Primary or premature ovarian insufficiency (POI) is the most common long-term complication experienced by girls and women with classic galactosemia; more than 80% and perhaps more than 90% are affected despite neonatal diagnosis and careful lifelong dietary restriction of galactose. In this review we explore the complexities of timing and detection of galactosemia-associated POI and discuss potential underlying mechanisms. Finally, we offer recommendations for follow-up care with current options for intervention.

Classic galactosemia is a potentially lethal disorder that results from profound deficiency of galactose-1-phosphate uridylyltransferase (GALT), the second enzyme in the Leloir pathway of galactose metabolism. Although early diagnosis and rigorous dietary restriction of galactose prevent or resolve the potentially lethal acute symptoms, patients are at markedly increased risk of long-term complications including significant cognitive, speech, and behavioral difficulties, among other problems. The mechanisms that underlie these long-term complications remain unclear, as do the factors that modify their severity. Here we explored the scholastic and behavioral outcomes experienced by a cohort of 54 school age children with classic galactosemia. Data collected included survey responses from parents and teachers, school records including standardized test scores, and GALT genotype data used to estimate predicted residual GALT activity based on a yeast expression system. As expected, many but not all of the children in our study demonstrated speech, scholastic, and behavioral difficulties. Perhaps most striking, we found that predicted cryptic residual GALT activity, often below the threshold of detection of clinical assays, appeared to modify scholastic outcome. These data raise the intriguing possibility that cryptic GALT activity might also influence the severity of other long-term complications in classic galactosemia.

Gene expression is controlled by RNA-binding proteins that modulate the synthesis, processing, transport and stability of various classes of RNA. Some RNA-binding proteins shuttle between the nucleus and cytoplasm and are thought to bind to RNA transcripts in the nucleus and remain bound during translocation to the cytoplasm. One RNA-binding protein that has been hypothesized to function in this manner is the Saccharomyces cerevisiae Scp160 protein. Although the steady-state localization of Scp160 is cytoplasmic, previous studies have identified putative nuclear localization (NLS) and nuclear export (NES) signals. The goal of this study was to test the hypothesis that Scp160 is a nucleocytoplasmic shuttling protein. We exploited a variety of yeast export mutants to capture any potential nuclear accumulation of Scp160 and found no evidence that Scp160 enters the nucleus. These localization studies were complemented by a mutational analysis of the predicted NLS. Results indicate that key basic residues within the predicted NLS of Scp160 can be altered without severely affecting Scp160 function. This finding has important implications for understanding the function of Scp160, which is likely limited to the cytoplasm. Additionally, our results provide strong evidence that the presence of a predicted nuclear localization signal within the sequence of a protein should not lead to the assumption that the protein enters the nucleus in the absence of additional experimental evidence.

Objective To determine if girls with Duarte variant galactosemia (DG) have an increased risk of developing premature ovarian insufficiency based on prepubertal anti-Mullerian hormone (AMH) levels. Design Cross-sectional study. Setting University research laboratory. Patient(s) Study volunteers included 57 girls with DG, 89 girls with classic galactosemia (GG), and 64 control girls between the ages of < 1 month and 10.5 years. Intervention(s) Blood sampling. Main Outcome Measure(s) We determined AMH and FSH levels in study volunteers with and without Duarte variant or GG. Result(s) FSH levels were significantly higher and AMH levels significantly lower in girls with GG than in age-stratified control girls, but there was no significant difference between FSH and AMH levels in girls with DG and control girls. Conclusion(s) Although > 80% of girls with GG in this study demonstrated low to undetectable AMH levels consistent with diminished ovarian reserve, 100% of girls with DG in our study demonstrated no apparent decrease in AMH levels or increase in FSH levels, suggesting that these girls are not at increased risk for premature ovarian insufficiency.

Study objective To determine whether premature ovarian insufficiency (POI) associated with classic galactosemia results from a true deficiency of ovarian function or from aberrant follicle stimulating hormone (FSH). Design Cross-sectional study Setting University research laboratory Patients or other participants Study subjects included 35 girls and women with galactosemia and 43 control girls and women between the ages of <1 yrs to 51 yrs. Interventions Blood sampling and medical and reproductive histories were obtained. Main outcome measures We determined FSH and anti-Müllerian hormone (AMH) levels in subjects with and without classic galactosemia. FSH bioactivity was measured in a subset of girls and women with and without galactosemia who were not on hormone therapy. Results FSH levels were significantly higher and AMH levels were significantly lower in our galactosemic cases relative to controls. FSH bioactivity did not significantly differ between cases and controls. Conclusions Close to 90% of girls and women with classic galactosemia have a profound absence of ovarian function, a deficit which is evident shortly after birth, if not before. These patients have no evidence of abnormally functioning FSH. AMH levels can be assessed prior to menarche or after initiation of hormone therapy and may supplement FSH as a useful blood biomarker of ovarian function for patients with classic galactosemia.

Classic galactosemia (CG) is an inherited metabolic disorder that affects about 1 in 50,000 live births in the United States and many other countries. With the benefit of early detection by newborn screening and rapid dietary restriction of galactose, generally achieved by removing dairy from the diet, most affected infants are spared the acute and potentially lethal symptoms of disease. Despite early detection and life-long dietary intervention, however, most patients grow to experience a constellation of long-term complications that include premature ovarian insufficiency in the vast majority of girls and young women. Our goal in the study reported here was to define the presentation, progression, and predictors of ovarian insufficiency in a cohort of 102 post-pubertal girls and women with CG. To our knowledge, this is the largest cohort studied to date. We found that 68% of the girls and women in our study achieved spontaneous menarche, while 32% achieved menarche only after starting hormone replacement therapy (HRT). Of those who achieved spontaneous menarche, fewer than 50% were still cycling regularly after 3 years, and fewer than 15% were still cycling regularly after 10 years. Of factors tested for possible association with spontaneous menarche, only detectable (≥ 0.04 ng/mL) plasma anti-Müllerian hormone (AMH) level was significant. These results extend substantially from prior studies and confirm that detectable plasma AMH is a useful predictor of ovarian function in girls and women with CG.

Type I galactosemia is a genetic disorder that is caused by the impairment of galactose-1-phosphate uridylyltransferase (GALT; EC 2.7.7.12). Although a large number of mutations have been detected through genetic screening of the human GALT (hGALT) locus, for many it is not known how they cause their effects. The majority of these mutations are missense, with predicted substitutions scattered throughout the enzyme structure and thus causing impairment by other means rather than direct alterations to the active site. To clarify the fundamental, molecular basis of hGALT impairment we studied five disease-associated variants p.D28Y, p.L74P, p.F171S, p.F194L and p.R333G using both a yeast model and purified, recombinant proteins. In a yeast expression system there was a correlation between lysate activity and the ability to rescue growth in the presence of galactose, except for p.R333G. Kinetic analysis of the purified proteins quantified each variant's level of enzymatic impairment and demonstrated that this was largely due to altered substrate binding. Increased surface hydrophobicity, altered thermal stability and changes in proteolytic sensitivity were also detected. Our results demonstrate that hGALT requires a level of flexibility to function optimally and that altered folding is the underlying reason of impairment in all the variants tested here. This indicates that misfolding is a common, molecular basis of hGALT deficiency and suggests the potential of pharmacological chaperones and proteostasis regulators as novel therapeutic approaches for type I galactosemia.

In both humans and Drosophila melanogaster, UDP-galactose 4′-epimerase (GALE) catalyzes two distinct reactions, interconverting UDP-galactose (UDP-gal) and UDP-glucose (UDP-glc) in the final step of the Leloir pathway of galactose metabolism, and also interconverting UDP-N-acetylgalactosamine (UDP-galNAc) and UDP-N-acetylglucosamine (UDP-glcNAc). All four of these UDP-sugars serve as vital substrates for glycosylation in metazoans. Partial loss of GALE in humans results in the spectrum disorder epimerase deficiency galactosemia; partial loss of GALE in Drosophila melanogaster also results in galactose-sensitivity, and complete loss in Drosophila is embryonic lethal. However, whether these outcomes in both humans and flies result from loss of one GALE activity, the other, or both has remained unknown. To address this question, we uncoupled the two activities in a Drosophila model, effectively replacing the endogenous dGALE with prokaryotic transgenes, one of which (Escherichia coli GALE) efficiently interconverts only UDP-gal/UDP-glc, and the other of which (Plesiomonas shigelloides wbgU) efficiently interconverts only UDP-galNAc/UDP-glcNAc. Our results demonstrate that both UDP-gal and UDP-galNAc activities of dGALE are required for Drosophila survival, although distinct roles for each activity can be seen in specific windows of developmental time or in response to a galactose challenge. By extension, these data also suggest that both activities might play distinct and essential roles in humans.

SUMMARY UDP-galactose 4′ epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose in the final step of the Leloir pathway; human GALE (hGALE) also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. GALE therefore plays key roles in the metabolism of dietary galactose, in the production of endogenous galactose, and in maintaining the ratios of key substrates for glycoprotein and glycolipid biosynthesis. Partial impairment of hGALE results in the potentially lethal disorder epimerase-deficiency galactosemia. We report here the generation and initial characterization of a first whole-animal model of GALE deficiency using the fruit fly Drosophila melanogaster. Our results confirm that GALE function is essential in developing animals; Drosophila lacking GALE die as embryos but are rescued by the expression of a human GALE transgene. Larvae in which GALE has been conditionally knocked down die within days of GALE loss. Conditional knockdown and transgene expression studies further demonstrate that GALE expression in the gut primordium and Malpighian tubules is both necessary and sufficient for survival. Finally, like patients with generalized epimerase deficiency galactosemia, Drosophila with partial GALE loss survive in the absence of galactose but succumb in development if exposed to dietary galactose. These data establish the utility of the fly model of GALE deficiency and set the stage for future studies to define the mechanism(s) and modifiers of outcome in epimerase deficiency galactosemia.

SUMMARY Classic galactosemia is a potentially lethal disorder that results from profound impairment of galactose-1-phosphate uridylyltransferase (GALT). Despite decades of research, the underlying pathophysiology of classic galactosemia remains unclear, in part owing to the lack of an appropriate animal model. Here, we report the establishment of a Drosophila melanogaster model of classic galactosemia; this is the first whole-animal genetic model to mimic aspects of the patient phenotype. Analogous to humans, GALT-deficient D. melanogaster survive under conditions of galactose restriction, but accumulate elevated levels of galactose-1-phosphate and succumb during larval development following galactose exposure. As in patients, the potentially lethal damage is reversible if dietary galactose restriction is initiated early in life. GALT-deficient Drosophila also exhibit locomotor complications despite dietary galactose restriction, and both the acute and long-term complications can be rescued by transgenic expression of human GALT. Using this new Drosophila model, we have begun to dissect the timing, extent and mechanism(s) of galactose sensitivity in the absence of GALT activity.