Arterial Calcification Of Infancy Research Articles

Generalized Arterial Calcification of Infancy (GACI)

Generalized arterial calcification of infancy (GACI) is an ultra-rare autosomal recessive disorder associated with pathogenic variants in ENPP1, the major gene involved in this condition, and in ABCC6, which is involved in a small fraction of affected individuals. Loss-of-function pathogenic variants of ENPP1 and ABCC6 lead to perturbations in the PPi/Pi ratio, thereby promoting hydroxyapatite mineralization in peripheral tissues. GACI is initially characterized by an abnormal ectopic mineralization process in arteries and soft tissue. Nearly half of the patients die within the first 6 months of life from cardiovascular complications, hence the poor prognosis associated with an early diagnosis. In recent years, progress has been made in our understanding of the long-term natural history of GACI, the intricate symptoms due to vascular calcifications, the overmineralization of soft tissues, of hypophosphatemia designated as ARHR2, and of the consequences such as undermineralization of the skeleton, but also of the features possibly seen in pseudoxanthoma elasticum (PXE). Indeed, GACI, PXE, and ARHR2 share common pathophysiological pathways and clinical features beyond the vascular calcifications. Treatment options for severe forms of GACI are mostly based on symptomatic management, including the option of starting bisphosphonates early after birth, such as etidronate and pamidronate, analogues of PPi. Follow-up within an expert and coordinated multidisciplinary team includes treatment of arterial hypertension, calcitriol and phosphorus adjustments, hearing aids, and early detection of possible angioid streaks. It is hoped that ongoing basic and clinical research will lead to the development of effective therapies that specifically target the abnormal PPi regulation and the other mechanisms involved in this disorder.

Autosomal recessive hypophosphatemic rickets type 2 due to ENPP1 deficiency (ARHR2)

Autosomal recessive hypophosphatemic rickets type 2 (ARHR2; MIM #613312) is a very rare disorder caused by biallelic loss-of-function mutations in the ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) gene. ENPP1 deficiency encompasses a spectrum of phenotypes that includes, in addition to ARHR2, generalized arterial calcification of infancy (GACI), ossification of the posterior longitudinal ligament (OPLL), and pseudoxanthoma elasticum. ARHR2 can be found in GACI survivors, but it may also be the first manifestation of ENPP1 deficiency. Although the precise mechanisms are not fully elucidated, patients with GACI and ARHR2 have elevated serum FGF23 levels, leading to renal phosphate wasting and hypophosphatemia. As a result, the clinical and radiological phenotype of ARHR2 patients is very similar to that of patients affected with other forms of hypophosphatemic rickets, such as X-linked hypophosphatemia. Patients show signs of rickets (abnormal mineralization of growth plates in children) and osteomalacia (abnormal bone mineralization in children and adults) of varying severity. Clinical manifestations specific to ENPP1 loss-of-function mutations and common to GACI, such as ectopic calcifications (valvular, arterial, or periarticular), deafness, OPLL, and PXE, may also be found. Genetic confirmation of the disease is important so as to ensure that patients receive the appropriate treatment or have the opportunity to participate in clinical trials to evaluate the safety and efficacy of novel and promising recombinant enzyme therapies.

Biology of bone mineralization and ectopic calcifications: the same actors for different plays

Bone has several crucial functions. It is essential for locomotion and allows our body to stand erect against gravity. A mismatch between the mechanical stresses applied to it and its mechanical resistance leads to fractures. Bone also has numerous endocrine functions. It acts as a reservoir for minerals such as calcium and phosphorus, making it the target of calciotropic hormones that mobilize these minerals, particularly calcium, according to the body's needs. Additionally, bone secretes hormones, notably fibroblast growth factor 23 (FGF23), which regulates urinary excretion of phosphate and the bioavailability of active vitamin D. Bone mineralization is the process that facilitates the organized deposition of minerals in the bone matrix, providing rigidity and appropriate mechanical resistance. This process is compromised in genetically related bone mineralization disorders, such as those causing hypophosphatemia or hypophosphatasia. Conversely, calcification can be pathological, affecting soft tissues like the blood vessels, as seen in generalized arterial calcification of infancy (GACI) or arterial calcification due to CD73 deficiency (ACDC). The aim of this article is to first present the composition and structure of the mineralized bone matrix, to review the current understanding of the molecular mechanisms of mineralization, and finally to discuss the conditions associated with ectopic calcification and the underlying mechanisms.

