Keywords
NLRP3 - periodontitis - gene polymorphisms
Introduction
Periodontitis is a complex multifactorial disease caused by several risk factors,
including genetic risk factors that account for nearly half of the population's diversity
in periodontitis risk.[1] Additionally, genetic factors account for the heterogeneity in disease location,
severity, and dissemination in susceptible individuals.[2]
Pathogenic microorganisms activate the host immune system.[3]
[4] Particularly, inflammasomes are intracellular pattern recognition receptors that
become active when several signals are recognized as pathogen-associated molecular
patterns (PAMPs).[5] The most intricate of them all, the nod-like receptor pyrin domain-containing protein
3 (NLRP3) inflammasomes react to PAMP.[6] Increased secretion of mature interleukin (IL)-1β, caspase-1, and IL-18, which have
a critical role in the host innate immune reactions during periodontitis, is brought
on by the NLRP3 inflammasomes' formation and activation.[6]
[7]
Additionally, due to the influence of genetic polymorphisms, various ethnic groups
may have different degrees of disease susceptibility. Some immunological genetic variations
have been associated with periodontitis depending on the candidate gene approach and
genome-wide association studies.[2]
[8] Many association studies with the candidate gene approach are not recommended as
a standard technique to evaluate genetic involvement in periodontitis because they
produce varying and frequently conflicting results due to a lack of statistical power
and the absence of multiple testing corrections. Moreover, the phenotyping of periodontitis
and control individuals has not been consistent among investigations. Furthermore,
many studies have failed to account for lifestyle variables that may influence the
periodontitis phenotype or the prevalence of comorbidities.[1]
[2] Thus, sufficient sample size, pilot studies, and inclusion and exclusion criteria
in these studies are critical for determining genetic involvement.
The single nucleotide polymorphism (SNP) is the most common type of polymorphism,
in which a single nucleotide is substituted by another nucleotide, especially those
that are in the vicinity of protein-coding genes that able to affect or modify the
corresponding gene expression or protein synthesis with influences on the periodontium's
structural elements or the host's immunological reaction to a microbial attack. Polymorphisms
can stimulate or retard the production of different cytokines. Furthermore, various
studies have discovered that genotypic polymorphisms are connected with phenotypic
variety, such as periodontitis severity and onset.[9]
[10]
[11]
[12]
[13] One such polymorphism is the NLRP3, there are a few studies that have looked at
the NLRP3 polymorphism as it relates to periodontitis.[14] Hence, the purpose of this study was to explore the association of polymorphisms
in the NLRP3 gene with periodontitis in the Arabs populations of Iraq.
Materials and Methods
Patient Selection
The design of our study was an observational case–control study. Each participant
received thorough explanations of the study and its methods, and their informed consent
was obtained using a form that had been approved by the College of Dentistry, University
of Baghdad ethics committee, reference number: 453.
The potential patients were recruited consecutively from the attendants to the teaching
hospital of the College of Dentistry, Baghdad University and the Iraqi National Centre
for blood donation during the period from March to August 2022.
The study included similar ethnic backgrounds of Iraqi Arabs subjects, systemically
healthy males or females without over- or underweight and height (18.5–29.9) according
to body mass index[15] with the presence of at least 20 or more natural teeth, willingness to sign a form
of informed consent, no antibiotics therapy has been received in the last 3 months,
and during the last 6 months, there was no periodontal therapy. Any pregnant, lactating
women and individuals with dental implants and orthodontic or prosthodontic appliances
were excluded.
The selected participants were classified into two groups based on their periodontal
clinical conditions. The control group with healthy intact periodontium, who have
no gingival inflammation signs at all “bleeding on probing (BOP) <10%, probing pocket
depth (PPD) ≤ 3 mm, and no probing or clinical attachment loss (CAL).”[16] In addition, the periodontitis group, which is defined, following the 2017 classification
of periodontal diseases and conditions, as the interdental CAL is identified at ≥
2 teeth (not adjacent), or oral CAL ≥ 3 mm and PPD > 3 mm is identified at ≥ 2 teeth.[17] Periodontitis causes must exhibit unstable status (PPD ≥ 5 mm or PPD 4 mm with BOP)
with no risk factors (diabetes mellitus [DM] and/or smoking).
Pilot Study and Sample Size
A pilot study was conducted using the first samples collected from each group following
an allocation ratio of 1:2 (periodontal health:periodontitis, respectively) (three
periodontal health and six periodontitis). A total of nine samples were analyzed in
the laboratory by polymerase chain reaction (PCR)-sequencing methods to validate the
primers and if any preparation was required for blood samples. The values obtained
from this pilot study were then added to the final data of the main study.
