Supplementary table

Table-S1: List of primers, genotyping and RFLP conditions

Marker / Primer sequence / Primer annealing conditions / RE / Alleles / Product size (bp) / db SNP
Leptin-2548G/A / F 5′-TTTCCTGTAATTTTCCCGTGAG-3′ / 50 °C for 45 s / C / 242 / rs7799039
R 5′-AAAGCAAAGACAGGCATAAAAA-3′ / A / 181,61
UCP2I/D / F 5′-CAGTGAGGGAAGTGGGAGG-3′ / 62 °C for 30 s / D / 457
R 5′-GGGGCAGGACGAAGATTC-3′ / I / 417
Ghrelin Leu72Met / F 5’-TCCAGCCTGCCACTTAGC-3’
R5’-GGACCCTGTTCACTGCCAC-3’ / 62°C for 30 s, 5 cycles
61°C for 30 s, 25 cycles
60°C for 30 s, 25 cycles / BsrI / Leu(C) / 272,101 / rs696217
Met(A) / 373
Ghrelin Arg51Gln / SacI / Arg(G) / 210, 163 / rs34911341
Gln(A) / 373

RE- restriction enzyme, bp- base pair

Table-S2: Demographic and biochemical profile of patients and controls

Variables / Controls
(N=200) / Patients
(N=257) / P-Value
Age (years) / 38.8±13.17 / 39.3±12.6 / 0.7
BMI(wt\ht2) / 22.06±2.183 / 21.2±3.9 / 0.001*
Period of dialysis(months) / NA / 12.87±16.34 / --
Systolic B.P. (mmHg) / 115.9±11.7 / 140.27±24.6 / <0.001*
Diastolic B. P. (mmHg) / 76.7±5.7 / 81.8±13.7 / <0.001*
S. Creatnine(mg/dl) / 0.80±0.23 / 6.44±3.11 / <0.001*
S. Albumin (g/dl) / 3.83±0.23 / 3.63±0.7 / <0.001
S. Protein (g/dl) / 6.84±0.33 / 6.72±2.98 / 0.524
S.BUN(mg/dl) / 10.39±2.98 / 50.17±25.23 / <0.001*
Triglyceride(mg/dl)
Total cholesterol(mg/dl)
HDL(mg/dl)
LDL(mg/dl)
VLDL(mg/dl) / 143.43±62.4
131.5±34.7
23.45±7.12
80.7±26.8
29.7±12.7 / 124.5±58.35
144.63±37.8
38.97±11.2
81.6±32.4
28.23±22.8 / <0.001*
<0.001*
<0.001*
0.75
0.3
S. Sodium (mmol/l) / 138.52±2.5 / 135.18±12.5 / <0.001*
S.Potassium(mmol/l) / 3.78±0.24 / 4.67±1.42 / <0.001*
S. Calcium(mg/dl) / 9.56±0.34 / 7.89±1.78 / <0.001*
S.Phosphorus(mg/dl) / 3.68±0.29 / 6.17±2.02 / <0.001*
S.ALKP(U/L) / 87.35±16.75 / 227.01±193.5 / <0.001*
S. Bil (T) (mg/dl) / 0.80±0.21 / 0.59±0.33 / <0.001*
S. Bil (c) (mg/dl) / 0.2±0.04 / 0.3±0.27 / 0.001*
SGOT(U/L) / 24.66±9.7 / 32.31±42.15 / 0.01*
SGPT(U/L) / 25.7±8.3 / 30.6±39.8 / 0.09
Haemoglobin (g/dl) / 12.3±1.34 / 9.1±2.2 / <0.001*
Ferritin (ng/ml) / 72.9±34.6 / 219.61±211.10 / <0.001*
S.Iron (µg/ml) / 73.01±7.99 / 84.89±81.8 / 0.04*
TIBC (µg/dl) / 330.86±33.9 / 296.64±49.5 / <0.001*
TSAT (% ) / 22.08±1.47 / 32.12±44.26 / <0.001
PTH (pg/ml) / 36.7±13.3 / 219.68±219.9 / <0.001*
CRP (mg/dl) / 0.57±0.22 / 2.44±2.86 / <0.001*

*p≤0.05 significant, NA- not applicable, Students T test was applied, data expressed in form of Mean±S.D.