Clinical presentation and burden of ENPP1 deficiency in adults

While the clinical consequences of severe ENPP1 deficiency leading to the rare disorders generalized arterial calcification of infancy (GACI) and autosomal recessive hypophosphatemic rickets type 2 (ARHR2) are well defined and understood, much less is known about how this evolves into adulthood and how moderate ENPP1 deficiency can first manifest in adulthood. Moreover, growing evidence substantiates an association of genetic variants in the ENPP1 gene with a wide range of further clinical manifestations including early-onset osteoporosis, osteoarthritis, and different forms of spinal ligament calcifications, i.e., diffuse idiopathic skeletal hyperostosis (DISH) and ossification of the posterior/anterior longitudinal ligament (OPLL/OALL). Furthermore, conditions with primarily extraskeletal signs and symptoms such as Cole disease, coagulopathies, and metabolic syndrome can seemingly result from ENPP1 variants. The causality and the pathophysiology behind these different clinical presentations appear complex and require further research, especially since the coincidence of these different phenotypes is rarely described and available evidence suggests that part of the aforementioned manifestations may result from ENPP1 effects beyond the catalytic activity of processing ATP to AMP and inorganic pyrophosphate (PPi). Growing awareness of the additional ENPP1-related manifestations across the lifespan will advance our understanding of this complex condition and help to standardize diagnostic approaches and develop individually tailored treatment concepts.

Inhibition of Vascular Smooth Muscle Cell Proliferation by ENPP1: The Role of CD73 and the Adenosine Signaling Axis.

The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.

Genetic analysis of a child with Generalized arterial calcification of infancy due to variant of ABCC6 gene

To explore the clinical manifestations and genetic basis for a rare case of Generalized arterial calcification of infancy (GACI). A 44-day-old female infant who was treated at Baoding Hospital of Beijing Children's Hospital Affiliated to Capital Medical University on August 26, 2022 was selected as the study subject. Clinical data of the child was collected, and Trio-whole exome sequencing (Trio-WES), whole genome copy number variation sequencing (CNV-seq) and minigene splicing assay were carried out to analyze the pathogenicity of the variants. The child had presented with fever and high inflammatory indicators, for which treatment with various antibiotics was ineffective. Ultrasound had revealed extensive arterial calcification and arterial wall thickening. The child was suspected for GACI with arteritis related to the primary disease. Her fever was relieved by treatment with glucocorticoid and biological agents. Trio-WES revealed that she has harbored compound heterozygous variants of the ABCC6 gene, namely c.4404-1G>A and c.4041+5G>T, for which the latter was unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics, the variants were classified as likely pathogenic (PVS1+PM2_Supporting) and variant of unknown significance (PM2_Supporting+PM3+PP3), respectively. The result of CNV-seq was negative. And the minigene splicing assay has further verified that both variants can result in alternative splicing. For pyrexia with unknown causes and refractory to conventional treatment, it is necessary to recommend early genetic testing to avoid missed diagnosis of GACI.

Natural history of ENPP1 deficiency: Nationwide Turkish Cohort Study of autosomal-recessive hypophosphataemic rickets type 2.

Autosomal-recessive hypophosphataemic rickets type 2 (ARHR2) is a rare disease that is reported in survivors of generalized arterial calcification of infancy (GACI). The objective of this study was to characterize a multicenter paediatric cohort with ARHR2 due to ectonucleotide pyrophosphatase/phosphodiesterase family member 1(ENPP1) deficiencyand with a diagnosis of GACI or GACI-related findings. The clinical, biochemicaland genetic characteristics of the patients were retrospectively retrieved. We identified 18 patients from 13 families diagnosed with ARHR2. Fifteen of the patients had an ENPP1 variation confirmed with genetic analyses, and three were siblings of one of these patients, who had clinically diagnosed hypophosphataemic rickets (HRs) with the same presentation. From nine centres, 18 patients, of whom 12 (66.7%) were females, were included in the study. The mean age at diagnosis was 4.2 ± 2.2 (1.6-9) years. The most frequently reported clinical findings on admission were limb deformities (66.6%) and short stature (44.4%). At diagnosis, the mean height SD was -2.2 ± 1.3. Five of the patients were diagnosed with GACI in the neonatal period and treated with bisphosphonates. Other patients were initially diagnosed with ARHR2, but after the detection of a biallelic variant in the ENPP1 gene, it was understood that they previously had clinical findings associated with GACI. Three patients had hearing loss, and two had cervical fusion. After the treatment of HRs, one patient developed calcification, and one developed intimal proliferation. ARHR2 represents one manifestation of ENPP1 deficiency that usually manifests later in life than GACI. The history of calcifications or comorbidities that might be associated with GACI will facilitate the diagnosis in patients with ARHR2, and patients receiving calcitriol and phosphate medication should be carefully monitored for signs of calcification or intimal proliferation.

Bone Marrow–Derived ABCC6 Is an Essential Regulator of Ectopic Calcification In Pseudoxanthoma Elasticum

Physiological calcification of soft tissues is a common occurrence in aging and various acquired and inherited disorders. ABCC6 sequence variations cause the calcification phenotype of pseudoxanthoma elasticum (PXE) as well as some cases of generalized arterial calcification of infancy, which is otherwise caused by defective ENPP1. ABCC6 is primarily expressed in the liver, which has given the impression that the liver is central to the pathophysiology of PXE/generalized arterial calcification of infancy. The emergence of inflammation as a contributor to the calcification in PXE suggested that peripheral tissues play a larger role than expected. In this study, we investigated whether bone marrow-derived ABCC6 contributes to the calcification in PXE. In Abcc6‒/‒ mice, we observed prevalent mineralization in several lymph nodes and surrounding connective tissues and an extensive network of lymphatic vessels within vibrissae, a calcified tissue in Abcc6‒/‒ mice. Furthermore, we found evidence of lymphangiogenesis in patients with PXE and mouse skin, suggesting an inflammatory process. Finally, restoring wild-type bone marrow in Abcc6‒/‒ mice produced a significant reduction of calcification, suggesting that the liver alone is not sufficient to fully inhibit mineralization. With evidence that ABCC6 is expressed in lymphocytes, wesuggest that the adaptative immune system and inflammation largely contribute to the calcification in PXE/generalized arterial calcification of infancy.

Clinical and Molecular Characterization of a Patient with Generalized Arterial Calcification of Infancy Caused by Rare ABCC6 Mutation.

Generalized arterial calcification of infancy (GACI) is a rare autosomal-recessive disease characterized by extensive arterial calcification in infancy, with clinical manifestations such as arterial stenoses and heart failure. The ENPP1 inactivation mutation has been identified as a potential defect in most of the cases of GACI, while mutations in ABCC6 are demonstrated in patients who are genotyped as pseudoxanthoma elasticum and only limited cases of GACI are reported. Whole-exome sequencing was applied for the detection of pathogenic variants. Copy-number variants of pathogenic genes were also evaluated through a bioinformatic process and were further validated by real-time quantitative PCR. In this report, we described the clinical information and treatment of a patient with extensive arterial calcification. We have identified the underlying cause as biallelic mutations in ABCC6 (NM_00117: exon30, c.4223_4227dupAGCTC p.(Leu1410Serfs*56)) and a unique exonic deletion that spans from the first to the fourth exons of ABCC6 (chr16:16313388-16330869)). This discovery was made by utilizing a combined genetic testing approach. With the review of previously reported GACI patients with ABCC6 mutation, our work contributed to enriching the mutation spectrum of GACI and providing further information on this rare form of inherited disorder.

Abstract #1399545: Calcification of Left Atrial Appendage in Autosomal Recessive Hypophosphatemic Rickets Type 2 (ENPP1 Mutation)

