A. Blood smear demonstrating poikilocytosis and polychromasia as well as irregularly contracted cells (1) and a “ghost” cell (2), suggesting hemolysis due to G6PD deficiency [18]. B. Hydrophobicity map of the G6PD tetramer visualized by the 3D-structure viewer application Protein Workshop (http://www.pdb.org/robohelp/viewers/proteinworkshop.htm) [19], with the arrows pointing to the two amino-acid sites mutated in the case discussed. Inset shows enlarged the site of asparagine (N) at the position 346 in a hydrophilic loop at the dimer interface, which in our patient has been substituted by a hydrophobic isoleucine (I).

A. G6PD protein sequences for the species shown were obtained from the NCBI nucleotide database (accession numbers NP_001035810, NP_032088, NP_001080019, XP_699168, P12646, AAT93017, and NP_416366 respectively). Sequence alignment was performed using the ClustalW protein sequence alignment tool (http://www.ebi.ac.uk/Tools/clustalw2/index.html); the N346 position is highlighted. Overall sequence homology to the human protein sequence was 93% for M.musculus, 77% for X.laevis, 75% for D.rerio, 61% for D.melanogaster, 44% for S.cerevisiae, and 34% for E.coli. B. Immunoblotting for G6PD after running hemoglobin-depleted blood samples from the patient (P) and a control (C) in native gel electrophoresis. Bands at the expected molecular weights for the monomer (52 kDa), dimer (105 kDa), and tetramer (210 kDa) are seen in the control specimen. Densitometry was performed using the ImageJ program (http://rsbweb.nih.gov/ij) and the relative quantitation of the bands is shown. The tetrameric form consists approximately 52% of the total enzyme in the control sample and 37% of the total enzyme in the patient's sample. GAPDH at the molecular weight of a tetramer (145 kDa) as expected in a native gel was detected in the same two lanes as loading control.