Publications by authors named "Silvia Giliani"

100 Publications

Case Report: Hypomorphic Function and Somatic Reversion in DOCK8 Deficiency in One Patient With Two Novel Variants and Sclerosing Cholangitis.

Front Immunol 2021 16;12:673487. Epub 2021 Apr 16.

Pediatric Hematology Outpatient Clinic, Department of Pediatrics, Fondazione MBBM, Monza, Italy.

DOCK8 deficiency is a combined immunodeficiency due to biallelic variants in dedicator of cytokinesis 8 () gene. The disease has a wide clinical spectrum encompassing recurrent infections (candidiasis, viral and bacterial infections), virally driven malignancies and immune dysregulatory features, including autoimmune (cytopenia and vasculitis) as well as allergic disorders (eczema, asthma, and food allergy). Hypomorphic function and somatic reversion of has been reported to result in incomplete phenotype without IgE overproduction. Here we describe a case of DOCK8 deficiency in a 8-year-old Caucasian girl. The patient's disease was initially classified as autoimmune thrombocytopenia, which then evolved toward a combined immunodeficiency phenotype with recurrent infections, persistent EBV infection and lymphoproliferation. Two novel variants (one deletion and one premature stop codon) were characterized, resulting in markedly reduced, but not absent, DOCK8 expression. Somatic reversion of the deletion was identified in T cells. Hypomorphic function and somatic reversion were associated with restricted T cell repertoire, decreased STAT5 phosphorylation and impaired immune synapse functioning in T cells. Although the patient presented with incomplete phenotype (absence of markedly increase IgE and eosinophil count), sclerosing cholangitis was incidentally detected, thus indicating that hypomorphic function and somatic reversion of may delay disease progression but do not necessarily prevent from severe complications.
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http://dx.doi.org/10.3389/fimmu.2021.673487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085392PMC
April 2021

Selective Laser Melting and Electron Beam Melting of Ti6Al4V for Orthopedic Applications: A Comparative Study on the Applied Building Direction.

Materials (Basel) 2020 Dec 7;13(23). Epub 2020 Dec 7.

Department of Mechanical and Industrial Engineering, University of Brescia, via Branze 38, 25123 Brescia, Italy.

The 3D printing process offers several advantages to the medical industry by producing complex and bespoke devices that accurately reproduce customized patient geometries. Despite the recent developments that strongly enhanced the dominance of additive manufacturing (AM) techniques over conventional methods, processes need to be continually optimized and controlled to obtain implants that can fulfill all the requirements of the surgical procedure and the anatomical district of interest. The best outcomes of an implant derive from optimal compromise and balance between a good interaction with the surrounding tissue through cell attachment and reduced inflammatory response mainly caused by a weak interface with the native tissue or bacteria colonization of the implant surface. For these reasons, the chemical, morphological, and mechanical properties of a device need to be designed in order to assure the best performances considering the in vivo environment components. In particular, complex 3D geometries can be produced with high dimensional accuracy but inadequate surface properties due to the layer manufacturing process that always entails the use of post-processing techniques to improve the surface quality, increasing the lead times of the whole process despite the reduction of the supply chain. The goal of this work was to provide a comparison between Ti6Al4V samples fabricated by selective laser melting (SLM) and electron beam melting (EBM) with different building directions in relation to the building plate. The results highlighted the influence of the process technique on osteoblast attachment and mineralization compared with the building orientation that showed a limited effect in promoting a proper osseointegration over a long-term period.
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http://dx.doi.org/10.3390/ma13235584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729448PMC
December 2020

Case Report: A Case of X-Linked Agammaglobulinemia With High Serum IgE Levels and Allergic Rhinitis.

Front Immunol 2020 5;11:582376. Epub 2020 Nov 5.

Department of Pediatrics, Policlinico Umberto I, Sapienza University of Rome, Rome, Italy.

X-linked Agammaglobulinemia (XLA) is a rare genetic disorder of B-lymphocyte differentiation, characterized by the absence or paucity of circulating B cells, markedly reduced levels of all serum immunoglobulin isotypes and lack of specific antibody production. Bruton Tyrosine Kinase () gene encodes a cytoplasmic tyrosine kinase involved in the B cell maturation and its mutation, blocking B cell differentiation at the pre-B cell stage, and is responsible for XLA. All domains may be affected by the mutation, and the many genotypes are associated with a wide range of clinical presentations. Little is known about genotype-phenotype correlation in this disorder, and factors influencing the phenotype of XLA are not clearly understood. In this report we present a unique case of a young patient affected by XLA. The disease was genetically diagnosed at birth due to a family history of XLA, but during follow up, it was characterized by a CD19+ B cell percentage consistently greater than 2%. He never suffered severe infections, but at two years of age, he developed persistent rhinitis. Thus, total serum IgE levels were measured and detected over the normal range, and specific allergic investigations showed sensitization to dust mites. Further immunological tests (BTK expression, functional "" B cell proliferation upon CpG stimulation, B cell subset analysis) explained these findings as possible manifestations of a mild XLA phenotype. XLA patients rarely present with allergic manifestations, which could warrant further investigation. High serum IgE levels could be a sign of a mild phenotype, but their role and the mechanisms underlying their production in XLA need to be clarified.
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http://dx.doi.org/10.3389/fimmu.2020.582376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674281PMC
November 2020

