TITLE: Prevalence of CYP2C19 Polymorphism in Bogotá, Colombia: The first report of allele *17
AUTHORS: Azucena Arevalo Gavis, William  A. Otero-Regino (Unidad de Gastroenterología Facultad de Medicina, Universidad Nacional de Colombia Bogotá D.C., Colombia), Gloria Natalia  Ovalle Celis , Eliana Rodriguez, Alba  Trespalacios-Rangel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>The aim of this protocol is to share useful information to determine the main polymorphisms of </span><span style = "font-style:italic;">CYP2C19</span><span>. </span><span style = "font-style:italic;">CYP2C19 </span><span>genotyping was performed on gastric biopsy samples. Polymorphisms *1, *2, and*3 were analyzed by real-time PCR (Roche®), and PCR-RFLP was used to determine the presence of allele *17. </span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-weight:bold;">Protocol scope:</span><span> This protocol can be used when someone want to replicate the techniques to analyze </span><span style = "font-style:italic;">CYP2C19</span><span> polymorphisms. In addition,  the protocol provides information on how to transport, properly preserve gastric biopsies and the steps for DNA extraction from gastric biopsies. </span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-weight:bold;">About </span><span style = "font-weight:bold;font-style:italic;">CYP2C19</span><span style = "font-weight:bold;">  *2, *3 and *17 polyporphisms:</span><span> they are a single nucleotide polymorphisms (SNP) in three different regions of </span><span style = "font-style:italic;">CYP2C19 </span><span>gen that affect the characteristic of its protein and it impacts in the metabolism of some drugs such as protom pumps inhibitors (PPIs).  The SNP in allele *2 occurs in exon 5 by a shif of guanine to adenine (G>A). The SNP in allele *3 occurs in exon 4 by a shif of guanine to adenine (G>A). This two SPNs reduce the ability to metabolize PPI. In the case of the SNP of allele *17 it occurs in promoter region by a shif of cytosine to thymine at -3404 and -806 positions (– 3402 C>T y - 806 C>T). The SNP in allele*17 may to recluit many transcription factors  and consequently large amounts of CYP2C19 enzyme are produced, giving to the subject the ability to metabolize very quickly the PPIs. ect will be ultrarapid metabolizer.  Therefore, the determination of </span><span style = "font-style:italic;">CYP2C19</span><span> polymorphisms  are so important for precision medicine.</span></div><style>
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[STEPS]
?. Ethics aprovalThe ethics committees from participant institutes approved the study protocol.  Since the study included human samples It was performed in agreement with Good Clinical Practice guidelines and the ethical principles of the Declaration of Helsinki.
?. Participants1. Inclusion and exclusion criteria: This study included subjects between 19 and 70 years old who were referred for functional dyspepsia or peptic ulcers and recommended to have endoscopy and who agreed to participate in the study. The protocol study excluded pregnant women; subjects with concomitant diseases such as diabetes, mental disorders, gastric atrophy or intestinal metaplasia; subjects with previous gastric cancer; and subjects with previous gastric surgery.2. Subjects recruitment: The subjects were enrolled randomly in the study during gastroenterology consultation after a gastroenterologist explained to them that the information derived from this research could help to select in a better way the PPIs that are used for patients with peptic acid diseases. Written informed consent was obtained from subjects who met the inclusion and exclusion criteria by the endoscopic service.The protocol study excluded pregnant women; subjects with concomitant diseases such as diabetes, mental disorders, gastric atrophy or intestinal metaplasia; subjects with previous gastric cancer; and subjects with previous gastric surgery.2. Subjects recruitment: The subjects were enrolled randomly in the study during gastroenterology consultation after a gastroenterologist explained to them that the information derived from this research could help to select in a better way the PPIs that are used for patients with peptic acid diseases. Written informed consent was obtained from subjects who met the inclusion and exclusion criteria by the endoscopic service.2. Subjects recruitment: The subjects were enrolled randomly in the study during gastroenterology consultation after a gastroenterologist explained to them that the information derived from this research could help to select in a better way the PPIs that are used for patients with peptic acid diseases. Written informed consent was obtained from subjects who met the inclusion and exclusion criteria by the endoscopic service.
?. Samples1. Samples obtention: The sample used was gastric biopsy.Endoscopy was performed by an expert gastroenterologist with an Exera Olympus CV 145 video endoscope. Endoscopy was performed after six hours of fasting with the standard methodology and with sedation depending on the tolerance of the procedure (on demand).  During the procedure, biopsies of the upper digestive tract were obtained according to established protocols independent of visible pathologies, and a gastric body biopsy sample was taken for the molecular analysis of CYP2C19 genetic polymorphisms. Endoscopy was performed by an expert gastroenterologist with an Exera Olympus CV 145 video endoscope. Endoscopy was performed after six hours of fasting with the standard methodology and with sedation depending on the tolerance of the procedure (on demand).  During the procedure, biopsies of the upper digestive tract were obtained according to established protocols independent of visible pathologies, and a gastric body biopsy sample was taken for the molecular analysis of CYP2C19 genetic polymorphisms. 2. Transport and store of samples for molecular analysis: The biopsy samples was transported in 500 µL of Brucella broth (Becton Dickenson®) plus 20% (v/v) glycerol (Invitrogen®) and was kept refrigerated until it was processed. 
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?. Molecular Analisys
?. DNA ExtrationThe  was used to obtain DNA from gastric biopsy samples according to the manufacturer’s instructions  with small variation in steps 1, 2, 9 and 10  as follow:1. Take a gastric biopsy  (antrum and /or body stomach tissue) of each patient, and resuspend in eppendorf with 2. With the activated carbon swab, macerate the biopsy completely and again add 3. Add   and incubate at. Centrifugate 4. Add . Mix thoroughly by vortexing for   and briefly centrifuge the tube to remove drops from the lid.5. Add . Vortex for . Briefly centrifuge the tube to remove drops from the lid. 6. Pipet the mixture onto the QIAamp Mini spin column (in a ). Centrifuge at or 7. Place the QIAamp Mini spin column in a new  Centrifuge at  or  . Discard the flow-through and collection tube.8. Place the QIAamp Mini spin column in a new   Centrifuge at full speed ( or )  Discard the flow-through and collection tube.9. Recommended: Place the QIAamp Mini spin column in a new   min. This eliminates the chance of possible Buffer AW2 carryover.10. Place the QIAamp Mini spin column in a new   and incubate at . Centrifuge at  or 10. Optional: Repeat step 9 for increased DNA yield with a further  .
[of PBS 1X]
[of PBS 1X]
[of Buffer ALT]
[ with shaking overnight]
Centrifuge: 1500 33, 1 min
[Buffer AL]
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[for 10 minutes]
[ethanol 96-100%)]
[collection tube]
Centrifuge: 6000 34, 1 min
Centrifuge: 8000 33, 1 min
[collection tube and add]
[of buffer AW1]
Centrifuge: 6000 34, 1 min
Centrifuge: 8000 33, 1 min
[collection tube and add]
[Buffer AW2.]
Centrifuge: 20.000 34, 3 min
Centrifuge: 14000 33, 3 min
[collection tube (not provided) and centrifugate at full speed for]
[microcentrifuge tube (not provided), add]
[warm molecular grade water]
0 Room temperature
Centrifuge: 6000 34
Centrifuge: 8000 33, 1 min
[to elute the DNA.]
[warm molecular grade water]
?. CYP2C19 *1, *2, *3 GenotypingCYP2C19 *1, *2, and *3 genetic polymorphisms were determined by real-time polymerase chain reaction (RT-PCR) using the LightMix® kit for human CYP2C19*2 and CYP2C19*3 (Roche®). PCR was performed according to the manufacturer’s instructions. CYP2C19 *2 and CYP2C19 *3 Experimental Protocol Roche1. Each PCR was carried out in a final volume of  MgCl2, 2. PCR was performed in a LightCycler 1.5. Before initial analysis, color compensation in reading channels was performed to guarantee good results, and quality control for each allele (wild type and mutant alleles 2 and 3) was included for every test. 
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							3. Allelic classification was analyzed by differences in melting temperatures (Tm) (curves obtained) in channel 530 for allele 2, with Tm between 48.6°C and 54.4°C, and in channel 640 for allele 3, with Tm between 53.4°C and 60.8°C, according to the manufacturer’s suggestion as follow:
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[as follows:]
[of molecuar-grade water (Tib Molbiol),]
1.6 µl
[primers,]
[probes,]
[Master mix and]
[of DNA.]
?. CYP2C19 *17 Genotyping PCR and RFLP were used to determine the CYP2C19 *17 genetic polymorphism. This PCR was standardized according to previous reports by Baldwin et al. 2008 
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							This nested PCR and RFLP consisted of two PCRs and a final enzymatic digestion from the product of the second PCR.
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							1. First, PCR was carried out in a final volume of  as follows:  (Sigma®),  (10 µM),  and 2. The primer pair used in this first PCR and the cycling conditions  are showing in table 1. Primers were synthesized by Invitrogen USA and amplified a 473 bp fragment, corresponding to CYP2C19 allele 1. 
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							3. The second PCR was performed using   It was carried out in a final volume of  as follows: (10 µM),   and of DNA and using another set of primers synthesized by Invitrogen USA listed in table 1 with the PCR cycling conditions.
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							4.  Next, at 
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							5. Subsequently, the PCR digestion productwas revealed on  to verify the presence of the 116 bp and 143 bp bands, corresponding to the CYP2C19*1 and CYP2C19*17 alleles, respectively (Figure: CYP2C19*17  PCR-RFLP Gel Eectrophoresis Results) .
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							6. In addition, to confirm the presence of CYP2C19*17 by nested PCR and RFLP, 18.4% of samples were selected randomly and sequenced (Macrogen, Korea).
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[as follows:]
[of molecular grade water]
[primers]
[Master mix]
[DNA.]
[of the first PCR product.]
[as follows:]
[of molecuar-grade water (Sigma),]
[primers]
[Masater mix]
[of DNA]
[of the second PCR reaction was incubated with]
[of Nsil restriction enzyme ]
[for ]
[.]
[agarosa gel and stained with]
?. Statistical AnalysisCharacteristics of the population and CYP2C*19 genetic polymorphism frequencies were analyzed using descriptive statistics, employing the SPSS v.24 statistics program.  Bioinformatic analysis of sequences for allele CYP2C19*17 was performed with the BLAST-N tool Nucleotide BLAST. The alignments were performed with the wild-type reference sequences (GenBank Access: AL583836 and NG_008384.3.18)