Year : 2008 | Volume
: 26 | Issue : 1 | Page : 18--21
Comparison of allele frequency for HLA-DR and HLA-DQ between patients with ECC and caries-free children
A Bagherian1, H Nematollahi2, JT Afshari3, N Moheghi3,
1 Department of Pediatric Dentistry, School of Dentistry, Rafsanjan University of Medical Science, Rafsanjan, Iran
2 Department of Pediatric Dentistry, Dental Research Center, Dental School, Mashhad University of Medical Science, Mashhad, Iran
3 Department of Immunogenetics, Immunology Research Center, Bu-Ali Research Centre, Medical School, Mashhad University of Medical Science, Mashhad, Iran
Department of Pediatric Dentistry, School of Dentistry, Rafsanjan University of Medical Science, Rafsanjan
Background: Early childhood caries (ECC) is one of the most common diseases of childhood. The etiology of ECC is multifactorial and both genetic and environmental factors play important roles in the pathogenesis of the disease. Genetic variations in the hosts may contribute to changes in the risk for dental caries. Genetic factors such as human leukocyte antigen (HLA) have recently been suggested as a predisposing factor.
Aim: The aim of this study was to look for an association between HLA-DRB1 and HLA-DQB1 with ECC for developing new strategies for the diagnosis as well as the prevention of the disease.
Design: In this study, we extracted the genomic DNAs from whole blood samples of 44 patients with ECC and 35 caries-free children by the salting-out method. We amplified the genomic DNA by PCR-SSP and then HLA-typing was performed for all alleles.
Results: The results revealed a significant increase in the frequency of HLA-DRB1*04 in the patient group (P = 0.019). The odds ratio for this allele was detected to be 10. The frequency of HLA-DQB1 alleles was not significantly different between the two groups.
Conclusion: The above results suggest that HLA-DRB1*04 is associated with the susceptibility to ECC. Thus HLA-DRB1*04 detection as a molecular marker for early diagnosis of ECC may be recommended.
|How to cite this article:|
Bagherian A, Nematollahi H, Afshari J T, Moheghi N. Comparison of allele frequency for HLA-DR and HLA-DQ between patients with ECC and caries-free children.J Indian Soc Pedod Prev Dent 2008;26:18-21
|How to cite this URL:|
Bagherian A, Nematollahi H, Afshari J T, Moheghi N. Comparison of allele frequency for HLA-DR and HLA-DQ between patients with ECC and caries-free children. J Indian Soc Pedod Prev Dent [serial online] 2008 [cited 2020 Aug 6 ];26:18-21
Available from: http://www.jisppd.com/text.asp?2008/26/1/18/40316
Dental caries is the single most common chronic childhood disease.  Despite the general global decline in dental caries in the past decades, early childhood caries (ECC) remains a significant problem in many developing countries and in a few industrialized nations. 
The American Academy of Pediatric Dentistry (AAPD) defines ECC as the presence of one or more decayed (noncavitated or cavitated), missing (due to caries), or filled tooth surfaces in any primary tooth in a child 71 months of age or younger.  Like other types of caries, ECC is caused by mutans streptococci that ferment dietary carbohydrates to produce acid on susceptible teeth, leading to caries over a period of time. However, while the general etiology of ECC appears similar to that of other types of caries, the predisposing factors are still unclear. 
Both genetic and environmental factors have influences on dental caries, and because of genetic differences certain environmental factors are potentially more cariogenic for some people than for others.  Microorganisms such as mutans streptococci are one of the most important etiological factors of dental caries and the individual's immune system, which defends the body against microorganisms, can have an important role.
Human leukocyte antigen (HLA) or major histocompatibility complex (MHC) molecules have important roles in the immune responsiveness. If an extracellular organism like a bacteria enters the body, antigen-presenting cells like macrophages first process it; the processed antigen (as a peptide) is linked to the HLA class II molecule and this complex is presented to the T-helper cells of the immune system, which activates the immune response.  Differences in MHC molecules may cause some variations in immune responses against microorganisms and may influence childrens' susceptibility to ECC.
To characterize the genetic contribution to both etiology and susceptibility to ECC and for developing new strategies for the diagnosis, management, risk assessment, as well as prevention in the future, we decided to compare the frequencies of DR and DQ HLA class II alleles in patients with ECC and in caries-free children.
Materials and Methods
This is a cross-sectional study, comparing the frequency of HLA-DR and HLA-DQ alleles in ECC with that in caries-free children. It is the equivalent of a retrospective cohort study since the alleles are present from birth.
The children were selected with a convenience sampling procedure from children referred to the emergency department of Dr. Sheikh Hospital in Mashhad, Iran.