Activated Phosphoinositide 3-Kinase Delta Syndrome 1: Clinical and Immunological Data from an Italian Cohort of Patients.

J Clin Med 2020 Oct 17;9(10). Epub 2020 Oct 17.

Pediatrics Clinic and "A. Nocivelli" Institute for Molecular Medicine, Department of Clinical and Experimental Sciences, University of Brescia, ASST Spedali Civili of Brescia, 25123 Brescia, Italy.

Activated phosphoinositide 3-kinase delta syndrome 1 (APDS-1) is a recently described inborn error of immunity caused by monoallelic gain-of-function mutations in the gene. We reviewed for the first time medical records and laboratory data of eight Italian APDS-1 patients. Recurrent sinopulmonary infections were the most common clinical feature at onset of disease. Seven patients presented lymphoproliferative disease, at onset or during follow-up, one of which resembled hemophagocytic lymphohistiocytosis (HLH). Genetic analysis of the gene revealed three novel mutations: functional testing confirmed their activating nature. In the remaining patients, the previously reported variants .E1021K ( = 4) and .E525A ( = 1) were identified. Six patients were started on immunoglobulin replacement treatment (IgRT). One patient successfully underwent hematopoietic stem cell transplantation (HSCT), with good chimerism and no GVHD at 21 months post-HSCT. APDS-1 is a combined immune deficiency with a wide variety of clinical manifestations and a complex immunological presentation. Besides IgRT, specific therapies targeting the PI3Kδ pathway will most likely become a valid aid for the amelioration of patients' clinical management and their quality of life.
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http://dx.doi.org/10.3390/jcm9103335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603210PMC
October 2020

Generation of induced pluripotent stem cell (iPSC) lines from a Joubert syndrome patient with compound heterozygous mutations in C5orf42 gene.

Stem Cell Res 2020 12 22;49:102007. Epub 2020 Sep 22.

Angelo Nocivelli Institute for Molecular Medicine, Department of Molecular and Translational Medicine, University of Brescia, Italy.

We have generated new disease-specific induced pluripotent stem cell (iPSC) lines from skin fibroblasts obtained from a female patient with Joubert syndrome (JS) caused by compound heterozygous mutations in C5orf42 gene. The generated iPSCs offer an unprecedented opportunity to obtain iPSC-derived neurons to investigate the pathogenesis of JS in vitro and to develop therapeutic strategies.
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http://dx.doi.org/10.1016/j.scr.2020.102007DOI Listing
December 2020

Human inborn errors of immunity caused by defects of receptor and proteins of cellular membrane.

Minerva Pediatr 2020 Oct 22;72(5):393-407. Epub 2020 Sep 22.

Department of Molecular and Translational Medicine, A. Nocivelli Institute for Molecular Medicine, University of Brescia, Brescia, Italy.

Inborn errors of immunity are diseases of the immune system resulting from mutations that alter the expression of encoded proteins or molecules. Total updated number of these disorders is currently 406, with 430 different identified gene defects involved. Studies of the underlying mechanisms have contributed in better understanding the pathophysiology of the diseases, but also the complexity of the biology of innate and adaptive immune system and its interaction with microbes. In this review we present and briefly discuss Inborn Errors of Immunity caused by defects in genes encoding for receptors and protein of cellular membrane, including cytokine receptors, T cell antigen receptor (TCR) complex, cellular surface receptors or receptors signaling causing predominantly antibody deficiencies, co-stimulatory receptors and others. These alterations impact many biological processes of immune-system cells, including development, proliferation, activation and down-regulation of the immunological response, and result in a variety of diseases that present with distinct clinical features or with overlapping signs and symptoms.
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http://dx.doi.org/10.23736/S0026-4946.20.06000-4DOI Listing
October 2020

Paediatric MAS/HLH caused by a novel monoallelic activating mutation in p110δ.

Clin Immunol 2020 10 16;219:108543. Epub 2020 Jul 16.

Paediatrics Clinic and Institute for Molecular Medicine A. Nocivelli, Department of Clinical and Experimental Sciences, University of Brescia, ASST- Spedali Civili of Brescia, Brescia, Italy.

