Summary of Biology doctoral thesis: Study on polymorphisms mutations of CYP2C29, CYP2C19, CYP3A5 và CYP2D6 cytochrome P450 in Kinh vietnamese

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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.

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MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING 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
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 1
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 2
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 3
trials, inclusion of pharmacogenetics data can be used to predict individual drug response such as: good drug response, risk of ADR, non-response (failure to achieve efficacy of treatment outcome). One of the challenges facing the pharmacogenetics filed is the need to develop high-throughput methods that can functionally analyze the large number of unknown functional variations. This can be done using next generation sequencing approach in large sample size. In recent years, several Genome wide associated studies focused on drug response have been performed. 1.4. Methodologies applied in study of CYP450 genetic variations Molecular biology methods have been widely applied in the study of genetic diversity of CYP450 genes such as Sanger sequencing, PCR- Restriction fragment length polymorphism (PCR-RFLP), and SNP array for detection of single nucleotide polymorphisms (SNPs) and insertion/deletions (indels). Methods commonly used to study CYP450 CNV include of real-time PCR, multiplex ligation-dependent probe amplification (MLPA), Comparative Genomic Hybridization (CGH), digital PCR. 1.5. Study on genetic variation of CYP450 in Vietnamese and research scope For the CYP450 gene that encodes enzymes involved in human drug metabolism, the research team from Hanoi Medical University and Central K Hospital focused on the common polymorphism of CYP2D6 (CYP2D6*10). In 2018, the research group of the School of Medicine and Pharmacy, Hanoi National University initially studied the relationship between polymorphism of CYP2C19 and platelet aggregation in patients with acute coronary syndrome in Vietnam. Up to now, only 6 international publications revealed the frequencies of known allele that responsible for PM, IM and UM phenotypes of CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A5 genes. However, there is no information about all variants in CYP2C9, CYP2C19, CYP2D6 and CYP3A5 genes in Vietnamese. The thesis focuses on the distribution of all variants of CYP2C9, CYP2C19, CYP2D6 and alleles with known function of CYP3A5 (*3, *5, *6 and *8) in the Kinh Vietnamese using direct sequence method. Additionally, the thesis also aims to analyze CNVs of the 4 studied genes in Kinh subjects. 4
CHAPTER 2. MATERIALS AND METHODS 2.1. Study subjects One hundred and thirty-six of unrelated healthy Kinh volunteers were recruited in the study (55 males, 81 females) with age ranging from 18 to 35. 2.2. Instruments and equipment All instruments and equipment used in this study were provided by Institute of Genome Research, Vietnam Academy of Science and Technology. 2.3. Methodologies 2.3.1. Total DNA extraction Peripheral blood from donors were collected into EDTA tubes and stored at -20oC. Genomic DNA was extracted from subject’s peripheral blood using ExgeneTMBlood SV mini Kit (GeneAll, Korea) according to manufacturer’s protocol. Subsequently, total genomic DNA were analyzed on 0.8% agarose gel. 2.3.2. DNA quantification 2.3.3. Amplification of CYP2C9, CYP2C19, CYP2D6 and CYP3A5 Primers specific for CYP2C9, CYP2C19, CYP2D6 and CYP3A5 were designed by Primer 3 (v.0.4.0) according to reference gene sequences in NCBI. Promoters, all exons and flanking regions of CYP2C9, CYP2C19, CYP2D6 and regions of CYP3A5 consisted of *3, *6, *8 and *9 were amplified. Afterwards, all PCR products were purified following the manufacturer instruction. 