title: Targeted next-generation sequencing: a novel diagnostic tool for primary immunodeficiencies.
abstract: BACKGROUND
  Primary immunodeficiency (PID) disorders are a heterogeneous group of inherited disorders caused by a variety of monogenetic immune defects. Thus far, mutations in more than 170 different genes causing PIDs have been described. A clear genotype-phenotype correlation is often not available, which makes a genetic diagnosis in patients with PIDs complex and laborious.
  
  
  OBJECTIVE
  We sought to develop a robust, time-effective, and cost-effective diagnostic method to facilitate a genetic diagnosis in any of 170 known PID-related genes by using next-generation sequencing (NGS).
  
  
  METHODS
  We used both targeted array-based and in-solution enrichment combined with a SOLiD sequencing platform and a bioinformatic pipeline developed in house to analyze genetic changes in the DNA of 41 patients with PIDs with known mutations and 26 patients with undiagnosed PIDs.
  
  
  RESULTS
  This novel NGS-based method accurately detected point mutations (sensitivity and specificity >99% in covered regions) and exonic deletions (100% sensitivity and specificity). For the 170 genes of interest, the DNA coverage was greater than 20× in 90% to 95%. Nine PID-related genes proved not eligible for evaluation by using this NGS-based method because of inadequate coverage. The NGS method allowed us to make a genetic diagnosis in 4 of 26 patients who lacked a genetic diagnosis despite routine functional and genetic testing. Three of these patients proved to have an atypical presentation of previously described PIDs.
  
  
  CONCLUSIONS
  This novel NGS tool facilitates accurate simultaneous detection of mutations in 161 of 170 known PID-related genes. In addition, these analyses will generate more insight into genotype-phenotype correlations for the different PID disorders.
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Isaac J
authors.initials: IJ
authors.lastname: Nijman
authors.affiliation: Department of Pediatric Immunology and Infectious Diseases, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Joris M
authors.initials: JM
authors.lastname: van Montfrans
authors.affiliation: Department of Medical Oncology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Marlous
authors.initials: M
authors.lastname: Hoogstraat
authors.affiliation: Department of Pediatric Immunology and Infectious Diseases, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Marianne L
authors.initials: ML
authors.lastname: Boes
authors.affiliation: Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Lisette
authors.initials: L
authors.lastname: van de Corput
authors.affiliation: Department of Immunology, University Children's Hospital, Ludwig-Maximilians-Universität, Munich, Germany.
authors.forename: Ellen D
authors.initials: ED
authors.lastname: Renner
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Patrick
authors.initials: P
authors.lastname: van Zon
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Stef
authors.initials: S
authors.lastname: van Lieshout
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Martin G
authors.initials: MG
authors.lastname: Elferink
authors.affiliation: Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands.
authors.forename: Mirjam
authors.initials: M
authors.lastname: van der Burg
authors.affiliation: Department of Pediatric Infectious Diseases, Immunology and Stem Cell Transplantation, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Clementien L
authors.initials: CL
authors.lastname: Vermont
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Bert
authors.initials: B
authors.lastname: van der Zwaag
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Esther
authors.initials: E
authors.lastname: Janson
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Edwin
authors.initials: E
authors.lastname: Cuppen
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: Johannes K
authors.initials: JK
authors.lastname: Ploos van Amstel
authors.affiliation: Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address: m.e.vangijn@umcutrecht.nl.
authors.forename: Marielle E
authors.initials: ME
authors.lastname: van Gijn
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D020022:Genetic Predisposition to Disease; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D009154:Mutation; D017422:Sequence Analysis, DNA
keywords: ALPS; Autoimmune lymphoproliferative syndrome; CID; Combined immunodeficiency; HLH; Hemophagocytic lymphohistiocytosis; IUIS; International Union of Immunodeficiency Societies; NGS; Next-generation sequencing; PID; Primary immunodeficiency; SNV; Single nucleotide variant; diagnosis; genetics; next-generation sequencing