"G6PD" Enzyme Deficiency among Children Suffering from Hemolytic Anemia in Gaza

71 Pages·2013·1.11 MB·English

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Islamic University-Gaza
Deanship of post Graduate Studies
Biological Sciences Master Program

Occurrence of "G6PD" Enzyme Deficiency
among Children Suffering from Hemolytic
Anemia in Gaza - Palestine

Submitted in Partial Fulfillment for the Master Degree of Science in
Biological Sciences/Medical Technology

BY
Lina Nimer Aboud

Prof Dr. Mohammad E. Shubair Dr. Mahmoud M. Sirdah
Professor of Medical Technology Associate Professor of Blood Pathophysiology
The Islamic University of Gaza Al (cid:150) Azhar University - Gaza
DECLARATION

I hereby declare that this submission is my own original work and that, to the best of my
knowledge and belief, it contains no materials previously published or written by
another person nor materials which to a substantial extent has been accepted for award
of any other degree of the university or other institute, except where due
acknowledgement has been made in the text.

Signature Name Date

LINA Lina N. Aboud

I
Dedication

To my Father(cid:146)s Pure Soul,
To my Mother,

To my Husband,
To my Sons,
Jawad, Mohammed
and,

To my daughter, Hala,

last but not least,
To the Palestinian people who are
steadfast and patient

on the beloved land of Palestine

II
Acknowledgement

The DNA Extraction and purification as well as well as the hematological and
biochemical parts of this work were carried out at the Islamic University of Gaza
Laboratories, while the molecular characterization of the G6PD gene was carried out at
the Associated Regional and University Pathologists, Inc., (ARUP Laboratories),
University of Utah School of Medicine Salt Lake City, Utah, USA.
My deepest and profound acknowledgments are to my supervisors Prof Dr.
Mohammad E. Shubair and Dr. Mahmoud Sirdah for their continuous support,
generous helps, and fruitful and constructive suggestions. I could not have imagined
having a better supervisors and mentors for my master thesis.
My special deep and sincere gratitude are to Prof Dr. Josef Prchal (Professor of
Medicine, Pathology, and Genetics , University of Utah School of Medicine, USA), Dr.
N. Scott Reading (associate Professor of Genetics ARUP Laboratories Utah, USA), for
their efforts and helps in the molecular tests performed in the present work and also for
their continuous support, helps, and encouragement.
I am especially indebted to the outstanding staff of the Genetics Laboratory at
the Islamic University: Mr. Nasser Abu Shaban and Mr. Mohammed Ashour, both
were very valuable in many parts of my work.
I am especially grateful to the ARUP Laboratories and University of Utah School of
Medicine Salt Lake City, UT, USA for performing the molecular tests of my study free
of charge.
I would like to thank the clinical and laboratory staff of Al-Naser pediatric
Hospital for patient's recruitment and enrollment to this work.
My thanks also go to Eng. Hammam Al-Rayes who supplied us with the materials and
reagents for our work at Gaza on time.
My appreciations are extended to all the patients and their parents for their
understanding and cooperation during the study and thereafter.
Finally, not to forget any one, I express my thanks to everyone who supported me
through finishing this work.

