title: Diagnostics of Primary Immunodeficiencies through Next-Generation Sequencing.
abstract: BACKGROUND
  Recently, a growing number of novel genetic defects underlying primary immunodeficiencies (PIDs) have been identified, increasing the number of PID up to more than 250 well-defined forms. Next-generation sequencing (NGS) technologies and proper filtering strategies greatly contributed to this rapid evolution, providing the possibility to rapidly and simultaneously analyze large numbers of genes or the whole exome.
  
  
  OBJECTIVE
  To evaluate the role of targeted NGS and whole exome sequencing (WES) in the diagnosis of a case series, characterized by complex or atypical clinical features suggesting a PID, difficult to diagnose using the current diagnostic procedures.
  
  
  METHODS
  We retrospectively analyzed genetic variants identified through targeted NGS or WES in 45 patients with complex PID of unknown etiology.
  
  
  RESULTS
  Forty-seven variants were identified using targeted NGS, while 5 were identified using WES. Newly identified genetic variants were classified into four groups: (I) variations associated with a well-defined PID, (II) variations associated with atypical features of a well-defined PID, (III) functionally relevant variations potentially involved in the immunological features, and (IV) non-diagnostic genotype, in whom the link with phenotype is missing. We reached a conclusive genetic diagnosis in 7/45 patients (~16%). Among them, four patients presented with a typical well-defined PID. In the remaining three cases, mutations were associated with unexpected clinical features, expanding the phenotypic spectrum of typical PIDs. In addition, we identified 31 variants in 10 patients with complex phenotype, individually not causativeper seof the disorder.
  
  
  CONCLUSIONS
  NGS technologies represent a cost-effective and rapid first-line genetic approach for the evaluation of complex PIDs. WES, despite a moderate higher cost compared to targeted, is emerging as a valuable tool to reach in a timely manner, a PID diagnosis with a considerable potential to draw genotype-phenotype correlation. Nevertheless, a large fraction of patients still remains without a conclusive diagnosis. In these patients, the sum of non-diagnostic variants might be proven informative in future studies with larger cohorts of patients.
authors.affiliation: Department of Translational Medical Sciences, Federico II University , Naples , Italy.
authors.forename: Vera
authors.initials: V
authors.lastname: Gallo
authors.affiliation: Department of Clinical and Experimental Medicine, "Angelo Nocivelli" Institute for Molecular Medicine, University of Brescia , Brescia , Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Dotta
authors.affiliation: Department of Translational Medical Sciences, Federico II University , Naples , Italy.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Giardino
authors.affiliation: Department of Translational Medical Sciences, Federico II University , Naples , Italy.
authors.forename: Emilia
authors.initials: E
authors.lastname: Cirillo
authors.affiliation: Department of Clinical and Experimental Medicine, "Angelo Nocivelli" Institute for Molecular Medicine, University of Brescia , Brescia , Italy.
authors.forename: Vassilios
authors.initials: V
authors.lastname: Lougaris
authors.affiliation: Department of Translational Medical Sciences, Federico II University , Naples , Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: D'Assante
authors.affiliation: Department of Clinical and Experimental Medicine, "Angelo Nocivelli" Institute for Molecular Medicine, University of Brescia , Brescia , Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Prandini
authors.affiliation: Department of Clinical and Experimental Medicine, University of Pisa , Pisa , Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Consolini
authors.affiliation: Center for Pediatric Genomic Medicine, Children's Mercy Hospital , Kansas City, MO , USA.
authors.forename: Emily G
authors.initials: EG
authors.lastname: Farrow
authors.affiliation: Center for Pediatric Genomic Medicine, Children's Mercy Hospital , Kansas City, MO , USA.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Thiffault
authors.affiliation: Center for Pediatric Genomic Medicine, Children's Mercy Hospital , Kansas City, MO , USA.
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Saunders
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, Federico II University , Naples , Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Leonardi
authors.affiliation: Department of Clinical and Experimental Medicine, "Angelo Nocivelli" Institute for Molecular Medicine, University of Brescia , Brescia , Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Plebani
authors.affiliation: Department of Clinical and Experimental Medicine, "Angelo Nocivelli" Institute for Molecular Medicine, University of Brescia , Brescia , Italy.
authors.forename: Raffaele
authors.initials: R
authors.lastname: Badolato
authors.affiliation: Department of Translational Medical Sciences, Federico II University , Naples , Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Pignata
keywords: genetic diagnosis; genotype–phenotype correlation; primary immunodeficiencies; targeted next-generation sequencing; whole exome sequencing