Heterozygosity and repeat numbers of the selected STR markers in the tested Chinese Han population.

<p>*Expected PCR product sizes are predicted according to human genome hg19 assembly.</p><p>**Heterozygosity for DXS1053, DXS981, DXS6809, DXS1187 and DXS8377 was calculated using Female samples, while heterozygosity for X22, D21S11 and D13S305 was calculated using all tested samples.</p><p>Heterozygosity and repeat numbers of the selected STR markers in the tested Chinese Han population.</p

Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for Rapid Diagnosis of Sex Chromosome Aneuploidies

<div><p>Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR). Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY), five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377), one X/Y-common STR (X22), and two autosomal STRs (D13S305 and D21S11). Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.</p></div

Representative examples for a normal female (A), a normal male (B), a trisomy X (47, XXX) (C), a Klinefelter syndrome (47, XXY) (D), a Jacob’s syndrome (47, XYY) (E) and a Turner syndrome (45, X) (F) obtained through the 10-plex QF-PCR assay.

<p>The results are produced using the GeneMapper software, showing full ranges and all sizes of the detected peaks on the vertical and horizontal axes respectively for intuitive intra-sample visual comparison of the peaks. Fragment sizes are shown in bp on the horizontal axis. Arbitrary fluorescence units are shown on the vertical axis.</p

Graphic scheme of the chromosomal localization of the eight sex chromosomal markers used in the newly designed multiplex PCR assay.

<p>The markers were located according to Human Genome Browser – hg38 assembly.</p

Capillary electrophoresis results for three samples with sex chromosomal aneuploidies identified in the prospective examination, including (A) a suspected 23, X finally confirmed by karyotyping, (B) a case of triploidy ‘69, XXX’, and (C) a case of triploidy with an additional chromosome 21 (70, XXX,+21).

<p>Capillary electrophoresis results for three samples with sex chromosomal aneuploidies identified in the prospective examination, including (A) a suspected 23, X finally confirmed by karyotyping, (B) a case of triploidy ‘69, XXX’, and (C) a case of triploidy with an additional chromosome 21 (70, XXX,+21).</p

The loci of active HERV-Hs in colon tumor and adjacent normal tissues.

<p>*Active elements individually contributing to more than 10% of the transcripts in tumor or adjacent normal samples are highlighted in boldface and their transcript abundances are indicated in the ‘Abundance’ column (individual and total, respectively).</p><p>**HERV-HX is the colon cancer-related HERV-H element identified by us previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029950#pone.0029950-Liang2" target="_blank">[14]</a>. Inserts of PCR product clones were all HERV-HX fragments but not spliced sequences, in concordance with our previous finding that no spliced transcripts were produced from HERV-HX in colon tumor samples.</p><p>***The element located at 1q31.3 consists of 5′LTR and 3′LTR, with the entire protein coding region (<i>gag-pro-pol-env</i>) missing.</p><p>****Open reading frames (ORFs) were predicted by the online program ORF Finder and putative peptide sequences were subjected to Blastp search against the Non-redundant protein sequences at <a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>. Only predicted ORFs≥303 nt (peptide sequence ≥100 aa) and with Blastp matches are included. RT, Reverse transcriptase; CREB5, cAMP response element-binding protein 5; Gag, group-specific antigen.</p

RT-PCR detection of HERV-H spliced transcripts in colon cancer cell lines.

<p><b>A.</b> Schematic demonstration of the primer locations. TSS, transcription start site; TTS, transcription termination site. <b>B.</b> RT-PCR was performed to detect HERV-H spliced transcripts in colon cancer cell lines with/without demethylation/histone acetylation treatment using the DNA demethylation agent DAC and the histone deacetylase inhibitor TSA. H2O and genomic DNA mixture (gDNA mix) were used as controls. Genomic DNA mixture produced bands distinct from cDNA samples, which were reverse transcribed from DNase-treated RNA.</p

The loci of active HERV-H elements in colon cancer cell lines.

<p>*Two adjacent HERV-H elements at 1p32.3 were combinedly active by making use of 5′ LTR of the first HERV-H and 3′ LTR of the second one (representative transcript sequence JK017392).</p><p>**The elements located at 16q24.1 and 19q13.31 are also actively transcribed in both tumor and adjacent normal colon tissues, while the one located at 20p12.1 is active in tumor tissue.</p><p>***Open reading frames (ORFs) were predicted by the online program ORF Finder and putative peptide sequences were subjected to Blastp search against the Non-redundant protein sequences at <a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>. Only predicted ORFs≥303 nt (peptide sequence ≥100 aa) and with Blastp matches are included. Gag, group-specific antigen; RT, Reverse transcriptase; CREB5, cAMP response element-binding protein 5.</p

Characterization of the active HERV-H elements in colon cancer cell lines and their spliced transcripts.

<p><b>A.</b> Schematic of the six commonly deleted regions in the active HERV-H elements in colon cancer cell lines. <b>B.</b> Schematics of the two extraordinarily short HERV-H elements and their transcripts. Pair-wise alignments for each HERV-H element were performed with the HERV-H consensus constructed by Jern P, <i>et al</i>. The shortened alignment results were shown to indicate the missing regions precisely. Color density represents the extent of homology with the HERV-H consensus. Gray areas represent deleted regions in the HERV-H elements as compared with the HERV-H consensus. Spliced transcripts are shown above the alignment results accordingly. Thick bars represent exons, and lines represent introns. Regions of LTRs, pre-gag, <i>gag</i>, <i>pro</i>, <i>pol</i> and <i>env</i> are labeled below. <b>C.</b> Schematics of the two combinedly active HERV-H elements located at 1p32.3 in HT29.</p

Gel electrophoresis of RT-PCR products of HERV-H-related transcripts in colon tumor and adjacent normal tissues.

<p>M, DL2000 ladder; T, tumor; N, normal.</p