
 cervical intraepithelial neoplasia ( cin ) is a premalignant disease , often leading to invasive cervical cancer .
it is widely known that cin is caused by an infection with high - risk human papillomavirus ( hpv ) types [ 3 , 4 ] .
interestingly , most hpv infections are transient and do not lead to cin . genetic , immunologic , and socioeconomic factors play a role in the natural history of hpv infection and development of cervical intraepithelial neoplasia .
lifestyle factors , such as cigarette smoking , are well known to be a risk factor for hpv persistence and development of cervical intraepithelial neoplasia , the precursor lesion of cervical cancer . compared to women who have never smoked , current cigarette smokers have a significantly increased risk of high - grade cervical intraepithelial neoplasia and cervical cancer .
host factors such as the immune systems and genetic factors also seem to be important factors in hpv persistence and subsequent cervical cancer carcinogenesis [ 8 , 9 ] .  
the transporter associated with antigen processing ( tap ) belongs to the superfamily of atp - binding cassette ( abc ) transporter that is essential for peptide loading onto class i major histocompatibility complex ( mhc - i ) molecules [ 10 , 11 ] .
transporter associated with antigen processing is composed of two integral membrane proteins , tap1 and tap2 , which have one hydrophobic region and one atp - binding region each .
tap 1 functions by providing a supply of candidate peptides to the mhc i molecules within the peptide loading complex and by transporting antigen peptides from the cytoplasm into the endoplasmic reticulum ( er ) [ 12 , 13 ] .
mhc - i molecules play a major role in the immune response against viral infections and transformed cells by presenting peptide antigens to cytotoxic t lymphocytes ( ctl ) [ 14 , 15 ] .
the loss of mhc class i expression is observed commonly in various tumours and is possibly the mechanism underlying the ability of neoplastic cells to evade the immune system . regarding cervical cancer ,
mhc - i antigen has been reported to be downregulated in hpv-16 and -18 positive cervical malignancies .
tap facilitates the detection of hpv by mhc - i molecules and contributes to successful detection and eradication of hpv despite various immunoevasion mechanisms of the virus .
therefore tap gene polymorphisms have been investigated in women with cervical cancer precursor lesions and in women with cervical cancer [ 1922 ] .
einstein et al . ascertained a reduced risk for high - grade cin at the presence of tap1 i333v and tap1 d637 g .   in the present study
, we evaluated the association between five common tap gene polymorphisms in tap1 and tap2 : tap1 1341 ( rs1057141 ) , tap1 2254 ( rs1135216 ) , tap2 1135 ( rs1800454 ) , tap2 1693 ( rs2228396 ) , and tap2 1993 ( rs241447 ) and the risk of cin in a total number of 822 women .
women , who were referred to the department 's outpatient clinic for genital dysplasia between 2004 and 2009 because of a cytological result  atypical cells of undetermined significance ( asc - ucs )  or higher , were asked to participate at the present study . in total ,
616 caucasian women with histologically proven cin 1 - 3 , treated at the department of gynaecology and gynaecological oncology , medical university of vienna , comprehensive cancer centre , vienna , austria , were included in this study .
the examination in our outpatient clinic comprised ecto- and endocervical cytology , human papilloma virus ( hpv ) dna testing ( hybrid capture 2-test , digene corporation , gaithersburg , md , usa ) , application of 3% acetic acid , colposcopy , and colposcopically guided biopsy .
after the women agreed to participate at the study by signing informed consent , they were asked about their smoking status ( yes including previous smokers versus no ) , and the clinical examination was performed .
the patients , in whom no cin 1 - 3 was diagnosed after colposcopically guided biopsy , were excluded of further analysis ( n = 60 patients ) .   as a control group ,
we included 206 consecutive caucasian women , who presented at the department 's gynaecologic outpatient clinic between 2004 and 2009 with vaginal infection , pelvic pain or similar symptoms , and without a history of conization or histologically proven cin 1 - 3 .
patients , who agreed to participate , signed informed consent and had in addition to their particular examination , ecto- and endocervical cytology and one cervical smear to collect the dna samples .
patients with known hiv , hepatitis b or c infection , or a history of gynaecologic malignancy were excluded from the study .
