Non-inflammatory destructive periodontal disease: a clinical,\nmicrobiological, immunological and genetic investigation
The patient, a healthy Caucasian 46-year-old lady, dental surgeon, presenting normal\nweight and regular physical activities, from the southeastern region of Brazil, promptly\nidentified the beginning of teeth mobility and gingival recession on her own mouth, and\nlooked for periodontal health care. First, she was unsuccessfully advised to intensify\ntoothbrushing procedures as an attempt to control the bone resorption. However, in spite\nof intense toothbrushing and relevant oral hygiene, the gingival recession and teeth\nmobility were continued and the patient came to evaluation by a periodontist from our\nresearch group. The history related by the patient, in this circumstance a dentist,\nsuggested an intriguing case of periodontal disease without visible signs of local\ninflammation. Then, we investigated this unique condition by performing a complete\nevaluation of the patient to provide the pathology's diagnosis, its mechanisms of\ndevelopment and to establish the most suitable treatment for this case.\nThe patient denied any pre-existing dental problems, neither was taking any medication\nfor at least 2 months prior to this study nor had a smoking history. Anamnesis was\nperformed, as well as clinical examination and a score for bleeding on probing, probing\ndepth, and clinical attachment loss at six sites per tooth, by the\nperiodontist, as described previously. Radiographic examination and occlusion movement data were also\nobtained and finally the patient was definitely diagnosed as presenting periodontal\ndisease. First, upon clinical examination, the patient did not present bleeding on\nprobing or probing depth ≥3 mm ();\nhowever, an extensive clinical attachment loss ranging from one third to two thirds of\nperiodontal support was verified in both arches, as well as in anterior and posterior\nteeth, which was confirmed by full-mouth radiographic survey (). The patient also showed gingival recession of 1-3 mm\naffecting mainly facial tooth surfaces ()\nand some teeth presented abnormal mobility. However, despite this broad attachment and\nbone loss, the patient presented satisfactory oral hygiene, with no apparent plaque\nformation or accumulation of gross calculus and the absence of gingivitis or other\ndetectable manifestations of gingival inflammation (). It is of note that this patient had never used tobacco products\nand reported vigorous and intensive toothbrushing, ranging from 5 up to 8 times a day.\nMoreover, regarding the dental occlusion conditions, no premature or abnormal dental or\nrestoration contact on occlusion contacts were verified, as well no laterality or\nprotrusion movements were found to be defective ().\nThe patient was questioned about the presence of systemic conditions that would require\nphysician consult, impact on the oral health or influence on periodontal status, but no\nsignificant findings were revealed and she reported to be generally in good health, as\nconfirmed by her physician. Nevertheless, complementary exams were still required for\ndeeper investigation, and the patient was submitted to standard protocols for evaluation\nof hematological, immunological and endocrine markers. Results from complete blood count\ndemonstrate a minor leukopenia (in special neutropenia) and lymphocytosis, both\nconsidered non significant by her physician (data not shown). Additionally, the levels\nof total hydroxyproline, ALP, calcium, calcitonin, urine calcium, PTH (parathyroid\nhormone), DHEA (dehydroepiandrosterone), FSH (follicle-stimulating hormone), LH\n(luteinizing hormone), glycemia, total cholesterol, HDL (high-density lipoprotein), LDL\n(low-density lipoprotein), triglycerides, type I urine, CRP (C reactive protein) and\nbone densitometry were completely normal.