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Prepublished online as a Blood First Edition Paper on August 15, 2002; DOI 10.1182/blood-2001-12-0306.
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Section on Genomic Variation, Pediatric
Oncology Branch, National Cancer Institute, National Institutes of
Health, Gaithersburg, MD; the Molecular and Clinical Hematology Branch,
NIDDK, and the Department of Transfusion Medicine, Clinical Center,
National Institutes of Health, Bethesda, MD; and MRC Laboratories,
University of the West Indies, Kingston, Jamaica.
Stroke is a major cause of morbidity and mortality in sickle cell
(SS) disease. Genetic risk factors have been postulated to contribute
to this clinical outcome. The human genome project has substantially
increased the catalog of variations in genes, many of which could
modify the risk for manifestations of disease outcome in a monogenic
disease, namely SS. VCAM1 is a cell adhesion molecule
postulated to play a critical role in the pathogenesis of SS disease.
We identified a total of 33 single nucleotide polymorphisms (SNPs) by
sequencing the entire coding region, 2134 bp upstream of the 5' end of
the published cDNA, 217 bp downstream of the 3' end of the cDNA, and
selected intronic regions of the VCAM1 locus. Allelic
frequencies for selected SNPs were determined in a healthy population.
We subsequently analyzed 4 nonsynonymous coding, 2 synonymous coding,
and 4 common promoter SNPs in a genetic association study of clinically
apparent stroke in SS disease conducted in a cohort derived from a
single institution in Jamaica (51 symptomatic cases and 51 matched
controls). Of the 10 candidate SNPs analyzed in this pilot study, the
variant allele of the nonsynonymous SNP, VCAM1 G1238C, may
be associated with protection from stroke (odds ratio [OR] 0.35, 95%
confidence interval [CI] 0.15-0.83, P = .04). Further
study is required to confirm the importance of this variant in
VCAM1 as a clinically useful modifier of outcome in SS disease.
(Blood. 2002;100:4303-4309) Stroke is a major complication of sickle cell (SS)
disease and is associated with significant morbidity and mortality. It is estimated that the lifetime risk for stroke is between 8% and 10%.1,2 Associated risk factors include peripheral
leukocytosis,1,2 the rate of acute chest syndrome (ACS)
episodes,1 relative hypertension,3 abnormal
cerebral blood flow detected by transcranial doppler
ultrasonography,4 and selected genetic
variants.5,6 The search for predictive markers has direct
clinical implications, specifically the institution of early
interventional strategies, including close monitoring and preventative
therapies.7
Currently, controversy exists among geneticists as to the best means
for identifying the genetic determinants of multigenic or complex
traits like stroke in SS disease.8 Traditionally, studies
have only proceeded to linkage analysis on multigenerational pedigrees
once a familial pattern of trait aggregation has been established. Once
localized by linkage to a genetic marker, a disease gene is identified
from candidates mapping to a chromosomal region. Recently, the draft
sequence of the human genome has generated an extraordinary resource
for using single nucleotide polymorphisms (SNPs) as physical markers in
either a candidate gene or eventually the entire genome to identify
complex traits and modifiers of monogenic diseases.9 While
traditional linkage studies are still attractive, some have generated
controversy by suggesting that such studies may require unrealistically
large pedigrees for localizing complex disease determinants of modest
effect, and suggesting that association studies are a reasonable
alternative approach.8,10 As a result, availability of
human genomic sequence has facilitated successful association studies
testing SNPs in candidate genes, selectively chosen on the basis of
existing knockout animal models or in vitro data implicating the gene
in disease pathogenesis, even in the absence of genetic
linkage.11-14
The cell adhesion molecules (CAMs) of the immunoglobulin superfamily
adhere with high affinity to integrins expressed on inflammatory and
endothelial cells. A critical member, the vascular cell adhesion molecule 1 (VCAM-1), coordinates the inflammatory response by recruiting leukocytes and in turn, activating
lymphocytes.15 VCAM-1 is a cell surface sialoglycoprotein
highly expressed on endothelial cells following cytokine stimulation