the collected pap smears and histological samples were analyzed by a board certified pathologist specialized in gynaecological oncology .
the institutional review board at the medical university of vienna approved this study ( reference : ek number 017/2004 ) .
dna was extracted from patients ' cervical smear using qiaamp dna blood mini kit ( qiagen , germany ) according to the instruction of manufacturer .
dna was amplified with polymerase chain reaction ( pcr ) and dna pyrosequencing was performed according to established protocols as described previously . after researching dbsnp , the snp database from national center for biotechnology information , and pubmed , the five most common and clinically relevant tap gene polymorphisms ( i.e. , tap 1341 , tap 2254 , tap 1135 , tap 1693 , and tap 1993 ) were chosen for investigation .
primers used for the amplification of the fragments including the polymorphic sites , the length of the pcr products , and the sequencing primers were shown in table 1 .
pcr was carried out in a total volume of 25  l including 25  ng total dna , 5  pmol of each sense and antisense primers and 12.5  l jumpstart redtaq readymix reaction mix ( sigma , missouri , usa ) , which contains 20  mm  tris - hcl , ph 8.3 , 100  mm kcl , 4  mm mgcl2 , 0.002% gelatin , 0.4  mm each dntp , inert dye , stabilizers , 0.06  unit/l taq dna polymerase , jumpstart taq antibody .
the reaction was performed on a perkin - elmer geneamp pcr system 9600 with 40 cycles at 94c for 30 seconds , at the corresponding annealing temperature ( see table 1 ) for 30 seconds and 72c for 30 seconds .
the reaction was preceded by a primary denaturation step at 94c for 1 minute and incubated at 72c for 7  min at last .
25  l pcr product was used for pyrosequencing according to the instruction of the manufactory .
hardy - weinberg equilibrium was tested by chi - square tests comparing observed and expected genotype frequencies .
differences in gene polymorphism frequencies and smoking between women with cin and controls were assessed by chi - square tests ; results are given as p value and odds ratio ( or ) ( 95% confidence interval ( 95%  ci ) ) . in a multivariate logistic regression model , we assessed the independent association between tap gene polymorphisms , smoking , and risk of cin .
differences in gene polymorphism frequencies between women with cin 1 and cin 2/3 were assessed by chi - square tests ; results are given as p value and odds ratio ( or ) ( 95% confidence interval ( 95%  ci ) ) .   
haplotypes can only be estimated and then probability values between 0 and 1 are assigned , expressing the likelihood for each haplotype to be present in one allele of a patient .
these probabilities sum up to 2 in each patient ( for the two alleles ) and were included as continuous covariates ( one for each haplotype ) in the logistic regression model .
a logistic regression model was estimated to evaluate whether the presence of one or more copies of each particular haplotype in a patient is associated with a higher or lower risk of cin , compared to a patient with two copies of the wild - type haplotype .
women with cin presented with the following histological results : cin i n = 206/616 ( 33.4% ) , cin ii n = 205/616 ( 33.3% ) , and cin iii n = 205/616 ( 33.3% ) .
sixty - one of 422 ( 14.5% ) women with cin were tested negative and 361 of 422 ( 85.5% ) positive for high - risk hpv infection . of note ,
patients with cin ( 58.8% ) were smoking significantly more often than controls ( 35.8% ) ( p = 0.0001 ) ( table 2 ) .
the tap snps 1341 , 2254 , 1135 , 1693 , and 1993 were genotyped in 616 patients with cin and 206 controls .
the genotype frequencies for all the studied snp 's were in hardy - weinberg equilibrium in both controls and patients with cin .
distribution of genotypes for the five snp 's did not differ significantly between patients with cin and controls ( table 3 ) . in a multivariate model , smoking , but none of the investigated five snps ,
was independently associated with risk of cin ( table 4 ) . moreover , in 616 patients with cin the risk of high - grade cin was investigated .
no significant association was found between the investigated tap genotypes and risk for high - grade cin ( p = 0.08 for tap 1341 ; p = 0.3 for tap 2254 ; p = 0.8 for tap 1135 ; p = 0.3 for tap 1693 ; p = 0.2 for tap 1993 ) ( table 3 ) .
