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Prospective phenotyping of long-term survivors of generalized arterial calcification of infancy (GACI)

      Abstract

      Purpose

      Generalized arterial calcification of infancy (GACI), characterized by vascular calcifications that are often fatal shortly after birth, is usually caused by deficiency of ENPP1. A small fraction of GACI cases result from deficiency of ABCC6, a membrane transporter. The natural history of GACI survivors has not been established in a prospective fashion.

      Methods

      We performed deep phenotyping of 20 GACI survivors.

      Results

      Sixteen of 20 subjects presented with arterial calcifications, but only 5 had residual involvement at the time of evaluation. Individuals with ENPP1 deficiency either had hypophosphatemic rickets or were predicted to develop it by 14 years of age; 14/16 had elevated intact FGF23 levels (iFGF23). Blood phosphate levels correlated inversely with iFGF23. For ENPP1-deficient individuals, the lifetime risk of cervical spine fusion was 25%, that of hearing loss was 75%, and the main morbidity in adults was related to enthesis calcification. Four ENPP1-deficient individuals manifested classic skin or retinal findings of PXE. We estimated the minimal incidence of ENPP1 deficiency at ~1 in 200,000 pregnancies.

      Conclusion

      GACI appears to be more common than previously thought, with an expanding spectrum of overlapping phenotypes. The relationships among decreased ENPP1, increased iFGF23, and rickets could inform future therapies.

      Keywords

      Introduction

      Generalized arterial calcification of infancy (GACI), an autosomal recessive disorder characterized by calcification of large- and medium-sized vessels, has a mortality of 55% within the first six months of life due to myocardial or cerebral infarctions.
      • Rutsch F.
      • Böyer P.
      • Nitschke Y.
      • et al.
      Hypophosphatemia, hyperphosphaturia, and bisphosphonate treatment are associated with survival beyond infancy in generalized arterial calcification of infancy.
      Only ~200 patients have been reported.
      • Chong C.R.
      • Hutchins G.M.
      Idiopathic infantile arterial calcification: the spectrum of clinical presentations.
      In 67% of cases, GACI is caused by biallelic inactivating variants in ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1),
      • Nitschke Y.
      • Baujat G.
      • Botschen U.
      • et al.
      generalized arterial calcification of infancy and pseudoxanthoma elasticum can be caused by mutations in either ENPP1 or ABCC6.
      which encodes an enzyme that cleaves adenosine triphosphate (ATP) into adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi) at the cell surface. Vascular calcification is thought to occur as a result of a deficiency of PPi, the main inhibitor of physiological calcification. In 9% of cases, GACI results from biallelic inactivating variants in ABCC6, which encodes a plasma membrane transporter highly expressed in the liver;
      • Jansen R.S.
      • Küçükosmanoglu A.
      • de Haas M.
      • et al.
      ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release.
      biallelic variants in ABCC6 typically cause pseudoxanthoma elasticum (PXE), a disorder characterized by calcification and fragmentation of elastic fibers in the skin (leading to coalescing papules in neck and flexures), retina (with peau d’orange, angioid streaks, and choroidal neovascularization), and cardiovascular system (with consequent adult-onset arterial calcification).
      • Ringpfeil F.
      • Lebwohl M.G.
      • Christiano A.M.
      • Uitto J.
      Pseudoxanthoma elasticum: mutations in the MRP6 gene encoding a transmembrane ATP-binding cassette (ABC) transporter.
      While the exact molecule transported by ABCC6 into the extracellular space is uncertain, one candidate is ATP, a substrate for ENPP1 and thus a source of plasma PPi. Accordingly, levels of PPi are decreased in affected humans and animal models of ABCC6 deficiency.
      • Jansen R.S.
      • Duijst S.
      • Mahakena S.
      • et al.
      ABCC6-mediated ATP secretion by the liver is the main source of the mineralization inhibitor inorganic pyrophosphate in the systemic circulation-brief report.
      Further evidence of genetic heterogeneity in the pathogenesis of GACI is based on identification of affected individuals who lack variants in either ENPP1 or ABCC6.
      • Nitschke Y.
      • Baujat G.
      • Botschen U.
      • et al.
      generalized arterial calcification of infancy and pseudoxanthoma elasticum can be caused by mutations in either ENPP1 or ABCC6.
      In a previous study, 5 of 19 children who survived GACI beyond infancy developed hypophosphatemia.
      • Rutsch F.
      • Böyer P.
      • Nitschke Y.
      • et al.
      Hypophosphatemia, hyperphosphaturia, and bisphosphonate treatment are associated with survival beyond infancy in generalized arterial calcification of infancy.
      Subsequently, ENPP1 deficiency was associated with autosomal recessive hypophosphatemic rickets type 2, or ARHR2; elevated or high-normal plasma intact FGF23 concentrations were documented and considered to be the cause of renal phosphate wasting in these patients.
      • Lorenz-Depiereux B.
      • Schnabel D.
      • Tiosano D.
      • Häusler G.
      • Strom T.M.
      Loss-of-function ENPP1 mutations cause both generalized arterial calcification of infancy and autosomal-recessive hypophosphatemic rickets.
      ,
      • Levy-Litan V.
      • Hershkovitz E.
      • Avizov L.
      • et al.
      Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene.
      Other complications (hearing loss,
      • Brachet C.
      • Mansbach A.L.
      • Clerckx A.
      • Deltenre P.
      • Heinrichs C.
      Hearing loss is part of the clinical picture of ENPP1 loss of function mutation.
      PXE-like skin and retinal changes,
      • Le Boulanger G.
      • Labrèze C.
      • Croué A.
      • et al.
      An unusual severe vascular case of pseudoxanthoma elasticum presenting as generalized arterial calcification of infancy.
      and cervical spine fusion
      • Nitschke Y.
      • Baujat G.
      • Botschen U.
      • et al.
      generalized arterial calcification of infancy and pseudoxanthoma elasticum can be caused by mutations in either ENPP1 or ABCC6.
      ,
      • Gopalakrishnan S.
      • Shah S.
      • Apuya J.S.
      • Martin T.
      Anesthetic management of a patient with idiopathic arterial calcification of infancy and fused cervical spine.
      ) have been described in case reports.
      Here we report the results of clinical, laboratory, and molecular evaluations of 20 individuals with GACI. The results expand the phenotypic and mutational spectra of the disease and provide a basis for developing outcome measures for future clinical trials.

      Materials and Methods

      Subjects

      We enrolled 20 subjects aged 9 months to 58 years who had a clinical diagnosis of GACI plus at least one pathogenic variant in ENPP1 or ABCC6.

      Ethics statement

      Affected individuals were co-enrolled in three institutional review board (IRB)–approved protocols at the National Institutes of Health (NIH): “Diagnosis and Treatment of patients with Inborn Errors of Metabolism and Other Genetic Disorders” (NCT00369421), “Study of People With generalized arterial calcification of infancy (GACI) or Autosomal Recessive Hypophosphatemic Rickets Type 2 (ARHR2)” (NCT03478839), and “Evaluation and Treatment of Bone and Mineral Disorders” (NCT00024804). All subjects or their guardians provided informed consent and, when appropriate, minors provided assent.

      Data collection

      All study participants underwent specialty consultations, echocardiograms, and computed tomography (CT) of the chest, abdomen, and pelvis with extension to extremities. Blood phosphate and alkaline phosphatase were measured on a Roche Cobas 6000 platform; parathyroid hormone was measured by electrochemiluminescence immunoassay on a Roche Cobas e601 analyzer; 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were measured by chemiluminescent immunoassay. Intact FGF23 (iFGF23) and C-terminal FGF23 were measured by enzyme-linked immunosorbent assay (ELISA) (Immutopics International, San Clemente, CA). Genomic DNA was extracted from peripheral blood mononuclear cells and sequenced by commercial laboratories.

