The emerging role of dipeptidyl peptidase 3 in pathophysiology.

Dipeptidyl peptidase 3 (DPP3), a zinc-dependent aminopeptidase, is a highly conserved enzyme among higher animals. The enzyme cleaves dipeptides from the N-terminus of tetra- to decapeptides, thereby taking part in activation as well as degradation of signalling peptides critical in physiological and pathological processes such as blood pressure regulation, nociception, inflammation and cancer. Besides its catalytic activity, DPP3 moonlights as a regulator of the cellular oxidative stress response pathway, e.g., the Keap1-Nrf2 mediated antioxidative response. The enzyme is also recognized as a key modulator of the renin-angiotensin system. Recently, DPP3 has been attracting growing attention within the scientific community, which has significantly augmented our knowledge of its physiological relevance. Herein, we review recent advances in our understanding of the structure and catalytic activity of DPP3, with a focus on attributing its molecular architecture and catalytic mechanism to its wide-ranging biological functions. We further highlight recent intriguing reports that implicate a broader role for DPP3 as a valuable biomarker in cardiovascular and renal pathologies and furthermore discuss its potential as a promising drug target.

Sanger Validation of High-Throughput Sequencing in Genetic Diagnosis: Still the Best Practice?

Next-generation sequencing (NGS)'s crucial role in supporting genetic diagnosis and personalized medicine leads to the definition of Guidelines for Diagnostic NGS by the European Society of Human Genetics. Factors of different nature producing false-positive/negative NGS data together with the paucity of internationally accepted guidelines providing specified NGS quality metrics to be followed for diagnostics purpose made the Sanger validation of NGS variants still mandatory. We reported the analysis of three cases of discrepancy between NGS and Sanger sequencing in a cohort of 218 patients. NGS was performed by Illumina MiSeq® and Haloplex/SureSelect protocols targeting 97 or 57 or 10 gene panels usually applied for diagnostics. Variants called following guidelines suggested by the Broad Institute and identified according to MAF <0.01 and allele balance >0.2 were Sanger validated. Three out of 945 validated variants showed a discrepancy between NGS and Sanger. In all three cases, a deep evaluation of the discrepant gene variant results and methodological approach allowed to confirm the NGS datum. Allelic dropout (ADO) occurrence during polymerase chain or sequencing reaction was observed, mainly related to incorrect variant zygosity. Our study extends literature data in which almost 100% "high quality" NGS variants are confirmed by Sanger; moreover, it demonstrates that in case of discrepancy between a high-quality NGS variant and Sanger validation, NGS call should not be a priori assumed to represent the source of the error. Actually, difficulties (i.e., ADO, unpredictable presence of private variants on primer-binding regions) of the so-called gold standard direct sequencing should be considered especially in light of the constantly implemented and accurate high-throughput technologies. Our data along with literature raise a discussion on the opportunity to establish a standardized quality threshold by International Guidelines for clinical NGS in order to limit Sanger confirmation to borderline conditions of variant quality parameters and verification of correct gene variant call/patient coupling on a different blood sample aliquot.

Torque teno virus microRNA detection in cerebrospinal fluids of patients with neurological pathologies.

BACKGROUND: Torque teno virus (TTV) is a widespread anellovirus that establishes persistent infections in humans and represents the most abundant component of the human virome. TTV encodes microRNAs (miRNA) which are found both in viremic and not viremic subjects being potentially ideal tools for the virus to evade the immune system response and to maintain chronic infection in the host. OBJECTIVE: To investigate TTV-DNA loads and TTV-miRNAs expression in cerebrospinal fluids (CSF) from subjects under analysis for the assessment of neurological diseases. STUDY DESIGN: Detection of TTV-DNA and TTV-miRNAs (e. g. miRNA t1a, t3b, and tth8) were carried out from CSF samples of 93 subjects with neurological diseases by using universal real-time PCR, real-time RT-PCR, and next-generation sequencing (NGS) analyses. RESULTS: TTV-DNA was detected in 11 of 93 (12 %) CSFs with a mean TTV load of 155 copies/mL. Conversely, 29 CSF samples (31 %) were positive for at least one TTV-miRNA, while 15 (16 %) CSFs contained all the TTV-miRNAs examined. Overall, TTV-miRNA tth8 was detected in 62 % of samples, followed by TTV miRNA t3b (56 %), and t1a (29 %). Interestingly, TTV-miRNAs were found in CSF samples that were negative for the presence of TTV-DNA. Next-generation sequencing analysis carried out from 4 TTV-DNA negative CSF samples detected reads mapped in TTV-miRNA sequences region. CONCLUSIONS: These results shed novel light on the relationship between TTV and the central nervous system and make compelling furthered studies for investigating the potential role of TTV-miRNAs in neurological disorders.