red blood cell (rbc) membrane and enzyme disorders … · red blood cell (rbc) membrane and enzyme...
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RED BLOOD CELL (RBC) MEMBRANE
AND ENZYME DISORDERS
Joan Lluis Vives CorronsJoan-Lluis Vives CorronsRed Cell Pathology Unit
Hospital Clinic i Provincial Universty of Barcelona
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Anemia reticulocytosis elevated bilirubin and LD
HEREDITARY HAEMOLYTIC ANAEMIA- GENERAL FEATURES -
Anemia, reticulocytosis, elevated bilirubin and LD levels, decreased haptoglobin levels, directantiglobulin test (Coombs) negative
Chronic hemolysis vs periodic hemolytic episodes
Palor,icterus,gall stones,splenomegaly,dark urines
HAEMOLYTIC ANAEMIA- SIMPLIFIED FLOWCHART -
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Red blood cell membrane disordersRBC MEMBRANE/CYTOSKELETON DISORDERS- Morphological abnormalities-
Red blood cell membrane and enzymedisorders – why discriminate?
� Diagnosis
RBC MEMBRANE AND ENZYME DISORDERS-WHY DISCRIMINATE ?-
� Therapy- prevention of- splenectomy
certain food & medications
� (Genetic) counseling- membrane disorders: autosomal dominant
enzyme disorders: autosomal recessive some X linked- enzyme disorders: autosomal recessive, some X-linked
� To better understand pathophysiology- future (gene) therapies
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complexes in the red cellMultiprotein complexes in the RBC membrane
Plasma membrane &Integral proteins
Anchoring proteins
Cytoskeletal proteins
Salomao M et al. PNAS 2008;105:8026-8031
A deficiency or dysfunction in any one of these membrane proteins can weakenor destabilize the cytoskeleton, resulting in membrane surface area loss and areduced life span: hemolysis
Red blood cell membrane disordersHorizontal Interactions in the RBC membrane
Spectrin-Actin-Protein 4.1 junctional complex
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Red blood cell membrane disordersHorizontal Interactions in the RBC membrane
Hereditary elliptocytosisHEREDITARY ELLIPTOCYTOSIS(normal osmotic fragility)
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Hereditary pyropoikilocytosisHEREDITARY PYROPOIKILOCYTOSIS(decrased membrane heat stability)
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Severe form of elliptocytosisPresentation usually in neonatal period(severe anemia) Homozygous/compound heterozygousfor spectrin mutations
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Red blood cell membrane disordersVertical Interactions in the RBC membrane
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Hereditary spherocytosisHEREDITARY SPHEROCYTOSIS
� Vesiculation of unsupported surfacereduction in membrane surface area
components cause a progressive
� Red cell shape changes from a flexible, deformable biconcave disc to aspherical, poorly deformable (fragile) red cell – the spherocyte
Hereditary spherocytosis
� Most common cause of inherited chronichemolysis in Northern Europe and NorthAmerica Prevalence EU: 1:3000
HEREDITARY SPHEROCYTOSIS
America. Prevalence EU: 1:3000Inheritance:Dominant: 75%, recessive: 25% Ankyrin (50-60%), spectrin (20%), and Band 3 (15-20%),Protein 4,2 defectsSeverity of hemolysis depends on theextent of decrease in membrane surfacearea (contents of spectrin)
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area (contents of spectrin)20% mild, 60% moderate HS, 10%moderate severe, 3-5% severe HSExcellent response to splenectomy�
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Laboratory diagnosis of red cell membranedisorders
Laboratory diagnosis of RBC membrane disorders
1.Screening testsRBC MorphologyRBC Morphology
RBC Osmotic fragility test
Increased RBC OFIncreased RBC OF
HERDITARY SPHEROCYTOSISOSMOTIC FRAGILITY TEST
↑ NaCl Water
NORMALCONTROL
HEREDITARYHEREDITARYSPHEROCYTOSIS
HYPOTONIC LYSIS OF RBCs
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Laboratory diagnosisdisorders