with interleukin 1 alpha (IL-1 Variants of the VCAM1 gene could be informative genetic
modifiers of phenotypic differences in SS disease. In vitro, sickle erythrocytes adhere to cytokine-stimulated or -transfected VCAM-1 on
endothelial or COS cells via VLA-4 ( Screening population for VCAM1 polymorphisms
Genotyping
Confirmation of candidate SNPs Allelic distributions of selected VCAM1 SNPs were determined in 130 healthy African American blood donors at the NIH and in 100 Centre d'Etude du Polymorphisme Humain (CEPH) African American donors (Human Variation Panel HD100AA; Coriell Cell Repositories, Camden, NJ).Patients with sickle cell disease Patients were recruited at the Sickle Cell Clinic of the University Hospital of the West Indies, Kingston, Jamaica. There were 51 patients with SS disease identified with a history of clinical stroke and 51 patients with SS disease without stroke (controls) who were matched by sex and age at the time of study recruitment to index cases. Radiographic evaluation of the head was not performed on control subjects. Study participants provided informed consent under the auspice of an institutional review board-approved study at the University of the West Indies. A symptomatic stroke was defined as an acute clinical neurologic syndrome with localizing symptoms of more than 24 hours duration. In addition, most stroke cases were documented by computed tomography or angiogram.2 Overall, all evidence (clinical and radiographic) suggested that the great majority of cases were symptomatic infarctive strokes. Patients with transient ischemic attacks or fatal stroke events were not included in this study.Clinical and laboratory values for this population have been previously reported including a mean age of 17.1 years for stroke cases at recruitment.31 Mean age at stroke was 10.3 years (standard deviation 6.1 years). Steady state white blood cell (WBC) counts were determined after the age of 5 years from an average of 10.9 measurements in the control group (range 1-43) and 9.7 measurements among stroke cases (range 1-37). Statistical analysis Statistical analysis used Instat 2.0 (Graph Pad Software, San Diego, CA), EpiInfo 6.04 (Centers for Disease Control, Atlanta, GA), and DnaSP 3.5 (http://www.bio.ub.es/julio/DnaSP.html). The standardized linkage disequilibrium coefficient D' was calculated using the method of Lewontin.32 was estimated as
S/a1 (where S is the
number of segregating sites;
a1 =    or the average heterozygosity per nucleotide site
( = k/[1  (1/n)], divided by the number of
nucleotides sequenced; where k =  pj),
pj is the observed frequency of the
jth SNP and n is 80 chromosomes). The
Tajima D statistic was determined from the difference between
these 2 estimates of nucleotide diversity ( and , Tajima
D =   2 and paired P values) including odds ratios
(ORs) and 95% confidence intervals (CIs) (using the approximation of
Woolf). The protective potential of the observed association
was determined as follows: (percent reduction in the prevalence of
stroke = 1 observed OR) × (observed genotype frequencies
for carriers of the informative allele).35 Continuous data
were analyzed by paired t test or Mann-Whitney test with
P values where indicated. In this pilot study, P
values are presented without correction, and were considered significant for an  less than .05.
Screening for common polymorphisms Sequencing of indicated regions of VCAM1 in 40 healthy individuals identified 33 biallelic SNPs: 17 upstream of the ATG, 4 nonsynonymous coding, 2 synonymous coding, 5 3' untranslated regions (UTRs), and 5 additional noncoding SNPs (Table 1). Allelic distributions of selected variants were determined in a healthy African American control population (Table 2), and estimated gene frequencies were calculated and found to be consistent with Hardy-Weinberg equilibrium (data not shown). Within the open reading frame of VCAM1, 4 nonsynonymous SNPs alter the predicted amino acid sequence and 2 are in linkage: C953T (S318F in domain 4 of VCAM-1) and A1150G (T360K, also in domain 4) (D' = 1.0 for 2-locus combination in 230 healthy controls by subcloning 10 of 15 variant allele carriers, data not shown). There are 2 other SNPs, G1238C (G413A in domain 5) and A2146T (I716L in the transmembrane domain), that appear to segregate independently. VCAM1 G-14C, G1238C, C2079T, A2146T, and A2208G are expressed at the transcriptional level in lymphocytes based on sequence analysis of first strand cDNA from 16 of 40 screening donors.
