ENPP1 deficiency: A clinical update on the relevance of individual variants using a locus‐specific patient database

Loss‐of‐function variants in the ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) cause ENPP1 Deficiency, a rare disorder characterized by pathological calcification, neointimal proliferation, and impaired bone mineralization. The consequence of ENPP1 Deficiency is a broad range of age dependent symptoms and morbidities including cardiovascular complications and 50% mortality in infants, autosomal recessive hypophosphatemic rickets type 2 (ARHR2) in children, and joint pain, osteomalacia and enthesopathies in adults. Recent research continues to add to the growing clinical presentation profile as well as expanding the role of ENPP1 itself. Here we review the current knowledge on the spectrum of clinical and genetic findings of ENPP1 Deficiency reported in patients diagnosed with GACI or ARHR2 phenotypes using a comprehensive database of known ENPP1 variants with associated clinical data. A total of 108 genotypes were identified from 154 patients. Of the 109 ENPP1 variants reviewed, 72.5% were demonstrably disease‐causing, a threefold increase in pathogenic/likely pathogenic variants over other databases. There is substantial heterogeneity in disease severity, even among patients with the same variant. The approach to creating a continuously curated database of ENPP1 variants accessible to clinicians is necessary to increase the diagnostic yield of clinical genetic testing and accelerate diagnosis of ENPP1 Deficiency.


| INTRODUCTION
The expanding role of ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (ENPP1) in the pathogenesis of ectopic mineralization and paradoxical impairment of bone mineralization continues to garner interest. Loss-of-function variants in the ENPP1 gene cause ENPP1 Deficiency, a rare disorder characterized by low pyrophosphate (inorganic pyrophosphate [PPi]) levels, excessive soft tissue calcification, arterial stenoses, and hypomineralization of bone Nitschke et al., 2018;Rutsch et al., 2008). Early mortality, cardiac complications, hearing loss, impaired bone mineralization and ligament calcification are the clinical consequences of ENPP1 Deficiency resulting in significant morbidity in these patients (Ferreira, Ansh, et al., 2022;Ferreira, Kintzinger, et al., 2021;Saito et al., 2011;Theng et al., 2022).
Current research in ENPP1 continues to elucidate the heterogeneity of clinical presentation and morbidity across the age spectrum.
These infants often first present with severe on-specific CV symptoms such as hypertension and heart failure, for which numerous etiologies exist are often the first presentation (Ferreira, Kintzinger, et al., 2021;Rutsch et al., 2008). Adults and children who survive the early phase of the disease or do not present at the early phase infant stage of the disease may go on to develop hypophosphatemic rickets often reported as autosomal recessive hypophosphatemic rickets type 2 (ARHR2) and hearing loss Ferreira, Kintzinger, et al., 2021;Theng et al., 2022). The clinical and biochemical characteristics of ARHR2 closely resemble X-linked hypophosphatemia (XLH) and other genetic forms of rickets, including short stature, bone deformities and pain, gait abnormalities, renal phosphate wasting, and elevated FGF23 (Haffner et al., 2019;Levy-Litan et al., 2010). Adults can go on to present with nonspecific symptoms such as joint pain, osteomalacia, and enthesopathies due to ENPP1 Deficiency (Ferreira, Ansh, et al., 2022;Oheim et al., 2020). Variants in the ENPP1 gene have been identified in adult patients presenting with ossification of the posterior longitudinal ligament (OPLL) (Ferreira, Ansh, et al., 2022;Nakamura et al., 1999;Saito et al., 2011). While ENPP1 Deficiency is defined as an autosomal recessive disorder, there is growing evidence from case reports of adults with monoallelic ENPP1 variants who presented with early-onset osteoporosis and fractures (H. Kato et al., 2022;Oheim et al., 2020).
