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From the Cardiovascular Research Laboratories, Division of Cardiology,
University of Pittsburgh Medical Center, Pittsburgh, Pa (T.K., D.M.M., J.J.W.,
M.T., C.F.M., A.M.F.), and the Cardiology Section of the Department of
Medicine, Veterans Administration Medical Center and Baylor College of
Medicine, Houston, Tex (D.L.M.).
Correspondence to Dennis M. McNamara, MD, Division of Cardiology, University of Pittsburgh Medical Center, 200 Lothrop St, 558 Scaife Hall, Pittsburgh, PA 15213. E-mail mcnama{at}msx.upmc.edu
Methods and Results—TNFA and TNFB genotypes were
determined by the polymerase chain reaction–restriction fragment
length polymorphism technique. There were no differences in the TNF
allele frequencies between CHF (n=229; TNFA1/2=0.84/0.16,
TNFB1/2=0.33/0.67) and control subjects (n=139; TNFA1/2=0.84/0.16,
TNFB1/2=0.32/0.68). In 211 patients with CHF, circulating levels of
TNF-
Conclusions—Despite their association with other inflammatory
diseases, neither TNFA nor TNFB polymorphisms are related to the
presence of CHF or the elevation of circulating TNF-
The genes for TNF-
To examine the effects of TNF polymorphisms on circulating levels
of TNF-
Genotyping of TNF Polymorphisms
Primers 5'-CCGTGCTTCGTGGTTTGGACTA-3' and 5'-AGAGGGGTGGATGCTTGGGTTC-3'
were used to amplify a DNA fragment of 782 bp containing the
polymorphic NcoI site of the human TNF-ß first intron
(+252).11 Conditions of PCR and NcoI
digestion were the same as for TNFA typing. Digested DNA was subjected
to electrophoresis in 1% agarose gel (SeaKem LE, FMC BioProducts).
TNFB1 allele gives 2 fragments of 586 and 196 bp, and TNFB2
allele a single 782-bp fragment.
Measurements of Circulating TNF-
Statistical Analysis
The Figure
The disparity between the associations of TNF polymorphisms
and autoimmune diseases and the lack of association between TNF
polymorphisms and CHF points out a major limitation in performing
genetic studies in a population of patients referred to a tertiary
hospital because of CHF. Unlike autoimmune diseases, CHF is associated
with a high mortality, with as many as 50% of patients dying within
the first year after referral. Furthermore, the denominator in CHF
patients is undefined, because it is generally believed that patients
with myopathies are asymptomatic for variable periods
of time before presenting with signs and symptoms of CHF.
Therefore, we cannot exclude the possibility that patients homozygous
for the TNFA2 or TNFB2 alleles had a more malignant clinical course
and died before their presentation at a tertiary referral
center and that the results were therefore inherently biased. Indeed,
this possibility may explain the enormous disparity among the large
number of studies that have assessed the relevance of ACE
polymorphisms in patients with CHF. One additional limitation of
this study was the slightly greater proportion of women and nonwhite
individuals in the control group compared with patients. However,
genotype analysis stratified by sex and race (data not
shown) did not differ from the group as a whole, arguing that these
demographic differences did not mask a true underlying association.
This study confirms previous reports of linkage disequilibrium
between specific TNF alleles, TNFA1 and TNFB2. This association
appears to be inconsistent with the finding of previous
studies10 11 that TNFB2 and TNFA2 but not TNFA1
were associated with increased TNF-
In conclusion, the present study does not define an
association between the genotypes of TNFA or TNFB
polymorphisms and the presence of CHF or circulating levels of
TNF-
Received February 10, 1998;
revision received April 22, 1998;
accepted May 3, 1998.
2.
Ferrari R, Bachetti T, Confortini R, Opasich C, Febo
O, Corti A, Cassani G, Visioli O. Tumor necrosis factor soluble
receptors in patients with various degrees of congestive heart failure.
Circulation. 1995;92:1479–1486.
3.
Torre-Amione G, Kapadia S, Benedict C, Oral H, Young
JB, Mann DL. Proinflammatory cytokine levels in patients with
depressed left ventricular ejection fraction: a report from
the studies of left ventricular dysfunction (SOLVD).
J Am Coll Cardiol. 1996;27:1201–1206.[Abstract]
4.
Nozaki N, Yamaguchi S, Shirakabe M, Nakamura H,
Tomoike H. Soluble tumor necrosis factor receptors are elevated in
relation to severity of congestive heart failure. Jpn Circ
J. 1997;61:657–664.[Medline]
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Torre-Amione G, Kapadia S, Lee J, Durand JB, Bies RD,
Young JB, Mann DL. Tumor necrosis factor-
6.
Kubota T, McTiernan CF, Frye CS, Slawson SE, Lemster
BH, Koretsky AP, Demetris AJ, Feldman AM. Dilated
cardiomyopathy in transgenic mice with
cardiac-specific overexpression of tumor necrosis factor-
7.
Wilson AG, di Giovine FS, Duff GW. Genetics of
tumor necrosis factor-
8.
Wilson AG, di Giovine FS, Blakemore AIF, Duff GW.
Single base polymorphism in the human tumor necrosis factor alpha
(TNF
9.
Messer G, Spengler U, Jung MC, Honold G, Blömer
K, Pape GR, Riethmüller G, Weiss EH. Polymorphic structure of
the tumor necrosis factor (TNF) locus: an NcoI polymorphism in the
first intron of the human TNF-ß gene correlates with a variant
amino acid in position 26 and a reduced level of TNF-ß
production. J Exp Med. 1991;173:209–219.
10.
Wilson AG, Symons JA, McDowell TL, di Giovine FS, Duff
GW. Effects of a tumor necrosis factor (TNF
11.
Stüber F, Petersen M, Bokelmann F, Schade U. A
genomic polymorphism within the tumor necrosis factor locus
influences plasma tumor necrosis factor-
12.
Stuber F, Udalova IA, Book M, Drutskaya LN, Kuprash D,
Turetskaya RL, Shade FU, Nedospasov SA. -308 tumor necrosis
factor (TNF) polymorphism is not associated with survival in severe
sepsis and is unrelated to lipopolysaccharide inducibility of
the human TNF promoter. J Inflamm. 1996;46:42–50.
13.
Danis VA, Millington M, Hyland V, Lawford R, Huang Q,
Grennan D. Increased frequency of the uncommon allele of a tumor
necrosis factor alpha gene polymorphism in rheumatoid arthritis and
systemic lupus erythematosus. Dis
Markers. 1994;12:127–133.
14.
McGuire W, Hill AVS, Allsopp CEM, Greenwood BM,
Kwjatkowski D. Variation in the TNF-
15.
Fugger L, Morling N, Ryder LP, Georgsen J,
Jakobsen BK, Svejgaard A, Andersen V, Oxholm P, Pedersen FK, Friis J,
Halberg P. NcoI restriction fragment length polymorphism (RFLP) of
the tumor necrosis factor (TNF
© 1998 American Heart Association, Inc.
Brief Rapid Communications
Effects of Tumor Necrosis Factor Gene Polymorphisms on Patients With Congestive Heart Failure
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
Appendix 1
References
Background—Tumor
necrosis factor-
(TNF-) is known to be elevated in
patients with congestive heart failure (CHF). Two biallelic
polymorphisms have been identified in the TNF gene locus: one in
the promoter region of TNF- (TNFA1/2), and the other in the first
intron of TNF-ß (TNFB1/2). Both TNFA2 and TNFB2 alleles are
associated with high TNF- production in vitro and
susceptibility to inflammatory diseases. Given the importance of
TNF- in the pathogenesis of CHF, we studied the prevalence of TNF
gene polymorphisms in CHF patients and the correlation of
genotypes to in vivo TNF- levels. and the soluble receptors type I and type II were measured by
ELISA: 6.18±3.59 pg/mL, 1768±761 pg/mL, and 4484±1750 pg/mL,
respectively. There were no correlations between TNFA or TNFB
genotypes and circulating levels of TNF- or its soluble
receptors in the CHF patients. . Thus, other
factors may be more important in determining the circulating levels of
TNF- in CHF.
Key Words: genetics • heart failure • immunology
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
Appendix 1
References
Tumor necrosis
factor- (TNF-) is a proinflammatory cytokine with
pleiotropic biological effects. Numerous studies have demonstrated that
circulating levels of TNF- are elevated in patients with congestive
heart failure (CHF).1 2 3 4 There is a significant
correlation between the plasma levels of TNF- and the severity of
CHF.2 3 Furthermore, TNF- is present in
the failing but not the nonfailing human heart.5
In experimental animals, TNF- depresses cardiac
contractility, induces cardiomyocyte
apoptosis, and produces dilated
cardiomyopathy.6 Therefore,
TNF- might be a key mediator in the development of CHF. and TNF-ß are located in tandem on the short
arm of chromosome 6. Genetic polymorphisms in the TNF locus are
known to be related to several autoimmune, infectious, and neoplastic
diseases.7 Two biallelic polymorphisms have
been studied extensively: a G-to-A transition at position -308 in the
promoter region of the TNF- gene (G=TNFA1,
A=TNFA2)8 and a G-to-A transition at position
+252 in the first intron of the TNF-ß gene (G=TNFB1,
A=TNFB2).9 Both TNFA2 and TNFB2 alleles are
associated with high TNF-
production,10 11 although contradictory
reports exist.9 12 The frequency of the TNFA2
allele increases in rheumatoid arthritis and systemic lupus
erythematosus,13 and
homozygotes for the TNFA2 allele have a higher risk for death due
to cerebral malaria.14 In contrast, the TNFB2
allele decreases in systemic lupus
erythematosus,15 and
homozygotes for the TNFB2 allele in severe sepsis have a higher
mortality rate with higher circulating levels of
TNF-.11 Given the importance of TNF- in the
pathogenesis of CHF, we hypothesized that these genetic variations
might affect susceptibility to and severity of CHF.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
Appendix 1
References
Subjects
Two patient populations were studied. To examine the
effects of TNF polymorphisms on susceptibility to CHF, we studied a
series of 229 patients referred to the University of Pittsburgh Heart
Failure Service from April 1996 to April 1997 and 139 age-matched
control subjects without a history of coronary disease or heart
failure. Controls were recruited from spouses of patients in the study.
Given the female predominance of patient spouses, additional healthy
male volunteers were recruited through a university advertisement. The
population profiles are summarized in the
Table
.
View this table:
[in a new window]
Table 1. Patient Profiles and TNF Genotypes , we studied 211 patients in the VEST for TNF
Genotype Analysis, which is a substudy of the
Vesnarinone Survival Trial (VEST, directed by Jay N. Cohn, MD). All
patients in the VEST study had New York Heart Association class III to
IV CHF and a left ventricular ejection fraction 
35%. The
study was approved by the review board at each institution, and
subjects gave written informed consent for genetic
analysis.
Genomic DNA was extracted from peripheral blood with
a Puregene kit (Gentra Systems Inc). Primers
5'-AGGCAATAGGTTTTGAGGGCCAT-3' and 5'-TCCTCCCTGCTCCGATTCCG-3' were used
to amplify a DNA fragment of 107 bp containing the variable -308
nucleotide of the human TNF-
promoter.8 The sense primer was modified to
incorporate the polymorphic site into an NcoI
restriction site. Genomic DNA (100 ng) was amplified with 1.25 U
Thermus aquaticus DNA polymerase (Gibco BRL) in 50 µL of
20 mmol/L Tris-HCl containing 50 mmol/L KCl, 1.5 mmol/L
MgCl2, 200 µmol/L of each dNTP, and
0.2 µmol/L of each primer. Polymerase chain reactions were run
for 35 cycles: 1 minute at 94°C, 30 seconds at 60°C, and 1 minute
at 72°C. The product (10 µL) was digested with 5 U
NcoI at 37°C for >4 hours, subjected to electrophoresis
in 3.5% agarose gel (NuSieve GTG, FMC BioProducts), and stained
with ethidium bromide. TNFA1 allele gives 2 fragments of 87 and 20
bp, and TNFA2 allele a single 107-bp fragment. Therefore, a
homozygote for the TNFA1 allele (TNFA11) gives 2 bands, whereas a
homozygote for the TNFA2 allele (TNFA22) gives 1 band and the
heterozygote (TNFA12) 3 bands. and Its Soluble
Receptors
Plasma samples were collected at the entry of the VEST study and
stored frozen at -80°C. Commercially available ELISA kits
(Quantikine, R&D Systems) were used to measure plasma levels of human
TNF-, human soluble TNF receptor type I (sTNFRI), and human soluble
TNF receptor type II (sTNFRII) as we described
previously.5
The results are presented as mean±SD. The
2 test was used to compare allele
frequencies between groups. ANOVA and Kruskal-Wallis test were used to
examine effects of TNF genotypes on circulating levels of
TNF- and its soluble receptors. Differences were considered
significant at a value of P<0.05.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
Appendix 1
References
TNF genotypes in each population are summarized in the
Table
. The observed allele frequencies were similar to those
reported in the previous studies,8 11 12 13 14 15 and no
differences were found between CHF and controls: TNFA1/TNFA2=0.84/0.16
(CHF) or 0.84/0.16 (control) and TNFB1/TNFB2=0.33/0.67 (CHF) or
0.32/0.68 (control). Furthermore, there were no differences in
functional classes, ejection fraction, or cause of CHF by TNFA or TNFB
genotypes (data not shown). The TNFB2 allele was
significantly linked to the TNFA1 allele (P<0.00001,
data not shown), as reported previously.12 summarizes the circulating levels of
TNF-, sTNFRI, and sTNFRII in the VEST patients. Plasma levels of
TNF- were elevated in patients with CHF (n=211, 6.18±3.59 pg/mL)
compared with the healthy control subjects in our previous study (n=14,
0.8±0.2 pg/mL, P<0.001).3 Similarly,
CHF patients had elevated plasma levels of sTNFRI and sTNFRII
(1768±761 and 4484±2049 pg/mL). These values are nearly identical to
that previously reported for CHF patients in studies using identical
methods and reagents.2 3 4 Neither
parametric (ANOVA) nor nonparametric
(Kruskal-Wallis test) statistics could detect significant associations
between the TNFA or TNFB genotypes and circulating levels of
TNF-, sTNFRI, or sTNFRII.
