Manton Center for Orphan Disease Research, Division of Genetics and Genomics, Boston Children's Hospital, Harvard Medical School, Boston, MA, United States.
Manton Center for Orphan Disease Research, Division of Genetics and Genomics, Boston Children's Hospital, Harvard Medical School, Boston, MA, United States.
Mol Genet Metab. 2019 Apr;126(4):368-376. doi: 10.1016/j.ymgme.2019.01.016. Epub 2019 Jan 22.
GALT deficiency is a rare genetic disorder of carbohydrate metabolism. Due to the decreased activity or absence of the enzyme galactose-1-phosphate uridylyltransferase (GALT), cells from affected individuals are unable to metabolize galactose normally. Lactose consumption in the newborn period could potentially lead to a lethal disease process with multi-organ involvement. In contrast to the newborn-stage disease, however, a galactose-restricted diet does not prevent long-term complications such as central nervous system (CNS) dysfunction with speech defects, learning disability and neurological disease in addition to hypergonadotropic hypogonadism or primary ovarian insufficiency (POI) in females. As the literature suggests an association between GALT enzyme activity and the long-term complications, it is of importance to have a highly sensitive assay to quantify the GALT enzyme activity. To that end, we had developed a sensitive and accurate LC-MS/MS method to measure GALT enzyme activity. Its ability to predict outcome is the subject of this report.
The GALT enzyme activity in erythrocytes from 160 individuals, in which 135 with classic, clinical variant or biochemical variant galactosemia, was quantified by LC-MS/MS. Individuals with GALT deficiency were evaluated for the long-term complications of speech defects, dysarthria, ataxia, dystonia, tremor, POI, as well as intellectual functioning (full scale IQ). The LC-MS/MS results were compared to a variety of assays: radioactive, [C]-galactose-1-phosphate, paper chromatography with scintillation counting, enzyme-coupled assays with spectrophotometric or fluorometric readout or high-pressure liquid chromatography with UV detection of UDP-galactose.
The LC-MS/MS method measured GALT activity as low as 0.2%, whereas other methods showed no detectable activity. Largely due to GALT activities that were over 1%, the LC-MS/MS measurements were not significantly different than values obtained in other laboratories using other methodologies. Severe long-term complications were less frequently noted in subjects with >1% activity. Patients with a p.Q188R/p.Q188R genotype have no residual enzyme activity in erythrocytes.
Our LC-MS/MS assay may be necessary to accurately quantify residual GALT activities below 5%. The data suggest that patients with >1% residual activity are less likely to develop diet-independent long-term complications. However, much larger sample sizes are needed to properly assess the clinical phenotype in patients with residual enzyme activities between 0.1 and 5%.
GALT 缺乏症是一种罕见的碳水化合物代谢遗传疾病。由于酶半乳糖-1-磷酸尿苷酰转移酶（GALT）活性降低或缺失，受影响个体的细胞无法正常代谢半乳糖。新生儿期摄入乳糖可能会导致多器官受累的致命疾病过程。然而，与新生儿期疾病不同，限制半乳糖饮食并不能预防长期并发症，如中枢神经系统（CNS）功能障碍伴有言语缺陷、学习障碍和神经系统疾病，以及女性的高促性腺激素性性腺功能减退或原发性卵巢功能不全（POI）。由于文献表明 GALT 酶活性与长期并发症之间存在关联，因此拥有一种高度敏感的测定法来定量 GALT 酶活性非常重要。为此，我们开发了一种灵敏准确的 LC-MS/MS 方法来测量 GALT 酶活性。本报告探讨了其预测结果的能力。
通过 LC-MS/MS 定量测定了 160 名个体（其中 135 名患有经典、临床变异或生化变异型半乳糖血症）红细胞中的 GALT 酶活性。对 GALT 缺乏症个体进行了评估，以了解言语缺陷、构音障碍、共济失调、肌张力障碍、震颤、POI 以及智力功能（全量表智商）等长期并发症的发生情况。将 LC-MS/MS 结果与各种测定法进行了比较：放射性测定法、[C]-半乳糖-1-磷酸、纸层析结合闪烁计数、酶偶联测定法（分光光度法或荧光法读数）或高压液相色谱法（UV 检测 UDP-半乳糖）。
LC-MS/MS 方法可测量低至 0.2%的 GALT 活性，而其他方法则未检测到活性。由于 GALT 活性大多超过 1%，因此 LC-MS/MS 测量值与其他实验室使用其他方法学获得的值没有显著差异。在活性>1%的个体中，严重的长期并发症较少见。具有 p.Q188R/p.Q188R 基因型的患者红细胞中无残留酶活性。
我们的 LC-MS/MS 测定法可能是准确测定低于 5%的残留 GALT 活性所必需的。数据表明，活性>1%的患者不太可能发生与饮食无关的长期并发症。然而，需要更大的样本量才能正确评估残留酶活性在 0.1 至 5%之间的患者的临床表型。
