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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-0429
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 1 345-352
Copyright © 2007 by The Endocrine Society

Association of {alpha}2-Heremans-Schmid Glycoprotein Polymorphisms with Subclinical Atherosclerosis

Allison B. Lehtinen, Kathryn P. Burdon, Joshua P. Lewis, Carl D. Langefeld, Julie T. Ziegler, Stephen S. Rich, Thomas C. Register, J. Jeffrey Carr, Barry I. Freedman and Donald W. Bowden

Departments of Biochemistry (A.B.L., K.P.B., D.W.B.), Center for Human Genomics (A.B.L., K.P.B., J.P.L., D.W.B.), Molecular Genetics (J.P.L., D.W.B.), Public Health Sciences (C.D.L., J.T.Z., S.S.R.), Comparative Medicine (T.C.R.), Radiology (J.J.C.), and Internal Medicine (B.I.F., D.W.B.), Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

Address all correspondence and requests for reprints to: Donald W. Bowden, Ph.D., Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157. E-mail: dbowden{at}wfubmc.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Cardiovascular disease is significantly increased in individuals with type 2 diabetes mellitus (T2DM), especially in the presence of calcified atherosclerotic plaque. Fetuin A is an important mineralization inhibitor, and polymorphisms in the corresponding {alpha}2-Heremans-Schmid glycoprotein (AHSG) gene have been shown to be associated with serum fetuin A levels and free phosphate levels, as well as cardiovascular disease death.

Objective: This study investigated whether polymorphisms in AHSG contribute to the development of calcified atherosclerotic plaque in the coronary and carotid arteries and to carotid artery intima-media thickness.

Design: Eleven single nucleotide polymorphisms (SNPs) in AHSG were genotyped and evaluated for association with quantitative measures of subclinical atherosclerosis.

Participants: Subjects were 829 T2DM-affected European Americans from 368 families in the Diabetes Heart Study.

Main Outcome Measures: Participants were phenotyped for cardiovascular risk factors and atherosclerosis traits. The extent of coronary artery calcified plaque (CorCP) and carotid artery calcified plaque (CarCP) was measured using quantitative computed tomography, and carotid artery intima-media thickness was measured using high-resolution B mode ultrasonography.

Results: Four SNPs in AHSG were nominally associated with CorCP in European Americans with T2DM (P < 0.05). Two 3-SNP haplotypes in the exon 6–7 region were associated with CorCP in European Americans with T2DM (P < 0.06).

Conclusions: Sequence variants in the AHSG gene affect the extent of CorCP in T2DM-affected European Americans, consistent with the known biological role of AHSG in vascular calcification. These data implicate AHSG in the development of vascular calcified plaque in diabetic subjects.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
CARDIOVASCULAR DISEASE (CVD) is the leading cause of death in industrialized nations, and a worldwide increase in type 2 diabetes mellitus (T2DM), a major risk factor for CVD, will contribute to increasing CVD deaths. Although diabetes increases the risk for heart attack and stroke (1), CVD is even more frequent among diabetic subjects with additional risk factors such as hyperlipidemia, albuminuria, hypertension, chronic kidney disease, and smoking (1). A genetic contribution to CVD is suggested by the familial aggregation of coronary artery disease (2, 3, 4, 5, 6), as well as ethnic-specific differences in the rates of myocardial infarction (7, 8, 9).

Computed tomography (CT)-based imaging modalities have provided the capability to detect arterial calcified plaque, even in asymptomatic individuals (10). Data suggest that the burden of calcified atherosclerotic plaque is an important predictor of subsequent CVD events (11, 12, 13). The understanding of the pathogenesis of atherosclerosis has been rapidly evolving and includes the study of factors that either promote or inhibit calcium deposition in the systemic vasculature. Calcified atherosclerotic plaques often contain cells manifesting an osteoblastic phenotype (14). Finally, an inverse relationship has been observed between calcium content in the skeleton and the vasculature, with osteopenic and osteoporotic individuals having greater levels of vascular calcified plaque (15, 16).

