Igor Petrovich Oscorbin , Maria Alexandrovna Gordukova , Nataliia Vladimirovna Davydova , Natalia Valentinovna Zinovieva , Elena Fedorovna Kovzel , Lucia Andries , Dmitry Anatolyevich Kudlay , Maxim Leonidovich Filipenko
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Diagnosis of DGS relies on phenotypic examination, qPCR, ultrasound, FISH, MLPA and NGS which can be relatively inaccurate, time-consuming, and costly.</p></div><div><h3>Materials and methods</h3><p>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 — <em>TUPLE1</em>, <em>ZNF74</em>, D22S936 — within the deletion areas and one reference target — <em>RPP30</em> — as an internal control.</p></div><div><h3>Results</h3><p>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.</p></div><div><h3>Conclusion</h3><p>The designed ddPCR assay is suitable for diagnosing DGS using whole blood and blood spots.</p></div>","PeriodicalId":10205,"journal":{"name":"Clinica Chimica Acta","volume":null,"pages":null},"PeriodicalIF":3.2000,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Multiplex droplet digital PCR for 22q11.2 microdeletions screening and DiGeorge syndrome diagnostics\",\"authors\":\"Igor Petrovich Oscorbin , Maria Alexandrovna Gordukova , Nataliia Vladimirovna Davydova , Natalia Valentinovna Zinovieva , Elena Fedorovna Kovzel , Lucia Andries , Dmitry Anatolyevich Kudlay , Maxim Leonidovich Filipenko\",\"doi\":\"10.1016/j.cca.2024.119903\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Background and aims</h3><p>DiGeorge syndrome (DGS) is a genetic disorder manifesting in polymorphic symptoms related to developmental abnormalities of various organs including thymus. 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引用次数: 0
摘要
背景和目的：迪乔治综合征（DGS）是一种遗传性疾病，表现为与胸腺等多个器官发育异常有关的多形性症状。DGS是由22q11.2区域几个低拷贝重复序列（LCR）之间的微缺失引起的，大约每4000个活产婴儿中就有1例。DGS 的诊断依赖于表型检查、qPCR、超声波、FISH、MLPA 和 NGS，这些方法可能相对不准确、耗时且昂贵：设计、优化并验证了一种新型多重液滴数字 PCR（ddPCR）检测方法，通过同时扩增缺失区内的三个靶标（TUPLE1、ZNF74、D22S936）和一个参考靶标（RPP30）作为内部对照，检测并绘制 22q11.2 微缺失图：当每次反应的模板浓度大于 32 个拷贝时，该检测方法能可靠地识别微缺失，并在 153 名有免疫缺陷症状的患者的临床样本中成功检测出 LCR22A-B、LCR22A-C、LCR22A-D 和 LCR22B-C 缺失。在微缺失患者中，流式细胞术检测到 B 细胞和自然杀伤细胞的数量和百分比显著增加，而 T 细胞百分比和 T 细胞受体切割圈（TREC）数量减少：结论：设计的 ddPCR 检测方法适用于使用全血和血斑诊断 DGS。
Multiplex droplet digital PCR for 22q11.2 microdeletions screening and DiGeorge syndrome diagnostics
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.
期刊介绍：
The Official Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)
Clinica Chimica Acta is a high-quality journal which publishes original Research Communications in the field of clinical chemistry and laboratory medicine, defined as the diagnostic application of chemistry, biochemistry, immunochemistry, biochemical aspects of hematology, toxicology, and molecular biology to the study of human disease in body fluids and cells.
The objective of the journal is to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management. Reports of an applied clinical character are also welcome. Papers concerned with normal metabolic processes or with constituents of normal cells or body fluids, such as reports of experimental or clinical studies in animals, are only considered when they are clearly and directly relevant to human disease. Evaluation of commercial products have a low priority for publication, unless they are novel or represent a technological breakthrough. Studies dealing with effects of drugs and natural products and studies dealing with the redox status in various diseases are not within the journal''s scope. Development and evaluation of novel analytical methodologies where applicable to diagnostic clinical chemistry and laboratory medicine, including point-of-care testing, and topics on laboratory management and informatics will also be considered. Studies focused on emerging diagnostic technologies and (big) data analysis procedures including digitalization, mobile Health, and artificial Intelligence applied to Laboratory Medicine are also of interest.
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