Due to the heterogeneity and nonspecific symptoms associated with ENPP1 Deficiency, an accurate diagnosis relies on genetic confirmation (Ferreira, Ansh, et al., 2022;Ferreira, Kintzinger, et al., 2021). However, the small number of patients diagnosed with this ultra-rare disease (genetic prevalence estimated as 1/200,000 pregnancies) , coupled with the relatively high frequency of private variants-found in single families or a small number of individuals-pose a challenge to variant interpretation by clinicians and geneticists. To flatten the variants of uncertain significance (VUS) curve, it is critical to capture all clinical and functional evidence available to support variant reclassification. The aim of this review is to summarize current knowledge on the spectrum of clinical with genetic findings in patients with ENPP1 Deficiency. Specifically, this analysis included patients with ENPP1 variant(s) and a diagnosis of GACI or ARHR2 as this currently represents the most common phenotypes in the literature, with particular interest in pathogenic variants shaping the clinical presentation.

| Data sources and variant classification
We integrated data from a comprehensive, retrospective literature review with results from two natural history studies of GACI and ARHR2 patients to produce the most complete patient and variant database for ENPP1 Deficiency. Joining data from both sources is especially valuable for rare diseases given the difficulty in identifying and studying patients in numbers sufficient to be statistically meaningful. The variant database includes single nucleotide variants, small and large insertions/deletions (indels), as well as larger structural variants including exon-level copy number variants and larger.
We identified and analyzed all published cases of ENPP1 Deficiency, specifically selecting for patients with a recorded diagnosis of GACI and/or ARHR2. The data was collated and all associated genetic variants in ENPP1 were interpreted using a novel approach to systematic curation of genetic evidence. This comprehensive literature review was performed using the data content in Mastermind, a database of variants with evidence cited in the medical literature (Chunn et al., 2020)  In addition to published case reports, data was also collected from two recently published natural history studies, "Study of People with GACI or ARHR2" (identifier: NCT03478839) performed at the US National Institutes of Health (NIH), and "Natural History of GACI With or Without ARHR2 or PXE" (identifier: NCT03758534) performed in Germany's Münster University Children's Hospital (Ferreira, Kintzinger, et al., 2021). Data was also collected from two additional patients who were seen at the NIH but were not included in the natural history study.  (Nykamp et al., 2017;Richards et al., 2015). The ACMG variant interpretations were based on manual review of curated evidence from the literature as well as data extracted from external databases for computational predictions (PolyPhen2 [Adzhubei et al., 2010] and SIFT [Sim et al., 2012]) and population frequency (gnomAD [Karczewski et al., 2020]).

| Patient and variant database composition
The combined approach described 154 total patients-111 individual GACI and/or ARHR2 patients harboring one or more ENPP1 variant were initially identified through the literature review (72.1%) to which an additional 41 patients (26.6%) were included from the two natural history studies, including 27 from the study conducted in Germany and 14 conducted at the NIH. Five patients, two of which were not previously published (1.3%), were seen at the NIH but were not included in either of the two natural history studies. Of note, 45 out of the 111 patients (40.5%) initially identified in the literature analysis were catalogued in either or both natural history studies (including 17 identified in the German study, 26 identified in the NIH study and 2 identified in both studies).
An additional 4 patients with monoallelic ENPP1 Deficiency were identified, but were excluded from the analysis on the basis that they did not have a recorded diagnosis of GACI or ARHR2. Three of these patients were instead diagnosed with early-onset osteoporosis, and one patient was diagnosed with pseudoxanthoma elasticum.
In our literature review, extensive effort was expended to ensure patients were not double counted so it is presumed that the unpublished patients identified in the natural history studies would not have otherwise been encountered. A summary of the origin of each patient is depicted in Figure 1 underscoring the benefit of taking a combined approach to patient identification and characterization to maximize understanding of this rare disease. The combined results of these two approaches are presented in Table 1 including 108 unique genotypes and a high-level summary of the clinical diagnostic data for each patient identified in both the retrospective and the natural history studies.

