Glucose-6-Phosphate Dehydrogenase (G6PD)Deficiency AnemiaThe gene coding for G6PD enzyme is located on the X chromosome . Glucose-6-Phosphate Dehydrogenase (G6PD) DeficiencyAnemia

G6PD

Glucose-6-PhosphateDehydrogenase(G6PD) DeficiencyAnemia

Glucose-6-PhosphateDehydrogenaseproducesNADPH

The gene coding for G6PD enzyme islocated on the X chromosome.

Glucose-6-Phosphate Dehydrogenase(G6PD) Deficiency Anemia is a X-linked recessive disease

Glucose-6-PhosphateDehydrogenase(G6PD) DeficiencyAnemia

G6PDdeficient

Glucose-6-PhosphateDehydrogenase(G6PD) DeficiencyAnemia

•Reductive biosynthesis e.g., fatty acid

biosynthesis

•Antioxidant (part of glutathione system)

•Oxygen-dependent phagocytosis by WBCs

•Synthesis of nitric oxide (NO)

Uses of NADPHGlucose-6-PhosphateDehydrogenaseproducesNADPH

NADPH:Nicotinamide adeninedinucleotide phosphateNADPHprovidesthereducingequivalentsforbiosyntheticreactionsandtheoxidation-reductioninvolvedinprotectingagainstthetoxicityofreactiveoxygenspecies(ROS),

DETAILLEDINFO:https://ghr.nlm.nih.gov/condition/glucose-6-phosphate-dehydrogenase-deficiency

If mutations in the G6PD gene reduce the amount of glucose-6-phosphate dehydrogenase or alter itsstructure, this enzyme can no longer play its protective role. As a result, reactive oxygen species canaccumulate and damage red blood cells. A a consequence, red blood cells to be destroyed faster than thebody can replace them = hemolysis. A reduction in the number of red blood cells causes the signs andsymptoms of hemolytic anemia.

Oxidativedamageto:DNAProteinsLipids(unsaturatedfattyacids)

Oxidativestressanddiseases:Inflammatoryconditionse.g.,RheumatoidarthritisAtherosclerosisandcoronaryheartdiseasesObesityCancersG6PDdeficiencyhemolyticanemia

Oxidativestress:imbalancebetweenoxidantproductionandantioxidantmechanisms

Biochemical basis ofG6PD Deficiency Hemolytic Anemia, continued…

Oxidation of sulfhydryl (SH) groups of proteins inside red blood cells (erythrocytes) causes protein denaturation and formation of insoluble masses (Heinz bodies) that attach to red blood cell membranes

AlthoughG6PDdeficiencyaffectsallcells,butitismostsevereinredbloodcells……Why?

OthercellshaveothersourcesforNADPHproduction:e.g.,Malicenzymethatconvertsmalateintopyruvate

Biochemical basis ofG6PD Deficiency Hemolytic Anemia, continued…

G6PD deficient patients will develop hemolytic attack upon:

1.Intake of oxidant drugs (AAA):Antibiotics e.g., sulfa preparationAntimalarial: e.g., PrimaquineAntipyretics

2.Exposure to infection3.Ingestion of fava beans (favism, Mediterranean variant)

Chronic nonspherocytic anemia: Hemolytic attack in absence of precipitating factors. Severe form due to class I mutation

CarriersofG6PDdonotnecessarilydevelopanemia

..Diseaseistriggerdbyincreasedincreasedreactiveoxygenspecies(ROS)levels

Different Classes ofG6PD Deficiency Hemolytic Anemia

G6PD Mediterranean (II)Enzyme with decreased stability and activity (severe).Affect all RBCs(both young and old)

G6PDA- (classIII):Moderate,youngRBCscontainenzymaticactivity.Unstableenzyme,butkineticallynormal

Different Classes ofG6PD Deficiency Hemolytic Anemia

Diagnosis ofG6PD Deficiency Hemolytic Anemia

Diagnosis of hemolytic anemiaComplete Blood Count (CBC) & reticulocytic count

Screening: Qualitative assessment of G6PD enzymatic activity(UV-based test)

Confirmatory test: Quantitative measurement of G6PD enzymatic activity

Molecular test: Detection of G6PD gene mutation

G6PD Mutations linlked with hemolytic anemiaG6PDgeneonXchromosome

Exon6

Allelefrequency:%oftotalXchromosomescarryingtheG6PDdeficiencyallele

Typeofmutations

G6PD Mutations linlked with hemolytic anemia

Detection of Mediterranean G6PD mutation by PCR-RFLP

RFLP = Restriction fragment length polymorphism

wt G6PDExon6nucleotidesaroundcodon563region:

XXXXXXXXXXXCATCTCCTCXXXXXXXXXXXXXXXXXXXXGTAGAGGAGXXXXXXXXX

MediterraneanG6PDExon6nucleotidesaroundcodon563:

Mbo IIRestrictionenzyme

MboII isatypeIIrestrictionenzymesrecognizeasymmetricDNAsequencesandcleaveoutsideoftheirrecognitionsequence

NewMbo IIsite

PCR

PCR

PCR

PCR5’

5’3’

3’

5’5’3’

3’cut

cut

In molecular biology, restriction fragment length polymorphism (RFLP) is a technique that exploits variations in homologous DNAsequences, known as polymorphisms, in order to distinguish individuals, populations, or species or to pinpoint the locations ofgenes within a sequence. The term may refer to a polymorphism itself, as detected through the differing locations of restrictionenzyme sites, or to a related laboratory technique by which such differences can be illustrated. In RFLP analysis, a DNA sample isdigested into fragments by one or more restriction enzymes, and the resulting restriction fragments are then separated by gelelectrophoresis according to their size.

PCR-RFLP: 1° step PCR amplicifation of DNA 2° step restiction digest -à mapping of sequence changes in PCR products derivedformdifferent sources of DNA (forexample differentpatients)

G6PD563CàTvariantPCRoligos amplify region ofinterest inExon 6oftheG6PDgene(around aminoacid position563)

XXXXXXXXXXXCATCTTCTCXXXXXXXXXXXXXXXXXXXXGTAGAAGAGXXXXXXXXX

R =sitoperenzimadirestrizioneMboII

563C

MboIIcuts 1x

MboIIcuts 2x

563Tmutationcreates newMboII site

G6PD563CàTvariant INEXON6

1. G6PDExon 6specific primers2. PCRamplify specific region of

students3. Purify PCRproduct4. DigestpurifiedDNAusing

MboII5. Run agarose gel6. 563CàTvariants results anew

MboII site inthePCRfragment7. Additional bandappears ingel

Detection of Mediterranean G6PD mutation

PCRfromwt G6PDallele

PCRproductfromMediterranean,mutant

G6PDalleleG6PD563C wild-type INEXON6Usedprimers amplify region thatcontains 1MboII site

Usedprimers amplify region thatcontains 1+1MboII sites

3901MboII35

PCR

PCR

5’

3’

3’

5’

Position 3901MboII35

PCR

PCR

5’

3’

3’

5’

PositionMboII133

35bp

355bp

35bp

98bp

257bp

Detection of Mediterranean G6PD mutationControlforPCR– MUSTGIVE

AMPLFICATION

DNAamplified fromhumanDNAwithwt G6PD(Taq)andclonedviaTA-cloning intopCR-TOPOII

à Make PCR-RFLPwitholigosà Run gel

à PCRgivesbandforwt allele

ControlforPCR–MUSTGIVEAMPLFICATION

DNAamplified fromhumanDNAwithwt G6PD(Taq)andclonedviaTA-cloning intopCR-TOPOII;

G6PD563CàTvariation inserted (newMboII site)

à Make PCR-RFLPwitholigosà Run gel

à PCRgivesbandformutant allele

R:MboII

wtG6PDalleleregion

wtG6PDalleleregion

USEPLASMIDSas positve andnegativecontrolforG6PDallelestatus!!PCRsetup:- Amplificationwtallelefromplasmid- Amplifcationof563CàTallelefromplasmid- DNAfromidividuals

R R R