A PRELIMINARY STUDY ON CYTOCHROME P450

2B6 SINGLE NUCLEOTIDE POLYMORPHISM

AMONG FULANI ETHNIC GROUP OF
NORTHWEST NIGERIA-IMPLICATION FOR
ARTEMISININ METABOLISM
M.Sc. Research Proposal
BY
Yasin Abdullahi Gada
(20210708002)
Department of Pharmacology and Therapeutics
Faculty of Basic Clinical Sciences
College Of Health Sciences
Usmanu Danfodiyo University, Sokoto.
December, 2022. 1
 SUPERVISORY TEAM
DR U.M TUKUR- Major Supervisor
PROF. AMINU CHIKA- Co-Supervisor I
DR MURTALA B ABUBAKAR- Co-Supervisor II

2
 OUTLINE
• INTRODUCTION
• STATEMENT OF RESEARCH PROBLEM
• JUSTIFICATION FOR THE RESEARCH
• HYPOTHESIS
• AIM
• OBJECTIVES
• MATERIALS AND METHODOLOGY
• EXPECTED OUTCOME
• TIME FRAME
• BUDGET
• REFERENCES 3
 Introduction
• Malaria is still a major public health concern and endemic in
Nigeria.

• In 2019, WHO established that, 227 million cases and 409,000

malaria deaths occurred globally .

• Africa accounted for 94% (213 million cases) of the cases and
94% (386,000 deaths) of the deaths.

• Although malaria is preventable and treatable, Nigeria

accounted for 27% (61.8 million cases) of the global burden
and 23% (94, 070 deaths) of global malaria deaths in 2019.

( Amede et al.,2022)
 Introduction Contd.
• Nigeria, is among the six countries that accounted for
51% of all malaria cases globally in 2019.

• Pregnant women , children under the age of 5 and

population with low immunity are the most vulnerable
populations in Nigeria.

• Malaria is responsible for 60% of all outpatient visits and

30% of all admissions in Nigeria. It is responsible for
about 11% of maternal fatalities and 30% of child
mortality among children under the age of five.
( Amede et al.,2022)
 Introduction Contd.
• Prevention and treatment of malaria are mainly
based on the use of drugs.

• Treatment response in Plasmodium falciparum

malaria depends on various factors such as
parasite resistance, host natural immunity, drug
quality and the pharmacokinetics of the
administered drug.
(Obua et al., 2008)
6
 Introduction Contd.
• Although malaria is preventable and easily treated, it still
remains one of the leading causes of morbidity and mortality
in Nigeria.

• Chloroquine (CQ), which has been used in Africa since the

1940s as the drug of choice in the treatment of uncomplicated
malaria, has become ineffective because of the development
of widespread resistance of the Plasmodium falciparum
parasite.

• In 2001 WHO approved the use of Artemisinin Combination

Therapy(ACT) in areas with proven Chloroquine,
Amodiaquine, Sulfadoxine-pyrimethamine resistance.

( WHO, 2005) 7
 Introduction Contd.
CYTOCHROME P450 2B6

• It belongs to a set of important hepatic drug-metabolizing

enzymes (Turpeinem and Zanger.,2012)

• It makes up roughly 3–6% of total hepatic CYP content and

metabolizes several pharmaceuticals including bupropion,
efavirenz, cyclophosphamide, pethidine, ketamine and
propofol . (Turpeinem and Zanger.,2012).

• It is also involve in Metabolism of Antimalarial drug

Artemisinin. (Zanger et al., 2007).
8
 Introduction Contd.
• In addition to drugs, CYP2B6 is able to both detoxify and
bioactivate a number of procarcinogens and
environmental agents including pesticides and
herbicides.
• There is an extensive interindividual variability in the
expression of CYP 2B6, which is in part explained by
extensive genetic polymorphism.

• CYP2B6 is one of the most polymorphic CYP genes in

humans with over 100 described Single Nuclueotide
Polymorphisms(SNPs), numerous complex haplotypes
and distinct ethnic and racial frequencies.
(Turpeinem and Zanger.,2012)
9
 Introduction Contd.
• Various studies have been carried out on molecular
mechanisms for resistance in malaria chemotherapy.
• However limited efforts have been made to determine
genetic polymorphisms in cytochrome 2B6 enzymes.

• CYP 2B6 polymorphisms may also cause toxicity (in poor

metabolizers) due to high drug concentrations and
treatment failure (in extensive metabolizers) as a result of
sub therapeutic drug concentrations.

• Recent studies have also associated drug resistant

selection with poor metabolizer (PM) phenotype.

