Vidal-Folch, Noemi

AbstractGlycosylation is a critical post/peri‐translational modification required for the appropriate development and function of the immune system. As an example, abnormalities in glycosylation can cause antibody deficiency and reduced lymphocyte signaling, although the phenotype can be complex given the diverse roles of glycosylation. Human MGAT2 encodes N‐acetylglucosaminyltransferase II, which is a critical enzyme in the processing of oligomannose to complex N‐glycans. Complex N‐glycans are essential for immune system functionality, but only one individual with MGAT2‐CDG has been described to have an abnormal immunologic evaluation. MGAT2‐CDG (CDG‐IIa) is a congenital disorder of glycosylation (CDG) associated with profound global developmental disability, hypotonia, early onset epilepsy, and other multisystem manifestations. Here, we report a 4‐year old female with MGAT2‐CDG due to a novel homozygous pathogenic variant in MGAT2, a 4‐base pair deletion, c.1006_1009delGACA. In addition to clinical features previously described in MGAT2‐CDG, she experienced episodic asystole, persistent hypogammaglobulinemia, and defective ex vivo mitogen and antigen proliferative responses, but intact specific vaccine antibody titers. Her infection history has been mild despite the testing abnormalities. We compare this patient to the 15 previously reported patients in the literature, thus expanding both the genotypic and phenotypic spectrum for MGAT2‐CDG.

AbstractBACKGROUNDDroplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need.METHODSWe validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies.RESULTSFor the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals.CONCLUSIONSStandard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.