The distribution of DR4 C626 > G and A683 > C genetic variants matched the Hardy-Weinberg equilibrium in all analysed groups and subgroups. HCV-infected patients without HCC revealed a slightly higher allele frequency of the DR4 626C allele (47.2%) - corresponding to an increased prevalence of 626C carriers (70.4%) - than our healthy controls (allele frequency 41.5%; frequency of 626C carriers 65.3%; Figure ). These differences within the HCV-infected patient groups were apparently independent from the presence of cirrhosis and did not reach statistically significance.
Figure 1 Frequencies of DR4 risk alleles in HCV-infected patients with and without HCC. Figure A shows the frequency of carriers with a DR4 626 C risk allele, and figure B the frequency of patients with the homozygous DR4 683AA genotype. Figure C shows the frequency (more ...)
Prevalence of carriers with a 626C allele (genotypes 626CG and CC) (79.9%) and 626C allele frequency (52.5%) were significantly increased in the patients with HCV-associated HCC (Figure ). The 626C allele frequency in patients with HCV-associated HCC was significantly different only from that in healthy controls (OR = 1.556, 95%CI: 1.192-2.031, p = 0.001).
Importantly, the increased prevalence of carriers with a 626C allele reached statistical significance both with respect to healthy controls (OR = 2.106, 95% CI: 1.348-3.290, p = 0.001) and to HCV-infected patients without HCC (OR = 1.670, 95% CI: 1.034-2.696, p = 0.034) (Figure ).
The distribution of A683 > C variants did not differ between healthy controls and HCV-infected patients without HCC irrespective from the presence of cirrhosis (Table ). Patients with HCV-associated HCC had the highest frequency of the 623A allele (86.5%), which was significantly increased as compared to healthy controls (A allele frequency 77.3%, OR = 1.856 95% CI: 1.291-2.667, p = 0.0006) and HCV-infected patients without HCC (A allele frequency 77.6%, OR = 1.828, 95% CI: 1.243-2.686, p = 0.002). This deviation was strong enough to achieve statistical significance even when HCV-infected subgroups with and without cirrhosis were compared separately (cirrhosis: 77.3% A allele frequency, OR = 1.852, 95% CI: 1.126-3.045, p = 0.013; without cirrhosis: 77.7% A allele frequency, OR = 1.816, 95% CI: 1.201-2.746, p = 0.004).
These changes were due to a significant increase in the proportion of patients with the homozygous 683AA genotype in patients with HCV associated HCC (76.1%) as compared to healthy controls (59.9%, OR = 0.477, 95% CI: 0.314-0.725, p = 0.0003) and HCV-infected patients without HCC (59.4%, OR = 0.468, 95% CI: 0.300-0.730, p = 0.0006), irrespective whether cirrhosis was present (60.0%, OR = 0.480, 95% CI: 0.267-0.862, p = 0.011) or not (59.1%, OR = 0.462, 95% CI: 0.286-0.747, p = 0.001) (Figure ). Interestingly, HCV viral loads tended to be increased in patients who carried the genetic risk variants associated with polymorphisms C626G (2.44 ± 0.28 × 106 IU/ml vs. 1.85 ± 0.30 × 106 IU/ml, n.s.) and A683C (2.30 ± 0.27 × 106 IU/ml vs. 1.94 ± 0.26 × 106 IU/ml, n.s.), and this effect reached statistical significance in patients who simultaneously carried both genetic risk factors (2.69 ± 0.36 × 106 IU/ml vs. 1.81 ± 0.23 × 106 IU/ml, p = 0.049) (Figure ).
Figure 2 HCV viral loads in patients with and without genetic DR4 risk variants. This Figure shows HCV viral loads (Means ± SEM) in patients who carry the DR4 626CG or 626CC risk variant (left columns), the DR4 683AA risk variant (middle columns) and both (more ...)
Next, we checked whether variants in the TRAIL receptor I (DR4) gene were confirmed as independent HCC risk factors in HCV-infected patients when known risk factors such as age, sex, obesity, diabetes, HCV viral load and HBV co-infection were also taken into account (Table ). Univariate analysis identified age (OR = 1.076 per year, 95% CI: 1.053-1.098, p < 0.0001), carriage of a 626C allele (OR = 1.670, 95% CI: 1.034-2.696, p = 0.035), the homozygous 683AA genotype (OR = 2.176, 95% CI: 1.390-3.407, p = 0.001) and the simultaneous presence of both genetic variants (OR = 1.940, 95% CI: 1.280-2.940, p = 0.002) as risk factors of HCC (Table ). In the multivariate regression analysis age was found to be an independent risk factor for the development of HCC in chronic hepatitis C (OR = 1.074, 95% CI: 1.052-1.097, p ≤ 0.001). More importantly, we could confirm that the simultaneous presence of a 626C allele in combination with the homozygous 683AA genotype was significantly associated with the occurrence of HCC in this continuative analysis (OR = 1.975, 95% CI: 1.205-3.236, p = 0.007) and could therefore identify the combined carriage of a DR4 risk variant as new independent predictor for the development of HCC in chronic hepatitis C.
Regression analysis for risk factors of HCC among patients with hepatitis C genotype 1
To check, if our findings were specific for hepatitis C, we performed a similar analysis in our group of HCC patients, who had chronic hepatitis B. However, unlike hepatitis C-associated HCC the distributions of genetic DR4 variants in HBV-associated HCC, healthy controls and HCV-positive patients without HCC were not significantly different (Table ). In particular, the simultaneous combination of a 626C allele with the 683AA genotype was observed in rather similar frequencies in the groups with HBV-associated HCC (50.0%), hepatitis C without HCC (49.4%) and healthy controls (47.0%) while the difference in frequency between HBV-and HCV-associated HCC (65.4%) was significant (OR = 1.891, 95% CI: 1.020-3.506, p = 0.042).