Table-S3: Demographic profile of patients sub-group based on SGA

Variables / Normal(N=78) / Mild(N=38) / Moderate(N=100) / Severe(N=41) / P-value
BMI (wt\ht2) / 21.6±3.35 / 20.6±3.55 / 20.8±3.25 / 19.7±3.5 / 0.05*
Systolic.B.P.(mmHg) / 134.2±19.49 / 141.07±30.86 / 140.7±29.8 / 144.14±22.53 / 0.3
Diastolic.B.P.(mmHg) / 82.49±11.6 / 84.35±15.21 / 82.52±14.20 / 80.6±15.6 / 0.7
Triglyceride(mg/dl) / 125.6±63.4 / 126.3±56.3 / 131.3±58.78 / 125.6±66.22 / 0.9
Total cholesterol(mg/dl) / 141.17±36.21 / 130.8±25.7 / 145.45±34.79 / 150.7±56.05 / 0.2
HDL(mg/dl) / 38.2±13.23 / 38.10±8.9 / 37.65±9.13 / 36.5±10.5 / 0.8
LDL(mg/dl) / 76.9±28.87 / 73.8±27.8 / 83.10±28.52 / 89.41±48.46 / 0.1
VLDL(mg/dl) / 31.9±30.7 / 27.4±15.7 / 27.82±23.38 / 25.5±11.9 / 0.5
S.Creatnine(mg/dl) / 6.47±3.05 / 6.15±2.7 / 7.0±3.4 / 6.48±2.7 / 0.3
S.Albumin(g/dl) / 3.7±0.56 / 3.5±0.77 / 3.5±0.7 / 3.5±0.77 / 0.3
S.Protein(g/dl) / 6.6±0.89 / 6.4±1.2 / 6.4±1.09 / 6.4±1.1 / 0.1
BUN(mg/dl) / 50.6±25.8 / 47.7±20.7 / 50.36±23.67 / 53.45±25.57 / 0.8
Bil.(T) (mg/dl) / 0.55±0.18 / 0.61±0.19 / 0.57±0.22 / 0.74±0.58 / 0.02*
Bil.(C) (mg/dl) / 0.26±0.12 / 0.26±11.9 / 0.26±0.14 / 0.29±0.17 / 0.8
SGOT(U/L) / 25.17±14.97 / 25.6±12.4 / 23.8±12.35 / 22.0±13.7 / 0.6
SGPT(U/L) / 23.3±16.8 / 22.29±16.0 / 23.15±13.9 / 22.82±13.7 / 0.9
ALKP(U/L) / 199.65±119.7 / 300.7±361.55 / 211.62±179.65 / 240.3±191.4 / 0.09
S. Sodium(mmol/l) / 136.36±5.9 / 133.3±22.76 / 135.8±5.2 / 135.2±7.0 / 0.5
S. Potassium(mmol/l) / 4.5±1.0 / 4.6±1.8 / 4.5±0.9 / 4.4±0.8 / 0.9
S. Phosphorus(mg/dl) / 5.4±1.8 / 4.7±1.5 / 5.8±1.7 / 5.4±1.6 / 0.02*
S. Calcium(mg/dl) / 8.5±1.3 / 8.6±0.8 / 8.4±1.39 / 8.3±0.9 / 0.8
Ferritin (ng/ml) / 202.48±188.37 / 168.5±127.28 / 218.83±235.72 / 269.6±263.03 / 0.2
S.Iron(µg/ml) / 84.4±37.7 / 73±30.6 / 81.02±35.7 / 76.9±60.1 / 0.5
TIBC(µg/dl) / 306.67±44.6 / 293.5±52.27 / 292.7±42.5 / 319.74±164.31 / 0.3
TSAT (% ) / 274.7±10.6 / 255.3±10.8 / 281.6±12.6 / 254.01±16.5 / 0.6
Haemoglobin(g/dl) / 9.6±2.1 / 8.7±2.1 / 8.46±2.04 / 8.9±2.6 / 0.006*
TLC(*1000/μl) / 8.2±3.07 / 7.5±3.6 / 7.6±2.7 / 7.8±3.2 / 0.6
Neutrophil(%) / 71.6±11.5 / 70.5±7.6 / 70.14±8.7 / 74±8.4 / 0.1
Lymphophil(%) / 24.4±9.1 / 25.8±11.4 / 25.07±7.7 / 22.09±7.9 / 0.2
Eosinophil(%) / 4.5±5.7 / 4.2±3.4 / 4.8±4.39 / 3.2±2.3 / 0.7
Monocyte(%) / 3.73±1.8 / 3.2±1.22 / 3.05±1.78 / 2.8±1.5 / 0.3
ESR(mm/h) / 49.25±17.9 / 49.18±17.98 / 53.3±17.9 / 58.06±15.71 / 0.1
Platelet(*1000/cr) / 188.77±66.69 / 182.33±90.86 / 195.3±84.27 / 203.9±128.5 / 0.7
PTH(pg/ml) / 238.48±267.8 / 224.4±227.4 / 219.7±207.95 / 232.2±225.8 / 0.9
CRP(mg/dl) / 1.43±1.36 / 3.02±4.84 / 2.23±2.85 / 2.16±2.4 / 0.1
No. of dialysis / 49.14±39.0 / 39.7±60.4 / 40.5±34.5 / 69.7±110.5 / 0.1
Period on Dialysis(months) / 7.85±7.0 / 9.92±18.9 / 6.6±6.5 / 9.09±12.2 / 0.4
Urine output(ml) / 1042.2±1044.8 / 968.1±912.46 / 735.4±766.31 / 595.8±409.0 / 0.03*
Dialysis duration(hours) / 4.2±0.46 / 4.1±0.34 / 4.2±0.36 / 4.2±0.25 / 0.7