| ENPP1 DEFICIENCY
3.1 | ENPP1 protein structure and function ENPP1 is expressed in numerous tissues including osteoblasts and osteocytes, chondrocytes, vascular smooth muscle cells, renal proximal tubule epithelial cells, mature plasma cells, and skin fibroblasts . The ENPP1 gene product is a type II transmembrane glycoprotein consisting of a small intracellular F I G U R E 1 Source of GACI/ARHR2 patient information for the present study. Retrospective analysis of the published literature was performed using the Genomenon database of genetic evidence. Two distinct prospective natural history studies of GACI/ARHR2 patients were independently performed at University Children's Hospital Münster and at the National Institute of Health (NIH). The overlap of the origin of each patient is depicted. Care was taken to ensure no duplication of patient records occurred. ARHR2, autosomal recessive hypophosphatemic rickets type 2; GACI, generalized arterial calcification of infancy domain, a single transmembrane domain, and a larger extracellular domain containing a catalytic site . The cytoplasmic domain (residues 1-76) and transmembrane domain (residue 77-97) localize the protein in the plasma membrane while two extracellular somatomedin B-like domains (SMB1, residue 104-144) and SMB2, residues 145-188) are implicated in homodimerization (Bello et al., 2001;Vaingankar et al., 2004). The ENPP1 catalytic domain comprises a phosphodiesterase (residues 191-591) which is N-terminally flanked by the SMB domains, and C-terminally linked to a nuclease-like domain (residues 654-925) (Jansen et al., 2012).
The ENPP1 catalytic domain cleaves the phosphodiester bonds of nucleotides, preferentially hydrolyzing extracellular ATP into PPi and adenosine monophosphate (AMP) (K. Kato et al., 2012;Zhou et al., 2012). PP i acts as the main physiologic inhibitor of calcification by antagonizing hydroxyapatite formation and deposition (Fleisch & Bisaz, 1962;Goding et al., 2003;Zhou et al., 2012). AMP is further metabolized into adenosine, an inhibitor of neointimal proliferation (Albayrak et al., 2015;Dubey et al., 1998). Biallelic loss-of-function variants in the ENPP1 gene are associated with low levels of PP i and adenosine, leading to pathologic ectopic vascular calcification and neointimal proliferation, respectively (Nitschke et al., 2018).

| Animal models of ENPP1 deficiency
Mouse models that recapitulate the clinical phenotypes of patients with ENPP1 Deficiency have been a valuable tool in understanding the disease mechanism and potential therapies targeting ENPP1 mutations. The first mouse model of ENPP1 deficiency was the Enpp1 ttw/ttw ("tiptoe walking") mouse, harboring a homozygous p.Gly568Ter variant and characterized by ectopic mineralization of the spinal ligaments (Okawa et al., 1998). This mouse model has also demonstrated that ENPP1 functions to inhibit neointima formation by producing AMP, together linking the role of ENPP1 to regulating PP i levels and arterial thickness (Nitschke et al., 2018). A mouse harboring a homozygous deletion of Enpp1 had a phenotype essentially identical to that of the Enpp1 ttw/ttw mouse (Johnson et al., 2003). Enpp1 asj/asj ("ages with stiffened joint") mice harboring the p.Val246Asp variant display low plasma PPi levels and vascular mineralization . When fed a diet high in phosphate but low in magnesium, these Enpp1 asj/asj mice showed accelerated mineralization and shorter lifespans. A fourth mouse model is the Enpp1 asj-2J mouse, harboring a large 40 kilobase deletion combined with a 74 bp insertion, and leading to more extensive mineralization than seen in the Enpp1 asj/asj mice   (Albright et al., 2015;Cheng et al., 2021;Ferreira, Ansh, et al., 2022;Ferreira, Kavanagh, et al., 2021;Maulding et al., 2021). A mutant zebrafish model also displays ectopic mineralization in soft tissues (Apschner et al., 2014).

| ENPP1 deficiency clinical presentation and phenotypes
ENPP1 Deficiency is associated with significant morbidity and mortality across the age spectrum. There is considerable heterogeneity in the age of symptom onset, clinical presentation, and severity.