79 subjects were selected for the study (44 in the ECC group and 35 in the caries-free group). Only children fulfilling the following conditions were included in this study: Age between 12 and 71 months, no history of congenital and genetic problems, parental permission. The protocol was reviewed and approved by the Medical Research Ethics Committee of Mashhad University.
Examinations for dental caries were carried out using conventional dental chairs, artificial light, flat mirror, and explorer; it was done according to the World Health Organization criteria and methods.  The children were divided into ECC and caries free groups after examination.
DNA HLA typing
DNA was extracted from 2 cc whole blood samples, using the Biogen DNA extraction kit (Mashhad, Iran) by the salting-out method.  Extracted DNAs were amplified with PCR-SSP (polymerase chain reaction-sequence specific primer). 
In this method, we used four sets of primers for HLA-DRB1*04 (DR4) alleles, two sets of primers for HLA-DQB1*02 (DQ2), two sets of primers for HLA-DQB1*03 (DQ3), two sets of primers for HLA-DQB1*05 (DQ5), and two sets of primers for HLA-DQB1*06 (DQ6).
Following amplification, the products of PCR-SSP were run in the electrophoresis tank of agarose gel 1.5% with ethidium bromide. A marker, positive control, negative control, and internal control (β-action) were used in each run. Documentation of positive and negative cases was performed by gel documentation device (Imago, Germany). A sample of gel documented photographs of HLA-DR4 is shown in [Figure 1].
To determine DR4 subtype alleles we used PCR-RFLP (restriction fragment length polymorphism) method. 
Mean age (±SD) was calculated for the two groups. The statistical significance of the difference between the means was tested by Student's t -test.
Fisher's exact test and the chi-square test were used to establish the statistical significance of the differences in the HLA alleles frequency between the ECC and the caries-free groups.
Strength of association of alleles was examined by calculation of odds ratios (OR) with a confidence interval (CI) of 95% in a logistic regression model. A P -value less than 0.05 was considered as statistically significant
The difference in the mean ages (±SD) of the caries-free group (46.5 ± 11.7 months) and the ECC group (53.3 ± 16.5 months) was significant ( t = 2.15 and P = 0.03).
The frequency of the DRB1*o4 allele was significantly increased in the ECC group (22.7%) compared with the caries-free group (2.9%). Fisher's exact test indicated a significant difference between the two groups ( P = 0.019) [Table 1]. Since the mean age was significantly different between the two groups, we used a logistic regression model [Table 2]. The results showed that being positive for the DRB1*o4 allele increases the risk for ECC by ten times (OR = 10) with a confidence interval of 95%. The genotype of the only caries-free child who was HLA-DR4 positive was (0409/0403).
The genotype frequencies in the ECC group are shown in [Table 3]. Our results indicated that the most frequent genotype in this group was also (0409/0403).
[Table 4] shows the frequencies of DQ2, DQ3, DQ5, and DQ6 alleles in the caries-free and the ECC groups. There were no statistically significant differences between these two groups for HLA-DQB1 alleles [Table 4].
To the best of our knowledge, this is the first reported study on the possible association between MHC alleles and ECC. ECC is one of the most common chronic childhood diseases. Like many medical and dental diseases, it depends on a complex interaction between the genetic structure of an individual and the superimposed environmental factors, a combination of nature and nurture. 
One aspect of genetic effects is genetic modification in immune response. Individuals with either inherited or acquired immune deficiency are subject to increased risk for dental caries.  HLA class II molecules play an important role in immune responsiveness and different hypotheses can explain their role. When cariogenic microorganisms enter the body, antigen-presenting cells first process them. These processed antigens (as peptides) are linked to the HLA class II molecules and this complex is presented to T-helper cells of the immune system, which activates the immune response.
Since mutans streptococci are found in almost all individuals, the large differences in oral colonization levels between individuals can be explained by variations in the immune response. Some types of MHC molecules stimulate B-lymphocyte cells less effectively and produce low salivary IgA activity against mutans streptococci.  A likely explanation might be that these alleles have an amino acid sequence that binds poorly to the antigenic peptides of the mutans streptococcus.
HLA variation can also cause alterations in enamel structure. These effects of HLA molecules have been studied in celiac disease. Celiac disease is a digestive disorder characterized by permanent gluten intolerance in genetically susceptible individuals. The cause of the dental lesion associated with celiac disease is uncertain. The low calcium and phosphorus absorption from the intestine in the first years of life, when the disease is still undiagnosed, may cause enamel hypoplasia.  Some studies have shown a relationship between some types of HLA and susceptibility to celiac disease. Different HLA molecules have different effects on the gastrointestinal malabsorption and produce different kinds of enamel defects that predispose an individual to dental caries. ,
In this study, we compared the frequency of DR and DQ HLA class II alleles in patients with ECC and in caries-free children.
