Publications by authors named "Bartolomeo Augello"

40 Publications

Pro-Fibrotic Phenotype in a Patient with Segmental Stiff Skin Syndrome via TGF-β Signaling Overactivation.

Int J Mol Sci 2020 Jul 20;21(14). Epub 2020 Jul 20.

Division of Medical Genetics, Fondazione IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy.

Transforming growth factor β (TGF-β) superfamily signaling pathways are ubiquitous and essential for several cellular and physiological processes. The overexpression of TGF-β results in excessive fibrosis in multiple human disorders. Among them, stiff skin syndrome (SSS) is an ultrarare and untreatable condition characterized by the progressive thickening and hardening of the dermis, and acquired joint limitations. SSS is distinct in a widespread form, caused by recurrent germline variants of encoding a key molecule of the TGF-β signaling, and a segmental form with unknown molecular basis. Here, we report a 12-year-old female with segmental SSS, affecting the right upper limb with acquired thickening of the dermis evident at the magnetic resonance imaging, and progressive limitation of the elbow and shoulder. To better explore the molecular and cellular mechanisms that drive segmental SSS, several functional studies on patient's fibroblasts were employed. We hypothesized an impairment of TGF-β signaling and, consequently, a dysregulation of the associated downstream signaling. Lesional fibroblast studies showed a higher phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2), increased levels of nuclear factor-kB (NFkB), and a nuclear accumulation of phosphorylated Smad2 via Western blot and microscopy analyses. Quantitative PCR expression analysis of genes encoding key extracellular matrix proteins revealed increased levels of , , , and , while zymography assay reported a reduced metalloproteinase 2 enzymatic activity. In vitro exposure of patient's fibroblasts to losartan led to the partial restoration of normal transforming growth factor β (TGF-β) marker protein levels. Taken together, these data demonstrate that in our patient, segmental SSS is characterized by the overactivation of multiple TGF-β signaling pathways, which likely results in altered extracellular matrix composition and fibroblast homeostasis. Our results for the first time reported that aberrant TGF-β signaling may drive the pathogenesis of segmental SSS and might open the way to novel therapeutic approaches.
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http://dx.doi.org/10.3390/ijms21145141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404389PMC
July 2020

Expanding the phenotype associated to KMT2A variants: overlapping clinical signs between Wiedemann-Steiner and Rubinstein-Taybi syndromes.

Eur J Hum Genet 2021 Jan 8;29(1):88-98. Epub 2020 Jul 8.

Genetica Medica e Biologia Applicata, Dipartimento di Scienze della Salute, Università degli Studi di Milano, Milano, Italy.

Lysine-specific methyltransferase 2A (KMT2A) is responsible for methylation of histone H3 (K4H3me) and contributes to chromatin remodeling, acting as "writer" of the epigenetic machinery. Mutations in KMT2A were first reported in Wiedemann-Steiner syndrome (WDSTS). More recently, KMT2A variants have been described in probands with a specific clinical diagnosis comprised in the so-called chromatinopathies. Such conditions, including WDSTS, are a group of overlapping disorders caused by mutations in genes coding for the epigenetic machinery. Among them, Rubinstein-Taybi syndrome (RSTS) is mainly caused by heterozygous pathogenic variants in CREBBP or EP300. In this work, we used next generation sequencing (either by custom-made panel or by whole exome) to identify alternative causative genes in individuals with a RSTS-like phenotype negative to CREBBP and EP300 mutational screening. In six patients we identified different novel unreported variants in KMT2A gene. The identified variants are de novo in at least four out of six tested individuals and all of them display some typical RSTS phenotypic features but also WDSTS specific signs. This study reinforces the concept that germline variants affecting the epigenetic machinery lead to a shared molecular effect (alteration of the chromatin state) determining superimposable clinical conditions.
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http://dx.doi.org/10.1038/s41431-020-0679-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852672PMC
January 2021

Customised next-generation sequencing multigene panel to screen a large cohort of individuals with chromatin-related disorder.

J Med Genet 2020 11 13;57(11):760-768. Epub 2020 Mar 13.

Division of Medical Genetics, IRCCS Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy

Background: The regulation of the chromatin state by epigenetic mechanisms plays a central role in gene expression, cell function, and maintenance of cell identity. Hereditary disorders of chromatin regulation are a group of conditions caused by abnormalities of the various components of the epigenetic machinery, namely writers, erasers, readers, and chromatin remodelers. Although neurological dysfunction is almost ubiquitous in these disorders, the constellation of additional features characterizing many of these genes and the emerging clinical overlap among them indicate the existence of a community of syndromes. The introduction of high-throughput next generation sequencing (NGS) methods for testing multiple genes simultaneously is a logical step for the implementation of diagnostics of these disorders.

Methods: We screened a heterogeneous cohort of 263 index patients by an NGS-targeted panel, containing 68 genes associated with more than 40 OMIM entries affecting chromatin function.

Results: This strategy allowed us to identify clinically relevant variants in 87 patients (32%), including 30 for which an alternative clinical diagnosis was proposed after sequencing analysis and clinical re-evaluation.

Conclusion: Our findings indicate that this approach is effective not only in disorders with locus heterogeneity, but also in order to anticipate unexpected misdiagnoses due to clinical overlap among cognate disorders. Finally, this work highlights the utility of a prompt diagnosis in such a clinically and genetically heterogeneous group of disorders that we propose to group under the umbrella term of chromatinopathies.
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http://dx.doi.org/10.1136/jmedgenet-2019-106724DOI Listing
November 2020

Novel Variants in Two Italian Patients with Classical-Like Ehlers-Danlos Syndrome.

