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Mutations in the amino terminus of ANKH in two US families with calcium pyrophosphate dihydrate crystal deposition disease
Abstract
To analyze ANKH in families with calcium pyrophosphate dihydrate crystal deposition disease (CPPD) for disease-causing mutations.
Two US families (one of British ancestry and the other of German/Swiss ancestry) with autosomal-dominant CPPD, whose disease phenotypes were found to be linked to chromosome 5p15.1 (locus symbol CCAL2), were screened by direct sequencing for mutations in ANKH, a gene in the CCAL2 candidate interval that has been shown to harbor mutations in other families with CPPD. Observed sequence variants were confirmed by antisense sequencing, and expression of the mutant allele was verified by reverse transcriptase-polymerase chain reaction amplification of messenger RNA followed by direct sequencing.
The two US families displayed the same mutation at position 5 of the ANKH gene product (P5T). All affected members were heterozygous for the P-to-T variant, and the mutation was not seen in 204 control alleles. The two families displayed distinct disease haplotypes, suggesting that they were unrelated to each other.
These observations represent the fourth and fifth families with heritable CPPD whose disease phenotypes are linked to the CCAL2 locus and who have missense mutations in the amino terminus of ANKH. This same position (P5) was the site of a missense mutation in an Argentine family of northern Italian ancestry; however, the sequence variant in that family generated a P5L mutation. The distinct disease haplotypes among the 3 families with P5 mutations suggest that the mutations arose independently and that the evolutionarily conserved P5 position of ANKH may represent a hot spot for mutation in families with autosomal-dominant CPPD.
... ANKH encodes a multiple-pass transmembrane protein that functions in PP i channelling [39][40][41][42] (Figure 20-2) and also appears to directly or indirectly promote ATP release. 42 ANKH also appears to regulate P i metabolism and uptake of P i by the type III sodium-dependent P i cotransporter Pit-1. ...
... 1 This is consistent with the concept of differing functional effects of ANKH mediated by specific regions of the molecule that cause either chondrocalcinosis (largely the N-terminal ANKH domain) or CMD (certain cytosolic regions). Most N-terminal ANKH mutations identified, to date, in association with familial chondrocalcinosis (see Table 20-1) appear to elevate PP i transport, 40 but some of ANKH mutations have differing effects on chondrocyte differentiation. 44 Moreover, the M48T ANKH mutant, originally characterized in a French kindred, appears to have unique effects; first, it is linked with increased intracellular PP i , 54 and, second, it interrupts ANKH interaction of with the sodium-dependent P i cotransporter Pit-1. ...
... In humans, mutations in the channel core of ANK cause craniometaphyseal dysplasia (CMD) [91,92], and mutations in the N-and C-terminus of the ANK protein cause chondrocalcinosis (CC) [95][96][97]. CMD is a rare skeletal condition of abnormal bone formation characterized by an increased density of craniofacial bones and abnormal modeling of the metaphyses of the tubular bones. CC is a disease of articular cartilage that is radiographically characterized by the deposition of calcium pyrophosphate dihydrate crystals in the joint. ...
... CC is a disease of articular cartilage that is radiographically characterized by the deposition of calcium pyrophosphate dihydrate crystals in the joint. Like mouse progressive ankylosis, CMD is associated with a decrease in extracellular PPi levels [91,92] whereas CC is associated with an increase in the amount of PPi in the extracellular space which induce the spontaneous formation of calcium pyrophosphate crystals [95][96][97]. ...
Elevated serum phosphorus has emerged as a key risk factorfor pathologic calcification of cardiovascular structures, or vascular calcification (VC). To prevent the formation of calciumphosphate deposits (CPD), the body uses adenosine-5'-triphosphate (ATP) to synthesize inhibitors of calcification, including proteins and inhibitors of low molecular weight. Extracellular pyrophosphate (PPi) is a potent inhibitor of VC,which is produced during extracellular hydrolysis of ATP. Loss of function in the enzymes and transporters that are involved in the cycle of extracellular ATP, including Pi transporters, leads to excessive deposition of calcium-phosphate salts. Treatment of hyperphosphatemia with Pi-binders and Injection of exogenous PPi are the effective treatments to prevent CPD in the aortic wall. The role of sodium phosphate cotransporters in ectopic calcification is contradictory and not well defined, but their important role in the control of intracellular Pi levels and the synthesis of ATP make them an important target to study.
... Extracellular PPi can derive either from the breakdown of adenosine triphosphate (ATP), wherein ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) enzymatically releases pyrophosphate [14], or it can be directly secreted from chondrocytes into the extracellular milieu by a transmembrane protein highly expressed in chondrocytes, the ankylosis human protein (ANKH) [15]. Gain-of-function mutations of the ANKH have been observed in individuals with familial CPPD disease [16], and ANKH up-regulation may also occur secondarily in the setting of cartilage damage, thereby promoting CPP crystal formation [15]. As previously mentioned, the amount of extracellular ATP influences the levels of extracellular PPi, and, consequently, CPP crystal formation; a significant amount of ATP is generated in response to mechanical loading and chondrocyte stress and injury [17], suggesting a role of articular degeneration on CPPD pathogenesis. ...
Citation: Filippou, G.; Sirotti, S.; Cipolletta, E.; Filippucci, E. Optimizing the Use of Ultrasound in Calcium Pyrophosphate Deposition (CPPD): A Review from the Ground Up. Gout Urate Cryst. Depos. Dis. 2024, 2, 17-33. https://doi.
Abstract: Ultrasound is a pivotal exam in calcium pyrophosphate deposition (CPPD) identification. It has been demonstrated to be feasible, accurate, and reliable for CPPD diagnosis. Even if standardized definitions and a scoring system for CPPD have been established by the OMERACT ultrasound working group, ultrasound is still considered one of the most operator-dependent techniques. This is because in ultrasound, both the acquisition and the interpretation phases of the diagnostic process are in the hands of one operator and are performed simultaneously, in contrast to what happens with other imaging exams, where the acquisition process is standardized and independent from the interpretation process. Therefore, the scanning technique and machine setting acquire a central role, almost as important as the interpretation of the images, as erroneous scanning may lead to interpretative mistakes. In this review, we will delve into the appearance of CPPD on ultrasound, based on the latest research findings, passing through its pathogenesis, and focusing on machine settings and ultrasound scanning techniques, providing some tips and tricks to facilitate accurate CPPD recognition in the most frequently affected sites.
... 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.
... Calcium pyrophosphate deposition disease is typically a condition of later adulthood but is occasionally seen in younger patients with predisposing conditions such as hypophosphatasia, hyperparathyroidism or hemochromatosis [56], as well as in children with familial variants, in whom an autosomal-dominant pattern of inheritance has been documented [57]. These children usually present acutely with inflammatory symptoms lasting weeks to months, with subsequent intermittent flares. ...
Hand involvement can assume an outsized role in the perception and presentation of disease as a result of functional impairment, visual conspicuity and susceptibility to early structural damage. Rheumatologic referral for inflammatory conditions can be delayed because of assumptions of a traumatic, infectious or neoplastic etiology; conversely, initial rheumatologic evaluation might be pursued for many of the same non-inflammatory causes. This pictorial essay highlights inflammatory conditions affecting the pediatric hand, including juvenile idiopathic arthritis, infectious arthritis, systemic connective tissue disorders, and a variety of less common inflammatory diseases, as well as non-inflammatory congenital, vascular, neoplastic and metabolic differential considerations.
... 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
... 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.
... Interest in the field of genetics appears to increase as more studies of ANKH protein in familial CPPD diseases have been published in the last years. It was established that mutation in CCAL 2 locus on chromosome 5 was linked to an autosomal-dominant form of CPPD, but mutation on chromosome 8 (CCAL 1) was also related to CPPD 72,73 . A subsequent study revealed that mutation in TNFRSF11B gene encoding OPG might lead to an association of OA and chondrocalcinosis 74 ; in the study conducted by William et al. 75 in 2018, CCAL1 locus on chromosome 8 was identified as TNFRSF11B (OPG) gene. ...
Bone endures a lifelong course of construction and destruction, with bone marker (BM) molecules released during this cycle. The field of measuring BM levels in synovial fluid and peripheral blood is a cardinal part of bone research within modern clinical medicine and has developed extensively in the last years. The purpose of our work was to convey an up-to-date overview of synovial fluid and serum BMs in the most common arthropathies.
... A higher age is strongly associated with CPPD [1][2][3][4] and CPPD is uncommon under age 60 years [1,2]. An onset of CPPD before age 45 is usually associated with genetic (i.e., familial CPPD) or metabolic factors [1][2][3][4][5][6][7][8][9]. Chondrocalcinosis (CC), a usually asymptomatic CPPD commonly visualized on radiographs, supports the diagnosis but the identification of rhomboid, mostly intracellular calcium pyrophosphate crystals in the synovial fluid, is necessary for confirmation of diagnosis [1][2][3][4]. ...
Objective:
To estimate the prevalence and incidence of non-gout crystal arthropathy in relation to socioeconomic factors in southern Sweden.
Methods:
All patients (age ≥ 18 years) with at least one visit to a physician with the diagnosis of interest in the Skåne region (population of 1.3 million) in 1998-2014 were identified. Non-gout crystal arthropathy (ICD-10 codes M11.0-M11.9) was subclassified in four different groups: calcium pyrophosphate crystal deposition related arthropathy (CPPD), unspecified non-gout arthropathies, chondrocalcinosis, and hydroxyapatite crystal deposition disease. The crude and age-adjusted point prevalence on December 31, 2014, and the cumulative incidence during 2014 were calculated for all non-gout crystal arthropathies, CPPD, and other unspecified non-gout arthropathies overall and in relation to occupation, income, and level of education.
Results:
The crude 2014 point prevalence (95% CI) and 2014 cumulative incidence (95% CI) of all non-gout crystal arthropathies were 0.23% (0.23-0.24) and 21.5 (19-25) cases/100,000 persons. Mean age (range) among all prevalent cases in 2014 was 71 (20-102) years and 56% were males. The point prevalence and cumulative incidence of CPPD were 0.09% (0.08-0.09) and 8 (7-10)/100,000 persons, respectively. The corresponding data for unspecified non-gout crystal deposition disease was 0.16% (0.16-0.17) and 15.6 (13-18)/100,000 persons, respectively. The prevalence and incidence of CPPD and unspecified non-gout crystal arthropathies were slightly higher in men and increased with age irrespective of gender. Unspecified non-gout crystal arthropathy but not CPPD was less prevalent in persons with ≥ 15 years of education, whereas there were no clear associations with occupation and income.
Conclusion:
The prevalence of all diagnosed non-gout crystal arthropathies was 0.23%, thus considerably less prevalent than gout in southern Sweden. CPPD and other unspecified non-gout crystal arthropathies are the predominant diagnoses, increasing with age and in men. With the exception for unspecified non-gout crystal arthropathies being inversely correlated to a higher level of education, no convincing association with the socioeconomic factors was found.
... Although most forms of CPPD disease are sporadic, multiple autosomal dominant forms of CPPD disease have been reported. Mutations in two distinct genetic loci, originally termed CCAL1 (found in chromosome 5p) and CCAL2 (found in chromosome 8q), have been observed in these families 18,19 . The disease-associated mutations exhibit an autosomal dominant pattern of inheritance 19 . ...
Chondrocytes in osteoarthritis undergo a phenotype shift leading to increased production of cartilage-degrading enzymes. There are similarities between the phenotype of osteoarthritic chondrocytes and those of growth plate chondrocytes. Hydroxyapatite can promote chondrocyte differentiation in the growth plate. Basic calcium phosphate (BCP) crystals (which consist of hydroxyapatite, octacalcium apatite and tricalcium phosphate) are frequently found in osteoarthritic joints. The objective of this study was to determine whether BCP crystals induce disease-associated changes in phenotypic marker expression in chondrocytes. Primary human chondrocytes isolated from macroscopically normal cartilage were treated with BCP for up to 48 h. Expression of indian hedgehog (IHH), matrix metalloproteinase 13 (MMP13), interleukin-6 (IL-6) and type X collagen (COLX) were higher, and expression of sry-box 9 (SOX9) lower, in BCP-treated chondrocytes (50 µg/mL) compared to untreated controls. COLX protein was also present in BCP-treated chondrocytes. Intracellular calcium and levels of phosphorylated and total calcium/calmodulin kinase 2 (CaMK2) were elevated following BCP treatment due to BCP-induced release of calcium from intracellular stores. CaMK2 inhibition or knockdown ameliorated the BCP-induced changes in SOX9, IHH, COLX, IL-6 and MMP13 expression. BCP crystals induce osteoarthritis-associated changes in phenotypic marker expression in chondrocytes by calcium-mediated activation of CaMK2. The presence of BCP crystals in osteoarthritic joints may contribute to disease progression.
... Although most forms of CPPD disease are sporadic, multiple autosomal dominant forms of CPPD disease have been reported. Mutations in two distinct genetic loci, originally termed CCAL1 (found in chromosome 5p) and CCAL2 (found in chromosome 8q), have been observed in these families 18,19 . The disease-associated mutations exhibit an autosomal dominant pattern of inheritance 19 . ...
The most common types of calcium-containing crystals that are associated with joint and periarticular disorders are calcium pyrophosphate dihydrate (CPP) and basic calcium phosphate (BCP) crystals. Several diverse but difficult-to-treat acute and chronic arthropathies and other clinical syndromes are associated with the deposition of these crystals. Although the pathogenic mechanism of calcium crystal deposition is partially understood, much remains to be investigated, as no drug is available to prevent crystal deposition, permit crystal dissolution or specifically target the pathogenic effects that result in the clinical manifestations. In this Review, the main clinical manifestations of CPP and BCP crystal deposition are discussed, along with the biological effects of these crystals, current therapeutic approaches and future directions in therapy.
... Another aspect to consider is the possible genetic pathogenesis of CPPD. More than one member of a family could be affected, particularly noted when it occurs before 60 years old, as a genetic mutation may result in a gain of function of the ANKH protein that regulates inorganic pyrophosphate levels (Williams et al., 2003). Since CPPD affected the two skeletons studied here, they could have been genetically related. ...
The odontoid process of the axis can be affected by congenital or acquired pathologies. While abnormalities such as os odontoideum, agenesis, and fractures are reported in archaeological remains, the abnormality of an elongated length of the odontoid process has not been described in the paleopathological literature. The aim of this paper is to evaluate two individuals with elongated odontoid processes from a skeletal assemblage from the B6 archaeological site (Mendoza, Argentina), and to discuss the possible etiologies of the condition, with particular attention given to the relation to trauma and Crowned Dens Syndrome (CDS), a condition characterized by the ossification of ligaments of the odontoid process of the second cervical vertebra.
... Although most forms of CPPD disease are sporadic, multiple autosomal dominant forms of CPPD disease have been reported. Mutations in two distinct genetic loci, originally termed CCAL1 (found in chromosome 5p) and CCAL2 (found in chromosome 8q), have been observed in these families 18,19 . The disease-associated mutations exhibit an autosomal dominant pattern of inheritance 19 . ...
Gout and osteoarthritis are known to coexist in certain joints, with urate and calcium crystals being associated with the development of osteoarthritis. Now, research is shedding light on the depth of this association and bringing surprising observations to the fore.
... Two genetic loci are associated with familial CPPD. Mutations in the CCAL2 locus on chromo-some 5p produce an autosomal dominant pattern of inheritance (probably resulting from a gain of function of the ANKH protein) 44 and provide additional support for a key role of ANKH in the pathogenesis of CPPD disease. The CCAL1 locus on chromosome 8 has not yet been fully characterized. ...
CPPD disease (also called pseudogout) is common, especially among persons older than 60 years of age, but it is underrecognized and undertreated. This review summarizes the diagnosis and treatment of the acute and chronic forms of this crystal-induced arthritis.
... Familial CPPDD is a rare human dominant disorder characterized by the deposition of CPPD crystals. Heterozygous ANKH mutations have been detected in at least five multiplex CPPDD families [37][38][39][40]. In three different multiplex families (one Argentinean of northern Italian descent and two US families), mutation occurred in amino acid 5 (proline), which was changed to either leucine or threonine. ...
... Many of the genes displaying lower expression levels in the hTERT-OA 13A FLSs have been previously implicated in the negative regulation of biomineral formation. For example, SRGN inhibited the growth of hydroxyapatite crystals [50]; PTHLH and FGF9 inhibited terminal differentiation of chondrocytes and mineralization [51][52][53]; mutation of ANKH caused chondrocalcinosis [54,55]; CTGFtreated mesenchymal stem cells (MSCs) lost the ability to differentiate into chondrocytes and osteoblasts [56]; EGFR signaling suppressed osteoblast differentiation [57,58]; and PTGER4 mediated the inhibition of mineralization in mature cementoblasts by prostaglandin E2 [59]. In contrast, many of the genes displaying higher expression levels have been previously implicated in the positive regulation of biomineral formation. ...
Calcium crystals are present in the synovial fluid of 65%-100% patients with osteoarthritis (OA) and 20%-39% patients with rheumatoid arthritis (RA). This study sought to investigate the role of fibroblast-like synoviocytes (FLSs) in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla) protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s). The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.
... A condrocalcinose hereditária ou familiar é uma forma menos comum de condrocalcinose, sendo previamente descrita em países e grupos étnicos distintos, incluindo nações como Inglaterra (1) , Tunísia (2) , Chile (3) , Canadá (4) , Suíça (5) , Suécia (6) , EUA (7,8) e Espanha (9,10,11) . Relatamos uma família com essa entidade nosológica no Brasil e descrevemos as características clínicas de sua apresentação. ...
Familial articular chondrocalcinosis is a disorder characterized by deposition of calcium pyrophosphate dihydrate crystal in synovial fluid and articular cartilage that can cause joint pain and arthritis. We have identified three members of the same family with chondrocalcinosis. The clinical features of the disease were intermittent episodes of arthritis in two patients and polyarthritis resembling rheumatoid arthritis in one member. The radiological evaluation showed calcification in several joints, particularly in cartilages of the knees. Therapy with colchicine was enough to prevent arthritic crisis in two patients and continous NSAID use was necessary to control symptoms in the last one. Familial chondrocalcinosis seems to be rare in Brazil, but we do not exclude the possibility that this figure is underestimated and suggest that in cases of sporadic chondrocalcinosis other members of the family should be fully investigated.
... Overexpression of wild-type ANK in mutant ank/ank mouse fibroblasts reversed the alterations in ePPi and iPPi levels, indicating an important role for ANK in regulating PPi trafficking 14 . Subsequently, mutations of ANKH, the human homolog of the ank gene, were documented in patients with familial and sporadic CPPD 16,17 . ...
Accumulation of excess extracellular inorganic pyrophosphate leads to calcium pyrophosphate dihydrate (CPPD) crystal formation in articular cartilage. CPPD crystal formation occurs near morphologically abnormal chondrocytes resembling hypertrophic chondrocytes. The ANK protein was recently implicated as an important factor in the transport of intracellular inorganic pyrophosphate across the cell membrane. We characterized ANK in joint tissues from patients with and without CPPD deposition and correlated the presence of ANK with markers of chondrocyte hypertrophy.
Articular tissues were obtained from 24 patients with CPPD crystal deposition disease, 11 patients with osteoarthritis (OA) without crystals, and 6 controls. We determined the number of ANK-positive cells in joint tissues using immunohistochemistry and in situ hybridization, and correlated ANK positivity with markers of chondrocyte hypertrophy including Runx2, type X collagen, osteopontin (OPN), and osteocalcin (OCN).
ANK was detected in synoviocytes, chondrocytes, osteoblasts, and osteocytes. ANK was seen extracellularly only in the matrix of cartilage and meniscus. The number of ANK-positive cells was significantly higher in CPPD than in OA or normal joint tissues. The amount and intensity of ANK immunoreactivity reached maximum levels in the large chondrocytes around crystal deposits. ANK was similarly distributed to and significantly correlated with Runx2, type X collagen, OPN, and OCN.
ANK levels were higher in articular tissues from patients with CPPD deposition. ANK was concentrated around crystal deposits and correlated with markers of chondrocyte hypertrophy. These findings support a role for ANK in CPPD crystal formation in cartilage.
... MSR1 and SCARA5 are involved in both lipid and phosphate transport [17,18]. ANKH is a multipass transmembrane protein, which regulates transport of pyrophosphate ions across the plasma membrane [19]. ENTPD1 is an integral membrane ectoapyrase that hydrolyzes extracellular ATP/ADP. ...
This study sought to examine the expression of genes implicated in phosphate transport and pathological calcification in osteoarthritis (OA) and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and investigate the biological effects of phosphate. Results revealed that several genes, which were implicated in phosphate transport and pathological calcification, were differentially expressed in OA FLS and RA FLS. Phosphate stimulated the expression of matrix metalloproteinse-1, matrix metalloproteinse-3, cyclooxygenase-2, and interleukin-1
β
in a dose-dependent manner. Phosphate also induced OA FLS cell death but not RA FLS cell death at higher concentration. Calcification inhibitors, phosphocitrate (PC), and ethane-1-hydroxy-1,1-diphosphonate (EHDP), effectively inhibited these detrimental biological effects of phosphate. These findings suggest that abnormal expression of genes implicated in phosphate transport and pathological calcification may contribute to the progression of OA through the induction of extracellular matrix-degrading enzymes, proinflammatory cytokines, cell death, and calcium deposits. Calcification inhibitors such as PC and EHDP are potent inhibitors of these detrimental biological effects of phosphate.
... 63 Moreover, numerous studies have shown that mutations in ANK could result in abnormal dystrophic mineralization in joints and bone. 64,65 Oxemic status may influence ANK expression which may be mediated by HIF-1α. 63 Skubutyte, et al. 31 examined the expression and localization of ANK in the IVDs of mature and neonate rats and found that the expression of ANK in the NP was significantly higher in mature rats than in neonate rats. ...
Intervertebral disc (IVD) degeneration is implicated as a major cause of low back pain. The alternated phenotypes, reduced cell survival, decreased metabolic activity, loss of matrix production and dystrophic mineralization of nucleus pulposus (NP) cells may be key contributors to progressive IVD degeneration. IVD is the largest avascular structure in the body, characterized by low oxygen tension in vivo. Hypoxia-inducible factor (HIF) is a master transcription factor that is induced upon hypoxia and directs coordinated cellular responses to hypoxic environments. This review summarizes relevant studies concerning the involvement of HIF in the regulation of biological behaviors of NP cells. We describe current data on the expression of HIF in NP cells and further discuss the various roles that HIF plays in the regulation of the phenotype, survival, metabolism, matrix production and dystrophic mineralization of NP cells. Here, we conclude that HIF may be a promising target for the prevention and treatment of IVD degeneration.
Calcium pyrophosphate deposition disease is defined by the presence of calcium pyrophosphate (CPP) crystals in articular cartilage and is the fourth most common type of arthritis in adults. Despite its high prevalence, the etiology of CPPD disease remains unclear and no specific therapies currently exist. It has been known for several decades that abnormalities of cartilage pyrophosphate metabolism are common in patients with CPPD disease, and this classic work will be reviewed here. Recent studies of rare familial forms of CPPD disease have provided additional novel information about its pathophysiology. This work suggests that CPPD disease occurs through at least two unique and potentially intertwined biomolecular pathways. We are hopeful that a detailed understanding of the components and regulation of these pathways will lead to improved therapies for this common disease.
Objective
The gene TNFRSF11B encodes for osteoprotegerin (OPG) and was recently identified as the CCAL1 locus associated with familial calcium pyrophosphate deposition disease (CPDD). While the CCAL1 OPG mutation (OPG‐XL) was originally believed to be a gain‐of‐function mutation, loss of OPG activity causes arthritis‐associated osteolysis in mice, which is likely related to excess subchondral osteoclast formation and/or activity. The purpose of the present study was to further explore the effect of OPG‐XL in osteoclastogenesis.
Methods
The effects of recombinant OPG‐XL and wild‐type (WT) OPG were determined in monoculture and coculture models of RANKL‐induced osteoclastogenesis. The effects of OPG‐XL on osteoclast survival as well as on TRAIL‐induced apoptosis were determined using standard in vitro assays and compared to WT OPG. The ability of OPG‐XL and WT OPG to bind to osteoblasts was measured with enzyme‐linked immunosorbent assay and flow cytometry using the osteoblastic MC3T3‐E1 cell line.
Results
OPG‐XL was less effective than WT OPG at blocking RANKL‐induced osteoclastogenesis in monoculture and coculture models. Osteoclast survival and inhibition of TRAIL‐induced apoptosis were similar in the presence of OPG‐XL and WT OPG. Compared to WT OPG, considerably less OPG‐XL bound to cells.
Conclusion
These findings indicate that OPG‐XL is a loss‐of‐function mutation as it relates to RANKL‐mediated osteoclastogenesis, and thus may permit increased osteoclast numbers and heightened bone turnover. Further studies are necessary to demonstrate how this mutation contributes to arthritis in individuals carrying this mutation.
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.
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).
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).
Many birth defects, although rare individually, are encountered in clinical practice and have now become treatable if properly diagnosed. In the Atlas of Genetic Diagnosis and Counseling, Harold Chen, MD, shares his almost 40 years of clinical genetics practice in a comprehensive pictorial atlas of 203 genetic disorders, malformations, and malformation syndromes. The author provides a detailed outline for each disorder, describing its genetics, basic defects, clinical features, diagnostic tests, and counseling issues, including recurrence risk, prenatal diagnosis, and management. Numerous color photographs of prenatal ultrasounds, imagings, cytogenetics, and postmortem findings illustrate the clinical features of patients at different ages, patients with varying degrees of severity, and the optimal diagnostic strategies. The disorders cited are supplemented by case histories and diagnostic confirmation by cytogenetics, biochemical, and molecular techniques, when available. Also available in a CD-ROM edition (ISBN: 1-58829-974-5).
Authoritative and up-to-date, the Atlas of Genetic Diagnosis and Counseling will help all physicians to understand and recognize genetic diseases and malformation syndromes, and consequently better evaluate, counsel, and manage affected patients.
Introduction Recent years have witnessed major advances in our understanding of the genetic basis for many skeletal disorders. In several instances, particularly with the less common Mendelian diseases, the responsible gene has been mapped, mutations identified, and their functional significance determined, contributing significantly to our understanding of the molecular basis for pathogenesis. For more common disorders where susceptibility is complex, a complete picture of the responsible genetic polymorphisms and how they influence pathogenesis has been more difficult to achieve. In this chapter we focus on our current understanding of the genetic basis and pathogenic mechanisms for disorders of bone homeostasis. For readers interested in the genetic basis of developmental disorders of the skeleton, we recommend a recent review (Kornak and Mundlos, 2003). Bone resorption and bone formation are ongoing processes in both the developing and mature skeleton. Even during growth, where the balance favors bone formation, bone resorption is necessary to remove calcified cartilage prior to the formation of mature bone. In the adult skeleton, there continues to be a dynamic balance between these processes that serves both metabolic and mechanical needs of the individual. Although bone mass continues to increase during childhood, it peaks between the ages of 25 and 35 years, and then begins a steady decline that becomes most prominent after age 50 years when bone formation does not fully compensate bone loss. The delicate balance between bone formation and bone resorption is maintained largely by the coordinated actions of two cell types, osteoblasts and osteoclasts. © Cambridge University Press 2007 and Cambridge University Press, 2009.
The cytokine TRAIL is a promising cancer therapeutic candidate as it induces apoptosis selectively in transformed cells. TRAIL-induced clustering of its receptors (DR) is essential for the DISC complex formation, which induces cell death. The mechanism for TRAIL’s tumour selective effect is largely unknown. We identified the cytoskeleton proteins non-muscle myosin heavy chain IIa, IIb (NMHCIIa, NMHCIIb), myosin regulatory light chain (MLC2) and ß-actin as novel DR-interactors. An initially weak and TRAIL-induced abrogation of NMHCII/DR interaction correlated with efficient DISC formation in tumour cells. In contrast, a robust NMHCII/DR interaction that was sustained upon TRAIL stimulus was accompanied by incomplete DISC arrangement. Weakening the NMHCII/DR interaction in normal cells using chemical inhibitors enhanced TRAIL-induced apoptosis. Intriguingly, siRNA-mediated NMHCIIa- but not NMHCIIb depletion potently released TRAIL resistance in normal cells and influenced DISC composition. Reduced NMHCII/DR interaction in transformed cells was characterised by diminished MLC2 phosphorylation and altered protein expression of upstream regulatory kinases. Our results suggest that normal cell resistance to TRAIL-apoptosis is based on the interaction of cytoskeleton components with DR that is impaired upon transformation. Since NMHCII function in cell adhesion and migration, it will be interesting to study possible roles of the interaction in cell detachment and altered TRAIL sensitivity; moreover this link may provide clues as to the cause of TRAIL resistance in some cancers.
This chapter discusses the major products of articular cartilage metabolism with special emphasis on those that are relevant as body fluid markers. It provides a brief review of the structure-function relationships of the major matrix building blocks. The cartilage matrix contains several molecules that are found in much higher concentrations than in other tissues. The attention that aggrecan and collagen type II are currently receiving as potential markers of specific metabolic alterations in cartilage disease will likely extend to less abundant molecules found to play key roles in the maintenance of matrix homeostasis. As the understanding of the function and metabolism of these molecules improves, so will their potential as markers of the irreversible destabilization of the collagenous meshwork or of other specific alterations in cartilage matrix organization.
Familial calcium pyrophosphate dihydrate deposition (CPPD) disease is a chronic condition in which CPPD microcrystals deposit in the joint fluid, cartilage, and periarticular tissues. Two forms of familial CPPD disease have been identified: CCAL1 and CCAL2. The CCAL1 locus is located on the long arm of chromosome 8 and is associated with CPPD and severe osteoarthritis. The CCAL2 locus has been mapped to the short arm of chromosome 5 and identified in families from the Alsace region of France and the United Kingdom. The ANKH protein is involved in pyrophosphate metabolism and, more specifically, in pyrophosphate transport from the intracellular to the extracellular compartment. Numerous ANKH gene mutations cause familial CCAL2; they enhance ANKH protein activity, thereby elevating extracellular pyrophosphate levels and promoting the formation of pyrophosphate crystals, which produce the manifestations of the disease. Recent studies show that growth factors and cytokines can modify the expression of the normal ANKH protein. These results suggest a role for ANKH in sporadic CPPD disease and in CPPD associated with degenerative disease.
Calcium pyrophosphate dihydrate and basic calcium phosphate crystals are commonly found in patients with calcium deposition diseases. Deposits of these crystals are common in the elderly and they have been implicated in the pathogenesis of osteoarthritis. The presence of crystal deposits is often asymptomatic, although it can be intermittently symptomatic and elicit acute and chronic arthritis. Acute intra- and peri-articular inflammation are clinically similar to cellulitis, gout and septic arthritis. Plain radiography of the affected joints is usually the first and most valuable diagnostic method to evaluate these conditions. Ultrasonography is highly sensitive for the detection of crystals in the synovial fluid and of crystals deposited in cartilage and soft tissue. Joint-fluid analysis is far more sensitive than radiologic study to detect calcium pyrophosphate dihydrate and basic calcium phosphate crystal deposits, and arthrocentesis is indicated in some patients to rule out septic arthritis.
Purpose of review: As calcium-containing crystals are present in up to 70% of osteoarthritis cases, understanding the nature of these crystals and the events triggered by crystals is important to orthopedic science and is critical for future drug development.
Recent findings: The discovery of ank (progressive ankylosis locus) mutations in mice and subsequent ank gene mutations in familial human osteoarthritis is the most significant finding in the recent years. The finding that phosphocitrate inhibits osteoarthritis in Hartley guinea pigs is one of the most significant findings in the past year. These studies provide direct evidence supporting a pathogenic role of pathological calcification in osteoarthritis. Two recent studies indicate, however, that the presence of calcium pyrophosphate dihydrate crystals is associated with a protective effect against cartilage loss and another study suggests that the presence of calcium pyrophosphate dihydrate crystals is an aging phenomenon.
Summary: Crystal-associated osteoarthritis is a complex and heterogeneous disease. In some cases, pathological calcification may be pathogenic; in others, crystals may be bystanders. In some instances, pathological calcification and osteoarthritis may be two intimately linked processes. Inhibition of an early event in either process may inhibit or deter the progression of both processes.
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.
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.
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.
In calcium pyrophosphate dihydrate (CPPD) crystal deposition disease, metabolic abnormalities favoring extracellular inorganic pyrophosphate (PPi) accumulation have been suspected. Elevations of intracellular PPi in cultured skin fibroblasts from a single French kindred with familial CPPD deposition (19) and elevated nucleoside triphosphate pyrophosphohydrolase activity (NTPPPH), which generates PPi in extracts of CPPD crystal-containing cartilages (14) favor this suspicion. To determine whether NTPPPH activity or PPi content of cells might be a disease marker expressed in extraarticular cells, human skin-derived fibroblasts were obtained from control donors and patients affected with the sporadic and familial varieties of CPPD (CPPD-S and CPPD-F) deposition. Intracellular PPi was elevated in both CPPD-S (P less than 0.05) and CPPD-F (P less than 0.01) fibroblasts compared with control fibroblasts. Ecto-NTPPPH activity was elevated in CPPD-S (P less than 0.01) but not CPPD-F. Intracellular PPi correlated with ecto-NTPPPH (P less than 0.01). Elevated PPi levels in skin fibroblasts may serve as a biochemical marker for patients with familial or sporadic CPPD crystal deposition disease; ecto-NTPPPH activity further separates the sporadic and familial disease types. Expression of these biochemical abnormalities in nonarticular cells implies a generalized metabolic abnormality.
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.
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.
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.
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.
Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5pl5. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition. © 2002 by The American Society of Human Genetics. All rights reserved.
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.
Arthritis and allied conditions
- Ryan LM
- McCarty DJ