Fig 2: A homozygous splice site (c.2230 + 5G > A; p.Asp701Asnfs*2) in a family. a The family pedigree illustrates a patient carrying a pathogenic variant in ENPP1 (c.2230 + 5G > A). Open symbols represent unaffected individuals, filled symbols represent affected individual, triangles represent spontaneous abortions, and symbols with a diagonal line indicate deceased individuals. The genotypes of the ENPP1 variant are shown below each examined member. b Representative sequence electropherograms show the wild-type sequence and the (c.2230 + 5G > A) heterozygous (III-2) and homozygous (IV-1) sequences. c Schematic of the human ENPP1 gene and protein domain structure. The ENPP1 protein consists of the N-terminal cytoplasmic domain (CD), transmembrane domain (TM), tandem somatomedin-B-like domains (SMB1-2), a phosphodiesterase catalytic domain (PCD), L1 and L2 linkers, and a C-terminal nuclease-like domain (NLD). Position of (c.2230 + 5G > A; p.Asp701Asnfs*2) is indicated

Fig 3: Plasma PPi concentrations in individuals with GACI or PXE and family members.Individuals with biallelic ENPP1 pathogenic variants, regardless of the diagnosis of GACI or PXE, had barely detectable PPi plasma concentrations. ENPP1 heterozygotes had intermediate PPi plasma concentrations. The physiologic PPi plasma concentrations were obtained from 9 healthy control volunteers. Data represent mean ± SEM.

Fig 4: 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 5: 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.