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Development and application of molecular screening test to detect genetic polymorphisms within the G6PD gene in malaria infected individuals

Elias Hanania

University of Sydney Student ID 500578836

A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of Science (Honours)

December 2020

Supervised by: Associate Professor Rogan Lee and Professor Wieland Meyer

Parasitology Laboratory NSW Pathology, ICPMR, Westmead Hopsital

Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology

The Westmead Institute for Medical Research

THE UNIVERSITY OF SYDNEY Honours Thesis 3

Acknowledgements

First and foremost, I would like to express my sincere gratitude and appreciation for my supervisors, Professor Wieland Meyer, and Associate Professor Rogan Lee, for their invaluable support and expertise throughout my honours year; without them, completion would not have been possible.

I would also like to express my appreciation for the remarkable molecular mycology research lab at the Westmead Institute for Medical Research, including Alex Kan, Christianne Prosser,

Krystyna Maszwsla, and Dr. Laszlo Irinyi, for their extensive assistance, encouragement, and support.

I want to thank my colleagues for their advice, company, and humour, making my honours journey pleasurable.

Lastly, I want to thank my friends and family for their steadfast love and encouragement, which kept me motivated throughout the year. Thank you all. 4

Table of Contents

Compliance Statement 2

Acknowledgements 3

List of Figures 7

List of Tables 9

List of Abbreviations 11

Abstract 14

Chapter 1: Introduction 17 1.1 Malaria overview 18 1.1.2 Causative agents of malaria and their taxonomy 18 1.1.2 Epidemiology of malaria 20 1.1.3 Plasmodium vivax life cycle 22 1.1.4 Clinical disease 23 1.2 Treatment of malaria 24 1.2.1 Primaquine and Tafenoquine 25 1.3 What is Glucose-6-Phosphate Dehydrogenase? 27 1.3.1 Glucose-6-Phosphate Dehydrogenase deficiency 27 1.3.2 Global presence of G6PD mutations 28 1.4 Testing for G6PD deficiency 30 1.4.1 Current methodology of testing for G6PD deficiency 30 1.4.2 Current typing-based methods for detection of G6PD mutations 32 1.4.3 Proposed methodology for G6PD mutation typing: PCR 33 1.5 Project significance and problem statements 34 1.6 Hypothesis 35 1.7 Aims 35

Chapter 2: Materials and Methods 36 2.1 Materials 37 2.2 Methods 37 2.2.1 DNA Extraction 37 2.2.1.1 Extraction of genomic DNA from whole blood 37 2.2.1.2 Extraction of genomic DNA from dried blood spots 38 2.2.2 Extracted DNA quality control 38 2.2.3 Preparation of primer solution 39 2.2.4 Protocol development 39 2.2.4.1 PCR protocol 39 2.2.5 Gel Electrophoresis 41 2.2.6 DNA extraction and purification from gel 41 5

2.2.7 Primer dilution for sequencing 42 2.2.8 PCR sequencing and variant analysis 42

Chapter 3: Molecular Assay Optimisation 44 3.1 Primer design selection 45 3.2 Optimisation of the PCR amplification conditions 45 3.2.1 G6PD locus 1 45 3.2.2 G6PD locus 2 48 3.2.3 G6PD locus 3 53 3.2.5 G6PD locus 5 57 3.3 Summary of the final optimised amplification conditions 62

Chapter 4: Application of the designed molecular assay to samples from Southern Thailand 64 4.1 Plasmodium vivax isolates from Southern Thailand 65 4.2 Results of the application of the newly designed PCR protocol to samples from Southern Thailand 65 4.3 Summary 69

Chapter 5: Application of the new molecular assay to samples from Australian travellers 71 5.2 Blood samples from Australian travellers infected with Plasmodium falciparum 72 5.2 Results obtained from the application of the new PCR protocol to samples from Australian travellers 72 5.3 Summary 76

Chapter 6: Discussion 77 6.1 Study importance 78 6.2 Aims 78 6.3 Results 79 6.4 DNA extraction 79 6.5 PCR optimisation 80 6.6 Sequence analysis 81 6.7 Pilot molecular surveillance results of G6PD genotypes from Southern Thailand and Australian travellers 82 6.8 Direction for future research 84 6.9 Concluding remarks 85

References 87

Appendices 102 6

Appendix I. Reagent list and chemical manufacturers 103 Appendix II. List of equipment and materials used and their product manufacturers 104 Appendix III. List of commercial kits and manufacturers 105 Appendix IV. List of bioinformatics programs and databases used 106 Appendix V. Reference G6PD GenBank sequence with attached designed primers for all 5 loci and Asian reported G6PD mutations 107 Appendix VI. Patients from Southern Thailand information summary 121 Appendix VII. Reference G6PD GenBank sequence aligned to sequences of Southern Thailand and Australian travellers samples and mutation sequences covering all reported Asian G6PD mutations 122 Appendix VIII. Patients from Australian travellers information summary 131

List of Figures

Chapter 1 Introduction

Figure 1. Global endemicity of P. vivax. 21 Figure 2. The life cycle of P. vivax. 22 Figure 3. Molecular structure of chloroquine 25

Chapter 2 Materials and Methods

Figure 4. Primer dilution calculation 42 Figure 5. DNA electrophoresis image of the amplification products of the G6PD locus 1. 46

Chapter 3 Molecular Assay Optimisation

Figure 6. DNA electrophoresis image of the amplification products of the G6PD locus 1. 46 Figure 7. DNA electrophoresis image of the amplification products of the G6PD locus 1. 47 Figure 8. DNA electrophoresis image of the amplification products of the G6PD locus 1. 48 Figure 9. DNA electrophoresis image of the amplification products of the G6PD locus 2. 49 Figure 10. DNA electrophoresis image of the amplification products of the G6PD locus 2.1. 50 Figure 11. DNA electrophoresis image of the amplification products of the G6PD locus 2.1. 51 Figure 12. DNA electrophoresis image of the amplification products of the G6PD locus 2.2. 52 Figure 13. DNA electrophoresis image of the amplification products of the G6PD locus 2.2. 53 Figure 14. DNA electrophoresis image of the amplification products of the G6PD locus 3. 54 Figure 15. DNA electrophoresis image of the amplification products of the G6PD locus 3. 55 Figure 16. DNA electrophoresis image of the amplification products of the G6PD locus 4. 56 Figure 17. DNA electrophoresis image of the amplification products of the G6PD locus 4. 57 Figure 18. DNA electrophoresis image of the amplification products of the G6PD locus 5. 58 Figure 19. DNA electrophoresis image of the amplification products of the G6PD locus 5. 59 Figure 20. DNA electrophoresis image of the amplification products of the G6PD locus 5. 59 8

Figure 21. DNA electrophoresis image of the amplification products of the G6PD locus 5.1. 60 Figure 22. DNA electrophoresis image of the amplification products of the G6PD locus 5.1. 61

Chapter 4 Application of the designed molecular assay to samples from Southern Thailand

Figure 23. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 1. 66 Figure 24. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 2. 66 Figure 25. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 3. 67 Figure 26. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 4. 67 Figure 27. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 5. 68 Figure 28. Alignment of sequenced DNA samples from Southern Thailand to G6PD gene (NG_009015) and the consensus sequences of all possible mutations of interest in Asia 69

Chapter 5 Application of the designed molecular assay to samples from Australian travellers

Figure 29. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 1. 73 Figure 30. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 2. 73 Figure 31. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 3. 74 Figure 32. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 4. 74 Figure 33. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 5. 75 Figure 34. Alignment of sequenced DNA samples from Australian travellers to the G6PD gene (NG_009015) and the consensus sequences of all possible mutations of interest. 76

List of Tables

Chapter 1 Introduction

Table 1. Subgenera of the Plasmodium species. 19

Table 2. Classification of human protozoa of the genus Plasmodium. 20

Table 3. Chloroquine treatment regimen for uncomplicated P. vivax. 25

Table 4. Primaquine and Tafenoquine treatment regimens for uncomplicated P. vivax relapses.

26

Table 5. Clinical severity classes of G6PD enzyme variants. 28

Table 6. Estimated G6PDd population by WHO region. 29

Table 7. Most common mutation variants of the G6PD gene in Thailand and their clinical severities classes. 29

Table 8. Limitations of phenotypic assays proposed by the WHO. 31

Table 9. SEA countries which test and routinely test before PQ/TQ administration. 31

Table 10. Initial amplification conditions of each primer set. 40

Table 11. Design parameters of primer pairs. 45

Table 12. G6PD locus 1 primer information. 45

Table 13. G6PD locus 2 primer information. 48

Table 14. Second new G6PD locus 2 primer information. 49

Table 16. Third G6PD locus 2 primer information. 51

Table 17. G6PD locus 3 primer information. 53

Table 18. G6PD locus 4 primer information. 55

Table 19. G6PD locus 5 primer information. 57

Table 20. G6PD locus 5.1 primer information. 60

Table 21. Summary of the final optimised PCR conditions for all 5 G6PD loci. 62 10

Table 22. Common G6PD SNP mutations found in Asia. 69

List of Abbreviations

µL Microlitre

µM Micrometres

°C Degrees Celsius

1kb STD Dna ladder

8-AQ 8-amino-quinoline

A Absorbance

ACT Artemisinin combination therapy bp Base pair cDNA Complementary DNA

CQ Chloroquine

CYP 2D6 Cytochrome P450 2D6

CYP 450 Cytochrome 450

DARC Duffy-antigen chemokine receptor

DBS Dried blood spots

DGGE Denaturing gradient gel electrophoresis

DNA Deoxyribonucleic acid dNTPs Dinucleotide triphosphates

FDA Food and drug administration g Gram

G6PD Glucose 6 phosphate dehydrogenase

G6PDd Glucose 6 phosphate dehydrogenase deficiency

GC Guanine and Cysteine gDNA Genomic deoxyribonucleic acid

GSH Gluthione 12

HREC Human research ethics committee

HRMA High resolution melt analysis

ICPMR Institute of clinical pathology and medical research

Kg Kilogram

MAO Monoamine oxidise mg Milligram ml Millilitre

MM1 Master mix

MW Molecular weight

NADPH Nicotinamide adenine dinucleotide phosphate

NC Negative control ng Nanogram

PCR Polymerase chain reaction

Pmol Picomol

PPP Pentose phosphate pathway

PQ Primaquine

RBC Red blood cell

RE Restriction enzyme

RFLP Restriction fragment length polymorphism

RNA Ribonucleic acid

SEA South-East Asia

SNP Single nucleotide polymorphism

TBE Tris/Borate/EDTA

TM Melting temperature

TQ Tafenoquine 13

UV Ultraviolet

WHO World health organization

WSLHD Western sydney local health district

X g Times gravity 14

Abstract

Background: The overall burden of malaria has declined over the past 15 years in South East

Asian (SEA) countries, including Thailand. Plasmodium falciparum (P. falciparum) was once the most prevalent malaria parasite for most of the country; however, it is now surpassed by

Plasmodium vivax (P. vivax). Thailand plans to revise or introduce new guidelines for effective and safe elimination of P. vivax using primaquine (PQ) and tafenoquine (TQ) (the only antimalarials registered for the radical cure of P. vivax) to eradicate malaria by 2024.

Individuals with Glucose 6 Phosphate dehydrogenase deficiency (G6PDd) can have adverse side affects with PQ and TQ and testing for this genetic deficiency isn't readily available in all parts of Thailand. These adverse drug in G6PDd individuals will hinder reaching malaria elimination status in Thailand. Surveillance of the genetic frequency and severity of Glucose 6

Phosphate dehydrogenase (G6PD) genotypes present in Asia is therefore vital to minimise the threat of haemolysis in G6PDd individuals infected with P. vivax. To achieve this a standardised, simple, and accurate method for detecting the prevalence and severity of G6PDd within a population is required. One such technology is polymerase chain reaction (PCR) that can amplify genomic DNA samples, followed by sequencing, which is a more accurate and rapid method than the currently used techniques to detect G6PDd in Thailand. Nevertheless this method requires the design of new loci specific primers that are used in optimised PCR protocols. Generated specific products containing the G6PD gene can be sequenced and single nucleotide polymorphisms (SNPs) of interest can be identified.

Aims: To develop a molecular assay covering the G6PD mutations present in SEA that will monitor the genetic frequency and severity of G6PD genotypes present in this region. Apply 15

the new molecular assay to malaria-infected blood samples from Southern Thailand and

Australian travellers to set the stage to survey the genetic frequency and severity of G6PD genotypes.

Methods: PCR protocols wer designed and optimised, facilitating amplification of the most commonly reported SNP mutations in Asia from human genomic DNA samples pre-extracted from 15 dried blood spots from Southern Thai patients infected with P. vivax and whole blood from 10 Australian travellers infected with P. falciparum. Commercial bidirectional Sanger sequencing was subsequently used to sequence these regions. The presence of SNPs in the sequenced regions was then examined for each sample.

Results: A PCR sceening assay for the most commonly reported SNP mutaions in Asia was established. Eleven out of the 15 Southern Thailand samples amplified and produced high- quality sequence data for all five regions of interest. All 10 samples from Australian travellers produced high-quality sequence data. SNPs were not identified in the G6PD gene for all tested samples for both sample groups.

Conclusion: The introduction of our PCR assay to identify G6PDd individuals that can be applied to genomic DNA samples will improve future surveillance studies investigating the frequency and severity of known G6PD genotypes in South-East Asia by providing a more informative molecular method than the currently used methods to detect G6PDd in Thailand.

Although no SNP mutations were found in our sample groups, the current study provides an assay that may later be applied to larger sample sizes, such as regional populations in Thailand, in order to evaluate whether there is a significant portion of the population with G6PDd at risk with the widespread administration of antimalarials, such as primaquine. Combination of this 16

assay with long-read seqeucning could be considered for the application of this assay in future surveillance studies. 17 Chapter 1: Introduction

Chapter 1: Introduction

18 Chapter 1: Introduction

1.1 Malaria overview

Malaria is a vector-borne disease caused by members of the genus Plasmodium. It is among the most infectious parasitic diseases in humans globally (1), and is one of the leading causes of death in children under five years of age (2). The disease incidences are highest within tropical and subtropical countries, costing more than $2 billion annually to governments of endemic countries to control and eliminate the infection (3, 4).

1.1.2 Causative agents of malaria and their taxonomy

The Plasmodium parasite was first described in the blood of birds by Vassily

Danilewsky as 'pseudovacuoles' (5). By 1890 it was known that a protozoan parasite causes malaria and that three species were responsible for benign tertian (Plasmodium vivax), malignant tertian (Plasmodium falciparum) and quartan (Plasmodium malariae) malaria (5).It took over 30 years before a fourth species (Plasmodium ovale) was identified (5).

Today, nearly 250 Plasmodium species are known to be capable of infecting vertebrate hosts, identified based on their morphology and categorised into 14 subgenera (Table 1) (6, 7).

A few species are zoonotic, such as P. cynomolgi, P. bastianelli, P. simiovale, P. brasilianum,

P. schwetzi, P. inui, and P. knowlesi (8). It is however traditionally recognised that P. falciparum, P. malariae, P. vivax, P. ovale curtisi and P. ovale wallikeri are the species responsible for human malaria. P. ovale curtisi and P. ovale wallikeri are morphologically identical, but regions of their conserved genes show distinct differences. P. falciparum and P. vivax are the most common species that cause human malaria (1, 4, 8, 9). 19 Chapter 1: Introduction

Table 1. Subgenera of the Plasmodium species

Sub-genus Primary host(s) Reference

Asiamoeba Reptiles (10)

Bennettinia Birds (11)

Carinamoeba Reptiles (12)

Giovannolaia Birds (13)

Haemamoeba Birds (13)

Huffia Birds (13)

Lacertamoeba Reptiles (10)

Laverania Great apes, Humans (14)

Novyella Birds (13)

Ophidiella Reptiles (10)

ParaPlasmodium Reptiles (10)

Plasmodium Monkeys and apes (15)

Sauramoeba Reptiles (12)

Vinckeia Mammals including Primates (16)

The genus Plasmodium belongs to the Apicomplexa phylum (see Table 2), a group of single-celled parasites that evolved from the Coccidian stem (8). Within Apicomplexa,

Plasmodium resides within the order Haemosporida; the group of apicomplexans which live in blood cells (Table 2) (8). This order is then split into four distinct families based on asexual reproduction and the presence of the pigment hemozoin, assigning Plasmodium to the family

Plasmodiidae (Table 2) (8). 20 Chapter 1: Introduction

Table 2. Classification of human protozoa of the genus Plasmodium (from Antinori et al., 2012) (8)

Domain Eukaryota

Kingdom Chromalveolata

Superphylum Alveolata

Phylum Apicomplexa

Class Aconoidasida

Order Haemosporida

Sub-order Haemosporidiidea

Family Plasmodiidae

Genus Plasmodia

Sub-genus Plasmodium; Laverania

Species P. falciparum, P. malariae. P. ovale curtisi and P.ovale wallikeri, P. vivax, and P. knowlesi

1.1.2 Epidemiology of malaria

In 2018, The World Health Organization (WHO) estimated that globally, there was approximately 228 million malaria cases (4). Of these cases, pregnant women and children under five years old within malaria-endemic regions are the most susceptible groups as they lack adequate immunity against the disease (17). Additionally, malaria is currently accountable for approximately 405,000 deaths annually, with nearly half the world's population at risk of the disease (4). P. falciparum is responsible for the majority of the global malaria burden, accounting for approximately 90% of deaths and half of all incidences of the disease (17). Until recently, P. vivax was thought to be 'benign' and 'non-fatal,' receiving much lower priority from policymakers and researchers in the past (18). However, this parasite should not be underestimated, as an increasing body of evidence has revealed that P. vivax is capable of causing severe malaria and death, with 3100 mortalities attributed to this species in 2015 according to the WHO (19). Furthermore, P. vivax encompasses a broader geographical distribution compared to P. falciparum. P. vivax frequently causes relapses of human malaria, 21 Chapter 1: Introduction

and is the second-highest contributor to annual malaria cases following P. falciparum

(approximately 14.3 million cases in 2017) (20, 21). Over 2.5 billion people are currently estimated to be at risk of being infected by P. vivax (20), and by far, the highest clinical burden of P. vivax is in South-East Asia (50%) and the Americas (75.4%) (Figure 1) (4).

Recent studies in Thailand have indicated that P. falciparum cases have declined, whereas P. vivax has become the dominant species in many parts of the country and presents particular challenges for elimination (22). This challenge is attributed primarily to P. vivax's biological ability to become dormant in the liver in a form known as hypnozoites, which cannot be detected, causing a subsequent relapse of infection weeks to months later (20). These relapses cause patients to become recurrently infectious, allowing for disease transmission, particularly in endemic countries like Thailand, where a substantial portion of P. vivax incidences may be attributed to relapses (23). Additionally, countries that are co-endemic for

P. falciparum and P. vivax have described co-infections with these two species being the cause of an increased risk of severe malaria (24).

Figure 1. Global endemicity of P. vivax. (P. vivax cases as a percentage of total malaria cases by World Health Organisation (WHO) region in 2018, (4)

22 Chapter 1: Introduction

1.1.3 Plasmodium vivax life cycle

P. vivax has a complex life cycle requiring the definitive host, a female Anopheline mosquito, to transmit the malaria parasite to the human intermediate host during blood-feeding

(25). The life cycle of the parasite comprises of several distinct stages, progressing through asexual stages within the human host and sexual stage within the mosquito vector (Figure 2)

(26).

Stage 1

Stage 2

Stage 4

Stage 3

Figure 2. The life cycle of P. vivax (modified from Mueller et al., 2009) (28) Stage 1) Anopheline mosquitoes inoculate their salivary fluid containing sporozoites into the bloodstream of human hosts through the dermis and migrate to the liver. Stage 2) Motile sporozoites infect liver cells known as hepatocytes and multiply asexually (schizogony), developing large quantities of unnucleated merozoites where some may develop directly into the dormant hypnozoite stage. Stage 3) Mature schizonts bursts out of the hepatocyte, discharging merozoites into the bloodstream. Stage 4) Merozoites invade immature red blood cells (RBCs) known as reticulocytes and mature into early trophozoites. These early trophozoites then mature and undergo schizogony to produce merozoites. Merozoites rupture from the infected cell and go on to attack new RBCs. Alternatively, they may enter the sexual reproductive stage through differentiating in the male and female forms known as gametocytes, which are ingested by a blood-feeding anopheline mosquito and delivered to another human host. 23 Chapter 1: Introduction

The life cycle is initiated when Anopheline mosquitoes inoculate their saliva containing sporozoites (an infective malaria stage in the mosquito's salivary glands) into the human host through the dermis, which then rapidly move to the liver via the bloodstream. Motile sporozoites then infect liver cells and multiply asexually (schizogony), developing significant quantities of unnucleated merozoites. Some of these parasites in the liver develop directly into the dormant hypnozoite stage and do not undergo schizogony. The mature schizont then bursts out of the hepatocyte, discharging merozoites into the bloodstream.

Merozoites go on to invade immature red blood cells (RBCs) known as reticulocytes

(28, 29). Invasion of RBCs is achieved through the presence of the Duffy-Antigen Chemokine

Receptor (DARC) on the surface of RBCs to mediate the entry of the P. vivax parasite. These young ring forms (early trophozoites) then mature and undergo schizogony,forming more merozoites (27). These merozoites rupture from the infected cell and go on to attack new RBCs.

Alternatively, trophozoites may enter the sexual reproductive stage through differentiating in the male and female forms of the Plasmodium parasite known as gametocytes which mediate parasite transmission from its mammalian host, which are then ingested by a blood-feeding

Anopheline mosquito (28, 29).

1.1.4 Clinical disease

Blood-stage parasites are responsible for the pathogenesis of malaria (Figure 2), causing periodic attacks of chills, fever and sweats known as "paroxysms" (30). P. vivax manifests either as asymptomatic or uncomplicated malaria (31). Symptoms of uncomplicated malaria are characteristically low in their severity with many symptoms being analogous to febrile diseases (e.g., influenza) (32). Typical symptoms include sweats, chills, malaise, and headaches, which appear approximately two weeks post-infection (32). Reports of severe P. 24 Chapter 1: Introduction

vivax infections have also been described, which can lead to clinical jaundice, splenomegaly, renal failure, coma, and even death, although this is uncommon (33).

1.2 Treatment of malaria

The current frontline antimalarial drug for P. vivax treatment in most countries is the 4- amino-quinoline chloroquine (CQ) (Figure 3) (34). CQ inhibits β-hematin formation in the digestive vacuole of Plasmodium parasites and hinders their nucleic acid synthesis, thus eliminating blood-stage parasites (34, 35). CQ converts readily and rapidly to its active form desethylchloroquine, which has a long half-life in vivo (3-4 days in plasma) (36). The current recommended regimen of CQ for treatment of uncomplicated P. vivax can be seen in Table 3

(34). Since the discovery of CQ resistance in P. vivax, drug regimens have been altered in response to the availability and emergence of newer drugs, which are more efficacious.

According to the WHO, artemisinin combination therapies (ACT) are currently the recommended protocol for chloroquine-resistant P. vivax (34).

ACTs comprise of a combination of artemisinin derivatives (artesunate/artemether) with a partner drug that contains a longer half-life and different mechanism of action, typically mefloquine, piperaquine, or lumefantrine (34, 37). ACTs have exhibited high efficacy against the blood stages of P. vivax and are safe for use in both children and adults. It is not recommended for women in their first trimester of pregnancy (34, 38).

Although, both CQ and ACTs are comparable and have high efficacies against all P. vivax blood stages, neither of these treatments target the hypnozoites in the liver, thus allowing for future relapse of malaria. Partnering CQ or ACTs with a second therapeutic agent, typically

PQ, has been recommended by the WHO as a combination treatment for this dormant liver- stage (34). 25 Chapter 1: Introduction

Figure 3: Molecular structure of chloroquine (from Ballestero et al., 2005) (39)

Table 3. Chloroquine treatment regimen for uncomplicated P. vivax (as recommended by World Health Organization., 2015) (34)

Drug Name Recommended Treatment Regimen

Chloroquine An oral dose of 10 mg/kg of CQ given on days 1 and 2, followed by 5 mg/kg given on day 3

1.2.1 Primaquine and Tafenoquine

In 1925, the first 8-AQ drug pamaquine was developed to treat P. vivax. Pamaquine was useful in preventing relapse affiliated with P. vivax. However, it could not be clinically used due to its high toxicity (40). Nevertheless, pamaquine was a springboard for the development of safer antimalarial 8-AQ analogues, culminating in 1946 with the synthesis of primaquine. This drug produced the highest efficacy against all forms of malaria, and the lowest levels of relative toxicity compared to all other 8-AQ analogues developed during this time (41). The mechanism of action of PQ is not well understood. It is thought that the build- up of methaemoglobin caused by the metabolites of primaquine damages the cell membrane of the parasite; however, its specific pathway remains unclear (42). PQ is metabolised by cytochrome P450 (CYP 450) and monoamine oxide (MAO) enzymes located in the liver to its active metabolite carboxyprimaquine (43). Fundamentally, PQ is a well-tolerated drug for 26 Chapter 1: Introduction

therapeutic applications, showing minimal side effects and additionally has a short half-life in vivo (4-6hrs in plasma) (5).

Subsequently, the 8-AQ primaquine analogue, TQ, was released in 1978. Comparative to PQ, TQ exhibits greater activity against blood and liver-stage P. vivax. TQ also possesses a longer half-life (14-28 days in plasma) and has a shorter treatment regimen compared to PQ, thus ensuring patient compliance (44, 45, 46). The mechanism of action of TQ isn't well understood, although it is theorised that the drug inhibits the parasites’ DNA and RNA production and protein synthesis (47). TQ is metabolised by Cytochrome P450 2D6 (CYP 2D6) microsomal enzymes in the liver to its active form, 5,6 ortho quinone tafenoquine (48).

Currently, these are the only drugs registered by the food and drug administration of the United

States to eliminate these dormant stages of P. vivax.

Unlike CQ, no clinically relevant drug resistance against PQ or TQ has been observed at this time; however, these drugs are both contraindicated in individuals with G6PDd as they induce oxidative lysis of red blood cells (RBC), which could be potentially fatal. This risk has prompted the WHO to advocate for G6PD screening before administering these drugs and this is particularly important in endemic malaria regions, such as SEA, which has a high frequency of G6PDd individuals (46). Recommended dose regimens with PQ and TQ can be seen in

Table 4.

Table 4. Primaquine and Tafenoquine treatment regimens for uncomplicated P. vivax relapses (as recommended by Commons, McCarthy & Price., 2020 and World Health Organization., 2015) (34, 50)

Drug Name Recommended Treatment Regimen

Primaquine 15 mg of PQ per day administered over 14 days or 30 mg/day in Asia or Oceania

Tafenoquine A single dose (300 mg) of TQ co-administered with a blood schizonticide agent e.g.,

chloroquine

27 Chapter 1: Introduction

1.3 What is Glucose-6-Phosphate Dehydrogenase?

Glucose-6-phosphate dehydrogenase is an enzyme containing 514 amino acid residues and is located on the X chromosome’s long arm at the Xq28 locus (51). This enzyme plays a role in the pentose phosphate pathway (PPP), catalysing the production of the coenzyme, nicotinamide adenine dinucleotide phosphate (NADPH) which functions to regulate the antioxidant, glutathione (GSH) (52). GSH protects the haemoglobin and cell membrane of

RBC from oxidative stress by highly reactive unstable molecules (52). There is currently substantial evidence suggesting that either the absence or reduced functioning of this enzyme, known as G6PDd, can manifest with clinical symptoms including kidney failure, acute haemolytic anaemia, neonatal jaundice, and chronic haemolysis (53, 54, 55).

1.3.1 Glucose-6-Phosphate Dehydrogenase deficiency

G6PD defiency is the most common human enzyme deficiency, affecting over 400 million people globally (56). This condition is primarily caused by SNP mutations within the

G6PD gene, which has been confirmed by molecular technologies in vitro (56).

Currently, 180 allelic variant mutations of the G6PD gene are affiliated with varying levels of deficiency (49). They are categorised into five distinct classes ranging from moderate to severe predicated on their residual enzyme activity, which determines the severity of the side effects (Table 5) (58). However, most individuals with G6PDd are asymptomatic, only exhibiting symptoms when exposed to oxidative agents including specific foods such as fava beans or drugs (e.g., 8-AQ's or some antibiotics) (55). When these oxidative agents are ingested, they induce oxyhaemoglobin generation and GSH depletion, causing RBC's to become damaged, thus manifesting clinical symptoms (59). Individuals with more severe forms of G6PDd are at higher risk of haemolytic anaemia when exposed to oxidative agents 28 Chapter 1: Introduction

compared to moderate to mild deficiency (49). Mutations within the G6PD gene is, therefore, the principal determinant on whether the 8-AQ's, PQ and TQ, can be used against P. vivax (60).

Table 5. Clinical severity classes of G6PD enzyme variants (from Chowdhry, Bisoyi & Mishra, 2012) (58)

Class Level of Enzyme Activity Clinical Symptoms

Deficiency

1 Severe <10% Chronic Haemolytic Anaemia

2 Severe <10% Acute Haemolytic Anaemia

3 Moderate 10-60% Occasional Acute Haemolytic Anaemia

4 Mild-None 60 -150% Asymptomatic

5 None >150% Asymptomatic

1.3.2 Global presence of G6PD mutations

The highest reported prevalence of G6PDd is primarily within sub-Saharan Africa and the Arabian Peninsula, with allelic frequencies peaking at 32.5% in males. At the same time, the Americas have the lowest reported prevalence of G6PDd (≤1%) (49). Conversely, SEA contains the majority of G6PDd individuals due to the large population sizes in these countries

(Table 6) (49). Although G6PD allelic frequencies in SEA don't typically exceed 20%, it is considered the most important G6PDd endemic region due to the high incidence of P. vivax

(56). Additionally, it's reported that there is a substantial geographical overlap between these

G6PDd endemic regions and the prevalence of P. vivax (49). This is particularly concerning for countries like Thailand, which reported 13 000 cases of P. vivax in 2016 and that 13% of males and 10% of females had G6PDd (61). Moreover, the most severe variants of G6PDd are endemic in populations in regions where P. vivax is the most prevalent species of malaria, such as in Thailand (Table 7) (49). 29 Chapter 1: Introduction

Table 6. Estimated G6PDd population by WHO region (from World Health Organization., 2018) (49)

WHO region G6PDd allele G6PDd males G6PDd females

frequency (%)

Africa 12.1 53,267 33,792

America 2.6 9,081 5,225

Eastern Mediterranean 9.0 27,620 16,536

Europe 2.9 2,080 1,149

South-East Asia 5.8 68,588 38,525

Western Pacific 9.2 41,793 23,250

World 8.0 2,02,428 1,18,476

Table 7. Most common mutation variants of the G6PD gene in Thailand and their clinical severities classes (from Kletzien et al., 1994; Mehta et al., 2000; Cappellini et al., 2008 and Lee et al., 2018) (57, 62, 63, 64)

Name of Mutation Country/Region of Origin Clinical Severity Class

Variant

Gaohe China 3

Quing Yuan China 3

Mahidol Thailand 3

Taiwan-Hakka 2 China 2

Coimbra Portugal 2

Viangchan Laos 3

Mahidol-like China 3

Union Philippines 2

Canton China 3

Kaiping China 2

Mediterranean Mediterranean / Middle East 2

Vanua Lava Vanuatu 2

Notes: Clinical Severity Classes: Class 1: Severe G6PDd associated with chronic haemolytic anaemia. Class 2: Severe G6PDd, associated with acute haemolytic anaemia. Class 3: Moderate G6PDd associated with occasional acute haemolytic anaemia. Class 4: Mild to absent G6PDd with no clinical symptoms. Class 5: No G6PDd with no clinical symptoms. (58) 30 Chapter 1: Introduction

1.4 Testing for G6PD deficiency

1.4.1 Current methodology of testing for G6PD deficiency

Detection of G6PDd can be done using genotypic or phenotypic assays. However, the preferred method is the latter, which assesses the NADPH production capacity of the G6PD enzyme in RBCs (65). These tests may either be quantitative (a precise measure of G6PD enzyme activity) or qualitative (determine whether enzyme activity is normal or abnormal).

While phenotypic assays are well suited for case management and decision-making, they are less appropriate for population studies such as that of the current study as these phenotypic tests are unable to characterise the G6PD mutations. Additionally, there are significant limitations to the use of these methods (Table 8) (49).

Genetic analysis is apt for use in population studies, unlike phenotypic assays, which are reliant on intact enzyme activity and require rapid sample processing. Post-collection, genetic studies can use samples that are stored to preserve the DNA, e.g., dried blood spots

(DBS) for later analysis (49, 55). Furthermore, genotypic assays can identify the presence or absence of known genotype mutations/SNPs in the G6PD gene against a known reference panel (55, 65). Thus, these assays are considered optimal for population studies of G6PDd in malaria-endemic regions to establish the prevalence and severity class of G6PDd genotypes in most Southeast Asian countries that don't test for G6PDd before PQ/TQ administration or on a routine basis or where only phenotypic testing is done routinely (Table 9), to prevent potentially dangerous administration of PQ/TQ. 31 Chapter 1: Introduction

Table 8. Limitations of phenotypic assays proposed by the WHO

Spectrophotometric Assay Quantitative Require training and specialised (66, 67)

(Reference Method) equipment

BinaxNOW G6PD test Qualitative Temperature-sensitive (operative 18 to (68)

(Alere) 25°C) requires venous blood, costly

(~$25/test)

CareStartTM G6PD rapid Qualitative Can only be used in male patients, No (69, 70, 71)

diagnostic test (G6PD RDT) control

Fluorescent Spot Test Qualitative Requires specialised training and (66, 72)

infrastructure

Cytochemical Assay Quantitative Complex, hard to interpret, biohazardous (49)

procedure, expensive and long turn around

time

Table 9. SEA countries which test and routinely test before PQ/TQ administration

Country Testing for G6PDd before Routinely testing for G6PDd References

PQ/TQ administration before PQ/TQ administration

Thailand Yes, but poor No (56, 73)

implementation

Cambodia Yes Yes (56, 73)

Indonesia No No (56, 73)

Lao People’s Democratic Yes, but not available at the No (56, 73)

Republic village level

Malaysia Yes Yes (56, 73)

Myanmar No No (56, 73)

Philippines Yes, but poor No (56, 73)

implementation

Timor-Leste No No (56, 73)

Vietnam No No (56, 73) 32 Chapter 1: Introduction

1.4.2 Current typing-based methods for detection of G6PD mutations

Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis is a common technique for screening G6PD mutations through SNPs analysis. This method involves amplifying DNA regions using PCR. The DNA of the amplified products are then recognised and cleaved in the region of the point mutation using restriction enzymes. The

SNP type is then identified through gel electrophoresis confirming and separating smaller sized

DNA fragments generated by endonuclease digestion (74). The main advantages of using this technique for detecting SNP in the G6PD gene is its inexpensiveness, simplicity, rapidness, and accuracy. Additionally, PCR-RFLP does not require additional sophisticated equipment

(74, 75). However, as this technique is confined to the restriction enzyme recognition site, other potential G6PD mutations in the sequence may be ignored unless double digestion is used with another restriction enzyme. Additionally, PCR-RFLP has a limited ability in achieving precise genotyping of G6PD SNPs when there is more than one nucleotide variation in a restriction enzyme recognition site as this method only detects the SNPs present at the exact site of clevage of the restriction enzyme. (76, 77).

Another method that can be used is polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE), where denaturing buffers, such as formamide or urea, are used to separate DNA fragments during electrophoresis. The ability to excise DNA bands from gels for further amplification and sequencing are the primary advantages of using DGGE (74).

Additionally, PCR-DGGE is a sensitive and reliable technique in the screening of gene mutations such as G6PD SNPs. Yet, this method is labour intensive and provides low reproducibility regarding banding patterns and intensity of DNA post-separation using electrophoresis, which is not optimal for pilot studies (78). 33 Chapter 1: Introduction

The PCR-high-resolution melt analysis (PCR-HRMA) is another genetic test that assesses the melting curve of DNA fragments post-PCR amplification (79). While this method has high specificity, discriminatory power, and reproducibility (80, 81), heterozygous variants in genes, such as G6PD, can produce almost identical melting curves if the genetic homology of DNA fragments is analysed, making them indiscernible (82).

1.4.3 Proposed methodology for G6PD mutation typing: PCR

The methods mentioned above have the added limitation of being low throughput and, as a result, are not appropriate for epidemiological or large-scale mutation screenings (83).

PCR followed by sequencing is considered the gold standard and among the most widely used genotyping techniques for screening G6PD SNPs (49). This approach enables simple, accurate, reliable, and effective detection of SNPs with high throughput at the population scale (49, 84).

It has already been extensively and successfully used in various small and large-scale studies for G6PDd, often involving many allelic variations of the G6PD gene, indicating its optimal use in population studies (85, 86). This technique's limitation is that it is labour intensive, expensive, and prone to contamination due to its high sensitivity (49, 84). However, the major advantage of PCR is that it provides precise estimates of the prevalence and severity of G6PDd within a population as they identify specific mutations in the G6PD gene (55, 65).

