The predictive value of PNH clones, 6p CN-LOH, and clonal TCR gene rearrangement for aplastic anemia diagnosis.

Acquired aplastic anemia (AA) is a life-threatening bone marrow aplasia caused by the autoimmune destruction of hematopoietic stem and progenitor cells. There are no existing diagnostic tests that definitively establish AA, and diagnosis is currently made via systematic exclusion of various alternative etiologies, including inherited bone marrow failure syndromes (IBMFSs). The exclusion of IBMFSs, which requires syndrome-specific functional and genetic testing, can substantially delay treatment. AA and IBMFSs can have mimicking clinical presentations, and their distinction has significant implications for treatment and family planning, making accurate and prompt diagnosis imperative to optimal patient outcomes. We hypothesized that AA could be distinguished from IBMFSs using 3 laboratory findings specific to the autoimmune pathogenesis of AA: paroxysmal nocturnal hemoglobinuria (PNH) clones, copy-number-neutral loss of heterozygosity in chromosome arm 6p (6p CN-LOH), and clonal T-cell receptor (TCR) γ gene (TRG) rearrangement. To test our hypothesis, we determined the prevalence of PNH, acquired 6p CN-LOH, and clonal TRG rearrangement in 454 consecutive pediatric and adult patients diagnosed with AA, IBMFSs, and other hematologic diseases. Our results indicated that PNH and acquired 6p CN-LOH clones encompassing HLA genes have ∽100% positive predictive value for AA, and they can facilitate diagnosis in approximately one-half of AA patients. In contrast, clonal TRG rearrangement is not specific for AA. Our analysis demonstrates that PNH and 6p CN-LOH clones effectively distinguish AA from IBMFSs, and both measures should be incorporated early in the diagnostic evaluation of suspected AA using the included Bayesian nomogram to inform clinical application.

An expanded immunohistochemical profile of osteoclast-rich undifferentiated carcinoma of the urinary tract.

Osteoclast-rich undifferentiated carcinoma of the urinary tract (ORUCUT) is a rare tumor composed of ovoid to spindle-shaped mononuclear cells with intermixed or focally clustered osteoclast-like giant cells. Previous studies have demonstrated that the mononuclear cells are neoplastic cells, while the giant cells are reactive cells of histiocytic lineage. The association between these tumors and classic urothelial carcinomas suggest that the mononuclear cells are derived from urothelial cells; however, no studies have been conducted to assess the immunohistochemical profile of ORUCUT with more specific urothelial markers. This study identified 21 cases of ORUCUT and performed immunohistochemistry for GATA3, uroplakin II, and thrombomodulin along with pancytokeratin (AE1/3) on all cases. Mononuclear cells stained positive in 20 cases (95%) for GATA3 and 19 cases (90%) for thrombomodulin. None of the mononuclear cells were positive for uroplakin II and only three cases showed focal positivity for AE1/3. The osteoclast-like giant cells were negative for GATA3, uroplakin II, thrombomodulin, and AE1/3, providing additional support to a reactive origin for these cells. Additionally, 15 cases (71%) were associated with either in situ or invasive urothelial carcinoma. This study provides an expanded immunohistochemical profile for ORUCUT and more definitively supports a urothelial origin for this tumor.

The Influenza A PB1-F2 and N40 Start Codons Are Contained within an RNA Pseudoknot.

Influenza A is a negative-sense RNA virus with an eight-segment genome. Some segments encode more than one polypeptide product, but how the virus accesses alternate internal open reading frames (ORFs) is not completely understood. In segment 2, ribosomal scanning produces two internal ORFs, PB1-F2 and N40. Here, chemical mapping reveals a Mg(2+)-dependent pseudoknot structure that includes the PB1-F2 and N40 start codons. The results suggest that interactions of the ribosome with the pseudoknot may affect the level of translation for PB1-F2 and N40.

Secondary structure of a conserved domain in the intron of influenza A NS1 mRNA.

Influenza A virus is a segmented single-stranded (-)RNA virus that causes substantial annual morbidity and mortality. The transcriptome of influenza A is predicted to have extensive RNA secondary structure. The smallest genome segment, segment 8, encodes two proteins, NS1 and NEP, via alternative splicing. A conserved RNA domain in the intron of segment 8 may be important for regulating production of NS1. Two different multi-branch loop structures have been proposed for this region. A combination of in vitro chemical mapping and isoenergetic microarray techniques demonstrate that the consensus sequence for this region folds into a hairpin conformation. These results provide an alternative folding for this region and a foundation for designing experiments to probe its functional role in the influenza life cycle.