      Statistical analysis

      Data were analyzed using descriptive statistics (conducted in SAS 9.4), with medians and ranges for continuous variables, and numbers and proportions for categorical variables. Kaplan–Meier curves were created with Prism version 6.0c (Graphpad Software Inc, La Jolla, CA). To model serum phosphate as a function of age, a generalized mixed model with an identity link function was used to account for unbalanced observations and intrasubject correlation using random intercepts and scaled identity covariance structure (IBM SPSS Statistics Subscription for Windows, IBM Corp., Armonk, NY, USA). Because serum phosphate concentrations vary with age in healthy children, we expressed phosphate values as Z-scores relative to published age-matched values.
      • Lockitch G.
      • Halstead A.C.
      • Albersheim S.
      • MacCallum C.
      • Quigley G.
      Age- and sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer.
      A quadratic model was fitted using age and change in age as covariates.

      Incidence calculation

      The predicted incidence of ENPP1 deficiency was estimated by searching for all published ENPP1 pathogenic variants. The Exome Aggregation Consortium (ExAC)
      • Lek M.
      • Karczewski K.J.
      • Minikel E.V.
      • et al.
      Analysis of protein-coding genetic variation in 60,706 humans.
      provided allele frequencies and allowed identification of variants predicted to damage ENPP1 function, but not reported in the literature before March 2017. ExAC was queried for canonical splice site, missense, frameshift, nonsense (stopgain), and stop loss variants. None of the cohorts or consortia in ExAC include patients ascertained for the presence of GACI, so we considered them to be unbiased with respect to variation in the ENPP1 gene. Variants were excluded if (1) they had a minor allele frequency of >0.5%; (2) they were found in sites covered in fewer than 80% of individuals, as this may indicate a low-quality site; (3) they were only present in a noncanonical transcript; (4) they were in an untranslated region; or (5) they fell in the SMB2 domain, corresponding to amino acids 145–189, since variants in this domain are associated with a different phenotype, Cole disease.
      • Chourabi M.
      • Liew M.S.
      • Lim S.
      • et al.
      ENPP1 mutation causes recessive Cole disease by altering melanogenesis.
      Variants that passed these filters were evaluated for their potential to alter protein function by using in silico prediction models, i.e., PolyPhen-2,
      • Adzhubei I.A.
      • Schmidt S.
      • Peshkin L.
      • et al.
      A method and server for predicting damaging missense mutations.
      SIFT,
      • Kumar P.
      • Henikoff S.
      • Ng P.C.
      Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.
      and the Combined Annotation-Dependent Depletion (CADD) score.
      • Kircher M.
      • Witten D.M.
      • Jain P.
      • O’Roak B.J.
      • Cooper G.M.
      • Shendure J.
      A general framework for estimating the relative pathogenicity of human genetic variants.
      Unpublished variants predicted to be benign by one or more in silico models were not considered for the calculation of incidence. For likely pathogenic variants, such as frameshift, stopgain (nonsense), and canonical splice site variants, only the CADD Phred-scaled score was provided.
      The carrier frequency was calculated as the number of individuals having an ENPP1 variant known or predicted to alter protein function, divided by the total number of individuals ascertained. The incidence of the disease was calculated based on the carrier frequency and/or the allele frequency using Hardy–Weinberg equilibrium.

      Results

      Subjects

      Twenty individuals (16 with biallelic ENPP1 variants and 4 with ABCC6 variants) survived infancy and underwent extensive evaluations at the NIH Clinical Center at ages ranging from 9 months to 58 years. Their family pedigrees are presented in Fig. S1 and their clinical and molecular findings are described in Table 1. Five siblings of the 16 individuals in our cohort with ENPP1 variants died within the first 5 months of life with findings typical of GACI. The location of each variant in the ENPP1 or ABCC6 protein is presented in Fig. S2; six novel variants in ENPP1 are described. For the full cohort, the median ages of onset of symptoms and diagnosis were 2.5 and 23 days, respectively. Individuals with ABCC6 variants did not differ from those with ENPP1 variants with respect to these parameters.
      Table 1Patient summary and clinical presentation of the full cohort.
      FamilyPatientPathogenic variants
      Novel variants in bold.
      Age at presentationAge at diagnosisHearing loss
      Type of hearing loss, age of onset specified.
      Age at last examinationComorbidities
      cDNAProteinGACIARHR2PXE
      ENPP1 (transcript: NM_006208.2) deficiency
      11c.1441C>Tp.(Arg481Trp)44 days47 days8 years 3 monthsConductive, 8 years 3 months8 years 3 monthsHypertension
      c.2312-5_2313del
      12c.1441C>Tp.(Arg481Trp)4 years9 years 2 monthsNone13 years 2 monthsHypertension
      c.2312-5_2313del
      23c.1438T>Cp.(Cys480Arg)8 months4 years 4 months4 years 7 monthsMixed, 3 years 10 months8 years 11 monthsLoeys–Dietz syndrome, chronic lung disease, hypertension
      c.2414G>Tp.(Gly805Val)
      24c.1438T>Cp.(Cys480Arg)Prenatally (38 wga)Prenatally (38 wga)3 years 4 monthsNone4 years 5 monthsNone
      c.2414G>Tp.(Gly805Val)
      35c.2735T>Cp.(Leu912Ser)0 days21 daysConductive, 1 year 9 months4 years 2 monthsNeurological disorder (developmental delay)
      3.4kb deletion of exon 6 (delIVS5_IVS6)
      46c.1441C>Tp.(Arg481Trp)Prenatally (20 wga)Prenatally (20 wga)8 monthsConductive, 4 years 1 month5 years 1 monthNone
      c.2713_2717delp.(Lys905Alafs*16)
      57c.1538A>Gp.(Tyr513Cys)2 years2 years 8 months2 years 2 monthsMixed, 1 year 4 months7 years 11 monthsCerebrovascular disease, congestive heart failure, neurological disorder (cerebral palsy, developmental delay, epilepsy, hypotonia), hypertension
      c.1538A>Gp.(Tyr513Cys)
      68c.749C>Tp.(Pro250Leu)12 days25 days8 years 2 months26 years 4 monthsNone26 years 4 monthsNone
      c.913C>Ap.(Pro305Thr)
      79c.749C>Tp.(Pro250Leu)Prenatally (31 wga)Prenatally (31 wga)32 daysConductive, 6 months1 year 8 monthsNone (some developmental delay)
      c.749C>Tp.(Pro250Leu)
      810c.1652A>Gp.(Tyr551Cys)10 days
      Patient presented with diffuse vascular stenosis/fibromuscular dysplasia shortly after birth, confirmed ENPP1 deficiency at 2 years of age.
      Conductive, 5 years 1 month5 years 2 monthsBrugada syndrome type 2, cerebrovascular disease (seizures), hypertension
      c.2330A>Gp.(His777Arg)
      911c.1412A>Gp.(Tyr471Cys)29 days71 days14 years 7 monthsConductive, 7 years 6 months25 years 7 monthsHypertension
      c.1442G>Ap.(Arg481Gln)
      1012c.1652A>Gp.(Tyr551Cys)0 days4 monthsNone2 years 1 monthHypertension
      c.1737G>Cp.(Leu579Phe)
      1113c.913C>Ap.(Pro305Thr)0 days6 days6 years 7 monthsMixed, 3 years8 years 11 monthsNone
      c.1499A>Cp.(His500Pro)
      1214c.913C>Ap.(Pro305Thr)Prenatally (32 wga)1 daysNone9 monthsRenal disease, hypertension
      c.2662C>Tp.(Arg888Trp)
      1315c.2320C>Tp.(Arg774Cys)Prenatally (31 wga)Prenatally (31 wga)2 years 6 monthsConductive, 3 years 6 months6 years 4 monthsPhenylketonuria
      c.2662C>Tp.(Arg888Trp)
      1416c.803A>Gp.(Tyr268Cys)4 years56 years 2 months43 yearsNone58 years 3 monthsRenal disease, diabetes mellitus, hypertension, hyperlipidemia
      c.2596G>Ap.(Glu866Lys)
      ABCC6 (transcript: NM_001171.5) deficiency
      1517c.3940C>Tp.(Arg1314Trp)Prenatally (16 wga)1 dayNone9 yearsCerebrovascular disease (intrauterine stroke), renal disease, neurological disorder
      c.3940C>Tp.(Arg1314Trp)
      1518c.3940C>Tp.(Arg1314Trp)5 days6 daysNone5 years 10 monthsCerebrovascular disease, congestive heart failure
      c.3940C>Tp.(Arg1314Trp)
      1619c.2861_286delp.(Phe954_Leu955del)3 months5 monthsNone3 years 5 monthsCongestive heart failure, developmental delay
      Deletion of exons 2–31
      1720c.1171A>Gp.(Arg391Gly)0 days15 daysNone11 years 9 monthsNone
      ARHR2 autosomal recessive hypophosphatemic rickets type 2, cDNA complementary DNA, GACI generalized arterial calcification of infancy, PXE pseudoxanthoma elasticum, wga weeksʼ gestational age.
      a Novel variants in bold.
      b Type of hearing loss, age of onset specified.
      c Patient presented with diffuse vascular stenosis/fibromuscular dysplasia shortly after birth, confirmed ENPP1 deficiency at 2 years of age.

      Ectopic calcification

      Representative histopathological findings of deceased individuals are shown (Fig. 1a–b). Sixteen of the 20 survivors initially had extensive arterial calcifications, most prominently involving the aorta and other large vessels (Fig. 1c), as well as the coronary arteries (Fig. 1d). The median age at first observation of calcification was 0.5 days, but at the latest NIH evaluation, the arterial calcification was no longer visible on imaging in all but five subjects (Fig. 2); these residual arterial calcifications were of minimal severity. Fifteen of the 16 individuals also exhibited organ calcifications, most frequently in the kidney and cardiac valves.
      Fig. 1
      Fig. 1Clinical presentation of ENPP1 deficiency.
      (a) Histopathology of the aorta of the first brother of patient 6 (deceased at 49 days), showing pronounced thickening of the tunica intima (indicated by white line, both in affected aorta and in the insert depicting a normal aorta) with consequent luminal narrowing, as well as internal elastic lamina distorted by dystrophic calcification (black arrows). (b) Histopathology of the heart of the second brother of patient 6 (deceased at 38 days), revealing deposition of calcium along the internal elastic lamina (disparate elastic fibers with severe degenerative changes separating the media from the intima), accompanied by fibrous thickening of the intima that results in luminal narrowing of the right coronary artery (hematoxylin and eosin [H&E]). (c) Coronal computed tomography (CT) of patient 6 at 4 weeks of life, showing calcification of the distal abdominal aorta and proximal bilateral iliac arteries (yellow arrows). (d) Axial CT scan of patient 6 at 4 weeks of life showing calcification (yellow arrows) of the left main (LM), left anterior descending (LAD) and left circumflex (LCX) coronary arteries. (e) Three-dimensional CT reconstruction of patient 10 at 5 years old revealing bilateral external iliac artery occlusion (yellow arrows) with prominent collaterals.
      Fig. 2
      Fig. 2Calcification of arteries, joints and organs in patients with generalized arterial calcification of infancy (GACI).
      The total length of each bar represents the frequency of calcification in affected patients, while the hatched bars represent the percentage of patients that showed resolution of calcification at last examination.
      Joint calcifications, most often affecting the shoulders (Fig. 3a), hips, and wrists, occurred in half of patients and were first observed at a median age of 4 months (Fig. 2). By the time of the NIH evaluation, the calcifications had resolved in more than half of the involved joints. Cervical spine fusion was present in four survivors; patient 3 had fusions of C3–C4 and C5–C6 during infancy and patient 7 had fused laminae of C3–C6 at 7.6 years. Patient 8 exhibited fusion of cervical vertebral bodies and neural arches at 15 years and was diagnosed with Klippel–Feil syndrome (Fig. 3b). Patient 11 had fused C2–C3 and C4–C5 posterior vertebral bodies and articular processes.
      Fig. 3
      Fig. 3Rickets in ENPP1 deficiency.
      (a) Three-dimensional computed tomography (CT) reconstruction showing periarticular calcification of the shoulder of patient 9 at 4 days of life. (b) Fusion of the C2–C3 and C4–C5 posterior vertebral bodies, articular processes, and laminae (patient 11, 25.5 years). (c) Calcification of the posterior longitudinal ligament enthesis (patient 16, 56.2 years). (d) Metaphyseal irregularities of the lateral distal femora as a result of untreated rickets (patient 13, 6.6 years). (e) Serum phosphate decreased with age; dashed line represents lower limit of normal (Z-score −1.96). (f) Correlation of serum phosphate and intact FGF23 levels. (g) Kaplan–Meier curve showing the probability of remaining free of hypophosphatemic rickets in the subpopulation of patients with generalized arterial calcification of infancy (GACI) due to ENPP1 variants. (h) Neck skin in patient 3 at 8.9 years showing classical findings of pseudoxanthoma elasticum (PXE). (i) Fundus photography in patient 16 at 56 years of age, demonstrating known retinal complications of PXE, i.e., macular hemorrhage (arrows), angioid streaks (carets), and peau d’orange (oval). (j) Kaplan–Meier curve showing the probability of remaining free of hearing loss in the subpopulation of patients with GACI due to ENPP1 variants. ARHR2 autosomal recessive hypophosphatemic rickets type 2.
      Calcification of the tendons or ligaments at contact sites with bones (enthesis) was present in all three adults and was associated with local musculoskeletal pain. The calcification of patient 8 affected the common extensor origin of the right lateral epicondyle at 25 years of age. Patient 11 had calcification of the Achilles tendon, leading to unilateral spontaneous rupture at age 25. Patient 16 had ossification of the posterior longitudinal ligament at vertebrae C2–C3 and C3–C4 (Fig. 3c).
      In general, the 16 individuals with ENPP1 variants did not differ in the frequency or location of ectopic calcification compared with those having ABCC6 variants.

      Rickets/osteomalacia

      Clinical rickets was diagnosed in children based on classic signs such as bowing, gait disturbance, metaphyseal flaring, etc., and/or metaphyseal irregularities on radiographs. In adults, osteomalacia was presumed based on a previous diagnosis of rickets during childhood and/or persistent hypophosphatemia in adulthood. There was no evidence of rickets/osteomalacia in the four patients with ABCC6 deficiency. By contrast, in individuals with ENPP1 deficiency, Kaplan–Meier analysis (Fig. 3g) estimated a 20% probability of developing rickets by 2 years of age, and a 100% probability of developing it by 13.6 years of age. In fact, 11 of the 16 ENPP1-deficient subjects had already developed hypophosphatemic rickets/osteomalacia at the time of NIH evaluation (Fig. 3d). Table 2
      • Lockitch G.
      • Halstead A.C.
      • Albersheim S.
      • MacCallum C.
      • Quigley G.
      Age- and sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer.
      ,
      • Estey M.P.
      • Cohen A.H.
      • Colantonio D.A.
      • et al.
      CLSI-based transference of the CALIPER database of pediatric reference intervals from Abbott to Beckman, Ortho, Roche and Siemens Clinical Chemistry Assays: direct validation using reference samples from the CALIPER cohort.
      ,
      • Stark H.
      • Eisenstein B.
      • Tieder M.
      • Rachmel A.
      • Alpert G.
      Direct measurement of TP/GFR: a simple and reliable parameter of renal phosphate handling.
      summarizes laboratory findings pertaining to mineral balance. Longitudinal blood phosphate data (a total of 131 observations, mean: 11/patient) were available for 12 subjects during childhood prior to onset of treatment. Age significantly influenced serum phosphate. At birth, the mean phosphate Z-score was 0, indicating serum phosphate equal to the normal population mean. However, after birth a sharp decline was observed (mean rate = −1.45 SD per year; 95% confidence interval [CI] = −1.90 to −1.00) that slowed over time (mean change in rate = 0.12 SD per year,
      • Chong C.R.
      • Hutchins G.M.
      Idiopathic infantile arterial calcification: the spectrum of clinical presentations.
      95% CI = 0.06–0.18) with an average onset of hypophosphatemia (serum phosphate Z-score < −1.96) at 1.6 years of age (Fig. 3e). iFGF23 concentrations were frankly elevated (>50 pg/mL) in 14 of 16 patients with ENPP1 deficiency, and were significantly and inversely correlated with blood phosphate (Fig. 3f). The values of 1,25-dihydroxyvitamin D were inappropriately suppressed in patients with elevated iFGF23 concentrations, with a mean value of 57.4 pg/mL as compared with 120.3 pg/mL in those with normal iFGF23 values (two-tailed P value 0.014 by unpaired t test).
      Table 2Biochemical findings of the full cohort.
      FamilyPatientAge at data collectioniFGF23C-FGF23Ionized calciumSerum phosphateAlkaline phosphataseParathyroid hormone25-OH-Vit D1,25-diOH-Vit DTRPTmP/GFR
      Normal values (units):≤50 (pg/mL)≤230 (RU/mL)1.15–1.27 (mmol/L)Age dependent,
      Age-dependent reference range as per Lockitch et al.13
      mmol/L (mg/dL)
      Age dependent,
      Age-dependent reference range as per Estey et al.18
      U/L (μkat/L)
      10–65 ng/L14–60 ng/mL (35–150 nmol/L)25–45 pg/mL (60–108 pmol/L)>85%Age dependent,
      Age-dependent reference range as per Stark et al.19
      mmol/L (mg/dL)
      118 years, 3 months751761.240.71 (2.2)544 (9.1)28.732 (79.9)37 (96.2)85.90.61 (1.89)
      213 years, 2 months901801.190.68 (2.1)301 (5.0)6213 (32.4)61 (158.6)83.90.57 (1.76)
      234 years, 4 months891391.121.10 (3.4)314 (5.2)53.622 (54.9)38 (98.8)85.90.94 (2.91)
      4 years, 10 months
      Calcitriol and phosphate.
      --1.181.07 (3.3)261 (4.4)51.337 (92.4)46 (119.6)82.90.88 (2.72)
      5 years, 11 months
      Calcitriol and phosphate.
      ---0.94 (2.9)302 (5.0)42.332 (79.9)22 (57.2)84.90.75 (2.31)
      6 years, 11 months
      Calcitriol and phosphate.
      -1671.150.97 (3.0)278 (4.6)42.437 (92.4)28 (72.8)86.90.84 (2.60)
      41 years, 10 months
      Ergocalciferol.
      461771.201.49 (4.6)204 (3.4)63.518 (44.9)152 (395.2)90.71.47 (4.56)
      2 years, 4 months
      Ergocalciferol and phosphate.
      --1.251.10 (3.4)191 (3.2)5238 (94.8)37 (96.2)81.80.90 (2.79)
      3 years, 5 months
      Ergocalciferol and phosphate.
      ---1.07 (3.3)322 (5.4)29.540 (99.8)42 (109.2)87.80.94 (2.92)
      4 years, 6 months
      Calcitriol and phosphate.
      -1581.151.10 (3.4)333 (5.6)34.445 (112.3)49 (127.4)82.00.90 (2.79)
      351 year, 3 months731981.301.55 (4.8)317 (5.3)29.836 (89.9)-87.81.37 (4.25)
      2 years, 2 months--1.321.39 (4.3)368 (6.1)31.534 (84.9)75 (195.0)90.61.37 (4.25)
      4 years, 2 months-1501.221.29 (4.0)414 (6.9)65.830 (74.9)32 (83.2)92.91.41 (4.35)
      464 months
      Cholecalciferol.
      391501.301.26 (3.9)392 (6.5)28.2-146 (379.6)93.21.38 (4.28)
      11 months
      Cholecalciferol.
      147--1.10 (3.4)----88.51.00 (3.10)
      1 years, 3 months
      Cholecalciferol.
      1092530.911.13 (3.5)513 (8.6)72.742 (104.8)-83.30.94 (2.91)
      1 years, 8 months
      Cholecalciferol.
      1481981.120.81 (2.5)387 (6.5)4460 (149.8)63 (163.8)72.70.59 (1.82)
      2 years, 1 months
      Cholecalciferol.
      80-0.700.87 (2.7)453 (7.6)42.740 (99.8)96 (249.6)90.00.84 (2.60)
      3 years, 1 month
      Calcitriol and phosphate.
      7088347.381.23 (3.8)293 (4.9)10.733 (82.4)69 (179.4)84.11.03 (3.20)
      4 years, 1 month
      Calcitriol and phosphate.
      -6305.551.07 (3.3)335 (5.6)18.743 (107.3)41 (106.6)66.30.71 (2.19)
      5 years, 1 month
      Calcitriol and phosphate.
      1291841.980.84 (2.6)463 (7.7)29.559 (147.3)18 (46.8)72.70.61 (1.89)
      577 years, 11 months291041.200.90 (2.8)173 (2.9)85.919 (47.4)63 (163.8)88.90.83 (2.58)
      6826 years
      Alfa-calcidol and phosphate.
      1281801.210.74 (2.3)96 (1.6)67.934 (84.9)45 (117.0)57.60.43 (1.32)
      791 years, 8 months
      Cholecalciferol and phosphate.
      924161.200.81 (2.5)505 (8.4)76.938 (94.8)49 (127.4)--
      8105 years, 2 months611251.171.23 (3.8)147 (2.5)36.6--86.01.06 (3.27)
      91121 years
      Calcitriol and phosphate.
      2263391.230.74 (2.3)180 (3.0)11.5-57 (148.2)73.30.55 (1.69)
      25 years
      Calcitriol.
      781241.230.61 (1.9)121 (2.0)30.625 (62.4)-72.10.44 (1.37)
      10122 years, 3 months69561.351.07 (3.3)201 (3.4)7.531 (77.4)135 (351.0)85.60.91 (2.83)
      11136 years, 7 months1164471.281.03 (3.2)349 (5.8)62.618 (44.9)29 (75.4)78.30.81 (2.50)
      8 years, 3 months
      Calcitriol and phosphate.
      --1.231.07 (3.3)322 (5.4)26.323 (57.4)58 (150.8)64.10.68 (2.12)
      8 years, 11 months
      Calcitriol and phosphate.
      1332191.200.84 (2.6)371 (6.2)43.431 (77.4)31 (80.6)63.10.53 (1.64)
      12149 months72217-1.36 (4.2)568 (9.5)42.335 (87.4)65 (169.0)--
      13155 years, 3 months96-1.190.78 (2.4)482 (8.0)40.361 (152.3)38 (98.8)97.71.04 (3.22)
      6 years, 4 months
      Calcitriol and phosphate.
      -1121.220.71 (2.2)511 (8.5)22.262 (154.8)70 (182.0)94.40.82 (2.55)
      7 years, 11 months
      Calcitriol and phosphate.
      -2071.170.90 (2.8)462 (7.7)25.663 (157.2)69 (179.4)--
      1644 years---0.94 (2.9)78 (1.3)-----
      56 years1683291.170.68 (2.1)114 (1.9)61.521 (52.4)77 (200.2)82.30.77 (2.39)
      15178 years501131.211.32 (4.1)91 (1.5)26.736 (89.9)146 (379.6)97.21.74 (5.38)
      185 years, 10 months891231.251.45 (4.5)269 (4.5)28.223 (57.4)116 (301.6)92.21.53 (4.75)
      16192 years, 3 months534201.31.13 (3.5)976 (16.3)-49 (122.3)56 (145.6)88.61.03 (3.19)
      3 years, 5 months601571.31.26 (3.9)275 (4.6)33.243 (107.3)59 (153.4)74.60.94 (2.91)
      17201 year, 3 months---2.07 (6.4)452 (7.5)10.2----
      1 year, 6 months---1.87 (5.8)419 (7.0)12.2----
      1 year, 10 months---1.84 (5.7)327 (5.5)8.827 (67.4)31 (80.6)--
      2 years, 4 months---1.20 (3.7)357 (6.0)15.720 (49.9)57 (148.2)--
      3 years, 7 months---1.42 (4.4)282 (4.7)2357 (142.3)64 (166.4)99.72.10 (6.49)
      5 years---1.29 (4.0)271 (4.5)2242 (104.8)-94.01.47 (4.56)
      6 years, 7 months4193-1.45 (4.5)381 (6.4)37.832 (79.9)-93.81.64 (5.07)
      10 years, 3 months1373-1.26 (3.9)410 (6.8)47.221 (52.4)76 (197.6)--
      12 years, 5 months---1.71 (5.3)529 (8.8)-----
      Bold values were measured while patients were taking medications as noted by superscripts a–g.
      a Calcitriol and phosphate.
      b Ergocalciferol.
      c Ergocalciferol and phosphate.
      d Cholecalciferol.
      e Alfa-calcidol and phosphate.
      f Cholecalciferol and phosphate.
      g Calcitriol.
      h Age-dependent reference range as per Lockitch et al.
      • Lek M.
      • Karczewski K.J.
      • Minikel E.V.
      • et al.
      Analysis of protein-coding genetic variation in 60,706 humans.
      i Age-dependent reference range as per Estey et al.
      • Estey M.P.
      • Cohen A.H.
      • Colantonio D.A.
      • et al.
      CLSI-based transference of the CALIPER database of pediatric reference intervals from Abbott to Beckman, Ortho, Roche and Siemens Clinical Chemistry Assays: direct validation using reference samples from the CALIPER cohort.
      j Age-dependent reference range as per Stark et al.
      • Stark H.
      • Eisenstein B.
      • Tieder M.
      • Rachmel A.
      • Alpert G.
      Direct measurement of TP/GFR: a simple and reliable parameter of renal phosphate handling.

