title: Next-generation sequencing and its initial applications for molecular diagnosis of systemic auto-inflammatory diseases.
abstract: Systemic auto-inflammatory disorders (SAIDs) are a heterogeneous group of monogenic diseases sharing a primary dysfunction of the innate immune system. More than 50% of patients with SAID does not show any mutation at gene(s) tested because of lack of precise clinical classification criteria and/or incomplete gene screening. To improve the molecular diagnosis and genotype interpretation of SAIDs, we undertook the development of a next-generation sequencing (NGS)-based protocol designed to simultaneous screening of 10 genes.
  
  
  
  Fifty patients with SAID, already genotyped for the respective causative gene(s), were massively sequenced for the coding portions of MEFV, MVK, TNFRSF1A, NLRP3, NLRP12, NOD2, PSTPIP1, IL1RN, LPIN2 and PSMB8. Three different bioinformatic pipelines (Ion Reporter, CLC Bio Genomics Workbench, GATK-based in-house workflow) were compared.
  
  
  
  Once resulting variants were compared with the expected mutation list, no workflow turned out to be able to detect all the 79 variants known in the 50 DNAs. Additional variants were also detected, validated by Sanger sequencing and compared to assess true and false positive detection rates of the three workflows. Finally, the overall clinical picture of 34 patients was re-evaluated in the light of the new mutations found.
  
  
  
  The present gene panel has resulted suitable for molecular diagnosis of SAIDs. Moreover, genotype-phenotype correlation has confirmed that the interpretation of NGS data in patients with an undefined inflammatory phenotype is remarkably difficult, thus supporting the need of evidence-based and validated clinical criteria to be used concurrently with the genetic analysis for the final diagnosis and classification of patients with SAIDs.
authors.affiliation: UOC Medical Genetics, IRCCS Istituto Giannina Gaslini, Genoa, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Rusmini
authors.affiliation: UOC Pediatric Rheumatology, Lab Immunology Rheumatic Diseases, IRCCS Istituto Giannina Gaslini, Genoa, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Federici
authors.affiliation: UOC Medical Genetics, IRCCS Istituto Giannina Gaslini, Genoa, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Caroli
authors.affiliation: UOC Medical Genetics, IRCCS Istituto Giannina Gaslini, Genoa, Italy.
authors.forename: Alice
authors.initials: A
authors.lastname: Grossi
authors.affiliation: Laboratory of Human Genetics, Ospedali Galliera, Genoa, Italy.
authors.forename: Maurizia
authors.initials: M
authors.lastname: Baldi
authors.affiliation: Amyloidosis Research and Treatment Centre, Biotechnology Research Laboratories, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Obici
authors.affiliation: Division of Rheumatology, Department of Pediatric Medicine, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Insalaco
authors.affiliation: Department of Pediatrics, Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Tommasini
authors.affiliation: UOC Pediatric Rheumatology, Lab Immunology Rheumatic Diseases, IRCCS Istituto Giannina Gaslini, Genoa, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Caorsi
authors.affiliation: Dipartimento di Salute Pubblica e Pediatria, Universita' di Torino, Torino, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Gallo
authors.affiliation: Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica, Seconda Universita' degli Studi di Napoli, Napoli, Italia.
authors.forename: Alma Nunzia
authors.initials: AN
authors.lastname: Olivieri
authors.affiliation: UO Dermatologia, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico-Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milan, Italy.
authors.forename: AngeloValerio
authors.initials: A
authors.lastname: Marzano
authors.affiliation: Laboratory of Human Genetics, Ospedali Galliera, Genoa, Italy.
authors.forename: Domenico
authors.initials: D
authors.lastname: Coviello
authors.affiliation: UOC Medical Genetics, IRCCS Istituto Giannina Gaslini, Genoa, Italy Dipartimento Neuroscienze, Riabilitazione, Oftalmologia, Genetica e Scienze Materno-Infantili (DiNOGMI), Università degli Studi di Genova, Genova, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Ravazzolo
authors.affiliation: UOC Pediatric Rheumatology, Lab Immunology Rheumatic Diseases, IRCCS Istituto Giannina Gaslini, Genoa, Italy Dipartimento Neuroscienze, Riabilitazione, Oftalmologia, Genetica e Scienze Materno-Infantili (DiNOGMI), Università degli Studi di Genova, Genova, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Martini
authors.affiliation: UOC Pediatric Rheumatology, Lab Immunology Rheumatic Diseases, IRCCS Istituto Giannina Gaslini, Genoa, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Gattorno
authors.affiliation: UOC Medical Genetics, IRCCS Istituto Giannina Gaslini, Genoa, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Ceccherini
mesh_terms: D019295:Computational Biology / Q000379:methods; D010505:Familial Mediterranean Fever / Q000175:diagnosis / Q000235:genetics; D005787:Gene Frequency; D005838:Genotype; D056660:Hereditary Autoinflammatory Diseases / Q000175:diagnosis* / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D010641:Phenotype
keywords: Fever Syndromes; Gene Polymorphism; Inflammation