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Variability of Serum Soluble Intercellular Adhesion Molecule-1 Measurements Attributable to a Common Polymorphism, Thomas C. Register,1* Kathryn P. Burdon,2,3 Leon Lenchik,4 Donald W. Bowden,2,3,5 Gregory A. Hawkins,2 Barbara J. Nicklas,5 Kurt Lohman,6 Fang-Chi Hsu,6 Carl D. Langefeld,6 and John J. Carr4,6 (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, and 6 Department of Public Health Sciences, Wake Forest University School of Medicine, Winston-Salem, NC; * address 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
[email protected]) 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 (1–9 ). 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 sys-
tems 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
Table 1. Biomarker values for the K29M ICAM-1 homozygous individual and the entire study group.a K29M individualb
Group Serum marker
Mean
Range
SD
Measured value
SD from group mean
Sample rank (1–80)
sICAM-1,c g/L sICAM-1,d g/L sVCAM-1, g/L sE-selectin, g/L IL-6,e ng/L MCP-1, ng/L sLeptin receptor kU/L Leptin, g/L ADPN, mg/L CICP, g/L BALP, U/L OC, g/L Ntx, nmol/L
266 217 598 71.7 4.30 341 24.0 16.8 12.2 101 22.6 8.9 10.8
0–626 133–434 222–1786 17–384 1.15–11.7 143–654 9.0–80.8 0.8–89.5 3.1–46.5 41–311 14.2–63.5 5.3–29.0 3.7–26.1
98 55 241 51 2.33 108 11 17 9.06 40 8 3 4
0 434 1786 384 7.41 345 80.8 8.3 18.1 168 36.9 5.7 8.7
⫺2.72 3.95 4.93 6.12 1.34 0.04 5.19 ⫺0.50 0.66 1.68 1.71 ⫺0.95 ⫺0.48
80 1 1 1 10 47 7 30 67 4 5 78 24
a
K29M ICAM-1 was not detected by assay BBE1B but was by assay BMS-201INST. Note the extreme values for sICAM-1, sVCAM-1, sE-selectin, and soluble leptin receptor in the K29M ICAM-1 homozygous individual. c sICAM-1 was measured by assay BBE1B. d sICAM-1 was measured by assay BMS-201INST. e IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1; ADPN, adiponectin; CICP, collagen type I C-terminal propeptide; BALP, bone-specific alkaline phosphatase; OC, osteocalcin; Ntx, N-telopeptide cross-link of type I collagen. b
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Technical Briefs
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 (12–18 ) 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. 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). 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 AfricanAmerican 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
Fig. 1. Distributions of values for serum markers by genotype. Data were recalculated as a fraction of the highest value for each particular assay. For ICAM-1, Assay 1 was the BBE1B ELISA and Assay 2 was the BMS201INST ELISA. AA, AT, and TT represent the genotypes at nucleotide 100 of exon 2 of ICAM-1. All Caucasians (C) were of the AA genotype.
Clinical Chemistry 50, No. 11, 2004
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.
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 1. Hwang SJ, Ballantyne CM, Sharrett AR, Smith LC, Davis CE, Gotto AM Jr, et al. Circulating adhesion molecules VCAM-1, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases: the Atherosclerosis Risk In Communities (ARIC) study. Circulation 1997;96:4219 –25. 2. Blann AD, Seigneur M, Steiner M, Miller JP, McCollum CN. Circulating ICAM-1 and VCAM-1 in peripheral artery disease and hypercholesterolaemia: relationship to the location of atherosclerotic disease, smoking, and in the prediction of adverse events. Thromb Haemost 1998;79:1080 –5. 3. Ridker PM, Hennekens CH, Roitman-Johnson B, Stampfer MJ, Allen J. Plasma concentration of soluble intercellular adhesion molecule 1 and risks of future myocardial infarction in apparently healthy men. Lancet 1998;351: 88 –92. 4. Ridker PM, Hennekens CH, Buring JE, Rifai N. C-Reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 2000;342:836 – 43. 5. Greenland P, Smith SC Jr, Grundy SM. Improving coronary heart disease risk assessment in asymptomatic people: role of traditional risk factors and noninvasive cardiovascular tests. Circulation 2001;104:1863–7. 6. Pradhan AD, Rifai N, Ridker PM. Soluble intercellular adhesion molecule-1, soluble vascular adhesion molecule-1, and the development of symptomatic peripheral arterial disease in men. Circulation 2002;106:820 –5. 7. Ridker PM, Rifai N, Rose L, Buring JE, Cook NR. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med 2002;347:1557– 65. 8. Ridker PM. Clinical application of C-reactive protein for cardiovascular disease detection and prevention. Circulation 2003;107:363–9. 9. Pearson TA, Mensah GA, Alexander RW, Anderson JL, Cannon RO 3rd, Criqui M, et al. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association. Circulation 2003;107:499 –511. 10. Wagenknecht LE, Bowden DW, Carr JJ, Langefeld CD, Freedman BI, Rich SS. Familial aggregation of coronary artery calcium in families with type 2 diabetes. Diabetes 2001;50:861– 6. 11. Lenchik L, Register TC, Hsu FC, Lohman K, Nicklas BJ, Freedman BI, et al. Adiponectin as a novel determinant of bone mineral density and visceral fat. Bone 2003;33:646 –51. 12. Fernandez-Reyes D, Craig AG, Kyes SA, Peshu N, Snow RW, Berendt AR, et al. A high frequency African coding polymorphism in the N-terminal domain of ICAM-1 predisposing to cerebral malaria in Kenya. Hum Mol Genet 1997;6:1357– 60. 13. Zimmerman PA, Wieseman M, Spalding T, Boatin BA, Nutman TB. A new intercellular adhesion molecule-1 allele identified in West Africans is prevalent in African-Americans in contrast to other North American racial groups. Tissue Antigens 1997;50:654 – 6. 14. Bellamy R, Kwiatkowski D, Hill AV. Absence of an association between intercellular adhesion molecule 1, complement receptor 1 and interleukin 1 receptor antagonist gene polymorphisms and severe malaria in a West African population. Trans R Soc Trop Med Hyg 1998;92:312– 6. 15. Adams S, Turner GD, Nash GB, Micklem K, Newbold CI, Craig AG. Differen-
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tial binding of clonal variants of Plasmodium falciparum to allelic forms of intracellular adhesion molecule 1 determined by flow adhesion assay. Infect Immun 2000;68:264 –9. 16. Craig A, Fernandez-Reyes D, Mesri M, McDowall A, Altieri DC, Hogg N, et al. A functional analysis of a natural variant of intercellular adhesion molecule-1 (ICAM-1Kilifi). Hum Mol Genet 2000;9:525–30. 17. Ohashi J, Naka I, Patarapotikul J, Hananantachai H, Looareesuwan S, Tokunaga K. Absence of association between the allele coding methionine at position 29 in the N-terminal domain of ICAM-1 (ICAM-1Kilifi) and severe malaria in the northwest of Thailand. Jpn J Infect Dis 2001;54:114 – 6. 18. Vijgen L, Van Essche M, Van Ranst M. Absence of the Kilifi mutation in the rhinovirus-binding domain of ICAM-1 in a Caucasian population. Genet Test 2003;7:159 – 61. DOI: 10.1373/clinchem.2004.036806
Detection of Fetal DNA and RNA in Placenta-Derived Syncytiotrophoblast Microparticles Generated in Vitro, Anurag Kumar Gupta,1 Wolfgang Holzgreve,1 Berthold Huppertz,2 Antoine Malek,3 Henning Schneider,3 and Sinuhe Hahn1* (1 Laboratory for Prenatal Medicine, University Women’s Hospital/Department of Research, University of Basel, Basel, Switzerland; 2 Department of Anatomy, University Hospital, RWTH, Aachen, Germany; 3 University Women’s Hospital, Inselspital, Bern, Switzerland; * address correspondence to this author at: Laboratory for Prenatal Medicine, University Women’s Hospital/Department of Research, Spitalstrasse 21, CH4031 Basel, Switzerland; fax 41-61-265-9399, e-mail
[email protected]) Fetal DNA and RNA can be readily detected in maternal plasma samples (1– 4 ). Most of this material appears to be of placental origin (5 ), and it appears to be in a predominantly cell-free form (2 ), whereas circulatory mRNA is membrane-encapsulated (6 ). Pregnancy is associated with the release of microparticles by the syncytiotrophoblast membrane into the maternal circulation (7 ). These particles, frequently termed STBM, are released by turnover of the syncytiotrophoblast monolayer covering the entire villous tree (8 –11 ). This process of normal physiologic syncytiotrophoblast turnover involves the release of apoptotic material into the maternal circulation by the extrusion of syncytial knots and the associated release of STBM (8 –11 ). The amount of material that is released by apoptotic shedding of syncytial knots (and STBM) is several grams per day (9 ), and the circulating concentrations are increased significantly in preeclampsia (11 ). STBM particles have been suggested to evoke the mild maternal inflammatory response accompanying normal pregnancies (12 ), and increased release has been proposed to play a role in the etiology of preeclampsia by triggering maternal endothelial cell damage (13, 14 ). As these particles are difficult to detect and prepare from maternal blood samples, use is frequently made of in vitro-prepared particles to study their physiologic activity (13 ). In this context, we have recently extensively examined three different modes of STBM preparation: mechanical dissection of fresh placental villous tissues; in vitro
