Disorders of the glycolytic pathway

Mutations of most of the enzymes in the glycolytic pathway have been described in association with congenital non-spherocytic haemolytic anaemia (CNSHA). However, deficiency of pyruvate kinase (PK) is far and away the most common defect. The haemolysis is the result of a failure to produce sufficient ATP.

Pyruvate kinase

PK catalyses the final steps of the glycolytic pathway with the conversion of phosphoenolpyruvate (PEP) to pyruvate, with the concomitant phosphorylation of ADP to ATP, leading to overall net gain of ATP from this pathway. There are four types of PK

in different tissues derived from two separate genes. The PKM gene, on chromosome 15, produces PKM1 and PKM2 through differences in post-transcriptional splicing. PKM1 is present in skeletal muscles, PKM2 in leucocytes, kidneys, adipose tissue and lungs. The second PK gene, on chromosome 1, gives rise to PKL in the liver and PKR in red cells. The isoenzymes are transcribed differently through the influence of tissue-specific promoters. The active enzymes are homotetramers. PKM2, PKR and PKL all demonstrate marked allosteric reactions with several ligands. PKM1, on the other hand, has no allosteric interactions.

The main ligands that are involved in the allosteric control of PKR in the erythrocyte are shown in Figure 9.3. PK is one of the dominant controlling steps in glucose metabolism (together with hexokinase), exerting its effect through major feedback loops, especially through the requirement of activation by fructose 1,6-diphosphate.

Pyruvate kinase deficiency

PK deficiency leads to a chronic CNSHA with extravascular haemolysis.

Molecular biology

PK deficiency is the commonest of the enzymopathies of the glycolytic pathway, at least 300 times more common than any other. Best estimates based on gene frequency in the white population suggest a prevalence of about 50 per million. Well over 100 different mutations have been described in PK deficiency, the majority involving point mutations or deletions in the transcribed gene, but a substantial number involving the promoter region. Haemolytic anaemia due to PK deficiency is an autosomal recessive disorder. Many individuals are compound heterozygotes. Not surprisingly, there is enormous genetic heterogeneity between affected individuals reflected in the multiplicity of quantitative and kinetic defects detected (Figure 9.3). Because PKR is a homotetramer composed of four chains derived from the products of the two alleles, enzyme kinetic variations are many and genotype/phenotype correlates are not predictable. Within families, however, the severity of the haemolysis tends to be similar in affected individuals. PK catalyses the ultimate step in the glycolytic pathway. Deficient activity leads to accumulation of substrates further up the pathway, including 2,3-DPG. The increased concentration of 2,3-DPG in PK-deficient red cells shifts the oxygen dissociation curve to the right, indicating low oxygen affinity. Patients with PK deficiency tolerate apparent anaemia well because the lower Hb content will deliver the same amount of oxygen to tissues as the normal Hb, at least under normal conditions, although oxygen reserve would be limited.

Reticulocytes have alternative means of producing energy in the form of ATP, through oxidative respiratory pathway of the remaining mitochondria. They can also synthesize enzyme in those defects where lack of stability is the major cause of enzyme deficiency in the mature cell. Reticulocytes thus have a metabolic advantage over mature red cells in PK deficiency.

Hexokinase Glucose <->-Glucose-6-P

Glucosephosphate isomerase

Glucose-6-P dehydrogenase

6-PG

Phosphofructo-kinase

ADP^j. Fructose-1,6-dP

.Aldolase

Lactic dehydrogenase ate

Dihydroacetone-P-

Triose phosphate isomerase

>- Glyceraldehyde-3-P

reductase NADH MetHb NADH

Pyruvate

ATPX Pyruvate

Phosphoenolpyruvate Enolase

NADP+

Glutathione reductase GSH Glutathione peroxidase H2O2 Catalase

Glyceraldehyde-3-P dehydrogenase

Diphosphoglycerate mutase

1,3-Diphosphoglycerate < ► 2,3-Diphosphoglycerate snolpy

ADP ATP

2-Phosphoglycerate

Phosphoglycerate mutase

Phosphoglycerate kinase

Diphosphoglycerate phosphatase 3-Phosphoglycerate <

Figure 9.2 The glycolytic pathway and its interactions with the pentose phosphate pathway and the Rapoport-Luebering shunt.

Figure 9.3 The reactions of pyruvate kinase, showing the main ligands that influence activity and the sites affected by various mutations encountered in PK deficiency.

