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SECTION I: SYMPOSIUM: Genetics in Orthopaedics

Cuff Tear Arthropathy

Evidence of Functional Variation in Pyrophosphate Metabolism Genes

Peach, Chris A MBBS, MRCS; Zhang, Yun DPhil; Dunford, James E PhD; Brown, Matthew A MD, FRACP; Carr, Andrew J MA, ChM, FRCS

Editor(s): Dobbs, Matthew B MD

Author Information
Clinical Orthopaedics and Related Research: September 2007 - Volume 462 - Issue - p 67-72
doi: 10.1097/BLO.0b013e31811f39de
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Abstract

The term cuff tear arthropathy was first used by Neer15 to describe the clinical and pathological findings in 26 patients undergoing total shoulder replacement arthroplasty. The condition is characterized by chronic massive rotator cuff tear, proximal humeral migration, recurrent joint effusions, and destruction of the joint with collapse of the humeral head. Neer15 suggested that abnormal kinematics leads to subchondral osteopenia and altered articular cartilage structure with consequent arthritis and bone destruction. Other authors3,11,12 have described similar features (Milwaukee shoulder syndrome) and have identified the presence of basic calcium phosphate and calcium pyrophosphate dihydrate crystals on synovial fluid analysis of the recurrent effusions. Basic calcium phosphate crystals are mainly partially carbonate-substituted hydroxyapatite, but also comprise octacalcium phosphate and tricalcium phosphate. It is unknown whether these crystals are a primary pathogen or an epiphenomenon. Calcium crystals can damage tissue in several ways, including induction of mitogenesis, up-regulation of matrix metalloproteinases (MMPs), stimulation of cyclooxygenase 1 and 2 and prostaglandin E2 production, and stimulation of cytokine production such as IL-1β and IL-8.13

Genetic studies of crystal joint diseases have focused on patients and families with chondrocalcinosis in whom the most common crystal types are calcium pyrophosphate dihydrate and basic calcium phosphate crystals. Genetic investigations of genes encoding the extracellular matrix proteins were inconclusive. A heterozygous mutation in the COL2A1 gene was identified in a family with multiple joint chondrocalcinosis but the phenotype suggested a secondary role of the chondrocalcinosis.18,25 The first gene found associated with primary chondrocalcinosis was the ANKH gene,7,16,27 the human homologue of the mouse progressive ankylosis gene (ank). A number of reports have identified mutations and polymorphisms in both familial and sporadic chondrocalcinosis confirming the role of ANKH in disease susceptibility.16,26,27,29

Studies of chondrocyte nucleoside triphosphate pyrophosphohydrolase (NTPPPH) have suggested that inorganic pyrophosphate (PPi) concentrations have a crucial role in calcium crystal deposition.6,14,20 Change in the extracellular concentration of PPi leads to crystal deposition, with a rise causing calcium pyrophosphate dihydrate deposition and a fall causing hydroxyapatite deposition.21 The alteration in concentrations could be due to increase or decrease in the function of proteins governing the homeostasis of PPi concentration. ANKH encodes a multipass transmembrane protein transporting PPi from inside chondrocytes.5TNAP encodes the protein Tissue-Nonspecific Alkaline Phosphatase which hydrolyses PPi to Pi and an increase or decrease in concentrations of the enzyme will decrease or increase PPi concentration levels respectively (Fig 1).2 Mutations in TNAP cause hypophosphatasia, characterized by defective bone mineralization and pathological fractures.4 Several cases have associated crystal arthropathy with hypophosphatasia.1,24

Fig 1
Fig 1:
Schematic diagram showing the proteins ANKH and TNAP and their relationship with pathways controlling PPi concentrations (iPPi = intracellular PPi; ePPi = extracellular PPi).

The only previous investigation of genetic association in Milwaukee shoulder reported on an Italo-Argentinian family with familial osteoarthritis and Milwaukee shoulder.17 Loci on chromosomes 8q, 5p15 (the ANKH locus), and COL2A1 were excluded as having a role in disease susceptibility.

We hypothesized crystal deposition is an integral part of disease pathogenesis. Therefore we looked for mutations in ANKH or TNAP and any association with cuff tear arthropathy and variations in the gene sequence. We also asked whether any associated variants in ANKH could alter PPi concentrations in human chondrocyte cells and whether patients with cuff tear arthropathy had abnormal serum concentrations of TNAP.

