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G6PD
TESTING FOR G6PD DEFICIENCY: CLINICAL AND LABORATORY
ASPECTS
George J. Reclos Ph.D.
CONGRESSO BRASILEIRO DE TRIAGEM NEONATAL – Sao Paulo
NOVEMBER 16-19, 2005
• Pythagoras (fl. 530 BCE ) warned his pupils of favism, something mentioned also by Herodotus ( 5th cent. BC). They had learned that from Egyptian priests who didn’t allow males to consume Fava beans (Vicia faba)
• Oldest known hereditary metabolic disorder
• Possibly the first genetic adaptation of the human species (Plasmodium falciparum; malaria)
Prevelance of G6PD deficiency
• More efficient phagocytosis of deficient infected red blood cells
G6PD deficiency
G6PD
Glucose-6-Phosphate + NADP+ 6-Phosphogluconate + NADPH
• EC 1.1.1.49
• Either a tetramer or a dimer
• G6PD gene 18 Kb, coded for 531 amino acids
• Chromosome Xq28 (long arm)
• Catalyzes the reaction:
G6PD • Importance of NADPH+
Peroxide detoxification (diagram)
Stability and preservation of catalase and GSH (reduced form of
Glutathione)
Scriver et al., The Metabolic & Molecular basis of in herited disease
G6PD
Things to know..
• First step in the hexose monophosphate pathway (but alternatives exist)
• Peroxide detoxification (but alternatives exist e.g. catalase)
• Catalase has a much weaker affinity to H2O2 but.. it is able to destruct more than 50% of the hemolyzing concentration
However..
• Red blood cells do not have catalase.
• Red blood cells can’t synthesize more G6PD enzyme after they mature, but have a very long life.
G6PD deficiency
What causes the deficiency?
Variation in the number of G6PD molecules
• 1-50% in RBCs, 1-90% in granulocytes
Variation in the activity of the enzyme
• Affinity for G6P and NADP
• (Thermo)stability of enzyme - especially for RBCs
• pH dependance
• Utilization of substrate analogs
G6PD
Many variants.. but mainly affect :
• Affinity for the substrate (mainly G6P)
• Instability of enzyme in the red blood cell
• Possibly altered protease activity in
RBCs
G6PD Variants
More than 400 variants; 130 different point mutations.
Class I Associated with chronic non-spherocytic
hemolytic anemia (CNSHA)
Class II < 10% residual activity
Class III 10-60% residual activity
Class IV Normal activity
Class V Increased activity
African type and Mediterranean/Asian type (Class II and
III)
Luzzatto L et al. WHO Tech Rep Ser 1967;366:53p
G6PD deficiency
• X-linked recessive disorder ? (both alleles are
expressed).
• Most common hemolytic disease in human
G6PD deficiency causes Neonatal Jaundice and Acute Hemolytic Anemia
Naphthalene (moth balls) Fava beans
Others: toluene, nitric volatile substances; drugs inducing hemolysis;
cosmetics (henna). Adults: Tonic water (quinine).
Hyperbilirubinemia in
neonates • Newborns have increased bilirubin
load and deficient enzyme systems
• Jaundice present in 60% full term
infants and 80% pre-term infants
• Bilirubin neurotoxicity leads to brain
damage (kernicterus)
WHO working group. Bull WHO 1989;67:601
G6PD deficiency
Worldwide Distribution
G6PD deficiency in Greece • Incidence
– Male 4.7% (less than 2.5 U/g Hb)
– Female 2.5% (less than 2.5 U/g Hb) – disputed**
• Dried blood spot screening program started in 1977
– Fluorescence spot test
– Coverage > 90% (1985); > 96% (2004)
• Results
– 1977: 1/302 pediatric hospital admission (94% need exchange transfusion)
– 1984: 1/1200 pediatric hospital admission
– 2004: 1/ 3000 pediatric hospital admission
**Reclos G.J, Hatzidakis CJ & Schulpis KE, J Med Screening, 2000
G6PD deficiency in Brazil • Incidence (Brazil)
– 1.65 - 9.7 % (less than 2.6 U/g Hb)
– 2.6 – 6.5 % (less than 6.0 U/g Hb)
• Incidence (RS)*
– 1.65 % (less than 2.5 U/g Hb)
– 2.6 % (less than 6.0 U/g Hb)
• Incidence (RS)**
– 1.4 % (less than 2.6 U/g Hb)
– 6.5 % (less than 7.6 U/g Hb)
• Incidence (MG)***
– 1.9 % (less than 2.6 U/g Hb)
– 6.5% (less than 7.6 U/g Hb)
• Incidence (Amazonas)****
– 9.7 % (less than 2.1 U/g Hb)
– 6.5 % (less than 6.3 U/g Hb)
* Weber L and E.C. Neto Abstract, 1st Brazilian Neonatal screening Meeting, Curitiba, 2001.
** Castro S. (personal communication).*** Lizia M.F.R. Campos et al. **** Maria Salete Girao MitosoG-6-PD 4th
Intercientifica Workshop on Neonatal Screening, Sao Paulo, 2001
G6PD deficiency
Clinical manifestations = Severe clinical expression but still compatible with life.
• Neonatal jaundice (even kernicterus)
• Drug hemolysis
• Infection induced hemolysis
• Favism
• Chronic non spherocytic hemolytic anemia (CNSHA) – may not be hereditary (de novo mutation)
• Low birth weight incidence is high
Some of them may lead to mental retardation or even death.
G6PD deficiency
To make things worse:
• Nobody can tell if a G6PD deficient person will
express symptoms, which symptoms and their
severity.
• Thus, heterozygotes with class II and III
variants are equally at risk.*
Prevention is all we have.
*Kaplan M, Beutler E. et al., Padiatrics, 1999, 104:68-74; Kaplan M, Hammermann C. et al., Pediatrics, 2001, 139:137-140
Multicentric study
A total of 657 pregnant women and their newborn children from
diferent races from Brazil and Greece were enrolled in this study. The
following parameters were tested vs. G6PD activity: race, age,
gestation age, placenta weight, newborn weight, concomitant drug
intake, history of abortion, Cu and Zn levels.
Chi -square test evaluated by Fisher’s exact significance showed that :
G6PD deficiency in newborns causes
Low Birth Weight (P=0,041)
Normal placenta weight (P=0,235)
G6PD deficiency
WEIGTH
4400,00
4200,00
4000,00
3800,00
3600,00
3400,00
3200,00
3000,00
2800,00
2600,00
2400,00
2200,00
2000,00
1700,00
1500,00
1000,00
Pe
rce
nt
30
20
10
0
B2.G6PD
homozi got
heterozigot + normal
G6PD deficiency
4936N =
Baby G6PD
heterozigot + normalhomozigot
BIR
TH
WG
HT
5000
4000
3000
2000
1000
0
G6PD deficiency
G6PD ASSAYS
Qualitative Fluorescence Screening Test *
One of the simplest tests in newborn
screening:
• Spot reaction mixture on filter paper
• Observe NADPH fluorescence under long
wave length UV light.
*Developed by Prof. Ernest Beutler
G6PD deficiency
*Picture by Prof. Hsiao, Taiwan
G6PD ASSAYS Quantitative tests
Measure production of NADPH+
• either in the UV or
• Colorimetrically in kinetic mode.
• Easy
• Quick
• Affordable
• No exotic equipment needed
H +
N A D P N A D P H
G 6 P 6 PG
+ oxidized yellow NBT reduced violet NBT + NADP
Diaphorase
Enzyme G-6-PD
G6PD Quantitative Assay Principle
G6PD Assay
What should be considered for the method performance ?
Reference values
Cut off determination
Long term CV,
precision
Accuracy
Normal P-P Plot of G6PD
Observed Cum Prob
1,00,75,50,250,00
Exp
ecte
d C
um P
rob
1,00
,75
,50
,25
0,00
Reference values :
Detrended Normal P-P Plot of INT
Observed Cum Prob
1,0,8,6,4,20,0
De
via
tio
n fro
m N
orm
al
,08
,06
,04
,02
0,00
-,02
-,04
-,06
G6PD deficiency
Nam
e of
the
subj
ects
AA
AS
FC
MG
MO
MU
NB
OB
Oı
OT
PY
RK
SD
SU
TE
TM
TT
UG
YAT
YET
95% Confidence Interval G6PD (U/gHb)
11,010,09,08,07,06,05,0
G6PD values of each individual and dispersion around their own mean
ROC Curve
1 - Specificity
1,00,75,50,250,00
Sen
siti
vity
1,00
,75
,50
,25
0,00
Coordinates of the Curve
Test Result Variable(s): G6PD
1,8550 1,000 ,550
2,1150 1,000 ,500
2,1650 1,000 ,450
2,2100 1,000 ,400
2,2900 1,000 ,350
2,3850 1,000 ,300
2,5900 1,000 ,250
2,7800 1,000 ,150
2,7950 1,000 ,100
2,9000 1,000 ,050
3,1250 1,000 ,000
3,2550 ,996 ,000
3,2950 ,991 ,000
3,3750 ,987 ,000
3,4400 ,982 ,000
3,5900 ,978 ,000
Positive if
Greater Than
or Equal Toa
Sensitiv ity 1 - Specif icity
The smallest cutof f v alue is the minimum
observed test value minus 1, and the largest cutof f
value is the maximum observed test v alue plus 1.
All the other cutof f values are the averages of two
consecutive ordered observ ed test v alues.
a.
Diagnostic accuracy is high
G6PD
CuSum Chart of G6PD
-20
-15
-10
-5
0
5
10
15
20
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52
Analytical run number
Cu
mu
lati
ve a
bso
lute
devia
tio
n
G6PD deficiency
-20
-15
-10
-5
0
5
10
15
20
25
30
1 5 9 13 17 21 25 29 33 37 41 45 49 53
analytical runs
CV
% d
istr
ibu
tio
n
Sigma Control 10,34
U/gHb
G6PD deficiency
METHOD DECISION CHART for G6PD
TEa
Precision/TEa ratio
,7,6,5,4,3,2,10,0
Bia
s / T
Ea
Rat
io
14
12
10
8
6
4
2
0
Excellent
Good
Marginal
Poor*
Unacceptable
Heat inactivation
Instability of the enzyme
Humidity
Storage time
Different filter papers
Matrix effect
G6PD
Main pitfalls of this test
G6PD • Instability of enzyme
*Reclos G.J, Hatzidakis CJ & Schulpis KE, J Med Screening, 2000
Standard Deviation Indexes Obtained from
Survey Materials in 2003
-3,5
-3
-2,5
-2
-1,5
-1
-0,5
0
0,5
1
1,5
0 5 10 15
10 measurements in each survey
SD
I allo
wa
ble
(+
/- 2
SD
I)
Survey 12
Survey 09
Survey 07
Survey 05
* Reclos GJ, Tanyalcin T. Accred. Qual. Assur., (2004) 10:27-31
G6PD
Tanyalcin T, Reclos GJ. 9th AEWIEM Stará Lesná, Slovakia, September 27 , 2004
CDC QA
Taiwan
Tanyalcin S&S 903
R&D
DGKC S&S2992
DGKC S&S 903
EWS2003
AARM
(J) NBSCARD
AARM
CDC QA
Taiwan
Tanyalcin S&S 903
R&D
DGKC S&S2992
DGKC S&S 903
EWS2003
(I) NBSCARD
Mean Di fference (I-J)
Std. Error
Sig .
95% Confidence Interval Lower Bound
95% Confidence Interval Upper Bound
Statistics
0,00000
0,25000
0,50000
0,75000
1,00000
Valu
es
Multiple Comparisons
Dependent Variable : ABSNBSTest : Bonferroni
Different filter papers showed diferent detection limits in galactose measurement .
Do they also present a remarkable Difference in enzyme activities?
• Despite all those sources of error, you can still get reliable results !
Parkes Error Grid for G6PD
0
5
10
15
20
25
30
0 5 10 15 20 25 30
Taiwan reference method for G6PD
G6
PD
0S
MM
R-2
00
0D
R&
D D
iag
no
stic
s
D
C
A
BDE
C
A
B
Region A: No effect on clinical action (clinically accurate)
Region B: Altered clinical action-little or no effect on clinical outcome (clinically acceptable)
Region C: Altered clinical action- likely to affect clinical outcome
Region D: Altered clinical action – could have significant medical risk
Region E: Altered clinical action –could have dangerous consequences
* Reclos GJ, Tanyalcin T. Accred. Qual. Assur., (2004) 10:27-31
G6PD deficiency Correlation with molecular analysis
• All DNA positive samples are also found
positive by G6PD quantitative kits.* **
• A 10% of kit – detected positive samples was
found DNA negative when only G6PD A- and
Med mutations were tested.*
• Heterozygous females showed as little as
30% residual enzymatic activity.*
* Lin Z, Fontaine JM, Freer DE, Naylor EW. Mol Genet Metab. 2005 Sep-Oct;86(1-2):212-9. Epub 2005 Jun
29.
** Dra Simone Castro et al. in preparation
G6PD deficiency
Head : Prof. Hsiao, Dept. of Medical Research & Education, Taipei Veterans General Hospital Taipei 112, Taiwan, R.O.C.
External Quality Assurance Program for G6PD
An external quality assurance (QA) program for the neonatal screening of
G6PD deficiency was developed to assess the reliability of the screening
tests.
