Project description:Twin studies are consistent with substantial heritability in anorexia nervosa (AN), although detecting and confirming individual risk alleles has been difficult, due to small effects of single alleles and limited samples in prior studies. Methods: 1105 AN DNA samples obtained in a multisite study and 3733 pediatric patients of normal body weight were genotyped using the Illumina 610Q chip at The Center for Applied Genomics at The Children's Hospital of Philadelphia. Cases and controls were of European ancestry. Single nucleotide polymorphism (SNP) genotypes were subjected to quality control procedures, including elimination of SNPs with: call rates < 95%, minor allele frequency < 5%, and if Hardy-Weinberg equilibrium testing yielded p < 0.0001. Samples were removed if not of European ancestry or if showing cryptic relatedness. Eight AN DNA samples were deleted for low call rate, and 51 AN samples were deleted for cryptic relatedness. The statistical program, PLINK, was used to calculate a P value for each SNP passing quality control using the Cochran-Armitage trend test. The PennCNV program was used to define copy number variation (CNV). Results: We performed a genome-wide association study (GWAS) using with 1033 AN cases and 3733 controls (genomic inflation factor = 1.08). There were 11 SNPs with P<1X10-5 [(rsID, chromosome location, nearest gene, distance to nearest gene (base-pair), P value) rs6959888, 7q21.13, ZNF804B, 0, 1.63E-06; rs17725255, 20p11.23, CSRP2BP, 39396, 1.72E-06; rs10494067, 1p13.3, NTNG1, 0, 5.83E-06; rs2383378, 14q12, AKAP6, 0, 6.41E-06; rs410644, 5q14.1, SSBP2, 40462, 6.74E-06; rs4479806, 5p14.1, CDH9, 156926, 7.79E-06; rs957788, 13q33.3, FAM155A, 0, 8.11E-06; rs830998, 2q31.1, LRP2, 0, 8.68E-06; rs6782029, 3p25.3, VGLL4, 0, 9.04E-06; rs512089, 9p21.3, ELAVL2, 47984, 9.85E-06; rs3808986, 11q24.3, APLP2, 29217, 9.92E-6]. CNV analyses were performed on 1015 AN cases and 3532 controls. Overall frequencies of common or rare CNVs were similar between cases and controls. Specific rare and large (>500 kb, <1%) CNVs of interest included deletion of a large (~1.4Mbp) region of chr13q12 found in 2 AN samples and at a 10 fold lower frequency in a large sample (72,918) of subjects containing the genes SGCG, SACS, TNFRSF19, MIPEP, PCOTH, C1QTNF9B, SPATA13, MIR2276, and C1QTNF9, and an overlapping deletion and a duplication observed in 3 AN samples overlapping the CNTN6 and CNTN4 genes. Partially overlapping ~ 30 kb duplications were seen in 12 AN cases and in 1 control at the CDC5L gene on chromosome 6. Partially overlapping deletion of the 3' half of the PSTPIP1 gene was seen in 5 AN cases and no controls. Finally, a ~ 60 kb deletion of the PLEC1 gene was observed in 5 AN cases and in none of 3532 controls. Discussion: This is the first GWAS of AN. These preliminary results are consistent with absence of a major gene effect in AN. These results are consistent with genetic risk conveyed by multiple common alleles of small effect, as well as some uncommon CNVs of potentially larger effect. None of the implicated genes has a clear role in eating behavior. It must be emphasized that these results are preliminary and an additional ~ 400 AN cases will be analyzed as time and resources permit. These results require confirmation through study of independent populations of AN women.

Project description:This experiment contains the RNA-Seq samples only. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5 % success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80 % of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.

