Red cell Enzymopathies

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• Red cell Enzymopathies

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The RBC challenge

• RBC lifespan is approx 120 days
• 1.7 x 105 circulatory cycles
• Enormous stress, both external internal
• Massive energy requirement to counter this stress
  for survival and function
• Expressed as ATP and redox equivalents
  continuously regenerated by metabolic processes

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RBC challenge Maintain

• Structural integrity
• Biconcave shape
• Membrane flexibility
• Glutathione and membrane components.

• Functional integrity
• Specific intracellular cation concentrations
• Reduced state of Hb with a Fe2
• Sulfhydryl groups of enzymes
• GSSG membrane components

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RBC energy resources

• Major resources
• Glycolysis Embden-Meyerhoff pathway
• Oxidative pentose phosphate pathway (OPPP)

• Minor resources
• Glutathione cycle
• MetHb reductase
• Nucleotide metabolism

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RBC glycolysis non-oxidative, ATP generating
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Glycolysis (Embden-Myerhoff Pathway) details
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The Oxidative pentose pathway (OPP) Hexose
Monophosphate Shunt (HMPS)
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Oxidative pentose pathway (OPP)
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THE CLINICAL PHENOTYPES IN INHERITED RBC
ENZYMOPATHIES

Those entirely normal with normally functioning
mutant genes Those with essentially normal
hematological features but with abnormal
mutations detected by special techniques Those
associated with overt hemolysis or other RBC
abnormality.

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Enzyme pathologies

• Enzyme deficiencies in both pathways have been
  identified
• Some cause non-spherocytic hemolytic anemia
• Others cause Methemogobinemia and erythrocytosis
• Frequencies differ markedly in
• Nature of enzymes
• Geographic distribution.

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The most common enzymopathies

• G6PD deficiency most common of all.
• Estimated to affect 400 million people worldwide.
  
• PK deficiency is the most common in the
  Embdem-Myerhoff pathway resulting in CHA.
• PK Class I G6PD deficiencies comprise the two
  most common erythroenzymopathies associated with
  CHA.

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Frequencies of RBC enzymopathies
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The G-6PD Gene

• Locus Xq28
• Gene Size (kb) 17.5
• Total number of exons 13
• Coding exons 12
• OMIM 305900

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G6PD RNA
Size in nucleotides 2269 5
untranslated 69 Coding region
1545 3 untranslated 655
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G6PD PROTEIN
Amino Acids, number 515(514) Molecular
Weight 59kD Subunits per molecule of
active enzyme 2 or 4 Molecules of
tightly bound NADP per subunit
1
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Molecular image of G-6PD
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MODIFICATION PROCESSES

• Susceptibility to proteolysis
• Deamidation
• Oxidation of sulphydryl residues
• Glycosilation
• Others

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Factors affecting clinical severity

• The importance of the affected enzyme
• Its rate of expression
• The stability of the mutant enzyme against
  proteolytic degradation and functional
  abnormalities
• The possibility to compensate the deficiency by
  an over-expression of the corresponding isoenzyme
  or by the use of an alternative metabolic
  pathway.

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THE REMAINING ACTIVITY OF THE OTHER GENE

• A silent innocuous variant
• Completely absent
• Unstable variant
• Kinetically aberrant or otherwise
• abnormal or defective

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G6PD (EC1.1.1.49, OMIM 305900 ) deficiency

• Most common human enzymopathy
• Worldwide 400 m/5.54B 0.07
• Southern Italy 0.01-0.07
• Southern China 0.02-0.16
• Tropical Africa 0.26
• Middle East 0.30
• Kurdish Jews 0.7

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Prevalence of G6PD deficiency

• G6PD frequency is much lower in Middle and
  Northern Europe with a frequency of 0.0005
  approximately
• Comparable with the frequency of pyruvate kinase
  (PK) enzymopathies, the most frequent enzyme
  deficiency in this area

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Saudi frequencies of G6PD deficiency
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Inactivation of X-chromosome

• Males are susceptible because they are XY
• Females can be susceptible, even though they are
  XX
• Turners can be susceptible because they are XO

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An X-chromosome is randomly inactivated
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G6PD deficiency the most common RBC enzymopathy

• Why?

