Current and Future Treatments for Classic Galactosemia

Type I (classic) galactosemia, galactose 1-phosphate uridylyltransferase (GALT)-deficiency is a hereditary disorder of galactose metabolism. The current therapeutic standard of care, a galactose-restricted diet, is effective in treating neonatal complications but is inadequate in preventing burdensome complications. The development of several animal models of classic galactosemia that (partly) mimic the biochemical and clinical phenotypes and the resolution of the crystal structure of GALT have provided important insights; however, precise pathophysiology remains to be elucidated. Novel therapeutic approaches currently being explored focus on several of the pathogenic factors that have been described, aiming to (i) restore GALT activity, (ii) influence the cascade of events and (iii) address the clinical picture. This review attempts to provide an overview on the latest advancements in therapy approaches.

Fluorinated Galactoses Inhibit Galactose-1-Phosphate Uridyltransferase and Metabolically Induce Galactosemia-like Phenotypes in HEK-293 Cells

Genetic defects of human galactose-1-phosphate uridyltransferase (hGALT) and the partial loss of enzyme function result in an altered galactose metabolism with serious long-term developmental impairment of organs in classic galactosemia patients. In search for cellular pathomechanisms induced by the stressor galactose, we looked for ways to induce metabolically a galactosemia-like phenotype by hGALT inhibition in HEK293 cells. In kinetic studies, we provide evidence for 2-fluorinated galactose-1-phosphate (F-Gal-1-P) to competitively inhibit recombinant hGALT with a KI of 0.9 mM. Contrasting with hepatic cells, no alterations of N-glycoprofiles in MIG (metabolic induction of galactosemia)-HEK293 cells were revealed for an inducible secretory netrin-1 probe by MALDI-MS. Differential fluorescence-activated cell sorting demonstrated reduced surface expression of N-glycosylated CD109, EGFR, DPP4, and rhMUC1. Membrane raft proteomes exhibited dramatic alterations pointing to an affection of the unfolded protein response, and of targeted protein traffick. Most prominent, a negative regulation of oxidative stress was revealed presumably as a response to a NADPH pool depletion during reduction of Gal/F-Gal. Cellular perturbations induced by fluorinated galactoses in normal epithelial cells resemble proteomic changes revealed for galactosemic fibroblasts. In conclusion, the metabolic induction of galactosemia-like phenotypes in healthy epithelial/neuronal cells could support studies on the molecular pathomechanisms in classic galactosemia, in particular under conditions of low galactose stress and residual GALT activity.

Galactose-1-Phosphate Uridyltransferase Activities in Different Genotypes: A Retrospective Analysis of 927 Samples

Background Classic galactosemia is an inherited disorder of galactose metabolism caused by the impaired activity of galactose-1-phosphate uridyltransferase (GALT). Untreated galactosemia is life-threatening; however, early dietary intervention prevents mortality and reduces morbidity associated with this disease. The diagnosis of galactosemia includes the measurement of GALT activity in red blood cells (RBC) and GALT gene analysis. In this study, we evaluate GALT activity in different genotypes using the results of combined biochemical and molecular testing in 927 samples. Methods GALT activity in RBC was measured by LC-MS/MS. The analysis of the GALT gene was performed by targeted gene analysis and/or full gene sequencing. Samples were assigned based on the presence of pathogenic (G) or Duarte 2 (D) variants, or their absence (Neg), to G/G, D/G, G/Neg, D/D, D/Neg, and Neg/Neg genotypes. Finite mixture models were applied to investigate distributions of GALT activities in these genotypes. The reference ranges were determined using the central 95% of values of GALT activities. Results The ranges of GALT activity in G/G, D/G, G/Neg, D/D, D/Neg, and Neg/Neg genotypes are 0.0 to 0.7 μmol·h−1 gHb−1, 3.1 to 7.8 μmol·h−1 gHb−1, 6.5 to 16.2 μmol·h−1 gHb−1, 6.4 to 16.5 μmol·h−1 gHb−1, 12.0 to 24.0 μmol·h−1 gHb−1, and 19.4 to 33.4 μmol·h−1 gHb−1, respectively. Conclusions The GALT activity ranges established in this study are in agreement with the expected impact of the genotype on the enzymatic activity. Molecular findings should be interpreted in view of biochemical results to confirm genotype–phenotype correlation.