2.3.4. Sanger sequencing PCR products were purified using Multiscreen PCR 96 Filter Plate. The purified PCR products were then sequenced using ABI Prism BigDye Terminator Cycle Sequencing Kit Version 3.1on an ABI genetic analyzer 3500. 2.3.5. MLPA 5
To identify deletions/duplications of CYP2C9, CYP2C19, CYP2D6 and CYP3A5, MLPA technique was performed using the commercial SALSA MLPA P128-C1 Cytochrome P450 Probemix kit following the manufacturer’s protocol. MLPA probemix were specifically designed for exons of CYP450 genes. After denaturing at 98oC, denatured DNA were hybridized with SALSA probemix by incubation at 60oC for 16h. Subsequently, the annealed probes were ligated using Ligase65 at 540 for 15min following by heating at 98oC in 5 min. In the next step, complete ligation reactions were used for the PCR. The amplicons were then separated by capillary gel electrophoresis on Genetic Analyzer 3500. 2.3.6. Longrange (LR) PCR To identify CYP2D6*5, LR-PCR reactions were performed with volume of 25µl in total. Primer pairs for LR-PCR were described previously. All amplified fragments were subsequently analyzed on 0.8% agarose gel. 2.3.7. Real-time PCR Real-time PCR reaction was performed using Luna Universal qPCR Master Mix (NEB). Additionally, primers were designed for specific detection of exon 4 and exon 7 of CYP2C9. Reference sample were chosen randomly which showed 2 copies form MLPA data. 2.3.8. Data analysis Bioedit software was used for initial analysis of the sequences. The Hardy-Weinberg equilibrium of genetics variants was evaluated by HAPLOVIEW. Inter-ethnic comparison of alleles frequencies and difference of allele frequencies between Kinh Vietnames and other populations in global were assessed by Chi-square (χ2) and Fisher exact test. P
CHAPTER 3. RESULTS AND DISCUSSION 3.1. Total DNA extraction Total genomic DNA was extracted from peripheral blood and subsequently analyzed by on 0.8% agarose gel. The total DNA yield with high purity ranged from 19,4 to 139,6 ng/µl. 3.2. Amplification of CYP2C9, CYP2C19, CYP2D6, CYP3A5 and sequencing We successfully amplified the promoter region, all 9 exons and flanking regions of CYP2C9, CYP2C19 and CYP3A5 regions bearing variants *3, *6, *8 and *9 from 100 subjects (40 males, 60 females). For CYP2D6, promoter with all 9 exons and flanking regions from 136 subjects were successfully amplified (55 males, 81 females). All amplified fragments had expected sizes and subsequently purified for sequencing in the next steps. 3.3. Genetic variations of CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in Kinh 3.3.1. Genetic variants of CYP2C9 For CYP2C9, the results identified total 14 variants. Notably, 6 variants have not been reported in the PharmVar database NCBI dbSNP as well as 1000 Genomes data. These genetic variants included 4 single nucleotide substitutions: intron 2 (3451C>T), 6 (33622T>C, 38658A>G) and 7 (42801A>G), 01 deletion in intron 8 (47543delT) and a single nucleotide substitution exon 7 (42627C>A) (Fig.3.6). MLPA method was performed to determine CNVs of CYP2C9. It was shown that 3/100 subjects carried only one copy of CYP2C9. Two real-time PCR assays were used to confirm the MLPA and the results were 100% in agreement with MLPA. However, CYP2C9 duplication alleles were not determined in all 100 subjects. 7
