lumbar disc degeneration ( lddg ) is a degenerative change in the lumbar disc that occurs with aging , affecting from 30% in people younger than 30 years to 50% in those 30 years and older . lddg also occurs in competitive athletes and is one of the causes of lumbar disc diseases , including lower back pain and discopathy . lddg is caused by genetic and environmental factors . cartilage layer intermediate protein ( cilp ) is abundantly expressed in the intervertebral discs . cilp interacts directly with transforming growth factor-1 ( tgf-1 ) , controlling the tgf-1-mediated construction of extracellular matrix proteins . several recent reports have shown that the cilp gene polymorphism ( 1184t / c ) is significantly associated with lumbar disc diseases in japanese subjects . mutated c allele was reported to increase the binding of tgf-1 and to disturb the regulation of tgf-1 signaling . previous studies have also shown that asporin ( aspn ) alleles with 14 aspartic acid repeats ( designated d14 ) in the n - terminal region are associated with osteoarthritis ( oa ) of the knee . recently , aspn d14 was also shown to be associated with lddg in the asian population . high aspn mrna and protein expression were also observed in degenerated lumbar discs , especially in the outer annulus . nakajima et al . reported that aspn inhibits tgf-1/smad signaling by co - localization with tgf- and blocking the interaction between tgf-1 and its receptors . showed that the aspn d14 allele results in greater inhibition of tgf- activity in atdc5 cells . as both cilp and aspn proteins in some way affect tgf- signaling , these two susceptible polymorphisms for lddg may mutually or independently affect the inhibition of tgf- signaling and lddg occurrence . recently , we showed that there was a significant association between the cilp 1184t / c polymorphism and lddg occurrence in japanese collegiate male athletes . in this study , we first examined whether aspn d14 is a significant risk factor for lddg occurrence in collegiate athletes . as a result , we found that aspn d14 and lddg occurrence were significantly associated . we then hypothesized that the evaluation of multiple risk alleles of aspn and cilp shows higher statistical predictability in comparison to only one risk allele , because these two susceptible polymorphisms have similar inhibitory effects on tgf- signaling . we analyzed the relationship between genetic factors and lddg in 516 ( male , 341 ; female , 175 ) collegiate athletes from seven types of sports ( wrestling , judo , gymnastics , swimming , soccer , american football , and track and field ) , with an average of more than 5 years of experience . we defined wrestling , judo , soccer , and american football as collision sports and gymnastics , swimming , and track and field as noncollision sports . mri evidence of lddg was defined as decreased signal intensity of the intervertebral discs at l1/l2 and l5/s1 in the t-2 weighted midsagittal fast spin - echo images ( repetition time , 5,000 ms ; echo time , 125 ms ) . all mri scans were analyzed and graded independently by two experienced orthopedic surgeons who were blinded to the subject s status . the grading system for the assessment of lddg was based on the study of pfirrmann et al . we defined grades 3 , 4 , and 5 as the lddg group and grades 1 and 2 as the control group , as reported by kaneoka et al . if the athlete had multiple degenerations , the highest grade and its location were determined . in the case of any discrepancy in the grading of lddg between the two orthopedic surgeons , the d - repeat region was amplified using the forward primer 5-tcctagactggtcttctacact-3 and the reverse primer 5-tctgagcaatgtacaactcgtg-3. the forward primer was 5 labeled with fam . the polymerase chain reaction ( pcr ) cycling reactions , using a thermal cycler ( mj mini , biorad , hercules , ca ) , were performed at 94c for 10 minutes , 30 cycles at 94c for 30 seconds , at 64c for 30 seconds , and at 72c for 30 seconds , followed by one cycle at 72c for 10 minutes . after amplification , the alleles were discriminated using the abi prism 310 genetic analyzer ( applied biosystems , foster city , ca ) and the fragment analysis genemapper v4.0 software ( applied biosystems ) . the genotype - specific risks were estimated as odds ratios ( ors ) and 95% confidence intervals ( cis ) . logistic regression analysis was performed to investigate the relationships between lddg and the two susceptibility genes , as well as with body weight , gender , and collision sport . we analyzed the relationship between genetic factors and lddg in 516 ( male , 341 ; female , 175 ) collegiate athletes from seven types of sports ( wrestling , judo , gymnastics , swimming , soccer , american football , and track and field ) , with an average of more than 5 years of experience . we defined wrestling , judo , soccer , and american football as collision sports and gymnastics , swimming , and track and field as noncollision sports . mri evidence of lddg was defined as decreased signal intensity of the intervertebral discs at l1/l2 and l5/s1 in the t-2 weighted midsagittal fast spin - echo images ( repetition time , 5,000 ms ; echo time , 125 ms ) . all mri scans were analyzed and graded independently by two experienced orthopedic surgeons who were blinded to the subject s status . the grading system for the assessment of lddg was based on the study of pfirrmann et al . we defined grades 3 , 4 , and 5 as the lddg group and grades 1 and 2 as the control group , as reported by kaneoka et al . if the athlete had multiple degenerations , the highest grade and its location were determined . in the case of any discrepancy in the grading of lddg between the two orthopedic surgeons , the d - repeat region was amplified using the forward primer 5-tcctagactggtcttctacact-3 and the reverse primer 5-tctgagcaatgtacaactcgtg-3. the forward primer was 5 labeled with fam . the polymerase chain reaction ( pcr ) cycling reactions , using a thermal cycler ( mj mini , biorad , hercules , ca ) , were performed at 94c for 10 minutes , 30 cycles at 94c for 30 seconds , at 64c for 30 seconds , and at 72c for 30 seconds , followed by one cycle at 72c for 10 minutes . after amplification , the alleles were discriminated using the abi prism 310 genetic analyzer ( applied biosystems , foster city , ca ) and the fragment analysis genemapper v4.0 software ( applied biosystems ) . the genotype - specific risks were estimated as odds ratios ( ors ) and 95% confidence intervals ( cis ) . logistic regression analysis was performed to investigate the relationships between lddg and the two susceptibility genes , as well as with body weight , gender , and collision sport . the height , weight , and body mass index of the lddg group were significantly higher than that of the control group . lddg = subjects with lumbar disc degeneration ; control = subjects without lumbar disc degeneration ; bmi = body mass index . unpaired t - test , p < 0.01 . the frequency of the aspn d14 allele in the lddg group was higher than that in the control group . we found a significant association between the aspn d14 allele and lddg ( p < 0.01 ) with an or of 2.02 ( 95% ci = 1.21 - 3.36 ) in all athletes ( table 2a ) . by using logistic regression analysis concomitant with the interaction term and the wald test , we found that weight ( or = 1.03 ; 95% ci = 1.01 - 1.05 ) and aspn d14 allele ( or = 2.14 ; 95% ci = 1.1 - 4.18 ) were significant risk factors for lddg ( table 2b ) . we also confirmed that the effect of d14 was significant only for male athletes ( table 2c ) . a. frequency of lddg and aspn d14 allele in 516 athletes . or = odds ratio ; ci = confidence interval . b. logistic regression analysis . or = odds ratio ; ci = confidence interval ; d14 = allele of aspn gene . lddg = exp(0 + 1w + 2 g + 4col + 5d14 + 6 g  d14 ) , where w , g , col , and d14 denote weight , gender , collision , and aspn d14 allele , respectively . * * * p < 0.001 . the null hypothesis is composed of the following restrictions : d14:5 + d14:gender:6 = 0 . 