Based on data for 80 chromosomes, estimates of
VCAM1 genotypes in stroke and SS disease Selected coding SNPs and common promoter SNPs (defined as a variant allele frequency of > 0.10) were then studied in the SS stroke population. The promoter SNP, T-1599G, was also selected for further study because it maps to position 5 of a putative AP-1 consensus-binding sequence36 (identified by NSITE software at http://genomic.sanger.ac.uk). Of the 10 SNPs analyzed in the pilot study, one is informative in this well-characterized Jamaican SS population. The wild-type VCAM1 G1238 allele was more common in the stroke group compared with the control group (P = .04; Table 4), suggesting that the C variant could be protective against stroke in SS disease. Analysis by allelic frequency showed C1238 alleles to be significantly reduced among stroke cases versus controls (8/102 C alleles versus 20/102 C alleles, respectively;2 = 5.70, OR 0.35; 95% CI 0.15-0.83;
P = .02; Table 5). The strength of this
association by analysis of matched pairs yielded comparable, but not
statistically significant, results (VCAM1 1238 GC/CC versus GG genotypes, McNair-corrected 2 = 3.37, OR 0.36, 95%
CI 0.11-1.06, paired P = .07). Based on genotype
frequencies for the VCAM1 C1238 allele (derived from Table
2; GC 39/229 individuals = 0.1703, and CC 3/229
individuals = 0.0131) and an observed 65% reduction in the
prevalence of stroke (1 observed 0.35 OR), the estimated proportion
of symptomatic stroke cases that might be prevented for carriers of
this genetic marker could be as high as 11.9%
[(0.65 × 0.1703) + (0.65 × 0.0131) = 11.9%].35 There were 7 additional
SNPs in the VCAM1 gene, presented in Table
4, that were not significantly
associated with stroke, as was also
observed for 2 synonymous coding SNPs in exon 9 (C2079T,
P = NS and A2208G, P = NS; data not
shown).
Steady state WBC count and VCAM1 genotype Previously, an elevated steady state WBC count has been identified as a risk factor for stroke in Jamaica (in this population stroke mean WBC count 15.3 × 106/µL versus control WBC count 13.6 × 106/µL; P = .006).2,31 Steady state WBC count was significantly higher among stroke cases with the VCAM11599 TT genotype compared with TG strokes (P = .02) and
TT controls (P = .002, Table
6). Comparison of steady state
WBC count by VCAM1 G1238C genotype demonstrates
that leukocyte counts are significantly elevated for stroke cases with
the GG genotype versus controls with the GG genotype
(P = .01, Table 7).
SS disease is a monogenic disorder characterized by a mutation
resulting in a single amino acid change in the In the present study, we have characterized sites of genetic variation
in selected regions of the candidate gene VCAM1, which has
previously been implicated in the pathogenesis of both SS disease19-22,25 and ischemic stroke without underlying SS
disease.26-28 To assess whether the SNPs identified might
be associated with selective function as opposed to neutral mutation,
we have estimated VCAM1 nucleotide diversity and tested the
population genetic hypothesis that all SNPs in VCAM1 are
selectively neutral (Tajima D). The nucleotide diversity values (ie,
Selected variants were then evaluated in a small SS disease population with clinically overt stroke.2,31 Significantly, the C allele of VCAM1 G1238C (domain 5) could be associated with protection from stroke in SS disease. Thus, as many as 11.9% of stroke cases might be prevented35 in association with the C1238 allele, implying a moderate-to-strong genetic effect in modifying the clinical outcome of SS disease. Lastly, subanalysis of steady state WBC count and genotype suggests that some of these variants could influence leukocyte counts, a known risk factor for stroke, adverse complications, mortality, and possibly a determinant of vascular occlusion in SS disease.1,2,44-46 This final observation is preliminary but intriguing in light of experimental data supporting a functional role for VCAM-1 in the release of hematopoietic cells from bone marrow, given recent observations of potentially fatal SS complications associated with intense leukocytosis after administration of G-CSF, and an association between duration of leukocyte infiltration and both infarct size and the severity of neurologic outcome in non-SS disease adults with CNS infarcts.17,18,47-49 Although this study indicates that a VCAM1 SNP could be associated with stroke in a homogeneous SS disease population, we recognize that confounding variables inherent to case control studies might also influence our preliminary observation. A limitation of our control group is the unknown prevalence of undetected silent cerebral infarcts, owing to the fact that neuroimaging was not performed on control subjects. Patients with SS disease may have silent lesions more frequently than previously appreciated.50 However, our study is defined on the basis of clinically documented