Giovanna Marchese

Timed regulation of P‐element‐induced wimpy testis–interacting RNA expression during rat liver regeneration

Small noncoding RNAs comprise a growing family of molecules that regulate key cellular processes, including messenger RNA (mRNA) degradation, translational repression, and transcriptional gene silencing. P‐element‐induced wimpy testis (PIWI)‐interacting RNAs (piRNAs) represent a class of small RNAs initially identified in the germline of a variety of species, where they contribute to maintenance of genome stability, and recently found expressed also in stem and somatic cells, where their role and responsiveness to physiopathological signals remain elusive. Here, we investigated piRNA expression in rat liver and its response to the stimuli exerted by regenerative proliferation of this organ. Quantitative polymerase chain reaction analysis identify in the liver the RNAs encoding PIWIL2/HILI, PIWIL4/HIWI2, and other components of the piRNA biogenesis pathways, suggesting that this is indeed functional. RNA sequencing before, during, and after the wave of cell proliferation that follows partial hepatectomy (PH) identified ∼1,400 mammalian germline piRNAs expressed in rat liver, including 72 showing timed changes in expression 24‐48 hours post‐PH, a timing that corresponds to cell transition through the S phase, returning to basal levels by 168 hours, when organ regeneration is completed and hepatocytes reach quiescence. Conclusion: The piRNA pathway is active in somatic cells of the liver and is subject to regulation during the pathophysiological process of organ regeneration, when these molecules are available to exert their regulatory functions on the cell genome and transcriptome, as demonstrated by the identification of several liver mRNAs representing candidate targets of these regulatory RNAs. (Hepatology 2014;60:798–806)

Characterization of two de novoKCNT1 mutations in children with malignant migrating partial seizures in infancy.

The KCNT1 gene encodes for subunits contributing to the Na(+)-activated K(+) current (KNa), expressed in many cell types. Mutations in KCNT1 have been found in patients affected with a wide spectrum of early-onset epilepsies, including Malignant Migrating Partial Seizures in Infancy (MMPSI), a severe early-onset epileptic encephalopathy characterized by pharmacoresistant focal seizures migrating from one brain region or hemisphere to another and neurodevelopment arrest or regression, resulting in profound disability. In the present study we report identification by whole exome sequencing (WES) of two de novo, heterozygous KCNT1 mutations (G288S and, not previously reported, M516V) in two unrelated MMPSI probands. Functional studies in a heterologous expression system revealed that channels formed by mutant KCNT1 subunits carried larger currents when compared to wild-type KCNT1 channels, both as homo- and heteromers with these last. Both mutations induced a marked leftward shift in homomeric channel activation gating. Interestingly, the KCNT1 blockers quinidine (3-1000μM) and bepridil (0.03-10μM) inhibited both wild-type and mutant KCNT1 currents in a concentration-dependent manner, with mutant channels showing higher sensitivity to blockade. This latter result suggests two genotype-tailored pharmacological strategies to specifically counteract the dysfunction of KCNT1 activating mutations in MMPSI patients.

Specific patterns of PIWI-interacting small noncoding RNA expression in dysplastic liver nodules and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the result of a stepwise process, often beginning with development within a cirrhotic liver of premalignant lesions, morphologically characterized by low- (LGDN) and high-grade (HGDN) dysplastic nodules. PIWI-interacting RNAs (piRNAs) are small noncoding RNAs (sncRNAs), 23–35 nucleotide-long, exerting epigenetic and post-transcriptional regulation of gene expression. Recently the PIWI-piRNA pathway, best characterized in germline cells, has been identified also in somatic tissues, including stem and cancer cells, where it influences key cellular processes. Small RNA sequencing was applied to search for liver piRNAs and to profile their expression patterns in cirrhotic nodules (CNs), LGDN, HGDN, early HCC and progressed HCC (pHCC), analyzing 55 samples (14 CN, 9 LGDN, 6 HGDN, 6 eHCC and 20 pHCC) from 17 patients, aiming at identifying possible relationships between these sncRNAs and liver carcinogenesis. We identified a 125 piRNA expression signature that characterize HCC from matched CNs, correlating also to microvascular invasion in HCC. Functional analysis of the predicted RNA targets of deregulated piRNAs indicates that these can target key signaling pathways involved in hepatocarcinogenesis and HCC progression, thereby affecting their activity. Interestingly, 24 piRNAs showed specific expression patterns in dysplastic nodules, respect to cirrhotic liver and/or pHCC. The results demonstrate that the PIWI-piRNA pathway is active in human liver, where it represents a new player in the molecular events that characterize hepatocarcinogenesis, from early stages to pHCC. Furthermore, they suggest that piRNAs might be new disease biomarkers, useful for differential diagnosis of dysplastic and neoplastic liver lesions.