Also, as the G6PD gene is located on the X chromosome, males will solely inherit a single gene copy (hemizygotes). Contrastingly, females inherit two gene copies and, therefore, may carry two wild-type alleles or two deficient alleles. Females may also carry a combination of one wild-type and one deficient gene (heterozygotes) (49). The latter is particularly problematic as heterozygous females may exhibit lyonization (random X chromosome inactivation), leading to a partial deficiency that won't be reliably detected with most screening tests (87, 49). 34 Chapter 1: Introduction

1.5 Project significance and problem statements

Over the last 15 years, the overall burden of malaria has declined in SEA countries, including Thailand (90). While P. falciparum was once the most prevalent malaria parasite for most of Thailand, P. vivax has now become the dominant species (61). Thailand plans to revise or introduce new guidelines for effective and safe elimination of P. vivax using primaquine and tafenoquine to eradicate malaria from its territory by 2024 (61). However, in areas where

G6PDd prevalence is high, and testing isn't available, contraindications with haemolysis may hinder this progress.

Additionally, there are no clear guidelines for the treatment of imported cases of P. vivax for individuals who are co-diagnosed with G6PDd returning to Australia. However, the

WHO has recommended a lower oral dose of 0.75 mg/kg PQ for eight weeks under supervision by a medical expert for individuals with less than 30% G6PD activity (34).

Molecular surveillance is a useful tool for identifying allelic mutations; therefore, it can be useful in monitoring G6PD variants as a risk assessment guide in mapping the safety of implementing primaquine and tafenoquine for the radical cure of P. vivax. Genotype classes established by the WHO allows for universal and straightforward categorisation of SNPs to determine their connection to the severity in reducing the enzyme activity (58).

35 Chapter 1: Introduction

1.6 Hypothesis

The development of a PCR assay for the detection of G6PD mutations will enable monitoring of the frequency and allelic variant types of the G6PD gene present in Southern

Thailand and travellers returning to Australia, which will identify G6PD deficient individuals for which the usage of primaquine and tafenoquine may not be safe.

1.7 Aims

Aim 1: Developing a molecular assay covering the G6PD mutations present in Asia

that will allow the surveillance of the genetic frequency and severity of G6PD

genotypes present in South East Asia.

Aim 2: Apply the new molecular assay to malaria infected blood samples from

Southern Thailand to set the stage to survey the genetic frequency and severity

of G6PD genotypes.

Aim 3: Apply the new molecular assay to malaria infected blood samples from

travellers returning to Australia to provide a proof of principle to identify the

genetic frequency and severity of G6PD genotypes 36 Chapter 2: Methods and Materials

Chapter 2: Materials and Methods

37 Chapter 2: Methods and Materials

2.1 Materials

Various molecular biology grade reagents, equipment, and materials were used in this project; they are listed in Appendix (I), Appendix (II), and Appendix (III), as well as bioinformatics databases and web resources, which are listed in Appendix (IV).

2.2 Methods

2.2.1 DNA Extraction

2.2.1.1 Extraction of genomic DNA from whole blood

Five anonymous human whole blood (negative for malaria on blood films) samples, previously stored at the Institute of Clinical Pathology and Medical Research (ICPMR) at -

20°C were used to optimise the PCR protocols for all five G6PD loci through trial assays.

These samples had previously been defrosted and homogenised through centrifugation. When not in use, samples were stored at -20°C.

The QIAamp DNA Extraction Mini Kit (QIAGEN and Cat No./ID: 51306) was used in correspondence with the manufacturer’s instructions to extract genomic DNA from human whole blood diagnostic samples. Aliquots (200µl) of each whole blood sample were placed into a 1.5ml microcentrifuge tube and vortexed with 20µl of Proteinase K solution. Samples were then incubated at 56°C in a dry block heater for 10 minutes before the addition of 200µl of 96-10% ethanol to each sample. The solution was then transferred into a spin column and centrifuged for 1 minute at 6000 x g. The column filter was then washed with 500µl of AW1 and 500µl of AW2 buffer and centrifuged for 3 minutes at 20,000 x g. Finally, the DNA was eluted to a total volume of 200µl. When not in use, samples were stored at -20°C. 38 Chapter 2: Methods and Materials

2.2.1.2 Extraction of genomic DNA from dried blood spots

A QIAamp DNA Extraction Mini Kit (QIAGEN and Cat No./ID: 51306) was used in correspondence with the manufacturer’s instructions to extract genomic DNA from 15 human dried blood samples on filter paper. Blood spots were cut into small fragments and sterilised with 70% ethanol before being placed into a 1.5 ml microcentrifuge tube. 180µl of buffer ATL was added to each sample which were then incubated at 85°C for 10 minutes. Samples were then vortexed with 20µl of Proteinase K solution and incubated at 56°C for 1 hour. Samples were then vortexed with 200 µl Buffer AL and incubated at 70°C for 10 minutes. Samples were then centrifuged with 200µl ethanol (96-100%) and transferred into a spin column where they underwent centrifugation for 1 minute at 6000 x g. The column filter was then washed with

500µl of AW1 and 500µl of AW2 buffer before centrifugation for 3 minutes at 20,000 x g.

Finally, the DNA was eluted to a total volume of 200µl. When not in use, samples were stored at -20°C.

2.2.2 Extracted DNA quality control

Total DNA concentrations were measured by a DeNovix DS-11 + Spectrophotometer at 260 nm and 280 nm. These measurements were compared to reference absorbance ratios at

260 and 280 nm (A260/280) and 230nm and 260nm (A230/260). Ratios outside of the standard ranges of 2-2.2 and 1.7-2, respectively, were excluded. Also, DNA quality was confirmed through gel electrophoresis (Section 2.2.5) and the visualisation of high molecular weight singular bands with minimal shearing under ultraviolet (UV) light. Stock DNA was diluted to a 10ng/µl working solution and refrigerated at 4°C for short term use. Additionally, stock concentrations were stored at -20°C. 39 Chapter 2: Methods and Materials

2.2.3 Preparation of primer solution

Lyophilised primer pellets were resuspended with sterile water to a stock concentration of 100 µM. Stock concentrations were diluted to a 10ng/µl working solution by a with sterile water and refrigerated at 4°C for short term use. Additionally, stock concentrations were stored at -20°C.

2.2.4 Protocol development

Since PCR was being used in this laboratory with newly designed primer sets, experiments to optimise PCR conditions were essential before application to samples from

Southern Thailand and Australian travelllers. To optimise these conditions, trial assays were conducted using recommended MgCl2, dNTPs, BioTAQ DNA polymerase, and DNA template concentrations to determine the optimal PCR cycle number for each primer set, indicated by sharp bands at the expected size of the amplicon of interest. Templates were then subjected to varying gradient PCR assays (intervals of 1 or 2°C) and cycle numbers, to identify optimal conditions for amplification which were later confirmed by the visualisation of PCR products as clear single bands via gel electrophoresis. PCR product size was determined by comparing products to a 1kb DNA ladder.

2.2.4.1 PCR protocol

PCR was used to amplify mutations in the G6PD gene with the oligonucleotide primers described in Tables 12, 13, 17, 18, and 19. A primary PCR master mix (MM1) with a total volume of 39.5µl per PCR reaction was prepared with the following reagents: 5µl 10 x PCR reaction buffer, 5µl dinucleotide triphosphates (dNTPs), containing 2mM dATP, dCTP, dGTP, dTTP, 5µl forward primers (10ng/µl), 5µl reverse primers (10ng/µl), 3µl of 50mM MgCl2,

16.5µl of sterile water, 0.5µl of 5U/µl BioTAQ DNA polymerase. 1 x PCR MM1 and 10µl 40 Chapter 2: Methods and Materials

from each genomic DNA sample were then added to individual 0.2mL PCR reaction tubes. A negative control accompanied all reactions, comprised of 1 x PCR MM1 in lieu of DNA template. The PCR was performed using a Sensoquest Labcycler thermal cycler for each loci specific primer set corresponding with the cycling conditions seen in Table 10. To confirm that the amplified DNA products were of the correct size, they underwent gel electrophoresis as per Section 2.2.5. Successfully amplified PCR products were sent to Macrogen (South

Korea) for bidirectional Sanger sequencing.

Table 10. Initial amplification conditions of each primer set. Primer sets and their respective PCR cycling conditions

Primer Set Conditions

1 Initial denaturation of 94°C for 3 minutes; followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 60 seconds, and a final extension at 72°C for 7 minutes and then hold step at 4°C

2 Initial denaturation of 94°C for 3 minutes; followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 59°C for 45 seconds, and extension at 72°C for 60 seconds, and a final extension at 72°C for 7 minutes and then hold step at 4°C

3 Initial denaturation of 94°C for 3 minutes; followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 60 seconds, and a final extension at 72°C for 7 minutes and then hold step at 4°C

4 Initial denaturation of 94°C for 3 minutes; followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 45 seconds, and extension at 72°C for 60 seconds, and a final extension at 72°C for 7 minutes and then hold step at 4°C

5 Initial denaturation of 94°C for 3 minutes; followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 60 seconds, and a final extension at 72°C for 7 minutes and then hold step at 4°C

41 Chapter 2: Methods and Materials

2.2.5 Gel Electrophoresis

All PCR products underwent gel electrophoresis. Gels were prepared by adding

Tris/Borate/EDTA (TBE) buffer (1X) to powdered agarose into a conical flask and made to a

1.5% agarose proportion. The solution was heated using a microwave oven until agarose was dissolved and then cooled to approximately 55°C. Ethidium bromide was added to the solution at 1µL per 100ml of agarose. The solution was poured into casting moulds comprising of combs and was allowed to set at room temperature. Gels were then placed into an electrophoresis chamber containing TBE buffer. 10µL of DNA sample was combined with 5µL of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, and 30% glycerol in water) and loaded into wells formed within the gel. All gels comprised of a lane for a 1kb plus Invitrogen

DNA ladder (Cat. No.10787-018). Loaded gels were run at a constant voltage of 80V for approximately 40 minutes. The gels were then illuminated under UV light using a Uniequip

UV transilluminator TM40E (λ=302nm) to visualise the presence of DNA products and then photographed. Product size was determined by comparing sample products to the DNA ladder.

2.2.6 DNA extraction and purification from gel

Following amplification of test DNA templates by the G6PD locus 2.1 primers in Table

13 and their amplification conditions listed in Section 3.2.2 produced multiple bands of varying sizes Figure 10. These unexpected bands required further investigation to identify whether or not they were specific regions to the G6PD gene. To address this, following gel electrophoresis, these bands were cut out of the gel, and DNA was extracted and purified using the Wizard®

SV Gel and PCR Clean-Up System (Promega and cat No./ID: a9281) according to the manufacturer's protocol. Briefly, DNA bands were excised from the gel vortexed and incubated at 60°C with 10μl of membrane binding solution per 10mg of gel slice until dissolved. The 42 Chapter 2: Methods and Materials

dissolved DNA solution was transferred to a mini-column assembly, incubated for 1 minute at room temperature, and centrifuged at 16,000 x g for 1 minute. The mini-column was then washed with 700μl of membrane wash solution + ethanol and centrifuged for 1 minute at

16,000 x g. This step was then repeated with 500μl of membrane wash solution and centrifuged for 5 minutes. DNA was eluted with warm nuclease-free water to a volume of 50μl and incubated for 1 minute at room temperature. Finally, the DNA solution was concentrated in a vacufuge concentrator (5301) or approximately 15 minutes.

2.2.7 Primer dilution for sequencing

Macrogen (South Korea) requires primer sets to be diluted to 5pmol/µl and sent with amplified PCR products for bidirectional Sanger sequencing. To do this, the volume of the primer in dilution was calculated using the formula in Figure 4. Sterile water was then added to make up the difference to a total volume of 1000µl

5 1 %"#&2$#3* 4&+5ℎ. "( )*+%&* !"#$%& "( )*+%&* +, -+#$.+", = 7",2&,.*3.+", "( )*+%&* 8."29

Figure 4. Primer dilution calculation

2.2.8 PCR sequencing and variant analysis

The program Sequencher version 5.3 (http://www.genecodes.com) was used to combine forward and reverse sequences for each sample to form a sample consensus sequence

(contig) and crop low-quality end base pairs (bp) of amplified G6PD region sequences.

BLASTN searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi) were used to validate that the sequence data generated was within the G6PD gene. Finally, the program MEGA ver.10 (using 43 Chapter 2: Methods and Materials

the integrated ClustalW algorithm) was used to align the obtained sample sequence contigs to the G6PD reference gene NG_009015 sequence (http://www.ncbi.nlm.nih.gov) and the consensus sequences of all possible mutations containing the SNPs of interest to determine the presence or absence of the respective G6PD mutations. 44 Chapter 3: Molecular Assay Optimisation

Chapter 3: Molecular Assay Optimisation

45 Chapter 3: Molecular Assay Optimisation

3.1 Primer design selection

The location of all frequently reported mutations in the G6PD gene (GenBank sequence accession number NG_009015) found in Asia are depicted in Tables 12, 13, 17, 18, and 19.

Five oligonucleotide primer sets capable of amplifying products capturing all reported

Asian G6PD mutations were designed (Appendix V) using the web-based program Primer-

Blast with the default parameters and design properties listed in (Table 11). The five loci and primer set characteristics can be visualised in Tables 12, 13, 17, 18, and 19. Optimisation for each loci specific primer set was completed using up to five anonymous human whole blood diagnostic samples (#1-5) extracted as per the protocol detailed in Section 2.2.1.1.

Table 11. Design parameters of primer pairs.

Base length between 18-24 GC content between 40-60% TM between 50-60°C A maximum variation of 5°C in TM between primer pairs No complementary regions between primer pairs

3.2 Optimisation of the PCR amplification conditions

3.2.1 G6PD locus 1

Position in the G6PD gene: 6339 bp – 6634 bp

Table 12. G6PD locus 1 primer information. Forward and reverse primer sequence, primer length, PCR product size, and name of mutations being detected by the amplification product.

Primer name and sequence Length GC Product Mutation name/s and (bp) (%) Size their location (bp) Forward Primer: G6PD (1)-F 20 55 336 Gaohe (6512 A>G) 5' TGTGAGACCCCAGAGGAACT 3' Reverse Primer: G6PD (1)-R 20 55 5' GGGAGGAGGAGCTCAACTTA 3'

Initial amplification conditions: - Initial denaturation of 94°C for 3 minutes; 46 Chapter 3: Molecular Assay Optimisation

- 35 cycles: denaturation at 94°C for 30 seconds, annealing at 59°C for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 5. DNA electrophoresis image of the amplification products of the G6PD locus 1. #1, #2, and #3 human blood DNA samples. NC = Negative control, 1kb STD = DNA ladder.

Outcome: Multiple bands (400bp and above) in addition to the expected band for the G6PD locus 1 at 336 bp

First round of altered amplification conditions: - Initial denaturation of 94°C for 3 minutes; - 30 cycles: denaturation at 94°C for 30 seconds, annealing at 59°C for 45 seconds, extension at 72°C for 60 Seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 6. DNA electrophoresis image of the amplification products of the G6PD locus 1. #1, #2, #3 #4, and #5 human blood DNA samples. NC = Negative control, 1kb STD = DNA ladder.

Outcome: No amplification products were obtained.

47 Chapter 3: Molecular Assay Optimisation

Second round of altered amplification conditions: A gradient PCR was run for 32 cycles with annealing temperatures of 57°C, 57.9°C, 59.2°C, 60.1°C, and 61° using sample (#1) as shown in each lane for calculating the mean melting temperature (TM) of the primer pair and subtracting 5°C to be set as the middle temperature. Annealing temperatures were then set in intervals of 1°C to two temperatures higher and lower than the median.

- Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at temperature gradient between 57°C, 57.9°C, 59.2°C, 50.1°C and 61°C, respectively, for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 7. DNA electrophoresis image of the amplification products of the G6PD locus 1. obtained from one human blood samples (#1) at 57°C, 57.9°C, 59.2°C, 60.1°C, and 61°C. NC = Negative control, 1kb STD = DNA ladder.

Outcome: None, products were amplified at all tested annealing temperatures.

Third round of altered amplification conditions: - Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at 61°C, for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

48 Chapter 3: Molecular Assay Optimisation

Results:

Figure 8. DNA electrophoresis image of the amplification products of the G6PD locus 1. #1, #2, #3, and #4 human blood DNA samples. NC = Negative control, 1kb STD = DNA ladder.

Outcome: The targeted G6PD locus 1 region amplified for all samples!

Final optimised amplification conditions for the G6PD locus 1: - Initial denaturation of 94°C for 3 minutes; - 32 cycles of denaturation at 94°C for 30 seconds, annealing at 61°C for 45 seconds, and extension at 72°C for 60 seconds - final extension at 72°C for 7 minutes, followed by a holding step at 4°C

3.2.2 G6PD locus 2

Position in the G6PD gene: 17138 bp – 17456 bp

Table 13. G6PD locus 2 primer information. Forward and reverse primer sequence, primer length, PCR product size, and name of mutations being detected by the amplification product.

Primer name and sequence Length GC Product Mutation name/s and (bp) (%) Size their location (bp) Forward Primer: G6PD (2)-F 20 55 299 Vanua Lava (17303 T>C), 5' GCAGAACACACACGGACTCA 3' Quing Yuan (G>T) Reverse Primer: G6PD (2)-R 20 55 5' CTCATAGAGTGGTGGGAGCA 3'

Initial amplification conditions: A gradient PCR was run for 32 cycles with annealing temperatures of 57°C, 57.9°C, 59.2°C, 60.1°C, and 61°C using sample (#1) as shown in each lane for calculating the mean melting temperature (TM) of the primer pair and subtracting 5°C to be set as the middle temperature. Annealing temperatures were then set in intervals of 1°C to two temperatures higher and lower than the median.

49 Chapter 3: Molecular Assay Optimisation

- Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at temperature gradient between 57°C, 57.9°C, 59.2°C, 60.1°C, and 61°C, respectively, for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 9. DNA electrophoresis image of the amplification products of the G6PD locus 2. obtained from one human blood sample (#1) at 57°C, 57.9°C, 59.2°C, 60.1°C, and 61°C. NC = Negative control, 1kb STD = DNA ladder.

Outcome: Non-specific bands (850bp and above) in addition to the expected band for the G6PD locus 2 at 359 bp at all tested temperatures.

G6PD locus 2.1 (second new primer set for locus 2)

Position in the G6PD gene: 17156 bp – 17678 bp

Table 14. Second new G6PD locus 2 primer information. Forward and reverse primer sequence, primer length, PCR product size, and name of mutations being detected by the amplification product.

Primer name and sequence Length GC Product Mutation name/s and (bp) (%) Size their location (bp)

Forward Primer: G6PD (2.1)-F 20 55 563 Vanua Lava (17303 T>C), 5' CAAAGAGAGGGGCTGACATC 3' Quing Yuan (G>T) Reverse Primer: G6PD (2.1)-R 20 50 5' AAAACCAGCCAGAGGACAAG 3'

Initial amplification conditions: A gradient PCR was run for 32 cycles with annealing temperatures of 51°C, 51.9°C, 53.2°C, 54.1°C, and 55°C using sample (#1) as shown in each lane for calculating the mean melting temperature (TM) of the primer pair and subtracting 5°C to be set as the middle temperature. 50 Chapter 3: Molecular Assay Optimisation

Annealing temperatures were then set in intervals of 1°C to two temperatures higher and lower than the median.

- Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at temperature gradient between 51°C, 51.9°C, 53.2°C, 54.1°C, and 55°C, respectively, for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 10. DNA electrophoresis image of the amplification products of the G6PD locus 2.1. obtained from one human blood sample (#1) at 57°C, 57.9°C, 59.2°C, 60.1°C, and 61°C. NC = Negative control, 1kb STD = DNA ladder.

Outcome: Multiple bands (850bp and above) in addition to the expected band for the G6PD locus 2.1 at 563 bp at all tested temperatures.

DNA extraction and purification from gel:

Human blood DNA samples #1, 2, and 3 were subjected to the PCR protocol described in Section 2.2.4.1 for 32 cycles at 51.9°C set as the annealing temperature Figure 11, producing various sized bands Table 15. All bands were extracted and purified as per the protocol in Section 2.2.6. Bands were subjected to bidirectional Sanger sequencing as described in Section 2.2.8.

51 Chapter 3: Molecular Assay Optimisation

Results:

Figure 11. DNA electrophoresis image of the amplification products of the G6PD locus 2.1. #1, #2 and #3 human blood DNA samples. NC = Negative control, 1kb STD = DNA ladder.

Table 15. Band sizes produced by DNA samples following amplification by the G6PD 2.1 locus. Human whole blood DNA samples and their band sizes

DNA samples

#1 #2 #3

Size/s of 560, 750 and 900 560, 750, 900 and 2000+ 560 and 900

bands

amplified

(bp)

Outcome: The sequenced DNA quality was significantly low, and therefore, the bands' data could not be analysed by primer-blast.

G6PD locus 2.2 (Third new primer set for locus 2)

Position in the G6PD gene: 16799 bp – 17555 bp

Table 16. Third G6PD locus 2 primer information. Forward and reverse primer sequence, primer length, PCR product size, and name of mutations being detected by the amplification product.

Primer name and sequence Length Product Mutation name/s (bp) GC Size and their location (%) (bp) Forward Primer: G6PD (2.2)-F 20 55 797 5' TTGAGACCCCCATTACCAGC 3' 52 Chapter 3: Molecular Assay Optimisation

Reverse Primer: G6PD (2.2)-R 20 55 Vanua Lava (17303 5' GAAAGGCGGTGTTTCGTGGA 3' T>C), Quing Yuan (G>T)

Initial amplification conditions: A gradient PCR was run for 32 cycles with annealing temperatures of 63.5°C, 64.3°C, 65.7°C, 66.3°C, and 67.5°C using sample (#1) as shown in each lane for calculating the mean melting temperature (TM) of the primer pair and subtracting 5°C to be set as the middle temperature. Annealing temperatures were then set in intervals of 1°C to two temperatures higher and lower than the median.

- Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at temperature gradient between 63.5°C,64.3°C, 65.7°C, 66.3°C, and 67.5°C, respectively, for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 12. DNA electrophoresis image of the amplification products of the G6PD locus 2.2. obtained from one human blood sample (#1) at 63.5°C, 64.3°C, 65.7°C, 66.3°C, and 67.5°C. NC = Negative control, 1kb STD = DNA ladder.

Outcome: Products were amplified at all tested annealing temperatures at the expected band for the G6PD locus 2.2 at 336 bp.

Second round of altered amplification conditions: - Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at 65.7°C, for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

53 Chapter 3: Molecular Assay Optimisation

Results:

Figure 13. DNA electrophoresis image of the amplification products of the G6PD locus 2.2. #1, #2, #3, and #4 human blood DNA samples. NC = Negative control, 1kb STD = DNA ladder.

Outcome: The targeted G6PD locus 2.2 region amplified for all samples!

Final optimised amplification conditions for the G6PD locus 2: - Initial denaturation of 94°C for 3 minutes; - 32 cycles of denaturation at 94°C for 30 seconds, annealing at 65.7°C for 45 seconds, and extension at 72°C for 60 seconds - final extension at 72°C for 7 minutes, followed by a holding step at 4°C

3.2.3 G6PD locus 3

Position in the G6PD gene: 17918 bp – 18327 bp

Table 17. G6PD locus 3 primer information. Forward and reverse primer sequence, primer length, PCR product size, and name of mutations being detected by the amplification product.

Primer name and sequence Length GC Product Mutation name/s and (bp) (%) Size (bp) their location Forward Primer: G6PD (3)-F 20 55.6 448 Mahidol (18078 G>A), 5' TTCCCGGAAGGTGTTGAG Taiwan-Hakka 2 (18084 3' A>G), Mediterranean Reverse Primer: G6PD (3)-R 20 55 (18154 C>T), Coimbra 5' (18183 C>T) GGGTCACCCTTGTCTGAGTT 3'

Initial amplification conditions: A gradient PCR was run for 32 cycles with annealing temperatures of 57°C, 57.9°C, 59.2°C, 60.1°C, and 61°C using sample (#1) as shown in each lane for calculating the mean melting temperature (TM) of the primer pair and subtracting 5°C to be set as the middle temperature. Annealing temperatures were then set in intervals of 1°C to two temperatures higher and lower than the median. 54 Chapter 3: Molecular Assay Optimisation

- Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at temperature gradient between 57°C, 57.9°C, 59.2°C, 60.1°C, and 61°C, respectively, for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 14. DNA electrophoresis image of the amplification products of the G6PD locus 3. obtained from one human blood sample (#1) at 57°C, 57.9°C, 59.2°C, 60.1°C, and 61°C. NC = Negative control, 1kb STD = DNA ladder.

Outcome: Products were amplified at all tested annealing temperatures at the expected band for the G6PD locus 3 at 336 bp

Second round of altered amplification conditions: - Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at 59°C for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

55 Chapter 3: Molecular Assay Optimisation

Results:

Figure 15. DNA electrophoresis image of the amplification products of the G6PD locus 3. #1, #2, #3, and #4 human blood DNA samples. NC = Negative control, 1kb STD = DNA ladder.

Outcome: The targeted G6PD locus 3 regions amplified for all samples!

Final optimised amplification conditions for the G6PD locus 3: - Initial denaturation of 94°C for 3 minutes; - 32 cycles of denaturation at 94°C for 30 seconds, annealing at 61°C for 45 seconds, and extension at 72°C for 60 seconds - final extension at 72°C for 7 minutes, followed by a holding step at 4°C

3.2.4 G6PD locus 4

Position in the G6PD gene: 19285 bp – 19723 bp

Table 18. G6PD locus 4 primer information. Forward and reverse primer sequence, primer length, PCR product size, and name of mutations being detected by the amplification product.

Primer name and sequence Length GC Product Mutation name/s and (bp) (%) Size (bp) their location Forward Primer: G6PD (4)-F 18 66.6 477 Viangchan (19453 5' ACCAGGGTGGTCCTGGAG G>A), Kerala-Kalyan 3' (19529 G>A), Mahidol- Reverse Primer: G6PD (4)-R 20 60 like (19604 C>T) 5' ACCAGCTCTCTCAGGGTGTG 3'

Initial amplification conditions: A gradient PCR was run for 32 cycles with annealing temperatures of 60°C, 60.9°C, 62.2°C, 63.1°C, and 64°C using sample (#1). The annealing temperatures as shown in each lane were 56 Chapter 3: Molecular Assay Optimisation

determined by using the mean of the melting temperature (TM) provided by the manufacturer of the primer pair and subtracting 5°C to be set as the middle temperature. 2 other temperatures higher and lower than the median were then tested with intervals of 1°C degree.

- Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at temperature gradient between 60°C, 60.9°C, 62.2°C, 63.1°C, and 64°C, respectively, for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 16. DNA electrophoresis image of the amplification products of the G6PD locus 4. obtained from one human blood sample (#1) at 60°C, 60.9°C, 62.2°C, 63.1°C, and 64°C. NC = Negative control, 1kb STD = DNA ladder.

Outcome: Multiple bands and smearing (300bp) in addition to the expected band for the G6PD locus 4 at 477 bp for all tested temperatures other than 64°C.

Second round of altered amplification conditions: - Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at 64°C for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

57 Chapter 3: Molecular Assay Optimisation

Results:

Figure 17. DNA electrophoresis image of the amplification products of the G6PD locus 4. #1, #2, #3, and #4 human blood DNA samples. NC = Negative control, 1kb STD = DNA ladder.

Outcome: The targeted G6PD locus 4 regions amplified for all samples!

Final optimised amplification conditions for the G6PD locus 4: - Initial denaturation of 94°C for 3 minutes; - 32 cycles of denaturation at 94°C for 30 seconds, annealing at 64°C for 45 seconds, and extension at 72°C for 60 seconds - final extension at 72°C for 7 minutes, followed by a holding step at 4°C

3.2.5 G6PD locus 5

Position in the G6PD gene: 20010 bp – 20434 bp

Table 19. G6PD locus 5 primer information. Forward and reverse primer sequence, primer length, PCR product size, and name of mutations being detected by the amplification product.

Primer name and sequence Length Product Mutation name/s (bp) GC Size and their location (%) (bp) Forward Primer: G6PD (5)-F 20 50 465 Union (20183 C>T), 5' ACGGCAACAGATACAAGGTG 3' Canton (20304 Reverse Primer: G6PD (5)-R 20 60 G>T) Kaping 5' TGAGGTAGCTCCACCCTCAC 3' (20316 G>A)

Initial amplification conditions: A gradient PCR was run for 32 cycles with annealing temperatures of 56°C, 56.9°C, 58.2°C, 59.1°C, and 60°C using sample (#1) as shown in each lane for calculating the mean melting temperature (TM) of the primer pair and subtracting 5°C to be set as the middle temperature. Annealing temperatures were then set in intervals of 1°C to two temperatures higher and lower than the median. 58 Chapter 3: Molecular Assay Optimisation

- Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at temperature gradient between 56°C, 56.9°C, 58.2°C, 59.1°C, and 60°C, respectively, for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 18. DNA electrophoresis image of the amplification products of the G6PD locus 5. obtained from one human blood sample (#1) at 56°C, 56.9°C, 58.2°C, 59.1°C, and 60°C. NC = Negative control, 1kb STD = DNA ladder.

Outcome: Non-specific bands (400bp,1000bp and above) in addition to the expected band for the G6PD locus 5 at 465 bp at all tested temperatures.

Second round of altered amplification conditions: A gradient PCR was run for 32 cycles with annealing temperatures of 58°C, 58.9°C, 60.2°C, 61.1°C, and 62°C, using sample (#1) as shown in each lane for calculating the mean melting temperature (TM) of the primer pair and subtracting 5°C to be set as the middle temperature. Annealing temperatures were then set in intervals of 1°C to two temperatures higher and lower than the median.

- Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at temperature gradient between 58°C, 58.9°C, 60.2°C, 61.1°C, and 62°C, respectively, for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

59 Chapter 3: Molecular Assay Optimisation

Results:

Figure 19. DNA electrophoresis image of the amplification products of the G6PD locus 5. obtained from one human blood sample (#1) at 58°C, 58.9°C, 60.2°C, 61.1°C, and 62°C. NC = Negative control, 1kb STD = DNA ladder.

Outcome: Non-specific bands (400bp,1000bp and above) in addition to the expected band for the G6PD locus 5 at 465 bp at all tested temperatures other than 62°C.

Third round of altered amplification conditions: - Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at 62°C for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 20. DNA electrophoresis image of the amplification products of the G6PD locus 5. #1, #2, #3, and #4 human blood DNA samples. NC = Negative control, 1kb STD = DNA ladder.

Outcome: Non-specific bands (400bp,1000bp and above) in addition to the expected band for the G6PD locus 5 at 465 bp at all tested temperatures.

60 Chapter 3: Molecular Assay Optimisation

G6PD locus 5.1 (New primer set for locus 5)

Position in the G6PD gene: 20009 bp – 20434 bp

Table 20. G6PD locus 5.1 primer information. Forward and reverse primer sequence, primer length, PCR product size, and name of mutations being detected by the amplification product.

Primer name and sequence Length GC Product Mutation name/s (bp) (%) Size and their location (bp)

Forward Primer: G6PD (5.1)-F 20 50 652 Union (20183 C>T), 5' ACGGCAACAGATACAAGGT 3' Canton (20304 Reverse Primer: G6PD (5.1)-R 20 50 G>T) Kaping 5' TGAGGTAGCTCCACCCTCAC 3' (20316 G>A)

Initial amplification conditions: A gradient PCR was run for 32 cycles with annealing temperatures of 59.5°C, 60.7°C, 61.3°C, 62.6 °C, and 63.5°C using sample (#1) as shown in each lane for calculating the mean melting temperature (TM) of the primer pair and subtracting 5°C to be set as the middle temperature. Annealing temperatures were then set in intervals of 1°C to two temperatures higher and lower than the median.

- Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at temperature gradient between 59.5°C, 60.7°C, 61.3°C, 62.6 °C, and 63.5°C, respectively, for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 21. DNA electrophoresis image of the amplification products of the G6PD locus 5.1. obtained from one human blood sample (#1) at 59.5°C, 60.7°C, 61.3°C, 62.6 °C, and 63.5°C. NC = Negative control, 1kb STD = DNA ladder.

61 Chapter 3: Molecular Assay Optimisation

Outcome: No amplification products were obtained at the expected band for the G6PD locus 5.1 at 563 bp at all tested temperatures other than at 62.6°C.

Second round of altered amplification conditions: - Initial denaturation of 94°C for 3 minutes; - 32 cycles: denaturation at 94°C for 30 seconds, annealing at 62.6°C for 45 seconds, extension at 72°C for 60 seconds - Final extension at 72°C for 7 minutes, followed by a holding step at 4°C

Results:

Figure 22. DNA electrophoresis image of the amplification products of the G6PD locus 5.1. #1, #2, and #3 human blood DNA samples. NC = Negative control, 1kb STD = DNA ladder.

Outcome: The targeted G6PD locus 5.1 region amplified for all samples!

Final optimised amplification conditions for the G6PD locus 5: - Initial denaturation of 94°C for 3 minutes; - 32 cycles of denaturation at 94°C for 30 seconds, annealing at 62.6°C for 45 seconds, and extension at 72°C for 60 seconds - final extension at 72°C for 7 minutes, followed by a holding step at 4°C 62 Chapter 3: Molecular Assay Optimisation

3.3 Summary of the final optimised amplification conditions

The conditions of five loci specific oligonucleotides primers capable of amplifying products capturing all reported Asian G6PD mutations were successfully established and optimised according to the steps described in Section 3.2.

Not all primer pairs were effective at generating their specific targets without forming non-specific bands, e.g., G6PD locus 2 and 5, which are likely attributed to these primer pairs annealing to other homologous regions outside of the G6PD gene. In these instances, primer pairs were re-designed and optimised, and checked for specific band formation. The final conditions for all 5 loci (see Table 21) enabled successful amplification of all tested samples resulting in single distinct bands without smearing. These conditions were selected as optimal for these primer sets and could now be applied to the pilot study for samples from Southern

Thailand and Australian travellers.

Table 21. Summary of the final optimised PCR conditions for all 5 G6PD loci

Primer Set Forward/reverse primer sequences Conditions

G6PD locus 1 Forward Primer: G6PD (1)-F Initial denaturation of 94°C for 3 5' TGTGAGACCCCAGAGGAACT 3' minutes; followed by 32 cycles of Reverse Primer: G6PD (1)-R denaturation at 94°C for 30 seconds, 5' GGGAGGAGGAGCTCAACTTA 3' annealing at 61°C for 45 seconds, and extension at 72°C for 60 seconds, and a final extension at 72°C for 7 minutes and then hold step at 4°C

G6PD locus 2 Forward Primer: G6PD (2)-F Initial denaturation of 94°C for 3 5' TTGAGACCCCCATTACCAGC 3' minutes; followed by 32 cycles of Reverse Primer: G6PD (2)-R denaturation at 94°C for 30 seconds, 5' GAAAGGCGGTGTTTCGTGGA 3' annealing at 65.7°C for 45 seconds, and extension at 72°C for 60 seconds, and a final extension at 72°C for 7 minutes and then hold step at 4°C

G6PD locus 3 Forward Primer: G6PD (3)-F Initial denaturation of 94°C for 3 5' TTCCCGGAAGGTGTTGAG 3' minutes; followed by 32 cycles of Reverse Primer: G6PD (3)-R denaturation at 94°C for 30 seconds, 5' GGGTCACCCTTGTCTGAGTT 3' annealing at 61°C for 45 seconds, and extension at 72°C for 60 seconds, and a 63 Chapter 3: Molecular Assay Optimisation

final extension at 72°C for 7 minutes and then hold step at 4°C

G6PD locus 4 Forward Primer: G6PD (4)-F Initial denaturation of 94°C for 3 5' ACCAGGGTGGTCCTGGAG 3' minutes; followed by 32 cycles of Reverse Primer: G6PD (4)-R denaturation at 94°C for 30 seconds, 5' ACCAGCTCTCTCAGGGTGTG 3' annealing at 64°C for 45 seconds, and extension at 72°C for 60 seconds, and a final extension at 72°C for 7 minutes and then hold step at 4°C

G6PD locus 5 Forward Primer: G6PD (5)-F Initial denaturation of 94°C for 3 5' ACGGCAACAGATACAAGGT 3' minutes; followed by 32 cycles of Reverse Primer: G6PD (5)-R denaturation at 94°C for 30 seconds, 5' TGAGGTAGCTCCACCCTCAC 3' annealing at 62.6°C for 45 seconds, and extension at 72°C for 60 seconds, and a final extension at 72°C for 7 minutes and then hold step at 4°C

Notes: Primer sets 2 and 5 are the final optimised primer sets for G6PD locus 2.2 and 5.1, respectively. 64 Chapter 4: Application of the designed molecular assay to samples from Southern Thailand

Chapter 4: Application of the designed molecular assay to samples from Southern Thailand

65 Chapter 4: Application of the designed molecular assay to samples from Southern Thailand

4.1 Plasmodium vivax isolates from Southern Thailand

This study used 15 whole blood samples (dried blood spots) positive for Plasmodium vivax obtained from the Faculty of Microbiology, Prince of Songkhla University, Thailand, with ethics approval obtained from the ethical review committee at Prince of Songkla

University, Thailand (Reference no. HSc-HREC-61-002-02-1). To protect patient privacy, unique sample names (listed in Appendix VI) were assigned to tubes. When not in use, samples were stored at -20°C.

