Left ventricular global transcriptional profiling in human end-stage dilated cardiomyopathy

AbstractWe employed ABI high-density oligonucleotide microarrays containing 31,700 sixty-mer probes (representing 27,868 annotated human genes) to determine differential gene expression in idiopathic dilated cardiomyopathy (DCM). We identified 626 up-regulated and 636 down-regulated genes in DCM compared to controls. Most significant changes occurred in the tricarboxylic acid cycle, angiogenesis, and apoptotic signaling pathways, among which 32 apoptosis- and 13 MAPK activity-related genes were altered. Inorganic cation transporter, catalytic activities, energy metabolism and electron transport-related processes were among the most critically influenced pathways. Among the up-regulated genes were HTRA1 (6.9-fold), PDCD8(AIFM1) (5.2) and PRDX2 (4.4) and the down-regulated genes were NR4A2 (4.8), MX1 (4.3), LGALS9 (4), IFNA13 (4), UNC5D (3.6) and HDAC2 (3) (p<0.05), all of which have no clearly defined cardiac-related function yet. Gene ontology and enrichment analysis also revealed significant alterations in mitochondrial oxidative phosphorylation, metabolism and Alzheimer's disease pathways. Concordance was also confirmed for a significant number of genes and pathways in an independent validation microarray dataset. Furthermore, verification by real-time RT-PCR showed a high degree of consistency with the microarray results. Our data demonstrate an association of DCM with alterations in various cellular events and multiple yet undeciphered genes that may contribute to heart muscle disease pathways

Renal Failure Associated with APECED and Terminal 4q Deletion: Evidence of Autoimmune Nephropathy

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disorder caused by mutations in the autoimmune regulator gene (AIRE). Terminal 4q deletion is also a rare cytogenetic abnormality that causes a variable syndrome of dysmorphic features, mental retardation, growth retardation, and heart and limb defects. We report a 12-year-old Saudi boy with mucocutaneous candidiasis, hypoparathyroidism, and adrenocortical failure consistent with APECED. In addition, he has dysmorphic facial features, growth retardation, and severe global developmental delay. Patient had late development of chronic renal failure. The blastogenesis revealed depressed lymphocytes' response to Candida albicans at 38% when compared to control. Chromosome analysis of the patient revealed 46,XY,del(4)(q33). FISH using a 4p/4q subtelomere DNA probe assay confirmed the deletion of qter subtelomere on chromosome 4. Parental chromosomes were normal. The deleted array was further defined using array CGH. AIRE full gene sequencing revealed a homozygous mutation namely 845_846insC. Renal biopsy revealed chronic interstitial nephritis with advanced fibrosis. In addition, there was mesangial deposition of C3, C1q, and IgM. This is, to the best of our knowledge, the first paper showing evidence of autoimmune nephropathy by renal immunofluorescence in a patient with APECED and terminal 4q deletion

p16INK4A Positively Regulates Cyclin D1 and E2F1 through Negative Control of AUF1

/pRB/E2F pathway, a key regulator of the critical G1 to S phase transition of the cell cycle, is universally disrupted in human cancer. However, the precise function of the different members of this pathway and their functional interplay are still not well defined. -dependent manner, and several of these genes are also members of the AUF1 and E2F1 regulons. We also present evidence that E2F1 mediates p16-dependent regulation of several pro- and anti-apoptotic proteins, and the consequent induction of spontaneous as well as doxorubicin-induced apoptosis. is also a modulator of transcription and apoptosis through controlling the expression of two major transcription regulators, AUF1 and E2F1

Phenotypical spectrum of cerebellar ataxia associated with a novel mutation in the CA8 gene, encoding carbonic anhydrase (CA) VIII

We define the neurological characteristics of familial cases from multiple branches of a large consanguineous family with cerebellar ataxia, mental retardation (MR), and dysequilibrium syndrome type 3 caused by a mutation in the recently cloned CA8 gene. The linkage analysis revealed a high logarithm of the odds (LOD) score region on 8q that harbors the CA8 in which a novel homozygous c.484G&gt;A (p.G162R) mutation was identified in all seven affected members. The patients had variable cerebellar ataxia and mild cognitive impairment without quadrupedal gait. The brain MRI showed variable cerebellar volume loss and ill-defined peritrigonal white matter abnormalities. The Fluorodeoxyglucose Positron Emission Tomography (FDG PET) revealed hypometabolic cerebellar hemispheres, temporal lobes, and mesial cortex. This report expands the neurological and radiological phenotype associated with CA8 mutations. CA8 involvement should be considered in the differential diagnosis of other genetically unresolved autosomal recessive cerebellar ataxias

Schematic representation of the p16-related regulation of AUF1,

<p>E2F1 and CyclinD1. See text for details.</p

List of genes under the control of both p16 and AUF1.

