Hexokinase (EC 220.127.116.11; HK) is a regulatory enzyme of the Embden-Meyerhof pathway, which catalyzes the phosphorylation of glucose to glucose-6-phosphate (G-6-P) by ATP (see Fig. 182-1). There are four major isozymes of human HK, each encoded by a separate gene (Table 182-2). HK-I is a monomer of 108 kDa that is ubiquitously expressed in many tissues including erythrocytes. It is strongly inhibited by G-6-P, glucose-1,6-diphosphate, and 2,3-DPG, and partially activated by Pi. HK-II and -III are major forms in muscle and fat tissues, and liver, lung and spleen, respectively. The liver isozyme HK-IV, or glucokinase, has higher Km for glucose and is not inhibited by G-6-P. The HK-I gene is located at 10q22193 and its 18 exons span more than 75 kb.194 Human red cells have another isozyme HKR that appears to be specific for erythrocyte.195 Both HK-I and HKR show similar kinetics for glucose and ATP. HKR, which has a half-life of about 10 days, is the major form in reticulocytes, whereas HK-I is more stable with a half-life of about 66 days, and predominates in mature erythrocytes.195 The different half-life of each isozyme may explain the biphasic decay196,197 of total HK activity during erythrocyte maturation.195 HKR cDNA198 is identical to HK-I cDNA199 except for the 5′ end of the coding sequence and upstream noncoding sequence. Human HK-I and HKR thus appears to be produced from a single gene using different tissue-specific promoters.194
Since the first description by Valentine et al.200 in 1967, HK deficiency has been reported in 18 cases from 17 families (see Table 182-1).200– 216 The clinical manifestations are characterized by chronic hemolytic anemia of variable severity (median Hb: 9.7 g/dl, range: 6.5 to 13.8; n = 14). Splenectomy is beneficial and may increase hemoglobin concentrations by 2 to 3 g/dl in association with marked reticulocytosis. Three propositi also showed multiple malformations204,208 and/or psychomotor retardation.208,214 Causal relationship between these symptoms and HK deficiency is unclear. The inheritance of HK deficiency is autosomal recessive.
Because HK is an age-dependent enzyme, it shows much higher activity in reticulocytes than in mature cells. HK activities in affected erythrocytes are 42 to 179 percent of normal controls, but are only 16 to 75 percent when compared with controls with a similar degree of reticulocytosis. This makes diagnosis of HK deficiency difficult in some situations. Calculation of the HK/PK ratio or HK/G-6-PD ratio is useful to avoid the false negative result.200 Reduced levels of 2,3-DPG and G-6-P in erythrocytes are the common metabolic features. ATP is modestly decreased if patients' reticulocytosis is taken into account.
The molecular study of HK deficiency has just begun. A missense mutation 1667T→C (Leu529Ser), and a 96-bp (577 to 672) deletion resulting in deletion of 32 amino acids (166 to 197) have been identified in a compound heterozygous Italian patient with HK “Melzo”.217 The latter is possibly caused by aberrant splicing. An expression study revealed that Leu529Ser completely abolished the HK activity, and the 32-residue deletion drastically reduced the activity.218 The molecular defect of a HK deficient fetus has been reported.216 The fetus showed intrauterine growth retardation and severe hemolytic anemia (Hb: 3.7 g/dl; reticulocyte counts: 42.1 percent) with marked reduction of erythrocyte HK activity (17 percent of a normal fetus), and died in utero at 37 weeks of gestation. Analysis of cDNA revealed a 0.5-kb and a 1.8-kb deletion in each transcript, which corresponded to exons 5 to 8, and 3 to 14 in the HK-1 gene, respectively.
Glucosephosphate Isomerase Deficiency
Glucosephosphate isomerase (EC 18.104.22.168; GPI) catalyzes the interconversion of G-6-P and fructose-6-phosphate (F-6-P) (see Fig. 182-1). Tissue-specific isozymes of GPI do not exist. A homodimer composed of two identical 63-kDa subunits is expressed in all human tissues (Table 182-2). The gene encoding human GPI located on the long arm of chromosome 19219 spans in excess of 40 kb including 18 exons.220,221 The cDNA sequence coding 558 amino acids has been published (GenBank K03515). It is noteworthy that GPI may also function as a bioactive polypeptide. Neuroleukin,222 a neurotrophic factor and a lymphokine, is known to be identical with GPI monomer.223,224 Interestingly, dimeric GPI, which is active as an enzyme, has no neuroleukin activity.102 Recently, a tumor-secreted cytokine that stimulates cell migration and metastasis (autocrine motility factor) in vivo,225,226 and a maturation mediator227 that is capable of differentiating human myeloid leukemic cells to terminal monocytic cells have been reported to be closely related to GPI.
