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Article ; Online: Next-Generation Sequencing Approaches to Define the Role of the Autophagy Lysosomal Pathway in Human Disease: The Example of LysoPlex.

Di Fruscio, Giuseppina / Banfi, Sandro / Nigro, Vincenzo / Ballabio, Andrea

Methods in molecular biology (Clifton, N.J.)

2017 Volume 1594, Page(s) 227–241

Abstract: Next-Generation Sequencing (NGS) technologies have deeply changed the throughput of genetic testing allowing analyzing millions of DNA fragments in parallel. One key application is the understanding of genetically heterogeneous and complex diseases where ...

Abstract	Next-Generation Sequencing (NGS) technologies have deeply changed the throughput of genetic testing allowing analyzing millions of DNA fragments in parallel. One key application is the understanding of genetically heterogeneous and complex diseases where 50-100 different genes may converge to control the same pathways. These disorders cannot be studied using traditional approaches, based on gene-by-gene Sanger sequencing. We have set up an NGS protocol based on a specific selection of DNA regions belonging to about 900 genes of the autophagy-lysosomal (ALP) pathway. We here specify all the technical steps and challenges of our protocol, named LysoPlex. This is based on the Haloplex technology and together with high-coverage sequencing empowers a high and uniform coverage of ALP genes. LysoPlex outplays other NGS applications in sensitivity and specificity, providing an accurate picture of all variations in ALP genes.
Language	English
Publishing date	2017
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-6934-0_15
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Article: KRAS Mutational Regression Is Associated With Oligo-Metastatic Status and Good Prognosis in Metastatic Colorectal Cancer.

Ottaiano, Alessandro / Nasti, Guglielmo / Santorsola, Mariachiara / Altieri, Vincenzo / Di Fruscio, Giuseppina / Circelli, Luisa / Luce, Amalia / Cossu, Alessia Maria / Scognamiglio, Giosuè / Perri, Francesco / Correra, Marco / Belli, Andrea / Delrio, Paolo / Botti, Gerardo / Caraglia, Michele

Frontiers in oncology

2021 Volume 11, Page(s) 632962

Abstract: Background: We previously reported that loss of : Material and methods: Patients were treated and followed-up according to European Society of Medical Oncology guidelines. Primary CRC FFPE tissue and metastatic circulating-free DNA were extracted ... ...

Abstract	Background: We previously reported that loss of Material and methods: Patients were treated and followed-up according to European Society of Medical Oncology guidelines. Primary CRC FFPE tissue and metastatic circulating-free DNA were extracted using the QIAamp DNA specific kits (Qiagen, Hilden, Germany). Samples were sequenced with the Oncomine Solid Tumour DNA kit (Thermo Fisher Scientific, Waltham, MA, USA). Plasma collection for liquid biopsy was done from 1 to 14 days before starting first-line chemotherapy. Analysis of the prognostic power of Results: One-hundred-fourteen patients were enrolled. Sixty-three patients presented with mutated Conclusions: Our data provide evidence that the evolutionary trajectories of
Language	English
Publishing date	2021-03-29
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2649216-7
ISSN	2234-943X
ISSN	2234-943X
DOI	10.3389/fonc.2021.632962
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Article ; Online: Are all the previously reported genetic variants in limb girdle muscular dystrophy genes pathogenic?

Di Fruscio, Giuseppina / Garofalo, Arcomaria / Mutarelli, Margherita / Savarese, Marco / Nigro, Vincenzo

European journal of human genetics : EJHG

2015 Volume 24, Issue 1, Page(s) 73–77

Abstract: Hundreds of variants in autosomal genes associated with the limb girdle muscular dystrophies (LGMDs) have been reported as being causative. However, in most cases the proof of pathogenicity derives from their non-occurrence in hundreds of healthy ... ...

