Target enriched sequencing was designed to cover 356 IEI genes as previously described [35 (link)] and used to diagnose P1 and P6. WES was perfomed for P4, P5, and P12 as described [24 (link), 35 (link)]. For Sanger sequencing of IL21R in P2 and P3, genomic DNA was extracted from whole blood (QIamp DNA Blood Kit, Qiagen). Targeted Sanger sequencing of IL21R in P7–P10 has been reported [22 (link), 24 (link)]. Primer sequences used to identify IL21R variants in P2, P3, P11, and P13 and validate NGS findings in P1, P4, P5, and P6 are described in Supplementary Table I . Pathogenicity of genetic variants was evaluated based on the allele frequency in public databases including ExAC and gnomAD and prediction tools such as Polyphen-2, SIFT and the combined annotation dependent depletion (CADD) score.
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Genetic Variant Identification in Primary Immunodeficiency
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Corresponding Organization : Hacettepe University Hospital
Other organizations : Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Marmara University, Garvan Institute of Medical Research, St Vincent's Clinic, Sağlık Bilimleri Üniversitesi, Hacettepe University, Istinye University, Istanbul University, Medical University of Vienna, National Institutes of Health, National Institute of Allergy and Infectious Diseases, University Medical Center Freiburg, University of Freiburg, Hôpital Necker-Enfants Malades, Institut des Maladies Génétiques Imagine, Université Paris Cité, Inserm, Hadassah Medical Center, Hebrew University of Jerusalem, CeMM Research Center for Molecular Medicine, Austrian Academy of Sciences
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Variable analysis
independent variables
- Target enriched sequencing to cover 356 IEI genes
- Whole-exome sequencing (WES) for P4, P5, and P12
- Sanger sequencing of IL21R in P2, P3, P7-P10, P11, and P13
- Diagnosis of P1 and P6
- Identification of IL21R variants in P2, P3, P11, and P13
- Validation of NGS findings in P1, P4, P5, and P6
- Genomic DNA extraction from whole blood using QIamp DNA Blood Kit (Qiagen)
- No positive or negative controls were explicitly mentioned in the provided information.
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