the participants were examined between january and march 2012 , and all of them gave informed consent for participation .
the study comprised patients and control participants between 25 and 65 years of age who were divided into : group one ( 2535 years ) , group two ( 3645 year ) , group three ( 4655 years ) , and group four ( 5665 years ) .
the diagnosis of periodontal disease was established according to the clinical parameters of the periodontal disease index ( pdi ) , also known as ramfjord index . for the pdi assessment , six teeth were evaluated : the upper left central , first premolar and right first molar ; and the lower right central , first premolar , and left first molar were measured .
examination method was applied by using university of michigan  0 probe with william 's markings at 1 , 2 , 3 , 5 , 7 , 8 , 9 , and 10 mm .
each sextant was designed as either healthy ( score 0 ) ; mild to moderate gingivitis , but changes not extending all around the tooth ( score 1 ) ; mild to moderate gingivitis extending all around the tooth ( score 2 ) ; severe gingivitis ( marked redness , bleeding tendency , and ulceration ) ( score 3 ) ; a probing depth less than 3 mm ( score 4 ) ; a pocket depth from 3 to 6 mm ( score 5 ) ; or a pocket depth exceeding 6 mm ( score 6 ) , according to the scores recorded at the index teeth .
cp was defined as oral health status with a pdi score of 5 and 6 . oral health status with pdi scores between 0 and 4
genomic dna was extracted from buccal swabs using the protocol of isohelix buccal dna isolation kit , which contained solution ls ( lysis buffer ) , solution pk ( proteinase k ) 1 ml , solution ct ( capture buffer ) , and solution te ( tris - edta ) , ( rehydration buffer ) ( 24 ) .
agarose gel electrophoresis was applied to examine the integrity of the genomic dna ( 25 ) .
about 0.75 m of each forward and reverse primers were added to a reaction mixture containing 1x ( 1.5 mm mgcl2)2 gotaq reaction buffer , pcr nucleotide mix 0.2 mm each dntp , 1.25 l of gotaq dna polymerase ( 5 u/l ) , and nuclease - free water to final volume 50 l .
the pcr program was performed for 35 cycles and consisted of an initial denaturation for 2 min at 95c , 35 sec at 95c for denaturation , 52c for 35 sec for annealing , and 72c for 35 sec for extension , followed by a final extension for 7 min at 72c ( 26 ) .
agarose gel electrophoresis was applied to the pcr products ( 25 ) , then pcr products were purified using the protocol of the qiaquick purification kit to remove excess salts , pcr primers , and dntps ( 27 )
. main characteristics of vdr gene polymorphisms and primers sequencesa 
  from naito et al .
after purification of the pcr products , dna cycle sequencing was performed using of bigdye terminator v1.1 cycle sequencing protocol ; 0.75 m of forward apai and bsmi , and 0.75 m of reverse foki primers were prepared . in the reaction mixture ,
6.6 l of nuclease - free water was placed , followed by 4 l of ready reaction premix , 2 l of bigdye terminator v1.1 , sequencing buffer ( 5x ) and 6.4 l of apai , bsmi , and foki .
the pcr program was performed for 25 cycles and consisted of an initial denaturation for 1 min at 96c , 10 sec at 96c for denaturation , 50c for 5 sec for annealing , and 60c for 4 min for extension .
sanger sequencing method was applied for capillary electrophoresis ; the run was performed on applied biosystem ( 3500xl genetic analyzer ) .
all statistical analyses were performed using the spss for windows , version 15.0 ( spss inc . ,
frequency differences of apai , bsmi , and foki polymorphisms and departure from hardy  weinberg equilibrium were tested by pearson 's chi - square test which was also used to quantify the association between cp and these polymorphisms .
the level of significance ( p value ) less than 0.05 was considered statistically significant for all analyses .
in total , 196 male and female libyan citizens from many governmental and private dental clinics in tripoli participated in the study .
the participants were examined between january and march 2012 , and all of them gave informed consent for participation .
the study comprised patients and control participants between 25 and 65 years of age who were divided into : group one ( 2535 years ) , group two ( 3645 year ) , group three ( 4655 years ) , and group four ( 5665 years ) .
the diagnosis of periodontal disease was established according to the clinical parameters of the periodontal disease index ( pdi ) , also known as ramfjord index . for the pdi assessment , six teeth were evaluated : the upper left central , first premolar and right first molar ; and the lower right central , first premolar , and left first molar were measured .
examination method was applied by using university of michigan  0 probe with william 's markings at 1 , 2 , 3 , 5 , 7 , 8 , 9 , and 10 mm .
each sextant was designed as either healthy ( score 0 ) ; mild to moderate gingivitis , but changes not extending all around the tooth ( score 1 ) ; mild to moderate gingivitis extending all around the tooth ( score 2 ) ; severe gingivitis ( marked redness , bleeding tendency , and ulceration ) ( score 3 ) ; a probing depth less than 3 mm ( score 4 ) ; a pocket depth from 3 to 6 mm ( score 5 ) ; or a pocket depth exceeding 6 mm ( score 6 ) , according to the scores recorded at the index teeth .
cp was defined as oral health status with a pdi score of 5 and 6 . oral health status with pdi scores between 0 and 4
genomic dna was extracted from buccal swabs using the protocol of isohelix buccal dna isolation kit , which contained solution ls ( lysis buffer ) , solution pk ( proteinase k ) 1 ml , solution ct ( capture buffer ) , and solution te ( tris - edta ) , ( rehydration buffer ) ( 24 ) .
agarose gel electrophoresis was applied to examine the integrity of the genomic dna ( 25 ) .
about 0.75 m of each forward and reverse primers were added to a reaction mixture containing 1x ( 1.5 mm mgcl2)2 gotaq reaction buffer , pcr nucleotide mix 0.2 mm each dntp , 1.25 l of gotaq dna polymerase ( 5 u/l ) , and nuclease - free water to final volume 50 l .
the pcr program was performed for 35 cycles and consisted of an initial denaturation for 2 min at 95c , 35 sec at 95c for denaturation , 52c for 35 sec for annealing , and 72c for 35 sec for extension , followed by a final extension for 7 min at 72c ( 26 ) .
agarose gel electrophoresis was applied to the pcr products ( 25 ) , then pcr products were purified using the protocol of the qiaquick purification kit to remove excess salts , pcr primers , and dntps ( 27 ) .
after purification of the pcr products , dna cycle sequencing was performed using of bigdye terminator v1.1 cycle sequencing protocol ; 0.75 m of forward apai and bsmi , and 0.75 m of reverse foki primers were prepared . in the reaction mixture ,
6.6 l of nuclease - free water was placed , followed by 4 l of ready reaction premix , 2 l of bigdye terminator v1.1 , sequencing buffer ( 5x ) and 6.4 l of apai , bsmi , and foki .
the pcr program was performed for 25 cycles and consisted of an initial denaturation for 1 min at 96c , 10 sec at 96c for denaturation , 50c for 5 sec for annealing , and 60c for 4 min for extension .
sanger sequencing method was applied for capillary electrophoresis ; the run was performed on applied biosystem ( 3500xl genetic analyzer ) .
all statistical analyses were performed using the spss for windows , version 15.0 ( spss inc . ,
frequency differences of apai , bsmi , and foki polymorphisms and departure from hardy  weinberg equilibrium were tested by pearson 's chi - square test which was also used to quantify the association between cp and these polymorphisms . the level of significance ( p value ) less than 0.05 was considered statistically significant for all analyses .
the distribution of the participants according to age groups between group one ( 2535 years ) and group four ( 5665 years ) was 15.5 , 23.7 , 30.9 , and 29.9% , respectively . according to pdi , 99 participants ( 50.0% )
were diagnosed with cp and 97 participants ( 49.5% ) were diagnosed as healthy controls .
the distribution of the pdi scores between 0 and 6 was 1.0 , 2.6 , 4.6 , 11.2 , 26.5 , 32.7 , and 21.4% , respectively .
cross - tabulation statistical test , which is based on pearson 's chi - square test , was used to analyze the frequency distribution , in order to find relationship between cp and both sex and age , as well as to find an association between cp and apai , bsmi , and foki vdr snps .
statistically significant association between cp and both sex ( p=0.000 ) and age ( p=0.001 ) was detected ( table 2 ) .
association between chronic periodontitis and age and sex the level of significance ( p<0.05 ) .
the flanking regions by apai , bsmi , and foki primers were analyzed ; the lengths of the pcr products were 156 , 106 , and 146 bases , respectively .
figure 1a shows a part of the sequence of the region flanked by apai primer including g / t snp rs#7975232 . in this study , the variant ,
c / t rs#731236 snp was detected in the region flanked by apai primers ( fig .
figure 1c shows part of the sequence of the region flanked by bsmi primers including a / g snp rs#1544410 .
figure 1d shows part of the sequence of the region flanked by foki primers including a / g snp rs#2228570 .
( a ) apai g / t snp ( highlighted in black , ambiguity code : k ) .
( b ) apai c / t snp ( highlighted in black , ambiguity code : y ) .
( c ) bsmi a / g snp ( highlighted in black , ambiguity code : r ) .
( d ) foki a / g snp ( highlighted in black , ambiguity code : r ) .
the analysis of genotypic and allelic frequencies of the analyzed polymorphisms showed that a significant association p = 0.022 and p=0.023 , respectively , was foundbetween cp and apai c / t snp rs#731236 , whereas no statistically significant associations were observed in genotypic frequencies between cp and apai g / t rs#7975232 ( p=0.939 ) , bsmi a / g rs#1544410 ( p=0.466 ) , and foki a / g rs#2228570 ( p=0.239 ) snps .
tables 37 
 show the numbers of the pcr products ( varied between 93 and 122 samples ) that have been successfully sequenced and read for the analyzed snps .
genotypic frequencies of vitamin d receptor gene apai g / t rs#7975232 snp the level of significance ( p<0.05 ) .
genotypic frequencies of vitamin d receptor gene apai c / t rs#731236 snp the level of significance ( p<0.05 ) .
allelic frequencies of vitamin d receptor gene apai c / t rs#731236 snp the level of significance ( p<0.05 ) .
genotypic frequencies of vitamin d receptor gene bsmi a / g rs#1544410 snp the level of significance ( p<0.05 ) .
genotypic frequencies of vitamin d receptor gene foki a / g rs#2228570 snp the level of significance ( p<0.05 ) .
cp is frequently seen among family members and across different generations within a family , suggesting a genetic basis for the susceptibility to periodontal disease ( 1 ) . in addition , studies of twins suggest a genetic contribution to cp in general , but the effects of bacterial transmission and environmental influences make it difficult to explain a complex interaction ( 1 , 29 ) . for instance ,
snps of interleukin ( il- ) 1 , il-1 , located in different regions of cytokine genes have also been shown to affect the risk of the disease in several populations ( 30 , 31 ) .
because cp is a multifactorial disease ( gene  environment interaction ) , several genetic factors may play a role in the disease development by alteration of the structural component of the periodontal tissues , whereas other genetic factors play a role in regulation of inflammatory response ; many gene products interact with each other with environmental factors and the mechanism in which this interaction can increase the risk of cp is incompletely interpreted ( 32 ) .
a finding by naito et al . ( 17 ) suggests that the chemical reactions involving bone mineral density - mediated effects are essential for the development of cp , but the effect of the vdr gene polymorphisms in total on cp have not been confirmed ( 2 ) .
our results showed a significant association between cp and newly detected apai c / t snp # rs731236 , which is located in intron 8 of the vdr gene .
this finding suggests an essential parameter that generally has not been considered on the prevention of diseases related to bone mineral density such as cp . in the present study no significant association
was found between cp and apai g / t snp rs#7975232 , bsmi a / g snp rs#1544410 , and foki a / g snp rs#2228570 .
our findings agreed with studies on brazilian , turkish , japanese , and chinese populations by de souza et al .
( 6 ) , gunes et al . ( 11 ) , naito et al .
( 34 ) , respectively , whereas other studies showed significant association between these vdr gene polymorphisms and the periodontal disease ( 19 , 35 ) . to our knowledge
, the present study is the first study to examine the association between cp and vdr gene polymorphisms in north africa . in this study
our results showed a significant association between cp and age , which agrees with a study on by amarasena et al .
it is suggested that as the length of time increases , the chronic plaque accumulation increases , followed by increase in periodontal tissues destruction ( 1 ) .
the causes of these sex differences have not been explained clearly , but suggest that men have poorer oral hygiene , less positive thinking toward oral health and dental - visit interaction , which agrees with a study by nazish et al . on the other hand ,
women still have varied periodontal problems due to hormonal disturbance in various decades of life .
in addition , not enough studies have been done in developing countries which might have different results as compared to developed countries ( 38 ) .
other studies of hormonal effect on periodontal diseases suggested that estrogen hormone in females can affect tooth retention by preventing the alveolar bone resorption 
 ( 3941 ) 
 .
17-estradiol , the primary female sex hormone , may also play a role in promoting periodontal regeneration in an experimental periodontitis model ( 42 ) . therefore , diagnostic periodontal risk assessments like vdr polymorphisms may be useful in the detection of the individuals susceptible for cp .
more genetic studies in the future , including analysis of vdr haplotype alleles , are necessary in the prediction of the disease . clearly , studies with enough funding , larger sample size , and in more geographic regions in the country will be beneficial due to the importance of the vdr gene and its relation to the cp and many other human diseases .
in the present study , we investigated apai c / t snp rs#731236 that confirmed the importance of the vdr gene in the libyan population . instead of restriction enzyme technique , which was performed in previous studies , we used the sequencing method which is more advanced , more expensive , and capable of revealing more snps in the vdr gene .
 backgroundchronic periodontitis ( cp ) is a common oral disease characterized by inflammation in the supporting tissue of the teeth  
 the periodontium , periodontal attachment loss , and alveolar bone loss . 
 the disease has a microbial etiology ; however , recent findings suggest that the genetic factors , such as vitamin d receptor ( vdr ) gene polymorphisms , have also been included.aiminvestigation of the relationship between vdr gene polymorphisms and cp among libyans.materials and methodsin this study , we examined 196 unrelated libyans between the ages of 25 and 65 years , including 99 patients and 97 controls . an oral examination based on ramfjord index 
 was performed at different dental clinics in tripoli and information were collected using a self - reported questionnaire . 
 dna was extracted from buccal swabs ; the vdr apai , bsmi , and foki polymorphisms were genotyped using polymerase chain reaction and were sequenced using sanger method.resultsa significant difference in the newly detected apai snp c / t rs#731236 was found ( p=0.022 ) , whereas no significant differences were found in apai snp g / t rs#7975232 , bsmi snp a / g rs#1544410 , and foki snp a / g rs#2228570 between patients and controls ( p=0.939 , 0.466 , 0.239 ) , respectively.conclusionvdr apai snp c / t rs#731236 may be related to the risk of cp in the libyan population . 
Materials and methods
Participants
Genotype determination
Statistical analysis
Results
Discussion
Conclusions
Conflict of interest and funding