Supplementary Digital Content - TABLE S1. Primer sequences of MC4R and HNF4α genes for pyrosequencing.
Gene / No. of CpG sites / Sequences (5’ – 3’) / Region / Length
(base pair)
MC4R / 1 / Forward : GAAGGAAGTAGAGGAGGAGTTAT
Reverse : biotin- TTTCCTTAAAACTACCATACCATTAAA
Sequencing : TAGGTTGGAAATAGAAGT / Promoter / 193 bp
2
3
HNF4α _P1 / 1 / Forward : GTGAGTTAGGGTTTTAGTAGTTGTAAT
Reverse : biotin- ATACCCTCTCTACCTTCCTTTCAA
Sequencing : AATAAATAGTTAAATTTTTGTAGT / 226 bp
2
3
4
HNF4α _P2 / 1 / Forward : GATAATGGGGAGGAGGTAGAA
Reverse : biotin - TTCTAACCCTACATCTAAATTTCA
Sequencing : GGAGGAGGTAGAAAG / 96 bp
3
4
MC4R = melanocortin 4 receptor, HNF4α = hepatocyte nuclear factor 4 alpha, P = promoter region.
Supplementary Digital Content - TABLE S2. Primer sets and Tm for quantitative-real time PCR
Gene / Primer sequences (5’ – 3’) / Tm (°C)
MC4R / Forward / ACACCCACTCCTCCACCTTT
TGCTGTAGCCAAATTCGTTG / 60°C
Reverse
HNF4α _P1 / Forward / GGTGAGCGTTTCAAATGGAT
TGCAAGCAAGGAGCTACAGA / 60°C
Reverse
HNF4α _P2 / Forward / AGAATGCGACTCTCCAAAAC
AACACCTGCACATTCTCAAA / 60°C
Reverse
MC4R = melanocortin 4 receptor, HNF4α = hepatocyte nuclear factor 4 alpha, P = promoter region.
Supplementary Digital Content - TABLE S3. DNA methylation levels of the MC4R and HNF4α in cord blood cells.
Gene / CpG sites / Distance relative to TSS (bp) / Genomic position
(GRCh38) / Average methylation levels (%)
(mean ± SD)
MC4R / 1 / -792 to -775 / Chr 18 : 60371318 - 60371336 / 94.88 ± 2.61
2 / 89.65 ± 2.16
3 / 80.95 ± 3.48
HNF4α _P1 / 1 / -115 to -88 / Chr 20 : 44401140 - 44401168 / 88.19 ± 5.79
2 / 70.65 ± 8.09
3 / 74.78 ± 7.71
4 / 74.84 ± 7.95
HNF4α _P2 / 1 / -44,920 to -44,903 / Chr 20 : 44356336- 44356353 / 97.12 ± 1.69
3 / 94.55 ± 1.88
4 / 90.75 ± 1.91
MC4R = melanocortin 4 receptor, HNF4α = hepatocyte nuclear factor 4 alpha, P = promoter region, TSS = transcription start site, SD = standard deviation.
Values are expressed as mean ± SD.
Supplementary Digital Content - TABLE S4. Mean methylation levels of MC4R and HNF4α in cord blood cells between term and preterm infants
Genes / Term
(N = 85) / Preterm
(N = 85)
Mean (SD) / Median (IQR) / Mean (SD) / Median (IQR) / P
MC4R
MC4R–CpG 1 / 95.24 (2.49) / 94.51 (2.69) / 0.07
MC4R–CpG 2 / 90.66 (89.12 - 91.21) / 89.44 (87.83 - 91.04) / 0.01
MC4R–CpG 3 / 81.42 (79.15 – 84.71) / 79.62 (78.30 – 80.79) / <0.001
HNF4α_P1
HNF4α–CpG 1 / 90.70 (87.74 – 92.47) / 88.46 (85.71 – 90.93) / <0.01
HNF4α–CpG 2 / 72.98 (68.59 – 77.17) / 70.62 (65.41 – 73.83) / 0.01
HNF4α–CpG 3 / 77.70 (73.89 – 80.65) / 74.71 (70.28 – 78.11) / <0.01
HNF4α–CpG 4 / 78.20 (72.78 – 80.77) / 75.33 (70.26 – 79.13) / 0.01
HNF4α_P2
HNF4α–CpG 1 / 97.34 (96.84 – 97.92) / 97.43 (96.74 – 97.79) / 0.78
HNF4α–CpG 3 / 94.82 (1.23) / 94.28 (2.33) / 0.06
HNF4α–CpG 4 / 90.91 (1.88) / 90.60 (1.94) / 0.31
MC4R = melanocortin 4 receptor, HNF4α = hepatocyte nuclear factor 4 alpha, P = promoter region, SD = Standard deviation, IQR = Interquartile range.
Normally distributed variables are expressed as mean (SD). Otherwise, variables are presented as median (IQR).