Summary of validation of somatic variation in the <i>IFNAR1</i> locus.

<p>Summary of Illumina SNP genotyping, which suggested structural variation within the hypevariable region and results from subsequent confirmation using Sanger sequencing and agarose gel electrophoresis. One (*) and two (**) asterisks after the subject ID indicate patients with breast cancer and pairs of monozygotic twins, respectively. BL, UM and PT stand for DNA from peripheral blood cells, healthy morphologically normal breast tissue from a patient affected with breast cancer and primary breast tumor, respectively. “Seq” indicate that the somatic mosaicism was verified by Sanger sequencing while “Gel” shows that it was confirmed by estimation of allele sizes from agarose gel.</p

Graphical summary of variation in a presumptive regulatory VNTR containing region.

<p>Panel <b>A</b> shows an overview of approximately 2 Mb locus on 21q, around four genes encoding functionally related receptors; <i>IFNAR2</i>, <i>IL10Rβ</i>, <i>IFNAR1</i> and <i>IFNGR2</i>. Panel <b>B</b> is zooming on the position of the hypervariable region (HVR, red box), which is located approximately 4 kb upstream from the transcription start site of the <i>IFNAR1</i> gene and is flanked by CpG-islands (green boxes). The last three and the first three exons of <i>IL10Rβ</i>, and <i>IFNAR1</i>, respectively, are shown as grey boxes. Panel <b>C</b> is showing the size and position of HVR according to the most common allele (HVR1098, see below panel D) in relation to the CpG island. Positions of PCR and sequencing primers used in the analysis of the locus are also displayed. Yellow boxes indicate the position of the non-repetitive anchor 1 (A1) and anchor 2 (A2) sequences, that are immediately flanking the repeated segments and were used for alignments of sequence reads. Panel <b>D</b> shows a summary of eight HVR-alleles from the studied samples, which were identified based on Sanger sequencing results of PCR fragments sub-cloned in plasmids. The displayed alleles are ordered from longest to shortest according to size from anchor 1 (A1) to anchor 2 (A2) sequences. Summary of sizes for all 14 different HVR-alleles is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067752#pone-0067752-t001" target="_blank">Table 1</a>. Sizes of fragments (in base pairs) are given between non-repetitive A1 and A2 sequences and between primers p1 and p8, which were used for PCR amplification from genomic DNA. Asterisk (*) indicates the most frequent allele (HVR1098), which is in agreement with the reference sequence according to NCBI sequence build 36.3. The allele frequency shown here is taking into account only the nine alleles, where the entire sequence could be unequivocally determined using Sanger sequencing. The most common variation encompasses the variable number of 32 base pair segments; i.e. indel 2, indel 3, indel 4, and indel 5. The latter indel 5 is composed of 6 repeated 32 base pair segments (HVR1066). However, there are also indels containing shorther segments; e.g. indel 1, indel 6 and indel 7. Panel <b>E</b> illustrates the positions of two of the four probes from Illumina beadchips, which are aligned onto the NCBI reference sequence for this locus (top sequence with an asterisk, representing HVR1098). The two probes shown here are from Illumina 610 SNP array; cnvi0010761 (green) and cnvi0010759 (blue). All four Illumina probes from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067752#pone-0067752-g001" target="_blank">Figure 1</a>, which were used for initial identification of variation in this region are located within hypervariable region. As shown here for two of these four probes, the probeA sequences (as called by Illumina and used for capturing of genomic DNA on beadchips) are shifted only by two bases. The core 32 bp repeat motif is shown in brackets.</p

Size and distribution of 14 HVR-alleles identified by sequencing and gel electrophoresis.

<p>The •/× indicate that the size of the allele was determined by both agarose gel images and sequencing, whereas filled circles (•) denote that the allele size was estimated from agarose gel images.</p><p>The two most common alleles (HVR1098 and HVR1700) are highlighted in bold and underlined text.</p><p>The sample indicated by a single asterisk (*) are from breast cancer patients. BL, PT and UM indicate peripheral blood DNA, primary breast tumor and healthy morphologically normal breast tissue from a patient affected with breast cancer, respectively.</p><p>The samples indicated by two asterisks (**) are monozygotic twin pairs.</p

Variable length of alleles within hypervariable region showing post-zygotic variation.

<p>Panel <b>A</b> shows post-zygotic mosaicism in healthy and phenotypically concordant monozygotic twin pair 148341/148342, with five alleles observed in twin 148341, and three alleles present in co-twin 148342. Similarly, panel <b>B</b> displays post-zygotic variation in another monozygotic twin pair 004_01/004_02. In total 5 different alleles are shown on this gel and only one of them is overlapping between both twins. Panel <b>C</b> illustrates post-zygotic mosaicism in breast cancer patient SK58. There are three different alleles in DNA from morphologically normal breast tissue (UM), two alleles in blood cells (BL) and three alleles in primary tumor (PT). In panels <b>A</b>, <b>B</b> and <b>C</b>, Taq DNA polymerase was used for initial PCR amplification from genomic DNA, as indicated by suffix “T” in the ID of each plasmid clone. In panel <b>D</b>, Phusion DNA polymerase confirmed post-zygotic mosaicism in monozygotic twin pair 148341/148342, as indicated by suffix “Ph” in the ID of each plasmid clone. The length of inserts in all plasmid clones was estimated after EcoRI digestion releasing the insert, and using 1% agarose gel. BL, PT and UM indicate peripheral blood DNA, primary breast tumor and healthy morphologically normal breast tissue from a patient affected with breast cancer, respectively.</p

The frequency distribution of the 14 alleles identified for the hypervariable region.

<p>The white bars represent alleles defined by both agarose gels and Sanger sequencing. The grey bars denote alleles that were characterized by estimation of their sizes from agarose gels. All alleles were defined based on the analysis between primers 1 and 8 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067752#pone-0067752-g002" target="_blank">Fig. 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067752#pone-0067752-t001" target="_blank">Table 1</a>).</p

Indications of post-zygotic copy number variation in a region between <i>IL10Rβ</i> and <i>IFNAR1</i>.

<p>Results from eight Illumina genotyping experiments are shown using blood DNA from two pairs of monozygotic twins (panel A for twin 012_01 versus co-twin 012_02, and in panel B for twin 159201 versus co-twin 159202) and two unrelated individuals, where two different tissues were analyzed from each subject (Panels C and D; subjects ML36 and SK58, respectively). Abbreviations BL, PT and UM indicate peripheral blood DNA, primary breast tumor and healthy morphologically normal breast tissue from a patient affected with breast cancer, respectively. Illumina 610 or 660W SNP arrays were used, which also contain so called “intensity only probes” (often with cvni-prefix), only useful for copy number analyses. Therefore, only Log R Ratio (LRR) windows of Illumina experiments are shown here, since the B Allele Frequency (BAF) values are not informative for this type of probes. LRR values below and above zero suggest a deletion or a gain, respectively. The four array probes showing variation between the studied samples are labeled as red dots in yellow fields.</p