Background
A significant challenge is faced for the genetic diagnosis of inherited platelet disorders in which candidate genetic variants can be found in more than 100 bleeding, thrombotic, and ...platelet disorder genes, especially within families in which there are both normal and low platelet counts. Genetic variants of unknown clinical significance (VUS) are found in a significant proportion of such patients in which functional studies are required to prove pathogenicity.
Objective
To identify the genetic cause in patients with a suspected platelet disorder and subsequently perform a detailed functional analysis of the candidate genetic variants found.
Methods
Genetic and functional studies were undertaken in three patients in two unrelated families with a suspected platelet disorder and excessive bleeding. A targeted gene panel of previously known bleeding and platelet genes was used to identify plausible genetic variants. Deep platelet phenotyping was performed using platelet spreading analysis, transmission electron microscopy, immunofluorescence, and platelet function testing using lumiaggregometry and flow cytometry.
Results
We report rare conserved missense variants (p.R182C and p.A183V) in TPM4 encoding tromomyosin‐4 in 3 patients. Deep platelet phenotyping studies revealed similar platelet function defects across the 3 patients including reduced platelet secretion, and aggregation and spreading defects suggesting that TPM4 missense variants impact platelet function and show a disordered pattern of tropomyosin staining.
Conclusions
Genetic and functional TPM4 defects are reported making TPM4 a diagnostic grade tier 1 gene and highlights the importance of including TPM4 in diagnostic genetic screening for patients with significant bleeding and undiagnosed platelet disorders, particularly for those with a normal platelet count.
Rpp4 (Resistance to Phakopsora pachyrhizi 4) confers resistance to Phakopsora pachyrhizi Sydow, the causal agent of Asian soybean rust (ASR). By combining expression profiling and virus induced gene ...silencing (VIGS), we are developing a genetic framework for Rpp4-mediated resistance. We measured gene expression in mock-inoculated and P. pachyrhizi-infected leaves of resistant soybean accession PI459025B (Rpp4) and the susceptible cultivar (Williams 82) across a 12-day time course. Unexpectedly, two biphasic responses were identified. In the incompatible reaction, genes induced at 12h after infection (hai) were not differentially expressed at 24 hai, but were induced at 72 hai. In contrast, genes repressed at 12 hai were not differentially expressed from 24 to 144 hai, but were repressed 216 hai and later. To differentiate between basal and resistance-gene (R-gene) mediated defence responses, we compared gene expression in Rpp4-silenced and empty vector-treated PI459025B plants 14 days after infection (dai) with P. pachyrhizi. This identified genes, including transcription factors, whose differential expression is dependent upon Rpp4. To identify differentially expressed genes conserved across multiple P. pachyrhizi resistance pathways, Rpp4 expression datasets were compared with microarray data previously generated for Rpp2 and Rpp3-mediated defence responses. Fourteen transcription factors common to all resistant and susceptible responses were identified, as well as fourteen transcription factors unique to R-gene-mediated resistance responses. These genes are targets for future P. pachyrhizi resistance research.
Crude solvent extracts were prepared from three sediment sites in Ireland namely Bull Lagoon, Dunmore East and Dublin Port. These were assayed with
Tisbe battagliai and the Microtox
® system. The ...extracts were chemically characterised using a variety of analytical techniques for a suite of organic contaminants. Metals and organic contaminant concentration data are reported for the three sites. On the basis of determined toxicity and chemical analysis of these crude extracts, a further bioassay-directed fractionation (BDF) employing the Dunmore East crude organic extract was conducted in addition to chemical analysis. For the crude extracts,
T. battagliai and Microtox
® system demonstrated an order of decreasing toxicity for each of the three sites to be Dublin Port
>
Dunmore East
>
Bull Lagoon. Microtox
® system EC
10 values after 30
min exposure were 1.08%, 11.6% and 26.9% solvent extract for these sites, respectively. Fractionation of the Dunmore East extract revealed that fraction 1 was the most toxic fraction to both the
T. battagliai and the Microtox
® system demonstrating EC
50’s after 48
h and 30
min of 44.7% and 16.8% solvent extract for the
T. battagliai and Microtox
® assays, respectively.
T. battagliai however did show increased sensitivity to fraction 3 when comparing EC
10 values and demonstrated an EC
10 value of 17.8% solvent extract after 48
h. Fraction 1 was shown to contain the highest quantity of the butyltins, in particular TBT in relation to fractions 2 and 3. A useful BDF technique was developed and employed in this study.
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