This study provides evidence for the first time for APDS-1 presenting as MAS/HLH, with evident clinical implications in patient's management and prognosis.
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http://dx.doi.org/10.1016/j.clim.2020.108543DOI Listing
October 2020

Transient Decrease of Circulating and Tissular Dendritic Cells in Patients With Mycobacterial Disease and With Partial Dominant IFNγR1 Deficiency.

Front Immunol 2020 26;11:1161. Epub 2020 Jun 26.

Department of Clinical and Experimental Sciences, Department of Pediatrics, A. Nocivelli Institute for Molecular Medicine, University of Brescia, Brescia, Italy.

Interferon-γ receptor 1 (IFNγR1) deficiency is one of the inborn errors of IFN-γ immunity underlying Mendelian Susceptibility to Mycobacterial Disease (MSMD). This molecular circuit plays a crucial role in regulating the interaction between dendritic cells (DCs) and T lymphocytes, thus affecting DCs activation, maturation, and priming of T cells involved in the immune response against intracellular pathogens. We studied a girl who developed at the age of 2.5 years a infection characterized by disseminated necrotizing granulomatous lymphadenitis, and we compared her findings with other patients with the same genetic condition. The patient carried a heterozygous 818del4 mutation in the gene responsible of autosomal dominant (AD) partial IFNγR1 deficiency. During the acute infection blood cells immunophenotyping showed a marked reduction in DCs counts, including both myeloid (mDCs) and plasmacytoid (pDCs) subsets, that reversed after successful prolonged antimicrobial therapy. Histology of her abdomen lymph node revealed a profound depletion of tissue pDCs, as compared to other age-matched granulomatous lymphadenitis of mycobacterial origin. Circulating DCs depletion was also observed in another patient with AD partial IFNγR1 deficiency during mycobacterial infection. To conclude, AD partial IFNγR1 deficiency can be associated with a transient decrease in both circulating and tissular DCs during acute mycobacterial infection, suggesting that DCs counts monitoring might constitute a useful marker of treatment response.
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http://dx.doi.org/10.3389/fimmu.2020.01161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7333364PMC
April 2021

Partial T cell defects and expanded CD56 NK cells in an SCID patient carrying hypomorphic mutation in the IL2RG gene.

J Leukoc Biol 2020 08 11;108(2):739-748. Epub 2020 May 11.

Unit of Immune and Infectious Diseases, Academic Department of Pediatrics, Bambino Gesù Childrens' Hospital-Scientific Institute for Research and Healthcare (IRCCS), Rome, Italy.

X-linked severe combined immunodeficiency (X-SCID) caused by full mutation of the IL2RG gene leads to T B NK phenotype and is usually associated with severe opportunistic infections, diarrhea, and failure to thrive. When IL2RG hypomorphic mutation occurs, diagnosis could be delayed and challenging since only moderate reduction of T and NK cells may be present. Here, we explored phenotypic insights and the impact of the p.R222C hypomorphic mutation (IL2RG ) in distinct cell subsets in an 8-month-old patient with atypical X-SCID. We found reduced CD4 T cell counts, a decreased frequency of naïve CD4 and CD8 T cells, and an expansion of B cells. Ex vivo STAT5 phosphorylation was impaired in CD4 CD45RO T cells, yet compensated by supraphysiological doses of IL-2. Sanger sequencing on purified cell subsets showed a partial reversion of the mutation in total CD3 cells, specifically in recent thymic emigrants (RTE), effector memory (EM), and CD45RA terminally differentiated EM (EMRA) CD4 T cells. Of note, patient's NK cells had a normal frequency compared to age-matched healthy subjects, but displayed an expansion of CD56 cells with higher perforin content and cytotoxic potential, associated with accumulation of NK-cell stimulatory cytokines (IL-2, IL-7, IL-15). Overall, this report highlights an alteration in the NK-cell compartment that, together with the high disease-phenotype variability, should be considered in the suspicion of X-SCID with hypomorphic IL2RG mutation.
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http://dx.doi.org/10.1002/JLB.5MA0220-239RDOI Listing
August 2020

Cutaneous barrier leakage and gut inflammation drive skin disease in Omenn syndrome.

J Allergy Clin Immunol 2020 11 18;146(5):1165-1179.e11. Epub 2020 Apr 18.

Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Md.

Background: Severe early-onset erythroderma and gut inflammation, with massive tissue infiltration of oligoclonal activated T cells are the hallmark of Omenn syndrome (OS).

Objective: The impact of altered gut homeostasis in the cutaneous manifestations of OS remains to be clarified.