Abstract	Hundreds of variants in autosomal genes associated with the limb girdle muscular dystrophies (LGMDs) have been reported as being causative. However, in most cases the proof of pathogenicity derives from their non-occurrence in hundreds of healthy controls and/or from segregation studies in small families. The limited statistics of the genetic variations in the general population may hamper a correct interpretation of the effect of variants on the protein. To clarify the meaning of low-frequency variants in LGMD genes, we have selected all variants described as causative in the Leiden Open Variation Database and the Human Gene Mutation Database. We have systematically searched for their frequency in the NHLBI GO Exome Sequencing Project (ESP) and in our internal database. Surprisingly, the ESP contains about 4% of the variants previously associated with a dominant inheritance and about 9% of those associated with a recessive inheritance. The putative disease alleles are much more frequent than those estimated considering the disease prevalence. In conclusion, we hypothesize that a number of disease-associated variants are non-pathogenic and that other variations are not fully penetrant, even if they affect the protein function, suggesting a more complex genetic mechanisms for such heterogeneous disorders.
MeSH term(s)	Age of Onset ; Alleles ; Cytoskeletal Proteins/genetics ; Databases, Genetic ; Dystrophin-Associated Proteins/genetics ; Exome ; Female ; Gene Frequency ; Genes, Dominant ; Genes, Recessive ; Genetic Variation ; Humans ; Male ; Muscular Dystrophies, Limb-Girdle/diagnosis ; Muscular Dystrophies, Limb-Girdle/genetics ; Muscular Dystrophies, Limb-Girdle/pathology ; Mutation ; Nerve Tissue Proteins/genetics ; Nuclear Matrix-Associated Proteins/genetics ; Penetrance
Chemical Substances	Cytoskeletal Proteins ; Dystrophin-Associated Proteins ; Nerve Tissue Proteins ; Nuclear Matrix-Associated Proteins
Language	English
Publishing date	2015-04-22
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1141470-4
ISSN	1476-5438 ; 1018-4813
ISSN (online)	1476-5438
ISSN	1018-4813
DOI	10.1038/ejhg.2015.76
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Article ; Online: Prediction of rare single-nucleotide causative mutations for muscular diseases in pooled next-generation sequencing experiments.

Ferraro, Maria Brigida / Savarese, Marco / Di Fruscio, Giuseppina / Nigro, Vincenzo / Guarracino, Mario Rosario

Journal of computational biology : a journal of computational molecular cell biology

2014 Volume 21, Issue 9, Page(s) 665–675

Abstract: Next-generation sequencing (NGS) is a new approach for biomedical research, useful for the diagnosis of genetic diseases in extremely heterogeneous conditions. In this work, we describe how data generated by high-throughput NGS experiments can be ... ...

Abstract	Next-generation sequencing (NGS) is a new approach for biomedical research, useful for the diagnosis of genetic diseases in extremely heterogeneous conditions. In this work, we describe how data generated by high-throughput NGS experiments can be analyzed to find single nucleotide polymorphisms (SNPs) in DNA samples of patients affected by neuromuscular disorders. In particular, we consider untagged pooled NGS data, where DNA samples of different individuals are combined in a single experiment, still providing information with an uncertainty limited to only two patients. At the moment, only few publications address the problem of SNPs detection in pooled experiments, and existing tools are often inaccurate. We propose a computational procedure consisting of two parts. In the first, data are filtered by means of decision rules. The second phase is based on a supervised classification technique. In the present work, we compare different de facto standard supervised and unsupervised procedures to identify and classify variants potentially related to muscular diseases, and we discuss results in terms of statistical and biological validation.
MeSH term(s)	Algorithms ; Amino Acid Substitution ; Genetic Association Studies/methods ; High-Throughput Nucleotide Sequencing ; Humans ; Muscular Diseases/diagnosis ; Mutation ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA
Language	English
Publishing date	2014-07-16
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2030900-4
ISSN	1557-8666 ; 1066-5277
ISSN (online)	1557-8666
ISSN	1066-5277
DOI	10.1089/cmb.2014.0037
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Article: Clinical and Genetic Evaluation of a Cohort of Pediatric Patients with Severe Inherited Retinal Dystrophies.

Di Iorio, Valentina / Karali, Marianthi / Brunetti-Pierri, Raffaella / Filippelli, Mariaelena / Di Fruscio, Giuseppina / Pizzo, Mariateresa / Mutarelli, Margherita / Nigro, Vincenzo / Testa, Francesco / Banfi, Sandro / Simonelli, Francesca

Genes

2017 Volume 8, Issue 10

Abstract: We performed a clinical and genetic characterization of a pediatric cohort of patients with inherited retinal dystrophy (IRD) to identify the most suitable cases for gene therapy. The cohort comprised 43 patients, aged between 2 and 18 years, with severe ...

Abstract	We performed a clinical and genetic characterization of a pediatric cohort of patients with inherited retinal dystrophy (IRD) to identify the most suitable cases for gene therapy. The cohort comprised 43 patients, aged between 2 and 18 years, with severe isolated IRD at the time of presentation. The ophthalmological characterization also included assessment of the photoreceptor layer integrity in the macular region (ellipsoid zone (EZ) band). In parallel, we carried out a targeted, next-generation sequencing (NGS)-based analysis using a panel that covers over 150 genes with either an established or a candidate role in IRD pathogenesis. Based on the ophthalmological assessment, the cohort was composed of 24 Leber congenital amaurosis, 14 early onset retinitis pigmentosa, and 5 achromatopsia patients. We identified causative mutations in 58.1% of the cases. We also found novel genotype-phenotype correlations in patients harboring mutations in the
Language	English
Publishing date	2017-10-20
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527218-4
ISSN	2073-4425
ISSN	2073-4425
DOI	10.3390/genes8100280
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Article ; Online: Dominant muscular dystrophy with a novel SYNE1 gene mutation.

Fanin, Marina / Savarese, Marco / Nascimbeni, Anna C / Di Fruscio, Giuseppina / Pastorello, Ebe / Tasca, Elisabetta / Trevisan, Carlo P / Nigro, Vincenzo / Angelini, Corrado

Muscle & nerve

2014 Volume 51, Issue 1, Page(s) 145–147

MeSH term(s)	Adult ; Cytoskeletal Proteins ; Female ; Humans ; Lysosomal-Associated Membrane Protein 2/genetics ; Male ; Muscular Dystrophies/genetics ; Muscular Dystrophies/pathology ; Mutation/genetics ; Nerve Tissue Proteins/genetics ; Nuclear Proteins/genetics
Chemical Substances	Cytoskeletal Proteins ; LAMP2 protein, human ; Lysosomal-Associated Membrane Protein 2 ; Nerve Tissue Proteins ; Nuclear Proteins ; SYNE1 protein, human
Language	English
Publishing date	2014-11-24
Publishing country	United States
Document type	Letter
ZDB-ID	438353-9
ISSN	1097-4598 ; 0148-639X
ISSN (online)	1097-4598
ISSN	0148-639X
DOI	10.1002/mus.24357
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Zs.A 1449: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: [Next Generation Sequencing and ADPKD].

Restivo, Arianna / Di Fruscio, Giuseppina / Masella, Cristina / Rinaldi, Luca / Capolongo, Giovanna / Raiola, Ilaria / Capasso, Giovambattista

Giornale italiano di nefrologia : organo ufficiale della Societa italiana di nefrologia

2015 Volume 35, Issue Suppl 71

Abstract: Autosomal Dominant Polycistic Kidney Disease (ADPKD) is the most common inherited genetic disorder in the word, caused by mutations in PKD1 gene in 85% of cases and PKD 2 gene in the remaining 15%. Although diagnosis is usually based on ultrasound, MRI ... ...

