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702 Part VI: The Erythrocyte Chapter 47: Erythrocyte Enzyme Disorders 703

Table 47–3 includes deficiencies that may cause hemolytic anemia but c.202G>A mutation has been found in a patient to cause deficiency
for which a cause-and-effect relationship has not been clearly estab- without the presence of the mutation at cDNA nucleotide (nt) 376. 274
lished, such as those of phosphogluconolactonase, enolase, 115,116 and Variants in the Mediterranean Region Among white popula-
163
glutathione-S-transferase. 228 tions, G6PD deficiency is most common in Mediterranean countries.
Patients with unstable hemoglobins (Chap. 49) may present with The most common enzyme variant in this region is G6PD Mediterra-
the clinical picture of hereditary nonspherocytic hemolytic anemia. nean. 270,277 The enzyme activity of the red cells of individuals who have
Hemolytic anemia resulting from abnormalities in the lipid composi- inherited this abnormal gene is barely detectable. Other variants are
tion of the red cell membrane, particularly increased phosphatidyl cho- also prevalent in the Mediterranean region, including G6PD A– and
line, occurs rarely (Chap. 46). G6PD Seattle (see Table 47–4).
Variants in Asia A great many different variants have been
Glucose-6-Phosphate Dehydrogenase described in Asian populations. Some of these proved to be identical
The “normal” or wild-type enzyme is designated as G6PD B. Many vari- at a molecular level (e.g., G6PD Gifu-like, Canton, Agrigento-like,
ants of G6PD have been detected all over the world, associated with a and Taiwan-Hakka all have the same mutation at cDNA nt 1376 [see
wide range of biochemical characteristics and phenotypes. Accordingly, Table 47–4]). DNA analysis shows that more than 100 different muta-
five classes of G6PD variants can be distinguished based on enzymatic tions are found in various Asian populations. 160,278
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activity and clinical manifestations (Table 47–4). Before it became Variants Producing Hereditary Nonspherocytic Hemolytic
possible to characterize G6PD variants at the DNA level, they were dis- Anemia Some mutations of G6PD result in chronic hemolysis with-
tinguished from each other on the basis of biochemical characteristics, out, but exacerbated by, precipitating causes. These variants are class I
265
such as electrophoretic mobility, K for NADP and glucose-6-P, ability mutants (World Health Organization [WHO] class 1). From a func-
m
to use substrate analogues, pH activity profile, and thermal stability. To tional point of view, these mutations are more severe than the more
facilitate comparison of variants characterized in different laboratories, commonly occurring polymorphic forms of the enzyme, such as G6PD
international standards for the methodology were established. In the Mediterranean and G6PD A–. On a molecular level, such variants are
266
case of the common G6PD A– and G6PD Mediterranean mutations, the often caused by mutations located in exons 10 and 11, encoding the sub-
abnormal enzyme may be synthesized at normal or near-normal rates unit interface, or affect residues that bind the structural NADP mole-
267
but has decreased stability in vivo. The amount of enzyme antigen in cule. 143,158 There are, however, exceptions to this rule. 28,279–281 The clinical
the red cells declines concurrently with enzyme activity. This suggests severity of these variants can be quite variable. 282
268
that the mutant protein in these variants is rendered unusually sensitive G6PD deficiency has also been encountered in the rat, dog,
283
to proteolysis in the environment of the erythrocyte. Other mutations mouse, and horse. G6PD deficiency in mice has been rescued by
284
269
285
also result in the formation of enzyme molecules with decreased enzyme stable in vivo expression of the human G6PD gene in hematopoietic tis-
activity and with altered kinetic properties, some of which may ren- sues by a gene transfer approach. 271,286
270
268
der them functionally inadequate. By far the majority of mutations
158
(85 percent) are missense mutations causing the substitution of a single Pyruvate Kinase
amino acid. More severe mutations such as frameshift and nonsense PK deficiency is the second most common enzyme disorder in glycoly-
158
mutations have not been found, indicating that some residual activity is sis and the most common cause of nonspherocytic hemolytic anemia.
287
required for survival. In agreement with this, targeted deletion of G6PD Like G6PD deficiency, the disease is genetically heterogeneous, with
in the mouse causes embryonic lethality. 271 different mutations causing different kinetic changes in the enzyme
Detailed biochemical and genetic characteristics of some 400 puta- that is formed. There are even cases in which the activity of PK as mea-
tively distinct G6PD variants and more than 200 different mutations sured in vitro is higher than normal, but a kinetically abnormal enzyme
288
have been tabulated. 155,160 Table 47–4 lists common G6PD variants that is responsible for the occurrence of hemolytic anemia. Kinetic char-
have reached polymorphic frequencies in certain populations. acterization and analysis of PK mutants is considerably more complex
African Variants Among persons of African descent, a mutant than analysis of G6PD mutants. Most PK-deficient patients are com-
enzyme G6PD A+, with normal activity is polymorphic. It migrates pound heterozygous for two different (missense) mutations, rather than
electrophoretically more rapidly than the normal B enzyme, has sub- homozygous for one. Assuming that stable mutant monomers are syn-
stitution of Asn to Asp at codon 126, resulting from nucleotide change thesized, up to seven different tetrameric forms of PK may be present
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c.376A>G. G6PD A– is the principal deficient variant found among in compound heterozygous individuals, each with distinct structural
people of African origin. The red cells contain only 5 to 15 percent of the and kinetic properties. This complicates genotype-to-phenotype cor-
normal amount of enzyme activity; however because of the instability of relations in these individuals as it is difficult to infer which mutation
the enzyme, the age-dependent decline of the activity renders old red is primarily responsible for deficient enzyme function and the clinical
cells severely deficient and susceptible to hemolysis. These two electro- phenotype. 289,290 More than 230 mutations in the PKLR gene encoding
phoretically rapid variants are common in African populations have in the red cell PK have been identified. Seventy percent of these mutations
common a nucleotide substitution at cDNA nucleotide 376 that pro- are missense mutations affecting conserved residues in structurally and
duces the amino acid substitution responsible for the rapid electropho- functionally important domains of PK. There appears to be no direct
retic mobility. Most samples with G6PD A– manifest an additional in cis relationship between the nature and location of the substituted amino
G>A mutation at cDNA nucleotide 202 (c.202G>A; p.Val68Met), which acid and the type of molecular perturbation. Hence, the nature of the
124
273
accounts for its in vivo instability. Less commonly, the additional mutation has relatively little predictive value with respect to the severity
273
mutation is at a different site (c.680G>T or c.968T>C). Thus G6PD of the clinical course and the phenotypic expression of identical muta-
A– arose in an individual who already had the G6PD A+ mutation. tions can be strikingly different in patients. 29,289–291
However, the ancestral human sequence has been deduced to be that Apart from decreased red blood cell survival ineffective erythro-
of G6PD B, both by showing that this is the sequence of the chimpan- poiesis because of increased numbers of apoptotic cells is implicated as
274
zee, our nearest relative, and by analysis of linkage dysequilibrium. one of the pathophysiologic features of PK deficiency. 292,293 In particular,
275
Although it has been suggested that only the interaction of p.Val68Met glycolytic inhibition by mutation of PKLR has been suggested to aug-
276
and p.Asn126Asp invariably results in G6PD A– deficiency, the ment oxidative stress, leading to proapoptotic gene expression. 293

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