of red cell membrane
2. Special diagnostic tests- Spectrin (RIA)- EMA (Eosine-5-maleimide)
Laboratory diagnosis of RBC membrane disorders
(binds Lys430 of Band 3, Rh(AG), CD47)- SDS-PAGE- Laser-assisted Optical Rotational CellAnalyzer (LORCA)- Viscometry-
Example: Spectrin: 100% (ref: 85-115%)EMA: 40% relative fluorescence (ref:> 80%)
Red blood cell membrane disorder due to band 3 deficiency
Hereditary pyropoikilocytosis
3. Molecular studies
Laboratory diagnosis of RBC membrane disorders
Example: SLC4A1 analysis (Anion exchanger 1 or Band 3 gene)
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Southeast Asian Ovalocytosis (SAO)
� Dominant inheritance
� Very common in malaria-endemic areas in Asia 5-25%
SOUTHEAST ASIAN OVALOCYTOSIS (SAO)
� Very common in malaria-endemic areas in Asia, 5-25%
� SAO SLC4A1 gene is due to a 27 bp deletion (amino acids 400-408)in Band 3, at the boundary of the cytoplasmic and membrane domains in cis with the Lys56Glu substitution
� SAO cells are rigid and hyperstable
� Hemolysis is mild or absent
Child, 2 months of age (Molukkan)Hb 5.1 mmol/L, MCV 81, Retics N
Hereditary Stomatocytosis
� A group of very rare, dominantly inheritedhemolytic anemias with abnormal membrane
HEREDITARY STOMATOCYTOSIS
hemolytic anemias with abnormalpermeability to univalent cations
membrane
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HydrocytosisXerocytosisCryohydrocytosisFamilial pseudohyperkaliemiap yp
� Splenectomy is contra-indicatedrisk of trombotic complications !
because of increased
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Hereditary red blood cell enzyme disorders
� A red blood cell enzyme disorder should be assumed in
HEREDITARY RBC ENZYME DISORDERS
A red blood cell enzyme disorder should be assumed in cases of persistent normocytic hemolytic anemia in which hemoglobin abnormalities and antiglobulin reactions have been cluded,spherocytes are absent, and osmotic fragility is normal
� Red blood cell enzymopathies cause hereditary nonspherocytic hemolytic anemia (HNSHA)
GLUCOSE
LactateEnzymes
METABOLIC PATHWAYS OF THE ERYTHROCYTE
Glycolytic pathway
ATP
Haemoglobin
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Hereditary pyropoikilocytosisRED BLOOD CELL ENZYMES
6-Phosphogluconate dehydrogenase6-PhosphogluconolactonaseAcetylcholinesterase
Glucose-6-phosphate dehydrogenase*Gluthathione peroxidase Gluthathione reductase Glutathione synthetase
Adenine phophoribosyl transferaseAdenosine deaminase Adenylate kinase AldolaseAMP deaminaseBisphosphoglycerate mutase Carbonic anhydrase I Carbonic anhydrase II CatalaseCytochrome b5 reductaseδ ALA d h d
yGlutathione-S-transferaseGlyceraldehyde 3-phosphate dehydrogenaseGlyoxalase I Hexokinase *Hypoxanthine-guanine phosphoribosyl transferaseITPaseLactate dehydrogenase NADPH di h δ-ALA dehydrase
Enolase GalactokinaseGalactose-1-P-uridyltransferaseγ-Glutamylcysteine synthetaseGlucose phosphate isomerase
NADPH diaphorase Phosphofructokinase Phosphoglucomutase Phosphoglycerate kinase Pyrimidine-5’-nucleotidase *Pyruvate kinase *Triosephosphate isomerase
* Enzymatic activity strongly age-dependent
Hereditary pyropoikilocytosisENZYMOPATHIES WITH CLEAR CAUSE EFFECT RELATIONSHIP
6-Phosphogluconate dehydrogenase6-PhosphogluconolactonaseAcetylcholinesterase
Glucose-6-phosphate dehydrogenase *Gluthathione peroxidase Gluthathione reductaseGlutathione synthetase Acetylcholinesterase
Adenine phophoribosyl transferase Adenosine deaminase (hyperactivity)Adenylate kinaseAldolaseAMP deaminase Bisphosphoglycerate mutase Carbonic anhydrase I Carbonic anhydrase II CatalaseCytochrome b5 reductase
Glutathione synthetaseGlutathione-S-transferaseGlyceraldehyde 3-phosphate dehydrogenaseGlyoxalase I Hexokinase *Hypoxanthine-guanine phosphoribosyl transferaseITPaseLactate dehydrogenase Cytochrome b5 reductase
δ-ALA dehydrase Enolase GalactokinaseGalactose-1-P-uridyltransferaseγ-Glutamylcysteine synthetaseGlucose phosphate isomerase