Abnormal substrate kinetics

Phosphoenolpyruvate

Abnormal activating ligand binding

Fructose 1,6-diphosphate, K+

Pyruvate

Mg2+-ADP

Pyruvate kinase A

Abnormal cofactor kinetics

Mg2+-ATP

Transcription failure

Non-functional protein Abnormal promoter Decreased stability

Abnormal feedback inhibition

Clinical features

The genetic heterogeneity is reflected in the wide variation of the phenotype. The presenting features may vary from severe neonatal jaundice and anaemia (Figure 9.4), rarely even presenting with hydrops fetalis, severe CNSHA requiring repeated transfusions, moderate haemolysis with exacerbation during infections or pregnancy, to symptomless compensated haemolysis with only a minor apparent anaemia. The majority of reported cases have presented in childhood. PK deficiency does not usually have an adverse effect on the outcome of pregnancy, although occasionally transfusion may be required to compensate for the added dilutional anaemia (Figure 9.5).

Jaundice, as with other congenital haemolytic anaemias, may be exacerbated by co-inheritance of other genes, for example, of Gilbert's syndrome. The haemolysis is nearly always extravascu-lar, although rare examples with some intravascular haemolysis

Splenectomy Transfusions V Uti uwwuumuii i i

Age (years)

Figure 9.4 Effect of splenectomy in a child with severe CNSHA due to PK deficiency. Splenectomy was delayed until the child was 3 years old. Note the marked reticulocytosis post splenectomy (V, viral infection; Uti, urinary tract infection). The child grew normally despite Hb of around 8 g/dL (see text).

Splenectomy

14 12 10 8

us ppppppp stuu

80 icu 60 locyt

Figure 9.5 Multiple successful pregnancies in a splenectomized patient with PK deficiency. Transfusion was required during pregnancy (p) and before surgery (st, sterilization) and during infection (u, urinary tract infection; s, sepsis).

have been detected. Gallstones are common in PK deficiency and may lead to bouts of cholecystitis and biliary colic (Figure 9.6). Jaundice may also be increased by administration of drugs that affect bile excretion. As with other cases of congenital haemolytic anaemias with extravascular haemolysis, excess iron accumulation may occasionally develop even in the absence of transfusion or co-inheritance of a haemochromatosis gene.

The spleen is usually palpable in cases where there is significant haemolysis, although in milder cases, it may be evident only by using ultrasound or other imaging techniques.

Pyruvate kinase deficiency is not associated with abnormalities of other tissues, as the deficient enzyme is unique to the red cells, but occasionally individuals are seen with associated abnormalities due to deletions within chromosome 1, close to the PK gene.

Laboratory diagnosis

The blood count reveals a normochromic anaemia with reti-culocytosis, sometimes producing a slight macrocytosis. An increased mean cell Hb concentration (MCHC) is occasion ally seen in severe cases as a result of dehydration brought about by ATP deficiency. This dehydration also produces the characteristic spur cells or acanthocytes seen on the blood film (Figure 9.7a and b). Apart from the spur cells (which are not specific) and the very large reticulocytosis post splenectomy (Figure 9.7c), there is nothing to distinguish PK deficiency from other causes of CNSHA.

The autohaemolysis test, involving the incubation of defib-rinated red cells with and without glucose for 48 h at 37°C, has traditionally been used for screening tests. The haemolysis in the unfortified aliquot is uncorrected by the addition of glucose (type II). The test is not specific and has mainly been abandoned in favour of the enzyme assay.

The definitive diagnosis depends on the analysis of enzyme activity and kinetics, although the finding of an elevated 2,3-DPG level (2-3 times normal) may be a useful pointer. Characterization of the enzyme requires measurement of maximal activation under optimal conditions (Vmax), kinetic studies to determine substrate(s) concentration at which the enzyme shows 50% Vmax (Km), thermal stability, pH optimum and

Figure 9.6 Plain radiograph of a patient with PK deficiency , showing a calcified gall bladder with mixed calcified pigment stones as a result of repeated attacks of inflammatory cholecystitis.

Figure 9.6 Plain radiograph of a patient with PK deficiency , showing a calcified gall bladder with mixed calcified pigment stones as a result of repeated attacks of inflammatory cholecystitis.

electrophoretic mobility. Meaningful enzyme levels can be achieved only after the total removal of leucocytes, which have up to 300 times the PK activity of red cells. The effect of reticulocytes also has to be taken into account. International guidelines for investigation of PK deficiency have been published. Typically, homozygotes have about 25% of the normal Vmax activity, allowing for the age of the red cells and heterozygotes 50-60%, but the range is great and there is much overlap in the Vmax between the groups, emphasizing the need for kinetic and other studies (Figure 9.8).

