Molecular Genetics and Modifier Genes in Pseudoxanthoma Elasticum, a Heritable Multisystem Ectopic Mineralization Disorder

Abstract

In the past two decades, there has been great progress in identifying the molecular basis and pathomechanistic details in pseudoxanthoma elasticum (PXE), a heritable multisystem ectopic mineralization disorder. While the identification of pathogenic variants in ABCC6 has been critical for understanding the disease process, genetic modifiers have been disclosed that explain the phenotypic heterogeneity of PXE. Adding to the genetic complexity of PXE are PXE-like phenotypes caused by pathogenic variants in other ectopic mineralization-associated genes. This review summarized the current knowledge of the genetics and candidate modifier genes in PXE, a multifactorial disease at the genome/environment interface.

INTRODUCTION

Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare genetic disease with a slight female predominance. It is identified by elastorrhexis and progressive ectopic mineralization affecting multiple organs, such as the skin, eyes, and the arterial blood vessels (Li et al., 2009b; Neldner, 1988). The primary cutaneous manifestations are small yellowish papules that progressively coalesce to make a leathery plaque on flexor areas. Histopathologic evaluation of cutaneous lesions reveals accumulation of elastotic material, which becomes progressively occupied by calcium deposits (Neldner, 1988). The ophthalmologic manifestations progressively comprise of peau d’orange, angioid streaks, neovascularization, hemorrhage, and central vision loss and blindness (Gliem et al., 2013). The involvement of arterial blood vessels is considered as a cardinal manifestation because it leads to cardiovascular complications with high morbidity and occasional mortality (Leftheriotis et al., 2013).

PXE is inherited in an autosomal recessive manner (Ringpfeil et al., 2006). The prevalence of PXE is estimated at 1:50,000 with complete penetrance without ethnic predilection (Li et al., 2019a). The rate of progression of PXE seems to have no discernible pattern, and the disease displays a broad spectrum of phenotypic severity: normal life span versus premature death due to life-threatening complications, depending on the extent of extra-cutaneous involvement. Therefore, its clinical diagnosis is challenging due to inter-familial and intra-familial phenotypic heterogeneity regarding the age of onset, organ involvement, and disease severity. A Phenodex score has been introduced as an international standard to evaluate the severity of manifestations in different organ systems (Pfendner et al., 2007; Legrand et al., 2017; Iwanaga et al., 2017). The high intra-familial and inter-individual clinical variability of PXE, similar to a number of other Mendelian disorders, suggests that secondary genetic co-factors exist. Disease variability in patients in a family bearing the same combination of ABCC6 pathogenic variants emphasizes the role of genetic background (Le Boulanger et al., 2010).

GENETICS OF PXE

Pathogenic variants in the ABCC6 gene

Identification of the causative gene implicated in PXE initially utilized positional cloning approaches which revealed evidence for linkage to the short arm of chromosome 16, the critical interval consisting of approximately 500 kb (Le Saux et al., 1999; Cai et al., 2000). Systematic sequencing of genes in this region identified ABCC6 as the gene harboring pathogenic variants in PXE (Ringpfeil et al., 2000; Le Saux et al., 2000; Bergen et al., 2000; Struk et al., 2000). The ABCC6 gene encodes a putative transmembrane efflux transporter protein, ABCC6, a member of the family of ATP-binding cassette proteins. The ABCC6 gene is primarily expressed in the liver and at a very low level in tissues affected in PXE (Belinsky and Kruh, 1999; Scheffer et al., 2002). Increasing evidence suggests that PXE is a metabolic disorder caused by defective ABCC6 transporter activity in the liver resulting in mineralization of affected peripheral tissues. Hepatic ABCC6 mediates ATP release from hepatocytes to the blood stream, albeit with unknown mechanism. ATP is subsequently converted by an ectonucleotidase, ENPP1, to inorganic pyrophosphate (PPi), the principal physiological inhibitor of hydroxyapatite crystal formation in soft tissue (Jansen et al., 2013; Jansen et al., 2014). PPi acts as a potent inhibitor of mineralization through high affinity binding to the surface of nascent or growing hydroxyapatite crystals, thereby blocking their ability to act as a nucleation site for mineralization and therefore preventing crystal growth (Orriss et al., 2016). Reduced plasma PPi levels explain, at least in part, the pathophysiology of PXE caused by ABCC6 pathogenic variants.