Genes (Basel) 2019 11 25;10(12). Epub 2019 Nov 25.

Division of Medical Genetics, Fondazione IRCCS-Casa Sollievo della Sofferenza, 71043 San Giovanni Rotondo (Foggia), Italy.

-related classical-like Ehlers-Danlos syndrome (-clEDS) is an ultrarare type of Ehlers-Danlos syndrome due to biallelic variants in , encoding tenascin-X. Less than 30 individuals have been reported to date, mostly of Dutch origin and showing a phenotype resembling classical Ehlers-Danlos syndrome without atrophic scarring. -clEDS is likely underdiagnosed due to the complex structure of the locus, a fact that complicates diagnostic molecular testing. Here, we report two unrelated Italian women with -clEDS due to compound heterozygosity for alleles in . Both presented soft and hyperextensible skin, generalized joint hypermobility and related musculoskeletal complications, and chronic constipation. In addition, individual 1 showed progressive finger contractures and shortened metatarsals, while individual 2 manifested recurrent subconjunctival hemorrhages and an event of spontaneous rupture of the brachial vein. Molecular testing found the two previously unreported c.8278C > T p.(Gln2760*) and the c.(2358 + 1_2359 - 1)_(2779 + 1_2780 - 1)del variants in Individual 1, and the novel c.1150dupG p.(Glu384Glyfs*57) and the recurrent c.11435_11524+30del variants in Individual 2. mRNA analysis confirmed that the c.(2358 + 1_2359 - 1)_(2779 + 1_2780 - 1)del variant causes a frameshift leading to a predicted truncated protein [p.(Thr787Glyfs*40)]. This study refines the phenotype recently delineated in association with biallelic alleles in , and adds three novel variants to its mutational repertoire. Unusual digital anomalies seem confirmed as possibly peculiar of -clEDS, while vascular fragility could be more than a chance association also in this Ehlers-Danlos syndrome type.
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http://dx.doi.org/10.3390/genes10120967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947605PMC
November 2019

Generation of the induced human pluripotent stem cell lines CSSi009-A from a patient with a GNB5 pathogenic variant, and CSSi010-A from a CRISPR/Cas9 engineered GNB5 knock-out human cell line.

Stem Cell Res 2019 10 22;40:101547. Epub 2019 Aug 22.

Division of Medical Genetics, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy. Electronic address:

GNB5 loss-of-function pathogenic variants cause IDDCA, a rare autosomal recessive human genetic disease characterized by infantile onset of intellectual disability, sinus bradycardia, hypotonia, visual abnormalities, and epilepsy. We generated human induced pluripotent stem cells (hiPSCs) from skin fibroblasts of a patient with the homozygous c.136delG frameshift variant, and a GNB5 knock-out (KO) line by CRISPR/Cas9 editing. hiPSCs express common pluripotency markers and differentiate into the three germ layers. These lines represent a powerful cellular model to study the molecular basis of GNB5-related disorders as well as offer an in vitro model for drug screening.
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http://dx.doi.org/10.1016/j.scr.2019.101547DOI Listing
October 2019

Mutational spectrum and clinical signatures in 114 families with hereditary multiple osteochondromas: insights into molecular properties of selected exostosin variants.

Hum Mol Genet 2019 07;28(13):2133-2142

Division of Medical Genetics, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy.

Hereditary multiple osteochondromas (HMO) is a rare autosomal dominant skeletal disorder, caused by heterozygous variants in either EXT1 or EXT2, which encode proteins involved in the biogenesis of heparan sulphate. Pathogenesis and genotype-phenotype correlations remain poorly understood. We studied 114 HMO families (158 affected individuals) with causative EXT1 or EXT2 variants identified by Sanger sequencing, or multiplex ligation-dependent probe amplification and qPCR. Eighty-seven disease-causative variants (55 novel and 32 known) were identified including frameshift (42%), nonsense (32%), missense (11%), splicing (10%) variants and genomic rearrangements (5%). Informative clinical features were available for 42 EXT1 and 27 EXT2 subjects. Osteochondromas were more frequent in EXT1 as compared to EXT2 patients. Anatomical distribution of lesions showed significant differences based on causative gene. Microscopy analysis for selected EXT1 and EXT2 variants verified that EXT1 and EXT2 mutants failed to co-localize each other and loss Golgi localization by surrounding the nucleus and/or assuming a diffuse intracellular distribution. In a cell viability study, cells expressing EXT1 and EXT2 mutants proliferated more slowly than cells expressing wild-type proteins. This confirms the physiological relevance of EXT1 and EXT2 Golgi co-localization and the key role of these proteins in the cell cycle. Taken together, our data expand genotype-phenotype correlations, offer further insights in the pathogenesis of HMO and open the path to future therapies.
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http://dx.doi.org/10.1093/hmg/ddz046DOI Listing
July 2019

Dissecting KMT2D missense mutations in Kabuki syndrome patients.

Hum Mol Genet 2018 11;27(21):3651-3668

Division of Medical Genetics, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo, Italy.

Kabuki syndrome is a rare autosomal dominant condition characterized by facial features, various organs malformations, postnatal growth deficiency and intellectual disability. The discovery of frequent germline mutations in the histone methyltransferase KMT2D and the demethylase KDM6A revealed a causative role for histone modifiers in this disease. However, the role of missense mutations has remained unexplored. Here, we expanded the mutation spectrum of KMT2D and KDM6A in KS by identifying 37 new KMT2D sequence variants. Moreover, we functionally dissected 14 KMT2D missense variants, by investigating their impact on the protein enzymatic activity and the binding to members of the WRAD complex. We demonstrate impaired H3K4 methyltransferase activity in 9 of the 14 mutant alleles and show that this reduced activity is due in part to disruption of protein complex formation. These findings have relevant implications for diagnostic and counseling purposes in this disease.
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http://dx.doi.org/10.1093/hmg/ddy241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488975PMC
November 2018

Correction: MYC-containing amplicons in acute myeloid leukemia: genomic structures, evolution, and transcriptional consequences.

Leukemia 2018 10;32(10):2304

Department of Biology, University of Bari "Aldo Moro", Bari, Italy.

In the original version of this Article, the affiliation details for Giovanni Martinelli were incorrectly given as 'Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, 40138, Bologna, Italy' and it should have been given as 'Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy and not Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, 40138, Bologna, Italy.'Furthermore, the original version of this Article contained an error in the spelling of the authors Alberto L'Abbate and Pietro D'Addabbo, an acute accent was used instead of an apostrophe for these authors names.These errors have now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41375-018-0177-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608236PMC
October 2018

TRIM50 regulates Beclin 1 proautophagic activity.

Biochim Biophys Acta Mol Cell Res 2018 Jun 29;1865(6):908-919. Epub 2018 Mar 29.

Division of Medical Genetics, IRCCS Casa Sollievo Della Sofferenza, Viale Cappuccini, 71013 San Giovanni Rotondo, Italy. Electronic address:

Autophagy is a catabolic process needed for maintaining cell viability and homeostasis in response to numerous stress conditions. Emerging evidence indicates that the ubiquitin system has a major role in this process. TRIMs, an E3 ligase protein family, contribute to selective autophagy acting as receptors and regulators of the autophagy proteins recognizing endogenous or exogenous targets through intermediary autophagic tags, such as ubiquitin. Here we report that TRIM50 fosters the initiation phase of starvation-induced autophagy and associates with Beclin1, a central component of autophagy initiation complex. We show that TRIM50, via the RING domain, ubiquitinates Beclin 1 in a K63-dependent manner enhancing its binding with ULK1 and autophagy activity. Finally, we found that the Lys-372 residue of TRIM50, critical for its own acetylation, is necessary for its E3 ligase activity that governs Beclin1 ubiquitination. Our study expands the roles of TRIMs in regulating selective autophagy, revealing an acetylation-ubiquitination dependent control for autophagy modulation.
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http://dx.doi.org/10.1016/j.bbamcr.2018.03.011DOI Listing
June 2018

MYC-containing amplicons in acute myeloid leukemia: genomic structures, evolution, and transcriptional consequences.

Leukemia 2018 10 22;32(10):2152-2166. Epub 2018 Feb 22.

Department of Biology, University of Bari "Aldo Moro", Bari, Italy.

Double minutes (dmin), homogeneously staining regions, and ring chromosomes are vehicles of gene amplification in cancer. The underlying mechanism leading to their formation as well as their structure and function in acute myeloid leukemia (AML) remain mysterious. We combined a range of high-resolution genomic methods to investigate the architecture and expression pattern of amplicons involving chromosome band 8q24 in 23 cases of AML (AML-amp). This revealed that different MYC-dmin architectures can coexist within the same leukemic cell population, indicating a step-wise evolution rather than a single event origin, such as through chromothripsis. This was supported also by the analysis of the chromothripsis criteria, that poorly matched the model in our samples. Furthermore, we found that dmin could evolve toward ring chromosomes stabilized by neocentromeres. Surprisingly, amplified genes (mainly PVT1) frequently participated in fusion transcripts lacking a corresponding DNA template. We also detected a significant overexpression of the circular RNA of PVT1 (circPVT1) in AML-amp cases versus AML with a normal karyotype. Our results show that 8q24 amplicons in AML are surprisingly plastic DNA structures with an unexpected association to novel fusion transcripts and circular RNAs.
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http://dx.doi.org/10.1038/s41375-018-0033-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170393PMC
October 2018

Clinical and Neurobehavioral Features of Three Novel Kabuki Syndrome Patients with Mosaic KMT2D Mutations and a Review of Literature.

Int J Mol Sci 2017 Dec 28;19(1). Epub 2017 Dec 28.

Division of Medical Genetics, IRCSS Casa Sollievo della Sofferenza Hospital, viale Cappuccini, 71013 San Giovanni Rotondo, Italy.

Kabuki syndrome (KS) is a rare disorder characterized by multiple congenital anomalies and variable intellectual disability caused by mutations in and , two interacting chromatin modifier responsible respectively for 56-75% and 5-8% of the cases. To date, three KS patients with mosaic deletions in blood lymphocytes have been described. We report on three additional subjects displaying gene mosaics including one in which a single nucleotide change results in a new frameshift mutation (p.L1199HfsX7), and two with already-known nonsense mutations (p.R4484X and p.R5021X). Consistent with previously published cases, mosaic mutations may result in mild KS facial dysmorphisms and clinical and neurobehavioral features, suggesting that these characteristics could represent the handles for genetic testing of individuals with slight KS-like traits.
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http://dx.doi.org/10.3390/ijms19010082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5796032PMC
December 2017

A New Split Hand/Foot Malformation with Long Bone Deficiency Familial Case.

J Pediatr Genet 2017 Jun 31;6(2):98-102. Epub 2016 Aug 31.