Since the first report by Baughan et al.,228 hereditary hemolytic anemia associated with GPI deficiency has been described in 74 cases from 60 kindreds.228– 274 It is the third most common erythroenzymopathy after G-6-PD deficiency and PK deficiency. The inheritance of GPI deficiency is autosomal recessive (Table 182-1). The GPI activity of erythrocytes of affected patients is reduced to 7 to 60 percent (median: 22.5 percent; n = 58) of normal. The G-6-P/F-6-P ratio is significantly elevated in affected erythrocytes. Benign polymorphic variants of GPI have also been reported.275
The main clinical manifestation of GPI deficiency is chronic hemolytic anemia of variable severity (median Hb: 9.8 g/dl, range: 4.5 to 16.8; n = 49). Affected individuals often exhibit hemolytic crises triggered by viral or bacterial infections.273 It has been proposed that these episodes might be due to the metabolic block at the F-6-P→G-6-P step, which prevents pentose from cycling more than once through the HMP shunt.228 Impaired recycling was confirmed by in vitro experiments using [2-14C] glucose in three cases,228,229,242 but in another study this defect could not be confirmed.276 Unlike G-6-PD deficiency, which also causes impaired shunt activity, drug-induced hemolysis is quite rare in GPI deficiency and was observed in only two cases.242,243 Hydrops fetalis seems more common in GPI deficiency than other enzyme deficiencies.255,263,277 Splenectomy results in significant clinical improvement particularly in childhood. The enzyme deficiency is also found in nonerythroid tissues. A few patients showed manifestations suggestive of dysfunction of such tissues, including mental retardation,243,257 neuromuscular symptoms,243,257,261,278 and impaired granulocyte function.261,264,278
Molecular analysis has been performed in 22 propositi (10 homozygotes and 12 compound heterozygotes), and 23 different mutations, scattered throughout the coding sequence, have been identified.266,268-272,279 So far overall heterogeneity of GPI mutations is remarkable compared with PK or triosephosphate isomerase (TPI) mutations. Even the most frequent mutation, 1039C→T (Arg347Cys), has been found in no more than five unrelated patients.268,272 What is more surprising is that there is evidence indicating that the same mutation has arisen independently more than once.272,280 In 6 of 12 compound heterozygous variants, including Elyria,238,272 Nordhorn,102,239,271,281 Bari,269 Mola,269 and Zwickau,102,271 one allele is inactive bearing either a nonsense mutation or a splicing mutation. In GPI Nordhorn, the existence of an inactive allele had been predicted from its biochemical properties.239,281 A study using recombinant mutants suggests that the impaired kinetics as well as the instability may cause metabolic perturbations in affected cells.282 An animal model of GPI deficiency has been developed in mice.283
Phosphofructokinase (EC 22.214.171.124; PFK) catalyzes the phosphorylation of F-6-P to fructose-1,6-diphosphate (F-1,6-DP2) by ATP. PFK activity is considered to play a major role in the regulation of the rate of glycolysis. PFK is a typical allosteric enzyme that is inhibited by various metabolic intermediates, including ATP, citrate, and 2,3-DPG.284 Human PFK is a tetramer of 340 kDa with variable combinations of three unique subunits, muscle (M), liver (L), and platelet (P) types (Table 182-2).285– 288 These subunits are encoded by separate loci (M: 1cen-q32;289 L: 21q22.3;290 and P: 10p15291), and are ubiquitously expressed, but in a tissue-specific manner. Human muscle and liver express homotetramers M4 and L4, respectively, while erythrocytes express M and L subunits composing five tetramers, M4, M3L, M2L2, ML3, and L4.289 M4 is less sensitive to inhibition by ATP and manifests a greater affinity for F-6-P than does L4,288 which might be advantageous in muscle and probably in erythrocytes. The cDNAs for the M292 and L293 subunits, both encoding 779 amino acids have been cloned. The 24 exons of the human M subunit gene spans 30 kb,294 and the L subunit gene spans more than 28 kb with 22 exons.295 Several alternative splicing products of the human M subunit gene have been found in a tissue-specific manner.296,297 Mammalian PFK is considered to have evolved by duplication of a gene that originated in prokaryotes.298
PFK deficiency (glycogen storage disease type VII, Tarui disease; see Chap. 71) has been reported at least 64 cases from 49 kindreds, since the first description by Tarui et al. in 1965.299– 334 Major clinical manifestations of PFK deficiency are myopathy and fully compensated hemolytic anemia. Many patients experience fatigue and pain with exercise from early childhood. In typical cases, strenuous exercise causes severe muscle cramps and myoglobinuria associated with abdominal pain and nausea. The severe muscle symptoms often make patients avoid vigorous exercise, and myopathy is prone to be overlooked in those cases.316 Although hemolysis is evident (median reticulocyte counts: 5.2 percent; n = 23), hemoglobin levels are normal or increased in most patients (median: 13.7 g/dl, range: 9.8 to 17.4; n = 18). PFK deficiency is transmitted as an autosomal recessive trait. One-third of reported patients are Jewish origin.
Muscle PFK activities are almost nil in most patients, while the erythrocyte exhibits partial deficiency (median: 51 percent, range: 8 to 75; n = 21), reflecting the complete loss of M subunit. Affected muscle shows a metabolic block at the PFK step and accumulation of glycogen to twice normal levels. Although ischemic forearm exercise causes no appreciable increase of venous lactate in patients, marked increases in plasma ammonia, inosine, and hypoxanthine levels are observed. The accelerated degradation of muscle purine nucleotides may subsequently lead to hyperuricemia particularly in males.335 Affected erythrocytes, which express only L4 isozyme, also show a metabolic block at the PFK step with a reduced 2,3-DPG level. Because of its effect on the oxygen dissociation curve the low 2,3-DPG level may account for the fully compensated or often overcompensated hemolysis.