The sample size was determined from the following equation[18]:
Sample size = (r + 1)/r × P (1 − P) (Z
1 − β + Z
1 − α/2)2/(P1 − P2)2
where r is the ratio of cases to controls equal to 2; P is the proportion of population equal to (P1 + P2)/2; Z
1 − β is the desired power (0.84 for 80% power); Z
1 − α/2 is the statistical value that corresponds to the level of confidence (95%) that is
equal to (1.96); P1 is the proportion in cases, which was estimated at 66% of genetic polymorphisms
of the NLRP3 at rs10925024 in the pilot study; and P2 is the proportion in controls, which was estimated at 33% of genetic polymorphisms
of the NLRP3 at rs10925024 in the pilot study. Then, after substituting the values
into the above equation:
Sample size = (2 + 1)/2 × 0.5 (1 − 0.5) (0.84 + 1.96)2/(0.66 − 0.33)2
Sample size = 26.98 = 27 + 3 (taking into account the 10% dropout rate of participants).
Thus, the total sample size for the groups must be 90 at a minimum (60 for cases and
30 for control with an allocation ratio of 2:1).
Clinical Examination
Before conducting any periodontal examination and the collection of blood samples,
every precaution needed to avoid coronavirus disease 2019 infection was followed.
Then, the clinical parameters to be measured in logic sequence included (plaque index
[PI, BOP, PPD, CAL, gingival recession indices, and the number of missing teeth) except
the third molars. PI and BOP were identified as present (1) or absent (0).[19]
[20] The periodontal examination was achieved by using a periodontal probe (Michigan
0 probe) at six sites/teeth except for plaque scores of only four surfaces for all
teeth.
The accuracy, validity, and reproducibility of the researcher were assessed by carrying
out an inter- and intraexaminer calibration; categorical variables (PI and BOP) were
assessed by using a kappa-coefficient assay. The targeted level was kappa value ≥75%
to decide a good level of agreement was present. For continuous variables (PPD and
CAL), the level of agreement rounded to the nearest millimeter should be >0.9 as determined
by interclass coefficient assay.
Sample Collection and DNA Extraction
Each individual had a venipuncture performed using a 5-mL disposable syringe and a
20-gauge needle to obtain 2 mL of blood from the antecubital fossa under an aseptic
technique with 70% ethanol alcohol. The collected blood was placed into ethylenediaminetetraacetic
acid tube (1.5 mg/mL) and stored at −40°C for the (NLRP3) genetic analysis by PCR-sequencing
methods. The processes include deoxyribonucleic acid (DNA) extraction, PCR amplification,
sequencing, and data analysis.
During the first step of the study, blood samples were used to isolate genomic DNA
following the instructions provided by the ABIOpure extraction kit (United States),
then the extracted DNA was quantified by Quantus Fluorometer (Promega, United States).
Polymorphism Detection
In detail, primer preparation and optimization were the first step in the PCR amplification
process. The primers in this study used for the detection of the NLRP3 gene were designed by PrimerQuest from Integrated DNA Technology. The primers covered
the majority of the gene to identify any potential alterations that might have been
present in the study groups and to compare them to typical sequences. The Macrogen
Company provided these primers in lyophilized form as a forward primer (5-TGTAAAACGACGGCCAGTCTAGACTCGAAAGGGCAAATAC-3)
and reverse primer (5-CAGGAAACAGCTATGACCTGCACCATCTCTAAGGTTTC-3) with 964 product size
(bp) at 60 annealing temperatures.
To prepare primer stock solution, lyophilized primers were diluted in nuclease-free
water (NFW) to a total of 100 pmol/μL for both reverse and forward tubes followed
by vertexing. Then, a primer working solution containing 10 pmol/μL was prepared by
adding 10 µL of stock solution and 90 µL of NFW for reverse primer and forward primer
in a 1.5-mL tube (stored at freezer −40°C).
To assess the ideal annealing temperature for primers, the DNA sample was replicated
with the same primers at treatment temperatures of 55, 58, 60, 63, and 65°C. After
that, the PCR amplifications were completed. The PCR cycling procedure was carried
out by PCR express (Thermal Cycler, Bio-Rad, United States) with the following scheme,
denatured at 94°C/4 minutes, then 30 denaturation cycles at 94°C/30 seconds, followed
by annealing at 55, 58, 60, 63, or 65°C/30 seconds, and extension at 72°C/30 seconds.
Following a final extension incubation at 72°C/7 minutes, the processes were stopped
by incubating for 10 minutes at 4°C.
Agarose gel electrophoresis (OWL, Thermo, United States) supplemented with 10 mg/mL
ethidium bromide staining (EBS) (Promega, United States) was used to verify the existence
of amplification following PCR amplification. Then, 5 μL of the PCR cycling product
was added straight to the well; 100 volts and 50 amps of electricity were turned on
for 60 minutes. From the negative (cathode) to the positive (anode) poles, DNA travels.
