Polymorphism in Tumor Necrosis Factor Genes
Associated with Mucocutaneous Leishmaniasis
By Maira Cabrera,*l: Marie-Anne Shaw,* Claire Sharpies,*
Hazel Williams,* Marianella Castes, ~ Jacinto Convit,:l:
and Jenefer M. Blackwell*
From the *Department of Medicine, University of Cambridge Clinical School, Addenbrooke's
Hospital, Cambridge CB2 2QQ, United Kingdom; and the ~Instituto de Biomedicina, Caracas
Recent studies have shown that mucocutaneous leishmaniasis (MCL), a severe and debilitating
form of American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis infection, is
accompanied by high circulating levels of tumor necrosis factor (TNF)-oL. Analysis of TNF
polymorphisms in Venezuelan ACL patients and endemic unaffected controls demonstrates a
high relative risk (RR) of 7.5 (P <0.001) of MCL disease in homozygotes for allele 2 of a
polymorphism in intron 2 of the TNF-[3 gene, especially in females (RR = 9.5; P <0.001)
compared with males (RR = 4; P <0.05). A significantly higher frequency (P <0.05) of allele
2 at the -308-basepair TNF-c~ gene polymorphism was also observed in MCL patients (0.18)
compared with endemic control subjects (0.069), again associated with a high relative risk of
disease (RR = 3.5; P <0.05) even in the heterozygous condition. Because both the TNF-c~
and TNF-[3 polymorphisms have previously been linked with functional differences in TNF-oL
levels, these data suggest that susceptibility to the mucocutaneous form of disease may be di-
rectly associated with regulatory polymorphisms affecting TNF-~x production.
niasis (MCL), I sometimes months to years after a simple lo-
calized cutaneous lesion, is not clearly understood. Clini-
cally advanced MCL has traditionally been associated with
strong skin test delayed-type hypersensitivity to leishmanial
antigens in vivo, and with high IFN-'y/IL-2-producing T
cell proliferative responses in vitro (1-3). Analysis of MCL
lesions has demonstrated mRNA for a mixture of Thl
(IFN-% IL-2, TNF-[3) and Th2 (IL-4, IL-5, IL-10) cyto-
kines (4, 5), with TNF-oL mRNA also present. Recently
we observed that active MCL disease was also associated
with high circulating levels of TNF-ot (6), which might, as
has now been demonstrated for cerebral malaria (7), be re-
lated to genetic regulation of cytokine production. The
TNF-ot gene itself lies in the class III region of the MHC
(8). Although previous studies (9) had demonstrated associ-
ation between class II DR[3 alleles and MCL disease, it was
possible that these associations had arisen through linkage
disequilibrium between class II genes and variable genetic
elements in the class III region known to control TNF-o~
he reason some individuals infected with Leishmania
braziliensis go on to develop mucocutaneous leishma-
IAbbreviations used in this paper: ACL, American cutaneous leishmaniasis;
df, degree of freedom; LCL, localized cutaneous leishmaniasis; MCL, mu-
cocutaneous leishmaniasis; RR, relative risk.
production. Two such polymorphisms have been described,
at position -308 in the promoter region of the TNF--cx
gene (10), for which allele 2 is associated with higher con-
stitutive and inducible levels of TNF-~x (11), and in intron
2/exon 3 of the TNF-[3 gene (12), with the two polymor-
phic alleles variably associated with high and low levels of
TNF-cx secretion by mononuclear cells, depending on the
population under investigation (12-14). The latter poly-
morphism involves an NcoI polymorphism in intron 2 and
is also always associated with a substitution at amino acid
position 26 encoded in exon 3 of the TNF-[3 gene (12).
Precisely how this polymorphism influences TNF-oL secre-
tion is not known, but may be caused by linkage disequi-
librium with other elements within the TNF-~x gene itself.
To determine whether these polymorphisms at the TNF
loci influence susceptibility to different forms of American
cutaneous leishmaniasis (ACL), case/control analysis was
undertaken comparing MCL patients or localized cutane-
ous leishmaniasis (LCL) patients with healthy endemic con-
trol subjects. Our results demonstrate a high relative risk
(RR = 7.5; P <0.001) of MCL disease in homozygotes for
allele 2 (=TNFB*I [12-14]) of the intron 2/exon 3 poly-
morphism in the TNF-[3 gene, especially in females (RR =
9.5; P <0.001) compared with males (RR = 4; P <0.05).
A significantly higher frequency (P <0.05) of allele 2 at the
1259 J. Exp. Med. y The Rockefeller University Press i 0022-1007/95/11/1259/06 $2.00
Volume 182 November 1995 1259-1264
Address correspondence to J. M. Blackwell, Department of Medicine, Level 5, Addenbrooke's Hospital,
Cambridge CB2 2QQ, UK.
Received for publication 7 March 1995 and in revised form 19June 1995.
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1264 Polymorphism in TNF Genes Associated with Mucocutaneous Leishmaniasis