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Collection of Genomic DNA by the Noninvasive Mouthwash Method for Use in Pharmacogenetic Studies
Abstract
To determine long-term stability, quantity, and quality of genomic DNA samples collected in buccal cells by the mouthwash method, for use in pharmacogenetic studies.
Prospective analysis.
University pharmacogenomics center in Florida and medical centers in Puerto Rico and the United States participating in a multicenter international trial.
Ten volunteers at the pharmacogenomics center and 201 participants in the ongoing multicenter clinical trial.
Stability of genomic DNA was determined by measuring DNA yield from mouthwash samples obtained from six volunteers and stored at room temperature over 90 days and from 201 clinical trial samples that were stored and shipped at room temperature. Whether DNA yield was higher with three 5-ml mouthwash rinses versus one 10-ml rinse was evaluated in four volunteers. Quality of genomic DNA was assessed on 32 randomly selected samples from the six volunteers in the stability study, by determining the success rate of DNA amplification with polymerase chain reaction (PCR) testing and by genotyping.
For the stability studies, the quantity of genomic DNA decreased over time with storage at room temperature (overall p < 0.01), with the largest declines occurring at 60 and 90 days. Median DNA recovery at 30 and 90 days was 59% and 28% of that at baseline, respectively. Mean +/- standard deviation, median, and range for recovery of genomic DNA from the 201 samples were 45.2 +/- 55 microg, 25.2 microg, and 1-330 microg, respectively. Median recoveries of DNA from the one-rinse and three-rinse methods were not statistically significantly different (9.1 vs 10.5 microg). All samples were amplified successfully by PCR and genotyped, indicating quality of the DNA samples.
The mouthwash method for collection of genomic DNA is a simple, inexpensive, and noninvasive method that poses less risk than venipuncture and may be used in a variety of settings. Genomic DNA in mouthwash is stable for prolonged periods at room temperature, and the quantity of DNA recovered from this method is more than sufficient for pharmacogenetic studies. Such an approach should be valuable to pharmacogenetic researchers and others who are conducting genetic research.
... Many of these cohort studies received the oral wash specimens by mail where the sample remained at ambient temperature over the course of a few days prior to processing and freezing. The impact of ambient temperature on human DNA from the oral wash sample has been considered (10)(11)(12)(13), but the impact of ambient temperature on microbial DNA from an oral wash sample is not well understood. ...
... Saliva samples from 4 adults stored in liquid dental transport medium or in an OMNIgene Kit had similar bacterial diversity after room temperature storage for 2-7 days (26). For oral wash samples, human DNA appeared stable at room temperature for variable lengths of time (10)(11)(12)(13) and as seen in this study, a number of microbial metrics were relatively stable at room temperature, but there were some significant differences for the relative abundances of taxa between the samples frozen immediately and those left at room temperature for 4 days. ...
Background:
A number of cohort studies have collected Scope mouthwash samples by mail which are being used for microbiota measurements. We evaluated the stability of Scope mouthwash samples at ambient temperature and determined the comparability of Scope mouthwash with saliva collection using the OMNIgene ORAL kit.
Methods:
Fifty-three healthy volunteers from Mayo Clinic and fifty cohort members from Bangladesh provided oral samples. One aliquot of the OMNIgene ORAL and Scope mouthwash were frozen immediately and one aliquot of the Scope mouthwash remained at ambient temperature for four days and then was frozen. DNA was extracted and the V4 region of the 16S rRNA gene was PCR amplified and sequenced using the HiSeq. Intraclass correlation coefficients (ICC) were calculated.
Results:
The overall stability of the Scope mouthwash samples was relatively high for alpha and beta diversity. For example, the meta-analyzed ICC for the Shannon Index was 0.86 (95% CI: 0.76, 0.96). Similarly, the ICCs for the relative abundance of the top 25 genera were generally high. The comparability of the two sample types was relatively low when measured using ICCs, but were increased by using a Spearman correlation coefficient (SCC) to compare the rank order of individuals.
Conclusions:
Overall, the Scope mouthwash samples appear to be stable at ambient temperature which suggests that oral rinse samples received by the mail can be used for microbial analyses. However, Scope mouthwash samples were distinct compared to OMNIgene ORAL samples.
Impact:
Studies should try to compare oral microbial metrics within one sample collection type.
... The method of alcohol-based mouthwash-expectorated buccal cell sampling that we employ is less commonly used than the cytobrush-exfoliated buccal cell collection method [9]. However, there have been a number of recent studies in the literature that use this method of mouthwash-mediated buccal cell collection, in particular those of Lum and Le Marchand [55], Garcia-Closas et al. [56], and Andrisin et al. [57]. As highlighted by Garcia-Closas et al. [56] and Lum and Le Marchand [55] the use of alcohol-based mouthwash has certain decisive advantages over cytobrush-exfoliated buccal cell collection in the proportion of high molecular weight DNA extracted and its stability, and the much lower proportion of non-human (bacterial) DNA content. ...
... As highlighted by Garcia-Closas et al. [56] and Lum and Le Marchand [55] the use of alcohol-based mouthwash has certain decisive advantages over cytobrush-exfoliated buccal cell collection in the proportion of high molecular weight DNA extracted and its stability, and the much lower proportion of non-human (bacterial) DNA content. As shown by Andrisin et al. [57] the method also produces samples with stable DNA content, that can be kept at room temperature for up to 90 days, unlike cytobrushexfoliated buccal cell samples, that require processing within a matter of days; as shown by Walsh et al. [58] there is significant bacterial contamination on cytobrush-collected buccal samples within 4 days of sample collection. The method is also more acceptable to study subjects than PBL samples obtained via phlebotomy or cytobrush-exfoliated buccal cell collection, being less invasive; it is also preferable to salinemouthwash collected buccal samples because of the more agreeable aftertaste. ...
Background: Ionizing radiation is a well-known carcinogen. Chromosome aberrations, and in particular micronuclei represent an early biological predictor of cancer risk. There are well-documented associations of micronuclei with ionizing radiation dose in some radiation-exposed groups, although not all. That associations are not seen in all radiation-exposed groups may be because cells with micronuclei will not generally pass through mitosis, so that radiation-induced micronuclei decay, generally within a few years after exposure.
Methods: Buccal samples from a group of 111 male workers in Ukraine exposed to ionizing radiation during the cleanup activities at the Chornobyl nuclear power plant were studied. Samples were taken between 12 and 18 years after their last radiation exposure from the Chornobyl cleanup. The frequency of binucleated micronuclei was analyzed in relation to estimated bone marrow dose from the cleanup activities along with a number of environmental/occupational risk factors using Poisson regression adjusted for overdispersion.
Results: Among the 105 persons without a previous cancer diagnosis, the mean Chornobyl-related dose was 59.5 mSv (range 0–748.4 mSv). There was a borderline significant increase in micronuclei frequency among those reporting work as an industrial radiographer compared with all others, with a relative risk of 6.19 (95% CI 0.90, 31.08, 2-sided p = 0.0729), although this was based on a single person. There was a borderline significant positive radiation dose response for micronuclei frequency with increase in micronuclei per 1000 scored cells per Gy of 3.03 (95% CI -0.78, 7.65, 2-sided p = 0.1170), and a borderline significant reduction of excess relative MN prevalence with increasing time since last exposure (p = 0.0949). There was a significant (p = 0.0388) reduction in MN prevalence associated with bone X-ray exposure, but no significant trend (p = 0.3845) of MN prevalence with numbers of bone X-ray procedures.
Conclusions: There are indications of increasing trends of micronuclei prevalence with Chornobyl-cleanup associated dose, and indications of reduction in radiation-associated excess prevalence of micronuclei with time after exposure. There are also indications of substantially increased micronuclei associated with work as an industrial radiographer. This analysis adds to the understanding of the long-term effects of low-dose radiation exposures on relevant cellular structures and methods appropriate for long-term radiation biodosimetry.
... In contrast to blood collection, buccal cell collection is noninvasive and can be self-administered, and samples can be transported via the postal system [11][12][13][14]. This method has been found to provide adequate DNA yield and quality [11,12,[14][15][16][17][18]. ...
... Subjects were asked to record the time and date of rinsing on the instruction sheet, and to mail the instruction sheet to the laboratory with the sample. The study initially requested one-swish, but based on data from other studies [12,17], the protocol was amended in October 2004, to request two consecutive swishes to be spit into two separate containers. ...
Purpose:
Although African-Americans experience higher cancer morbidity and mortality rates compared to their White counterparts, their participation in biospecimen research is lower than that of their white peers. This study investigated the prevalence and predictors of biospecimen donation in a large, cohort study of Black women.
Methods:
The BWHS is a follow-up study of U.S. Black women aged 21-69 years enrolled through postal health questionnaires. Between January 2004 and December 2007, participants were sent a consent form with a postage-paid return envelope, and a mouthwash collection kit. Univariate and age- and educational status-adjusted logistic regression models were used to estimate the association of socio-demographic, lifestyle and medical factors with donation of biospecimens.
Results:
Buccal cells with consent forms were obtained from 26,790 women, for a response rate of 51 %. The strongest predictors of biospecimen donation were age: response increased from 48.6 % among those aged <40 to 63.1 % among those aged 60 and older [RR 1.30 (95 % CI 1.27, 1.34)]; multivitamin use [RR (95 % CI) 1.32 (1.30, 1.34)]; physician visit in the previous 2 years [RR (95 % CI) 1.61 (1.58, 1.65)], and a history of breast [RR (95 % CI) 1.59 (1.56, 1.63)], colon [RR (95 % CI) 1.18 (1.16, 1.20)], and cervical [RR (95 % CI) 1.63 (1.60, 1.67)] cancer screening.
Conclusions:
We found that 51 % of women in the geographically-dispersed Black Women's Health Study cohort were willing to provide mouthwash samples to be used for genetic analyses. The response in this study is encouraging given published findings of low overall participation rates of African-Americans in genetic studies.
... Participants were asked to abstain from smoking and caffeine during the study time period. Blood samples for the measurement of pioglitazone, M-III, and M-IV plasma concentrations were collected predose, and 0.5, 1, 2, 3,4,5,7,9,12,18,24, and 48 hours post-pioglitazone dose. Plasma was harvested within 30 minutes of each blood draw and stored at -80°C for later bioanalytical processing. ...
... For the genetic screening process, a buccal cell sample was collected from each participant using a published mouthwash method. 12 Genomic DNA was isolated from buccal cells using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). Subjects were genotyped for CYP2C8*2 (Ile269Phe, rs11572103), CYP2C8*3 (Arg139Lys, rs11572080; Lys399Arg, rs10509681), and CYP2C8*4 (Ile264Met, rs1058930) using PCR-pyrosequencing assays (PSQ 96 MA, Qiagen, Valencia, CA, USA), according to standard manufacturer protocol. ...
Study objectives:
To determine the influence of the Cytochrome P450 (CYP) 2C8*2 polymorphism on pioglitazone pharmacokinetics in healthy African-American volunteers.
Design:
Prospective, open-label, single-dose pharmacokinetic study.
Setting:
University of Colorado Hospital Clinical and Translational Research Center.
Participants:
Healthy African-American volunteers between 21 and 60 years of age were enrolled in the study based on CYP2C8 genotype: CYP2C8*1/*1 (9 participants), CYP2C8*1/*2 (7 participants), and CYP2C8*2/*2 (1 participant).