Figure 3.6. Novel variants identified in introns and exon of CYP2C9 Novel variants located from intron 2 to intron 8 of CYP2C9, including 5 variants in intron and 1 variant in exon 7. a) 3415C>T (intron 2), b) 33622T>C (intron 6), c) 38658A>G (intron 6), e) 42801A>G (intron 7), f) 47543delT (intron 8), and d) 42627C>A (exon 7). Nucleotide substitution positions were shown by arrows. Totally, 3 CYP2C9 genotypes were identified among 100 studied subjects (Table 3.3). Among these, the most frequent genotype is wild-type CYP2C9*1/*1 (90%) with normal enzyme function. Other two heterozygous genotypes showing lower frequency are CYP2C9*1/*3 (7%) and CYP2C9*1/Del with decreased and unknown function, respectively. As 8
a result, 3 CYP2C9 alleles were determined in total, which are CYP2C9*1, CYP2C9*3 and CYP2C9Del (Table 3.4). Of these, *1 is wild-type allele with highest frequency (95%), following by *3 (3.5%) and Del (only 1.5%). Table 3.3. Genotype frequencies of CYP2C9 in Kinh Vietnamese Phenotype Total Frequency Genotype (CPIC) (N=100) (%) *1/*1 EM 90 90 *1/*3 IM 7 7 *1/Del - 3 3 N: number of subjects Table 3.4. Allele frequencies of CYP2C9 in Kinh Vietnamese Allele Total (n=200) Frequency (%) *1 190 95 *3 7 3.5 Del 3 1.5 n: number of alleles 3.3.2. Genetic variants of CYP2C19 Overall, 14 different CYP2C19 variants were identified in our study group, including 3 novel variants. The novel variants include of 12637C>G (intron 2), 57637delG (intron 5) and 90008C>T (intron 8) (Fig.3.9). We also detected 6 genotypes of CYP2C19 in Vietnamese Kinh ethnic group with the percentage varies from 1% to 58% (Table 3.7). Among of these, the most abundant genotypes were the wildtype *1/*1 (58%) and the heterozygous *1/*2 (32%), while the heterozygous *2/*3 showed lowest prevalence (1%). Totally, 4 CYP2C19 alleles were detected in studied subjects, containing CYP2C19*1, CYP2C19*2, CYP2C19*3 and CYP2C19*17 (Table 3.8). The wild-type allele CYP2C19*1 showed highest frequency (76%) in the study subjects. Meanwhile, the remaining variant alleles CYP2C19*2, CYP2C19*3 and CYP2C19*17 accounted for 20.5%, 2.5% and 1%, 9
respectively. There are no CNV of CYP2C19 detected by MLPA in 100 subjects. Figure 3.9. Novel variants detected in introns of CYP2C19. Three novel variants were identified in introns of CYP2C19: a) 2637C>G (intron 2), b) 57637delG (intron 5) and c) 90008C>T (intron 8)-reverse primer was used for sequencing. Nucleotide changes were shown by arrows. Table 3.7. Genotype frequencies of CYP2C19 in Kinh Vietnamese Phenotype Frequency Grnotype Total (%) (N=100) (CPIC) *1/*1 58 EM 58 *1/*2 32 IM 32 *1/*3 4 IM 4 *2/*2 3 PM 3 *2/*3 1 PM 1 *2/*17 2 IM 2 N: number of subjects 10
Table 3.8. Allele frequencies of CYP2C19 in Kinh Vietnamese Allele Total Frequency % (n=200) *1 154 76 *2 41 20.5 *3 5 2.5 *17 2 1 n: number of alleles 3.3.3. Genetic variants of CYP2D6 The Sanger sequencing analysis identified totally 30 SNPs including 7 novel variants. Of these, 3 located in the promoter (-498C>A, -184A>T, - 175A>T) (Fig.3.10), 2 in the intron 4 and 6 (2137G>C, 2988G>A), 2 in the exon 7 and 8 (3157G>T, 3851G>A) of the CYP2D6 gene (Fig.3.11). Figure 3.10. Novel variants identified in promoter of CYP2D6 Three novel variants were found in promoter (reverse primers were used for sequencing): a) -175A>T, b) -184A>T, c) -498C>A. Nucleotide substitution positions were indicated by arrows. 11
