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Supplemental material
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Table S1
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Details of primer sequences, PCR conditions, the targeted regions and analyzed CpG
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positions of the pyrosequencing assays for the imprinted loci PLAGL1, IGF2R DMR2,
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GRB10, H19 3CTCF binding site and 6 CTCF binding site, IGF2, MEG3, NDN, SNRPN,
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NESP and NESPAS. Primer sequences of the MS-PCR experiments for the DLK1/MEG3 IG-
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DMR.
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Table S2
Details on the 10 evaluable pyrosequencing assays used for DNA methylation analysis.
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Locus
Chromosomal
region
Methylated
allele
Targeted
region
Reference
PLAGL1
6q24
maternal
Putative ICR
overlapping
ZAC/HYMAI
Arima et al 34
GRB10
7p12
maternal
DMR
Hikichi et al 35
IGF2
11p15
maternal
DMR0 within
intron 2
Liu et al 36
H19
11p15
paternal
6CTCF and
3CTCF binding sites
Takai et al 37
NESP
20q13
paternal
DMR
Liu et al 38
NESPAS
20q13
maternal
DMR
Liu et al 38
NDN
15q11
maternal
promoter CpG island
Lau et al 39
SNRPN
15q11
maternal
DMR overlapping
exon 1
Zeschnigk et al 40
MEG3
14q32
paternal
promoter DMR
Temple et al 9
IGF2R
6q25
maternal
DMR2 in intron 2
Smrzka et al 41
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Table S3
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Raw data on pyrosequencing results of the analyzed patients born SGA and the controls.
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Methylation values are provided in % methylation per studied CpG position. For each of the
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10 evaluated loci single CpG positions and their corresponding position in bp (hg19) is
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displayed. SGA= small for gestational age. MC= commercial in vitro methylated DNA
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(positive control). Pool= pooled DNA from 20 healthy donors (10 males, 10 females). UC=
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whole genome amplification DNA (illustra GenomiPhi V2 DNA Amplification Kit, GE
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Healthcare, Freiburg, Germany) used as negative control during assay optimization (i.e. in
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independent control series). N/A= not analyzable.
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For all established assays except IGF2R (see below) we calculated the thresholds for hyper-
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and hypomethylation as follows:
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First, per locus the mean methylation value of the tested CpGs (called “mean(locus)”) was
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determined in all samples and for all loci. Second, the mean methylation level of the
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“mean(locus)” and its standard deviation (SD) was calculated over all 50 controls analyzed
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per locus (called “mean(locus/controls)”). Each assay specific “normal methylation range”
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(=NMR) was then defined as “mean(locus/controls)” +/- 3SD. Next we compared the
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“mean(locus)” values for the patient samples to the respective locus-specific NMR. If the
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“mean(locus)” in a sample exceeded the locus-specific NMR the locus was called
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“hypermethylated” in this sample. If the “mean(locus)” in a sample was below the locus-
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specific NMR the locus was called “hypomethylated” in this sample. Finally, for such “hyper-
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“ and “hypomethylated” loci we determined for each single CpG in this locus whether its
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methylation level was outside the NMR of the same CpG in the 50 controls.
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Hypermethylation of the IGF2R DMR2 in healthy probands has repeatedly been reported in
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the recent literature
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defined differently. Thus, the methylation results for IGF2R of the commercially available
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fully methylated controls (MC) carried through all IGF2R experiments was used as reference.
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We built “mean(IGF2R/MC)” by calculating the mean methylation value over all 3 tested
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CpG positions and all MC samples analyzed in the same runs with patient and control
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samples. In the next step the hypermethylation cutoff for IGF2R was defined as
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“mean(IGF2R/MC)” – 3 SD. In an alternative approach we excluded the fully methylated
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samples in the control group with the method explained above (2 samples met the criteria)
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and calculated in a second step “mean(IGF2R/controls)” +/- 3SD from the remaining control
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samples. With this approach, that raises the test sensitivity, the identical cases turned out to be
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classified “hypermethylated” in the patient cohort, while 3 additional cases were labeled
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“hypermethylated” in the control cohort. No hypomethylated samples were detected with the
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latter approach.
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49
50
51
52
3
12,22
. Therefore, the hypermethylation cutoff for this assay needed to be