Besides the classical Tarui disease, which manifests both early onset myopathy and hemolysis, several variant types of PFK deficiency have been reported.316 The isolated hemolytic type has been described in three probands.303,305,306 Although myopathic symptoms are possibly masked by the patients' aversion to exercise, these cases have unusual features including moderate to severe hemolytic anemia,305 a normal ischemic exercise test,303 very-low erythrocyte PFK activity,303 and/or normal muscle PFK activity.306 The fatal infantile type is a variant characterized by severe myopathy and early death before the age of 4.309,314,318,320,321,333 Unfortunately, molecular analysis has not been performed in both of these types at the time of this writing. Although some investigators have proposed that a distinct late onset type exists,313,319,325,330,332 recent molecular studies have provided evidence that the mutations in the late onset type are indistinguishable from those found in the classical type.325,330,336 Hereditary deficiency of the L subunit, documented through chromatographic analysis of the erythrocytes, has been found in two asymptomatic individuals.316,337 Family studies indicate that these individuals are heterozygous for L subunit deficiency.316,337
Molecular abnormalities causing PFK deficiency have been studied in more than 30 patients and 15 different mutations have so far been identified.322-328,331,334,336,338,339 In the Jewish population, a splicing mutation IVS5 + 1G→A causing a deletion of the exon 5 sequence,322,323,325,331 and a single nucleotide deletion 2003delC leading to a premature stop codon at 684323,331 are the most frequent. IVS15 + 1G→T causing deletion of 25 residues (423 to 447) has been identified in the first Japanese family described by Tarui et al.,299,338 and IVS19 + 1G→A which results in skipping of the exon 19 was found in another Japanese family.324 IVS6 − 2A→C documented in an Italian patient326 is an interesting mutation because it activates two different cryptic splice sites and leads to the production of 2 different species, one with a deletion of 4 amino acids (codons 143 to 146) and the other with premature termination at 145 amino acid residues. Four subjects in a Swedish family are compound heterozygotes for an intronic mutation IVS16 − 64A→G and an exonic splicing site mutation 1127G→A,339 each of which generates a truncated peptide with 376 residues and 488 residues, respectively. A nonsense mutation 283C→T predicting a polypeptide of only 94 residues has been described in three homozygous individuals in a Jewish kindred.336 All of the above mutations may produce completely inactive M subunits.
Missense mutations 116G→T (Arg39Leu), and 116G→C (Arg39Pro) have been identified in a Jewish323 and two unrelated Italian patients,326,334 respectively. Arg39 may be one of the essential residues for ATP binding.298 The complete loss of muscle PFK activity that was found in homozygous individuals with Arg39Pro326,334 is consistent with the importance of this residue. The mutant with Arg39Leu expressed in yeast showed marked instability.328 Gly209Asp caused by 626G→A was found in a homozygous French Canadian patient.328 Gly209 is a highly conserved residue, and it is located adjacent to the Met208 that is essential for F-6-P binding,298 suggesting that a Gly209Asp might be quite deleterious. Indeed, the mutant enzyme expressed in yeast was completely inactive.328 Asp543Ala caused by 1628A→C, and Trp686Cys by 2058G→T have been found in an Italian326 and a Japanese patient,327 respectively. Both Asp543 and Trp686 are conserved residues, but neither are involved in F-6-P or ATP binding. It is of interest that the patient with Trp686Cys exhibited a small but positive lactate response in forearm exercise test.327 Although the patient's muscle PFK activity was virtually nil, the mutant enzyme may function to a very minor extent. The other allele of the patient with Asp543Ala was not expressed for some unknown reason.326 A pair of mutant alleles, 299G→A and 2087G→A, causing Arg100Gln and Arg696His substitutions, were found in a compound heterozygous Swiss patient. Neither of Arg100 and Arg696 is phylogenetically conserved and neither is involved in active site regions. The mutants expressed in yeast were functional but slightly thermolabile.328 The less deleterious nature of these mutations may account for the detectable PFK activity (8 percent of normal) in the patient's muscle. Hereditary PFK M-subunit deficiency has also been found in dogs.340
Aldolase (EC 126.96.36.199) catalyzes a reversible reaction, cleaving fructose 1,6-diphosphate (F-1,6-P2) into glyceraldehyde-3-phosphate (GA-3-P) and dihydroxyacetone phosphate (DHAP) (see Fig. 182-1). In mammals, there are three distinct isozymes, A, B, and C, each of which is encoded by a separate gene (Table 182-2). Erythrocytes, muscle, brain, and developing embryos express aldolase-A, which is a tetramer composed of four identical subunits of 36 kDa. Deficiency of liver aldolase-B causes hereditary fructose intolerance (see Chap. 70). Aldolase-C is expressed in brain and other nerve tissues. The cDNA for aldolase-A coding 364 amino acids has been cloned.341 The aldolase-A gene, located at 16q22-q24342 extends over 7.5 kb and encompasses 12 exons.343 Several pseudogenes have been identified.344
Hereditary deficiency of aldolase-A has been reported in only four patients from three kindreds.345– 347 The common manifestation is moderate chronic hemolytic anemia. The initial Canadian-Jewish boy reported in 1973,345 showed mild mental retardation and diverse malformations, and a German boy in the third kindred347 exhibited psychomotor retardation and myopathy in addition to hemolytic anemia. Two Japanese boys in the second kindreds346 showed no symptoms other than anemia. Erythrocyte aldolase activities in affected individuals were reduced to 4 to 16 percent of normal. Muscle aldolase was assayed in the German patient, and found to be reduced to 11 percent of normal.347 Accumulation of F-1,6-P2 in affected erythrocytes was remarkable in the Japanese patients (632- and 878-fold),346 but only modest in the German patient (1.8-fold).347 Interestingly, the former patients showed mild but overt hemolytic anemia (Hb: 9.5 g/dl and 11.3 g/dl), while hemolysis was fully compensated in the latter (Hb: 14.6 g/dl). It is not clear whether the modest metabolic block accounts for the mild anemia of the latter.
Molecular analysis has revealed that the Japanese patients are homozygous for the missense mutation 386A→G (Asp129Gly)348 (note that amino acids are counted from the methionine coded by an initiation codon). The patient's enzyme expressed in E. coli showed marked instability, indicating that the phylogenetically conserved Asp129 is important for maintaining enzyme stability.348 Another mutation 619G→A (Glu207Lys) was identified in the German kindred.347 Glu207 is located in a helix essential for the subunit interaction, and the Glu207Lys mutation may disturb this interaction leading to the unstable quaternary structure,347 a conclusion consistent with the marked instability of the patient's enzyme. The inheritance of aldolase deficiency is autosomal recessive mode, and it is of interest that the parents of the first case reported345 had normal enzyme activity. This finding was interpreted as indicating that the mutant subunit may function normally when incorporated into a tetramer with normal subunits.
Triosephosphate Isomerase Deficiency
Triosephosphate isomerase (EC 188.8.131.52; TPI) catalyzes the interconversion of GA-3-P and DHAP (see Fig. 182-1). The equilibrium favors the formation of DHAP by 20:1. Human TPI is a homodimer of 54 kDa comprised of two identical subunits of 248 amino acids. The cDNA has been cloned from a human liver library.349 The TPI gene, which extends over 5 kb, is divided into seven exons,350 and has been localized at 12p13 (Table 182-2).351 Three processed pseudogenes have been identified.350
TPI is expressed in all tissues, and the deficiency is a multisystem disorder that is characterized by chronic hemolytic anemia, progressive neuromuscular dysfunction, and increased susceptibility to infection. The mode of inheritance is autosomal recessive (Table 182-1). Since the first description by Schneider et al.352 in 1965, it has been reported in nearly 50 cases from 38 kindreds.352– 371
Hemolytic anemia is the first symptom and is often associated with neonatal hyperbilirubinemia requiring exchange transfusion. Although hemolysis is precipitated by the frequent episodes of infections, anemia in the chronic phase is rather mild (median Hb: 10.4 g/dl; n = 18). Splenectomy was performed in one patient with no improvement of the anemia.354
Detailed neurologic information is available in 18 propositi.352-354,357-362,365-368,370 Retardation of motor development usually appears within a year after birth (median: 7 months, range: 1 to 24). Progressive weakness and hypotonia were the cardinal symptoms in 15 cases, often preceded by spasticity. Other neurologic manifestations included dystonic-dyskinetic syndrome,358 tremor,354 opisthotonus,357 and optic nerve atrophy.370 The neuromuscular symptoms are progressive in early infantile period, but may stabilize later in surviving patients.354,366,368 Sensory impairment is absent. Intellectual status has been described in 12 kindreds,353,354,357-360,362,366,368,370 and 5 propositi were mentally retarded.353,360,362,366,370 Neuropathologic findings are available only in one patient.353,372 These were characterized by olivary and cerebellar neuron loss, and axonal spheroids in midbrain and cerebellum. The latter might reflect a nonspecific axonal reaction to various factors including toxins.372 Cardiac complications have been reported in three propositi.353,360,362
Increased susceptibility to infection is observed in most patients. Granulocyte function was impaired in one propositus,359 but normal in two.357,358 The patients with TPI deficiency rarely survive beyond childhood (median survival: 20.5 months, range 11 months to 12 years; n = 16). Although the reported causes of death include respiratory failure, cardiac failure, and severe infections, patients often die suddenly without obvious cause. It is noteworthy that several patients have shown an unusually mild clinical course with stable neuromuscular symptoms354,366,368 and/or absence of increased susceptibility to infection.366,368 The most striking is a Hungarian family.368 The 15-year-old propositus had exhibited no neurologic symptoms until the age of 12, and his 23-year-old brother is free of any clinical signs and symptoms except for well-compensated chronic hemolysis.368 An additional case without neuromuscular symptom has been described briefly in an abstract.371 Prenatal diagnosis has been performed in several cases.361,365,367,373
TPI activities of the erythrocytes of affected individuals are reduced to 2 to 33 percent (median: 12 percent; n = 24) of normal. Markedly reduced enzyme activities are also found in other tissues. The striking increase in DHAP concentration (eighteen- to ninety-sixfold) that has been documented357-360,365– 368 is consistent with a metabolic block at the TPI step. Except in one patient, the residual TPI has been found to be thermolabile,371 and in most cases the residual enzyme has normal kinetics for GA-3-P. The mechanism of TPI deficiency causing multisystem dysfunction is poorly understood. A hypothesis that the accumulation of DHAP might be toxic for tissues and responsible for the dysfunction is attractive,374 but there has been no sufficient experimental evidence to support it. In the two Hungarian brothers with markedly discordant neurologic phenotypes, there were roughly 100 percent and 30 percent more 16:0/20:4 and 18:0/20:4 diacyl-phosphatidylcholine species in the erythrocytes than in normal controls, and the membrane fluidity was increased, more so in the mildly affected brother than in the one with severe neurologic impairment. This led to the speculation that differences in membrane fluidity and enzyme activities between the erythrocytes from the phenotypically differing TPI-deficient might reflect the state of the membranes elsewhere, as in the nervous system and thus be related to the neurologic disorder that was observed.375
Molecular analysis of TPI variants has been performed in 19 patients, and 9 different mutations have been identified.363,364,367,369-371,375– 377 The most frequent is a missense mutation 315G→C (Glu105Asp) (note that nucleotide numbers are for cDNA sequence, and that amino acids are counted from the methionine coded by an initiation codon), that was identified in 17 of 19 patients353,363,364,367,369-371,376,377 and accounted for 74 percent of the total pathogenic TPI mutations. An extensive haplotypic analysis showed that there had been no crossovers over a considerable physical distance, suggesting that all subjects with 315G→C are descendants of a common ancestor in northern Europe who probably lived more than 1000 years ago.378 This is consistent with the fact that almost all TPI-deficient individuals are of European origin and none of Asian or African origin have ever been reported. A Glu105Asp mutant expressed in cultured Chinese hamster ovary cells as well as the erythrocyte enzyme from the patients was thermolabile.363 Daar et al.363 and Mande et al.379 suggested that the molecular mechanism of the Glu105Asp mutation was perturbation of the local structure of the active site. Glu105 is located near the active site and shields Arg99 from Lys113.379 Arg99 is very important residue involved in intersubunit binding and is also in contact with the catalytic His96. The shortening of the Glu105 side chain by the substitution to aspartic acid may lead to an unfavorable interaction between Arg99 and Lys113, reducing the stability of the mutant enzyme. All homozygotes for the Glu105Asp variant and compound heterozygotes for Glu105Asp and another allele coding an inactive mutation exhibit progressive neuromuscular symptoms, indicating the deleterious nature of this mutation.
Phe241Leu caused by 721T→C, which has been identified in Hungarian patients with mild phenotype,368 is another mutation studied molecularly in detail.369,379 In the crystal structure, Phe241 is located proximal to the active site.379 The phenyl ring of Phe241 contacts with residues on a helix, which is important for the phosphate binding. Although the substitution of phenylalanine by leucine is conservative, it may thus alter the direction or the position of the helix and lead to a change in the substrate binding property.
Val232Met caused by 694G→A has been found in a homozygous patient with typical neuromuscular symptoms.367 Val232 is one of a series of seven highly conserved residues (codons 231 to 237). Cys42Tyr caused by the 125G→A mutation has been found in three unrelated compound heterozygous patients in combination with Glu105Asp.371,378 Cys42 is not highly conserved, but it is located in the β barrel structure.379 Ile171Val produced by the 511A→G mutation is of great interest because this mutation was found in a compound heterozygote who did not manifest neurologic symptoms.371 Moreover, the patient's enzyme is unique in that its thermostability is normal.371 Considering that the patient has another allele coding a drastic Glu105Asp mutation,371 the stable enzyme must be the one with the Ile171Val substitution, and one might then expect that this mutation is less deleterious than the common Glu105Asp substitution. However, Ile171 is located in the highly conserved active site loop including the catalytic Glu166,379 indicating that the residue should be important for the catalytic function. Two nonsense mutations, 568C→T (Arg190Ter) and 436G→T (Glu146Ter), have been found in two compound heterozygotes together with Glu105Asp353,363,376 or Phe241Leu,368,369,375 respectively. These nonsense mutations may not only produce inactive premature polypeptides but also reduce the abundance of nuclear mRNA.376,380 Another type of premature termination caused by a two-nucleotide deletion 86delGT that produces a frameshift results in the deduced production of a truncated chain with only 70 residues has been described.377 This deletion mutation was found in a compound heterozygote together with Glu105Asp.377
Four coding mutations have been identified in asymptomatic heterozygous individuals: 367G→A (Gly123Arg),381 218G→C (Gly73Ala),382 463G→A (Val155Met),382 315G→C (Glu105Asp).382,378 Gly123Arg is known to cause a thermolabile variant TPI Manchester. In 1982, using a large-scale automated enzyme estimations, Mohrenweiser383 reported that he found seven putative heterozygotes, infants with reduced enzyme activity, among 146 African-Americans. The absence of clinically affected homozygotes in the African-American population suggested that it was likely that the defect would prove to be a null allele, incompatible with life in the homozygous state. This suggestion is consistent with a subsequent observation in mice of a series of induced mutations with nucleotide substitutions; null alleles were found to be lethal in homozygotes at an early postimplantation stage of embryonic development (see