Restoring galactose metabolism without restoring GALT rescues both compromised survival in larvae and an adult climbing deficit in a GALT-null D. Melanogaster model of classic galactosemia.

Classic galactosemia (CG) is an autosomal recessive disorder that results from profound deficiency of galactose-1-phosphate uridylyltransferase (GALT), the middle enzyme in the highly conserved Leloir pathway of galactose metabolism. That galactose metabolism is disrupted in patients with CG, and in GALT-null microbial, cell culture, and animal models of CG, has been known for many years. However, whether the long-term developmental complications of CG result from disrupted galactose metabolism alone, or from loss of some independent moonlighting function of GALT, in addition to disrupted galactose metabolism, has been posed but never resolved. Here, we addressed this question using a GALT-null Drosophila melanogaster model of CG engineered to express uridine diphosphate (UDP)-glucose/galactose pyrophosphorylase (UGGP), a plant enzyme that effectively bypasses GALT in the Leloir pathway by converting substrates uridine triphosphate (UTP) plus galactose-1-phosphate (gal-1P) into products UDP-galactose plus pyrophosphate (PPi). While GALT and UGGP share one substrate (gal-1P) and one product (UDP-galactose), they are structurally and evolutionarily unrelated enzymes. It is therefore extremely unlikely that they would also share a moonlighting function. We found that GALT-null flies expressing UGGP showed not only partial rescue of metabolic abnormalities and acute larval sensitivity to dietary galactose, as expected, but also full rescue of an adult motor deficit otherwise seen in this model. By extension, these results may offer insights to the underlying bases of at least some acute and long-term complications experienced by patients with CG.

Health and well-being of maturing adults with classic galactosemia.

Long-term outcomes in classic galactosemia (CG) have been studied previously, but all prior studies have relied on cohorts of patients that were small in number, or heavily skewed toward children and young adults, or both. Here, we extend what is known about the health and well-being of maturing adults with CG by analyzing the results of anonymous custom surveys completed by 92 affected individuals, ages 30-78, and 38 unaffected sibling controls, ages 30-79. The median age for patients was 38.5 years and for controls was 41 years. These study participants hailed from 12 different countries predominantly representing Europe and North America. Participants reported on their general life experiences and outcomes in seven different domains including: speech/voice/language, cognition, motor function, cataracts, bone health, psychosocial well-being, and gastrointestinal health. We also queried women about ovarian function. Our results indicated a prevalence of long-term complications across all outcome domains that aligned with levels previously reported in younger cohorts. Given the sample size and age range of participants in this study, these findings strongly suggest that the adverse developmental outcomes commonly linked to CG are not progressive with age for most patients. We also tested four candidate modifiers for possible association with each of the outcomes followed, including: days of neonatal milk exposure, rigor of dietary galactose restriction in early childhood, current age, and home continent. We observed no associations that reached even nominal significance, except for the following: cataracts with neonatal milk exposure (p = 2.347e-04), cataracts with age (p = 0.018), and bone health with home continent (p = 0.03).

Racial and ethnic diversity of classic and clinical variant galactosemia in the United States.

Classic and clinical variant galactosemia (CG/CVG) are allelic, autosomal recessive disorders that result from deficiency of galactose-1-P uridylyltransferase (GALT). CG/CVG has been reported globally among patients of diverse ancestries, but most large studies of outcomes have included, almost exclusively, patients categorized as White or Caucasian. As a first step to explore whether the cohorts studied are representative of the CG/CVG population at large, we sought to define the racial and ethnic makeup of CG/CVG newborns in a diverse population with essentially universal newborn screening (NBS) for galactosemia: the United States (US). First, we estimated the predicted racial and ethnic distribution of CG/CVG by combining the reported demographics of US newborns from 2016 to 2018 with predicted homozygosity or compound heterozygosity of pathogenic, or likely pathogenic, GALT alleles from the relevant ancestral groups. Incorporating some simplifying assumptions, we predicted that of US newborns diagnosed with CG/CVG, 65% should be White (non-Hispanic), 23% should be Black (non-Hispanic), 10% should be Hispanic, and 2% should be Asian (non-Hispanic). Next, we calculated the observed racial and ethnic distribution of US newborns diagnosed with CG/CVG using available de-identified data from state NBS programs from 2016 to 2018. Of the 235 newborns in this cohort, 41 were categorized as other or unknown. Of the remaining 194, 66% were White (non-Hispanic or ethnicity unknown), 16% were Black (non-Hispanic or ethnicity unknown),15% were Hispanic, and 2% were Asian (non-Hispanic or ethnicity unknown). This observed distribution was statistically indistinguishable from the predicted distribution. To the limits of our study, these data confirm the racial and ethnic diversity of newborns with CG/CVG in the US, demonstrate an approach for estimating CG/CVG racial and ethnic diversity in other populations, and raise the troubling possibility that current understanding of long-term outcomes in CG/CVG may be skewed by ascertainment bias of the cohorts studied.

