Somatic Pairing of Chromosome 19 in Renal Oncocytoma Is Associated with Deregulated ELGN2-Mediated Oxygen-Sensing Response

Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase ELGN2, a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of ELGN2 in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma

SWI/SNF Antagonism of PRC2 Mediates Estrogen-Induced Progesterone Receptor Expression

Endometrial cancer (EC) is characterized by high estrogen levels unopposed by progesterone. Treatment with progestins is standard for early EC, but the response to progestins is dependent on progesterone receptor (PGR) expression. Here, we show that the expression of PGR in endometrial epithelial cells is dependent on ARID1A, a DNA-binding subunit of the SWI/SNF chromatin-remodeling complex that is commonly mutated in EC. In endometrial epithelial cells with estrogen receptor overexpression, we find that ARID1A promotes estrogen signaling and regulates common gene expression programs. Normally, endometrial epithelial cells expressing estrogen receptors respond to estrogen by upregulating the PGR. However, when ARID1A expression is lost, upregulation of PGR expression is significantly reduced. This phenomenon can also occur following the loss of the SWI/SNF subunit BRG1, suggesting a role for ARID1A- and BRG1-containing complexes in PGR regulation. We find that PGR is regulated by a bivalent promoter, which harbors both H3K4me3 and H3K27me3 histone tail modifications. H3K27me3 is deposited by EZH2, and inhibition of EZH2 in the context of ARID1A loss results in restoration of estrogen-induced PGR expression. Our results suggest a role for ARID1A deficiency in the loss of PGR in late-stage EC and a therapeutic utility for EZH2 inhibitors in this disease

Partial Deletion of the Sulfate Transporter <em>SLC13A1</em> Is Associated with an Osteochondrodysplasia in the Miniature Poodle Breed

<div><p>A crippling dwarfism was first described in the Miniature Poodle in Great Britain in 1956. Here, we resolve the genetic basis of this recessively inherited disorder. A case-control analysis (8∶8) of genotype data from 173 k SNPs revealed a single associated locus on <em>CFA14</em> (P_raw <10^–8). All affected dogs were homozygous for an ancestral haplotype consistent with a founder effect and an identical-by-descent mutation. Systematic failure of nine, nearly contiguous SNPs, was observed solely in affected dogs, suggesting a deletion was the causal mutation. A 130-kb deletion was confirmed both by fluorescence <em>in situ</em> hybridization (FISH) analysis and by cloning the physical breakpoints. The mutation was perfectly associated in all cases and obligate heterozygotes. The deletion ablated all but the first exon of <em>SLC13A1</em>, a sodium/sulfate symporter responsible for regulating serum levels of inorganic sulfate. Our results corroborate earlier findings from an <em>Slc13a1</em> mouse knockout, which resulted in hyposulfatemia and syndromic defects. Interestingly, the metabolic disorder in Miniature Poodles appears to share more clinical signs with a spectrum of human disorders caused by <em>SLC26A2</em> than with the mouse <em>Slc13a1</em> model. <em>SLC26A2</em> is the primary sodium-independent sulfate transporter in cartilage and bone and is important for the sulfation of proteoglycans such as aggregan. We propose that disruption of <em>SLC13A1</em> in the dog similarly causes undersulfation of proteoglycans in the extracellular matrix (ECM), which impacts the conversion of cartilage to bone. A co-dominant DNA test of the deletion was developed to enable breeders to avoid producing affected dogs and to selectively eliminate the mutation from the gene pool.</p> </div

Evaluation of whole-genome DNA methylation sequencing library preparation protocols

Abstract Background With rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols currently exist. We have performed 22 whole-genome DNA methylation sequencing experiments on snap frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfite-based protocols and a new enzyme-based protocol. In addition, different input DNA quantities were compared for two kits compatible with a reduced starting quantity. In addition, we also present bioinformatic analysis pipelines for sequencing data from each of these library types. Results An assortment of metrics were collected for each kit, including raw read statistics, library quality and uniformity metrics, cytosine retention, and CpG beta value consistency between technical replicates. Overall, the NEBNext Enzymatic Methyl-seq and Swift Accel-NGS Methyl-Seq kits performed quantitatively better than the other two protocols. In addition, the NEB and Swift kits performed well at low-input amounts, validating their utility in applications where DNA is the limiting factor. Results The NEBNext Enzymatic Methyl-seq kit appeared to be the best option for whole-genome DNA methylation sequencing of high-quality DNA, closely followed by the Swift kit, which potentially works better for degraded samples. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Biosciences’s Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence

Cytogenetic confirmation of a deletion on chromosome 14 by <i>FISH</i>.