Methods: We analyzed a cohort of 15 patients with OS and the 129Sv/C57BL/6 knock-in Rag2 (Rag2) mouse model. Homing phenotypes of circulating lymphocytes were analyzed by flow cytometry. Inflammatory cytokines and chemokines were examined in the sera by ELISA and in skin biopsies by immunohistochemistry and in situ RNA hybridization. Experimental colitis was induced in mice by dextran sulfate sodium salt.

Results: We show that memory/activated T cells from patients with OS and from the Rag2 mouse model of OS abundantly express the skin homing receptors cutaneous lymphocyte associated antigen and CCR4 (Ccr4), associated with high levels of chemokine C-C motif ligands 17 and 22. Serum levels of LPS are also elevated. A broad T1/T2/T17 inflammatory signature is detected in the periphery and in the skin. Increased Tlr4 expression in the skin of Rag2 mice is associated with enhanced cutaneous inflammation on local and systemic administration of LPS. Likewise, boosting colitis in Rag2 mice results in increased frequency of Ccr4 splenic T cells and worsening of skin inflammation, as indicated by epidermal thickening, enhanced epithelial cell activation, and dermal infiltration by T1 effector T cells.

Conclusions: These results support the existence of an interplay between gut and skin that can sustain skin inflammation in OS.
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http://dx.doi.org/10.1016/j.jaci.2020.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7649331PMC
November 2020

Biomarkers and Precision Therapy for Primary Immunodeficiencies: An In Vitro Study Based on Induced Pluripotent Stem Cells From Patients.

Clin Pharmacol Ther 2020 Aug 9;108(2):358-367. Epub 2020 May 9.

Department of Life Sciences, University of Trieste, Trieste, Italy.

Ataxia telangiectasia (AT) and Aicardi-Goutières syndrome (AGS) are inherited disorders of immunity with prevalent neurological phenotype. Available treatments are only partially effective, and the prognosis is poor. Induced pluripotent stem cells (iPSCs) are obtained by reprogramming patient somatic cells, preserving the donor individual genetic heritage and creating patient-specific disease models, useful to investigate pathogenesis and drug effects and to develop precision therapies. The aim is to investigate the cytotoxicity of a panel of immunomodulators using iPSCs of patients with AT or different forms of AGS (AGS1, AGS2, and AGS7). iPSCs were obtained by reprogramming AT and AGS patients' cells and, as a control, the BJ normal human fibroblast line, using Sendai virus. Cytotoxic effects of two drugs proposed to treat respectively AT and AGS (dexamethasone and mepacrine) were tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after 72 hours' exposure. Data were obtained also for other immunomodulatory drugs (thioguanine, mercaptopurine, thalidomide, and lenalidomide). Relative expression of genes involved in the tested drug pathways was analyzed. AGS7-derived iPSCs displayed altered viability when treated with a low dose of mepacrine and higher expression of cyclic guanosine monophosphate-adenosine monophosphate synthase, which is the main target for mepacrine action. AGS7-derived iPSCs were also more sensitive to thioguanine, while AGS2 and AT iPSCs were less sensitive to this medication than the BJ-iPSC. All iPSCs were equally sensitive to mercaptopurine and resistant to dexamethasone, thalidomide, and lenalidomide. This work establishes an innovative in vitro model that is useful to investigate the mechanisms of drugs potentially effective in AT and AGS.
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http://dx.doi.org/10.1002/cpt.1837DOI Listing
August 2020

Long-term follow-up of 168 patients with X-linked agammaglobulinemia reveals increased morbidity and mortality.

J Allergy Clin Immunol 2020 08 10;146(2):429-437. Epub 2020 Mar 10.

University Department of Pediatrics, Unit of Immune and Infectious Diseases, Bambino Gesù Children's Hospital, University of Rome Tor Vergata, and the Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy.

Background: X-linked agammaglobulinemia (XLA) is the prototype of primary humoral immunodeficiencies. Long-term follow-up studies regarding disease-related complications and outcome are scarce.

Objective: Our aim was to describe the natural history of XLA.

Methods: A nationwide multicenter study based on the Italian Primary Immunodeficiency Network registry was established in 2000 in Italy. Affected patients were enrolled by documenting centers, and the patients' laboratory, clinical, and imaging data were recorded on an annual base.

Results: Data on the patients (N = 168) were derived from a cumulative follow-up of 1370 patient-years, with a mean follow-up of 8.35 years per patient. The mean age at diagnosis decreased after establishment of the Italian Primary Immunodeficiency Network registry (84 months before vs 23 months after). Respiratory, skin, and gastrointestinal manifestations were the most frequent clinical symptoms at diagnosis and during long-term follow-up. Regular immunoglobulin replacement treatment reduced the incidence of invasive infections. Affected patients developed chronic lung disease over time (47% after 40 years of follow-up) in the presence of chronic sinusitis (84%). Malignancies were documented in a minority of cases (3.7%). Overall survival for affected patients was significantly reduced when compared with that for the healthy male Italian population, and it further deteriorated in the presence of chronic lung disease.

