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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 9  |  Issue : 1  |  Page : 6-12

Collagen 1 alpha 2 gene polymorphism and dental fluorosis: A polymerase chain reaction study


Department of Periodontics, College of Dental Sciences, Davangere, Karnataka, India

Date of Web Publication16-Mar-2018

Correspondence Address:
Dr. Kharidhi Laxman Vandana
Department of Periodontics, College of Dental Sciences, Room No 4, Davangere - 577 004, Karnataka
India
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DOI: 10.4103/srmjrds.srmjrds_84_16

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  Abstract 


Background and Objectives: The objective of this study is to assess the association of collagen 1 alpha 2 (COL1A2) gene polymorphism in participants with dental fluorosis and periodontitis. Subjects and Methods: Atotal of 50 age- and sex-matched participants, thirty participants with dental fluorosis with or without periodontitis and 20 nonfluorosis participants with or without periodontitis, participated in this study to find the association of COL1A2 gene polymorphism, dental fluorosis, and periodontitis. Clinical parameters including plaque index, gingival bleeding index, modified gingival index, community periodontal index, and clinical attachment loss were recorded. The PvuII polymorphisms in the COL1A2 gene were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real-time PCR. Urinary fluoride level estimation was done with fluoride ion selective electrode. Results: DNA was genotyped at the PvuII RFLP (in exon 25) marker inside the COL1A2 gene. For the PvuII RFLP, a 584-base pair (bp) DNA fragment was produced. In the gel picture after restriction digestion, only one band of 584 bp was seen in all the 50 samples. This indicates that this gene polymorphism is rare in Indian population as compared to Chinese population. Results for clinical parameters were explained using mean, standard deviation, and unpaired t-test. In addition, fluoride levels in urine were found to be significantly lower in nonfluorosis (0.48 mg/L) controls compared to cases with dental fluorosis (1.17 mg/L). Interpretation and Conclusion: This preliminary study provides no evidence of association between polymorphisms in the COL1A2 gene with dental fluorosis in high-fluoride-exposed populations. Further studies with large sample size are needed to confirm the association and the findings of this preliminary study.

Keywords: Collagen 1 alpha 2 gene polymorphism, dental fluorosis, polymerase chain reaction, periodontitis, real-time polymerase chain reaction


How to cite this article:
Pragya G, Vandana KL. Collagen 1 alpha 2 gene polymorphism and dental fluorosis: A polymerase chain reaction study. SRM J Res Dent Sci 2018;9:6-12

How to cite this URL:
Pragya G, Vandana KL. Collagen 1 alpha 2 gene polymorphism and dental fluorosis: A polymerase chain reaction study. SRM J Res Dent Sci [serial online] 2018 [cited 2018 Oct 22];9:6-12. Available from: http://www.srmjrds.in/text.asp?2018/9/1/6/227772




  Introduction Top


Periodontal diseases are chronic infectious disorders caused primarily by bacteria. The various risk factors associated with periodontal diseases are oral hygiene level, local factors, bacterial specificity, viruses, host response factors, smoking, gender, genetic factors, osteopenia, osteoporosis, socioeconomic status, psychological factor, race-ethnicity, and geographic region.[1]

Endemic fluorosis remains a challenging and extensively studied national health problem in India.[2] In dental fluorosis, various hard tissue effects seen are hypomineralization of enamel [3],[4] and dentin,[4] hypercementosis, recession of the alveolar crest,[5] root resorption,[6] and hypermineralization of cementum.[7] The soft-tissue changes include inhibition of Type I collagen synthesis, degree of cross-linking,[8] fibroblast growth inhibition and morphologic changes,[9] and lethal effects on fibroblasts.[10]

Epidemiological studies concerning the prevalence of periodontal disease in relation to dental fluorosis have given inconsistent results.

The inconsistency in relationship of fluorosis to periodontal disease could be attributed to genetic reasons such as variation in occurrence of gene polymorphism among different population globally. Various gene polymorphisms have been investigated as putative risk factors for periodontitis.[1]

Genetic polymorphism has racial variation, and it varies population wise globally. Identifying genes that contribute to the pathogenesis of periodontitis can have significant public health, therapeutic, and scientific repercussions.[11]

Two gene polymorphism studies in literature are association of collagen 1 alpha 2 (COL1A2) and estrogen receptor (ER-RsaI) gene polymorphism with occurrence of dental fluorosis.

The association of COL1A2 gene polymorphism with dental fluorosis has been studied in Chinese children carrying that the homozygous genotype had a significantly increased risk of dental fluorosis compared to children carrying the homozygous genotype pp in an endemic fluorosis village.[12] Another study to evaluate the relationship of COL1A2 PvuII gene polymorphism in high-fluoride area concluded that there was no significant difference of dental fluorosis between the high-fluoride area and the high-fluoride and iodine area.[13] A recent study done to decide the role of COL1A2 gene polymorphism and vulnerability to dental fluorosis in Iowa City gave confirmation of a relationship between the presence dental fluorosis and collagen, Type I, alpha 2 polymorphism. They also bolster earlier reports suggestive of connection between fluoride consumption and certain genetic polymorphisms.[14] Another Chinese study reported the association of erythrocyte sedimentation rate-RsaI gene polymorphism with dental fluorosis;[15] incidentally, ER-alpha gene polymorphisms were found to be associated with alveolar bone loss in patients with periodontitis.[16]

The occurrence of dental fluorosis in around Davangere district is possibly an environmental causative factor for dental and periodontal diseases of these areas. The water fluoride level ranges from 0.2 mg/L to 2.41 mg/L,[17] and the occurrence of periodontitis was found to be more with the increased degree of fluorosis.[18] The interaction of environmental factors with periodontal diseases is poorly understood. Unfortunately, the effects of fluorosis as an environmental factor is not evaluated and researched to designate it as a risk factor similar to smoking.

The COL1A2 gene provides instructions for making part of a large molecule called Type I collagen. Type I collagen is the most abundant form of collagen in the human body [19] and tooth-supporting structures.[20] The soft-tissue changes induced by fluoride such as inhibition of collagen Type I synthesis and impairment of cross-linking could be induced by COL1A2 gene polymorphism. These collagen alterations may be responsible for susceptibility of participants from high-fluoride areas to periodontitis. The possibility of fluorosed participants to be more susceptible to periodontitis could be attributed to easy early breakdown or destruction of weakly formed Type I collagen in the periodontitis by the bacterial and inflammatory products.

Since COL1A2 gene polymorphism significantly increased the risk of dental fluorosis in China,[13] Iowa City,[15] and considering the higher prevalence of periodontitis in dental fluorosis participants [18] of endemic fluoride areas of Davangere district; an attempt has been made in this preliminary study to establish a correlation between COL1A2 gene polymorphism, dental fluorosis and periodontitis for better understanding the genetic aspects of “periodontitis” per se in endemic fluorosis areas (EFAs). This study was the first attempt from Indian scene to assess such kind of association. Medline search using the keywords Fluorosis, collagen gene polymorphism, and periodontitis revealed no study.

Hence, this case–control study was conducted in Davangere, Karnataka, India, to investigate the COL1A2 (PvuII) polymorphism in dental fluorosis participants with periodontitis.


  Subjects and Methods Top


Location and population

A case–control study conducted in Davangere district, Karnataka, India, in 2012–2013. It is EFA, and EFA was defined as an area with fluoride levels exceeding 0.5–3 mg/L in drinking water. A total of fifty participants aged 18–50 years, born and raised in the Davangere District, were selected from the Outpatient Department, Department of Periodontics, College of Dental Sciences, Davangere. Out of which, a total of 30 dental fluorosis cases with periodontitis and 20 control periodontitis participants without dental fluorosis were selected. The participants with dental fluorosis were confirmed from the history of their permanent residence in EFAs and dental fluorosis stains using Jackson's fluorosis index. From each participant, about 3 ml of fasting blood and 10 ml of instant urine samples were collected. The study was approved by the Institutional Ethical Review Board.

Detection of urine fluoride

The fluoride levels in the samples of urine from both the fluorosed and nonfluorosed (NF) groups were detected by fluoride ion selective electrode (Thermo Fisher Scientific Inc., USA). Participants with a fluoride concentration exceeding 1.5 mg/L in the urine were classified as highly exposed.

Clinical parameters

The following clinical parameters were recorded for both the fluorosed and NF participants:

  • Jackson's fluorosis index (Vandana, 2007)[18]
  • Plaque index (PI) (Silness and Loe, 1964)[21]
  • Modified gingival index (MGI)[23]
  • Gingival bleeding index (GBI) (Ainamo and Bay, 1975)[22]
  • Community periodontal index (CPI)[24] (WHO 1997)[25]
  • Clinical attachment loss.[25]


Genotyping

Blood (3 ml) was drawn and stored at 4°C until DNA extraction. DNA was extracted using whole blood genomic DNA miniprep kits (FlexiGene DNA kit, Qiagen); DNA was genotyped at the following markers: the PvuII restriction fragment length polymorphism (RFLP) (in exon 25) inside the COL1A2 gene. For the PvuII RFLP, the forward primer (5-GGA AAT ATC GGC CCC GCT GGA AAA-3) and the reverse primer (5-GTC CAG CCA ATC CAA TGT TGC C-3) (obtained from Imperial Life Sciences Private Limited, Gurgaon, Haryana, India) were used in polymerase chain reaction (PCR) to produce a 584-base pair (bp) DNA fragment. The PCR amplification was conducted in reaction mixtures each containing 35.5 μl of H2O,

5 μl of 10 PCR buffer, 1.5 mM of magnesium chloride, deoxynucleoside triphosphate (200 μM each), 2.5 U of Taq polymerase (TaKaRa, Japan), 0.4 μM each of the two primers for each marker, and 300 ng of genomic DNA. The PCR was performed on T-gradient thermocyclers (GeneAmp PCR System; Whatman Biometra, Gottingen, Germany). DNA Amplification using multiplex PCR PCR was done for 30- 40 cyles in the following three steps; Before the first cycle, during initiation, a step is performed at a temperature of 94°C–96°C for 4–10 min. This first hold is employed to ensure that most of the DNA template and primers are denatured and further subjected to annealing in the temperature range of 50°C–60°C for 20–40 s.

A final elongation step of 5–15 min (depending on the length of the DNA template) after the last cycle may be used to ensure that any remaining single-stranded DNA is fully extended at the temperature of 72°C.

After PCR amplification, 7 μl of the respective PCR products were removed and digested with 10 U of PvuII (Imperial Life Sciences Private Limited, Gurgaon, Haryana, India) at 37°C for 3 h. The samples were electrophoresed in 1.8% Metaphor agarose gels in ×1 Tris-acetate-ethylenediaminetetraacetic acid buffer and 0.3 μg/ml of ethidium bromide and visualized on a transilluminator under ultraviolet (UV) light and photographed for the presence and absence of the PvuII restriction sites of the COL1A2 gene. To reconfirm the DNA formed in PCR, real-time PCR RTPCR was also performed for all the 50 samples.

Amplified (uncut) PCR product is 584 bp. This 584-bp PCR product is digested with PvuII restriction enzyme. When there is no polymorphism present in PvuII, there is the presence of single band as found in this study indicated by 584 bp in gel picture, wild (Wt±)/normal COL1A2 genotype [Figure 1] and [Figure 2]. If there was the presence of polymorphism, there would have been an extra band of lower molecular weight appreciated on gel picture [Figure 3] and [Figure 4] as an example.
Figure 1: Wild (Wt±)/normal collagen 1 alpha 2 genotype

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Figure 2: Schematic representation of Wild (Wt±)/normal collagen 1 alpha 2 genotype

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Figure 3: Gene polymorphism

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Figure 4: Restriction site present, multiple bands are seen proves presence of polymorphism

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Statistical analysis

Data were analyzed using SPSS 17.0 (SPSS version 17, SPSS, Chicago, IL, USA) for clinical parameters and urinary fluoride levels. Means of fluorosed (F) and NF groups for age, MGI, CPI, Loss of Attachment, and Urinary Fluoride Level were compared using unpaired t-test. Comparison of fluorosed (F) and NF group for PI and GBI was done using Mann–Whitney U-test. With respect to the occurrence of COL1A2 gene polymorphism, statistical analysis was not possible as none of the sample expressed gene polymorphism.


  Results Top


A total of 50 age- and sex-matched participants, thirty participants with dental fluorosis (mean age: 23.63 ± 7.01) with periodontitis and 20 nonfluorosis participants (mean age: 27.30 ± 6.02) with periodontitis, participated in this study to find the association of COL1A2 gene polymorphism.

The results of the study are interpreted in [Table 1], [Table 2], [Table 3] and Graph 1.
Table 1: Comparison of demographic data

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Table 2: Comparision of clinical and biochemical parameters of fluorosed group (f) and non fluorosed group (nf) with numerical values

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Table 3: Comparision of clinical parameters with discrete ( in%) values

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Demographic data

A total of 50 age- and sex-matched participants, 30 dental fluorosis participants (mean age: 23.63 ± 7.01) and 20 NF participants (mean age: 27.30 ± 6.02), participated in the study [Table 1]. The P value was found to be not significant (P = 0.62) between the fluorosed and the NF group. The means of the two groups were compared using unpaired t-test since the data were numerical. In the fluorosed group, 3% of the participants had Type A dental fluorosis with gingivitis and 10 (33%) participants had Type B dental fluorosis, out of which 23% had periodontitis and 10% had gingivitis. Type F dental fluorosis was found in 19 (63%) of total participants, and in them, 20% had periodontitis and 43% had gingivitis.

Results of clinical parameters

Results for clinical parameters were explained using mean, standard deviation, and unpaired t-test. The clinical parameters were found to be nonsignificant between the cases and controls [Table 2] and [Table 3].

Biochemical parameter urinary fluoride levels

On comparison of mean urinary fluoride level of fluorosed and NF groups, it was found that P value was highly significant (P = 0.00) with the mean urinary fluoride level of 1.17 ± 0.14 mg/L in fluorosed group and 0.48 ± 0.19 mg/L for NF group [Table 2].

Collagen 1 alpha 2 polymorphism (PvuII)

Variations in the nucleotide sequence at a locus (allele) in COL1A2 (PvuII) gene were studied in this preliminary study. No extra band of any molecular weight was observed indicating that all the samples collected were wild type in both control and exposed groups. [Figure 5].
Figure 5: Gel picture of all 50 samples after restriction enzyme digestion

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  Discussion Top


The occurrence of periodontitis in high water fluoride areas has shown a global variation [18] and also fluorosis plays as an environmental factor in causing periodontitis through its effects on hard and soft tissues of the periodontium.[20] Environmental factors play an important role in the expression of periodontitis which include oral hygiene/bacterial plaque, smoking, stress, and systemic factors that may exacerbate the inflammatory pathology associated with periodontitis.[26] The role of smoking as an environmental risk factor [26] demonstrates variability in causing periodontitis, i.e. all smokers do not suffer from periodontitis and could be attributed to genetic polymorphism.[27] Based on this view, fluorosis which is possibly considered as environmental factor, fluorosed participants may or may not suffer from periodontitis can be attributed to putative genetic factors. To analyze this issue, the role of genetic polymorphism requires to be probed in dental fluorosis participants with periodontitis.

However, the association of gene polymorphism in the participants suffering from dental fluorosis and periodontitis has not being focused so far, and hence, the current study was conducted.

The study results are discussed as follows. The baseline values for all the parameters, i.e. age, sex, PI, GBI, MGI, and CPI were matched in this study.[28]

On comparing urinary fluoride level of fluorosed and NF groups, the mean urinary fluoride level (1.17 ± 0.14 mg/L) was highly significant in the fluorosed than the NF group (0.48 ± 0.19 mg/L). A study stated that highly significant differences were present between the mean urinary F, plasma F, and serum F levels of participants from fluorosed and NF areas.[29] Another study correlated serum fluoride levels with high fluoride in drinking water.[30]

In the current study, COL1A2 gene polymorphism association has been attempted in dental fluorosis participants with periodontitis, for the first time as per the lack of studies from Medline search using words: periodontitis, dental fluorosis, COL1A2 gene polymorphism. Two genes, COL1A1 and COL1A2 provide instructions for making components of collagen. Specifically, the COL1A1 gene produces a component of Type I collagen called the pro-alpha (I) chain, whereas the COL1A2 gene produces the pro-alpha (2) chain.

The chains combine to make a molecule of Type I procollagen. The “rope-like” procollagen molecules arrange themselves into long, thin fibrils that cross-link to one another in the spaces around cells. The cross-links result in the formation of very strong mature Type I collagen fibers, which play important role in bone formation and soft-tissue formation.[15],[20] Type I collagen forms 78.06% of periodontal ligament fibers, 73.09% in cementum, and 30.50% in alveolar bone [20] building the attachment apparatus of the tooth. PvuII is derived from Proteus vulgaris (Pvu) gene coding for the PvuII restriction enzyme.[31],[32] “II” stands for the type of restriction endonuclease enzyme activity. Type II restriction endonucleases are components of restriction modification systems that protect bacteria and archaea against invading foreign DNA. Most are homodimeric or tetrameric enzymes that cleave DNA at defined sites of 4–8 by in length and require Mg2+ ions for catalysis.[33] PvuII recognizes the sequence: 5' C A G + C T G 3' forward primer; 3' G T C + G A C 5' reverse primer and cleaves at the site indicated by +.[32] If there was the presence of polymorphism in the studied participants after restriction enzyme PvuII digestion in the PCR product (584 bp) of the COL1A2 gene, there would have been presence extra bands of smaller molecular weight seen under UV camera, after gel electrophoresis (gel picture). The present study result using PCR observed; no extra band of any molecular weight was observed indicating that all the samples collected were wild type in both fluorosed and NF group.

The reason for the absence of this gene polymorphism could be rarity of this gene polymorphism in Indian population, or difference in geographical areas could be an attributable factor for negative result compared to Chinese [12] and Iowa [14] study. Since there are contradictory reports are there in literature from two China studies,[12],[13] the DNA product formed in multiplex PCR was reconfirmed using RTPCR in the present study. The present study result using PCR observed; no extra band of any molecular weight was observed indicating that all the samples collected were wild type in both fluorosed and NF group [Figure 5]. This indicates that this genotype is possibly rare in Indian population as compared to Chinese Han population. However, the possibility of sample size to be small cannot be ruled out.

There were no correlation between COL1A2 PvuII gene polymorphism and the dental fluorosis in three geographical areas of China.[13] Another study done to investigate the relationship between dental fluorosis, COL1A2 gene polymorphism, and serum calciotropic hormone levels provided the first evidence of an association of this gene polymorphism with dental fluorosis in high-fluoride-exposed Chinese population.[12] A recent study done in Iowa City gave confirmation of a relationship between the presence dental fluorosis and collagen, type I, alpha 2 polymorphism. They also bolster earlier reports suggestive of connection between fluoride consumption and certain genetic polymorphisms and fortify enthusiasm in the role of genetics in the development of dental fluorosis.[34]

The sorts of genetic variants that add to the occurrence of complex diseases are generally called “genetic polymorphisms” in light of the fact that they are predominant in the population than the genetic mutations. The environmental factors too add to the etiology of most common diseases.[35]

Both environmental and genetic factors contribute to individual variations in the risk for periodontal disease. Several studies were performed to elucidate the inter-individual variations of severity and progression of periodontitis.

An important environmental factor such as dental fluorosis from endemic fluoride area could serve as risk factor in causing periodontitis through several influences on hard and soft tissue of periodontium. Although direct local effects are not obvious, its systemic effect is pathogenic through its complex interactions. A synergistic risk pattern may explain the genetic–environmental interaction.

There is an individual variability in response to external stimuli, and this may influence some people likely to be affected by the sequel of periodontitis than others. Periodontal diseases occur with a wide range of clinical variability and severity. Genetic susceptibility and environmental factors such as smoking and dental fluorosis seems to be of importance in determining its manifestation and progression.

In periodontal literature, the association of various genetic risk factors has been confirmed in numerous studies, sometimes with controversial conclusions. Most of the studies published so far consist of less participants who are mostly convenient sample, an obstacle in the present study.[35] However, large studies will be needed to fully understand these interactions. Hence, numerous clinical and genetic studies are required to explain the correlation between dental fluorosis and periodontitis.

The main limitation of this study was small sample size, due to self-funding without external financial support. The larger sample size would provide better insight regarding COL1A2 gene polymorphism association with dental fluorosis and periodontitis. Further study should involve dental fluorosis participants with and without periodontitis.


  Conclusion Top


There was no association between polymorphisms in the COL1A2 gene (PvuII) with dental fluorosis in high water fluoride-exposed participants. There was no association between polymorphisms in the COL1A2 gene (PvuII) with periodontitis in fluorosed and NF participants. The urinary fluoride levels were found to be significantly lower in nonfluorosis controls compared to cases with dental fluorosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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