Association of Surfactant Protein D Single Nucleotide Polymorphisms rs721917, rs2243639, rs3088308 with Recurrent Aphthous Stomatitis in Pakistani Population

Recurrent aphthous stomatitis (RAS) is a benign ulcerative condition, defined by the recurrent formation of non-contagious mucosal ulcers. Surfactant protein D (SP-D) is secreted frequently at surfaces exposed directly to body fluids. This study aims to investigate the association of SP-D single nucleotide polymorphisms (SNPs) with the onset of RAS. Blood samples from 212 subjects (106 cases/controls each) were collected during 2019 and genotyped for SP-D SNPs (rs721917, rs2243639, rs3088308) by polymerase chain reaction and restriction fragment length polymorphism followed by 12% polyacrylamide gel electrophoresis. Minor aphthous (75.5%) was the commonly observed ulcer type as compared to herpetiform (21.7%) and major aphthous ulcers (2.8%). A family history of RAS was reported in 70% of cases. RAS was found significantly associated with rs3088308 genotypes T/A (95% (Cl): 1.57–5.03, p = 0.0005), A/A (95% (Cl): 1.8–6.7, p = 0.0002), T-allele (95% (Cl): 1.09–2.36, p = 0.01), A-allele (95% (Cl): 1.42–3.91, p = 0.01), rs721917 genotype T/T (95% (Cl): 1.15–25.35, p = 0.03), and T-allele (95% (Cl): 1.28–3.10, p = 0.002). Female gender and obese body mass index (BMI) were significantly associated with rs3088308 genotypes T/A (95% (CI): 1.89–15.7, p = 0.001), T/T (95% (Cl): 1.52–11.9, p = 0.005), A-allele (95% (Cl): 1.65–7.58, p < 0.001), and T-allele (95% (Cl): 1.4–10.1, p <0.001) and rs721917 genotype T/T (95% (CI) = 1.3–33, p = 0.02), respectively. This study describes the association of SP-D SNPs (rs721917, rs3088308) with RAS in the Pakistani population.


Introduction
The oral cavity is a unique anatomical site consisting of different parts that work together to safeguard the oral mucosa from the harmful environment. Oral mucosal lesions constitute a broad spectrum of clinical manifestations in the form of different signs and symptoms that can be challenging for general physicians and dentists to diagnose accurately. These lesions can be a source of pain and uneasiness for patients, thereby preventing them from carrying out routine oral activities, such as mastication, swallowing, fluid intake, and speech [1][2][3][4]. An oral ulcerative lesion is defined as a breach in the epithelium and defect in connective tissue lying underneath the mucosa or epithelium [5]. Three main classes of ulcerative lesions of the oral cavity have been identified; acute, chronic, and recurrent ulcers [1,5].
Recurrent aphthous stomatitis (RAS) is a painful localized mucosal ulcerative condition, characterized by recurring episodes of tender, isolated, or abundant ulcerations limited to the mucosa of the oral cavity [6,7]. RAS is categorized into different groups on

Sample Collection
A total of 212 blood samples (106 RAS cases, 106 controls) were collected during 2019 from the routine diagnostic facility of Punjab Dental Hospital, Lahore, Pakistan. Data collection proforma was developed to establish RAS diagnosis as described previously [55]. Briefly, types of RAS (minor, major, and herpetiform) were recorded in the form of number, size, and site of oral mucosa. Study subjects with smoking histories were assessed by number of cigarette packs consumed per day. Body mass index (BMI) was calculated manually using Quetelet's Index. Weight was measured (to the nearest to 0.5 kg after correcting zero error) using a standard spring balance and ensuring no shoes and the least amount of clothing were worn (without jackets and sweaters) by participants. Height was measured in the standing position by using a stadiometer without footwear, measuring to the nearest 0.1 cm and then units were converted to meters. BMI was taken as normal (18.5-24.9), underweight (<18.5), and obese (>25) [56]. Study subjects with COPD, asthma, acute infection, inflammatory bowel disease, and severe anemia and patients taking systemic steroids were excluded due to the potential role of SP-D in the onset of diseases. The data collection proforma is given in Table S1.

SP-D SNP Genotyping by Restriction Fragment Length Polymorphism (RFLP)
Total genomic DNA was isolated from whole blood using the GeneJET Whole blood genomic DNA purification kit (catalog # K0781, Thermo Fischer Scientific, Waltham, MA, USA). SNPs of SP-D gene (rs3088308, rs721917, rs2243639) were investigated by RFLP. The characteristics of SP-D SNPs are given in Table 1. SNPs were amplified by polymerase chain reaction (PCR) in a thermal cycler (ProFlex PCR system, Thermo Fischer Scientific, Waltham, MA, USA). PCR reaction mixture of 50 µL consisted of 25 µL of 2 X PCR Master Mix (catalog # K0171, Thermo Fischer Scientific, Waltham, MA, USA), 10 pM of each primer, 2 ng of DNA, and dH2O up to 50 µL. The sequence of primers used for PCR amplification, amplicon size, and annealing temperature were as follows [57]: rs3088308 forward primer: 5 -ACG GAG GCA CAG CTG CTG-3 , reverse primer: 5 -GGA AAG CAG CCT CGT TCT-3 , 115 bp, 52 • C, rs721917 forward primer: 5 -CCC CAT AGC AGA GGA CAG AA -3 , 238 bp, 55 • C, reverse primer: 5 -CCA GGG TGC AAG CAC TGG AC-3 and rs2243639 forward primer: 5 -CCC CAC TTC TCT CTC TGA CC-3 , reverse primer: 5 -CTG CTC ACC TGC TGC CCC CG-3 , 238 bp, 50 • C. Sterile nuclease-free water was used as the negative control. Agarose gel (1.5-2%) was used to resolve and analyze the PCR products. PCR products were purified using the GeneJET PCR purification kit (catalog # K0701, Thermo Fischer Scientific, Waltham, MA, USA). The purified PCR product was further subjected to RFLP analysis. Specified restriction enzymes and conditions used for SNP detection are given in Table 2. Restriction fragments were resolved using 12% polyacrylamide gel electrophoresis (PAGE) followed by staining with SYBR safe DNA gel stain (Thermo Fischer Scientific, Waltham, MA, USA). Gel was run at 80 V for 2 h and visualized in a gel documentation system (Bio-Rad, Hercules, CA, USA).