Conclusions: This is the first detailed long-term follow-up study for patients with XLA, revealing that although immunoglobulin replacement treatment reduces the incidence of invasive infections, it does not appear to influence the development of chronic lung disease. The overall survival of affected patients is reduced. Further studies are warranted to improve patients' clinical management and increase awareness among physicians.
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http://dx.doi.org/10.1016/j.jaci.2020.03.001DOI Listing
August 2020

PAX1 is essential for development and function of the human thymus.

Sci Immunol 2020 02;5(44)

Laboratory of Clinical Immunology and Microbiology, NIAID, NIH, Bethesda, MD 20892, USA.

We investigated the molecular and cellular basis of severe combined immunodeficiency (SCID) in six patients with otofaciocervical syndrome type 2 who failed to attain T cell reconstitution after allogeneic hematopoietic stem cell transplantation, despite successful engraftment in three of them. We identified rare biallelic rare variants in all patients. We demonstrated that these mutant PAX1 proteins have an altered conformation and flexibility of the paired box domain and reduced transcriptional activity. We generated patient-derived induced pluripotent stem cells and differentiated them into thymic epithelial progenitor cells and found that they have an altered transcriptional profile, including for genes involved in the development of the thymus and other tissues derived from pharyngeal pouches. These results identify biallelic, loss-of-function mutations as the cause of a syndromic form of SCID due to altered thymus development.
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http://dx.doi.org/10.1126/sciimmunol.aax1036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189207PMC
February 2020

Generation of 3 clones of induced pluripotent stem cells (iPSCs) from a patient affected by Autosomal Recessive Osteopetrosis due to mutations in TCIRG1 gene.

Stem Cell Res 2020 01 20;42:101660. Epub 2019 Nov 20.

A. Nocivelli Institute for Molecular Medicine, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy.

Autosomal recessive osteopetrosis (ARO) is a rare inherited disorder leading to increased bone density with impairment in bone resorption. Among the genes responsible for ARO, the TCIRG1 gene, coding for the a3 subunit of the osteoclast proton pump, is mutated in more than 50% of the cases, increasing the importance of TCIRG1-iPSCs as disease model. We generated 3 iPSC clones derived from Peripheral Blood Mononuclear Cells (PBMCs) of a patient carrying the heterozygous mutations p.Y512X and c.2236 + 1G > A. A Sendai virus-based vector was used and the iPSCs were characterized for genetic identity to parental cells, genomic integrity, pluripotency, and differentiation ability.
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http://dx.doi.org/10.1016/j.scr.2019.101660DOI Listing
January 2020

Persistent Infection with Rotavirus Vaccine Strain in Severe Combined Immunodeficiency (SCID) Child: Is Rotavirus Vaccination in SCID Children a Janus Face?

Vaccines (Basel) 2019 Nov 16;7(4). Epub 2019 Nov 16.

Institute of Microbiology, Department of Molecular and Translational Medicine, University of Brescia-Spedali Civili, 25123 Brescia, Italy.

We report the first case, to our knowledge, in Italy, of a severe combined immunodeficiency patient with a persistent rotavirus infection due to a vaccine derived strain. Rotavirus was detected by enzyme immunoassays and RT-PCR in stool specimens for five months. The persistent infection was resolved after complete immune reconstitution achieved by hematopoietic stem cell transplantation. This case underlines the importance of neonatal SCID_screening.
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http://dx.doi.org/10.3390/vaccines7040185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963346PMC
November 2019

Generation of three isogenic induced Pluripotent Stem Cell lines (iPSCs) from fibroblasts of a patient with Aicardi Goutières Syndrome carrying a c.2471G>A dominant mutation in IFIH1 gene.

Stem Cell Res 2019 12 22;41:101623. Epub 2019 Oct 22.

Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy; "Angelo Nocivelli" Institute for Molecular Medicine, ASST Spedali Civili, Brescia, Italy.

Aicardi-Goutières syndrome (AGS) is an early-onset monogenic encephalopathy characterized by intracranial calcification, leukodystrophy and cerebrospinal fluid lymphocytosis. To date, seven genes have been related to AGS. Among these, IFIH1 encodes for MDA5, a cytosolic double-stranded RNA receptor, and is responsible for AGS type 7. We generated three isogenic iPSC clones, using a Sendai virus-based vector, starting from fibroblasts of a patient carrying a dominant mutation in IFIH1. All lines were characterized for genomic integrity, genetic uniqueness, pluripotency, and differentiation capability. Our clones might offer a good model to investigate AGS7 pathophysiological mechanism and to discover new biomarkers for this condition treatment.
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http://dx.doi.org/10.1016/j.scr.2019.101623DOI Listing
December 2019

Incontinentia Pigmenti Associated with Aplasia Cutis Congenita in a Newborn Male with Klinefelter Syndrome: Is the Severity of Neurological Involvement Linked to Skin Manifestations?

Dermatol Ther (Heidelb) 2020 Feb 6;10(1):213-220. Epub 2019 Nov 6.

Dermatologic Clinic, Department of Medicine and Aging Science, Università degli Studi "G. d'Annunzio" Chieti-Pescara, Chieti, Italy.

We report a rare case of a newborn male affected by incontinentia pigmenti, Klinefelter syndrome, and aplasia cutis congenita, who developed severe cutaneous, neurological, and ophthalmological manifestations. Genetic analysis showed the presence of the common mutation of NEMO (exon 4-10 deletion), Klinefelter syndrome karyotype (47 XXY), and random X inactivation. This is in accordance with the severity of involvement of the affected tissues (skin, central nervous system, and retina). Indeed, the patient developed typical skin lesions all over the body, except the head. Equally, multiple lesions diffusely involving both the cortical grey matter and subcortical white matter of the cerebellum and cerebral hemispheres were observed. Discussing current knowledge about the etiopathogenesis of skin and brain lesions in incontinentia pigmenti, our case seems to support the proapoptotic origin of central nervous system involvement. Possibly, incontinentia pigmenti patients suffer an impaired protection against apoptosis at the level of cerebral endothelial cells of small vessels, leading to vascular damage and subsequent ischemic brain lesions.
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http://dx.doi.org/10.1007/s13555-019-00336-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994639PMC
February 2020

Establishment of three iPSC lines from fibroblasts of a patient with Aicardi Goutières syndrome mutated in RNaseH2B.

Stem Cell Res 2019 12 22;41:101620. Epub 2019 Oct 22.

Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy; Angelo Nocivelli Institute for Molecular Medicine, ASST Spedali Civili, Brescia, Italy.

We report the generation of three isogenic iPSC clones (UNIBSi007-A, UNIBSi007-B, and UNIBSi007-C) obtained from fibroblasts of a patient with Aicardi Goutières Syndrome (AGS) carrying a homozygous mutation in RNaseH2B. Cells were transduced using a Sendai virus based system, delivering the human OCT4, SOX2, c-MYC and KLF4 transcription factors. The resulting transgene-free iPSC lines retained the disease-causing DNA mutation, showed normal karyotype, expressed pluripotent markers and could differentiate in vitro toward cells of the three embryonic germ layers.
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http://dx.doi.org/10.1016/j.scr.2019.101620DOI Listing
December 2019

Generation of three iPSC lines from fibroblasts of a patient with Aicardi Goutières Syndrome mutated in TREX1.

Stem Cell Res 2019 12 14;41:101580. Epub 2019 Sep 14.

"Angelo Nocivelli" Institute for Molecular Medicine, Department of Molecular and Translational Medicine, University of Brescia, Italy, ASST Spedali Civili, Brescia, Italy.

Fibroblasts from a patient with Aicardi Goutières Syndrome (AGS) carrying a compound heterozygous mutation in TREX1, were reprogrammed into induced pluripotent stem cells (iPSCs) to establish isogenic clonal stem cell lines: UNIBSi006-A, UNIBSi006-B, and UNIBSi006-C. Cells were transduced using the episomal Sendai viral vectors, containing human OCT4, SOX2, c-MYC and KLF4 transcription factors. The transgene-free iPSC lines showed normal karyotype, expressed pluripotent markers and displayed in vitro differentiation potential toward cells of the three embryonic germ layers.
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http://dx.doi.org/10.1016/j.scr.2019.101580DOI Listing
December 2019

Generation of 3 clones of induced pluripotent stem cells (iPSCs) from a patient affected by Crohn's disease.

Stem Cell Res 2019 10 23;40:101548. Epub 2019 Aug 23.

A. Nocivelli Institute for Molecular Medicine, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy.

Crohn's disease is a debilitating and incurable chronic inflammatory bowel disease, affecting millions of individuals worldwide, with an increasing frequency. Surgery must be applicable in half of the cases often with a disabling course, and pharmacological treatments may have adverse complications. We generated three isogenic clones of iPSCs from peripheral blood mononuclear cells (PBMCs) of a patient with Crohn's Disease under pharmacological treatment without adverse effects. Sendai virus based vector was used and the iPSCs were characterized for genetic uniqueness, genomic integrity, pluripotency, and differentiation ability. These iPSCs will be a powerful tool to develop tailored therapies.
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http://dx.doi.org/10.1016/j.scr.2019.101548DOI Listing
October 2019

Clinical, Immunological, and Molecular Features of Typical and Atypical Severe Combined Immunodeficiency: Report of the Italian Primary Immunodeficiency Network.

Front Immunol 2019 13;10:1908. Epub 2019 Aug 13.

Department of Pediatric Hematology and Oncology, Bambino Gesù Children's Hospital, Rome, Italy.

Severe combined immunodeficiencies (SCIDs) are a group of inborn errors of the immune system, usually associated with severe or life-threatening infections. Due to the variability of clinical phenotypes, the diagnostic complexity and the heterogeneity of the genetic basis, they are often difficult to recognize, leading to a significant diagnostic delay (DD). Aim of this study is to define presenting signs and natural history of SCID in a large cohort of patients, prior to hematopoietic stem cell or gene therapies. To this purpose, we conducted a 30-year retro-prospective multicenter study within the Italian Primary Immunodeficiency Network. One hundred eleven patients, diagnosed as typical or atypical SCID according to the European Society for Immune Deficiencies criteria, were included. Patients were subsequently classified based on the genetic alteration, pathogenic mechanism and immunological classification. A positive relationship between the age at onset and the DD was found. SCID patients with later onset were identified only in the last decade of observation. Syndromic SCIDs represented 28% of the cohort. Eight percent of the subjects were diagnosed in Intensive Care Units. Fifty-three percent had an atypical phenotype and most of them exhibited a discordant genotype-immunophenotype. Pre-treatment mortality was higher in atypical and syndromic patients. Our study broadens the knowledge of clinical and laboratory manifestations and genotype/phenotype correlation in patients with SCID and may facilitate the diagnosis of both typical and atypical forms of the disease in countries where newborn screening programs have not yet been implemented.
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http://dx.doi.org/10.3389/fimmu.2019.01908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700292PMC
September 2020

Targeted NGS Platforms for Genetic Screening and Gene Discovery in Primary Immunodeficiencies.

Front Immunol 2019 11;10:316. Epub 2019 Apr 11.

Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy.

Primary Immunodeficiencies (PIDs) are a heterogeneous group of genetic immune disorders. While some PIDs can manifest with more than one phenotype, signs, and symptoms of various PIDs overlap considerably. Recently, novel defects in immune-related genes and additional variants in previously reported genes responsible for PIDs have been successfully identified by Next Generation Sequencing (NGS), allowing the recognition of a broad spectrum of disorders. To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes. Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: and . The Ion Torrent sequencing platform was used in 73 patients. Among these, 18 selected patients without a molecular diagnosis and 32 additional patients were analyzed by Haloplex enrichment technology. The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% ( = 30) of patients. Twenty-two out of 73 (34.6%) patients were diagnosed by Ion Torrent. In this group 20 were included in the SCID/CID category. Eight out of 50 (16%) patients were diagnosed by Haloplex workflow. Ion Torrent method was highly successful for those cases with well-defined phenotypes for immunological and clinical presentation. The Haloplex approach was able to diagnose 4 SCID/CID patients and 4 additional patients with complex and extended phenotypes, embracing all three PID categories in which this approach was more efficient. Both technologies showed good gene coverage. NGS technology represents a powerful approach in the complex field of rare disorders but its different application should be weighted. A relatively small NGS target panel can be successfully applied for a robust diagnostic suspicion, while when the spectrum of clinical phenotypes overlaps more than one PID an in-depth NGS analysis is required, including also whole exome/genome sequencing to identify the causative gene.
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http://dx.doi.org/10.3389/fimmu.2019.00316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470723PMC
September 2020

Pseudomonas aeruginosa severe skin infection in a toddler with X-linked agammaglobulinemia due to a novel BTK mutation.

Infez Med 2019 Mar;27(1):73-76

Infectious Disease Unit, Istituto Giannina Gaslini, University of Genoa, Italy.

Agammaglobulinemia is a congenital deficit of humoral immunity characterized by a decreased level or complete absence of immunoglobulins and profound reduction of B-lymphocytes associated with an increased risk of life-threatening bacterial infection. We report a case of invasive Pseudomonas aeruginosa severe skin and soft tissue infection treated with vacuum-assisted closure and antibiotics in a toddler with a previously unreported mutation of the Bruton tyrosin kinase gene.
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March 2019

Generation of induced pluripotent stem cells (iPSCs) from patient with Cri du Chat Syndrome.

Stem Cell Res 2019 03 26;35:101393. Epub 2019 Jan 26.

A. Nocivelli Institute for Molecular Medicine, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy.

The Cri du Chat Syndrome (CdCS) is a genetic disease resulting from variable size deletion occurring on the short arm of chromosome 5. The main clinical features are a high-pitched monochromatic cry, microcephaly, severe psychomotor and mental retardation with characteristics of autism spectrum disorders such as hand flapping, obsessive attachments to objects, twirling objects, repetitive movements, and rocking. We reprogrammed to pluripotency peripheral blood mononuclear cells derived from a patient carrying large deletion on the short arm of chromosome 5, using a commercially available non-integrating expression system. The iPSCs expressed pluripotency markers and differentiated in the three embryonic germ layers.
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http://dx.doi.org/10.1016/j.scr.2019.101393DOI Listing
March 2019

Sine causa tetraparesis: A pilot study on its possible relationship with interferon signature analysis and Aicardi Goutières syndrome related genes analysis.

Medicine (Baltimore) 2018 Dec;97(52):e13893

Department of Clinical and Experimental Sciences, University of Brescia.

Tetraparesis is usually due to cerebral palsy (CP), inborn errors of metabolism, neurogenetic disorders and spinal cord lesions. However, literature data reported that about 10% of children with tetraparesis show a negative/non-specific neuroradiological findings without a specific etiological cause. Aicardi Goutières Syndrome (AGS) is a genetic encephalopathy that may cause tetraparesis. Interferon signature is a reliable biomarker for AGS and could be performed in sine-causa tetraparesis. The aim of the study was to examine the type I interferon signature and AGS related-genes in children with sine causa tetraparesis, to look for misdiagnosed AGS. A secondary aim was to determine which aspects of the patient history, clinical picture and brain imaging best characterize tetraparesis due to an interferonopathy.Seven out of 78 patients affected by tetraparesis, characterized by unremarkable pre-peri-postnatal history and normal/non-specific brain magnetic resonance imaging (MRI) were selected and underwent anamnestic data collection, clinical examination, brain imaging review, peripheral blood interferon signature and AGS-related genes analysis.At our evaluation time (mean age of 11.9 years), all the 7 patients showed spastic-dystonic tetraparesis. At clinical onset brain MRI was normal in 4 and with non-specific abnormalities in 3; at follow-up 3 patients presented with new white-matter lesions, associated with brain calcification in 1 case. Interferon signature was elevated in one subject who presented also a mutation of the IFIH1 gene.AGS should be considered in sine-causa tetraparesis. Core features of interferonopathy-related tetraparesis are: onset during first year of life, psychomotor regression with tetraparesis evolution, brain white-matter lesions with late calcifications. A positive interferon signature may be a helpful marker to select patients with spastic tetraparesis who should undergo genetic analysis for AGS.
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http://dx.doi.org/10.1097/MD.0000000000013893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314769PMC
December 2018

Next Generation Sequencing Analysis in Early Onset Dementia Patients.

J Alzheimers Dis 2019 ;67(1):243-256

Genetics Unit, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy.

Background: Early onset dementias (EOD) are rare neurodegenerative dementias that present before 65 years. Genetic factors have a substantially higher pathogenetic contribution in EOD patients than in late onset dementia.

Objective: To identify known and/or novel rare variants in major candidate genes associated to EOD by high-throughput sequencing. Common-risk variants of apolipoprotein E (APOE) and prion protein (PRNP) genes were also assessed.

Methods: We studied 22 EOD patients recruited in Memory Clinics, in the context of studies investigating genetic forms of dementia. Two methodological approaches were applied for the target-Next Generation Sequencing (NGS) analysis of these patients. In addition, we performed progranulin plasma dosage, C9Orf72 hexanucleotide repeat expansion analysis, and APOE genotyping.

Results: We detected three rare known pathogenic mutations in the GRN and PSEN2 genes and eleven unknown-impact mutations in the GRN, VCP, MAPT, FUS, TREM2, and NOTCH3 genes. Six patients were carriers of only common risk variants (APOE and PRNP), and one did not show any risk mutation/variant. Overall, 69% (n = 9) of our early onset Alzheimer's disease (EAOD) patients, compared with 34% (n = 13) of sporadic late onset Alzheimer's disease (LOAD) patients and 27% (n = 73) of non-affected controls (ADNI, whole genome data), were carriers of at least two rare/common risk variants in the analyzed candidate genes panel, excluding the full penetrant mutations.

Conclusion: This study suggests that EOD patients without full penetrant mutations are characterized by higher probability to carry polygenic risk alleles that patients with LOAD forms. This finding is in line with recently reported evidence, thus suggesting that the genetic risk factors identified in LOAD might modulate the risk also in EOAD.
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http://dx.doi.org/10.3233/JAD-180482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398561PMC
March 2020