Autosomal Recessive Hypophosphatemic Rickets type 2 (ARHR2) is a rare genetic condition secondary to a loss-of-function mutation in ENPP1 gene. ARHR2 is associated with renal phosphate wasting and suppressed 1,25-dihydroxyvitamin D due to increased FGF-23 levels and causing abnormal bone mineralization. Furthermore, as ENPP1 is implicated in the generation of PPi (inorganic pyrophosphate) which inhibits hydroxyapatite deposition, some ARHR2 patients present with life-treating generalized arterial calcification of infancy (GACI). We reported the case of a 32-year-old woman with a known diagnosis of ARHR2 since the age of seven. At that time, she presented a growth failure, bowed legs but no GACI history. She was treated from the age of 7 to 18 with phosphate supplements and calcitriol. She had ten surgeries for her leg deformities and had a subtotal parathyroidectomy at age 13 for primary hyperparathyroidism. She was referred to a tertiary centre for management of hypophosphatemia and diffuse bone pain. The alkaline phosphatase was at the upper limit of normal but imagings, including computed tomography scans and bone scintigraphy, did not show pseudofractures. Her chronic pain was associated with osteoarthritis and periarticular calcifications. She was treated for hypertension, and had a history of preeclampsia at 33 weeks of pregnancy. At the physical exam, a 30 mm Hg systolic blood pressure differential was noted between the right and left arms, suspect of severe subclavian artery stenosis. Additional investigations demonstrated a normal cardiac ultrasound (US), an Agatson score of 0 (coronary calcium scan) but extensive calcifications of the left atrial appendage, dense calcifications of the mitral valve and numerous calcifications in the ascending aortic wall. The spectrum of clinical manifestations associated with ENPP1-related conditions (GACI and ARHR2) is still unclear. In some patients who survive GACI, resolution of vascular calcifications has been reported. Furthermore, this case raises the possibility of undetected vascular calcifications in the infancy uncovered only after the diagnosis of ARHR2. Previous cases reported pulmonary stenosis, thickening of the aortic valve, and increased carotid intima-media thickness in patients with ARHR2 phenotype. Left atrial calcifications have been described in mechanical valve replacement, mitral stenosis, post-radiotherapy and in chronic kidney disease. Recommendations of cardiac US and kidney US have been proposed at the diagnosis of AHRH2. We further suggest vascular screening with cardiac scans to examine large vessels, and calcium score in these patients.

Prenatal diagnosis of generalized arterial calcification of infancy in the second trimester.

Generalized arterial calcification of infancy (GACI) is a rare autosomal recessive disorder characterized by subintimal fibrous proliferation and deposition of calcium salts in the internal elastic lamina, leading to extensive arterial calcification and stenosis of large and medium-sized arteries. Prenatal diagnosis is usually made in the third trimester by detection of aortic and pulmonary calcification with associated nonimmune hydrops; earlier prenatal diagnosis is rare. This study was performed to examine the prenatal ultrasound and genetic features of fetuses with GACI. We retrospectively reviewed the ultrasound findings, their progression in utero, and the clinical features in three fetuses with GACI ascertained using ultrasound in the second trimester. GACI was subsequently confirmed through pathological examination and/or molecular genetic testing. All three fetuses had hyperechogenic valves or annuli as the first detectable manifestation in the second trimester, followed by relatively rapid progression to arterial wall calcification. Three novel mutations of the ENPP1 gene associated with GACI were found in two of the cases (c.26dupG, c.1454A>G, and c.263C>G). GACI should be suspected when hyperechogenic cardiac valves, annuli, or arterial walls are noted after ruling out other causes of arterial calcification. Genetic testing is important for prenatal and future preimplantation genetic diagnosis.

Characterization of hearing-impairment in Generalized Arterial Calcification of Infancy (GACI)

Background and importanceHearing loss (HL) has been sporadically described, but not well characterized, in Generalized Arterial Calcification of Infancy (GACI), a rare disease in which pathological calcification typically presents in infancy.ObjectivesThis study aims to describe the clinical audiologic and otologic features and potential etiology of hearing impairment in GACI and gain pathophysiological insight from a murine model of GACI.DesignCross-sectional cohort study of individuals with GACI. Murine ossicle micromorphology of the ENPP1asj/asj mutant compared to wild-type.SettingClinical research hospital; basic science laboratory.ParticipantsNineteen individuals with GACI who met clinical, biochemical, and genetic criteria for diagnosis.Main outcomes and measuresClinical, biochemical, and radiologic features associated with hearing status.ResultsPure-tone thresholds could be established in 15 (n = 30 ears) of the 19 patients who underwent audiological assessments. The prevalence of HL was 50% (15/30) of ears, with conductive HL in 80% and sensorineural HL in 20%. In terms of patients with HL (n = 8), seven patients had bilateral HL and one patient had unilateral HL. Degree of HL was mild to moderate for 87% of the 15 ears with hearing loss. Of those patients with sufficient pure-tone and middle ear function data, 80% (8/10) had audiometric configurations suggestive of ossicular chain dysfunction (OCD). Recurrent episodes of otitis media (ROM) requiring pressure-equalizing tube placement were common. In patients who underwent cranial CT, 54.5% (6/11) had auricular calcification. Quantitative backscattered electron imaging (qBEI) of murine ossicles supports an OCD component of auditory dysfunction in GACI, suggesting loss of ossicular osteocytes without initiation of bone remodeling.Conclusions and relevanceHearing loss is common in GACI; it is most often conductive, and mild to moderate in severity. The etiology of HL is likely multifactorial, involving dysfunction of the ossicular chain and/or recurrent otitis media. Clinically, this study highlights the importance of early audiologic and otologic evaluation in persons with GACI. Novel findings of high rates of OCD and ROM may inform management, and in cases of unclear HL etiology, dedicated temporal bone imaging should be considered.

