Dr. S. Ismat Bukhari

Introduction

• G6PD deficiency is the most common disease producing enzyme abnormalities in humans, affecting more than 200 million individuals worldwide.

• The highest prevalence in the Middle East, tropical Africa & Asia.

• G6PD Deficiency is caused by 400 different mutations in gene coding for G6PD, only few of them causes the clinical symptoms of the disease.

What is G6PD?

• G6PD is an metabolic enzyme is involved in pentose phosphate pathway, especially important in red blood cell metabolism

• It also protects red blood cells from the effects of potentially harmful molecules called REACTIVE OXYGEN SPECIES

G6PD Deficiency

• The G6PD gene is located on the telomeric region of the long arm of X-chromosome (band Xq28).

• Mutation of the X-linked G6PD gene (approximately 127 have been reported with a single base substitute leading to amino acid replacements) results in many variants of protein with varying enzyme activity that result in different patterns of clinical manifestations.

Male to Female ratio

• Male cases are overrepresented compared with female cases.

• Males are hemizygous for the G6PD gene; therefore the expression is either normal or deficient.

• In contrast, in females who have two copies of the gene on each chromosome, the gene expression can be normal or heterozygous.

• Homozygous inheritance in females can occur; whereas, heterozygous females have genetic mosaicism secondary to X-chromosome inactivation and can have similar manifestation as male neonates.

Inheritance

What happens in G6PD deficiency?

Prevalence of G6PD worldwide

What is favism?

• Favism is formally defined as hemolytic response to the consumption of broad beans

• Favism is disorder characterized by hemolytic reaction to the consumption of broad beans

• All individual with favism show G6PD deficiency

• However not all individuals with G6PD deficiency show favism

Decreased amounts of glutathionedue to decreased production of NADPH

• Reduction of amounts of NADPH in RBCs in G6PD deficiency causes reduction of oxidized glutathione .

Role of reduced glutathione in RBCs:

• Reduced glutathione gets rid of Reactive oxygen species including hydrogen peroxide.

• Reduced Glutathione helps to keep sulfhydryl groups of haemoglobin protein in the reduced state.

Reduced production of reduced glutathione results in:

1- A decrease in detoxication of peroxides. This causes damage to RBCs membrane and hemolysis (ending in hemolytic anemia).

2- Hemoglobin protein is denatured forming insoluble masses (Heinz bodies). Heinz bodies attach to red cell membranes. Membrane proteins are also oxidized.Accordingly, red cells become rigid and removed from the circulation by macrophages in the spleen and liver ending in anemia

Although Deficiency of G6PD occurs in all cells of affected individual. It is severe in RBCs because the only pathway to form NADPH in RBCs is pentose phosphate pathway (using G6PD).

Individuals who have inherited one of the many G6PD mutations do not show clinical manifestation.

Some of patients with G6PD develop hemolytic anemia if they are exposed or ingest any oxidizing drugs

G6PD VARIANTS

• Most G6PD variants are caused by point mutations in the G6PD gene.

• Some of these point mutations do not disturb the structure of the enzyme's active site and hence, do not affect enzyme activity.

• Other point mutations may lead to production of mutant enzymes with one or more of the following:

– altered catalytic activity

– decrease stability

– an alteration of binding affinity for NADP+ or Glucose 6-phosphate.

G6PD variants can be classified into three categories

Class I Mutations

severe deficiency with no or

minimal detected enzyme activity

often associated with chronic non-

spherocytic anemia

(occurs even in absence of oxidative

stress)

Class II Mutations

(G6PD Mediterranean)

severe deficiency

with 1% to 10% residual enzyme

actiity

G6PD enzyme shows normal stability but, very low activity in

all RBCs

Class III

(G6PD Group A-)

moderate deficiency

with 10% to 60% residual enzyme

activity,

RBCs contain unstable G6PD

enzyme, but normal activity in younger

RBCs and reticulocytes

Class IV

Normal enzyme activity at 60% to

150%

Class V

increased

enzyme activity at >150%

Both G6PD Mediterranean and G6PD A- represent mutant enzymes that differ from the normal variants by a single amino acid. This change is due to DNA changes in the form of point mutations or missense mutations.

Frame shift mutations or large deletions have not been identified indicating that the complete absence of G6PD is lethal.

Mutation causing non spherocytic hemolytic anemia are clustered near the carboxyl end of the enzyme, whereas mutations causing milder forms of the disease tend to be located at the amino end of the enzyme.

Care of G6PD patients

• The most important measure is prevention – avoidance of the drugs and foods that cause hemolysis.

• Vaccination against some common pathogens (e.g. hepatitis A and hepatitis B) may prevent infection-induced attacks.

• In the acute phase of hemolysis, blood transfusions might be necessary, or even dialysis in acute renal failure.

• Blood transfusion is an important symptomatic measure, as the transfused red cells are generally not G6PD deficient and will live a normal lifespan in the recipient's circulation.

• Some patients may benefit from splenectomy as this is an important site of red cell destruction.

• Folic acid should be used in any disorder featuring a high red cell turnover.

• Although vitamin E and selenium have antioxidant properties, their use does not decrease the severity of G6PD deficiency.

Hemolytic Anemia

• G6PD deficiency cause impraired red blood cells’

transport of oxygen effectively throughout the

body resulting in stress conditions and hence

leading to hemolysis.

• There are other conditions that also caused by

G6PD deficiency- neonatal jaundice, abdominal

back pain, dizziness, headache, irregular breathing,

and palpitations.

ASSOCIATED CONDITIONS OF G6PD DEFICIENCY

Neonatal hyperbilirubinemia in G6PD

• Newborns with G6PD deficiency have a 9% incidence of hyperbilirubinemias.

• Estimated at 3.4% incidence, the condition ranges by infant race/ethnicity (12.2% in African American male infants to nearly 0% in white female infants).

• Oxidant stressors, sepsis, and delay in bilirubin elimination (such as co-inheritance with Gilbert’s disease or persistent enterohepatic recirculation) add to total plasma or serum bilirubin (TSB) rise, need for phototherapy, and risk for exchange transfusion.

Vinod K. Bhutani, MD,jaundice due to glucose-6-phosphate dehydrogenase deifciency, NeoReviews Vol.13 No.3 March 2012

Clinical patterns of hyperbilirubinemia in G6PD deficient neonates

• Early-onset hyperbilirubinemia (ie, TSB >75th percentile and increased bilirubin production)

• Pre-discharge TSB <75th percentile track exacerbated by starvation, unrecognized sepsis or late prematurity;

• Slow postnatal rise with natural decline

• Slow postnatal rise with persistent prolonged unconjugatedhyperbilirubinemia, >2 weeks age

• Complicated by acute-onset, dramatic hyperbilirubinemia with TSB rise >1 mg/dL per hour (“favism”).

Vinod K. Bhutani, MD,jaundice due to glucose-6-phosphate dehydrogenase deifciency, NeoReviews Vol.13 No.3 March 2012

Elevation of COHb in G6PD deficient neonates

• Within the reticuloendothelial system, hemoglobin releases globin and heme, which in turn undergo further degradation to release equimolar amounts of bilirubin and CO.

• The CO released then binds strongly to hemoglobin, forming carboxyhemoglobin (COHb).

Cathy Hammerman, MD and Michael Kaplan, MB, ChB; Recent Developments in the Management of Neonatal Hyperbilirubinemia.

• Because endogenous CO production in the newborn occurs almost exclusively by this pathway, hemolysis can be quantitated by determining blood COHb levels.

• Elevated COHb levels have been correlated with increased hemolysis in fetuses and neonates suffering from immune hemolytic disease and even with kernicterus and death in G6PD-deficient infants.

Cathy Hammerman, MD and Michael Kaplan, MB, ChB; Recent Developments in the Management of Neonatal Hyperbilirubinemia.

Risk of Sepsis

• Increased risk of sepsis has also been reported among preterm infants with G6PD deficiency.

• The prevalence of G6PD deficiency among 170 infants with birthweight <2.0 kg admitted to a neonatal intensive care nursery was 5.3%.

• Stage 2 necrotizing enterocolitis was 6.9-fold higher (95% CI: 2–23.5) compared with matched cohort.

Schutzman DL, Porat R. Glucose-6-phosphate dehydrogenase deficiency: another risk factor for necrotizing enterocolitis? J Pediatr. 2007;151(4):435–437

Neonatal screening for G6PD deficiency

• Newborn screening relies on the accurate (phenotypic) identification of deficient enzyme activity.

• However, variations due to partial genotypic manifestations, postnatal age, and population of younger, high enzyme activity RBCs are significant confounding factors.

Vinod K. Bhutani, MD,jaundice due to glucose-6-phosphate dehydrogenase deifciency, NeoReviews Vol.13 No.3 March 2012

• The definitive biochemical test is the spectrophotometricquantitative enzyme assay based on rate of NADPH formation (mmoles/min/gHb) with absorbance at 340 nm wavelength and expressed as IU/ gHb.

• A useful semi-quantitative screening test, Fluorescent spot test (FST) relies on the ability of NADPH to fluoresce intensely with exposure to long wave ultraviolet light.

• It lacks the sensitivity to diagnose infants with partial enzyme activity (20%–60%) including female heterozygotes.

• Other tests that detect NADPH such as methylene blue reduction, cresyl blue dye decolorization, cytochemical staining are also available.

• Identification of specific mutations by DNA/polymerase chain reaction (PCR) screening, ideal for identification of female heterozygotes, is limited by the diversity of known mutations, time-intensive process and occasional mismatch with phenotype expression of enzyme activity.

• In patients with acute hemolysis, testing for G6PD deficiency may be falsely negative because older erythrocytes with a higher enzyme deficiency have been hemolyzed.

• Female heterozygotes may be hard to diagnose because of X-chromosome mosaicism leading to a partial deficiency that will not be detected reliably with screening tests.

• Thus, a two-step approach to measure enzyme functional assay with concomitant DNA verification seems to be the most accurate and practical approach to screen, monitor and diagnose neonatal G6PD deficiency.

Drugs to avoid in

G6PD deficiency

Food to avoid in G6PD deficiency

Thank you