Figure 1. Clinical and biochemical analyses of rare variant alleles of ABCB6 in porphyric patients.

(a) Flow chart describing strategies to identify variants associated with severe porphyria symptoms. (b) Urinary porphyrin levels normalized to creatinine. ^#Patients with only defective ABCB6 alleles were included. The asymptomatic patient with ABCB6 R192Q variant allele showed a low porphyrin levels compared with patients with other ABCB6 variant alleles. n for WT and variant are 17 and 7, respectively. (c) Ribbon diagram showing the location of amino acids encoded by rare variant ABCB6 alleles (for clarity, only a monomer is shown). The variant R192Q is not shown because high-resolution structural data are not available as this residue lies in a non-conserved region among ABC transporters. (d) Immunoblot of transient transfection of ABCB6 wild-type (WT) and variant alleles (the epitope tag was previously shown to not disrupt function¹⁸). (e) The ATP-binding capacity measured from ATP-agarose beads pull-down was plotted (n=2). (f) ATP-dependent CPIII transport into membrane vesicles prepared from indicated MEL cell lines is shown (n=2). (g) ABCB6 WT and variant expression at the cell surface in Abcb6^−/− mouse embryonic fibroblast was determined by cell surface biotinylation assays. FT, flow through. (h) ABCB6 pulled down by streptavidin-agarose beads was quantified by densitometry. Two independent experiments were performed. Only the representative results shown. (i) Immunoblot of ABCB6 WT and variants when co-transfected with V5-tagged WT ABCB6. The red arrow shows R192Q is stabilized by WT ABCB6. Two independent experiments were performed. Only the representative results shown. (j) Co-immunoprecipitation assay to assess interaction between ABCB6 WT and variant alleles. *P<0.05; **P<0.01; ***P<0.001 using Student t-test with error bars showing s.d.