A Copernican revolution of multigenic analysis: A retrospective study on clinical exome sequencing in unclear genetic disorders.

Despite the inevitable shift in medical practice towards a deeper understanding of disease etiology and progression through multigenic analysis, the profound historical impact of Mendelian diseases cannot be overlooked. These diseases, such as cystic fibrosis and thalassemia, are characterized by a single variant in a single gene leading to clinical conditions, and have significantly shaped our medical knowledge and treatments. In this respect, the monogenic approach inevitably results in the underutilization of Next-Generation Sequencing (NGS) data. Herein, a retrospective study was performed to assess the diagnostic value of the clinical exome in 32 probands with specific phenotypic characteristics (patients with autoinflammation and immunological dysregulation, N = 20; patients diagnosed with Hemolytic uremic syndrome N = 9; and patients with Waldenström macroglobulinemia, N = 3). A gene enrichment analysis was performed using the *. VCF file generated by SOPHiA-DDM-v4. This analysis selected a subset of genes containing pathogenic or likely pathogenic variants with autosomal dominant (AD) inheritance. In addition, all variants of uncertain significance (VUS) were included, filtered by AD inheritance mode, the presence of compound heterozygotes, and a minor allele frequency (MAF) cutoff of 0.05 %. The aim of the pipeline described here is based on a perspective shift that focuses on analyzing patients' gene assets, offering new light on the complex interplay between genetics and disease presentation. Integrating this approach into clinical practices could significantly enhance the management of patients with rare genetic disorders.

Hydrodistension plus Onabotulinumtoxin A in bladder pain syndrome refractory to conservative treatments. / Hidrodistensión bajo anestesia más inyección de Onabotulinumtoxin A en pacientes con síndrome de dolor vesical refractario a tratamiento conservador.

INTRODUCTION: For bladder pain syndrome (BPS) refractory to conservative treatment, the European guidelines consider bladder hydrodistention (HD) under anaesthesia and the injection of Onabotulinumtoxin A (OnabotA) jointly. The objective of this study was to assess our experience in implementing this technique. MATERIAL AND METHODS: A prospective study of 25 patients with BPS who underwent HD plus a submucosal injection of 100 U of OnabotA in trigone. The Hunner lesions were treated endoscopically using resection or electrocoagulation. Thirty-eight procedures were performed (25 first interventions and 13 reoperations). To study the clinical change, we evaluated the subjective improvement (Treatment Benefit Scale [TBS] and Patient Global Impression of Change [PGIC] scales), the visual analogue scale (VAS) for pain, the Bladder Pain/Interstitial Cystitis Symptom Score (BPIC-SS) questionnaire and the voiding diary for 3 days. For the data analysis, we employed the Wilcoxon, Kruskal-Wallis, Kaplan-Meier and log-rank tests. RESULTS: We observed subjective improvement in 21 patients (84%), which was significant in 47% of these patients, moderate in 41.2% and slight in 11.8%. Four patients did not improve. A post-treatment reduction in the pain VAS (from 7.1 to 1.8 points; P=.001), in daytime (from 11.8 to 7.5; P=.012) and night-time (from 5.9 to 3.6; P=.003) voiding frequency and in the BPIC-SS (from 27.9 to 11.2 points; P=.042). The degree of improvement was not related to age, the presence of bladder lesions or the treatment of relapses. The median duration of improvement was 7 months (95% CI 5.69-8.31), although this duration was somewhat longer for the patients younger than 65 years. Mild complications occurred in 23.7% of the cases. CONCLUSIONS: The joint implementation of HD plus OnabotA is a valid therapeutic option in refractory BPS, which provides good clinical results and maintains its effectiveness in retreatments.

The activity of hsp90 alpha promoter is regulated by NF-kappa B transcription factors.

Heat-shock proteins (HSP) 90 exert a relevant role in the survival and response to therapy of many neoplastic cell types. Here, we show that the promoter of hsp90alpha gene, that encodes the inducible form of HSP90, is regulated by nuclear factor-kappaB (NF-kappaB) activity. Indeed, we found that NF-kappaB factors bound to one of the two putative consensus sequences present in the hsp90alpha-flanking region; mutation of such motif hampered the phorbol-myristate-13-acetate-stimulated expression of a luciferase reporter gene under the control of the hsp90alpha promoter. Furthermore, the downmodulation of NF-kappaB (p65) levels by a specific small interfering (si) RNA resulted in reducing the levels of endogenous HSP90alpha protein. These findings disclose a previously unrecognized mechanism that contributes to connect NF-kappaB factors and HSPs in cell defence machinery.

An homologue of the human 100-kDa protein (p100) is differentially expressed by Histoplasma capsulatum during infection of murine macrophages.