immediately below). Almost 15 years later382 the DNA from all of these infants was reported to have base substitutions in each of two sites, −5 and −8 base pairs upstream of the transcription start site. The substitutions were A→G at position −5, G→A at position −8 (−40A→G and −43G→A, counting backward from the A of the methionine start codon), mutations that were in the cap site of the TPI gene. Three subjects were found to have an additional T→G substitution −24 base pairs upstream of the start of transcription, in the TATA-box. It was suggested that the −3 and −8 mutations markedly reduced TPI activity and were lethal in the homozygous state. Further studies have shown that this not to be the case. All three of these mutations are polymorphisms in the African-American population. Three haplotypes (−5G), (−5G, −8A), and (−5G, −8A, −24G) with gene frequencies of 0.111, 0.101, and 0.0047, respectively, were found to be associated with a statistically significant, but very modest, reduction in enzyme activity. Indeed, among 424 African-American adults, no cases of one-half normal activity were found as one would have expected if a severe deficiency gene were polymorphic in this population. None of the haplotypes were restricted to subjects with reduction of enzyme activity. Normal homozygotes for the −5G, −8A haplotype were found, ruling out the possibility that these mutations were responsible for lethal TPI deficiency.384
An animal model of TPI deficiency has been developed in 1-ethyl-1-nitrosourea-treated mice. All the homozygous mutations are lethal at embryonic stage. Three missense mutations and one terminal codon mutation (Ter → Cys) have been identified.385
Phosphoglycerate Kinase Deficiency
Phosphoglycerate kinase (EC 184.108.40.206; PGK) catalyzes the interconversion of 1,3-bisphosphoglycerate (1,3-DPG) and 3-phosphoglycerate. ATP is generated in the forward reaction 1,3-DPG → 3-phosphoglycerate (see Fig. 182-1). In addition to a ubiquitous enzyme PGK-1, that is a 48-kDa monomer, a tissue-specific intronless isoform PGK-2 has been found in spermatozoa (Table 182-2).386 The human PGK-1 gene is located on the X chromosome (Xq13.3),387 11 exons spanning 23 kb.388 Several pseudogenes have been identified.388,389 The cDNA sequence for PGK-1, encoding 417 amino acids, has been cloned from a human fetal liver library.390 Structural investigations have revealed that PGK consists of two domains (the N- and C-terminal domains) connected by a well-conserved hinge region.391 The binding site for ATP/ADP is located on the C-terminal domain, while the site for the phosphoglycerates is on the opposite domain. These binding sites are distant from each other in the native enzyme or in the presence of only one substrate. However, when both substrates are present, the enzyme undergoes a conformational rearrangement, bending the hinge to bring the substrates close enough for a direct phosphorylation.392
Since the first description by Kraus et al.393 in 1968, symptomatic deficiency of PGK-1 has been reported in 26 patients from 21 kindreds.393– 415 The mode of inheritance is X-linked, and most of the affected individuals are hemizygous males (Table 182-1). Due to random inactivation of the X-chromosome,416,417 heterozygous females may exhibit various degrees of enzyme deficiency, and sometimes clinical symptoms as well. PGK deficiency is a multisystem disorder characterized by three major clinical manifestations: chronic hemolytic anemia, central nervous system dysfunction, and myopathy.
Chronic hemolytic anemia was found in 12 of 19 hemizygous propositi,394,397,399-403,407,409-411,413 usually mild (median Hb: 10 g/dl; n = 9), and often fully compensated. Splenectomy was beneficial in three hemizygous patients,394,399,400 but ineffective in one affected heterozygote.393
Slowly progressive central nervous system dysfunction was apparent in 11 of 19 propositi.394,397,400-403,408,410– 412 Speech difficulties in early childhood are often the first sign. Mild to moderate mental retardation has been found in all the reported cases with central nervous system abnormalities. Emotional instability,394,401,410 convulsion,394,402,403,408,411 ataxia,401,402 and tremor401,402 are also found in some patients, but sensory impairment is absent. Neuropathologic findings are not available.
Myopathy characterized by recurrent episodes of exercise intolerance, cramps, and myoglobinuria has been reported in 9 of 19 propositi.405,406,408,409,411,412,414,415 Strangely, no PGK-deficient case with myopathy had been described until the first report of such a patient by Rosa et al.404 in 1979, although it has been reported frequently thereafter. The typical episode with massive myoglobinuria is triggered by a short but vigorous exercise including running and dancing, and in one case, by a generalized tonic-clonic convulsion.408 The episode is often associated with nausea, vomiting, abdominal pain, and dizziness, and may lead to acute renal failure. Although the first typical episode occurs usually in adolescence, patients often have mild myalgia or muscle weakness since childhood. Muscle biopsy specimens have shown only nonspecific findings.405,406,408,412,414 Muscle glycogen levels were found to be increased in two patients,405,408 but normal in two others.406,414 Most of the patients with myopathy show no increase of lactate after ischemic forearm exercise.
