1. The Clinical Aspects of Enzyme Deficiencies in Haematology Dr Pasquale Barbaro Paediatric Haematologist The Children’s Hospital at Westmead

2. Summary Red cell enzyme deficiencies Enzyme deficiencies in platelet function defects Enzyme deficiencies implicated in Neutropenia Enzyme deficiencies implicated in Bone Marrow failure

3. Red Cell Enzyme deficiencies Red Cell Metabolism During maturation, RCs lose their nucleus, organelles and mitochondria Require cellular metabolic pathways to ensure red cell membrane and haemoglobin integrity Require ATP to drive K/Na pump essential for membrane integrity Require NADH to protect from oxidative stress Require 2,3 bisphophoglycerate to regulate oxygen affinity Red cell enzyme deficiencies lead to non-spheroctyic haemolytic anaemia Chronic or intermittent haemolysis Pallor, jaundice Anaemia, reticulocytosis, hyperbilirubinaemia Splenomegaly Gall stone production Iron overload

4. Source of ATP Required to drive K/Na Pump Essential for Red cell membrane flexibility and integrity Source of NADPH Ensures adequate reduced glutathione Protects from oxidative stress Involved in ATP production

5. Embden-Meyerhof pathway ATP production Enzyme deficiencies lead to chronic non spherocytic haemolysis of various severities Many also have non haematological manifestations Neurological Myopathy Most common: Pyruvate Kinase deficiency Glucose-6-phosphate isomerase deficiency Phosphofructokinase deficiency All have autosomal recessive inheritance Except Phosphoglycerate kinase deficiency – X-Linked

6. Pyruvate Kinase deficiency Frequency is approximately 1:20,000 in the general white population Intracellular Lack of ATP Excess production of 2,3-bisphosphoglycerate Clinical manifestations Chronic non-spherocytic haemolytic anaemia Stabilises in adulthood, and post-splenectomy Pallor, jaundice Splenomegaly Iron overload Treatment Supportive – Transfusions Splenectomy Iron chelation therapy

7. Glucose phosphate isomerase (GPI) deficiency More than 50 cases described Second most common enzyme defect in the red cell anaerobic glycolysis Clinical features More severe haemolysis Hydrops foetalis is more common than in other enzymopathies May be associated with neurological complications Granulocyte dysfunction has also been described

8. Others Phosphofructokinase Mild or fully compensated haemolytic anaemia Myopathy may be more pronounced symptom Aldolase Moderate to severe haemolysis Neurological and muscle dysfunction Recurrent rhabdomyolysis Triose-phosphate Most severe enzyme defect in EM pathway Hydrops foetalis or death in early childhood are common Severe neurological complications

9. Oxidative pathway deficiencies RCs continuously exposed to oxidative stress Iron is required to be maintained in its active, reduced state Oxidised Haemoglobin denatures, and deposits on the red cell membrane leading to damage. Damaged Red cells are then removed in the spleen. Deficiencies in these enzymes lead to acute intermittent haemolysis when there is increased oxidative stress Glucose-6-phosphate dehydrogenase deficiency most common Glutathione reductase deficiency Very rare (2 families reported to date) Symptoms are similar to that seen in G6PD deficiency

10. Nucleotide metabolism disorders Purine metabolism is important in RCs to maintain adequate ATP within red cells Pyrimidine-5’-nucleotidase deficiency is the third most prevalent red cell enzymopathy >60 families have been described Leads to build up of pyrimidine nucleotides which are visible as insoluble aggregates (basophilic stippling) Mild to moderate chronic haemolysis, splenomegaly and jaundice Deficiencies in adenylate kinase and adenosine deaminase have been described associated with haemolysis.

11. Platelet enzyme defects Platelets rely on a number of enzymes to ensure adequate supply of agonists in granules to ensure platelet aggregation occurs Thromboxame pathway Cyclo-oxygenase -1 Thromboxane synthase Cytosolic phospholipase A2 Bleeding phenotype is mild in almost all cases.

12. Enzyme defects causing Congenital Neutropenia Marked neutropenia (<0.2x10^9/L) Presents in the first year of life with recurrent infections and neutropenia May be isolated or syndromic Isolated forms are associated with mutations in Neutrophil Elastase (ELANE), HAX1, GFI1. Many syndromic forms associated with chronic neutropenia Glycogen storage disease Type 1b - Glucose-6-phosphate translocase Glucose-6-phosphatase catalytic subunit 3 (G6PC3) deficiency Barth Syndrome – tafazzin (a mitochondrial phopspholipid transacylase) deficiency

14. Enzyme deficiencies causing Aplastic Anaemia Telomere defects Ends of linear chromosomes Cap the chromosome, forming a buffer, so that Genomic DNA is not lost during each cell division. When telomeres reach a critically short length, the DNA Damage response mechanisms are initiated leading to cellular senescence or apoptosis.

15. DNA DAMAGE RESPONSE APOPTOSIS / SENESCENCE GENETIC INSTABILITY TELOMERASE T-LOOP

16. Short telomere syndromes Dyskeratosis congenita Mucocutaneous triad Nail dystrophy Oral leukoplakia Skin pigmentation abnormalities Bone Marrow failure Lung fibrosis Predisposition to malignancy

17. Short telomere syndromes Deficiencies in the specialised polymerase, Telomerase is found in the majority of cases Telomerase is a multiunit holoenzyme made up of Telomerase Reverse Transcriptase (TERT), Telomerase RNA component (TERC) and Dyskerin. Deficiencies in the helicase RTEL1 is also found in a minority of cases of DC RTEL1 catalyses the unfolding of the T-loop so that the telomere can be elongated.

18. Enzyme deficiencies causing Aplastic Anaemia Fanconi Anaemia Inherited bone marrow failure syndrome caused by defective DNA break repair mechanisms Other congenital anomalies Typical facies Short stature Café-au-lait Radial ray abnormalities Renal Cardiac Predisposition to cancer

19. Fanconi Anaemia

20. Conclusion Enzymes are essential for function of all haematopoietic cell lineages Defects in enzymes can lead to a large variety of haematological conditions Haemolytic anaemias Bleeding disorders Neutropenia Bone marrow failure Treatment is mostly supportive, with bone marrow transplantation able to cure defect in more severe cases.