Study population
This cross-sectional study was conducted among patients seeking dental care at Khon Kaen University (KKU) Dental Hospital in Khon Kaen, Thailand, from June 2017 to May 2019. Inclusion criteria were patients aged 18 years and older. Exclusion criteria were current smoker, xerostomia, diabetes mellitus, immunocompromised condition, autoimmune diseases, antibiotics use in the past three months, anti-inflammatory drugs use in the past six months, and significant cognitive or communication problems that may affect the ability to answer the questions. Participants had no symptoms of cold, flu, sinusitis, pharyngitis, salivary gland infections or other related condition during the data collection visits. The prevalence of PAV/PAV genotype was reported to be about 17% among all race/ethnicity groups and ranges between 16–22% in study subgroups [21]. We thus made a recruitment target of 250 participants to be able to detect the prevalence of 20% or lower.
This research was conducted in accordance with the Declaration of Helsinki. The study obtained ethical approval from the Institutional Review Board of the University of Washington, Seattle, USA (STUDY00002762), and KKU Ethics Committee in Human Research (HE592279). All participants provided written, informed consent for participation in the study.
Data collection
Enrollment, written consent process, interview, and saliva sample collection were done at KKU Dental Hospital. Information recorded from an interview included demographic data and personal oral self-care information (Fig. 1).
One calibrated dentist performed clinical examinations on all teeth, excluding third molars, in a clinical setting. The dentist was blinded to patient genotype at the time of the examination. Dental caries was determined according to the World Health Organization criteria and decayed, missing, and filled teeth (DMFT) index was calculated [22,23,24]. For periodontal examination, the researcher had been trained by a certified periodontist over 4 clinical sessions to achieve ≥ 80% agreement, also any difference was ≤ 1 millimetre (mm). We used a CP-15 periodontal probe to measure and record periodontal measures in 6 locations around each tooth, including probing depth (PD) and gingival margin position (GM) in mm. Clinical attachment loss (CAL) was calculated using PD and GM.
Saliva collection
We used Oragene-DNA kits (OG-500, DNA Genotek Inc., ON, Canada) to collect 2 ml of whole saliva samples by following the manufacturer’s instructions. Samples were mailed to Monell Chemical Senses Center, PA, USA for TAS2R38 genotyping assays.
TAS2R38 genotyping assay
DNA extraction was done following the protocol for samples collected with the OG-500 kits, using prepIT®•L2P (PT-L2P) (prepIT® L2P, DNA Genoteck Inc., ON, Canada). Genetic variation of TAS2R38 (NCBI Reference Sequence: NM_176817.5) was explored at 3 SNPs: rs713598 (C/G), rs1726866 (G/A), and rs10246939 (C/T) (C___8876467_10, C___9506827_10, and C___9506826_10, respectively; TaqMan®, ThermoFisher Scientific, CA, USA), using real-time polymerase chain reaction (PCR) single nucleotide polymorphism assays [25, 26]. Then, haplotypes and diplotypes were identified and recorded, using Applied Biosystem™ StepOnePlus® Real-Time PCR systems (Applied Biosystems® by Life Technologies™, ThermoFisher Scientific, CA, USA) for genotyping experiments. The genotyping was performed blind to the clinical status of the patients.
Data analysis
We analyzed continuous variables as mean and standard deviation. For categorical variables, we reported counts and percentages. We used one-way ANOVA to compare means. Proportion was tested using chi-square tests. Kruskal–Wallis test was used to compare the difference among groups for skewed data and to test the proportion trend. For the disease association study, we used logistic regression, and the results were presented as odds ratios (OR) and 95% confidence interval (CI).
Participants with three common genotypes were classified according to the data from the oral examinations. Dental caries measures included mean number of decayed, missing and filled teeth due to dental caries (mean DMFT) and prevalence of having DMFT ≥ 1. Various case definitions of periodontal disease have been employed in previous studies. Therefore, we explore the association between the genotypes and periodontal disease using different cut-offs, including the prevalence and extent (% of sites) of PD ≥ 4, 5 mm and CAL ≥ 3, 4, 5 mm.
StataIC 16 (StataCorp LLC, Texas, USA) was used for data analysis. The significance level was set at 5%.