It should be noted that PGK-deficient patients seldom manifest all three cardinal clinical feature of the disease, chronic hemolytic anemia, central nervous system dysfunction, and myopathy.418 Most patients show only one or two of them in various combinations. The lack of myopathy in several patients may be explained by the fact that patients who are very young or severely limited by mental retardation are unable to perform muscular exercise sufficiently intense to trigger myoglobinuria.405 It is also possible that symptoms associated with myopathy have been overlooked or misdiagnosed. Symptoms such as “black-colored urine associated with convulsion.”402 “intense fatigue.”397 or “dizziness associated with nausea and vomiting.”410 which were originally interpreted as symptoms associated with hemolytic episodes, may also be consistent with myopathic episodes. Another possibility is that patients' aversion to exercise masks the myopathic symptoms. Such situations are not rare in PFK deficiency.316
Erythrocyte PGK activities in hemizygous individuals are decreased to 0 to 23 percent (median: 6.5 percent; n = 18) of normal. Heterozygous females may exhibit various degrees of enzyme deficiency. Muscle PGK activities in hemizygotes are 1.5 to 25 percent (median: 5 percent; n = 9) of normal. There is no evidence that clinical severity is related to the degree of residual activity. The reduced ATP level and the accumulation of intermediates above PGK step, particularly DHAP (five- to eighteenfold) and F-1,6-dP2 (four- to twenty-onefold), are observed in affected erythrocytes. The increased levels of DHAP (twenty-onefold) and F-1,6-dP2 (seventy-fivefold) have also been found in the muscle of a hemizygous individual.419 The 2,3-DPG content in affected erythrocytes is almost doubled, probably due diversion of 1,3-DPG from the PGK reaction to the bisphosphoglycerate mutase reaction. The right shift in the oxygen dissociation curve that results may improve oxygen delivery to body tissues thereby compensating, to some extent, for the decreased hemoglobin level of the blood.395 As in the case of other glycolytic enzymopathies, the mechanism of hemolysis in PGK deficiency is unclear. A hypothesis has been proposed that hemolysis is caused by impaired HMP shunt activity resulting from inhibition of 6-phosphogluconate dehydrogenase by the high concentration of 2,3-DPG.397 However, in one study,396 no decrease in the HMP shunt activity of affected erythrocytes was found.
Amino acid sequence alterations have been documented in 12 disease-causing variants, 2 by peptide mapping420,421 and the others by nucleotide sequencing.409,410,412,413,415,422– 425 The mutations are highly heterogeneous, and 11 different mutations have been identified. A 492A→T (Asp164Val) substitution has been found in a Chinese boy with PGK New York394,424 and a French patient with PGK Amiens403,423 (note that amino acids are counted from the methionine coded by an initiation codon). Although both probands show moderate hemolytic anemia associated with mental retardation and convulsions, the clinical course is much more progressive with the Asp164Val mutation. Most of the affected males in this family have shown frequent episodes of infection-induced hemolytic crisis and have died before reaching adulthood,394– 396 while the French patient has survived for more than 20 years despite the marked mental retardation and frequent seizures.403,423 It is of interest that granulocyte function was impaired in one hemizygote of the Chinese family,426 but appeared intact in the French propositus.403 Asp164 is located proximal to the binding site for the phosphoglycerates on the N-terminal domain. The replacement of the negatively charged aspartic acid by a hydrophobic valine may cause drastic structural alteration of the active site.423,424 Gly158Val found in a Japanese male with PGK Shizuoka409 is another substitution that was identified in the vicinity of Asp164. Although neither Asp164Val nor Gly158Val is a conservative substitution, the latter is located in a random coil apart from the active site.409 This may explain the much milder clinical consequence of PGK Shizuoka.
The amino acid substitution, Arg206Pro, causing PGK Uppsala397,420 is of special interest because its possible effect on enzyme function has been studied in detail by using a recombinant yeast mutant.427 Although the overall three-dimensional structure was not modified, and neither the specific activity nor the kinetic parameters was greatly affected, the Arg206Pro mutant showed a decreased stability. Tertiary structural analysis suggested that the decreased stability may result from the loss of the interaction between Arg206 and Lys229, which might be crucial for stability of the enzyme.427
A missense mutation 755A→C, which changes a consensus 5′ splice sequence AGgt to a nonconsensus CGgt, found in a Belgian patient with PGK Antwerp,415 is interesting because it causes not only an amino acid substitution Glu252Ala but also aberrant splicing leading to the production of an inactive enzyme lacking most of the C-terminal domain. Because Glu252 is not a conserved residue and is located in the region that is involved in neither substrate nor ATP binding of PGK, the aberrant splicing should be responsible for the reduced activity of PGK Antwerp.415 Indeed, the PGK mRNA content in patient's lymphoblastoid cells was only 10 percent of that of normal,415 a finding that would be unusual in a variant caused solely by a missense mutation. Ile253 which is replaced by a threonine in PGK Hamamatsu408,425 is located next to the Glu252. Although both PGK Antwerp and PGK Hamamatsu cause myopathy and lack hemolytic anemia, only the latter is associated with central nervous system dysfunction.