Lactate dehydrogenase NADPH diaphorase Phosphofructokinase Phosphoglucomutase Phosphoglycerate kinasePyrimidine-5’-nucleotidase * Pyruvate kinase * Triosephosphate isomerase
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Hereditary red blood cell enzymopathies
In general: two groups
I Enzyme deficiencies of glycolysis and nucleotide metabolism
HEREDITARY RBC ENZYMOPATHIES
I. Enzyme deficiencies of glycolysis and nucleotide metabolismcontinuously impaired ATP generation: → lack of sufficient energy
ATP is required for maintenance of:- glycolysis- electrolyte red cell/plasma gradient- glutathione synthesis- membrane phospholipid asymmetryp p p y y- purine/pyrimidine metabolism
→ chronic haemolytic anaemia Most common cause: pyruvate kinase (PK) deficiency
Hereditary red blood cell enzymopathies
II. Enzyme deficiencies of the hexose monophosphate shuntglutathione metabolism
I d t l l f d d l t thi (GSH)
and
HEREDITARY RBC ENZYMOPATHIES
Inadequate levels of reduced glutathione (GSH):inability to withstand oxidative stress
GSH is required for:
- maintenance of hemoglobinʼs iron in itʼs functional (ferrous) state
- protection of metabolic enzymes and b t i f id ti d t timembrane proteins from oxidative denaturation
→ acute hemolytic crisisinduced by oxidant drugs, food (favism), infection, physiologic stress
Most common cause: glucose-6-phosphate dehydrogenase (G6PD) deficiency
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Hereditary red blood cell enzymopathies
� Metabolic dysfunction depends on:
HEREDITARY RBC ENZYMOPATHIES
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Importance of the affected enzyme (rate-limiting enzymes)Functional abnormalities (kinetic properties) of the mutant enzymeStability of the mutant enzyme (RBC lacks biosynthesis) Possibility of compensation for the deficiency by isoenzymeoverexpression or use of alternative pathways
� Most enzymopathies are associated with extravascular hemolysis
Hereditary red blood cell enzymopathies
� Clinically highly heterogeneous!Fully compensated hemolysis (without anemia) to severe hemolytic anemia
HEREDITARY RBC ENZYMOPATHIES
requiring regular transfusions
� Presentation at later age vs neonatal death (hydrops fetalis)
� No correlation between residual enzyme activity and clinical severity
� Exacerbation of hemolysis during infection� Exacerbation of hemolysis during infection
� In some cases also non-hematological symptoms:e.g. myopathy in phosphofructokinase (PFK) deficiency,severe neuropathy in triosephosphate isomerase (TPI) deficiency
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Pyruvate kinase (PK)
� Key enzyme of glycolysis: sole source of energy for the red blood cell
PYRUVATE KINASE DEFICIENCY
� Catalyses the irreversible phosphoryl group transfer fromphosphoenolpyruvate to ADP, yielding pyruvate + ATP
� Mammals: 2 genes – 4 isozymesPKM PK-M1 (muscle, heart, brain)
PK-M2 (leukocytes, platelets, erythroblasts)PKLR PK-L (liver)
PK-R (red blood cell)
� Clinical symptoms confined to the red blood cell
� Most common cause of hereditary chronic non-spherocytic hemolytic anemia
PYRUVATE KINASE DEFICIENCY
Rare, but most frequently occuring abnormality of glycolysis
Autosomal recessive disorderEstimated prevalence: 1:20,000 (white population)Worldwide distribution
Highly variable phenotypic expression
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severe: diagnosed at birth (hydrops fetalis)
mild fully compensated hemolytic anemiadiagnosed later in life
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Effects of pyruvate kinase deficiency
� Biochemical features of the PK-R deficient red blood cell:
- PK enzymatic activity usually decreased
EFFECTS OF PYRUVATE KINASE DEFICIENCY
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ATP levels usually decreased2,3-DPG levels increased due to metabolic block at???
PK step
� Red cell destruction- Precise mechanism of red cell destruction is unknown- ATP depletion initiates a series of events leading to premature destruction of (young!) PK deficient red cells in the spleendestruction of (young!) PK-deficient red cells in the spleen
� Premature destruction as well as apoptosis of RBCprogenitors may contribute to the pathogenesis of PK- R deficiency
11‐‐ X CHROMOSOME LINKED HEREDITARY TRANSMISSIONX CHROMOSOME LINKED HEREDITARY TRANSMISSION
GLUCOSE 6 PHOSPHATE DEHYDROGENASE
(G6PD) DEFICIENCY
11 X CHROMOSOME LINKED HEREDITARY TRANSMISSION X CHROMOSOME LINKED HEREDITARY TRANSMISSION Women (heterozygous o homozygous)
Men (hemicygotes)
22‐‐ PREVALENCE 1/10.000PREVALENCE 1/10.000
33‐‐ NO CLINICAS MANIFESTATIONS NO CLINICAS MANIFESTATIONS
44‐‐ HAEMOLYTIC ANAEMIAHAEMOLYTIC ANAEMIA44 HAEMOLYTIC ANAEMIA HAEMOLYTIC ANAEMIA Acute (crisis) in case of oxidative effectChronic (sporadic forms)
55‐‐ PROTECTION AGAINST THE MALARIA PARASITEPROTECTION AGAINST THE MALARIA PARASITE
66‐‐ HIGH GENETIC POLYMORPHISMHIGH GENETIC POLYMORPHISM
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G6PD DEFICIENCY ↓ GSH
SKISTOCYTES“BITTED” CELLSEXCCENTROCYTES
G6PD DEFICIENCY AND MALARIA
Geographical areas with endemic Malaria (present or past)
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1. Drug ingestionA ti l i l d
G6PD DEFICIENCY: HAEMOLYTIC TRIGGERS
• Antimalarial drugs• Sulphonamides• Phenacetin• Vitamin K
2. Fava beans ingestion (favism)g ( )3. Infections4. Metabolic acidosis
Diagnostics and pitfalls
� Demonstration of the specific enzyme defect by measuring red blood cell enzyme activitiesMolecular characterization�
ENZYMOPATHIES: DIAGNOSIS AND PITFALS
Diagnosis
PitfallsBlood transfusions�
� Reticulocytosis: some red cell enzymes are red cell age dependent (HK, PK, G6PD, aldolase, P5N)→ density gradient separation
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� Leucocyte and platelet contamination� Enzyme deficiencies may be less
pronounced under optimized in vitroconditions → low [S] assays
� Storage and shipment conditions
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Hereditary pyropoikilocytosisFAMILY SCREENING
COINHERITANCE OF TWO DIFFERENT RBC MTATIONS
AKNOWLEDGEMENTS
To all the members of the
Biological Haematology Department and Red Cell Pathology Unit
Hospital Clinic i Provincial.University of BarcelonaAssumpciò Pujades
Josep-Lluis Aguilar
Dolors Colomer
Montserrat Corbella
Mar Mañu Pereira
Nuria Radó