Periodically (1~2 month), 10 specimens were distributed to each neonatal
screening center by speed post.
Reports of the analytical results were requested to be returned by fax or e-
mail within 3 days. (7 days for overseas screening centers)
The results of each screening center were compared with the quantitative
reference value, determined by the QA laboratory.
In addition to the three screening centers of Taiwan, seven overseas
screening centers have also participated in this QA program.
G6PD deficiency
• External Quality Assurance Program for G6PD
http://www.g6pd.org.tw
G6PD deficiency
Things to consider • Cytoplasmic analyte (not serum)
• Lysis of cells
• Enzyme (not amino acid)
• Heat inactivated
• Kinetic mode
– UV lamp, 340 nm,
– 550 nm (amplification step)
• Appropriate units (Units / g Hemoglobin)
• Normalization of samples
• In situ* or Hematology lab (2nd test)
*Reclos G.J, Hatzidakis CJ & Kruithof RA Pharmakeftiki, , 2000
G6PD deficiency Confirmatory Tests
• Quantitative determination of erythrocyte G6PD activity using maleimide as the inhibitor. Deutsch J. Clin Chem 1978;24:885
• Determination of G6PD activities of the parents and siblings for pedigree analysis.
• Identification of the G6PD mutations by analysis of the DNA amplified by PCR methods.
G6PD deficiency Current Screening Programs
Routine screening
– Cyprus, Greece, Hong Kong, Philippines
Singapore,Taiwan
Selective screening
– Brazil, Germany, Italy, Lebanon, Thailand, Turkey,
Vietnam, USA (Missouri, Pennsylvania,
Washington DC), Virgin Islands (NYS)
G6PD deficiency
To screen or not to screen ? March of Dimes / ISNS criteria
Serious disorder affecting newborns
High incidence
Treatable disorder
Simple, reliable, available test
G6PD deficiency
Serious disorder affecting newborns ? • “It is important to look for G6PD deficiency in infants
with significant hyperbilirubinemia, because some develop a sudden increase in TSB”
• “It is also recognized that immediate laboratory determination of G6PD is generally not available in most US hospitals and thus, translation of the above information into clinical practice is currently difficult”
Subcommittee on Hyperbilirubinemia, Pediatrics, 2004, 114: 297-316.
G6PD deficiency
High incidence ? • G6PD deficiency is the most common
inherited metabolic disease.
• Even the lowest incidence (1%) results in 2.000.000 patients in Brazil or 3.000.000 in USA.
• The “isolated island” theory does not exist. Immigration and intermarriage have made G6PD a world problem.
G6PD deficiency
Treatable disorder ? • In the strict sense of the word, this deficiency
is not “treatable” – apart from blood
transfusion and some drugs (?).
• However: Prevention is better than treatment
(Hippocrates ).
• Additional advantage: No post-screening cost
for Health Authorities.
G6PD deficiency
Simple, reliable, available tests ? Many manufactures offering simple, fast and reliable
tests today.
• The UV lamp method,
• The color reduction dye
• The fully quantitative methods either at UV or visible
• The DNA array methods for some variants
Their reliability has been tested both by the users as well as the external quality assurance programs.
G6PD deficiency
Some math..
Even if only 1% of the positive samples in a very low risk country with 1% incidence for this deficiency shows mental retardation or dies..
►.. there is no reason for this country to screen for PKU or Galactosemia !
►More newborns are “lost” from G6PD than saved from PKU or Gal !!
G6PD deficiency
G6PD screening should be enrolled at least
for the high risk population..
.. which includes people from the Far East,
Mediterranean, Africa …
… plus the CNSHA cases ..
So, why not screen everybody ?
G6PD deficiency
Conclusions & Discussion • The incidence of G6PD deficiency is estimated to be
about 1% - 25% worldwide.
• Kernicterus and exchange transfusion due to neonatal hyperbilirubinemia were dramatically decreased after screening of G6PD deficiency has been started in Greece, Italy (Sicily) and Far East.
• Although the screening program was effective in prevention of the sequelae of G6PD deficiency, the preventive education was especially important for the patient during the newborn period before they were informed with the positive screening result.
G6PD deficiency Acknowledgments
During those years I was honored to work with several scientists all over the world. However, there are some who made the difference:
Simone Castro – Brazil
Salete Maria Girao Mitoso – Brazil
Eurico C. Neto – Brazil
Evangelos Papaconstantinou – Greece
Kenneth A. Pass, USA
Claudio Sampaio JR – Brazil
Kleopatra H. Schulpis – Greece
Tijen Tanyalcin – Turkey