Fetuin A [also known as {alpha}2-Heremans-Schmid glycoprotein (AHSG)] (17, 18) is a potent inhibitor of vascular calcium deposition. AHSG gene polymorphisms have been associated with serum levels of the associated fetuin A protein (19, 20), which are reduced in individuals with kidney failure and are associated with increased risk of vascular calcified plaque, inflammation, and CVD mortality and all-cause mortality (21, 22). These findings suggest that levels of calcified atherosclerotic plaque may vary based on genetic predisposition due to AHSG gene variation.

The Diabetes Heart Study (DHS) population (3, 23) is at high risk for clinical CVD based upon longstanding T2DM and associated risk factors and has significant subclinical CVD. We have assessed AHSG gene polymorphisms for association with coronary artery calcified plaque (CorCP) and carotid artery calcified plaque (CarCP) and carotid artery intima-media thickness (IMT). In addition, we examined the relationships between the AHSG gene and serum levels of fetuin A, calcium, phosphate, and the calcium x phosphate ion product in subjects with relatively well-preserved renal function.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study subjects

The study sample consisted of 829 European-American T2DM-affected individuals in 368 families comprising 478 diabetic sibling pairs from the DHS. Ascertainment and recruitment have been described previously (3, 23). Briefly, siblings concordant for T2DM lacking advanced renal insufficiency were recruited. T2DM was clinically defined as diabetes developing after the age of 35 yr and treated with insulin and/or oral agents, in the absence of historic evidence of ketoacidosis. The general absence of nephropathy was defined as a serum creatinine concentration less than or equal to 1.3 mg/dl (women) or less than or equal to 1.5 mg/dl (men) after a minimum diabetes duration of 5 yr.

All protocols were approved by the Institutional Review Board of Wake Forest University School of Medicine, and all participants gave informed consent. Participant examinations were conducted in the General Clinical Research Center of the Wake Forest University Baptist Medical Center and included interviews for medical history and health behaviors, anthropometric measures, resting blood pressure, fasting blood sample, and spot urine collection. Laboratory assays included urine albumin and creatinine, total cholesterol, low-density lipoprotein -cholesterol (calculated), high-density lipoprotein-cholesterol, triglycerides, hemoglobin A1c,fasting glucose, calcium, and inorganic phosphate. Serum fetuin A levels were measured by a commercially available sandwich ELISA (Epitope Diagnostics, Inc., San Diego, CA). For fetuin A analyses, standards and quality control samples were run with each individual ELISA plate. Interassay deviation from the fetuin A quality control sample supplied with each kit was 7.1% for the low control (20.5 ng/ml) and 14.2% for the high control (200 ng/ml). In a resampling of a subset of subjects across the study, mean fetuin A values (mean ± SEM) were in the same range (1113 ± 51 for the original dataset vs. 1192 ± 28 for the resampled dataset). Carotid artery IMT was measured by high-resolution B-mode ultrasonography with a 7.5-MHz transducer and a Biosound Esaote (AU5) ultrasound machine as previously described (23). CorCP and CarCP were measured using fast-gated helical CT scanners and calcium scores were calculated as previously described (24, 25). Data from subjects who had undergone vascular surgery (carotid endarterectomy or coronary artery bypass surgery) were excluded from analyses of calcification at the relevant site. Not all measurements were available for all participants.

Genetic analysis

Total genomic DNA was purified from whole-blood samples obtained from subjects using the PUREGENE DNA isolation kit (Gentra, Inc., Minneapolis, MN). DNA was quantitated using standardized fluorometric readings on a Hoefer DyNA Quant 200 fluorometer (Hoefer Pharmacia Biotech, Inc., San Francisco, CA). Each sample was diluted to a final concentration of 5 ng/µl.