      PXE

      PXE-like complications appeared in four patients with ENPP1 variants. Skin manifestations of PXE (Fig. 3h) were documented in two children and typical retinal findings were observed in two adults (peau d’orange) and one child (peau d’orange, optic nerve head drusen); this child also had PXE-like skin involvement. One patient with ENPP1 deficiency received a diagnosis of PXE at 43 years of age, after presenting with an acute retinal hemorrhage and angioid streaks (Fig. 3i). Signs of PXE were not seen in any of the ABCC6-deficient patients.

      Other complications

      Ten of 16 patients with ENPP1 deficiency manifested hearing loss (7 conductive; 3 mixed) at a median age of 3.7 years. The estimated probability of developing hearing loss was 20% by 2 years of age, 50% by 4 years, and 75% over a lifetime (Fig. 3j). Hearing loss was not observed in any of the ABCC6-deficient patients.
      One subject (patient 10) presented in the neonatal period with cardiac failure due to multiple stenoses within the systemic vasculature, with no evidence of calcification of these vessels on CT. He was initially given a diagnosis of fibromuscular dysplasia (Figs. 1e and S3).
      Hematochezia occurred in 3 of the 20 GACI survivors during the newborn period (patient 13), at 2.5 weeks (patient 4), and at 4 months of age (patient 10).
      Four of 17 families (23%) experienced recurrent pregnancy loss involving 4–6 spontaneous abortions each (Fig. S1). Factor V Leiden was found in one of those families (family 4), but in the other families the etiology remained unexplained.

      Treatment

      Fifteen patients received some form of bisphosphonate; 12 received etidronate, 8 pamidronate, and 1 risedronate. The median age at initiation of bisphosphonate use was 32 days, with ten patients beginning treatment before 2 months of age (in four of those cases, within the first week of life). The mean length of treatment duration was 480 days (range: 60–835 days). Only one of the deceased siblings received bisphosphonates, from birth until the time of death at 1 month of age. Eight patients received oral phosphate supplementation and/or an active form of vitamin D for the management of hypophosphatemia; after at least 7 months of therapy, there was no significant worsening of vascular calcification by CT. The median age at initiation of rickets treatment was 5.5 years (range: 1.5 months–14.6 years). Medications for heart failure were used in 13 patients and antihypertensives were employed for 12 patients.

      Phenotypic variability

      In family 1, patient 2 presented in childhood with bone pain and deformities related to ARHR2, while her younger brother (patient 1) presented at 7 weeks with severe GACI leading to cardiac arrest, resuscitation, and extracorporeal membrane oxygenation. In family 2, patient 3 presented with periarticular calcifications of both shoulders in the absence of vascular calcification, while his younger sister (patient 4) had cardiovascular calcifications diagnosed in utero. In family 15, two siblings had one of the five most common missense variants associated with PXE,
      • Legrand A.
      • Cornez L.
      • Samkari W.
      • et al.
      Mutation spectrum in the ABCC6 gene and genotype–phenotype correlations in a French cohort with pseudoxanthoma elasticum.
      a condition that typically does not present with vascular calcification until adulthood. Despite this, the older brother presented in utero with strokes leading to devastating neurologic sequelae, and the younger sister experienced neonatal stroke with residual contralateral paraparesis.

      Incidence

      Table S1 lists all ENPP1 variants included and excluded from the calculation of the expected incidence of GACI. The allele counts for known and predicted pathogenic variants were 93 and 179, respectively, for a total of 272 over 121,412 alleles. The pathogenic allele frequency is thus 0.224%, or 1 in 446, yielding a carrier frequency of 1 in 223 individuals in the general population and a predicted disease incidence of 1 in 199,244 pregnancies.

      Discussion

      Our comprehensive evaluations of GACI survivors has revealed several significant new findings.

      Rickets

      The development of rickets in survivors of ENPP1-GACI appears universal by age 14 years. The inverse relationship between serum phosphate and iFGF23 and inappropriately normal 1,25-dihydroxyvitamin D suggest that the hypophosphatemia is FGF23-mediated.
      Burosumab is a recently approved human monoclonal antibody against FGF23. Since rickets related to ENPP1 deficiency is FGF23-dependent, at least in principle one might consider the use of burosumab for the treatment of these patients. However, FGF23 suppresses alkaline phosphatase,
      • Erben R.G.
      Physiological actions of fibroblast growth factor-23.
      so that FGF23 inhibition might lead to alkaline phosphatase upregulation, which in turn would cause a further decrease in PPi. Thus, there is a theoretical concern that burosumab use could lead to worsening of ectopic calcifications in ENPP1-deficient patients.

      Ectopic calcification

      The presence or pathologic effects of ectopic calcification in GACI may not be fully appreciated. Because 80% of subjects showed evidence of very early-onset of arterial calcifications, cardiovascular calcification or intimal proliferation during fetal development could be responsible for the high frequency of recurrent pregnancy loss (≥4 miscarriages per family), i.e., 23%, compared with the general population rate of 1–2%.

      Bashiri A, Borick JL. Recurrent pregnancy loss: definitions, epidemiology, and prognosis. In: Bashiri A, Harlev A, Agarwal A, editors. Recurrent pregnancy loss: evidence-based evaluation, diagnosis and treatment. Heidelberg: Springer; 2016. p. 3–18.