4.2 Results of the application of the newly designed PCR protocol to samples from Southern Thailand

All DNA samples from Southern Thailand were subjected to the PCR protocol with their optimised conditions and the gel electrophoresis protocol detailed in Section 2.2.4.1,

Table 21, and, Section 2.2.5, respectively. Primer pairs for G6PD locus 1, 2, and 3 successfully amplified their respective products in most samples (where sufficient DNA was obtained through the extraction method described in Section 2.2.1.2), as per the gel photographs in

Figures 23, 24, and 25. Unlike primer pairs for G6PD locus 1, 2, and 3, the G6PD 4 and 5 locus primer pairs did not successfully amplify most of their respective products resulting in faint amplification of most samples Figures 26 and 27. As there was insufficient extracted genomic DNA or blood samples from which the DNA could have been re-extracted, the PCR’s could not be repeated. As such, the products were sent for bidirectional Sanger sequencing in their current state. 66 Chapter 4: Application of the designed molecular assay to samples from Southern Thailand

Figure 23. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 1. PCR products were generated by subjecting DNA samples extracted from whole human blood to the PCR protocol with their optimised conditions and gel electrophoresis protocol described in Section 2.2.4.1, Table 21 and Section 2.2.5, respectively, alongside a negative control (NC). The 1kb DNA ladder (1kb STD) was run on each gel for molecular size estimations. Successful amplification of the target region can be seen as single bands at the amplicons' expected size (approximately 340 bp).

Figure 24. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 2. PCR products were generated by subjecting DNA samples extracted from whole human blood to the PCR protocol with their optimised conditions and gel electrophoresis protocol described in Section 2.2.4.1, Table 21 and Section 2.2.5, respectively, alongside a negative control (NC). The 1kb DNA ladder (1kb STD) was run on each gel for molecular size estimations. Successful amplification of the target region can be seen as single bands at the amplicons' expected size (approximately 780 bp).

67 Chapter 4: Application of the designed molecular assay to samples from Southern Thailand

Figure 25. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 3. PCR products were generated by subjecting DNA samples extracted from whole human blood to the PCR protocol with their optimised conditions and gel electrophoresis protocol described in Section 2.2.4.1, Table 21 and Section 2.2.5, respectively, alongside a negative control (NC). The 1kb DNA ladder (1kb STD) was run on each gel for molecular size estimations. Successful amplification of the target region can be seen as single bands at the amplicons' expected size (approximately 450 bp).

Figure 26. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 4. PCR products were generated by subjecting DNA samples extracted from whole human blood to the PCR protocol with their optimised conditions and gel electrophoresis protocol described in Section 2.2.4.1, Table 21 and Section 2.2.5, respectively, alongside a negative control (NC). The 1kb DNA ladder (1kb STD) was run on each gel for molecular size estimations. Successful amplification of the target region can be seen as single bands at the amplicons' expected size (approximately 480 bp).

68 Chapter 4: Application of the designed molecular assay to samples from Southern Thailand

Figure 27. Gel image of DNA electrophoresis of DNA from Southern Thailand amplified using G6PD locus 5. PCR products were generated by subjecting DNA samples extracted from whole human blood to the PCR protocol with their optimised conditions and gel electrophoresis protocol described in Section 2.2.4.1, Table 21 and Section 2.2.5, respectively, alongside a negative control (NC). The 1kb DNA ladder (1kb STD) was run on each gel for molecular size estimations. Successful amplification of the target region can be seen as single bands at the amplicons' expected size (approximately 650 bp).

Following bidirectional Sanger sequencing 11 samples amplified and produced high- quality sequence data for all 5 G6PD loci in the location of the G6PD SNPs of interest, with all bases within the target SNPs having a quality value >40 (i.e., error probability less than one in 10000) as assessed by Sequencher version 5.3. The successful sequences generated were aligned to the reference G6PD gene (GenBank sequence accession number NG_009015) and the consensus sequences of all possible mutations containing the SNPs of interest using MEGA ver. 10 (Appendix VII). No mutations/SNPs within the G6PD gene were found (Figure 28). 69 Chapter 4: Application of the designed molecular assay to samples from Southern Thailand

Primer < G6PD1 >< G6PD2 >< G6PD3 >< G6PD4 >< G6PD5 > bp position 6512 17303 17312 18078 18084 18154 18183 19453 19529 19604 20183 20304 20316 Consensus A T G G A C C G G C C G G Mutation G C T A G T T A A T T T A T1 A T G G A C C G G C C G G T2 A T G G A C C G G C - - - T3 A T G G A C C ------T4 A T G - - - - G G C - - - T5 A T G G A C C G G C C G G T6 A T G G A C C G G C C G G T7 A T G G A C C G G C C G G T8 A T G G A C C G G C C G G T9 A T G G A C C G G C C G G T10 A T G G A C C G G C C G G T11 A T G G A C C G G C C G G T12 A T G G A C C G G C C G G T13 A T G G A C C G G C - - - T14 A T G G A C C G G C C G G T15 A T G G A C C G G C C G G

Notes: The base pair positions above correlate with the G6PD SNPs detailed in Table 21 and Appendix V and VII. Base pairs of samples with poor quality sequencing results are highlighted in grey.

Figure 28. Alignment of sequenced DNA samples from Southern Thailand to G6PD gene (NG_009015) and the consensus sequences of all possible mutations of interest in Asia

Table 22. Common G6PD SNP mutations found in Asia. Base pair positions and their respective nucleotide change

SNP name bp position Nucleotide change Gaohe 6512 A>G Vanua lava 17303 T>C Quing yuan 17312 G>T Mahidol 18078 G>A Taiwan-hakka 2 18084 A>G Mediterranean 18154 C>T Coimbra 18183 C>T Viangchan 19453 G>A Kerala-kalyan 19529 G>A Mahidol-like 19604 C>T Union 20183 C>T Canton 20304 G>T Kaiping 20316 G>A

4.3 Summary

The optimised PCR protocols for the five designed loci specific primer sets were applied for all 15 DNA samples from Southern Thailand. G6PD loci’s 1, 2 and 3 amplified most of their respective products successfully. While the G6PD 4 and 5 loci’s did amplify for most of their respective products, they produced faint bands, which is likely attributed to 70 Chapter 4: Application of the designed molecular assay to samples from Southern Thailand

genomic DNA being extracted from DBS as opposed to whole blood, resulting in lower DNA concentrations. Genomic DNA samples were also exposed to mechanical shearing through pipetting and vortexing; the combination of these practices likely resulted in DNA degradation

(see Section 6.4). Following bidirectional Sanger sequencing 11 out of 15 samples produced viable sequence data for all 5 G6PD loci in the location of the G6PD SNPs of interest. It should be noted that the majority of samples (5 out of 6) which did not produce high-quality sequence data were those amplified using the G6PD locus 4 and 5 primer sets. Following examination of the G6PD SNPs of interest from the sequence data no mutations were found. 71 Chapter 5: Application of the designed molecular assay to samples from Australian travellers.

Chapter 5: Application of the new molecular assay to samples from Australian travellers

72 Chapter 5: Application of the designed molecular assay to samples from Australian travellers.

5.2 Blood samples from Australian travellers infected with Plasmodium falciparum

This study used 10 clinical blood samples from patients positive for Plasmodium falciparum obtained from the Institute of Clinical Pathology and Medical Research (ICPMR) at Westmead

Hospital in NSW, with ethics approval obtained from the human ethics committee of Western

Sydney Local Health District (WSLHD) (HREC reference No. LNR/18/WMEAD/139). These clinical samples were used for an earlier malaria study in 2018. To protect patient privacy, unique sample names (listed in Appendix VIII) were assigned to tubes. When not in use, samples were stored at -20°C.

5.2 Results obtained from the application of the new PCR protocol to samples from Australian travellers

All DNA samples from Australian travellers were subjected to the PCR protocol with their optimised conditions and the gel electrophoresis protocol detailed in Section 2.2.4.1,

Table 21, and Section 2.2.5, respectively. The optimised protocols for all G6PD primer pairs detailed in Table 21 successfully amplified their respective products in 100% of samples per the gel photographs in Figure 29, 30, 31, 32 and 33.

73 Chapter 5: Application of the designed molecular assay to samples from Australian travellers.

Figure 29. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 1. PCR products were generated by subjecting DNA samples extracted from whole human blood to the PCR protocol with their optimised conditions and gel electrophoresis protocol described in Section 2.2.4.1, Table 21 and Section 2.2.5, respectively, alongside a negative control (NC). The 1kb DNA ladder (1kb STD) was run on each gel for molecular size estimations. Successful amplification of the target region can be seen as single bands at the amplicons' expected size (approximately 340 bp).

Figure 30. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 2. PCR products were generated by subjecting DNA samples extracted from whole human blood to the PCR protocol with their optimised conditions and gel electrophoresis protocol described in Section 2.2.4.1, Table 21 and Section 2.2.5, respectively, alongside a negative control (NC). The 1kb DNA ladder (1kb STD) was run on each gel for molecular size estimations. Successful amplification of the target region can be seen as single bands at the amplicons' expected size (approximately 780 bp).

74 Chapter 5: Application of the designed molecular assay to samples from Australian travellers.

Figure 31. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 3. PCR products were generated by subjecting DNA samples extracted from whole human blood to the PCR protocol with their optimised conditions and gel electrophoresis protocol described in Section 2.2.4.1, Table 21 and Section 2.2.5, respectively, alongside a negative control (NC). The 1kb DNA ladder (1kb STD) was run on each gel for molecular size estimations. Successful amplification of the target region can be seen as single bands at the amplicons' expected size (approximately 450 bp).

Figure 32. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 4. PCR products were generated by subjecting DNA samples extracted from whole human blood to the PCR protocol with their optimised conditions and gel electrophoresis protocol described in Section 2.2.4.1, Table 21 and Section 2.2.5, respectively, alongside a negative control (NC). The 1kb DNA ladder (1kb STD) was run on each gel for molecular size estimations. Successful amplification of the target region can be seen as single bands at the amplicons' expected size (approximately 480 bp).

75 Chapter 5: Application of the designed molecular assay to samples from Australian travellers.

Figure 33. Gel image of DNA electrophoresis of DNA from Australian travellers amplified using G6PD locus 5. PCR products were generated by subjecting DNA samples extracted from whole human blood to the PCR protocol with their optimised conditions and gel electrophoresis protocol described in Section 2.2.4.1, Table 21 and Section 2.2.5, respectively, alongside a negative control (NC). The 1kb DNA ladder (1kb STD) was run on each gel for molecular size estimations. Successful amplification of the target region can be seen as single bands at the amplicons' expected size (approximately 650 bp).

Following bidirectional Sanger sequencing, all 5 loci specific primer pairs for the 10 samples produced high-quality sequence data in the location of the G6PD SNPs of interest, with all bases within the target SNPs having a quality value >40 (i.e., error probability less than one in

10000) as assessed by Sequencher version 5.3. The successful sequences generated were aligned to the referenceG6PD gene (GenBank sequence accession number NG_009015) and the consensus sequences of all possible mutations containing the SNPs of interest using MEGA ver. 10 (Appendix VII). No mutations/SNPs within the G6PD gene were found (Figure 34).

76 Chapter 5: Application of the designed molecular assay to samples from Australian travellers.

Primer < G6PD1 >< G6PD2 >< G6PD3 >< G6PD4 >< G6PD5 > bp position 6512 17303 17312 18078 18084 18154 18183 19453 19529 19604 20183 20304 20316 Consensus A T G G A C C G G C C G G Mutation G C T A G T T A A T T T A W1 A T G G A C C G G C C G G W2 A T G G A C C G G C C G G W3 A T G G A C C G G G C G G W4 A T G G A C C G G C C G G W5 A T G G A C C G G C C G G W6 A T G G A C C G G C C G G W7 A T G G A C C G G C C G G W8 A T G G A C C G G C C G G W9 A T G G A C C G G C C G G W10 A T G G A C C G G C C G G

The base pair positions listed above correlate with the G6PD SNPs detailed in Table 21 and Appendix V and VII

Figure 34. Alignment of sequenced DNA samples from Australian travellers to the G6PD gene (NG_009015) and the consensus sequences of all possible mutations of interest.

5.3 Summary

The optimised PCR protocols for the five designed loci specific primer sets were applied to all

10 DNA samples from Australian travellers. The 5 G6PD loci’s were successfully amplified in 100% of the samples. Following bidirectional Sanger sequencing 10 out of 10 samples produced acceptable sequence data for all 5 G6PD loci in the location of the G6PD SNPs of interest. 77 Chapter 6: Discussion

Chapter 6: Discussion

78 Chapter 6: Discussion

6.1 Study importance

In order for Thailand to achieve safe and effective malaria elimination (zero indigenous case status) by 2024, the antimalarial's PQ or TQ are mandated, particularly for P. vivax which has now become the most prevalent malaria parasite for most of the country (61). In areas with high prevalence of G6PDd, and G6PD testing isn't readily available, the adverse effects of these 8AQ's (PQ and TQ) in G6PDd individuals poses a hindrance to reaching malaria elimination status in Thailand (49). To evaluate the risk of adverse effects in the population, a standardised, simple, and accurate method for identifying G6PDd genotypes within a population is required. Such an assay could be used in population surveillance studies to establish the genetic frequency and severity of G6PD genotypes present in Thailand and South

East Asia. This would support policy makers in determining whether wide-spread administration of PQ and TQ is safe for the population or whether routine screening for G6PDd is required prior to 8AQ therapy (56). PCR followed by sequencing is a well-documented and feasible option (49). The herein newly developed PCR assay can be standardised in various testing laboratories to survey populations for G6PD genotypes and provide a more informative molecular method than current phenotypic methods used to detect G6PDd in Thailand (61).

6.2 Aims

Firstly, this project aimed to develop a PCR assay for SNP detection covering the most common G6PD mutations present in Asia, and secondly to apply the newly designed molecular assay to pilot studies to screen for genetic mutations in G6PD genotypes from Southern

Thailand, and Australian travellers returning from Africa. Genomic DNA was extracted from blood samples from patients infected with malaria. These samples were previously obtained 79 Chapter 6: Discussion

from the Prince of Songkla University, Thailand, and the parasitology laboratory at ICPMR at

Westmead Hospital in NSW.

6.3 Results

The PCR protocols for all five designed loci specific primer sets were optimised, facilitating the amplification of the five G6PD gene regions inclusive of all commonly reported mutations in Asia (see Figures 23-27 and 29-33).

Fifteen (from Southern Thailand) and 10 (from Australian travellers) genomic DNA samples (2012-2018) pre-extracted from DBS and whole human blood, respectively, were examined in this study. Epidemiologically important patient information was recorded, enabling a basic profile of patients to be established (see Appendix VI and VIII). Despite non-ideal genomic DNA sample qualities, PCR products of all targeted loci were successfully generated from most Southern Thailand samples and all Australian traveller's samples. All successful PCR products gave reliable sequence data.

The G6PD genotype investigation in Southern Thailand samples found no SNPs in the known mutation hotspots (see Table 7) of the G6PD gene for all tested samples following bidirectional Sanger sequencing. In the 10 Australian travellers, SNPs in the G6PD gene were also not detected.

6.4 DNA extraction

Human genomic DNA in this study was initially used in previous investigations. These samples were pre-extracted using the QIAGEN blood extraction kit from 15 dried blood spots

(DBS) on filter paper from patients infected with P. vivax from Southern Thailand. The same extraction kit was used on whole blood samples from the 10 Australian travellers infected with 80 Chapter 6: Discussion

P. falciparum. The genomic DNA samples, all of which were used in previous studies, would have been kept at 4°C for short-term storage during these earlier studies and then stored at -

20°C for longer term storage. During the earlier study period, the DNA extracts may have been exposed to mechanical shearing (e.g., vigorous pipetting and vortexing) and fluctuations in temperature as well as prolonged exposure to ambient temperatures, resulting in lower DNA quality as found in some of our samples and subsequently poor DNA amplification results (88).

Genomic DNA samples from Southern Thailand were pre-extracted from blood eluted in DBS. The literature has suggested that using this technique may result in low sample volume and therefore low concentration of genetic material (89). This was particularly evident when measuring DNA concentrations from Southern Thailand samples, which was possibly the reason for the faint band amplification, and poor sequencing result for some samples facilitated by loci's G6PD 1, 3, 4 and 5 (see Figure 23, 25, 26, 27, 28 and Appendix VII).

In contrast, the five loci successfully amplified and led to high sequence data quality for all regions of interest in samples from Australian travellers (see Figure 29, 30, 31, 32, 33,

34 and Appendix VII). All these samples had high DNA concentrations due to being extracted from whole human blood.

While additional template DNA from Southern Thailand was inaccessible, repeating this protocol with genomic DNA samples extracted from fresh whole blood would likely result in successful amplification of all five targeted loci in preparation for genotyping using this protocol.

6.5 PCR optimisation

This project's primers were designed to amplify regions of interest containing all commonly reported Asian G6PD mutations as identified within the G6PD reference sequence 81 Chapter 6: Discussion

(Appendix V) using genomic DNA samples. As this protocol has not yet been established and validated for G6PD genotyping, PCR optimisation was necessary to assure assay specificity and efficiency. In some cases, redesign of primer pairs was required because non-specific bands were produced. Altering the cycle number and annealing temperature, particularly the latter, had the most pronounced effect on generating the desired target sequence. Employment of gradient PCR in increments of 1°C was effective at excluding non-specific products in this study. Resulting protocols capable of generating clear banding via gel electrophoresis of specific products across eleven of the fifteen samples from Southern Thailand and all ten samples from Australian travellers were articulated (described in Chapter 3).

6.6 Sequence analysis

Sanger chain-termination sequencing has been recognised as an appropriate method for detecting G6PD mutations (90). Despite newer technologies, such as Next Generation

Sequencing (NGS), being used for whole-genome sequencing of the G6PD gene (91). Single nucleotide polymorphism investigations and smaller-scale projects primarily utilise the Sanger sequencing method (92).

A limitation to the Sanger method when genetic regions are sequenced this way is the potential of low-grade base reads towards the start and end of the sequence (within the first 10-

20bp). To prevent this, documented G6PD mutations prevalent in Asia were identified on the

G6PD reference sequence (GenBank sequence accession number NG_009015) and primer locations were chosen, assuring a buffer zone was present before and after the region of interest.

The Sequencher version 5.3 algorithm was used to score the likelihood of an inaccurate base call, ensuring high quality and reliable forward and reverse reads to be combined to form a 82 Chapter 6: Discussion

contig for each sample. Thus, SNPs found from the G6PD reference sequence post alignment using MEGA ver. 10 can be determined as true with a high confidence level.

6.7 Pilot molecular surveillance results of G6PD genotypes from

Southern Thailand and Australian travellers

This newly designed and optimised PCR assay protocol produced successful amplification and sequencing data in regions of interest for all five loci when applied to DNA samples from Southern Thailand and Australian travellers. No SNP mutations were observed in either sample group. This is contrary to previous studies on G6PDd conducted within

Thailand and specifically Southern Thailand using the fluorescent spot test, which showed a prevalence of approximately 14-17% of this region's population (93, 94). Additional studies on

G6PD SNPs within Southern Thailand have concluded that the G6PD mutations Viangchan and Mahidol are the most common variants making up around 74-86% of the G6PDd population (95, 64). These genotypes are regarded as class 3 (Table 5) and are at potential risk of developing acute haemolytic anaemia when exposed to either PQ or TQ due to reduced activity levels of the G6PD enzyme (10-60%) (58). However, it is considered an acceptable risk to administer normal therapeutic doses of PQ and TQ (Table 4) to individuals with a G6PD enzyme activity at levels of over 30% (96). As class 3 variants encompass a range of 10-60%

G6PD enzyme activity, phenotypic testing methods, such as the spectrophotometric assay and fluorescent spot test, become necessary as they provide a precise measure of G6PD enzyme activity (64). Once G6PD activity is known, then prescribing either PQ or TQ to prevent P. vivax relapses in G6PDd patients can be done at lower dose levels to minimise the risk of induced haemolytic anaemia (96). Alternatively, when identifying individuals with class 3 83 Chapter 6: Discussion

G6PD variants in the absence of phenotypic methods, PQ and TQ may be administered to individuals under close medical supervision to monitor potential side effects (96).

