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Extended report
Next-generation sequencing and its initial applications for molecular diagnosis of systemic auto-inflammatory diseases
  1. Marta Rusmini1,
  2. Silvia Federici2,
  3. Francesco Caroli1,
  4. Alice Grossi1,
  5. Maurizia Baldi3,
  6. Laura Obici4,
  7. Antonella Insalaco5,
  8. Alberto Tommasini6,
  9. Roberta Caorsi2,
  10. Eleonora Gallo7,
  11. Alma Nunzia Olivieri8,
  12. AngeloValerio Marzano9,
  13. Domenico Coviello3,
  14. Roberto Ravazzolo1,10,
  15. Alberto Martini2,10,
  16. Marco Gattorno2,
  17. Isabella Ceccherini1
  1. 1UOC Medical Genetics, IRCCS Istituto Giannina Gaslini, Genoa, Italy
  2. 2UOC Pediatric Rheumatology, Lab Immunology Rheumatic Diseases, IRCCS Istituto Giannina Gaslini, Genoa, Italy
  3. 3Laboratory of Human Genetics, Ospedali Galliera, Genoa, Italy
  4. 4Amyloidosis Research and Treatment Centre, Biotechnology Research Laboratories, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
  5. 5Division of Rheumatology, Department of Pediatric Medicine, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy
  6. 6Department of Pediatrics, Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
  7. 7Dipartimento di Salute Pubblica e Pediatria, Universita’ di Torino, Torino, Italy
  8. 8Dipartimento della Donna, del Bambino e di Chirurgia Generale e Specialistica, Seconda Universita’ degli Studi di Napoli, Napoli, Italia
  9. 9UO Dermatologia, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico—Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milan, Italy
  10. 10Dipartimento Neuroscienze, Riabilitazione, Oftalmologia, Genetica e Scienze Materno-Infantili (DiNOGMI), Università degli Studi di Genova, Genova, Italy
  1. Correspondence to Dr Isabella Ceccherini, UOC Genetica Medica, Istituto Giannina Gaslini, Genova 16148, Italy; isa.c{at}


Objectives 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.

Methods 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.

Results 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.

Conclusions 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.

  • Fever Syndromes
  • Inflammation
  • Gene Polymorphism

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