Signature of ABCC6 pathogenic variants

Over 300 distinct pathogenic variants in ABCC6 have been described (Li et al., 2019a). The types of pathogenic variants include missense, nonsense, intronic variants causing mis-splicing, small deletions and insertions, as well as large deletions. The p.R1141* variant is the most common, particularly in Caucasian individuals, with a prevalence of approximately 30% (Pfendner et al., 2007; Legrand et al., 2017). AluI-mediated deletion of exons 23–29, the second most common variant, has been found in up to 20% of the US and 12% of European patients (Pfendner et al., 2008; Le Saux et al., 2001; Legrand et al., 2017; Ringpfeil et al., 2001). By contrast, the Chinese and Japanese PXE populations harbor a unique ABCC6 variant profile distinct from that found in Caucasian patients (Jin et al., 2015; Iwanaga et al., 2017).

Several population-based studies reported variant hotspots in some geographic areas. For example, variants p.E1400K and p.R1314W are common in cases originating from North Africa, with the prevalence of approximately 10% and 9%, respectively (Legrand et al., 2017). Arginine codon 518 is a recurrently mutated amino acid, and two variants, p.R518Q and p.R518*, correspond to about 10% of the mutant alleles in North Africa and a French cohort (Legrand et al., 2017), and account for 16% of the alleles in an Italian PXE cohort (Gheduzzi et al., 2004). The missense substitution, p.R1339C, is a founder variant in PXE patients in South Africa, present in 41% of all ABCC6 alleles (Ramsay et al., 2009). The missense variant, p.R1138W, occurs at a prevalence of 46% in patients of French descent including French Canadians (LaRusso et al., 2010). Although most of these studies don’t have clearly defined ethnic background of the patient cohorts, knowledge of recurring variants in different geographic regions can facilitate an efficient and time-saving screening strategy.

ABCC6 genotype-phenotype correlation

Several attempts have been made to identify a correlation between the types of ABCC6 variants and phenotypic manifestations. In a large international PXE cohort consisting of 270 patients with Caucasians representing 94%, no correlation was established between the nature or the location of the variants in ABCC6 and the phenotype (Pfendner et al., 2007). Similarly, no obvious genotype-phenotype correlations were identified in a Japanese PXE cohort (Iwanaga et al., 2017). In contrast, in 220 French PXE cases, more severe eye and vascular phenotype in patients with loss-of-function variants was reported (Legrand et al., 2017).

Implications of ABCC6 genetic testing

ABCC6 genetic testing can be used to confirm a clinically suspected diagnosis of PXE. In 2010, biallelic pathogenic ABCC6 variants were incorporated in the updated classification system as a major diagnostic criteria (Plomp et al., 2010). As the clinical manifestations of PXE are of late onset, usually not recognized until at teen or early adulthood, ABCC6 variant analyses can be used at secondary prevention level for carrier detection and identification of PXE patients before clinical symptoms appear (Li et al., 2010; Akoglu et al., 2014). Moreover, although prenatal testing is feasible, it is rarely contemplated by the parents who consider the risk for recurrence (Sharon Terry, PXE International; personal communication).

The knowledge of specific ABCC6 variants may make the application of allele-specific precision medicine feasible. For example, premature termination codon (PTC) variants in ABCC6 account for approximately 35% of all mutant alleles. PTC124, a prototypic PTC read-through molecule, demonstrated synthesis of full-length ABCC6 protein in cell culture systems, although at low levels (Zhou et al., 2013). Amlexanox, another PTC read-through molecule that also counteracts nonsense-mediated mRNA decay (Atanasova et al., 2017), has the potential to read through PTC variants in ABCC6. Missense variants in ABCC6 often result in altered intracellular trafficking and subcellular mislocalization of the mutant protein, yet with the synthesis of the full-length protein. A chemical chaperone, 4-phenylbutyrate, has been shown to be capable of correcting the trafficking defect (Jin et al., 2015; Le Saux et al., 2011; Pomozi et al., 2014; Pomozi et al., 2017)

GENETIC FACTORS MODIFYING PXE PHENOTYPE

Modifier effects are remarkably common in humans and model organisms (Nadeau, 2001). The identification of modifier genes would help explain the clinical variability of PXE. Here, we summarize current knowledge of the genetic factors that modify PXE phenotype based on human and animal studies. It should be noted, however, that many of the studies associating variants with PXE phenotypes were performed in relatively small cohorts from restricted ethnic backgrounds. Therefore, to validate these associations, larger multiethnic cohorts need to be examined.

