Download red blood cell (rbc) membrane and enzyme disorders

RED BLOOD CELL (RBC) MEMBRANE
AND ENZYME DISORDERS
Joan Lluis Vives Corrons
Joan-Lluis
Red Cell Pathology Unit
Hospital Clinic i Provincial
Universty of Barcelona
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HEREDITARY HAEMOLYTIC ANAEMIA
- GENERAL FEATURES Anemia, reticulocytosis,
Anemia
reticulocytosis elevated bilirubin and LD
levels, decreased haptoglobin levels, direct
antiglobulin 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 disorders DISORDERS
RBC
MEMBRANE/CYTOSKELETON
- Morphological abnormalities-
Red blood cell membrane and enzyme
RBC MEMBRANE
AND ENZYME DISORDERS
disorders
– why discriminate?
-WHY DISCRIMINATE ?Diagnosis
Therapy
- prevention of certain food & medications
- splenectomy
(Genetic) counseling
- membrane disorders: autosomal dominant
- enzyme disorders: autosomal recessive
recessive, some X
X-linked
linked
To better understand pathophysiology
- future (gene) therapies
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thethe
red cell
Multiprotein complexes
complexesin in
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 weaken
or destabilize the cytoskeleton, resulting in membrane surface area loss and a
reduced life span: hemolysis
Horizontal
the RBC membrane
Red bloodInteractions
cell membrane in
disorders
Spectrin-Actin-Protein 4.1 junctional complex
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Horizontal
the RBC membrane
Red bloodInteractions
cell membrane in
disorders
HEREDITARY
ELLIPTOCYTOSIS
Hereditary
elliptocytosis
(normal osmotic fragility)
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Hereditary
pyropoikilocytosis
HEREDITARY
PYROPOIKILOCYTOSIS
(decrased membrane heat stability)
Severe form of elliptocytosis
Presentation usually in neonatal period
(severe anemia)
Homozygous/compound heterozygous
for spectrin mutations
Vertical
Interactions
in disorders
the RBC membrane
Red blood
cell membrane
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Hereditary
spherocytosis
HEREDITARY
SPHEROCYTOSIS
Vesiculation of unsupported surface components cause a progressive
reduction in membrane surface area
Red cell shape changes from a flexible, deformable biconcave disc to a
spherical, poorly deformable (fragile) red cell – the spherocyte
Hereditary
spherocytosis
HEREDITARY
SPHEROCYTOSIS
Most common cause of inherited chronic
hemolysis in Northern Europe and North
America Prevalence EU: 1:3000
America.
Inheritance:Dominant: 75%, recessive:
25%
Ankyrin (50-60%), spectrin (20%),
and Band 3 (15-20%),Protein 4,2 defects
Severity of hemolysis depends on the
extent of decrease in membrane surface
area (contents of spectrin)
20% mild, 60% moderate HS, 10%
moderate severe, 3-5% severe HS
Excellent response to splenectomy
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Laboratorydiagnosis
diagnosisofofRBC
red cell
membrane
Laboratory
membrane
disorders
disorders
1. Screening tests
RBC Morphology
RBC Osmotic fragility test
Increased RBC OF
HERDITARY SPHEROCYTOSIS
OSMOTIC FRAGILITY TEST
↑ NaCl
Water
NORMAL
CONTROL
HEREDITARY
SPHEROCYTOSIS
HYPOTONIC LYSIS OF RBCs
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Laboratorydiagnosis
diagnosisofofRBC
red cell
membrane
Laboratory
membrane
disorders
disorders
2. Special diagnostic tests
- Spectrin (RIA)
- EMA (Eosine-5-maleimide)
(binds Lys430 of Band 3, Rh(AG), CD47)
- SDS-PAGE
- Laser-assisted Optical Rotational Cell
Analyzer (LORCA)- Viscometry-
Example:
Spectrin: 100% (ref: 85-115%)
EMA: 40% relative fluorescence (ref:> 80%)
Red blood cell membrane disorder due to band 3 deficiency
Laboratory
of RBC membrane disorders
Hereditarydiagnosis
pyropoikilocytosis
3. Molecular studies
Example: SLC4A1 analysis (Anion exchanger 1 or Band 3 gene)
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Southeast Asian
Ovalocytosis
(SAO)
SOUTHEAST
ASIAN
OVALOCYTOSIS
(SAO)
Dominant inheritance
Very common in malaria-endemic areas in Asia
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
HEREDITARY
STOMATOCYTOSIS
A group of very rare, dominantly inherited
hemolytic anemias with abnormal membrane
permeability to univalent cations
- Hydrocytosis
- Xerocytosis
- Cryohydrocytosis
- Familial pseudohyperkaliemia
p
yp
Splenectomy is contra-indicated because of increased
risk of trombotic complications !
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Hereditary
red blood
cell ENZYME
enzyme disorders
HEREDITARY
RBC
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)
METABOLIC PATHWAYS OF THE ERYTHROCYTE
Glycolytic pathway
GLUCOSE
Enzymes
Lactate
ATP
Haemoglobin
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Hereditary
pyropoikilocytosis
RED
BLOOD CELL ENZYMES
Glucose-6-phosphate dehydrogenase*
Gluthathione peroxidase
Gluthathione reductase
Glutathione synthetase
y
Glutathione-S-transferase
Glyceraldehyde 3-phosphate
dehydrogenase
Glyoxalase I
Hexokinase *
Hypoxanthine-guanine phosphoribosyl
transferase
ITPase
Lactate dehydrogenase
NADPH di
diaphorase
h
Phosphofructokinase
Phosphoglucomutase
Phosphoglycerate kinase
Pyrimidine-5’-nucleotidase *
Pyruvate kinase *
Triosephosphate isomerase
6-Phosphogluconate dehydrogenase
6-Phosphogluconolactonase
Acetylcholinesterase
Adenine phophoribosyl transferase
Adenosine deaminase
Adenylate kinase
Aldolase
AMP deaminase
Bisphosphoglycerate mutase
Carbonic anhydrase I
Carbonic anhydrase II
Catalase
Cytochrome b5 reductase
δ ALA d
δ-ALA
dehydrase
h d
Enolase
Galactokinase
Galactose-1-P-uridyltransferase
γ-Glutamylcysteine synthetase
Glucose phosphate isomerase
* Enzymatic activity strongly age-dependent
ENZYMOPATHIES
WITH CLEAR CAUSE
Hereditary
pyropoikilocytosis
EFFECT RELATIONSHIP
Glucose-6-phosphate dehydrogenase *
Gluthathione peroxidase
Gluthathione reductase
Glutathione synthetase
Glutathione-S-transferase
Glyceraldehyde 3-phosphate
dehydrogenase
Glyoxalase I
Hexokinase *
Hypoxanthine-guanine phosphoribosyl
transferase
ITPase
Lactate dehydrogenase
NADPH diaphorase
Phosphofructokinase
Phosphoglucomutase
Phosphoglycerate kinase
Pyrimidine-5’-nucleotidase *
Pyruvate kinase *
Triosephosphate isomerase
6-Phosphogluconate dehydrogenase
6-Phosphogluconolactonase
Acetylcholinesterase
Adenine phophoribosyl transferase
Adenosine deaminase (hyperactivity)
Adenylate kinase
Aldolase
AMP deaminase
Bisphosphoglycerate mutase
Carbonic anhydrase I
Carbonic anhydrase II
Catalase
Cytochrome b5 reductase
δ-ALA dehydrase
Enolase
Galactokinase
Galactose-1-P-uridyltransferase
γ-Glutamylcysteine synthetase
Glucose phosphate isomerase
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HEREDITARY
RBC
Hereditary
red blood
cellENZYMOPATHIES
enzymopathies
In general: two groups
Enzyme deficiencies of glycolysis and nucleotide metabolism
I
I.
continuously impaired ATP generation: → lack of sufficient energy
ATP is required for maintenance of:
- glycolysis
- electrolyte red cell/plasma gradient
- glutathione synthesis
phospholipid
p
p asymmetry
y
y
- membrane p
- purine/pyrimidine metabolism
→ chronic haemolytic anaemia
Most common cause: pyruvate kinase (PK) deficiency
HEREDITARY
RBC
Hereditary
red blood
cellENZYMOPATHIES
enzymopathies
II.
Enzyme deficiencies of the hexose monophosphate shunt and
glutathione metabolism
IInadequate
d
t levels
l
l off reduced
d
d glutathione
l t thi
(GSH)
(GSH):
inability to withstand oxidative stress
GSH is required for:
- maintenance of hemoglobinʼs iron in itʼs
functional (ferrous) state
- protection of metabolic enzymes and
membrane
b
proteins
t i ffrom oxidative
id ti
d
denaturation
t ti
→ acute hemolytic crisis
induced by oxidant drugs, food (favism), infection, physiologic stress
Most common cause: glucose-6-phosphate dehydrogenase (G6PD) deficiency
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Hereditary
red blood
cellENZYMOPATHIES
enzymopathies
HEREDITARY
RBC
Metabolic dysfunction depends on:
- Importance of the affected enzyme (rate-limiting enzymes)
- Functional abnormalities (kinetic properties) of the mutant enzyme
- Stability of the mutant enzyme (RBC lacks biosynthesis)
- Possibility of compensation for the deficiency by isoenzyme
overexpression or use of alternative pathways
Most enzymopathies are associated with extravascular hemolysis
Hereditary
red blood
cellENZYMOPATHIES
enzymopathies
HEREDITARY
RBC
Clinically highly heterogeneous!
Fully compensated hemolysis (without anemia) to severe hemolytic anemia
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
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 DEFICIENCY
Pyruvate
kinase (PK)
Key enzyme of glycolysis: sole source of energy for
the red blood cell
Catalyses the irreversible phosphoryl group transfer from
phosphoenolpyruvate to ADP, yielding pyruvate + ATP
Mammals: 2 genes – 4 isozymes
PKM
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
PYRUVATE KINASE DEFICIENCY
Most common cause of hereditary chronic non-spherocytic hemolytic anemia
Rare, but most frequently occuring abnormality of glycolysis
Autosomal recessive disorder
Estimated prevalence: 1:20,000 (white population)
Worldwide distribution
Highly variable phenotypic expression
severe:
mild
diagnosed at birth (hydrops fetalis)
fully compensated hemolytic anemia
diagnosed later in life
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EFFECTS
PYRUVATE
Effects ofOF
pyruvate
kinase KINASE
deficiencyDEFICIENCY
Biochemical features of the PK-R deficient red blood cell:
-
PK enzymatic activity usually decreased
ATP levels usually decreased
2,3-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
PK deficient red cells in the spleen
Premature destruction as well as apoptosis of RBC
progenitors may contribute to the pathogenesis of PK- R deficiency
GLUCOSE 6 PHOSPHATE DEHYDROGENASE
(G6PD) DEFICIENCY
1‐ X CHROMOSOME LINKED HEREDITARY TRANSMISSION
Women (heterozygous o homozygous)
Men (hemicygotes)
2‐ PREVALENCE 1/10.000
3‐ NO CLINICAS MANIFESTATIONS
4‐ HAEMOLYTIC ANAEMIA
Acute (crisis) in case of oxidative effect
Chronic (sporadic forms)
5‐ PROTECTION AGAINST THE MALARIA PARASITE
6‐ HIGH GENETIC POLYMORPHISM
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G6PD DEFICIENCY
↓ GSH
SKISTOCYTES
“BITTED” CELLS
EXCCENTROCYTES
G6PD DEFICIENCY AND MALARIA
Geographical areas with endemic
Malaria (present or past)
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G6PD DEFICIENCY: HAEMOLYTIC TRIGGERS
1. Drug ingestion
• Antimalarial
A ti l i l d
drugs
• Sulphonamides
• Phenacetin
• Vitamin K
g
(favism)
(
)
2. Fava beans ingestion
3. Infections
4. Metabolic acidosis
ENZYMOPATHIES: DIAGNOSIS AND PITFALS
Diagnostics and pitfalls
Diagnosis
Demonstration of the specific enzyme defect by measuring red blood cell
enzyme activities
Molecular characterization
Pitfalls
Blood transfusions
Reticulocytosis: some red cell enzymes are red cell age dependent
(HK, PK, G6PD, aldolase, P5N) → density gradient separation
Leucocyte and platelet contamination
Enzyme deficiencies may be less
pronounced under optimized in vitro
conditions → low [S] assays
Storage and shipment conditions
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Hereditary pyropoikilocytosis
FAMILY 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 Barcelona
Assumpciò Pujades
Josep-Lluis Aguilar
Dolors Colomer
Montserrat Corbella
Mar Mañu Pereira
Nuria Radó
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