Under ultraviolet light, EBS bands in the gel were viewed, and then gel imaging equipment
was used to take a photograph (Major Science, Taiwan) ([Fig. 1]).
Fig. 1 Electrophoresis of the amplification of the NLRP3 gene at 964 bp for some samples. M, 100bp DNA ladder marker.
Then, PCR products were sent to Macrogen Company, Korea for Sanger sequencing using
ABI3730XL, automated DNA sequences, after the gene's fragments appear in the PCR with
the anticipated size to identify the nucleotide sequences of these fragments. The
nucleotide sequences of the samples were received by e-mail and compared with the
source nucleotide sequence and then analyzed by the software program Basic Local Alignment
Search Tool (BLAST).
Statistical Methods' Outcomes
Statistical Methods' Outcomes
Data description and analysis were achieved by Statistical Package for Social Science
(SPSS version 28, IBM, United States). Mean, standard deviation, and median were used
for the continuous data, while frequency (number) and percentage were used for categorical
variables.
The normality of distribution was checked by the Shapiro–Wilk and D'agostino's tests.
Mann–Whitney's U tests determined the significant difference between the examined groups. In addition,
categorical variables were analyzed by using the chi-square test. The correlation
between clinical parameters and NLRP3 polymorphisms genes was identified by Spearman's
correlation test. The significant difference will be revealed if p < 0.05.
The intergroup differences in the distributions of the SNPs were analyzed using the
chi-square test. Frequency distributions for selected variables were done first. Hardy–Weinberg
equilibrium (HWE) is used to calculate the expected alleles from the observed genotypes.
The odds ratio (OR) measures the strength of association between the presence of certain
polymorphisms and disease status.
Results
A total of 94 subjects were enrolled out of the 940 subjects whose eligibility was
determined. In brief, the average age was 41.66 ± 8.176 (median 40.5) and 38.66 ± 6.553
(median 37) with a range between 30 and 55 years for the periodontitis and control
groups, respectively. In addition, distribution according to sex and family history
is summarized in [Table 1].
Table 1
Statistics of demographic and clinical parameters of the studied population
|
Parameters
|
Healthy (32)
|
Periodontitis (62)
|
p-Value
|
|
Age
|
Mean ± SD
|
38.66 ± 6.553
|
41.66 ± 8.176
|
0.091 NS[a]
|
|
Median
|
37
|
40.50
|
|
Sex
[b]
|
Male
|
19 (59.4%)
|
37 (59.7%)
|
0.977 NS
|
|
Female
|
13 (40.6%)
|
25 (40.3%)
|
|
Family history
[b]
|
Positive
|
2 (6.3%)
|
10 (16.1%)
|
0.174 NS[a]
|
|
Negative
|
30 (93.8%)
|
52 (83.9%)
|
|
PI
|
Mean ± SD
|
0.156 ± 0.100
|
0.634 ± 0.223
|
0.000[c]
|
|
Median
|
0.122
|
0.676
|
|
BOP
|
Mean ± SD
|
0.055 ± 0.032
|
0.472 ± 0.180
|
0.000[c]
|
|
Median
|
0.056
|
0.450
|
|
PPD
|
Mean ± SD
|
0.000 ± 0.000
|
4.524 ± 0.614
|
0.000[c]
|
|
Median
|
0.000
|
4.3150
|
|
CAL
|
Mean ± SD
|
0.000 ± 0.000
|
3.458 ± 0.992
|
0.000[c]
|
|
Median
|
0.000
|
3.300
|
|
GR
|
Mean ± SD
|
0.000 ± 0.000
|
2.230 ± 1.153
|
0.000[c]
|
|
Median
|
0.000
|
2.230
|
|
Teeth
[b]
|
Lost teeth
|
16 (1.8%)
|
198 (11.4%)
|
0.000[d]
|
|
Mobile teeth
|
0 (0.0%)
|
46 (2.6%)
|
|
Present teeth
|
880 (98.2%)
|
1,538 (85.9%)
|
Abbreviations: BOP, bleeding on probing, CAL, clinical attachment loss, GR, gingival
recession; NS, nonsignificant; PI, plaque index, PPD, probing pocket depth, SD, standard
deviation.
a Nonsignificant at p ≥ 0.05 by Mann–Whitney's test and for the chi-square test.
b Number (%).
c Significant at p ≤ 0.001 by Mann–Whitney's test.
d Significant at p ≤ 0.001 for the chi-square test.
Regarding the result of the clinical examination, the periodontitis subjects had significantly
greater PI, BOP, and a greater number of missing and mobile teeth at p < 0.001 ([Table 1]). As well, the mean of PPD in the periodontitis group was 5.32 ± 0.687 and the mean
of CAL was 6.52 ± 0.667, whereas the control group was free from PPD and CAL.
