Bruch's membrane abnormalities in PRDM5-related brittle cornea syndrome.

BACKGROUND: Brittle cornea syndrome (BCS) is a rare, generalized connective tissue disorder associated with extreme corneal thinning and a high risk of corneal rupture. Recessive mutations in transcription factors ZNF469 and PRDM5 cause BCS. Both transcription factors are suggested to act on a common pathway regulating extracellular matrix genes, particularly fibrillar collagens. We identified bilateral myopic choroidal neovascularization as the presenting feature of BCS in a 26-year-old-woman carrying a novel PRDM5 mutation (p.Glu134*). We performed immunohistochemistry of anterior and posterior segment ocular tissues, as expression of PRDM5 in the eye has not been described, or the effects of PRDM5-associated disease on the retina, particularly the extracellular matrix composition of Bruch's membrane. METHODS: Immunohistochemistry using antibodies against PRDM5, collagens type I, III, and IV was performed on the eyes of two unaffected controls and two patients (both with Δ9-14 PRDM5). Expression of collagens, integrins, tenascin and fibronectin in skin fibroblasts of a BCS patient with a novel p.Glu134* PRDM5 mutation was assessed using immunofluorescence. RESULTS: PRDM5 is expressed in the corneal epithelium and retina. We observe reduced expression of major components of Bruch's membrane in the eyes of two BCS patients with a PRDM5 Δ9-14 mutation. Immunofluorescence performed on skin fibroblasts from a patient with p.Glu134* confirms the generalized nature of extracellular matrix abnormalities in BCS. CONCLUSIONS: PDRM5-related disease is known to affect the cornea, skin and joints. Here we demonstrate, to the best of our knowledge for the first time, that PRDM5 localizes not only in the human cornea, but is also widely expressed in the retina. Our findings suggest that ECM abnormalities in PRDM5-associated disease are more widespread than previously reported.

Diagnostic Mutation Profiling and Validation of Non-Small-Cell Lung Cancer Small Biopsy Samples using a High Throughput Platform.

BACKGROUND: A single platform designed for the synchronous screening of multiple mutations can potentially enable molecular profiling in samples of limited tumor tissue. This approach is ideal for the assessment of advanced non-small-cell lung cancer (NSCLC) diagnostic specimens, which often comprise small biopsies. Therefore, we aimed in this study to validate the mass spectrometry-based Sequenom LungCarta panel and MassARRAY platform using DNA extracted from a single 5 µM formalin-fixed paraffin-embedded tissue section. METHODS: Mutations, including those with an equivocal spectrum, detected in 90 cases of NSCLC (72 lung biopsies, 13 metastatic tissue biopsies, three resections, and two cytology samples) were validated by a combination of standard sequencing techniques, immunohistochemical staining for p53 protein, and next-generation sequencing with the TruSight Tumor panel. RESULTS: Fifty-five mutations were diagnosed in 47 cases (52%) in the following genes: TP53 (22), KRAS (15), EGFR (5), MET (3), PIK3CA (3), STK11 (2), NRF-2 (2), EPHA5 (1), EPHA3 (1), and MAP2K1 (1). Of the 90 samples, one failed testing due to poor quality DNA. An additional 7 TP53 mutations were detected by next-generation sequencing, which facilitated the interpretation of p53 immunohistochemistry but required 5 × 10 µM tumor sections per sample tested. CONCLUSIONS: The LungCarta panel is a sensitive method of screening for multiple alterations (214 mutations across 26 genes) and which optimizes the use of limited amounts of tumor DNA isolated from small specimens.

Differential sub-nuclear localisation of repressive and activating histone methyl modifications in P. falciparum.

Post-translational histone modifications and sub-nuclear organization epigenetically influence gene regulation, especially those implicated in antigenic variation of Plasmodium falciparum. Here we screened for histone methylation modifications and determined, for the first time, their spatial nuclear localisation. Differential enrichment in sub-nuclear compartments, suggesting high local concentrations of particular methyl marks was observed. H3-K79me3 particularly, does not co-localise with already defined nuclear compartments, thus apparently defining a compartment not observed previously in other eukaryotes. Our data show the presence of discrete sub-nuclear zones enriched for histone methyl marks, pointing to the existence of specialized transcription factories and repressive regions in Plasmodium.