*p≤0.05 significant, ANOVA for comparison of groups, data expressed in form of Mean±S.D.

Table-S4: Demographic profile of patients sub-group based on period of dialysis

Variables / Period< 1 year / Period ≥1 year / P -value
Age / 39.4±14.0 / 39.1±12.2 / 0.876
Weight / 55.8±9.8 / 52.0±7.9 / 0.01*
BMI / 20.7±3.3 / 19.8±3.16 / 0.07
Urineoutput / 680.4±464.0 / 454.0±475.66 / 0.002
Triglyceride / 134.5±66.2 / 122.7±58.6 / 0.228
TC / 142.2±35.9 / 139.9±33.8 / 0.674
HDL / 36.0±9.8 / 39.2±10.8 / 0.04*
LDL / 78.3±28.5 / 80.2±28.6 / 0.679
VLDL / 29.6±22.7 / 28.5±30.1 / 0.781
Systolic BP / 139.9±27.9 / 146.3±25.7 / 0.152
Diastolic BP / 82.3±14.36 / 84.3±13.4 / 0.357
S.creatnine / 7.2±2.8 / 7.9±2.9 / 0.07
S.albumin / 3.4±0.6 / 3.8±0.7 / 0.003*
S.protein / 6.5±2.8 / 6.6±0.9 / 0.813
BUN / 53.0±25.5 / 54.3±22.4 / 0.726
Bil.T / 0.58±0.35 / 0.63±0.26 / 0.379
Bil.C / 0.27±0.16 / 0.26±0.08 / 0.691
SGOT / 24.6±14.0 / 23.6±11.5 / 0.649
SGPT / 22.8±15.4 / 22.8±13.9 / 0.996
ALKP / 227.1±230.2 / 222.3±181.6 / 0.881
Na / 134.4±13.0 / 137.0±5.7 / 0.04*
K / 4.4±1.1 / 4.8±0.9 / 0.04*
P / 5.9±1.6 / 5.7±1.8 / 0.391
Ca / 8.3±1.3 / 8.7±0.8 / 0.04*
Ferritin / 234.1±193.9 / 266.3±277.6 / 0.374
Iron / 82.6±47.3 / 80.8±33.9 / 0.799
TIBC / 306.7±99.0 / 290.8±57.8 / 0.251
TSAT / 273.0±12.9 / 288.5±13.6 / 0.463
Haemoglobin / 8.5±1.9 / 8.7±1.8 / 0.59
TLC / 8.0±2.8 / 7.5±3.2 / 0.308
Neutrophil / 71.6±8.1 / 70.9±12.1 / 0.642
Lymphocyte / 24.35±8.8 / 25.0±9.4 / 0.617
Eosinophil / 4.3±3.4 / 5.4±6.6 / 0.384
Monocytes / 2.8±1.1 / 3.8±2.4 / 0.01*
ESR / 53.9±16.9 / 50.6±20.3 / 0.284
Platelet / 199.1±95.5 / 185.2±82.4 / 0.323
PTH / 255.3±237.3 / 241.3±254.3 / 0.717
CRP / 2.02±2.2 / 2.3±3.5 / 0.514

*p≤0.05 significant,Students T test was applied,data expressed in form of Mean±S.D.