      Similarly, the hematochezia seen in 15% of GACI survivors could be related to mesenteric ischemia; the frequency of cow’s milk allergy—the most common cause of hematochezia in infancy—in the general population approximates 2%.
      • Høst A.
      • Halken S.
      A prospective study of cow milk allergy in Danish infants during the first 3 years of life. Clinical course in relation to clinical and immunological type of hypersensitivity reaction.
      Extravascular ectopic calcification also occurred commonly in the joints, organs, and other tissues of GACI survivors. Fusion of the cervical spine, reported only twice previously,
      • Nitschke Y.
      • Baujat G.
      • Botschen U.
      • et al.
      generalized arterial calcification of infancy and pseudoxanthoma elasticum can be caused by mutations in either ENPP1 or ABCC6.
      ,
      • Gopalakrishnan S.
      • Shah S.
      • Apuya J.S.
      • Martin T.
      Anesthetic management of a patient with idiopathic arterial calcification of infancy and fused cervical spine.
      affected 25% of our ENPP1-GACI patients and involved mainly the posterior vertebral bodies and neural arches. Painful calcification of the attachments of tendons or ligaments was present in all three adults in our cohort, and appear to represent a late complication of GACI. Calcifications of the entheses were previously described in a 53-year-old woman
      • Kotwal A.
      • Ferrer A.
      • Kumar R.
      • et al.
      Clinical and biochemical phenotypes in a family with ENPP1 mutations.
      and 62-year-old woman
      • Chen J.
      • Song D.
      • Wang X.
      • Shen X.
      • Li Y.
      • Yuan W.
      Is ossification of posterior longitudinal ligament an enthesopathy?.
      with ARHR2. In X-linked hypophosphatemia (XLH), the most common genetic form of FGF23-mediated hypophosphatemic rickets, calcification of the enthesis also develops with increasing age
      • Polisson R.P.
      • Martinez S.
      • Khoury M.
      • et al.
      Calcification of entheses associated with X-linked hypophosphatemic osteomalacia.
      and impairs quality of life.
      • Che H.
      • Roux C.
      • Etcheto A.
      • et al.
      Impaired quality of life in adults with X-linked hypophosphatemia and skeletal symptoms.
      The pathophysiology of enthesis calcification remains speculative, but it is likely directly related to FGF23 since it is present in other forms of FGF23-mediated hypophosphatemia
      • Karaplis A.C.
      • Bai X.
      • Falet J.-P.
      • Macica C.M.
      Mineralizing enthesopathy is a common feature of renal phosphate-wasting disorders attributed to FGF23 and is exacerbated by standard therapy in hyp mice.
      but absent from patients with SLC34A3 variants, a genetic form of hypophosphatemic rickets not mediated by FGF23.
      • Chen A.
      • Ro H.
      • Mundra V.R.R.
      • et al.
      Description of 5 novel SLC34A3/NPT2c mutations causing hereditary hypophosphatemic rickets with hypercalciuria.
      A mouse model of ENPP1 deficiency recapitulates the phenotype of calcification of fibrocartilage (present in entheses), tendons, and ligaments.
      • Zhang J.
      • Dyment N.A.
      • Rowe D.W.
      • et al.
      Ectopic mineralization of cartilage and collagen-rich tendons and ligaments in Enpp1asj-2J mice.
      Even the hearing loss of GACI might be attributable to calcification. A mouse model of ENPP1 deficiency develops progressive conductive hearing loss, with otitis media, aseptic effusion, fusion of malleus and incus, and thickening and overcalcification of the stapedial artery.
      • Tian C.
      • Harris B.S.
      • Johnson K.R.
      Ectopic mineralization and conductive hearing loss in Enpp1asj mutant mice, a new model for otitis media and tympanosclerosis.
      Although hearing loss was previously described in only four of more than 50 patients with confirmed ENPP1 deficiency,
      • Brachet C.
      • Mansbach A.L.
      • Clerckx A.
      • Deltenre P.
      • Heinrichs C.
      Hearing loss is part of the clinical picture of ENPP1 loss of function mutation.
      the majority of our ENPP1-GACI patients exhibited hearing loss. Similar to the mouse model, our patients’ hearing loss appeared to be progressive, since most patients passed a newborn hearing screen. Thus, we recommend audiologic assessment on an annual basis, as decreased hearing, when unaddressed, can affect school performance.

      PXE-like changes

      While it is widely recognized that individuals with biallelic ABCC6 pathogenic variants can also manifest GACI, we found that patients with biallelic ENPP1 variants can present with findings of PXE after surviving infancy. Angioid streaks, a typical retinal finding of PXE, were previously described in a 5-year-old child with ENPP1 deficiency,
      • Nitschke Y.
      • Baujat G.
      • Botschen U.
      • et al.
      generalized arterial calcification of infancy and pseudoxanthoma elasticum can be caused by mutations in either ENPP1 or ABCC6.
      but one of our patients developed retinal hemorrhage with subsequent macular scarring and legal blindness. The shared phenotypes associated with ENPP1 and ABCC6 variants suggest that modifying genes are in play.

      Incidence

      We estimate the incidence of ENPP1-GACI, based upon the frequency of pathogenic variants, as ~1 in 200,000. The disorder might be more common than previously thought and calls for greater recognition by obstetricians and neonatologists. The database we utilized accounts for diverse populations, including 55% non-Finnish European individuals, 13.6% South Asians, 9.5% individuals of Latino descent, 8.6% individuals of African or African American descent, 7.1% East Asians, and 5.4% persons of Finnish heritage;
      • Lek M.
      • Karczewski K.J.
      • Minikel E.V.
      • et al.
      Analysis of protein-coding genetic variation in 60,706 humans.
      notwithstanding, the incidence of GACI will vary depending on the specific population studied.

      Genotype–phenotype correlation

      Comparison of clinical and molecular findings in our cohort suggests significant phenotypic heterogeneity, even among siblings with identical genotypes. Five siblings of the 20 individuals who survived GACI as infants had severe enough disease to be fatal; four of the five deceased patients were born prior to the birth of the surviving sibling, which makes it possible that subsequent siblings survived due to earlier recognition and management of their disease. Despite this, surviving sibling pairs manifested substantial differences in the severity of their disease, arguing strongly against a genotype–phenotype correlation in GACI.

      Unresolved issues

      Despite the new understanding of some features of GACI, several issues remain unresolved. Is there a pathophysiological link between the resolution of ectopic calcification in GACI and the later development of hypophosphatemic rickets? It is known that PPi inhibits mineralization and inorganic phosphate (Pi) stimulates it. ENPP1 deficiency reduces PPi, and this deficit is exacerbated during the second half of pregnancy by increases in placental alkaline phosphatase, which cleaves PPi to Pi;
      • Whyte M.P.
      • Landt M.
      • Ryan L.M.
      • et al.
      Alkaline phosphatase: placental and tissue-nonspecific isoenzymes hydrolyze phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5’-phosphate. Substrate accumulation in carriers of hypophosphatasia corrects during pregnancy.
      this drastically increases the Pi/PPi ratio in utero. After birth, however, two compensatory mechanisms ensue. First, the overriding influence of placental alkaline phosphatase vanishes, allowing an increase in PPi. Second, renal glomerular function matures postnatally, increasing Pi clearance.
      • Bistarakis L.
      • Voskaki I.
      • Lambadaridis J.
      • Sereti H.
      • Sbyrakis S.
      Renal handling of phosphate in the first six months of life.
      This may promote resolution of vascular calcification and possibly contribute to survival in some. Later, excess FGF23 production further increases phosphate excretion, causing hypophosphatemia and rickets. However, the exact mechanism by which ENPP1 induces FGF23 excess, which may be beneficial to patients, remains unknown.
      This study also highlights the controversy regarding therapy of ARHR2. It is reasonable to be concerned that treating GACI survivors with calcitriol and phosphorus might induce progression or recurrence of vascular calcification. In addition, phosphorus supplementation could lead to even higher concentrations of FGF23, an independent cardiovascular risk factor.
      • Stöhr R.
      • Schuh A.
      • Heine G.H.
      • Brandenburg V.
      FGF23 in cardiovascular disease: innocent bystander or active mediator?.
      Moreover, although standard therapy of hypophosphatemic rickets apparently does not worsen the enthesopathy in XLH patients,
      • Connor J.
      • Olear E.A.
      • Insogna K.L.
      • et al.
      Conventional therapy in adults with X-linked hypophosphatemia: effects on enthesopathy and dental disease.
      it can be associated with hyperparathyroidism
      • Schmitt C.P.
      • Mehls O.
      The enigma of hyperparathyroidism in hypophosphatemic rickets.
      and exacerbate nephrocalcinosis.
      • Verge C.F.
      • Lam A.
      • Simpson J.M.
      • Cowell C.T.
      • Howard N.J.
      • Silink M.
      Effects of therapy in X-linked hypophosphatemic rickets.
      Consequently, some GACI survivors remain untreated and experience bone pain, deformities, and short stature from their rickets. Hence, we previously reported one subject (patient 11 in the current cohort) in whom vascular calcification did not recur after years of vitamin D and phosphate treatment for ARHR2,
      • Ferreira C.R.
      • Ziegler S.G.
      • Gupta A.
      • Groden C.
      • Hsu K.S.
      • Gahl W.A.
      Treatment of hypophosphatemic rickets in generalized arterial calcification of infancy (GACI) without worsening of vascular calcification.
      and our current cohort includes another seven individuals who received judicious treatment of rickets without noticeable worsening of vascular calcification.
      Another GACI mystery involves the relationship between ENPP1 and ABCC6 deficiencies, since the phenotypes of these two disorders overlap. Biochemically, ENPP1 deficiency results in reduced PPi and AMP, and the common occurrence of vascular calcification in ENPP1-GACI and in PXE has led to the interpretation that reduced PPi levels are responsible for the pathology of both diseases. However, AMP deficiency could be the pivotal parameter shared by ENPP1-GACI and PXE,
      • Ziegler S.G.
      • Ferreira C.R.
      • MacFarlane E.G.
      • et al.
      Ectopic calcification in pseudoxanthoma elasticum responds to inhibition of tissue-nonspecific alkaline phosphatase.
      since AMP has protean biochemical effects. Specifically, intimal proliferation results from AMP or adenosine deficiency
      • Nitschke Y.
      • Yan Y.
      • Buers I.
      • Kintziger K.
      • Askew K.
      • Rutsch F.
      ENPP1-Fc prevents neointima formation in generalized arterial calcification of infancy through the generation of AMP.
      and comprises part of the ENPP1-GACI phenotype. In fact, one of our patients (patient 10) with ENPP1 deficiency did not develop any vascular calcification, but did manifest multivessel narrowing and was diagnosed with pediatric fibromuscular dysplasia (FMD). Intimal fibroplasia is common in FMD, and affected individuals might benefit from ENPP1 sequencing.

      Strengths and limitations

      The strengths of our study include the size of the cohort, its molecular characterization, and the uniform and comprehensive evaluation of patients in a prospective fashion. Nevertheless, we were unable to address whether bisphosphonates are helpful in GACI, since we evaluated only survivors of the disease. However, 5 of the 20 survivors did not receive bisphosphonates, suggesting that survival of bisphosphonate-treated patients may not be due to the treatment.

      Conclusion

      With a predicted incidence of 1 in 200,000 pregnancies, there should be 20 new cases of GACI every year in the United States alone. As new therapies are developed to treat GACI and ARHR2, additional prospective studies to elucidate the natural history of these disorders will help identify drug targets and outcome parameters for clinical trials.

      Ethics declarations

      Disclosure

      C.R.F., R.I.G., W.A.G., and M.E.H. report a collaboration with Inozyme Pharma as part of a Cooperative Research and Development Agreement (CRADA). Inozyme is developing ENPP1 as therapy for ARHR2 and GACI. S.W. and K.M. are employees of ICON plc, a contract research organization. The other authors declare no conflicts of interest.

      Acknowledgements

      We thank the patients and their families for their kind cooperation. This work was supported by the Intramural Research Program of the National Human Genome Research Institute and the National Institute of Dental and Craniofacial Research.

      Additional information

      Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

      Supplementary information

      References

        • Rutsch F.
        • Böyer P.
        • Nitschke Y.
        • et al.
        Hypophosphatemia, hyperphosphaturia, and bisphosphonate treatment are associated with survival beyond infancy in generalized arterial calcification of infancy.
        1:CAS:528:DC%2BD1MXhtV2is7fJ
        10.1161/CIRCGENETICS.108.797704
        Circ Cardiovasc Genet. 2008; 1: 133-140
        • Chong C.R.
        • Hutchins G.M.
        Idiopathic infantile arterial calcification: the spectrum of clinical presentations.
        10.2350/07-06-0297.1
        Pediatr Dev Pathol. 2008; 11: 405-415
        • Nitschke Y.
        • Baujat G.
        • Botschen U.
        • et al.
        generalized arterial calcification of infancy and pseudoxanthoma elasticum can be caused by mutations in either ENPP1 or ABCC6.
        1:CAS:528:DC%2BC38XovFGgsg%3D%3D
        10.1016/j.ajhg.2011.11.020
        Am J Hum Genet. 2012; 90: 25-39
        • Jansen R.S.
        • Küçükosmanoglu A.
        • de Haas M.
        • et al.
        ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release.
        1:CAS:528:DC%2BC3sXhvFKmsL%2FI
        10.1073/pnas.1319582110
        Proc Natl Acad Sci U S A. 2013; 110: 20206-20211
        • Ringpfeil F.
        • Lebwohl M.G.
        • Christiano A.M.
        • Uitto J.
        Pseudoxanthoma elasticum: mutations in the MRP6 gene encoding a transmembrane ATP-binding cassette (ABC) transporter.
        1:CAS:528:DC%2BD3cXjvFars7k%3D
        10.1073/pnas.100041297
        Proc Natl Acad Sci U S A. 2000; 97: 6001-6006
        • Jansen R.S.
        • Duijst S.
        • Mahakena S.
        • et al.
        ABCC6-mediated ATP secretion by the liver is the main source of the mineralization inhibitor inorganic pyrophosphate in the systemic circulation-brief report.
        1:CAS:528:DC%2BC2cXhtl2hsb%2FN
        10.1161/ATVBAHA.114.304017
        Arterioscler Thromb Vasc Biol. 2014; 34: 1985-1989
        • Lorenz-Depiereux B.
        • Schnabel D.
        • Tiosano D.
        • Häusler G.
        • Strom T.M.
        Loss-of-function ENPP1 mutations cause both generalized arterial calcification of infancy and autosomal-recessive hypophosphatemic rickets.
        1:CAS:528:DC%2BC3cXlt1Kmu7Y%3D
        10.1016/j.ajhg.2010.01.006
        Am J Hum Genet. 2010; 86: 267-272
        • Levy-Litan V.
        • Hershkovitz E.
        • Avizov L.
        • et al.
        Autosomal-recessive hypophosphatemic rickets is associated with an inactivation mutation in the ENPP1 gene.
        1:CAS:528:DC%2BC3cXlt1Kmu7c%3D
        10.1016/j.ajhg.2010.01.010
        Am J Hum Genet. 2010; 86: 273-278
        • Brachet C.
        • Mansbach A.L.
        • Clerckx A.
        • Deltenre P.
        • Heinrichs C.
        Hearing loss is part of the clinical picture of ENPP1 loss of function mutation.
        1:CAS:528:DC%2BC2cXksFyjtrs%3D
        10.1159/000354661
        Horm Res Paediatr. 2014; 81: 63-66
        • Le Boulanger G.
        • Labrèze C.
        • Croué A.
        • et al.
        An unusual severe vascular case of pseudoxanthoma elasticum presenting as generalized arterial calcification of infancy.
        10.1002/ajmg.a.33162
        Am J Med Genet A. 2010; 152A: 118-123
        • Gopalakrishnan S.
        • Shah S.
        • Apuya J.S.
        • Martin T.
        Anesthetic management of a patient with idiopathic arterial calcification of infancy and fused cervical spine.
        10.1111/j.1460-9592.2008.02585.x
        Paediatr Anaesth. 2008; 18: 1006-1007
        • Lockitch G.
        • Halstead A.C.
        • Albersheim S.
        • MacCallum C.
        • Quigley G.
        Age- and sex-specific pediatric reference intervals for biochemistry analytes as measured with the Ektachem-700 analyzer.
        1:CAS:528:DyaL1cXlsVGkurk%3D
        10.1093/clinchem/34.8.1622
        Clin Chem. 1988; 34: 1622-1625
        • Lek M.
        • Karczewski K.J.
        • Minikel E.V.
        • et al.
        Analysis of protein-coding genetic variation in 60,706 humans.
        1:CAS:528:DC%2BC28XhtlOnsbbP
        10.1038/nature19057
        Nature. 2016; 536: 285-291
        • Chourabi M.
        • Liew M.S.
        • Lim S.
        • et al.
        ENPP1 mutation causes recessive Cole disease by altering melanogenesis.
        1:CAS:528:DC%2BC1cXkslOhsQ%3D%3D
        10.1016/j.jid.2017.08.045
        J Invest Dermatol. 2018; 138: 291-300
        • Adzhubei I.A.
        • Schmidt S.
        • Peshkin L.
        • et al.
        A method and server for predicting damaging missense mutations.
        1:CAS:528:DC%2BC3cXjvFKqu78%3D
        10.1038/nmeth0410-248
        Nat Methods. 2010; 7: 248-249
        • Kumar P.
        • Henikoff S.
        • Ng P.C.
        Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.
        1:CAS:528:DC%2BD1MXovVyns78%3D
        10.1038/nprot.2009.86
        Nat Protoc. 2009; 4: 1073-1081
        • Kircher M.
        • Witten D.M.
        • Jain P.
        • O’Roak B.J.
        • Cooper G.M.
        • Shendure J.
        A general framework for estimating the relative pathogenicity of human genetic variants.
        1:CAS:528:DC%2BC2cXhs1Sjt7g%3D
        10.1038/ng.2892
        Nat Genet. 2014; 46: 310-315
        • Estey M.P.
        • Cohen A.H.
        • Colantonio D.A.
        • et al.
        CLSI-based transference of the CALIPER database of pediatric reference intervals from Abbott to Beckman, Ortho, Roche and Siemens Clinical Chemistry Assays: direct validation using reference samples from the CALIPER cohort.
        10.1016/j.clinbiochem.2013.04.001
        Clin Biochem. 2013; 46: 1197-1219
        • Stark H.
        • Eisenstein B.
        • Tieder M.
        • Rachmel A.
        • Alpert G.
        Direct measurement of TP/GFR: a simple and reliable parameter of renal phosphate handling.
        1:STN:280:DyaL2s%2FjvFGnuw%3D%3D
        10.1159/000184216
        Nephron. 1986; 44: 125-128
        • Legrand A.
        • Cornez L.
        • Samkari W.
        • et al.
        Mutation spectrum in the ABCC6 gene and genotype–phenotype correlations in a French cohort with pseudoxanthoma elasticum.
        1:CAS:528:DC%2BC2sXht1yit77O
        10.1038/gim.2016.213
        Genet Med. 2017; 19: 909-917
        • Erben R.G.
        Physiological actions of fibroblast growth factor-23.
        10.3389/fendo.2018.00267
        Front Endocrinol (Lausanne). 2018; 9: 267
      1. Bashiri A, Borick JL. Recurrent pregnancy loss: definitions, epidemiology, and prognosis. In: Bashiri A, Harlev A, Agarwal A, editors. Recurrent pregnancy loss: evidence-based evaluation, diagnosis and treatment. Heidelberg: Springer; 2016. p. 3–18.

        • Høst A.
        • Halken S.
        A prospective study of cow milk allergy in Danish infants during the first 3 years of life. Clinical course in relation to clinical and immunological type of hypersensitivity reaction.
        10.1111/j.1398-9995.1990.tb00944.x
        Allergy. 1990; 45: 587-596
        • Kotwal A.
        • Ferrer A.
        • Kumar R.
        • et al.
        Clinical and biochemical phenotypes in a family with ENPP1 mutations.
        1:CAS:528:DC%2BB3cXms1Smt7w%3D
        10.1002/jbmr.3938
        J Bone Miner Res. 2020; 35: 662-670
        • Chen J.
        • Song D.
        • Wang X.
        • Shen X.
        • Li Y.
        • Yuan W.
        Is ossification of posterior longitudinal ligament an enthesopathy?.
        10.1007/s00264-010-1163-9
        Int Orthop. 2011; 35: 1511-1516
        • Polisson R.P.
        • Martinez S.
        • Khoury M.
        • et al.
        Calcification of entheses associated with X-linked hypophosphatemic osteomalacia.
        1:STN:280:DyaL2M3htlOntA%3D%3D
        10.1056/NEJM198507043130101
        N Engl J Med. 1985; 313: 1-6
        • Che H.
        • Roux C.
        • Etcheto A.
        • et al.
        Impaired quality of life in adults with X-linked hypophosphatemia and skeletal symptoms.
        1:CAS:528:DC%2BC28Xntlantrs%3D
        10.1530/EJE-15-0661
        Eur J Endocrinol. 2016; 174: 325-333
        • Karaplis A.C.
        • Bai X.
        • Falet J.-P.
        • Macica C.M.
        Mineralizing enthesopathy is a common feature of renal phosphate-wasting disorders attributed to FGF23 and is exacerbated by standard therapy in hyp mice.
        1:CAS:528:DC%2BC38XhvVahtbjJ
        10.1210/en.2012-1551
        Endocrinology. 2012; 153: 5906-5917
        • Chen A.
        • Ro H.
        • Mundra V.R.R.
        • et al.
        Description of 5 novel SLC34A3/NPT2c mutations causing hereditary hypophosphatemic rickets with hypercalciuria.
        10.1016/j.ekir.2019.05.004
        Kidney Int Rep. 2019; 4: 1179-1186
        • Zhang J.
        • Dyment N.A.
        • Rowe D.W.
        • et al.
        Ectopic mineralization of cartilage and collagen-rich tendons and ligaments in Enpp1asj-2J mice.
        10.18632/oncotarget.7455
        Oncotarget. 2016; 7: 12000-12009
        • Tian C.
        • Harris B.S.
        • Johnson K.R.
        Ectopic mineralization and conductive hearing loss in Enpp1asj mutant mice, a new model for otitis media and tympanosclerosis.
        10.1371/journal.pone.0168159
        PLoS One. 2016; 11: e0168159
        • Whyte M.P.
        • Landt M.
        • Ryan L.M.
        • et al.
        Alkaline phosphatase: placental and tissue-nonspecific isoenzymes hydrolyze phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5’-phosphate. Substrate accumulation in carriers of hypophosphatasia corrects during pregnancy.
        1:CAS:528:DyaK2MXkslyksLc%3D
        10.1172/JCI117814
        J Clin Invest. 1995; 95: 1440-1445
        • Bistarakis L.
        • Voskaki I.
        • Lambadaridis J.
        • Sereti H.
        • Sbyrakis S.
        Renal handling of phosphate in the first six months of life.
        1:CAS:528:DyaL28XltlWiurk%3D
        10.1136/adc.61.7.677
        Arch Dis Child. 1986; 61: 677-681
        • Stöhr R.
        • Schuh A.
        • Heine G.H.
        • Brandenburg V.
        FGF23 in cardiovascular disease: innocent bystander or active mediator?.
        10.3389/fendo.2018.00351
        Front Endocrinol (Lausanne). 2018; 9: 351
        • Connor J.
        • Olear E.A.
        • Insogna K.L.
        • et al.
        Conventional therapy in adults with X-linked hypophosphatemia: effects on enthesopathy and dental disease.
        1:CAS:528:DC%2BC28Xjt1Ggt7s%3D
        10.1210/JC.2015-2199
        J Clin Endocrinol Metab. 2015; 100: 3625-3632
        • Schmitt C.P.
        • Mehls O.
        The enigma of hyperparathyroidism in hypophosphatemic rickets.
        10.1007/s00467-004-1443-y
        Pediatr Nephrol. 2004; 19: 473-477
        • Verge C.F.
        • Lam A.
        • Simpson J.M.
        • Cowell C.T.
        • Howard N.J.
        • Silink M.
        Effects of therapy in X-linked hypophosphatemic rickets.
        1:STN:280:DyaK38%2Fnt1Kgtg%3D%3D
        10.1056/NEJM199112263252604
        N Engl J Med. 1991; 325: 1843-1848
        • Ferreira C.R.
        • Ziegler S.G.
        • Gupta A.
        • Groden C.
        • Hsu K.S.
        • Gahl W.A.
        Treatment of hypophosphatemic rickets in generalized arterial calcification of infancy (GACI) without worsening of vascular calcification.
        10.1002/ajmg.a.37574
        Am J Med Genet A. 2016; 170A: 1308-1311
        • Ziegler S.G.
        • Ferreira C.R.
        • MacFarlane E.G.
        • et al.
        Ectopic calcification in pseudoxanthoma elasticum responds to inhibition of tissue-nonspecific alkaline phosphatase.
        10.1126/scitranslmed.aal1669
        Sci Transl Med. 2017; 9: eaal1669.
        • Nitschke Y.
        • Yan Y.
        • Buers I.
        • Kintziger K.
        • Askew K.
        • Rutsch F.
        ENPP1-Fc prevents neointima formation in generalized arterial calcification of infancy through the generation of AMP.
        10.1038/s12276-018-0163-5
        Exp Mol Med. 2018; 50