III
Abstract
Background: The Glucose-6-phosphate dehydrogenase (G6PD) deficiency has been
considered as the commonest enzymopathic inherited disorder of red blood cells, which
affecting more than 500 million people worldwide. The G6PD gene in human is X-
linked so males are more affected than females. The mutation in the G6PD gene may
lead to the production of a G6PD enzyme that has diminished functionality and/or
stability, leading to wide range of biochemical and clinical presentations principally
neonatal jaundice and acute hemolytic anemia triggered by an exogenous agent in most
cases. More than 176 mutations, and 500 different variants have been described to date
for the G6PD gene, however, the Mediterranean mutation c.563 C>T, whether
associated with c.1311 C>T polymorphism or not, is among most common variants that
lead to enzyme deficiency and often associated with Favism.
Objectives: The main objective of this study is to determine the frequency of the
Mediterranean mutation (c.563 C>T) and its association with c.1311 C>T
polymorphism among Palestinian G6PD deficient children admitted to Al Nasser
pediatric hospital at Gaza due to hemolytic crises.
Methodology: In this cross- sectional descriptive study, 80 children (2-8 years old)
presenting with hemolytic anemia were included and they represent the period from
April 2010 and March 2011. Venous blood samples (2.5 ml) were withdrawn at hospital
admission from each hemolytic child and were collected in K -EDTA tubes and were
3
used for performing complete blood count (CBC), G6PD enzyme assay, DNA
extraction and, mutation analysis. Forty school-aged children without G6PD deficiency
were selected as the non-G6PD deficient control group for the hematological studies.
Additionally, 40 X-chromosomes from apparently healthy, male subjects served as
controls tested for the c.1311 C>T polymorphism.
Results: The results showed that 65 (60 males & 5 females) out of the 80 children
were found to be G6PD deficient. In most G6PD deficient cases (67.7%) hemolytic
crisis occurred in early childhood (≤ 40 months), and it was totally (100 %) due to
ingestion of fava beans, either green (96.9 %) or dried (3.1 %). Most (98.5%) of G6PD
deficient cases went through neonatal jaundice after birth, which last 1-2 weeks. The
Mediterranean c.563 C>T and the c.1311 C>T polymorphism were encountered,
respectively, in 35.4 % (allele frequency 0.33) and 41.8 % (allele frequency 0.42) of
G6PD deficient children. Also the c.1311 C>T polymorphism was identified in 17.5 %.
IV
(allele frequency 0.18) of control blood samples. However, the incidence of c.1311 C>T
polymorphism with patients carrying the Mediterranean c.563 C>T mutation was 95.2
percent (allele frequency 0.96) compared to 11.4 percent (allele frequency 0.08) in
G6PD deficient individuals without the Mediterranean c.563 C>T mutation. The
comparisons of G6PD enzyme activity and erythrogram revealed that the G6PD
deficient subjects were admitted to the hospital with significantly lower hematological
parameters except for MCHC. In addition, no significant hematological or enzymatic
activity differences were reported between the G6PD deficient children carrying the
Mediterranean c.563 C>T mutation and those G6PD deficient children carrying non-
Mediterranean mutation.
Conclusions: It is concluded that the Mediterranean mutation c.563 C>T is common
among G6PD deficient Gaza Strip Palestinians and is highly associated with the c.1311
C>T polymorphism. This work could be foundational for further investigations of
G6PD molecular studies with emphasis on ancestral origin of these variants. The
present study also emphasizes the predominant existence of the G6PD deficiency
among Palestinian population, which justifies the necessity of the Palestinian Health
policy leaders establishing nationwide programs of newborn screening for G6PD
deficiency.

Key words
Hemolysis and G6PD deficiency, Mediterranean G6PD mutationc.563T,
c.1311T G6PD polymorphism.

V

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ƇƒűŪƄż
ΓΰϏ
ŗŪŒŧťƃŒŭŤƄƆ
ŚƛƜśŷŔũŝƄŌƉƈ ¿ŪśŦƈƅŔŚŕſŬƏſƅŔƓŬŔŧŬŪƏƄƏƆŠƇƔŪƊŏůƂƊũŗśŸƔŗƆťƀƆ
G6pD
Ƒ»Ɔŷűũ»ƈƅŔŔŨƎŗŖŕŰƈůŦŮƉƏƔƆƈ ƉƈũŝƄŌƉŌŜƔţžŕćŷƏƔŮřƔŝŔũƏƅŔŚŕƈƔŪƊƛŔ

