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Clinical Chemistry 52: 227-234, 2006. First published December 29, 2005; 10.1373/clinchem.2005.059253
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Right arrow Proteomics and Protein Markers
(Clinical Chemistry. 2006;52:227-234.)
© 2006 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Role of Serum Fetuin-A, a Major Inhibitor of Systemic Calcification, in Pseudoxanthoma Elasticum

Doris Hendig1, Veronika Schulz1, Marius Arndt1, Christiane Szliska2, Knut Kleesiek1 and Christian Götting1,a

1 Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
2 Dermatologische Klinik, Krankenhaus Bethesda, Freudenberg, Germany.

aAddress correspondence to this author at: Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstrasse 11, 32545 Bad Oeynhausen, Germany. Fax 49-5731-972013; e-mail cgoetting{at}hdz-nrw.de.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: Pseudoxanthoma elasticum (PXE) is a hereditary disorder of the connective tissue affecting the skin, retina, and cardiovascular system and characterized by progressive calcification of abnormal and fragmented elastic fibers in the extracellular matrix. The aim of the present study was to investigate the association of fetuin-A, a major systemic inhibitor of calcification, with PXE.

Methods: Fetuin-A was measured by quantitative sandwich enzyme immunoassay in sera from 110 German patients with PXE, 53 unaffected first-degree family members, and 80 healthy blood donors. We determined the distribution of the fetuin-A polymorphisms c.742C>T (p.T248M) and c.766C>G (p.T256S) in these same 3 groups. The occurrences of the frequent ABCC6 gene mutations c.3421C>T (p.R1141X) and c.EX23_EX29del were also assessed.

Results: Serum fetuin-A concentrations in male and female PXE patients were lower than in unaffected first-degree relatives and controls [mean (SD) concentrations, 0.55 (0.11) g/L in patients; 0.70 (0.23) g/L in relatives; and 0.80 (0.23) g/L in controls (P <0.0001)]. Serum fetuin-A was higher in female PXE patients with cardiovascular involvement than in the corresponding male group (P <0.05). The fetuin-A polymorphism frequencies did not differ among PXE patients, family members, and blood donors.

Conclusion: A deficiency of multidrug resistance-associated protein 6 leads to alteration of circulating substrates, e.g., inhibitors of calcification as fetuin-A, leading to progressive mineralization of elastic fibers in PXE.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Pseudoxanthoma elasticum (PXE; OMIM 264800) is an autosomally heritable disorder of the connective tissue (1). The age of onset of PXE and the clinical expression of the disease are highly variable. Skin lesions are the most prevalent characteristics of PXE and generally its first physical signs (1)(2). Angioid streaks in the retina arise from fractures in the calcified elastic lamina of Bruch’s membrane and are associated with subretinal neovascularization; bleeding from the newly formed vessels may impair vision (3). The increasing arteriosclerosis may lead to hypertension, intermittent claudication, gastrointestinal bleeding, and myocardial infarction at a young age (4)(5)(6)(7)(8).

We and others have identified more than 100 PXE-associated mutations in the PXE candidate gene ABCC6 (9)(10)(11)(12)(13)(14)(15)(16). Marked phenotypic variations were observed in affected siblings bearing the same ABCC6 genotype (7)(17). Thus, other genes and environmental factors are assumed to contribute to the expression and severity of PXE (7)(17)(18). ABCC6 belongs to the ATP-binding cassette transporter subfamily C genes and encodes a 165-kDa transmembrane protein that is termed multidrug resistance-associated protein 6 (MRP6) (19). MRP proteins are involved in hepatic detoxification, drug distribution, and signal transduction by ATP-dependent transport of a variety of compounds (20). MRP6 is highly expressed in the liver and kidneys and, to a lesser extent, in tissues affected by PXE (19)(21). MRP6 has been shown to transport the endothelin-1 receptor antagonist BQ123 and glutathione conjugates under in vitro conditions (22). Organic anions, such as benzbromarone, specifically inhibit MRP6-mediated glutathione conjugate transport (23), but the physiologic function and molecules transported by MRP6 in vivo are still unknown.

PXE is histologically characterized by progressive calcification of elastic fibers and massive accumulation of proteoglycans in the extracellular matrix (24)(25)(26). Because of the minor amount of MRP6 produced in tissues frequently affected, PXE was considered a metabolic disorder with undetermined circulating molecules leading to the mineralization of elastic fibers in the skin, Bruch’s membrane, and vessel walls (4)(14)(18). Indeed, the authors of several studies reported pathologic alterations in clinical chemistry features, e.g., plasma lipoproteins and components of vitamin D metabolism (27)(28). The recent generation of an ABCC6-deficient mouse with altered HDL-cholesterol and increased creatinine concentrations supported these observations (29). The latter pointed toward impaired kidney function. Maccari et al. (30) reported abnormal excretion of glycosaminoglycans in the urine of patients affected by PXE. Further investigations revealed enhanced elastin degradation leading to high amounts of demosines in the urine and plasma of PXE patients (31). We recently found increased xylosyltransferase I activity in the sera of PXE patients, which reflects the extracellular matrix alterations (32). Despite these findings, the implication of MRP6 in calcium–phosphate metabolism remains unknown.

Although most PXE patients, as well as the ABCC6-deficient mice, have been shown to have serum calcium and phosphate concentrations within the reference intervals, the authors of several case reports observed important alterations to serum calcium and phosphate in patients with PXE (4)(27)(29)(33). Increased serum phosphate concentrations and increases in the calcium x phosphate ion product induce mineralization and are a risk factor for pathologic calcification (34). Interestingly, serum concentrations of calcium and phosphate exceed their solubility product in biological fluids and must be prevented from precipitation. The regulation of unwanted extraosseus calcification is a complex process in which several components are involved. Numerous disorders characterized by pathologic calcification have pointed at altered expression of calcification inhibitors (35). The question is whether an MRP6 deficiency has an effect on the production of proteins that are actively involved in inhibiting calcification. Fetuin-A, also known as {alpha}2-Heremans–Schmid glycoprotein, was recently found to be a major systemic inhibitor of calcification (36). This glycoprotein is synthesized by hepatocytes and occurs in all extracellular fluids, where its concentration ranges from 0.5 to 1.5 g/L. Fetuin-A was shown to form soluble colloidal complexes with calcium and phosphate, referred to as "calciprotein particles" (37). Low amounts of fetuin-A are associated with extraosseus, in particular, vascular calcification (36)(38)(39). Specific polymorphisms of the fetuin-A gene were demonstrated to influence circulating fetuin-A concentrations (40).

In the present study, we evaluated the association between serum fetuin-A, calcium and phosphate, and the PXE phenotype to investigate the link between an absence or functional insufficiency of MRP6 and mineralization of elastic fibers in PXE.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
patient characteristics
The study cohort comprised 110 German patients with PXE and 53 unaffected first-degree family members from 96 nonconsanguineous families with an apparently autosomal recessive or sporadic mode of inheritance of the PXE phenotype. In 12 of the 96 families, more than 1 person was affected by PXE. The diagnosis of PXE in all patients was consistent with the reported consensus criteria (41)(42). PXE status was determined by the presence of ocular findings and dermal lesions and was histologically confirmed by the observation of calcified elastic fibers in skin biopsies after von Kossa staining. The diagnoses of PXE were determined before patient recruitment to this study, without knowledge of the fetuin-A assay results. All members of the study were thoroughly questioned about their personal diseases, organ involvements, and their family history by 1 medical specialist to minimize interobserver variability. Thirty-one male [mean (SD) age, 50.4 (14.8) years] and 79 female [47.2 (14.2) years] PXE patients and 21 male [43.4 (19.8) years] and 32 female [49.2 (18.7) years] relatives were included in this study. The mean (SD) number of first-degree members per family was 1.89 (1.19). Patients were recruited between 2001 and 2004.

We used blood samples from 80 Westphalian blood donors [41 males; mean (SD) age, 45.1 (10.4) years] for analysis of fetuin-A concentrations, and blood samples from 175 [100 males; mean age, 41.8 (4.2) years] served as control cohort for analysis of the fetuin-A polymorphisms c.742C>T and c.766C>G. The clinical characteristics of the patients, relatives, and controls in this retrospective study are summarized in Table 1 . The study was approved by the Institutional Review Board, and all patients gave informed consent.


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  		Table 1. Clinical characteristics of the PXE patients, unaffected first-degree relatives, and healthy controls.

collection of dna and serum samples
Genomic DNA was extracted by use of the QIAamp blood reagent set (Qiagen). For the determination of serum fetuin-A, calcium, and phosphorus concentrations, venous blood samples were collected in serum Monovettes (Sarstedt). After clotting and centrifugation at 4000g for 15 min, sera were stored at –70 °C until assayed as described below.