the combined effect of the five tap gene polymorphisms on the risk for cervical intraepithelial neoplasia was investigated by haplotype analysis ( table 5 ) .
haplotype analysis revealed that women with haplotype mut - wt - wt - wt - wt ( tap polymorphisms t1135-t1341-t1693-t1993-t2254 ) had a significantly lower risk for cin , compared to women with the haplotype wt - wt - wt - wt - wt ( p = 0.006 ; or 0.5 [ 0.350.84 ] ) .
the aim of the present study was to evaluate the association between five common tap gene polymorphisms and risk for cin .
the haplotype combination mut - wt - wt - wt - wt ( tap polymorphisms t1135-t1341-t1693-t1993-t2254 ) was associated with a reduced risk for the presence of cervical intraepithelial neoplasia ( or 0.5 ( 0.40.8 ) ) .   
this finding seems biologically plausible , as tap facilitates the detection of hpv by mhc - i molecules and contributes to successful detection and eradication of hpv despite various immunoevasion mechanisms of the virus .
although the biological implication of the observed haplotype combination is not fully elucidated , this haplotype combination might facilitate the detection of hpv and subsequently reduce the risk for cin development .  
our observation is in line with studies investigating the role of tap gene polymorphisms and risk for high - grade cin and cervical cancer [ 7 , 13 ] .
einstein et al . ascertained a reduced risk for high - grade cin at the presence of tap1 i333v ( p = 0.02 ) and tap1 d637 g ( p = 0.01 ) . in women with cervical cancer ,
a tap2 gene polymorphism has been associated with a reduced risk for cervical cancer . of note , this study was performed in an indian cohort although no population diversities for these gene polymorphisms are described .   
interestingly , we were not able to ascertain any association between five common tap polymorphisms and risk of cin in a relatively large number of women . as we did not aim to evaluate the exact biological mechanisms exerted by the five tap gene polymorphisms or their haplotype combinations , we did not investigate mhc - i serum levels and we did not obtain tissue of the patients .
moreover , patients in the present study represent a highly selected group of women , as all patients were of caucasian origin and have been recruited at a single tertiary care center in vienna , austria .   in a multivariate model , smoking , but none of the investigated five snps , was independently associated with risk of cin .
it is well known that women smoking cigarettes are at higher risk for development of cin . due to these facts patients
should be advised to quit smoking to reduce the risk for high - grade cin development and increase spontaneous regression .   in the present study the protective effect of a tap1 and
tap2 haplotype combination for the presence of cin has been observed in a relatively large cohort of caucasian women .
although its further implication has to be elucidated , this haplotype combination could be used to identify women , less susceptible for development of cin following an infection with high - risk human papillomavirus .
 
 background . transporter associated with antigen processing ( tap ) is responsible for peptide loading onto class i major histocompatibility complex ( mhc - i ) molecules . 
 tap seems to facilitate the detection of hpv by mhc - i molecules and contributes to successful eradication of hpv . 
 tap polymorphisms could have an important impact on the course of hpv infection . 
 objective . 
 the aim of this study is to evaluate the association between five tap gene polymorphisms and the risk of cin . 
 methods . 
 this case - control study investigated five common tap polymorphisms in tap1 ( 1341 and 2254 ) and tap2 ( 1135 , 1693 , and 1993 ) in 616 women with cin and 206 controls . 
 associations between gene polymorphisms and risk of cin were analysed by univariate and multivariable models . 
 the combined effect of the five tap gene polymorphisms on the risk for cin was investigated by haplotype analysis . 
 results . 
 no significant difference in genotype distribution of the five tap polymorphisms was observed in women with cin and controls . 
 haplotype analysis revealed that women with haplotype mut - wt - wt - wt - wt ( tap polymorphisms t1135-t1341-t1693-t1993-t2254 ) had a significantly lower risk for cin , compared to women with the haplotype wt - wt - wt - wt - wt ( p = 0.006 ; or 0.5 [ 0.350.84 ] ) . conclusion . 
 identification of this haplotype combination could be used to identify women , less susceptible for development of cin following hpv infection . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion