993:
12 Turner G, Fletcher J, Elber J, et al. Molecular defect of a 990-1022.
phosphoglycerate kinase variant associated with haemolytic 19 Hirono A, Iyori H, Sekine I, et al. Three cases of hereditary
anaemia and neurological disorders in a large kindred. BrJ7 nonspherocytic hemolytic anemia associated with red
Haematol 1995;91:60-5. blood cell glutathione deficiency. Blood 1996;87:2071-4.