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Galactosemia is an inborn disorder of carbohydrate metabolism of which early detection can prevent severe illness. Although the assay for galactose-1-phosphate uridyltransferase (GALT) enzyme activity has been available since the 1960s, many issues prevented it from becoming universal. In order to develop the Israeli newborn screening pilot algorithm for galactosemia, flow injection analysis tandem mass spectrometry measurement of galactose-1-phosphate in archived dried blood spots from newborns with classical galactosemia, galactosemia variants, epimerase deficiency, and normal controls, was conducted. Out of 431 330 newborns screened during the pilot study (30 months), two with classical galactosemia and four with epimerase deficiency were identified and confirmed. Five false positives and no false negatives were recorded. Following this pilot study, the Israeli final and routine newborn screening algorithm, as recommended by the Advisory Committee to the National Newborn Screening Program, now consists of galactose-1-phosphate measurement integrated into the routine tandem mass spectrometry panel as the first-tier screening test, and GALT enzyme activity as the second-tier performed to identify only newborns suspected to be at risk for classical galactosemia. The GALT enzyme activity cut-off used in the final algorithm was lowered in order to avoid false positives., (© 2022 SSIEM.)

Inherited deficiencies of UDP-galactose 4-epimerase (GALE) have been associated with two distinct phenotypes. The vast majority of North American patients are clinically asymptomatic, are identified through newborn screening programmes for classical galactosaemia, and are of African-American descent. At least two symptomatic patients have been reported, one Pakistani and the other Asian Muslim, both with severe complications in the neonatal period and subsequent mental retardation. Through newborn screening, we have identified a GALE-deficient patient who is of mixed Pakistani/caucasian ancestry. He was clinically well in the neonatal period on a lactose-containing diet, and biochemical studies, including urine reducing sugars and galactitol, were consistent with a diagnosis of peripheral GALE deficiency. Although early developmental milestones were met normally, he now shows significant developmental delays in both motor and language skills. Mutational analysis revealed this patient to be a compound heterozygote at the GALE locus, with mutations N34S and L183P identified in the patient and confirmed in the parents. This report represents the first characterization of specific mutations in a GALE-deficient patient in conjunction with biochemical and clinical phenotype, and facilitates further studies of the GALE enzyme and its role in the different clinical forms of epimerase-deficiency galactosaemia.

UDP-galactose-4-epimerase (GALE) is a highly conserved enzyme that catalyzes the interconversion of UDP-galactose and UDP-glucose. Impairment of this enzyme in humans results in one of two clinically distinct forms of epimerase-deficiency galactosemia-one benign, the other severe. The molecular and biochemical distinction between these disorders remains unknown. To enable structural and functional studies of both wild-type and patient-derived alleles of human GALE (hGALE), we have developed and applied a null-background yeast expression system for the human enzyme. We have demonstrated that wild-type hGALE sequences phenotypically complement a yeast gal10 deletion, and we have biochemically characterized the wild-type human enzyme isolated from these cells. Furthermore, we have expressed and characterized two mutant alleles, L183P-hGALE and N34S-hGALE, both derived from a patient with no detectable GALE activity in red blood cells but with approximately 14% activity in cultured lymphoblasts. Analyses of crude extracts of yeast expressing L183P-hGALE demonstrated 4% wild-type activity and 6% wild-type abundance. Extracts of yeast expressing N34S-hGALE demonstrated approximately 70% wild-type activity and normal abundance. However, yeast coexpressing both L183P-hGALE and N34S-hGALE exhibited only approximately 7% wild-type levels of activity, thereby confirming the functional impact of both substitutions and raising the intriguing possibility that some form of dominant-negative interaction may exist between the mutant alleles found in this patient. The results reported here establish the utility of the yeast-based hGALE-expression system and set the stage for more-detailed studies of this important enzyme and its role in epimerase-deficiency galactosemia.