The clinical presentation of ENPP1 Deficiency in infants, typically described as GACI, is characterized by arterial calcification, stenosis of large and medium-sized vessels and severe CV problems such as systemic hypertension, pulmonary hypertension, heart failure, cardiomyopathy or myocardial ischemia/infarction Rutsch et al., 2008). Infants may also present with seizures or stroke, attributed to calcification of the cerebral arteries Ferreira, Kintzinger, et al., 2021;Mulcahy et al., 2019). Calcification of organs and joints are also observed . Approximately 50% of infants with ENPP1 Deficiency die within the first 6 months of life despite receiving standard of care treatments (Ferreira, Kintzinger, et al., 2021;Rutsch et al., 2008).
Patients who survive infancy or who first exhibit symptoms of ENPP1 Deficiency in childhood typically present with FGF23mediated hypophosphatemic rickets, described as ARHR2 . This might represent a compensatory mechanism to mitigate vascular calcification, as increased FGF23 expression reduces renal phosphate reabsorption and promotes its excretion. In a prospective natural history study of patients with ENPP1 Deficiency a Kaplan-Meier analyses estimated that 90% of patients would develop hypophosphatemic rickets by age 15 . Additionally, a large portion of patients with ENPP1 Deficiency will also develop and present with hearing loss, joint calcification, or enthesopathies (Brachet et al., 2014;Ferreira, Ansh, et al., 2022;Kotwal et al., 2020;Theng et al., 2022;Thumbigere-Math et al., 2018).
The CV and skeletal complications of ENPP1 Deficiency may continue into adulthood. Adult patients with ENPP1 Deficiency often present with symptoms of osteomalacia or late-onset musculoskeletal complications, including bone and joint pain and enthesopathies impacting daily function (Ferreira, Ansh, et al., 2021;. CV or renal disease associated with vascular calcification or vessel wall thickening has also been described in some adults with ENPP1 Deficiency Kotwal et al., 2020;Lorenz-Depiereux et al., 2010). Variants in the ENPP1 gene may also lead to OPLL, as demonstrated in mouse model studies (Nakamura et al., 1999) and patient case reports (Ferreira, Ansh, et al., 2022;Saito et al., 2011). These patients may suffer from nerve root compression resulting in radiculopathy and myelopathy, that may severely limit mobility and negatively affect patients'quality of life.
One patient with OPLL due to ENPP1 Deficiency was originally diagnosed with diffuse idiopathic skeletal hyperostosis (DISH) before genetic testing (H. Kato et al., 2022). Findings of low/ normal serum phosphorous with elevated FGF-23 in some of these patients suggest that cervical ligament ossifications may represent a complication of ARHR2 (H. Kato et al., 2022;Saito et al., 2011).
While ENPP1 Deficiency is considered an autosomal recessive disorder, there are case reports of adults with monoallelic ENPP1 variants who presented with early-onset osteoporosis and fractures (Oheim et al., 2020 Interestingly, one heterozygous patient who was diagnosed with GACI was found to have the benign ENPP1 p.Arg821His polymorphism (Patel et al., 2004;Stella et al., 2016) in addition to the ABCC6 variant p.Arg1314Trp. This ABCC6 variant is a well-characterized pathogenic variant having been found in at least 2 families with demonstrated segregation Li et al., 2014) or otherwise in multiple patients within a large cohort (Legrand et al., 2017;Nitschke et al., 2012;Pfendner et al., 2007;Ramsay et al., 2009) and confirmed to be functionally consequential in multiple empirical studies (Le Saux et al., 2011;Letavernier et al., 2018;Pomozi et al., 2014;Pomozi, Brampton, Szeri, et al., 2017;Ran & Thibodeau, 2017 These results are depicted in Figure 2d. When examining the distribution of these variants across the We, therefore, next sought to better understand the distribution of these variants across the domain structure of the ENPP1 protein based not solely on the list of unique variants but rather based on their appearance across all alleles. Figure 3a depicts the distribution of all alleles across the ENPP1 linear protein structure based on the variant type relative to the exon structure of the fully spliced canonical ENPP1 mRNA transcript NM_006208.3 and the canonical ENPP1 protein sequence with its functional domains (NP_006619.2 isoform). These regions are depicted in Figure 3b,c, respectively.