<p>*: The variation in the expression of these genes was less than 2 fold in microarray analysis data. However, the variation was significant when RT-PCR was used for assessment, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021111#pone-0021111-g001" target="_blank">figure 1D</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021111#pone-0021111-g007" target="_blank">figure 7</a>.</p

Involvement of ARE in the E2F1 3′UTR and response to AUF1 and p16.

<p>(A) Schematic diagram of the E2F1 3′UTR, ARE region sequences, and locations. (B) The E2F1 3′UTR sequences in different species (C) Sequences from the E2F1 3′UTR (<i>ARE regions 1 to 3</i>), IL-8 3′UTR (<i>ARE control</i>), and a control that lacks ARE were inserted in BamHI/XbaI sites in EGFP expression vector as shown. The Huh7 cell line (2.10⁴ cells per well) in 96-well black clear-bottom microplates were transfected with the different 3′UTR constructs. The reporter activity was assessed after 24 hr using BD bio-imaging apparatus and software. The non-ARE 3′UTR was used as control and its fluorescence activity was taken as 100%. Data are presented as Mean±SEM (n = 4) of % of the control. *** denote <i>p</i> values of <0.005 (student t- test) when compared to non-ARE control. (D) Huh7 cells (left panel) or U2OS (right panel) in 96-well microplates were co-transfected with siRNA against AUF1 or scrambled control (50 ng per well) and reporter constructs (25 ng per well) as indicated. Reporter activity was assessed at 48 hr post-transfection. Data (Mean±SEM, n = 4) were presented as % increase in reporter fluorescence due to AUF1 silencing when compared to the fluorescence in control-siRNA-treated cells. * and *** denote <i>p</i> <0.05 and <0.005, respectively (student t-test) when compared to non-ARE control. (E) U2OS and EH1 cells (2.10⁴ cells per well) were seeded in 96-well black clear-bottom microplates and then transfected with the different 3′UTR constructs. Reporter activity was assessed as described in (D). ANOVA was performed to compare between U2OS and EH1 data groups.</p

p16 controls the mRNA levels of <i>cyclin D1</i> and <i>E2F1</i> through AUF1.

<p>(A) HFSN1 cells were transfected with plasmids expressing either AUF1-siRNA (pSILENCER-AUF15) or control-siRNA (pSILENCER). 3 days after transfection total RNA was purified from both cells, and RT-PCR (top panel) as well as real time RT-PCR (histogram) using specific primers for the indicated genes were performed. Error bars indicate standard errors of 3 different experiments (B) RNAs bound to the AUF1 protein were isolated by immunoprecipitation from HFSN1 cells expressing either p16-siRNA or control-siRNA using anti-AUF1 antibody or anti-IgG (as control), and then target transcripts were amplified by RT-PCR visualized on Ethidium bromide stained 1% agarose gels. Amplification of the highly abundant GAPDH transcript, which bound IP materials at background levels, was used as loading control. (C) HFSN1 whole cell lysate was immunoprecipitated using the indicated antibodies and then used for immunostaining analysis. (D) Total RNA was purified from p16 proficient (HFSN1 and EH1) and p16-deficient (U2OS and HFSN1 expressing p16-siRNA) cells, expressing either <i>AUF1</i>-siRNA or control-siRNA. Transcripts for the indicated genes were detected by RT-PCR, and the corresponding products were visualized on ethidium bromide stained 1% agarose gels. The numbers below the bands indicate the corresponding expression levels. GAPDH was used as internal control. These experiments were repeated several times and representative ones are shown.</p

p16 modulates apoptosis through E2F1.

<p>(A) Western blots showing the expression of the indicated pro-and anti-apoptotic proteins in U2OS and EHI. (B) Western blots showing the expression of the indicated proteins in U2OS, U2OS cells stably transfected with plasmids encoding either scrambled sequence (ctl) or E2F1 (+). The numbers below the bands indicate the corresponding expression levels. (C) U2OS, EH1 and E2F1-expressing U2OS cells were either mock-treated or challenged with doxorubicin (2 µM) and then re-incubated for 72 hrs. Cells were then divided into two groups; one was used to analyze cell death by annexinV/PI flow cytometry. The numbers in the charts indicate the proportions of early and late apoptosis. (D) Histogram showing the proportions of apoptosis (early+late) induced by different doses of doxorubicin. The error bars represent standard deviation of three different experiments. (E) The second group of cells was used to assess the level of the indicated proteins by immunoblotting. (F) Graph showing the Bax/Bcl-2 ratio in the indicated cells after treatment with doxorubicin. Error bars represent standard deviation of at least three different experiments.</p