MATERIALS AND METHODS

Twenty-two patients with sporadic cuff tear arthropathy with no family history were recruited from those attending the shoulder and elbow clinic at the Nuffield Orthopaedic Centre in Oxford between 2000 and 2004. All patients were diagnosed clinically and radiographically by the senior author (AC). Six-hundred healthy control subjects who were blood donors and were not receiving any medications were recruited from the national blood service (Oxford, UK). Peripheral blood genomic DNA was obtained from peripheral blood leukocytes from the two populations, who were all of British Caucasian origin. The study had ethics committee approval and all patients gave written informed consent to participate.

Mutations were screened for by direct DNA sequencing of exons, untranslated regions and intron-exon boundaries of ANKH and TNAP after DNA amplification by polymerase chain reaction (details of primers on request). DNA from all 22 patients with cuff tear arthropathy was sequenced. The study had a 90% power to detect polymorphisms with a minor allele frequency of 5%. DNA sequencing was carried out using ABI Big Dye chemistry on an ABI 3100 automated sequencing machine (Applied Biosystems, UK). The single nucleotide polymorphisms (SNPs) identified were subsequently genotyped by PCR/restriction fragment-length polymorphism analysis or by Taqman (KBiosciences, UK).

The 3′UTR SNP construct was generated using the Stratagene QuickChange site-directed mutagenesis protocol (Stratagene, La Jolla, CA). Forward (3′-gccatgggcactgcagggacGgtcagtcaggatgacacttc-5′) and reverse (5′-gaagtgtcatcctgactgacCgtccctgcagtgcccatggc-3′) primers were designed to incorporate the sequence change. Dpn1 was used to digest the original template, and the circular PCR product was transformed into DH5A supercompetent cells. The construct was sequenced across the entire open reading frame to ensure that there were no other mutations introduced other than the one intended. Immortalized CH8 cells (previously generated using the adenoviral-driven SV40 early gene28) were transfected with wild-type ANKH cDNA, +31 3′UTR ANKH SNP construct, and vector-only control. Samples prepared for the transfection were equalized for DNA concentration, and ANKH-HA and vector only controls were used. ANKH expression was tested by western blot analysis after each transfection. We observed the protein expression by using anti-HA antibody before carrying out the PPi assay.

The concentrations of intracellular and extracellular inorganic pyrophosphate were determined by a UDPG pyrophosphorylase reaction as previously described.9 Concentration of PPi was measured in cells transfected. The PPi assay was chosen as a means of investigating whether cases with the detected variant demonstrated altered PPi concentrations in vitro.

Serum TNAP levels in 16 patients (12 female and four male) with cuff tear arthropathy were measured by TNAP enzyme-linked immunosorbent assay (Metra Biosystems, Inc). Serum TNAP levels were taken to ascertain whether there was an increased level in cases of cuff tear arthropathy. This would indicate an increase in function of TNAP and predict a reduction in extracellular PPi (ePPi) concentrations predisposing to hydroxyapatite crystal deposition.

The genotype frequency of each SNP identified in the patients with cuff tear arthropathy was compared to those in the healthy control subjects (Tables 1 and 2). Genotype and allele frequencies in patients and controls were compared by standard χ2 contingency table analysis. Genotype relative risks were estimated using the Lathrop method.8 Comparison of mean concentrations of PPi and TNAP was assessed by Student's t test with a level of significance of < 0.05.

TABLE 1
TABLE 1:
Genotyping of Variants in ANKH in Patients with Cuff Tear Arthropathy versus Controls
TABLE 2
TABLE 2:
Genotyping of Variants in TNAP in Patients with Cuff Tear Arthropathy versus Controls

RESULTS

Direct sequencing of ANKH revealed no novel mutations or SNPs not previously identified in the literature or published in the public SNP databases (www.ensembl.org; www.ncbi.nlm.nih.gov; or genome.ucsc.edu). However, we identified six SNPs: two synonymous-coding-region SNPs, two untranslated-region SNPs, and two intronic SNPs (Table 3). All SNPs identified in ANKH were in Hardy-Weinberg equilibrium.

TABLE 3
TABLE 3:
Variants Identified from DNA Sequencing of ANKH in Cases with Cuff Tear Arthropathy

We observed an association with the disease in those patients who carry the SNP identified in the 3′UTR of ANKH, 31 bases after the stop codon (Table 1). The heterozygous genotype was seen more frequently (p = 0.0003; Relative Risk (RR) = 2.78) in patients with cuff tear arthropathy (45%) than in controls (20%).

We identified 16 SNPs from the sequence analysis of TNAP (Table 4), six of which were not published on public databases. One of these is a nonsynonymous SNP that causes an amino acid change from glutamine to arginine at amino acid position 292 in Exon 8. In addition we detected a coding SNP documented previously in Exon 6 changing amino acids from tyrosine to histidine as well as a synonymous SNP in exon 4. All SNPs identified in TNAP were in Hardy-Weinberg equilibrium.

TABLE 4
TABLE 4:
Variants Identified from DNA Sequencing of TNAP in Cases with Cuff Tear Arthropathy

We observed an association between cases with cuff tear arthropathy and carriers of a SNP in TNAP, 46 bases into intron 10 (Table 2). The heterozygous genotype was seen more commonly (p = 0.007; RR = 2.85) in patients with cuff tear arthropathy (32%) then in healthy control subjects (9%).

The intracellular PPi concentrations in cells transfected with the SNP detected in the 3′UTR of ANKH (Fig 2) were reduced (p = 0.034) when compared with those cells transfected with wild-type ANKH cDNA. We detected no difference in extracellular concentrations of PPi.

Fig 2
Fig 2:
Effects of transfected ANKH wild type and mutants on intracellular PPi concentrations are shown. The intracellular PPi concentration in 3′UTR mutant cells was reduced (p = 0.034) compared with wild-type ANKH and control cells suggesting that this rise, caused by the mutant, could predispose cases with the SNP to calcium pyrophosphate dihydrate crystal formation.

Mean serum TNAP levels (Fig 3) for the 16 patients were elevated (p = 0.00003) to 46.8 U/L (SD 16.4; range, 21-82); control mean 24.5 U/L. The mean serum TNAP level for male patients was not raised at 30 U/L (SD 10.5; range, 21-42); normal range is 15-41 U/L. The mean level for female patients was elevated (p = 0.000001) at 52.3 U/L (SD 12.1; range, 31-82); normal range is 11-31 U/L.

Fig 3
Fig 3:
Serum TNAP concentrations were elevated (p = 0.00003) in patients with cuff tear arthropathy. The female patients had an elevated serum TNAP concentration (p = 0.000001) whereas there was no rise in the mean concentration in male patients. Elevated TNAP concentrations predispose patients to hydroxyapatite crystal deposition.

DISCUSSION

This study was designed to investigate whether there were discrete mutations in genes controlling extracellular pyrophosphate concentrations in patients with cuff tear arthropathy. It also aimed to find any association between cases and variants in the two genes ANKH and TNAP. Furthermore, we attempted to confirm that these variants altered function of the protein encoded by the genes.

There are limitations of our study particularly in relation to the small numbers of cases available for investigation due to the relative rarity of the condition. The PPi assay is a technically difficult method and failure to detect appreciable differences in extracellular PPi levels after cell culture may have been methodological. Alternatively, CH-8 cells, which produce high levels of TNF-α which reduces ePPi, may intrinsically be resistant to ANKH gain of function. Future studies will need a larger patient cohort.

We identified one SNP in the 3′UTR of ANKH associated with patients with cuff tear arthropathy. This has not previously been associated with chondrocalcinosis. SNPs in ANKH have been shown to cause an increase in ePPi concentration, which is thought to lead to calcium pyrophosphate dihydrate crystal deposition. We were unable to ascertain any change in the ePPi concentrations, which may represent a failure of the assay. Alternatively this may indicate that ANKH functions not just as a PPi transporter, but may have other roles influencing PPi synthesis and breakdown. However, there was a considerable reduction in intracellular PPi concentrations in the cells transfected with the ANKH cDNA with the 3′UTR polymorphism construct when compared with wild-type cDNA. ANKH encodes a multipass transmembrane protein which transports PPi, and this reduction in intracellular concentration may represent an up-regulation of the transporter which would increase extracellular concentration and therefore predispose the carrier to calcium pyrophosphate dihydrate deposition. This would concur with previous reports of ANKH polymorphisms.16,29 Sequence-altering variants of the UTRs have been well documented to contribute to serious diseases, for example expanded CTG repeats in the DMPK gene in mytonic dystrophy22 and the antitermination mutation in α-thalassaemia.23 The 3′UTR contains regulatory sequences including polyadenylation sequences, which mediate the cleavage of the transcript and addition of adenine residues preventing degradation of the mRNA. The region also contains binding sites for micro RNAs, which are thought to regulate the expression of other genes.

TNAP hydrolyses PPi and an increase or decrease in the function of the protein could lead to crystal deposition. There was a positive association between a SNP in TNAP and cases of cuff tear arthropathy. The serum levels of TNAP in patients with cuff tear arthropathy were substantially elevated and when subset analysis was performed, the serum concentrations in women with cuff tear arthropathy were significantly raised whereas the male subset of only four patients was within normal limits. An increase in TNAP activity would increase hydrolysis of PPi and therefore reduce extracellular concentrations. This will predispose to hydroxyapatite crystal formation (Fig 1). Although there were few male cases to analyze in our cohort, the normal levels of TNAP detected may account for the female preponderance of cuff tear arthropathy. There are clues in subtle differences in clinical features of the disease between male and female patients that might suggest two distinct pathological processes. Women appear to have more bilateral disease, multiple joint arthropathies, and soft tissue calcification whereas men often have a single shoulder affected with the more classical biomechanical and kinematic abnormalities that Neer15 first described. These genetic variations appear to have conflicting effects on PPi concentrations. Perhaps the altering increases and decreases in protein function causes a flux in PPi concentrations that account for the dual populations of basic calcium phosphate and calcium pyrophosphate dihydrate crystals encountered in the disease.

Better understanding of the underlying biology may assist surgical decision making and the relative roles of surgical and medical treatment of the crystal joint disease. Probenecid is known to inhibit transport by the ANKH protein and decrease the amount of PPi released by cultured chondrocytes19 and misoprostol, a prostaglandin E1 analogue, has been shown to inhibit basic calcium phosphate crystal-induced mitogenesis and collagenase accumulation in human fibroblasts.10 When the pathological processes leading to calcium crystal deposition and their effects are better explained, other therapeutic pathways could be pursued.

We have shown that variations in ANKH and TNAP are associated with patients with cuff tear arthropathy. These variants may contribute toward disease development by altering the extracellular concentrations of PPi thereby predisposing the patient to calcium crystal formation. Genetic influences will be only part of a patient's disease susceptibility with environmental factors likely to play an important role.

