Stephen J. Welna(1), Patricia L. Hall(2), Coleman T. Turgeon(1), Dimitar K. Gavrilov(1), Silvia Tortorelli(1), Devin Oglesbee(1), Piero Rinaldo(1), Kimiyo M. Raymond(1), Dietrich Matern(1)
(1)Biochemical Genetics Laboratory, Mayo Clinic; (2)Emory Genetics Laboratory, Emory University
Galactosemias are autosomal recessive disorders that results from a deficiency of one of three enzymes catalyzing the conversion of galactose to glucose: galactose-1-phosphate uridyltransferase (GALT), galactokinase (GALK), and uridine diphosphate galactose-4-epimerase (GALE). We implemented LC-MS/MS assays for each enzyme that can be performed from the same specimen. 843 controls and samples from known patients (GALT: n=161; GALK: n=1; GALE: n=6) and asymptomatic mutation carriers (GALT: n=92) were analyzed to determine reference ranges. These tests provide a comprehensive and cost-effective laboratory evaluation to quickly resolve abnormal NBS results for galactosemia or a diagnosis of other at-risk patients.
Introduction: Galactosemias are autosomal recessive disorders that results from a deficiency of one of three enzymes catalyzing the conversion of galactose to glucose: galactose-1-phosphate uridyltransferase (GALT), galactokinase (GALK), and uridine diphosphate galactose-4-epimerase (GALE). GALT deficiency is the most common cause of galactosemia and is often referred to as classic galactosemia. The complete or near-complete deficiency of GALT enzyme is life threatening if left untreated. Individuals with GALK deficiency have a milder clinical presentation that is limited to the development of bilateral cataracts early in life. GALE deficiency comprises three variants: generalized, peripheral, and intermediate. Infants with generalized GALE deficiency present a phenotype similar to that of GALT deficiency, while infants with peripheral or intermediate GALE deficiency remain clinically unaffected. While newborn screening (NBS) aims to identify GALT deficiency, many NBS programs measure total galactose in NBS samples which is elevated in GALT deficiency as well as GALK and GALE deficiencies. We implemented assays for each enzyme that can be performed from the same specimen and therefore provide a comprehensive and cost-effective laboratory evaluation to quickly resolve abnormal NBS results for galactosemia.
Methods: Red blood cells (RBS) are isolated from EDTA blood and lysed. The lysate is then incubated at 37°C for 15 (GALT) or 60 minutes (GALK, GALE) in the presence of the respective enzyme substrates (13C2-Galactose-1-P, 13C6-galactose, UDP galactose) and cofactors. The concentration of each enzyme’s reaction product is determined independently by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis employing a normal phase LC separation of the enzyme product from residual substrate and matrix compounds. The MS/MS is operated in multiple reaction monitoring (MRM) negative mode with a total analysis time of 7.5, 5, and 10 minutes per sample for GALT, GALK, and GALE respectively. The concentrations of enzyme products 13C2-UDP-galactose (for GALT), 13C6-galactose-1-phosphate (for GALK), and UDP-glucose (for GALE) are established by comparison of their respective ion intensities to that of an internal standard. The final enzyme activities are normalized to incubation time and hemoglobin (Hb) concentration (nmol/h/mg of Hb). 843 controls and samples from known patients (GALT: n=161; GALK: n=1; GALE: n=6) and asymptomatic mutation carriers (GALT: n=92) were analyzed to determine reference ranges.
Results: Accuracy, precision, reportable range, analytic sensitivity, and analytic specificity met their pre-defined criteria. Unaffected patients had enzyme activities greater than 24.5, 0.8, and 5.0 nmol/h/mg Hb while deficient patients had enzyme activities of less than 1.0, 0.1, and 3.0 nmol/h/mg Hb for GALT, GALK, and GALE respectively. Milder variants and carriers of GALT deficiency are distinguished from classic galactosemia but – as expected - not consistently from each other. Acceptable anticoagulants for sample collection are EDTA, sodium Heparin, lithium Heparin, and acid-citrate dextrose.
Conclusion: By utilizing 13C-labeled substrates enzymatic products can be distinguished from potentially interfering endogenous analytes for GALT and GALK. A novel LC separation of the unlabeled enzyme product UDP-glucose allows for the use of an inexpensive unlabeled UDP-galactose substrate. Acceptable anticoagulants for all three assays are the same, allowing for ease of testing from a single patient draw. These tests are useful for rapid confirmation of abnormal newborn screening results and diagnosis of other at-risk patients.
Chen J et al. An interference-free two-step enzyme assay with UPLC-tandem mass spectrometric product measurement for the clinical diagnosis of uridine diphosphate galactose-4-epimerase deficiency. J Chromatogr B Analyt Technol Biomed Life Sci. 2014; 959: 5-9.
Ko DH et al. Multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry. Clin Chem. 2010; 56: 76471.
Li Y et al. Liquid chromatography-tandem mass spectrometry enzyme assay for UDP-galactose 4'-epimerase: use of fragment intensity ratio in differentiation of structural isomers. Clin Chem. 2014; 60: 783-90.
Li Y et al. Ultra fast and sensitive liquid chromatography tandem mass spectrometry based assay for galactose-1-phosphate uridylyltransferase and galactokinase deficiencies. Mol Gen and Metab. 2011; 33: 102.
Marquart et al. Enhanced Interpretation of Newborn Screening Results without Analyte Cutoff Values. Genet Med. 2012; 14: 648-55.