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Technical Briefs |
1 Department of Pathology, Section on Comparative Medicine,2 Center for Human Genomics,3 Department of Biochemistry,4 Department of Radiology,5 Department of Internal Medicine, and6 Department of Public Health Sciences, Wake Forest University School of Medicine, Winston-Salem, NC;
aaddress correspondence to this author at: Section on Comparative Medicine, Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1040; fax 336-716-1515, e-mail register{at}wfubmc.edu
Circulating soluble intercellular adhesion molecule-1 (sICAM-1) and other novel markers have been correlated with a variety of cardiovascular outcomes, including the likelihood of future clinical cardiovascular events (1)(2)(3)(4)(5)(6)(7)(8)(9). We assessed circulating markers to explore relationships between subclinical atherosclerosis, bone density and metabolism, and fat distribution and metabolism in relation to type 2 diabetes mellitus. Serum sICAM-1, soluble vascular cell adhesion molecule-1 (sVCAM-1), sE-selectin, monocyte chemoattractant protein-1, and interleukin-6 were evaluated as markers of inflammation; adiponectin, leptin, and soluble leptin receptor as markers of adiposity; and collagen type I C-terminal propeptide, bone-specific alkaline phosphatase, osteocalcin, and N-telopeptide cross-link of type I collagen as markers of bone metabolism in a random sample of 80 participants from 53 families in the Diabetes Heart Study. The Diabetes Heart Study is a study of the cardiovascular and skeletal systems in families with siblings concordant for type 2 diabetes mellitus (10)(11) as well as unaffected family members. This study population included 42 men and 38 women, ranging in age from 39 to 81 years. Sixty-nine participants (86%) had type 2 diabetes mellitus, and 19 (24%) were African American.
Serum was obtained from a morning fasted blood sample and stored at –70 °C before analysis. Commercially available assays from R&D Systems were used for measuring serum sE-selectin, sVCAM-1, monocyte chemoattractant protein-1, and sICAM-1 (assay BBE1B). Adiponectin was measured by RIA and leptin by ELISA with reagents and protocols from LINCO Research, Inc. Soluble leptin receptor was measured by an ELISA from ALPCO Diagnostics. Collagen type I C-terminal propeptide, bone-specific alkaline phosphatase, and osteocalcin were measured by ELISAs with reagents and protocols from Quidel, and N-telopeptide cross-link of type I collagen was measured by an ELISA from Wampole Laboratories. Intraassay CVs were <7%.
One of the African-American individuals, the focus of this report, had undetectable concentrations of sICAM-1, as measured by a common monoclonal antibody-based sandwich ELISA (BBE1B), but at the same time had the highest concentrations of sE-selectin, sVCAM-1, and soluble leptin receptor of the 80 individuals investigated (Table 1
). Lack of serum immunoreactivity could result from complete absence of ICAM-1 expression, impaired release of ICAM-1 from the plasma membrane into the bloodstream, enhanced degradation or clearance of ICAM-1, or a missing or altered epitope in the ICAM-1 protein. To investigate these possibilities, we performed DNA sequence analysis of the coding and promoter regions of the ICAM-1 gene in this individual and family members. All coding regions of the ICAM-1 gene and 1.4 kb of the promoter were amplified from genomic DNA by PCR (primer sequences available on request), and cleaned PCR products were sequenced with Big Dye Terminators on an ABI 3730 (Applied Biosystems). The individual with undetectable serum sICAM-1 was found to be homozygous for an A>T substitution at nucleotide 100 of exon 2, which encodes a lysine-to-methionine substitution at position 29 in the mature ICAM-1 protein (residue 56 of the precursor peptide, which contains a 27-amino acid leader sequence). Originally identified in Kilifi, Kenya, by Fernandez-Reyes et al. (12), this K29M ICAM-1 variant (also designated ICAM-1Kilifi) has been the subject of some investigation (12)(13)(14)(15)(16)(17)(18) and is represented in dbSNP by rs5491. The frequency of this allele in Kenya was 33.2%. In our study, based in Forsyth County, NC, this allele was present in 37 African-American probands at a frequency of 20% compared with a frequency of 0.4% in a group of 255 Caucasian probands.
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Because monoclonal antibody specificity was a primary suspect in the failure to detect the K29M variant, we used an alternative commercial human sICAM-1 ELISA (BMS201INST; Bender MedSystems) to reevaluate the 80 study samples. With this alternative assay, sICAM-1 was detected in the individual homozygous for K29M ICAM-1 at the highest concentration among the sample population, consistent with the findings for the other adhesion molecules for this individual. Comparison of the sICAM-1 values obtained from the two assays in the remaining 18 African Americans (13 AA genotype and 5 AT heterozygotes) showed that sICAM-1 in AT heterozygotes was recognized to a greater extent by BMS201INST than by BBE1B (Fig. 1
).
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The results demonstrate that commercially available sICAM-1 assays vary markedly in their ability to recognize a common ICAM-1 variant (20% allele frequency) in African-American populations. The serum concentration of K29M sICAM-1, undetectable with one assay, was extremely high with the other, an effect presumably related to limited ability of a monoclonal antibody in the BBE1B assay to recognize the K29M region of the ICAM-1 protein. The binding characteristics of several monoclonal antibodies for this variant have been investigated (12), and one antibody in particular (BBA4; R&D Systems) was found to have markedly reduced ability to interact with the K29M variant. The antibodies used in the BBE1B ELISA and their recognition domains are proprietary information and not public knowledge. Regardless of the specifics of the antibody used, based on these studies it appears that the BBE1B ELISA has little or no ability to recognize the K29M ICAM-1. These findings have important implications for investigations into the relationships between serum concentrations of sICAM-1 and cardiovascular and other disease outcomes, particularly in African-American populations.
K29M is located in domain 1 near the NH2 terminus of the ICAM-1 protein, a region critical for binding of the malarial organism Plasmodium falciparum, the human rhinovirus LFA-1, and fibrinogen. In vitro studies have demonstrated that K29M ICAM-1 has an altered ability to bind to P. falciparum (15)(16), reduced affinity for LFA-1, and no apparent affinity for fibrinogen (16). Important insights into the function of ICAM-1 may be obtained from future investigations of relationships in additional individuals homozygous and heterozygous for K29M ICAM-1.
In summary, previous studies exploring sICAM-1 as a marker for cardiovascular and other diseases may need to be reevaluated in light of the demonstration that commercial sICAM-1 ELISAs vary markedly in their ability to recognize this ICAM-1 variant, which is common (20–35% allele frequency) in African-American populations.
Acknowledgments
This study was supported in part by the General Clinical Research Center of the Wake Forest University School of Medicine (Grant M01 RR07122), and National Heart, Lung, and Blood Institute Grants R01 HL67348 (to D.W.B.) and R01 AR48797 (to J.J.C.). K.P.B. was supported by an American Diabetes Association Mentor-based Fellowship.
References
The following articles in journals at HighWire Press have cited this article:
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  D. I. Chasman, G. Pare, and P. M Ridker Population-Based Genomewide Genetic Analysis of Common Clinical Chemistry Analytes Clin. Chem., January 1, 2009; 55(1): 39 - 51. [Abstract] [Full Text] [PDF]
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M. Khaleghi, Z. Ali, T. H. Mosley Jr., S. T. Turner, and I. J. Kullo Association of Soluble Cell Adhesion Molecules with Ankle-Brachial Index in a Biethnic Cohort of Predominantly Hypertensive Individuals Clin. Chem., November 1, 2008; 54(11): 1788 - 1795. [Abstract] [Full Text] [PDF]
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