NEONATAL G-6PD DEFICIENCY

Murtaza KamalMBBS, MD, DNBDivision of NeonatologyDepartment of PediatricsSafdarjung Hospital & VMMC, New DelhiDOP- 04/05/2016

Overview Neonatal Hyperbilirubemia

Why are neonates prone to it Pathological hyperbilirubemia Approach to neonatal hyperbilirubemia

G6PD & its deficiency What is it and its deficiency Epidemiology, genetics and pathogenesis Clinical presentation, diagnosis and management

Neonatal screening program

A common, mostly benign problem60% terms, 80% preterms

Neonatal Hyperbilirubemia

Why neonates are prone to hyperbilirubemia?

1. Increased bilirubin productionDecrease RBC survival, Increased mechanical fragility, More

prone to oxidant damage

2. Increased enterohepatic circulationHigh beta- glucuronidase, Decreased intestinal bacteria and

motility

3. Defective uptake of bilirubin from plasmaDecreased ligandin

4. Immaturity of liverDefective conjugation, Decreased hepatic excretion

When does this turn pathological?

Onset in first 24 hours of life Rate of rise of serum bilirubin >0.2mg/dl/hr Jaundice requiring phototherapy Jaundice staining palms/soles Persistence beyond 8 (term) and 14 days

(preterms)

How to approach a case of NNJ?

Increased direct bilirubinIncreased indirect bilirubin

SepsisIntrauterine infections Coomb’s test +ve Coomb’s test -veBile duct paucity IsoimmunisationBiliary atresia Rh, ABO, Mi BGCholedochal cystTyrosenemia etc Hemoglobin

Jaundice in a neonateSerum bilirubin

Hemoglobin

Normal/ Low High (polycythemia) Twin-twin transfusion

Reticulocyte count Maternal-fetal transfusion Delayed cord clamping

Increased SGA babies

Red cell morphology

Characteristic Nonspecific NormalSpherocytosis G6PD defi.G6PD defi. Enclosed hemorrhageElliptocytosis PK defi. Increased E-H circu.Stomatocytosis Inadequate calorie intakeFragmented cells Neonatal asphyxia

Approach (cont…)Normal red cell morphology +

Increased/ normal reticulocyte count

Prolonged hyperbilirubemiaGilbert syndromeDown syndromeHypothyrodismCrigler-Najjar syndrome

Nelson’s Textbook of Pediatrics- 20th Edition

When to suspect hemolysis in a neonate?

Clinical features: Jaundice, anemia, hepatosplenomegaly

Laboratory evidence : Peripheral smear showing evidence of hemolysis, Indirect hyperbilirubemia, Increase in reticulocyte count, DCT positivity

Causes of hemolysisIntracorpuscular defects:

RBC membrane defects Hereditary spherocytosis,

elliptocytosis,stomatocytosis, pyknocytosis

RBC enzyme abnormalities G6PD deficiency, pyruvate

kinase deficiency Hemoglobinopathies

Alpha thalassemia

Extracorpuscular defects:• Immune mediated• Rh isoimmunization, ABO or Mi

BG incompatibility• Macro/microangiopathic

• Large vessel thrombi, Cavernous hemangioma, Renal artery stenosis

• Infections • Sepsis, TORCH ,Parvovirus

B19 (anemia due to RBC aplasia) ,Malaria

• Drugs- ibuprofen, penicillin, • Galactosemia• Acidosis- metabolic/ respiratory

Glucose-6-Phosphate Dehydrogenase (G6PD)

and its deficiency

What is G6PD? A house keeping enzyme critical in

redox metabolism of all aerobic cells Role in RBCs very critical- The only

source of reduced NADP NADPH directly or via reduced

glutathione defends RBCs against oxidative stress

G6PD Deficiency The most prevalent RBC enzymes

deficiency 400 million people are affected worldwide

(4.9%) [1]

Coincides with the geographic distribution of endemic malaria

1.Frank JE. Diagnosis and management of G6PD deficiency. Am Fam Physician 2005;72:1277-82

Distribution

Highest prevalence in Sub-Saharan Africa, Middle East, Mediterranean Europe, and Southeast Asia

The Indian scenario… Incidence: 2-27%[2]

MC variant: G6PD Mediterranean Mostly in:[3]

Vataliya prajapatis of north india Parsis Punjab, Kerala, Andhra Pradesh

[2]. Mohanti D, Mukherjee MB, Colah RB. G6PD deficiency in India. Indian J Pediar 2004;71:525-9.[3]. Pao M, Kulkarni A, Gupta V, Kaul S, Balan S. Neonatal screening for G6PD deficiency. Indian J Pediatr 2005;72:835-7.

Genetics Gene located on X-chromosome (sex

linked recessive) Disease fully expressed in hemizygous

males and homozygous females Variable intermediate expression by

heterozygous females

Clinical implications of molecular basis of G6PD

deficiency A house keeping gene- Deletions of G6PD genes incompatible with

life (hydeletions); death in utero Point mutations:

Sporadic: No geographical specificity No causal relationship with malaria selection Manifests with CNSHA

Polymorphic: Resulted from malaria selection Correlate with specific geographical areas

WHO Classification of G6PD variants

WHO Class Level of deficiency

Enzyme activity Severity of hemolysis

1 Severe <10% CNSHA

2 Severe <10% Intermittent hemolysis

3 Moderate 10-60% Intermittent hemolysis with stressors

4 Mild to none 60-150% No hemolysis

5 None >150% No hemolysis

Pathogenesis As red cells age, G6PD activity falls rapidly and

prematurely

So, diminished NADPH/NADP and GSH/GSSG ratios

Impaired elimination of oxidants (H202)

Oxidation of hemoglobin and sulfhydrl groups in membrane

Pathogenesis (cont.) Red cell integrity impaired (on exposure

to oxidant drugs/ oxidant response to infections and chemicals)

Oxidized hemoglobin precipitates to form Heinz bodies, plucked out of the cell leading to hemolysis and bite cell blister cell morphology

Pathogenesis (cont…)

When to suspect G6PD deficiency in a neonate?