Clinical, Biochemical and Molecular Profile of Variant Galactosemia in Children Detected by National Newborn Screening: A Pilot Study

Objective. The observed irregularities in the biochemical profile and the limited information on long-term outcomes among patients with Duarte variant (D/G) galactosemia have led to patient management variability. This study examined the molecular characteristics of Filipino patients with presumed variant galactosemia for confirmation of diagnosis. It also aimed to describe the corresponding biochemical, clinical and neurodevelopmental profiles in order to gain a better understanding of the patients with normal galactose metabolites in spite of low to absent GALT activity detected by the local newborn screening program. Methods. Thirteen (13) patients who were presumed to have a variant form of galactosemia by national newborn screening between 2002 and 2010, and who previously underwent physical and neurodevelopmental assessment were included in the study. Repeat clinical, ophthalmologic and neurodevelopmental evaluations were done upon recruitment of participants. Direct sequence analysis of the coding region of the GALT gene was conducted to determine the patients’ genotypes. Results. None of the patients’ genotypes were consistent with Duarte variant (D/G) galactosemia. Their genotypes reflect the normal total blood galactose levels in patients, but were inconsistent with the absent or trace GALT activity. Conclusion. Molecular testing for the entire cohort of presumed “variant” galactosemia Filipino patients will provide better profiling of this condition. Re-evaluation and assessment of the current guidelines used by national newborn screening in classifying variant galactosemia are recommended.

The Incidence and Clinical Presentations of Galactosemia in Fars province, South West of Iran

Background: In this survey we studied the incidence and clinical presentations of galactosemia in Fars province, in south west of Iran. Galactosemia is a rare genetic metabolic disorder ofgalactose. Its metabolism can be performed through 3 pathways. Although enzymes deficiencyof each of them can lead to galactose accumulation in plasma, the term galactosemia is specifically used for UDP-galactose uridyl transferase (GALT) deficiency. Classical galactosemia (G/G) is mostly manifested by poor growth, irritability, lethargy, vomiting, poor feeding, and jaundice.Material and method: 337000 newborns were screened for galactosemia by measuring total galactose level. Blood samples were collected from the heel on the Gauthriepaper, and thencalorimetric test with enzyme was performed to determine total galactose level. Blood galactoselevel below 4mg/dl was considered as normal and it was repeated if it was more than 4mg/dl in the first stage. The test was considered as abnormal if it was more than 5mg/dl, then blood samples were collected on filter paper and dried for 3-4 hours at room temperature and shipped frozen to laboratory for detection of GALT activity and galactose and galactose-1-phosphate.Results: From those who were gone for screening, 105 newborns had total galactose level more than 5mg/dl, among them, 37 patients had galactose level more than 15 mg/dl. Overall 12 cases were considered as classic galactosemia with an incidence rate of 1/28000, in Fars province.Conclusion: Although all of our patients were symptomatic and were admitted byhyperbilirubinemia before receiving the results, neonatal screening had an important role in the early diagnosis and management of this disease.

Quantification of Galactose-1-Phosphate Uridyltransferase Enzyme Activity by Liquid Chromatography–Tandem Mass Spectrometry

Background: The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based assay for GALT enzyme activity measurement. Method: Our assay used stable isotope-labeled α- galactose-1-phosphate ([13C6]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([13C6]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [13C6]-Glu-1-P (265 > 79) as an internal standard. Results: The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) μmol · (g Hgb)−1 · h−1 in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 μmol · (g Hgb)−1 · h−1 (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent Km of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity. Conclusions: This LC-MS/MS–based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities.