adjuvant radiotherapy ( rt ) after conservative surgery for early stage breast cancer is a standard of care that is well tolerated by the patients in terms of compliance and risk of adverse effects . in most cases , breast cancer patients have a long life expectancy with a risk of late normal tissue complications that may continue to accumulate years after treatment concludes . for this reason , late effects remain important health concerns for long - term survivors . common late toxicity manifestations in the breast include fibrosis , cutaneous atrophy and skin telangiectasia . in particular , fibrosis is the development of excess fibrous connective tissue due to fibroblasts proliferation because of tissue injury . there is considerable interindividual variability in the development of late toxicity , which depends on rt parameters such as total dose , dose per fraction , irradiated volume , and dose inhomogeneity , as well as on patient related factors such as age and life style factors . in the last decade , several studies have suggested that development of late normal tissue complications in the breast after rt is at least in part genetically determined . currently , clinical radiosensitivity is nowadays regarded as a complex polygenic trait resulting from the combined effects of multiple factors , each with relatively modest effects . therefore , an approach based on the combination of multiple genetic factors into a genetic risk score ( grs ) may be an attractive strategy for prediction of adverse rt effects . dna methylation is an epigenetic mechanism established and maintained by a family of dna methyltransferase ( dnmt ) enzymes that catalyze the addition of a methyl group to cytosine residues , using s - adenosylmethionine as the methyl group donor . dna methylation generally occurs on cytosine residues located in cpg dinucleotides within the promoter region , resulting in chromatin compaction and gene silencing . three active dnmts have been described in mammals : dnmt1 is required for maintenance of methylation through generations , while dnmt3a and dnmt3b are mainly involved in establishment of de novo dna methylation patterns . in vitro and in vivo experimental evidence suggests that enzymes of the methylation machinery play a role in fibrogenesis and radiation response . for instance , upregulation of dnmt1 has been detected in the fibrotic tissue of the skin , kidneys , lungs , and liver , whereas activation of myofibroblasts or hepatic stellate cells can be reversed via inhibition of dnmt1 by dna - demethylating drugs or by specific sirna shutdown . in addition , reduction of global methylation levels has been reported after irradiation , probably due to decreased expression of dnmt1 , dnmt3a , and dnmt3b . despite evidence of involvement of dna methylating enzymes in fibrogenesis and radiation response , no information is currently available regarding whether common genetic variants of dnmts genes contribute to the development of radiation - induced fibrosis in cancer patients . however , recent in vitro studies suggest that mitochondria are the primary loci of rt effects , and that mitochondrial dna haplogroups differently affect mrna expression of dnmt1 , dnmt3a , and dnmt3b ,   as well as global dna methylation levels . in the present study , we assessed the role of four single nucleotide polymorphisms ( snps ) of dnmt genes ( dnmt1 rs2228611 , dnmt3a rs1550117 , dnmt3a rs7581217 , and dnmt3b rs2424908 ) as risk factors for subcutaneous fibrosis in a cohort of italian breast cancer patients who received rt after breast conserving surgery . in addition to dnmt snps , we evaluated the predictive role of xrcc1 rs2682585 , which was previously reported to be associated with the standardized total average toxicity ( stat ) score , an index of overall toxicity combining skin toxicities and fibrosis of the breast . we also assessed the ability of the aforementioned snps to improve prediction accuracy when combined with mitochondrial haplogroup h , which we recently found to be independently associated with a lower hazard of radiationinduced fibrosis in breast cancer patients . this study included 286 caucasian patients affected by histologically confirmed breast cancer who underwent conservative surgery and adjuvant rt from 1989 to 2010 at our department of radiotherapy . briefly , rt consisted of two opposite tangential wedged beams , followed by a boost on the tumor bed . patients underwent whole breast rt with conventional fractionation to a total dose of 50 gy followed by boost dose on the tumor bed in cases of invasive tumors . at the time of patient recruitment , a peripheral blood sample was taken and stored at 4c until analysis . during annual follow - up visits ( last update on january 2015 ) , radiation oncologists evaluated the appearance of subcutaneous and cutaneous late toxicities , with particular attention to the onset of fibrosis toxicity was scored according to the late effects of normal tissue - subjective objective management analytical ( lent - soma )   scale . patients with moderate to severe fibrosis (  grade 2 ) were referred to as the " radiosensitive group " and compared to patients with no or minimal fibrotic reactions ( grade 0 - 1 , control group ) . this study was approved by the local ethics committees of our university hospital and met the requirements of the declaration of helsinki . determination of snps was conducted on genomic dna by real - time polymerase chain reaction ( pcr ) using the following taqman pre - designed snp genotyping assays ( applied biosystems , milan , italy ) : c_27838930_10 ( dnmt1 rs2228611 ) ; c_8722920_10 ( dnmt3a rs1550117 ) ; c_7863728_10 ( dnmt3a rs7581217 ) ; c_16013055_10 ( dnmt3b rs2424908 ) ; and c_16269889_10 ( xrcc1 rs2682585 ) . real - time pcr amplification and detection was performed in 96-well pcr plates using a cfx connect real - time pcr detection system ( bio - rad , milan , italy ) . each polymorphism was tested for deviation from the hardy - weinberg equilibrium ( hwe ) by use of pearson s chisquared test as implemented in finetti s program ( http://ihg.gsf.de/cgi-bin/hw/hwa1.pl ) . for the selected polymorphisms , we considered the co - dominant , dominant , and recessive modes of inheritance . the time to event end - point ( grade  2 fibrosis ) was calculated from the first session of rt , and patients not experiencing the end - point were censored at the last follow - up performed . the cumulative incidence of grade  2 fibrosis was calculated by the kaplan - meier method and comparisons between genotype groups were performed using the log - rank test . univariate cox regression analyses were performed to calculate the hazard ratio ( hr ) and the 95% confidence interval ( ci ) to evaluate the influence of genotypes on grade  2 fibrosis risk . multivariate adjustments were made for the mitochondrial haplogroup ( h vs. non - h ) and clinical variables having a cut - off p - value of < 0.20 in univariate cox analysis ( body mass index , breast diameter , adjuvant treatment , dose per fraction , radiation quality , acute skin toxicity , and postsurgical complications ) . to avoid the risk of over - fitting biases , only significant genetic factors were considered for construction of a grs . to accomplish this , the cox proportional hazard regression coefficient of each genetic factor was converted into an integer risk score by rounding the quotient and dividing the regression coefficient by a single constant . discrimination capabilities of genetic models were assessed using the area under the receiver operating characteristic curve ( roc ) . the area under the roc curve and comparisons between roc curves were calculated using the method described by delong et al . . because of the exploratory nature of this study , we reported nominal statistical associations ( p < 0.05 ) . adjusted p - values based on the bonferroni correction were also considered to avoid chance findings due to multiple testing of five snps , and the significance was lowered to p < 0.01 . this study included 286 caucasian patients affected by histologically confirmed breast cancer who underwent conservative surgery and adjuvant rt from 1989 to 2010 at our department of radiotherapy . briefly , rt consisted of two opposite tangential wedged beams , followed by a boost on the tumor bed . patients underwent whole breast rt with conventional fractionation to a total dose of 50 gy followed by boost dose on the tumor bed in cases of invasive tumors . at the time of patient recruitment , a peripheral blood sample was taken and stored at 4c until analysis . during annual follow - up visits ( last update on january 2015 ) , radiation oncologists evaluated the appearance of subcutaneous and cutaneous late toxicities , with particular attention to the onset of fibrosis toxicity was scored according to the late effects of normal tissue - subjective objective management analytical ( lent - soma )   scale . patients with moderate to severe fibrosis (  grade 2 ) were referred to as the " radiosensitive group " and compared to patients with no or minimal fibrotic reactions ( grade 0 - 1 , control group ) . this study was approved by the local ethics committees of our university hospital and met the requirements of the declaration of helsinki . determination of snps was conducted on genomic dna by real - time polymerase chain reaction ( pcr ) using the following taqman pre - designed snp genotyping assays ( applied biosystems , milan , italy ) : c_27838930_10 ( dnmt1 rs2228611 ) ; c_8722920_10 ( dnmt3a rs1550117 ) ; c_7863728_10 ( dnmt3a rs7581217 ) ; c_16013055_10 ( dnmt3b rs2424908 ) ; and c_16269889_10 ( xrcc1 rs2682585 ) . real - time pcr amplification and detection was performed in 96-well pcr plates using a cfx connect real - time pcr detection system ( bio - rad , milan , italy ) . each polymorphism was tested for deviation from the hardy - weinberg equilibrium ( hwe ) by use of pearson s chisquared test as implemented in finetti s program ( http://ihg.gsf.de/cgi-bin/hw/hwa1.pl ) . for the selected polymorphisms , we considered the co - dominant , dominant , and recessive modes of inheritance . the time to event end - point ( grade  2 fibrosis ) was calculated from the first session of rt , and patients not experiencing the end - point were censored at the last follow - up performed . the cumulative incidence of grade  2 fibrosis was calculated by the kaplan - meier method and comparisons between genotype groups were performed using the log - rank test . univariate cox regression analyses were performed to calculate the hazard ratio ( hr ) and the 95% confidence interval ( ci ) to evaluate the influence of genotypes on grade  2 fibrosis risk . multivariate adjustments were made for the mitochondrial haplogroup ( h vs. non - h ) and clinical variables having a cut - off p - value of < 0.20 in univariate cox analysis ( body mass index , breast diameter , adjuvant treatment , dose per fraction , radiation quality , acute skin toxicity , and postsurgical complications ) . to avoid the risk of over - fitting biases , only significant genetic factors were considered for construction of a grs . to accomplish this , the cox proportional hazard regression coefficient of each genetic factor was converted into an integer risk score by rounding the quotient and dividing the regression coefficient by a single constant . discrimination capabilities of genetic models were assessed using the area under the receiver operating characteristic curve ( roc ) . the area under the roc curve and comparisons between roc curves were calculated using the method described by delong et al . . 13.3.3 ( medcalc software , mariakerke , belgium ) software . because of the exploratory nature of this study , we reported nominal statistical associations ( p < 0.05 ) . adjusted p - values based on the bonferroni correction were also considered to avoid chance findings due to multiple testing of five snps , and the significance was lowered to p < 0.01 . overall , 51 of the 286 participants ( 17.8% ) experienced moderate to severe fibrosis ( lent - soma  grade 2 ) , while 235 patients ( 82.2% ) had no or minimal fibrosis ( lentsoma grade 0 - 1 ) . detailed demographic and clinical data in the whole cohort of breast cancer patients and following stratification according to their radiosensitive status have been reported elsewhere . the distributions of genotype frequencies for the polymorphisms analyzed were in hwe ( all p > 0.05 ) . for the entire set of breast cancer patients , frequencies of minor variant alleles were 0.48 ( dnmt1 rs2228611 g ) , 0.09 ( dnmt3a rs1550117a ) , 0.35 ( dnmt3a rs7581217 t ) , 0.14 ( dnmt3b rs2424908 t ) , and 0.22 ( xrcc1 rs2682585 ) . kaplan - meier curves for dnmt1 rs2228611 showed differences among genotypes in the cumulative incidence of grade  2 subcutaneous fibrosis ( log - rank test p - value=0.018 ) ( fig . univariate cox regression analysis revealed that dnmt1 rs2228611 was associated with a lower risk of grade  2 fibrosis under either the codominant ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.70 ; p=0.007 ) or the recessive contrast ( gg vs. aa+ag : hr , 0.31 ; 95% ci , 0.12 to 0.78 ; p=0.013 ) ( table 1 ) . multivariate cox regression analysis adjusted for mitochondrial haplogroup ( h vs. non - h ) and clinical confounding factors revealed dnmt1 rs2228611 as an independent protective factor for moderate to severe radiation - induced fibrosis ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.71 ; p=0.009 ; gg vs. aa+ag : hr , 0.29 ; 95% ci , 0.12 to 0.75 ; p=0.011 ) ( table 1 ) . conversely , none of the other dnmt snps ( table 1 ) , nor xrcc1 rs2682585 ( table 2 ) were associated with radiation - induced fibrosis of the breast upon both univariate or multivariate cox regression analysis . it is worth noting that mitochondrial haplogroup h emerged as a significant protective factor in these multivariate models when dnmt1 rs2228611 was considered under the codominant ( hr , 0.48 ; 95% ci , 0.26 to 0.88 ; p=0.018 ) or the recessive mode of inheritance ( hr , 0.48 ; 95% ci , 0.26 to 0.89 ; p=0.019 ) . to understand the joint effects of genetic markers on individual risk of grade 2 fibrosis , we built a grs for grade  2 fibrosis based on different combinations of dnmt1 rs2228611 and/or mitochondrial haplogroups according to five cox regression models , as shown in table 3 . model 1 included mitochondrial haplogroups only ( h vs. non - h ) , model 2 and model 3 comprised the codominant or the recessive contrast of rs2228611 , respectively , model 4 combined mitochondrial haplogroup h with the codominant contrast of dnmt1 rs2228611 and model 5 incorporated mitochondrial haplogroup h with the recessive contrast of dnmt1 rs2228611 . we then performed roc curve analyses to determine the performance of the five models at discriminating patients with or without grade  2 fibrosis ( fig . the lowest discrimination accuracy , quantified by area under the receiver operating characteristic curves ( auc ) , was obtained for models 2 and 3 , which were based on the two contrasts of rs2228611 ( both auc , 0.587 ; 95% ci , 0.28 to 0.645 ) , and the highest was observed for model 5 , which included the combination of mitochondrial haplogroup h with the recessive contrast of rs2228611 ( auc , 0.655 ; 95% ci , 0.597 to 0.710 ) . 2 , adding the recessive contrast of dnmt1 rs2228611 to haplogroup h determined an increase of the discrimination accuracy from 0.595 ( 95% ci , 0.536 to 0.653 ) to 0.655 ( 95% ci , 0.597 to 0.710 ) . when applying the score developed in model 5 , the proportion of patients with grade  2 radiation - induced fibrosis for each score group showed an increasing trend from lower to higher sum risk scores : 3.4% ( score 0 ) , 10.0% ( score 1 ) , 14.0% ( score 2 ) , and 27.3% ( score 2 ) . the kaplan - meier analysis of model 5 revealed that patients with an increasing sum risk score showed a trend towards higher frequencies of grade  2 fibrosis ( p trend=0.0005 ) ( fig . overall , 51 of the 286 participants ( 17.8% ) experienced moderate to severe fibrosis ( lent - soma  grade 2 ) , while 235 patients ( 82.2% ) had no or minimal fibrosis ( lentsoma grade 0 - 1 ) . detailed demographic and clinical data in the whole cohort of breast cancer patients and following stratification according to their radiosensitive status have been reported elsewhere . the distributions of genotype frequencies for the polymorphisms analyzed were in hwe ( all p > 0.05 ) . for the entire set of breast cancer patients , frequencies of minor variant alleles were 0.48 ( dnmt1 rs2228611 g ) , 0.09 ( dnmt3a rs1550117a ) , 0.35 ( dnmt3a rs7581217 t ) , 0.14 ( dnmt3b rs2424908 t ) , and 0.22 ( xrcc1 rs2682585 ) . kaplan - meier curves for dnmt1 rs2228611 showed differences among genotypes in the cumulative incidence of grade  2 subcutaneous fibrosis ( log - rank test p - value=0.018 ) ( fig . univariate cox regression analysis revealed that dnmt1 rs2228611 was associated with a lower risk of grade  2 fibrosis under either the codominant ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.70 ; p=0.007 ) or the recessive contrast ( gg vs. aa+ag : hr , 0.31 ; 95% ci , 0.12 to 0.78 ; p=0.013 ) ( table 1 ) . multivariate cox regression analysis adjusted for mitochondrial haplogroup ( h vs. non - h ) and clinical confounding factors revealed dnmt1 rs2228611 as an independent protective factor for moderate to severe radiation - induced fibrosis ( gg