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ENPP1 variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification

['Douglas Ralph', 'Department Of Dermatology', 'Cutaneous Biology', 'Sidney Kimmel Medical College', 'Jefferson Institute Of Molecular Medicine', 'Thomas Jefferson University', 'Philadelphia', 'Pennsylvania', 'United States Of America', 'Genetics']

Date: 2022-06

Abstract Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are clinically distinct genetic entities of ectopic calcification associated with differentially reduced circulating levels of inorganic pyrophosphate (PPi), a potent endogenous inhibitor of calcification. Variants in ENPP1, the gene mutated in GACI, have not been associated with classic PXE. Here we report the clinical, laboratory, and molecular evaluations of ten GACI and two PXE patients from five and two unrelated families registered in GACI Global and PXE International databases, respectively. All patients were found to carry biallelic variants in ENPP1. Among ten ENPP1 variants, one homozygous variant demonstrated uniparental disomy inheritance. Functional assessment of five previously unreported ENPP1 variants suggested pathogenicity. The two PXE patients, currently 57 and 27 years of age, had diagnostic features of PXE and had not manifested the GACI phenotype. The similarly reduced PPi plasma concentrations in the PXE and GACI patients in our study correlate poorly with their disease severity. This study demonstrates that in addition to GACI, ENPP1 variants can cause classic PXE, expanding the clinical and genetic heterogeneity of heritable ectopic calcification disorders. Furthermore, the results challenge the current prevailing concept that plasma PPi is the only factor governing the severity of ectopic calcification.

Author summary Biallelic inactivating mutations in the ENPP1 gene cause generalized arterial calcification of infancy (GACI), a frequently fatal disease characterized by infantile onset of widespread arterial calcification and/or narrowing of large and medium-sized vessels often resulting in the early demise of affected individuals. Significantly reduced, almost zero plasma levels of a potent and endogenous calcification inhibitor, inorganic pyrophosphate (PPi), is thought to be the underlying cause of vascular calcification in GACI. Mutations in ENPP1 have not been found in patients with pseudoxanthoma elasticum (PXE), another genetic multisystem ectopic calcification disorder caused by mutations in the ABCC6 gene. This study reports that ENPP1 mutations can also cause PXE with more favorable clinical outcomes. In addition, it was previously thought that plasma PPi levels correlate with vascular calcification severity. However, we here show that vascular calcification severity does not correlate with plasma PPi levels. The results suggest that in addition to PPi, the long-believed determinant of ectopic calcification, additional mechanisms may be at play in regulating ectopic calcification.

Citation: Ralph D, Nitschke Y, Levine MA, Caffet M, Wurst T, Saeidian AH, et al. (2022) ENPP1 variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification. PLoS Genet 18(4): e1010192. https://doi.org/10.1371/journal.pgen.1010192 Editor: Melissa Wasserstein, Children’s Hospital at Montefiore, UNITED STATES Received: November 26, 2021; Accepted: April 5, 2022; Published: April 28, 2022 Copyright: © 2022 Ralph et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by PXE International and the National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases grants R01AR072695 (to JU and QL) and R21AR077332 (to QL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction ABCC6 and ENPP1 encode proteins that are required for the generation of inorganic pyrophosphate (PPi), a potent endogenous inhibitor of calcification [1], and pathogenic variants in both genes have been associated with syndromes of ectopic calcification [2]. Biallelic inactivating variants in ENPP1 or ABCC6 cause generalized arterial calcification of infancy type 1 (OMIM 208000) and type 2 (OMIM 614473), respectively, rare autosomal recessive disorders that are nearly indistinguishable and often diagnosed by prenatal vascular calcification [3–5]. Arterial calcification and intimal hyperproliferation frequently lead to stenoses and early demise of affected infants by six months of age [6,7]. Loss-of-function variants in ABCC6 also cause pseudoxanthoma elasticum (PXE; OMIM 264800), an autosomal recessive disorder characterized by late-onset yet progressive ectopic calcification in the skin, eyes, and arterial blood vessels [8]. In contrast to GACI, the clinical manifestations of PXE are usually not recognized until early adulthood or at adolescence, either diagnosed by practicing dermatologists finding yellowish papules of the skin that progressively coalesce to make a leathery plaque on flexor areas, or by the patient presenting with a retinal bleed. The skin manifestations usually signify later development of vascular complications [8]. The ENPP1 gene encodes a type II transmembrane glycoprotein, the principal enzyme that generates extracellular PPi by hydrolysis of adenosine triphosphate (ATP). Reduced plasma PPi concentration, at approximately 0–10% of control subjects, is the basis for vascular calcification in ENPP1-deficiency [9]. ABCC6, a hepatic plasma membrane transporter, works upstream of ENPP1 by facilitating the extracellular release of ATP, the substrate of ENPP1, thus contributing to PPi generation as well [10,11]. As a result, plasma PPi levels in patients with PXE and Abcc6 knockout murine models of PXE are reduced to approximately 30–50% of controls [10,12–14]. While GACI and PXE are considered PPi deficiency disorders, the plasma PPi concentrations, reduced to different extents, were thought to correlate with the onset and disease severity in these conditions [15,16]. Natural history studies of patients with GACI due to ENPP1-deficiency indicate that many who survive the critical first year of life experience some resolution of arterial calcification but also can later develop autosomal recessive hypophosphatemic rickets type 2 (ARHR2; OMIM 613312) [5]. In addition, some GACI patients with ENPP1-deficiency, diagnosed prenatally or neonatally with vascular calcification, have been reported to develop skin lesions and/or angioid streaks, features that occur in PXE [3,5,17]. These manifestations, however, appear later in adult life. Despite the considerable genotypic and phenotypic overlap between PXE and GACI, ENPP1 variants have not been associated with classic PXE. Here we report the results of clinical, laboratory, and molecular evaluations of ten patients with GACI1 in five distinct families and two patients with PXE in two unrelated families, all carrying biallelic variants in ENPP1. The results show that in addition to GACI, ENPP1 variants can also cause PXE, expanding the phenotypic and genotypic overlap between GACI and PXE.

Discussion GACI, regardless of ABCC6- or ENPP1-deficiency, is a life-threatening arterial calcification disease with a poor prognosis. Although there are several reports of long-term survivors into their third to fifth decade [3,6,24], a large proportion of children with GACI die within the first six months of life. Death is related to cardiovascular collapse, including myocardial infarction, congestive heart failure, persistent pulmonary hypertension, and multi-organ failure. GACI survivors with ENPP1-deficiency develop ARHR2 with short stature and skeletal deformities [3,18]. It was suggested that ARHR2 is FGF23-mediated [3], and the elevated serum FGF23 levels in several GACI patients in the current study support this hypothesis. Elevated serum FGF23 levels may also be a response of cells to circulating calciprotein particles, which are associated with vascular calcification [25]. ABCC6-associated PXE has a different clinical course with late-onset and more favorable clinical outcomes than GACI. Though fully penetrant, clinical features of PXE, including skin, eye, and vasculature lesions, do not usually present until adolescence or early adulthood. In contrast to GACI, individuals affected by PXE have normal life expectancy in the overwhelming number of cases, without evidence of skeletal anomalies. Studies over the past few years have indicated that ENPP1- and ABCC6-deficiency are associated with considerable clinical pleiotropy. Specifically, some patients with ENPP1 variants develop hypophosphatemic rickets without a prior clinical history of GACI [26,27], while nearly all patients with ABCC6 variants present with PXE without a history of GACI [2]. In the current study, while all the patients harbor biallelic ENPP1 variants, their varied clinical expression was highlighted by them seeking support from different advocacy organizations. Among the 10 GACI patients enrolled in GACI Global, eight presented with extensive calcifications of large and medium-sized arteries in the prenatal or neonatal period, while two GACI patients, #7 and #10, had arterial stenosis without evidence of vascular calcification. This is not surprising as regression of vascular calcification has been documented in patients with GACI [24,28]. By contrast, two adult patients #11 and #12 had classical PXE, and both were enrolled in the PXE International registry. From a clinical point of view, these patients do not differ from the typical PXE patients with ABCC6-deficiency. Our studies demonstrate that in addition to GACI and rickets, ENPP1-deficiency can also present as classical PXE, a finding that extends the clinical spectrum of ENPP1-associated diseases. Moreover, because the two ENPP1 variants identified in PXE patient #12 had previously been described in patients diagnosed as GACI [20,23], there does not appear to be a genotype-phenotype explanation for the variation in these two clinical presentations. In this study, we identified five previously unreported ENPP1 variants. We also report original findings of UPD inheritance of a previously unreported homozygous variant, c.1530G>C (p.L510F). The c.241G>T and c.715+5G>T variants were functionally determined to cause aberrant splicing, although c.241G>T (p.V81L) was initially thought to be a missense variant. In contrast to splicing variants, different ENPP1 coding variants have different outcomes on the protein’s functionality. These include reduced protein abundance, impaired cellular localization, compromised stability and/or conformational changes, reduced enzyme activity, and ability to generate PPi. Although the c.1530G>C (p.L510F) mutant had reduced enzyme activity and PPi generation, its residual activity was probably attributed to the partial localization on the plasma membrane. Therefore, the p.L510F variant appears to be a candidate for allele-specific therapy in correcting the misfolded, otherwise functional protein, as previously demonstrated for ABCC6 [29–31]. While plasma PPi concentrations in patients with ABCC6-deficiency were reduced to approximately 30–50% of controls, patients with ENPP1-deficiency had a further reduction to approximately 0–10% of controls. It is not clear why the same ENPP1 variants can result in different diagnoses of either PXE or GACI since our patients’ plasma PPi concentrations were equally low. Several potential mechanisms may explain the phenotypic heterogeneity and the poor correlation between plasma PPi concentrations and disease severity. First, environmental factors and genetic modifiers may influence the disease severity of ectopic calcification [2,32]. Second, circulating PPi may be a poor proxy of the local PPi concentrations which may be more important in preventing tissue calcification. Unfortunately, we cannot currently measure extracellular PPi levels in tissues. Third, in addition to PPi, ENPP1-mediated hydrolysis of ATP also produces adenosine monophosphate. The pathophysiologic role of adenosine monophosphate in the disease process of GACI was recently reported [9]. Furthermore, the potential dysregulation of extracellular nucleotide metabolism, for example, ENPP1-mediated disruption of pyrimidine synthesis known to regulate tissue repair, may play a role in ectopic tissue calcification [33]. The concept of ENPP1-deficiency has evolved dramatically over the past several decades: what once considered an exclusively fatal arterial disease with poor prognosis is now recognized as a complex, multi-systemic process with a broad phenotypic spectrum spanning from infantile vascular calcification associated with early demise, to hypophosphatemic rickets in survivors, and as indicated in this study, to typical late-onset PXE with more favorable prognosis and normal life expectancy. In conclusion, the phenotypic spectrum of ENPP1-deficiency is much broader than was previously anticipated. In addition to GACI, we show that the late-onset skin and ocular phenotypes of patients with ENPP1-deficiency can be indistinguishable from typical PXE with ABCC6-deficiency. The divergent phenotypes in patients with ENPP1-deficiency cannot be explained exclusively by plasma concentrations of PPi which were reduced to the same extent. The results suggest that although PPi is a major determinant of ectopic calcification, additional yet unidentified mechanisms may play a role in the regulation of ectopic calcification.

Materials and methods Ethics statement All patients were enrolled with written or verbal informed consent/assent into this study with approval from the institutional review board at Children’s Hospital of Philadelphia (Approval number 12–008863) or Genetic Alliance (Approval number PXE001 for PXE International). Formal consent was obtained from the parent/guardian for child participants. Patients with GACI and PXE were registered in the databases of GACI Global and PXE International, advocacy organizations for GACI and PXE, respectively. We used the Phenodex score, an international standard to assess phenotypes in five organ systems: skin (S), eyes (E), gastrointestinal (G), cardiac (C), and vasculature (V), to determine the clinical severity of PXE [19]. Variant detection and bioinformatics Genomic DNA was extracted from saliva (DNA Genotek Inc) or peripheral blood leukocytes (Qiagen, Valencia, CA). Variant detection was performed by Sanger sequencing of the entire coding region and intron/exon boundaries of the ABCC6 and ENPP1 genes, exome sequencing (MyGenostics, Beijing, China), next-generation sequencing of hypophosphatemic rickets-targeted genes including ENPP1 (Exeter Clinical Laboratory, Leeds, UK), or next-generation sequencing of ectopic calcification-associated 29 gene panel including ABCC6 and ENPP1 [34]. Exome sequencing and stepwise bioinformatics were performed according to previously reported approaches [35,36]. The kinship analysis was done using VCFtools—relatedness2 on the merged Variant Call Format files [21]. The screening for Runs of Homozygosity (ROH) of more than 4 Mb and establishment of patterns of UPD in trio samples were performed according to the previously described method, with slight modifications [37]. ENPP1 variant nomenclature was based on NC_000006.12 (NM_006208). The variant nomenclature followed the recommendations of the Human Genome Variation Society (http://www.hgvs.org/mutnomen/). The number of individuals carrying the specific variant as homozygous and the minor allele frequency in the general population was extracted from Genome Aggregation Database (gnomAD) (gnomad.broadinstitute.org) and BRAVO (https://bravo.sph.umich.edu/freeze8/hg38/) consisting of over 120,000 and 130,000 apparently healthy individuals, respectively. The recommended ENPP1 gene-specific MAF threshold is 0.1% (https://franklin.genoox.com/clinical-db/home). Various in silico prediction programs (https://franklin.genoox.com/clinical-db/home) and the Combined Annotation Dependent Depletion (CADD) score were used to assess the effects of variants on the protein function [38,39]. Classification of variants follows the latest guidelines of the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP), which classifies variants as benign (B), likely benign (LB), variants of unknown significance (VUS), likely pathogenic (LP), and pathogenic (P) [40,41]. The ENPP1 gene-specific CADD score within the 95% confidence interval was calculated using the mutation significance cutoff method [22]. RNA analysis of the c.241G>T (p.V81L) variant in ENPP1 Total RNA was extracted from patient #7’s peripheral blood cells after venous blood collection in a PAXgene Blood RNA tube (BD Diagnostics, Franklin Lakes, NJ) followed by RT-PCR and Sanger sequencing. In vitro mini-gene splicing assay of the c.715+5G>T variant in ENPP1 WT and c.715+5G>T mutant mini-gene segments spanning from exon 5 to exon 7 of human ENPP1 were cloned into the pCMV-3Tag-3a vector (Genscript, Piscataway, NJ). Human embryonic kidney (HEK293) cells were transfected with WT or mutant constructs using FuGENE HD (Promega, Madison, WI). Cells were collected 48 hours post-transfection for RNA extraction and RT-PCR followed by bidirectional Sanger sequencing of different transcript isoforms. Functional assessment of c.656G>A (p.G219E), c.1530G>C (p.L510F), and c.876_880delTAAAG variants in ENPP1 The full-length WT cDNA and mutant human ENPP1 cDNA entailing each of the three variants, c.656G>A, c.1530G>C, and c.876_880delTAAAG, were cloned into a pcDNA3.1(+) vector (GenScript). HEK293 and Cercopithecus aethiops kidney (COS7) cells were transfected using jetPEI (Illkirch, France). We measured the activity of nucleotide phosphodiesterase (NPP) 24 hours after transfection of HEK293 cells using pNP-TMP as substrate [7,20]. Expression of human ENPP1 protein was detected by Western blot using a rabbit anti-human ENPP1 antibody, #5342, 1:1,000 (Cell Signaling, Boston, MA). An anti-human β-actin antibody, #4970, 1:1,000, was used to normalize protein loading (Cell Signaling). The cellular localization of the ENPP1 protein was analyzed in transfected COS7 cells using a monoclonal anti-human ENPP1 antibody, 1:100 (3E8, kind gift from Dr. Fabio Malavasi, Torino, Italy). Cells were stained with fluorescein isothiocyanate labeled phalloidin, #P5282, 1:40 (Sigma-Aldrich, Taufkirchen, Germany), to visualize plasma membrane localization. The PPi concentration in the medium of transfected HEK293 cells was measured 20 min after incubation with 20 μM GTP. PPi was quantified as previously described [10,12]. Biochemical analyses The serum concentrations of calcium, phosphorus, alkaline phosphatase, fibroblast growth factor 23, parathyroid hormone, and 25-hydroxyvitamin D3 were retrieved from patients’ medical records. Whole blood was collected into CTAD and transferred to EDTA tubes (BD Diagnostics), followed by depletion of platelets by filtration through a Centrisart I 300-kDa mass cutoff filter (Sartorius, New York, NY). Determination of PPi concentration in platelet-free plasma was performed as previously described [10,12]. Statistical analysis Statistical analyses were performed using ordinary one-way ANOVA. Statistical significance was considered with P < 0.05. All statistical analyses were completed using Prism 8 (GraphPad, San Diego, CA).

Acknowledgments We thank all the affected individuals and families for their participation. We thank Dr. Hansel J. Otero at Children’s Hospital of Philadelphia, and Dr. Meisam Sargazi in Alzahra Eye Hospital Research Center at Zahedan University of Medical Sciences, Zahedan, Iran, for interpretations of clinical images. We thank Mary Peckiconis at PXE International for assistance in obtaining the participants’ medical records, and Dr. Talat Mushtaq at Leeds Children’s Hospital in UK for assistance with the clinical findings in family #4.

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