References

1. Eade AW, Swannell AJ, Williamson N. Pyrophosphate arthropathy in hypophosphatasia. Ann Rheum Dis. 1981;40:164-170.
2. Fallon MD, Whyte MP, Teitelbaum SL. Stereospecific inhibition of alkaline phosphatase by L-tetramisole prevents in vitro cartilage calcification. Lab Invest. 1980;43:489-494.
3. Halverson PB, Carrera GF, McCarty DJ. Milwaukee shoulder syndrome. Fifteen additional cases and a description of contributing factors. Arch Intern Med. 1990;150:677-682.
4. Henthorn PS, Whyte MP. Missense mutations of the tissue-nonspecific alkaline phosphatase gene in hypophosphatasia. Clin Chem. 1992;38:2501-2505.
5. Ho AM, Johnson MD, Kingsley DM. Role of the mouse ank gene in control of tissue calcification and arthritis. Science. 2000;289:265-270.
6. Howell DS, Martel-Pelletier J, Pelletier JP, Morales S, Muniz O. NTP pyrophosphohydrolase in human chondrocalcinotic and osteoarthritic cartilage. II. Further studies on histologic and subcellular distribution. Arthritis Rheum. 1984;27:193-199.
7. Hughes AE, McGibbon D, Woodward E, Dixey J, Doherty M. Localisation of a gene for chondrocalcinosis to chromosome 5p. Hum Mol Genet. 1995;4:1225-1228.
8. Lathrop GM. Estimating genotype relative risks. Tissue Antigens. 1983;22:160-166.
9. Lust G, Seegmiller JE. A rapid, enzymatic assay for measurement of inorganic pyrophosphate in biological samples. Clin Chim Acta. 1976;66:241-249.
10. McCarthy GM, Mitchell PG, Cheung HS. Misoprostol, a prostaglandin E1 analogue, inhibits basic calcium phosphate crystal-induced mitogenesis and collagenase accumulation in human fibroblasts. Calcif Tissue Int. 1993;52:434-437.
11. McCarty DJ, Halverson PB, Carrera GF, Brewer BJ, Kozin F. Milwaukee shoulder”-association of microspheroids containing hydroxyapatite crystals, active collagenase, and neutral protease with rotator cuff defects. I. Clinical aspects. Arthritis Rheum. 1981;24:464-473.
12. McCarty DJ, Lehr JR, Halverson PB. Crystal populations in human synovial fluid: identification of apatite, octacalcium phosphate, and tricalcium phosphate. Arthritis Rheum. 1983;26:1220-1224.
13. Molloy ES, McCarthy GM. How crystals damage tissue. Curr Rheumatol Rep. 2004;6:228-234.
14. Muniz O, Pelletier JP, Martel-Pelletier J, Morales S, Howell DS. NTP pyrophosphohydrolase in human chondrocalcinotic and osteoarthritic cartilage: I: Some biochemical characteristics. Arthritis Rheum. 1984;27:186-192.
15. Neer CS, Craig EV, Fukada H. Cuff-tear arthropathy. J Bone Joint Surg Am. 1983;65:1232-1244.
16. Pendleton A, Johnson MD, Hughes A, Gurley KA, Ho AM, Doherty M, Dixey J, Gillet P, Loeuille D, McGrath R, Reginato A, Shiang R, Wright G, Netter P, Williams C, Kingsley DM. Mutations in ANKH cause chondrocalcinosis. Am J Hum Genet. 2002;71:933-940.
17. Pons-Estel BA, Gimenez C, Sacnun M, Gentiletti S, Battagliotti CA, de la Pena LS, Williams CJ, Reginato AJ. Familial osteoarthritis and Milwaukee shoulder associated with calcium pyrophosphate and apatite crystal deposition. J Rheumatol. 2000;27:471-480.
18. Reginato AJ, Passano GM, Neumann G, Falasca GF, Diaz-Valdez M, Jimenez SA, Williams CJ. Familial spondyloepiphyseal dysplasia tarda, brachydactyly, and precocious osteoarthritis associated with an arginine 75->cysteine mutation in the procollagen type II gene in a kindred of Chiloe Islanders: I: Clinical, radiographic, and pathologic findings. Arthritis Rheum. 1994;37:1078-1086.
19. Rosenthal AK, Ryan LM. Probenecid inhibits transforming growth factor-beta 1 induced pyrophosphate elaboration by chondrocytes. J Rheumatol. 1994;21:896-900.
20. Ryan LM, Wortmann RL, Karas B, Lynch MP, McCarty DJ. Pyrophosphohydrolase activity and inorganic pyrophosphate content of cultured human skin fibroblasts. Elevated levels in some patients with calcium pyrophosphate dihydrate deposition disease. J Clin Invest. 1986;77:1689-1693.
21. Terkeltaub RA. Inorganic pyrophosphate generation and disposition in pathophysiology. Am J Physiol Cell Physiol. 2001;281:C1-C11.
22. Timchenko LT. Myotonic dystrophy: the role of RNA CUG triplet repeats. Am J Hum Genet. 1999;64:360-364.
23. Weiss IM, Liebhaber SA. Erythroid cell-specific mRNA stability elements in the alpha 2-globin 3′ nontranslated region. Mol Cell Biol. 1995;15:2457-2465.
24. Whyte MP, Murphy WA, Fallon MD. Adult hypophosphatasia with chondrocalcinosis and arthropathy. Variable penetrance of hypophosphatasemia in a large Oklahoma kindred. Am J Med. 1982;72:631-641.
25. Williams CJ, Considine EL, Knowlton RG, Reginato A, Neumann G, Harrison D, Buxton P, Jimenez S, Prockop DJ. Spondyloepiphyseal dysplasia and precocious osteoarthritis in a family with an Arg75->Cys mutation in the procollagen type II gene (COL2A1). Hum Genet. 1993;92:499-505.
26. Williams CJ, Pendleton A, Bonavita G, Reginato AJ, Hughes AE, Peariso S, Doherty M, McCarty DJ, Ryan LM. Mutations in the amino terminus of ANKH in two US families with calcium pyrophosphate dihydrate crystal deposition disease. Arthritis Rheum. 2003;48:2627-2631.
27. Williams CJ, Zhang Y, Timms A, Bonavita G, Caeiro F, Broxholme J, Cuthbertson J, Jones Y, Marchegiani R, Reginato A, Russell RG, Wordsworth BP, Carr AJ, Brown MA. Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH. Am J Hum Genet. 2002;71:985-991.
28. Yoshimatsu T, Saitoh A, Ryu JN, Shima D, Handa H, Hiramoto M, Kawakami Y, Aizawa S. Characterization of immortalized human chondrocytes originated from osteoarthritis cartilage. Int J Mol Med. 2001;8:345-351.
29. Zhang Y, Johnson K, Russell RG, Wordsworth BP, Carr AJ, Terkeltaub RA, Brown MA. Association of sporadic chondrocalcinosis with a -4-basepair G-to-A transition in the 5′-untranslated region of ANKH that promotes enhanced expression of ANKH protein and excess generation of extracellular inorganic pyrophosphate. Arthritis Rheum. 2005;52:1110-1117.
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