While the investigation conducted was a pilot study with a small sample size, it was used as a proof of concept and showed that the specific loci could be targeted, amplified and sequenced for the detection of gene mutations. With this new assay, expansion of the sample size would create statistically more significant surveillance information on SNP mutations found in malaria endemic populations.

G6PD genotyping has already been successfully used within small- and large-scale studies within Thailand and Southern Thailand populations (93, 94, 95 and 64). However,

G6PD genotyping was previously conducted with optimised PCR protocols based on complementary DNA (cDNA) G6PD reference sequences. Presumably, this was done to avoid large-sized introns between exons so that primer pairs can amplify multiple large exons (4-5) at one time to screen for many G6PD SNP mutations that are solely found in exon (90).

Nonetheless, the advantage of using a genomic DNA reference sequence for loci design rather than cDNA is that several polymorphic sites in introns have been identified. While previous studies suggest that only G6PD SNP mutations located within exon regions lead to clinical symptoms, it has now been suggested that G6PD mutations within non-coding regions can also manifest clinical features of G6PDd, including jaundice and anaemia (90, 51). In addition, cDNA cannot be directly extracted from blood. RNA must first be extracted, then using the enzyme reverse transcriptase, cDNA is synthesised for PCR, which is laborious, costly, and time-consuming. Considering this molecular assay was designed for surveillance studies, the ability to directly extract genomic DNA from samples and produce PCR products with good sequence outcomes allows for simple and accurate surveillance of G6PD genotypes, 84 Chapter 6: Discussion

particularly when applied to samples from SEA, as the majority of mutant genotypes of G6PD are most frequently reported in this region.

Although this molecular method is appropriate for population studies, it is time- consuming and expensive compared to phenotypic methods, which are the primary diagnostic techniques used to determine G6PD status in Southern Thailand. However, the detailed genotyping of G6PD gene targets in a population will provide information for logistical and financial decisions to be made when planning elimination programs for areas such as Southern

Thailand. The widespread use of phenotypic testing methods, such as the CareStartTM G6PD rapid diagnostic test, may be a necessary part of the malaria elimination program if populations show genetic predisposition to having severe adverse reactions to PQ usage.

Also, as PCR-sequencing based SNP analysis is considered the gold standard for G6PD population studies (49), this method would be suitable as a universal and standardised method for uniform testing when applied to national studies in all countries planning on eliminating P. vivax, that are also co-endemic for G6PDd, including Thailand and Cambodia. Adopting additional genotypic screening (e.g., PCR and sequencing) in conjunction with phenotypic point of care assays (e.g., CareStartTM G6PD rapid diagnostic test) will help achieve accelerated elimination of P. vivax in endemic areas such as Southern Thailand.

6.8 Direction for future research

The current study provides a basic platform for surveillance of the genetic frequency and severity of known G6PD genotypes present in Asia by enabling a protocol for multiplex

PCR-target next-generation sequencing to be developed. Developing this protocol will enable

DNA sequencing libraries to be pooled and allow for the use of several primers (such as those designed herein) in an optimised PCR protocol that will enable specific amplification of 85 Chapter 6: Discussion

targeted DNA regions. Finally, next-generation long-read sequencing technologies, such as the nanopore MinION, can enable simultaneous sequencing of multiple targeted regions to identify the presence of SNPs (97). Though it should be noted that next-generation sequencing technologies face issues with SNP detection algorithms, most notably base pair inaccuracies which may hinder the integrity of results. Therefore, technological advancement and optimisation of next-generation technologies are required before application to G6PD mutation detection studies (97).

Future application of this molecular assay would preferentially require samples extracted from whole blood as opposed to DBS. As indicated within the study, amplification and sequencing results of DNA samples extracted from whole blood are much more optimal than those extracted from DBS. Furthermore, the limited sample size does not allow for this generalisation; instead, an independent investigation should be conducted to determine whether

DBS is suitable for G6PD genotyping. Nonetheless, future investigations using this protocol would benefit from researchers extracting DNA directly from blood samples rather than utilising pre-extracted DNA to avoid the possibility of mechanical shearing of DNA, which may lead to poor amplification results.

6.9 Concluding remarks

This study had two primary research outcomes: 1) A PCR assay covering all commonly reported Asian G6PD SNP mutations found in SEA was successfully developed. 2) Successful application of this assay into pilot studies to screen for genetic mutations in G6PD genotypes from Southern Thailand and Australian travellers returning from Africa. These outcomes satisfy the project aims and substantiate the hypotheses that developing a PCR assay for the 86 Chapter 6: Discussion

detection of G6PD mutations will enable monitoring of the frequency and allelic variant types of the G6PD gene present in Southern Thailand and travellers returning to Australia.

Thailand plans to revise or introduce new guidelines for the effective and safe elimination of P. vivax using primaquine and tafenoquine to eradicate malaria from its territory by 2024. Thailand would require a universal method for standardised testing of G6PDd surveillance at the national scale to guide policymakers for logistical and financial decisions when planning malaria elimination programmes in all countries co-endemic for G6PDd that plan on eliminating P. vivax, including Thailand and Cambodia. A molecular PCR assay, such as that detailed within this study, would meet that criterion. However, further testing would be required on larger sample sizes to validate this molecular assay. Nevertheless, the method described is suitable for population studies but not for routine diagnostics as it is time- consuming and expensive.

Finally, guidelines for the treatment of imported cases of P. vivax for individuals who are co-diagnosed with G6PDd returning to Australia is lacking. More epidemiological information is required and application of the PCR assay described in this project to detect

G6PD genotypes will help determine levels of mutant genes found in travellers to malaria endemic areas. Furthermore, Australian travellers may not need to know the specific mutation of the G6PD gene whilst living in a non-endemic country, but future clinical situations may arise where knowledge of their G6PDd status is vital so as to avoid certain food or drug types e.g. fava beans and some antibiotics, which are contraindicated in these individuals.. 87 References

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Appendices

103 Appendices

Appendix I. Reagent list and chemical manufacturers

Product Manufacturer (Location)

10 x NH4 PCR Buffer Bioline Australia Pty. Ltd., Alexandria, NSW, Australia

1kb Plus DNA ladder (Cat. No.10787-018) Invitrogen Australia Pty. Ltd., Mulgrave, VIC, Australia

Agarose, Molecular Grade (Cat. No.BIO-41025) Bioline Australia Pty. Ltd., Alexandria, NSW, Australia

Deoxynucleotides (dNTP) (Cat. No.BIO-39025) Bioline Australia Pty. Ltd., Alexandria, NSW, Australia

Ethidium Bromide (EtBr) Solution 10 mg/ml (Cat. Bio-Rad Laboratories Pty., Ltd., Gladesville, No. 161-0433) NSW, Australia

Milli-Q® ultrapure water Sigma – Aldrich Pty. Ltd., Castle Hill, NSW, Australia

Oligonucleotide primers Sigma – Aldrich Pty. Ltd., Castle Hill, NSW, Australia

TBE Buffer (Tris/borate/EDTA) Bioline Australia Pty. Ltd., Alexandria, NSW, Australia

104 Appendices

Appendix II. List of equipment and materials used and their product manufacturers

Product Manufacturer (Location)

Automatic pipettes (P10, P20, P200, P1000) Pipetman, Gilson Medical Electronics, Viliers- le-Bel, France

Electrophoresis Chamber Bio-Rad Laboratories Pty., Ltd., Gladesville, NSW, Australia

PCR Thermal cycler (LabCycler D-37085) SensoQuest, Göttingen, Germany

Spectrophotometer (DeNovix DS-11+) DeNovix Inc., Wilmington, DE, USA

UV Transilluminator TM40E (λ=302nm) UniEquip, Martinreid, Munich, Germany

Vortex mixer Cole-Parmer, Vernon Hills, Illinois, USA

0.2ml PCR tubes Integrated Sciences Pty. Ltd., Willoughby, NSW, Australia

Pipette tips (P10, P20, P200, P1000) Interpath Services Pty. Ltd., Heidelberg West, VIC, Australia

1.5ml Microcentrifuge tubes Eppendorf-Netherler-Hinz, GmbH, Hamburg, Germany

Vacufuge concentrator (5301) Eppendorf-Netherler-Hinz, GmbH, Hamburg,

Germany

105 Appendices

Appendix III. List of commercial kits and manufacturers

Commercial Kit Manufacturer (Location)

Wizard® SV Gel and PCR Clean-Up System (Cat. No. A9281) Sigma-Aldrich (MO, USA)

BIOTAQTM PCR Kit (Cat. No. BIO-21040) Bioline Australia Pty. Ltd., Alexandria, NSW, Australia

106 Appendices

Appendix IV. List of bioinformatics programs and databases used

Program/Database Website/Software

MEGA ver. 10 http://www.megasoftware.net

NCBI http://www.ncbi.nlm.nih.gov

Primer-Blast https://www.ncbi.nlm.nih.gov/tools/primer-blast/

Sequencher 5.3 http://www.genecodes.com Chapter 1: Introduction

Appendix V. Reference G6PD GenBank sequence with attached designed primers for all 5 loci and Asian reported G6PD mutations

>NG_009015 | Homo sapiens glucose-6-phosphate dehydrogenase | Chromosome X | 23182bp

G6PD reference sequence with attached primers and all reported G6PD SNP mutations found in Asia including: G6PD reference sequence aligned to sequences of Southern Thailand and Australian Traveller samples and all sequences of reported G6PD SNP mutations found in Asia including: Gaohe (98), Vanua lava (99), Quing yuan (100), Mahidol (100), Taiwan-Hakka 2 (101), Mediterranean (102), Coimbra (103), Viangchan (104), Kerala kelyan (105), Mahidol-like (100), Union (106), Canton (107) and Kaiping (108).

1 tgccctgtcc caggatggac agcacttcat gagtgctcag tgcagggaca cagaggcctg 61 gacatgttgg tccaacctgg aaggttcatt ccagctccag agctccctgt aagatccagc 121 aaggctgtcc tggagcctac ccggcagcgt gcttctccct ctgccctccc tggcttcttc 181 ccctgtccct cctttccctt cccagtactg gtcctgagag catgctaaac ttcctccccg 241 ctaatctgca tctcagagtc tacttcctga aaccaagagg gagggcagca catccccacc 301 ccaggcccag gaaacgcaga ggtgcaggtg gggagaagac gccttcccct cagcagggct 361 ggatcccctg actctttcct cacctcggag ctcttgattc tcctccaggc agcgctggag 421 ggtctcagga gcgccctgtt ctgaaggcag gtgcagcatg gctggcttcc ccagaggaga 481 ctcttcgccc agtacgtcct gatctgctgc cgggccacca ctgggctgca ccatctcaca 541 cagttggctc ttccagaggt gcctattcat ccaacagggc aagggctgaa agagaaaggc 601 agcaggggag tcacaggaga cccacatcct tacccagcag aaaaagccta tgagttctgg 661 ggctacattt taaacagaat gctgataatt cacctagtgt atcaggatgc taaagtgggt 721 tttcagtcta aggggtagca gatactccca ttccaaaggg aggtgctgga ccacccatgt 781 gggacttgaa aaggcctagc tccaggctgg aaatagaaga atttcaccag agatgggaga 841 gtcgggacaa gactgtttgt ggagggagat gtcagtatgc tgggaatcct gatcccctac 901 acacctccaa gcaggagaag tggagggtac cggcccctca tccagtgtag tgggctgaat 961 ggtagccccc cgaaaagata tgcccatgtt ctaacccctg aaacatatgt gattcgggaa 1021 aagggtcttt gcagatgtag gatctcaaga tgagatcatt ctgggtgacg tgggtgggcc 1081 ctaaatccaa caacgtgtct ttatgagaga cagaagagag aagccagagg gagacgcaag 1141 gggaggaggc catgtcggga ctgcctcgga gactggagtg aggcagccac aagcccagga 1201 ataaatgggg cagaagcttg ggagaggcaa gggaggattg ccctcttaga gcctttgagg 1261 gtgtgtgctt gctaaccctt tggttttgga cttctagtct ccagaactgt gagattaaaa 1321 tgtctggccg ggcgcagtgg ctcacacctg taatcccagc actttgggag gccgaggcgg 1381 gccaatcatg aggtcaggag atcgagacca gcctggctaa cacggtgaaa ccctgcctct 1441 actaaaaaaa aaaaaaaaaa ttgccggtca cggtggctca cgcctgtaat cccagcactt 1501 tgggaggccg acacgggtgg atcatgaggt caggagattg agaccatcct ggctaacaca 1561 gtgaaaccct gtctctacta aaaatacaaa aaattagcca ggcatggtag cgggcgcctg 108 Appendices

1621 tagccccagc tacccaggag gctgaggcag gagaatggcg tgaacccggg aggcaggaga 1681 atggcgtgaa cccgggatcg cgccactgca ctccagcctg ggcaacagag cgagactcca 1741 tctcaaaaaa aaaaaataca aaaaattagc agagtgtggt gacacgcgcc tgtagtccca 1801 gctactcggg aggccgaagc aagagaatcg cttgaaccca ggaggcggac gttgcagtga 1861 gctgagattg tgccactgca ctccaccctt ggctttcaga acagcccctt ctcctggttt 1921 cctccctatc ttgctggcta ctcctgccca ggcactctca ctgggttccc atccttcatg 1981 ttgggctgcc ccagggctct gtcctaggac atcttcttct ctgtaacttc attcactccc 2041 acaggaattc tgtccagtct catgccttga actccaatct tgatgcttaa ttagacctct 2101 tttctaaact ccacattcat ttatcaaact cgacatttct acttggttat ctagtagcct 2161 tctcaaaacg aacctctcta agacctccga cagtccccaa aacccagatc tacccaaggc 2221 cttctttgtt atcagctgat ggcgaatcca tcctccagct gtcaaaagtc ttttttgatt 2281 ctctctctct ctctcacaca cacacacaca catacacaca cacacacaca cacacacaag 2341 agcacatcta atccatcaag aaatcctgtt aaaatgtatc ccccaaccga tctgatcaca 2401 ctgagcaatt aacattaact attttaggtg tcataatgat attgtggttg tttttcagag 2461 tcctgtctta gagagaggca cacttaaatg tttaaagatg atgtctgaat tcacctccaa 2521 ataatgggaa aactcctagg tatataccgg agagaaatga aaacacatgt gtccacatga 2581 atgcttgtag cagcattact catagcagcc agagagtgga aacaacccaa atgtccatca 2641 actatgaata aacaaaatct agtacatcca catgctataa catgaacaaa tcttgaaaat 2701 atgctgagaa ataaacaagt cacaaaagat taaacattgt atgagtctat ttatataaga 2761 tgtctacaac aggcaaatcc acagagacat aaagtggatt aaagattgct ggggagtgcc 2821 acttagggct gaggggaaga taagctgagc taaagggtat ggggtttctt tgaggggtga 2881 cgaaaatgtc ctgtgaatat acatacaaca aaccattgaa ttgtacactt taaatggatg 2941 aactgtgtga catgctaatg atatctcaag ctgttattga aaataatagt ggtggccagg 3001 catggtggtt cacacctgta atcccagcac tttgggaggt catggcaagt ggatcacttg 3061 aggtcaggaa ttcaagacca gcctggccaa catgacaaaa ccctgtattt accaaaaata 3121 caaaaaaaaa aaaaatagct gggagtgatg gtgcatgcct gtaatcccag ctatttggga 3181 agctgaggca ggagaatcgc tcaaacctgg gagatggagg ttgcggtgag ccaagatcgt 3241 gccactgcac tccattctgg gcgacagagt gagactgggt ctcaaaaaaa gaggccgggc 3301 gcggtggctc atgcctgtaa tcccagcact ttgggaggcc gaggcgggcg gatgacaagg 3361 tcaagagatc aagaccatcc tggccaacat gatgaaaccc catctctact aaaaatacaa 3421 aaattagctg ggcgtggtgg tgcacaactg tagtcccagc ggctgaggca gaagaatcgc 3481 ttgaatccag gaggcggaag ttgcagtggg tggagatcac accactgcac tccagcctgg 3541 tgacagagcg agattctgtc tcaaaaaaaa aaaaaaaaga aagaaagaaa gaaaaagaaa 3601 taaaataata atgggagagg attgcgaggg atataaacaa aattagtcag aagttgatac 3661 cttcaattgt tgaagttggt agctgtgaat gtaagattta ctatactggc cgagcgcagt 3721 ggctcacacc tgtaatccca gcactttggg agtctgaggc aggcagatca cctgaggtca 3781 ggagttcaag accagactgg ccaacatata gtgaaacccc gtctctacta aaaatacaaa 109 Appendices

