Gene and expression analysis. (A) Sequence chromatograms showing part of the AP4M1 exon 3 (NM_004722.3) obtained from a healthy control (top), a heterozygous parent (middle) and the affected individual IV:3 (bottom). The two nucleotides in AP4M1 (c.194_195delAT, p.Y65Ffs*50) deleted on the mutated allele are indicated by asterisks in the top panel. The arrow denotes a synonymous SNP (rs367614875) in the middle panel. Codons corresponding to the two alleles are indicated with the altered reading frame in the bottom chromatogram. (B) Schematic representation of the AP4M1 protein with its functional domains (http://www.ebi.ac.uk/interpro/protein/O00189) and all reported mutations (top). The degree of conservation of the Tyr65 residue (shaded) is shown across different species (bottom). (C) Quantitative RT-PCR of AP4M1 mRNA expression in fibroblasts cells from cases (n = 2) and healthy controls (n = 2). Data are normalized to β-actin mRNA and presented as cases relative to controls ± standard deviation. The relative expression of AP4M1 in controls is set to one. The AP4M1 expression levels in patients are reduced three-fold when compared to controls (P = 0.0026, two sided Student’s t-test assuming equal variance).