Multiplex droplet digital PCR for 22q11.2 microdeletions screening and DiGeorge syndrome diagnostics
Oscorbin I.P. Gordukova M.A. Davydova N.V. Zinovieva N.V. Kovzel E.F. Andries L. Kudlay D.A. Filipenko M.L.
15 September 2024Elsevier B.V.
Clinica Chimica Acta
2024#563
Background and aims: DiGeorge syndrome (DGS) is a genetic disorder manifesting in polymorphic symptoms related to developmental abnormalities of various organs including thymus. DGS is caused by microdeletions in the 22q11.2 region between several low copy repeats (LCR) occurring in approximately 1 in 4000 live births. Diagnosis of DGS relies on phenotypic examination, qPCR, ultrasound, FISH, MLPA and NGS which can be relatively inaccurate, time-consuming, and costly. Materials and methods: A novel multiplex droplet digital PCR (ddPCR) assay was designed, optimized and validated for detection and mapping 22q11.2 microdeletions by simultaneous amplification of three targets — TUPLE1, ZNF74, D22S936 — within the deletion areas and one reference target — RPP30 — as an internal control. Results: The assay reliable identified microdeletions when the template concentration was >32 copies per reaction and successfully detected LCR22A-B, LCR22A-C, LCR22A-D, and LCR22B-C deletions in clinical samples from 153 patients with signs of immunodeficiency. In patients with the microdeletions, flow cytometry detected a significant increase in B-cell and natural killer cell counts and percentages, while T-cell percentages and T-cell receptor excision circle (TREC) numbers decreased. Conclusion: The designed ddPCR assay is suitable for diagnosing DGS using whole blood and blood spots.
22q11.2 microdeletions , DiGeorge syndrome , Droplet digital PCR, ddPCR , Multiplex ddPCR
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Laboratory of Pharmacogenomics, The Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences (ICBFM SB RAS), Novosibirsk, 630090, Russian Federation
G.N. Speransky Childrens Hospital No. 9, Moscow, Russian Federation
Clinical Immunology, Allergology, Pulmonology Program, Corporate Fund “University Medical Center” of Nazarbayev University, Astana, Kazakhstan
Laboratory of Clinical Immunology and Allergology, Nicolae Testemitanu State University of Medicine and Pharmacy of the Republic of Moldova, Chișinău, Moldova
The Department of Pharmacology, Faculty of Medicine, I.M. Sechenov First Moscow State Medical University, Pogodinskaya St. 1, Moscow, 119991, Russian Federation
Laboratory of Pharmacogenomics
G.N. Speransky Childrens Hospital No. 9
Clinical Immunology
Laboratory of Clinical Immunology and Allergology
The Department of Pharmacology
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