Medical Genetics Unit, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG), Italy.

Split hand/foot malformation with long bone deficiency (SHFLD) is a congenital limb anomaly where hands and/or feet cleft and syndactyly are associated with long bone defects, usually involving the tibia. Previously published data reported that 17p13.3 chromosomal duplication, including the gene, has been associated with the distinct entity, termed SHFLD3 (OMIM 612576), inherited as an autosomal dominant trait. Here, we present a family with three members affected by SHFLD harboring duplication. We exploited in vitro differentiation system to promote proband's skin fibroblasts toward osteoblastic lineage, and we observed a slight but consistent delay in the mineralization pattern. This result possibly suggests an impairment of the osteogenic process in the affected members.
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http://dx.doi.org/10.1055/s-0036-1588029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423807PMC
June 2017

Clock-genes and mitochondrial respiratory activity: Evidence of a reciprocal interplay.

Biochim Biophys Acta 2016 Aug 7;1857(8):1344-1351. Epub 2016 Apr 7.

Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy. Electronic address:

In the past few years mounting evidences have highlighted the tight correlation between circadian rhythms and metabolism. Although at the organismal level the central timekeeper is constituted by the hypothalamic suprachiasmatic nuclei practically all the peripheral tissues are equipped with autonomous oscillators made up by common molecular clockworks represented by circuits of gene expression that are organized in interconnected positive and negative feed-back loops. In this study we exploited a well-established in vitro synchronization model to investigate specifically the linkage between clock gene expression and the mitochondrial oxidative phosphorylation (OxPhos). Here we show that synchronized cells exhibit an autonomous ultradian mitochondrial respiratory activity which is abrogated by silencing the master clock gene ARNTL/BMAL1. Surprisingly, pharmacological inhibition of the mitochondrial OxPhos system resulted in dramatic deregulation of the rhythmic clock-gene expression and a similar result was attained with mtDNA depleted cells (Rho0). Our findings provide a novel level of complexity in the interlocked feedback loop controlling the interplay between cellular bioenergetics and the molecular clockwork. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
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http://dx.doi.org/10.1016/j.bbabio.2016.03.035DOI Listing
August 2016

Deregulated expression of cryptochrome genes in human colorectal cancer.

Mol Cancer 2016 Jan 15;15. Epub 2016 Jan 15.

Division of Epidemiology and Health Statistics, IRCCS Scientific Institute and Regional General Hospital "Casa Sollievo della Sofferenza", San Giovanni Rotondo, FG, Italy.

Background: Circadian disruption and deranged molecular clockworks are involved in carcinogenesis. The cryptochrome genes (CRY1 and CRY2) encode circadian proteins important for the functioning of biological oscillators. Their expression in human colorectal cancer (CRC) and in colon cancer cell lines has not been evaluated so far.

Methods: We investigated CRY1 and CRY2 expression in fifty CRCs and in the CaCo2, HCT116, HT29, SW480 cell lines.

Results: CRY1 (p = 0.01) and CRY2 (p < 0.0001) expression was significantly changed in tumour tissue, as confirmed in a large independent CRC dataset. In addition, lower CRY1 mRNA levels were observed in patients in the age range of 62-74 years (p = 0.018), in female patients (p = 0.003) and in cancers located at the transverse colon (p = 0.008). Lower CRY2 levels were also associated with cancer location at the transverse colon (p = 0.007). CRC patients displaying CRY1 (p = 0.042) and CRY2 (p = 0.043) expression levels over the median were hallmarked by a poorer survival rate. Survey of selected colon cancer cell lines evidenced variable levels of cryptochrome genes expression and time-dependent changes in their mRNA levels. Moreover, they showed reduced apoptosis, increased proliferation and different response to 5-fluorouracil and oxaliplatin upon CRY1 and CRY2 ectopic expression. The relationship with p53 status came out as an additional layer of regulation: higher CRY1 and CRY2 protein levels coincided with a wild type p53 as in HCT116 cells and this condition only marginally affected the apoptotic and cell proliferation characteristics of the cells upon CRY ectopic expression. Conversely, lower CRY and CRY2 levels as in HT29 and SW480 cells coincided with a mutated p53 and a more robust apoptosis and proliferation upon CRY transfection. Besides, an heterogeneous pattern of ARNTL, WEE and c-MYC expression hallmarked the chosen colon cancer cell lines and likely influenced their phenotypic changes.

Conclusion: Cryptochrome gene expression is altered in CRC, particularly in elderly subjects, female patients and cancers located at the transverse colon, affecting overall survival. Altered CRY1 and CRY2 expression patterns and the interplay with the genetic landscape in colon cancer cells may underlie phenotypic divergence that could influence disease behavior as well as CRC patients survival and response to chemotherapy.
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http://dx.doi.org/10.1186/s12943-016-0492-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4714521PMC
January 2016

Clock genes-dependent acetylation of complex I sets rhythmic activity of mitochondrial OxPhos.

Biochim Biophys Acta 2016 Apr 28;1863(4):596-606. Epub 2015 Dec 28.

Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy. Electronic address:

Physiology of living beings show circadian rhythms entrained by a central timekeeper present in the hypothalamic suprachiasmatic nuclei. Nevertheless, virtually all peripheral tissues hold autonomous molecular oscillators constituted essentially by circuits of gene expression that are organized in negative and positive feed-back loops. Accumulating evidence reveals that cell metabolism is rhythmically controlled by cell-intrinsic molecular clocks and the specific pathways involved are being elucidated. Here, we show that in vitro-synchronized cultured cells exhibit BMAL1-dependent oscillation in mitochondrial respiratory activity, which occurs irrespective of the cell type tested, the protocol of synchronization used and the carbon source in the medium. We demonstrate that the rhythmic respiratory activity is associated to oscillation in cellular NAD content and clock-genes-dependent expression of NAMPT and Sirtuins 1/3 and is traceable back to the reversible acetylation of a single subunit of the mitochondrial respiratory chain Complex I. Our findings provide evidence for a new interlocked transcriptional-enzymatic feedback loop controlling the molecular interplay between cellular bioenergetics and the molecular clockwork.
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http://dx.doi.org/10.1016/j.bbamcr.2015.12.018DOI Listing
April 2016

TRIM8 downregulation in glioma affects cell proliferation and it is associated with patients survival.

BMC Cancer 2015 Jun 16;15:470. Epub 2015 Jun 16.

Medical Genetics Unit, IRCCS Casa Sollievo della Sofferenza, Poliambulatorio Giovanni Paolo II, I-71013, San Giovanni Rotondo (FG), Italy.

Background: Human gliomas are a heterogeneous group of primary malignant brain tumors whose molecular pathogenesis is not yet solved. In this regard, a major research effort has been directed at identifying novel specific glioma-associated genes. Here, we investigated the effect of TRIM8 gene in glioma.

Methods: TRIM8 transcriptional level was profiled in our own glioma cases collection by qPCR and confirmed in the independent TCGA glioma cohort. The association between TRIM8 expression and Overall Survival and Progression-free Survival in TCGA cohort was determined by using uni-multivariable Cox regression analysis. The effect of TRIM8 on patient glioma cell proliferation was evaluated by performing MTT and clonogenic assays. The mechanisms causing the reduction of TRIM8 expression were explored by using qPCR and in vitro assays.

Results: We showed that TRIM8 expression correlates with unfavorable clinical outcome in glioma patients. We found that a restored TRIM8 expression induced a significant reduction of clonogenic potential in U87MG and patient's glioblastoma cells. Finally we provide experimental evidences showing that miR-17 directly targets the 3' UTR of TRIM8 and post-transcriptionally represses the expression of TRIM8.

Conclusions: Our study provides evidences that TRIM8 may participate in the carcinogenesis and progression of glioma and that the transcriptional repression of TRIM8 might have potential value for predicting poor prognosis in glioma patients.
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http://dx.doi.org/10.1186/s12885-015-1449-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468980PMC
June 2015

7q11.23 dosage-dependent dysregulation in human pluripotent stem cells affects transcriptional programs in disease-relevant lineages.

Nat Genet 2015 Feb 15;47(2):132-41. Epub 2014 Dec 15.

1] Department of Experimental Oncology, European Institute of Oncology (Istituto di Ricovero e Cura a Carattere Scientifico, IRCCS), Milan, Italy. [2] Department of Health Sciences, University of Milan, Milan, Italy.

Cell reprogramming promises to make characterization of the impact of human genetic variation on health and disease experimentally tractable by enabling the bridging of genotypes to phenotypes in developmentally relevant human cell lineages. Here we apply this paradigm to two disorders caused by symmetrical copy number variations of 7q11.23, which display a striking combination of shared and symmetrically opposite phenotypes--Williams-Beuren syndrome and 7q-microduplication syndrome. Through analysis of transgene-free patient-derived induced pluripotent stem cells and their differentiated derivatives, we find that 7q11.23 dosage imbalance disrupts transcriptional circuits in disease-relevant pathways beginning in the pluripotent state. These alterations are then selectively amplified upon differentiation of the pluripotent cells into disease-relevant lineages. A considerable proportion of this transcriptional dysregulation is specifically caused by dosage imbalances in GTF2I, which encodes a key transcription factor at 7q11.23 that is associated with the LSD1 repressive chromatin complex and silences its dosage-sensitive targets.
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http://dx.doi.org/10.1038/ng.3169DOI Listing
February 2015

DPP6 gene disruption in a family with Gilles de la Tourette syndrome.

Neurogenetics 2014 Oct 17;15(4):237-42. Epub 2014 Aug 17.

Medical Genetics Unit, Department of Surgical and Biomedical Sciences, University of Perugia, Hospital "S. M. della Misericordia", Via E. dal Pozzo, 06123, Perugia, Italy,

Gilles de la Tourette syndrome (TS) is a neurodevelopmental disorder characterized by multiple motor and vocal tics, frequently associated with psychiatric co-morbidities. Despite the significant level of heritability, the genetic architecture of TS still remains elusive. Herein, we investigated an Italian family where an 8-year-old boy, his father, and paternal uncle have a diagnosis of TS. Array-CGH and high resolution SNP-array analyses revealed a heterozygous microdeletion of ∼135 kb at the 7q36.2 locus in the proband and his father. Fluorescent in situ hybridization and quantitative PCR (qPCR) analyses confirmed the presence of the alteration also in the paternal uncle. The deletion selectively involves the first exon of the DPP6 gene, leading to a down-regulation of its expression, as demonstrated by the reduced messenger RNA (mRNA) levels assessed by RT-qPCR. The DPP6 gene encodes for a type II membrane glycoprotein expressed predominantly in the central nervous system. To date, a de novo DPP6 exonic duplication, of uncertain significance, was reported in one patient with TS. Moreover, the DPP6 gene has been implicated in the pathogenesis of autism spectrum disorder (ASD) and, notably, in haloperidol-induced dyskinesia. This first familial case provides evidence for association between DPP6 haploinsufficiency and TS, further suggesting a plausible molecular link between TS and ASD, and might shed some light on the efficacy and tolerability profiles of antidopaminergic agents used for tic management, thus prompting further studies on a larger cohort of patients.
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http://dx.doi.org/10.1007/s10048-014-0418-9DOI Listing
October 2014

Molecular analysis, pathogenic mechanisms, and readthrough therapy on a large cohort of Kabuki syndrome patients.

