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HEMOLYTIC ANEMIAgsia.tums.ac.ir/Images/Download/9549/G6PD__PK_deficiency.pdf · Five classes of...
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HEMOLYTIC ANEMIA Enzymopathies Deficiencies in Hexose Monophosphate Shunt Glucose 6-Phosphate Dehydrogenase Deficiency Deficiencies in the EM Pathway Pyruvate Kinase Deficiency
Transcript of HEMOLYTIC ANEMIAgsia.tums.ac.ir/Images/Download/9549/G6PD__PK_deficiency.pdf · Five classes of...
HEMOLYTIC ANEMIAEnzymopathies
Deficiencies in Hexose Monophosphate
Shunt
Glucose 6-Phosphate Dehydrogenase
Deficiency
Deficiencies in the EM Pathway
Pyruvate Kinase Deficiency
G6PD
Five classes of G-6-PD deficiency exist based on enzyme activity levels, as follows:
Enzyme deficiency with chronic nonspherocytic hemolytic anemia
Severe enzyme deficiency (<10%)
Moderate-to-mild enzyme deficiency (10-60%)
Very mild-to-no enzyme deficiency (60%)
Increased enzyme activity
> 200 million people in the world
Genetic heterogeneity (over 400 variants)
Severity of the problem can vary from hemolysis even in the absence of oxidative stress to hemolysis only on exposure to mild to marked oxidant stress
G6PD
Glucose-6-phosphate dehydrogenase
Gene symbol : G6PD
Location : Xq28
The G6PD gene is located on the X chromosome
Thus the deficiency state is an sex-linked trait
seen only in hemizygote males
Most female carriers are asymptomatic
Molecular data on human G6P
DNA
Size of gene (in kilobases) 18.5
Number of exons 13
Number of introns 12
mRNA Size (in nucleotides) 2269
Protein
Number of amino acids 515
Molecular weight (in Daltons) 59,265
Subunits per molecule of active enzyme 2 or 4
The G6PD gene is located on the X
chromosome
Thus the deficiency state is an sex-linked
trait seen only in hemizygote males
Most female carriers are asymptomatic
Structure of G6PD
Glucose-6-Phosphate Dehydrogenase, is comprised of a dimer or tetramer of identical polypeptide chains
Each unit consists of 515 amino acids
The single G6PD locus in humans is located on the telomeric region of the long arm of the X-chromosome
Females have two X chromosomes, hence two copies of G6PD, while males have only one X chromosome and one copy of G6PD
The normal G6PD is called as Type B
Type A- has two base substitutions and is seen in people from central Africa
A second variant is seen among the people of the Mediterranian and is more severe than Type A-
The third variant that is relatively more common but less severe is seen in southern China
Familial Genetics of G6PD
Five genotypes can form from combinations of
one normal (GdB) and one deficient form (e.g.,
GdA- or GdMed) of G6PD
Females GdB/GdB, Homozygous Normal; "Normal"
GdB /GdA-, Heterozygous; "Heterozygote"
GdA-/GdA-, Homozygous Deficient; "G6PD Deficient"
Males
GdB, Hemizygous Normal; "Normal"
GdA-, Hemizygous Deficient; "G6PD Deficient
Four most common variants out of 300+ known
GdB Normal Activity All World Populations
GdANormal Activity; Aspartic acid substituted for
asparagine at position 126, Guanine for
adenine at DNA position 376
Africa (most common variant)
GdA-
8 - 20% Normal Activity; Methionine for
Valine at position 67 and Aspartic Acid for
Asparagine at position 126, Adenine for
Guanine at position 202 and Guanine for
Adenine at position 376
Africa
GdMed< 5% Normal Activity; Phenylalanine for
Serine at position 188; Thymine for Cytosine
at position 563
Iran, Iraq, India, Pakistan,
Greece, Sardinia
Glucose 6-Phosphate
DehydrogenaseDifferent Isozymes
0
0.2
0.4
0.6
0.8
1
0 20 40 60 80 100 120
RBC Age (Days)
G6P
D A
cti
vit
y (
%)
Normal (GdB) Black Variant (GdA-)
Mediterranean (Gd Med)
Level needed for protection vs ordinary oxidative stress
G6PD Heterozygotes
Because of the random inactivation of one X chromosome in each female body cell, heterozygotes have two kinds of Red Blood Cells
G6PD Normal
G6PD Deficient
Depending on which X chromosome was inactivated in the stem cell giving rise to the particular RBC
The Pentose Phosphate Pathway. Note the importance of G6PD in the production of reduced G-SH, ribose, and NADPH
NADP+ = nicotinamide adenine dinucleotide phosphate
NADPH = reduced nicotinamide adenine dinucleotide phosphate
GS-SG = oxidized glutathione
G-SH = reduced glutathione
G6PD DEFICIENCYFunction of G6PD
G6PD
GSSG 2 GSH
NADPH NADP
2 H2O H2O2
6-PG G6P
Hgb
Sulf-Hgb
Heinz bodies
Hemolysis
InfectionsDrugs
Function of G6PD
present in the cytoplasm of all cells
In (RBC), which lack nuclei, mitochondria, and other organelles, G6PD is particularly significant
G6PD is involved in the first step of the Pentose Phosphate Shunt