Single nucleotide polymorphisms (SNPs) in the AHSG gene were chosen from public databases to comprehensively cover the 9.75-kb genomic region containing AHSG. Polymorphisms affecting the protein coding sequence were preferentially selected for evaluation. Of the 11 SNPs evaluated, nine were selected from the HapMap database. Eight of the HapMap SNPs evaluated in this study had a minor allele frequency (MAF) more than or equal to 0.15 in the Centre d’Etude du Polymorphisme Humain Utah residents with ancestry from northern and western Europe (CEU population), with the remaining HapMap SNP (rs1029353) being monomorphic in the CEU population but polymorphic in the Yoruba population from Ibadan, Nigeria (YRI population). The remaining two SNPs were selected in an effort to maintain consistent SNP spacing (average spacing of 1 SNP/975 bp).

Genotypes were determined using a MassARRAY SNP Genotyping System (Sequenom, Inc., San Diego, CA) as previously described (26). This genotyping system uses single-base extension reactions to create allele-specific products that are separated automatically and scored in a matrix-assisted laser desorption ionization/time of flight mass spectrometer. Primers for PCR amplification and extension reactions were designed using the MassARRAY Assay Design Software (Sequenom, Inc.).

Statistical analysis

Allele and genotype frequencies for each SNP were calculated from unrelated probands and tested for departure from Hardy-Weinberg equilibrium using a {chi}2 test. Estimates of linkage disequilibrium (LD) between SNPs were determined by calculating pair-wise D' and r2 statistics in unrelated individuals. To test for an association between each SNP and each phenotype, a series of generalized estimating equations (27) were computed. The correlation between subjects within a family was adjusted for by assuming exchangeable correlation among siblings within a pedigree and computing the sandwich estimator of the variance (28). The sandwich estimator is also denoted the robust or empirical estimator of the variance because it is robust to misspecification of the correlation matrix because it estimates the within-pedigree correlation matrix from the first and second moments of the data.

For each SNP, the 2 df test of genotypic association with each phenotype was performed. If there was evidence of a significant association (P < 0.05), three individual contrasts defined by the a priori genetic models (dominant, additive, and recessive) were computed. This is consistent with the Fisher’s protected least significant difference multiple comparison procedure. All effects were estimated while adjusting for age, gender, smoking status (current/past/never), and use of lipid-lowering medications (yes/no). Phenotypes included in these analyses were transformed to approximate conditional normality and to reduce heterogeneity of residual phenotypic variance across SNP genotypes.

Within each trait, a sequential Bonferroni multiple comparison adjustment was computed (29). This conservative multiple testing adjustment rank orders the observed P values, divides the a priori threshold for statistical significance (i.e. 0.05) by the P value rank, and declares significance if the observed P value is less than the rank-adjusted threshold for significance. This multiple testing correction was applied to the number of SNPs within each trait examined but did not take into account the number of traits examined. Because this investigation contains a strong a priori hypothesis, we report the unadjusted P values and indicate which SNPs retain statistical significance after adjustment by the conservative sequential Bonferroni correction.

The quantitative pedigree disequilibrium test (QPDT) (30) using one-, two-, three-, and four-marker moving windows was performed to assess for association between alleles (or haplotypes) and CorCP. A likelihood ratio test was used, implementing the expectation maximization algorithm to account for haplotype ambiguities. The analyses were conducted adjusting for the clinical covariates age, gender, smoking status, and use of lipid-lowering medications. Unlike the population-based generalized estimating equation approach, the family-based QPDT analysis is modestly conservative in the presence of population stratification and remains valid even under population admixture. These exhaustive haplotype analyses were not corrected for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The clinical characteristics of the 829 European-American T2DM-affected subjects are presented in Table 1Go. The average age of these participants was 62 yr, and 51% were female. The median carotid artery IMT was 0.66 mm, the median CarCP score was 93.3, and the median CorCP score was 594. Participants had fairly well preserved renal function after a mean 10.5-yr diabetes duration, evidenced by a median Modification of Diet in Renal Disease glomerular filtration rate of 64.9 ml/min and median urinary albumin:creatinine ratio of 14.3 mg/g, both based on a single laboratory result obtained at the time of each participant’s visit.


