Summary of sample characteristics from the retrospective study.

<p>Summary of sample characteristics used in the retrospective study. Undet.: Undetermined (e.g. no Y chromosomal sequences were detected); Pos: positive; Y: Y chromosomal sequences were detected; Y-PAP: Y-chromosomal specific PAP-assay; Karyo: Full karyotyping performed on these samples; birth; fetal gender confirmed at birth.</p

Sequences after Methprimer prediction.

<p>Predicted sequences of the <i>RASSF1A</i> for Bisulfite Specific Primers (BSP) design using Methprimer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084051#pone.0084051-Li1" target="_blank">[31]</a>. BSP primers are located outside differentially methylated regions. Methylated nucleotides are indicated with +, unmethylated nucleotides with: and other nucleotides with |. A: The predicted sequence of the BisB forward primer (indicated as >>>). B: The predicted sequence of the BisB reverse primer (indicated as <<<).</p

Sample characteristics from the prospective study.

<p>Sample characteristics of clinical samples (prospective study). Undet.: Undetermined (e.g. no Y chromosomal sequences were detected); IF: Informative; Pols: Polymorphisms; Pos: positive; QF PCR: Quantitative Fluorescent PCR; US: Ultrasound; birth; fetal gender confirmed at birth.</p><p>^a No informative polymorphisms detected/inherited,</p><p>^b no informative polymorphisms present,</p><p>^c results did not meet our quality criteria used in diagnostics (only 1/3 Ct values ≤40).</p

Predicted and confirmed sequences for PAP-primer design.

<p>Sequences of the <i>RASSF1A</i> gene were analyzed after bisulfite sequencing of maternal gDNA and fetal gDNA derived from CVS. Differentially methylated regions of the BisB region predicted by MethPrimer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084051#pone.0084051-Li1" target="_blank">[31]</a> could be confirmed using bisulfite sequencing. Both forward (A, upper panel, underlined) and reverse PAP-primer (B, upper panel, underlined) are specific for fetal sequences (middle panels) after bisulfite conversion and both primers have several mismatches to the maternal sequences (lower panels). Mismatches between fetal specific PAP-primers and maternal sequences are indicated with an * for each primer.</p

<i>mRASSF1A</i>-PAP serial dilution range of gDNA from CVS.

<p>Serial dilutions were performed with gDNA from CVS in a background of 1000(pg) mentioned is the total amount of fetal gDNA. M = 50 bp marker, 1 = 1000 pg, 2 = 500 pg, 3 = 250 pg, 4 = 125 pg, 5 = 60 pg, 6 = 30 pg, 7 = 15 pg, 8 = 7 pg, 9 =  positive control for bisulfite conversion, 10 = NTC for bisulfite conversion, 11 =  negative control for bisulfite conversion (non-bisulfite converted fetal gDNA), 12 =  positive control for <i>mRASSF1A</i>-PAP, 13 = NTC for <i>mRASSF1A</i>-PAP. A 110 bp product (arrow) is obtained in cases where <i>mRASSF1A</i> sequences could be detected using <i>mRASSF1A</i>-PAP.</p

Bisulfite sequencing primers and PAP primers.

<p>Primer sequences. M13 tag used for Sanger sequencing is depicted in bold. BSP: Bisulfite Specific Primer, PAP: Pyrophosphorolysis-activated Polymerization.</p><p>*Product sizes for BSP primers are including the M13 tags.</p

Differentially methylated regions after bisulfite sequencing.

<p>Sanger sequencing results for <i>RASSF1A</i> of a fully methylated control cell line (A), maternal gDNA (B) and fetal gDNA derived from CVS (C) after bisulfite sequencing. A representative part of the complete sequence is shown. All unmethylated cytosines are converted to uracil after bisulfite sequencing. Differences between maternal and fetal (methylated) sequences are indicated with an *.</p

Malan syndrome: Sotos-like overgrowth with de novo NFIX sequence variants and deletions in six new patients and a review of the literature

Genetics of disease, diagnosis and treatmen