Dmitri V. Gnatenko

Transcript profiling of human platelets using microarray and serial analysis of gene expression

Platelets are anucleated cells that are generated from megakaryocytes via thrombopoiesis. They lack genomic DNA but have a pool of individual mRNA transcripts. Taken together, these mRNAs constitute a platelet transcriptome. Platelets have a unique and reproducible transcript profile, which includes approximately 1,600-3,000 individual transcripts. In this chapter, we will focus on platelet purification and on transcript profiling using an Affymetrix microarray platform and serial analysis of gene expression (SAGE). Platelet purification is described in detail. Large-scale platelet purification schema is designed to purify platelets from apheresis platelet bags (approximately 3-5 x 10(11) platelets/bag). Modification of this schema --small-scale platelet purification--is designed to isolate platelets from 20 ml of peripheral blood. This chapter provides detailed protocols for microarray and SAGE transcript profiling. We also discuss peculiarities of platelet purification, RNA isolation, and transcript profiling.

Aristolochic acid-associated urothelial cancer in Taiwan

Aristolochic acid, a potent human carcinogen produced by Aristolochia plants, is associated with urothelial carcinoma of the upper urinary tract (UUC). Following metabolic activation, aristolochic acid reacts with DNA to form aristolactam (AL)-DNA adducts. These lesions concentrate in the renal cortex, where they serve as a sensitive and specific biomarker of exposure, and are found also in the urothelium, where they give rise to a unique mutational signature in the TP53 tumor-suppressor gene. Using AL-DNA adducts and TP53 mutation spectra as biomarkers, we conducted a molecular epidemiologic study of UUC in Taiwan, where the incidence of UUC is the highest reported anywhere in the world and where Aristolochia herbal remedies have been used extensively for many years. Our study involves 151 UUC patients, with 25 patients with renal cell carcinomas serving as a control group. The TP53 mutational signature in patients with UUC, dominated by otherwise rare A:T to T:A transversions, is identical to that observed in UUC associated with Balkan endemic nephropathy, an environmental disease. Prominent TP53 mutational hotspots include the adenine bases of 5′AG (acceptor) splice sites located almost exclusively on the nontranscribed strand. A:T to T:A mutations also were detected at activating positions in the FGFR3 and HRAS oncogenes. AL-DNA adducts were present in the renal cortex of 83% of patients with A:T to T:A mutations in TP53, FGFR3, or HRAS. We conclude that exposure to aristolochic acid contributes significantly to the incidence of UUC in Taiwan, a finding with significant implications for global public health.

The platelet proteome.

Purpose of reviewThe proteome is the pool of proteins expressed at a given time and circumstance. The word ‘proteomics’ summarizes several technologies for visualization, quantitation and identification of these proteins. Recent advances in these techniques are helping to elucidate platelet processes which are relevant to bleeding and clotting disorders, transfusion medicine and regulation of angiogenesis. Recent findingsOver 1100 platelet proteins have been identified using proteomic techniques. Various subproteomes have been characterized, including platelet releasates (the ‘secretome’), alpha and dense granules, membrane and cytoskeletal proteins, platelet-derived microparticles, and the platelet ‘phosphoproteome’. Proteomic data about platelets have become increasingly available in integrated databases. SummaryProteomic experiments in resting and activated platelets have identified novel signaling pathways and secreted proteins which may represent therapeutic targets, as well as potential cancer biomarkers.

Class prediction models of thrombocytosis using genetic biomarkers

Criteria for distinguishing among etiologies of thrombocytosis are limited in their capacity to delineate clonal (essential thrombocythemia [ET]) from nonclonal (reactive thrombocytosis [RT]) etiologies. We studied platelet transcript profiles of 126 subjects (48 controls, 38 RT, 40 ET [24 contained the JAK2V(617)F mutation]) to identify transcript subsets that segregated phenotypes. Cross-platform consistency was validated using quantitative real-time polymerase chain reaction (RT-PCR). Class prediction algorithms were developed to assign phenotypic class between the thrombocytosis cohorts, and by JAK2 genotype. Sex differences were rare in normal and ET cohorts (< 1% of genes) but were male-skewed for approximately 3% of RT genes. An 11-biomarker gene subset using the microarray data discriminated among the 3 cohorts with 86.3% accuracy, with 93.6% accuracy in 2-way class prediction (ET vs RT). Subsequent quantitative RT-PCR analysis established that these biomarkers were 87.1% accurate in prospective classification of a new cohort. A 4-biomarker gene subset predicted JAK2 wild-type ET in more than 85% patient samples using either microarray or RT-PCR profiling, with lower predictive capacity in JAK2V(617)F mutant ET patients. These results establish that distinct genetic biomarker subsets can predict thrombocytosis class using routine phlebotomy.

