The ability of an LC-MS/MS-based erythrocyte GALT enzyme assay to predict the phenotype in subjects with GALT deficiency

Background: GALT deficiency is a rare genetic disorder of carbohydrate metabolism. Due to the decreased activity or absence of the enzyme galactose-l-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. Materials and methods: 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-14]-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. Results: 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. Conclusion: 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%.