High quality and quantity Genome-wide germline genotypes from FFPE normal tissue

<p>Abstract</p> <p>Background</p> <p>Although collections of formalin fixed paraffin embedded (FFPE) samples exist, sometimes representing decades of stored samples, they have not typically been utilized to their full potential. Normal tissue from such samples would be extremely valuable for generation of genotype data for individuals who cannot otherwise provide a DNA sample.</p> <p>Findings</p> <p>We extracted DNA from normal tissue identified in FFPE tissue blocks from prostate surgery and obtained complete genome wide genotype data for over 500,000 SNP markers for these samples, and for DNA extracted from whole blood for 2 of the cases, for comparison.</p> <p>Four of the five FFPE samples of varying age and amount of tissue had identifiable normal tissue. We obtained good quality genotype data for between 89 and 99% of all SNP markers for the 4 samples from FFPE. Concordance rates of over 99% were observed for the 2 samples with DNA from both FFPE and from whole blood.</p> <p>Conclusions</p> <p>DNA extracted from normal FFPE tissue provides excellent quality and quantity genome-wide genotyping data representing germline DNA, sufficient for both linkage and association analyses. This allows genetic analysis of informative individuals who are no longer available for sampling in genetic studies.</p

MS

thesisHematopoietic stem cell and progenitor populations (HSPG) within the bone marrow compartment consist of multiple groups of cell that vary in function when transplanted. The subsets of HSPG are beginning to be separated to define their engraftment characteristics, with the aim to improve current transplant management for patients with malignant disease. In this study, methods were developed to identify hemoglobin (Hbb) and glucose phosphate isomerase (Gpi) allelic variants in lysates of mouse peripheral blood cells, in order to facilitate evaluation of erythrocyte and platelet engraftment after bone marrow transplantation. The long-term repopulation assay (LTRA) was then used to demonstrate the efficacy of these techniques to distinguish donor-derived engraftment of bone marrow transplanted into lethally irradiated mice. Donor-derived Hbb[d] isoforms in recipient hemolysates, when pretreated with a cystamine lysing agent, could be separated by agarose gel electrophoresis, then stained and quantitated by densitometric analysis. A detection limit of 5% was established when mixtures to known hemolysates were assayed. As a second approach, transplant hemolysates were evaluated by HPLC. This cost-effective assay was demonstrated to have a direct correlation with the electrophoresis assay (r=0.998). Agarose gel electrophoresis and a specific stain for Gpi distinguished the platelet variants, Gpi-1a and Gpi-1b, with a limit of detection corresponding to 100,000 total platelets. Two contamination assays were performed to evaluate the contribution of residual leukocytes and erythrocytes in platelet preparations to the platelet Gpi signals. The results indicated that the separation protocol utilized in these studies was sufficient to exclude contaminating leukocytes and erythrocytes from platelet preparations. Platelets were also stained with monoclonal antibodies detecting allelic variants of Ly5 and analyzed by flow cytometry to detect post-transplant chimerism. The absence of fluorescence indicated a down-regulation of Ly5 during platelet maturation precluding the use of this method. The techniques developed in this project successfully measured erythrocyte and platelet engraftment kinetics using the LTRA. One million transplanted morrow cells repopulated lethally irradiated mice with multi-lineage progeny and sustained donor-derived hematopoiesis was observed one year post-transplant. In conclusion, the assays developed in this study can successfully evaluate erythrocyte and platelet repopulation of transplanted HSPG in mouse models. Defining HSPG will establish a better understanding of bone marrow transplantation in humans

Mepolizumab as a corticosteroid-sparing agent in lymphocytic variant hypereosinophilic syndrome

Background: Mepolizumab, a monoclonal anti-IL-5 antibody, is an effective corticosteroid-sparing agent for patients with Fip1-like 1/platelet-derived growth factor receptor α fusion (F/P)-negative hypereosinophilic syndrome (HES). Lymphocytic variant hypereosinophilic syndrome (L-HES) is characterized by marked overproduction of IL-5 by dysregulated T cells. Objective: To determine whether patients with L-HES respond to mepolizumab in terms of corticosteroid tapering and eosinophil depletion to the same extent as corticosteroid-responsive F/P-negative patients with HES and a normal T-cell profile. Methods: Patients enrolled in the mepolizumab trial were evaluated for L-HES on the basis of T-cell phenotyping and T-cell receptor gene rearrangement patterns, and their serum thymus-and-activation-regulated chemokine (TARC) levels were measured. Response to treatment was compared in patient subgroups based on results of these analyses. Results: Lymphocytic variant HES was diagnosed in 13 of 63 patients with HES with complete T-cell assessments. The ability to taper corticosteroids on mepolizumab was similar in patients with L-HES and those with a normal T-cell profile, although a lower proportion of patients with L-HES maintained eosinophil levels below 600/μL. Increased serum TARC levels (>1000 pg/mL) had no significant impact on the ability to reduce corticosteroid doses, but a lower proportion of patients with elevated TARC achieved eosinophil control on mepolizumab. Conclusion: Mepolizumab is an effective corticosteroid-sparing agent for patients with L-HES. In some cases however, eosinophil levels remain above 600/μL, suggesting incomplete neutralization of overproduced IL-5 or involvement of other eosinophilopoietic factors. © 2010 American Academy of Allergy, Asthma & Immunology.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

EGO, a novel, noncoding RNA gene, regulates eosinophil granule protein transcript expression

Gene expression profiling of early eosinophil development shows increased transcript levels of proinflammatory cytokines, chemokines, transcription factors, and a novel gene, EGO (eosinophil granule ontogeny). EGO is nested within an intron of the inositol triphosphate receptor type 1 (ITPR1) gene and is conserved at the nucleotide level; however, the largest open reading frame (ORF) is 86 amino acids. Sucrose density gradients show that EGO is not associated with ribosomes and therefore is a noncoding RNA (ncRNA). EGO transcript levels rapidly increase following interleukin-5 (IL-5) stimulation of CD34+ hematopoietic progenitors. EGO RNA also is highly expressed in human bone marrow and in mature eosinophils. RNA silencing of EGO results in decreased major basic protein (MBP) and eosinophil derived neurotoxin (EDN) mRNA expression in developing CD34+ hematopoietic progenitors in vitro and in a CD34+ cell line model. Therefore, EGO is a novel ncRNA gene expressed during eosinophil development and is necessary for normal MBP and EDN transcript expression