Intervention:
Participants received a single 15-mg dose of pioglitazone in the fasted state, followed by a 48-hour pharmacokinetic study.
Measurements and main results:
Plasma concentrations of pioglitazone and its M-III (keto) and M-IV (hydroxy) metabolites were compared between participants with the CYP2C8*1/*1 genotype and CYP2C8*2 carriers. Pioglitazone area under the plasma concentration-time curve (AUC)0-∞ and half-life (t1/2 ) did not differ significantly between CYP2C8*1/*1 and CYP2C8*2 carriers (AUC0-∞ 7331 ± 2846 vs 10431 ± 5090 ng*h/ml, p=0.15, t1/2 7.4 ± 2.7 vs 10.5 ± 4.0 h, p=0.07). M-III and M-IV AUC0-48 also did not differ significantly between genotype groups. However, the M-III:pioglitazone AUC0-48 ratio was significantly lower in CYP2C8*2 carriers than CYP2C8*1 homozygotes (0.70 ± 0.15 vs 1.2 ± 0.37, p=0.006). Similarly, CYP2C8*2 carriers had a significantly lower M-III:M-IV AUC0-48 ratio than participants with the CYP2C8*1/*1 genotype (0.82 ± 0.26 vs 1.22 ± 0.26, p=0.006).
Conclusion:
These data suggest that CYP2C8*2 influences pioglitazone pharmacokinetics in vivo, particularly the AUC0-48 ratio of M-III:parent drug, and the AUC0-48 ratio of M-III:M-IV. Larger studies are needed to further investigate the impact of CYP2C8*2 on the pharmacokinetics of CYP2C8 substrates in individuals of African descent.
... Previous studies have investigated the stability and comparability of fecal and oral microbiota samples using 16S rRNA gene sequencing and shotgun sequencing [5,8,10,[19][20][21][22][23][24]. For fecal samples after 4 days at room temperature, there were no substantial differences in relative abundance for samples collected by FOBT card [5,8]. ...
Background
To initiate fecal and oral collections in prospective cohort studies for microbial analyses, it is essential to understand how field conditions and geographic differences may impact microbial communities. This study aimed to investigate the impact of fecal and oral sample collection methods and room temperature storage on collection samples for studies of the human microbiota.
Results
We collected fecal and oral samples from participants in two Iranian cohorts located in rural Yazd ( n = 46) and urban Gonbad ( n = 38) and investigated room temperature stability over 4 days of fecal (RNA later and fecal occult blood test [FOBT] cards) and comparability of fecal and oral (OMNIgene ORAL kits and Scope mouthwash) collection methods. We calculated interclass correlation coefficients (ICCs) based on 3 alpha and 4 beta diversity metrics and the relative abundance of 3 phyla. After 4 days at room temperature, fecal stability ICCs and ICCs for Scope mouthwash were generally high for all microbial metrics. Similarly, the fecal comparability ICCs for RNA later and FOBT cards were high, ranging from 0.63 (95% CI: 0.46, 0.75) for the relative abundance of Firmicutes to 0.93 (95% CI: 0.89, 0.96) for unweighted Unifrac. Comparability ICCs for OMNIgene ORAL and Scope mouthwash were lower than fecal ICCs, ranging from 0.55 (95% CI: 0.36, 0.70) for the Shannon index to 0.79 (95% CI: 0.69, 0.86) for Bray-Curtis. Overall, RNA later , FOBT cards and Scope mouthwash were stable up to 4 days at room temperature. Samples collected using FOBT cards were generally comparable to RNA later while the OMNIgene ORAL were less similar to Scope mouthwash.
Conclusions
As microbiome measures for feces samples collected using RNA later , FOBT cards and oral samples collected using Scope mouthwash were stable over four days at room temperature, these would be most appropriate for microbial analyses in these populations. However, one collection method should be consistently since each method may induce some differences.
... All rights reserved As previously described, either a buccal cell or venous blood sample was collected from each patient for genotyping. 7, 15 Clinical data were collected from the electronic medical record or through patient interview. Genotypes for VKORC1 rs9923231 and CYP2C variants were available from previous efforts. ...
VKORC1 and CYP2C9 genotypes explain less variability in warfarin dose requirements in African Americans (AAs) compared to Europeans. Variants in BCKDK and GATA-4 gene regions, purported to regulate VKORC1 and CYP2C9 expression, have been shown to play an important role in warfarin dose requirements in Europeans and Asians, respectively. We sought to determine whether rs56314408 near BCKDK or GATA-4 rs2645400 influence warfarin dose requirements in 200 AAs. Unlike the strong linkage disequilibrium (LD) between rs56314408 and VKORC1 rs9923231 in Europeans, they were not in LD in AAs. No associations were found on univariate analysis. On multivariable analysis, rs56314408 was associated (p=0.027) with dose in a regression model excluding VKORC1 rs9923231, and GATA-4 rs2645400 was associated (p=0.032) with dose in a model excluding CYP2C variants (CYP2C9*2, *3, *5, *6, *8, *11, CYP2C rs12777823). Neither variant contributed to dose in the model including both VKORC1 rs9923231 and CYP2C variants. Our results do not support contributions of the studied variants to warfarin dose requirements in AAs. However, they illustrate the value of studies in African descent populations, who have low LD in their genome, in teasing out genetic variation underlying drug response associations. They also emphasize the importance of confirming associations in persons of African ancestry.
... It is known that the proportion of samples with successful multiplex PCR amplification is an indicator of the effectiveness of DNA extraction as regards DNA integrity and purity. (35)(36)(37)(38)(39) Effect of DNA quantity and purity on sex determination: ...
BACKGROUND:
Determination of sex using DNA typing is one of the most important procedures in the forensic field. The aim of the present work was evaluation of the simultaneous use of sex- determining region Y (SRY) and androgen receptor (AR) genes in sex determination from dried blood stains.
METHODS:
The dried blood samples (n=30) were divided into two groups: dried blood samples on glass slide and stains on filter paper. The dried blood samples were stored at room temperature till DNA extraction using QIAamp DNA investigator kit.
The quantity and purity of DNA extracted from each group was measured using Nano Drop-2000 spectrophotometry. High purity DNA has an A260/A280 ratio of 1.7–2.0. Lower ratios indicate presence of protein contaminants.
For sex determination, the samples were subjected to multiplex PCR amplification of the SRY and AR gene. Separation and detection of the amplified product were performed using 2% Agarose gel electrophoresis and U/V transillumination.
RESULTS:
Successful DNA extraction from all samples was done within a conventional period of time. No significant difference was observed in DNA extracted from the dried blood scraped from glass slide and stains on filter paper samples regarding quantity and purity of DNA extracted.
The sex typing results were based on the PCR amplification product of the SRY gene locus in the Y chromosome (779 bp) for male samples only and AR gene locus in the X chromosome (292 bp) for male and female samples.
Successful sex determination was achieved in 66.7% of dried blood samples. The quantity and purity of DNA extracted from dried blood samples were significantly lower in undetermined than determined sex samples.
CONCLUSION:
From this study it was concluded that QIAamp DNA investigator Kit provides a fast, easy, and reproducible method for genomic DNA isolation from samples on difficult substrates like filter paper. Genomic DNA was of sufficient quantity and quality suitable for demanding PCR-based genetic assays especially DNA sex typing.
There was no effect of storage periods on DNA quality and quantity. Multiplex PCR targeting SRY and AR genes is a rapid, accurate, sensitive, reliable and easily manipulated method for determination of sex of dried blood samples and provided no false positive results. The low quantity and poor purity of extracted DNA affect the success of sex determination. As the sample that showed successful sex determination has significantly higher DNA quantity and purity than samples with undetermined sex.
Keywords: Sex determination, Multiplex PCR, SRY gene, AR gene, Dried blood stains, NanoDrop 2000 spectrophotometer
... Several chemicals and compounds have been reported to preserve nucleic acids at room temperature for weeks to months. DNA-binding protein from starved cells (Dps) and poly(A) binding protein (Pab1p) were reported to stabilize DNA and mRNA, respectively [12,[23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. Commercial products such as RNAlater and Trizol (Life Technologies) are based on this approach and have been documented to stabilize nucleic acids at room temperature for long periods of time [11,27,30,[39][40][41]. ...
This effort focused on development of a combined storage and delivery system intended to offer much-needed stability to biomolecules, especially DNA and RNA. The goal was to provide stabilization methods for reagents and targets in order to allow for a wider range of applications through utilization of organized porous materials as scaffolds for their encapsulation. This report details the synthesis of solid support materials, selection of stabilization components, and development of methods for their application. Design considerations focused on control of interactions with the nucleic acids that result in degradation. Over the course of the effort, the potential for adsorption of RNA, DNA, and ssDNA onto porous organosilicate sorbents with and without additional stabilizing reagents was demonstrated. Improved binding capacities were achieved with sorbents using chemical functionalities rather than proteins and sugars. These sorbents were found to provide similar improvements in stability to the traditional stabilization compounds. The materials were further shown to provide capture and subsequent stabilization of targets from a complex solution.
... Several chemicals and compounds have been reported to preserve nucleic acids at room temperature from periods of weeks to months. DNA-binding protein from starved cells (Dps) and poly(A) binding protein (Pab1p) were reported to stabilize DNA and mRNA, respectively [3,[15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]. Commercial products, such as RNAlater and Trizol (ThermoFisher Scientific, Waltham, MA, USA), are based on this approach and have been documented to stabilize nucleic acids at room temperature for long periods of time [16,26,[31][32][33][34]. ...
... Samples of DNA for analysis of enzymes may now be obtained through blood or buccal cells. Collection of buccal cells via the mouthwash method or buccal swab is a cost-effective, stable, noninvasive, and reliable method of collecting samples of DNA for genetic research (Andrisin, Humma, & Johnson, 2002). Buccal swab tests are found to be as effective as traditional blood specimen collection (McMichael et al., 2009). ...
Background:
Despite extensive scholastic and professional training, medication management in psychiatry is often relegated to trial-and-error prescribing. Pharmacogenetic testing (PGT) may expedite identification of medications with maximal efficacy and minimal side effects by recognizing individual genetic variability in drug response.
Objectives:
This article outlines the background of PGT, explains drug metabolism, and evaluates the impact of PGT.
Design:
A review of the literature since 2010 found 42 articles regarding PGT in clinical nursing settings on PubMed and ProQuest.
Results:
Despite continuing rises in health care costs, new biotechnology has led to a decrease in the cost of genetic sequencing and application of PGT to practice.
Conclusion:
As PGT becomes increasingly prevalent, nurses should be knowledgeable of its purpose, possibilities, and potential limitations to provide accurate and up-to-date patient information.
... In the genetic substudy (INVEST-GENES), gDNA was collected from 5,979 patients using buccal cells from mouthwash samples. 33 All patients provided written informed consent, as approved by the University of Florida Institutional Review Board. The present case-control study focused on the 258 INVEST-GENES patients who experienced a primary outcome event during study follow-up (cases), frequency matched 3:1 for age, sex, and race/ethnicity with 774 individuals who were event-free during study follow-up (controls) (baseline characteristics in Table 4). ...
... The commonly used methods employ polyethylene swabs or brushes, treated Guthrie-type cards, rinses with saline solution or 3% sucrose. [4][5][6][7][8][9][10][11][12][13] However, these techniques also have their disadvantages since the sample must be frozen or processed immediately so as to assure the quantity and quality of DNA. This requirement represents a difficulty for their use in studies that require transportation of the sample and its maintenance at room temperature. ...
