Fogorvosi szemle, 2007 (100. évfolyam, 1-6. szám)

2007-10-01 / 5. szám

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FOGORVOSI SZEMLE ■ 100. évf. 5. sz. 2007.

population depending on the population and wether deciduous teeth or adult ones were studied [7], [8]. Around 16% of the adult Hungarian population suffers from hypodontia in adult teeth [9]. Missing tooth germs frequently coincide with other disorders such as ectodermal displasia, which can be variably autosomal dominant, autosomal recessive or linked to X chromosome. The disease is usually characterized by nail anomalies, dry skin, thin hair and oligodontia [10], The etiology of hypodontia is complex, it can be related to both genetic and environmental factors [3]. Gene polymorphism studies and gene mutation experiments on animals revealed the importance of genetic factors in tooth development [11]. The exact importance of the individual components is not known, but the complexity of tooth formation is underlined by the fact that at least 200 individual genes have been identified during the study of murine tooth development [12],

The studies performed up till now in both periodontitis and hypodontia yielded highly variable results depending of the ethnic groups investigated [13]. Therefore, it is important to accomplish a study with the Central and Eastern European population. This study may reveal the genetic factors besides the classic epidemiological factors such as oral hygiene. As the first part of our long term, high sample number investigation, in the present work we developed and optimized the detection of gene polymorphisms, which have high potential to be involved as risk factors in the initiation of periodontitis and hypodontia [14] [15].

Materials and methods

Tissue samples

For our investigations oral mucosal scrapings were collected from patients of the Clinics of Semmelweis University. Ethical permission was received before starting the studies. Before sample collection, fully informed written consent was received from each individual participating in the study. In the periodontitis study we have the following groups: (1) gingivitis, disorders localized exclusively in the gingiva; (2) chronic periodontitis; (3) aggressive periodontitis; (4) necrotizing periodontitis; (5) control group consisting of healthy volunteers with healthy, normal denture and without gingival or periodontal disease or craniofacial abnormality.

In the hypodontia and oligodontia studies the primary criteria was at least one missing adult tooth germ excluding the wisdom teeth, and also the lack of craniofacial malformation and any systemic disease. The control group involved healthy volunteers with healthy, normal deciduous and adult denture and without any craniofacial abnormalities.

DNA isolation

For the gene polymorphism studies DNA was isolated

from oral mucosal scrapings using the QIAmp DNA Blood Mini Kit (Qiagen GmbH, Hilden), and the Nucleo- Spin Tissue kit (Macherey-Nagel GmbH & Co. KG, Düren), according to the manufacturers’ protocols. Integrity of the DNA was checked by electrophoresis on 1% agarose gel, concentrations were measured by a Quibit fluorometer (Invitrogen, Carlsbad, CA).

PCR-RFLP

To identify the individual alleles polymerase chain reaction (PCR) was performed. The specific parameters initially applied in the periodontitis studies are based on the previous work of Brett and coworkers [14] but we have made a considerable number of changes. The oligonucleotide sequences of the primers are given in Table 1. In the hypodontia series the initial parameters followed the work of Peres and co-investigators [15], but again, we have made modifications and the final conditions are given in Table 1.

PCR reactions were performed with GoTaq Flexi polymerase (Promega, Madison Wl) in 25 pi final volume, where the reaction mixture was as follows: 50 mM Tris-HCI (pH=9.0), 50 mM NaCI, 200 pM of all four dNTPs in the buffer, 1.5 mM MgCI2, 1.25 Unit GoTaq Flexi polymerase, and 5-5 pM of forward and reverse primers. Following the amplification 1 pg PCR product was digested with the selected restriction endonucleases for the restriction fragment length polymorphism analysis (RFLP). The restriction endonucleases are such microbial enzymes that are able to recognize a 4-8 nucleotide long DNA sequence and to cut through the DNA chain at the given sequence.

The alleles were separated by 2-2.5% agarose gel electrophoresis and ethidium-bromide staining. The separated bands were visualized under ultraviolet light and the picture obtained was scanned by a digital gel documentation system.

Results

To underline the importance of our studies, first we show epidemiological data representative for the Hungarian population in respect of the occurrence of periodontitis and hypodontia. Recently a study has been carried out at Semmelweis University, in accordance with the guidelines of the World Health Organization, including more than 4100 volunteers. This study clearly shows that only a very small percent of the adult Hungarian population is not affected by periodontal anomalies, and the sum of these anomalies is practically independent of age (Figure 1A). On the other hand, the severity of the disorder gradually increases with age (Figure 1B). Hypodontia also affects a considerable part of the population according to another study performed at Semmelweis University, in which almost 1900 children and adolescents were included. This representative investigation demonstrates that