Hum Mutat 2014 Jul 9;35(7):841-50. Epub 2014 Apr 9.

Medical Genetics Unit, IRCCS Casa Sollievo Della Sofferenza Hospital, San Giovanni Rotondo, Italy.

Kabuki syndrome (KS) is a multiple congenital anomalies syndrome characterized by characteristic facial features and varying degrees of mental retardation, caused by mutations in KMT2D/MLL2 and KDM6A/UTX genes. In this study, we performed a mutational screening on 303 Kabuki patients by direct sequencing, MLPA, and quantitative PCR identifying 133 KMT2D, 62 never described before, and four KDM6A mutations, three of them are novel. We found that a number of KMT2D truncating mutations result in mRNA degradation through the nonsense-mediated mRNA decay, contributing to protein haploinsufficiency. Furthermore, we demonstrated that the reduction of KMT2D protein level in patients' lymphoblastoid and skin fibroblast cell lines carrying KMT2D-truncating mutations affects the expression levels of known KMT2D target genes. Finally, we hypothesized that the KS patients may benefit from a readthrough therapy to restore physiological levels of KMT2D and KDM6A proteins. To assess this, we performed a proof-of-principle study on 14 KMT2D and two KDM6A nonsense mutations using specific compounds that mediate translational readthrough and thereby stimulate the re-expression of full-length functional proteins. Our experimental data showed that both KMT2D and KDM6A nonsense mutations displayed high levels of readthrough in response to gentamicin treatment, paving the way to further studies aimed at eventually treating some Kabuki patients with readthrough inducers.
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http://dx.doi.org/10.1002/humu.22547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4234006PMC
July 2014

TBC1D7 mutations are associated with intellectual disability, macrocrania, patellar dislocation, and celiac disease.

Hum Mutat 2014 Apr;35(4):447-51

Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland; Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland.

TBC1D7 forms a complex with TSC1 and TSC2 that inhibits mTORC1 signaling and limits cell growth. Mutations in TBC1D7 were reported in a family with intellectual disability (ID) and macrocrania. Using exome sequencing, we identified two sisters homozygote for the novel c.17_20delAGAG, p.R7TfsX21 TBC1D7 truncating mutation. In addition to the already described macrocephaly and mild ID, they share osteoarticular defects, patella dislocation, behavioral abnormalities, psychosis, learning difficulties, celiac disease, prognathism, myopia, and astigmatism. Consistent with a loss-of-function of TBC1D7, the patient's cell lines show an increase in the phosphorylation of 4EBP1, a direct downstream target of mTORC1 and a delay in the initiation of the autophagy process. This second family allows enlarging the phenotypic spectrum associated with TBC1D7 mutations and defining a TBC1D7 syndrome. Our work reinforces the involvement of TBC1D7 in the regulation of mTORC1 pathways and suggests an altered control of autophagy as possible cause of this disease.
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http://dx.doi.org/10.1002/humu.22529DOI Listing
April 2014

HDAC6 mediates the acetylation of TRIM50.

Cell Signal 2014 Feb 2;26(2):363-9. Epub 2013 Dec 2.

Medical Genetics Unit, IRCCS Casa Sollievo Della Sofferenza Hospital, 71013 San Giovanni Rotondo, Italy; Scienze della Riproduzione e dello Sviluppo, University of Trieste, Italy. Electronic address:

The E3 Ubiquitin ligase TRIM50 promotes the formation and clearance of aggresome-associated polyubiquitinated proteins through HDAC6 interaction, a tubulin specific deacetylase that regulates microtubule-dependent aggresome formation. In this report we showed that TRIM50 is a target of HDAC6 with Lys-372 as a critical residue for acetylation. We identified p300 and PCAF as two TRIM50 acetyltransferases and we further showed that a balance between ubiquitination and acetylation regulates TRIM50 degradation.
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http://dx.doi.org/10.1016/j.cellsig.2013.11.036DOI Listing
February 2014

Genetic variants of membrane metallopeptidase genes in inflammatory bowel diseases.

Dig Liver Dis 2013 Dec 1;45(12):1003-10. Epub 2013 Jul 1.

Department of Surgery, IRCCS "Casa Sollievo Della Sofferenza" Hospital, San Giovanni Rotondo, Italy.

Background: The substance P pathway modulates neuroimmune interactions during intestinal inflammation.

Aims: To analyse mucosal expression and genetic variants of the genes coding for substance P, neurokinin-1 receptor and neutral endopeptidase in patients with inflammatory bowel disease.

Methods: qRT-PCR was used to analyse mRNA levels in matched, paired samples of inflamed colonic mucosa and adjacent non-inflamed endoscopic tissue from 26 Crohn's disease and 25 ulcerative colitis patients. Allele and genotype frequencies of tag-SNPs were determined in 908 Crohn's disease, 929 ulcerative colitis, and 853 controls. Expression levels and genotype distributions were examined within patients' clinical sub-phenotypes.