3841 aattagctgg gcgtagtggc gcacgcctgc agtcccagct acctgggaag ctgaggcagg 3901 agaatcgctt gaacccagga ggaggaggtt agagtgagct gagatcatgc cactgcactc 3961 cagcctgggc gacagagcga gactccatct ctcaaaaaca aacaaaccaa aaagatttac 4021 tatactcatt tttctacttt cgtacatttt tgaaatttcc aataataaaa tgctcaaagc 4081 actgtctctg agctggatta ctgcaattac ctcttaccac gccacccttc ttttaccctt 4141 gcccactatg gtctgttctc cccaccactg ctagaacgat cagtgtaaga cgtaattccg 4201 atcacattcc tcctctactg agaaccctac catggatccc cgttgccttc tgcgtaaaag 4261 ccaatctccc tacaacggcc tacaaggccc tgtaccatcg ggttccccat cccttcctct 4321 ccaactgtgc tcccctccct cactctgctc cagctatagt gacctccgtg ctattcctct 4381 aatgtgccag gagagcccat tcattctcag atcacagaaa cagtatgacg ataggcagat 4441 gttgtcttgc aaaacgagca aacaggcata tgaaatcaga tacctagctg ccaaactata 4501 tgtgaggcat ttccgaagaa tttaacgacc tcgatagagc gcagtcaagt ttggtgaaca 4561 gaatatgtct ctgaactaga ggagtcctca cacaaggagt agggtcagac cccgcagtgg 4621 aggaggaggg aggagtagaa acagtccagc tcgccgccca agtaacctgg gtcctgaatc 4681 ggcccgcctt ggccagtgct ccagaagcgc ggagcaggaa cgggctgggg cccaaaaaag Exon 1 4741 aggggggagc ctgaacgtcc gggggaagtt tcggaggcgg cggaacgccc acggatggaa 4801 ccctgtcttt ggggaaaagg accacacctg tcagcagagt ccgtcagacg tgagaagggt 4861 gggagcggcg gactgtgaac gctggtaggg ccccggcgct ccgagaaagt cccagtttcg 4921 cggtcgccct tccctaccac gcttccggct tccggtgtca tagctgtggg atccggaagt 4981 aaaaacacaa gccccgcccc cgagaactcg ggaagccggc gagaagtgtg aggccgcggt 5041 agggccgcat cccgctccgg agagaagtct gagtccgcca ggctctgcag gcccgcggaa 5101 gctcggtaat gataagcacg ccggccactt tgcagggcgt caccgcctac acgccccctc 5161 gtctctcgga cggcggcgtc tagcctcggg gcgctcggcc gccccgccct ctccggggga 5221 ggaatcaaga agagactgcc caatagggcc ggcttgaccc gcgaacaggc gagggttccc 5281 gggggagtgg cgcggcagaa ggccccgccc aggagccgag ggacagccca gaggaggcgt 5341 ggccacgctg ccggcggaag tggagccctc cgcgagcgcg cgaggccgcc ggggcaggcg 5401 gggaaaccgg acagtagggg cggggccggg ccggcgatgg ggatgcggga gcactacgcg 5461 gagctgcacc cgtgcccgcc ggaattgggg atgcagagca gcggcagcgg gtatggcagg 5521 cagccggcgg gccggcctcc agcgcaggtg cccgagaggc aggggctggc ctgggatgcg Exon 2 5581 cgcgcacctg ccctcgcccc gccccgcccg cacgaggggt ggtggccgag gccccgcccc 5641 gcacgcctcg cctgaggcgg gtccgctcag cccaggcgcc cgcccccgcc cccgccgatt 5701 aaatgggccg gcggggctca gcccccggaa acggtcgtac acttcggggc tgcgagcgcg 5761 gagggcgacg acgacgaagc gcaggtaacc ggccgggcgg gcgccgcgca ggcggaggag 5821 cgtactgtcc cgcgctgcgc cgcgcggcgg taaaatacac gctgtttgtt gtgcttgaga 5881 accgagcaga atcgagaggg tcttaaccaa tccctttata ccccgcacct cctctcttga 5941 gcccctgaga ccccgagagc gaaggggact tgccgaccgg ggtcacccag cttggcaagg 6001 ggagggctgg agctgaactc cagcatctgc accatctccc atgctccagg tcattgtgga 110 Appendices

6061 gttcccgcta cagtcgggaa tgagatggtc ctgggcacgc agttccatgc cccacaagga 6121 ttttactcgg ttgtccagaa ttgatgctgt agtcggaata caccaatgct ttgagtaatt 6181 ttgtaatgta caccctgaat gaaggctgcc taggagagag tggctggagc ccagagccag 6241 cagtttctaa cccatcaacc actccccaat gcccagccgt tcacaaggag tgatttgggc

G6PD (1) Mutation Location of Nucleotide SNP/s (bp) change G6PD Gaohe Exon 3: 6512 a>g

Primer set Forward/reverse

G6PD (1) Forward

6301 aatcaggtgt caccctggtg tgagacccca gaggaactct caagaaaggg gctaacttct 6361 caatgctctc ctgttcttct gccttgttaa cgagcctttc ttccaccaga cagcgtcatg Exon 3 6421 gcagagcagg tggccctgag ccggacccag gtgtgcggga tcctgcggga agagcttttc 6481 cagggcgatg ccttccatca gtcggataca cacatattca tcatcatggg tgcatcggtg 6541 agtatctccc aggccccaat cttaaaagcc aggaagtgcc tgctccatgc ctcagctttt

Primer set Forward/reverse

G6PD (1) Reverse

6601 ccaactaatt gttgcagggc cccacaggct ctgctaagtt gagctcctcc tcccgctcct 6661 gctagtgcgc caggatcatc ccggctctac ctggtgtgca tatgcacttg caagactttt 6721 tttttttttt ttttttggag acggagtttt gctctggtcg cccaggatgg ggtgcagtgg 6781 cgccatcttg gctcactgca acctcggcct cctgggttca agcgattctc ctgcctcagc 6841 cccccaagta gctgagatta caggcatgca ccaccacacc cggctaattt ttgtattgtt 6901 agtagagacg gagtttcacc atgttgacca ggctggtctc aaactcctga cctcaagtga 6961 tccgcccgcc tcggcctccc aaagtgctgg gatgacaggc atgagccacc acgcccggct 7021 gcaggacgtt tctgaaccct gtgcctagcc gctgaatgta gcccttaatc ctggagttgc 7081 aggctcttta gggactgtct agatggactt cttggggaaa acagggaatg ctattcattc 7141 tgtagttttt tcagacctgt tcatgcctta acaaatgagt tccaaaggca ggatctgagg 7201 cctgatctct gcctcttcac taacacttca agttccgtcg atacaaacac ttagcttttc 7261 tttgaacact gtctctttgc ctcaatttcg aaagcctacc aaggagctct gcctcacccc 7321 acctggcccc aattgtccag cttgtagaac cgccattctg cactcatagg aaagacaaaa 111 Appendices

7381 gaatagtata gcatgaggat ttccatggga ggggagggag gggtggtcct aggacagcca 7441 tcgcccgaga ctagagcacc ctcccctgtg tcccatgggg cagtgagact gtgcaggggc 7501 tggtggaaca agggcttcat ctcaaattac acgtggtctc agttttcact tttaaccctg 7561 gatgcactca agaatctctc tgctcattac tgtatgacca tctttgccat taacttacct 7621 tgtaaccttg agcaggagct ctatcaaaac tagttagttc ttccacatcg tgcgcccctc 7681 agtgctggct gagctcagtg gttctttgtt cacatagaac aacgcgctgt cacactttgt 7741 caaacctact ccttgacacc tttgtacttc tgatcagaac ttagaaaagg agagccaaaa 7801 gggaatgagg aggcagccga gggagaaggg ctctgccagt ggcagagtaa ggaggggcca 7861 ttgtcctctg actccccata agcctgagac ccaggagagg taccaggtgg agcccacagt 7921 agattgggct tggcctcagc cccagcccca gcctggtccc ctcagaagag ccatacctgg 7981 ccgggcacgg tggctcacgc ctgtattccc agcactttgg gaggccgagg caggtggatc 8041 gcctgaggtc aggagtttga caccagcctg gccaacatgg cgaaacccag tctctactta 8101 aaatacaaaa attggctggg cgcagtggct cacatctgta atcccagcac tttggggggc 8161 caaggtgggc agatcacaag gtcaagagat cgagaccatc ctggccaaca tggtgaaacc 8221 ccatctctac taaaaataca aaaattagct gggcgtggtg gtgcgtgcct gtagtcccag 8281 ctactcagta ggctgaggca gtagaatcgc ttgaatcagg gagtcagagg ttgcagtgag 8341 ctgagatcgc gccactgcac tccagcctgg cgacagagcg agactctgtc tcaaaacaaa 8401 acaaaacaaa aaggccgggc gcagtggctc acgtctgtaa tcccagcact ttgggaggct 8461 gaagcgggtg gatcatgagg tcaggagatt gagaccatcc tggctaacat ggtgaaatcc 8521 cgtctctact aaataaaaaa ttagccaggc ctggtgtggg cacctgtagt ccagctactc 8581 aggaggctga ggcaggagaa aggcgtgaac tcgggaggcg gagcttgcag tgagcagaaa 8641 tcgcaacact gcattccagc ctgggtgaca gagtgagact ccatctcaaa aaaaaaaaaa 8701 aaataacaaa aattagttag gtgtagtggc acccatctgt aatcccagct acttgggagg 8761 ctgaggcagg aaaatcgctt gaacccagga ggtggaggtt gcagtgagct gagatggcac 8821 cattgcactc catcctgggt gacggagtga gtgagactcc atctcaaaaa aataaaaata 8881 aaaagagcca tacctgcatc agccacaaac cttaacagcc aggactctga ccgttaaacc 8941 cttagtccaa gaagcagact ggaggagaag gtaactgagg ttgctttctc catccccacc 9001 ccatgctgtc acaaggcact gcaacagagc aacccaggcc acaatgatag cttatgactc 9061 ctggccttca gctcttatta gacacctcat tgagagcatc caagcttgct gtgtgagttg 9121 gagcaagtgt cttccttttc ctgaacctca gcatttcgtg tagcaaatga cagcttttga 9181 aacgagggcc cagggtcctt aggcttttcc aagctgattt tgtcatttca ctgcctccgc 9241 ctgtggccag cttcgggttg ggcaactttg ttttgttgaa atcagtaggg ttcccctttg 9301 ctctcctctg ttctgagagt gacaacctga ggctttcaaa gcagaaatag gaagtgccag 9361 ctccagcact gtgggtaggg gacaaggggt cctctctctg aagagcctcg tggtttgttt 9421 tttcctcttc ctgctgtggg ggagatggga ggtggcgtgc tcaggcagca cctggaacag 9481 ctccggctcc caggcttctt ccacgcctga tgcctcactc ggtgcgggag gtaggcaggg 9541 tggcacttga gggcacgtga tcctagaatc tgctgttcca ggaaagcagc ccattctccg 112 Appendices

9601 ttcattgagc agacagcgat tgggtacttc tgggccacag cagccagtcc ctgccctcct 9661 catgggacct atgtgctatt gcggccaggg aaccacggca agtaagcaga ccagcaagag 9721 ttcaggctga cgtgtgtttc agaagataca catgggccct gtgtccccag tcccccaaag 9781 cgcatagatc ccacaaagag ggccgggaga gcacaaacac agaaggaaga gcaaatgccc 9841 ggagccccga gttgggaaca agctcagcat gacctagaaa ctgaaagcag gcaagtggct 9901 ggcagaccca cagcctgggc aagcaggaca ccctggggca ggctggaccc acgtggcctt 9961 cctctgtccc ttcctcctcc ctggccagct tacctcacac ttgcccacac acccagcttc 10021 cttcctgggg gccctgatgg cacctcagag ttaccaagtg ggccacttcc tgccttctca 10081 ggactcccac ccctaccaga aagggagccg aggtacttac tgcccccttc cagaaagctc 10141 tggccttgta gactagtgtg cgcctggaac agcatctttc cccacctgat gggacctcga 10201 tgatgctgga gagcttcaca gggagtgagg ggaagagact gggattgata gtctgcgggg 10261 tcctcacttc tctgggcctt agtttcccca cctggatcac aagggccatg ggcttgatgg 10321 gcgttcagga cgcatcccaa tgatgggcct gtgggaggga tgacccagcg gcccccgcct 10381 cccctttcct cagaacattt ctgggctgac ttccccattg gtctgataag attcttgctg 10441 tgtcatgatg atgttggcac tcccccaaag ccccggtgac tcatgagccc ccgggtatag 10501 cagggaggcc cttttgttgg tttttcagct gaactaactc agcagccccc aggttctctg 10561 gccccagtgg ccaggaacca gctgggcccc tgccaaggcc cagggctcca cccagggagt 10621 ctgggcaggt ccttaacccc agtgaccctt ggggatggga tggcccaccc ccgctggccc 10681 tccagcaggc atcgtgctct accatgaaac cctcatgcca agacaagggc tgcagcgatt 10741 tcggtttata aaacagacag gtcatggccc aggtcccttt gccctggcct agccagatgt 10801 tggtggctag agatcttcat catgccctgc ttctactaag tcaccttgtg tccttgggca 10861 gatccctccc ctgctgtggt cctcaggaaa gaggagggcg tggggaagat gagaagctcc 10921 caactgcttt tcactaagga ccccttgaat tgcccccacc cacatggcac gagtagactc 10981 ttgaccaagc tgtcaggttt cccgatgtct tcccatgttt gttcctccag aaagtcccac 11041 tagagcatcg cagccagctg ggagggcagg gctacccctg agtcctgagg agagcagctc 11101 agtccggctc agctcagctt agctccctgt caccatggga gagagcggcc tgccttaccc 11161 cacccctact gtccgtctta cctcacctcg cctctcaggt gacttggagc ccagcagacg 11221 gtcggtcggt tgcagggcct gagctgaaac aaggtgagcc ggttcaatct ctagttaatc 11281 ccaaagggag cagcaggaac aaacggcctg gagcgtgggg ttgctgtgac tcagcagacc 11341 agtcccctgc caccagtacc tttctggagt ctgtcccgat gcctcccagt cttccggaat 11401 ttggtcatga gcaaacatga cctgctttga gtgaggctta gaggtatttg caggctgtgg 11461 ttctttccgt ttatttcagg gtccagttag ttcccagtaa gctgaccctt tctgtgtggg 11521 gccagccgtg agttgatgtg acatgggcaa cgttcccctc cccagcccca ctgatggcag 11581 gtgctgtgag gagctgagta gaggagctcc taccccaggg cgaggggttg tctgctggac 11641 tttgaagggt gcagcagtga gaggtccgtg tcgctgtagt cctggcaccg agctcacctg 11701 aggagcatca gaagtggagc agggcccctg aagagcgcaa gcacacaagg cacccacacc 11761 atagcagttg ccctcctgga agccctgtgg tggcttccta actgcctggc tctgggtgag 113 Appendices

11821 ggcgtttgac ctggtggcat ggcccccatt ctgtaatggt ttgtagaaat ggacccatcc 11881 atggttgcag tgcctgattg cttcgaagag catgtgtttt tttttttttt ttttcgtgac 11941 atggagtctt gctgtcaccc aggctggagt ttggtggcgc aatcttggct cactgcaacc 12001 tccgtctcct ggattcaagt gattctcctg ccttagcctc ctgagtagct gggattccag 12061 gtgcctgcca ccacatccag ctaatttttg ttttttcttt tttttgagat ggagtcttgc 12121 tctttcacca ggctggagta cagtggcgca atcttggctc actgcaatct ccacctgcga 12181 ggttcaagca attcccctgc ctcagcctcc cgagtagctg ggactacagg cgcgcaccac 12241 cacatccagc taattttttg tattttagta gaaacggggt ctcaccatgt tggccaggat 12301 ggtctcaatc tcctgacctc gtgatccacc cacctcaacc tcccaaagtg ctaggattac 12361 aggcatgagc caccgcgcct ggcctgtatt tttagtagag acggggtttc accatgtttg 12421 ccaggctggt ctcgaactcc tgacctcagg tgatccaccc tcctcagcct cccaaagtgc 12481 tggggttaca ggcatgagcc accacgccca gactattagt ttttaccatg ttttattgaa 12541 atatatgtca tgggccggcc acggtggctc acgcctgtaa tgccagcact ttgggaggcc 12601 acggcaggtg gatcacaagg tcaggagttt gaggccagcc tggccaacat ggcgaaaccc 12661 catctctagt aaaaatacaa aaattagccg ggcgtggtgg caggtgcctg gagtcccagc 12721 tactcaggag gctgaggcag gagaatggct tgaacccagg aggtggagat ttcagtgagc 12781 caagatcacg ccactgcact ccagcctggc cgacagagtg agactccgtc tcaaaaaaaa 12841 aaagaaatac atgtcatgaa gaacacagat ggtgcaggtc tagcttgttg atgacttttc 12901 acaaactcag cacagccaca gaaactgctc ctagatcaag aaacagaata tgctctgcac 12961 ccaggaagcc ccctcacctg gggctctcac agtcactggc cctcaaaggg tcatttctga 13021 cccactcccc accctccgct ccattgttgg ttttctcgcc gtcttttgtt ggtgcgcaaa 13081 tcttaactgt acaattggct gtacaccctc atcacaataa aggccatcgt catctcccca 13141 gaaagttcct gggtacccat tcccagccaa tctgccctgc tccaggcaac cgctgatctg 13201 atttctgtca caatgggttg catctctgat gctgagattt cccagtgttg ctgagtagta 13261 tccgttgtat gaatgcacag tttgccgggt cacatgttaa ttcaagtgtt tgacttgttt 13321 ccagtttggg gccgctatga accaaggctg ctgtgaccat tcgtgtgcag gtctttgtgt 13381 ggacttgcac tttcatttct cttggggtgg aacggccagg tcacaggata ggtgtgtgtt 13441 tcacttgatc agagggagca cccgcaccac ctcacagctc aacttcagcc attttcattt 13501 tcttgcctta ctgcactttt cttgttcctg attgcttaaa gggatgatta atatttcagc 13561 atttcctata agtgtgatat ttgtagtatt tgtaaggatg atggtttggg ggtttttttg 13621 ttctttgttt tttggtggtg gttttttttt ttgagacgac gtctagctct gttgctcagg 13681 ctacagtggt gtgatctcgg ctcactccag tctctccctc ctggttcaag cgattctcct 13741 gcctcagtct cctgaatagc tgggattaca ggcatacacc accatgttca gctaagtttt 13801 gtatttttag tagaaatggg gtttcaccat gttggtcagg ctggtctcaa actcctgacc 13861 tcaagcgatt ccccgcctcg gcctcccaaa gtgctgggat tacaggcttg agccaccacg 13921 cccagccaga atggtgtttt atagatgccc tttatcaagt tagtgaagtt cccttgcatt 13981 ccccctctgc taagagtttt agcgtgaata ggtgatttct gaaacacttg atctacatct 114 Appendices