\nTo deeper explore the local modification of periodontal status, the biofilm samples\nobtained from the patient periodontal crevice/pocket were analyzed by Real-time PCR for\nperiodontopathogens detection. The gingival crevicular fluid and biofilm samples from\nthe periodontal crevice/pocket were collected with sterile paper point ISO #40\n(Dentsply, DeTrey, Konstanz, Germany) from at least 5 different bone\nloss sites in the inferior and superior arches, before treatment recommendations, as\ndescribed previously. Subgingival\nbiofilm samples were used for PCR assays for periodontopathogens detection. Bacterial or\nviruses DNA was extracted as previously described, and submitted to Real-time PCR analyses using SybrGreen\n(Invitrogen, Carlsbad, CA, USA) system, specific primers (the primer sequences are\ndepicted in ) and ng of DNA in each reaction. The positivity of bacteria or viruses\ndetection in each sample was determined based on the comparison with positive and\nnegative controls. As expected due to the oral hygiene condition of the patient, no\npositivity was observed for Aggregatibacter actinomycetemcomitans, Porphyromonas\ngingivalis, Fusobacterium nucleatum or Treponema\ndenticola, in any of the sites evaluated from both the inferior or superior\narches. Similarly, there was no DNA detection for viruses related to periodontal\ndiseases like Epstein-Barr virus type 1 (EBV-1), Herpes Simplex virus type 1 (HSV-1) or\nHuman Cytomegalovirus (HCMV) in the same sites ().\nGingival fluid was used for ELISA and enzymatic tests for quantification of the bone\nmetabolism markers acid phosphatase, alkaline phosphatase and RANKL, and inflammatory\ncytokines IL-1β and TNF-a, performed as previously described. These results were compared to 12 control and 12\nchronic periodontitis subjects who also had their gingival fluid collected from healthy\nor diseased sites respectively, as selected before. Although systemic bone metabolism markers were in the\nnormality levels, we observed elevated production of ACP, ALP and RANKL in the gingival\ncrevicular fluid (GCF) samples, in concentration similar to those found in chronic\nperiodontitis patients (-). Conversely, the levels of IL-1β and TNF-a were\nfound to be similar in the samples collected from the patient and the controls subjects,\nwhile higher levels of inflammatory mediators were detected in the chronic periodontitis\nsites (-). These results suggested that, although systemic features were not\naltered, the local bone metabolism was indeed modified, as observed when periodontal\nsites were evaluated.\nIn order to verify whether features like HLA or genetic polymorphisms could also\naccomplish for the extensive bone loss observed, the patient was typed for HLA molecules\nand single nucleotide polymorphisms (SNPs) in gene coding for cytokines associated to\nperiodontal disease susceptibility, as described previously. We also verified if the genetic status\nof the patient could account for the differential clinical condition observed. When the\nIL-1β 3954 SNP was analyzed, we observed the non-polymorphic CC genotype. The present\ndata also demonstrated that the patient carried the IL6-174 GC genotype (polymorphic\nheterozygous), the AA polymorphic genotype for TNFA-308 SNP, the MMP1-1607 - genotype\n1G/2G (polymorphic heterozygous), the non-polymorphic CC genotype of IL-10 592 SNP and\nTGFB1 -509 - genotype CT (polymorphic heterozygous) (). The genetic analysis also demonstrated that the patient was\npositive for HLA-DR4 (*0404) and HLA-B27 alleles, described to be associated with bone\nresorptive diseases like rheumatoid arthritis and periodontal diseases. Interestingly,\nthe patient reported a familial history of rheumatoid arthritis too.\nAltogether, the data obtained in the present case showed that the periodontal disease\npresented by the patient was not a direct consequence of the usual causes like\nmicrobiological factors. Instead, it could involve genetic findings and, in special,\nperiodontal trauma possibly due to excessive toothbrushing and oral hygiene, similar to\nsome gingival recession cases. In view of these results, a less vigorous and diminished\nfrequency of toothbrushing was recommended to the patient. At 2- and 6-month\nre-evaluations, the clinical periodontal condition, including bone loss was stabilized.\nFive years of follow-up revealed that the clinical scenario was stable without further\nevidence of disease progression, since no redness or bleeding on probing, increased\nprobing depth or attachment loss, or tooth mobility were observed. In fact, the clinical\nimages and the panoramic radiography (),\nafter 5 years of follow-up, showed no progression in the generalized horizontal alveolar\nbone loss when compared to the initial findings. Moreover, after controlling the\nexcessive toothbrushing, the levels of ALP, ACP and RANKL in gingival crevicular fluid\nin diseased sites were also greatly reduced, reaching nearby the control levels (-).