Results: All 3 evaluated genes were overexpressed in inflamed tissues from Crohn's disease (P=0.033, P=4×10(-5), P=0.001), while in ulcerative colitis only higher levels of the gene coding for neutral endopeptidase were statistically significant (P=2.5×10(-5)). Smoking habit and perianal disease were significantly associated with substance P (P=0.002) and neurokinin-1 receptor levels (P=0.02) in Crohn's disease. Neutral endopeptidase rs701109 variant was associated with inflammatory bowel disease (Crohn's disease: P=0.022; ulcerative colitis: P=0.045), and with the need for colectomy in ulcerative colitis (P=0.008, OR=2.46, 95% CI=1.27-4.76).

Conclusions: Genetic variants of the gene coding for neutral endopeptidase might affect the neuroimmune interaction during intestinal inflammation and influence clinical sub-phenotypes in patients with inflammatory bowel disease.
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http://dx.doi.org/10.1016/j.dld.2013.05.010DOI Listing
December 2013

Smaller and larger deletions of the Williams Beuren syndrome region implicate genes involved in mild facial phenotype, epilepsy and autistic traits.

Eur J Hum Genet 2014 Jan 12;22(1):64-70. Epub 2013 Jun 12.

1] Medical Genetics Unit, IRCCS Casa Sollievo Della Sofferenza Hospital, San Giovanni Rotondo, Italy [2] Reproduction and Development Sciences, University of Trieste, Italy.

Williams Beuren syndrome (WBS) is a multisystemic disorder caused by a hemizygous deletion of 1.5 Mb on chromosome 7q11.23 spanning 28 genes. A few patients with larger and smaller WBS deletion have been reported. They show clinical features that vary between isolated SVAS to the full spectrum of WBS phenotype, associated with epilepsy or autism spectrum behavior. Here we describe four patients with atypical WBS 7q11.23 deletions. Two carry ~3.5 Mb larger deletion towards the telomere that includes Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxigenase activation protein gamma (YWHAG) genes. Other two carry a shorter deletion of ~1.2 Mb at centromeric side that excludes the distal WBS genes BAZ1B and FZD9. Along with previously reported cases, genotype-phenotype correlation in the patients described here further suggests that haploinsufficiency of HIP1 and YWHAG might cause the severe neurological and neuropsychological deficits including epilepsy and autistic traits, and that the preservation of BAZ1B and FZD9 genes may be related to mild facial features and moderate neuropsychological deficits. This report highlights the importance to characterize additional patients with 7q11.23 atypical deletions comparing neuropsychological and clinical features between these individuals to shed light on the pathogenic role of genes within and flanking the WBS region.
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http://dx.doi.org/10.1038/ejhg.2013.101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3865388PMC
January 2014

Associations between genetic polymorphisms in IL-33, IL1R1 and risk for inflammatory bowel disease.

PLoS One 2013 25;8(4):e62144. Epub 2013 Apr 25.

Division of Gastroenterology, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy.

Background: Recent evidence suggests that the IL-33/IL1RL1 axis plays a critical role in several autoimmune and inflammatory disorders; however, its mechanistic role in inflammatory bowel disease (IBD) has not been clearly defined. We investigated the contribution of IL-33 and IL1RL1 polymorphisms to IBD risk, and possible correlations with phenotype in an Italian cohort of adult and pediatric patients.

Methods: We evaluated the association of six SNPs in IL-33 and IL1RL1 genes, in 805 Crohn's disease (CD), 816 ulcerative colitis (UC), and 752 controls, using Taqman. IL-33 and IL1RL1 mRNA expression was also analyzed.

Results: Significant allele and genotype associations with IL-33 rs3939286 were found in CD (P = 0.004; P = 0.035) and UC patients (P = 0.002; P = 0.038). After stratifying the cohort for age at diagnosis, the differences remained significant only in the IBD adult-onset. Significant associations were also obtained in CD patients with two IL1RL1 polymorphisms (rs13015714 and rs2058660, P<0.015). By combining homo- and heterozygous carriers of the rs13015714 risk allele, differences were still significant for both CD adult- and pediatric-onset. Upon genotype-phenotype evaluation, an increased frequency of extensive colitis in adult UC (P = 0.019) and in steroid-responsive pediatric patients (P = 0.024) carrying the IL-33 rs3939286 risk genotype, was observed. mRNA expression of IL-33 and IL1RL1 in inflamed IBD biopsy samples was significantly increased.

Conclusions: Common IL-33 and IL1RL1 polymorphisms contribute to the risk of IBD in an Italian cohort of adult and pediatric patients, with some influence on sub-phenotypes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062144PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636262PMC
November 2013

Interplay between SOX9, β-catenin and PPARγ activation in colorectal cancer.

Biochim Biophys Acta 2013 Aug 11;1833(8):1853-65. Epub 2013 Apr 11.

Department of Medical Sciences, IRCCS Scientific Institute and Regional General Hospital, Italy.

Colorectal carcinogenesis relies on loss of homeostasic mechanisms regulating cell proliferation, differentiation and survival. These cell processes have been reported to be influenced independently by transcription factors activated downstream of the Wnt pathway, such as SOX9 and β-catenin, and by the nuclear receptor PPARγ. The purpose of this study was to explore the expression levels and functional link between SOX9, β-catenin and PPARγ in the pathogenesis of colorectal cancer (CRC). We evaluated SOX9, β-catenin and PPARγ expression levels on human CRC specimens by qPCR and immunoblot detection. We tested the hypothesis that PPARγ activation might affect SOX9 and β-catenin expression using four colon cancer cell lines (CaCo2, SW480, HCT116, and HT29 cells). In CRC tissues SOX9 resulted up-regulated at both mRNA and protein levels when compared to matched normal mucosa, β-catenin resulted up-regulated at protein levels, while PPARG mRNA and PPARγ protein levels were down-regulated. A significant relationship was observed between high PPARG and SOX9 expression levels in the tumor tissue and female gender (p=0.005 and p=0.04, respectively), and between high SOX9 expression in the tumor tissue and age (p=0.04) and microsatellite instability (MSI), in particular with MSI-H (p=0.0002). Moreover, treatment with the synthetic PPARγ ligand rosiglitazone induced different changes of SOX9 and β-catenin expression and subcellular localization in the colon cancer cell lines examined. In conclusion, SOX9, β-catenin and PPARγ expression levels are deregulated in the CRC tissue, and in colon cancer cell lines ligand-dependent PPARγ activation unevenly influences SOX9 and β-catenin expression and subcellular localization, suggesting a variable mechanistic role in colon carcinogenesis.
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http://dx.doi.org/10.1016/j.bbamcr.2013.04.004DOI Listing
August 2013

A fish-specific transposable element shapes the repertoire of p53 target genes in zebrafish.

PLoS One 2012 31;7(10):e46642. Epub 2012 Oct 31.

Medical Genetics Unit, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy.

Transposable elements, as major components of most eukaryotic organisms' genomes, define their structural organization and plasticity. They supply host genomes with functional elements, for example, binding sites of the pleiotropic master transcription factor p53 were identified in LINE1, Alu and LTR repeats in the human genome. Similarly, in this report we reveal the role of zebrafish (Danio rerio) EnSpmN6_DR non-autonomous DNA transposon in shaping the repertoire of the p53 target genes. The multiple copies of EnSpmN6_DR and their embedded p53 responsive elements drive in several instances p53-dependent transcriptional modulation of the adjacent gene, whose human orthologs were frequently previously annotated as p53 targets. These transposons define predominantly a set of target genes whose human orthologs contribute to neuronal morphogenesis, axonogenesis, synaptic transmission and the regulation of programmed cell death. Consistent with these biological functions the orthologs of the EnSpmN6_DR-colonized loci are enriched for genes expressed in the amygdala, the hippocampus and the brain cortex. Our data pinpoint a remarkable example of convergent evolution: the exaptation of lineage-specific transposons to shape p53-regulated neuronal morphogenesis-related pathways in both a hominid and a teleost fish.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046642PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485254PMC
May 2013

Absence of deletion and duplication of MLL2 and KDM6A genes in a large cohort of patients with Kabuki syndrome.

Mol Genet Metab 2012 Nov 6;107(3):627-9. Epub 2012 Jul 6.

Unita' Operativa di Genetica Medica, Azienda Ospedaliera Bianchi-Melacrino-Morelli, Reggio Calabria, Italy.

Kabuki syndrome is a rare, multiple congenital anomaly/mental retardation syndrome caused by MLL2 point mutations and KDM6A microdeletions. We screened a large cohort of MLL2 mutation-negative patients for MLL2 and KDM6A exon(s) microdeletion and microduplication. Our assays failed to detect such rearrangements in MLL2 as well as in KDM6A gene. These results show that these genomic events are extremely rare in the Kabuki syndrome, substantiating its genetic heterogeneity and the search for additional causative gene(s).
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http://dx.doi.org/10.1016/j.ymgme.2012.06.019DOI Listing
November 2012

The E3-ubiquitin ligase TRIM50 interacts with HDAC6 and p62, and promotes the sequestration and clearance of ubiquitinated proteins into the aggresome.

PLoS One 2012 9;7(7):e40440. Epub 2012 Jul 9.

Medical Genetics Unit, IRCCS Casa Sollievo Della Sofferenza Hospital, San Giovanni Rotondo, Italy.

In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040440PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392214PMC
April 2013

TRIM8 modulates p53 activity to dictate cell cycle arrest.

Cell Cycle 2012 Feb 1;11(3):511-23. Epub 2012 Feb 1.

Institute for Biomedical Technologies, National Research Council, Bari, Italy.

p53 is a central hub in controlling cell proliferation. To maintain genome integrity in response to cellular stress, p53 directly regulates the transcription of genes involved in cell cycle arrest, DNA repair, apoptosis and/or senescence. An array of post-translational modifications and protein-protein interactions modulates its stability and activities in order to avoid malignant transformation. However, to date it is still not clear how cells decide their own fate in response to different types of stress. We described here that the human TRIM8 protein, a member of the TRIM family, is a new modulator of the p53-mediated tumor suppression mechanism. We showed that under stress conditions, such as UV exposure, p53 induced the expression of TRIM8, which in turn stabilized p53 leading to cell cycle arrest and reduction of cell proliferation through enhancement of CDKN1A (p21) and GADD45 expression. TRIM8 silencing reduced the capacity of p53 to activate genes involved in cell cycle arrest and DNA repair, in response to cellular stress. Concurrently, TRIM8 overexpression induced the degradation of the MDM2 protein, the principal regulator of p53 stability. Co-immunoprecipitation experiments showed that TRIM8 physically interacted with p53, impairing its interaction with MDM2. Altogether, our results reveal a previously unknown regulatory pathway controlling p53 activity and suggest TRIM8 as a novel therapeutic target to enhance p53 tumor suppressor activity.
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http://dx.doi.org/10.4161/cc.11.3.19008DOI Listing
February 2012