vs. aa : hr , 0.26 ; 95% ci , 0.10 to 0.71 ; p=0.009 ; gg vs. aa+ag : hr , 0.29 ; 95% ci , 0.12 to 0.75 ; p=0.011 ) ( table 1 ) . conversely , none of the other dnmt snps ( table 1 ) , nor xrcc1 rs2682585 ( table 2 ) were associated with radiation - induced fibrosis of the breast upon both univariate or multivariate cox regression analysis . it is worth noting that mitochondrial haplogroup h emerged as a significant protective factor in these multivariate models when dnmt1 rs2228611 was considered under the codominant ( hr , 0.48 ; 95% ci , 0.26 to 0.88 ; p=0.018 ) or the recessive mode of inheritance ( hr , 0.48 ; 95% ci , 0.26 to 0.89 ; p=0.019 ) . to understand the joint effects of genetic markers on individual risk of grade  2 fibrosis , we built a grs for grade  2 fibrosis based on different combinations of dnmt1 rs2228611 and/or mitochondrial haplogroups according to five cox regression models , as shown in table 3 . model 1 included mitochondrial haplogroups only ( h vs. non - h ) , model 2 and model 3 comprised the codominant or the recessive contrast of rs2228611 , respectively , model 4 combined mitochondrial haplogroup h with the codominant contrast of dnmt1 rs2228611 and model 5 incorporated mitochondrial haplogroup h with the recessive contrast of dnmt1 rs2228611 . we then performed roc curve analyses to determine the performance of the five models at discriminating patients with or without grade  2 fibrosis ( fig . the lowest discrimination accuracy , quantified by area under the receiver operating characteristic curves ( auc ) , was obtained for models 2 and 3 , which were based on the two contrasts of rs2228611 ( both auc , 0.587 ; 95% ci , 0.28 to 0.645 ) , and the highest was observed for model 5 , which included the combination of mitochondrial haplogroup h with the recessive contrast of rs2228611 ( auc , 0.655 ; 95% ci , 0.597 to 0.710 ) . 2 , adding the recessive contrast of dnmt1 rs2228611 to haplogroup h determined an increase of the discrimination accuracy from 0.595 ( 95% ci , 0.536 to 0.653 ) to 0.655 ( 95% ci , 0.597 to 0.710 ) . when applying the score developed in model 5 , the proportion of patients with grade  2 radiation - induced fibrosis for each score group showed an increasing trend from lower to higher sum risk scores : 3.4% ( score 0 ) , 10.0% ( score 1 ) , 14.0% ( score 2 ) , and 27.3% the kaplan - meier analysis of model 5 revealed that patients with an increasing sum risk score showed a trend towards higher frequencies of grade  2 fibrosis ( p trend=0.0005 ) ( fig . in the present study , we tested whether the occurrence of radiation - induced fibrosis in breast cancer patients could be at least partially explained by genetic variability in dnmt genes , which encode for epigenetic enzymes known to be involved in fibrogenesis and radiation response . to accomplish this , we investigated the association between common polymorphisms in dnmt1 , dnmt3a , and dnmt3b genes and the risk of subcutaneous fibrosis in a cohort of breast cancer survivors who received rt after breast - conserving surgery . multivariate cox analysis revealed dnmt1 rs2228611gg genotype as an independent predictor of lower risk for radiation - induced skin fibrosis . to the best of our knowledge , this is the first study to suggest that genetic variability at the dnmt1 locus contributes to the development of late normal tissue complications after radiation therapy , which further supports the importance of dnmt1 on fibrogenesis and wound healing after radiation exposure . it is worth noting that xrcc1 rs2682585 , previously reported to be associated with an overall toxicity score combining skin toxicities and fibrosis of the breast , was not found to be correlated with fibrosis alone in the present study . snps in dnmts genes have primarily been investigated as risk factors for cancer susceptibility given that aberrant dna methylation is one of the earliest molecular events during carcinogenesis . in addition , since snps in dnmts genes influence dna methylation at global and gene - specific levels , they have also been also investigated as susceptibility factors for common complex diseases other than cancer . among the snps investigated in the present study , dnmt3a rs1550117 and dnmt3b rs2424908 have been associated with a lower cancer risk , as shown by recent meta - analyses . with regard to dnmt1 rs2228611 , the