Background:
Obtaining high quality genomic DNA safely and economically is vital for diverse studies of large populations aimed at evaluating the role of genetic factors in susceptibility to disease.
Aim:
This study was to test a protocol for the extraction of high quality genomic DNA from saliva samples obtained with mouthwash and taken from patients with periodontal disease.
Methods:
Saliva samples were taken from 60 patients and then stored at room temperature. DNA extraction was carried out at distinct post-sampling times (10, 20 and 30 days). Evaluation of genomic DNA was performed with spectrophotometry, electrophoresis, and PCR genotyping and sequencing.
Results:
The greatest concentration of DNA obtained was 352 μg at 10 days post-sampling, followed by 121.025 μg and 19.59 μg at 20 and 30 days, respectively. When determining the purity of DNA with the spectrophotometric ratio of 260/230, the relations of 1.20, 1.40 and 0.781 were obtained for 10, 20 and 30 days, respectively. In all samples, it was possible to amplify the product of 485 bp and the sequence of the amplicons showed 95% similarity to the reference sequence.
Conclusion:
The present protocol represents an easy, safe and economical technique for obtaining high quality genomic DNA.
... Genomic DNA was isolated either from buccal cells obtained from mouth wash samples (European cohort) or leukocytes in peripheral blood samples (Egyptian cohort) using the manufacturers' guidelines. 32 Genotyping for GGCX C>T, rs12714145; STX1B A>G, rs4889606; FPGS A>G, rs7856096; VKORC1 -1639 G>A (rs9923231); CYP2C9*2 430 C>T (rs1799853); and CYP2C9*3 1075 A>C (rs1057910) was performed by polymerase chain reaction (PCR) and pyrosequencing 33 according to the manufacturer's recommendations (Qiagen, Valencia, CA). The PCR and sequencing primers for PCR and pyrosequencing reactions are shown in Supporting Table S1. ...
Genotype-based algorithms that include VKORC1 and CYP2C9 genotypes are less predictive of warfarin dose variability in Africans as opposed to Europeans. Polymorphisms in GGCX, FPGS, or STX1B are associated with warfarin dose requirements in African-Americans. We sought to determine if they influenced warfarin dose in European-Americans, and another African population, specifically Egyptians. We genotyped 529 adults (n = 325 European-Americans, 204 Egyptians) on a stable warfarin dose for GGCX rs12714145 and rs10654848, FPGS rs7856096, and STX1B rs4889606. Rs12714145, rs10654848, and rs7856096 were not associated with warfarin dose, whereas STX1B rs4889606 was a significant determinant in univariate analysis (P < 0.0001) in both cohorts. However, STX1B rs4889606 was in high linkage disequilibrium with VKORC1-1639 G>A, and was no longer significant after including VKORC1-1639 G>A in the regression model. Based on these data, the polymorphisms do not appear to influence, in a clinically important way, warfarin dose requirements in European-Americans and Egyptians. This article is protected by copyright. All rights reserved.
... 4 Genotyping INVEST Genomic DNA was extracted from buccal cells collected in mouthwash samples according to standard protocols. 18 All 1741 INVEST-GENES participants were successfully genotyped on the HumanCVD Genotyping BeadChip (Illumina, San Diego, CA), a gene-centric array containing %50 000 SNPs in %2100 genes associated with cardiovascular, inflammatory, and metabolic processes. 13 Genotypes were called using GenomeStudio software version 2011.1 and Genotyping Module version 1.9 calling algorithm (Illumina, San Diego, CA). ...
Background
Resistant hypertension (RHTN), defined by lack of blood pressure (BP) control despite treatment with at least 3 antihypertensive drugs, increases cardiovascular risk compared with controlled hypertension. Yet, there are few data on genetic variants associated with RHTN.
Methods and Results
We used a gene‐centric array containing ≈50 000 single‐nucleotide polymorphisms (SNPs) to identify polymorphisms associated with RHTN in hypertensive participants with coronary artery disease (CAD) from INVEST‐GENES (the INnternational VErapamil‐SR Trandolapril STudy—GENEtic Substudy). RHTN was defined as BP≥140/90 on 3 drugs, or any BP on 4 or more drugs. Logistic regression analysis was performed in European Americans (n=904) and Hispanics (n=837), using an additive model adjusted for age, gender, randomized treatment assignment, body mass index, principal components for ancestry, and other significant predictors of RHTN. Replication of the top SNP was conducted in 241 European American women from WISE (Women's Ischemia Syndrome Evaluation), where RHTN was defined similarly. To investigate the functional effect of rs12817819, mRNA expression was measured in whole blood. We found ATP2B1 rs12817819 associated with RHTN in both INVEST European Americans (P‐value=2.44×10−3, odds ratio=1.57 [1.17 to 2.01]) and INVEST Hispanics (P=7.69×10−4, odds ratio=1.76 [1.27 to 2.44]). A consistent trend was observed at rs12817819 in WISE, and the INVEST‐WISE meta‐analysis result reached chip‐wide significance (P=1.60×10−6, odds ratio=1.65 [1.36 to 1.95]). Expression analyses revealed significant differences in ATP2B1 expression by rs12817819 genotype.
Conclusions
The ATP2B1 rs12817819 A allele is associated with increased risk for RHTN in hypertensive participants with documented CAD or suspected ischemic heart disease.
Clinical Trial Registration
URL: www.clinicaltrials.gov; Unique identifiers: NCT00133692 (INVEST), NCT00000554 (WISE).
... United States and Puerto Rico. Genomic DNA was collected using buccal cells from mouthwash samples as previously described (Andrisin et al., 2002). All patients provided written informed consent for participation in the genetic substudy and the study was approved by the University of Florida IRB. ...
... Presently, visual inspection is the only proven screening method regularly used in clinical settings. Collection of oral rinse (mouthwash) samples is a simple, noninvasive method for obtaining DNA from the upper aerodigestive tract epithelium [269,270]. ...
... Se realizó la actividad en dos sesiones de 3 horas de duración cada una. En la primera de ellas se realizó el aislamiento de ADN de las diferentes muestras biológicas siguiendo protocolos previamente descritos, escogiéndose varios métodos sencillos y que no involucran el manejo y uso de sustancias toxicas 9,10,11,12,13 . Los métodos utilizados permitieron la visualización de ADN de alto peso molecular al ser analizados mediante electroforesis, y suficiente material genético de óptima calidad para ser amplificado mediante PCR. ...
Recent progress of scientific research has notably increased the basic knowledge in genetic and molecular biology, required by medical doctors. Biochemistry educators in medical schools are challenged not only, to provide students with a strong and updated base with this new information, but also to engage them into the scientific process. One of the newest educational tools that could provide this achievement is the acquisition of information by learning-doing, like laboratory activities. In order to evaluate the impact of laboratory work over medical development a pilot experience was held at the Biochemistry Department of Luis Razetti Medical School, at Universidad Central de Venezuela from 2005 to 2006. Basic techniques in molecular biology were introduced to registered students and they were able to isolate DNA from different human samples, perform a PCR reaction and visualize the PCR product in agarose electrophoresis gel. We also included information about ethical issues regarding information for genetic and genomic research, such an informed consent and donation of biological samples to biobank, very actual and important information for medical doctor.At the end of the activity, the students were asked, by using a voluntary and anonymous survey, about the impact of the laboratory knowledge acquisition. The result of the survey reported, that the students considered these laboratory activities important and useful tools in their learning process. We received their suggestion to include more laboratory activities in the Biochemistry curricular program.
... The majority of studies use whole blood as a source of DNA, since it supplies genomic DNA and is easy to obtain in a clinical setting. Alterna tive sample types include buccal swabs or saliva, both of which are ideal for use in nonclinical settings and for shipping by mail [1,3]. ...
Background & aim:
Results from different pharmacogenetic association studies in colorectal cancer are often conflicting. Both peripheral blood and formalin-fixed, paraffin-embedded (FFPE) tissue are routinely used as DNA source. This could cause bias due to somatic alterations in tumor tissue, such as loss of heterozygosity. We therefore compared genotypes in DNA from peripheral blood and FFPE colorectal tumor samples for SNPs with putative influence on the cytotoxicity of chemotherapy.
Materials & methods:
Eleven SNPs in nine genes involved in anticancer drug metabolism or efficacy were determined in matched samples from blood and FFPE tissue of colorectal tumors by pyrosequencing and TaqMan(®) techniques. The κ-statistic was calculated to assess concordance.
Results:
A total of 149 paired FFPE tissue and EDTA blood DNA samples were available for comparison. Overall, 20 out of 1418 genotypes were discordant (1.4%); in ten cases, loss of heterozygosity could not be ruled out. Only GSTP1 showed significant discordance between FFPE tissue and blood genotype (κ = 0.947; 95% CI: 0.896-0.998).
Conclusion:
FFPE tissue-derived DNA can be used as a valid proxy for germline DNA for a selection of SNPs in (retrospective) pharmacogenetic association studies in colorectal cancer. However, for future studies, genotyping of blood-derived DNA is preferred.
... For the screening genetic sample, a buccal cell sample was collected via a mouthwash method [29]. Genomic DNA was isolated using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). ...
Objectives:
The objectives of this study were to determine if ABCB1 polymorphisms are associated with interindividual variability in sitagliptin pharmacokinetics and if atorvastatin alters the pharmacokinetic disposition of sitagliptin in healthy volunteers.
Methods:
In this open-label, randomized, two-phase crossover study, healthy volunteers were prospectively stratified according to ABCB1 1236/2677/3435 diplotype (n = 9, CGC/CGC; n = 10, CGC/TTT; n = 10, TTT/TTT). In one phase, participants received a single 100 mg dose of sitagliptin; in the other phase, participants received 40 mg of atorvastatin for 5 days, with a single 100 mg dose of sitagliptin administered on day 5. A 24-h pharmacokinetic study followed each sitagliptin dose, and the study phases were separated by a 14-day washout period.
Results:
Sitagliptin pharmacokinetic parameters did not differ significantly between ABCB1 CGC/CGC, CGC/TTT, and TTT/TTT diplotype groups during the monotherapy phase. Atorvastatin administration did not significantly affect sitagliptin pharmacokinetics, with geometric mean ratios (90 % confidence intervals) for sitagliptin maximum plasma concentration, plasma concentration-time curve from zero to infinity, renal clearance, and fraction of sitagliptin excreted unchanged in the urine of 0.93 (0.86-1.01), 0.96 (0.91-1.01), 1.02 (0.93-1.12), and 0.98 (0.90-1.06), respectively.
Conclusions:
ABCB1 CGC/CGC, CGC/TTT, and TTT/TTT diplotypes did not influence sitagliptin pharmacokinetics in healthy volunteers. Furthermore, atorvastatin had no effect on the pharmacokinetics of sitagliptin in the setting of ABCB1 CGC/CGC, CGC/TTT, and TTT/TTT diplotypes.
... Genomic DNA was isolated from whole blood or buccal cells using previously described methods [35]. CXCL5 genotypes were determined by polymerase chain reaction (PCR) and pyrosequencing (Qiagen, Valencia, CA, USA) as we have previously described [36]. ...
Objective
Leukocyte count has been associated with blood pressure, hypertension, and hypertensive complications. We hypothesized that polymorphisms in the CXCL5 gene, which encodes the neutrophilic chemokine ENA-78, are associated with blood pressure in cardiovascular disease (CVD)-free adults and that these polymorphisms are functional.