Candidate modifier genes in humans

Regulation of biological mineralization

Ectopic mineralization is an active process regulated by the balance of protective and inhibitory factors. The inhibitory role of matrix Gla protein (MGP) in pathological mineralization was supported by the vascular and cartilage mineralization in the Mgp knockout mice as well as in patients with Keutal syndrome carrying loss-of-function variants in MGP (Luo et al., 1997; Munroe et al., 1999). Analysis of the MGP promoter variant frequencies in a cohort of 101 patients revealed one MGP haplotype correlating with later disease onset (Hendig et al., 2008). Secreted phosphoprotein 1 (SPP1) is another mineralization inhibitor, and inactivation of SPP1 enhances vascular calcification in Mgp knockout mice (Speer et al., 2002). Three SPP1 promoter variants, c.−1748A>G, c. −155_156insG, and c.244_245insTG, were significantly more frequent in 93 PXE patients than in 93 age- and sex-matched healthy controls, suggesting that SPP1 is a secondary genetic risk factor contributing to PXE susceptibility (Hendig et al., 2007).

Pyrophosphate metabolism

Reduced plasma PPi levels underlie ectopic mineralization in PXE. It has been suggested that defects in any of the proteins involved in the formation, transport, and hydrolysis of PPi can have profound effects on the level of mineralization. A study evaluated sequence variants in alkaline phosphatase (ALP), ectonucleotide pyrophosphatase 1 (ENPP1) and ankylosis (ANKH) genes encoding pyrophosphate metabolizing proteins TNAP, ENPP1, and ANKH. The c.1190–65C>A in ALP, c.313+9G>T in ENPP1, and c.294C>T in ANKH, were significantly more frequent in 190 German PXE patients than that of 190 age- and sex-matched healthy controls (Dabisch-Ruthe et al., 2014).

Extracellular matrix remodeling and oxidative stress

Oxidative stress, elevated proteolytic activity, and increased remodeling of the extracellular matrix have been reported in PXE (Hendig et al., 2013). Variants in three genes, c. −262C>T in catalase (CAT), c.47C>T in superoxide dismutase 2 (SOD2), and c.593C>T in glutathione peroxidase 1 (GPX1), encoding essential antioxidant enzymes, were found to correlate with earlier disease onset in 117 German PXE patients (Zarbock et al., 2007). The promoter variants, c. −1575G (new nomenclature c. −1855G), c. −1306C (new nomenclature c. −1586C), c. −790T (new nomenclature c. −1070T), and the haplotype c. −1855G/c. −1586C/c. −1070T/c. −1015C/c. −448G in matrix metallopeptidase 2 (MMP2), were more abundant in 168 German PXE patients (Zarbock et al., 2010). Variations in the XYLT genes were genetic co-factors influencing the severity of PXE (Schon et al., 2006). Specifically, c.343G>T in XYLT1 was associated with higher serum xylosyltransferase activity in 65 German PXE patients. Three sequence variants in XYLT2, c.166G>A, c.1569C>T, and c.2402C>G, were found to be more frequent in PXE patients with higher organ involvement.

Angiogenesis

The choroidal neovascularization in PXE patients is mediated by vascular endothelial growth factor (VEGF) signaling, a molecule encoded by the VEGFA gene. Five VEGFA sequence variants, c. −152G>A, c. −460C>T, c. −1540A>C, c.674C>T, and c.1032C>T, showed significant association with severe retinopathy in 163 German PXE patients (Zarbock et al., 2009). Three of these variants, c. −152G>A, c. −460C>T, and c.674C>T, were replicated in an independent PXE cohort (De Vilder et al., 2020).

Cardiovascular risk

In a cohort of 119 Italian PXE patients, the association between apolipoprotein E (APOE) and methylenetetrahydrofolate reductase (MTHFR) gene variants and the severity of the cardiovascular phenotype was evaluated (Boraldi et al., 2014). The ε2 allele in APOE2 conferred a protection against the age-related increase of cardiovascular manifestations. In addition, PXE patients had a high frequency of the c.677C>T genotype in MTHFR. With age, cardiovascular manifestations were more severe in patients with c.677C>T variant. Moreover, the c.1721G>A variant in low density lipoprotein receptor (LDLR) combined with biallelic ABCC6 pathogenic variants was thought to result in severe vascular complications in a PXE patient (Pisciotta et al., 2010).

