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 recent study demonstrated that children with Duarte galactosemia (DG) do not show increased prevalence of detectable developmental complications when 6-12 years old. However, that study left unanswered whether infants with DG might be at increased risk for acute problems when drinking milk or whether children with DG younger than 6 years might show increased prevalence of perhaps transient developmental challenges. Here, we have addressed both of these questions by analyzing parent/guardianreported data collected retrospectively for 350 children, 206 with DG and 144 unaffected siblings from the same families. The variables analyzed included whether each child had experienced (1) acute complications in infancy, (2) early intervention services when <3 years old, and/or (3) special educational services when 3-5 years old. For each case-control comparison, or case-by-diet comparison, we used logistic regression that included the following potential covariates: age, sex, race, family income, and parent education, as appropriate. We found that none of the three outcome variables tested showed significant differences between cases and controls, or among cases as a function of galactose exposure in infancy. To the limits of our study, we therefore conclude that regardless of whether a child with DG drinks milk or low-galactose formula as an infant, they are not at increased risk for acute complications or early childhood developmental challenges that require intervention.

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 longterm 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 evi-

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.

Here, we describe DAB-quant, a novel, open-source program designed to facilitate objective quantitation of immunohistochemical (IHC) signal in large numbers of tissue slides stained with 3,3′-diaminobenzidine (DAB). Scanned slides are arranged into separate folders for negative controls and test slides, respectively. Otsu’s method is applied to the negative control slides to define a threshold distinguishing tissue from empty space, and all pixels deemed tissue are scored for normalized red minus blue (NRMB) color intensity. Next, a user-defined tolerance for error is applied to the negative control slides to set a NRMB threshold distinguishing stained from unstained tissue and this threshold is applied to calculate the fraction of stained tissue pixels on each test slide. Results are recorded in a spreadsheet and pseudocolor images are presented to document how each pixel was categorized. Slides can be analyzed in full, or sampled using small boxes scattered randomly and automatically across the tissue area. Quantitation of sampling boxes enables faster processing, reveals the degree of heterogeneity of signal, and enables exclusion of problem areas on a slide, if needed. This system should prove useful for a broad range of applications. The code, usage instructions, and sample data are freely and publicly available on GitHub (https://github.com/sarafridov/DAB-quant) and at protocols.io (dx.doi.org/10.17504/protocols.io.dm6gpb578lzp/v1).