(Marwa et al., 2014)

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 Statement of the Problem
• Nigeria has the highest burden of malaria
worldwide and contributes 31.3% of the total
global burden. ( WHO, 2022)

• The increasing use of Artemisinin-based Combination

Therapy as an effective treatment of resistant malaria
demands determination of genetic polymorphisms in
the metabolism of these drugs and its influence on
pharmacokinetic profiles between individuals, adverse
drug reactions and clinical outcome.
(Marwa et al.,2014)
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 Justification
• CYP2B6 enzyme plays an important role in the
biotransformation of several therapeutic drugs in
humans, including Antiretrovirals, Antidepresants,
Cytotoxics, Narcotics, Anaesthetics and Anti-malarial
drug Artemisinin. (Ward et al., 2003).

• A study conducted in Nigeria has demonstrated Single

Nucleotide Polymorphism for genes encoding for CYP 2B6
enzyme among Yoruba, Igbo and Hausa ethnic group
(Benjamin et al.,2011)

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 Justification Contd.
• Fulani constitutes a major ethnic group not only in
Nigeria but the whole of West Africa where malaria
is endemic and poses major health challenge.

• So knowledge gained from this study would be

important to the patients, healthcare providers and
policy makers.

• Currently, to the best of our knowledge, there is no

such study conducted among Fulani Ethnic group in
Northwestern Nigeria, hence this study.
 HYPOTHESIS
• Null: There is no difference in genotype and
allelic frequencies of CYP 2B6 among Fulani
ethnic group.

• Alternative: There is difference in genotype

and allelic frequency of CYP 2B6 among Fulani
Ethnic group.

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 AIM OF THE STUDY
• To determine the CYP 2B6 Single
Nucleotide Polymorphism among Fulani
ethnic group in Northwest Nigeria.

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 OBJECTIVES OF THE STUDY
• To determine the socio-demographic
characteristics of study population.

• To determine genotype of CYP 2B6 among

Fulani ethnic group.

• To determine the allelic frequencies of CYP

2B6 among Fulani ethnic group.

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 MATERIALS AND METHOD
• Consumables: EDTA containers, disposable
hand gloves, syringes,cotton wool, spirit and
stationery.
• DNA Extraction kits, PCR kit.
 MATERIALS AND METHOD
Study Population
Volunteers from Fulani extractions will be
recruited from Gidan Hillani and Dabagin Ardo
Dange Shuni in Sokoto state Northwest Nigeria
through their informed consent.

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 Study Site
• Blood sample collection and labeling will be done in
the Laboratory of Department of Pharmacology &
Therapeutics of College of Health Sciences.

• Storage, DNA extractions, PCR and Gel electrophoresis

will be done in Centre for Advanced Medical Research
& Training (CAMRET) Usmanu Danfodiyo University.

• PCR samples will be send to Inqaba Biotech for

Sequencing.

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 Inclusion and Exclusion Criteria
Inclusion Criteria
• The participant must be confirmed Fulani Ethnic
group.
• Healthy individuals confirmed by physical examination
and vital signs.
• Must be tested negative for HBsAg and Anti-HCV and
RVS
• Packed Cell Volume of >30%.
Exclusion Criteria
• Individuals less than 18 years and more than 70 years.
• Pregnant women
• Breast Feeding mothers
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 MATERIALS AND METHOD CONTD.
• Sample Size: The sample size of 40(20 men and 20
females )participants was based on similar studies.
• Ethical Consideration
• The study shall be carried out according to the
Declaration of Helsinki.
• All participants must sign infromed consent form
before participating in the study procedures.
• The study protocol, study site, informed consent
form, and recruiting materials will be presented for
approval to the Ethics Committee of Sokoto State
Ministry of Health.

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 MATERIALS AND METHOD Contd.
• A trained personel will collect 2mls of whole
blood from each participant through veno-
puncture at cubital vein using a syringe into a
labeled EDTA vacuum container.
• After the collection, participants will be
discharged and blood samples will be stored
at -20℃ till DNA Extraction.

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 MATERIALS AND METHOD Contd.
DNA Extraction
• The DNA extraction should be done using the
QIAamp DNA blood mini kit.
The frozen samples will be equilibrated to room
temperature.

20µl of Proteinase K will be aliquot into the

bottom of each 1.5ml microcentrifuge tubes.
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200µl will be added to blood sample and vortex for
15 seconds.

200µl of lysis buffer (AL buffer) will be added and

vortexed for another 15 seconds.

mixture will then be Incubated at 56 degree

Celsius

Centrifuge Briefly

200µl ethanol (96-100%) will be added to the

mixture and pulse vortex for 15seconds 24
Mixture will be loaded to QIAamp spin column in
a 2ml collection tube and centrifuge at
8000rpm for minute

Then add 500µl of buffer (AW2) and spin at

14,000rpm for 3 minutes

Add 200µl of buffer (AE) and incubate at room

temperature for 5 minutes

Centrifuge at 8000rpm for 1 minute

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 MATERIALS AND METHOD Contd.
• Polymerase Chain Reaction
PCR conditions be set up according to
manufacturer’s recommendation followed by
optimization to select the optimum conditions
using ' forward 5 -CCAGTCGAGTCTACATTGTCA-3'
and reverse 5'- TTCATTCTGTCTTCTAACTGG-3'
primers.

