The assessment of differentially expressed microRNAs in patients and healthy controls is important to identify potential tumor biomarkers. Recently, it has been shown that the microRNA levels in exosomes are more correlated with the clinical-pathological variables than vesicle-free microRNAs (miRNAs) in biofluids; therefore, there is an increasing interest in these specific evaluations. However, these measurements can be affected by experimental problems that not always are evaluated and/or by inadequate procedural choices. In particular, exosome isolation and miRNA extraction procedures are crucial to avoid contaminations, and even the choice of the most suitable purity controls is important. Moreover, a stable endogenous RNA should be used for normalization of miRNA expression obtained by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) in order to make these measures comparable among different samples. A rushed choice of the endogenous control can bias study conclusions without revealing inconsistencies. Unfortunately, a few studies systematically identified the best normalizer for their specific experimental context. Instead, sometimes, the normalization procedures were performed in a disputable way or the normalizer choices simply based on the previous literature. Here, we reviewed the studies where the exosomal miRNA profiling was assessed in human biofluids to point out the adopted procedures and the specific endogenous controls chosen for normalization.

Cerebral cavernous malformations (CCMs) are lesions affecting brain microvessels. The pathogenesis is not clearly understood. Conventional classification criterion is based on genetics, and thus, familial and sporadic forms can be distinguished; however, classification of sporadic cases with multiple lesions still remains uncertain. To date, three CCM causative genes have been identified: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. In our previous mutation screening, performed in a cohort of 95 Italian patients, with both sporadic and familial cases, we identified several mutations in CCM genes. This study represents further molecular screening in a cohort of 19 Italian patients enrolled by us in the few last years and classified into familial, sporadic and sporadic with multiple lesions cases. Direct sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis were performed to detect point mutations and large genomic rearrangements, respectively. Effects of detected mutations and single-nucleotide polymorphisms (SNPs) were evaluated by an in silico approach and by western blot analysis. A novel nonsense mutation in CCM1 and a novel missense mutation in CCM2 were detected; moreover, several CCM2 gene polymorphisms in sporadic CCM patients were reported. We believe that these data enrich the mutation spectrum of CCM genes, which is useful for genetic counselling to identify both familial and sporadic CCM cases, as early as possible.

Clear cell Renal Cell Carcinoma (ccRCC) is due to loss of von Hippel–Lindau (VHL) gene and at least one out of three chromatin regulating genes BRCA1-associated protein-1 (BAP1), Polybromo-1 (PBRM1) and Set domain-containing 2 (SETD2). More than 350, 700 and 500 mutations are known respectively for BAP1, PBRM1 and SETD2 genes. Each variation damages these genes with different severity levels. Unfortunately for most of these mutations the molecular effect is unknown, so precluding a severity classification. Moreover, the huge number of these gene mutations does not allow to perform experimental assays for each of them. By bioinformatic tools, we performed predictions of the molecular effects of all mutations lying in BAP1, PBRM1 and SETD2 genes. Our results allow to distinguish whether a mutation alters protein function directly or by splicing pattern destruction and how much severely. This classification could be useful to reveal correlation with patients’ outcome, to guide experiments, to select the variations that are worth to be included in translational/association studies, and to direct gene therapies.

Ciliopathies are a group of multi-organ diseases caused by the disruption of the primary cilium. This event leads to a variety of kidney disorders, including nephronophthisis, renal cystic dysplasia, and renal cell carcinoma (RCC). Primary cilium contributes to the regulation of the cell cycle and protein homeostasis, that is, the balance between protein synthesis and degradation by acting on the ubiquitin-proteasome system, autophagy, and mTOR signaling. Many proteins are involved in renal ciliopathies. In particular, fibrocystin (PKHD1) is involved in autosomal recessive polycystic kidney disease (ARPKD), while polycystin-1 (PKD1) and polycystin-2 (PKD2) are implicated in autosomal dominant polycystic kidney disease (ADPKD). Moreover, primary cilia are associated with essential signaling pathways, such as Hedgehog, Wnt, and Platelet-Derived Growth Factor (PDGF). In this review, we focused on the ciliopathies associated with kidney diseases, exploring genes and signaling pathways associated with primary cilium and the potential role of cilia as therapeutic targets in renal disorders.

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