Classification of RBC enzyme deficiencies

(Except G6PD deficiency which is X-Linked recessive disorder, all are autosomal recessive conditions)

• Enzymes of EmbdenMayerhof pathway
  • Pyruvate kinase
  • Glucose phosphate isomerase
  • Hexokinase
  • Phosphoglycerate Kinase
  • Triose phosphate isomerase
• Hexosemonophosphate shunt
  • Glucose 6 phosphate dehydrogenase
  • Glutathione synthetase
  • Glutamylcystienesynthetase
• Nucleotide metabolism
  • Pyrimidine 5’ nucleotidase.

Glucose 6Phosphate Dehydrogenase Deficiency

Introduction:

• G6PD deficiency is the most common genetically determined enzyme deficiency.

Epidemiology:

• In some populations >20% people are affected
• Overall prevalence- 4.9%

Etiology:

• G6PD deficiency is inherited in X-Linked recessive pattern.
• Gene is located on Xq28, which has 13 exons and 12 introns.
• This gene produces monomer of 514 amino acids which come together to form dimer and tetramer.
• Mutations affect the stability of the transcribed enzyme.
• Total 127 different variants of G6PD deficiency are described.
• Classes of mutant G6PDisoenzymes

Pathogenesis:

• Normally intracellular reduced glutathione (GSH) inactivates oxidants which tend to damage cell membrane.
• This GSH is produced in hexosemonophosphate shunt which needs G6PD
• So decreased G6PD levels lead to decreased protective capacity against oxidant injury
• In presence of oxidative stress there is failure to maintain hemoglobin in reduced state (oxidation of -SH groups by H2O2), which leads to precipitation of hemoglobin (Heinz bodies). 
• Precipitated hemoglobin damages red cell membrane and subsequently there is both intravascular hemolysis and removal of RBCs by spleen.

Agents causing oxidative damage

• Antimalarials-Primaquine 
• Sulphonamides- Dapsone, Septran
• Other antibacterials-Nitrofurans,Nalidixic acid
• Analgesics- Aspirin
• Miscellaneous: Vitamin K analogues, Naphthalene (mothballs), Probenecid, Dimercaprol (BAL), Methylene blue, Toluidine blue, Fava beans (broad beans), Glabenclamide, Rasburicase
• Note: Infections and diabetic acidosis increase the hemolytic action of drugs

Clinical Features:

• Features of intravascular hemolysis, 2-3 days after exposure to oxidant injury/ following infections.
• It is self limiting as only older RBCs are affected. 
• Neonatal jaundice

Investigations:

• Peripheral smear
  • Abnormalities are seen only during hemolytic crisis
  • Microspherocytes and small hypochromatic cells
  • Polychromasia
  • Erythrocyte fragments
  • Bite cells-chunk of cell removed from one side
  • Eccentrocyte- Irregularly contracted cell, erythrocyte hemighosts, double colored erythrocyte, cross bonded erythrocytes seen
  • Hemoglobin is confined to one side of cell and other side is transparent. Transparent side contains Heinz bodies, which can be highlighted by staining with brilliant cresyl blue.
• Reticulocyte count – 8-12%- 5-15 days after hemolytic episode
• Indices –MCV-Decreased, MCH increased
• Indirect bilirubin – Raised
• LDH – Raised
• Haptoglobulin – Decreased
• Tests for presence of G6PD
  • These tests should not be done during the period of acute hemolysis. (Reticulocytes have normal enzyme levels, because of which G6PD levels can be falsely normal)
  • Brilliant cresyl blue reduction test
    • Hemolysate of patient’s blood, glucose 6 phosphate, NADP and brilliant cresyl blue are added in a test tube and incubated.
    • If NADPH is formed, it reduces blue dye to its colorless form.
    • Time taken for this change is inversely proportional to the amount of G6PD present.
  • Fluorescent spot test
    • Whole blood, glucose 6 phosphate, NADP and saponin are mixed in a test tube and drop of this mixture is placed on a filter paper.
    • Now this is examined under UV light for fluorescence
    • NADPH is fluorescent.
    • Absence of fluorescence indicates deficiency of G6PD.
  • Spectrophotometric measurement of enzyme.
    • Erythrocyte hemolysate is incubated with glucose 6 phosphate and NADP.
    • Rate of reduction of NADP to NADPH is measured at 340 nm
  • Ascorbate cyanide test
    • Patient’s blood is incubated with sodium ascorbate, sodium cyanide & glucose
    • H₂O₂ is generated due to interaction of Hb with cyanide(Cyanide inhibits catalase which inhibits formation of H₂O₂)
    • H₂O₂ is not reduced by G6PD deficient cells
    • H₂O₂ converts Hb to metHb which imparts brown color to solution
    • It is positive also in case of PNH, PK deficiency and unstable Hb

