1 Department of Epidemiology, University of Iowa College of Public Health, Iowa City, IA.
2 Veterans Affairs Medical Center, Iowa City, IA.
3 Center for Perinatal, Pediatric and Environmental Epidemiology, Department of Epidemiology and Public Health, Yale School of Medicine, New Haven, CT.
Received for publication September 25, 2003; accepted for publication January 21, 2004.
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ABSTRACT |
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DNA; HLA antigens; mouth mucosa; specimen handling
Abbreviations: Abbreviations: HLA, human leukocyte antigen; SD, standard deviation.
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INTRODUCTION |
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Among adults, studies suggest that mouthwash collections provide buccal cell DNA of higher quantity and purity than cytobrushes, with the alcohol content serving as a preservative to retard the growth of bacterial and fungal contaminants (1, 3, 8). However, mouthwash collection is not an option for infants or toddlers or for adults from societies unaccustomed to its use.
Studies of human leukocyte antigen (HLA) genotyping generally require 1,000 times more DNA than studies testing for specific gene polymorphisms (1). Thus, optimization of DNA yield is essential for investigators conducting HLA genotyping. We are conducting a case-control study of maternal-fetal HLA sharing and risk of preeclampsia that relies on self-collected, mailed cytobrush collections of buccal cell DNA from mothers and infants. There have been few methodological studies, particularly among infants, to guide our efforts to optimize buccal cell DNA yield for HLA genotyping (1). Thus, we designed and conducted a series of pilot studies among women and infants to identify methods of collection, storage, and packaging that would maximize DNA yields from cytobrushes. In this paper, we present findings from those studies. We hypothesized that buccal cell DNA yields would be improved by using an alternate collection method that maximized surface area contact between the cytobrush and the buccal mucosa and by optimizing storage and mailing conditions.
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MATERIALS AND METHODS |
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Cytobrush collection methods
Samples were collected using the Cytosoft Brush (catalog no. CP-5B; Medical Packaging Corporation, Camarillo, California). Study subjects were instructed to brush and twirl each cytobrush for 30 seconds over the specified regions of the mouth (12). Areas of the mouth designated for collection were: 1) the left inner and right inner cheeks, that is, the standard method described by Richards et al. (12), and 2) the upper and lower "gutters," that is, the alternate method. The term "gutters" refers to the two semicircular crevice areas of the mouth located between the upper gum line and the mucosa of the upper lip and cheek and between the lower gum line and the mucosa of the lower lip and cheek (figure 1).
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Our study was approved by the University of Iowa Institutional Review Board to ensure the protection of research subjects and compliance with federal regulations, as well as by the Iowa Department of Public Health. Subjects for each pilot study were recruited among staff employed by the study investigators or their colleagues or among mother-infant pairs identified from Iowa state birth records.
Pilot studies
In our preliminary testing, it became apparent that the standard procedure for packaging and storing collected samples (i.e., returning brushes to the plastic tubes in which they were originally packaged) resulted in some moldy samples and inadequate DNA yields. Thus, subsequent pilot testing involved methods that attempted to reduce the moisture present in collected samples. Table 1 describes the objectives, subjects, and procedures used for each of these pilot studies.
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Based on the results of pilot study 1, in pilot study 2 we did not include the hole-in-tube approach and instead set out to determine whether the mouthwash and paper envelope approaches would continue to provide adequate results under actual mailing conditions. However, to prevent leakage and to comply with US Postal Service policies, we placed the mouthwash and envelope samples in Ziploc plastic bags (S. C. Johnson and Son, Inc., Racine, Wisconsin) before inserting them into brown Kraft paper envelopes (Quality Park Products, St. Paul, Minnesota).
Pilot study 3 was designed to compare storage in the paper business envelope with storage in a Ziploc bag (we eliminated the mouthwash method from further pilot testing on the basis of the comparative cost and inconvenience for subjects). To meet postal standards, we identified a bubble-lined Tyvek envelope (Quality Park Products) to serve as the outer mailing envelope. The plastic bubble lining in this envelope appeared to provide more air circulation than would a smaller, smooth-surfaced plastic bag. Based on the results of this pilot study, we adopted the paper envelope approach, with a bubble-lined Tyvek outer envelope, for packaging and storage in all subsequent pilot studies.