14041 gttgggttga gcaagattgc tagatttaaa aaatcaaaac atagcagtag gcagagagtt 14101 cttagaggtt ttttttaatc tcttaatatg gtgaatttca atgatttttt tctgttaata 14161 tcaaaccaac tttttattta cagatgtatt tattctgtaa ataaaaaacc taccgttttt 14221 aacattgcag gatttggttt gctgatgttt tcttcttttt ttttaaagag acagggtcag 14281 tgggtcgtcg tggctcacgc ctgtaatccc aacactttgg gaggctgagg cgggcagatc 14341 acttgagccc aggagttcaa gagcagcctg gacaacatgg ggaaaccctg tctctacaaa 14401 aaatacaaaa aattagctgg gtgtgttggc atgtacctgt agtcccagct actccagagg 14461 ctgaggtggg aggattgctt gagcccagag gcagaggttg cagtgaactg agatcgtgcc 14521 actgcactcc agcctaggtg acagagtgag accatgtctt taaaaaaaaa aaaaaaaaaa 14581 aacctcctga tttcaagcaa tcttcctccc accttggtct cccaaagtgc tggggtgtgt 14641 gagttgtgcc cggcctgctg atatttcgtg tgggatttct gcatctgcat gtgtgtgtga 14701 atgaggcgta tgggttttcc catgtgcctt tctcatcagg tttccgtacc aaggctctgt 14761 aggccccata aggtggcttg ggaagtgttc ccctttcttc cttctgcagg aagagtttgc 14821 ttcaggctgg aattctcttg tgcttgcatt ttggtagaac ccactggcaa cactctctat 14881 gccagggggt tgtttctgta ggaagatttg tggccactgt cccctctcag gtccattgct 14941 ctgttttttg tttgtttgag acggagtttc gcttttgtag cccaggctgg agtgcagtgg 15001 cttgatcttg gctcactgca acctccgcct cccaggttca agcgattctc ctgcctcagc 15061 ctcctgagta gctgggatta caggcatggg ccaccatgcc cagccaattt tgtattttta 15121 gtagaaacgg tttctccatg ttggtcaggc tggtcttgaa ctcccgaact caggtaatcc 15181 atccgcctcg gccttccaaa gtgctgggat tataggcttg agccaccgcg tccagctgtt 15241 tagttgtttt tttgtttttt gttttttgag acggagtttc actcttgtcg cccaggctgg 15301 ggtgcaatgg tgcaatcttg gctcactgca acctccatct cccgggttca agtgattctc 15361 ctacctcagc cttccgagta gctgggacta cagacacccg ccaccacgcc tggctaattt 15421 tttgtatttt tggtagaggc ggggtttcac cgtgtcagcc aggatggtct cgatctcctg 15481 acctcttgat ccgcctgcct cggcctctca aagtgctggg attacaggag taagccactg 15541 tgcccggcct atgccctttt aaaaaagcaa ccatgccagg gggttgtctg tttctattcg 15601 tctttttcaa acaccaattt ccagctttgt tcatcttttc tttgggttag cttttaattt 15661 tattaatttt acttgtcatt atttcctatt ttcttcctgg taatttggct tgcagttcta 15721 tctttttctc actttttttt tggagatgaa gtctcactct tgtcacccag gctggagtgc 15781 agtggcacga tctcggctca ctgcaacctc tgcctcccgg attcaagtga ttctcctgcc 15841 tcagcctcct gagtaactgg gattacaggt gcctgccacc acacctggct aatttttgta 15901 tttttaatag agatggggtt ttgccacatt ggctggtctt gaactcctga cttcaggtga 15961 tctgcctgcc tcagcctccc aaagtgctgg gattacaggt gtgagccacc gcatctggcc 16021 ctttttctca cttcttaagt ggaatgctta gctccttatt tttgaaccac tcttttttgc 16081 taatgtaagc acttgaggtc acgtctcaca aatgtggcta catggtactt ctgtcgtccc 16141 ttggttctac tgaattgaat tttcaggttt tctcctggaa cgcaggagat gcgtttatgt 16201 cttctgggtc agggatggag ggctggcaca ggcttgaatc tcggggctct tctgtctgta 115 Appendices

16261 tatcaggcaa gacagacatg cttgtggccc agtagtgatc ctgagtagtg cccagatcac 16321 caagggtgga ggatgatgta tgtaggtcgt gtccccagcc acttctaacc acacacctgt 16381 tccctctgcc acagggtgac ctggccaaga agaagatcta ccccaccatc tggtaagtgt Exon 4 16441 gtcccaccac tgcccctgtg acctcccgcc agggacaggc ctggtcctgc cctgcccgca 16501 ctggttacag ctgtgccctg ccctcaggtg gctgttccgg gatggccttc tgcccgaaaa Exon 5 16561 caccttcatc gtgggctatg cccgttcccg cctcacagtg gctgacatcc gcaaacagag 16621 tgagcccttc ttcaaggtgg gtggtgtcag ggcctccccc agcctggttc tgccctctct 16681 accagccccc agcatggcca gcttcgggga cctcccccca tcccatcccg ggatgctctc

G6PD (2.2) Mutation Location Nucleotide of SNP/s change (bp) G6PD Vanua lava Exon 6: t>c 17303 G6PD Quing Yuan Exon 6: g>t 17312

Primer set Forward/reverse G6PD (2.2) Forward

16741 ctcctctcct gccccgcccc gcctgctctc gtacttcctt gagaccccca ttaccagccc 16801 ccgtgaccag gacccacagg tcccctcctg ctgtgctctg ctgcgttttc tccgccaatc 16861 atagttgggt gtcatgattt tggagagaga gctttctcca gtgtatttct cccaggtcaa 16921 aatatcctga aatctggcct ctgtcctaag gcacaggggt cccagcctgg ggcagtgtct 16981 gtgctgcctg ctttggcctc cctccctctg gatgtgcaga gctgctaaga tggggctgaa 17041 cccagtgtgg gacggggaca ctgacttctg agggcaccct ccctggacct ccagggaaga 17101 ccctccactc ccctggggca gaacacacac ggactcaaag agaggggctg acatctgtct 17161 gtgtgtctgt ctgtccgtgt ctcccaggcc accccagagg agaagctcaa gctggaggac Exon 6 17221 ttctttgccc gcaactccta tgtggctggc cagtacgatg atgcagcctc ctaccagcgc 17281 ctcaacagcc acatgaatgc cctccacctg gggtcacagg ccaaccgcct cttctacctg 17341 gccttgcccc cgaccgtcta cgaggccgtc accaagaaca ttcacgagtc ctgcatgagc 17401 cagatgtaag gcttgccgtt gccctccctt cccgcctgcc aggctggccc aggcagtgct 17461 cccaccactc tatgagcgtg tccggggccg gggatctggg cagcatccat ggtgccgggg

116 Appendices

Primer set Forward/reverse G6PD (2.2) Reverse

17521 ccatccccag cgggaccaca aggtggcagc gttgctccac gaaacaccgc ctttccgctc 17581 tgcttcccca aaggcccggc caggccgcag ggtggcagcc ttgctctgcg aatgcagcat 17641 ggcccgcgct gggtggtttc ccaacccagc cagaggctct tgtcctctgg ctggttttga 17701 atgcgggggt agtaaagcaa aggtcctctt ctcattttca aaaccaatga ggaagccatg 17761 gcttggatgc ctcctccccc tgctccccta caggccttca ggccactcag acccaccggg 17821 gacccagcat gaggcaggag gggaacgggc ccccggcagc atgccagcaa tgccaccctg

G6PD (3) Mutation Location of Nucleotide SNP/s (bp) change G6PD Mahidol 18078 g>a

G6PD Taiwan- 18084 a>g Hakka 2 G6PD Exon 7: c>t Mediterranean 18154 G6PD Coimbra Exon 7: c>t 18183

Primer set Forward/reverse

G6PD (3) Forward

17881 gcacccaggg tgggaaggct tcccggaagg tgttgagcca gagggtcatc tgggaacaca 17941 aggcacggga ggtggccacg ggggcgagga ggttctggcc tctactcccc tgggagggcg 18001 tctgaatgat gcagctctga tcctcactcc ccgaagaggg gttcaagggg gtaacgcagc 18061 tccgggctcc cagcagaggc tggaaccgca tcatcgtgga gaagcccttc gggagggacc Exon 7 18121 tgcagagctc tgaccggctg tccaaccaca tctcctccct gttccgtgag gaccagatct 18181 accgcatcga ccactacctg ggcaaggaga tggtgcagaa cctcatggtg ctgaggtggg 18241 gccaagcctg ggccggggga ccagggtggg ggtggtactc aggagcctca cctggcccac Primer set Forward/reverse

G6PD (3) Reverse 117 Appendices

18301 tgcctccccg aggacgaatt cctccagaac tcagacaagg gtgacccctc acatgtggcc 18361 cctgcaccac agaggcccaa ggtcagttcc tccaccttgc ccctccctgc agatttgcca Exon 8 18421 acaggatctt cggccccatc tggaaccggg acaacatcgc ctgcgttatc ctcaccttca 18481 aggagccctt tggcactgag ggtcgcgggg gctatttcga tgaatttggg atcatccggt 18541 gagagctctt cctctctcct gggaggctgg cacagggtgg cagagccagt caccctgcag 18601 ggctactctt ccctatcttg ggggagctcc tcctcaccct gcagttcaaa acctaagtgt 18661 ctgagctatc agaccgggct ggaaagggct ggacccctac acagccaagc accccacggt 18721 tttatgattc agtgatagca tcaccatgtc cttccttgat ttaaggggac ctggaagaca 18781 agggggatca ggaagtgagt cttgcagctt gtcactagga agccttgttt ggggtcccca 18841 tgcccttgaa ccaggtgaac agggcgggga gctaaggcga gctctggcct cttccgtccc 18901 cagggacgtg atgcagaacc acctactgca gatgctgtgt ctggtggcca tggagaagcc Exon 9 18961 cgcctccacc aactcagatg acgtccgtga tgagaaggta gggggtgcac cccagtcccc 19021 aggagcatgc cctgtcgcag gcccatctgt gacgaggcac tgagctgggg tgtgcatgca 19081 gagcaggtgt cctcaacccc ggagaagtca ccacctctga gcacagcgtg gcctcccgga 19141 ggtgacctgg actggcagtc atgaagccca agttgtcatg tcccaggcct gacagtcact 19201 atgtgaccag ggaaggccat tgcctctctg ggcctcagct tgttcatcag aatagactcg

G6PD (4) Location of SNP/s Nucleotide Mutation (bp) change G6PD Exon 10: 19453 g>a Viangchan G6PD Kerala- Exon 10: 19529 g>a kelyan G6PD Mahidol- Exon 10: 19604 c>t like

Primer set Forward/reverse G6PD (4) Forward

19261 agatggacca gggtggtcct ggagggtcct cagggagggg ccctgagctg ggcctctggc 19321 agggtgagca gagccaagca ggggcctcct cctgccctga gggctgcaca tctgtggcca 19381 cagtcatccc tgcaccccaa ctcaacaccc aaggagccca ttctctccct tggctttctc 19441 tcaggtcaag gtgttgaaat gcatctcaga ggtgcaggcc aacaatgtgg tcctgggcca Exon 10 118 Appendices

19501 gtacgtgggg aaccccgatg gagagggcga ggccaccaaa gggtacctgg acgaccccac 19561 ggtgccccgc gggtccacca ccgccacttt tgcagccgtc gtcctctatg tggagaatga 19621 gaggtgggat ggtaggtgat gccttcgagg cccagcaagg cagaactggg catgccctgt

Primer set Forward/reverse Primer set Forward/reverse G6PD (5.1) Forward G6PD (4) Reverse

19681 gtgcgggcac tggagctccc actgagacac tcacgcactg gtccacaccc tgagagagct 19741 ggtgctgagg ctgccctttc cgccacgtag gggtgccctt catcctgcgc tgcggcaagg Exon 11 19801 ccctgaacga gcgcaaggcc gaggtgaggc tgcagttcca tgatgtggcc ggcgacatct 19861 tccaccagca gtgcaagcgc aacgagctgg tgatccgcgt gcagcccaac gaggccgtgt 19921 acaccaagat gatgaccaag aagccgggca tgttcttcaa ccccgaggag tcggagctgg 19981 acctgaccta cggcaacaga tacaaggtgc cctacagaga aggagcagtg tggagggtgg 20041 gcggcctggg cccgggggac tccacatggt ggcaggcagt ggcatcagca agacactctc 20101 tccctcacag aacgtgaagc tccctgacgc ctatgagcgc ctcatcctgg acgtcttctg Exon 12 20161 cgggagccag atgcacttcg tgcgcaggtg aggcccagct gccggcccct gcatacctgt 20221 gggctatggg gtggcctttg ccctccctcc ctgtgtgcca ccggcctccc aagccatacc

G6PD (5.1) Location of Nucleotide Mutation SNP/s (bp) change Exon 12: G6PD Union c>t 20183 Exon 13: G6PD Canton g>t 20304 Exon 13: G6PD Kaiping g>a 20316

Primer set Forward/reverse

G6PD (5.1) Reverse

20281 atgtcccctc agcgacgagc tccgtgaggc ctggcgtatt ttcaccccac tgctgcacca Exon 13 20341 gattgagctg gagaagccca agcccatccc ctatatttat ggcaggtgag gaaagggtgg 20401 gggctgggga cagagcccag cgggcagggg cggggtgagg gtggagctac ctcatgcctc Exon 14 119 Appendices

20461 tcctccaccc gtcactctcc agccgaggcc ccacggaggc agacgagctg atgaagagag 20521 tgggtttcca gtatgagggc acctacaagt gggtgaaccc ccacaagctc tgagccctgg 20581 gcacccacct ccacccccgc cacggccacc ctccttcccg ccgcccgacc ccgagtcggg 20641 aggactccgg gaccattgac ctcagctgca cattcctggc cccgggctct ggccaccctg 20701 gcccgcccct cgctgctgct actacccgag cccagctaca ttcctcagct gccaagcact 20761 cgagaccatc ctggcccctc cagaccctgc ctgagcccag gagctgagtc acctcctcca 20821 ctcactccag cccaacagaa ggaaggagga gggcgcccat tcgtctgtcc cagagcttat 20881 tggccactgg gtctcactcc tgagtggggc cagggtggga gggagggacg agggggagga 20941 aaggggcgag cacccacgtg agagaatctg cctgtggcct tgcccgccag cctcagtgcc 21001 acttgacatt ccttgtcacc agcaacatct cgagccccct ggatgtcccc tgtcccacca 21061 actctgcact ccatggccac cccgtgccac ccgtaggcag cctctctgct ataagaaaag 21121 cagacgcagc agctgggacc cctcccaacc tcaatgccct gccattaaat ccgcaaacag 21181 cccctcctgt ccccttgtca tttgcttccc tgaggaccca cttccttgtc ccactcccca 21241 agtcacccgg gtgcttcctc tgtggccact gaggagcctc ctcacttctg gttctgaccc 21301 catgtccagc catgaccatg tttggtgtca gagacacctc ctgagtcctc ctccgctcca 21361 cgcatattgt cattgtgaga ccagcttgag gtgcaaggat gtggggtcac cttccgctct 21421 tactgtccat gtccctgccc cacccaagat gacttccagc aacactgcca gtccacagaa 21481 gtatttagga aggagagggg atgggttttg tgactgatgt cagaggccaa gggagagggg 21541 acagtccaca gtgatgcaca gccaggccca gccacccctt ccaggcagaa gggccacaca 21601 ctgggcagct ctcaacatag gctgattctc ccagttctgg aggccagagt ccaagatcca 21661 gcactcccgc cagaggccca aggggagggt ccttcctgcc ctctccagct cctggggaac 21721 caggcatctt gggctggtgg tcacctcact ccactcactc tgcctgtctt ctcgtggcct 21781 ccccatggtg tgacttctat gtcctaactc agcgacatct gcagacctcc tatttccaaa 21841 taaggccaca tcaggaactc ccaggagaca tgtatttggg ggccgctgtt cagcccctgc 21901 actgctcccc acacagcccc tgggtgtttc agtgcagcat gtgtcctgcc agggagatgg 21961 cgaaaccaga cactatggtc cctgcacttg tggcccgggt tctaggaggt gaggccggca 22021 gggaacaaaa taaccaggtg gtgctgggat ttgtcctttg cggagagcaa accagtggcc 22081 tggaaagtgg ccagggagga gtgttccacc cgcagcacag gtgaggacgc gcaccctggg 22141 cgctgcaggt agcagggccc gatgtcgtag aatgaaggag gcctctggtg agacctgtgg 22201 caacagggac acatgctgca tcccggtgtt caattcttgc tcttcctgtg ccctgaatgg 22261 ggagtggcac ctcttgagtt ttcagaggga ggaagtatgc caagttcacc tggaccctag 22321 atagggaaag gagcttgatg ggggccaagg acaggtcaca ggctcagtgg gctctgtgtg 22381 cacaggattg ggggccaggc ttcggccaca ggggcagtgg ttttagtcct cttgtcctgg 22441 ttcctgtggc ctccagaagg ggctgtggca aggctgagag tcgggttggg ctgggggctg 22501 atggggtgtg gtaaggctaa gcagggtagg ttccagggca gggaacaaca cccaagtaag 22561 gccaggcgca gtggctcaag cgtgtaatct cagcacttta ggaagccaag atgggcagat 22621 cacttgaggc cagaagttca agaccagcct gaccaacatg acgaaaacat gtccctacca 120 Appendices

22681 aaaaaaatac aaaaatttgc caggcgtggt ggcgggcacc tgtggtccca gctactcagg 22741 aggctgaggc aggacaatcg cttgaacccg ggaggtggaa gaccatccac tggcacctga 22801 acccatcagg caactgtccc ttcagctctg ggcgcctact tgggtggagg caggaggtcc 22861 caggtgagga cagcactggg tccacaatgg gagagaatga gagatcaggt cagggcattc 22921 cactgtcacc tcagttgcgg gtgtccacac tgagacatgg acataagccc gagaaaacaa 22981 cacgagctgt accctgaaca agaaggcagt cctccccagc cccttggagc tcacaggagc 23041 actgaggccc ttgcctgtga gcccagatcc accctggccc attccatagg gtgactcact 23101 cagcctccct gacccctgct gaccggttca aggctggagc cctgttttca ccccttctct 23161 gcctgcaatg ccaaagaatt ca 121 Appendices

Appendix VI. Patients from Southern Thailand information summary

Specimen Nationality Age Sex Location Date of ID (years) collection T1 Thai 30 F Chumphon 2014 T2 Thai 24 F Chumphon 2014 T3 Thai 47 M Chumphon 2014 T4 Thai 47 M Chumphon 2014 T5 Thai Yala 2017 T6 Thai 39 M Yala 2017 T7 Thai 34 M Yala 2017 T8 Thai 14 F Yala 2017 T9 Thai 36 M Yala 2017 T10 Thai 27 F Yala 2017 T11 Thai 20 M Yala 2017 T12 Thai 51 F Yala 2017 T13 Yala 2018 T14 Yala 2018 T15 Yala 2018

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Appendix VII. Reference G6PD GenBank sequence aligned to sequences of Southern Thailand and Australian travellers samples and mutation sequences covering all reported Asian G6PD mutations

>NG_009015 | Homo sapiens glucose-6-phosphate dehydrogenase | Chromosome X | 23182bp G6PD reference sequence aligned to sequences of Southern Thailand and Australian Traveller samples and all sequences of reported G6PD SNP mutations found in Asia including: Gaohe (98), Vanua lava (99), Quing yuan (100), Mahidol (100), Taiwan-Hakka 2 (101), Mediterranean (102), Coimbra (103), Viangchan (104), Kerala kelyan (105), Mahidol-like (100), Union (106), Canton (107) and Kaiping (108).

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Chapter 1: Introduction

Appendix VIII. Patients from Australian travellers information summary

Specimen Nationality Age Sex Date of ID (years) collection W1 Kenya 21 F 2017 W2 Sumatra 49 M 2017 W3 Kenya 32 F 2018 W4 Sierra Leone 32 M 2018 W5 Sudan 17 F 2018 W6 Cambodia 47 F 2018 W7 South Sudan 1 F 2018 W8 Africa 54 M 2012 W9 Africa 48 M 2012 W10 Sudan 41 M 2016 Chapter 1: Introduction