Based on this analysis of 308 patient alleles, we identified an incidence of 178 phosphodiesterase and 102 nuclease domain variants, suggesting a more significant selection bias for damaging effects to these domains leading to GACI and/or ARHR2 phenotypes but a small one. This result should underscore the conclusion that variants in other domains of ENPP1 should not be discounted. Overall, there were 109 unique ENPP1 variants found in patients.
These variant data are shown in Table 2   To gain more insight into the relative prevalence of missense that are likely to be seen in diagnostic laboratories, we also considered the distribution of each variant type across the multiple different genotypes for the total number of patients identified in this work.

| Splice variants
Twelve canonical splice site variants (11.0%, 12/109) were reported, all predicted to be pathogenic. As depicted in Figure 3a, seven of these are in the phosphodiesterase region, three in the nuclease-like domain, and one in each of the SMB domains.

| Indels and large deletions
Whole gene, exon or partial exon deletions or indels comprised 8.3%

| Polymorphisms
Two ENPP1 variants were deemed benign polymorphisms (p.Glu668Lys and p.Arg821His) and two were found to have conflicting evidence of pathogenicity with insufficient evidence to make either a benign or a pathogenic designation. These include the p.Leu611Val variant and the polymorphism p.Arg774Cys. The latter p.Arg774Cys variant was found in 3.3% of alleles in gnomAD (as common as 9.7% in the Finnish population) and was, therefore, deemed too common to be disease-causing according to clinical guidelines. However, at least one functional study examined the effect of p.Arg774Cys in SaOS2 osteosarcoma cell lines and showed 30-40% of NPP activity indicating a possible damaging effect (Rutsch et al., 2003). A separate study in HEK293 cells showed no effect on PPi generation and localization resulting from this variant (Stella et al., 2016). The true nature of the effect of this variant on GACI or ARHR2 remains unclear. The less frequently encountered p.Leu611-Val variant is present in gnomAD at 0.75% (and as commonly as 4.9% in African-Americans) in an unselected population as well as being predicted to be functionally inert by SIFT, PolyPhen and Mutation-Taster. However, in a study of a family with the complex genotype p.Gln792Ser-p.Glu668Lys-p.Leu611Val, functional testing of NPP activity for SaSO2 osteosarcoma cell lines bearing the missense variant p.Glu668Lys were normal and the patient's phenotype was therefore attributed to the p.Leu611Val variant (Rutsch et al., 2003).
Given that there were several affected patients with heterozygous ENPP1 variants, it is unclear whether this interpretation is accurate.
Based on these findings overall, the p.Arg774Cys and p.Leu611Val variants should, therefore, be interpreted cautiously within the context of the patient's overall clinical presentation and molecular results.

| ENPP1 variants in ClinVar
We next sought to determine whether any ENPP1 variants not identified in our approach were catalogued in ClinVar. In total there were 341 ENPP1 variants described in ClinVar at the time of A summary of the source of ENPP1 variants deemed to be pathogenic or likely pathogenic for GACI/ARHR2 is depicted in Over half of the individuals harbored two missense variants, which raises to question the mechanism of missense variant pathogenicity. Jansen et al. (2012) analyzed the location and stability of the ENPP1 protein product associated with disease-associated ENPP1 missense variants. Of the 26 missense variants located in the PDE or nuclease domain, the majority were predicted to cause protein destabilization (e.g., buried within cell membrane) or aggregation (Jansen et al., 2012).