In the caries-free group, only one case (2.9%) had HLA-DR4 but in the ECC group, ten cases (22.7%) had HLA-DR4; the difference between the two groups was significant ( P = 0.019).
Wallengren et al . have also shown that high levels of oral cariogenic microorganisms are present in saliva among DRB1*o4 positive dental students and staff in Sweden. 
Acton et al . demonstrated that high levels of S. mutans were positively associated with the presence of DR3 and DR4 alleles in African-American women. However, no significant associations were observed between any DRB1 and DQB1 allele and the DMFS index. 
In a population of Caucasians from London, a higher dose of streptococcal antigen was required to release T-helper activity in DR4 positive individuals than in those who were DR4 negative. 
We did not observe an association between any of the DQB1 alleles and ECC, but Ozawa et al . have shown that HLA-DQB1*0601 was associated with the number of mutans streptococci in the saliva and DQ03 was weakly associated with the numbers of lactobacilli in the saliva among young adults in Japan. 
Our data and those of others provide evidence that genes within the MHC, especially the DR4 group, can influence susceptibility to dental caries.
HLA class II molecules may play an important role as a predisposing factor for ECC.Positive HLA-DRB1*04 may increase the risk of ECC ten times.
The researchers would like to express their gratitude to Mr Habib Esmaeli and Mr Mohsen Rezaeian for helping with the statistical analysis. We would also like to thank the research center of Mashhad University of Medical Science (MUMS) for funding this research and the Bou-Ali Research Center of Mashhad for their cooperation.
|1||McDonald RE, Avery DR, Dean JA. Dentistry for the child and adolescent. 8 th ed. St. Louis: Mosby Co; 2004. p. 105,209.|
|2||Bowen H. Response to seow: Biological mechanism of early childhood caries. Community Dent Oral Epidemiol 1998;26:8-27.|
|3||Shuler CF. Inherited risk for susceptibility to dental caries. J Dent Educ 2001;65:1038-45.|
|4||Lechler R, Warrens A. HLA in health and diseases. 2 nd ed. London: Harcourt Co; 2000. p. 104.|
|5||World Health Organization. Oral health surveys, basic methods. 3 rd ed. Geneva: WHO; 1987.|
|6||Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215.|
|7||Cannava AA, Olerup O. HLA-DQB1 low resolution typing by PCR amplification with sequence- specific primers(PCR-SSP). Eur J Immun 1994;21:447-55.|
|8||Mitsunaga S, Oguchi T, Tokunaga K, Akaza T, Tadokoro K, Juji T. High-resolution HLA-DQB1 typing by combination of group-specific amplification and restriction fragment length polymorphism. Hum Immunol 1995;42:307-14.|
|9||Verbin S. Genetic influence in women's oral health. Dent Clin North Am 2001;45:443-67.|
|10||Madigan A, Murray PA, Houpt M, Gatalanotto F, Feuerman M. Caries experience and cariogenic markers in HIV-positive children and their siblings. Pediat Dent 1996;18:129-36.|
|11||Wallengren ML, Hamberg K, Ericson D, Nordberg J. Low salivary IgA activity to cell-surface antigens of mutans streptococci related to HLA-DRB1*04. Oral Microbiol Immunol 2005;20:73-81.|
|12||Aguirre JM, Rodriguez R, Oribe D, Vitorial JC. Dental enamel defects in celiac patients. Oral Surg Oral Med Oral pathol Oral Radiol Endod 1997;84:646-50.|
|13||Mariani P, Mazzili MC, Margutti G, Lionetti P, Triglione P, Petronzelli F, et al . Celiac disease, enamel defect and HLA typing. Acta Paediatr 1994;83:1272-5.|
|14||Tighe MR, Hall MA, Barbado M, Cardi E, Welsh KI, Ciclitira PJ. HLA Class II alleles associated with celiac disease susceptibility in a southern European population. Tissue Antigens 1992;40:90-7.|
|15||Wallengren ML, Johnson U, Ericson D. HLA-DR4 and number of mutans streptococci in saliva among dental students and staff. Acta Odontol Scand 1997;55:296.|
|16||Acton RT, Dasanoyake AP, Harrison RA, Li Y, Roseman JM, Go RC, et al . Associations of MHC genes with levels of caries-inducing organisms and caries severity in African-American women. Hum Immunol 1999;60:984-9.|
|17||Lehner T, Lamb JR, Welsh KI, Batchelor J. Association between HLA-DR antigens and helper cells activity in the control of dental caries. Nature 1981;292:770.|
|18||Ozawa Y, Chiba J, Sakamoto S. HLA Class II alleles and salivary numbers of mutans streptococci and lactobacilli among young adults in Japan. Oral Microbiol Immunol 2001;16:353-7.|