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 MATERIALS AND METHOD Contd.
• PCR Conditions

Cycle Time Operation Temperature(

Celcious)
1 20-40 seconds Denaturation 95
30 30-60 seconds Annealing 55
1 minute Elongation 72
5 minutes Final Extension 72

PCR product will then divided into two aliquots. One for gel
electrophoresis, the other for sequencing.
 DATA ANALYSIS
• Data will be analyzed using SPSS version 20
• Participants characteristics will be described in
frequencies and proportions for categorical variables,
while numeric variables will be described by median
or mean depending on the distribution.
• The frequency distribution of the alleles and
observed frequency of the genotypes will be
tabulated.
• Bioedit software for analysis of Sequencing result.
• The genotype frequency distribution in the studied
population will be compared with the Hardy–
Weinberg equilibrium based on the Chi‑square (χ2 )
test of observed versus expected. 28
 WORKFLOW AND TIMELINE
METHODS TIMELINE
1. Purchase of consumables and 4 weeks
reagents
2. Recruitment of participants and 4 weeks
sensitization.
Serology Tests, Retroviral screening
and Packed Cell Volume(PCV)
3. Blood sample collection, storage,DNA 6 weeks
extraction, PCR, Genotyping Gel
Electrophoresis and Sequencing

4. Dissertation Writing 12 weeks

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 BUDGET
S/ Item Cost(Naira) Subtotal
no.
1. Consumables( EDTA containers, 45,000 45000
disposable hand gloves,
syringes,cotton wool, spirit and
stationery

2. Lab Investigation( HBsAg, Anti 3000 for 120,000

HCV, RVS and PCV each
participant
x40
3. QIAamp DNA Blood KIT 350,000 350,000
4. Genotyping 60,000 X4 240,000
5. Sequencing for 40 participants 6000x 40 480,000
( Forward and Reverse)

6. Logistic fee for participant 500 x 40 20000

7. Miscelleneous 50,000 50,000

TOTAL: 1,305,000 Naira
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 REFERENCES
• Amede, P.O., Umeokonkwo, C.D., Abege, S., Akawe, J., Derek, J., Adedire, E.
and Balogun, M.S., 2022. Evaluation of malaria surveillance system in Benue
State, Nigeria. Malaria Journal, 21(1), pp.1-14.
• Obua, C., Hellgren, U., Ntale, M., Gustafsson, L.L., Ogwal‐Okeng, J.W., Gordi,
T. and Jerling, M., 2008. Population pharmacokinetics of chloroquine and
sulfadoxine and treatment response in children with malaria: suggestions
for an improved dose regimen. British journal of clinical
pharmacology, 65(4), pp.493-501.
• Turpeinen, M. and Zanger, U.M., 2012. Cytochrome P450 2B6: function,
genetics, and clinical relevance. Drug metabolism and drug
interactions, 27(4), pp.185-197.

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 References Contd.
• Marwa, K.J., Schmidt, T., Sjögren, M., Minzi, O., Kamugisha, E. and Swedberg,
G., 2014. Cytochrome P450 single nucleotide polymorphisms in an indigenous
Tanzanian population: a concern about the metabolism of artemisinin-based
combinations. Malaria Journal, 13(1), pp.1-7.
• White, N.J., 2008. Qinghaosu (artemisinin): the price of
success. Science, 320(5874), pp.330-334.
• Ismail, S. and Essawi, M., 2012. Genetic polymorphism studies in
humans. Middle East Journal of Medical Genetics, 1(2), pp.57-63.
• Abraham, B.K. and Adithan, C., 2001. Genetic polymorphism of CYP2D6. Indian
journal of pharmacology, 33(3), pp.147-169.
• Mpye, K.L., Matimba, A., Dzobo, K., Chirikure, S., Wonkam, A. and Dandara, C.,
2017. Disease burden and the role of pharmacogenomics in African
populations. Global health, epidemiology and genomics, 2.
• World Health Organization, 2006. WHO briefing on Malaria Treatment
Guidelines and artemisinin monotherapies. Geneva: WHO, pp.1-28.

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 Reference Contd.
• Benjamin, U.E., Oluseye, O.B. and Collen, M.M., 2011. Allele and genotype
frequencies of cytochrome P450 2B6 and 2C19 genetic polymorphisms in
the Nigerian populations: Possible implication on anti-retroviral and anti-
malarial therapy. International Journal of Medicine and Medical
Sciences, 3(6), pp.193-200.
• Ward BA, Gorski JC, Jones DR, Hall SD, Flockhart DA, Desta Z (2003). The
cytochrome P450 2B6 (CYP2B6) is the main catalyst of efavirenz primary
and secondary metabolism: implication for HIV/AIDS therapy and utility of
efavirenz as a substrate marker of CYP2B6 catalytic activity. J. Pharmacol.
Exp. Ther., 306: 287-300.

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