Besides the mutations causing clinical symptoms, two non-disease-causing mutations have been identified: Asp268Asn in PGK München428 and Thr352Asn in PGK II.429 Electrophoretic variants of PGK with normal enzyme activity have also been found.430,431,432
Bisphosphoglycerate Mutase Deficiency
Bisphosphoglycerate mutase (diphosphoglycerate mutase) [EC 220.127.116.11; bisphosphoglycerate mutase (BPGM)] is a multifunctional enzyme that catalyzes the synthesis and the degradation of 2,3-DPG in the erythrocyte by virtue of its bisphosphoglycerate mutase and bisphosphoglycerate phosphatase activities433,434 (Rapoport-Luebering shunt). This erythrocyte specific enzyme also has a phosphotransferase activity for the interconversion of 3-phosphoglycerate and 2-phosphoglycerate.433,434 However, 95 percent of the phosphoglyceromutase activity in erythrocytes may be attributed to another glycolytic enzyme monophosphoglycerate mutase (see Fig. 182-1). Bisphosphoglycerate mutase is a dimer of 60 kDa composed of two identical subunits each of which consists of 258 residues. The cDNA has been cloned from reticulocyte mRNA.435 The human bisphosphoglycerate mutase gene has been assigned to region 7q34-7q22,436 where its 3 exons spans more than 22 kb (Table 182-2).437
Complete deficiency of bisphosphoglycerate mutase has been found in only a French family reported by Rosa et al.438 in 1978. The affected individuals, a 42-year-old propositus and his three sisters, had no clinical symptoms except for ruddy cyanosis. They had no hemolytic anemia, but rather erythrocytosis with the hemoglobin levels 17 to 19 g/dl. Bisphosphoglycerate mutase and bisphosphoglycerate phosphatase activities were undetectable in erythrocytes. The patients also showed severe depletion of 2,3-DPG (<3 percent of normal), increased ATP (twofold), and marked accumulation of triosephosphates (fifteenfold) and F-1,6-P2 (tenfold). These metabolic alterations are consistent with a metabolic block at the bisphosphoglycerate mutase step. The increased ATP level may reflect the increased utilization of 1,3-DPG by PGK in the absence of bisphosphoglycerate mutase. The erythrocytosis is presumably due to the decreased oxygen affinity of hemoglobin caused by the depletion of 2,3-DPG. Immunochemical analysis revealed that the patients' bisphosphoglycerate mutase was very thermolabile if 2,3-DPG is absent.439 Erythrocytes of heterozygotes showed half-normal enzyme activities (bisphosphoglycerate mutase: 41 to 46 percent ; bisphosphoglycerate phosphatase: 44 to 63 percent) and 2,3-DPG levels (41 to 75 percent).438,440 Some of them exhibited abnormal oxygen dissociation curves and erythrocytosis.438,440
Molecular analysis revealed that the affected patients are compound heterozygous for a missense mutation 268C→T and a deletion of cytidine (60delC)441 (note that nucleotide numbers are for cDNA sequence, and amino acids are counted from the methionine coded by an initiation codon). The missense mutation generates a variant enzyme with Arg90Cys (bisphosphoglycerate mutase Créteil I), while the deletion causes a frameshift and produces a truncated protein with only 47 residues (bisphosphoglycerate mutase Créteil II). Although Arg90 has not been described as part of the active site, its functional importance may be expected from the strict conservation of the residue.441 Garel et al.442 have revealed that the guanidinium group of Arg90 may play an essential role both in the stability and in the catalytic functions of the enzyme, probably by maintaining the correct three-dimensional structure in this region of the molecule. The loss of the guanidinium group associated with the Arg90Cys substitution may thus perturb the local structure and abolish bisphosphoglycerate mutase and bisphosphoglycerate phosphatase activities.
Other Enzyme Abnormalities of the Glycolytic Pathway
Partial deficiency of erythrocyte enolase (EC 18.104.22.168) (see Fig. 182-1) has been documented in association with hemolytic anemia in two unrelated individuals.443,444 Although the enzyme deficiency was found in three generations in the second family, no one but the propositus showed apparent signs of hemolytic anemia.444
Hereditary deficiency of the H (B) subunit of lactate dehydrogenase (EC 22.214.171.124; LDH) (see Fig. 182-1) causes a remarkable reduction of LDH activity (<10 percent of normal) and accumulation of glycolytic intermediates above GA-3-PD step in affected erythrocytes.445 The latter is considered to be due to the inhibition of GA-3-PD step by a high concentration of NADH associated with LDH deficiency. Despite the remarkable metabolic alteration, affected individuals do not show any symptoms including hemolytic anemia, although in one case minimal reticulocyte count elevations were noted.446 Patients with LDH M (A) subunit deficiency are known to manifest myopathy and skin lesions, but no hemolysis.447 It is of interest that, in contrast to human, mice homozygous for LDH M subunit deficiency exhibit chronic hemolytic anemia.448
Human monophosphoglycerate mutase (EC 126.96.36.199; MPGM) is composed of two distinct subunits, M and B. Muscle contains the homodimer MM, and many tissues, including erythrocytes, express BB. Hereditary deficiency of the M subunit causes myopathy,449 but deficiency of B subunit has not been reported.