<p>Dual-color FISH probe results are shown for representative metaphase spreads obtained from (A) wildtype, (B) obligate carrier, and (C) affected Miniature Poodle dogs, respectively. The green LR-PCR 14–63.6 g FISH signal is within the deleted region and shows some centromeric background staining. The red BAC probe 14–59.1or signal distinguishes <i>CFA14</i> within the canine metaphase spread. Wildtype shows co-localization of red (control) and green (experimental) probes. This co-localization is absent in the affected dog (homozygous for deletion), and is heterozygous in the carrier dog.</p

Gross presentation of Miniature Poodle osteochondrodysplasia.

<p>(A) An affected dog (foreground) shows dwarfism relative to an age-matched normal (wildtype) dog (background). (B) The misshapen paw of an affected dog (Aff) relative to that of a wildtype (WT) dog. (C) Close-up of the affected foot, which is similar to talipes varus, or clubfoot, a distinguishing feature of human osteochondrodsyplasias.</p

DNA test of large scale deletion.

<p>(A) A three-primer, dual reporter design exploits known breakpoints to create differentially dye-labeled and differentially-sized PCR products for a co-dominant DNA test readout. (B) The assay converted to a high-throughput format by analysis with a semi-automated DNA sequencing instrument for fluorescence-based fragment analysis. The electropherogram shows standard fragments for sizing (red peaks), product corresponding to the deletion-specific allele (blue peak), and product corresponding to the wildtype reference allele (green peak). Test outcomes are shown for each of the three genotypes possible. There is a size difference in the products corresponding to the wildtype allele in the clear dog and the carrier dog. This is attributable to a small insertion/deletion in the interval that segregates in the Miniature Poodle population.</p

Human Biosample Authentication Using the High-Throughput, Cost-Effective SNPtrace^TM System

<div><p>Cell lines are the foundation for much of the fundamental research into the mechanisms underlying normal biologic processes and disease mechanisms. It is estimated that 15%–35% of human cell lines are misidentified or contaminated, resulting in a huge waste of resources and publication of false or misleading data. Here we evaluate a panel of 96 single-nucleotide polymorphism (SNP) assays utilizing Fluidigm microfluidics technology for authentication and sex determination of human cell lines. The SNPtrace Panel was tested on 907 human cell lines. Pairwise comparison of these data show the SNPtrace Panel discriminated among identical, related and unrelated pairs of samples with a high degree of confidence, equivalent to short tandem repeat (STR) profiling. We also compared annotated sex calls with those determined by the SNPtrace Panel, STR and Illumina SNP arrays, revealing a high number of male samples are identified as female due to loss of the Y chromosome. Finally we assessed the sensitivity of the SNPtrace Panel to detect intra-human cross-contamination, resulting in detection of as little as 2% contaminating cell population. In conclusion, this study has generated a database of SNP fingerprints for 907 cell lines used in biomedical research and provides a reliable, fast, and economic alternative to STR profiling which can be applied to any human cell line or tissue sample.</p></div

Genetic mapping of the osteochondrodysplasia locus.

<p>(A) Manhattan plot of genome-wide results displaying a statistically significant peak of association on the terminal end of <i>CFA14</i>. Plotted <i>p</i>-values were calculated by Fisher’s Exact Test. The p-values for the six most strongly associated SNPs (overlapping on the graph), adjusted by permutation, were p = 0.0006. (B) Genotypic pattern of SNPs across the interval of interest on <i>CFA14</i> (62.5–64.0 Mb). Red, major allele in cases; orange, heterozygous SNP genotypes; yellow, homozygous minor allele in cases. Genotype data are shown horizontally for each of seven controls and eight cases. The arrow notes an historical crossover that bounds the ancestral haplotype shared among cases, upon which the founder mutation is predicted to have occurred. Nine SNPs systematically failed in the case samples (white block with orange), suggesting a large deletion. (C) Raw fluorescence intensity readings from the SNP array for loci across the region of interest (62.5–64.0 Mb). Although several SNPs were initially scored heterozygous (orange, in B), the raw intensity data shows systematic failure in cases for all 12 SNPs in this interval (red bracket), relative to controls. (D) The potential deletion was bounded by two SNPs that were scored in cases. The putative deletion includes a single gene, <i>SLC13A1</i>, drawn to scale, with exon structure in red. The location of physical breakpoints that were subsequently cloned are shown as opaque boxes.</p