Methods and results
A total of 192 community-dwelling participants without CVD or risk equivalents were enrolled. Two CXCL5 polymorphisms (−156 G > C (rs352046) and 398 G > A (rs425535)) were tested for associations with blood pressure. Allele-specific mRNA expression in leukocytes was also measured to determine whether heterozygosity was associated with allelic expression imbalance. In −156 C variant carriers, systolic blood pressure (SBP) was 7 mmHg higher than in −156 G/G wild-type homozygotes (131 ± 17 vs. 124 ± 14 mmHg; P = 0.008). Similarly, diastolic blood pressure (DBP) was 4 mmHg higher in −156 C variant carriers (78 ± 11 vs. 74 ± 11 mmHg; P = 0.013). In multivariate analysis of SBP, age, sex, body mass index, and the −156 G > C polymorphism were identified as significant variables. Age, sex, and the −156 G > C SNP were further associated with DBP, along with white blood cells. Allelic expression imbalance and significantly higher circulating ENA-78 concentrations were noted for variant carriers.
Conclusion
CXCL5 gene polymorphisms are functional and associated with variable blood pressure in CVD-free individuals. The role of CXCL5 as a hypertension- and CVD-susceptibility gene should be further explored.
... A previously described, noninvasive mouthwash technique was used to collect DNA. 25 This collection method provides sufficient quantity and stability of genomic DNA when samples are stored at room temperature for up to 90 days. Subjects were told to refrain from any oral hygiene activity, such as brushing teeth, 30 minutes before collection of DNA to increase buccal cell collection. ...
Background: Polymorphisms of NAT-2 acetylation contribute to drug toxicity, efficacy and cancer risk. Predominance of slow acetylation (SA) phenotype in ethnically distinct populations may have clinical implications for drug selection and cancer risk. The purpose of this study was to determine the genetic basis of SA phenotype predominance in Minnesota Hmong.Methods: Urine and DNA were obtained for phenotype and genotype analysis from unrelated healthy Hmong 18 and 65 years of age. Urinary molar ratios (MR) of caffeine metabolites (AFMU/1X) identified rapid acetylators (RA) phenotypes with a MR >= 0.6 and SA with MR < 0.6. Direct sequencing of the NAT-2 coding-region followed by cloning techniques for ambiguous genotypes identified individuals homozygous or heterozygous with a *4 and *13 allele as RA and variants as SA by genotype.Results: From 61 subjects (3011 years, 27 male), analysis of 51 urine-DNA pairs identified 46 (90.2%) SA and 5 (9.8%) RA by phenotype. In contrast, genotypic analysis identified 5 (9.8%) SA and 46 (90.2%) RA. An 84% discordance between phenotype and genotype was observed. Direct sequencing did not reveal novel NAT-2 polymorphisms.Conclusions: Genotypic analysis appears to demonstrate considerable discordance with the phenotype in Hmong. Genotyping alone, without a metabolic probe, would not have accurately predicted acetylation phenotype.
... For DNA collection during the screening process, subjects were asked to swish vigorously 15 ml of Scope® mouthwash (Procter and Gamble, Cincinnati, OH, USA) for 1 min and then expectorate into a sterile collection tube [38]. Genomic DNA was isolated from buccal cells using a commercially available kit (QIAmp DNA Mini Kit, Qiagen, Valencia, CA, USA). ...
Aim:
The objective of this study was to determine the extent to which the CYP2C8*3 allele influences pharmacokinetic variability in the drug-drug interaction between gemfibrozil (CYP2C8 inhibitor) and pioglitazone (CYP2C8 substrate).
Methods:
In this randomized, two phase crossover study, 30 healthy Caucasian subjects were enrolled based on CYP2C8*3 genotype (n = 15, CYP2C8*1/*1; n = 15, CYP2C8*3 carriers). Subjects received a single 15 mg dose of pioglitazone or gemfibrozil 600 mg every 12 h for 4 days with a single 15 mg dose of pioglitazone administered on the morning of day 3. A 48 h pharmacokinetic study followed each pioglitazone dose and the study phases were separated by a 14 day washout period.
Results:
Gemfibrozil significantly increased mean pioglitazone AUC(0,∞) by 4.3-fold (P < 0.001) and there was interindividual variability in the magnitude of this interaction (range, 1.8- to 12.1-fold). When pioglitazone was administered alone, the mean AUC(0,∞) was 29.7% lower (P = 0.01) in CYP2C8*3 carriers compared with CYP2C8*1 homozygotes. The relative change in pioglitazone plasma exposure following gemfibrozil administration was significantly influenced by CYP2C8 genotype. Specifically, CYP2C8*3 carriers had a 5.2-fold mean increase in pioglitazone AUC(0,∞) compared with a 3.3-fold mean increase in CYP2C8*1 homozygotes (P = 0.02).
Conclusion:
CYP2C8*3 is associated with decreased pioglitazone plasma exposure in vivo and significantly influences the pharmacokinetic magnitude of the gemfibrozil-pioglitazone drug-drug interaction. Additional studies are needed to evaluate the impact of CYP2C8 genetics on the pharmacokinetics of other CYP2C8-mediated drug-drug interactions.
... Buccal tissue samples were obtained by mouthwash and genomic DNA was isolated as previously described [29]. We selected the following seven polymorphisms in the LXRA gene region (NR1H3 ±5kb) using a tag SNP approach in Blacks and Whites (CEU/CEPH and Yoruban) from the International HapMap project (Haploview software): rs11039149, rs12221497, rs2279238, rs7120118, rs326213, rs11039159 and rs10501321. ...
Liver X receptor-α (LXRA) is a nuclear receptor that regulates genes important in cholesterol homeostasis and inflammation. Several single nucleotide polymorphisms (SNPs) in the LXRA gene (NR1H3) have been earlier associated with metabolic phenotypes (dyslipidemia and elevated body mass index). Metabolic dysregulation is a major contributor to coronary disease; therefore, we assessed LXRA in International Verapamil Sustained Release SR Trandolapril Study Genetic Substudy (INVEST-GENES), a genetic-substudy of a large clinical trial in patients with hypertension and coronary artery disease.
Seven tag SNPs in the LXRA gene region (NR1H3) were selected for study: rs11039149, rs12221497, rs2279238, rs7120118, rs326213, rs11039159, and rs10501321. One thousand fifty-nine patients were genotyped from the INVEST-GENES case-control set (verapamil-sustained release-based or atenolol-based treatment strategies) that comprised of 297 cases frequency matched (approximately 2.5:1) with that of event-free controls by sex and race. The primary outcome was defined as first occurrence of all-cause death, nonfatal myocardial infarction, or nonfatal stroke. Adjusted odds ratios (ORs) were calculated using logistic regression.
Three of the seven SNPs were associated with significant effects on the primary outcome in nonBlacks. The variant G allele of rs11039149 and the variant A allele of rs12221497 were associated with reduced risk of experiencing the primary outcome [OR: 0.62, confidence interval (CI): 0.45-0.85, P=0.003 and OR: 0.60, CI: 0.39-0.91, P=0.016, respectively]. The rs2279238 genotype was associated with a significant increase in risk for the primary outcome (OR: 1.42, CI: 1.03-1.95, P=0.03). Furthermore, there was a significant genotype-treatment strategy interaction for carriers of the variant T allele of rs2279238 (OR for verapamil-sustained release strategy compared with atenolol strategy: 2.86, CI: 1.50-5.46, P=0.0015). Diplotype analyses showed that the SNPs are rarely coinherited and support the directionally opposite effects of the SNPs on the primary outcome.
LXRA genotypes were associated with variable risk for cardiovascular outcomes and pharmacogenetic effect in INVEST-GENES. These novel findings suggest that LXRA is a genetic/pharmacogenetic target that should be further explored.
... Genomic DNA was collected using buccal cells from mouthwash samples as previously described. 17 5,598 samples still have sufficient quality and quantity DNA and were tested here. All patients provided written informed consent for participation in the main INVEST and in the genetic substudy and both studies were approved by the University of Florida Institutional Review Board. ...
Single-nucleotide polymorphisms (SNPs) within the regulatory β2 subunit of the voltage-gated calcium channel (CACNB2) may contribute to variable treatment response to antihypertensive drugs and adverse cardiovascular outcomes.
SNPs in CACNB2 from 60 ethnically diverse individuals were identified and characterized. Three common SNPs (rs2357928, rs7069292, and rs61839258) and a genome-wide association study-identified intronic SNP (rs11014166) were genotyped for a clinical association study in 5598 hypertensive patients with coronary artery disease randomized to a β-blocker (BB) or a calcium channel blocker (CCB) treatment strategy in the INternational VErapamil SR-Trandolapril STudy GENEtic Substudy (INVEST-GENES). Reporter gene assays were conducted on the promoter SNP, showing association with clinical outcomes. Twenty-one novel SNPs were identified. A promoter A>G SNP (rs2357928) was found to have significant interaction with treatment strategy for adverse cardiovascular outcomes (P for interaction, 0.002). In whites, rs2357928 GG patients randomized to CCB were more likely to experience an adverse outcome than those randomized to BB treatment strategy, with adjusted hazard ratio (HR) (CCB versus BB) of 2.35 (95% CI, 1.19 to 4.66; P=0.014). There was no evidence for such treatment difference in AG (HR, 1.16; 95% CI, 0.75 to 1.79; P=0.69) and AA (HR, 0.63; 95% CI, 0.36 to 1.11; P=0.11) patients. This finding was consistent in Hispanics and blacks. CACNB2 rs11014166 showed similar pharmacogenetic effect in Hispanics, but not in whites or blacks. Reporter assay analysis of rs2357928 showed a significant increase in promoter activity for the G allele compared to the A allele.
These data suggest that genetic variation within CACNB2 may influence treatment-related outcomes in high-risk patients with hypertension.
... In contrast with previous reports, during our sample collection process, the cytobrush was not allowed to come into contact with teeth, in order to reduce bacterial contamination. This is a major difference with the other studies, in which the results were significantly influenced by the presence of bacteria (Feigelson et al., 2001), since the previous studies indicated that a high quantity of the DNA obtained was the result of oral microbial contamination (Feigelson et al., 2001; Andrisin et al., 2002). To minimize contamination with any remaining food and/or PCR inhibitors (e.g., lipstick ), as well as bacterial proliferation, participants were requested to rinse their mouth with water prior to sampling. ...
We evaluated the cost-effectiveness of using buccal swab brushes in comparison with blood samples for obtaining DNA for large epidemiological studies of the elderly population. The data reported here are from the third phase of the Integral Study of Depression among the Elderly in Mexico City's Mexican Institute of Social Security, conducted in 2007. The total cost of the two procedures was determined. The measurement of effectiveness was the quality and quantity of DNA measured in ng/μL and the use of this DNA for the determination of apolipoprotein E (APO E) polymorphism by PCR. Similar rates of amplification were obtained with the two techniques. The cost of the buccal swab brushes, including sample collection and DNA extraction, was US23.35. Using the buccal swab, the savings was US$6.72 per patient (P < 0.05). The effectiveness was similar. Quantity and quality of DNA obtained were similar for the oral and blood procedures, demonstrating that the swab brush technique offers a feasible alternative for large-scale epidemiological studies.