Figure 3.11. Novel variants identified in intron and exon of CYP2D6 There were 2 novel variants detected in intron and 2 novel variants detected in exon: a) 2137G>C (intron 4), b) 2988G>A (intron 6), c) 3157G>T (exon 7), d) 3851G>A (exon 8- reverse primers were used for sequencing). Nucleotide substitution positions were indicated by arrows. Totally, MLPA analysis revealed 12 different types of copy number chances. Among these, the subjects with only 1 copy number CYP2D6 were randomly validated by LR-PCR and the results were 100% in agreement with MLPA. Additionally, no duplication or multiplication of CYP2D6 normal function alleles were detected in 136 subjects. 12
a) b) Figure 3.15. Confirmation of CYP2D6*5 by LR-PCR. a) Diagram of primer set used for LR-PCR, CYP2D6 deletion (*5) would result in 2 fragments containing 3.2 kb product indicating *5 allele (primers Dup and Dlow) and 5.1 kb product indicating wild-type allele (primers DPKup and DPKlow). Samples with wild-type genotype would result in 1 fragment (5.1 kb). b) Lane 1: DNA ladder. Lane 2-3: samples homozygous for wild-type genotype. Lane 3-4: samples heterozygous for CYP2C6*5 allele. To determine the genotypes of CYP2D6, we combined the sequencing- based genotypes and copy number alterations identified by MLPA of each subject. In 136 subjects, 29 different genotypes with the frequency varied from 0.7% to 22.8% were assigned. Among these, the most abundant genotypes were the homozygous *10/*10 (22.8%) followed by the heterozygous *1/*10 (15.4%) and *10/*36-*10 (11%). For all population, there are total 9 CYP2D6 variant alleles (75.37%), 8 structural variants (SVs) including 3 hybrid structures and 5 tandem arrangements (16.54%) and a CNV (whole gene deletion) (8.09%) (Table 3.11). 13
Among 9 variant alleles, the CYP2D6*10, known for decreased function, showed highest frequency (43.75%), while the normal function alleles CYP2D6*1 and CYP2D6*2 accounted for 18.75% and 7.35%, respectively. Additionally, we identified 5 nonfunction alleles, of which percentage varied from 0.37-8.09% (*4, *5, *14, *15, *36). Three unknown function alleles were *60, *65 and *86 accounting for 0.74, 2.94 and 0.37%. Of the detected tandem arrangements, the most prominent variant was *36- *10 (12.13%). Table 3.11. Allele frequencies of CYP2D6 in Kinh Vietnamese Frequency Function Variant/Allele Type Number Variant/Allele (%) (CPIC) 1 *1 18.75 Normal 2 *2 7.35 Normal 3 *4 0.74 None 4 *10 43.75 Decreased SNP/indel 5 *14 0.37 None 6 *15 0.37 None 7 *60 0.74 Unknown 8 *65 2.94 Unknown 9 *86 0.37 Unknown CNV 1 *5 8.09 None 2 *13 1.547 None Hybrid 3 *36 0.37 None Structural 4 *67 0.37 Unknown variant 5 *13-*1 0.74 Normal (SV) 6 *13-*2 0.74 Normal Tandem 7 *36-*36-*10 0.37 Decreased arrangements 8 *36-*10 12.13 Decreased 9 *68-*4 0.37 None 14
The CYP2D6 SVs detected in this study were presented in Fig. 3.16 Figure 3.16. Graphic view of CYP2D6 SVs determined in Kinh subjects. a) Reference gene locus of CYP2D6 (red boxes) and pseudo genes CYP2D7(blue boxes), CYP2D8 (grey boxes). REP6 (dark purple boxes) and REP7 (black boxes) are repetitive sequences located downstream of CYP2D6 and CYP2D7, respectively. b) Deletion of entire CYP2D6 gene. c) Hybrid structures of CYP2D6 containing CYP2D6- 2D7 (the 5’ portion is derived from CYP2D6 and the 3’ portion is derived from CYP2D7) and CYP2D7-2D6 (the 5’ portion is derived from CYP2D7 and the 3’ portion is derived from CYP2D6). (d) CYP2D6 tandem arrangements including two or more gene copies that are different with “duplications/multiplications”. # Details of SVs were reported in Pharmacogene Variation (PharmVar) Consortium (https://www.pharmvar.org/gene/CYP2D6). ∆ Detail of structural variants were reported in Andrea Gaedigk’s work. 15
