Biochemical and computational analyses of two phenotypically related GALT mutations (S222N and S135L) that lead to atypical galactosemia

Galactosemia is a metabolic disorder caused by mutations in the GALT gene [1] and [2]. We encountered a patient heterozygous for a known pathogenic H132Q mutation and a novel S222N variant of unknown significance [3]. Reminiscent of patients with the S135L mutation, our patient had loss of GALT enzyme activity in erythrocytes but a very mild clinical phenotype [3], [4], [5], [6], [7] and [8]. We performed splicing experiments and computational structural analyses to investigate the role of the novel S222N variant. Alamut software data predicted loss of splicing enhancers for the S222N and S135L mutations [9] and [10]. A cDNA library was generated from our patient׳s RNA to investigate for splicing errors, but no change in transcript length was seen [3]. In silico structural analysis was performed to investigate enzyme stability and attempt to understand the mechanism of the atypical galactosemia phenotype. Stability results are publicly available in the GALT Protein Database 2.0 [11], [12], [13] and [14]. Animations were created to give the reader a dynamic view of the enzyme structure and mutation locations. Protein database files and python scripts are included for further investigation.