Development of hyperbilirubemia within 24 hours of life

History of neonatal jaundice in family members of siblings

Have bilirubin level >95th percentile Have evidence of hemolysis with

negative DCT

Clinical Presentation A. Neonatal hyperbilirubemia B. Drug induced hemolysis C. Favism D. Chronic non-spherocytic hemolytic

anemia

Neonatal hyperbilirubemia in G6PD deficient neonates Severe manifestation of G6PD deficiency and

source of potential morbidity from kernicterus Probably starts in utero, but clinical problem

becomes apparent on day 2/3 of life 10-50% deficient neonates affected Indian data: 13.3% of all jaundiced neonates

(and out of these 16% female neonates) [4]

[4]. Seema Kapoor et al. Newborn screening for G6PD deficiency; A 2-year data from north India. Indian J Public Health.2015.

Neonatal hyperbilirubemia in G6PD deficient neonates(cont.)

Mechanisms: Reduced glucuronidation of bilirubin due to

defective G6PD activity in hepatocytes Hemolysis triggered on exposure to oxidants like

naphthalene balls Co-inheritance of UDP-glucuronyltransferase 1

deficiency of Gilbert syndrome Pregnant women ingesting oxidant drugs,

transmitting it to her G6PD deficient fetus

Drugs carrying risk of hemolysis in G6PD

deficientsDrugs Definite risk Possible risk Doubtful riskAntimalarials Primaquine

DapsoneChloroquine Quinine

Sulfonamides Sulphametoxazole SulfasalizineSulfadimadine

SulfisoxazoleSulfadiazine

Antibiotics NitrofurantoimNalidixic acidCotrimoxazoleNiridazole

CiprofloxacinNorfloxacin

ChloramphenicolP-Aminosalicylic acid

Antipyretics/Analgesics

Acetanilide Aspirin(>3g/d) Aspirin(<3g/d)Acetaminophen

Others NaphthaleneNaphthaleneMethylene blue

Vit K analoguesAscorbic acid >1gRasburicase

DoxorubicinProbenecid

Harrison’s Textbook of Internal Medicine-19th edition

Laboratory diagnosis of G6PD deficiency

Screening/ Qualitative tests:Ultraviolet spot testMethaemoglobin reduction testBrilliant cresyl blue decolorisation test

Definitive/ Quantitative test:Spectrophotometric analysis

Molecular diagnostic testing and DNA analysis

Methord used in our case… Dried blood spots collected by heel prick on blood

sample collection card (Whatman 903 s & s, GE Healthcare)

Analysis done by fluroimmunoassey using Flurometer Principal: DBS allowed to react with substrate (G6P

+NADP) for 30 mins at ambient temperature Fluorescent measured using excitation wavelength of

355nm and emission wavelength of 460nm Values <16U/dl considered deficient

Importance of quantitative tests

Situation 1: During a hemolytic attack, old RBCs destroyed, therefore

surviving young RBCs and reticulocytes have relatively higher G6PD

Screening test can be false normalSituation 2:Heterozygous females diagnosis Probability of clinically significant hemolysis in

heterozygote roughly correlates with proportion of G6PD deficient cells

Hence, if a normal level of G6PD activity is found in a heterozygotye, she is unlikely to be at risk of G6PD related hemolysis

Prevention & Treatment Phototherapy and exchange transfusion for

neonatal jaundice Stop the offending drug Blood transfusion if severe anemia Folic acid supplementation

Initiated back in 1963…

Newborn Screening Programme

Wilson & Juegner criteria for disease screening

1. The condition sought should be an important health problem.2. There should be an accepted treatment for patients with recognized disease.3. Facilities for diagnosis and treatment should be available.4. There should be a recognizable latent or early symptomatic stage.5. There should be a suitable test or examination.6. The test should be acceptable to the population.

Wilson & Juegner criteria for disease screening

(cont.)7. The natural history of the condition, including development from latent to declared disease, should be adequately understood.8. There should be an agreed policy on whom to treat as patients.9. The cost of case-finding (including diagnosis and treatment of patients diagnosed) should be economically balanced in relation to possible expenditure on medical care as a whole.10. Case-finding should be a continuing process and not a “once and for all” project

Diseases for which neonatal screening is

being done Glucose 6 phosphate dehydrogenase

deficiency (G6PD levels) Congenital hypothyroidism (TSH) Congenital adrenal hyperplasia (17OHP) Galactosemia Biotinidase deficiency

Take home message Answer to neonatal hyperbilirubemia is not just photo-

therapy and exchange transfusion, Do investigate for its cause

G6PD deficiency is not very uncommon condition as approximately 5% of world’s population is affected, so should be kept in mind

In view of a high gene frequency for a disorder that is manageable with just elimination of a few drugs and foodstuffs, newborn screening is the need of the hour