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Paracchini V.,Medical Genetics Laboratory | Carbone A.,Medical Genetics Laboratory | Carbone A.,University of Foggia | Castellani S.,University of Foggia | And 5 more authors.
Journal of Biomedicine and Biotechnology | Year: 2012

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with lung and liver manifestations. Because of pitfalls of gene therapy, novel approaches for reconstitution of the airway epithelium and CFTR expression should be explored. In the present study, human amniotic mesenchymal stem cells (hAMSCs) were isolated from term placentas and characterized for expression of phenotypic and pluripotency markers, and for differentiation potential towards mesoderm (osteogenic and adipogenic) lineages. Moreover, hAMSCs were induced to differentiate into hepatocyte-like cells, as demonstrated by mixed function oxidase activity and expression of albumin, alpha1-antitrypsin, and CK19. We also investigated the CFTR expression in hAMSCs upon isolation and in coculture with CF airway epithelial cells. Freshly isolated hAMSCs displayed low levels of CFTR mRNA, which even decreased with culture passages. Following staining with the vital dye CM-DiI, hAMSCs were mixed with CFBE41o- respiratory epithelial cells and seeded onto permeable filters. Flow cytometry demonstrated that 33-50 of hAMSCs acquired a detectable CFTR expression on the apical membrane, a result confirmed by confocal microscopy. Our data show that amniotic MSCs have the potential to differentiate into epithelial cells of organs relevant in CF pathogenesis and may contribute to partial correction of the CF phenotype. Copyright 2012 Valentina Paracchini et al.


PubMed | Medical Genetics Laboratory, Cystic Fibrosis Center and University of Milan
Type: Journal Article | Journal: Journal of human genetics | Year: 2016

Despite extensive screening, 1-5% of cystic fibrosis (CF) patients lack a definite molecular diagnosis. Next-generation sequencing (NGS) is making affordable genetic testing based on the identification of variants in extended genomic regions. In this frame, we analyzed 23 CF patients and one carrier by whole-gene CFTR resequencing: 4 were previously characterized and served as controls; 17 were cases lacking a complete diagnosis after a full conventional CFTR screening; 3 were consecutive subjects referring to our centers, not previously submitted to any screening. We also included in the custom NGS design the coding portions of the SCNN1A, SCNN1B and SCNN1G genes, encoding the subunits of the sodium channel ENaC, which were found to be mutated in CF-like patients. Besides 2 novel SCNN1B missense mutations, we identified 22 previously-known CFTR mutations, including 2 large deletions (whose breakpoints were precisely mapped), and novel deep-intronic variants, whose role on splicing was excluded by ex-vivo analyses. Finally, for 2 patients, compound heterozygotes for a CFTR mutation and the intron-9c.1210-34TG


PubMed | Fondazione IRCCS Instituto Neurologico Carlo Besta, Medical Genetics Laboratory and University of Milan
Type: Case Reports | Journal: Congenital anomalies | Year: 2016

Deletions on chromosome 6q are rarely reported in the literature, and genotype-phenotype correlations are poorly understood. We report a child with a deletion of the 6q21-q22 chromosomal region, providing some intriguing results about the correlation between this region and acro-cardio-facial syndrome, congenital heart disease, split hand and foot malformation, and epilepsy.


PubMed | Ospedale Civile di Castel San Giovanni, Medical Genetics Laboratory, University of Genoa, Irccs Azienda Ospedaliera San Martino Instituto Nazionale Per La Ricerca Sul Cancro and University of Milan
Type: Journal Article | Journal: Journal of gastroenterology | Year: 2016

The ABCB4 gene encodes the MDR3 protein. Mutations of this gene cause progressive familial intrahepatic cholestasis type 3 (PFIC3) in children, but their clinical relevance in adults remains ill defined. The study of a well-characterized adult patient series may contribute to refining the genetic data regarding cholangiopathies of unknown origin. Our aim was to evaluate the impact of ABCB4 mutations on clinical expression of cholestasis in adult patients.We consecutively evaluated 2602 subjects with hepatobiliary disease. Biochemical evidence of a chronic cholestatic profile (CCP) with elevated serum gamma-glutamyltransferase activity or diagnosis of intrahepatic cholestasis of pregnancy (ICP) and juvenile cholelithiasis (JC) were inclusion criteria. The personal/family history of additional cholestatic liver disease (PFH-CLD), which includes ICP, JC, or hormone-induced cholestasis, was investigated. Mutation screening of ABCB4 was carried out in 90 patients with idiopathic chronic cholestasis (ICC), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), ICP, and JC.Eighty patients had CCP. PSC and ICC patients with PFH-CLD had earlier onset of disease than those without it (p = 0.003 and p = 0.023, respectively). The mutation frequency ranged from 50% (ICP, JC) to 17.6% (PBC). Among CCP patients, presence or absence of PFH-CLD was associated with ABCB4 mutations in 26.8 vs 5.1% (p = 0.013), respectively; in the subset of ICC and PSC patients, the corresponding figures were 44.4 vs 0% (p = 0.012) and 28.6 vs 8.7% (p = 0.173).Cholangiopathies attributable to highly penetrant ABCB4 mutant alleles are identifiable in a substantial proportion of adults that generally have PFH-CLD. In PSC and ICC phenotypes, patients with MDR3 deficiency have early onset of disease.


Solda G.,University of Milan | Robusto M.,University of Milan | Primignani P.,Medical Genetics Laboratory | Castorina P.,UO Audiology | And 5 more authors.
Human Molecular Genetics | Year: 2012

The miR-96, miR-182 and miR-183 microRNA (miRNA) family is essential for differentiation and function of the vertebrate inner ear. Recently, point mutations within the seed region of miR-96 were reported in two Spanish families with autosomal dominant non-syndromic sensorineural hearing loss (NSHL) and in a mouse model of NSHL. We screened 882 NSHL patients and 836 normal-hearing Italian controls and identified one putative novel mutation within the miR-96 gene in a family with autosomal dominant NSHL. Although located outside the mature miR-96 sequence, the detected variant replaces a highly conserved nucleotide within the companion miR-96*, and is predicted to reduce the stability of the pre-miRNA hairpin. To evaluate the effect of the detected mutation on miR-96/mir-96* biogenesis, we investigated the maturation of miR-96 by transient expression in mammalian cells, followed by real-time reverse-transcription polymerase chain reaction (PCR). We found that both miR-96 and miR-96* levels were significantly reduced in the mutant, whereas the precursor levels were unaffected. Moreover, miR-96 and miR-96* expression levels could be restored by a compensatory mutation that reconstitutes the secondary structure of the pre-miR-96 hairpin, demonstrating that the mutation hinders precursor processing, probably interfering with Dicer cleavage. Finally, even though the mature miR-96 sequence is not altered, we demonstrated that the identified mutation significantly impacts on miR-96 regulation of selected targets. In conclusion, we provide further evidence of the involvement of miR-96 mutations in human deafness and demonstrate that a quantitative defect of this miRNA may contribute to NSHL. © The Author 2011. Published by Oxford University Press. All rights reserved.


PubMed | Medical Genetics Laboratory and University of Milan
Type: Case Reports | Journal: American journal of medical genetics. Part A | Year: 2016

Interstitial deletions of the long arm of chromosome 6 are rare. Clinically, these deletions are considered to be part of a unique microdeletion syndrome associated with intellectual disability and speech impairment, typical dysmorphic features, structural anomalies of the brain, microcephaly, and non-specific multiple organ anomalies. The critical region for the interstitial 6q microdeletion phenotype was mapped to 6q24-6q25, particularly the 6q25.3 region containing the genes ARID1B and ZDHHC14. It has been hypothesized that haploinsufficiency of these genes impairs normal development of the brain and is responsible for the phenotype. This case report describes a girl presenting with typical features of 6q microdeletion syndrome, including global developmental delay, speech impairment, distinct dysmorphic features, dysgenesis of the corpus callosum, common limb anomalies, and hearing loss. Chromosome analysis by array-CGH revealed a small interstitial 6q deletion spanning approximately 1.1 Mb of DNA and containing only one coding gene, ARID1B. We suggest that ARID1B is the key gene behind 6q microdeletion syndrome, and we discuss its possible role in the phenotypic manifestations.


PubMed | Medical Genetics Laboratory and University of Milan
Type: | Journal: European journal of medical genetics | Year: 2016

7p22.1 microduplication syndrome is mainly characterized by developmental and speech delay, craniofacial dysmorphisms and skeletal abnormalities. The minimal critical region includes two OMIM genes: ACTB and RNF216. Here, we report on a girl carrying the smallest 7p22.1 microduplication detected to date, contributing to the delineation of the clinical phenotype of the 7p22.1 duplication syndrome and to the refinement of the minimal critical region. Our patient shares several major features of the 7p22.1 duplication syndrome, including craniofacial dysmorphisms and speech and motor delay, but she also presents with renal anomalies. Based on present and published dup7p22.1 patients we suggest that renal abnormalities might be an additional feature of the 7p22.1 microduplication syndrome. We also pinpoint the ACTB gene as the key gene affecting the 7p22.1 duplication syndrome phenotype.


PubMed | Medical Genetics Laboratory and University of Milan
Type: | Journal: Italian journal of pediatrics | Year: 2015

HOXA genes cluster plays a fundamental role in embryologic development. Deletion of the entire cluster is known to cause a clinically recognizable syndrome with mild developmental delay, characteristic facies, small feet with unusually short and big halluces, abnormal thumbs, and urogenital malformations. The clinical manifestations may vary with different ranges of deletions of HOXA cluster and flanking regions.We report a girl with the smallest deletion reported to date involving the entire HOXA cluster at 7p15.2-p14.3. The patient was the third child born to a healthy and non-consanguineous Italian couple. She was born at the 34th week of gestation by caesarean section due to cholestasis of pregnancy. Her birth weight, length, and occipitofrontal circumference were 2,140 g (25-50th centile), 46 cm (50th centile), and 33 cm (75-90th centile), respectively. The Apgar scores were 8 at both the 1st and 5th minutes. The patient presented with typical mild facial anomalies, hand and feet abnormalities, urinary anomalies, and mild speech delay. Unexpectedly, the patient demonstrated complex unusual features of multiple episodes of oxyhemoglobin desaturation, laryngeal stridor and a branchial cyst. Chromosome analysis of the patient revealed an apparently normal karyotype at the 550 band level. Based on array comparative genomic hybridization, a 2.5 Mb interstitial deletion was detected at 7p15.2p14.3 (chr7: 26,333,553-28,859,312), involving the entire HOXA cluster and a small number of other genes as SNX10, SKAP2, EVX1, HIBADH, TAX1BP1, JAZF1, and CREB5.This report improves our understanding of the genotype-phenotype correlations of HOXA genes cluster deletions via the identification and characterization of the smallest deletion (as well as critical region) reported to date. In particular we discuss the possible implications of preterm and haploinsufficiency in the pathogenesis of the unusual findings, furthermore opening new discussion and interpretation cues.


Carbone A.,Medical Genetics Laboratory | Carbone A.,University of Foggia | Paracchini V.,Medical Genetics Laboratory | Castellani S.,University of Foggia | And 4 more authors.
Current Stem Cell Research and Therapy | Year: 2014

Lung diseases represent a significant burden of morbidity and mortality worldwide. Current therapies have not proven adequate in the long term and are often associated with significant side effects. There has been recent interest in the regenerative/reparative potential of cell-based therapies, including cells derived from the placental tissues. Amnion-derived cells are fetal-derived and characterized by expression profile and differentiative capacity of pluripotent cells. Moreover, because placenta is discarded after delivery, they represent an ethical source for the purposes of regenerative medicine. Amnion-derived cells are endowed with immunomodulatory, anti-inflammatory, anti-scarring and antibacterial properties, which may explain many of the beneficial effects observed with administration of the cells in animal models for a large number of inflammatory diseases. Both human amniotic epithelial cells (hAEC) and mesenchymal stromal cells (hAMSC) have been shown to acquire in vitro and in vivo some characteristics of epithelial cells, i.e. CFTR (cystic fibrosis transmembrane conductance regulator) and surfactant proteins. Administration of hAEC or hAMSC in vivo in the bleomycin-induced lung injury model has proven their therapeutic effects in term of reduction of pulmonary fibrosis and inflammation, as well as recovery of lung mechanical function. Many biological and clinical information have to be gathered before proposing amnion-derived cells in the clinic for the treatment of acute and chronic lung diseases. © 2014 Bentham Science Publishers.


Conese M.,University of Foggia | Piro D.,University of Foggia | Carbone A.,University of Foggia | Carbone A.,Medical Genetics Laboratory | And 2 more authors.
The Scientific World Journal | Year: 2014

Chronic lung diseases, such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD) are incurable and represent a very high social burden. Stem cell-based treatment may represent a hope for the cure of these diseases. In this paper, we revise the overall knowledge about the plasticity and engraftment of exogenous marrow-derived stem cells into the lung, as well as their usefulness in lung repair and therapy of chronic lung diseases. The lung is easily accessible and the pathophysiology of these diseases is characterized by injury, inflammation, and eventually by remodeling of the airways. Bone marrow-derived stem cells, including hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal (stem) cells (MSCs), encompass a wide array of cell subsets with different capacities of engraftment and injured tissue regenerating potential. Proof-of-principle that marrow cells administered locally may engraft and give rise to specialized epithelial cells has been given, but the efficiency of this conversion is too limited to give a therapeutic effect. Besides the identification of plasticity mechanisms, the characterization/isolation of the stem cell subpopulations represents a major challenge to improving the efficacy of transplantation protocols used in regenerative medicine for lung diseases. © 2014 Massimo Conese et al.

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