Demographic and clinical characteristics of participants
The demographic characteristics and clinical parameters of the study subjects are summarized in Table 2. Three hundred and thirty control subjects included 165 male and 165 female with a mean age of 47.0 ± 13.3 years. The CKD group was composed of 92 adults and included 44 male and 48 female with a mean age of 49.9 ± 11.9 years. The distribution of gender of the subjects was not significantly different in the two groups. The levels of BMI, SBP, DBP, BUN, creatinine, uric acid, FBS, and TG in the CKD group were significantly higher than those of the control group (p < 0.05). The levels of eGFR, total protein, albumin, total cholesterol, HDL cholesterol, and LDL cholesterol in the CKD group were significantly lower than those of the control group (p < 0.05). Estimated glomerular filtration rate (eGFR) < 60 was found 18 controls (5.5%) and 90 CKD patients (97.8%). In the control and CKD group, the genotype distribution of the seventeen polymorphic SNPs were in the Hardy-Weinberg equilibrium (HWE) (p > 0.05).
Table 2
Demographic characteristics and clinical parameters for the study population
| Control | CKD | p-value |
(N = 330) | (N = 92) |
Age (years) | 47.0 ± 13.3 | 49.9 ± 11.9 | 0.02 |
Male | 47.3 ± 10.5 | 51.8 ± 9.5 | |
Female | 46.7 ± 10.1 | 48.2 ± 13.5 | |
Gender | | | |
Male / Female n (%) | 165 (50.0) / 165 (50.0) | 44 (47.8) / 48 (52.2) | 0.725 |
Etiology | | | |
Diabetes Mellitus n (%) | | 28 (30.4) | |
Hypertension n (%) | | 4 (4.3) | |
CGN n (%) | | 58 (63.0) | |
Others n (%) | | 2 (2.2) | |
LVH n (%) | | 18 (19.6) | |
Body Mass Index (kg/m2) | 22.5 ± 2.6 | 23.8 ± 3.6 | 0.002 |
Systolic Blood Pressure (mmHg) | 109.0 ± 7.2 | 139.2 ± 25.6 | < 0.001 |
Diastolic Blood Pressure (mmHg) | 68.7 ± 5.9 | 83.8 ± 12.5 | < 0.001 |
Blood Urea Nitrogen (mg/dl) | 14.1 ± 3.7 | 64.6 ± 29.9 | < 0.001 |
Creatinine (mg/dl) | 0.9 ± 0.2 | 7.2 ± 3.7 | < 0.001 |
eGFR (mL/min/1.73 m2) | 75.6 ± 12.0 | 12.5 ± 15.5 | < 0.001 |
eGFR < 60 (mL/min/1.73 m2, %) | 18 (5.5) | 90 (97.8) | < 0.0001 |
Uric acid (mg/dl) | 4.6 ± 1.3 | 8.3 ± 2.5 | < 0.001 |
Fasting blood sugar (mg/dl) | 85.8 ± 6.6 | 128.4 ± 63.8 | < 0.001 |
Total protein (g/dl) | 7.4 ± 0.4 | 6.3 ± 0.8 | < 0.001 |
Albumin (g/dl) | 4.4 ± 0.2 | 3.5 ± 0.5 | < 0.001 |
AST (IU/l) | 21.6 ± 5.2 | 19.1 ± 14.7 | 0.115 |
ALT (IU/l) | 17.8 ± 7.0 | 19.2 ± 22.4 | 0.558 |
Total Cholesterol (mg/dl) | 186.1 ± 25.6 | 170.9 ± 48.0 | 0.004 |
Triglyceride (mg/dl) | 88.6 ± 36.0 | 127.11 ± 77.8 | < 0.001 |
HDL Cholesterol (mg/dl) | 55.4 ± 11.1 | 44.7 ± 16.4 | < 0.001 |
LDL Cholesterol (mg/dl) | 113.0 ± 25.4 | 102.0 ± 38.1 | 0.031 |
p, Categorical variable is summarized as count (%) with statistical comparison using Chi-square |
Continuous variables are summarized as mean ± Standard Deviation with statistical comparison using T-test |
p value for different between non-obese and obese group |
Genotype and Allele Frequencies of IFNL3, IFNL2, IFNAR2, TLR9, IL-10RB, IL-22, IFNRA, IRF7, JAK2, and STAT3 Genes SNPs
