Frequent Promoter Hypermethylation of the APC and RASSF1A Tumour Suppressors in Parathyroid Tumours

BACKGROUND: Parathyroid adenomas constitute the most common entity in primary hyperparathyroidism, and although recent advances have been made regarding the underlying genetic cause of these lesions, very little data on epigenetic alterations in this tumour type exists. In this study, we have determined the levels of promoter methylation regarding the four tumour suppressor genes APC, RASSF1A, p16(INK4A) and RAR-beta in parathyroid adenomas. In addition, the levels of global methylation were assessed by analyzing LINE-1 repeats. METHODOLOGY/PRINCIPAL FINDINGS: The sample collection consisted of 55 parathyroid tumours with known HRPT2 and/or MEN1 genotypes. Using Pyrosequencing analysis, we demonstrate APC promoter 1A and RASSF1A promoter hypermethylation in the majority of parathyroid tumours (71% and 98%, respectively). Using TaqMan qRT-PCR, all tumours analyzed displayed lower RASSF1A mRNA expression and higher levels of total APC mRNA than normal parathyroid, the latter of which was largely conferred by augmented APC 1B transcription levels. Hypermethylation of p16(INK4A) was demonstrated in a single adenoma, whereas RAR-beta hypermethylation was not observed in any sample. Moreover, based on LINE-1 analyses, parathyroid tumours exhibited global methylation levels within the range of non-neoplastic parathyroid tissues. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that APC and RASSF1A promoter hypermethylation are common events in parathyroid tumours. While RASSF1A mRNA levels were found downregulated in all tumours investigated, APC gene expression was retained through APC 1B mRNA levels. These findings suggest the involvement of the Ras signaling pathway in parathyroid tumorigenesis. Additionally, in contrast to most other human cancers, parathyroid tumours were not characterized by global hypomethylation, as parathyroid tumours exhibited LINE-1 methylation levels similar to that of normal parathyroid tissues

PathogenMip Assay: A Multiplex Pathogen Detection Assay

The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Figure 4

<p>Bar-histograms representing fluorescence intensities from the in-house barcode-chip for genotyping genomic DNA extracts from tumor samples derived from four patients with cervical cancers. Seen in the figure are four examples of single HPV infections, one from each genotype, observed in the sample set. HPV-16 was genotyped in sample OM-1751, HPV-18 in OM-1452, HPV-45 in OM-2258 and HPV-59 in OM-1569. The signal-intensities were normalized to the intensity of the peak for the reference probe targeting human β-globin gene (rMIP). The remaining bars constitute of the reaction background signal.</p

Figure 6

<p>Schematic overview of the PathogenMip Assay. <b>A</b>) The 24 probes included in the assay are situated at their respective target sites on the approximately 8000 base pairs of double stranded HPV genomic DNA. Early genes (denoted E) code for virus integration and replication and late genes (denoted L) encode the viral capsule creation. The probes recognize ∼40 base pair fragments unique for each targeted genotype. <b>B</b>) Following enzymatic inversion of reacted probes and universal amplification, the amplicons are used for subsequent appropriate HPV genotype screening. <b>C</b>) Conventional HPV genotyping takes a different approach, in which the nested primer pairs PGMY09/11 and GP5+/6+ amplify respectively ∼450 base pair and ∼150 base pair fragments that, through an appropriate readout process, will make up the basis for genotyping. These primers are restricted to the highly conserved genomic regions, most commonly found in the L1 gene. <b>D</b>) Multiple-primer DNA Pyrosequencing of an incorporated ID-tag. The diagrams depict the complementary sequence of the investigated probes -16 and -18. Marked in the figure is the ID-tag for each probe and the point of ligation where the probes circularized, incorporation of a dGTP, seen here as the complementary “C”. <b>E</b>) The in-house barcode chips here used to detect one HPV-16 positive, and one HPV-18 positive in human genomic DNA presence as seen with a positive rMIP in both chips.</p

Figure 3

<p>Probe selectivity. The figure depicts superimposed pyrosequencing (dispensation time vs. light signal intensity) diagrams for two probes following the MIP reactions, targeting HPV-45 (black) and HPV-59 (red), mimicking a double HPV infection. <b>A</b>) The initial DNA amount of each contributing plasmid was 100 ng and equal levels of sequencing intensities are seen. <b>B</b>) The DNA amount of HPV-45 plasmid remained at 100 ng, while HPV-59 plasmid was set at 10 ng resulting in ∼2-fold lower signal intensity. <b>C</b>) DNA amounts were set at 100 ng for HPV-45 plasmid and 1 nanogram for HPV-59 plasmid, which was observed as a ∼3-fold decrease in signal intensity. <b>D</b>) DNA amounts of 100 ng of HPV-45 plasmid and 100 pg of HPV-59 plasmid resulted in signal from HPV-45-probe without a measurable signal from the probe for HPV-59.</p

Figure 1

<p>Schematic overviews of molecular inversion probe technology. <b>A</b>) Synthetic oligonucleotide containing following four regions; i) H1 and H2: homology regions comprised of unique continuous 40–50 base pair fragments for target recognition ii) BARCODE: molecular barcode comprised of a 20 base pair DNA tag for target identification iii) U1 and U2: universal primer regions for inverted probe amplification, and iv) R: restriction site for probe linearization. <b>B</b>) Upon target recognition, a DNA polymerase fills the missing gap in between the juxtaposition of the probes' flanking ends, and through the activity of a DNA ligase the probe is circularized. In all cases the missing nucleotide is a “G”. <b>C</b>) Circular DNA enrichment through DNA degradation by enzymes Exonuclease I and III. <b>D</b>) Probe linearization restriction site cleavage. <b>E</b>) All reacted and inverted probes are amplified with universal primers, of which one is biotinylated for subsequent amplicon validation.</p

Figure 2

<p>Ligase-based probe construction for the PathogenMip Assay. Seen in the figure is the sequence for probe-16 prior to inversion. Two shorter fragments were synthesized and hybridized to a bridge complementary to the universal primer regions, common for all probes used in the assay. The coupling of the shorter fragments was mediated by polymerase and ligase activity to achieve maximum yield.</p