ƓŬ»ƊŠƅŔƓżŗŰƅŕŗųŗśũƈƓţƊśƈƓŝŔũƏűũƈ ƇƔŪƊŏůƂƊűũƈƇƅŕŸƅŔƐƏśŬƈ
X G6PD
řŗŕŰŔũŬſƔŕƈŔŨƍƏ ƓŬƊŠƅŔƓżŗŰƅŔƑƆŷŧƏŠƏƈƉƔŠƇƔŪƊƛŔŔŨƍŶƔƊŰśƓžƇƄţśƔŜƔţ
X
ůƂƊƑƅŏŘũſųƅŔƉƔŠƅŔŔŨƍƓž¿ƆŦƒŔŜƏŧţƒŧŎƔƏűũƈƅŔŔŨƎŗŜŕƊƛŔƉƈũŝƄŔũƏƄŨƅŔ
ʼnŔũ»ƈţƅŔƇŧƅŔŚŕƔũƄƅ¿ƜţƊŔƏũŬƄśŜƏŧţƑƅŔƒŧŎƔŕƈƈžƇƔŪƊƛŔŔŨƍŶƔƊŰśƓž¿ƜśŷŔƏŔ
ũ»ŝƄŔŧ»ŠƏƔ Řŕ»ƔţƆƅĻŔŧŧƎƈƏĻŔŧƔŧŮƉƏƄƔŧƁƒŨƅŔƇŧƅŔũƂžŜƏŧţƓƅŕśƅŕŗƏƒƏƈŧƅŔ¿ƜţƊƛŔ
.
ų»ŬƏśƈƅŔűƔ»ŗƛŔũ»ţŗƅŔŘũ»ſųũŗśŸśƏűũƈƅŔŔŨƎƅŕſƆśŦƈŔũƔżśƈ ƏŘũſų Ɖƈ
 
ƉƔ»ŗƉ»ƈƓ»ƍƛƏŔ ƓƆƄŮ»ƅŔŧŧŸśƅŔřųŗśũƈŚƊŕƄʼnŔƏŬ
(c.1311 C>T (c.563C>T)
¿ƏſƅŔŕƔƈƔƊŕŗųŗśũƈƅŔ ƇƔŪƊŔůƂƊƑƅŔƒŧŎśƓśƅŔŕŷƏƔŮũŝƄƛŔŚŔũƔżśƈƅŔ
G6pD
ŗŪŒŧťƃŒƇƆŻťƌƃŒ
űƔ»ŗƛŔũ»ţŗƅŔŘũ»ſųũŕŮ»śƊŔƐŧ»ƈŖŕŬ»ţƏ»ƍřŬŔũŧƅŔƉƈƓŬƔœũƅŔŽŧƎƅŔ
ƇŧƅŔũƂſŗƉƔŗŕŰƈƅŔ¿ŕſųƛŔƓž ŘũſųƅŔŶƈŕƎųŕŗśũŔƏ ųŬƏśƈƅŔ
(c.1311 C>T ) (c.563C>T)
¿ŕſųƘƅũŰƊƅŔƑſŮśŬƈƓž ƇƔŪƊƛŔůƂƊŗƉƔŗŕŰƈƏƓƅƜţƊƛŔ
G6PD
VI
ƏřƊ»Ŭ  ƉƈƇƍũŕƈŷŔšƏŔũśśƜſų ŚƆƈŮřƔſŰƏƅŔřŬŔũŧƅŔƋŨƍŘŒƍťƕŒƍžŧűƃŒ
  
 ūũŕƈƓƅŔ ¿ƔũŗŔƉƈŘũśſƅŔƓžƓƅƜţƊƛŔƇŧƅŔũƂžřŠƔśƊƑſŮśŬƈƅŔƓžƉƔŠƅŕŸƈƅŔ
 
ƌ»ƅƏŦŧƇ»śƓƅƜţƊƛŔƇŧƅŔũƂſŗŖŕŰƈ¿ſų¿ƄƉƈ¿ƈ ƒŧƔũƏƇŧŚŕƊƔŷŖţŬƇśŧƂƅ

ř»ƆƈŕƄƇŧŘũƏ»ŰƏŔŧŔŧŸśʼnŔŧƗƇŧŦśŬśŚƊŕƄƏž ŖƔŗŕƊŌƓžŚŸƈŠƏƑſŮśŬƈƆƅ
EDTA-K3
¿»ƔƆţśƏžƒƏƏƊƅŔűƈţƅŔŞŔũŦśŬŔŕŲƔŔƇśƏ ƇƔŪƊŔųŕŮƊƏřƔƅŕŸžūŕƔƁƇśƏ 
G6pD (CBC
ř»ŷƏƈŠƈƄ ƇƔŪƊŔůƂƊŗƉƔŗŕŰƈũƔŻūũŔŧƈƅŔƉƈ¿ſų ũŕƔśŦŔƇśŧƁƏŚŔũſųƅŔ
G6pD 
řųŗŕ»ŲřƊƔŸƄŔƏƈŧŦśŬŔ¿ŕſųŔƉƈƓŬƊŠƓżŗŰ ƃƅŨƑƅŔřžŕŲƛŕŗƇŧƅŔŚŕŬŔũŧƅřųŗŕŲ