A study by Stella et al. further explored the impact of pathogenic missense variants on ENPP1 activity and PP i generation (Stella et al., 2016). Of the 13 analyzed missense variants, eight showed complete loss of enzyme activity with no PP i generation, four demonstrated reduced ENPP1 activity, and one variant had normal activity. Consistent with prior evidence, 5/8 of the missense variants with abolished ENPP1 activity were not localized in the plasma membrane. While the precise mechanism is still not fully elucidated, it has been hypothesized that loss of structural stability associated with missense variants may lead to protein misfolding, and reduced ENPP1 abundance (Jansen et al., 2012).
In addition to three LoF variants, four pathogenic or likely pathogenic missense variants in the SMB domains were identified in this review. Prior studies suggest that the SMB domains of ENPP1 are implicated in protein homodimerization (Bellacchio, 2012;Gijsbers et al., 2003), but do not interact with the catalytic domain (K. Kato et al., 2012). Of note, heterozygous variants in the SMB2 domain of ENPP1 underlie the autosomal-dominant skin disorder, Cole disease, which is characterized by abnormal keratinization and hypopigmentation (Eytan et al., 2013).
Our study identified four variants located in the cytosolic domain, all of which were LoF variants deemed pathogenic. The cytosolic domain contains a di-leucine motif (residues 49-50) that supports localization of ENPP1 to the basolateral surface, the region of the plasma membrane which buds off into matrix vesicles (Bello et al., 2001;Vaingankar et al., 2004). Mutations to one or both leucines were shown to direct ENPP1 expression to the apical surface. Functionally, these ENPP1 mutants had approximately 50% lower matrix-vesicle associated PP i levels and increased calcification, as compared to wild-type osteoblastic cells (SaOS-2) (Vaingankar et al., 2004). Thus, while cytosolic ENPP1 variants are uncommon, there is evidence supporting their potential for pathogenicity.
Other catalytically-independent functions of ENPP1 which have been proposed through in-vitro studies include prevention of osteoblast differentiation (Nam et al., 2011) and modulation of FGF-23 expression (Mackenzie et al., 2012;Maulding et al., 2021). including bowed extremities, metaphyseal irregularities and cupping.

| ENPP1 DEFFICIENCY PHENOTYPES
The second most common genotype, p.Gly186Arg/p.Gly186Arg, found in four patients, was associated with neonatal mortality in all cases. Polyhydramnios was detected in all four patients prenatally.
The three patients who were homozygous for the large partial deletion (c.2444+702_*868del/c.2444+702_*868del) showed some phenotypic similarities. All three patients presented with skeletal symptoms of ARHR2 (2 during childhood, 1 with unknown time of onset) including bowed extremities (n = 3), hypophosphatemia (n = 2) and rickets (n = 3). Though none had a medical history of GACI, one of these patients was found to have thickening of aortic valves on echocardiography, and a second had a reported heart valve defect. could benefit from gene sequencing with a panel including ENPP1.
Brightness of great vessels on prenatal or neonatal ultrasound or echocardiogram warrant ENPP1 Deficiency evaluation (Ziegler et al., 1993 (Maulding et al., 2021;Zimmerman et al., 2022). We note that our data focused on reported diagnosis of GACI or ARHR2 and it is possible that we have under-reported the prevalence of pathogenic variants in noncatalytic domains of ENPP1, possibly specific to patients diagnosed with OPLL or early-onset osteoporosis. More work is needed to elucidate the potential noncatalytic role of ENPP1 in GACI and ARHR2 patients.
We also found that some variants previously described as disease-causing are too frequent to be considered pathogenic. As an example, the recurrent variant p.Arg774Cys (found in 12 total alleles, with 6.5% or 10/154 patients harboring one or two of this variant) has an allele frequency almost as high as 10% in certain populations.
Similarly, the allele frequency of the p.Leu611Val variant is almost as high as 5% in certain populations. Although these two variants are unlikely to be pathogenic, it is still possible that they may lead to disease when in cis between each other or another common variant in combination with a rare pathogenic variant in trans in the other allele (monogenic triallelic inheritance).
Monoallelic inheritance has been associated with skeletal disease in the form of low bone mineral density, originally described as earlyonset osteoporosis as well as OPLL and DISH (H. Kato et al., 2022;Oheim et al., 2020;Saito et al., 2011). This phenotype has been ascribed to intermediate levels of PPi, and ENPP1 activity, supporting the possibility of an ENPP1 gene dosage effect when regulating bone mass, suggesting ENPP1 Deficiency may be autosomal dominant in specific states (H. Kato et al., 2022;Kotwal et al., 2020;Oheim et al., 2020). The mechanism by which monoallelic ENPP1 variants leads to skeletal disease is still being elucidated. Anecdotally, parents of most patients with GACI or ARHR2 do not have clinically significant osteomalacia, although it is possible that this complication of monoallelic ENPP1 deficiency depends on the severity of enzyme deficiency associated with each variant, and that thus only single variants associated with severe enzyme deficiency will lead to skeletal disease. Therefore, one possibility is that haploinsufficiency (leading to approximately 50% residual enzyme activity) versus a dominant negative effect (leading to <50% enzyme activity) would be a possible mechanism for monoallelic ENPP1 deficiency; further work is needed to assess whether this is a valid hypothesis. Another possibility is that a second variant was missed in these adults, such as a noncoding variant in the promoter or deep-intronic regions. Yet a third possibility is that common variants in trans with rare severe variants might lead to disease, but are not reported by clinical laboratories as they are assumed to be benign. As mentioned earlier, some common variants (p.Arg774Cys and p.Leu611Val) have been MERCURIO ET AL.
| 1701 shown to lead to decreased PDE activity in SaOS2 osteosarcoma cell lines. It is thus possible that although these variants will not lead to disease in homozygosity, they might when associated with other variants in the other allele. There is precedent for such mechanism, as in partial biotinidase deficiency, where a common variant (with an allele frequency of 3.9% in a European non-Finnish population, and up to 5.6% in a Finnish population) is not associated with disease in homozygosity, but leads to clinical manifestations in combination with a pathogenic variant in trans (Swango et al., 1998). Interestingly, we identified in the literature eight infants with a severe GACI phenotype and early mortality harboring with only a single heterozygous variant in ENPP1. This puts the gene dose effect at odds with severe vascular calcifications and high mortality observed in infants is largely ascribed to nearly undetectable PPi levels (Nitschke et al., 2018). How monoallelic ENPP1 Deficiency could result in a severe, highly fatal GACI phenotype is unclear, and merits further study as more GACI patients with a single ENPP1 variant are identified. Evaluation of PPi levels in infants may help discern whether this is true ENPP1 haploinsufficiency, or if a second undetectable variant was likely present.
It is not known, however, whether the severity of enzyme deficiency is associated with a more severe phenotype. We found that 8/24 (33.3%) of patients with two truncating variants and known survival outcome succumbed to their disease, while mortality was somewhat higher among patients with known survival outcomes who harbored two nontruncating variants (42/71, or 59.2%). This appears to confirm the fact described in prior smaller cohorts asserting the lack of genotype-phenotype correlation in ENPP1 Deficiency . It should also be noted that the extent to which phenotypes are described in the literature vary between authors. The relatively high prevalence of cardiovascular complications reported in patients with an ARHR2 only diagnosis (34.5%) affirms the understanding of ENPP1 Deficiency as a single spectrum of disease, and points to the importance of thorough cardiovascular workup and continued monitoring in children and adults. Therefore, the absence of a phenotype in literature-sourced patient information may be limited to individual authors' descriptions or assessments, rather than a confirmed lack of the trait.
Though this is the largest study of GACI and ARHR2 patients ever gathered, the statistical significance of these findings is uncertain. We hope that this work will serve as a starting point for continued aggregation of genetic and phenotypic data as new GACI and ARHR2 patients are identified and characterized.