Candidate modifier genes in mice

Ectopic mineralization in Abcc6^−/− mice is affected by genetic background

Following identification of ABCC6 as the gene harboring pathogenic variants in PXE, Abcc6 “knock-out” (Abcc6^−/−) mice were developed and these mice recapitulated features of human PXE by demonstrating ectopic mineralization in the skin, eyes, and the arterial blood vessels (Klement et al., 2005; Gorgels et al., 2005). When maintained on the C57BL/6J background, mineralization of the vibrissae dermal sheath in muzzle skin, a phenotypic hallmark of the overall progression of the calcification process, can be observed as early as 5–6 weeks postnatally in the Abcc6^−/− mouse model of PXE. By contrast, the onset of mineralization in Abcc6^−/− mice, bred on the 129X1 background and housed on the same environmental and high barrier conditions, was delayed until between 3 and 4 months of age, suggesting that the genetic background played a role in modifying the mineralization process (Li and Uitto, 2010).

Mouse inbred strains with spontaneous Abcc6 hypomorphic allele have various degrees of severity of PXE

In addition to genetically engineered Abcc6^−/− mouse model of PXE, four mouse strains developed various degrees of tissue mineralization (Berndt et al., 2013; Berndt et al., 2014; Li et al., 2012; Li et al., 2014b). KK/HlJ, and to a lesser extent 129S1/SvImJ mice, had mineralization in the vibrissae dermal sheath, similar to the Abcc6^−/− mice. KK strain has also systemic mineralization. C3H/HeJ and DBA/2J had very little or no mineralization. These four inbred mouse strains harbor the same non-synonymous coding variant (rs32756904) in Abcc6 resulting in alternative pre-mRNA splicing and reduced hepatic ABCC6 protein expression (Li et al., 2012; Li et al., 2014b). Quantitative trait locus analysis (QTL) identified eight organ-specific QTLs that modify the severity of the mineralization phenotype in these mice with the same Abcc6 hypomorphic allele (Li et al., 2019b). In addition to Abcc6 which was confirmed as a major determinant for ectopic mineralization in multiple tissues, a total of nine additional candidate modifier genes were identified. Specifically, Car2 and Postn were candidate modifier genes for heart mineralization; seven genes, Abcg3, Aldh2, Chek2, Dmp1, Hnf1a, Idua, and Ttc28, were candidate modifier genes for renal tubule mineralization. Further work is necessary to replicate these findings and validate their effects on the ectopic mineralization process when juxtaposed with ABCC6 pathogenic variants. Translation of these candidate modifier genes found in mice can be tested by genotyping cohorts of patients with PXE.

ECTOPIC MINERALIZATION GENES AND GENETIC INTERACTIONS

Genotypic and phenotypic overlap between PXE and GACI

In some cases, ABCC6 pathogenic variants can cause generalized arterial calcification of infancy type 2 (GACI2; OMIM 614473) (Nitschke et al., 2012; Li et al., 2014a). GACI type 1 is caused by pathogenic variants in ENPP1 (GACI1; OMIM 208000). GACI, regardless of the type, has overlapping phenotypic features with PXE (Table 1); however, GACI is an exceedingly severe disease often characterized by prenatal vascular calcification. Most children with GACI die from cardiovascular complications within six months of life (Rutsch et al., 2008). Studies of the genotypic and phenotypic overlaps between PXE and GACI facilitated the demonstration of a unifying pathomechanism of reduced plasma levels of PPi, a powerful inhibitor of mineralization.

Table 1.

Similarities and Differences between PXE, GACI, and PXE-like Diagnoses

Characteristics	PXE	GACI	PXE/VKCFD1
Clinical spectrum
Yellowish skin papules	Yes	Rare	Yes, cutis laxa-like
Mineralization in skin lesions	Yes	Yes	Yes
Retinal angioid streaks	Yes	Rare	Yes
Vascular calcification	Yes	Yes	No
Hearing loss	No	Yes	No
Coagulation defect	No	No	Yes or No
Hypophosphatemic rickets	No	Yes, mostly survivors	No
Age at onset	Late onset	Pre- and perinatal onset	Late onset
Life expectancy	Mostly normal	Demise usually <6 months	Mostly normal
Genetic basis
Gene involved	ABCC6, ENPP1	ENPP1, ABCC6	GGCX

PXE, pseudoxanthoma elasticum; GACI, generalized arterial calcification of infancy; PXE/VKCFD1, PXE-like disorder with or without multiple coagulation factor deficiency.

GGCX-associated PXE-like phenotypes

The patients with GGCX pathogenic variants display PXE-like disorder with multiple coagulation factor deficiency (OMIM 610842) (Table 1). The GGCX gene encodes a γ-glutamyl carboxylase catalyzing γ-carboxylation of glutamyl residues in several proteins, including blood coagulation factors and matrix Gla protein (MGP) (Berkner, 2008). Fully carboxylated form of MGP prevents unwanted mineralization under normal calcium and phosphate homeostasis (Shearer, 2000). As a result of reduced γ-glutamyl carboxylase activity due to pathogenic variants in GGCX, the degree of carboxylation of MGP is reduced, promoting aberrant mineralization (Li et al., 2009c). In addition to cutaneous lesions that are similar to those in classic PXE, most patients with GGCX pathogenic variants demonstrate loose and redundant skin with loss of recoil, characteristic of cutis laxa (Uitto et al., 2013). Despite these overlapping features, vitamin K-dependent multiple coagulation factor deficiencies have been reported in some but not all patients carrying GGCX variants (Vanakker et al., 2007; Li et al., 2009c; Kariminejad et al., 2014; Dordoni et al., 2018).

Ectopic mineralization-associated genes

Besides ABCC6, ENPP1, and GGCX with overlapping phenotypes (Table 1), literature review identified 39 genes which, when mutated, resulted in ectopic mineralization phenotypes in genetically engineered mice and/or human diseases (PXE genocopies; Table 2). Dysregulated interactions between genes known to cause ectopic mineralization have been encountered. The potential interaction and synergistic effects of these genes are discussed below.

Table 2. A list of 39 genes, which when mutated in humans and/or mice, result in ectopic mineralization phenotypes (PXE genocopies).

Some of these arose or were made in mouse inbred (congenic) strain backgrounds prone to these conditions, such as C3 or 129 substrains, which carry a hypomorphic allele of Abcc6 and therefore had lesions that could interfere with interpretation of the phenotype for the gene in question (Berndt et al., 2013; Li et al., 2014c). While there are many ectopic mineralization genocopies, few can be critically compared due to variations in genetic background or lack of consistency in phenotyping.