3.3.4. Genetic variants of CYP3A5 This study did not determine the existence of CYP3A5*6, *8 and *9 among 100 studied subjects. For CYP3A5*3, this allele was detected in both heterozygous state *1/*3 and homozygous state *3/*3 (Fig 3.17). In total, number of genotype carrying at least 1 allele CYP3A5*3 accounted for 90% (*1/*3, *3/*3). Meanwhile, the wildtype genotype (CYP3A5*1/*1) made up only 10%. Figure 3.17. Sanger sequencing of CYP3A5*3 a)Homozygous wildtype genotype CYP3A5*1/*1 (AA), b) Heterozygous genotype CYP3A5*1/*3 (AG), c) Homozygous variants CYP3A5*3/*3 (GG). The nucleotide substitutions position were indicated by arrows. Of the detected alleles, CYP3A5*3 made up 32.5% and CYP3A5*3 accounted for 67.5% (Table 3.12). According to CYP3A5 allele freqiencies identified, all observed variants were in Hardy-Weinberg equilibrium (p=0.9677). Table3.12. Genotype and allele frequencies of CYP3A5 in Kinh Vietnamese Frequency Genotype (N=100) Allele (n=200) *1/*1 *1/*3 *3/*3 *1 *3 Present study 10 45 45 65 135 10.56 43.88% Hardy-Weinberg (10%) (45%) 45.56% (45%) (32,5%) (67,5%) % p= 0.9677 N: number of subjects, n: number of alleles 16
There are no CNV of CYP3A5 detected by MLPA in 100 subjects. Final probe ratio of all subjects ranged from 0.8 to 1.2. 3.4. Linkage Disequilibrium of genetic variants found in CYP2C9, CYP2C19 and CYP2D6 Linkage Disequilibrium analysis was performed to examine the SNPs relationship by Haploview 4.2 software and D’ value. For CYP2C9, we identified two LD blocks and extended haplotypes in the sequenced data. The markers in these blocks were not tightly correlated with LOD< 2 and D’=1 (blue squares). For CYP2C19, only one LD block was determined from the sequenced data. This block spanned a 87 kb region from -98 in promoter (marker -98T>C) to 87106 in intron 7 (makrer 87106CT>C). For CYP2D6, Haploview analysis revealed only one LD block spanning a region from 214 in intron1 (marker 214G>C) to 245 in intron 1 (makrer 245G>C) and these markers showed tight correlation with LOD # 2 and D’≤ 1. 3.5. Functional prediction of novel variants detected in CYP2C9, CYP2C19 and CYP2D6 *Functional prediction of novel variants found in CYP2C9 Two pairs of datasets were used to train and test Polyphen-2 prediction models, including HumDiv and HumVar. As a consequence, both HumDiv and HumVar trained Polyphen-2 models prediction regarding to the novel variant 42627C>A (p.Pro363His) revealed that it was probably damaging with a score of 1. Similarly, PROVEAN result indicated that this amino acid substitution is predicted to be deleterious (score = -8.373) (Fig. 3.21). Moreover, Multiz aligment demonstrated that Proline at position 363 of CYP2C9 is conservative (Fig.3.22). Human Splicing Finder online tool predicted that all 5 novel variants in intron of CYP2C9 would not have impact on mRNA splicing. 17
Figure 3.21. Functional prediction of novel variants detected in CYP2C9 by Polyphen-2 (a) and PROVEAN (b) Figure 3.22. Multiz protein alignment showed conservative amino acid sequence of CYP2C9 Position of Proline 363 in CYP2C9 was marked with vertical line (red) *Functional prediction of novel variants found in CYP2C19 According to Human Splicing Finder analysis, none of 3 novel intronic mutations found in this study resulted in splicing defects of CYP2C19 mRNA. *Functional prediction of novel variants found in CYP2D6 To predict wherer the 3 novel variants in the promoter region of CYP2D6 have ability to regulate translation of gene, the promoter sequences containing each of novel variants (-498C>A, -184A>T, -175A>T) was analysed using MATCH bioinformatic tool. However, none of them shown to be overlapped with the transcriptional factor binding sites in CYP2D6 promoter. 18