Project description:In recent years, the use of FFPE tissue in gene expression microarray (GEM) studies has become a topic of increased interest, because pathology departments worldwide contain an invaluable source of biological disease-specific FFPE- tissue material. Thus far, some published studies on FFPE tissue consider the processed tissue amenable, whereas other studies assert that the tissue is not useful in GEM. In general, pancreatic tissue is known to contain large amounts of nucleases, leading to high turnover rates and degradation of RNA in the tissue. This characteristic, in combination with FFPE processing time and its effect on the tissue, does not make FFPE pancreatic tissue the obvious choice for GEM. The basis for using FFPE pancreatic tissue in RNA-based assays seems suboptimal. Previous GEM studies have used different FFPE tissue types, but GEM studies on FFPE pancreatic tissue have not been published. In most studies, the tissue was specially processed for use with GEMs. Here we demonstrate the usefulness of randomly archived FFPE pancreatic tissues for GEMs. For this purpose we included FFPE pancreatic tissue from patients with congenital hyperinsulinism or insulinoma; In these patients, pancreas resection is performed when medical treatment is not adequate to prevent hyperinsulinemic hypoglycemia or when patients do not respond to medical treatment. Although ribonuclease-rich, we obtained biologically relevant and disease-specific, significant genes; cancer-related genes and genes involved in a) the regulation of insulin secretion and synthesis, b) amino acid metabolism, and c) calcium ion homeostasis. This application may extend the possibilities of gene expression studies to many tissue types, especially in rare diseases, for which fresh frozen tissue is not readily available. A total of 15 samples were included in this study using a 58K chip. 29,134 human 60mer oligonucleotide targets were arrayed on the chips in duplicates. Each chip is hybridized with sample material (labelled with Cy5) from one patient and reference material (labelled with Cy3). Reference material contains pooled RNA from four different cancer cell lines; HeLa (cervical epithelium), SK-BR-3 (mammary gland), HT29 (colon) and A431 (skin) cells. Seven patients with congenital hyperinsulinism (five FFPE and two frozen samples), one patient with insulinoma and five controls were included in the study. Inclusion criteria for controls was that their cause of death was not pancreas-related. Samples were obtained from archived FFPE pancreatic material. The insulinoma sample was included in the study, because the biopsy was originally divided with one part being frozen and the other part being routinely formalin-fixed and paraffin-embedded. In the study we therefore include a frozen insulinoma sample and a FFPE insulinoma sample that was processed in duplicate.

Project description:We performed a biological and molecular characterization of the novel human multiple myeloma cell line CMA03/06, an IL-6-independent variant of CMA03 cell line previously established in our Institution. We showed that the CMA03/06 cells grows in the absence of IL-6 with a doubling time comparable to CMA03; the addition of IL-6 to the culture medium or co-culture with multipotent mesenchymal stromal cells does not induce an increase in proliferation rate. Interestingly, we provided evidence that IL-6 independence of CMA03/06 cells is not a consequence of the development of an autocrine IL-6 loop, even though the cells maintain the IL-6 signaling pathway responsiveness as demonstrated by STAT1 and STAT3 induction by IL-6. A slight constitutive activation of STAT3 has been observed in CMA03/06 cell line; however STAT3 silencing in CMA03/06 cells did not affect their viability and proliferation suggesting that this pathway is not the only responsible for the IL-6 independency of CMA03/06 cell line. The new cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA03 cells, whereas an increased induction of apoptosis was observed in CMA03/06 cell line after Bortezomib treatment which may suggest the involvement NF-kB pathway in the IL-6 independent growth and survival of CMA03/06 cells. Finally, global gene expression profiling analysis allowed the identification of a list of 308 modulated genes in CMA03/06 versus CMA03 cells, many of which particularly relevant for MM biology. Overall, the novel CMA03/06 cell line may represent a suitable model for studies investigating molecular mechanisms involved in clonal evolution towards IL-6 and/or stroma-independent growth and survival of myeloma cells. This series of microarray experiments contains the gene expression profiles of 3 independent replicas of CMA03 grown in in normal culture conditions and in absence of IL-6, respectively, and 3 independent replicas of CMA03/06 in normal culture conditions. Cell pellets were resuspended in TRIzol Reagent (Life Technologies, Inc., Rockville, MD, USA), total RNA was then purified using the RNeasy® total RNA Isolation Kit (Qiagen, Valencia, CA).5.5 micrograms of single-stranded DNA target obtained from 100 ng of purified total RNA was fragmented and then labeled using the WT Terminal Labeling Kit according to the standard Affymetrix protocol (GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual).The fragmented labeled single-stranded DNA target was hybridized for 16 hr 30 minutes at 45°C on GeneChip® Gene 1.0 ST array according to the standard Affymetrix protocol. The arrays were washed and stained in the Affymetrix Fluidics Station 450.

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EGAS00001002124-sc-20180606 - samples

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