The SNPs of IFNL3, rs148543092 (T > C) were significantly associated with CKD in the codominant and dominant model (T/T vs. T/C and T/T vs. T/C + C/C, p = 0.013, OR = 2.50, 95% CI = 1.21–5.15). The SNPs of IFNL2, rs8103362 (A > G) were significantly associated with CKD in the codominant, dominant and log-additive model (A/A vs. A/G, p = 0.013, OR = 2.50, 95% CI = 1.21–5.15; A/A vs. A/G + G/G, p = 0.018, OR = 2.37, 95% CI = 1.16–4.86; A/A vs. A/G vs. G/G, p = 0.036, OR = 2.14, 95% CI = 1.07–4.28, respectively). The SNP of IFNRA2, rs1051393 (G > T) was significantly associated with CKD in the codominant and log-additive model (G/G vs. T/T, p = 0.029, OR = 2.10, 95% CI = 1.08–4.09; G/G vs. G/T vs. T/T, p = 0.026, OR = 1.45, 95% CI = 1.04–2.02, respectively). The SNP of TLR9, rs187084 (T > C) was significantly associated with CKD in the codominant model (T/T vs. C/C, p = 0.016, OR = 2.26, 95% CI = 1.16–4.40), dominant model (T/T vs. T/C + C/C, p = 0.047, OR = 1.77, 95% CI = 1.01–3.10) and log-additive model (T/T vs. T/C vs. C/C, p = 0.015, OR = 1.50, 95% CI = 1.08–2.09). The SNP of IL-22, rs2227484 (G > A) was significantly associated with CKD in the codominant model (G/G vs. G/A, p = 0.040, OR = 1.95, 95% CI = 1.03–3.69), dominant model (G/G vs. G/A + A/A, p = 0.046, OR = 1.91, 95% CI = 1.01–3.62) (Table 3). There was no significant difference of genotype and allele frequencies between control and CKD in the IL-10RB gene polymorphisms (rs8178562 G > A, rs 2834167 A > G) and IRF7 gene polymorphism (Affix-52325648 T/del). There were no polymorphisms but only major allele homozygotes in the IFNRA (Affix-52347487), JAK2 (rs77375493), and STAT3 (rs113994139) (data not shown).
Table 3
Distribution of frequencies of Interferon Lambda-Related Genotype in controls and CKD patients in model of inheritance
Gene | SNP number | Model of Inheritance |
Co-dominamt genetic model | Dominamt genetic model | Recessivet genetic model | Log-Additivet genetic model |
OR (95% CI) | p-value | OR (95% CI) | p-value | OR (95% CI) | p-value | OR (95% CI) | p-value |
IFNL3 | rs148543092 | 2.50 (1.21–5.15) | 0.013 | 2.50 (1.21–5.15) | 0.013 | | | | |
Thr108Ala | - | | | | | | | |
IFNL2 | rs8103362 | 2.50 (1.21–5.15) | 0.013 | 2.37 (1.16–4.86) | 0.018 | | | 2.14 (1.07–4.28) | 0.036 |
Thr112Ala | - | | | | | | | |
IFNAR2 | rs1051393 | 1.52 (0.86–2.69) | 0.147 | 1.68(0.98–2.87) | 0.057 | 1.61 (0.93–2.79) | 0.094 | 1.45 (1.04–2.02) | 0.026 |
Phe10Ile | 2.10 (1.08–4.09) | 0.029 | | | | | | |
TLR9 | rs187084 | 1.56 (0.86–2.83) | 0.147 | 1.77 (1.01–3.10) | 0.047 | 1.69 (1.00-2.86) | 0.052 | 1.50 (1.08–2.09) | 0.015 |
T-1486C | 2.26 (1.16–4.40) | 0.016 | | | | | | |
IL-22 | rs2227513 | 3.72 (0.96–14.47) | 0.058 | 3.72 (0.96–14.47) | 0.058 | | | | |
T-111C | - | | | | | | | |
rs2227484 | 1.95 (1.03–3.69) | 0.04 | 1.91 (1.01–3.62) | 0.046 | | | 1.82 (0.98–3.39) | 0.065 |
G-701A | - | | | | | | | |
p, Chi-square test p value; OR, odds ratio; CI, confidence interval |
p value for different between control and CKD group |
Replication of IFNL3, IFNL2, TLR9, and IL-22 Genes SNPs
The comparison of genotypic frequencies between cases and controls for all the SNPs analysed achieved a nominal significant value in four polymorphisms located in four different genetic regions. Although none of them withstand Bonferroni correction, we tried to replicate associations involving IFNL3, IFNL2, TLR9 and IL22 using a second sample set (Table 4).