symptomatic events and not radiographic findings in asymptomatic patients with SS, as it is not presently known whether symptomatic strokes are different from silent infarcts. However, a difference is suggested by a multicenter evaluation of patients with SS disease who are asymptomatic for cerebrovascular disease, in which there was discordance between MRI and transcranial doppler results suggestive of CNS infarction.51 Finally, VCAM1 could be in linkage disequilibrium (LD) with another nearby locus affecting susceptibility to this SS disease complication, although this seems unlikely given the observation that LD does not appear to extend significantly beyond 5 kb in many regions of the genome, particularly in a population of African descent.52,53 Confirmatory testing of this association in a much larger independent SS disease population with attention to these and other factors is required before clinically meaningful conclusions can be made. Furthermore, future studies of genetic modifiers in SS disease will need to provide heritability estimates for the phenotypes or complications under study, so that the relative contribution of any single genetic determinant can be assessed. The specific mechanism for a modifying effect in SS disease by a single amino acid substitution in VCAM-1 could shed light on a critical structure/function relationship, especially in the context of previous in vitro mutational studies of immunoglobulin superfamily adhesion molecules.54 Indeed, adhesion receptors direct a variety of functions such as cell-cell interaction, leukocyte trafficking/signaling, and T-cell recognition.15 Immune responses generated by these interactions can be either protective or pathologic, as in the case of transplant rejection. The role of VCAM1 promoter and nonsynonymous SNPs, which could theoretically alter function and expression, await further investigation. This pilot genetic association study provides preliminary evidence that an individual VCAM1 genetic variant may influence risk for stroke in SS disease, especially in light of the reported role of VCAM-1 in the pathogenesis of SS disease.19-22,25 However, the results of this pilot association study must be confirmed in both larger population-based trials and family studies before its implications can be realized in either clinical care or the design of novel anti-inflammatory therapies such as monoclonal antibodies.55 Further examination of variation at the VCAM1 locus by haplotype may help to quantify the cumulative effects of all variants at this locus, elucidate the underlying pathogenesis of cerebrovascular events, both in the absence and presence of SS disease, and identify patients with SS disease who are candidates for therapies designed to prevent strokes.7
We thank Todd Brooks, Ed Miller, and Bernice Packer of the Genetic Annotation Initiative/NCI Cancer Genome Anatomy Project for providing VCAM1 cDNA PCR primers, equipment, advice, and assistance with software; and Drs EunWha Choi, Hans C. Erichsen, and Charles B. Foster for encouragement and useful discussions.
Submitted December 21, 2002; accepted July 4, 2002.
Prepublished online as Blood First Edition Paper, August 15, 2002; DOI 10.1182/blood-2001-12-0306.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stephen J. Chanock, Section on Genomic Variation, Pediatric Oncology Branch, Advanced Technology Center, National Cancer Institute, 8717 Grovemont Cir, Gaithersburg, MD 20877; e-mail: sc83a{at}nih.gov.
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© 2002 by The American Society of Hematology.
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  B. Bader-Meunier, S. Verlhac, M. Elmaleh-Berges, G. Ithier, F. Sellami, S. Faid, F. Missud, R. Ducrocq, C. Alberti, I. Zaccaria, et al. Effect of transfusion therapy on cerebral vasculopathy in children with sickle-cell anemia Haematologica, January 1, 2009; 94(1): 123 - 126. [Abstract] [Full Text] [PDF]
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 Q. Lan, L. Zhang, M. Shen, M. T. Smith, G. Li, R. Vermeulen, S. M. Rappaport, M. S. Forrest, R. B. Hayes, M. Linet, et al. Polymorphisms in Cytokine and Cellular Adhesion Molecule Genes and Susceptibility to Hematotoxicity among Workers Exposed to Benzene Cancer Res., October 15, 2005; 65(20): 9574 - 9581. [Abstract] [Full Text] [PDF]
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 C. Hoppe, W. Klitz, S. Cheng, R. Apple, L. Steiner, L. Robles, T. Girard, E. Vichinsky, and L. Styles Gene interactions and stroke risk in children with sickle cell anemia Blood, March 15, 2004; 103(6): 2391 - 2396. [Abstract] [Full Text] [PDF]
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 G. R. Buchanan, M. R. DeBaun, C. T. Quinn, and M. H. Steinberg Sickle Cell Disease Hematology, January 1, 2004; 2004(1): 35 - 47. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
1
integrin) expressed on sickle red cells.
4, 9.8 × 10