... After written informed consent and authorization for reviewing medical records had been obtained, a buccal cell sample was collected for genetic analysis, as previously described. 45 Demographic, clinical, and social history were assessed through patient interviews and reviews of the medical records. The study was approved by the institutional review board of each institution. ...
The objective of this study was to determine whether, in African-American patients, additional vitamin K oxidoreductase complex subunit 1 (VKORC1), cytochrome P450 2C9 (CYP2C9), CYP4F2, or apolipoprotein E (APOE) polymorphisms contribute to variability in the warfarin maintenance dose beyond what is attributable to the CYP2C9*2 and *3 alleles and the VKORC1 -1639G>A genotype. In a cohort of 226 African-American patients, weekly warfarin dose requirements were lower in those with the CYP2C9*8 allele (34 (30-47) mg; P = 0.023) and the CYP2C9 *2, *3, *5, *6, or *11 allele (33(28-40 mg); P < 0.001) as compared with those with the CYP2C9*1/*1 genotype (43 (35-56) mg). The combination of CYP2C9 alleles, VKORC1 -1639G>A genotype, and clinical variables explained 36% of the interpatient variability in warfarin dose requirements. By comparison, a model without the CYP2C9*5, *6, *8, and *11 alleles explained 30% of the variability in dose. No other VKORC1, CYP4F2, or APOE polymorphism contributed to the variance. The inclusion of additional CYP2C9 variants may improve the predictive ability of warfarin dosing algorithms for African Americans.
... Molecular epidemiologic studies of low prevalence diseases require access to patients who may not live within easy commuting range of the investigator. Fortunately, DNA can be reliably obtained from buccal mucosa by the mouthwash method without having the study participant travel to the study site [2, 8]. However, if the subject is not to be examined by the investigators, a reliable instrument is needed to confirm the presence of the disease process under study. ...
In order to determine the reliability of a self-administered instrument to diagnose excessive sweating conditions, including palmar hyperhidrosis (PH), we designed two successive questionnaires and compared responses with physical examination and sweat measurement in normal volunteers and a cohort of patients with documented PH. The reliable diagnosis of PH via questionnaire would enable molecular epidemiological studies without the need for physical examination or direct sweat measurement.
Subjects self identified as either normal or affected by PH. Each completed one or both questionnaires and underwent physical examination. Sweat production from the thenar eminence and forehead was measured at rest and following mental/emotional stress. Correlation among sweat measurement, physical examination, and questionnaire score was assessed.
Forty-seven subjects enrolled in the study, 29 of whom underwent sweat measurements. The participants' perception of whether they were affected agreed with the examiner's visual and tactile observation of PH in all cases (P < 0.0005). The mean peak sweat rate for those participants with PH was 1.59 mg/cm(2)/min, while that of the normal cohort was 0.37 mg/cm(2)/min (P = 0.001). The mean questionnaire #1 and #2 scores for those participants with PH and the normal cohort was 7.10 versus 0.36 (P = 0.0005) and 5.145 versus 0.045 (P = 0.0005), respectively. Peak sweat rate correlated with questionnaire score (Pearson correlation coefficient = 0.723).
Palmar hyperhidrosis can be accurately diagnosed via questionnaire. Molecular epidemiological studies of PH may be reliably conducted without the need for direct physical examination.
... After obtaining informed consent, a pre-screening genetic sample was collected from each potential subject via a mouthwash collection method [31]. Subjects were asked vigorously to swish 15 ml of Scope mouthwash (Procter & Gamble, Cincinnati, OH, USA) for one minute and then expectorate into a sterile collection tube. ...
Polymorphisms in drug transporter genes and/or drug-metabolising enzyme genes may contribute to inter-individual variability in rosiglitazone pharmacokinetics in humans. We sought to determine the joint effects of polymorphisms in the SLCO1B1 drug transporter gene and the cytochrome P450 ( CYP ) 2C8-metabolising enzyme gene on rosiglitazone pharmacokinetics in healthy volunteers. Healthy Caucasian subjects were prospectively enrolled on the basis of SLCO1B1 521 T > C genotype. Additionally, subjects were genotyped for SLCO1B1 -11187 G > A, -10499 A > C and 388 A > G polymorphisms, and the CYP2C8*3 polymorphism. SLCO1B1 haplotypes and diplotypes were computationally assigned. Rosiglitazone plasma concentrations were determined by high-performance liquid chromatography and analysed using non-compartmental methods. The study population consisted of 26 subjects, with a mean age of 33 +/- 9 years, and a mean weight of 66.6 +/- 11.7 kg. There were no significant differences in rosiglitazone pharmacokinetic parameters between SLCO1B1 diplotype groups. Subjects with the CYP2C8*1/*3 genotype ( n = 7), however, had significantly lower rosiglitazone area under the plasma concentration-time curve (AUC) and significantly higher rosiglitazone oral clearance, compared with CYP2C8 wild-type homozygotes ( n = 19). Stepwise linear regression analysis revealed that CYP2C8 genotype ( p = 0.006) and weight ( p = 0.022) were significant predictors of rosiglitazone AUC (overall p = 0.002; R 2 = 41.6 per cent). We concluded that polymorphisms in the CYP2C8 drug-metabolising enzyme gene, but not the SLCO1B1 drug transporter gene, significantly influence rosiglitazone disposition in humans. Future studies examining the influence of CYP2C8 genotypes and haplotypes on thiazolidinedione disposition and response in patients with type 2 diabetes are warranted.
... Buccal cells are commonly collected with a small cyto-brush or tongue depressor, which is then rinsed in conical centrifuge tubes containing stabilizing buffer [66]. Use of commercial mouthwash and simple mouth rinse for buccal cell collection has recently gained popularity [44,67,68]). Collection of buccal cells can also be done on pretreated cards [69,70]. ...
Molecular epidemiology uses biomarkers and advanced technology to refine the investigation of the relationship between environmental exposures and diseases in humans. It requires careful handling and storage of precious biological samples with the goals of obtaining a large amount of information from limited samples, and minimizing future research costs by use of banked samples. Many factors, such as tissue type, time of collection, containers used, preservatives and other additives, transport means and length of transit time, affect the quality of the samples and the stability of biomarkers and must be considered at the initial collection stage. An efficient study design includes provisions for further processing of the original samples, such as cryopreservation of isolated cells, purification of DNA and RNA, and preparation of specimens for cytogenetic, immunological and biochemical analyses. Given the multiple uses of the samples in molecular epidemiology studies, appropriate informed consent must be obtained from the study subjects prior to sample collection. Use of barcoding and electronic databases allow more efficient management of large sample banks. Development of standard operating procedures and quality control plans is a safeguard of the samples' quality and of the validity of the analyses results. Finally, specific state, federal and international regulations are in place regarding research with human samples, governing areas including custody, safety of handling, and transport of human samples, as well as communication of study results.Here, we focus on the factors affecting the quality and the potential future use of biological samples and some of the provisions that must be made during collection, processing, and storage of samples, based on our experience in the Superfund Basic Research Program and Children's Environmental Health Center, at the University of California, Berkeley.
... Buccal cells are commonly collected with a small cyto-brush or tongue depressor, which is then rinsed in conical centrifuge tubes containing stabilizing buffer [59]. Use of commercial mouthwash and simple mouth rinse606162 has recently gained popularity. Buccal cells can also be collected on pre-treated cards [63,64]. Exfoliated cells are easier to collect than blood and do not require highly trained personnel such as a pediatric phlebotomist. ...
In the last decade, molecular epidemiological studies have provided new perspectives on studying environmental risks in pediatric populations, based on the growing understanding that children may be more susceptible to toxicants than adults. Protecting children's health is a social priority, and specific research programs have been initiated with this purpose in the United States and Europe. These programs address the development of (i) less invasive methods for biological specimens collection, (ii) specific tools for interpretation and validation of biomarkers, (iii) methods for translating biomarker results into intervention strategies and for integrating them with environmental monitoring and health data, (iv) optimal ways to obtain consent and provide information to children and/or their parents participating in the studies and (v) techniques for the effective communication with policy makers and the public. Critical issues in children's environmental research discussed in this paper include specific needs of study design, exposure assessment, sample collection and ethics. Special consideration is given to the autonomy of the child in giving consent, the details and nature of the information provided, and the need to warrant controlled access to sensitive information. The use of incentives such as gifts and payment to ensure the participation of school-aged children is specifically discussed. Examples of field studies that are focused on the effects of pesticides, air pollution and formaldehyde are used to illustrate advantages and limitations of biomarker studies in children.
Single‐nucleotide polymorphisms (SNP) of ATP2B1 gene are associated with essential hypertension but their association with resistant hypertension (RHT) remains unexplored. The authors examined the relationship between ATP2B1 SNPs and RHT by genotyping 12 SNPs in ATP2B1 gene of 1124 Japanese individuals with lifestyle‐related diseases. Patients with RHT had inadequate blood pressure (BP) control using three antihypertensive drugs or used ≥4 antihypertensive drugs. Patients with controlled hypertension had BP controlled using ≤3 antihypertensive drugs. The association between each SNP and RHT was analyzed by logistic regression. The final cohort had 888 (79.0%) and 43 (3.8%) patients with controlled hypertension and RHT, respectively. Compared with patients homozygous for the minor allele of each SNP in ATP2B1, a significantly higher number of patients carrying the major allele at 10 SNPs exhibited RHT (most significant at rs1401982: 5.8% vs. 0.8%, p = .014; least significant at rs11105378: 5.7% vs. 0.9%, p = .035; most nonsignificant at rs12817819: 5.1% vs. 10%, p = .413). After multivariate adjustment for age, sex, systolic BP, and other confounders, the association remained significant for rs2681472 and rs1401982 (OR: 7.60, p < .05 and OR: 7.62, p = .049, respectively). Additionally, rs2681472 and rs1401982 were in linkage disequilibrium with rs11105378. This study identified two ATP2B1 SNPs associated with RHT in the Japanese population. rs1401982 was most closely associated with RHT, and major allele carriers of rs1401982 required significantly more antihypertensive medications. Analysis of ATP2B1 SNPs in patients with hypertension can help in early prediction of RHT and identification of high‐risk patients who are more likely to require more antihypertensive medications.
Recent progress of scientific research has notably increased the basic knowledge in genetic and molecular biology, required by medical doctors. Biochemistry educators in medical schools are challenged not only, to provide students with a strong and updated base with this new information, but also to engage them into the scientific process. One of the newest educational tools that could provide this achievement is the acquisition of information by learning-doing, like laboratory activities. In order to evaluate the impact of laboratory work over medical development a pilot experience was held at the Biochemistry Department of "Luis Razetti" Medical School, at Universidad Central de Venezuela from 2005 to 2006. Basic techniques in molecular biology were introduced to registered students and they were able to isolate DNA from different human samples, perform a PCR reaction and visualize the PCR product in agarose electrophoresis gel. We also included information about ethical issues regarding information for genetic and genomic research, such an informed consent and donation of biological samples to biobank, very actual and important information for medical doctor.At the end of the activity, the students were asked, by using a voluntary and anonymous survey, about the impact of the laboratory knowledge acquisition. The result of the survey reported, that the students considered these laboratory activities important and useful tools in their learning process. We received their suggestion to include more laboratory activities in the Biochemistry curricular program.
