National external quality assessment for next-generation sequencing-based diagnostics of primary immunodeficiencies


Dutch genome diagnostic centers (GDC) use next-generation sequencing (NGS)-based diagnostic applications for the diagnosis of primary immunodeficiencies (PIDs). The interpretation of genetic variants in many PIDs is complicated because of the phenotypic and genetic heterogeneity. To analyze uniformity of variant filtering, interpretation, and reporting in NGS-based diagnostics for PID, an external quality assessment was performed. Four main Dutch GDCs participated in the quality assessment. Unannotated variant call format (VCF) files of two PID patient analyses per laboratory were distributed among the four GDCs, analyzed, and interpreted (eight analyses in total). Variants that would be reported to the clinician and/or advised for further investigation were compared between the centers. A survey measuring the experiences of clinical laboratory geneticists was part of the study. Analysis of samples with confirmed diagnoses showed that all centers reported at least the variants classified as likely pathogenic (LP) or pathogenic (P) variants in all samples, except for variants in two genes (PSTPIP1 and BTK). The absence of clinical information complicated correct classification of variants. In this external quality assessment, the final interpretation and conclusions of the genetic analyses were uniform among the four participating genetic centers. Clinical and immunological data provided by a medical specialist are required to be able to draw proper conclusions from genetic data.

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Fig. 1: Overview of sample distribution within the project per site.
Fig. 2: Overview of sample workup per center.


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This publication and the underlying study have been made possible partly on the basis of the data that Hartwig Medical Foundation and Central Genome Analysis Laboratory—UMC St. Radboud have generated for the study. We thank our colleague Walter de Valk from the Erasmus Medical Center and Genomics Coordination Center—UMC Groningen for his support regarding bioinformatics.


The external quality assessment (EQA) is part of the Genetics First: next-generation sequencing for cost-effective PID diagnostics project. This project is funded by ZonMw (grant No. 846002001).

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Correspondence to Mariëlle E. van Gijn.

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Elsink, K., Huibers, M.M.H., Hollink, I.H.I.M. et al. National external quality assessment for next-generation sequencing-based diagnostics of primary immunodeficiencies. Eur J Hum Genet (2020).

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