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G6PD Mutations

• Polymorphic
• Sporadic

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G6PD mutations The Falciparum effect

• Endemic areas
• WHO Class II III v
• GdB- GdA-
• Frequencies of 1-50
• Mostly non-hemolytic

• Non-endemic areas
• WHO Class I
• Sporadic Gd-
• Very low frequencies
• Cause CNSHA

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Why G6PD deficiency is so highly prevalent

• The malaria hypothesis

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World distribution of P. falciparum
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World distribution of G6PD deficiency
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G6PD deficiency Fragmentation hemolysis
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The Hexosemonophosphate shunt

• For G6PD to work, there must be effective binding
  of G-6-P and NADP

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G6PD Molecule Binding sites

• G6P Lys-205
• NADP (Probable) Gly Ala-Ser-Gly-Asp-Leu-Ala

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MECHANISM OF HEMOLYSIS

• WHO Class I deficiency
• ? G6PD activity below threshold value
• Chronic NADPH insufficiency
• Chronic disulfide precipitation
• CCNSHA

• WHO Class II III deficiency
• G6PD activity above threshold
• External challenge
• Acute NADPH shortage
• Fall in GSH
• Massive HB formation
• Increased O-

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MECHANISM OF HEMOLYSIS
Inability to maintain adequate levels of GSH,
leads to disulfide aggregates attaching to RBC
membrane Neutralization of toxic free O- radicals
is impaired, and H2O2 accumulates. Hb oxidation
and molecular rupture lead to Heinz body
formation causing membrane alterations
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Heinz Bodies in acute oxidant hemolysis
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Inactivation of X-chromosome

• Males are susceptible because they are XY
• Females can be susceptible, even though they are
  XX
• Turners can be susceptible because they are XO

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G6PD deficiency Causes of Hemolysis

• Oxidative damage to RBC proteins by oxygen
  radicals produced by
• 1. Infections
• 2. Drugs
• 3. Fava bean ingestion

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INFECTIOUS PRECIPITANTS
Infectious hepatitis Pneumonia Typhoid

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CLINICAL SYNDROMES OF G6PD DEFICIENCY
Drug-induced hemolysis Infection-induced
hemolysis Favism Neonatal jaundice
(NNJ) Chronic non-spherocytic hemolytic anemia
(CNSHA)


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Sepsis as a cause of hemolysis in G6PD deficiency

• Covering the Umbilical Stump of Newborn
• Patient Infant boy 9 days old
• Source Hong Kong
• Presenting features 
• Increasing jaundice and lethargy for 2 days.
•  
• Been observed in hospital for the first 6 days,
  for being G6PD deficient.
• Transient mild jaundice around day 4 which
  resolved before sent home.

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DRUGS AND CHEMICALS CAUSING SIGNIFICANT HEMOLYSIS
IN G6PD DEFICIENT SUBJECTS
DRUGS DEFINITE POSSIBLE ASSOCIATION ASSOCI
ATION
Anti-malarials Primaquine
Pamaquine Pentaquine Chloroquine Sulfo
namides Sulfamethoxy-
Sulfanilamide pyridazine
Sulfacetamide Sulfadimidine
Sulfapyridine Sulfamethoxazole
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FACTORS INFLUENCING INDIVIDUAL SUSCEPTIBILITY
TO, AND SEVERITY OF, DRUG-INDUCED OXIDATIVE
HEMOLYSIS
INHERITED ACQUIRED Metabolic integrity of
the Dose, absorption, metabolism Erythrocyte
and excretion of drug Precise nature of
enzyme defect Presence of additional
oxidative Stress (e.g.....
infection) Genetic difference in
pharmacokinetics Effect of drug or
metabolite on enzyme activity
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FAVISM
Any age, but most in childhood Variable
expression in family members with the same Gd-.
Seasonal, but most commonly in Spring.
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WHY THIS UNPREDICTABLE EXPRESSION OF FAVISM?
? Variation in levels of glucosidases
responsible for releasing the pyrimidine
aglycones, divicine and isouramil But,
intestinal biopsies reveal normal levels of
glucosidases.
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FACTORS AFFECTING HEMOLYTIC TENDENCY
Concomitant administration of oxidant
drugs Pre-existing level of HB Hepatic
function Age Infection

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SUMMARY OF ERYTHROCYTIC DAMAGE IN G6PD DEFICIENCY
Agents that cause hemolysis increase HMP
activity Reduced GSH is observed in individuals
with hemolytic events Massive HB
formation Oxygen radicals lead to autoxidation
of hemoglobin
48
CNSHA WHO Class I deficiency
Chronic anemia with increased retics Probable
severe NNJ Variation in clinical
expression Enzyme mutation is sporadic, so
that each family usually has a different enzyme
defect
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Pyruvate Kinase (EC 2.7.1.40 OMIM 266200 )

• PK reversibly catalyzes phosphoenolpyruvate to
• Pyruvate in a heavily substrate-dependent
  reaction

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Reduction of pyruvate
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Pyruvate kinase

• There are four isoenzymes in mammalian tissues
• L-type Hepatic tissue
• R-type Erythrocytes
• M1-type Muscle brain
• M2-type Most fetal adult tissue
• Gene is located in chromosome 1q

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PK deficiency (PKD)

• In PKD, only the pyruvate kinase isozyme found in
  red blood cells, called PKR, is abnormal.
• Therefore, PKD only affects the red blood cells
  and does not directly affect the energy
  production in the other organs and tissues of the
  body.