Statistical Analysis
All statistical analyses were conducted using the Statistical Package for Social Sciences software (SPSS 26). Categorical data were presented as frequency and percentage. The Hardy-Weinberg equilibrium was used for the control and RAS groups. Chi-square test was applied to calculate significance between genotype distribution and clinical parameters. Odds ratio (OR) and 95% confidence interval (CI) were used to estimate the strength of association of parameters and calculated by MedCalc OR calculator (version 20.218; https: //www.medcalc.org/calc/odds_ratio.php, (accessed on 3 March 2021)). p-value ≤ 0.05 was considered to be statistically significant. The Adobe Illustrator CC 2018 program was used for labeling of PAGE gels.

Association Analysis of SP-D SNPs
SP-D SNPs (rs2243639, rs721917, and rs3088308) were detected by PCR-RFLP followed by PAGE. The PAGE results are given in Figure 1.

Discussion
RAS is a multifactorial chronic ulcerative disease with severely inflamed oral mucosa, and it is generally characterized by the presence of painful ulcerations of the oral mucosa. RAS pathogenesis remains unclear as multiple factors can cause its occurrence. Therefore, differential diagnosis of RAS is usually established for treatment regime [7]. Keeping in view that patients with positive family history are at higher risk to develop recurrent ulcerations [58,59] and not much data are available from Pakistan about the association of polymorphism with RAS, the current study was designed to investigate SP-D SNP association with RAS in the Pakistani population.
The results obtained are in agreement with the previous studies [60][61][62] that female subjects are at higher risk to develop oral ulcerations. In our study, most of the subjects were adults; older than 18 years of age with the mean age being 29 years. Another study from Pakistan reported mean age of 22 years for RAS onset [62]. Other studies reported the RAS onset at the peak age of 19 years and continued for their whole lives [63,64]. We observed that 35.9% of the RAS cases were either non-smokers or developed aphthous after quitting smoking, as previously described [65][66][67][68]. The buccal mucosa was the most common site involved for ulcer development. Similarly, buccal mucosa was reported to be the most common site for RAS development [64], while another study reported that the tongue was the predominant site followed by buccal mucosa and lower lip [69,70]. We noticed that 41.5% of participants had to use either a topical or systemic steroid for their aphthous ulcerations. However, it was shown that about 67.72% aphthous ulcers require no treatment, and stress was the major factor that contributed to RAS development [64,71]. Similarly, stress was considered a major factor in another study from Pakistan [26,62]. Normal Hb levels were detected in 78.8% of study participants. However, low Hb levels in the RAS group (42%) were reported from Pakistan [61,72], while normal Hb levels were suggested in another study [73]. During our research, hematinic deficiencies were seen in a small population of RAS cases that is lower as compared to other studies, such as ferritin deficiency [16,72,74,75], altered zinc levels [76][77][78][79][80][81], and B12 deficiency [82][83][84] in RAS patients. Moreover, a significant association was observed among Hb, iron, B12, and folic acid deficiencies and RAS occurrence [85]. Our study did not find any BMI differences in the RAS group; similarly, another study reported that all BMI groups have equal chances of RAS development [85][86][87][88][89]. However, in another study, the low BMI group has been found to be associated with RAS onset [90].
In the current investigation, the records of family history of RAS showed that 70% of cases had positive family history. Previously, 24% to 46% of RAS cases were reported with positive familial predisposition [91]. Genetic analysis of RAS showed that monozygotic twins had higher risks of developing the condition than dizygotic ones, thus indicating that genetic factors have the ability to modify the RAS susceptibility and severity, such as DNA polymorphism of interleukins (IL-2, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10) and TNFα [14,58,92]. In the Chinese population, a strong association of RAS with HLA-DRw9 has been documented [93].
Our results showed that SNP rs3088308 heterozygous variant T/A, homozygous variant A/A, T allele, A allele and SNP rs721917 homozygous variant T/T and T allele were significantly associated with the RAS onset. However, no association was observed for SNP rs2243639. On the other hand, SNP rs3088308 genotypes T/A, T/T, A allele, and T allele were significantly associated with female gender, while SNP rs721917 genotype T/T was associated with the obese BMI range. In conclusion, we are reporting, for the first time, the association of SP-D rs3088308 and rs721917 with RAS.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes14051119/s1, Table S1: Data collection proforma used during sampling; Table S2: SP-D SNPs genotype distribution in study population by HWE.  Informed Consent Statement: Informed consents were obtained from all subjects involved in the study.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding authors upon request.