Furthermore, the sequencing of NLRP3 SNPs by the Sanger method was described for some
samples in [Figs. 2] to [5] for rs10925024, rs4612666, rs34777555, and rs10754557, respectively, and there was
the substitution of allele cytosine (C) with thymine (T) in rs10925024 SNP, while
there was the substitution of allele T with C in rs4612666 SNP. In addition, there
was a substitution of allele guanine (G) with C in rs34777555 SNP and a substitution
of allele G with adenine (A) in rs10754557 SNP.
Fig. 2
NLRP3 gene's rs10925024 SNP analysis by Sanger sequencing. A homozygous “C” allele is indicated
by a single “C” peak. Homozygous T allele indicated by a single “T” peak. The “C”
and “T” peaks are a sign of the heterozygous C/T allele. SNP, single nucleotide polymorphism.
Fig. 3 Sanger sequencing analysis of the NLRP3 gene's rs4612666 SNP. Homozygous T allele denoted by a single “T” peak. A homozygous
“C” allele is denoted by a single “C” peak. The “T” and “C” peaks are a sign of the
T/C heterozygous allele. SNP, single nucleotide polymorphism.
Fig. 4 Sanger sequencing analysis of the NLRP3 gene's rs34777555 SNP. The G homozygous allele is denoted by a single “G” peak. A
homozygous “C” allele is denoted by a single “C” peak. The “G” and “C” peaks are a
sign of the G/C heterozygous allele. SNP, single nucleotide polymorphism.
Fig. 5 Sanger sequencing analysis of the NLRP3 gene's rs10754557 SNP. The G homozygous allele is shown by a single “G” peak. A homozygous
allele is shown by a single “A” peak. The “G” and “A” peaks are a sign of the G/A
heterozygous allele. SNP, single nucleotide polymorphism.
Analysis by HWE for studied groups was done for the NLRP3 SNPs to compare the observed
genotype with the expected genotype in each studied group as well as total samples.
The equilibrium was nonsignificant, as shown in [Table 2].
Table 2
The HWE for studied groups in NLRP3 polymorphism
|
NLRP3
|
All groups
|
Healthy
|
Periodontitis
|
|
rs
|
Genotype
|
Observed
|
Expected
|
Observed
|
Expected
|
Observed
|
Expected
|
|
10925024
|
C-C
|
49
|
49.2
|
22
|
21.1
|
27
|
28.5
|
|
C-T
|
38
|
37.6
|
8
|
9.8
|
30
|
27.1
|
|
T-T
|
7
|
7.2
|
2
|
1.1
|
5
|
6.5
|
|
HWE
|
0.009
|
1.030
|
0.711
|
|
p-Value
|
0.921 NS
|
0.309 NS
|
0.398 NS
|
|
4612666
|
T-T
|
7
|
6.9
|
3
|
3.4
|
4
|
3.6
|
|
T-C
|
37
|
37.2
|
15
|
14.1
|
22
|
22.7
|
|
C-C
|
50
|
49.9
|
14
|
14.4
|
36
|
35.6
|
|
HWE
|
0.0018
|
0.127
|
0.065
|
|
p-Value
|
0.965 NS
|
0.721 NS
|
0.797 NS
|
|
34777555
|
G-G
|
91
|
91.0
|
31
|
31.0
|
60
|
60.0
|
|
G-C
|
3
|
3.0
|
1
|
1.0
|
2
|
2.0
|
|
C-C
|
0
|
0.0
|
0
|
0.0
|
0
|
0.0
|
|
HWE
|
0.024
|
0.008
|
0.016
|
|
p-Value
|
0.875 NS
|
0.928 NS
|
0.897 NS
|
|
10754557
|
G-G
|
11
|
11.9
|
4
|
5.3
|
7
|
6.8
|
|
G-A
|
45
|
43.1
|
18
|
15.4
|
27
|
27.4
|
|
A-A
|
38
|
38.9
|
10
|
11.3
|
28
|
27.8
|
|
HWE
|
0.178
|
0.881
|
0.016
|
|
p-Value
|
0.672 NS
|
0.347 NS
|
0.898 NS
|
Abbreviations: A, adenine; C, cytosine; G, guanine; HWE, Hardy–Weinberg equilibrium;
NLRP3, nod-like receptor pyrin domain-containing protein 3; NS, p ≥ 0.05 nonsignificant; rs, reference single nucleotide polymorphism; T, thymine.
Concerning this, the distribution of NLRP3 SNPs according to their types in the studied
groups' samples is illustrated in [Table 3]. All the detected SNPs after sequencing were of single base substitution, 148 (98.7%)
of them were transition and 2 (1.3%) transversion SNPs in the periodontitis, while
in the healthy, 67 (98.5%) were transition and 1 (1.5%) transversion SNPs. The transition
mutations belong to rs10925024, rs4612666, and rs10754557, whereas transversion mutations
belong to rs34777555. There was a nonsignificant difference (p < 0.05) between the studied groups in SNPs type distribution.
Table 3
The types of NLRP3 SNPs in study groups
|
Types of SNPs
|
Healthy[a]
|
Periodontitis[a]
|
X2
|
p-Value
|
|
Transition
|
67
|
98.5%
|
148
|
98.7%
|
0.006
|
0.936 NS
|
|
Transversion
|
1
|
1.5%
|
2
|
1.3%
|
|
Total
|
3
|
1.4%
|
215
|
98.6%
|
Abbreviations: NLRP3, nod-like receptor pyrin domain-containing protein 3; NS, p ≥ 0.05 nonsignificant; SNPs, single nucleotide polymorphisms; X2, chi-square.
a Number (%).
The SNPs number and distribution in both groups are illustrated in [Table 4], as there were 35 SNPs in the periodontitis group for rs10925024 and 10 SNPs in
the control group for the same rs with significant differences.
Table 4
The number of SNPs for NLRP3 in periodontitis patients and control samples
|
NLRP3
|
Healthy[a]
|
Periodontitis[a]
|
OR (95% CI)
|
X2
|
p-Value
|
|
rs10925024
|
10
|
31.3%
|
35
|
56.5%
|
2.852 (1.159–7.018)
|
5.372
|
0.020[b]
|
|
rs4612666
|
29
|
90.6%
|
58
|
93.5%
|
1.500 (0.315–7.152)
|
0.262
|
0.609 NS
|
|
rs34777555
|
1
|
3.1%
|
2
|
3.2%
|
1.033 (0.090–11.847)
|
0.001
|
0.979 NS
|
|
rs10754557
|
28
|
87.5%
|
55
|
88.7%
|
1.122 (0.303–4.160)
|
0.030
|
0.863 NS
|
Abbreviations: CI, confidence interval; NLRP3, nod-like receptor pyrin domain-containing
protein 3; NS, p ≥ 0.05 nonsignificant; OR, odds ratio; SNPs, single nucleotide polymorphisms; X2, chi-square.
a Number (%).
b Significance level at p < 0.05.
While there were 58 SNPs in the periodontitis group and 29 SNPs in the healthy group
at rs4612666 with a nonsignificant difference between them. On the other hand, there
were two SNPs in the periodontitis and one SNP in the healthy group at rs34777555
with a nonsignificant difference between them. In addition, there were 55 SNPs in
the periodontitis and 28 SNPs in the healthy group at rs10754557 with a nonsignificant
difference between them.
Interestingly, the effect of NLRP3 SNPs on periodontitis distribution was assessed
by counting OR and it was found 2.852, 1.500, 1.033, and 1.122 for rs10925024, rs4612666,
rs34777555, and rs10754557, respectively.
The genetic analysis for NLRP3 SNPs in the present study revealed the genotype and
allele frequency in the studied groups as shown in [Table 5], the chi-square test showed a nonsignificant difference in their distribution between
studied groups at p-value ≥ 0.5 for all SNPs, except for the C-C and C-T genotypes of rs10925024 showed
a significant difference. In a more detailed description of the effect of genotype
on disease distribution, the study illustrates the effect of each genotype of NLRP3
SNPs on disease distribution. It was found that genotype C-T had a higher OR (2.813)
than other genotypes to develop the disease in rs10925024. While the genotype T-T
has a 1.378 chance to develop the disease in comparison with the other genotypes located
in the rs10925024.