Now that we had settled on a packaging and storage approach, we turned our attention to exploring collection methods that would maximize the yield and purity of DNA. We theorized that increasing the inner-mouth surface area brushed would result in higher yields. This led us to conduct pilot study 4, in which we compared the conventional cheek collection method with collection from the upper and lower gutter areas of the mouth. Brushing in the gutters achieves contact with two surfaces at once (the gum and the cheek) and provides cells from the inner lip area of the mouth. The results of pilot study 4 led us to adopt the gutter collection method for our subsequent pilot study.
Pilot study 5 was conducted to determine whether the gutter collection method and our mailing and storage procedures were sufficient under actual field conditions, in which eligible mothers collected the samples from a remote location. Mother-infant pairs were selected from birth certificate files, and mothers were recruited by letter and a follow-up telephone call to participate in a telephone interview and buccal cell collection.
Because pilot study 5 did not compare the DNA yields of cheek collections with those of gutter collections, we designed pilot study 6. For this pilot study, we wanted to control optimally for all variables not related to the particular site in the mouth from which samples were collected. We invited subjects to a "collection session" in which they were personally instructed in and observed using the procedures needed to collect samples from themselves and their babies. This method allowed us to control the collection procedures used and to maintain consistent handling and storage time for all samples.
DNA extraction and quantification
All cytobrush samples were extracted using Puregene DNA Tissue Kits (catalog no. D-70KA; Gentra Systems, Inc., Minneapolis, Minnesota) and the manufacturers protocol, with the following modifications. Before extraction, the bristle heads were snipped and placed in an Eppendorf tube (2.5 ml) with 550 µl of cell lysis solution (100 mM sodium chloride, 10 mM Tris-hydrochloric acid, 25 mM ethylenediaminetetraacetic acid, and 0.5 percent sodium dodecyl sulfate) and 3 µl of 20 mg/ml Proteinase K (Invitrogen Corporation, Carlsbad, California). The brushes were incubated overnight in a 55°C bath. Following incubation, brushes were centrifuged (13,000 x g for 1 minute), removed from the extract, and discarded. The extract solution was then incubated for 4560 minutes at 37°C with 3 µl of 4 mg/ml RNase A solution (Gentra Systems). Tubes were cooled to 4°C, vortexed for 20 seconds with 250 µl of Protein Precipitation Solution (Gentra Systems), incubated for 20 minutes at 4°C (or 10 minutes on ice), and then centrifuged for 5 minutes at 13,000 x g. The supernatant containing DNA was collected and transferred to a new tube containing 400 µl isopropanol and 2 µl glycogen (Gentra Systems). Tubes were rocked gently to mix the contents thoroughly and were incubated at room temperature for at least 15 minutes. Samples were centrifuged for 5 minutes at 13,000 x g, and the supernatant was discarded. DNA-containing pellets were treated with 70 percent ethanol to dry, spun at 13,000 x g for 1 minute, and then air-dried in the laminar flow hood for 1020 minutes. The DNA was rehydrated with 50 µl Tris-ethylenediaminetetraacetic acid (0.5 M) at room temperature for 2 days or more. DNA concentrations and purity were determined by absorbance measured with a Beckman DU-64 spectrophotometer (Beckman Coulter, Inc., Fullerton, California). Pure preparations of DNA have absorbance ratios (260:280 nm) of 1.8. Contamination of samples with protein or phenol reduces absorbance ratios, making DNA quantification less accurate.
Statistical analysis
To examine the effects of varying methods of buccal cell collection, storage, and mailing, we calculated summary statistics for DNA yield, including mean values, standard deviations, and ranges. We performed paired t tests on paired samples from individual subjects to test for statistically significant differences in DNA yield and purity, based on absorbance.
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RESULTS |
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To investigate further, we conducted pilot study 3 (n = 6) to compare DNA yields from mailed cytobrushes stored in Ziploc bags containing a paper absorbent with those from cytobrushes stored in a paper business envelope (figure 2, part C). Mailed samples stored in the paper envelope provided substantially higher mean yields than samples placed in a Ziploc bag with a paper absorbent (17.0 µg (SD, 7.0) for the envelope vs. 8.6 µg (SD, 4.1) for the Ziploc bag; paired t test: p < 0.06). Moreover, the DNA extracted from the brushes stored in Ziploc bags with a paper absorbent was of significantly lower purity than DNA extracted from comparable brushes stored in paper envelopes (data not shown). Some of the cytobrushes stored in the Ziploc bags showed signs of mold growth upon receipt.