Treatment:

• Remove inciting agent (by gastric lavage etc)
• Support with RBC transfusions if hemolysis is severe
• Folic acid supplementation
• Phototherapy/ exchange transfusion/ single dose of Sn-mesoporphyrin-hemeoxygenase inhibitor- for neonatal jaundice
• Avoid oxidant injury
• No use of splenectomy

Note: G6PD deficient patients are protected against Pl. falciparum infection as the parasite also requires HMP shunt for its survival

Pyruvate Kinase Deficiency

Introduction:

• 4 types of PK are present in different tissues, which are derived from 2 separate genes
  • PKM gene on Chromosome 15- Produces PKM 1(Skeletal muscle) and PKM2 (Leucocytes, Kidney, adipose tissue, Lungs)
  • PK gene on Chromosome 1-  ProducesPKL (Liver) and PKR (Red cells). 
  • Transcription is influenced by tissue specific promoters
• PK is dominant controlling enzyme in glucose metabolism, exerting its effect through major feed back loops.
• PK catalyses irreversible transfer of phosphate from phospoenolpyruvate to adenosine diphosphate.

Etiology:

• More than 190 mutations are described in PK deficiency
• Autosomal Recessive inheritance

Pathogenesis:

1.

Deficiency of Pyruvate Kinase

↓

Loss of 2 ATP per mole of glucose

↓

Alteration in erythrocyte membrane

↓

Potassium loss and dehydration

↓

Formation of echinocytes which have decreased deformability

↓

Sequestration in splenic cords and phagocytosis by macrophages

2.

Because of enzyme deficiency there is accumulation of substrates further up in pathway

↓

Increased 2, 3 D P G

↓

Shift to right of oxygen dissociation curve

↓

Decreased oxygen affinity

(So these patients tolerate apparent anemia well)

3.  Reticulocytes can produce ATP through oxidative respiratory pathway of remaining mitochondria & they can also synthesize enzyme. So they are safe in PK deficiency

Clinical features.

• Mild to moderate anemia
• Splenomegaly
• Rarely jaundice and gall stones

Investigations

• Hemoglobin – 6 – 12 g/dl
• Peripheral smear
  • Normocytic normochromic anemia
  • No Heinz bodies
  • Echinocytes/prickle cells are seen- Small dense crenated RBCs
• Reticulocyte count- 2-15%(After splenectomy it is > 40%)
• Bilirubin, LDH – Elevated
• Haptoglobulin – Diminished
• Osmotic fragility – Normal
• Autohemolysis – Increased at 48 hours and not corrected by addition of glucose (type II)
• 2, 3 DPG level in RBC - Increased
• Detection of enzyme activity (<25% of Vmax)
  • RBCs are separated from WBCs as they have increased PK activity.
  • RBC + Phosphoenolpyruvate + NADH + LDH+ADP+PEP+ADP Under action of Pyruvate kinase form Pyruvate +ATP
  • Pyruvate+ NADH+H⁺  under the action of LDH form  Lactate+ NAD⁺
  • NADH fluoresces under UV light
  • Normal RBCs (PK+)-Fluorescence disappears in 30 min
  • Presence of it for 45-60min indicates PK deficiency

Treatment

• Transfusion to maintain Hb above 8-10 g/dL
• Splenectomy in case of
  • Severe neonatal hemolysis
  • Chronic transfusion requirements
• Folic acid 5mg/week

Hexokinase Deficiency

• It is involved in phosphorylation of glucose to glucose 6 phosphate
• In RBCs hexokinase lacks porin binding domain that links the enzyme to mitochondrial membrane
• It provides major rate limiting step and has extensive allosteric interactions
• Total absence is lethal
• In hexokinase deficiency mutations affect stability of enzyme
• Patients present with variable non spherocytic hemolytic anemia
• There is decreased levels of 2, 3 DPG, so there is exercise intolerance

Glucose Phosphate Isomerase Deficiency

• Catalyses interconversion of glucose 6 phosphate to fructose 6 phosphate
• Presents with mild to moderate anemia
• Can be associated with neurologic dysfunction and granulocytic defects
• Jaundice may be treated with phenobarbital

Phosphofructokinase Deficiency

• Catalyses reaction- fructose 6 phosphate to fructose 1, 6 diphosphate
• Major rate limiting step in glycolysis
• Enzyme has 2 submits M & L both encoded by separate genes
• It is usually associated with muscle cramps and myoglobinuria

Fructose Diphosphate Aldolase A Deficiency

• This enzyme is needed in conversion of fructose 1, 6 diphosphate to glyceraldehyde 3 phosphate and dihydroxyacetone phosphate
• Presents with
  • CNSHA (congenital non spherocytic hemolytic anemia)
  • Mental retardation
  • Dysmorphic features.

Triose Phosphate Isomerase Deficiency

• Involved in interconversion of glyceraldehyde 3 phosphate &dihydroxyacetone phosphate
• Presents with (Since birth)
  • CNSHA
  • Progressive neurological disorder with spasticity and CNS degeneration
  • Cardiac failure due to arrhythmias
• Most common mutation – Gly 104 Asp
• Most children die in childhood due to neuromuscular problems 

Phosphate Glycerate Kinase

• Involved in interconversion of 1,3 DPG to 3 Phosphoglycerate
• Product of gene on X-chromosome
• Clinical features
  • CNSHA
  • CNS disorder
  • Myopathy with or without rhabdomyolysis

Glutathione (GSH) Deficiency

• Functions of GSH
  • Protecting cells against oxidative damage
  • Participation in detoxification of foreign compounds
  • Maintenance of protein suprahydryl groups in a reduced state
  • Transportation of amino acids.
• It is synthesized from glutamate, cystiene&glycine by link reaction of 2 enzymes- γ glutamylcystienesynthase and glutathione synthetase
• Deficiency results in
  • 5-Oxoprolinuria
  • Metabolic acidosis
  • Mental retardation
  • Hemolytic anemia, aggregated by oxidative stress

Enzymopathies of the glutathione cycle and synthetic pathways

Pyrimidine 5 Nucleotidase Deficiency

• This enzyme catalyses dephosphorylation of UMP and CMP
• In case of deficiency partially degraded mRNA and rRNA accumulate in RBC which leads to severe hemolytic anemia
• Peripheral smear shows basophilic stippling of RBCs(Lead inhibits this enzyme, so in case of lead poisoning also stippling is seen)
• Treatment: PRBC transfusions as and when required.

Other rare enzyme deficiencies which cause hematological disease

(They also present with CNSHA)

• Aldolase
• Triose phosphate isomerase
• Phosphoglycerate kinase
• Biphosphoglyceratemutase
• Adenylate kinase
• Adenosine deaminase

Recent advances:

Recent advances:

Mitapivat versus Placebo for Pyruvate Kinase Deficiency 

Pyruvate kinase deficiency is a rare, hereditary, chronic condition that is associated with haemolytic anemia. Mitapivat, is an oral, activator of erythrocyte pyruvate kinase. In a recently published trial it was shown that mitapivat increased the hemoglobin level in patients with pyruvate kinase deficiency. No new safety signals were identified in the patients who received mitapivat.

https://doi.org/10.1056/NEJMoa2116634

Comorbidities and complications in adult and paediatric patients with pyruvate kinase deficiency

Pyruvate kinase (PK) deficiency, a rare congenital hemolytic anemia caused by mutations in the PKLR gene, presents with a wide range of clinical manifestations. In the Peak Registry, a longitudinal study of 241 patients, 48.3% had undergone splenectomy, and 50.5% had received chelation therapy. Iron overload was common, affecting 52.5% of patients, including 20.7% of never-transfused individuals. Early-life complications included neonatal jaundice, splenomegaly, and hepatomegaly, with interventions needed in 41.5% of cases. In adulthood, osteopenia/osteoporosis and pulmonary hypertension were noted in 19.0% and 6.7% of patients, respectively. Complications, such as biliary events and bone health issues, were prevalent across PKLR genotypes

https://doi.org/10.1111/bjh.19601