The severity of hereditary porphyria is modulated by the porphyrin exporter and Lan antigen ABCB6

Introduction

The inherited porphyrias are metabolic disorders of haem biosynthesis caused by mutations in genes coding for enzymes in the haem biosynthetic pathway1,2. The intermediates of haem biosynthesis are toxic and their accumulation is particularly damaging to the liver, haematopoietic system, skin and neural tissues, accounting for the characteristic clinical symptoms. Most porphyrias are inherited as autosomal dominant mutations with marked variability in phenotypes that cannot be accounted for by the causal mutation. Indeed, a long-standing clinical mystery has been why some porphyria patients become more ill than others, even within the same pedigree3,4. We hypothesized that the variable penetrance and expressivity of porphyrias might be due to genetic modifiers3,5,6,7.

It is unknown if porphyrin transporters modify the severity of porphyria, especially those transporters that are expressed in red blood cells where most of haem biosynthesis occurs. Multiple porphyrins are exported from differentiating red blood cell model systems, but the identity of the transporter(s) in normal red blood cells remains unknown8,9. We hypothesized that porphyrin transporters might impact porphyria. Disruptions in porphyrin export might produce overaccumulation of toxic porphyrins (protoporphyrin IX (PPIX), coproporphyrins (CPs) and uroporphyrins (Uros))10 in red blood cells. The export of PPIX from red blood cells is mediated by the plasma membrane transporter ABCG2 (refs 11, 12, 13, 14), but it is unknown if this is the sole energy-dependent exporter of porphyrins. Another porphyrin transporter, ABCB6, was recently identified as the Lan blood group antigen in the plasma membrane of red blood cells15, but its function in the red cells as a porphyrin exporter has not been directly tested.

Here we show that variant alleles of ABCB6 gene that are identified in individuals with severe cases of porphyrias have poor expression and/or are defective. We also demonstrate that ABCB6 mediates the plasma membrane export of porphyrins in red cells. Imposing Abcb6deletion on the Fechm1Pas mouse model exacerbates porphyria symptoms, as evidenced by increased red blood cell and hepatic porphyrin concentrations, and enhanced liver injury. These studies show that rare ABCB6 variant alleles modify the severity of porphyria symptoms in patients. More generally, we show how complementary clinical observations, genetic and biochemical studies synergize to inform precision medicine.

Results

WES reveals ABCB6 variants in severely affected patients

At the Royal Prince Alfred Hospital in Australia, a cohort of 36 porphyria patients of European descent and their family members were evaluated for clinical history, biochemical profile and detection of pathogenic mutations in haem biosynthetic pathway genes (HMBS, CPOX, PPOX orFECH; Supplementary Table 1). We performed whole-exome sequencing (WES) on seven individuals with the most severe clinical phenotypes (symptomatic intensive care unit (ICU); for patient stratification, seeSupplementary Methods; Fig. 1a, Supplementary Fig. 1a). The WES data of these individuals were filtered to develop a prioritized candidate gene list, then enrichment analysis was performed using Database of Annotation, Visualization and Integrated Discovery (DAVID)16,17 (see Methods; Supplementary Fig. 1a; Supplementary Table 2). The ‘ABC transporters’ cluster defined by KEGG_PATHWAY and INTERPRO was significantly enriched among the candidate genes (Supplementary Table 3; enrichment score=1.93, false discovery rate P<0.20). ABC transporters mostly consist of four domains, two transmembrane domains, each composed of six membrane-spanning helices, and two nucleotide-binding domains (NBDs). While the NBDs are highly conserved, the transmembrane domain are diverse, a likely reflection of the wide variety of substrates. This ABC cluster cluster included two porphyrin transporters, ABCG2 and ABCB6 (refs 11, 12, 18). ABCG2 mediates PPIX export from red blood cells, the major site of haem synthesis11,12. Plasma membrane ABCB6 defines the Langereis (Lan) antigen15, which can cause severe haemolytic transfusion reactions and haemolytic disease of the fetus and newborn19. Lan(−) individuals are typically asymptomatic, thus the physiological function of plasma membrane ABCB6 is unknown.

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.