Down Syndrome covers a large number of pathologies affecting practically every bodily system or apparatus, including cardiovascular; haematological; skeletal; muscular; nervous; endocrine; ear, nose, and throat; ocular; and digestive. It affects to a large extent their oral health and, accordingly, their dental treatment. Periodontal diseases such as necrotizing gingivitis, periodontitis, and aggressive periodontitis are among the most common oral complications that usually affect this type of patients due to their high incidence. Aggressive periodontitis is particularly serious given that patients tend to lose almost all their teeth during bouts recurring every 7-9 years [
Although aggressive periodontitis shows characteristic clinical features similar to those of the population who do not suffer from the syndrome, special features in periodontal pathogens have been identified, such as an increase in
However, what appears to take on increasing importance in this disease is host response and individual susceptibility, since, in every case of aggressive periodontitis a delay occurs in the chemotactic response of the neutrophils, a specially impaired response in patients with this syndrome [
In this study, we have attempted to identify the statistically significant difference in inflammation-related gene expression by comparing patients with Down Syndrome with Periodontal Disease (DS+PD+) with patients with Down Syndrome without Periodontal Disease (DS+PD-). In this way, the aim is to show the relationship of periodontitis as a chronic oral inflammation with some of the 92 reference genes obtained from the results of the work of another research group [
A retrospective study was carried out on cases and controls which was approved by the Virgen del Rocío Hospital Ethics Committee. Patients or their corresponding guardians provided their consent given that the benefits are direct for the research patient.
The study groups are (1) Down Syndrome patients without Periodontal Disease (DS+PD-) and (2) Down Syndrome patients with Periodontal Disease (DS+PD+). The patients in the control group had not been diagnosed with periodontal disease (PD) at any time in their lives and had no bleeding to the catheter (BOP). The patients in the study group had been diagnosed with PD, although when a restoration with implants had to be carried out, the disease was inactive, or the patient had previously lost all his teeth. The criteria used to determine that a patient suffered from PD was the one developed by Bassani et al. [
The exclusion criteria are as follows: non-Down Syndrome patients, patients receiving treatment which could potentially affect bone metabolism, and patients with active periodontal disease or untreated. The existence or not of periodontal disease was confirmed using the patients’ clinical records. Seven patients not suffering from the disease were included compared to four patients who did, thus making a total of eleven patients in the study. Demographic and clinical variables were taken from medical records.
At the time when the patients who were selected and included were examined, a sample was taken comprising two blood collections from the medial cubital vein per person in PAXgene™ tubes (5 ml), reference 762165 (100 tubes), with the final aim of extracting RNA. Transfer of the samples to the processing centre was undertaken under refrigerated conditions (2-8°C) and never took more than three days.
RNA sample extraction was performed using a Qiagen PAXgene Blood miRNA Kit reference 763134 at a QIAcube automated station. Subsequently, a database was built for the samples which detailed, inter alia, RNA quantification data.
First, RNA concentrations were quantified using visible light spectroscopy employing NanoDrop 2000c equipment (Thermo Fisher Scientific) to ensure the correct processing of these before storage. Second, a much more precise measurement was taken, using fluorescence and using Qubit 3.0 equipment (Thermo Fisher Scientific) to study gene expression, the results of which were added to the database.
The RNA selected were amplified and hybridized using the “GeneChip® WT PLUS Reagent Kit” (Thermo Fisher Scientific, Santa Clara, CA, USA). Amplification was carried out from a total of 55 nanograms of initial RNA, and then the procedures described in the “GeneChip® WT PLUS Reagent Kit” were followed.
Amplification of the cDNA was quantified, fragmented, marked, and prepared for hybridization from the GeneChip® Clariom S Human Array (Thermo Fisher Scientific) for human, mouse, and rat, with more than 20,0000 genes annotated for expression level measurement using 5.5
The analysis of the different gene expression was undertaken using Transcriptome Analysis Console (TAC, Affymetrix) Software. The reference genes taken for the search in our study were those from the results used in a prior study carried out by another group [
92 inflammation-related genes and 4 reference genes.
18Sª | GAPDHª | HPRT1ª | GUSBª | A2M | ADRB1 | ADRB2 | ALOX12 | ALOX5 | ANXA1 | ANXA3 | ANXA5 |
---|---|---|---|---|---|---|---|---|---|---|---|
KLK3 | BDKRB1 | BDKRB2 | CACNA1C | CACNA1D | CACNA2D1 | CACNB2 | CACNB4 | CASP1 | CD40 | CD40LG | CES1 |
LTB4R | MAPK14 | NR3C1 | HPGD | HRH1 | HRH2 | HTR3A | ICAM1 | IL1R1 | AL2RA | IL2RB | IL2RG |
IL13 | ITGAL | ITGAM | ITGB1 | KTGB2 | KLK1 | KLK2 | KLKB1 | KNG1 | LTA4H | LTC4S | MC2R |
NFKB1 | NOS2 | PDE4A | PDE4B | PDE4C | PDE4D | PLA2G1B | PLA2G2A | PLA2G5 | PLCB2 | PLCB3 | PLCB4 |
PLCD1 | PLCG1 | PLCG2 | MAPK1 | MAPK3 | MAPK8 | PTAFR | PTGDR | PTGER2 | PTGER3 | PTGFR | PTGIR |
PTGIS | PTGS1 | PTGS2 | TBXA2R | TBXAS1 | TNF | TNFRSF1A | TNFRSF1B | VCAM1 | IL1R2 | PLA2G7 | PLA2G10 |
PLA2G4C | IL1RL1 | HTR3B | TNFSF13B | CYSLTR1 | HRH3 | PLA2G2D | IL1RAPL2 | KLK14 | PLCE1 | KLK15 | LTB4R |
ªFrom the 96-plex gene card used in the study used as a reference study [
Of the 92 inflammation-related genes selected from a prior study of another group [
Results of differential gene expression across the two study groups (Down Syndrome patients with Periodontal Disease (DS+PD+) and Down Syndrome patients without Periodontal Disease (DS+PD-)).
Gene | Gene ID-OMIM | Gene name | DS+PD+ AVG (log2) | DS+PD- | DS+PD+ standard deviation | DS+PD- standard deviation | Fold change | Chromosome | |
---|---|---|---|---|---|---|---|---|---|
TNFSF13B | Tumor necrosis factor ligand superfamily member 13B | 11.67 | 12.66 | 0.35 | 1.44 | -2 | 0.0448 | Chr13 | |
ITGB2 | Beta 2-integrin | 7.79 | 7.02 | 0.63 | 0.16 | 1.71 | 0.0033 | Chr21 | |
ANXA3 | Annexin 3 | 7.7 | 8.85 | 0.74 | 0.99 | -2.22 | 0.0479 | Chr4 | |
ANXA5 | Annexin 5 | 11.85 | 12.65 | 0.24 | 0.65 | -1.75 | 0.016 | Chr4 |
Each of the four genes that showed differential expression in the patients studied was analysed. The analysis of each of the genes was conducted using the National Center for Biotechnology Information (NCBI) and Online Mendelian Inheritance in Man® (OMIM®) databases. The metabolic pathways of the four genes were also examined using the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database and the reactome for those genes whose information was not available on the KEGG (Table
Summary of the cell routes in which each of the statistically significant inflammation-related genes take part in the patients studied.
Gene | Cellular pathways | Fold change | |
---|---|---|---|
TNFSF13B | (1) Cytokine-cytokine receptor interaction |
0.0448 | -2 |
ITGB2 | (1) Rap1 signalling pathway |
0.0033 | 1.71 |
ANXA3 | No data | 0.0479 | -2.22 |
ANXA5 | (1) Release of platelet cytosolic components | 0.016 | -1.75 |
In accordance with these databases, it was observed that none of the genes share routes, and that for all of them there is a defined participation, except for Annexin 3, for which no information is available concerning participation in cell pathways/metabolic routes.
Conversely, in the “Diseases” section on the KEGG, where disease routes have been collected and represented, each of the genes was searched for, in order to discover whether there was any direct relation with any disease. Only ITGB2 gave a positive result with the clinical features of leukocyte adhesion deficiency, related to the route of transendothelial migration of leukocytes (hsa04670 from KEGG).
Of the 92 inflammation-related genes which were included in our study, four showed statistically significant expression differences across the patients studied (Down Syndrome patients, one of them with positive Periodontal Disease (DS+PD+) compared to negative Periodontal Disease (DS+PD-)). Said genes were TNFSF13B, ITGB2, ANXA3, and ANXA5: three of them showed significantly diminished expression (TNFSF13B, ANXA3, and ANXA5) and one of them showed increased expression (ITGB2). This could be explained by its location, since the latter is the only one of the genes which is found on chromosome 21. The rest have a different cytogenetic location (TNFSF13B on chromosome 13, whilst ANXA3 and ANXA5 on chromosome 4), which seems to corroborate that Down Syndrome causes impairments to the complete genome.
TNFSF13B codifies a cytokine protein of the tumour necrosis factor superfamily, which acts as a ligand for receptors TNFRSF13B/TACI, TNFRSF17/BCMA, and TNFRSF13C/BAFR. TNFSF13B is also known as a lymphocyte B stimulator or B-cell activating factor (BAFF), in reference to its involvement in the activation, proliferation, and differentiation of this type of cell [
The beta 2 integrin chain gene (ITGB2) codifies an integrin of the beta chain which combines with other chains to form different integrins, proteins that mediate cell adhesion, as well as cell signalling. Defects in the gene have been related to impairments in the immune system linked to leukocyte adhesion, by affecting the CD18 leukocyte antigen [
ANXA3 and ANXA5 are part of the annexin family. Annexins, basically, are link proteins to calcium-dependent phospholipids. Whilst for the ANXA3 gene there is scarcely any information available in the human genome database [
In conclusion, this study highlights how despite the two groups of patients presenting the same genetic syndrome, chromosome 21 trisomy, they do not necessarily have to suffer certain immune and/or inflammatory impairments to the same degree. Differential expression of inflammation-related genes when comparing groups of Down Syndrome patients who suffer from periodontitis or not shows that certain patients present greater individual susceptibility to developing inflammatory symptoms, such as periodontitis, which may possibly be explained by impairments to metabolic/cell routes/pathways from impairment of the genes involved.
We must point to the fact that it will be necessary to undertake studies in greater depth for the validation of the expression of the genes identified in this study to be able to confirm the results obtained.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare no conflict of interest.
This study was funded by Consejería de Salud, Junta de Andalucía (Spain) (PI-0081-2016).