Table-S5: Haplotype frequency distribution of Ghrelin in patients and controls

Ghrelin Arg51Gln / Ghrelin Leu72met / Control / Patient / OR (95% CI) / P-value
G / C / 70.06% / 62.7% / 1.00 / ---
G / A / 14.19% / 15.71% / 1.19 (0.84 - 1.69) / 0.33
A / C / 12.94% / 16.48 % / 1.41 (0.95 - 2.08) / 0.088
A / A / 2.81% / 5.11 % / 1.82 (0.85 - 3.93) / 0.13

*p≤0.05 significant

Table-S6: Comparison of genotype among patients on the basis of CRP level.

Genotype / Non-inflamed / Inflamed / OR(95%CI) / P-value
UCP2-I\D
DD / 88(56.4%) / 61(60.4%) / Reference
ID / 46(29.5%) / 24(23.8%) / 0.7(0.4-1.3) / 0.38
II / 22(14.1%) / 16(15.8%) / 1.1(0.5-2.3) / 0.7
D / 222(71.2%) / 146(72.3%) / Reference
I / 90(28.8%) / 56(27.7%) / 0.9(0.6-1.4) / 0.8
Leptin-2548 G/A
GG / 57(36.5%) / 33(32.7%) / Reference
GA / 55(35.3%) / 44(43.6%) / 0.7(0.4-1.1) / 0.19
AA / 44(28.2%) / 24(23.7%) / 1.2(0.7-2.2) / 0.47
G / 169(54.2%) / 110(54.5%) / Reference
A / 143(45.8%) / 92(45.5%) / 0.9(0.6-1.4) / 1.0
Ghrelin-Arg51Gln
GG / 104(66.6%) / 60(59.4%) / Reference
GA / 38(24.5%) / 37(36.6%) / 1.8(1.1-3.1) / 0.03*
AA / 14(8.9%) / 4(4.0%) / 2.3(0.7-7.0) / 0.14
G / 246(78.8%) / 157(77.7%) / Reference
A / 66(21.2%) / 45(22.3%) / 1.0(0.6-1.6) / 0.8
Ghrelin-Leu72Met
CC / 112(71.8%) / 66(65.3%) / Reference
CA / 27(17.3%) / 24(23.8%) / 0.7(0.3-1.2) / 0.26
AA / 17(10.9%) / 11(10.9%) / 1.0(0.4-2.2) / 1
C / 251(80.4%) / 156(77.2%) / Reference
A / 61(19.6%) / 46(22.8%) / 1.2(0.7-1.8) / 0.4

*p≤0.05 significant, Logistic regression analysis was done.

Table-S7: Comparison of genotype among patients on the basis of period of dialysis.

Genotype / Period < 1 year / Period ≥1 year / OR(95%CI) / P-Value
UCP2 I/D
DD / 100(58.8) / 49(56.3) / Reference
ID / 46(27.0) / 24(27.6) / 1.0(0.5-1.8) / 1
II / 24(14.2) / 14(16.1) / 1.1(0.5-2.3) / 0.7
D / 246(72.4) / 122(70.1) / Reference
I / 94(27.6) / 52(29.9) / 1.3(0.9-1.9) / 0.2
Leptin-2548 G/A
GG / 68(40.0) / 22(25.3) / Reference
GA / 61(35.9) / 38(43.7) / 1.3(0.8-2.3) / 0.2
AA / 41(24.1) / 27(31.0) / 1.4(0.7-2.5) / 0.2
G / 197(57.9) / 82(47.1) / Reference
A / 143(42.1) / 92(52.9) / 1.5(1.0-2.2) / 0.02
Ghrelin-Arg51gln
GG / 107(62.9) / 57(65.5) / Reference
GA / 52(30.6) / 23(26.4) / 0.8(0.4-1.4) / 0.5
AA / 11(6.5) / 7(8.1) / 1.2(0.4-3.2) / 0.8
G / 266(78.2) / 137(78.7) / Reference
A / 74(21.8) / 37(21.3) / 0.9(0.6-1.5) / 1.0
Ghrelin-Leu72met
CC / 118(69.4) / 60(69.0) / Reference
CA / 36(21.2) / 15(17.2) / 0.7(0.4-1.5) / 0.5
AA / 16(9.4) / 12(13.8) / 1.5(0.7-3.4) / 0.3
C / 272(80.0) / 135(77.6) / Reference
A / 68(20.0) / 39(22.4) / 1.1(0.7-1.8) / 0.5

*p≤0.05 significant, Logistic regression analysis was done.