 ƓƆƄŮƅŔŧŧŸśƆƅ
(c.1311 C>T
ƉƔƅƏƈŮƈƅŔ¿ŕſųƛŔƉƈƜſų ¿ŰŔƉƈ ŜŕƊŏƏũƏƄŨ  ƉŔşœŕśƊƅŔŚũƎŴŌŝőœřƈƃŒ
   
Ƈŧ»ƅŔũƂſŗƏŗƔŰŔØ ƓƅŔƏţ ůƂƊŚƛŕţƓž ůƂƊŗƉƔŗŕŰƈřŬŔũŧƅŕŗ
 G6PD G6PD
¿ƏſƅŔŕƈŏž¿ƏſƅŔ¿ƏŕƊśŖŗŬŗØ ƉŕƄƏžŔũƎŮ  ŘũƄŗƈƅŔřƅƏſųƅŔřƆţũƈƓƅƜţƊƛŔ
  •
ŚƛŕţƉƈØ ƉƈŖũŕƂƔŕƈƉŔŕƈƄØ řŗŬƊŗŽſŠƈƅŔƏŌØ řŗŬƊŗũŲŦƗŔ
  
Ƈ»śŧ»ƂƅŵƏŗŬŔ  ƉƈũƈśŬŔƓśƅŔƏžŘŧƛƏƅŔŧŸŗƓŠƏƅƏƔŬſƅŔƉŕƁũƔƅŕŗƏŗƔŰŔ ůƂƊ
  G6PD
Ɠ»ž ƓƆƄŮƅŔŧŧŸśƅŔƏ ųŬƏśƈƅŔűƔŗƗŔũţŗƅŔŘũſųŧƔŧţś
( c.1311C >T) c.563 C> T
Ƒ»ƆŷŽũ»ŸśƅŔƇ»śŕƈƄ ůƂƊŗƉƔŗŕŰƈƅŔ¿ŕſųƗŔƓžƓƅŔƏśƅŔƑƆŷØ ƏØ
G6pD  
ƏřƔũƍƏŠƏřƔƏƁřƁƜŷƃŕƊƍƉŔŕƈƄ¿ƏũśƊƄƅŔƇŧƅŔŚŕƊƔŷƉƈØ Ɠž
 c.1311 C> T
ŜƔţ ƓƆƄŮƅŔŧŧŸśƅŔŶƈŕƎųŕŗśũŔƏųŬƏśƈƅŔŘũſųƓƆƈŕţƓŲũƈƅŔƉƔŗųŕŗśũŔ
c.1311 C> T
Ŷ»ƈřƊũŕƂƈřųŬƏśƈƅŔŘũƂųƅŔ¿ƈţśƓśƅŔűũƈƅŔŚƛŕţƉƈ ƓžƓƆƄŮƅŔŧŧŸśƅŔŧŠƏ

