Blood, 15 May 2003, Vol. 101, No. 10, pp. 4223-4224
CORRESPONDENCE
To the editor:
The 
1C>T mutation in the annexin A5
gene does not affect plasma levels of annexin A5
González-Conejero and coworkers describe how the 
1C>T
mutation, located in the Kozak sequence of the annexin A5
gene (formerly named annexin V), reduced the risk of myocardial
infarction in young patients.1 They postulated and showed
supporting evidence that 1C>T in the Kozak sequence of the
annexin A5 gene increased translation, resulting in higher
plasma levels of annexin A5 in T allele carriers. These findings do not
support the Kozak context rules as reported by Marilyn
Kozak.2,3 She furthermore had some practical comments
about the reliability of the in vitro translation assay used in the
study.2
Our concerns are related to the determination of the plasma annexin A5
levels in citrated plasma. González-Conejero and coworkers used
the enzyme-linked immunosorbent assay (ELISA) technique of Diagnostica Stago.1 This ELISA allows only the measurement of free nonbound annexin A5. As annexin A5 binds with high affinity to
negatively charged phospholipids in the presence of
calcium,4 addition of citrate will not disturb the binding
efficiently and thus, the levels of annexin A5 measured in citrated
plasma will be underestimated. The annexin A5 levels are significantly
higher in EDTA (ethylenediaminetetraacetic acid)
plasma.5 In all our studies performed so far, we measured
only increased annexin A5 levels in EDTA plasma. This might indicate
that the annexin A5 levels in citrated plasma are not biologically
active or are released out of the residual platelets due to
freeze-thawing the plasma.
To determine whether T allele carriers indeed have increased levels of
circulating annexin A5, we measured the annexin A5 levels in citrated
and EDTA plasma of 40 healthy volunteers and linked the results to the
1C>T mutation. All plasma samples were obtained by centrifugation of
citrated and EDTA blood for 10 minutes at 4000g. The plasma
samples were stored at 80°C.
Our results show significantly higher annexin A5 levels in EDTA plasma
compared with citrated plasma (1.15 ± 0.56 vs 0.95 ± 0.28 ng/mL).
Furthermore, among these 40 healthy volunteers, no
differences in annexin A5 levels (both in citrated and EDTA plasma)
were observed between T allele and C allele carriers (Figure 1). Our measurements are in contrast to the results of
González-Conejero et al, who showed increased levels of annexin
A5 in plasma of T allele carriers.1 The prevalence of the
mutation in our control group was comparable to the prevalence in the
Mediterranean population (26%).
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| Figure 1.
Annexin A5 levels in C allele carriers and carriers of
the 1C>T mutation.
Annexin A5 levels were measured with the Zymutest Annexin A5 ELISA
(Hyphen Biomed, Andresy, France), and the polymorphism was determined
by NcoI restriction fragment-length polymorphisms (RFLP).
The mean annexin A5 levels are expressed (± SEM).
|
|
We have shown that the 1C>T mutation does not result in elevated
plasma annexin A5 levels. More studies have to be performed to
understand the reduced risk of myocardial infarction in young patients
carrying the T allele in the Kozak sequence of the annexin A5 gene.
Waander L. van
Heerde, Heidi Kenis, Selene Schoormans, Paul Lap, and Chris P. M. Reutelingsperger
Correspondence: Waander L. van Heerde, University
Medical Center Nijmegen, Central Hematology Laboratory, CHL 539, PO Box
9101, 6500 HB Nijmegen, The Netherlands; e-mail:
w.vanheerde{at}chl.umcn.nl
References
1.
Gonzalez-Conejero R, Corral J, Roldan V, et al.
A common polymorphism in the annexin V Kozak sequence (1C>T) increases translation efficiency and plasma levels of annexin V, and decreases the risk of myocardial infarction in young patients.
Blood.
2002;100:2081-2086[Abstract/Free Full Text].
2.
Kozak M, Neufeld E.
Not every polymorphism close to the AUG codon can be explained by invoking context effects on initiation of translation.
Blood.
2003;101:1202[Free Full Text].
3.
Kozak M.
Pushing the limits of the scanning mechanism for initiation of translation.
Gene.
2002;299:1-34[CrossRef][Medline]
[Order article via Infotrieve].
4.
Andree HA, Reutelingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM.
Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.
J Biol Chem.
1990;265:4923-4928[Abstract/Free Full Text].
5.
van Heerde WL, de Groot PG, Reutelingsperger CP.
The complexity of the phospholipid binding protein Annexin V.
Thromb Haemost.
1995;73:172-179[Medline]
[Order article via Infotrieve].
Response:
Annexin V polymorphisms, plasma levels, and myocardial
infarction
Studies about common polymorphisms give conflicting results when
analyzing both their functional role and their clinical relevance. For
every study that finds a positive association with a particular polymorphism, there are several reporting no effect or the
opposite.1-4 As expected for genetic changes present in
more than 1% of the population, common polymorphisms would unlikely
have a strong effect. Thus, even minor modifications in selection of
samples, design of the study, environmental factors, or genetic
background of different populations might modify the functional or
clinical consequences associated with a single polymorphism. In a
recent study, we found the 1T allele of the annexin V (ANV; highly
prevalent in the white Mediterranean population [23%]) associated
with increased circulating levels of this protein in citrated samples.
This allele also related to increased translation of this protein when
using in vitro systems containing either whole ANV cDNA or mini-cDNA generated by genomic polymerase chain reaction. In agreement
with these results, we found that the 1T allele conferred protection against premature myocardial infarction.5 van Heerde and
coworkers have argued against some points of our findings. First, they
are concerned about the appropriateness of ANV analysis in citrated samples. Certainly it is necessary to use EDTA
(ethylenediaminetetraacetic acid) samples to determine total
circulating ANV. However, we consider that citrated values could give
more clinical information. As indicated by van Heerde et al, citrated
samples allow the measurement of free nonbound ANV, the molecule with
potential antithrombotic role, by binding to negatively charged
phospholipids exposed in procoagulant surfaces during pathologic
prothrombotic states. Moreover, although ANV levels in samples
anticoagulated with EDTA are higher than those obtained using citrated
samples, we observed a correlation between these samples in all tested individuals.
Second, these authors found no difference in the plasma level of ANV
associated with the 1C>T polymorphism in a Dutch population. The
association between the 1C>T polymorphism and circulating ANV levels in both studies were established with data from few C>T
individuals (7 Dutch and 10 Spanish subjects). Moreover, circulating ANV values displayed a wide interindividual variation independently of
the 1C>T genotype, especially in the Dutch study. Thus, further studies including more individuals with either genotype (C>C, C>T,
and T>T) would give definitive insight on whether the 1C>T polymorphism influences the plasma levels of ANV.
Our in vivo, in vitro, and clinical data consistently support that the
ANV 1 T allele plays a minor but significant role influencing the
levels of ANV in our population. The relevance of the Kozak sequence in
the control of translation led us to suggest that the 1C>T
polymorphism could effect its influence by affecting translation.
However, Dr Kozak recently has suggested that increase in plasma levels
of ANV associated with the 1T allele could reflect an effect on mRNA
stability or splicing, rather than an effect on
translation.6 Noteworthy, we also identified a second
polymorphism in the ANV gene: ISV2 + 18 C>G. This polymorphism is
located near the splicing signal of exon 1 and interestingly, is in
linkage disequilibrium with the 1 C>T polymorphism. The 1T allele
always associated with the ISV2 + 18G allele in the Spanish
population. It seems also reasonable to test whether this linkage is
also observed in other populations. Moreover, additional experiments
are required to clarify the mechanism controlling the levels of ANV
associated with the 1 C>T and ISV2 + 18 C>G polymorphisms.
Finally, it would also be necessary to perform further studies to
confirm the clinical relevance of this polymorphism, as we stated in
our original manuscript.
Rocio González-Conejero, Javier Corral, Vanessa Roldan, Constantino Martínez, Francisco Marín, José Rivera, Juan A. Iniesta, Maria L. Lozano, Pascual Marco, and Vicente Vicente
Correspondence: V. Vicente, University of Murcia,
Centro Regional de Hemodonacion, Murcia, Spain; e-mail:
vvg{at}um.es
References
1.
Humphries SE, Panahloo A, Montgomery HE, Green F, Yudkin J.
Gene-environment interaction in the determination of levels of haemostatic variables involved in thrombosis and fibrinolysis.
Thromb Haemost.
1997;78:457-461[Medline]
[Order article via Infotrieve].
2.
Ridker PM, Stampfer MJ.
Assessment of genetic markers for coronary thrombosis: promise and precaution.
Lancet.
1999;353:687-678[CrossRef][Medline]
[Order article via Infotrieve].
3.
Grant PJ, Humphries SE.
Genetic determinants of arterial thrombosis.
Baillieres Best Pract Res Clin Haematol.
1999;12:505-532[Medline]
[Order article via Infotrieve].
4.
Lane DA, Grant PJ.
Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease.
Blood.
2000;95:1517-1532[Free Full Text].
5.
González-Conejero R, Corral J, Roldán V, et al.
A common polymorphism in the annexin V Kozak sequence (1C>T) increases translation efficiency and plasma levels of annexin V, and decreases the risk of myocardial infarction in young patients.
Blood.
2002;100:2081-2086[Abstract/Free Full Text].
6.
Kozak M.
Not every polymorphism close to the AUG codon can be explained by involving context effects on initiation of translation.
Blood.
2003;101:1202.
