Unknown,Transcriptomics,Genomics,Proteomics

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A Gene Expression-based Blood Diagnostic for Symptomatic Transthyretin Amyloidosis Revealing Male and Female-specific Signatures

ABSTRACT: Early diagnosis of transthyretin (TTR) amyloid diseases remains challenging because of variable disease penetrance. Currently, patients must have an amyloid positive tissue biopsy to be eligible for disease modifying therapies. Early diagnosis is often difficult because the patient exhibits apparent symptoms of polyneuropathy or cardiomyopathy, but has a negative amyloid biopsy. Thus, there is a pressing need for more objective, quantitative diagnostics and biomarkers of TTR-aggregation-associated polyneuropathy and cardiomyopathy. This is especially true in the context of clinical trials demonstrating significant disease modifying effects, e.g. when the TTR tetramer stabilizer tafamidis was administered to familial amyloid polyneuropathy (FAP) patients early in the disease course. When asked if the findings of the tafamidis registration trial were âsufficiently robust to provide substantial evidence of efficacy for a surrogate endpoint that is reasonably likely to predict a clinical benefitâ the advisory committee said yes, but the FDA rejected the tetramer stabilization surrogate biomarker required for orphan tafamidis approvalâhence, acceptable biomarkers are badly needed. Herein, we explored whether peripheral blood cell mRNA expression profiles could differentiate symptomatic from asymptomatic V30M FAP patients, and if such a profile would normalize upon tafamidis treatment. We demonstrate that blood cell gene expression patterns reveal sex-independent as well as male and female specific inflammatory signatures in symptomatic FAP patients, but not in asymptomatic carriers, that normalize in FAP patients 6 months after tafamidis treatment. Thus these signatures have potential both as an early diagnostic and as a surrogate biomarker for measuring response to treatment in FAP patients. Study Subjects: Tafamidis-treated and untreated V30M FAP patients, asymptomatic V30M carriers, and healthy, age- and sex-matched controls without TTR mutations had whole blood drawn. RNA was extracted from a total of 309 peripheral blood samples collected in PAXgene tubes (Qiagen). RNA Extraction and Microarray Processing: RNA was extracted with the PAXgene RNA Whole Blood Kit. Each RNA sample (100ng) was amplified and labeled with the Ambion WT Express Kit and hybridized to Affymetrix HuGene 1.1 ST Arrays using the Affymetrix GeneTitan Multi-Channel (MC) platform. Normalized signals were generated using Robust Multichip Average (RMA) in Partek Genomics Suite software version 6.6. Probesets on the DNA microarrays with low expressed signals were determined using a kernel density plot, and signals

ORGANISM(S): Homo sapiens

SUBMITTER: Sunil Kurian

PROVIDER: E-GEOD-67784 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Immunosuppression is needed in HLA identical sibling renal transplantation. We conducted a tolerance trial in this patient cohort using Alemtuzumab induction, donor hematopoietic stem cells, tacrolimus/mycophenolate immunosuppression converted to sirolimus, planning complete drug withdrawal by 24 months post-transplantation. After an additional 12 months with no immunosuppression, normal biopsies and renal function, recipients were considered tolerant. Twenty recipients were enrolled. Of the first 10 (>36 months post-transplantation), 5 had immunosuppression successfully withdrawn for 16-36 months (tolerant), 2 had disease recurrence and 3 had subclinical rejection in protocol biopsies (non-tolerant). Microchimerism disappeared after 1 year, and CD4+CD25highCD127-FOXP3+ T cells and CD19+IgD/M+CD27- B cells increased to 5 years post-transplantation in both groups, whereas immune/inflammatory gene expression pathways in the peripheral blood and urine were differentially downregulated in tolerant compared to non-tolerant recipients. Therefore, in this HLA identical renal transplant tolerance trial, absent chimerism, Treg and Breg immunophenotypes were indistinguishable between tolerant and non-tolerant recipients, but global genomic changes indicating immunomodulation were observed only in tolerant recipients. A total of 46 PBMC samples representing blood draws from four time points in the first 9 recipients were processed for microarray analysis (The Scripps Research Institute, La Jolla, CA). The analyzed time points were: immediately pre-operatively in the absence of immunosuppression (n=9); post-operatively at 1 year (n=8, range 11-13 months); at 2 years (n=12, range 18-25 months); >3 years (n=17, range 32-48 months). (At year 2 and at > 3 years, repeated samples were obtained from individual subjects, and at one year, one subject had a technically unsatisfactory sample.) To discount the effects of immunosuppression on gene expression, microarray data were included on whole blood from 18 healthy human subjects (controls: GSE40586; NCBI Gene Expression Omnibus [GEO] repository).

Project description:Rationale: Interstitial fibrosis and tubular atrophy (IFTA) is found in ~25% of 1-year biopsies post-transplant(1, 2). It correlates with decreased graft survival when histological evidence of inflammation is present¬.(3-5) Identifying the etiology of IFTA is important because longterm graft survival has not changed as expected given improved therapies and a dramatically reduced incidence of acute rejection.(6-8) Methods: Gene expression profiles of 234 samples were obtained with matching clinical and outcome data (7 transplant centers). 81 IFTA samples were divided into subphenotypes by the degree of inflammation on histology: IFTA with acute rejection (AR), IFTA with inflammation and IFTA without inflammation. Samples with AR (n=54) and normally functioning transplants (TX; n=99) were used in comparisons. Conclusions: Gene expression profiling of all IFTA phenotypes were strongly enriched for cAR gene dysregulation pathways, including IFTA samples without histological evidence of inflammation. Thus, by molecular profiling we demonstrate that most IFTA samples have ongoing immune-mediated injury or chronic rejection that is more sensitively detected by gene expression profiling. We also found that the relative expression of AR-affiliated genes correlated with future graft loss in IFTA samples without inflammation. We conclude that undetected and/or undertreated immune rejection is leading to IFTA and graft failure. RNA was extracted from biopsy samples using the RNEasy kit (Qiagen), biotinylated cRNA prepared using Ambion MessageAmp Biotin II and hybridized to Affymetrix HG U133 Plus PM peg arrays (http://affymetrix.com/index.affx). Probe intensity data were log2–transformed and normalized using a custom-designed frozen Robust Multichip Average (fRMA). Low-variance and low signal probes were filtered according to industry standards, resulting in a filtered gene list with 17,564 transcripts. This dataset is part of the TransQST collection.

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A Gene Expression-based Blood Diagnostic for Symptomatic Transthyretin Amyloidosis Revealing Male and Female-specific Signatures

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