Identification of cytoplasmic proteins interacting with unliganded estrogen receptor α and β in human breast cancer cells

Estrogen receptor subtypes (ERα and ERβ) are transcription factors sharing a similar structure but exerting opposite roles in breast cancer cells. Besides the well‐characterized genomic actions of nuclear ERs upon ligand binding, specific actions of ligand‐free ERs in the cytoplasm also affect cellular functions. The identification of cytoplasmic interaction partners of unliganded ERα and ERβ may help characterize the molecular basis of the extra‐nuclear mechanism of action of these receptors, revealing novel mechanisms to explain their role in breast cancer response or resistance to endocrine therapy. To this aim, cytoplasmic extracts from human breast cancer MCF‐7 cells stably expressing tandem affinity purification‐tagged ERα and ERβ and maintained in estrogen‐free medium were subject to affinity‐purification and MS analysis, leading to the identification of 84 and 142 proteins associated with unliganded ERα and ERβ, respectively. Functional analyses of ER subtype‐specific interactomes revealed significant differences in the molecular pathways targeted by each receptor in the cytoplasm. This work, reporting the first identification of the unliganded ERα and ERβ cytoplasmic interactomes in breast cancer cells, provides novel experimental evidence on the nongenomic effects of ERs in the absence of hormonal stimulus. All MS data have been deposited in the ProteomeXchange with identifier PXD001202 (http://proteomecentral.proteomexchange.org/dataset/PXD001202).

Lack of pathogenic mutations in six patients with MMPSI

Sequencing of the KCNT1, PLCB1, SCN1A and TBC1D24 loci was performed in six children with typical features of malignant migrating partial seizures of infancy (MMPSI), to verify the presence of potential disease-causing mutations, including those already reported to be associated with the disease. Sanger sequencing failed to identify in these genes the previously reported pathogenic mutations in these patients, while a comprehensive mutational scanning analysis of these four loci by targeted re-sequencing led to detection of both intronic and exonic new variants. Based on the current knowledge, the sequence variants identified here do not allow to predict functional phenotypes that might explain, at least in part, MMPSI symptoms.

The nuclear receptor ERβ engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading

BackgroundThe RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease.ResultsApplying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ–AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ + vs ERβ − cells, and before and after AGO2 knock-down in ERβ + cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability.ConclusionsThese results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions.

Phenytoin neurotoxicity in a child carrying new STXBP1 and CYP2C9 gene mutations

• A new mutation in STXBP1 gene in a patient with a clinical history of Ohtahara syndrome and a severe adverse reaction to phenytoin, co-occurring with compound heterozygous mutations in CYP2C9 gene.

Decreased miRNA expression in Klinefelter syndrome

The widelyvariable phenotypic spectrum and the different severity of symptoms in men with Klinefelter syndrome (KS) suggest a role for epigenetic mediators. Therefore, the aim of this study is to evaluate the possible involvement of miRNAs in the clinical manifestations of KS. To accomplish this, we performed a transcriptome analysis in peripheral blood mononuclear cells (PBMCs) of 10 non-mosaic KS patients, 10 aged-matched healthy men and 10 aged-matched healthy female controls with normal karyotype. After RNA extraction from PBMC and the preparation of RNA libraries, the samples were sequenced using next generation high-throughput sequencing technology. Expression profiling analysis revealed a significant differential expression of 2 miRNAs in KS compared to male controls. In particular, MIR3648 resulted significantly (q-value < 0.0001) down-regulated by −19.084- fold, while MIR3687was strongly down-regulated (q-value < 0.0001) considering KS patients. These results were confirmed by qRT-PCR. The functional analysis of the two transcripts showed that they seem to play a role in breast cancer, hemopoietic abnormalities, immune defects and adipocyte differentiation and fat cell maturation. Therefore, we speculate that both miRNAs may play a role in the immune and metabolic disorders and in the risk of breast cancer development in men with KS.

