International Journal of Molecular Sciences · 16 authors, 4 centres
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This study reports a novel GALT missense variant (p.A303D) in a two-week-old female with classic galactosemia and acute liver failure. Computational analyses predict that the p.A303D substitution destabilizes the GALT protein structure, likely contributing to the severe phenotype. A literature review of 54 patients with the p.K285N variant found most had classic galactosemia and 61.9% had hepatic damage, emphasizing the need for integrating genetic analysis into clinical care.
**Background:** Classic galactosemia (CG) is an autosomal recessive disorder caused by deficient activity of galactose-1-phosphate uridylyltransferase (GALT), leading to accumulation of galactose and its metabolites. Symptoms appear in neonates after galactose ingestion and include jaundice, hepatomegaly, failure to thrive, hypoglycemia, and cataracts. Long-term complications such as cognitive impairment, ataxia, and ovarian dysfunction occur despite dietary galactose restriction. More than 350 GALT alterations have been reported, mostly missense variants affecting protein folding and stability. The c.855G>T (p.K285N) variant is the second most common in European patients, accounting for 26-34% of alleles, and is associated with complete loss of GALT activity in homozygotes.
**Methods:** The index case was a two-week-old female neonate presenting with hypotonia, poor feeding, projectile vomiting, hypoglycemia, and jaundice. Liver function tests showed total bilirubin 24 mg/dL (normal 0.3-1.2), direct bilirubin 2.5 mg/dL (normal <0.3), AST 269 U/L (normal <34), ALT 136 U/L (normal 10-49). Total galactose was 2680.37 µmol/L (normal <490), and GALT activity was <2.5 U/dL (normal >6.7). After transitioning to lactose-free nutrition, genetic analysis by Sanger sequencing identified two GALT variants: c.855G>T (p.K285N) inherited from the father, and a novel c.908C>A (p.A303D) inherited from the mother. The mother also carried the D1 Duarte variant (c.940A>G (p.N314D) and c.652C>T (p.L218L) in cis) but not the D2 variant. Variants were classified per ACMG/AMP criteria. In silico analyses using Missense3D, PROVEAN, mCSM, SDM, DUET, and PMut assessed structural impact of p.A303D. A literature review on Mastermind identified all GALT missense variants reported in homozygotes or compound heterozygotes with p.K285N, extracting clinical data.
**Key Results:** The p.K285N variant was classified as pathogenic; p.A303D as likely pathogenic. Missense3D predicted that p.A303D causes three damaging effects: replacing a buried hydrophobic alanine with hydrophilic aspartate, introducing a buried charge, and disrupting side-chain and main-chain H-bonds. Other tools confirmed a destabilizing effect. The literature review identified 54 patients with 16 missense variants in trans with p.K285N: 3/54 (5.6%) were p.K285N homozygotes, 51/54 (94.4%) were compound heterozygotes. The most common co-occurring variant was p.Q188R (31/54, 57.4%). Clinical subtype was reported for 36 patients: 33/36 (91.7%) had classic galactosemia, 3/36 (8.3%) had clinical variant. Liver function data were available for 21/54 patients, of whom 13/21 (61.9%) had hepatic damage. Variants were distributed throughout the gene: many near substrate-binding sites (e.g., p.Q188R, p.Q169K, p.R333L, p.Y339C) or zinc-binding sites (e.g., p.L195P, p.Y209S, p.A320T, p.P325L), and others elsewhere (e.g., p.R51L, p.H114P, p.S135L, p.R231H, p.P244S, p.E271D). Most were associated with molecular defects including altered substrate binding, impaired dimerization, or misfolding. The index case also presented with classic galactosemia and acute liver failure.
**Clinical Implications:** This study expands the mutational spectrum of GALT by characterizing a novel likely pathogenic variant (p.A303D) that computer models indicate destabilizes the protein, likely via disruption near the zinc-binding site (H301). The severe phenotype in the index case, consistent with classic galactosemia and acute liver failure, underscores the functional importance of this region. The literature review shows that the majority of patients with p.K285N and another missense variant develop classic galactosemia, with a high rate of acute hepatic damage. This highlights the need for early genetic diagnosis and suggests that variants affecting protein stability or substrate binding correlate with severe disease. Integrating computational prediction with clinical and genetic data may improve variant classification and guide patient management. However, the study is limited by incomplete clinical data in the literature review and the absence of functional validation of the p.A303D variant. Future work should include in vitro assays to confirm the predicted impact on GALT activity and explore potential therapies aimed at stabilizing mutant GALT protein.