Long-term complications in classic galactosemia are not progressive.

Classic galactosemia (CG) is a potentially lethal genetic disorder that results from profound deficiency of galactose-1-P uridylyltransferase. Despite early detection and life-long dietary restriction of galactose, which is the current standard of care, many patients with CG grow to experience a range of long-term developmental complications that can include difficulties with speech/voice/language, cognitive, motor, and psychosocial outcomes, among other problems. That these complications are common in CG is well-documented, but whether they are also progressive has been a point of controversy for decades. Here, we addressed the question of whether long-term outcomes in CG are progressive by analyzing a robust data set in each of 4 ways. First, we compared cross-sectional Vineland-3 Adaptive Behavior Scales scores for 101 cases and 65 unaffected sibling controls and found no evidence of consistently declining scores with age. Second, we analyzed longitudinal Vineland-3 subdomain scores for 45 cases and 34 controls to see if individual participants demonstrated developmental gains (positive slope) or losses (negative slope) over time. The changes in most growth scale value (GSV) scores, which are not normed, were positive for both cases and controls <10y, and either positive or near zero for participants ≥10y. In contrast, the slopes of most v-Scale scores, which are normed, were negative for many cases <10y, indicating that these children, while gaining milestones, were gaining them at a slower pace than their counterparts in the reference population. Third, we analyzed medical records from 76 cases, assigning ordinal scores for complications and gathering the quantitative results of relevant formal assessments where available. Both cross-sectional and longitudinal analyses of both ordinal and formal assessment scores confirmed that outcomes were mostly stable, albeit with some ups and downs in isolated cases. Finally, we analyzed data collected via custom family-response surveys from 124 cases and 67 controls regarding each participant's perceived symptom severity over time. Among cases, the percentages of respondents reporting worsening symptoms over time for speech, cognitive, motor, and psychosocial outcomes were 0.8%, 6.6%, 5.2%, and 9.8%, respectively. Among controls, the corresponding percentages were 0.0%, 1.5%, 1.5%, and 6.5%, respectively. These results provide compelling evidence that long-term developmental complications are not progressive for a majority of patients with CG.

Grip strength in patients with galactosemia and in a galactose-1-phosphate uridylyltransferase (GALT)-null rat model.

Classic galactosemia (CG) and clinical variant galactosemia (CVG) are allelic inborn errors of metabolism that result from profound deficiency, and near-profound deficiency, respectively, of galactose-1-P uridylyltransferase (GALT). Despite early detection and lifelong dietary restriction of galactose, which is the current standard of care, most patients with CG/CVG grow to experience a range of long-term developmental and other complications. One of the less well-understood complications of CG/CVG is decreased hand grip strength, as reported by Potter et al. (2013). Here, we confirm this phenotype in an independent cohort of 36 cases (4-18 years) and 19 controls (4-17 years), and further demonstrate that the grip strength deficit observed in cases may be secondary to growth delay. Specifically, we found that when grip strength of cases and controls in a new cohort recruited in 2022 was plotted by weight, rather than age, the difference between cases and controls for both sexes disappeared. Reanalyzing data from the original 2013 cohort, we found that differences in weight accounted for grip strength differences between cases and controls in girls and young women, but not in boys and young men. Finally, we tested whether a GALT-null rat model of CG also showed a grip strength deficit-it did-and again the difference between GALT-null and wild-type rats associated with differences in body mass. Combined, these results confirm that GALT deficiency is associated with a grip strength deficit in both young patients with CG/CVG and GALT-null rats, and further demonstrate that this phenotype may be secondary to growth delay, and therefore not evidence of a muscle abnormality.

Pathophysiology of long-term complications in classic galactosemia: What we do and do not know.

Despite many decades of research involving both human subjects and model systems, the underlying pathophysiology of long-term complications in classic galactosemia (CG) remains poorly understood. In this review, intended for those already familiar with galactosemia, we focus on the big questions relating to outcomes, mechanism, and markers, drawing on relevant literature where available, attempting to navigate inconsistencies where they appear, and acknowledging gaps in knowledge where they persist.

Neonatal GALT gene replacement offers metabolic and phenotypic correction through early adulthood in a rat model of classic galactosemia.

Classic galactosemia (CG) results from profound deficiency of galactose-1-P uridylyltransferase (GALT). Despite early detection by newborn screening and lifelong dietary restriction of galactose, most patients grow to experience a range of long-term complications. Recently, we developed and characterized a GALT-null rat model of CG and demonstrated that AAV9-hGALT, administered by tail vein injection to neonatal pups, dramatically improved plasma, liver, and brain galactose metabolites at 2 weeks posttreatment. Here we report a time-course study of GALT restoration in rats treated as neonates with scAAV9-hGALT and harvested at 8, 14, 30, and 60 days. Cohorts of rats in the two older groups were weaned to diets containing either 1% or 3% of calories from galactose. As expected, GALT activity in all treated animals peaked early and then diminished over time, most notably in liver, ostensibly due to dilution of the nonreplicating episomal vector as transduced cells divided. All treated rats showed dramatic metabolic rescue through 1 month, and those weaned to the lower galactose diet showed continued strong metabolic rescue into adulthood (2 months). Prepubertal growth delay and cataracts were both partially rescued by treatment. Finally, we found that UDP glucose pyrophosphorylase (UGP), which offers a metabolic bypass around missing GALT, was 3-fold more active in brain samples from adult rats than from young pups, offering a possible explanation for the improved ability of older GALT-null rats to metabolize galactose. Combined, these results document promising metabolic and phenotypic efficacy of neonatal GALT gene replacement in a rat model of classic galactosemia.

A multinational study of acute and long-term outcomes of Type 1 galactosemia patients who carry the S135L (c.404C > T) variant of GALT.

Patients with galactosemia who carry the S135L (c.404C > T) variant of galactose-1-P uridylyltransferase (GALT), documented to encode low-level residual GALT activity, have been under-represented in most prior studies of outcomes in Type 1 galactosemia. What is known about the acute and long-term outcomes of these patients, therefore, is based on very limited data. Here, we present a study comparing acute and long-term outcomes of 12 patients homozygous for S135L, 25 patients compound heterozygous for S135L, and 105 patients homozygous for two GALT-null (G) alleles. This is the largest cohort of S135L patients characterized to date. Acute disease following milk exposure in the newborn period was common among patients in all 3 comparison groups in our study, as were long-term complications in the domains of speech, cognition, and motor outcomes. In contrast, while at least 80% of both GALT-null and S135L compound heterozygous girls and women showed evidence of an adverse ovarian outcome, prevalence was only 25% among S135L homozygotes. Further, all young women in this study with even one copy of S135L achieved spontaneous menarche; this is true for only about 33% of women with classic galactosemia. Overall, we observed that while most long-term outcomes trended milder among groups of patients with even one copy of S135L, many individual patients, either homozygous or compound heterozygous for S135L, nonetheless experienced long-term outcomes that were not mild. This was true despite detection by newborn screening and both early and life-long dietary restriction of galactose. This information should empower more evidence-based counseling for galactosemia patients with S135L.

A pilot study of neonatal GALT gene replacement using AAV9 dramatically lowers galactose metabolites in blood, liver, and brain and minimizes cataracts in GALT-null rat pups.

Classic galactosemia (CG) is a rare metabolic disorder that results from profound deficiency of galactose-1-P uridylyltransferase (GALT). Despite early detection by newborn screening and rapid and lifelong dietary restriction of galactose, which is the current standard of care, most patients grow to experience a broad constellation of long-term complications. The mechanisms underlying these complications remain unclear and likely differ by tissue. Here we conducted a pilot study testing the safety and efficacy of GALT gene replacement using our recently-described GALT-null rat model for CG. Specifically, we administered AAV9.CMV.HA-hGALT to seven GALT-null rat pups via tail vein injection on day 3 of life; eight GALT-null pups injected with PBS served as the negative control, and four GALT+ heterozygous pups injected with PBS served as the positive control. All pups were returned to their nursing mothers, weighed daily, and euthanized for tissue collection 2 weeks later. Among the AAV9-injected pups in this study, we achieved GALT levels in liver ranging from 64% to 595% wild-type, and in brain ranging from 3% to 42% wild-type. In liver, brain, and blood samples from these animals we also saw a striking drop in galactose, galactitol, and gal-1P. Finally, all treated GALT-null pups showed dramatic improvement in cataracts relative to their mock-treated counterparts. Combined, these results demonstrate that GALT restoration in both liver and brain of GALT-null rats by neonatal tail vein administration using AAV9 is not only attainable but effective.

Hand fine motor control in classic galactosemia.

Classic galactosemia (CG) is a rare inborn error of metabolism that results from profound deficiency of galactose-1-P uridylyltransferase (GALT). Despite early detection and rapid and lifelong dietary restriction of galactose, which is the current standard of care, most patients grow to experience a broad range of complications that can include motor difficulties. The goal of this study was to characterize hand fine motor control deficit among children and adults with classic galactosemia (CG). Specifically, we used Neuroglyphics software to collect digital Archimedes spiral drawings on a touch screen from 57 volunteers with CG (cases) and 80 controls. Hand fine motor control was scored as root mean square (RMS) of spirals drawn relative to an idealized template. Presence of tremor was defined as a peak in periodicity of changes in drawing speed or direction in the 4-8 Hz range. We observed a highly significant difference (P < .001) in RMS scores between cases and controls, with almost 51% of cases showing at least 1 of 4 spirals scoring outside the 95th percentile for controls. The corresponding prevalence for controls was 10%. Similarly, more than 35% of cases, and almost 14% of controls, showed at least 1 of 4 spirals with a tremor amplitude above the 95th % cutoff for controls. Our results both confirm and extend what is known about hand fine motor control deficit among children and adults with CG and establish digital assessment as a useful approach to quantify this outcome.

A galactose-1-phosphate uridylyltransferase-null rat model of classic galactosemia mimics relevant patient outcomes and reveals tissue-specific and longitudinal differences in galactose metabolism.

Classic galactosemia (CG) is a potentially lethal inborn error of metabolism, if untreated, that results from profound deficiency of galactose-1-phosphate uridylyltransferase (GALT), the middle enzyme of the Leloir pathway of galactose metabolism. While newborn screening and rapid dietary restriction of galactose prevent or resolve the potentially lethal acute symptoms of CG, by mid-childhood, most treated patients experience significant complications. The mechanisms underlying these long-term deficits remain unclear. Here we introduce a new GALT-null rat model of CG and demonstrate that these rats display cataracts, cognitive, motor, and growth phenotypes reminiscent of patients outcomes. We further apply the GALT-null rats to test how well blood biomarkers, typically followed in patients, reflect metabolic perturbations in other, more relevant tissues. Our results document that the relative levels of galactose metabolites seen in GALT deficiency differ widely by tissue and age, and that red blood cell Gal-1P, the marker most commonly followed in patients, shows no significant association with Gal-1P in other tissues. The work reported here establishes our outbred GALT-null rats as an effective model for at least four complications characteristic of CG, and sets the stage for future studies addressing mechanism and testing the efficacy of novel candidate interventions.

Metabolic perturbations in classic galactosemia beyond the Leloir pathway: Insights from an untargeted metabolomic study.

Classic galactosemia (CG) is an autosomal recessive disorder that impacts close to 1/50000 live births in the United States, with varying prevalence in other countries. Following exposure to milk, which contains high levels of galactose, affected infants may experience rapid onset and progression of potentially lethal symptoms. With the benefit of early diagnosis, generally by newborn screening, and immediate and lifelong dietary restriction of galactose, the acute sequelae of disease can be prevented or resolved. However, long-term complications are common, and despite many decades of research, the bases of these complications remain unexplained. As a step toward defining the underlying pathophysiology of long-term outcomes in CG, we applied an untargeted metabolomic approach with mass spectrometry and dual liquid chromatography, comparing thousands of small molecules in plasma samples from 183 patients and 31 controls. All patients were on galactose-restricted diets. Using both univariate and multivariate statistical methods, we identified 252 differentially abundant features from anion exchange chromatography and 167 differentially abundant features from C18 chromatography. Mapping these discriminatory features to putative metabolites and biochemical pathways revealed 14 significantly perturbed pathways; these included multiple redox, amino acid, and mitochondrial pathways, among others. Finally, we tested whether any discriminatory features also distinguished cases with mild vs more severe long-term outcomes and found multiple candidates, of which one achieved false discovery rate-adjusted q < 0.1. These results extend substantially from prior targeted studies of metabolic perturbation in CG and offer a new approach to identifying candidate modifiers and targets for intervention.

Presentation, progression, and predictors of ovarian insufficiency in 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.

Rigor of non-dairy galactose restriction in early childhood, measured by retrospective survey, does not associate with severity of five long-term outcomes quantified in 231 children and adults with classic galactosemia.

One of many vexing decisions faced by parents of an infant with classic galactosemia (CG) is how carefully to restrict non-dairy galactose from their growing child's diet. Until recently, many experts recommended vigorous lifelong dietary restriction of milk and all high-galactose dairy products as well as some non-dairy sources of galactose such as legumes and specific fruits and vegetables. Recently, experts have begun to relax their recommendations. The new recommendations, that restrict only high galactose dairy products, were made in the face of uncertainty, however, because no sufficiently powered study had been reported testing for possible association between rigor of non-dairy galactose restriction and severity of long-term outcomes in CG. Here we describe the largest study of diet and outcomes in CG reported to date, conducted using information gathered from 231 patients with CG and 71 unaffected sibling controls. We compared rigor of dietary galactose restriction, measured using a 4-point scale by a retrospective parent-response survey, with outcomes including growth, adaptive behaviors, receipt of speech therapy, receipt of special educational services, and for girls and women, a plasma marker of ovarian function (AMH). Our results confirmed the expected differences between patients and controls, but among patients showed no significant association between rigor of non-dairy galactose restriction in early childhood and any of the outcomes quantified. Indeed, some weak associations were seen suggesting that rigorous restriction of non-dairy galactose may be deleterious rather than beneficial. Despite limitations, these findings support the ongoing trend toward diet liberalization with regard to non-dairy sources of galactose for children and adults with classic galactosemia.

UDP-galactose 4'-epimerase activities toward UDP-Gal and UDP-GalNAc play different roles in the development of Drosophila melanogaster.

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.

Misfolding of galactose 1-phosphate uridylyltransferase can result in type I galactosemia.

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.

Liquid chromatography-tandem mass spectrometry enzyme assay for UDP-galactose 4'-epimerase: use of fragment intensity ratio in differentiation of structural isomers.

BACKGROUND: Distinction between asymptomatic and potentially clinically significant forms of galactosemia due to UDP-galactose 4'-epimerase (GALE) deficiency requires enzyme measurement in erythrocytes and other cells. We sought to develop a GALE assay using a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. METHODS: The reversible GALE assay was conducted with UDPGal as a substrate. The coeluting reaction product, uridine diphosphate glucose (UDPGlc), and its isomeric substrate, uridine diphosphate galactose (UDPGal), were detected by MS/MS at mass transitions 565 > 280, 565 > 241 and 565 > 403. The UDPGal was enriched in mass transition 565 > 403 compared with UDPGlc, whereas the UDPGlc was enriched in the mass transition 565 > 241 compared with UDPGal. The percentage of UDPGal in the reaction mixture was calculated by use of the ratio of ion intensities of the 2 daughter ions and a fourth-order polynomial calibrator curve. RESULTS: The method yielded a mean (SD) GALE activity of 9.8 (2.2) µmol · g(-1) hemoglobin · h(-1) in erythrocyte extracts from 27 controls. The apparent Km of the substrate, UDPGal, was 0.05 mmol/L. The GALE activity ranged from 433 to 993 µmol · g(-1) protein · h(-1) in control lymphoblast extracts. In a blinded test of 22 subjects suspected of GALE deficiency, we identified 6 individuals whose residual activities were below the range of controls, compatible with intermediate GALE deficiency. CONCLUSIONS: This assay can be used to distinguish the different forms of GALE deficiency. From an analytical standpoint, differentiating isomers on the basis of fragment intensity ratios should also prove useful for analogous enzymatic studies involving substrates and products that are structural isomers.

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection.

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 µL of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

Altered cofactor binding affects stability and activity of human UDP-galactose 4'-epimerase: implications for type III galactosemia.

Deficiency of UDP-galactose 4'-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and no ability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD(+). p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two α-helices that contain residues that interact with NAD(+). These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation.