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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