5 and 6 are coefficients in the multinomial logistic model as shown in table 2b . the logistic regression model was constructed with six variables , as shown in table 3 . lddg was significantly correlated with body weight , gender , aspn d14 , cilp ct , and cc . however , collision sports were not correlated with lddg ( table 3a ) . furthermore , the two genotypes had no effect on lddg occurrence in female athletes ( table 3b ) . logistic regression analysis for lddg in two susceptible genes a. logistic regression analysis . or = odds ratio ; ci = confidence interval ; d14 = allele of aspn gene ; tc = genotype of cilp gene ; cc = genotype of cilp gene . the formulated logistic model is as follows : lddg = exp(0 + 1w + 2 g + 3col + 4d14 + 5tc + 6cc + 7d14  g + 8tc  g + 9cc  g ) , where w , g , col , d14 , tc , and cc denote weight , gender , collision , aspn d14 allele , cilp tc genotype , and cilp cc genotype , respectively . p < 0.05 . * * * p < 0.001 . the null hypothesis is composed of the following restrictions : d14:4 + d14:gender : 7 = 0 tc:5 + tc : gender:8 = 0 cc:6 + tc : gender:9 = 0 4 to 9 are coefficients in the multinomial logistic model as shown in table 3a . we also calculated the ors of the susceptibility genes by using logistic regression analysis from the result of each genotype . the or of lddg occurrence was increased by the risk alleles of the two susceptibility genes ( table 4 ) . odds ratios by logistic regression analysis . or = odds ratio ; ci = confidence interval . although the proportion of lddg occurrence increased with the number of risk alleles , there was no significant interaction between these two susceptibility genes ( p > 0.05 ; fig . the height , weight , and body mass index of the lddg group were significantly higher than that of the control group . lddg = subjects with lumbar disc degeneration ; control = subjects without lumbar disc degeneration ; bmi = body mass index . unpaired t - test , p < 0.01 . the frequency of the aspn d14 allele in the lddg group was higher than that in the control group . we found a significant association between the aspn d14 allele and lddg ( p < 0.01 ) with an or of 2.02 ( 95% ci = 1.21 - 3.36 ) in all athletes ( table 2a ) . by using logistic regression analysis concomitant with the interaction term and the wald test , we found that weight ( or = 1.03 ; 95% ci = 1.01 - 1.05 ) and aspn d14 allele ( or = 2.14 ; 95% ci = 1.1 - 4.18 ) were significant risk factors for lddg ( table 2b ) . we also confirmed that the effect of d14 was significant only for male athletes ( table 2c ) . a. frequency of lddg and aspn d14 allele in 516 athletes . or = odds ratio ; ci = confidence interval . b. logistic regression analysis . or = odds ratio ; ci = confidence interval ; d14 = allele of aspn gene . the formulated logistic model is as follows : lddg = exp(0 + 1w + 2 g + 4col + 5d14 + 6 g  d14 ) , where w , g , col , and d14 denote weight , gender , collision , and aspn d14 allele , respectively . p < 0.05 . * * * p < 0.001 . the null hypothesis is composed of the following restrictions : d14:5 + d14:gender:6 = 0 . 5 and 6 are coefficients in the multinomial logistic model as shown in table 2b . the logistic regression model was constructed with six variables , as shown in table 3 . lddg was significantly correlated with body weight , gender , aspn d14 , cilp ct , and cc . however , collision sports were not correlated with lddg ( table 3a ) . furthermore , the two genotypes had no effect on lddg occurrence in female athletes ( table 3b ) . logistic regression analysis for lddg in two susceptible genes a. logistic regression analysis . or = odds ratio ; ci = confidence interval ; d14 = allele of aspn gene ; tc = genotype of cilp gene ; cc = genotype of cilp gene . the formulated logistic model is as follows : lddg = exp(0 + 1w + 2 g + 3col + 4d14 + 5tc + 6cc + 7d14  g + 8tc  g + 9cc  g ) , where w , g , col , d14 , tc , and cc denote weight , gender , collision , aspn d14 allele , cilp tc genotype , and cilp cc genotype , respectively . p < 0.05 . * * * p < 0.001 . the null hypothesis is composed of the following restrictions : d14:4 + d14:gender : 7 = 0 tc:5 + tc : gender:8 = 0 cc:6 + tc : gender:9 = 0 4 to 9 are coefficients in the multinomial logistic model as shown in table 3a . we also calculated the ors of the susceptibility genes by using logistic regression analysis from the result of each genotype . the or of lddg occurrence was increased by the risk alleles of the two susceptibility genes ( table 4 ) . odds ratios by logistic regression analysis . or = odds ratio ; ci = confidence interval . although the proportion of lddg occurrence increased with the number of risk alleles , there was no significant interaction between these two susceptibility genes ( p > 0.05 ; fig . we previously reported that cilp ( 1184t / c ) was significantly associated with lddg in collegiate male athletes . in this study , we also found a significant association between the aspn d14 allele and lddg in collegiate male athletes . except for gender difference , this finding is consistent with the results in the asian population as reported by song et al . although significant interactions of these two susceptibility genes have not been observed , we also found that the addition of the risk alleles cilp 1184t / c and aspn d14 increased the odds ratio . the d14 allele of aspn has been reported as a susceptibility gene for oa of the knee . recently , song et al . found that aspn d14 was also associated with lddg in a chinese and japanese cohort . we also found that aspn d14 was significantly associated with lddg occurrence in collegiate athletes . song et al . also showed that there was a trend for an association between aspn d14 and severe lddg . in their study , the chinese cohort was defined only by radiological abnormality , but the japanese cohort was dependent on painful disc herniations . in this study , the lddg group was defined by the mri findings and the definition was independent of pain . thus , we think that the aspn d14 allele is associated with lddg , independent of symptoms such as pain or discomfort . we previously found that cilp 1184t / c was significantly associated with lddg occurrence in collegiate athletes . in this study , we also found that there was a significant association between aspn d14 and lddg . as both these genes are related to tgf-1 signaling and their high expression was observed in degenerated lumbar discs , we hypothesized that there might be a significant association between them . thus , we believe that these two genes affect different portions of the tgf- signaling pathway . in addition , there is no evidence suggesting that these two molecules interact with each other . as we further confirmed that each additional risk allele increased the odds of lddg , we think that these two risk alleles have a cumulative effect on the occurrence of lddg . we previously reported that cilp 1184t / c was a lddg - susceptible gene for men , but not women . in this report , we also found that aspn d14 is only a significant risk factor for men . even in the logit model concomitant with two susceptibility genes , genetic variance was only a significant risk factor for men . as these two proteins interact with the tgf- pathway , a similar sex dependency might be observed . valdes et al . found that a haplotype of the cilp gene gave the strongest association in men with knee reported that there is a significant difference in the allele frequency of aspn d13 between female oa patients and female controls . although the molecular mechanisms of this phenomenon are ambiguous , we think that the effect of the cilp and aspn polymorphisms is gender dependent . we evaluated not only genetic factors but also intrinsic and extrinsic factors , such as gender , weight , and collision . as a result , we found that weight and gender were significant risk factors for lddg . we previously reported that high body weight is a risk factor for lddg occurrence in collegiate athletes . as high body weight causes greater stress on the lumbar disc , it is reasonable that high body weight is significantly associated with lddg occurrence . on the contrary , it is difficult to fathom why gender difference is a risk factor for lddg . there are several reports showing that more women complained of lower back pain and hip joint stress than men . although the molecular mechanisms are still unclear , we think that the occurrence of lddg might be gender - dependent after adjusting for body weight , collision , and several genetic factors , as discussed before . the predictive power of tests can be evaluated by the area under the receiver operating characteristic curve ( auc ) . in this study , we calculated auc for our logit model . a perfect model would have an auc of 1 , whereas a model with no discriminative power has an auc of 0.5 . cauchi et al . evaluated 15 polymorphisms associated with type 2 diabetes and found that a good predictive model would have an auc of 0.98 . as in type 2 diabetes , lddg is a polygenic disease caused by genetic and environmental factors . the present study demonstrated that the cilp and aspn polymorphisms are independent genetic factors for lddg in japanese male collegiate athletes . we also raise the possibility that the relationship between these genetic factors and lddg occurrence is a gender - dependent phenomenon .