Targeted next-generation sequencing: a novel diagnostic tool for primary immunodeficiencies

Isaac J Nijman, Joris M van Montfrans, Marlous Hoogstraat, Marianne L Boes, Lisette van de Corput, Ellen D Renner, Patrick van Zon, Stef van Lieshout, Martin G Elferink, Mirjam van der Burg, Clementien L Vermont, Bert van der Zwaag, Esther Janson, Edwin Cuppen, Johannes K Ploos van Amstel, Marielle E van Gijn

Research output: Contribution to journal/periodicalArticleScientificpeer-review

124 Citations (Scopus)


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.

CONCLUSION: 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.

Original languageEnglish
Pages (from-to)529-34
Number of pages6
JournalJournal of Allergy and Clinical Immunology
Issue number2
Publication statusPublished - Feb 2014


  • Adolescent
  • Adult
  • Child
  • Genetic Predisposition to Disease
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Immunologic Deficiency Syndromes
  • Male
  • Mutation
  • Sequence Analysis, DNA


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