Table 5
Analytic statistics of the genotype and allele distribution of NLRP3 polymorphism
|
NLRP3
|
Genotype/allele
|
Healthy[a]
|
Periodontitis[a]
|
OR (95% CI)
|
X2
|
p-Value
|
RR
|
|
rs10925024
|
C-C
|
22
|
68.8%
|
27
|
43.5%
|
0.351 (0.143–0.863)
|
5.372
|
0.020[a]
|
0.633
|
|
C-T
|
8
|
25%
|
30
|
48.4%
|
2.813 (1.096–7.218)
|
4.793
|
0.029[a]
|
1.935
|
|
T-T
|
2
|
6.3%
|
5
|
8.1%
|
1.316 (0.241–7.191)
|
0.101
|
0.751 NS
|
1.290
|
|
C
|
52
|
81.3%
|
84
|
67.7%
|
0.485 (0.233–1.008)
|
3.849
|
0.050 NS
|
0.834
|
|
T
|
12
|
18.8%
|
40
|
32.3%
|
2.063 (0.992–4.290)
|
3.849
|
0.050 NS
|
1.720
|
|
rs4612666
|
T-T
|
3
|
9.4%
|
4
|
6.5%
|
0.667 (0.140–3.179)
|
0.262
|
0.609 NS
|
0.688
|
|
T-C
|
15
|
46.9%
|
22
|
35.5%
|
0.623 (0.262–1.484)
|
1.147
|
0.284 NS
|
0.757
|
|
C-C
|
14
|
43.8%
|
36
|
58.1%
|
1.780 (0.752–4.213)
|
1.737
|
0.188 NS
|
1.327
|
|
T
|
21
|
32.8%
|
30
|
24.2%
|
0.653 (0.336–1.270)
|
1.586
|
0.208 NS
|
0.737
|
|
C
|
43
|
67.2%
|
94
|
75.8%
|
1.530 (0.788–2.973)
|
1.586
|
0.208 NS
|
1.128
|
|
rs34777555
|
G-G
|
31
|
96.9%
|
60
|
96.8%
|
0.968 (0.084–11.09)
|
0.001
|
0.979 NS
|
0.999
|
|
G-C
|
1
|
3.1%
|
2
|
3.2%
|
1.033 (0.09–11.847)
|
0.001
|
0.979 NS
|
1.032
|
|
C-C
|
0
|
0.0%
|
0
|
0.0%
|
–
|
–
|
–
|
–
|
|
G
|
63
|
98.4%
|
122
|
98.4%
|
0.968 (0.086–10.88)
|
0.001
|
0.979 NS
|
0.999
|
|
C
|
1
|
1.6%
|
2
|
1.6%
|
1.033 (0.092–11.61)
|
0.001
|
0.979 NS
|
1.032
|
|
rs10754557
|
G-G
|
4
|
12.5%
|
7
|
11.3%
|
0.891 (0.240–3.302)
|
0.030
|
0.863 NS
|
0.903
|
|
G-A
|
18
|
56.3%
|
27
|
43.5%
|
0.600 (0.254–1.418)
|
1.365
|
0.243 NS
|
0.774
|
|
A-A
|
10
|
31.3%
|
28
|
45.2%
|
1.812 (0.737–4.454)
|
1.696
|
0.193 NS
|
1.445
|
|
G
|
26
|
40.6%
|
41
|
33.1%
|
0.722 (0.387–1.347)
|
1.052
|
0.305 NS
|
0.814
|
|
A
|
38
|
59.4%
|
83
|
66.9%
|
1.385 (0.742–2.584)
|
1.052
|
0.305 NS
|
1.127
|
Abbreviations: A, adenine; C, cytosine; CI, confidence interval; G, guanine; NLRP3,
nod-like receptor pyrin domain-containing protein 3; NS, p ≥ 0.05 nonsignificant; OR, odds ratio; RR, relative reduction; T, thymine; X2, chi-square.
a Number (%).
a Significance level at p < 0.05.
Moreover, for rs10925024, there was a high frequency of the C allele in the healthy
group than in the periodontitis, while the periodontitis showed a higher T allele
than that in the healthy, conversely concerning rs4612666. On the other hand, the
frequency of the A allele was higher in the periodontitis group than that in the healthy,
while the healthy showed a higher G allele than that in the periodontitis for rs10754557.
Finally, the frequency of G and C alleles in the control and periodontitis groups
for rs34777555 was equal.
Surprisingly, the present study found the T allele and C-T genotype association with
periodontitis (OR = 2.063, confidence interval [CI] = 0.992–4.290, and relative reduction
[RR] = 1.720 and OR = 2.813, CI = 1.096–7.218, and RR = 1.935, respectively) for rs10925024.
The analytic statistic in this study found a nonsignificant association of NLRP3 SNPs
with demographic and clinical parameters in both periodontitis and control groups
at a p-value ≥ 0.05. However, there was a significant (positive) correlation between rs10925024
and CAL, as shown in [Table 6].