Management

The mainstay of management of patients who have severe enough haemolysis to warrant treatment is splenectomy. Assessment of the need for splenectomy should take into account the symptoms of the patient, not the Hb level. The low Hb (even as low as 7.0 g/dL) may be well tolerated because of the right-shifted oxygen dissociation curve. Indications for splenectomy include severe neonatal haemolysis and chronic transfusion

Figure 9.7 PK deficiency, peripheral blood. (a) Red cell anisocytosis and poikilocytosis presplenectomy. (b) Postsplenectomy showing acanthocytes or 'prickle' cells. (c) Gross reticulocytosis post splenectomy (supravital new methylene blue stain).

Figure 9.7 PK deficiency, peripheral blood. (a) Red cell anisocytosis and poikilocytosis presplenectomy. (b) Postsplenectomy showing acanthocytes or 'prickle' cells. (c) Gross reticulocytosis post splenectomy (supravital new methylene blue stain).

requirements. The dangers of splenectomy, particularly the risk of overwhelming sepsis, are discussed in Chapters 8 and 21. Splenectomy raises the effective Hb so that transfusion is not required but does not prevent hyperbilirubinaemia, so biliary complications may still arise after splenectomy.

Following splenectomy for PK deficiency, there is a huge elevation of the reticulocyte count, which may reach 80%

Figure 9.8 PK activity Vmax measured in patients, obligate heterozygotes and normal control subjects. Note the wide overlap of values between the two groups, indicating importance of kinetic studies as well as effect of reticulocytosis.

for other enzyme activities, when absolute levels may be difficult to interpret because of the age distribution of the red cells.

Hexokinase deficiency

Hexokinase deficiency has been recorded in fewer than 30 cases but, even within this small number, there is evidence of molecular and phenotypic heterogeneity. Complete hexokinase deficiency is probably lethal. Most patients have moderately reduced activity for the age of the red cell and, in the majority of cases, it is the stability of the enzyme that is affected by the mutation. Combined heterozygosity for complete gene deletion and a nonsense mutation affecting activity has been recorded as a typical example of compound genetic inheritance. In most recorded cases, the activity of the enzyme is reduced to between 10% and 20% of normal. As with other glycolytic enzyme deficiencies, there is variable non-spherocytic haemolytic anaemia, in this case mostly relatively mild. Obligate heterozygotes have reduced levels of the enzyme, but assays of the activity in patients with haemolytic anaemia due to hexokinase deficiency may be difficult to interpret because of the higher activity in reticulocytes and in young cells. Typically, the reduced hexokinase activity is associated with a fall in the concentration of 2,3-DPG within the cells. Patients have less exercise tolerance for a given level of Hb than would be expected because of the left shift in the oxygen dissociation curve.

or more of the peripheral blood cells (Figure 9.7c). Although pregnancy may exacerbate this anaemia, there is no indication that PK deficiency itself has an adverse effect on the pregnancy or outcome, as long as the anaemia is managed as necessary (Figure 9.5). As with all chronic haemolytic anaemias, folic acid, 5 mg per week or 400 |ig daily, is a sensible supplement.

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Responses

  • Ari-Pekka
    What disorders that can come out of defective glycolysis pathway?
    2 years ago
  • elisa michael
    What diseases are as a result of a defective glycolysis pathway?
    2 years ago
  • Candido
    What condition can arise from defective glycolysis pathway?
    2 years ago
  • carmen
    What are the disordors associated with glycolysis?
    2 years ago
  • asmait
    What are the disorder associated with glycolysis?
    2 years ago
  • goldilocks
    What are the disorders association with glycolysis?
    2 years ago
  • Dudo
    How does lack of some glycolytic enzyme lead to anemia.?
    2 years ago
  • iida
    What are the glycolytic disorder?
    2 years ago
  • LARA
    How hemolytic anaemia is associated with glycolysis?
    2 years ago
  • PROSERPINA MILANO
    What happens during glycolytic pathway defect?
    2 years ago
  • alfred
    What are the disorder of glycolytic pathway?
    2 years ago
  • girma selassie
    What are the metabollic disoders of glycolysis?
    1 year ago
  • alexander bey
    Which disease caused due to glycolysis disorder?
    1 year ago

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