ŚſŮƄųŬƏśƈƅŔűƔŗƗŔũţŗƅŔŘũſųŧƏŠƏƉƏŧŗ ůƂƊŗƉƔŗŕŰƈƅŔŧŔũžƗŔƓž 
G6PD 
ůƂƊ»ŗƉƔŗŕŰ»ƈƅŔ¿ŕ»ſųƘƅ ƇŧƅŔŕƔƜŦƅ¿ƈŕƄƅŔŧŸƅŔƏ ƇƔŪƊŔřƈƔƂƅŚŕƊũŕƂƈƅŔ
(CBC) G6PD
VII
¿ŧŸƈŗŽƜśŦŔŧŠƏƔƛƌƊŔřžŕŲƙŕŗ ŔŧŷŕƈřƆƔƆƁŕƔƜŦƆƅŧŸƅŔŚŔʼnŔũƁŶƔƈŠƉŔƇƔŪƊƛŔ
MCHC
ƋŨ»ƎŗƉƔŗŕŰ»ƈũ»ƔżƅŔƏųŬƏśƈƅŔűƔŗƛŔũţŗƅŔŘũſųŗƉƔŗŕŰƈƅŔƉƔŗŕƈƇƔŪƊƛŔųŕŮƊůƂƊŗ
ŘũſųƅŔ
ũŝƄƗŔƓƍ ųŬƏśƈƅŔűƔŗƗŔũţŗƅŔŘũſųƉŌřŬŔũŧƅŔƉƈşśƊśŬƊŘœŞœřƈřŪƙŒ
c.563 C> T
 ƓƆƄŮƅŔŧŧŸśƅŔŶƈũƔŗƄŧţƅřųŗśũƈƓƍƏ ƇƔŪƊŔůƂƊƑŲũƈŗŔũŕŮśƊŔ
c.1311 C>T G6PD
Ŷ»ƈ ƇƔŪƊŔůƂƊƅřƔœƔŪŠƅŔŚŕŬŔũŧƅŔƉƈŧƔŪƈƅřƈƎƈŘƏųŦũŗśŸƔŧƁƓƅŕţƅŔ¿ƈŸƅŔƉŔ
G6PD
ƇƔŪ»ƊŔƓžůƂƊŧƏŠƏƏŔũŕŮśƊŔŕŲƔŔŧƄŎśřŬŔũŧƅŔƋŨƍŚŔũƔżśƈƅŔƋŨƍ¿ŰŌƑƆŷŪƔƄũśƅŔ
Ŷ»ŲƏŗřƔƊƔųŬ»ƆſƅŔřţŰƅŔŘũŔŪƏƇŕƔƁŘũƏũŲũũŗƔƒŨƅŔũƈƗŔžƉƔųŬƆžƉŕƄŬƉƔŗ
G6PD
 ůƂƊƅŘŧƛƏƅŔƓŝƔŧţůţſƅşƈŔũŗ
G6PD
ŗƒšœřŽƆƃŒŘœƆƄƂƃŒ
ƓƆƄŮƅŔŧŧŸśƅŔž ųŬƏśƈƅŔűƔŗƛŔũţŗƅŔŘũſųž ƇƔŪƊŔƏ¿ƆţśƅŔ
(c.563C>T) G6PD
(c.1311 C>T)

VIII
Table of contents
Declaration (cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133)(cid:133).......I
Dedication..................................................................................................................II
Acknowledgement.....................................................................................................III
Abstract.....................................................................................................................IV
Δγ΍έΪϟ΍κΨϠϣ................................................................................................................VI
List of Table............................................................................................................XII
List of Figures........................................................................................................XIII
Abbreviations.........................................................................................................XIV
Chapter 1.....................................................................................................................1
Introduction................................................................................................................1
1.1Overview................................................................................................................1
1.2 Objectives..............................................................................................................2
1.2.1 General objectives.........................................................................................2
1.2.2 Specific objectives..........................................................................................2
1.3 Significance...........................................................................................................3
Chapter 2.....................................................................................................................4
Literature review........................................................................................................4
2.1 Hemolytic disease of children..............................................................................4
2.1.1 Immune hemolysis:.......................................................................................4
2.1.1.1 Rh incompatibility..................................................................................4
2.1.1.2 ABO hemolytic disease of the newborn................................................4
2.1.2 Non immune hemolysis.................................................................................5
2.1.2.1 RBCs structural membrane defects......................................................5
2.1.2.2 Hemoglobinopathies...............................................................................6
2.1.2.3 RBCs enzymatic defects.........................................................................7
2.2 Erythrocyte metabolism......................................................................................8
2.2.1 The Embden Meyerhof pathway.................................................................8
2.2.2 The hexose monophosphate pathway..........................................................9
2.2.3 Function of G6PD in red blood cells..........................................................11
2.2.4 Mechanism of hemolysis.............................................................................12
2.3 Structure of G6PD..........................................................................................13
2.3.1 Genetics and molecular biology of G6PD.............................................13
IX