DNA yield from cheek and gutter collections
To determine whether cytobrush collections from the upper and lower gutter areas of the mouth provided more DNA than the conventional method of brushing the left and right inner cheeks, we analyzed paired samples provided by 12 female staff volunteers (pilot study 4). As table 1 shows, the mean DNA yield from gutter collections (two brushes) was nearly double the yield from cheek swabs (21.7 µg (SD, 6.6) vs. 12.4 µg (SD, 7.1); paired t test: p < 0.01). In addition, none of the gutter samples yielded less than 6 µg of DNA, the amount required for HLA analyses, whereas one third of the cheek samples (four of 12) yielded amounts that were insufficient. DNA purity for the two collection methods was comparable.
DNA yield based on the gutter collection method was examined further in pilot study 5, a mail-out pilot study of 26 mother-infant subject pairs (table 2). The mean DNA yield from mothers samples from two cytobrushes was 17.8 µg (standard error, 9.6), within the range of the yield of pilot study 4, the unmailed staff pilot study described above. Only one of 26 samples provided less than 6 µg of DNA, and the mean absorbance ratio was 1.8 (260:268 nm).
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We conducted pilot study 6 to compare the DNA yield and purity of cheek collections with those of gutter collections among mother and infant subjects (table 2). The mean DNA yield from mothers was significantly higher using the gutter collection method (15.0 µg (SD, 6.4) for gutter collection vs. 7.6 µg (SD, 3.6) for cheek collection; paired t test: p < 0.001). Furthermore, only one of 15 gutter samples yielded less than 6 µg, as compared with six of 15 cheek samples among mothers.
In contrast, gutter and cheek sample yields from infants aged 34 months were fairly comparable (9.9 µg (SD, 10.7) vs. 11.4 µg (SD, 14.2)) (table 2). The mean yields among pilot study 6 infants tended to be higher than those for pilot study 5 infants, largely because two pilot study 6 infants had very high DNA yields. Without the data from these two infants (n = 13), the mean yields from gutters and cheeks were equivalent (7.1 µg (SD, 6.0) vs. 7.0 µg (SD, 4.3), respectively) and were comparable to yields obtained in pilot study 5.
Final protocol for field collection
On the basis of the collective results from our pilot studies, we established the final protocol for collecting, storing, and mailing buccal cell samples for the full study. Since starting our full study, we have collected samples from 32 mother and infant subjects (data not shown). Of these, three mothers returned their own and their babies samples in the original Puregene tubes instead of the Kraft envelopes. All three of the babies samples and two of the mothers samples that were returned in tubes resulted in inadequate yields (<6 µg). In contrast, of the 29 samples that were returned correctly (in Kraft envelopes), only one of the mothers samples and three of the babies samples yielded less than 6 µg of total DNA. To discourage subjects from reusing the tubes, we have begun placing a brightly colored label on each Kraft envelope directing women to return the samples in the envelopes provided. All samples containing at least 6 µg of total DNA that have been amplified thus far (n = 10) have been successfully genotyped for HLA-A, -B, and -DR.
Agreement to participate in a study involving collection of genetic material is another concern. Through November 2003, 97 subjects have agreed to participate in this study, while 37 have refused. Among refusers, seven explicitly named privacy concerns as their reason for refusing. The remainder expressed a lack of time (n = 5), a lack of interest or a nonspecific reason (n = 18), or other reasons (n = 7). Because participation also involves the completion of a 30-minute telephone interview and a medical release form granting access to prenatal/delivery charts, it is not possible to determine the extent to which these refusals are due solely to the buccal cell component of the study. To date, none of our interviewed subjects who have been sent collection kits has expressed objections or has refused to follow our protocol for collecting buccal cells from their infants.