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  		TABLE 1. Clinical characteristics of 829 European-American T2DM-affected DHS participants

 
Power calculations assuming 700 individuals with complete genotypic and phenotypic data suggest that a SNP with MAF of 0.20 provides approximately 80% power at {alpha} = 0.05 to observe a 0.22 change in SD (see supplemental table, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). This analysis indicates that the DHS has the power to detect clinically meaningful differences in quantitative measures of vascular calcification.

DNA from these participants was genotyped for 11 SNPs (seven noncoding SNPs, four protein-coding SNPs) selected to completely cover the 9.75-kb genomic region containing AHSG (Fig. 1Go). Of the 11 SNPs evaluated, nine were selected from the HapMap database (http://www.hapmap.org) (31), with eight of these HapMap SNPs having MAF ≥ 0.15 in the HapMap CEU population. The ability of these SNPs to capture genotypic and haplotypic variation was assessed using the greedy pair-wise tagging algorithm implemented in the Tagger program (32) of Haploview (33). The eight most polymorphic HapMap SNPs captured a very high proportion of the genetic variation within this region (mean r2 = 0.980, minimum r2 = 0.871). The proportion of genetic variation captured by these eight SNPs is higher than more parsimonious sets of SNPs that contain the four protein-coding SNPs evaluated in this study, two representing synonymous changes (L131L and T270T) and two representing nonsynonymous changes (T248M and T256S).


Figure 1
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  		FIG. 1. Genomic map of the AHSG gene with locations of the 11 genotyped SNPs. The full-length shaded regions are exons, numbered 1–7. The half-length shaded regions represent the 5'- and 3'-untranslated regions. The ruler along the bottom represents the relative location and spacing of exons and SNPs in kilobases within the 9.75-kb region containing AHSG.

 
The mean genotyping success rate for the 11 SNPs evaluated was 94.8%, with rs4917 having the lowest success rate (88.7%) and rs4831 having the highest success rate (98.3%). The genotyping consensus rate for duplicate DNA samples within and across DNA plates was 100% for all SNPs. Allele and genotype frequencies were consistent with those expected under Hardy-Weinberg equilibrium. Ten of the 11 AHSG SNPs evaluated were contained in a single block of LD (Fig. 2Go) as defined by the D' statistic using both the confidence interval (34) and solid spine algorithms implemented in Haploview (33). SNP rs6787344, located downstream of the AHSG gene, was located outside of the defined LD block.


Figure 2
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  		FIG. 2. LD between 11 SNPs in AHSG.

 
Genotype data were analyzed for association with measures of subclinical atherosclerosis, including carotid artery IMT, CarCP, and CorCP, using a 2 df test for overall genotypic association (Table 2Go). No statistically significant evidence of association was observed with IMT or CarCP. However, significant evidence of association was observed (P ≤ 0.05) for three noncoding SNPs (rs2248690, rs2593813, and rs2070632) and the T270T synonymous protein-coding SNP (rs1071592) with CorCP in the European-American T2DM subjects.


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  		TABLE 2. Association analysis: P values for overall genotypic associations of AHSGSNPs with CorCP in European-American T2DM-affected DHS participants

 
For the four SNPs exhibiting significant association with CorCP by the 2 df test, the dominant, additive, and recessive genetic models were evaluated (Table 3Go). The promoter SNP (rs2248690) was most strongly associated under an additive model (P = 0.0063). The mean CorCP scores support this model because the addition of copies of the minor allele (T) linearly increased the mean CorCP score. The two intronic SNPs were most strongly associated under a recessive model. The best evidence for association with the intron 1 SNP (rs2593813) was obtained under a recessive model with respect to the major allele (A; P = 0.0004), whereas the intron 2 SNP (rs2070632) was most strongly associated under a recessive model with respect to the minor allele (A; P = 0.0006). In both cases, the presence of the AA homozygote resulted in decreased CorCP scores. The exon 7 synonymous coding SNP, T270T (rs1071592), was also most strongly associated under a recessive model with respect to the minor allele (A; P = 0.0075). However, this latter association resulted in an increase in the mean CorCP score for the AA genotype when compared with the other genotypes.


