MINISTRY OF EDUCATION
AND TRAINING
VIETNAM ACADEMY
OF SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY
----------------------------
Vu Phuong Nhung
STUDY ON POLYMORPHISMS/MUTATIONS OF CYP2C9,
CYP2C19, CYP3A5 AND CYP2D6 CYTOCHROME P450
IN KINH VIETNAMESE
Major : Biotechnology
Code : 9420201
SUMMARY OF BIOLOGY DOCTORAL THESIS
Hanoi - 2020
The thesis has been completed at: Graduate University of Science and
Technology - Vietnam Academy of Science and Technology.
Supervisor 1: Dr. Nguyen Hai Ha
Supervisor 2: Prof. Dr. Nong Van Hai
Reviewer 1: ………………………
Reviewer 2: ………………………
Reviewer 3: ………………………
The thesis will be defended at the Board of Examiners of Graduate
University of Science and Technology-Vietnam Academy of Science and
Technology at………..on……………….
The thesis can be referred at:
- Library of Graduate University of Science and Technology
- National Library of Vietnam
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INTRODUCTION
1.The necessity of research
Interindividual differences in drug response are unavoidable during
treatments, including adverse drug reactions (ADRs). ADRs are one of the
main cause of hospitalizations, which could be lethal in some cases. Since
2009, the Clinical Pharmacogenetics Implementation Consortium (CPIC)
has been providing information on genetic testing that can be applied in drug
regimen optimization. For some gene-drug pairs, the CPIC has also given
specific dosage instructions. The four genes CYP2C9, CYP2C19, CYP2D6
and CYP3A5 are highly polymorphic CYP450 genes which polymorphism
could lead to ability to metabolize specific drugs very differently in patients.
Up to now, the understanding of genetic diversity of the CYP450 genes
involved in drug metabolism in Vietnam is still limited. Given that
pharmacogenetics plays a significant role in drug metabolism and drug
interactions in the clinic, it is noteworthy to pay more attention in this field.
This study focuses on the detection of known alleles as well as the
identification of novel allele in Kinh Vietnamese. Data obtained from the
study would provide scientific information on the genetic diversity of
CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in Vietnamese. These are also
significant basis which partly contribute to the development of the field of
pharmacogenetics and personalized medicine in the future.
2.Research objective
Establish the genetic variants database of 04 genes CYP2C9,
CYP2C19, CYP2D6 and CYP3A5 in Kinh Vietnamese.
Identification of genotype and allele frequencies of 04 genes CYP2C9,
CYP2C19, CYP2D6 and CYP3A5 in Kinh Vietnamese. Perform in silico
functional analysis of novel variants.
3.Research content
- Samples collection and extract total DNA from peripheral blood of
136 unrelated healthy Vietnamese Kinh.
- Identification of genetic variants in promoter, 9 exons and flanking
regions of 03 genes CYP2C9, CYP2C19 and CYP2D6 by Sanger
sequencing. For CYP3A5, sequencing also used to detect the variants that
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had been reported to influence enzyme activity, including: *3, *6, *8 and
*9.
- Determination of copy number variant (CNV) of 4 genes by
Multiplex Ligation-dependent Probe Amplification (MLPA) method.
- Determination of genotype and allele frequencies of 4 genes in Kinh
Vietnamese.
- Comparison of the allele frequencies and distribution of CYP450
genes found in Vietnam with other population in the world.
- Prediction of in silico function of novel genetic variants identified.
CHAPTER 1. LITERATURE OVERVIEW
1.1. Cytochrome P450 (CYP450) and drug metabolism in liver
The CYP450 enzymes involved in phase I drug metabolism are
proteins expressed mainly in either the mitochondrial inner membrane of
the smooth endoplasmic reticulum hepatocytes. These enzymes were also
found in intestinal epithelium, kidney, brain. In humans, there are 115
CYP450 genes including pseudo genes, named starting with CYP1A1 and
ending with CYP51P3. Proteins encoded by CYP450s are classified based
on the amino acid sequence homology: family (CYP1, CYP2…), subfamily
(CYP1A, CYP2B…) and individual enzymes (CYP1A1, CYP2D6…). Of
the CYP450s expressed in the liver, CYP3A4 had the strongest expression
level (22.1%), followed by other 2 enzymes CYP2E1 (15.3%) and CYP2C9
(14.6%) in total proteins in the liver. The CYP450 enzymes are involved in
catalysis for variety of oxidation, oxidation of sulpho (sulfur), aromatic
cyclic (nucleus) hydroxylation, cyclic compounds hydroxy, reduction
(separation) of N-alkyl groups, reduction of (separating) the O-alkyl
(alkoxy) group.
1.2. Genetic polymorphism of genes encoding for CYP450 enzyme
involved in drug metabolism
The different genetic polymorphisms of the CYP450 genes are
determined in terms of function and metabolic phenotype: Extensive
metabolizer-IM, Intermediate Metabolizer-IM, Poor Metabolizer-PM and
Ultra rapid Metabolizer-UM. The CYP450 involved in the metabolism of
various drugs on the market today are CYP2C9, CYP2C19, CYP2D6 and
CYP3A4/5. The five CYP450 genes encoding for above enzymes involved
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in the metabolism of approximately 60-80% of commonly prescribed drugs.
Among the proteins in the 18 CYP450 families, the ones with the highest
polymorphism are CYP2A6, CYP2B6, CYP2C9, CYP2D6 and CYP2C19.
In addition, there are some CYP450 proteins belonging to other subfamilies
with lower polymorphism such as CYP1A1, CYP1A2, and CYP2E1.
Concerning to drug metabolism function, CYP3A4/5 is involved in the
metabolism of many drugs on the market, following by the enzymes
CYP2D6, CYP2C9, CYP2C19 and CYP2E.
1.3. Effect of pharmacogenes variations on ADRs and variable drug
responses
An ADR is a response to drugs in the form of toxic and unwanted
reactions, even though the drug is used in normal doses for prophylaxis,
therapeutic, diagnostic purposes as well as modifications of physiological
functions (according to the World Health Organization).
There have been numerous reports of the effect of variants of the
CYP450 genes coding for phase I drug metabolizing enzymes on differences
in individual drug response. There is some evidence that the CYP2B6
genotypes that responsible for PM phenotypes (homozygous or double
heterozygous for 2 alleles *6 and/or *8) are associated with a decrease in
the amount of efavirenz metabolized, and an increase the risk of this drug
poisoning. For phenytoin, CYP2C9 is responsible for the metabolism of
90% of this drug, with the allele CYP2C9*2 and *3 decreasing the enzyme
activity in the phenytoin metabolism. In addition, alleles CYP2C9*4 and *
6 had been detected in patients with adverse reactions to phenytoin.
Polymorphisms of CYP2D6 have also been shown to affect drug metabolism
used in cancer treatment, cardiovascular disorders, psychosis, and analgesia.
In addition to phase I and II drug metabolizing enzymes, polymorphisms of
the genes coding for drug transporters also contribute to the formation of
ADR.
1.3. Applications and perspectives of pharmacogenetics in clinicals
In the future, further research on pharmacogenetics will require to
strengthen collaboration between research groups from different countries.
Pharmacogenetic research requires the collection and analysis of samples to
generate data in order to support the drug development process. In clinical