Influenza B virus has global ordered RNA structure in (+) and (-) strands but relatively less stable predicted RNA folding free energy than allowed by the encoded protein sequence.

BACKGROUND: Influenza A virus contributes to seasonal epidemics and pandemics and contains Global Ordered RNA structure (GORS) in the nucleoprotein (NP), non-structural (NS), PB2, and M segments. A related virus, influenza B, is also a major annual public health threat, but unlike influenza A is very selective to human hosts. This study extends the search for GORS to influenza B. FINDINGS: A survey of all available influenza B sequences reveals GORS in the (+) and (-)RNAs of the NP, NS, PB2, and PB1 gene segments. The results are similar to influenza A, except GORS is observed for the M1 segment of influenza A but not for PB1. In general, the folding free energies of human-specific influenza B RNA segments are less stable than allowable by the encoded amino acid sequence. This is consistent with findings in influenza A, where human-specific influenza RNA folds are less stable than avian and swine strains. CONCLUSIONS: These results reveal fundamental molecular similarities and differences between Influenza A and B and suggest a rational basis for choosing segments to target with therapeutics and for viral attenuation for live vaccines by altering RNA folding stability.

The influenza A segment 7 mRNA 3' splice site pseudoknot/hairpin family.

The 3' splice site of the influenza A segment 7 transcript is utilized to produce mRNA for the critical M2 ion-channel protein. In solution a 63 nt fragment that includes this region can adopt two conformations: a pseudoknot and a hairpin. In each conformation, the splice site, a binding site for the SF2/ASF exonic splicing enhancer and a polypyrimidine tract, each exists in a different structural context. The most dramatic difference occurs for the splice site. In the hairpin the splice site is between two residues that are involved in a 2 by 2 nucleotide internal loop. In the pseudoknot, however, these bases are canonically paired within one of the pseudoknotted helices. The conformational switching observed in this region has implications for the regulation of splicing of the segment 7 mRNA. A measure of stability of the structures also shows interesting trends with respect to host specificity: avian strains tend to be the most stable, followed by swine and then human.

The 3' splice site of influenza A segment 7 mRNA can exist in two conformations: a pseudoknot and a hairpin.

The 3' splice site of influenza A segment 7 is used to produce mRNA for the M2 ion-channel protein, which is critical to the formation of viable influenza virions. Native gel analysis, enzymatic/chemical structure probing, and oligonucleotide binding studies of a 63 nt fragment, containing the 3' splice site, key residues of an SF2/ASF splicing factor binding site, and a polypyrimidine tract, provide evidence for an equilibrium between pseudoknot and hairpin structures. This equilibrium is sensitive to multivalent cations, and can be forced towards the pseudoknot by addition of 5 mM cobalt hexammine. In the two conformations, the splice site and other functional elements exist in very different structural environments. In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop. The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site.

Influenza A virus coding regions exhibit host-specific global ordered RNA structure.

Influenza A is a significant public health threat, partially because of its capacity to readily exchange gene segments between different host species to form novel pandemic strains. An understanding of the fundamental factors providing species barriers between different influenza hosts would facilitate identification of strains capable of leading to pandemic outbreaks and could also inform vaccine development. Here, we describe the difference in predicted RNA secondary structure stability that exists between avian, swine and human coding regions. The results predict that global ordered RNA structure exists in influenza A segments 1, 5, 7 and 8, and that ranges of free energies for secondary structure formation differ between host strains. The predicted free energy distributions for strains from avian, swine, and human species suggest criteria for segment reassortment and strains that might be ideal candidates for viral attenuation and vaccine development.

Identification of potential conserved RNA secondary structure throughout influenza A coding regions.

Influenza A is a negative sense RNA virus of significant public health concern. While much is understood about the life cycle of the virus, knowledge of RNA secondary structure in influenza A virus is sparse. Predictions of RNA secondary structure can focus experimental efforts. The present study analyzes coding regions of the eight viral genome segments in both the (+) and (-) sense RNA for conserved secondary structure. The predictions are based on identifying regions of unusual thermodynamic stabilities and are correlated with studies of suppression of synonymous codon usage (SSCU). The results indicate that secondary structure is favored in the (+) sense influenza RNA. Twenty regions with putative conserved RNA structure have been identified, including two previously described structured regions. Of these predictions, eight have high thermodynamic stability and SSCU, with five of these corresponding to current annotations (e.g., splice sites), while the remaining 12 are predicted by the thermodynamics alone. Secondary structures with high conservation of base-pairing are proposed within the five regions having known function. A combination of thermodynamics, amino acid and nucleotide sequence comparisons along with SSCU was essential for revealing potential secondary structures.