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  		TABLE 3. Association analysis: untransformed, unadjusted summary statistics of AHSGSNPs with CorCP in European-American T2DM-affected DHS participants

 
A sequential Bonferroni correction was applied to all SNPs to account for multiple comparisons. The two intronic SNPs, rs2593813 and rs2070632, remained nominally significant after this adjustment. However, the promoter SNP (rs2248690) and the T270T synonymous coding SNP (rs1071592) were no longer significant.

To test whether a haplotype would exhibit stronger evidence for association than the individual SNP associations with CorCP, the QPDT evaluating one-, two-, three-, and four-marker moving windows across the 10 SNPs within the LD block was used to test for association of AHSG haplotypes with CorCP in the European-American T2DM-affected subjects. This moving window analysis resulted in a total of 150 alleles and haplotypes, of which a specific 3-SNP window containing three observed haplotypes exhibited nominal association with CorCP (global P = 0.035; Table 4Go). Two haplotypes spanning the genomic region from exons 6–7 (rs4917, rs1029353, and rs4918) were responsible for this observed association. The CGC haplotype was associated with increased CorCP scores (P = 0.024), whereas the TGG haplotype trended toward association with decreased CorCP scores (P = 0.058). The TGG association was strengthened (P = 0.028) with the addition of the neighboring T270T synonymous coding polymorphism (rs1071592), whereas the addition of this fourth polymorphism did not affect the magnitude of the CGC association (Table 4Go). These analyses were not corrected for the exhaustive number of haplotypic comparisons. Although the haplotype analyses did not provide stronger evidence for association, the associations with the original SNPs noted above remained comparably significant. This is important in that the pedigree disequilibrium test methodology employed is robust to population stratification.


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  		TABLE 4. QPDT haplotype analysis of AHSGSNPs with CorCP

 
Because of the known biological role of AHSG in vascular calcification, secondary analyses involved analyzing the genotype data for genotypic association with serum levels of fetuin A, calcium, inorganic phosphate, and the calcium x phosphate ion product (data not shown). No significant evidence of association was observed with serum fetuin A levels or serum calcium concentrations. However, nominally significant evidence of association was observed for the SNP just downstream of AHSG (rs6787344) with serum phosphate (P = 0.05) and the calcium x phosphate ion product (P = 0.03) in the European-American T2DM subjects. Neither of these associations remained significant after applying a sequential Bonferroni correction to account for multiple comparisons.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Genetic evaluation of 11 SNPs in the AHSG gene in European-American families enriched for T2DM reveals association between SNPs in AHSG and CorCP, independent of the clinical covariates age, gender, smoking status, and use of lipid-lowering medications (Table 1Go). The SNP tagging algorithm implemented in this study suggests that only eight of these 11 SNPs are necessary to capture all HapMap variants within the region; however, additional SNPs were evaluated to increase SNP density in regions that appeared sparse and to increase the likelihood of capturing genetic variation in the small African-American component of the study currently being explored. Four SNPs are nominally associated with CorCP in European-American T2DM families. The SNPs within AHSG are in high LD (Fig. 2Go). Although there is no widely accepted approach for correction for multiple comparisons in a study such as this where the SNPs are in LD with each other and the traits are correlated, a sequential Bonferroni correction has been implemented in this study. This multiple comparison approach is extremely conservative, and the study was based on an a priori hypothesis of AHSG association with subclinical atherosclerosis, suggesting that examination of the results with and without adjustment for multiple comparisons is appropriate. Results from the two intronic SNPs (rs2593813, rs2070632) and the T270T synonymous coding SNP should be viewed with caution because analyses suggested a recessive mode of inheritance, and the number of minor allele homozygotes for these SNPs is small (77 for rs2593813, 29 for rs2070632, and 30 for T270T).

Genetic association of AHSG has been reported with T2DM (35), insulin-mediated inhibition of lipolysis (36), stimulation of lipogenesis in adipocytes (36), plasma cholesterol levels (36), lower body fat (37), ß2-adrenoreceptor sensitivity in sc fat cells (38), and serum phosphate (19) and fetuin A levels (19, 20). The SNP rs4918, which codes for the Thr256Ser amino acid substitution in exon 7 (historically referred to as Thr238Ser), has been the most widely evaluated polymorphism in AHSG and has been shown to be associated with serum fetuin A levels (20), leanness (37), and T2DM (35). The SNP rs4917, which codes for the Thr248Met amino acid substitution in exon 6 (historically referred to as Thr230Met), has been shown to be associated with plasma cholesterol levels (36), leanness (37), and increased ß2-adrenoreceptor sensitivity in sc fat cells (38). Osawa et al. (19) reported that individuals carrying the rare alleles for both the Thr248Met and the Thr256Ser amino acid substitutions had significantly reduced serum fetuin A levels. Although both of these polymorphisms were evaluated in the present study, we observed no associations between the polymorphisms and quantitative measures of vascular calcification (CorCP, CarCP, and IMT), body mass index (data not shown), T2DM (data not shown), or serum fetuin A levels. Evaluation of these two polymorphisms under an additive genetic model suggests that these polymorphisms do not have an additive effect on CorCP in our study sample (P = 0.125 for T248M, P = 0.171 for T256S; data not shown). The observed differences may be attributed to the different populations (Scandinavian, French, Caucasian, Japanese, European American) or to the smaller sample sizes (58–364 subjects) compared with the 829 subjects in the study reported here. It is also possible that potential relationships could be masked by the influence of T2DM on the quantitative measures analyzed in the current study.

Of the four polymorphisms exhibiting nominal evidence of association with CorCP in this report (Table 2Go), three were associated with other phenotypes in other studies. The promoter SNP rs2248690 was significantly associated with plasma cholesterol in Scandinavian women (36) and T2DM in French Caucasians (35), the minor allele of the intron 1 SNP rs2593813 was significantly associated with body mass index in Swedish men (37), and the exon 7 synonymous coding SNP T270T (rs1071592) was significantly associated with T2DM in French Caucasians (35). Our study failed to replicate the associations observed in other studies, possibly due to the different populations evaluated or to variability in sample sizes.

In addition to single SNP analysis, haplotypes were evaluated for association with CorCP. Moving window haplotype analysis of the 10 SNPs within the defined LD block suggests that combinations of alleles at three SNPs (rs4917, rs1029353, and rs4918) are associated with CorCP in the European Americans with diabetes. None of the SNPs comprising the associated haplotypes were individually associated with CorCP. When a fourth SNP (rs1071592) representing the T270T synonymous coding polymorphism was added, these haplotypes remained associated with CorCP. The T270T polymorphism was positively associated with CorCP when evaluated alone but did not retain significance after applying a sequential Bonferroni correction for multiple comparisons. The associated haplotypes differ at multiple loci; thus, variation in CorCP cannot be attributed to a single SNP. In additional haplotype analyses, the two most associated single SNPs (rs2593813 and rs2070632) were included as covariates and generated the same haplotypes as the original analysis. Both global and haplotype-specific P values retained the same degree of significance (data not shown). Therefore, we hypothesize that haplotypes rather than individual SNPs may be driving the observed associations and that the CorCP phenotype is due to multiple variants working in combination.

The concentration of circulating fetuin A protein has been shown to predict risk of vascular calcified plaque, inflammation, and all-cause and CVD mortality (21, 22). This is especially true for patients with end-stage renal disease. AHSG polymorphisms, either as single SNPs or haplotypes, were not associated with fetuin A levels or serum calcium concentrations (data not shown). The majority of our subjects were evaluated for serum calcium (n = 777), serum phosphate (n = 773), and the calcium x phosphate ion product (n = 773), indicating that the general lack of associations with these measures was unlikely due to small sample size and/or lack of statistical power to detect an effect.

The lack of association with serum fetuin A levels in this study may be explained by the differences in measured fetuin A levels presented here and in previous reports (20, 21, 39), although it is interesting to note that a sandwich ELISA was used to measure fetuin A levels in all of these studies. The mean serum fetuin A level in the current study of European Americans with T2DM (1.15 g/liter) is greater than the 95% reference range for 70 healthy adults (0.35–0.95 g/liter) reported by the assay manufacturer, although this difference may be due to the complexity of the population in the current study (obese, T2DM, heavily medicated). It is therefore likely that environment could overrule genetic influences on a circulating biomarker in such situations. In addition, 80–100% of the European Americans with T2DM evaluated in the present study have serum fetuin A levels greater than the levels reported in previous studies, although it should be noted that a modest number of subjects had data available for serum fetuin A (n = 297). Therefore, it is possible that this reduced sample size for serum fetuin A does not provide adequate statistical power to detect genetic associations of modest effect. In addition, the individuals evaluated in the current study had a clinical diagnosis of T2DM in the absence of advanced renal insufficiency, making these individuals clinically different from the subjects of other studies who had end-stage renal disease (20) or were on hemodialysis (21). Because diabetes is a risk factor for cardiovascular calcification, it seems possible that the use of diabetes medications by our study participants could influence fetuin A levels. However, of the 297 individuals with serum fetuin A data available, only 77 were treated with thiazolidinediones, and 72 were treated with insulin. The sparseness of this dataset makes it difficult to assess whether diabetes medications influence serum fetuin A levels.

Although an association with CorCP was detected, no association was observed between AHSG SNPs and carotid artery IMT or CarCP. However, the correlation among CorCP, CarCP, and IMT is modest (40), suggesting that they each carry independent information about atherosclerotic disease. Therefore, it is not surprising that an association was observed exclusively with CorCP.

To our knowledge, this is the first study that has investigated the variation in AHSG in relation to measures of subclinical atherosclerosis. We present evidence that individual polymorphisms, as well as haplotypes, in this gene are associated with CorCP in European Americans with T2DM, independent of clinical covariates. These data implicate AHSG in the development of vascular calcified plaque in diabetic subjects without advanced kidney disease, extending the current literature to the predialysis patient with subclinical atherosclerosis. Confirmation of the effects of these gene variants on the extent of CorCP will strengthen our understanding of the regulation of vascular calcification in T2DM-affected individuals.


   Footnotes
 
This work was supported by the General Clinical Research Center of the Wake Forest University School of Medicine (Grant M01 RR07122) and by the National Heart, Lung, and Blood Institute (Grants R01 HL67348 to D.W.B. and R01 AR048797 to J.J.C.).

First Published Online October 24, 2006

Abbreviations: AHSG, {alpha}2-Heremans-Schmid glycoprotein; CarCP, carotid artery calcified plaque; CorCP, coronary artery calcified plaque; CT, computed tomography; CVD, cardiovascular disease; DHS, Diabetes Heart Study; LD, linkage disequilibrium; MAF, minor allele frequency; QPDT, quantitative pedigree disequilibrium test; SNP, single nucleotide polymorphism; T2DM, type 2 diabetes mellitus.

Received February 23, 2006.

Accepted October 13, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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G. Marhaug, V. Shah, R. Shroff, H. Varsani, L. R. Wedderburn, C. A. Pilkington, and P. A. Brogan
Age-dependent inhibition of ectopic calcification: a possible role for fetuin-A and osteopontin in patients with juvenile dermatomyositis with calcinosis
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