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Mutations in ANKH Cause Chondrocalcinosis
Abstract
Chondrocalcinosis (CC) is a common cause of joint pain and arthritis that is caused by the deposition of calcium-containing crystals within articular cartilage. Although most cases are sporadic, rare familial forms have been linked to human chromosomes 8 (CCAL1) or 5p (CCAL2) (Baldwin et al. 1995; Hughes et al. 1995; Andrew et al. 1999). Here, we show that two previously described families with CCAL2 have mutations in the human homolog of the mouse progressive ankylosis gene (ANKH). One of the human mutations results in the substitution of a highly conserved amino acid residue within a predicted transmembrane segment. The other creates a new ATG start site that adds four additional residues to the ANKH protein. Both mutations segregate completely with disease status and are not found in control subjects. In addition, 1 of 95 U.K. patients with sporadic CC showed a deletion of a single codon in the ANKH gene. The same change was found in a sister who had bilateral knee replacement for osteoarthritis. Each of the three human mutations was reconstructed in a full-length ANK expression construct previously shown to regulate pyrophosphate levels in cultured cells in vitro. All three of the human mutations showed significantly more activity than a previously described nonsense mutation that causes severe hydroxyapatite mineral deposition and widespread joint ankylosis in mice. These results suggest that small sequence changes in ANKH are one cause of CC and joint disease in humans. Increased ANK activity may explain the different types of crystals commonly deposited in human CCAL2 families and mutant mice and may provide a useful pharmacological target for treating some forms of human CC.
... Previous studies have also evaluated the role of anKH in heterotopic ossification (34,35). For instance, Gurley et al (34) demonstrated that the deletion of the anK gene caused progressive mineralization and joint disease, leading to stiffness of the spine, a symptom similar to AS in humans. ...
... For instance, Gurley et al (34) demonstrated that the deletion of the anK gene caused progressive mineralization and joint disease, leading to stiffness of the spine, a symptom similar to AS in humans. Moreover, loss of anKH function resulted in pathological hydroxyapatite formation (35). Consistent with these previous studies, the present findings demonstrated that the expression levels of anKH in ligaments from patients with aS were downregulated compared with the control subjects. ...
Ankylosis progressive homolog (ANKH) is associated with fibroblast ossification in ankylosing spondylitis (AS). As the human ANKH gene is poorly characterized relative to its murine counterpart, the aim of the present study was to examine ANKH expression in ligament tissue isolated from patients with AS and the role played by this gene in AS‑associated fibroblast ossification. Fibroblasts were isolated from ligament tissue collected from patients with AS and ligament tissue from individuals with spinal cord fractures, then cultured. Fibroblasts from patients with AS were subsequently transfected with an ANKH overexpression vector, while those collected from individuals with spinal cord fractures were transfected with small interfering RNA specific for ANKH. Cell viability, apoptosis and mineralization were analyzed using MTT assays, flow cytometry and Alizarin Red staining, respectively. Furthermore, ANKH mRNA and protein expression levels were analyzed using reverse transcription‑quantitative PCR and western blotting analysis, respectively. The expression levels of osteogenesis markers, including alkaline phosphatase, osteocalcin, Runt‑related transcription factor 2, c‑Myc, as well as the β‑catenin signaling protein, were also determined using western blotting. The results of the present study revealed that ANKH protein expression levels were downregulated in AS total ligament tissue extract, compared with spinal fracture ligament. Moreover, the fibroblasts derived from patients with AS exhibited an increased viability and reduced apoptosis rates, compared with the fibroblasts from patients with spinal fracture. Notably, ANKH overexpression inhibited viability, mineralization and ossification, increased the phosphorylation of β‑catenin and downregulated β‑catenin and c‑Myc protein expression levels in fibroblasts from patients with AS. In addition, ANKH overexpression increased the ratio of p‑β‑catenin/β‑catenin in fibroblasts from patients with AS. By contrast, ANKH silencing in fibroblasts from patients with spinal fracture resulted in the opposite effect. In conclusion, the findings of the present study suggested that ANKH may inhibit fibroblast viability, mineralization and ossification, possibly by regulating the Wnt/β‑catenin signaling pathway.
... Mutations in PC-1 abolish its activity in individuals with idiopathic infantile arterial calcification (Rutsch et al., 2003;Hasegawa et al., 2017). In idiopathic chondrocalcinosis, mean cartilage NTPPH activity increases to twice the normal level (Pendleton et al., 2002;Abhishek & Doherty, 2011). ANK is a transmembrane protein that transports intracellular PPi to the extracellular milieu; ANK expression is upregulated in OA cartilage (Beck, Moran & Knecht, 2003;Uzuki et al., 2014). ...
... The extracellular PPi originates either from the breakdown of nucleotide triphosphates by PC-1 or from PPi transport by the transmembrane protein ANK. Mutations in the human ANK gene have been shown to cause familial CPPD (Abhishek & Doherty, 2011), and mutations in the ANK gene are found in families with chondrocalcinosis, a disease that results in CPPD crystallite deposition in the articular cartilage (Pendleton et al., 2002;Zaka & Williams, 2005). These mutations may result in the activation of ANK and PPi transport activities, leading to increased extracellular PPi concentration and eventually CPPD formation. ...
In the progression of osteoarthritis, pathological calcification in the affected joint is an important feature. The role of these crystallites in the pathogenesis and progression of osteoarthritis is controversial; it remains unclear whether they act as a disease initiator or are present as a result of joint damage. Recent studies reported that the molecular mechanisms regulating physiological calcification of skeletal tissues are similar to those regulating pathological or ectopic calcification of soft tissues. Pathological calcification takes place when the equilibrium is disrupted. Calcium phosphate crystallites are identified in most affected joints and the presence of these crystallites is closely correlated with the extent of joint destruction. These observations suggest that pathological calcification is most likely to be a disease initiator instead of an outcome of osteoarthritis progression. Inhibiting pathological crystallite deposition within joint tissues therefore represents a potential therapeutic target in the management of osteoarthritis.
... This method had been used earlier in other studies for studying the ANKH gene mutation. [6] PCR appears to be an adequate method for studying the present mutation. We decided to choose the sample size of 25 Khatri patients of primary knee OA because in the previous study, [4] almost 4% of patients with clinically identified sporadic CC were homozygous for the similar mutation, which was 8-fold more common than in normal controls. ...
... In a study by Adrian Pendleton et al., a positive association was found between ANKH gene mutation and CC. [6] Although a very rare occurrence, a familial predisposition of ANKH gene mutation is reported from several countries and different ethnic groups. [7][8][9][10] Limitations of our study: First, the sample size was quite small to detect this mutation. ...
Method:
A cohort study was carried out for a year to evaluate the presence of G-to-A transition in 5'-untranslated region of ankylosis human (ANKH) gene in Indian Khatri patients (closely resembling Europeans of primary knee osteoarthritis (OA), residing in Lucknow, India.
Results:
In the total participants, 25 were Khatri primary knee OA patients (cases) residing in Lucknow and 101 were random blood donors' samples (controls) collected from a blood bank. All were studied for the abovementioned mutation using real-time polymerase chain reaction (RT-PCR). GG genotype was present in 72.3% of controls and 76% of Khatri knee OA patients. The studied G-to-A mutation was found to be positive in 24.8% of controls and 16% of cases, odds ratio (95% confidence interval) being 0.6 (0.19-1.98, P = 0.42). The frequency of AA (D) genotype found around 3% (cases) and 8% (controls) with P value of 0.70. The combined frequency of both homozygous and heterozygous mutation (GA and AA) in the studied population was 28 (27.7%) in controls and 6 (24%) in cases with the odds ratio (OD) ratio of 0.82 (0.29-2.27, P = 0.70). No significant differences were observed at both genotype and allelic level in the distribution of ANKH-4 G-to-A gene polymorphism in studied subjects.
Conclusion:
This study did not show any significant G to A mutation in the studied subjects.
... Their role in calcification has become clear in relation to calcification pathologies. Ankh mutations result in the development of chondrocalcinosis [10], while mutations in the Abcc6 result in development of pseudoxanthoma elasticum [11]. In contrast, Pi generation by chondrocytes is partially regulated by alkaline phosphatase (ALP). ...
The pathological relevance of articular cartilage calcification in osteoarthritis (OA) is becoming increasingly evident. We are only beginning to understand the pathobiological mechanisms that contribute to articular cartilage calcification in OA. How molecular environmental factors interact with calcification mechanisms is poorly explored. In this study, we developed an in vitro phosphate-driven calcification model for human OA articular chondrocytes, in which these cells are cultured in the presence of calcification medium containing adenosine triphosphate (ATP) and β-glycerophosphate (BGP). We employed this model to investigate the role of transforming growth factor β (TGF-β) in chondrocyte calcification. Chondrocyte culture in calcification medium resulted in mineral nodule formation over a time course of 7 days. The presence of calcium and phosphate deposition in these nodules was validated with von Kossa staining, scanning electron microscopy–energy-dispersive X-ray spectroscopy (SEM–EDX), and colorimetric calcium and phosphate assays. Supplementation of calcification medium with TGF-β resulted in enhanced nodule formation with a different morphology and changed the expression of extracellular matrix-related genes such as collagen type I and III. In conclusion, we developed a new in vitro model for human OA articular chondrocyte calcification, in which we demonstrated a pro-calcifying role for TGF-β. This in vitro model may be used as a basis to aid the investigation of the influence of environmental factors on chondrocyte calcification and the development of new anti-calcification disease-modifying osteoarthritis drugs.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00223-025-01365-x.
... 43 Patients with familial chondrocalcinosis type 2 (CCAL2) carrying mutations in ANKH suffer from gout-like joint pain due to calcium pyrophosphate dihydrate (CPPD) crystal deposits. 44 A missense mutation in ANKH (L244S) leads to mental retardation, spinal ankylosis, and periarticular calcifications of small joints in homozygous patients and a mild arthropathy in heterozygous individuals. 45 Ankylosing spondylitis, genetically linked to ANKH, is characterized by chronic joint and entheseal inflammation, and ankylosis of axial and peripheral joints. ...
Craniometaphyseal dysplasia (CMD) is a rare genetic bone disorder, characterized by progressive thickening of craniofacial bones and flared metaphyses of long bones. Craniofacial hyperostosis leads to the obstruction of neural foramina and neurological symptoms such as facial palsy, blindness, deafness, or severe headache. Mutations in ANKH (mouse ortholog ANK), a transporter of small molecules such as citrate and ATP, are responsible for autosomal dominant CMD. Knock-in (KI) mice carrying an ANKF377del mutation (AnkKI/KI) replicate many features of human CMD. Pyrophosphate (PPi) levels in plasma are significantly reduced in AnkKI/KI mice. PPi is a potent inhibitor of mineralization. To examine the extent to which restoration of circulating PPi levels may prevent the development of a CMD-like phenotype, we treated AnkKI/KI mice with the recombinant human ENPP1-Fc protein IMA2a. ENPP1 hydrolyzes ATP into AMP and PPi. Male and female Ank+/+ and AnkKI/KI mice (n ≥ 6/group) were subcutaneously injected with IMA2a or vehicle weekly for 12 wk, starting at the age of 1 wk. Plasma ENPP1 activity significantly increased in AnkKI/KI mice injected with IMA2a (Vehicle/IMA2a: 28.15 ± 1.65/482.7 ± 331.2 mOD/min; p <.01), which resulted in the successful restoration of plasma PPi levels (Ank+/+/AnkKI/KI vehicle treatment/AnkKI/KI IMA2a: 0.94 ± 0.5/0.43 ± 0.2/1.29 ± 0.8 μM; p <.01). We examined the skeletal phenotype by X-Ray imaging and μCT. IMA2a treatment of AnkKI/KI mice did not significantly correct CMD features such as the abnormal shape of femurs, increased bone mass of mandibles, hyperostotic craniofacial bones, or the narrowed foramen magnum. However, μCT imaging showed ectopic calcification near basioccipital bones at the level of the foramen magnum and on joints of AnkKI/KI mice. Interestingly, IMA2a treatment significantly reduced the volume of calcified nodules at both sites. Our data demonstrate that IMA2a is sufficient to restore plasma PPi levels and reduce ectopic calcification but fails to rescue skeletal abnormalities in AnkKI/KI mice under our treatment conditions.
... 30 Impairment of the ANKH gene leads to excessive mineralization, including calcification of arteries leading to joint pain, arthritis, atherosclerosis, and diabetes. 31,32 Together, this suggests that the prolonged cognitive health in centenarians may be supported by maintained vasculature and low pathology load in the brain. ...
BACKGROUND
Alzheimer's disease (AD) prevalence increases with age, yet a small fraction of the population reaches ages > 100 years without cognitive decline. We studied the genetic factors associated with such resilience against AD.
METHODS
Genome‐wide association studies identified 86 single nucleotide polymorphisms (SNPs) associated with AD risk. We estimated SNP frequency in 2281 AD cases, 3165 age‐matched controls, and 346 cognitively healthy centenarians. We calculated a polygenic risk score (PRS) for each individual and investigated the functional properties of SNPs enriched/depleted in centenarians.
RESULTS
Cognitively healthy centenarians were enriched with the protective alleles of the SNPs associated with AD risk. The protective effect concentrated on the alleles in/near ANKH, GRN, TMEM106B, SORT1, PLCG2, RIN3, and APOE genes. This translated to >5‐fold lower PRS in centenarians compared to AD cases (P = 7.69 × 10⁻⁷¹), and 2‐fold lower compared to age‐matched controls (P = 5.83 × 10⁻¹⁷).
DISCUSSION
Maintaining cognitive health until extreme ages requires complex genetic protection against AD, which concentrates on the genes associated with the endolysosomal and immune systems.
Highlights
Cognitively healthy cent enarians are enriched with the protective alleles of genetic variants associated with Alzheimer's disease (AD).
The protective effect is concentrated on variants involved in the immune and endolysosomal systems.
Combining variants into a polygenic risk score (PRS) translated to > 5‐fold lower PRS in centenarians compared to AD cases, and ≈ 2‐fold lower compared to middle‐aged healthy controls.
... PPi is produced either: (A) directly by ectoenzymes that generate Pi (pyrophosphatases and adenosine triphosphatases) and PPi [nucleoside triphosphate pyrophosphohydrolase (NTPPPH)] on apoptotic particles and matrix vesicles (MVs) membrane surfaces or (B) indirectly as a metabolic byproduct of multiple biochemical and biosynthetic reactions during synthesis and secretion of matrix proteins [38]. The three major regulators of PPi homeostasis are: (A) ectonucleotide pyrophosphatase/phosphodiesterase (PDE) 1 (ENPP1), an ubiquitous type II transmembrane glycoprotein with enzymatic activity, which produces PPi from ATP, inhibiting PPi related calcification and mineralization [39]; (B) tissue non-specific ALP (TNAP), an isozyme of a family of four homologous human ALP genes, presented in the MVs and with the ability to hydrolyze PPi [40]; and (C) ankylosis progressive homolog (ANKH), a multiple-pass transmembrane protein which is involved in the transport of PPi across plasma membrane to the extra cellular compartment. Mutations in ANKH can be the cause of the abnormal deposition of Ca PPi crystals in the cartilage [41]. ...
Bone formation is a complex process that occurs throughout life, and is normally limited to the skeletal system. In bone formation, osteoprogenitor cells follow several developmental stages, including differentiation in osteoblasts, proliferation, matrix maturation, and mineralization. The mechanisms involved in the mineralization process of bone, such as in the new bone formation, are extremely complex and have been under intense investigation for many years. Bone formation follows two distinct processes, intramembranous and endochondral ossification; both are regulated by signaling pathways involving numerous genes. Disturbance of these signaling pathways may cause a large spectrum of skeletal diseases characterized by new bone formation and bone growth anomalies. This review will only focus on the key genetic pathways involved in heterotopic bone formation. Wingless/integrated (Wnt), hedgehog (HH), and transforming growth factor beta (TGFβ)/bone morphogenetic protein (BMP) signaling pathways are described and illustrated; their relation with new bone formation is demonstrated through their involvement in bone formation disorders.
... [25] Impairment of the ANKH gene leads excessive mineralization, including calcification of arteries leading to joint pain, arthritis, atherosclerosis and diabetes. [26,27] Together, this suggests that the prolonged cognitive health in centenarians may be supported by maintained vasculature and low pathology load in brain. ...
Alzheimer's Disease (AD) prevalence increases with age, yet a small fraction of the population reaches ages beyond 100 years without cognitive decline. We aimed to uncover the genetic factors associated with such resilience against AD.
Genome-Wide-Association-Studies (GWAS) identified 86 single-nucleotide-polymorphisms (SNPs) associated with AD-risk. We studied each SNP in 2,281 AD-cases, 3,165 middle-aged population controls, and 346 cognitively healthy centenarians, and we combined SNPs into Polygenic Risk Scores (PRS) for each individual. Finally, we investigated the functional properties of the SNPs enriched/depleted in centenarians using snpXplorer.
Centenarians were depleted with risk-increasing AD-SNPs and enriched with protective AD-SNPs. The PRS was more than 5-fold lower in centenarians compared to AD cases (p=7.69x10-71) and almost 2-fold lower compared to middle-aged population controls (p=5.83x10-17). The strongest protection was found in ANKH, GRN, TMEM106B, SORT1, EPDR1, PLCG2, RIN3, CD2AP, and APOE associated alleles. As expected, the genetic protection was diluted in the offspring of the centenarians.
Becoming a cognitively healthy centenarian is associated with a complex genetic protection against AD, which concentrates on an advantageous functioning of the endo-lysosomal and immune systems, and their effect on amyloid-clearance.
... CPPD cases of genetic (familial) type are notably rare, autosomal dominant, and mostly associated with early onset, before the age of 55 years [10]. Multiple studies have identified familial CPPD to have genetic linkages to A1 B12 DR3 human leukocyte antigen (HLA) haplotype and chromosomal region 8q, previously dubbed chondrocalcinosis 1 or chondrocalcinosis with early-onset osteoarthritis (CCAL1) type, and chromosomal region 5p5.1 referred to as chondrocalcinosis 2 (CCAL2) type [13][14][15]. Specific HLA-associated gene mutations and chromosome 8q-linked CCAL1 are still unknown. However, CCAL2 is reportedly mediated by mutations in the progressive ankylosis protein homolog human gene (ANKH) [10,16]. ...
Background: The coexistence of calcium pyrophosphate dihydrate crystal deposition (CPP) and synovial chondromatosis (SC) in the temporomandibular joint (TMJ) is rarely reported. CPP disease (CPPD) is complex arthritis synonymous with excessive pyrophosphate production and variable aberrations in mineral and organic phase metabolism of the joint cartilage, leading to local inundated CPP and crystal deposition of partially deciphered predispositions. Meanwhile, SC is a rare benign synovial joint proliferative disease of unclear etiology and has a low risk of malignant
transformation. However, SC manifests severe joint disability and dysfunction because of connective tissue metaplasia of the synovial membrane, which forms cartilaginous nodules with or without calcifications or ossifications. These nodules often detach and form intra-articular loose bodies and very rarely within extraarticular spaces.
Case presentation: We report the case of a 61-year-old man to expand the body of literature on these unusual coexisting arthropathies of the TMJ. The patient presented to our hospital in 2020 with complaints of pain in the right TMJ
and trismus for over 6 months. Radiographic assessments of the TMJ provided a preoperative provisional diagnosis of SC. However, the histopathology of the open biopsy revealed tumor-like lesions comprising several deposits of rhomboid and rod-shaped crystals that displayed positive birefringence in polarized light, confirming a coexistence of CPPD. A second-stage operation was performed for the complete removal of the loose bodies and chalk-like lesions including synovectomy. No evidence of recurrence was recorded after a follow-up of nearly 1.5 years.
Conclusions: Isolated CPPD and SC of the TMJ are prevalent in the literature however, monoarticular coexistence of these diseases is rare, due to the lack of consistency in the diagnostic criteria in clinical practice. Moreover, optimal
treatment depends on several considerations. This report delineated the molecular etiopathology and underscored the need for continued deciphering of the causal mechanisms of coexisting CPPD and SC of the TMJ. In addition, the importance of confirmatory testing for accurate diagnosis, and appropriate management of these diseases were discussed.
... In the context of calcification, regulation of extracellular PP i and P i levels by ENPP1, progressive ankylosis protein homologue (ANK), TNAP, PiT-1 and PiT-2 ( Fig. 1, upper panel) has a major role. ENPP1 overactivity 124 , gain-of-function mutations in the gene encoding ANK [125][126][127][128] or ANK protein overexpression 129 were found in patients with chondrocalcinosis together with increased PP i levels. If, on the one hand, PP i is an inducer of CPP crystal formation, on the other hand it strongly inhibits the nucleation and growth of BCP crystals, by coating their surface and preventing further apposition of Ca 2+130 . ...
Pathological calcification of cartilage is a hallmark of osteoarthritis (OA). Calcification can be observed both at the cartilage surface and in its deeper layers. The formation of calcium-containing crystals, typically basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP) crystals, is an active, highly regulated and complex biological process that is initiated by chondrocytes and modified by genetic factors, dysregulated mitophagy or apoptosis, inflammation and the activation of specific cellular-signalling pathways. The links between OA and BCP deposition are stronger than those observed between OA and CPP deposition. Here, we review the molecular processes involved in cartilage calcification in OA and summarize the effects of calcium crystals on chondrocytes, synovial fibroblasts, macrophages and bone cells. Finally, we highlight therapeutic pathways leading to decreased joint calcification and potential new drugs that could treat not only OA but also other diseases associated with pathological calcification.
... This rare skeletal condition is characterized by abnormal bone formation, with a higher density of the craniofacial bones and abnormal modeling of the metaphyses of long bones, and is associated with lower extracellular pyrophosphate concentration. In contrast, mutations in sequences that encode the N-and C-termini of the ANK protein cause chondrocalcinosis [72]. This articular cartilage disorder is radiographically characterized by the deposition of calcium pyrophosphate dihydrate crystals in joints and is associated with a high extracellular pyrophosphate concentration. ...
Conventionally, ATP is considered to be the principal energy source in cells. However, over the last few years, a novel role for ATP as a potent extracellular signaling molecule and the principal source of extracellular pyrophosphate, the main endogenous inhibitor of vascular calcification, has emerged. A large body of evidence suggests that two principal mechanisms are involved in the initiation and progression of ectopic calcification: high phosphate concentration and pyrophosphate deficiency. Pathologic calcification of cardiovascular structures, or vascular calcification, is a feature of several genetic diseases and a common complication of chronic kidney disease, diabetes, and aging. Previous studies have shown that the loss of function of several enzymes and transporters involved in extracellular ATP/pyrophosphate metabolism is associated with vascular calcification. Therefore, pyrophosphate homeostasis should be further studied to facilitate the design of novel therapeutic approaches for ectopic calcification of cardiovascular structures, including strategies to increase pyrophosphate concentrations by targeting the ATP/pyrophosphate metabolism cycle.
... ANKH is one of the key regulators in the process of localized mineralization in tissues [23]. ANKH is associated with chondrocalcinosis [24] and heterotopic ossification [16]. In addition, the inhibition of ANKH facilitates pathological mineralization and has been associated with osteogenic differentiation [25,26]. ...
Background
MicroRNAs (miRNAs) may participate in the process of vascular calcification. However, the role of microRNA-17-5p in vascular calcification has not been clarified. In this study, we showed the effects of microRNA-17-5p on vascular calcification.
Materials and Methods
Vascular smooth muscle cells (VSMCs) were transfected with miR-17-5p mimics, an miR-17-5p inhibitor or a negative control (NC) using Lipofectamine 2000. Then the cells were induced by an osteogenic medium. Alkaline phosphatase (ALP) activity and mineralization were determined. Osteocalcin (OC), bone morphogenetic protein 2(BMP-2), Col1agren Ia (Colla), Runx2 and ankylosis protein homolog (ANKH) gene expressions were determined by reverse transcription-polymerase chain reaction. Vascular calcification was developed using a renal failure model.
Results
The ALP activity was increased when miR-17-5p mimics were transfected, whereas the miR-17-5p inhibitor reduced ALP activity (p < 0.05). The number and average area of mineral node in miR-17-5p mimics group were larger than those in corresponding control and NC groups (p < 0.05). The number and average area of the mineral nodes in the miR-17-5p inhibitor group were smaller than those in corresponding control and NC groups (p < 0.05). Bmp2, OC, Col1a and Runx2 were higher in the miR-17-5p mimics group compared to those in the control and NC groups. ANKH expression was decreased in VSMCs with the miR-17-5p mimics and increased in VSMCs with miR-17-5p inhibitor. ANKH siRNA intervention also promoted mineralization. The miR-17-5p expression was upregulated and ANKH was down-regulated in the aortic arteries with calcification.
Conclusion
Our data showed that miR-17-5p may promote vascular calcification by inhibiting ANKH expression.
... (7) Nonfunctional ecto-5 0 -nucleotidase results in arterial calcification due to deficiency of CD73 (ACDC), (30) and inactive TNAP causes hypophosphatasia (HOPS). (12) Absence of ANKH activity results in chondrocalcinosis type 2 (CCAL2) (31) or craniometaphyseal dysplasia (CMD). (32) small increase substantially contributed to the reduction in plasma PPi concentrations detected in ank mice, however, as a more than 30-fold increase in plasma alkaline phosphatase activity detected in TNAP transgenic mice was found to, surprisingly, not affect plasma PPi concentrations. ...
The plasma membrane protein Ankylosis Homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints. We recently showed that when overproduced in HEK293 cells, ANKH mediates release of large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. ATP is converted extracellularly into PPi and AMP by the ectoenzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANKH. We now addressed the question if PPi leaves cells via ANKH using HEK293 cells that completely lack ENPP1. Introduction of ANKH in these ENPP1‐deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi seen in ENPP1‐proficient cells. Ank‐activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1−/− mice contained <2.5% of the PPi found in bones of wild type mice, showing that Enpp1‐activity is also a prerequisite for Ank‐dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1‐mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60–70% of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification, therefore do so by extruding NTPs rather than PPi itself. This article is protected by copyright. All rights reserved.
... The best clinical examples of such a mechanism are gain-of-function mutations in the ANKH protein seen in some cases of familial CPPD. ANKH is present in normal chondrocytes, and ANKH gain of function increases levels of the PPi precursor, ATP in cartilage [12]. ...
Objectives
Arthritis is a common clinical manifestation of hereditary hemochromatosis (HH), and HH is one of a handful of conditions linked to calcium pyrophosphate deposition (CPPD) in joints. The connection between these two types of arthritis has not yet been fully elucidated. In light of new pathogenic pathways recently implicated in CPPD involving bone, we reviewed the literature on the etiology of hemochromatosis arthropathy (HHA) seeking shared pathogenic mechanisms.
Results
Clinical observations reinforce striking similarities between HHA and CPPD even in the absence of CPP crystals. They share a similar joint distribution, low grade synovial inflammation, and generalized bone loss. Excess iron damages chondrocytes and bone cells in vitro. While direct effects of iron on cartilage are not consistently seen in animal models of HH, there is decreased osteoblast alkaline phosphatase activity, and increased osteoclastogenesis. These abnormalities are also seen in CPPD. Joint repair processes may also be impaired in both CPPD and HHA.
Conclusions
Possible shared pathogenic pathways relate more to bone and abnormal damage/repair mechanisms than direct damage to articular cartilage. While additional work is necessary to fully understand the pathogenesis of arthritis in HH and to firmly establish causal links with CPPD, this review provides some plausible hypotheses explaining the overlap of these two forms of arthritis.
... Notably, in humans, mutations in the channel core of progressive ankylosis protein cause craniometaphyseal dysplasia, a rare skeletal condition of abnormal bone formation characterized by an increased density of craniofacial bones and abnormal modeling of the metaphysis of the tubular bones [79,80]. Moreover, mutations in the N-and C-terminus of the progressive ankylosis protein cause chondrocalcinosis, a disease of articular cartilage that is radiographically characterized by the deposition of calcium pyrophosphate dihydrate crystals in the joints [81][82][83]. Craniometaphyseal dysplasia is associated with decreased extracellular pyrophosphate levels, whereas chondrocalcinosis is associated with an increase in the amount of pyrophosphate in the extracellular space, which induces the spontaneous formation of calcium pyrophosphate crystals. ...
Cardiovascular complications due to accelerated arterial stiffening and atherosclerosis are the leading cause of morbimortality in Western society. Both pathologies are frequently associated with vascular calcification. Pathologic calcification of cardiovascular structures, or vascular calcification, is associated with several diseases (for example, genetic diseases, diabetes, and chronic kidney disease) and is a common consequence of aging. Calcium phosphate deposition, mainly in the form of hydroxyapatite, is the hallmark of vascular calcification and can occur in the medial layer of arteries (medial calcification), in the atheroma plaque (intimal calcification), and cardiac valves (heart valve calcification). Although various mechanisms have been proposed for the pathogenesis of vascular calcification, our understanding of the pathogenesis of calcification is far from complete. However, in recent years, some risk factors have been identified, including high serum phosphorus concentration (hyperphosphatemia) and defective synthesis of pyrophosphate (pyrophosphate deficiency). The balance between phosphate and pyrophosphate, strictly controlled by several genes, plays a key role in vascular calcification. This review summarizes the current knowledge concerning phosphate and pyrophosphate homeostasis, focusing on the role of extracellular pyrophosphate metabolism in aortic smooth muscle cells and macrophages.
... It is caused by gain of function mutations in the ANKH gene at location 5p15.2. The ANKH gene codes for a transmembrane protein that is expressed in joints and other tissues, its absence causes chondrocalcinosis [87]. Its function affects intra and extracellular levels of PPi. ...
Osteoarthritis (OA) is one of the most prevalent conditions in the world, particularly in the developed world with a significant increase in cases and their predicted impact as we move through the twenty-first century and this will be exacerbated by the covid pandemic. The degeneration of cartilage and bone as part of this condition is becoming better understood but there are still significant challenges in painting a complete picture to recognise all aspects of the condition and what treatment(s) are most appropriate in individual causes. OA encompasses many different types and this causes some of the challenges in fully understanding the condition.
There have been examples through history where much has been learnt about common disease(s) from the study of rare or extreme phenotypes, particularly where Mendelian disorders are involved. The often early onset of symptoms combined with the rapid and aggressive pathogenesis of these diseases and their predictable outcomes give an often-under-explored resource. It is these “rarer forms of disease” that William Harvey referred to that offer novel insights into more common conditions through their more extreme presentations. In the case of OA, GWAS analyses demonstrate the multiple genes that are implicated in OA in the general population. In some of these rarer forms, single defective genes are responsible.
The extreme phenotypes seen in conditions such as Camptodactyly Arthropathy-Coxa Vara-pericarditis Syndrome, Chondrodysplasias and Alkaptonuria all present potential opportunities for greater understanding of disease pathogenesis, novel therapeutic interventions and diagnostic imaging. This review examines some of the rarer presenting forms of OA and linked conditions, some of the novel discoveries made whilst studying them, and findings on imaging and treatment strategies.
... The CEP calcification associated with the severity of IVD degeneration [87] causes the permeability of CEP to decline, and nutrients and metabolic waste in the IVD may cause the occurrence and progression of IVD degeneration through an increase in HIF-1α [88]. Previous studies have shown that mutations in ankylosis protein homolog gene (ANK), a pyrophosphate transporter, could result in abnormal dystrophic mineralization in joints and bone [89,90] and the hypoxic status may influence ANK expression, which may be mediated by HIF-1α. HIF-1α controls the dystrophic mineralization of NP cells by suppressing ANK expression [29]. ...
The intervertebral disc (IVD) is a complex joint structure comprising three primary components—namely, nucleus pulposus (NP), annulus fibrosus (AF), and cartilaginous endplate (CEP). The IVD retrieves oxygen from the surrounding vertebral body through CEP by diffusion and likely generates ATP via anaerobic glycolysis. IVD degeneration is characterized by a cascade of cellular, compositional, structural changes. With advanced age, pronounced changes occur in the composition of the disc extracellular matrix (ECM). NP and AF cells in the IVD possess poor regenerative capacity compared with that of other tissues. Hypoxia-inducible factor (HIF) is a master transcription factor that initiates a coordinated cellular cascade in response to a low oxygen tension environment, including the regulation of numerous enzymes in response to hypoxia. HIF-1α is essential for NP development and homeostasis and is involved in various processes of IVD degeneration process, promotes ECM in NP, maintains the metabolic activities of NP, and regulates dystrophic mineralization of NP, as well as angiogenesis, autophagy, and apoptosis during IVD degeneration. HIF-1α may, therefore, represent a diagnostic tool for early IVD degeneration and a therapeutic target for inhibiting IVD degeneration
... Another genetic condition affecting pyrophosphate metabolism is a gain-of-function mutation of the progressive ankylosis protein homologue (ANKH) gene, which encodes the PP i transport regulator and causes several variants of familial autosomal dominant CPPD deposits [36]. ANKH gene mutations cause familial chondrocalcinosis 2 leading to high extracellular PP i resulting in CPPD [37,38]. The periarticular nature of periarticular calcifications in ACDC vs the intraarticular CPPD deposition in ANKH mutant patients highlights how mutations of various proteins controlling adenosine and phosphate metabolism may result in different clinical phenotypes. ...
Objective
Arterial calcification due to deficiency of CD73 (ACDC) is a hereditary autosomal recessive ectopic mineralization syndrome caused by loss-of-function mutations in the 5'-nucleotidase Ecto (NT5E) gene. Periarticular calcification has been reported but the clinical characterization of arthritis as well as the microstructure and chemical composition of periarticular calcifications and synovial fluid crystals has not been systematically investigated.
Methods
Eight ACDC patients underwent extensive rheumatological and radiological evaluation over a period of 11 years. Periarticular and synovial biopsies were obtained from four patients. Characterization of crystal composition was evaluated by compensated polarized light microscopy, Alizarin red staining for synovial fluid along with x-ray diffraction and x-ray micro tomosynthesis for periarticular calcification.
Results
Arthritis in ACDC patients has a clinical presentation of mixed erosive-degenerative joint changes with a median onset of articular symptoms at 17 years of age and progresses over time to the development of fixed deformities and functional limitations of small peripheral joints with eventually, larger joint and distinct axial involvement later in life. We have identified calcium pyrophosphate (CPP) and calcium hydroxyapatite (CHA) crystals in synovial fluid specimens and determined that CHA crystals are the principal component of periarticular calcifications.
Conclusion
This is the largest study in ACDC patients to describe erosive peripheral arthropathy and axial enthesopathic calcifications over a period of 11 years and the first to identify the composition of periarticular calcifications and synovial fluid crystals. ACDC should be considered among the genetic causes of early-onset osteoarthritis, as musculoskeletal disease signs may often precede vascular symptoms.
... Mitations in the human gene have t)een identified in individuals with chondrcx^alcinosis (Poidleton et al., 2002;Williams et al., 2002) which is caused by deposition of calciumcottaining crystals within articular cartilage and results in joint pain and arthritis. These mitations are found in families with autosomal dominantly inherited disease and in vitro stuiies show that this is a result of increased ANKH function (Pendleton et al., 2002). ...
Hereditary retinal disease is a significant cause of visual loss throughout the world. The underlying causes are, however, extremely varied. One group of these disorders preferentially affects the photoreceptors that are essential for colour vision and vision in normal daylight. These are the cone and cone-rod dystrophies which form part of a heterogeneous group of retinal dystrophies. Compared to diseases such as retinitis pigmentosa, which preferentially affect the rod photoreceptors, this group of disorders has been relatively poorly studied. Molecular genetic studies to date have identified mutations in eight different genes causing cone dystrophy. Many more, however, remain to be discovered. A number of families with different classes of cone, cone-rod and macular dystrophy were studied at a molecular genetic level. Mutation screening of genes previously implicated in achromatopsia identified new mutations and revealed a subset of individuals for whom no mutations could be found and, thus, a basis for further study of the heterogeneity of this disease. This has led to the identification of a novel gene for achromatopsia. A similar approach for a group of patients with blue cone monochromacy indicated the genetic basis of disease in all individuals studied. Candidate gene analysis in a group of patients with an X-linked cone dystrophy associated with colour vision deficiency suggested a novel locus for this disease. Linkage analysis in three families with macular disease identified three novel linkages. Screening for mutations in a novel candidate gene in a family with a cone-rod dystrophy (CORD7) identified a putative disease-causing mutation that implicates a new pathway in retinal disease. This thesis extends the breadth of knowledge on the molecular basis of retinal diseases. This is important since the clinical management of a particular disorder will benefit from a precise knowledge of the gene mutation involved and with the development of disease therapy, this research will lay the foundation for utilising novel treatment strategies in the future.
... It should be noted that over 20% symptoms in SCs share more than 90 genes on average. For example, symptoms in SC 7 (knee joint pain, shoulder pain, hip pain, elbow pain, joint pain, etc.) share ANKH [56], COL9A1 [57], ACAN [58], FBLN2 [59], NT5E [60], COMP [61], etc. The above results significantly show that the symptoms within SCs tend to share more genes than random trials. ...
... Diffuse ectopic calcifications, located in all kinds of soft tissues, such as the arteries, myocardium, joints, liver and brain, are often the result of mutations in genes encoding for/or involved in the production of calcification inhibitors (MGP [140], pyrophosphate [141][142][143]), or proteins involved in phosphate regulation [144][145][146]. Data regarding the role of sclerostin during this ectopic calcification process are very limited. ...
Sclerostin, a 22-kDa glycoprotein that is mainly secreted by the osteocytes, is a soluble inhibitor of canonical Wnt signaling. Therefore, when present at increased concentrations, it leads to an increased bone resorption and decreased bone formation. Serum sclerostin levels are known to be increased in the elderly and in patients with chronic kidney disease. In these patient populations, there is a high incidence of ectopic cardiovascular calcification. These calcifications are strongly associated with cardiovascular morbidity and mortality. Although data are still controversial, it is likely that there is a link between ectopic calcification and serum sclerostin levels. The main question, however, remains whether sclerostin exerts either a protective or deleterious role in the ectopic calcification process.
... E490del (p.Glu490del, NM_054027.4:c.1468_1470delGAG) deregulated TNAP activity 9,10 . CMD related mutants have largely been restricted to clinical case studies. ...
ANKH mutations are associated with calcium pyrophosphate deposition disease and craniometaphyseal dysplasia. This study investigated the effects of these ANKH mutants on cellular localisation and associated biochemistry. We generated four ANKH overexpression-plasmids containing either calcium pyrophosphate deposition disease or craniometaphyseal dysplasia linked mutations: P5L, E490del and S375del, G389R. They were transfected into CH-8 articular chondrocytes and HEK293 cells. The ANKH mutants dynamic differential localisations were imaged and we investigated the interactions with the autophagy marker LC3. Extracellular inorganic pyrophosphate, mineralization, ENPP1 activity expression of ENPP1, TNAP and PIT-1 were measured. P5L delayed cell membrane localisation but once recruited into the membrane it increased extracellular inorganic pyrophosphate, mineralization, and ENPP1 activity. E490del remained mostly cytoplasmic, forming punctate co-localisations with LC3, increased mineralization, ENPP1 and ENPP1 activity with an initial but unsustained increase in TNAP and PIT-1. S375del trended to decrease extracellular inorganic pyrophosphate, increase mineralization. G389R delayed cell membrane localisation, trended to decrease extracellular inorganic pyrophosphate, increased mineralization and co-localised with LC3. Our results demonstrate a link between pathological localisation of ANKH mutants with different degrees in mineralization. Furthermore, mutant ANKH functions are related to synthesis of defective proteins, inorganic pyrophosphate transport, ENPP1 activity and expression of ENPP1, TNAP and PIT-1.
... This rare skeletal condition is characterized by abnormal bone formation, with increased craniofacial bone density and abnormal modeling of the tubular bone metaphyses and is also associated with decreased extracellular pyrophosphate levels. By contrast, mutations in the sequences that encode the N-and C-termini of the ANK protein can cause chondrocalcinosis [78]. This articular cartilage disorder is characterized by deposition of calcium pyrophosphate dihydrate crystals in the joints, and is associated with increased pyrophosphate levels in the extracellular space [79,80]. ...
Cardiovascular complications due to accelerated atherosclerosis and arterial stiffening are the leading cause of morbidity and mortality in the Western society. Both pathologies are frequently associated with vascular calcification. Deposits of calcium phosphate salts, mainly in form of hydroxyapatite, is the hallmark of vascular calcification. Calcification is frequently observed in atherosclerotic lesions (intimal calcification), associated with vascular smooth muscle cells (VSMCs) and macrophages. By contrast, medial calcification, occurring in the elastic region of the arteries, is almost exclusively associated with VSMCs, and is common in arteriosclerosis related to aging, diabetes, and chronic kidney disease. In extracellular fluids, a range of endogenous low- and high-molecular weight calcification inhibitors are present, including osteopontin, matrix-Gla proteins and Fetuin A. Moreover, pyrophosphate deficiency plays a key role in vascular calcification. Pyrophosphate is produced by extracellular hydrolysis of ATP and is degraded to phosphate by tissue non-specific alkaline phosphatase. Loss of function in the enzymes and transporters involved in the extracellular pyrophosphate metabolism leads to excessive deposition of calcium-phosphate salts. This review summarizes the current knowledge about endogenous mechanisms of protection against calcification in the aortic wall, focusing on the role of extracellular pyrophosphate metabolism in vascular smooth muscle cells and macrophages.
... Progressive ankylosis CMDD cartilage cartilage (Pendleton et al., 2002), (Sweet and Green, 1981), (Villa-Bellosta et al., 2011), (Ho et al., 2000) Apoe ...
Pseudoxanthoma elasticum, a prototype of heritable multisystem ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter, ABCC6. The phenotypic spectrum of pseudoxanthoma elasticum varies, and the correlation between genotype and phenotype has not been established. To identify genetic modifiers, we performed quantitative trait locus analysis in inbred mouse strains that carry the same hypomorphic allele in Abcc6 yet with highly variable ectopic mineralization phenotypes of pseudoxanthoma elasticum. Abcc6 was confirmed as a major determinant for ectopic mineralization in multiple tissues. Integrative analysis using functional genomics tools that included GeneWeaver, String, and Mouse Genome Informatics identified a total of nine additional candidate modifier genes that could influence the organ-specific ectopic mineralization phenotypes. Integration of the candidate genes into the existing ectopic mineralization gene network expands the current knowledge on the complexity of the network that, as a whole, governs ectopic mineralization in soft connective tissues.
... Em humanos o gene ANKH foi associado a uma variedade de defeitos esqueléticos e articulares, incluindo espondilite anquilosante, artropatia rasgada do manguito, 30 displasia craniometafisária, 31 e condrocalcinose. 32 Distúrbios da ATM têm sido relacionados a alterações genéticas na tríade RANK/OPG/RANKL tanto em manifestações locais 33 como em associação com outras artropatias 34 conferindo o papel sistêmico e primordial da tríade molecular. ...
A anquilose da articulação temporomandibular é a fusão entre o côndilo mandibular e a cavidade glenóide, gerando restrição aos movimentos articulares, função mastigatória limitada além de consequências estéticas e psicossociais. A condição ainda apresenta
influência no desenvolvimento facial, erupção e posicionamento dentários quando ocorre em pacientes em crescimento. O estudo relata um caso clínico de AATM unilateral esquerda com infecção otológica prévia. A alteração evoluiu com completa impossibilidade
de abertura bucal e complicações odontológicas e psicossociais. Artroplastia interposicional foi a técnica de escolha com utilização de retalho do músculo temporal como material de interposição acompanhada de coronoidectomia ipsilateral. Após o procedimento
cirúrgico, obteve-se abertura bucal de 45 mm e o caso seguiu com tratamento multidisciplinar com fonoterapia e ortodontia. Dois anos após a cirurgia, o paciente apresentou abertura interincisal máxima de 37 mm e ausência de sinais de recidiva, o que confere estabilidade a conduta terapêutica proposta.
(Rev Port Estomatol Med Dent Cir Maxilofac. 2018;59(1):54-60)
... 4 In addition, in humans, mutations in the channel core of ANK cause craneometaphyseal dysplasia, 68,69 which is characterized by an increased density of craniofacial bones and abnormal modeling of the metaphyses of the tubular bones. Moreover, mutations in the N-and C-terminus of the ANK protein cause chondrocalcinosis, 70,71 which is characterized by the deposition of calcium pyrophosphate dehydrate crystals in the joint. ...
Vascular calcification is a pathology characterized by the deposition of calcium-phosphate in cardiovascular structures, mainly in the form of hydroxyapatite crystals, resulting in ectopic calcification. It is correlated with increased risk of cardiovascular disease and myocardial infarction in diabetic patients and in those with chronic kidney disease (CKD). Vascular smooth muscle cells are sensitive to changes in inorganic phosphate (Pi) levels. They are able to adapt and modify some of their functions and promote changes which trigger calcification. Pi is regulated by parathyroid hormone and 1,25-dihydroxyvitamin D. Changes in the transport of Pi are the primary factor responsible for the regulation of Pi homeostasis and the calcification process.
Synthesis of calcification inhibitors is the main mechanism by which cells are able to prevent vascular calcification. Extracellular pyrophosphate (PPi) is a potent endogenous inhibitor of calcium-phosphate deposition both in vivo and in vitro. Patients with CKD show lower levels of PPi and increased activity of the enzyme alkaline phosphatase. Numerous enzymes implicated in the metabolism of PPi have been associated with vascular calcifications. PPi is synthesized from extracellular ATP by nucleotide pyrophosphatase/phosphodiesterase from extracellular ATP hydrolysis. PPi is hydrolyzed into Pi by tissue-nonspecific alkaline phosphatase. ATP can be hydrolyzed to Pi via the ectonucleoside triphosphate diphosphohydrolase family. All these enzymes must be in balance, thereby preventing calcifications. However, diseases like CKD or diabetes induce alterations in their levels. Administration of PPi could open up new treatment options for these patients.
... PP i concentrations are elevated (about 2-fold) in SFs of patients with CPPD [810] and cartilage of ageing individuals generates increased levels of PP i [11]. Although gainof-function mutations in the ANKH gene have been demonstrated to cause rare syndromes of familial CPPD, and a functional polymorphism in the 5 0 -untranslated region of ANKH is associated with sporadic CPPD [12,13], eNPP1 has more influence on the levels of PP i [14]. eNPP1 (formerly termed PC-1) generates PP i by hydrolysing extracellular ATP, as has been demonstrated in ex vivo human articular tissue and cellular models [1417] and in osteoblasts and MVs derived from genetically altered murine models [18,19]. ...
Objectives:
Calcium pyrophosphate deposition (CPPD) is associated with osteoarthritis and is the cause of a common inflammatory articular disease. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (eNPP1) is the major ecto-pyrophosphatase in chondrocytes and cartilage-derived matrix vesicles (MVs). Thus, eNPP1 is a principle contributor to extracellular pyrophosphate levels and a potential target for interventions aimed at preventing CPPD. Recently, we synthesized and described a novel eNPP1-specific inhibitor, SK4A, and we set out to evaluate whether this inhibitor attenuates nucleotide pyrophosphatase activity in human OA cartilage.
Methods:
Cartilage tissue, chondrocytes and cartilage-derived MVs were obtained from donors with OA undergoing arthroplasty. The effect of SK4A on cell viability was assayed by the XTT method. eNPP1 expression was evaluated by western blot. Nucleotide pyrophosphatase activity was measured by a colorimetric assay and by HPLC analysis of adenosine triphosphate (ATP) levels. ATP-induced calcium deposition in cultured chondrocytes was visualized and quantified with Alizarin red S staining.
Results:
OA chondrocytes expressed eNPP1 in early passages, but this expression was subsequently lost upon further passaging. Similarly, significant nucleotide pyrophosphatase activity was only detected in early-passage chondrocytes. The eNPP1 inhibitor, SK4A, was not toxic to chondrocytes and stable in culture medium and human plasma. SK4A effectively inhibited nucleotide pyrophosphatase activity in whole cartilage tissue, in chondrocytes and in cartilage-derived MVs and reduced ATP-induced CPPD.
Conclusion:
Nucleotide analogues such as SK4A may be developed as potent and specific inhibitors of eNPP1 for the purpose of lowering extracellular pyrophosphate levels in human cartilage with the aim of preventing and treating CPPD disease.
... CCAL2, on chromosome 5p, is in a region syntenic with the human homolog of the progressive ankylosis gene coding for the ANKH protein 5 . Eight families with ANKH mutations show missense, insertion, and frameshift mutations in the 5' end of the ANKH gene generally believed to result in gain-of-function 6 . Current evidence supports a role for ANKH in transporting critical precursors of CPP crystal formation, including pyrophosphate (PPi) 5 and/or its precursor, ATP 7 , across the cell membrane. ...
Objective:
Mutations on chromosomes 5p (CCAL2) and 8q (CCAL1) have been linked to familial forms of calcium pyrophosphate deposition disease (CPDD). Mutations in the ANKH gene account for CCAL2, but the identity of CCAL1 has been elusive. Recently, a single Dutch kindred with a mutation in the Tumor Necrosis Factor Receptor Super Family member 11B (TNFRSF11B) gene coding for osteoprotegerin (OPG) was described as a gain-of-function mutation. Affected family members had premature generalized osteoarthritis (PGOA) and CPDD. As the TNFRSF11B gene is on 8q, we sought additional evidence that TNFRSF11B was CCAL1, and investigated potential disease mechanisms.
Design:
DNA from two novel PGOA/CPDD families was screened for sequence variants in the TNFRSF11B gene. Mutations were verified by genotype analysis of affected and unaffected family members. We also investigated effects of normal and mutant OPG on regulators of CPP crystal formation in porcine cartilage.
Results:
The identical TNFRSF11B mutation described in the Dutch family was present in two novel PGOA/CPDD families. ANKH was normal in affected patient fibroblasts. Exogenous OPG did not alter ANKH mRNA or protein levels, affect translocation of ANKH to the membrane, nor increase [PPi] or other key regulators of CPDD.
Conclusion:
We have firmly established the identity of CCAL1 as TNFRSF11B (OPG). Our findings suggest that this mutation produces disease in an ANKH-independent manner via novel mechanisms not primarily targeting cartilage. This work rationalizes further investigation of OPG pathway components as potential druggable targets for CPDD.
... 4 In addition, in humans, mutations in the channel core of ANK cause craneometaphyseal dysplasia, 68,69 which is characterized by an increased density of craniofacial bones and abnormal modeling of the metaphyses of the tubular bones. Moreover, mutations in the N-and C-terminus of the ANK protein cause chondrocalcinosis, 70,71 which is characterized by the deposition of calcium pyrophosphate dehydrate crystals in the joint. ...
Vascular calcification is a pathology characterized by the deposition of calcium-phosphate in cardiovascular structures, mainly in the form of hydroxyapatite crystals, resulting in ectopic calcification. It is correlated with increased risk of cardiovascular disease and myocardial infarction in diabetic patients and in those with chronic kidney disease (CKD). Vascular smooth muscle cells are sensitive to changes in inorganic phosphate (Pi) levels. They are able to adapt and modify some of their functions and promote changes which trigger calcification. Pi is regulated by parathyroid hormone and 1,25-dihydroxyvitamin D. Changes in the transport of Pi are the primary factor responsible for the regulation of Pi homeostasis and the calcification process.
Synthesis of calcification inhibitors is the main mechanism by which cells are able to prevent vascular calcification. Extracellular pyrophosphate (PPi) is a potent endogenous inhibitor of calcium-phosphate deposition both in vivo and in vitro. Patients with CKD show lower levels of PPi and increased activity of the enzyme alkaline phosphatase. Numerous enzymes implicated in the metabolism of PPi have been associated with vascular calcifications. PPi is synthesized from extracellular ATP by nucleotide pyrophosphatase/phosphodiesterase from extracellular ATP hydrolysis. PPi is hydrolyzed into Pi by tissue-nonspecific alkaline phosphatase. ATP can be hydrolyzed to Pi via the ectonucleoside triphosphate diphosphohydrolase family. All these enzymes must be in balance, thereby preventing calcifications. However, diseases like CKD or diabetes induce alterations in their levels. Administration of PPi could open up new treatment options for these patients.
... On the other hand, excess of extracellular PP i in articular joint tissue can lead to calcium pyrophosphate dehydrate (CPPD) deposition, as seen in familial chondrocalcinosis [23]. ...
Purpose of review:
We give an update on the etiology and potential treatment options of rare inherited monogenic disorders associated with arterial calcification and calcific cardiac valve disease.
Recent findings:
Genetic studies of rare inherited syndromes have identified key regulators of ectopic calcification. Based on the pathogenic principles causing the diseases, these can be classified into three groups: (1) disorders of an increased extracellular inorganic phosphate/inorganic pyrophosphate ratio (generalized arterial calcification of infancy, pseudoxanthoma elasticum, arterial calcification and distal joint calcification, progeria, idiopathic basal ganglia calcification, and hyperphosphatemic familial tumoral calcinosis; (2) interferonopathies (Singleton-Merten syndrome); and (3) others, including Keutel syndrome and Gaucher disease type IIIC. Although some of the identified causative mechanisms are not easy to target for treatment, it has become clear that a disturbed serum phosphate/pyrophosphate ratio is a major force triggering arterial and cardiac valve calcification. Further studies will focus on targeting the phosphate/pyrophosphate ratio to effectively prevent and treat these calcific disease phenotypes.
Background:
Rare diseases (RDs) affect less than 5/10,000 people in Europe and fewer than 200,000 individuals in the United States. In rheumatology, RDs are heterogeneous and lack systemic classification. Clinical courses involve a variety of diverse symptoms, and patients may be misdiagnosed and not receive appropriate treatment. The objective of this study was to identify and classify some of the most important RDs in rheumatology. We also attempted to determine their combined prevalence to more precisely define this area of rheumatology and increase awareness of RDs in healthcare systems. We conducted a comprehensive literature search and analyzed each disease for the specified criteria, such as clinical symptoms, treatment regimens, prognoses, and point prevalences. If no epidemiological data were available, we estimated the prevalence as 1/1,000,000. The total point prevalence for all RDs in rheumatology was estimated as the sum of the individually determined prevalences.
Results:
A total of 76 syndromes and diseases were identified, including vasculitis/vasculopathy (n = 15), arthritis/arthropathy (n = 11), autoinflammatory syndromes (n = 11), myositis (n = 9), bone disorders (n = 11), connective tissue diseases (n = 8), overgrowth syndromes (n = 3), and others (n = 8). Out of the 76 diseases, 61 (80%) are classified as chronic, with a remitting-relapsing course in 27 cases (35%) upon adequate treatment. Another 34 (45%) diseases were predominantly progressive and difficult to control. Corticosteroids are a therapeutic option in 49 (64%) syndromes. Mortality is variable and could not be determined precisely. Epidemiological studies and prevalence data were available for 33 syndromes and diseases. For an additional eight diseases, only incidence data were accessible. The summed prevalence of all RDs was 28.8/10,000.
Conclusions:
RDs in rheumatology are frequently chronic, progressive, and present variable symptoms. Treatment options are often restricted to corticosteroids, presumably because of the scarcity of randomized controlled trials. The estimated combined prevalence is significant and almost double that of ankylosing spondylitis (18/10,000). Thus, healthcare systems should assign RDs similar importance as any other common disease in rheumatology.
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. Although 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 summarizes the current knowledge of the genetics and candidate modifier genes in PXE, a multifactorial disease at the genome-environment interface.
ZUSAMMENFASSUNG
Als Chondrokalzinose bezeichnet man die Ablagerung von Kalziumphosphaten im chondralen Gewebe. Im Gelenk treten die Minerale sowohl im hyalinen Knorpel wie im Faserknorpel auf. Die Kalzifikationen sind teils amorph, teils kristallin. Es werden sowohl apatitische Kristalle wie Pyrophosphatkristalle beobachtet. Die Ätiologie ist unklar, möglicherweise nicht monokausal. Es mehren sich die Hinweise, dass es sich um ein systemisches Geschehen, d. h. alle Gelenke betreffend, handelt, das – in geringerer Ausprägung – schon in jungem Alter einsetzt. Die Chondrokalzinose lässt sich so nicht als Sekundärphänomen einer Arthrose verstehen. Die Chondrokalzinose ist wahrscheinlich viel häufiger als sie diagnostiziert wird. Im Röntgenbild ist die Kalzinose erst dann erkennbar, wenn stärkere Depositionen vorliegen und wenn sich die Knorpelgewebe ohne Knochenüberlagerung projizieren lassen. Apatitische Kristalle sind derart klein, dass sie weder im Punktat noch im histologischen Schnitt lichtmikroskopisch erkennbar sind. Das Serumlabor ist unergiebig.
Fractures of the humerus constitute 5% to 8% of fractures and most have an uneventful healing, but occasionally fracture with joint stiffness of shoulder and/or elbow and prolonged debilitating pain may be encountered. Predisposing conditions
usually are osteoporosis, obesity, alcoholism and smoking. Comminuted or segmental fractures, soft tissue interposition at the fracture
site, improper fixation and infection may also result in non-union.
A fracture of neck of the right humerus (dominant limb) in a young lady failed to unite following internal fixation and lead to atrophic
non-union with implant loosening, which initially was suspected to be due to poor fixation and or infection, but histopathological
evaluation on two separate occasions of the fracture site revealed a picture of Chondrocalcinosis (CC). CC is a condition wherein
deposition of calcium pyrophosphate dihydrate (CPPD) crystals occurs within articular cartilage and synovial tissue and is associated
with joint pain leading to arthritis. The presence of these CPPD crystal has not been mentioned in the bone or at fracture sites in the
literature, The histopathological evidence of CPPD at the fracture site in this case of humerus non-union did not establish whether the
fracture was due to the pathology or the CPPD got deposited at the fracture site and was a cause of non-union.
Keywords : Chondrocalcinosis, Calcium pyrophosphate dehydrate(CPPD) ANKH gene, PHILOS, Non union.
F
Diffuse idiopathic skeletal hyperostosis (DISH) is a skeletal syndrome that has been known for more than 70 years. Yet, its pathogenesis and treatment options are still under investigation. DISH and spondyloarthritidies may manifest itself clinically as very similar disorders causing impaired axial flexibility, axial pain and peripheral tendinopathies. On the other hand, these two processes are different in many ways, from different genetic and metabolic predispositions, to different clinical and imaging manifestations, and at last, a different attitude toward treatment. The knowledge of the similarities and differences between DISH and spondyloarthritidies can guide the clinician toward a better diagnostic and treatment approach. This review tries to emphasize these details.
Fractures of the humerus constitute 5% to 8% of fractures and most have an uneventful healing, but occasionally non-unions of the fracture with joint stiffness of shoulder and/or elbow and prolonged debilitating pain may be encountered. Predisposing conditions usually are osteoporosis, obesity, alcoholism and smoking. Comminuted or segmental fractures, soft tissue interposition at the fracture site, improper fixation and infection may also result in non-union.
A fracture of neck of the right humerus (dominant limb) in a young lady failed to unite following internal fixation and lead to atrophic non-union with implant loosening, which initially was suspected to be due to poor fixation and or infection, but histopathological evaluation on two separate occasions of the fracture site revealed a picture of Chondrocalcinosis (CC). CC is a condition wherein deposition of calcium pyrophosphate dihydrate (CPPD) crystals occurs within articular cartilage and synovial tissue and is associated with joint pain leading to arthritis. The presence of these CPPD crystal has not been mentioned in the bone or at fracture sites in the literature, The histopathological evidence of CPPD at the fracture site in this case of humerus non-union did not establish whether the fracture was due to the pathology or the CPPD got deposited at the fracture site and was a cause of non-union.
In 1954, Jackson et al. (1954) coined the term “craniometaphyseal dysplasia” for a hereditary bone disease with metaphyseal widening of the tubular bones and bony overgrowth of the facial and skull bones (leonteasis ossea).
Abstract Mutations in the progressive ankylosis protein (NP_473368, human ANKH) cause craniometaphyseal dysplasia (CMD), characterized by progressive thickening of craniofacial bones and widened metaphyses in long bones. The pathogenesis of CMD remains largely unknown, and treatment for CMD is limited to surgical intervention. We have reported that knock-in mice (Ank KI/KI) carrying a F377del mutation in ANK (NM_020332, mouse ANK) replicate many features of CMD. Interestingly, ablation of the Ank gene in Ank KO/KO mice also leads to several CMD-like phenotypes. Mutations causing CMD led to decreased steady-state levels of ANK/ANKH protein due to rapid degradation. While wild type (wt) ANK was mostly associated with plasma membranes, endoplasmic reticulum (ER), Golgi apparatus and lysosomes, CMD-linked mutant ANK was aberrantly localized in cytoplasm. Inhibitors of proteasomal degradation significantly restored levels of overexpressed mutant ANK, whereas endogenous CMD-mutant ANK/ANKH levels were more strongly increased by inhibitors of lysosomal degradation. However, these inhibitors do not correct the mislocalization of mutant ANK. Co-expressing wt and CMD-mutant ANK in cells showed that CMD-mutant ANK does not negatively affect wt ANK expression and localization, and vice versa. In conclusion, our finding that CMD mutant ANK/ANKH protein is short-lived and mislocalized in cells may be part of the CMD pathogenesis.
Several monogenic disorders are characterized by abnormal soft tissue mineralization. Human disorders of ectopic calcification can be caused by: (1) abnormal extracellular nucleotide metabolism, leading to adenosine or pyrophosphate deprivation; (2) excessive pyrophosphate export causing deposition in the joint space; (3) decreased FGF23 action producing excess inorganic phosphate, which in turn leads to an increased calcium-phosphate product; (4) abnormal phosphate homeostasis or PDGFR? signaling leading to ectopic brain calcification; (5) decreased activity of a known protein inhibitor of calcification; or (6) an excessive innate immune response against nucleic acids, such as viral particles or double-stranded RNA. We review the clinical manifestations, animal models, and pathogenesis of these disorders, characterized by a disrupted balance between known promoters and inhibitors of calcification. Knowledge of the homeostatic mechanisms regulating soft tissue mineral balance provides insight into the pathogenesis of common non-Mendelian etiologies of calcification, with the hope of improved therapeutic strategies.
Twelve families with exuberant and early-onset calcium pyrophosphate dehydrate chondrocalcinosis (CC) and diffuse idiopathic skeletal hyperostosis (DISH), hereafter designated DISH/CC, were identified in Terceira Island, the Azores, Portugal. Ninety-two (92) individuals from these families were selected for whole-genome-wide linkage analysis. An identity-by-descent (IBD) analysis was performed in 10 individuals from 5 of the investigated pedigrees. The chromosome area with the maximal logarithm of the odds score (1.32; P=0.007) was not identified using the IBD/identity-by-state (IBS) analysis; therefore, it was not investigated further. From the IBD/IBS analysis, two candidate genes, LEMD3 and RSPO4, were identified and sequenced. Nine genetic variants were identified in the RSPO4 gene; one regulatory variant (rs146447064) was significantly more frequent in control individuals than in DISH/CC patients (P=0.03). Four variants were identified in LEMD3, and the rs201930700 variant was further investigated using segregation analysis. None of the genetic variants in RSPO4 or LEMD3 segregated within the studied families. Therefore, although a major genetic effect was shown to determine DISH/CC occurrence within these families, the specific genetic variants involved were not identified.
In 1954, Jackson et al. (1954) coined the term “craniometaphyseal dysplasia” for a hereditary bone disease with metaphyseal widening of the tubular bones and bony overgrowth of the facial and skull bones (leonteasis ossea).
The progressive ankylosis protein (ANK) is a transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu. In this study we show increased fatty degeneration of the bone marrow of adult ank/ank mice, which lack a functional ANK protein. In addition, isolated bone marrow stromal cells (BMSCs) isolated from ank/ank mice showed a decreased proliferation rate and osteogenic differentiation potential, and an increased adipogenic differentiation potential compared to BMSCs isolated from wild type (WT) littermates. Wnt signaling pathway PCR array analysis revealed that Wnt ligands, Wnt receptors and Wnt signaling proteins that stimulate osteoblast differentiation were expressed at markedly lower levels in ank/ank BMSCs than in WT BMSCs. Lack of ANK function also resulted in impaired bone fracture healing, as indicated by a smaller callus formed and delayed bone formation in the callus site. Whereas 5 weeks after fracture, the fractured bone in WT mice was further remodeled and restored to original shape, the fractured bone in ank/ank mice was not fully restored and remodeled to original shape. In conclusion, our study provides evidence that ANK plays a critical role in the adipogenic/osteogenic fate decision of adult mesenchymal precursor cells. ANK functions in precursor cells are required for osteogenic differentiation of these cells during adult bone homeostasis and repair, whereas lack of ANK functions favors adipogenic differentiation.
Mutation at the mouse progressive ankylosis(ank) locus causes a generalized, progressive form of arthritis accompanied by mineral deposition, formation of bony outgrowths,
and joint destruction. Here, we show that theank locus encodes a multipass transmembrane protein (ANK) that is expressed in joints and other tissues and controls pyrophosphate
levels in cultured cells. A highly conserved gene is present in humans and other vertebrates. These results identify ANK-mediated
control of pyrophosphate levels as a possible mechanism regulating tissue calcification and susceptibility to arthritis in
higher animals.
It has been observed that the sign and the magnitude of the magnetoelectric conversion in a magnetoelectric composite material depended on the treatment used to achieve electrical polarization.The observed phenomena can be explained by the existence of a space charge field in the ferro-electric phase, which arises due to the high temperature poling procedure used. This model has been confirmed by the sign of the piezoelectric effect in the pure ferroelectric phase which has been poled under the same conditions.
Objective:
In subjects with idiopathic calcium pyrophosphate dihydrate (CPPD) deposition disease, cartilage chondrocytes elaborate increased amounts of PPi. The mechanism of the intracellular PPi elevation is not known. Plasma membrane 5'-nucleotide phosphodiesterase I/nucleotide pyrophosphohydrolase (NTPPPH) activity also is elevated in chondrocytes and dermal fibroblasts of patients with idiopathic CPPD deposition disease. NTPPPH, as an ecto-enzyme, could act within certain intracellular compartments. Thus, we hypothesized a potential causal link between increased NTPPPH activity and increased intracellular PPi.
Methods:
Transformed simian fibroblasts (COS cells) and human osteoblasts (U2OS cells) were transfected with the 5'-nucleotide phosphodiesterase I ecto-enzyme plasma cell membrane glycoprotein-1 (PC-1), recently shown to be expressed in cartilage, osteoblasts, and fibroblasts.
Results:
Transfection with PC-1 markedly up-regulated 5'-nucleotode phosphodiesterase I activity and increased intracellular PPi concentrations by increasing the capacity of cells to generate PPi. Importantly, this did not require supplementation with exogenous nucleotides.
Conclusion:
Cellular overexpression of PC-1 produces NTPPPH overactivity and increased intracellular PPi generation in vitro. These findings support the potential importance of NTPPPH overactivity in PPi generation, both inside and outside the cell, in some subjects with CPPD deposition disease.
Inorganic pyrophosphate (PP(i)) levels were estimated by radiometric assay in urine and in synovial fluid (SF) from asymptomatic, nonarthritic knees of patients with untreated metabolic disease and normal controls. SF PP(i) was significantly elevated in patients with hyperparathyroidism (mean +/- SEM 19 +/- 3-mu-M; n = 9), hemochromatosis (23 +/- 5-mu-M; n = 6), and hypomagnesemia (27 +/- 0.1-mu-M; n = 2) compared with normal subjects (10 +/- 0.5-mu-M, n = 50), and was low in patients with hypothyroidism (4.2 +/- 2.3-mu-M; n = 11) (P < 0.05 all comparisons). Urinary PP(i) was elevated only in those with hypophosphatasia. Local elevation of ionic PP(i) may be relevant to the mechanism of crystal formation in metabolic diseases predisposing to calcium pyrophosphate dihydrate (CPPD) crystal deposition. The finding of low SF PP(i) levels in patients with hypothyrodism further questions the association between this condition and CPPD.
The overproduction of inorganic pyrophosphate (PPi) by cartilage is thought to be a key element in the formation of calcium pyrophosphate dihydrate (CPPD) crystals in joints, and the subsequent development of pseudogout or chondrocalcinosis. We report herein that transforming growth factor β1 (TGFβ1), alone and in synergy with epidermal growth factor (EGF) or TGF, markedly stimulates PPi elaboration by porcine articular cartilage in organ culture and monolayer culture. This effect is not seen with platelet-derived growth factor, basic fibroblast growth factor, or insulin-like growth factor types 1 and 2, substances which also affect chondrocyte metabolism or are mitogenic. TGFβ1 produces only a modest increase in nucleoside triphosphate pyrophosphohydrolase (NTPPPH), a chondrocyte ectoenzyme that produces PPi; this implies the existence of other pathways for PPi elaboration. TGFβ1 is present in joint fluid and cartilage. TGFβ1, TGF, and EGF are the first known physiologic modifiers of cartilage PPi production. They provide a novel model for the study of CPPD crystal formation in cartilage, as well as new insights into the pathogenesis of this common affliction of aging.
Thirty-nine members of one family, covering three generations, were HLA-typed. Twenty-five suffered from primary diffuse articular chondrocalcinosis, and all had the same dominantly transmitted autosomally controlled disease. This was characterized by acute articular attacks, which always started before the age of 35, and radiologically by typical cartilaginous and fibrocartilaginous deposits associated with para-articular calcifications. The lesions were both peripherally and axially generalized. None of the 28 HLA antigens tested seemed related to the disease, nor did the disease segregate with an HLA haplotype.
Although many metabolic and endocrine diseases have been reported to predispose to calcium pyrophosphate dihydrate crystal deposition, the validity of many of these associations remains unclear. A critical review of the literature relating to these associations, with illustrative cases and data derived from the authors' own experience, is presented. It is concluded that there is good evidence to associate hypophosphatasia, hypomagnesemia, and hyperparathyroidism with chondrocalcinosis and acute attacks of "pseudogout." Meta-analysis also suggests a small but significant association between hypothyroidism and chondrocalcinosis. Hemochromatosis stands alone in clearly associating not only with chondrocalcinosis but also with structural change and chronic arthropathy. The biochemical mechanisms that may produce these various associations are discussed. Recommendations are made concerning appropriate screening for metabolic and endocrine disease in patients with chondrocalcinosis.
Familial predisposition to chondrocalcinosis (CC) due to calcium pyrophosphate dihydrate (CPPD) crystal desposition is described in five English kindreds. Two families were characterized by premature-onset polyarticular CC with little associated structural arthropathy. In one of these families, recurrent childhood fits were strongly associated with subsequent development of CC. Affected members of the other three families resembled sporadic disease in showing predominantly late-onset, oligoarticular CC with mild arthritis and destructive change in only one case. Knee synovial fluid levels of inorganic pyrophosphate (PPi) and nucleoside triphosphate pyrophosphatase (NTPP) did not differ from those of 59 sporadic cases of CC due to CPPD, although PPi and NTPP levels in both groups were higher than in normal knee synovial fluid (P<0.0001). Urinary PPi levels were not different from normal controls. Screening for other metabolic abnormality was negative in all cases.
This is the first report of familial CC in the UK, and the first to associate this condition with childhood fits. Absence of overt primary abnormality of PPi metabolism suggests that other factors relating to crystal nucleation/growth may be more relevant to predisposition in these cases.
Chondrocalcinosis and osteoarthritis (OA) both increase in prevalence with age, yet previous studies assessing their association have often been too small to adjust for age and have had biased selection of subjects. We assessed the prevalence of chondrocalcinosis and its link with OA in the Framingham Knee Osteoarthritis Study, a large population based study of the elderly. After controlling for age, we found a modestly increased rate of radiographic OA in those with chondrocalcinosis (relative risk = 1.52 (95% CI 1.22, 1.90). There was a similar link between chondrocalcinosis and severe radiographic OA (relative risk = 1.52). Chondrocalcinosis was positively associated with both symptomatic and asymptomatic radiographic OA. The proportion of radiographic OA potentially attributable to chondrocalcinosis was only 4.4%. Our results confirm that chondrocalcinosis is significantly associated with OA after controlling for age, but they also suggest that chondrocalcinosis and OA increase independently with age.
CPPD deposition occurs in a wide variety of clinical settings, most commonly as an age-related phenomenon in the absence of other joint abnormality. Although our knowledge of CPPD and other intra-articular particles has increased in the last decade, the role of CPPD remains unclear. The paradox of asymptomatic deposition of phlogistic crystals, the wide spectrum of clinical presentation, and the lack of disease specificity, however, have challenged recognition of pyrophosphate arthropathy (PA) as a separate, distinct disease entity and led to reappraisal of earlier concepts of crystal deposition disease. In this article, clinical aspects of PA will first be presented; the validity of PA as a discrete arthropathy will then be discussed.
Once thought of as a biosynthetic waste product, over the last 2 decades PPi has become understood as an entity with a variety of biologic roles (see Table 1). Documented roles include participation in intracellular Ca++ traffic, mediation of nucleotide and iron transport, storage of molecules in cellular granules, modification of enzyme function, and modulation of mineralization. Much has been established regarding plasma, urine, and synovial fluid levels (see Fig. 1) and urinary excretion in health and disease. Derangements in intracellular PPi content of skin fibroblasts have been noted in patients with CPPD deposition arthropathy (see Table 2). Mechanisms by which elevated PPi concentration develops in synovial fluid from joints with CPPD deposition and related arthropathies have come under scrutiny. The chondrocyte is now recognized as the probable cellular source of intra-articular extracellular PPi (see Figs. 3 and 4). Special attention has been focused on two basic pathways by which chondrocytes could generate extracellular PPi (see Fig. 2). In the first mechanism, chondrocytes demonstrate a set of ectoenzymes which could work in concert to directly produce extracellular PPi. The second pathway involves the major reactions by which PPi is formed within the cell and how intracellular PPi thus formed could be transported into the extracellular space. Much future research is needed regarding these two pathways and their relative importance in the pathogenesis of CPPD crystal deposition and related arthropathies.
Osteoarthritis (OA), which represents synovial joint failure, is usually defined radiologically. The characteristic features of joint space narrowing, subchondral bone sclerosis, cysts and osteophytosis reflect the pathology of articular cartilage destruction and altered activity of subchondral bone. These radiological and pathological findings are often associated with the presence of crystals and other particles in the cartilage, synovial fluid, synovium or capsule. Four different crystals have been identified in OA joints. The frequent finding of crystals in OA tissue raises several important questions revelant to the pathogenesis and clinical features of the disorder. The problem is complex since the identification of crystals is not wholly reliable, OA is clinically difficult to define or subdivide, and it takes a lifetime to examine its natural history. Therefore, most of the questions remain largely unanswered. To establish the relevance of crystal deposits in osteoarthritis, it is first necessary to establish the relative incidence of crystal deposition in OA, other joint diseases and normal control groups. Such data do not exist. Crystals are difficult to identify and almost impossible to exclude. The only available compromise is to use the existing methodology to compare findings in different patient groups.
Idiopathic articular Chondrocalcinosis was found in 22 of 108 adults belonging to two generations of one pedigree. An equal number of males and females were affected. In only 14 of the affected persons did acute joint attacks occur; six of these patients have not sought medical help to date. Degenerative arthropathy with conspicuous preservation of joint space, involving the spine and the larger peripheral joints, was encountered in affected members older than 60 years of age. Osteoarthritic joint complaints did not have any relationship to the occurrence of previous acute joint symptoms. Periarticular calcifications, especially of the hip and knee joints, and paravertebral calcifications were predominant features in affected members who had reached senescence. Amorphous deposits only were identified in articular material, except in one synovial fluid sample that contained an unidentified crystalline deposit. In this benign type of articular Chondrocalcinosis, a single autosomal dominant hereditary factor seems to be involved, because the disease occurred only in children of affected members, including sons of affected fathers.
Progressive ankylosis (ank), a murine autosomal recessive mutation, produces an inflammatory joint disorder associated with intraarticular calcium hydroxyapatite deposition and culminates in fusion of the joints. Joints in the feet become stiffened and swollen with milky white fluid containing polymorphonuclear leukocytes, large mononuclear macrophage-like cells, and calcium hydroxyapatite. The joints develop a proliferative synovitis, sometimes associated with marginal erosions of the articular cartilage and periosteal bone. Subsequently, cartilaginous osteophytes, extending outward from the subchondral bone, bridge the margins of the joint and undergo ossification. The progressive ankylosis mutation provides a useful system for investigating calcium hydroxyapatite-associated arthropathies.
The history of diphosphonates began with studies of inorganic pyrophosphate. This compound was found to occur in many biological fluids and inhibited the precipitation of calcium phosphates. It also slowed the transformation of amorphous calcium phosphate to its crystalline form, and inhibited crystal aggregation and dissolution. These observations suggested that it might be a compound of physiological or pathophysiological significance, perhaps in hypophosphatasia and in renal lithiasis. Diphosphonates are compounds where the P-O-P bond of pyrophosphate is replaced by a P-C-P bond. Many diphosphonates have been synthesized and tested and some relationship of their structure to the spectrum of biological effects has been observed. These analogues have similar properties to pyrophosphate, but unlike pyrophosphate they are resistant to enzymic degradation. Their experimental properties have led to their clinical development as bone scanning agents and in the treatment of disorders of ectopic mineralization and increased bone resorption.
In a study of 45 adults in a family of Mexican-Indian ancestry, it was found that 22 (49%) had joint symptoms resembling those of degenerative joint disease. Eleven family members had radiographic evidence of chondrocalcinosis, and 1 adult and 3 adolescents had clinical histories and examinations consistent with the familial arthropathy, but no radiographic evidence of disease. The cause of the arthritis in the affected family members is calcium pyrophosphate crystal deposition. The mode of inheritance appears to be autosomal dominant with a high degree of penetrance. The disease is characterized by onset in the second to fifth decades of either episodes of acute inflammatory arthritis or degenerative joint disease. A unique finding of this study was a "halo" surrounding chondrocytes in 1 patient's cartilage, demonstrating loss of the proteoglycans.
Progressive ankylosis, ank, a new recessive skeletal mutation causing a noninflammatory joint disease in mice is described. Preliminary observations suggest that three abnormal processes are involved: increased calcification of calcified cartilage and of joint tissues, hyperplasia of the cells and tissues of the joints, and degeneration within and around the tissues, tendons, and ligaments of the joints. The new mutation is closely linked, with about 4 percent recombination, to underwhite on chr 15.
The elaboration of excess extracellular inorganic pyrophosphate (ePPi) by cartilage contributes to calcium pyrophosphate dihydrate (CPPD) crystal deposition disease. Transforming growth factor-beta 1 (TGF beta 1) is the only defined physiologic stimulant of cartilage ePPi elaboration. The mechanism of ePPi generation by chondrocytes is unknown, but current evidence suggests that TGF beta 1 induced ePPi is made intracellularly. An active transport mechanism such as an anion transporter would then be necessary to export ePPi to the matrix where crystals form. We determined the effect of probenecid (PB), an anion transport inhibitor, on TGF beta 1 induced ePPi elaboration.
Porcine hyaline articular chondrocytes in high density monolayer cultures were exposed to serum-free media with and without TGF beta 1 and/or PB. ePPi was measured in the media after 48-96 h of exposure. Cell injury was measured by examining the release of 3H-deoxyglucose from chondrocytes. The activity of the ePPi generating ectoenzyme nucleoside triphosphate pyrophosphohydrolase (NTPPPH) and media lactate concentrations were measured with standard colorimetric assays. As PB may inhibit phosphodiesterase (PDE), its effects on ePPi generation were compared with isobutylmethylxanthine (IBMX), a specific PDE inhibitor.
PB inhibited TGF beta 1 induced ePPi elaboration by chondrocytes. PB did not cause membrane injury or decrease NTPPPH activity. Lactate production was decreased by PB but did not correlate with the effects of PB on ePPi elaboration. IBMX did not inhibit TGF beta 1 effect on ePPi elaboration.
PB blocks TGF beta 1 induced ePPi elaboration. This effect is independent of cell membrane injury, decreased NTPPPH activity, or PDE inhibition. Our data implicate a role for anion transport in TGF beta 1 induced ePPi elaboration, and suggest a potential therapy for CPPD disease.
Calcium pyrophosphate-deposition disease (CPDD), also called "chondrocalcinosis" or "pseudogout," is a disorder characterized by the deposition of calcium-containing crystals in joint tissue, which leads to arthritis-like symptoms. The presence of these crystals in joint tissue is a common finding in the elderly, and, in this population, there is a poor correlation with joint pain. In contrast, early-onset CPDD has been described in several large families in which the disease progresses to severe degenerative osteoarthritis (OA). In these families, an autosomal dominant mode of inheritance is observed, with an age at onset between the 2d and 5th decades of life. In this report, we describe a large New England family with early-onset CPDD and severe degenerative OA. We found genetic linkage between the disease in this family and chromosome 8q, with a multipoint lod score of 4.06. These results suggest that a defective gene at this location causes the disease in this family.
In subjects with idiopathic calcium pyrophosphate dihydrate (CPPD) deposition disease, cartilage chondrocytes elaborate increased amounts of PPi. The mechanism of the intracellular PPi elevation is not known. Plasma membrane 5'-nucleotide phosphodiesterase I/nucleotide pyrophosphohydrolase (NTPPPH) activity also is elevated in chondrocytes and dermal fibroblasts of patients with idiopathic CPPD deposition disease. NTPPPH, as an ecto-enzyme, could act within certain intracellular compartments. Thus, we hypothesized a potential causal link between increased NTPPPH activity and increased intracellular PPi.
Transformed simian fibroblasts (COS cells) and human osteoblasts (U2OS cells) were transfected with the 5'-nucleotide phosphodiesterase I ecto-enzyme plasma cell membrane glycoprotein-1 (PC-1), recently shown to be expressed in cartilage, osteoblasts, and fibroblasts.
Transfection with PC-1 markedly up-regulated 5'-nucleotode phosphodiesterase I activity and increased intracellular PPi concentrations by increasing the capacity of cells to generate PPi. Importantly, this did not require supplementation with exogenous nucleotides.
Cellular overexpression of PC-1 produces NTPPPH overactivity and increased intracellular PPi generation in vitro. These findings support the potential importance of NTPPPH overactivity in PPi generation, both inside and outside the cell, in some subjects with CPPD deposition disease.
Chondrocalcinosis is a common disorder which may associate with acute and chronic arthritis. A familial form, inherited as an autosomal dominant trait, has been mapped in a large family in which affected members also suffer recurrent fits in childhood. The gene which causes this disease shows linkage with several polymorphic markers on chromosome 5p with a maximum multipoint lod score of 4.6 between D5S810 and D5S416. Mapping a locus for chondrocalcinosis will allow the heterogeneity of the disorder to be assessed and may also be relevant to understanding the aetiology of osteoarthritis with which it commonly associates.
Familial calcium pyrophosphate dihydrate deposition disease (CPPDD) is a disease of articular cartilage that is radiographically characterized by chondrocalcinosis due to the deposition of calcium-containing crystals in affected joints. We have documented the disease in an Argentinean kindred of northern Italian ancestry and in a French kindred from the Alsace region. Both families presented with a common phenotype including early age at onset and deposition of crystals of calcium pyrophosphate dihydrate in a similar pattern of affected joints. Affected family members were karyotypically normal. Linkage to the short arm of chromosome 5 was observed, consistent with a previous report of linkage of the CPPDD phenotype in a large British kindred to the 5p15 region. However, recombinants in the Argentinean kindred have enabled us to designate a region<1 cM in length between the markers D5S416 and D5S2114 as the CPPDD locus.
Mutation at the mouse progressive ankylosis (ank) locus causes a generalized, progressive form of arthritis accompanied by mineral deposition, formation of bony outgrowths, and joint destruction. Here, we show that the ank locus encodes a multipass transmembrane protein (ANK) that is expressed in joints and other tissues and controls pyrophosphate levels in cultured cells. A highly conserved gene is present in humans and other vertebrates. These results identify ANK-mediated control of pyrophosphate levels as a possible mechanism regulating tissue calcification and susceptibility to arthritis in higher animals.
Craniometaphyseal dysplasia (CMD) is a rare skeletal disorder characterized by progressive thickening and increased mineral density of craniofacial bones and abnormally developed metaphyses in long bones. Linkage studies mapped the locus for the autosomal dominant form of CMD to an approximately 5-cM interval on chromosome 5p, which is defined by recombinations between loci D5S810 and D5S1954. Mutational analysis of positional candidate genes was performed, and we describe herein three different mutations, in five different families and in isolated cases, in ANK, a multipass transmembrane protein involved in the transport of intracellular pyrophosphate into extracellular matrix. The mutations are two in-frame deletions and one in-frame insertion caused by a splicing defect. All mutations cluster within seven amino acids in one of the six possible cytosolic domains of ANK. These results suggest that the mutated protein has a dominant negative effect on the function of ANK, since reduced levels of pyrophosphate in bone matrix are known to increase mineralization.
Craniometaphyseal dysplasia (CMD) is a bone dysplasia characterized by overgrowth and sclerosis of the craniofacial bones and abnormal modeling of the metaphyses of the tubular bones. Hyperostosis and sclerosis of the skull may lead to cranial nerve compressions resulting in hearing loss and facial palsy. An autosomal dominant form of the disorder (MIM 123000) was linked to chromosome 5p15.2-p14.1 (ref. 3) within a region harboring the human homolog (ANKH) of the mouse progressive ankylosis (ank) gene. The ANK protein spans the outer cell membrane and shuttles inorganic pyrophosphate (PPi), a major inhibitor of physiologic and pathologic calcification, bone mineralization and bone resorption. Here we carry out mutation analysis of ANKH, revealing six different mutations in eight of nine families. The mutations predict single amino acid substitutions, deletions or insertions. Using a helix prediction program, we propose for the ANK molecule 12 membrane-spanning helices with an alternate inside/out orientation and a central channel permitting the passage of PPi. The mutations occur at highly conserved amino acid residues presumed to be located in the cytosolic portion of the protein. Our results link the PPi channel ANK with bone formation and remodeling.
Polypeptide growth factors promote cellular proliferation by binding to specific plasma membrane-anchored receptors. This interaction triggers the phosphorylation of signal transducing molecules and the transcriptional activation of numerous genes. We have used a differential display approach to identify fibroblast growth factor (FGF)-1-inducible genes in murine NIH 3T3 fibroblasts. Here we report that one of these genes encodes ank, a type IIIa transmembrane protein reported to function in cells as an inorganic pyrophosphate transporter. FGF-1 induction of ank mRNA expression is first detectable at 2 h after growth factor addition and is dependent on de novo RNA and protein synthesis. Ank gene expression is also upregulated after treating quiescent fibroblasts with several other mitogenic agents (e.g., calf serum or platelet-derived growth factor-BB) or the tumor promoter phorbol 12-myristate 13-acetate. Furthermore, in comparison to parental NIH 3T3 cells, oncogene-transformed NIH 3T3 cells constitutively express elevated levels of ank mRNA. FGF-1 also increases ank gene expression in non-immortalized human embryonic lung fibroblasts. Finally, the murine and human ank genes are expressed in vivo in a tissue-specific manner, with highest levels of mRNA expression found in brain, heart, and skeletal muscle. These results indicate that ank is a growth factor-regulated delayed-early response gene in mammalian cells, and we propose that increased ank expression during cell cycle progression may be necessary to maintain proper intracellular pyrophosphate levels during conditions of high cellular metabolic activity.
Polypeptide growth factors and phorbol ester induce pro-gressive ankylosis (Ank) gene expression in murine and hu-man fibroblasts Hereditary joint disorder in progressive ankylosis (ank/ank) mice. I. Association of calcium hydrox-yapatite deposition with inflammatory arthropathy
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Transforming growth factor beta 1 stimulates inorganic pyrophosphate elaboration by porcine cartilage
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Refinement of the chromosome 5p locus for familial calcium pyrophosphate dihydrate deposition dis-ease Linkage of early-onset osteoarthritis and chondrocalcinosis to human chromosome 8q
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Heterozygous mutations in ANKH, the human ortholog of the mouse progressive ankylosis gene, result in craniometaphyseal dysplasia
- Nurnberg
Articular chondrocalcinosis in a Dutch pedigree
- van der Korst