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DISCUSSION |
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Prior epidemiologic studies of buccal cell collections using cytobrushes have reported total DNA yields averaging 12 µg/brush from the inner cheeks (1, 3). However, our total DNA yields from inner cheek collections are most consistent with the average yield of 4 µg/brush (i.e., 12 µg/three brushes) reported recently by Satia-Abouta et al. (2), based on a mail-out study among men and women aged 45 years or more. This study relied, as we did, on methods that optimize DNA extraction from cytobrushes. It is also the only published study to date that has directly compared DNA yields from cytobrush and mouthwash collections within the same persons (2). Although cytobrush samples provided 30 percent less total DNA and no long DNA fragments (7.8 kilobases), the authors concluded that cytobrush collections are generally preferable to mouthwash rinses because they are substantially cheaper and provide DNA of sufficient quantity and purity for most genotyping analyses (2). Our own findings suggest that implementation of the gutter collection method in large-scale epidemiologic studies of adults can significantly increase DNA yields and reduce total costs, by reducing the number of brushes required without sacrificing DNA purity.
In contrast, gutter collections of buccal cells from infants aged 34 months did not provide higher yields than collections taken from the inner cheeks. It may be that the gutter collection method showed no advantage because of the abundance of saliva and the absence of teeth in very young infants, allowing improved brush access to all parts of the mouth. For these reasons, this finding should not be generalized to studies of older babies or toddlers. In addition, fairly wide variability in DNA yield for infants cheek and gutter samples is observed. We speculate that factors such as mouth sores, stage of tooth eruption, timing of last feeding, or even recent use of a pacifier could influence buccal cell DNA yields in infants. However, our study did not collect data on these factors.
Our packaging, storage, and mail-out experiments indicate that cytobrush samples should not be stored or mailed in plastic bags or in the original Puregene tubes. Cytobrushes enclosed in regular paper envelopes before mailing provided DNA of significantly higher yield and purity than brushes stored in Ziploc bags, even with a paper absorbent enclosure. We speculate that humid conditions imposed by storage in a closed plastic system may have played a role. Some support for this comes from storage experiments performed by Richards et al. (12), who reported slightly lower DNA yields from swabs stored in tubes submerged in a 37°C water bath as compared with samples stored in an incubator at the same temperature. Consistent with this, we observed fungal growth on several samples that were mailed in closed plastic systems. It is also possible that storage in paper (no plastic) may inhibit the growth of anaerobic mouth flora and other contaminants, which can decrease DNA yield.
When conducting this series of pilot studies, it was not our intent to directly compare DNA yield and purity between pilot studies. Approximate comparisons can be drawn between some of them, taking into account the varied subject populations, handling procedures, and storage times. In addition, infants in pilot study 5 were substantially older than infants in pilot study 6, preventing direct comparisons between these pilot studies. As infants age, we can expect changes in their level of cooperation with collection procedures, available surface area, and degree of sloughing of mouth cells due to eating habits and teething to substantially alter collection conditions.
By systematically analyzing paired samples in our experiments, we were able to control for individual differences in saliva production and other differences between subjects that might have affected yield and/or purity. However, we did not conduct HLA genotyping on every sample collected in our pilot studies, nor did we measure percentages of human DNA versus bacterial DNA, because of cost limitations. Studies have shown that a substantial amount of buccal cell DNA can arise from bacterial sources (8, 9). We obtained HLA-A, -B, and -DR genotypes from amplification of DNA from in-house samples using sequence-specific primer amplification, which indicates that 6 µg of DNA would suffice for HLA analysis. We have further confirmed this notion in the field; all samples from our full study of at least 6 µg of total DNA amplified thus far (n = 10) have been successfully genotyped for HLA-A, -B, and -DR. Because we perform HLA genotyping by sequence-specific primer amplification with commercially available primers that are unique to human HLA genes and do not utilize probe hybridization, there is no chance of cross-hybridization to bacterial sequences in our HLA genotyping technique.
With the increased availability of kits that optimize DNA extraction from cytobrushes and use of whole genome amplification methodology (13), the appeal of cytobrush collections is on the rise. In addition, mouthwash rinses have the disadvantage of being more expensive and messy than cytobrush collections (1, 2). Our findings indicate that self-collected, mailed buccal cell samples of the mouths gutter areas provide DNA of sufficient quantity and purity for most epidemiologic studies of adult subjects. In future studies, researchers should examine the DNA yields of inner-cheek versus gutter samples in older infants, toddlers, and school-age children to determine further the efficacy of this sampling method.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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