Generalized Arterial Calcification of Infancy (GACI): Optimizing Care with a Multidisciplinary Approach.

It is very unusual to see evidence of arterial calcification in infants and children, and when detected, genetic disorders of calcium metabolism should be suspected. Generalized arterial calcification of infancy (GACI) is a hereditary disease, which is characterized by severe arterial calcification of medium sized arteries, mostly involving the media with marked intimal proliferation and ectopic mineralization of the extravascular tissues. It is caused by inactivating variants in genes encoding either ENPP1, in a majority of cases (70–75%), or ABCC6, in a minority (9–10%). Despite similar histologic appearances between ENPP1 and ABCC6 deficiencies, including arterial calcification, organ calcification, and cardiovascular calcification, mortality is higher in subjects carrying the ENPP1 versus ABCC6 variants (40% vs 10%, respectively). Overall mortality in individuals with GACI is high (55%) before the age of 6 months, with 24.4% dying in utero or being stillborn. Rare cases show spontaneous regression with age, while others who survive into adulthood often manifest musculoskeletal complications (osteoarthritis and interosseous membrane ossification), enthesis mineralization, and cervical spine fusion. Despite recent advances in the understanding of the genetic mechanisms underlying this disease, there is still no ideal therapy for the resolution of vascular calcification in GACI. Although bisphosphonates with anti-calcification properties have been commonly used for the treatment of CAGI, their benefit is controversial, with favorable results reported at one year and questionable benefit with delayed initiation of treatment. Enzyme replacement therapy with administration of recombinant form of ENPP1 prevents calcification and mortality, improves hypertension and cardiac function, and prevents intimal proliferation and osteomalacia in mouse models of ENPP1 deficiency. Therefore, newer treatments targeting genes are on the horizon. In this article, we review up to date knowledge of the understanding of GACI, its clinical, pathologic, and etiologic understanding and treatment in support of more comprehensive care of GACI patients.

Systematic characterization of enzyme activity on ENPP1 deficiency disease phenotype

ENPP1 codes for a type 2 transmembrane glycoprotein in the ectonucleotide pyrophosphatase/phosphodiesterase protein family. The enzyme’s catalytic activity hydrolyzes extracellular ATP to generate pyrophosphate and adenosine monophosphate (AMP), thereby contributing to downstream purinergic signaling pathways. Pathogenic variants in ENPP1 are associated with diseases such as Type 2 Diabetes, Generalized Arterial Calcification of Infancy (GACI), Ossification of the Posterior Longitudinal Ligament (OPLL), Autosomal Recessive Hypophosphatemic Rickets Type 2 (ARHR2), Cole Disease, and early onset osteoporosis. The human phenotypes associated with these diseases are varied, and include life threatening large arterial calcifications, phosphate wasting rickets due to FGF23 elevation, cutaneous hypopigmentation and punctate keratosis, ankylosis and ossification of spinal ligaments, premature osteoporosis, as well as insulin resistance and cardiovascular disease.To determine whether the above disease phenotypes correlate with ENPP1 enzyme activity we quantitated enzyme velocities of ENPP1 variants identified in 42 ENPP1‐deficient patients followed at the NIH, together with an additional 22 variants reported in the literature. We simultaneously evaluated catalytic velocity of all variants in triplicate, enabling a direct comparison of enzyme activity across disease phenotypes. Cole disease is induced by variants in the SMB domains of ENPP1, and variants associated with the Autosomal Dominant form reduced enzyme velocity to a greater extent than the Autosomal Recessive form – between 32% to 8% of wild type (WT) vs. 33% of WT levels, respectively. Further supporting the presence of a gene dose effect, monoallelic variants in ENPP1 associated with elevation of FGF23, phosphate wasting, and early onset osteoporosis, occurred in patients with variants that reduced enzyme velocity below 50% of WT, while the polymorphisms associated with insulin resistance and cardiovascular disease reduced enzyme velocity to 73% of WT levels. Additionally, variants associated with life‐threatening vascular calcifications in GACI, which only occur in autosomal recessive biallelic ENPP1 deficient patients, reduced ENPP1 enzyme activity over a surprisingly wide range, from 25% to 100% of WT levels. Finally, disease severity in GACI correlated with the more severely damaging ENPP1 variant rather than the average of both variant allele products, suggesting that the effects of biallelic ENPP1 variants on in vivo enzyme activity are dominated by the more damaging ENPP1 allele. In summary, comparison of enzyme velocities among various ENPP1 deficient disease phenotypes supports the notion that specific variants determine these variable phenotypes, and are sometimes, but not always, related to enzyme activity. ENPP1 gene dose and dominance of the severe isoform in biallelic disease are notable mechanisms for determination of phenotype in some forms of ENPP1 deficiency.