Abstract	Autosomal Dominant Polycistic Kidney Disease (ADPKD) is the most common inherited genetic disorder in the word, caused by mutations in PKD1 gene in 85% of cases and PKD 2 gene in the remaining 15%. Although diagnosis is usually based on ultrasound, MRI and CT scans, in some cases genetic testing is necessary, for example, in patients with atypical phenotype or with a negative family history, or in cases of donation from relatives. The presence of pseudogenes in PKD1, the size of the gene, the costs of the Sanger sequencing and genetic heterogeneity underlying kidney disease make genetic analysis particularly difficult to be performed. Next Generation Sequencing (NGS) represents the last frontier of innovation among diagnostic tools for molecular diagnosis of inherited cystic kidney disease thanks to the ability to analyze several genes at the same time. In this regard, we have developed a NGS platform, called Nephroplex, with the aim of identifying variations in 115 genes responsible for numerous kidney diseases, including cystic and polycystic disease, achieving, overall, a target region of 338.8 kbps. The technology used for the enrichment is HaloPlex system, based on the digestion of genomic DNA with restriction enzymes and the capture of the regions of interest with specific hybridization probes. With our platform, we have analyzed 9 patients with clinical diagnosis of ADPKD. We have obtained a depth coverage of 100x for 96.5% of the target, while the region not covered accounted for only 3% of the region of interest. In 6 patients, we found causative mutations in the genes PKD1 and PKD2, achieving a detection rate of 66%. In conclusion, the NephroPlex platform has proved to be an excellent device for molecular diagnosis of kidney disease and could clarify the mechanisms underlying genetic heterogeneity observed in kidney disease.
Language	Italian
Publishing date	2015-12
Publishing country	Italy
Document type	English Abstract ; Journal Article
ZDB-ID	1237110-5
ISSN	1724-5990 ; 0393-5590
ISSN (online)	1724-5990
ISSN	0393-5590
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Article ; Online: Mutations in the PCYT1A gene are responsible for isolated forms of retinal dystrophy.

Testa, Francesco / Filippelli, Mariaelena / Brunetti-Pierri, Raffaella / Di Fruscio, Giuseppina / Di Iorio, Valentina / Pizzo, Mariateresa / Torella, Annalaura / Barillari, Maria Rosaria / Nigro, Vincenzo / Brunetti-Pierri, Nicola / Simonelli, Francesca / Banfi, Sandro

European journal of human genetics : EJHG

2017 Volume 25, Issue 5, Page(s) 651–655

Abstract: Mutations in the PCYT1A gene have been recently linked to two different phenotypes: one characterized by spondylometaphyseal dysplasia and cone-rod dystrophy (SMD-CRD) and the other by congenital lipodystrophy, severe fatty liver disease, and reduced HDL ...

Abstract	Mutations in the PCYT1A gene have been recently linked to two different phenotypes: one characterized by spondylometaphyseal dysplasia and cone-rod dystrophy (SMD-CRD) and the other by congenital lipodystrophy, severe fatty liver disease, and reduced HDL cholesterol without any retinal or skeletal involvement. Here, we identified, by next generation sequencing, sequence variants affecting function in the PCYT1A gene in three young patients with isolated retinal dystrophy from two different Italian families. A thorough clinical evaluation of the patients, with whole skeleton X-ray, metabolic assessment and liver ultrasound failed to reveal signs of skeletal dysplasia, metabolic and hepatic alterations. This is the first report showing that the PCYT1A gene can be responsible for isolated forms of retinal dystrophy, particularly without any skeletal involvement, thus further expanding the phenotypic spectrum induced by mutations in this gene.
MeSH term(s)	Adolescent ; Child ; Choline-Phosphate Cytidylyltransferase/genetics ; Female ; Humans ; Male ; Mutation ; Phenotype ; Retinal Dystrophies/diagnosis ; Retinal Dystrophies/genetics
Chemical Substances	Choline-Phosphate Cytidylyltransferase (EC 2.7.7.15) ; PCYT1A protein, human (EC 2.7.7.15)
Language	English
Publishing date	2017-03-08
Publishing country	England
Document type	Case Reports ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1141470-4
ISSN	1476-5438 ; 1018-4813
ISSN (online)	1476-5438
ISSN	1018-4813
DOI	10.1038/ejhg.2017.23
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Article ; Online: MotorPlex provides accurate variant detection across large muscle genes both in single myopathic patients and in pools of DNA samples.

Savarese, Marco / Di Fruscio, Giuseppina / Mutarelli, Margherita / Torella, Annalaura / Magri, Francesca / Santorelli, Filippo Maria / Comi, Giacomo Pietro / Bruno, Claudio / Nigro, Vincenzo

Acta neuropathologica communications

2014 Volume 2, Page(s) 100

Abstract: Mutations in ~100 genes cause muscle diseases with complex and often unexplained genotype/phenotype correlations. Next-generation sequencing studies identify a greater-than-expected number of genetic variations in the human genome. This suggests that ... ...

Abstract	Mutations in ~100 genes cause muscle diseases with complex and often unexplained genotype/phenotype correlations. Next-generation sequencing studies identify a greater-than-expected number of genetic variations in the human genome. This suggests that existing clinical monogenic testing systematically miss very relevant information.We have created a core panel of genes that cause all known forms of nonsyndromic muscle disorders (MotorPlex). It comprises 93 loci, among which are the largest and most complex human genes, such as TTN, RYR1, NEB and DMD. MotorPlex captures at least 99.2% of 2,544 exons with a very accurate and uniform coverage. This quality is highlighted by the discovery of 20-30% more variations in comparison with whole exome sequencing. The coverage homogeneity has also made feasible to apply a cost-effective pooled sequencing strategy while maintaining optimal sensitivity and specificity.We studied 177 unresolved cases of myopathies for which the best candidate genes were previously excluded. We have identified known pathogenic variants in 52 patients and potential causative ones in further 56 patients. We have also discovered 23 patients showing multiple true disease-associated variants suggesting complex inheritance. Moreover, we frequently detected other nonsynonymous variants of unknown significance in the largest muscle genes. Cost-effective combinatorial pools of DNA samples were similarly accurate (97-99%). MotorPlex is a very robust platform that overcomes for power, costs, speed, sensitivity and specificity the gene-by-gene strategy. The applicability of pooling makes this tool affordable for the screening of genetic variability of muscle genes also in a larger population. We consider that our strategy can have much broader applications.
MeSH term(s)	Computer Simulation ; Female ; Genetic Variation ; Humans ; Male ; Muscle, Skeletal/physiopathology ; Muscular Diseases/genetics ; Sensitivity and Specificity ; Sequence Analysis, DNA/methods
Language	English
Publishing date	2014-09-11
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2715589-4
ISSN	2051-5960 ; 2051-5960
ISSN (online)	2051-5960
ISSN	2051-5960
DOI	10.1186/s40478-014-0100-3
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Article ; Online: Familial trisomy 6p in mother and daughter.

Savarese, Marco / Grandone, Anna / Perone, Lucia / Blanco, Francesca Del Vecchio / De Luca, Giuseppina / Di Fruscio, Giuseppina / Fogu, Giuseppina / Piluso, Giulio / Perrone, Laura / del Giudice, Emanuele Miraglia / Nigro, Vincenzo

American journal of medical genetics. Part A

2013 Volume 161A, Issue 7, Page(s) 1675–1681

Abstract: Several patients with partial trisomy 6p resulting from parental balanced translocations or from a de novo duplication or insertion have already been described. Here, we report on the first case of familial pure trisomy 6p as a result of interstitial ... ...

Abstract	Several patients with partial trisomy 6p resulting from parental balanced translocations or from a de novo duplication or insertion have already been described. Here, we report on the first case of familial pure trisomy 6p as a result of interstitial tandem duplication. The patient, an 11-year-old female, presented with mild dysmorphic features, moderate intellectual disability with behavioral disturbances, immunodeficiency, and epilepsy. Conventional cytogenetic analysis showed a duplication of the 6p region in the patient and in her mother presenting with a partially overlapping phenotype. The rearrangement was confirmed and defined by molecular cytogenetic analysis, including FISH and array CGH analysis showing a gain of ~13.8 Mb from 6p12.3 to 6p21.31. The phenotype of a pure partial trisomy 6p is extremely heterogeneous depending on the gene contents of the duplicated region. The clinical features of our patients have been compared with overlapping cases from the literature.
MeSH term(s)	Body Height/genetics ; Child ; Chromosomes, Human, Pair 6/genetics ; Epilepsy/genetics ; Face/abnormalities ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Intellectual Disability/genetics ; Mothers ; Nuclear Family ; Phenotype ; Trisomy/genetics ; Trisomy/physiopathology
Language	English
Publishing date	2013-05-17
Publishing country	United States
Document type	Case Reports ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2108614-X
ISSN	1552-4833 ; 0148-7299 ; 1552-4825
ISSN (online)	1552-4833
ISSN	0148-7299 ; 1552-4825
DOI	10.1002/ajmg.a.35928
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