Guangzhou Newborn Screening Center, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, Guangdong, China.
Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, Guangdong, China.
Clin Biochem. 2021 Jan;87:79-84. doi: 10.1016/j.clinbiochem.2020.10.011. Epub 2020 Nov 11.
Gaucher disease (GD) is caused by a deficiency of β-glucosidase (GCase), leading to accumulation of glucosylceramide (GlcC) and glucosylsphingosine (Lyso-Gb1). Lyso-Gb1 is a reliable biomarker for GD.
This study aims to develop a simple, effective and accurate method for the screening and diagnosis of GD using dried blood spot (DBS) samples.
Lyso-Gb1 in DBS was extracted by 50% acetonitrile aqueous solution containing isotope-labeled internal standard and analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). A reference interval was established by analyzing samples from 277 healthy controls. Lyso-Gb1 was detected in the residual DBS samples from 142 high-risk patients with splenomegaly and/or thrombocytopenia. Based on GCase activity in DBS, samples were classified into four groups: confirmed GD patients (n = 52), GD carriers (n = 5), false positive (n = 36) and negative (n = 49).
The optimized Lyso-Gb1 assay showed intra- and inter-assay variations ranged between 2.0%-8.2% and 3.8%-10.2%, respectively. Accuracies ranged from 93.5% to 112.6%. The lowest limit of quantification was 1 ng/mL. The normal reference interval of Lyso-Gb1 in DBS ranged from 2.1 to 9.9 ng/mL. Among the 142 subjects, except for one GD patient (Lyso-Gb1 > 2500 ng/mL), the Lyso-Gb1 concentrations in 51 GD patients ranged from 190.5 to 2380.6 ng/mL (the median 614.8 ng/mL). Also, one negative patient was found to have an elevated Lyso-Gb1 level (684.5 ng/mL), while the other patients were normal. The negative case was then confirmed to be an atypical GD patient with a c.1091A > G (p.Y364C) homozygous variant in PSAP gene by next generation sequencing.
The optimized method to determine Lyso-Gb1 in DBS was demonstrated as a useful tool for the screening and diagnosis of GD.
戈谢病（GD）是由于β-葡萄糖脑苷脂酶（GCase）缺乏引起的，导致葡萄糖脑苷脂（GlcC）和葡萄糖神经酰胺（Lyso-Gb1）积累。Lyso-Gb1 是 GD 的可靠生物标志物。
本研究旨在开发一种使用干血斑（DBS）样本进行 GD 筛查和诊断的简单、有效和准确的方法。
用含有同位素标记内标的 50%乙腈水溶液从 DBS 中提取 Lyso-Gb1，并用液相色谱串联质谱法（LC-MS/MS）进行分析。通过分析 277 名健康对照者的样本，建立参考区间。在 142 名脾肿大和/或血小板减少的高危患者的剩余 DBS 样本中检测 Lyso-Gb1。根据 DBS 中 GCase 活性，将样本分为四组：确诊 GD 患者（n=52）、GD 携带者（n=5）、假阳性（n=36）和阴性（n=49）。
优化的 Lyso-Gb1 分析方法的批内和批间变异分别在 2.0%-8.2%和 3.8%-10.2%之间。准确度范围为 93.5%-112.6%。最低定量下限为 1ng/mL。DBS 中 Lyso-Gb1 的正常参考区间为 2.1-9.9ng/mL。在 142 名受试者中，除了一名 GD 患者（Lyso-Gb1>2500ng/mL）外，51 名 GD 患者的 Lyso-Gb1 浓度范围为 190.5-2380.6ng/mL（中位数为 614.8ng/mL）。此外，还发现一名阴性患者的 Lyso-Gb1 水平升高（684.5ng/mL），而其他患者则正常。随后通过下一代测序证实该阴性病例为 PSAP 基因 c.1091A>G（p.Y364C）纯合变异的非典型 GD 患者。
优化的 DBS 中 Lyso-Gb1 测定方法可作为 GD 筛查和诊断的有用工具。