Catalyzes the oxidation of Glucose-6-Phosphate to 6-Phosphogluconolactone (Phosphogluconate)
Only source of NADPH and GSH, necessary for the reduction of hydrogen peroxide
Hydrogen Peroxide is a strong oxidant that will degrade the RBC and cause hemolysis if it is not reduced
Thiol-ester (-SH) groups found in many cellular enzymes are maintained in a reduced state by NADPH and GSH through the action of G6PD in the hexose monophosphate shunt (pentose phosphate pathway)
In the absence of an antioxidative mechanism, oxidant stress results in conversion of reactive thiol groups in haemoglobin to disulphides and sulphates, with ferriheme dissociation
sulfhaemoglobin accumulation, and finally large aggregates of precipitated haemoglobin (Heinz bodies)
G6PD Activity
Declines with age of RBC
GdB has 62 day half-life for decay of activity
Sustains GSH levels for 100 to 120 day RBC life span
GdA- has normal activity when new, but the activity half-life is only 13 days
Deficiency is due to instability of the enzyme
GdMed has greater instability with 8 day half-life
New cells already have reduced activity, and mature RBC have enzyme levels < 1% normal activity
Glucose 6-Phosphate
DehydrogenaseFunctions
Regenerates NADPH, allowing regeneration of glutathione
Protects against oxidative stress
Lack of G6PD leads to hemolysis during oxidative stress Infection
Medications
Fava beans
Oxidative stress leads to Heinz body formation
The diagnosis can be confirmed by G6PD
assay
The test may be negative during a hemolytic
crisis when the older and defective RBCs are
replaced by younger cells
In such cases, the test has to be repeated
Diagnosis.
Female heterozygotes may be difficult to detect as they have G6PD levels intermediate between those of hemizygous deficient males and normal individuals
G6PD screening during an acute attack may result in a false-normal result as reticulocytes have higher G6PD content than mature red cells
Heinz bodies may be detected during the early phase of haemolysis
Symptoms of G6PD deficiency
G6PD deficiency is manifested as anemia, with RBCs being prematurely destroyed
RBCs are also extremely susceptible to oxidative stress
Neonatal jaundice is a yellowish discoloration sclera, skin, and mucous membranes caused by deposition of bile salts in these tissues
The hemolytic crisis may manifest within hours of exposure to oxidant stress
In severe cases, hemoglobinuria and peripheral circulatory collapse can occur
Since only older red cells are affected most, the problem is usually self-limiting
There will be a rapid drop in hematocrit, rise in plasma hemoglobin and unconjugated bilirubin.
Heinz bodies can be seen on crystal violet staining. These are removed in the spleen in a day or two and 'bite cells', with loss of a portion of the periphery of the red cell, may be seen
Hemolytic episodes triggered by :
viral or bacterial infections
drugs or toxins that have an oxidating potential :
Antimalarials like primaquine, pamaquine, dapsone,
sulfonamides like sulfamethoxazole, nitrofurantoin,
vitamin K, doxorubicin, methylene blue, nalidixic acid,
furazolidone etc. and naphthalene balls can cause
hemolysis in defective individuals.
severe form of this is a direct result of
insufficient activity of the G6PD enzyme in the
liver
In some cases, the neonatal jaundice is severe
enough to cause death or permanent
neurologic damage
Acute haemolysis may occur
1. Drugs : aspirin, sulfonamides, antimalarials,
nitrofurantoin, para-aminosalicylic acid,
chloramphenicol, naphthalene, methylene blue
and vitamin K analogues.
2. Following infections
3. After ingestion of broad beans (favism)
4. In the neonatal period (Icterus Neonatorum)
chloroquine phenylhydrazine
hydroxychloroquine pyridium
mepacrine (quinacrine) quinine
pamaquine toluidine blue
pentaquine trinitrotoluene
primaquine urate oxidase
quinine vitamin K (water soluble)
quinocide CYTOTOXIC/ANTIBACTERIAL
CARDIOVASCULAR DRUGS chloramphenicol
procainamide co-trimoxazole
quinidine furazolidone
SULFONAMIDES/SULFONES furmethonol
dapsone nalidixic acid
sulfacetamide neoarsphenamine
sulfamethoxypyrimidine nitrofurantoin
sulfanilamide nitrofurazone
sulfapyridine PAS
sulfasalazine para-aminosalicylic acid
sulfisoxazole PLEASE READ DISCLAIMER
Infection trigger
Oxidative metabolites produced by bacterial,
viral, and rickettsial cause an anemic response
Viral hepatitis, pneumonia, and typhoid fever
are particularly likely to precipitate a hemolytic
episode in G6PD deficient individuals
Bite Cells
Heinz Bodies
G6PD Deficiency: Acute haemolysis
Reticulocytosis and "blister" or "bite"
Blood film shows anisocytosis with a single blister cell.