View larger version (27K):
[in a new window]
Figure 1. Plasma levels of TNF- and soluble receptors by TNF
genotypes. TNF indicates tumor necrosis factor; sTNFRI, soluble
TNF receptor type I; and sTNFRII, soluble TNF receptor type II. Values
are expressed as mean±SD.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
Appendix 1
References
This is the first report that has studied TNF
polymorphisms in patients with CHF. Despite their association with
other inflammatory diseases,7 neither TNFA nor
TNFB polymorphisms were related to the presence of CHF or the
elevation of circulating TNF-, thus suggesting that elevated levels
of TNF- in CHF patients are not necessarily linked to genetic
polymorphisms of TNF- or TNF-ß. production. In fact, our
data demonstrate no association of either TNFA2 or TNFB2 with increased
levels of proinflammatory cytokines in patients with CHF. In
most previous studies, however, an association was demonstrated by
assessing the ability of peripheral blood mononuclear cells
to respond to lipopolysaccharides or phytohemagglutinin,
whereas we examined baseline levels of plasma TNF- in patients with
severe but stable CHF. Furthermore, although TNF- is expressed in
the failing heart,5 TNF- levels in the
myocardium are known to be different from those in the
plasma.5 6 Because circulating levels of TNF-
are also affected by other factors, such as clearance and
production by nonmyocardial tissues, it may not reflect the
true cytokine burden to the failing heart. Finally, all
patients with TNF levels measured in this study had at least class III
heart failure. This may decrease the variance of TNF- levels within
the group and limit the power of the study to detect a correlation with
TNF genotypes. . Although we cannot exclude the possibility that other factors
are important in determining the risk for CHF, additional trials will
be needed to clarify the prognostic importance of polymorphisms in
the TNF genes. Specifically, studies in patients with compromised left
ventricular function after a first myocardial infarction
might provide a more appropriate population in which to test this
hypothesis.
Appendix 1
Top
Abstract
Introduction
Methods
Results
Discussion
Appendix 1
References
VEST Investigators for TNF Genotype Analysis
Evan Loh, MD, Kim Craig, RN, (University of Pennsylvania,
Philadelphia, Pa); Robert Bourge, MD, Melanie Smith, RN, Patti Smith,
RN, Amy Trimble, RN (University of Alabama, Birmingham); Sidney
Gottlieb, MD, Loriane Black, RN, BSN (Midatlantic
Cardiovascular Associates, Baltimore, Md); Michael
McIvor, MD, Liz Floden, RN (Cardiology
consultants, St Petersburg, Fla); Srinivas Murali, MD,
Warren Rosenblum, MD, Susan Loftus, RN, BSN (University of Pittsburgh,
Pittsburgh, Pa).
Acknowledgments
Dr Kubota is the recipient of a Japan Heart Foundation and a
Bayer Yakuhin Research Grant Abroad. This study was supported in part
by a grant from Otsuka America.
Footnotes
Guest editor for this article was Jeffrey M. Isner, St Elizabeth's Medical Center, Boston, Mass.
References
Top
Abstract
Introduction
Methods
Results
Discussion
Appendix 1
References
1.
Levine B, Kalman J, Mayer L, Fillit HM, Packer M.
Elevated circulating levels of tumor necrosis factor in severe chronic
heart failure. N Engl J Med. 1990;223:236–241. and tumor necrosis
factor receptors in the failing human heart. Circulation. 1996;93:704–711..
Circ Res. 1997;81:627–635. in autoimmune, infectious, and
neoplastic diseases. J Inflamm. 1995;45:1–12.[Medline]
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Hum Mol Genet. 1992;1:353.) promoter base
transition on transcriptional activity. Br J Rheumatol.
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