Table 6
Association of NLRP3 SNPs with demographic and clinical characteristics
|
Parameters
|
rs10925024
|
rs4612666
|
rs34777555
|
rs10754557
|
|
r
|
p-Value
|
r
|
p-value
|
r
|
p-Value
|
r
|
p-Value
|
|
Age
|
0.089
|
0.396 NS
|
0.036
|
0.729 NS
|
0.054
|
0.607 NS
|
0.122
|
0.242 NS
|
|
Tooth loss
|
0.111
|
0.287 NS
|
0.100
|
0.337 NS
|
0.027
|
0.793 NS
|
0.164
|
0.115 NS
|
|
Tooth mobile
|
0.030
|
0.773 NS
|
0.089
|
0.393 NS
|
0.088
|
0.400 NS
|
0.132
|
0.205 NS
|
|
Sex
|
−0.023
|
0.827 NS
|
0.047
|
0.654 NS
|
–0.150
|
0.150 NS
|
–0.186
|
0.073 NS
|
|
Family history
|
0.162
|
0.118 NS
|
0.090
|
0.388 NS
|
0.069
|
0.506 NS
|
0.023
|
0.828 NS
|
|
PI
|
0.109
|
0.298 NS
|
0.088
|
0.400 NS
|
0.081
|
0.435 NS
|
0.201
|
0.052 NS
|
|
BOP
|
0.160
|
0.124 NS
|
0.115
|
0.269 NS
|
0.001
|
0.991 NS
|
0.031
|
0.767 NS
|
|
PPD
|
0.176
|
0.090 NS
|
0.016
|
0.880 NS
|
–0.016
|
0.878 NS
|
0.039
|
0.706 NS
|
|
CAL
|
0.206
|
0.046[a]
|
0.063
|
0.546 NS
|
0.047
|
0.655 NS
|
0.002
|
0.985 NS
|
|
GR
|
0.143
|
0.168 NS
|
0.123
|
0.238 NS
|
–0.075
|
0.471 NS
|
0.054
|
0.603 NS
|
Abbreviations: BOP, bleeding on probing; CAL, clinical attachment loss; GR, gingival
recession; NLRP3, nod-like receptor pyrin domain-containing protein 3; NS, p ≥ 0.05 nonsignificant; PI, plaque index; PPD, probing pocket depth; r, Spearman's rank correlation; SNPs, single nucleotide polymorphisms.
a Significance level at p < 0.05.
Concerning the correlation among SNPs of NLRP3, the result showed a nonsignificant
association at a p-value ≥0.05 in the studied groups between rs10925024 and rs34777555 as well as between
rs10754557 and rs34777555. While there was a significant correlation among other SNPs
of NLRP3, as illustrated in [Table 7].
Table 7
Correlation of NLRP3 SNPs with each other
|
NLRP3
|
r
|
p-Value
|
|
rs10925024
|
rs4612666
|
0.332
|
0.001[a]
|
|
rs34777555
|
−0.060
|
0.565 NS
|
|
rs10754557
|
0.228
|
0.027[b]
|
|
rs4612666
|
rs34777555
|
−0.217
|
0.035[b]
|
|
rs10754557
|
0.374
|
0.000[c]
|
|
rs34777555
|
rs10754557
|
−0.066
|
0.525 NS
|
Abbreviations: NLRP3, nod-like receptor pyrin domain-containing protein 3; NS, p ≥ 0.05 nonsignificant; r, Spearman's rank correlation; SNPs, single nucleotide polymorphisms.
a Significance level at p ≤ 0.01.
b Significance level at p < 0.05.
c Significance level at p ≤ 0.001.
Discussion
In the present investigation, NLRP3 polymorphisms were discovered that may indicate
higher genetic susceptibility to periodontitis. This was supported by findings that
the NLRP3 rs10925024 was positively correlated with elevated CAL. The current study,
to the authors' knowledge, is the first study to investigate the relationship between
genetic variation of NLRP3 and periodontitis risk in an Iraqi Arab population.
Multiple variables, including genetics, influence the progression of periodontitis,
which is a complex disease.[21]
[22] As a potential pathogenic factor for periodontal disease, SNP of cytokines and inflammatory
mediators have drawn more attention in recent research.[14]
[23]
This research may contribute to the development of innovative treatment and preventive
approaches as well as a better understanding of the pathophysiology of periodontitis.[24] Pathogenic bacteria in the tooth biofilm are responsible for the onset and progression
of periodontitis because they induce an inflammatory immune reaction that causes tissue
damage in a susceptible host.[25]
[26] The pathogenic periodontal pockets biofilm in conjunction with the host's immune-inflammatory
reactions is known to interact in various ways to cause periodontal disease. Members
of the inflammasome family including NLRP3 have a crucial role in immune responses
in periodontitis, which results in elevated IL-1β and IL-18 secretion.[6] The role of NLRP3 polymorphism and greater genetic susceptibility to periodontitis
revealed contradictions and highlighted a knowledge gap in this field.[14]
[27]
Advances for studying periodontal inflammation have emerged with the development of
the inflammasome concept. Polymorphism of NLRP3 is one of the most studied SNPs of
all the inflammasomes in inflammatory and autoimmune diseases; NLRP3 has been implicated
as having a key role in the pathogenesis of several diseases in the general population,
including obesity, DM, rheumatoid arthritis,[28]
[29] all of which are clinical diseases known for their association with periodontitis.[14]
[23]
[30]
[31]
In our study, we detected a significant difference between the examined groups of
Iraqi subjects concerning the SNPs at position rs10925024 of NLRP3. As a result, 35
SNPs were found in the periodontitis cohort, while 10 SNPs were found in the healthy.
Thus, the current finding revealed a significantly higher frequency of CT + TT genotypes
of NLRP3 at rs10925024 was found in the periodontitis group accounting for 56.5% in
contrast to 31.3% in healthy controls and is associated with an increased likelihood
of having periodontitis compared with those with the CC genotype by (OR = 2.852) times.
On the other hand, allele frequencies in study groups appear to be more frequent in
the periodontitis group 32.3% versus 18.8% in controls. The allele and genotype frequencies
complied with HWE. Unfortunately, there are no available references to compare the
result of NLRP3 rs10925024 with it.
The CT genotype at rs10925024 was significantly higher in patients with periodontitis
with a percentage of 48.4% as compared with 25% in the healthy. In addition, we observed
a remarkable enhancement in the proportion of the T allele in rs10925024 of periodontitis,
whereas there was a significant drop in the proportion of the C allele at the same
location. This, in turn, increases the transcriptional activity of the NLRP3 gene, and overexpression of cytokine and caspase, which leads to an exacerbated inflammation
pathway in the periodontal tissue during the pathogenesis of the periodontal disease
and leads to bone loss.[14]
[27]
[32] This is corroborated by the significant association between NLRP3 polymorphism and
rising attachment loss.
Thus, it is biologically plausible that individuals with NLRP3 polymorphisms have
increased genetic susceptibility to periodontitis due to increased cytokine production
after NLRP3 activation.[32] The disparities in outcomes among various races or particular ethnicities, however,
might be explained by the presence of distinct alleles responsible for susceptibility
to the same disease.
While there was a slightly higher frequency of TC + CC genotypes of NLRP3 at rs4612666
in periodontitis which accounts for 93.5 versus 90.6% in healthy controls and is associated
with an increased likelihood of having periodontitis compared with those with the
TT genotype (OR = 1.500) times. On the other hand, the frequency of the genotypes
(GC + CC) was higher in the periodontitis 3.2% than in the healthy group 3.1% at rs34777555
with a nonsignificant difference between them. In addition, there was a slightly higher
frequency of GA + AA genotypes at rs10754557 in the periodontitis 88.7% than in the
healthy group 87.5% with a nonsignificant difference between them. Thus, this study
found no association between the NLRP3 polymorphism at positions rs4612666, rs34777555,
and rs10754557 with periodontitis. In this respect, some studies have also found no
relationship between these NLRP3 polymorphisms and inflammatory conditions,[33]
[34] although Isaza-Guzmán et al reported an association between NLRP3 rs4612666 and
chronic periodontitis in the Colombian population[35]; however, this study was lacking the ethnicity of chronic periodontitis patients
and healthy controls, which may have explained some confounding influence of population
stratification that could not be detected.
The results of a study on a Brazilian population indicated the reverse pattern, with
a greater frequency of the T-C genotype in periodontitis patients. These findings
were in contrast to those of this study.[32]
The inconsistent findings in the literature may be attributed to sampling size, racial
differences, case definitions of studied groups (periodontal disease and health),
and different environmental and pathological factors that influence the host response.
As a result, the proportion of polymorphism in a particular genotype may vary between
populations or racial groups.
Men have been thought to be more prone to periodontal disease than women because of
hormonal variables, poor personal hygiene practices, and poor health prevention behaviors,
according to various research.[36] In addition, age is one of the confounding factors for periodontitis[37]; thus, studied groups were matched with a nonsignificant difference in age and gender.
There were some limitations with the current study, it is important to investigate
other confounding factors such as environmental and pathological that may contribute
to or amplify a specific genetic variant to understand how genetics affect complex
diseases. Just for uniformity and to avoid bias, other periodontal disease risk variables
such as smoking, diabetes, and obesity were excluded from this study. Despite this,
they may have an impact on the susceptibility to periodontitis in the presence of
different polymorphisms. The genetic heterogeneity of periodontitis is an additional
limitation that prevents generalizing the findings. Moreover, observational study
results only provide an association, not causality, which must be further evaluated
through higher evidence-based trials. Yet, this research was one of the few to look
into the genetic basis of periodontitis in the Iraqi Arabs community with sufficient
sample size and pilot study to allow for multiple testing corrections, which prevented
many false-positive results associated with candidate gene approach studies.
Future trends are required to identify the association of periodontitis with genetic
polymorphisms of other influential mediators (inflammation-associated molecules) such
as ILs and the Fc receptor, especially in a sufficient sample size through higher
evidence-based trials.
Conclusion
The present study concluded that NLRP3 SNP may have a role in periodontitis genetic
susceptibility in the population. Furthermore, the positive association between CAL
and rs10925024 NLRP3 polymorphisms suggested a possible role in increasing the severity
of periodontal disease. The discovery of genetic markers for periodontitis susceptibility
will enable early detection of those at high risk and may ultimately aid through personalized
types of therapy. More research will be needed to evaluate NLRP3's contribution to
periodontitis in addition to other genetic, pathological, and environmental risk factors
because of its complicated regulatory system.