Platelets express steroidogenic 17β-hydroxysteroid dehydrogenases Distinct profiles predict the essential thrombocythemic phenotype

Human blood platelets have important, regulatory functions in diverse hemostatic and pathological disorders, including vascular remodeling, inflammation, and wound repair. Microarray analysis was used to study the molecular basis of essential thrombocythemia, a myeloproliferative disorder with quantitative and qualitative platelet defects associated with cardiovascular and thrombohemorrhagic symptoms, not infrequently neurological. A platelet-expressed gene (HSD17B3) encoding type 3 17beta-hydroxysteroid dehydrogenase (previously characterized as a testis-specific enzyme catalyzing the final step in gonadal synthesis of testosterone) was selectively down-regulated in ET platelets, with reciprocal induction of the type 12 enzyme (HSD17B12). Functional 17beta-HSD3 activity corresponding to approximately 10% of that found in murine testis was demonstrated in normal platelets. The induction of HSD17B12 in ET platelets was unassociated with a concomitant increase in androgen biosynthesis, suggesting distinct functions and/or substrate specificities of the types 3 and 12 enzymes. Application of a molecular assay distinguished ET from normal platelets in 20 consecutive patients (p < 0.0001). These data provide the first evidence that distinct subtypes of steroidogenic 17beta-HSDs are functionally present in human blood platelets, and that the expression patterns of HSD17B3 and HSD17B12 are associated with an uncommon platelet disorder manifest by quantitative and qualitative platelet defects.

Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to haemophilia A gene therapy.

Adeno‐associated virus (AAV) is a single‐stranded DNA parvovirus displaying several attractive features applicable to haemophilia A gene therapy, including non‐pathogenicity and potential for long‐term transgene expression from either integrated or episomal forms. We have generated and characterized two B‐domain‐deleted (BDD) fVIII mutants, deleted in residues Phe756 to Ile1679 (fVIIIΔ756–1679) or Thr761 to Asn1639 (fVIIIΔ761–1639). [35S]metabolic labelling experiments and immunoprecipitation demonstrated intact BDD‐fVIII of the predicted size in both lysates and supernatants (Mr ~ 155 kD for fVIIIΔ756–1679 and Mr ~ 160 kD for fVIIIΔ761–1639) after transient transfection into COS‐1 cells. Functional fVIII quantification appeared maximal using fVIIIΔ761–1639, as evaluated by Coatest and clotting assay (98 ± 20 mU/ml/1×106 cells and 118 ± 29 mU/ml/1×106 respectively, collection period 48 h). To bypass potential size limitations of rAAV/fVIII vectors, we expressed fVIIIΔ761–1639 using a minimal human 243 bp cellular small nuclear RNA (pHU1‐1) promoter, and demonstrated fVIII activity ~30% of that seen using CMV promoter. This BDD‐fVIII (rAAV(pHU1‐1) fVIIIΔ761–1639) can be efficiently encapsidated into rAAV (107% of wild type), as demonstrated by replication centre and DNAase sensitivity assays. A concentrated recombinant viral stock resulted in readily detectable factor VIII expression in COS‐1 cells using a maximally‐achievable MOI ~35 (Coatest 15 mU/ml; clotting assay 25 ± 2.0 mU/ml/1×106 cells). These data provide the first evidence that rAAV is an adaptable virus for fVIII delivery, and given the recent progress using this virus for factor IX delivery in vivo, provide a new approach towards definitive treatment of haemophilia A.

IQGAP1 and IQGAP2 are Reciprocally Altered in Hepatocellular Carcinoma

BackgroundIQGAP1 and IQGAP2 are homologous members of the IQGAP family of scaffold proteins. Accumulating evidence implicates IQGAPs in tumorigenesis. We recently reported that IQGAP2 deficiency leads to the development of hepatocellular carcinoma (HCC) in mice. In the current study we extend these findings, and investigate IQGAP1 and IQGAP2 expression in human HCC.MethodsIQGAP1 and IQGAP2 protein expression was assessed by Western blotting and immunohistochemistry. IQGAP mRNA was measured by quantitative RT-PCR. The methylation status of the Iqgap2 promoter was determined by pyrosequencing of bisulfite-treated genomic DNA.ResultsIQGAP1 and IQGAP2 expression was reciprocally altered in 6/6 liver cancer cell lines. Similarly, immunohistochemical staining of 82 HCC samples showed that IQGAP2 protein expression was reduced in 64/82 (78.0%), while IQGAP1 was present in 69/82 (84.1%). No IQGAP1 staining was detected in 23/28 (82.1%) normal livers, 4/4 (100.0%) hepatic adenomas and 23/23 (100.0%) cirrhosis cases, while IQGAP2 was increased in 22/28 (78.6%), 4/4 (100.0%) and 23/23 (100.0%), respectively. Although the Iqgap2 promoter was not hypermethylated in HCC at any of the 25 CpG sites studied (N = 17), IQGAP2 mRNA levels were significantly lower in HCC specimens (N = 23) than normal livers (N = 6).ConclusionsWe conclude that increased IQGAP1 and/or decreased IQGAP2 contribute to the pathogenesis of human HCC. Furthermore, downregulation of IQGAP2 in HCC occurs independently of hypermethylation of the Iqgap2 promoter. Immunostaining of IQGAP1 and IQGAP2 may aid in the diagnosis of HCC, and their pharmacologic modulation may represent a novel therapeutic strategy for the treatment of liver cancer.

In vivo gene transfer into rat arterial walls with novel adeno-associated virus vectors

PURPOSE We studied the ability of recombinant adeno-associated virus (rAAV) vectors to achieve gene transfer in vivo to intact rat carotid arteries. METHODS Isolated segments of uninjured rat carotid arteries were incubated with (1) rAAV vectors that expressed a beta-galactosidase gene, (2) a related vector with no promoter, or (3) a normal saline solution. Gene transfer was evaluated with in situ polymerase chain reaction (PCR). Transgene expression was assessed at intervals that ranged from 24 hours to 2 months by measurement of beta-galactosidase activity and protein mass in tissue extracts with fluorometric and enzyme-linked immunosorbent assays, respectively. Dose dependence of expression was determined for virus concentrations that ranged from 5 x 10(4) to 5 x 10(5) infectious units (iu)/ml. RESULTS Light microscopic analysis of in situ PCR-stained histologic sections of transduced vessel walls showed approximately 90% of intimal and medial cell nuclei contained the beta-galactosidase gene, compared with none in control arteries. In vivo beta-galactosidase expression was (1) highest 24 hours after gene transfer, (2) elevated for 1 month, and (3) dose responsive. CONCLUSIONS rAAV vectors can mediate focal gene transfer into the intact rat carotid artery with detectable levels of transgene expression for 1 month and are potentially useful agents for in vivo gene transfer into intact arteries.

Systematic analysis of microRNA fingerprints in thrombocythemic platelets using integrated platforms

Posttranscriptional and translational controls mediated by microRNAs (miRNA) regulate diverse biologic processes. We dissected regulatory effects of miRNAs relevant to megakaryocytopoiesis and platelet biology by analyzing expression patterns in 79 subjects with thrombocytosis and controls, and integrated data with transcriptomic and proteomic platforms. We validated a unique 21-miRNA genetic fingerprint associated with thrombocytosis, and demonstrated that a 3-member subset defines essential thrombocythemia (ET). The genetic signature includes functional guide and passenger strands of the previously uncharacterized miR 490 (5p and 3p), which displayed restricted, low-level expression in megakaryocytes/platelets (compared with leukocytes), and aberrant expression during thrombocytosis, most profound in ET. Overexpression of miR 490 in a bilineage differentiation model of megakaryocyte/erythroid progenitor formation was insufficient for hematopoietic colony differentiation and/or lineage specification. Integration of transcriptomic and mass spectrometric datasets with functional reporter assays identified dishevelled associated activator of morphogenesis 1 (DAAM1) as a miR 490 5p protein target demonstrating decreased expression in ET platelets, putatively by translational control (and not by mRNA target degradation). Our data define a dysregulated miRNA fingerprint in thrombocytosis and support a developmentally restricted function of miR 490 (and its putative DAAM1 target) to conditions associated with exaggerated megakaryocytopoiesis and/or proplatelet formation.

Computationally designed adeno-associated virus (AAV) Rep 78 is efficiently maintained within an adenovirus vector

Adeno-associated virus (AAV) is a single-stranded parvovirus retaining the unique capacity for site-specific integration into a transcriptionally silent region of the human genome, a characteristic requiring the functional properties of the Rep 78/68 polypeptide in conjunction with AAV terminal repeat integrating elements. Previous strategies designed to assemble these genetic elements into adenoviral (Ad) backbones have been limited by the general intolerability of AAV Rep sequences, prompting us to computationally reengineer the Rep gene by using synonymous codon pair recoding. Rep mutants generated by using de novo genome synthesis maintained the polypeptide sequence and endonuclease properties of Rep 78, while dramatically enhancing Ad replication and viral titer yields, characteristics indistinguishable from adenovirus lacking coexpressed Rep. Parallel approaches using domain swaps encompassing WT and recoded genomic segments, coupled with iterative computational algorithms, collectively established that 3′ cis-acting Rep genetic elements (and not the Rep 78 polypeptide) retain dominant-acting sequences inhibiting Ad replication. These data provide insights into the molecular relationships of AAV Rep and Ad replication, while expanding the applicability of synonymous codon pair reengineering as a strategy to effect phenotypic endpoints.