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

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.

Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) due to ENPP1-deficiency.

Awareness for hypophosphatemic rickets has increased in the last years, based on the availability of specific medical treatments. Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) is a rare form of hypophosphatemic rickets, which is known to develop in survivors of generalized arterial calcification of infancy (GACI). Both disorders are based on a deficiency of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and present with a high clinical variability and a lack of a phenotype-genotype association. ARHR2 is characterized by phosphate wasting due to elevated fibroblast growth factor 23 (FGF23) levels and might represent a response of the organism to minimize ectopic calcification in individuals with ENPP1-deficiency. This report reviews the recent clinical and preclinical data on this ultra-rare disease in childhood.

Oral supplementation of inorganic pyrophosphate in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes and the cardiovascular system. Skin findings often lead to early diagnosis of PXE, but currently, no specific treatment exists to counteract the progression of symptoms. PXE belongs to a group of Mendelian calcification disorders linked to pyrophosphate metabolism, which also includes generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Inactivating mutations in ABCC6, ENPP1 and NT5E are the genetic cause of these diseases, respectively, and all of them result in reduced inorganic pyrophosphate (PPi ) concentration in the circulation. Although PPi is a strong inhibitor of ectopic calcification, oral supplementation therapy was initially not considered because of its low bioavailability. Our earlier work however demonstrated that orally administered pyrophosphate inhibits ectopic calcification in the animal models of PXE and GACI, and that orally given Na4 P2 O7 is absorbed in humans. Here, we report that gelatin-encapsulated Na2 H2 P2 O7 has similar absorption properties in healthy volunteers and people affected by PXE. The sodium-free K2 H2 P2 O7 form resulted in similar uptake in healthy volunteers and inhibited calcification in Abcc6-/- mice as effectively as its sodium counterpart. Novel pyrophosphate compounds showing higher bioavailability in mice were also identified. Our results provide an important step towards testing oral PPi in clinical trials in PXE, or potentially any condition accompanied by ectopic calcification including diabetes, chronic kidney disease or ageing.

Abstract 16747: Rapidly Lethal Dilated Cardiomyopathy Secondary To Generalized Arterial Calcification Of Infancy

Case Presentation: A previously healthy 3-month-old infant presented with cardiorespiratory arrest, from which she was successfully resuscitated. A dilated cardiomyopathy with severely depressed systolic function was diagnosed. ECG showed high voltage QRS complexes and generalized alteration of repolarization. In the following 12 hours, she suffered 2 other cardiac arrests, recuperated with defibrillation. Hemodinamic stability was achieved but brain death was diagnosed 36 hours after the onset of symptoms. Autopsy showed striking calcification of the right and left coronary arteries, with narrowing of the arterial lumen, causing extensive subendocardial infarction. Calcification also affected the aorta, pulmonary arteries, thyroid, kidney and other splanchnic arteries. Generalized arterial calcification of infancy (GACI) was diagnosed and a genetic study found two biallelic variants in ABCC6 gene: p.Arg1114Cys and p.Trp38Ser, both previously described in elastic pseudoxanthoma (PXE), but not in GACI. Genotyping of the healthy parents confirmed genetic segregation with biallelic variants. Discussion: GACI is an extremely rare genetic disease characterized by widespread arterial calcification and narrowing of large and medium-sized vessels. The usual clinical presentation is heart failure in fetal life or in the first months of infancy. In most cases it is lethal, with death occurring within a few hours or days after the onset of symptoms, although clinical involvement is highly variable and cases with long survival have been described. GACI is an autosomal recessive disease secondary to biallelic variants in the ENPP1 gene (67% of cases) and in the ABCC6 gene (9%). The variants found in our patient had not been previously described in GACI, just in PXE, a much milder disease with usually normal lifespan. This case confirms that both entities reflect two extremes of a clinical spectrum of ectopic calcification instead of two different disorders. The aim of presenting this case is to remind clinicians of this rare etiology in neonates or infants with dilated cardiomyopathy. In case of death, autopsy should always be requested. When this condition is diagnosed, genetic study will be positive in 75% of cases, allowing prenatal counseling.