Heinz Bodies
Haemoglobin denaturation leads to its precipitation as Heinz bodies.
Heinz bodies bind to the red cell membrane and alter its rigidity, resulting in premature destruction in the spleen. The spleen also removes membrane-bound Heinz bodies from red cells resulting in "blister" or "bite" cells.
Favism
The Fava Bean (Vicia faba) is a favored cultigen in areas where the GdMed allele is common
Vicine and convicine make up approximately 0.5% of the wet weight of the Fava bean
These compounds metabolize to divicine and isouramil in the intestine
These metabolites decrease RBC reduced glutathione (GSH)
Produce hydrogen peroxide and free radicals
Creates a severe oxidant stress in G6PD deficient cells
The defect is known to provide partial
protection against malaria, by providing
defective environment in the affected red
cells
Plasmodium in the RBC
Plasmodium preferentially attack immature RBC but P.
falciparum can invade RBC of all ages
Plasmodium oxidizes RBC NADPH from the Pentose
Phosphate pathway for its metabolism
This results in a deficiency of RBC GSH, most severe
in G6PD deficient individuals, leading to peroxide-
induced hemolysis which curtails the development of
Plasmodium
After several cell cycles the Plasmodium can adapt to
produce its own G6PD, reducing the adaptive benefit
of G6PD deficiency
Fava Beans and Malaria
Recall that fava beans contain compounds that metabolize to powerful oxidants
In a cell that is oxidant-stressed by Plasmodium infection, the addition of another strong oxidant can lead a rapid build-up of peroxide
In vitro and in vivo (mouse) studies indicate a suppressant effect of divicine and isouramil on Plasmodium in G6PD normals
This effect would be expected to be even greater in G6PD deficient individuals
Fava bean cultivation is widespread, especially
throughout the circum-Mediterranean region
There is substantial overlap between the cultivation of
fava beans and the GdMed allele
About 1 in 12 cases of favism results in mortality
Mostly affects children (up to 95% of cases)
Responses to Favism
Mediterranean populations have developed several responses including food taboos, preparation techniques, and folk remedies
Highly susceptible groups including children and pregnant women are frequently forbidden to consume fava beans
Drying, soaking, and removing the skins appear to reduce toxicity
Increasing sugar consumption reduces the severity of an impending hemolytic crisis
World distribution of G6PD deficiency. "The
values shown by the different shadings are
gene frequencies in the different populations"
No specific treatment is needed since the
condition is usually self limiting
Rarely blood transfusions are indicated
Adequate urine output should be ensured
MALARIA and the RED CELL
A remarkable example of Darwinism in action.
All elements -- blood groups on the envelope
(e.g.Duffy), the hemoglobin, and the presence of
G6PD deficiency are all involved in guarding against
malaria.
PYROVATE KINAZE
Next most common enzymopathy after
G6PD deficiency, but is rare
It is inherited in a autosomal recessive
pattern and is the commonest cause of
the so-called :
"congenital non-spherocytic haemolytic
anaemias" (CNSHA)
Pathophysiology
PK catalyses the conversion of
phosphoenolpyruvate to pyruvate with
the generation of ATP
Inadequate ATP generation leads to
premature red cell death
There is considerable variation in the severity
of haemolysis
Most patients are anaemic or jaundiced in
childhood
Gallstones, splenomegaly and skeletal
deformities due to marrow expansion may
occur
Aplastic crises due to parvovirus have been
described.
Since the erythrocyte has no mitochondria, it
has no Krebs cycle. Its only source of ATP is
through the Embden-Myerhof pathway (the
hexose monophosphate shunt does not
generate any high-energy phosphate bonds,
thus no ATP)
ATP is needed primarily for the maintenance of
the ATP-dependent potassium/sodium pump
pyruvate kinase deficiencyPK deficiency differs from G-6-PD deficiency
Mode of inheritance is autosomal recessive
Hemolysis is chronic and ongoing, unlike
G-6-PD deficiency, which episodic and related
to environmental exposures
Blood film: PK deficiency:
Characteristic "prickle cells" may be seen
Other red cell enzyme
defects causing haemolysis
Glycolytic pathway enzymes e.g.
hexokinase, glucose phosphate isomerase, aldolase,
phosphoglycerate kinase etc
Glutathione metabolism pathway enzymes e.g.
glutathione synthetase, glutathione reductase etc.
Nucleotide metabolism pathway enzymes e.g.
adenylate kinase, adenosine deaminase, pyrimidine 5'
nucleotidase (associated with marked basophilic
stippling) etc.
All are extremely rare