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The common PK mutations

• Three mutations in the PKLR gene 1529A, 1456T,
  and 1468T are most frequently observed.
• 1529A is most frequently seen in Caucasians of
  northern and central European descent and is the
  most common mutation seen in PKD.
• 1456T is more common in individuals of southern
  European descent
• 1468T is more common in individuals of Asian
  descent.

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Pyruvate kinase deficiency Prevalence

• PK deficiency is the most common cause of CNSHA
• Most commonly seen in northern European
  populations
• There are at least 15 different mutations
  recognized.
• Clinical severity variable

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CAUSES OF REDUCED PYRUVATE KINASE ACTIVITY

• 1. Marked subunit instability
• 2. Kinetic abnormalities involving
• The substrate phospho-enol pyruvate cofactor ADP
  or
• Defective activation by the allosteric
• Modifier Fructose 1,6 diphosphate

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Clinical manifestations of PK deficiency

• The more severe the PKD, the earlier in life
  symptoms tend to be detected.
• Individuals with the more severe form of PKD
  often show symptoms soon after birth
• Most individuals with PKD begin to exhibit
  symptoms during infancy or childhood.
• The milder forms of PKD may not be diagnosed
  until late adulthood, after an acute illness, or
  during a pregnancy evaluation.

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CLINICAL FEATURES OF PYRUVATE KINASE DEFICIENCY
Chronic anemia of 60-120 g/L. and may be Tx
dependent Jaundice and increased indirect
bilirubin Gallstones Splenomegaly Increased
2,3 DPG Possible growth retardation

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PK deficiency blood smear post splenectomy
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Diagnosis of RBC enzymopathies

• Clinical evidence of hemolytic anemia not
  explained by other causes, eg
• Congenital spherocytosis
• Acquired spherocytosis
• Hemoglobinopathies
• Mechanical damage, etc

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Diagnosis of RBC enzymopathies

• Peripheral blood smear
• Heinz Body preparation
• Biochemical tests
• Screening
• Confirmatory

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Laboratory diagnosis of G6PD deficiency
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Determination of G6P-D levels Measuring rate of
OD change of NADPH

• Tris-HCl EDTA buffer
• MgCl2
• NADP
• Hemolysate
• Water
• Start reaction with GP

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Laboratory diagnosis of PK deficiency

• Screening test
• Detection in reduction of NADH
• Confirmatory assay
• Determination of rate of decay of NADH

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Reactions involving PK
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Determination of PK levels Measuring rate of OD
change of NADH

• Tris-HCl EDTA buffer
• KCl
• MgCl2
• NAD
• ADP
• LDH
• Hemolysate
• Water
• PEP

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Problems of enzyme quantitation

• High reticulocyte counts
• Contaminating leukocytes
• Mutants with high PEP affinities
• Recent transfusion

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G6PD ACTIVITY IN LEUKOCYTES COMPARED TO RBC IN
SOME VARIANTS

• GdB- GdA- GdBari
• RBC 0 10 lt1
• WBC 30N N 33N

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Treatment of anemia in CNSHA due to enzymopathy

• Supportive
• Blood transfusion
• Hematinics supplementation
• Splenectomy

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Acquired enzymopathies

• Causes
• Hematological malignancies
• Chemotherapy

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References

• The Metabolic Basis of Inherited Disease.
• Editor Charles R Scriver
• OMIM On Line Mendelian Genetics in Man
  http//www.ncbi.nlm.nih.gov/entrez/query.fcgi?dbO
  MIM

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Khalass!
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Effect of X-inactivation on expression of G6PD
and PGK iso-enzyme (Xq13)
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Why does X-inactivation take place?
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Phenotype-genotype correlations

• For most of the mutations, no correlation between
  the specific mutation and the severity of the
  disorder observed.
• Two of the mutations might affect severity.
• Homozygous 1456T occurring rarely, has resulted
  in very mild symptoms.
• Homozygotes for the 1529A mutation have been very
  severely affected.
• Therefore, the 1456T mutation might be associated
  with a milder form of the disease and the 1529A
  mutation is associated with a more severe form of
  the disease.
• It is not known how these mutations affect the
  severity of PKD when paired with different
  mutations