Table 4
Distribution of frequencies of TLR9 Genotype in replication
TLR9 | Original set* | Original set** | | Replication set# |
rs187084 | CKD | Controls | CKD with CGN | Controls | | CKD with CGN | Controls |
Genotype | (N = 92) | (N = 330) | (N = 58) | (N = 330) | | (N = 137) | (N = 446) |
TT | 19 (20.6) | 102 (30.9) | 10 (17.2) | 102 (30.9) | | 29 (21.2) | 141 (31.6) |
TC | 45 (48.9) | 157 (47.6) | 31 (53.4) | 157 (47.6) | | 72 (52.5) | 210 (47.1) |
CC | 28 (30.4) | 71 (21.5) | 17 (29.3) | 71 (21.5) | | 36 (26.3) | 95 (21.3) |
TC + CC genotype *: p = 0.047 OR = 1.77 95% CI 1.01–3.10; **: p = 0.018 OR = 2.27 95% CI 1.10–4.70; #: p = 0.017 OR = 1.71 95% CI 1.08–2.71. p, Categorical variable is summarized as count (%) with statistical comparison using Chi-square |
No significant associations involving IFNL3, IFNL2 and IL22 were observed in the replication set. Regarding the rs187084, in the TLR9 gene, a significant association was observed pooling the original and the replication sets (p = 0.017 OR = 1.71 95% CI 1.08–2.71) (Table 4).
Association of TLR9 SNP with clinical characteristics
After adjustment for age, gender, BMI, hypertension, diabetes mellitus, and dyslipidemia as covariates whether examined the genotype distribution of TLR9 gene polymorphism rs187084 is associated with clinical characteristics (creatinine, eGFR, uric acid, total protein, and albumin) in both original and replication set of CKD group.
In both original and replication set, total protein level was significantly higher in homozygous C/C genotype than T/T + T/C genotype of TLR9 gene, rs187048 (6.57 ± 0.65 vs. 6.16 ± 0.81, p = 0.025: 6.29 ± 0.78 vs. 6.03 ± 0.77, p = 0.049, respectively). In original set, albumin level were significantly higher in homozygous C/C genotype than T/T + T/C genotype (3.72 ± 0.45 vs. 3.42 ± 0.48, p = 0.009). In replication set, eGFR level was significantly lower in homozygous C/C genotype than T/T + T/C genotype (7.45 ± 5.13 vs. 10.28 ± 8.89, p = 0.007) (Table 5).
Table 5
Association of TLR9 SNP with clinical characteristics in replication
SNP | Parameter | Genotype | p-value* | Genotype | p-value# |
rs187084 | | T/T + T/C (N = 64) | C/C (N = 28) | | T/T + T/C (N = 154) | C/C (N = 46) | |
| Creatinine (mg/dl) | 7.17 ± 3.53 | 7.37 ± 4.09 | 0.809 | 6.96 ± 3.45 | 8.00 ± 3.21 | 0.069 |
| eGFR (mL/min/1.73 m2) | 12.94 ± 17.49 | 11.31 ± 9.87 | 0.643 | 10.28 ± 8.89 | 7.45 ± 5.13 | 0.007 |
| Uric acid (mg/dl) | 8.35 ± 2.26 | 8.23 ± 3.12 | 0.846 | 8.52 ± 2.58 | 8.73 ± 2.94 | 0.647 |
| Total protein (g/dl) | 6.16 ± 0.81 | 6.57 ± 0.65 | 0.025 | 6.29 ± 0.78 | 6.03 ± 0.77 | 0.049 |
| Albumin (g/dl) | 3.42 ± 0.48 | 3.72 ± 0.45 | 0.009 | 3.55 ± 0.55 | 3.39 ± 0.52 | 0.089 |
*: Original set |
#: Replication set |
p, Continuous variables are summarized as mean ± Standard Deviation with statistical comparison using T-test |
p value for different between genotype in CKD group |