Fig 2: Functional characterization of five previously unreported ENPP1 variants, c.241G>T (p.V81L), c.715+5G>T, c.656G>A (p.G219E), c.1530G>C (p.L510F) and c.876_880delTAAAG.(a) Sanger sequencing of RT-PCR products from leukocytes in patient #7 carrying the c.241G>T variant showed skipping of exon 2. (b) The splicing of ENPP1 mini-gene pre-mRNA from transfected HEK293 cells was assessed by RT-PCR using a forward primer specific to the mini-gene and a reverse primer targeting the vector’s Flag-tag sequence, which allows splicing of the mini-gene-produced transcripts to be studied. Sanger sequencing of RT-PCR products showed normal splicing for the wild-type construct and exon 6 skipping for mutant c.715+5G>T ENPP1 mini-gene. (c) Western blot of ENPP1 protein expression in transfected HEK293 cells. ENPP1 monomers migrate as doublets at 118 kDa and 128 kDa on SDS-PAGE. Beta-actin was used as internal loading control. n = 3 independent experiments. (d) ENPP1 localization (red fluorescence) in transfected COS7 cells. Phalloidin, a peptide specific to actin filaments which are frequently found attached to or near the plasma membrane, was stained in green fluorescence to define plasma membrane. n = 3 independent experiments. Scale bar = 50 µm. (e) ENPP1 enzyme activity in transfected HEK293 cells, three cultures per group. #P < 0.001, compared with the wild-type construct. n = 3 independent experiments. #P < 0.001, compared with the wild-type construct. Data were presented as mean ± SEM. (f) Extracellular PPi generation in medium of transfected HEK293 cells 20 min after adding 20 µM GTP. Data represent % of PPi generated by the WT ENPP1-transfected cells. n = 3 independent experiments. +P < 0.01, #P < 0.001, compared with the wild-type construct. Data were presented as mean ± SEM.

Fig 3: Nuclear pedigrees of families with a diagnosis of GACI or PXE.(a-e), GACI families #1–5; (f-g), PXE families #6–7. Patient identifier (#1 through 12) was placed above their symbol. The variants identified in the ENPP1 and ABCC6 genes in the individual family members are indicated below each individual: +/+, variants present in both alleles; +/-, heterozygous; -/-, homozygous for the wild-type allele. Unless otherwise noted, all variants were found in ENPP1. The stepwise bioinformatic filtering of exome sequencing data for variant detection in family #6 was narrowed to ENPP1 (panel f2). The uniparental inheritance of the c.1530G>C (p.L510F) variant in ENPP1 in patient #11 was shown in one Run of Homozygosity (ROH; blue region) on Chromosome 6 inherited from her mother (panel f3). d, died; a, alive.