Ectopic Calcification and Hypophosphatemic Rickets: Natural History of ENPP1 and ABCC6 Deficiencies.

ABSTRACTGeneralized arterial calcification of infancy (GACI) is a rare disorder caused by ENPP1 or ABCC6 variants. GACI is characterized by low pyrophosphate, arterial calcification, and high mortality during the first year of life, but the natural course and possible differences between the causative genes remain unknown. In all, 247 individual records for patients with GACI (from birth to 58.3 years of age) across 19 countries were reviewed. Overall mortality was 54.7% (13.4% in utero or stillborn), with a 50.4% probability of death before the age of 6 months (critical period). Contrary to previous publications, we found that bisphosphonate treatment had no survival benefit based on a start‐time matched analysis and inconclusive results when initiated within 2 weeks of birth. Despite a similar prevalence of GACI phenotypes between ENPP1 and ABCC6 deficiencies, including arterial calcification (77.2% and 89.5%, respectively), organ calcification (65.8% and 84.2%, respectively), and cardiovascular complications (58.4% and 78.9%, respectively), mortality was higher for ENPP1 versus ABCC6 variants (40.5% versus 10.5%, respectively; p = 0.0157). Higher prevalence of rickets was reported in 70.8% of surviving affected individuals with ENPP1 compared with that of ABCC6 (11.8%; p = 0.0001). Eleven affected individuals presenting with rickets and without a GACI diagnosis, termed autosomal recessive hypophosphatemic rickets type 2 (ARHR2), all had confirmed ENPP1 variants. Approximately 70% of these patients demonstrated evidence of ectopic calcification or complications similar to those seen in individuals with GACI, which shows that ARHR2 is not a distinct condition from GACI but represents part of the spectrum of ENPP1 deficiency. Overall, this study identified an early mortality risk in GACI patients despite attempts to treat with bisphosphonates, high prevalence of rickets almost exclusive to ENPP1 deficiency, and a spectrum of heterogenous calcification and multiple organ complications with both ENPP1 and ABCC6 variants, which suggests an overlapping pathology. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Generalized Arterial Calcification of Infancy Type 1 (GACI1): Identification of a Novel Pathogenic Variant (c.1715T>C (p.Leu572Ser)).

Generalized Arterial Calcification of Infancy (GACI) is a rare disease inherited in a recessive manner, with severe and diffuse early onset of calcifications along the internal elastic lamina in large and medium size arteries. The diagnosis results are from clinical manifestations, imaging, histopathologic exams, and genetic tests. GACI is predominantly caused by biallelic pathogenic variant in the ENPP1 gene (GACI1, OMIM#208000) and, to a lesser extent, by pathogenic variants in the ABCC6 gene (GACI2, OMIM#614473). We present a novel variation in the ENPP1 gene identified in a patient clinically diagnosed with GACI and confirmed by genetic investigation and autopsy as GACI type 1. The sequence analysis of the patient’s ENPP1 gene detected two heterozygous variants c.1412A>G (p.Tyr471Cys) and c.1715T>C (p.Leu572Ser). The variant c.1715T>C (p.Leu572Ser) has not been described yet in the literature and in mutation databases. A genetic analysis was also carried out for the parents of the newborn; the heterozygous pathogenic variant c.1412A>G (p.Tyr471Cys) was detected in the mother’s ENPP1 gene, and a sequence analysis of the father’s ENPP1 gene revealed the novel heterozygous variant c.1715T>C (p.Leu572Ser). Our results showed that the variant c.1715T>C (p.Leu572Ser) may have a pathogenic role in the development of GACI type1 (GACI1, OMIM#208000), at least when associated with the pathogenic c.1412A>G (p.Tyr471Cys) variant. The identification of novel mutations potentially enabled genotype/phenotype associations that will ultimately have an impact on clinical management and prognosis for the disease.