Kleefstra-variant syndrome with heterozygous mutations in EHMT1 and KCNQ2 genes: a case report

We report the results of genetic analysis by whole exome sequencing (WES) of an atypical case of Kleefstra syndrome (KS), with psychomotor delay and intellectual disability, muscle hypotonia and dysmorphic traits accompanied by clonic seizures beginning very early after birth. KS (OMIM #610253) is a genetic disorder characterized clinically by the phenotypical features of intellectual disability, severely limited or absent speech, hypotonia, synophrys, hypertelorism and microcephaly [1, 2]. In approximately 75 % of cases, KS is caused by heterozygous microdeletions in 9q34.3 or de novo point mutations in euchromatin histone methyltransferase 1 (EHMT1) gene, and point mutations in EHMT1 can cause also severe intellectual disability and behavioral disorders [3–6]. A young patient, the only child from healthy non consanguineous parents, whose birth weight was 2850 g and Apgar score was 8 and 10 at 10 and 50, respectively, showed a head circumference of 31.5 cm (\3 centile), associated with mild dysmorphic traits, including hypertelorism, prognathism, midfacial hypoplasia and synophrys. From the age of 2 days, he suffered clusters of clonic seizures, mainly interesting the left side though sometime shifting to the other side. EEG recordings showed multifocal sharpwave discharges over both hemispheres (Fig. 1a). A first brain MRI at 2 months of age showed a thin corpus callosum with a slight white matter reduction and mildly enlarged lateral ventricles. In the following years several anticonvulsant drugs, alone or in combination, were tried without achieving complete seizure control. At 3.5 years, his height was 91 cm (\3 centile), weight kg 12.200 (3 centile) and head circumference 46 cm ([3 centile), and he presented left cryptorchidism and cardiac mild ventricular septal defect. A second brain MRI confirmed the same features previously described. Suspecting a possible genetic cause of the disease, WES was performed on genomic DNA isolated from peripheral blood leukocytes of the patient as previously described [7]. Genetic analysis of his parents could not be performed, due to lack of consent on their part. A summary of the WES results is reported in Table 1. Data analysis revealed the presence of an heterozygous c.362G[A mutation in the EHMT1 gene, causing a glycine to serine (G121S) substitution in the protein (Fig. 1b, left). This prompted diagnosis of Kleefstra syndrome (KS). The patient, however, suffered also from a severe epileptic disorder, a finding to our knowledge rarely described in this syndrome. Interestingly, we identified also & Alessandro Weisz aweisz@unisa.it

Relations of gut liver axis components and gut microbiota in obese children with fatty liver: A pilot study

The prevalence of metabolic syndrome and its hepatic component — non-alcoholic fatty liver disease (NAFLD) — has increased alarmingly, paralleling the worldwide obesity epidemics. The pathophysiology of NAFLD is not clearly understood, but it has been proposed to be the result of multiple ‘‘hits’’[1]. A number of studies increasingly supports the pathogenetic role also of the gut microbiota (GM) both in NAFLD onset and progression. In this respect, GM would exert its noxious effects through the dysfunction of the gut-liver axis (GLA), which includes some or all of the following components: increased intestinal permeability (IP), endogenous ethanol (ETOH) and systemic endotoxin (LPS) concentrations [2,3]. As these players have hitherto not been simultaneously investigated in the same patient [reviewed in reference 4], the aim of our study was to explore the possible existence of reciprocal influences of several GLA components and GM composition in the same group of well characterized obese (Ob) children with and without fatty liver compared to normal-weight (NW) control peers. We studied 10 Ob Italian children, consecutively recruited at our center after parental agreement and written informed consent. The inclusion criteria were age 8—13 years, and a body mass index (BMI) > 97th percentile. Six non-Ob and non-overweight (BMI < 85th percentile), healthy normal-weight (NW) controls with normal anthropometric, clinical, laboratory and ultrasonographic (US) hepatic parameters and no other associated diseases were recruited among patients of the Pediatric Surgery Section listed for elective minor surgery. Lifestyle including eventual medications or alcohol exposition and total daily fructose and caloric intake and food preferences were investigated by multiple-choice questionnaires [5]. Weight, height, BMI values and percentiles,