Tóm tắt Luận án Study on polymorphisms / mutations of cyp2c9, cyp2c19, cyp3a5 and cyp2d6 cytochrome p450 in kinh Vietnamese

f samples to generate data in order to support the drug development process. In clinical 4 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. 5 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 6 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<0.05 was considered as statistically significant. 2.3.9. Functional prediction of novel variants The online tools were used to search potential transcriptional factor binding sites in promoter (MATCH), predict possible impact of non- synonymous SNPs in the coding regions (PROVEAN, Polyphen-2) and SNPs in introns (Human Splicing Finder). 7 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. 8 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 9 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 Genotype Phenotype (CPIC) Total (N=100) Frequency (%) *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 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%, Allele Total (n=200) Frequency (%) *1 190 95 *3 7 3.5 Del 3 1.5 10 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 Grnotype Total Phenotype (CPIC) Frequency (%) (N=100) *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 11 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. 12 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. 13 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). 14 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 Variant/Allele Type Number Variant/Allele Frequency (%) Function (CPIC) SNP/indel 1 *1 18.75 Normal 2 *2 7.35 Normal 3 *4 0.74 None 4 *10 43.75 Decreased 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 Structural variant (SV) CNV 1 *5 8.09 None Hybrid 2 *13 1.547 None 3 *36 0.37 None 4 *67 0.37 Unknown Tandem arrangements 5 *13-*1 0.74 Normal 6 *13-*2 0.74 Normal 7 *36-*36-*10 0.37 Decreased 8 *36-*10 12.13 Decreased 9 *68-*4 0.37 None 15 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. 16 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 (10%) 45 (45%) 45 (45%) 65 (32,5%) 135 (67,5%) Hardy-Weinberg 10.56 % 43.88% 45.56% p= 0.9677 N: number of subjects, n: number of alleles 17 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. 18 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. 19 The novel variant 3157G>T (exon 7) causing the amino acid substitution from arginine to leucine at the position 329 (p.Arg329Leu), was evaluated for the probable effect by using Polyphen-2 and PROVEAN online tools. As the consequence, both HumDiv and HumVar trained Polyphen-2 models predicted that this variant was benign (Fig.3.23a). In contrast, PROVEAN tool indicated that the Arg329Leu mutation would be deleterious (score = -4.326) (Fig.3.23b). Arginine at the position 329 was indicated that highly conservative among several vertebrates (Fig.3.24). For intronic variants, the novel variant 2988G>A located in the intron 6 was predicted to affect branching points and was capable to modify splicing motif (Fig.3.24c). Meanwhile, no impact was found when examining the remaining novel variant 2137G>C in the intron 4. Figure 3.23. Functional prediction of novel variants detected in CYP2D6 a)Functional prediction of 3157G>T by Polyphen-2, b)Functional prediction of 3157G>T by PROVEAN, c) Splicing effect prediction of variant 2988G>A in intron 6. Figure 3.24. Multiz protein alignment showed conservative amino acid sequence of CYP2D6 Position of Aginine 329 in CYP2D6 was marked with vertical line (red) 20 3.6. Comparison of allele frequencies of study genes among pupolations in global For CYP2C9, the CYP2C9*3 allele frequency of study population is comparable to those in other populations located in Asia and America. However, this was shown to be significantly different to the South (p<0.05 when compared with Indonesian) and West of Asia (p<0.001 when compared with Iranian and Turkish) as well as in Europe (p<0.001). For CYP2C19, the CYP2C19*2 allele frequency was significantly higher than those in other populations located in Western Asia and Europe. Meanwhile, it was shown that CYP2C19*3 statistically lower than Japan and Korea but higher than those in the South and West of Asia as well as in Europe. Concerning CYP2C19*17 percentage, this allele was shown to be significantly lower than those seen in European and African-American (p<0.001). For CYP2D6, among nonfunctional alleles, the CYP2D6*4 frequency was significantly lower than those in other populations located in Europe and Africa (p<0.0001). In Asian countries, the null allele CYP2D6*5 prevalence was found almost similar except Thailand (p<0.05). Other alleles having no enzyme activity (*13, *14 and *15) showed no significant difference compared with populations in global. Among decreased functional alleles, it was shown that CYP2D6*10 frequency statistically higher than that found in Europe and Africa as well as in other remaining parts of the world. For CYP3A5, frequency of the CYP3A5*3 allele in the Kinh population (67.5%) was statistically lower than those detected in the Caucasian populations (p<0.0001) and statistically higher compared with the black populations (p<0.0001). In Asia, this ratio is not greatly different from the average CYP3A5*3 frequency of South Asians (p>0.05) and East Asia (p> 0.05) but significantly lower than that in West Asia (p<0.0001) and whites (p<0.0001). 3.7. Discussion 3.7.1. Distribution of SNPs that influence protein function of CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in worldwide populations 21 The prevalence of CYP2C9*3 in this study subjects showed no significant difference from remaining parts of Asia. According to our results, CYP2C19*2 is the most common variant in Vietnamese Kinh, which is in consistent with previous findings of Vietnamese Kinh as well as in other Asian populations. Furthermore, the geographical diference could lead to the divergence distribution of CYP2C9 and CYP2C19 alleles in Asia. For CYP2D6, statistical analysis also demonstrated significant higher frequency of CYP2D6*10 in Kinh Vietnamese compared with other worldwide regions except for Asian countries. Our work further approved the high prevalence of CYP2D6*10 retricted to Asian populations. Meanwhile, the nonfunction allele CYP2D6*4 was very rare in our study subjects as well as in other Asian populations. This was significant difference compared with figure observed in Europe America and Middle East, where CYP2D6*4 was reported with high frequency. This result is in accordance with previous works, in which a gradual decrease of CYP2D6*4 from Europe to Asia countries was reported. These data further explain the frequent existence of IM phenotype in Asians as well as in Kinh. For CYP3A5, allele CYP3A5*3 was detected with highest frequency in Caucasian, West Asian (80-98%) and lowest in Blacks such as American- African, South African and Nigerian (14,5-27%). Regarding to geographical difference, the various natural selection pressure on certain variatns of CYP3A locus possibly responsible for the diversity in the genetic variants distribution. 3.7.2. Linkage analysis and significance in CYP450 genetic diversity study This derives from the meaning of variant association analysis that is to determine the non-random association of variations in different loci of a gene. If an association between a number of variants can be determined, it can be inferred that the variations in that tight linkage would tend to inherit together. Reagrading to CYP450 genetic variation study, a new variant that is predicted to negatively affect protein function would have many implications in case such variant is in strong linkage with reported variant. It would make sense in identifying allele that is actual cause of enzyme activity alteration. Linkage analysis of 14 CYP2C9 variants with 2 identified blocks of 5 kb and 32 kb, respectively. However, the SNPs in both of these 22 blocks are not closely linked. For CYP2C19 and CYP2D6, only one block was identified for each gên (87 kb and about 31 bp, respectively). None of the new variants of these two genes are strongly linked to other