Gene Symbol	Gene Description	Human isease‡	Sites of Ectopic Mineralization
			Human	Mouse	Reference
Systemic calcification
ABCC6	ATP-binding cassette subfamily C member 6	PXE, GACI2	skin, eye, kidney, heart, aorta	skin, muzzle skin, eye, kidney, heart, aorta	(Klement et al, 2005; Uitto et al, 2017)
AHSG	Alpha 2-HS glycoprotein	not reported	no lesions reported	skin, heart, kidney, muscle, lung, tongue	(Schafer et al, 2003)
CASR	Calcium-sensing receptor	HHC1	no lesions reported	muzzle skin, heart, aorta, kidney, muscle, tongue, testis, ovary, colon	(Hough et al, 2004)
ENPP1	Ectonucleotide pyrophosphatase/phosphodiesterase 1	GACI1	arteries	skin, muzzle skin, eye, kidney, heart, aorta, cartilage, ligament, tendon	(Rutsch et al, 2003; Sali et al, 1999)
FAM20A	FAM20A golgi associated secretory pathway pseudokinase	ERS	kidney, pulpal	eye, kidney, heart, lung, muscle, testis	(Vogel et al, 2012; Wang et al, 2013)
KL	Klotho	HFTC3	skin	skin, aorta, kidney, heart, lung, testis	(Kuro-o et al., 1997; Ichikawa et al, 2007)
TRIM24	Tripartite motif containing 24	not reported	no lesions reported	skin, muzzle skin, eye, heart, aorta, kidney, lung, tongue	(Ignat et al, 2008)
Brain calcification
CA2	Carbonic anhydrase 2	OPTB3	brain	arteries in multiple organs	(Sly et al, 1983; Spicer et al, 1989)
CTC1	CST telomere replication complex component 1	CRMCC1	brain	no lesions reported	(Anderson et al, 2012)
ISG15	ISG15 ubiquitin like modifier	Immunodeficiency 38 with basal ganglia calcification	brain	no lesions reported	(Zhang et al, 2015)
JAM2	Junction adhesion molecule 2	IBGC8	brain	no lesions reported	(Cen et al, 2020; Schottlaender et al, 2020)
JAM3	Junctional adhesion molecule 3	HDBSCC	brain	no lesions reported	(Mochida et al, 2010)
MYORG	Myogenesis regulating glycosidase	IBGC7	brain	brain	(Yao et al, 2018; Arkadir et al, 2019)
OCLN	Occludin	PTORCH1	brain	brain	(O’Driscoll et al, 2010; Saitou et al, 2000)
NRROS	Negative regulator of reactive oxygen species	SENEBAC	brain	no lesions reported	(Smith et al, 2020)
PCDH12	Protocadherin 12	DMJDS1	brain	no lesions reported	(Guemez-Gamboa et al, 2018)
PDGFB	Platelet-derived growth factor subunit B	IBGC5	brain	brain	(Keller et al, 2013)
PDGFRB	Platelet-derived growth factor receptor beta	IBGC4	brain	no lesions reported	(Nicolas et al, 2013)
SLC20A2	Solute carrier family 20 member 2	IBGC1	brain	brain	(Wang et al, 2012; Jensen et al, 2013)
SNORD118	Small nucleolar RNA, C/D box 118	LCC	brain	mouse model not available	(Jenkinson et al, 2016)
STN1	STN1 subunit of CST complex	CRMCC2	brain	mouse model not available	(Simon et al, 2016)
XPR1	Xenotropic and polytropic retrovirus receptor 1	IBGC6	brain	no lesions reported	(Legati et al, 2015)
Chondral calcification
ANKH	ANKH inorganic pyrophosphate transport regulator	CMDD	cartilage	cartilage	(Pendleton et al, 2002; Ho et al, 2000)
DDR2	Discoidin domain receptor tyrosine kinase 2	SMED-SL	larynx, trachea, choroid plexus	no lesions reported	(Bargal et al, 2009)
LBR	Lamin B receptor	GRBGD	cartilage	no lesions reported	(Waterham et al, 2003)
SLC29A1	Solute carrier family 29 member 1	not reported	no lesions reported	spinal tissues	(Warraich et al, 2013)
Vascular calcification
ADIPOQ	Adiponectin, C1Q and collagen domain containing	not reported	no lesions reported	aorta	(Luo et al, 2009)
APOE	Apolipoprotein E	Atherosclerosis	mitral valve	aorta	(Novaro et al, 2003; Sullivan et al, 1998)
ATF4	Activating transcription factor 4	Atherosclerosis	aorta	no lesions reported	(Masuda et al, 2016)
GBA	Glucosylceramidase beta	Gaucher disease, type IIIC	aorta, mitral valve	no lesions reported	(Bohlega et al, 2000)
NOTCH1	Notch receptor 1	Aortic valve disease	aortic valve	no lesions reported	(Garg et al., 2005)
SPP1	Secreted phosphoprotein 1	Atherosclerosis	aorta	aorta	(Speer et al., 2002)
TNFRSF11B	TNF receptor superfamily member 11b	PDB5	no lesions reported	aorta	(Bucay et al, 1998)
Chondral and vascular calcification
MGP	Matrix Gla protein	KTLS	aorta	aorta, cartilage	(Munroe et al., 1999; Luo et al, 1997)
NT5E	5’-nucleotidase ecto	CALJA	arteries, cartilage, ligament, tendon	cartilage, ligament, tendon	(St Hilaire et al, 2011; Li et al, 2014c)
Skin calcification
FGF23	Fibroblast growth factor 23	HFTC2	skin	heart, kidney	(Chefetz et al., 2005; Shimada et al., 2004)
GALNT3	Polypeptide N-acetylgalactosaminyltransferase 3	HFTC1	skin	no lesions reported	(Topaz et al, 2004)
GGCX	Gamma-glutamyl carboxylase	PXE/VKCFD1	skin	no lesions reported	(Vanakker et al, 2007)
SAMD9	Sterile alpha motif domain containing 9	NFTC	skin	no mouse equivalent gene	(Topaz et al, 2006)

^‡PXE, pseudoxanthoma elasticum; GACI2, generalized arterial calcification of infancy 2; HHC1, hypocalciuric hypercalcemia, type 1; GACI1, generalized arterial calcification of infancy 1; ERS, Enamel-renal syndrome; HFTC3, hyperphosphatemic familial tumoral calcinosis 3; OPTB3; osteopetrosis and renal tubular acidosis; CRMCC1, cerebroretinal microangiopathy with calcifications and cysts-1; IBGC8, idiopathic basal ganglia calcification-8; HDBSCC, hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts; IBGC7, idiopathic basal ganglia calcification-7; PTORCH1, pseudo-TORCH syndrome-1; SENEBAC, early-onset seizures with neurodegeneration and brain calcifications; DMJDS1, diencephalic-mesencephalic junction dysplasia syndrome-1; IBGC5, idiopathic basal ganglia calcification-5; IBGC4, idiopathic basal ganglia calcification-4; IBGC1, idiopathic basal ganglia calcification-1; LCC, Leukoencephalopathy with brain calcifications and cysts; CRMCC2, cerebroretinal microangiopathy with calcifications and cysts-2; IBGC6, idiopathic basal ganglia calcification-6; CMDD, craniometaphyseal dysplasia, autosomal dominant; SMED-SL, spondylo-meta-epiphyseal dysplasia, short limb-hand type; GRBGD, Greenberg skeletal dysplasia; PDB5, Paget disease of bone-5; KTLS, Keutel syndrome; CALJA, calcifications of arteries and joints; HFTC2, hyperphosphatemic familial tumoral calcinosis 2; HFTC1, hyperphosphatemic familial tumoral calcinosis 1; PXE/VKCFD1, PXE-like disorder with or without multiple coagulation factor deficiency; NFTC, normophosphatemic tumoral calcinosis.

Genetic interactions and cumulative effects of ectopic mineralization-associated genes

The possible digenic inheritance in some patients, displaying PXE-like skin features, has been noted. For example, patients carrying a heterozygous p.V255M variant in GGCX and a heterozygous p.R1141* variant in ABCC6, implied a role for genetic interaction in ectopic mineralization (Li et al., 2009a). GGCX may serve as a modifier gene that complements the loss-of-function variants in ABCC6, thus enhancing ectopic mineralization. The effects of GGCX juxtaposed to ABCC6 deficiency was further supported by experimental studies demonstrating accelerated mineralization in the Abcc6^−/−;Ggcx^+/− mice as compared to age-matched Abcc6^−/−;Ggcx^+/+ mice (Li and Uitto, 2010).

In one β-thalassemia patient presenting with severe early-onset PXE, heterozygous variants in ABCC6 and ENPP1 were identified (Omarjee et al., 2020). In three additional beta-thalassemia patients with ectopic mineralization phenotype similar to PXE, targeted exome sequencing in a panel of 19 genes involved in calcification diseases or calcium-phosphate homeostasis revealed possible genetic interactions (Boraldi et al., 2019). In addition to variants in the HBB gene causing beta-thalassemia, compound heterozygous variants were detected in ABCC6 and ENPP1, and variants were detected in other ectopic mineralization-associated genes. Thus, the genetic interaction and cumulative effects of the various variants may influence an uncommon PXE-like phenotype in these patients.

CONCLUSION

Genetic analysis in ABCC6 and other interacting genes is integral to the genetic complexity of PXE. Genetic factors in PXE are manifold and include multiple layers of biological complexity. Beyond the candidate gene approach, exome- and genome-wide next generation sequencing approaches can be used in an unbiased manner to uncover unrecognized genes for ectopic mineralization. Searching for modifier genes requires the precise clinical phenotypes to be defined to document the variability between affected individuals. Identification of genetic modifiers provides tools to improve prognosis, predict the onset and severity of PXE, and identify new targets for therapy that can be customized to the individual patients (personalized medicine). Like every disease, being able to identify people at risk before the disease appears provides the greatest opportunity to intervene and prevent the development of the life-threatening disease.

Abbreviations:

GACI

generalized arterial calcification of infancy

PPi

inorganic pyrophosphate

PTC

premature termination codon

PXE

pseudoxanthoma elasticum

QTL

quantitative trait locus