Identification of Somatic Mosaicism in the blood and uninvolved margin in breast cancer patients

Somatic mosaicism is the presence of genetically diverse lineage of somatic cells within an organism even though deriving from a single zygote. Mosaicism is known to occur in many diseases. Breast cancer is the second most cause of deaths in woman caused by malignant tumor. Breast cancer was a common malignancy and main cause of morbidity and disease related mortality among women. By studying the genetic profiles of the cells surrounding the primary tumor and blood of same patient may reveal the cause of tumor cells. The purpose of this study was to identify the presence of somatic mosaicism in healthy uninvolved margin of breast tissue surrounding the primary tumor and blood of breast cancer patient by using Illumina 1M BeadChip and Nimblegen 720K array. So far, all long and very rigorous research explains only 10% of the breast cancer. This indicates the complexity of cancer and the factors responsible for the development of tumor in healthy tissues. Results from whole genome genotyping by Illumina 1M beadchip of blood and cells surrounding the primary tumor of same patient indicate the presence of somatic mosaicism in chromosome 6, 8 and 18 of ML36B (uninvolved margin), ML36A2 and ML36A3 (primary tumor) which was confirmed by genotyping with Nimblegen 720K microarrays. Genotyping also showed the presence of genomic imbalance in genetic makeup of the healthy tissue surrounding the primary tumor. It is to be likely that rate of discovery of somatic mosaicism in the small proportion of studies cells might continue to accelerate the studies on genetic heterogeneity of breast malignancies. As well as studies by comparing the genetic profiles of tumours and tissue surrounding the primary tumour may lead to identify the genomic aberrations in context to cancer progression

Copy number variations as potential biomarkers for disease development in Breast Cancer

Breast cancer is second foremost cause of deaths in women caused by malignant tumor. Comparing genetic profiles of cells surrounding the tumor and blood from same patient may reveal de‐novo somatic aberrations that may predispose normal cells to cancer cells. Two major types of genetic variations were discovered in human genome: single nucleotide polymorphisms (SNPs) and more recently discovered copy number variations which are chromosomal segments ranging from kilobases to megabases. It is assumed that somatic cells are genetically identical. But it is unknown if CNVs arise in somatic cells. Somatic mosaicism is defined as genetic differences in the cells of a single individual which were developed from one fertilized egg. The purpose of this study was to identify and compare copy number variants in IFNAR1 loci of healthy uninvolved margin of breast tissue surrounding primary tumor and blood derived‐DNA of breast cancer patients by using Illumina SNPs array‐Human660W‐Quad. So far, long and very rigorous research on breast cancer explains only about 10% of all cases. This may signify unusual complexity of cancer and discovery of factors responsible for progression of cancer from healthy cells. Illumina SNPs genotyping of blood and cells surrounding tumor indicated the occurrence of changes in the region connected to IFNAR1 gene loci. Variations in IFNAR1 loci formed the base for detailed investigation of expected aberrations. The amplification of DNA fragments and deeper investigations were performed to identify and confirm the differences in genotype between blood and cells surrounding the tumor by cloning, restriction digestion and Sanger’s sequencing. The results noticeably confirmed the presence of somatic mosaicism. We have identified aberrations occurring as variable number of tandem repeats (VNTRs) with length of 32 nucleotides. Consequently, the significant progress in research on cause of cancer carries development of applied technologies (next generation sequencing) that allow examining more number of samples in fewer instances with better precision

Hot Nano-particles in Polar or Paramagnetic Liquids Interact as Monopoles.

When neutral nano-particles are heated or cooled in a polar liquid, they will interact with each other as if they carry an electrostatic charge that is proportional to the temperature difference between the particle and the surrounding fluid. The same should hold for suspensions liquids of asymmetric ferromagnetic particles, in which case the heated nano-particles should behave as magnetic monopoles. However, the analogy with electrostatics/magnetostatics is not complete: heated/cooled nano-particles do not move under the influence of an applied homogeneous field. They should, however, interact as monopoles with each other and should move in inhomogeneous fields.This is the author accepted manuscript. The final version is available from the American Chemical Society via https://doi.org/10.1021/acs.jpcb.6b0184

Draft genome of the brown-rot fungus Fomitopsis pinicola GR9-4

Basidiomycete brown-rot fungi have a huge importance for wood decomposition and thus the global carbon cycle. Here, we present the genome sequence of Fomitopsis pinicola GR9-4 which represent different F. pinicola clade than the previously sequenced North American isolate FP-58527 SS1. The genome was sequenced by using a paired-end sequence library of Illumina and a 2.5k and 5k mate-pair library (ABI SOLiD). The final assembly adds up to a size of 45 Mb (including gaps between contigs), with a GC-content of 56%. The gene prediction resulted in 13,888 gene models. The genome sequence will be used as a basis for understanding population genomics, genome-wide association studies and wood decay mechanisms of this brown-rot fungus. Keywords: Draft genome, Brown-rot, Fomitopsi

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

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

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

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