Using differential display reverse transcription-PCR (DDRT-PCR) we have identified several sequences that are specifically expressed by Histoplasma capsulatum during infection of murine macrophages (MPhi). Here, we report the characterization of a clone, pHc12, identified as a differentially expressed gene 1 hour after infection of MPhi. Screening of a cDNA library of H. capsulatum allowed us to isolate a clone, pHc12-E, that contains the complete coding sequence. We show that after infection the level of transcription of this gene increases about 5 fold. Analysis of its sequence revealed the presence of an open reading frame of 890 aa (ORF890) that shares respectively 30 and 33% identity with human and Caenorhabditis elegans p100 kD and rat p105 kD co-activator proteins. Using the two-dimensional Hydrophobic Cluster Analysis (HCA) method, we showed that H. capsulatum ORF890 and p100 kD co-activator proteins are clearly related. The H. capsulatum protein consists of a four-fold repeated module (domains I to IV) like the p100 kD co-activator proteins, whose three-dimensional (3D) structure is related to staphylococcal thermonuclease, followed by a modified fifth "hybrid" domain which partially resembles the structure of the tudor domain found in multiple copies in the Drosophila melanogaster tudor protein. These data strongly suggest that ORF890 is homologous to human p100 kD and that this protein, named Hcp100, may play an essential role during infection by co-activating the expression of specific genes.

The gene, protein and glycan structures of laccase from Pleurotus ostreatus.

A member of the laccase multigene family in Pleurotus ostreatus has been cloned and sequenced. The gene structure has been determined by comparison with the corresponding cDNA, synthesized by reverse transcription/PCR amplification. The gene encode a laccase isoenzyme of 533 amino acids which has already been purified and characterized [Palmieri, G., Giardina, P., Marzullo, L., Desiderio, B., Nitti, G., Cannio, R. & Sannia, G.(1993) Appl. Microbiol. Biotechnol. 39, 632-636]. More than 92% of the protein sequence, including the N and C termini, has been verified by fast-atom-bombardment mass spectrometry, thus confirming the correspondence between the gene and its protein product. The protein was N-glycosylated Asn444. Glycan analysis showed the presence of only a high-mannose structure containing varying numbers of mannose residues. The presence of O-linked oligosaccharides as well as other post-translational modification could be ruled out by the mass analysis.

Cloning and sequencing of a laccase gene from the lignin-degrading basidiomycete Pleurotus ostreatus.

The gene (pox1) encoding a phenol oxidase from Pleurotus ostreatus, a lignin-degrading basidiomycete, was cloned and sequenced, and the corresponding pox1 cDNA was also synthesized and sequenced. The isolated gene consists of 2,592 bp, with the coding sequence being interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA consensus sequences could be identified at positions -174 and -84, respectively. The isolation of a second cDNA (pox2 cDNA), showing 84% similarity, and of the corresponding truncated genomic clones demonstrated the existence of a multigene family coding for isoforms of laccase in P. ostreatus. PCR amplifications of specific regions on the DNA of isolated monokaryons proved that the two genes are not allelic forms. The POX1 amino acid sequence deduced was compared with those of other known laccases from different fungi.

Veratryl alcohol oxidase from Pleurotus ostreatus participates in lignin biodegradation and prevents polymerization of laccase-oxidized substrates.

Oxidative enzymes (laccases and peroxidases) isolated from the culture media of different fungi are involved in the basic mechanism of ligninolysis via radical intermediates. However, experiments aimed at reproducing natural biodegradation in vitro have been unsuccessful so far since the single biocatalysts alone are not able to solubilize lignins because of the simultaneous recondensation of these intermediates. FAD oxidases can prevent this side reaction in lignin depolymerization by reducing quinonoids and radical compounds. This study investigates the possible role of a laccase and a FAD-dependent aryl alcohol oxidase (veratryl alcohol oxidase, VAO) excreted by the basidiomycete Pleurotus ostreatus. In fact, we found that VAO is able to reduce synthetic quinones, laccase-generated quinonoids, and phenoxy radicals with concomitant oxidation of veratryl alcohol to veratryl aldehyde. This cooperative action of laccase and VAO also prevented the polymerization of phenolic compounds and reduced the molecular weight of soluble lignosulfonates to a significant extent.

Stability and activity of a phenol oxidase from the ligninolytic fungus Pleurotus ostreatus.

Three different phenol oxidases produced by the basidiomycete fungus Pleurotus ostreatus have been isolated and their main structural, enzymatic and physico-chemical properties characterized. Studies have focused on the most abundantly secreted of these proteins, a copper-enzyme specific towards ortho-diphenol substrates. This protein was purified to homogeneity and part of its primary structure determined by direct protein sequencing. The influence of pH, temperature and presence of water-soluble or water-insoluble organic solvents on the activity and stability of the enzyme were also investigated. These data can be used for applying bioreactors to problems of environmental concern such as waste-water treatment.