ABCC6 genotyping
Mutational analysis of the ABCC6 gene and the common c.3421C>T mutation were performed as described previously (13)(15)(16). A multiplex PCR was used to detect the frequent 16-kb deletion c.EX23_EX29del as reported by Hu et al. (43).

analysis of fetuin-a polymorphisms C.742C>T and C.766C>G by restriction length polymorphism analysis
PCR primers were designed based on the published sequence of human chromosome 3 (GenBank accession no. AB038689). Mutation numbering refers to the fetuin-A cDNA sequence (GenBank accession no. NM_0016622) according to den Dunnen and Antonarakis (44). Analysis of the fetuin-A c.742C>T polymorphism was performed with the primers 5'-CCTCCCACAAGCAGAAAC-3' and 5'-TGATGATTCCGCATACCC-3'. PCR was performed in a 50-µL reaction volume containing ~65 ng of genomic DNA, 25 pmol of each primer (Biomers), 1.5 U of HotStar Taq DNA polymerase (Qiagen) in 1x Reaction Buffer supplied with the enzyme, and 0.25 mM of each deoxynucleotide triphosphate (Promega). The PCR conditions were as follows: initial denaturation at 95 °C for 15 min; 35 cycles of denaturation at 94 °C for 1 min, annealing at 59 °C for 1 min, and extension at 72 °C for 1 min; and a final extension at 72 °C for 15 min. The obtained 366-bp fragments were digested at 37 °C with 2.5 U of NlaIII (New England Biolabs) overnight and separated on a 1.5% agarose gel. The c.742T allele yielded 165- and 201-bp fragments, whereas the c.742C allele remained undigested. The fetuin-A c.766C>G polymorphism was analyzed with use of the primers 5'-GTCACCCCTCCTTGTAAC-3' and 5'-CCCCAATGAGACCACA-3'. The reaction mixture and the amplification procedures were as described above. The amplified 405-bp products were digested at 37 °C with 5 U of SacI (New England Biolabs) overnight and separated on a 1.5% agarose gel. The c.766C allele remained undigested, whereas the c.766G allele yielded 193- and 212-bp fragments.

measurement of serum fetuin-a, calcium, and phosphorus
The total fetuin-A concentration in serum was measured with a commercially available ELISA (Epitope Diagnostics). All samples were tested in randomized order with controls and calibration curves included in each run. Technicians performing the assays were blinded with respect to the diagnoses. Identical fetuin-A assay reagents from the same lot were used for the determinations. The interassay CVs were calculated from results for samples measured in 11 assays and were 7.7% at 0.19 g/L and 6.5% at 1.8 g/L, respectively. The determined CVs were similar to those given by the manufacturer. Serum concentrations of calcium and phosphorus were measured by automated clinical chemistry techniques at our laboratory (Architect ci8200; Abbott Laboratories).

statistical analysis
All values are given as the mean (SD). Normality testing for gaussian distribution of values was performed with the Kolmogorov–Smirnov test. Statistical analysis was performed with the Student t-test and Mann–Whitney U-test, where appropriate. We tested the significance of the difference in the alleles observed between the groups by {chi}2 analysis; we also used the {chi}2 test to examine whether the genotype distributions were in Hardy–Weinberg equilibrium. We used Pearson and Spearman correlation coefficients to assess correlations between variables. P values <0.05 were considered significant. All tests were performed with GraphPad Prism 4.0 (GraphPad Software).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
ABCC6 genotyping
We determined the occurrence of the c.3421C>T mutation in PXE patients and their relatives as we have described previously (13)(32). We found 11 (10%) homozygous and 40 (36.4%) heterozygous carriers of the common c.3421C>T mutation in the patient group, whereas 13 (24.5%) relatives were heterozygous for this mutation. No homozygous carriers of the c.3421T allele were detected in this cohort. The allelic frequency of this mutation in Caucasian blood donors was previously shown to be 0.05% (13). The 16-kb deletion spanning exons 23 to 29 of the ABCC6 gene was identified in the heterozygous state in 9 PXE patients. In addition, 5 close family members were found to carry this deletion on 1 allele. Seventy-seven of the PXE patients examined were homozygous or compound heterozygous carriers of unique mutations, including missense, nonsense, splice-site mutations, or small deletions in different exons of the ABCC6 gene. Of the 53 family members, 27 were heterozygous carriers of 1 ABCC6 mutation.

determination of serum fetuin-a, calcium, and phosphorus
Fetuin-A was measured in serum samples obtained from 110 patients with PXE, 53 relatives, and 80 healthy blood donors (controls). Serum fetuin-A in PXE patients was significantly lower than in the relatives and the controls (P <0.0001). Scatter plots showing the serum fetuin-A concentrations in the 3 groups are shown in Fig. 1 . The mean values were 0.55 (0.11) g/L for PXE patients (~32.5% lower than in the controls) and 0.80 (0.23) g/L for the controls, respectively (Table 2 ). Serum fetuin-A concentrations in relatives were also significantly higher than in the PXE patients [mean, 0.70 (0.23) g/L; P <0.0001; Table 2 ]. Interestingly, the value observed in the group of relatives was intermediate between that of the patients and the controls (Fig. 1 ). Moreover, serum fetuin-A in the relatives was also lower than in the control group (~12.5% lower; P = 0.01). Because 27 of the 53 investigated relatives were identified as heterozygous carriers of 1 ABCC6 mutation, we divided this group into symptom-free carriers and symptom-free noncarriers and reanalyzed the obtained data. We found no difference in serum fetuin-A concentrations between these subgroups: the mean values were 0.70 (0.24) and 0.70 (0.22) g/L for carriers and noncarriers, respectively.


Figure 1
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  		Figure 1. Serum fetuin-A concentrations in PXE patients, unaffected relatives, and controls.

Shown are serum fetuin-A concentrations (g/L) in patients with PXE (n = 110), unaffected relatives (n = 53) from 96 nonconsanguineous families, and healthy controls (n = 80). Scatter plots illustrate the distribution of fetuin-A concentrations for the individuals in the 3 groups. The horizontal lines represent the mean value for each group. ***, P <0.0001; *, P = 0.01.


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  		Table 2. Values of analyzed variables in PXE patients and unaffected first-degree relatives.

We also determined serum calcium and inorganic phosphorus in our cohort of PXE patients and relatives. Interestingly, serum calcium was moderately increased in the patient group [101.9 (10.8) mg/L] compared with the group of relatives [98.3 (6.6) mg/L]. Serum phosphorus was lower in the PXE patients than in the family members [43.1 (15.7) and 48.6 (13.8) mg/L, respectively; P = 0.007]. The resulting calcium x phosphorus product was not different between the 2 cohorts analyzed (Table 2Up ).

analysis of the fetuin-a genotype
Analysis of the fetuin-A polymorphisms c.742C>T (p.T248M) and c.766C>G (p.T256S) in PXE patients, relatives, and healthy controls revealed 3 genotypes, as reported previously (40). Fetuin-A genotype 1 is characterized by the alleles c.742C and c.766C, whereas the fetuin-A genotypes 2-1 and 2 have a C->T transversion at position 742 and a C->G transition at position 766 in the heterozygous and homozygous states, respectively (Table 3 ). The frequencies of fetuin-A genotype 1 were 44.5% in the patient cohort, 41.5% in the relatives, and 38.3% in the control group (Table 3 ). Twelve (11.0%) PXE patients, 7 relatives (13.2%), and 27 controls (15.4%) carried fetuin-A genotype 2. Finally, 49 (44.5%) patients, 24 (45.3%) relatives, and 81 (46.3%) controls were carriers of fetuin-A genotype 2-1. The determined genotype frequencies for both polymorphisms did not differ among PXE patients, relatives, and controls. All genotype distributions were in Hardy–Weinberg equilibrium.


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  		Table 3. Frequency distributions of the fetuin-A polymorphisms p.T248M and p.T256S producing fetuin-A genotypes 1, 2, and 2-1, and association of serum fetuin-A and fetuin-A genotype.

correlation of serum fetuin-a to variables
As already known, there is an association between serum fetuin-A and 3 major fetuin-A genotypes (40). We observed that PXE patients and relatives carrying fetuin-A genotype 1 had significantly higher serum fetuin-A concentrations than the groups of patients and relatives carrying fetuin-A genotypes 2 and 2-1 (Table 3Up ). Serum fetuin-A concentrations of PXE patients carrying fetuin-A genotype 2 were not different from those in the corresponding group of relatives [0.49 (0.11) and 0.56 (0.18) g/L; Table 3Up ]. We found no correlation between serum fetuin-A and patient age, age at PXE onset, or serum calcium concentration. We did find a significant negative correlation between serum fetuin-A and phosphorus in PXE patients (n = 65) and unaffected relatives (r = –0.271 and –0.416 for patients and relatives, respectively; P <0.05; n = 29). Furthermore, linear regression analysis revealed a significant negative correlation between serum fetuin-A and calcium x phosphorus product in the studied groups (r = –0.254 and –0.384 for patients and relatives, respectively; P <0.05). We noticed decreasing fetuin-A in association with the number of organs involved (Fig. 2 ), although it did not reach significance. In addition, we observed significantly increased serum fetuin-A concentrations in female PXE patients with cardiovascular involvement compared with the corresponding male group [0.59 (0.06) and 0.51 (0.06) for female and male patients, respectively; P <0.05].


Figure 2
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  		Figure 2. Correlation of serum fetuin-A concentration and severity of clinical manifestations in PXE patients.

Shown are serum fetuin-A concentrations (g/L) in individuals without and with different clinical manifestation of PXE. PXE severity was categorized by the number of affected organs. None, unaffected first-degree relatives who showed no clinical manifestation of PXE [mean (SD), 0.70 (0.23) g/L]; Mild, observed clinical manifestations were primarily in the skin and eyes [mean, 0.56 (0.12) g/L]; Moderate, involvement of the skin and eyes and up to 2 other additional organs [mean, 0.54 (0.11) g/L]; Severe, involvement of more than 4 organs [mean, 0.53 (0.11) g/L]. Scatter plots illustrate the distribution of serum fetuin-A concentrations (g/L). The horizontal lines represent the mean values for each group. ***, P <0.0001; **, P = 0.0007.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Abnormalities in several extracellular matrix components have been observed in tissues affected by PXE (26)(30)(32)(45). Kornet et al. (8) found an accumulation of proteoglycans and calcium in addition to elastin fragmentation in PXE patients. The authors suggested that proteoglycans attract calcium ions, a mechanism already known (46); the accumulated calcium might promote elastin fragmentation. Aberrant glycosaminoglycan content has been observed in the skin and urine of PXE patients (4)(30). There is much debate on whether glycosaminoglycans inhibit or promote calcification. Whether the increase in glycosaminoglycan content inhibits calcification or whether mineralization is secondary to glycosaminoglycan accumulation remains to be investigated. The question also remains of what may be inducing calcification of connective tissues in PXE. The disorder is caused by mutations in the ABCC6 gene, which encodes for MRP6, a transmembrane transporter with an as yet unidentified function. MRP6 is localized to the basolateral side of human hepatocytes and membranes of kidney proximal tubules, indicating that MRP6 extrudes unknown substrates into the blood. These substrates are hypothesized as being involved in the production of other components, finally leading to pathologic alterations of the extracellular matrix in PXE (21)(47). Ectopic calcification in PXE might result from an imbalance of positive and negative regulatory factors.

To determine the link between MRP6 and the pathologic calcification in PXE, we investigated the production of fetuin-A in PXE patients. To the best of our knowledge, this is the first study of an association between systemic calcification inhibitors and PXE. We found decreased fetuin-A in our cohort of PXE patients compared with relatives and controls. These results indicate that MRP6 might be indirectly involved in calcium–phosphate metabolism; however, the underlying mechanism remains to be investigated. Lower serum fetuin-A concentrations have previously been shown to increase the risk of cardiovascular disease and to lead to systemic, especially vascular, calcification (36)(48). Moreover, fetuin-A–deficient mice developed severe soft tissue calcification (38). Price et al.(49) demonstrated that arterial calcification is also correlated with a decrease in serum fetuin-A in rats treated with vitamin D. Interestingly, in our study, the measured serum fetuin-A concentration in the group of relatives was intermediate between those of the PXE patients and controls. Most of these individuals had been identified as carriers of only 1 ABCC6 mutation but did not manifest any clinical signs of PXE. Subdivision of the group of relatives into symptom-free carriers and noncarriers revealed no difference in serum fetuin-A. These findings led us hypothesize that decreased fetuin-A in PXE patients might result from fetuin-A bound to the mineral deposits. However, we must take into account that we did not identify a minimal PXE phenotype and possible PXE-associated ABCC6 mutations in all relatives defined as noncarriers. A thorough mutational screening of both ABCC6 alleles and intensive clinical investigation of relatives might help to overcome this lack of information. Nevertheless, similar trends have been observed by other groups who evaluated elastin degradation by determining glycosaminoglycans and desmosines in the urine and plasma of PXE patients and close family members (30)(31). Authors of previous studies observed mild but distinct alterations of extracellular matrix components in the skin of healthy carriers (50). Taken together, these results underline the assumption that ABCC6 mutations on a single allele might determine a limited PXE phenotype (50)(51).

We observed the tendency of decreasing serum fetuin-A in association with the number of organs involved, although this association did not reach significance. These data agree with recent findings that the severity of clinical manifestations increases with time, because of disease progression (7)(26). Detailed analysis of affected organs and establishment of a validated PXE score might underscore our initial findings. Cardiovascular involvement is common in PXE, and patients typically present with arteriosclerosis (42). Fetuin-A deficiency might contribute to the reduction in vascular wall elasticity (36)(38). Measured serum fetuin-A was higher in female PXE patients with cardiovascular involvement than in the corresponding male group. A possible explanation for our observation might be a sex-specific response to decreasing serum fetuin-A. Our hypothesis is supported by the results of other groups who found lower fetuin-A in correlation with altered conditions of hormones (e.g., estradiol) and cytokines (52)(53).

The frequencies of the p.T242M and p.T256S polymorphisms did not differ among PXE patients, relatives, and controls; we therefore conclude that the difference in fetuin-A concentrations results from MRP6 deficiency and not from a genetic background. Fetuin-A concentrations in PXE patients carrying fetuin-A genotype 2 were not altered compared with the corresponding group of relatives. Carriers of fetuin-A genotype 2 have recently been shown to have lower serum fetuin-A concentrations (40)(48). It could be speculated that fetuin-A genotype 2 is an additional disease-promoting risk factor.

In this study, we investigated for the first time serum calcium and phosphorus in a large cohort of PXE patients. Serum phosphorus was decreased in the PXE patients. Moreover, we found a significant negative correlation between serum fetuin-A and phosphorus in the PXE patients and their relatives. This is in concordance with the results of Osawa et al. (40), who demonstrated that fetuin-A is a determinant of serum phosphate. Increased serum phosphate and urinary calcium and phosphate concentrations resulting from increased 1,25-dihydroxyvitamin D3 have been found in tumoral calcinosis (OMIM 211900). These patients were found to bear angioid streaks and skin lesions similar to those observed in PXE. Although the histologic findings differ from those in PXE, analogies might exist. Whether fetuin-A concentrations are altered in tumoral calcinosis patients is not known. We found decreased serum phosphorus in the PXE patient cohort, which might be associated with altered fetuin-A expression. However, urinary calcium and phosphate as well as serum parathyroid hormone and 1,25-dihydroxyvitamin D3 were not applicable for the PXE patients investigated.

Beck et al. (47) suggested that MRP6 fulfills multiple functions in different tissues. Our findings support the assumption that MRP6 deficiency might lead to dysregulation of substrates involved in the regulation of calcification inhibitors. MRP6 is produced in cells of the human immune response system, suggesting that MRP6 might also play a role in inflammation (47). Interestingly, fetuin-A has been discussed as a negative acute-phase protein and might be decreased because of inflammation (36). Therefore, it cannot be ruled out that PXE has an inflammatory component similar to those observed in many other calcification disorders.

In conclusion, we found decreased serum fetuin-A in PXE patients. We have not yet determined whether the lower fetuin-A concentrations are a result of impaired MRP6 transport or just secondary to progressive calcification. The lower fetuin-A concentrations found in the relative cohort may suggest that ABCC6 mutations are associated with increased calcification. Further studies are warranted to examine the biological relevance of fetuin-A in the pathology of PXE. The results of the present study support the hypothesis that MRP6 deficiency might be linked with an as yet unknown component required to prevent elastic fiber mineralization. However, the implication of MRP6 in elastic fiber mineralization and degradation remains unclear.


   Acknowledgments
 
We gratefully acknowledge financial support by the "Stiftung für Pathobiochemie und Molekulare Diagnostik" of the Deutsche Vereinte Gesellschaft für Klinische Chemie und Laboratoriumsmedizin. We are very grateful to all of the PXE patients and their relatives, whose cooperation made this study possible. We also thank Peter Hof, Chairman of the Selbsthilfe für PXE Erkrankte Deutschlands e.V., and the members of the clinical ambulance for PXE at the Bethesda Hospital in Freudenberg, Germany. We would like to thank Alexandra Adam and Marlen Ewald for excellent technical assistance and Sarah Kirkby for linguistic advice. Parts of this work (ABCC6 genotyping) were presented at the AACC annual meeting in 2005 and were acknowledged with the "Distinguished Abstract Award" of the National Academy of Clinical Biochemistry.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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