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

<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

Successful Noninvasive Trisomy 18 Detection Using Single Molecule Sequencing

<p>BACKGROUND: Noninvasive trisomy 21 detection performed by use of massively parallel sequencing is achievable with high diagnostic sensitivity and low false-positive rates. Detection of fetal trisomy 18 and 13 has been reported as well but seems to be less accurate with the use of this approach. The reduced accuracy can be explained by PCR-introduced guanine-cytosine (GC) bias influencing sequencing data. Previously, we demonstrated that sequence data generated by single molecule sequencing show virtually no GC bias and result in a more pronounced noninvasive detection of fetal trisomy 21. In this study, single molecule sequencing was used for noninvasive detection of trisomy 18 and 13.</p><p>METHODS: Single molecule sequencing was performed on the Helicos platform with free DNA isolated from maternal plasma from 11 weeks of gestation onward (n = 17). Relative sequence tag density ratios were calculated against male control plasma samples and results were compared to those of previous karyotyping.</p><p>RESULTS: All trisomy 18 fetuses were identified correctly with a diagnostic sensitivity and specificity of 100%. However, low diagnostic sensitivity and specificity were observed for fetal trisomy 13 detection.</p><p>CONCLUSIONS: We successfully applied single molecule sequencing in combination with relative sequence tag density calculations for noninvasive trisomy 18 detection using free DNA from maternal plasma. However, noninvasive trisomy 13 detection was not accurate and seemed to be influenced by more than just GC content.</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

Association between mutations in a thyroid hormone transporter and severe X-linked psychomotor retardation

Monocarboxylate transporter 8 (MCT8) is a thyroid hormone transporter, the gene of which is located on the X chromosome. We tested whether mutations in MCT8 cause severe psychomotor retardation and high serum triiodothyronine (T3) concentrations in five unrelated young boys. The coding sequence of MCT8 was analysed by PCR and direct sequencing of its six exons. In two patients, gene deletions of 2·4 kb and 24 kb were recorded and in three patients missense mutations Ala150Val, Arg171 stop, and Leu397Pro were identified. We suggest that this novel syndrome of X-linked psychomotor retardation is due to a defect in T3 entry into neurons through MCT8, resulting in impaired T3 action and metabolism

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