Mutational and phenotypic characterization of hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Care delivery for HHT patients is impeded by the need for laborious, repeated phenotyping and gaps in knowledge regarding the relationships between causal DNA variants in ENG, ACVRL1, SMAD4 and GDF2, and clinical manifestations. To address this, we analyzed DNA samples from 183 previously uncharacterized, unrelated HHT and suspected HHT cases using the ThromboGenomics high-throughput sequencing platform. We identified 127 rare variants across 168 heterozygous genotypes. Applying modified American College of Medical Genetics and Genomics Guidelines, 106 variants were classified as pathogenic/likely pathogenic and 21 as nonpathogenic (variant of uncertain significance/benign). Unlike the protein products of ACVRL1 and SMAD4, the extracellular ENG amino acids are not strongly conserved. Our inferences of the functional consequences of causal variants in ENG were therefore informed by the crystal structure of endoglin. We then compared the accuracy of predictions of the causal gene blinded to the genetic data using 2 approaches: subjective clinical predictions and statistical predictions based on 8 Human Phenotype Ontology terms. Both approaches had some predictive power, but they were insufficiently accurate to be used clinically, without genetic testing. The distributions of red cell indices differed by causal gene but not sufficiently for clinical use in isolation from genetic data. We conclude that parallel sequencing of the 4 known HHT genes, multidisciplinary team review of variant calls in the context of detailed clinical information, and statistical and structural modeling improve the prognostication and treatment of HHT.

Effect of Different Proteinase K Digest Protocols and Deparaffinization Methods on Yield and Integrity of DNA Extracted From Formalin-fixed, Paraffin-embedded Tissue.

DNA extracted from formalin-fixed, paraffin-embedded tissue sections is often inadequate for sequencing, due to poor yield or degradation. We optimized the proteinase K digest by testing increased volume of enzyme and increased digest length from the manufacturer's protocol using 54 biospecimens, performing the digest in centrifuge tubes. Doubling the quantity of proteinase K resulted in a median increase in yield of 96%. Applying the optimized proteinase K protocol to sections deparaffinized on microscope slides generated a further increase in yield of 41%, but only at >50,000 epithelial tumor cells/section. DNA yield now correlated with (χ2 = 0.84) and could be predicted from the epithelial tumor cell number. DNA integrity was assayed using end point multiplex PCR (amplicons of 100-400 bp visualized on a gel), quantitative PCR (qPCR; Illumina FFPE QC Assay), and nanoelectrophoresis (DNA Integrity Numbers [DINs]). Generally, increases in yield were accompanied by increases in integrity, but sometimes qPCR and DIN results were conflicting. Amplicons of 400 bp were almost universally obtained. The process of optimization enabled us to reduce the percentage of samples that failed published quality control thresholds for determining amenability to whole genome sequencing from 33% to 7%.

COPD and the gut-lung axis: the therapeutic potential of fibre.

Current management strategies for chronic obstructive pulmonary disease (COPD) incorporate a step-wise, multidisciplinary approach to effectively manage patient symptoms and prevent disease progression. However, there has been limited advancement in therapies to address the underlying cause of COPD pathogenesis. Recent research has established the link between the lungs and the gut-the gut-lung axis -and the gut microbiome is a major component. The gut microbiome is likely perturbed in COPD, contributing to chronic inflammation. Diet is a readily modifiable factor and the diet of COPD patients is often deficient in nutrients such as fibre. The metabolism of dietary fibre by gut microbiomes produces anti-inflammatory short chain fatty acid (SCFAs), which could protect against inflammation in the lungs. By addressing the 'fibre gap' in the diet of COPD patients, this targeted dietary intervention may reduce inflammation, both systemically and in the airways, and value-add to the paradigm shift in respiratory medicine, from reactive to personalised and participatory medicine.