Plasma Level of Pyrophosphate Is Low in Pseudoxanthoma Elasticum Owing to Mutations in the ABCC6 Gene, but It Does Not Correlate with ABCC6 Genotype

Abstract

Background: Pseudoxanthoma elasticum (PXE), a monogenic disorder resulting in calcification affecting the skin, eyes and peripheral arteries, is caused by mutations in the ABCC6 gene, and is associated with low plasma inorganic pyrophosphate (PP_i). It is unknown how ABCC6 genotype affects plasma PP_i.

Methods: We studied the association of ABCC6 genotype (192 patients with biallelic pathogenic ABCC6 mutations) and PP_i levels, and its association with the severity of arterial and ophthalmological phenotypes. ABCC6 variants were classified as truncating or non-truncating, and three groups of the 192 patients were formed: those with truncating mutations on both chromosomes (n = 121), those with two non-truncating mutations (n = 10), and a group who had one truncating and one non-truncating ABCC6 mutation (n = 61). The hypothesis formulated before this study was that there was a negative association between PP_i level and disease severity.

Results: Our findings confirm low PP_i in PXE compared with healthy controls (0.53 ± 0.15 vs. 1.13 ± 0.29 µM, p < 0.01). The PP_i of patients correlated with increasing age (β: 0.05 µM, 95% CI: 0.03-0.06 per 10 years) and was higher in females (0.55 ± 0.17 vs. 0.51 ± 0.13 µM in males, p = 0.03). However, no association between PP_i and PXE phenotypes was found. When adjusted for age and sex, no association between PP_i and ABCC6 genotype was found.

Conclusions: Our data suggest that the relationship between ABCC6 mutations and reduced plasma PP_i may not be as direct as previously thought. PP_i levels varied widely, even in patients with the same ABCC6 mutations, further suggesting a lack of direct correlation between them, even though the ABCC6 protein-mediated pathway is responsible for ~60% of this metabolite in the circulation. We discuss potential factors that may perturb the expected associations between ABCC6 genotype and PP_i and between PP_i and disease severity. Our findings support the argument that predictions of pathogenicity made on the basis of mutations (or on the structure of the mutated protein) could be misleading.

Keywords: PXE; ectopic mineralization; genotype-phenotype association; plasma pyrophosphate; pseudoxanthoma elasticum.

Figure 1

Inorganic pyrophosphate in PXE and controls. Plasma PP_i is approximately 60% lower in PXE patients than in controls (A). Plasma PP_i is correlated with age in PXE (B) and female PXE patients have slightly higher PP_i (C). Data were analyzed with Student’s t-test (categorical variables) or the Pearson correlation (continuous variables). A p-value < 0.05 was regarded as statistically significant.

Figure 2

Correlation of plasma PP_i and ophthalmological phenotypes. Plasma PP_i is correlated with the presence of choroidal neovascularizations (B). Patients with severe macular atrophy have higher PP_i than patients without macular atrophy (C), and worse visual acuity is weakly correlated with higher PP_i (D). No significant correlation with the length of angioid streaks (A) was found. Data were analyzed with Student’s t-test (categorical variables) or the Pearson correlation (continuous variables). A p-value < 0.05 was regarded as statistically significant.

Figure 3

Correlation ABCC6 genotype and plasma PP_i. (A) Plasma PP_i does not correlate with ABCC6 genotype. ABCC6 variants were classified as truncating or non-truncating. Accordingly, three groups of patients were formed: those with truncating mutations on both chromosomes (TR/TR); those with two non-truncating mutations (NTR/NTR), and a third group who had one truncating and one non-truncating ABCC6 mutation (TR/NTR). (B) Plasma PP_i levels of patients homozygous for R1141* vary between 0.35 and 0.85 μM. R1141* homozygotes are shown on the age vs. plasma PP_i plot as orange dots, R1141* homozygous patients from the same family are indicated by squares. (C) Patients with the non-truncating R1138Q mutation on one allele and TR on the other allele are highlighted in purple on the age vs. plasma PP_i plot; (D) patients with the non-truncating R1314Q mutation on one allele and TR on the other allele are highlighted in green. (E) Representative images of the subcellular localization of R1138Q, wt and R1314Q ABCC6 protein variants in vivo in the liver of Abcc6^−/− mice [10]; ABCC6 protein is shown in green, and nuclei stained by DAPI are shown in blue (scale bar = 20 µm).

Figure 4

Crucial players of plasma pyrophosphate homeostasis, and factors modifying plasma pyrophosphate level and disease severity in pseudoxanthoma elasticum. (A) The activity of ABCC6 protein and ENPP1 in the liver are responsible for approximately 60% of extracellular PP_i in the circulation. In extrahepatic tissues, human progressive ankylosis (ANKH) protein, similarly to ABCC6 protein, facilitates ATP release from cells, while tissue-nonspecific alkaline phosphatase (TNAP) cleaves PP_i, thus lowering its concentration. AMP is hydrolyzed by NT5E (CD73) to inorganic phosphate and adenosine, and the latter down-regulates TNAP expression, thus controlling its activity and increasing plasma PP_i. Ectonucleoside triphosphate diphosphohydrolase enzymes (eNTPDs) compete for ATP with ENPP1. (B) Left: the expression and activity of ABCC6 protein, ANK and ENPP1 all contribute to increasing plasma PP_i, while TNAP, on the other hand, decreases it. Competition for extracellular ATP in plasma by ENPP1 and eNTPDs may also influence pyrophosphate homeostasis. (B) Right: other calcification inhibitors, which exert their effect independent of plasma PP_i concentration, as well as dietary and environmental factors, can also impact calcification symptoms of PXE.