dnmt1 rs2228611gg genotype has been reported to be associated with a higher risk of ovarian cancer . although dnmt1 rs2228611 is a synonymous snp located in exon 17 , it has a possible splice regulatory function with a > g resulting in gaining of three exonic splicing enhancer binding motifs . in addition , dnmt1 rs2228611 has significant modifying effects on the inverse relationship between line-1 methylation , a marker of global methylation , and cadmium exposure in argentinean women , suggesting a possible role in regulating the methylation level in response to environmental cues . reactive oxygen species ( ros ) caused by radiation could be a common trigger mechanism in both fibrosis and radiation response . in oxygenated tumor and normal cells , mitochondria play a major role in the generation of ros and appear to be the primary loci of rt effects . conversely , mitochondrial polymorphisms defining major haplogroups have been suggested to alter oxidative phosphorylation coupling as well as ros production . in addition , cybrids containing mitochondrial haplogroup h have lower levels of dnmt1 , dnmt3a , and dnmt3b and global methylation than those with mitochondrial haplogroup j . based on our previous findings highlighting a protective role of mitochondrial haplogroup h on radiation - induced fibrosis , we built a grs based on its combination with dnmt1 rs2228611 . in line with the concern recently raised for high risk of over - fitting and false positive results of multifactorial genetic models , for the grs construction we only considered dnmt snps significantly associated with radiationinduced fibrosis , rather than using the total number of putative risk alleles . our results provided evidence of an increased discrimination accuracy when dnmt1 rs2228611 was combined with mitochondrial haplogroup h , suggesting potential usefulness of rs2228611 in genetic - based models for prediction of radiation - induced skin fibrosis . in addition , the method used for weighting predictors , which was based on cox hazard regression coefficients , can be easily applied for inclusion of clinical factors as well . unfortunately , none of the clinical or dosimetric variables analyzed emerged as independent predictors for grade  2 fibrosis , as we reported in our previous analysis . first , the lack of a second cohort of breast cancer patients precluded the possibility of validating dnmt1 rs2228611 as a predictive factor for radiation - induced fibrosis . thus , our results need to be interpreted cautiously because of the small sample size and risk of false positive findings given that very few radiogenomic studies with a candidate gene approach have been replicated in independent cohorts . second , in the present study we did not evaluate gene polymorphisms involved in enzymatic dna demethylation , such as the ten - eleven translocation ( tet ) protein family , which are known to play a role in fibrogenesis . therefore , a more comprehensive investigation of genes encoding epigenetic enzymes should be conducted to develop and validate a clinically useful geneticbased model for prediction of radiation - induced skin fibrosis . third , the predictive performance of the genetic model , based solely on rs2228611 and haplogroup h , is rather modest . however , this is expected for complex polygenic traits such as clinical radiosensitivity , which is probably the result of the combined effects of multiple polymorphic loci . in addition , further investigation in cellular and in vivo animal models is still required to provide mechanistic insights into the protective role of rs2228611gg on radiation - induced fibrosis . our findings support a protective effect of the dnmt1 rs2228611gg genotype on the risk of radiation - induced fibrosis in breast cancer patients . in addition , we provide evidence that inclusion of dnmt1 rs2228611 in a genetic - based model has the potential to increase prediction accuracy for radiation - induced fibrosis . however , further investigation to provide mechanistic insights into the role of dnmt1 rs2228611 in fibrogenesis and radiation response is warranted . in addition , a more comprehensive investigation of the role of germline polymorphisms should be conducted to develop and validate a clinically useful genetic - based model for prediction of radiation - induced fibrosis after treatment of breast cancer .