Fig. 2

Functional characterization of the identified HINT1 variants. A Western blot analysis of protein extract from HNT1-deleted yeast strain expressing human HINT1, either wildtype (hWT) or the p.Arg37Pro or p.Glu100Gly alleles. Equal loading was validated with mouse monoclonal anti-PGK1 antibody and relative HINT1 expression was normalized to hWT. The graph represents relative quantification of band intensities of four independent replicates. Note the severe reduction of HINT1 in the p.Arg37Pro-expressing yeast, and the modest reduction of HINT1 in the p.Glu100Gly expressing yeast. B Genetic complementation analysis in HNT1-deleted yeast strain performed by spot assay. Serial dilutions of the different yeast strains were spotted on minimal media without leucine, supplemented with either 2% glucose or 2% galactose, and incubated at 39 °C for 3 days. Note the reduction of growth of yeast expressing p.Arg37Pro or p.Glu100Gly compared to hWT under the restrictive conditions. C Western blot analysis of total protein extracts from HINT1 patient Lit2.4 (p.Glu100Gly/p.Glu100Gly) and patient Lit3.3 (p.Glu100Gly /p.Arg37Pro) cells or control lymphoblasts. Membranes were immunoblotted with polyclonal rabbit anti-human HINT1 antibody. Equal loading was validated with mouse monoclonal anti-β-actin antibody. The bar charts represent the means with standard error of the mean (s.e.m.) of the relative quantification of band intensities of three independent replicates. Note the severe reduction of HINT1 from the patient samples. Statistical one-way ANOVA analysis was performed. ns = not significant; ***p < 0.001; ****p < 0.0001. D Sanger sequencing traces of HINT1 cDNA isolated from peripheral blood mononuclear cells of patient Lit2.4 (p.Glu100Gly/p.Glu100Gly) and patient Lit3.3 (p.Glu100Gly/p.Arg37Pro) or a control individual. The c.299A>G transition is framed. Note the comparable intensity of the peaks at the c.299 position in the compound heterozygous patient