In the post-genomic era. It is necessary to gain a better understanding of the relationship between genes and oral disease. For example, single nucleotide polymorphisms (SNPs) are the most common form of DNA sequence variation and searching for SNPs covers many genes. To perform genetic testing such as testing for SNPs, DNA samples need to be obtained from patients. In the near future, genetic testing will become widespread as a chair-side procedure in general practice. It would be optimal to use a method that results in minimum bodily injury and that does not cause anxiety to patients. Here, we report a simple method of obtaining lingual mucosal cells with a toothbrush for extraction of DNA samples.
Comparative Diagnostic Pharmacology: Clinical and Research Applications in Living-System Models is the first evidence-based reference text devoted exclusively to the subject of applying pharmaceutical and biopharmaceutical agents as diagnostic probes in clinical medicine and investigative research.This unique and groundbreaking book is a versatile guide for clinicians and researchers interested in using pharmacologic agents to: Diagnose disease. Assess physiological processes. Identify the appropriateness of a therapeutic agent. Determine appropriate dosing for therapeutic use. Extensively referenced and organized by major body systems, individual topics are listed in an evidence-based format according to specific disease processes or physiological processes of interest. Each entry also includes information on the mechanism of action, administration, and diagnostic interpretation. Descriptions have been provided for the application of diagnostic pharmaceuticals to assess a wide spectrum of diseases and physiological processes relevant to the fields of veterinary and human medicine. Comparative Diagnostic Pharmacology is useful not merely for pharmaceutical-oriented research investigations, but it will also prove invaluable for the monitoring and evaluation of physiological responses and disease processes in animal models.
Background: Relative to blood samples, mouth swab samples are more beneficial for apolipoprotein E gene polymorphism analysis among large cohorts. However, agreement has not yet been reached about how to extract genomic DNA form mouth swab samples. Objective: To develop an appropriate method to extract genomic DNA form mouth swab samples, which are suitable for apolipoprotein E gene polymorphism analysis. Methods: Fifty mouth swab samples from patients with sporadic Alzheimer's disease were collected. Magnetic nanoparticles and PicoDNA trace nucleic acid extraction kit were used to extract genomic DNA form mouth swab samples. And the purity and concentration of the genomic DNA extracted by the two methods were analyzed. Then PCR amplifications and DNA electrophoresis were performed to confirm whether the genomic DNA was able to amplify desired DNA fragments. DNA sequencing was applied to analyze apolipoprotein E gene polymorphisms. Results and Conclusion: Genomic DNA extracted by the two methods was of high purity. The concentration of genomic DNA extracted by magnetic nanoparticles was higher than by PicoDNA trace nucleic acid extraction kit, and the difference had statistical significance (P < 0.05). All the genomic DNA were able to performed PCR amplifications to obtain desired PCR products, but results of DNA electrophoresis showed that DNA fragments were more clear by nanoparticles method. The results of DNA sequencing were the same by the two methods. The distribution of ε2, ε3, ε4 genotypes of apolipoprotein E gene was 6%, 71%, 23%, respectively. Magnetic nanoparticles were better than PicoDNA trace nucleic acid extraction kit for extracting genomic DNA form mouth swab samples for apolipoprotein E gene polymorphism analysis.
Aim:
The objective of this study was to determine the additional contribution of NQO1 and CYP4F2 genotypes to warfarin dose requirements across two racial groups after accounting for known clinical and genetic predictors.
Patients & methods:
The following were assessed in a cohort of 260 African-Americans and 53 Hispanic-Americans: clinical data; NQO1 p.P187S (*1/*2); CYP2C9*2, *3, *5, *6, *8 and *11; CYP4F2 p.V433M; and VKORC1 c.-1639G>A genotypes.
Results:
Both the CYP4F2 433M (0.23 vs 0.06; p < 0.05) and NQO1*2 (0.27 vs 0.18; p < 0.05) allele frequencies were higher in Hispanic-Americans compared with African-Americans. Multiple regression analysis in the Hispanic-American cohort revealed that each CYP4F2 433M allele was associated with a 22% increase in warfarin maintenance dose (p = 0.019). Possession of the NQO1*2 allele was associated with a 34% increase in warfarin maintenance dose (p = 0.004), while adjusting for associated genetic (CYP2C9, CYP4F2 and VKORC1) and clinical factors. In this population, the inclusion of CYP4F2 and NQO1*2 genotypes improved the dose variability explained by the model from 0.58 to 0.68 (p = 0.001), a 17% relative improvement. By contrast, there was no association between CYP4F2 or NQO1*2 genotype and therapeutic warfarin dose in African-Americans after adjusting for known genetic and clinical predictors.
Conclusion:
In our cohort of inner-city Hispanic-Americans, the CYP4F2 and NQO1*2 genotypes significantly contributed to warfarin dose requirements. If our findings are confirmed, they would suggest that inclusion of the CYP4F2 and NQO1*2 genotypes in warfarin dose prediction algorithms may improve the predictive ability of such algorithms in Hispanic-Americans.
Objectives
The objectives of this paper are to determine the storage stability of saliva at 37 °C over an 18-month period, and its influence on the DNA yield, purity, PCR protocols and genotyping efficacy.
Materials and methods
Of the 60 participants, blood samples were obtained from 10 and saliva from 50. Samples were subjected to different storage conditions: DNA extracted immediately; DNA extracted following storage at 37 °C for 1, 6, 12 and 18 months. Subsequently, DNA yield, OD260/280 and OD260/230 ratios were measured. The isolated DNA was used to amplify exons 0–7 of the RUNX2 gene and subsequently sequenced. Furthermore, 25 SNPs were genotyped.
Results
The mean DNA yield, OD260/280 and OD260/230 ratios obtained from blood were 67.4 ng/μl, 1.8 ± 0.05 and 1.8 ± 0.4 respectively. DNA yield obtained from saliva was significantly higher than blood (p < 0.0001), ranging from 97.4 to 125.8 ng/μl while the OD260/280 ratio ranged from 1.8 ± 0.13 to 1.9 ± 0.1. The success rates for the 25 SNPs ranged from 98 to 100 % for blood and 96–99 % for saliva samples with the genotype frequencies in Hardy–Weinberg equilibrium (>0.01).
Conclusions
Saliva can be stored at 37 °C for 18 months without compromising its quality and ability to endure genetic analyses.
Clinical relevance
Saliva is a viable source of human DNA to facilitate the feasibility of large-scale genetic studies.
Development of resistance to 1-β-arabinofuranosylcytosine (AraC) is a major obstacle in the treatment of patients with acute myeloid leukaemia (AML). Deficiency of functional deoxycytidine kinase (dCK) plays an important role in AraC resistance in vitro. We screened 5378 bp sequences of the dCK gene, including all exons and the 5′ flanking region, and identified two single nucleotide polymorphisms (SNPs) in the regulatory region (rSNPs) with high allele frequencies. These two rSNPs (-201C>T and -360C>G) formed two major haplotypes. Genotyping with sequencing and MassARRAY system among 122 AML patients showed that those with -360CG/-201CT and -360GG/-201TT compound genotypes (n = 41) displayed a favourable response to chemotherapy whereas those with -360CC/-201CC (n = 81) tended to have a poor response (P = 0.025). Moreover, real-time quantitative reverse transcriptase-polymerase chain reaction showed that patients with -360CG/-201CT and -360GG/-201TT genotypes expressed higher level of dCK mRNA compared to those with the -360CC/-201CC genotype (P = 0.0034). Luciferase-reporter assay showed that dCK 5′ regulatory region bearing -360G/-201T genotype alone had an eight-fold greater transcriptional activation activity compared to that with -360C/-201C genotype, whereas co-transfection of both -360G/-201T and -360C/-201C constructs mimicked the heterozygous genotype, which exhibited a four-fold greater activity compared to that with -360C/-201C. These results indicate that rSNP haplotypes of dCK gene may serve as a genetic marker for predicting drug responsiveness, which will be beneficial in establishing more effective AML chemotherapeutic regimens.
Health information technology (HIT) offers a resource for public empowerment through tailored information.
Use interactive community health events to improve awareness of chronic disease risk factors while collecting data to improve health.
Let's Get Healthy! is an education and research program in which participants visit interactive research stations to learn about their own health (diet, body composition, blood chemistry). HIT enables computerized data collection that presents participants with immediate results and tailored educational feedback. An anonymous wristband number links collected data in a population database. RESULTS AND LESSONS LEARNED: Communities tailor events to meet community health needs with volunteers trained to conduct research. Participants experience being a research participant and contribute to an anonymous population database for both traditional research purposes and open-source community use.
By integrating HIT with community involvement, health fairs become an interactive method for engaging communities in research and raising health awareness.
While Hispanics are the largest and most rapidly growing minority population in the United States, they are underrepresented in pharmacogenomic studies with warfarin. We sought to determine the combination of clinical and genetic influences of warfarin dose requirements in Hispanics. In addition, we tested the performance of published warfarin dosing algorithms derived from largely non-Hispanic cohorts in an inner-city U.S. Hispanic population. Genetic samples and clinical data were obtained from 50 Hispanics on a stable dose of warfarin. The contribution of cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex-1 (VKORC1) genotypes and clinical factors to warfarin dose requirements was determined. The correlation between the predicted dose using published algorithms and therapeutic dose was also assessed. Compared to the VKORC1-1639 GG genotype, warfarin dose requirements were 30% and 62% lower with the GA and AA genotypes, respectively (p=0.001). The combination of the VKORC1-1639G>A and CYP2C9 genotypes and clinical factors explained 56% of the inter-patient variability in warfarin dose. Warfarin dose predicted using algorithms derived from mostly non-Hispanic cohorts was significantly correlated with the therapeutic dose in our Hispanic cohort (r(2)=0.43 to 0.49; p<0.001); the predicted dose was within 1.0 mg/day of the therapeutic dose for 40% to 50% of patients. Our data suggest that factors influencing warfarin dose requirements in Hispanic Caucasians are similar to those previously described in European Caucasians and that dosing algorithms derived from non-Hispanic Caucasian cohorts are applicable to Hispanics living in the U.S.
The gene encoding the target of calcium channel blockers, the alpha1c-subunit of the L-type calcium channel (CACNA1C), has not been well characterized, and only small pharmacogenetic studies testing this gene have been published to date.
Resequencing of CACNA1C was performed followed by a nested case-control study of the INternational VErapamil SR/trandolapril STudy (INVEST) GENEtic Substudy (INVEST-GENES). Of 46 polymorphisms identified, 8 were assessed in the INVEST-GENES. Rs1051375 was found to have a significant interaction with treatment strategy (P=0.0001). Rs1051375 A/A genotype was associated with a 46% reduction in the primary outcome among those randomized to verapamil SR treatment, when compared with atenolol treatment (odds ratio 0.54 95% CI 0.32 to 0.92). In heterozygous A/G individuals, there was no difference in the occurrence of the primary outcome when randomized to verapamil SR versus atenolol treatment (odds ratio 1.47 95% CI 0.86 to 2.53), whereas homozygous G/G individuals had a greater than 4-fold increased risk of the primary outcome with verapamil treatment compared with those randomized to atenolol treatment (odds ratio 4.59 95% CI 1.67 to 12.67). We did not identify allelic expression imbalance or differences in mRNA expression in heart tissue by rs1051375 genotype.
Variation in CACNA1C is associated with treatment response among hypertensive patients with stable coronary artery disease. Our data suggest a genetically defined group of patients that benefit most from calcium channel blocker therapy, a group that benefits most from beta-blocker therapy, and a third group in which calcium channel blocker and beta-blocker therapy are equivalent.
Genetic variants of ACE are suspected risk factors in cardiovascular disease, but the alleles responsible for the variations remain unidentified. To search for regulatory polymorphisms, allelic angiotensin I-converting enzyme (ACE) mRNA expression was measured in 65 heart tissues, followed by genotype scanning of the ACE locus. Marked allelic expression imbalance (AEI) detected in five African-American subjects was associated with single-nucleotide polymorphisms (SNPs) (rs7213516, rs7214530, and rs4290) residing in conserved regions 2-3 kb upstream of ACE. Moreover, each of the SNPs affected transcription in reporter gene assays. SNPs rs4290 and rs7213516 were tested for associations with adverse cardiovascular outcomes in hypertensive patients with coronary disease (International Verapamil SR Trandolapril Study Genetic Substudy (INVEST-GENES), n = 1,032). Both SNPs were associated with adverse cardiovascular outcomes, largely attributable to nonfatal myocardial infarction in African Americans, showing an odds ratio of 6.16 (2.43-15.60) (P < 0.0001) for rs7213516. The high allele frequency in African Americans (16%) compared to Hispanics (4%) and Caucasians (<1%) suggests that these alleles contribute to variation between populations in cardiovascular risk and treatment outcomes.
An integrated portable genetic analysis microsystem including PCR amplification and capillary electrophoretic (CE) analysis coupled with a compact instrument for electrical control and laser-excited fluorescence detection has been developed. The microdevice contains microfabricated heaters, temperature sensors, and membrane valves to provide controlled sample positioning and immobilization in 200-nL PCR chambers. The instrument incorporates a solid-state laser and confocal fluorescence detection optics, electronics for sensing and powering the PCR reactor, and high-voltage power supplies for conducting CE separations. The fluorescein-labeled PCR products are amplified and electrophoretically analyzed in a gel-filled microchannel in <10 min. We demonstrate the utility of this instrument by performing pathogen detection and genotyping directly from whole Escherichia coli and Staphylococcus aureus cells. The E. coli detection assay consists of a triplex PCR amplification targeting genes that encode 16S ribosomal RNA, the fliC flagellar antigen, and the sltI shigatoxin. Serial dilution demonstrates a limit of detection of 2-3 bacterial cells. The S. aureus assay uses a femA marker to identify cells as S. aureus and a mecA marker to probe for methicillin resistance. This integrated portable genomic analysis microsystem demonstrates the feasibility of performing rapid high-quality detection of pathogens and their antimicrobial drug resistance.
To compare the accuracy, speed, and cost of two methodologies used for genotyping known variants in the cytochrome P450 (CYP) 2C9 metabolizing enzyme gene.
Comparative study.
University research center.
Fifteen-milliliter mouthwash samples collected from 253 subjects participating in a warfarin pharmacogenomic study.
Genotyping for the isoleucine-to-leucine change at codon 359 (Ile359Leu [*3] polymorphism) was performed by using the Pyrosequencing and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods in all 253 samples. Genotyping for the arginine-to-cysteine change at codon 144 (Arg144Cys [*2] polymorphism) was performed by using Pyrosequencing in all samples and by PCR-RFLP in a random subset of 136 samples.
Comparisons of genotyping success rates, time efficiency, and cost analyses were conducted for Pyrosequencing and PCR-RFLP at each variant site. Pyrosequencing and PCR-RFLP produced similar success rates on the first genotyping attempt for the Arg144Cys variant (93.3% vs 90.4%, respectively) and the Ile359Leu variant (83.8% vs 79.1%, respectively). With Pyrosequencing, genotyping 96 samples for either polymorphism could be performed in 1 hour. In contrast, genotyping 96 samples by RFLP took 10 hours for the Arg144Cys variant and 20 hours for the Ile359Leu variant. Total cost/sample for Arg144Cys genotyping was dollars 1.90 with PCR-Pyrosequencing and dollars 3.14 with PCR-RFLP. Total cost/sample for Ile359Leu genotyping was dollars 1.88 with PCR-Pyrosequencing and dollars 10.18 with PCR-RFLP CONCLUSION: Compared with RFLP, genotype determination by Pyrosequencing is a more time-efficient, cost-effective, and robust method for CYP2C9 genotyping. Because of its wide applicability and ease of use, Pyrosequencing is a promising technology for future pharmacogenomic investigations.
We developed a method for obtaining viable buccal cells from mouthwash samples for use as a source of mRNA and protein. Immunofluorescent analysis showed that most cells were derived from nonkeratinized parabasal epithelia, with a minor proportion of proliferative cells. Gene expression was detected in buccal cells using reverse transcription PCR, Western blot analysis, and immunofluorescence. Using a keratinocyte-specific medium, buccal cells could be cultured on Matrigel-coated permeable filters for up to 2 weeks while maintaining the expression of some epithelial-specific markers, including cytokeratin 13, cytokeratin 10, transferrin receptor, and beta-integrin. The basal marker cytokeratin 14 and Ki67, an indicator of cellular proliferation, were detected in a few cells. We show that buccal cells can be obtained from a noninvasive procedure for use as a source of material for biochemical analyses. A population of the buccal cells can be maintained in culture for up to 2 weeks using keratinocyte-specific medium in combination with extracellular matrix.
Development of resistance to 1-beta-arabinofuranosylcytosine (AraC) is a major obstacle in the treatment of patients with acute myeloid leukaemia (AML). Deficiency of functional deoxycytidine kinase (dCK) plays an important role in AraC resistance in vitro. We screened 5378 bp sequences of the dCK gene, including all exons and the 5' flanking region, and identified two single nucleotide polymorphisms (SNPs) in the regulatory region (rSNPs) with high allele frequencies. These two rSNPs (-201C>T and -360C>G) formed two major haplotypes. Genotyping with sequencing and MassARRAY system among 122 AML patients showed that those with -360CG/-201CT and -360GG/-201TT compound genotypes (n = 41) displayed a favourable response to chemotherapy whereas those with -360CC/-201CC (n = 81) tended to have a poor response (P = 0.025). Moreover, real-time quantitative reverse transcriptase-polymerase chain reaction showed that patients with -360CG/-201CT and -360GG/-201TT genotypes expressed higher level of dCK mRNA compared to those with the -360CC/-201CC genotype (P = 0.0034). Luciferase-reporter assay showed that dCK 5' regulatory region bearing -360G/-201T genotype alone had an eight-fold greater transcriptional activation activity compared to that with -360C/-201C genotype, whereas co-transfection of both -360G/-201T and -360C/-201C constructs mimicked the heterozygous genotype, which exhibited a four-fold greater activity compared to that with -360C/-201C. These results indicate that rSNP haplotypes of dCK gene may serve as a genetic marker for predicting drug responsiveness, which will be beneficial in establishing more effective AML chemotherapeutic regimens.
The authors assessed whether collection by mail of saliva and buccal cells for genetic analysis was feasible in participants recruited through the Internet. In 2003, 14,773 visitors of a smoking cessation website were invited by e-mail to take part in the study. Salivettes (plastic vials containing a cotton roll) were mailed to participants, for collection of saliva and buccal cells. Because of limited resources, the authors stopped recruitment when 392 participants (3% of 14,733) were registered. They received 315 saliva samples back (80% of 392). Salivary cotinine was analyzed in 145 daily smokers. Cotinine concentration could be assessed in 141 samples (97%) (range 0.7-899 ng/ml, median 260 ng/ml). DNA extraction was achieved in all the 285 samples in which it was attempted. Quality of DNA was assessed by optical density measurements and by polymerase chain reaction amplification of a gene coding for the alpha-4 nicotinic receptor, with the detection of a known polymorphism. Successful results were obtained in 235 samples (82% of 285). Thus collecting saliva by mail for cotinine and DNA analysis in participants recruited through the internet produced samples of good quality at a reasonable cost. This approach should be valuable for genetic epidemiology and pharmacogenetic research.
Polymorphisms of N-acetyltransferase 2 (NAT2) acetylation may influence drug toxicities and efficacy and are associated with a differential susceptibility to select cancers. Acetylation phenotype may have clinical implications. The purposes of this study were to determine the genetic basis of an apparent predominance of slow acetylation phenotype and to assess concordance with genotype in a population of Hmong residing in Minnesota. Urine and DNA obtained from unrelated Hmong 18 to 65 years of age were used to determine phenotype from caffeine metabolites, whereas direct nucleotide sequencing of the NAT2 coding region, followed by cloning, identified all known allelic variants. From 61 subjects (27 men, 30 +/- 11 years), analysis of 50 urine-DNA pairs identified 46 (92%) slow acetylators and 4 (8%) rapid acetylators by phenotype. Genotypic analysis inferred 5 (10%) slow acetylators and 45 (90%) rapid acetylators. There is 86% discordance between phenotype and genotype. A predominance of NAT2 slow acetylation phenotype in the Hmong is confirmed, and a significant discordance between NAT2 phenotype and genotype is identified. In this population, slow acetylation phenotype determined by a metabolic probe would not have been predicted by genotype alone. Environmental, genetic, or phenotypic anomalies that may contribute to this discordance should be considered and evaluated in future studies within this unique population.
Common laboratory methods used in pharmacogenomics studies are described.
The reliable and accurate determination of a person's genetic makeup at a particular locus in the DNA molecule, or genotype, is fundamental to pharmacogenomics. Whole blood cells and buccal cells are commonly collected to obtain a DNA sample. Once DNA is collected, the genomic DNA must be isolated from other cellular material. Next, a specific region of interest must be identified and amplified, performed via polymerase chain reaction (PCR). Gel electrophoresis is often performed after PCR to verify that PCR was successful and that the amplified target sequence is the correct size. Numerous methods are available to determine a person's genotype and differ based on allele discrimination and detection. PCR coupled with restriction fragment length polymorphism (RFLP) analysis, a conventional genotyping method, does not rely on automated technology and is practical for laboratories that genotype a limited number of samples. Pyrosequencing is an automated genotyping method in which the principal allele discrimination method is a primer extension reaction coupled with a luciferase-based enzyme reaction. TaqMan relies on the use of fluorescencelabeled probes, in addition to PCR primers, in the reaction mixture, enabling PCR amplification and allele discrimination in the same step. Mass spectrometry differentiates DNA molecules using a defined mass. Denaturing high-performance liquid chromatography (DHPLC) uses a reverse-phase ion-pair column to discriminate between variant and nonvariant alleles.
An understanding of the common genotyping methods used in pharmacogenomics studies, including PCR-RFLP analysis, pyrosequencing, TaqMan, mass spectrometry, and DHPLC, will aid pharmacy practitioners and students when interpreting the methods sections of such studies.
Blood samples are an excellent source of large amounts of genomic DNA. However, alternative sources are often needed in epidemiological studies because of difficulties in obtaining blood samples. This report evaluates the buccal cytobrush and alcohol-containing mouthwash protocols for collecting DNA by mail. Several DNA extraction techniques are also evaluated. The study was conducted in two phases. In phase 1, we compared cytobrush and mouthwash samples collected by mail in two different epidemiological studies: (a) cytobrush samples (n = 120) from a United States case-control study of breast cancer; and (b) mouthwash samples (n = 40) from a prospective cohort of male United States farmers. Findings from phase 1 were confirmed in phase 2, where we randomized cytobrush (n = 28) and mouthwash (n = 25) samples among participants in the breast cancer study to directly compare both collection methods. The median human DNA yield determined by hybridization with a human DNA probe from phenol-chloroform extracts was 1.0 and 1.6 microg/2 brushes for phases 1 and 2, respectively, and 27.5 and 16.6 microg/mouthwash sample for phases 1 and 2, respectively. Most (94-100%) mouthwash extracts contained high molecular weight DNA (>23 kb), in contrast to 55-61% of the brush extracts. PCR success rates for amplification of beta-globin gene fragments (268, 536, and 989 bp) were similar for cytobrush and mouthwash phenol-chloroform extracts (range, 94.4-100%). Also, we obtained high success rates in determining the number of CAG repeats in the androgen receptor gene, characterizing tetranucleotide microsatellites in six gene loci, and screening for mutations in the BRCA1/2 genes in a subset of phenol-chloroform DNA extracts. Relative to DNA extracted by phenol-chloroform from cytobrush samples, DNA extracted by NaOH had lower molecular weight, decreased PCR success rates for most assays performed, and unreliably high spectrophotometer readings for DNA yields. In conclusion, although DNA isolated from either mouthwash or cytobrush samples collected by mail from adults is adequate for a wide range of PCR-based assays, a single mouthwash sample provides substantially larger amounts and higher molecular weight DNA than two cytobrush samples.
Context.—To maximize the participation rate in population genetic studies, alternatives to invasive whole blood collection are increasing. One such alternative is buccal epithelial cell collection, which, in contrast to venipuncture and finger sticks, is painless. Buccal cells, if collected and purified efficiently, offer an acceptable source for DNA to be used in research and clinical applications.
Objective.—To develop a noninvasive sampling method for collecting cells for routine DNA testing in a clinical laboratory setting.
Design.—Five factors were used to evaluate several brands of mouthwash: (1) compatibility with the DNA purification chemistry, (2) DNA yield, (3) DNA quality, (4) DNA stability at room temperature, and (5) mouthwash taste. Next, an optimization study was undertaken to maximize DNA yield. Finally, a validation study was undertaken with the optimized protocol to test a panel of 14 donors for DNA yield and performance and to test for the stability of DNA held in mouthwash.
Setting.—Industrial research and development laboratory.
Results.—Of 5 mouthwashes tested, Scope brand mouthwash received the highest overall ranking. The addition of proteinase K and glycogen to the protocol significantly enhanced DNA yields, with a test panel (n = 14) giving a range of 12 to 60 μg of DNA per donor. In a 4-week room temperature stability study, the DNA in mouthwash samples was found to be stable for at least 2 weeks.
Conclusion.—A clinically validated DNA purification chemistry was adapted to a noninvasive specimen collection method. This method used a commercially available mouthwash, Scope, to collect buccal epithelial cells for the preparation of high-quality DNA in high yield.
To demonstrate the utility of a "swish and spit" technique as a nucleated cell source for transporter associated with antigen processing (TAP) haplotype determination by molecular methods in large-scale clinical trials.
Twenty normal volunteers were recruited for this prospective feasibility study. From each subject, buccal or blood cells (or both) were collected for use in TAP haplotype assignment by molecular methods and subjected to various storage conditions.
As an alternative to use of lymphocytes obtained by venipuncture, we developed a swish and spit technique for collecting buccal cells for assigning TAP haplotype. For this technique, the subject vigorously swishes isotonic saline in the mouth and expectorates it into a collection container. DNA is extracted from the buccal cells by proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. In addition, we compared DNA extracted from mouthwash specimens stored under various conditions to which a specimen might be exposed if mailed.
DNA extracted from buccal cells obtained by the swish and spit technique provided excellent templates for the polymerase chain reaction (PCR), and subject acceptance of this method was universal. In all cases, assigning TAP haplotype by PCR amplification of specific alleles with use of buccal or blood-derived specimens was successful. The integrity of the specimens was unaffected by storage at -20 degrees C, 4 degrees C, 25 degrees C, or 37 degrees C, and we were able to use the DNA from cells stored under any of these conditions for TAP haplotying.
We conclude that DNA from buccal cells collected by the swish and spit technique for TAP haplotype assignment is an excellent substitute for DNA obtained from nucleated blood cells, and the technique is useful for large-scale clinical studies that require DNA from subjects geographically distant from the research site.
Genomic DNA for genetic analyses has traditionally been derived from blood samples. With the availability of PCR techniques requiring only minute amounts of DNA and the current demand for high-volume testing, a less invasive, simpler to perform, and cheaper method to obtain DNA is desirable. We developed a method to obtain high-quality genomic DNA from buccal cells that has high acceptability and allows for a large number of PCR assays from a single sample. Sixty subjects vigorously swished 10 ml of undiluted commercial mouthwash in the mouth for 60 s and expelled the liquid into a collection container. DNA was isolated from the buccal cells with a rapid method using proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. Electrophoretic analysis of the extracted DNA showed detectable levels of high molecular weight genomic DNA in all samples. The DNA yields ranged from 0.2 to 134.0 microg, for an average of 49.7 microg. Using these samples, all 60 subjects were successfully genotyped by PCR-based assays for polymorphisms in the CYP1A1 (MspI and exon 7), CYP2E1 (RsaI), GSTM1, GSTT1, and NQO1 genes, confirming that the quality of DNA isolated from mouthwash samples was sufficient to reliably support PCR amplification. Storage of the (unprocessed) specimens at room temperature or at 37 degrees C for 1 week (temperature conditions that may be encountered when mailing samples) or at -20 degrees C for at least 6 months did not affect the DNA yield or ability to PCR amplify the samples. The results suggest that this mouthwash procedure may be suitable for large community-based studies of genetic susceptibility to disease in which samples can be collected by the participants themselves, mailed back to the study center, and stored for months prior to DNA analysis.
To maximize the participation rate in population genetic studies, alternatives to invasive whole blood collection are increasing. One such alternative is buccal epithelial cell collection, which, in contrast to venipuncture and finger sticks, is painless. Buccal cells, if collected and purified efficiently, offer an acceptable source for DNA to be used in research and clinical applications.
To develop a noninvasive sampling method for collecting cells for routine DNA testing in a clinical laboratory setting.
Five factors were used to evaluate several brands of mouthwash: (1) compatibility with the DNA purification chemistry, (2) DNA yield, (3) DNA quality, (4) DNA stability at room temperature, and (5) mouthwash taste. Next, an optimization study was undertaken to maximize DNA yield. Finally, a validation study was undertaken with the optimized protocol to test a panel of 14 donors for DNA yield and performance and to test for the stability of DNA held in mouthwash.
Industrial research and development laboratory.
Of 5 mouthwashes tested, Scope brand mouthwash received the highest overall ranking. The addition of proteinase K and glycogen to the protocol significantly enhanced DNA yields, with a test panel (n = 14) giving a range of 12 to 60 microg of DNA per donor. In a 4-week room temperature stability study, the DNA in mouthwash samples was found to be stable for at least 2 weeks.
A clinically validated DNA purification chemistry was adapted to a noninvasive specimen collection method. This method used a commercially available mouthwash, Scope, to collect buccal epithelial cells for the preparation of high-quality DNA in high yield.
This study assessed the feasibility of obtaining buccal cell DNA by mail from participants in a large, community-based cohort study in Hawaii. Mouthwash collection kits were sent to a total of 355 randomly selected Japanese, Caucasian, and Hawaiian cohort members. Subjects were requested to swish 10 ml of mouthwash in their mouth for 60 s and expel it into a collection cup, which they mailed back to our laboratory. Half of the subjects were requested to collect a second sample. After up to two mailings and two reminder phone calls, two-thirds of the subjects returned a sample. The participation rate was lower for Hawaiians (59.0%) than for Caucasians (68.1%) and Japanese (76.3%). Participation was not affected by requesting two specimens. Participants did not differ from the total sample in terms of education and smoking status. The mean DNA yield was lower in females (41.7 microg) than males (53.4 microg) and in Japanese (37.8 microg) as compared with Hawaiians (51.9 microg) and Caucasians (54.5 microg). For subjects who returned two samples, the DNA yields were similar when both specimens were extracted in the same batch. All samples were successfully genotyped for polymorphisms in the CYP1A1, CYP2E1, GSTM1, GSTT1, and NQO1 genes by PCR-RFLP. From these and previous data, we conclude that, in situations where blood samples cannot be obtained, mail collection of mouthwash samples should be considered because it yields substantial amounts of high-quality genomic DNA for large numbers of study subjects.
Buccal cells are becoming an important source of genomic DNA in epidemiological studies, but little is known about the effect of different sampling conditions on DNA quality and yield. We used a mouthwash protocol to collect six daily buccal cell samples from 35 healthy volunteers. Twenty-four individuals (six men and 18 women) correctly completed the protocol and were included in paired analyses to determine whether "swish" time (30 s versus 60 s), toothbrushing before collection, or lag time between collection and DNA extraction (1 day versus 5, 10, or 30 days at room temperature) would affect sample quality and yield. Total DNA, human-specific DNA (hDNA), degradation of DNA, and ability to amplify by PCR were determined. hDNA yield did not significantly vary by "swish" time. However, toothbrushing 1 h before sample collection reduced the amount of hDNA by nearly 40% (34 microg versus 21 microg; P = 0.06). Median hDNA yields for samples that were held for 1, 5, 10, and 30 days before extraction were 32 microg (range, 4-196), 32 microg (2-194), 23 microg (3-80), and 21 microg (5-56), respectively. The 10- and 30-day samples had significantly less hDNA than those processed after 1 day (P = 0.01). PCR success rates for beta-globin gene fragments of length 268 bp, 536 bp, and 989 bp were 94% or better, and high molecular weight DNA (>23 kb) was found in all but one sample. These results suggest that buccal cells should be collected before brushing teeth and processed within 5 days of collection to maximize hDNA yield.
It is well recognized that most medications exhibit wide interpatient variability in their efficacy and toxicity. For many medications, these interindividual differences are due in part to polymorphisms in genes encoding drug metabolizing enzymes, drug transporters, and/or drug targets (e.g., receptors, enzymes). Pharmacogenomics is a burgeoning field aimed at elucidating the genetic basis for differences in drug efficacy and toxicity, and it uses genome-wide approaches to identify the network of genes that govern an individual's response to drug therapy. For some genetic polymorphisms (e.g., thiopurine S-methyltransferase), monogenic traits have a marked effect on pharmacokinetics (e.g., drug metabolism), such that individuals who inherit an enzyme deficiency must be treated with markedly different doses of the affected medications (e.g., 5%-10% of the standard thiopurine dose). Likewise, polymorphisms in drug targets (e.g., beta adrenergic receptor) can alter the sensitivity of patients to treatment (e.g., beta-agonists), changing the pharmacodynamics of drug response. Recognizing that most drug effects are determined by the interplay of several gene products that govern the pharmacokinetics and pharmacodynamics of medications, pharmacogenomics research aims to elucidate these polygenic determinants of drug effects. The ultimate goal is to provide new strategies for optimizing drug therapy based on each patient's genetic determinants of drug efficacy and toxicity. This chapter provides an overview of the current pharmacogenomics literature and offers insights for the potential impact of this field on the safe and effective use of medications.
A significant association between beta (1)-adrenergic receptor codon 389 genotype and various hemodynamic parameters is reported for a population with symptoms of ischemic heart disease or other clinical indications for cardiovascular stress testing. This finding may have important implications with respect to cardiovascular disease and the efficacy of beta blockers, in cardiovascular disease.