Immunology Letters 118 (2008) 148–151
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/
The 3?UTR 1188 A/C polymorphism in the interleukin-12p40 gene (IL-12B)
is associated with lepromatous leprosy in the west of Mexico
Anabell Alvarado-Navarroa, Margarita Montoya-Buelnaa, Jos´ e Francisco Mu˜ noz-Valleb,
Rocio Ivette L´ opez-Roaa, Cecilia Guill´ en-Vargasa, Mary Fafutis-Morrisa,∗
aCentro de Investigaci´ on en Inmunolog´ ıa y Dermatolog´ ıa, Departamento de Fisiolog´ ıa, Centro Universitario de Ciencias de la Salud,
Universidad de Guadalajara, Guadalajara, Jalisco, Mexico
bInstituto de Investigaci´ on en Reumatolog´ ıa y del Sistema M´ usculo Esquel´ etico, Centro Universitario de Ciencias de la Salud,
Universidad de Guadalajara, Guadalajara, Jalisco, Mexico
a r t i c l ei n f o
Received 9 February 2007
Received in revised form 22 March 2008
Accepted 30 March 2008
Available online 28 April 2008
a b s t r a c t
Leprosy is a chronic infectious disease caused by Mycobacterium leprae. IL-12 participates in the immune
response against M. leprae by regulating T cell differentiation into the Th1-type response. Several single
nucleotide polymorphisms have been identified in the IL-12 gene such as 3?UTR 1188 A/C polymorphism,
which is associated with different diseases. However, the relationship of this polymorphism with the
immune response in leprosy has not been explored. In this case–control study, we evaluated 44 patients
with lepromatous leprosy (LL) and 51 healthy subjects (HS). We aimed to determine the relationship
between 3?UTR 1188 A/C polymorphism of IL-12 p40, mRNA expression, and soluble IL-12 concentration
in LL patients and HS. Genotype frequencies were 41% A/A, 36% A/C, and 23% C/C in LL patients, and 47%
A/A, 49% A/C, and 4% C/C in HS (p<0.05). LL patients had a lower mRNA expression of IL-12 p40 gene,
whereas HS had a higher expression level. Soluble IL-12 p40 concentration was higher in LL patients than
in HS (p<0.05). IL-12 p70 concentration did not differ between groups, and IL-12 p40 concentration was
1188 A/C polymorphism is associated with greater susceptibility to lepromatous leprosy in patients from
western Mexico, independently of IL-12 p40 and p70 expression levels.
© 2008 Elsevier B.V. All rights reserved.
Leprosy remains a public health problem despite global cov-
erage of multidrug therapy promoted by the World Health
Organization and the dramatic decrease in prevalence. However,
the number of new cases of leprosy has remained obstinately con-
by the intracellular pathogen Mycobacterium leprae, is character-
ized by several clinical manifestations determined presumably by
the host immune response to M. leprae. In the lepromatous pole
of patients with lepromatous leprosy (LL), the lack of effective cell-
mediated immunity to M. leprae increases the risk of progression of
infection to active disease, which is caused by reduced production
of Th1 cytokines. Instead, the tuberculoid pole (TT) is characterized
∗Corresponding author at: CIINDE, Av. Federalismo Norte 3102, Col. Atemajac
Zapopan, Jalisco, CP 44220 Guadalajara, Jalisco, Mexico. Tel.: +52 33 10 58 53 07;
fax: +52 33 10 58 53 07.
E-mail address: firstname.lastname@example.org (M. Fafutis-Morris).
ination of the mycobacteria . Tuberculoid leprosy lesions have a
predominance of CD4+T cells, and these cells produce the typical T
helper 1 (Th1) cytokine pattern. IL-12 mRNA is expressed at levels
10 times higher in tuberculoid lesions than in lepromatous lesions
IL-12 secretion is regulated by negative feedback through Th2
cells and their secretion of IL-10. The active cytokine is a 70kDa
heterodimer comprising two covalently linked proteins of 35kDa
(p35) and 40kDa (p40) ; each subunit is encoded on different
chromosomes. In humans, the IL-12 p35 subunit is encoded on
chromosome 3p12–3q13.2, whereas the IL-12 p40 subunit gene is
located on chromosome 5q31–33. The p40 subunit is a component
The p35 subunit can be secreted only in association with the p40
IL-12 is a cytokine produced by macrophages and dendritic cells
in response to intracellular pathogens and bacterial products. The
IL-12 receptor (IL-12R) is expressed in natural killer and activated
T cells, and is made up of two chains called IL-12R?1 and IL-12R?2.
0165-2478/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
A. Alvarado-Navarro et al. / Immunology Letters 118 (2008) 148–151
The coexpression of both chains allows binding of IL-12 with high
affinity, initiating the physiological response to this cytokine .
Among many biological activities, IL-12 provides an obligatory sig-
nal for the differentiation of Th1 effectors cells and IFN-? and IL-2
Several polymorphisms have been described in the promoter
region [9,10], and in the 3?untranslated region (UTR) of IL-12 p40
gene, IL-12B [11,12]. The presence of a single nucleotide polymor-
phism (SNP) at 3?UTR 1188 A/C (TaqI) is associated with IL-12
production in different pathologies that affect the delicate Th1/Th2
balance . However, the relationship of this polymorphism with
the immune response in leprosy has not been explored. We ana-
lyzed the polymorphic region in IL-12 p40 (3?UTR 1188 A/C) and its
possible association with susceptibility to lepromatous leprosy in
patients from the west of Mexico. In addition, we assessed IL-12
p40 mRNA expression and the concentrations of soluble IL-12 p40
and p70 in patients and healthy individuals.
2. Materials and methods
2.1. Study population
We recruited 44 LL patients (20–65 years old) from the Der-
matological Institute, Guadalajara, M´ exico, who were diagnosed
according to the international criteria of Ridley and Jopling . All
patients received during 3–5 years multidrug therapy treatment as
DNA was demonstrated  independently of the negative bacil-
lary charge. Fifty-one healthy subjects (HS), matched for age and
sex with the patients, were included as a control group. Informed
consent according to the Helsinki Declaration was obtained in all
individuals included in the study.
2.2. Screening of 3?UTR 1188 A/C polymorphism in the IL-12p40
Genomic DNA was extracted from leukocytes obtained from
whole blood samples according to the Miller method . TaqI
polymorphism genotyping in the 3?UTR of the IL-12 p40 gene (Gen-
Bank accession number: AY008847) was performed as described
previously . Amplification was performed by PCR in a thermal
cycler (Gene Amp PCR System 9700, Applied Biosystems), using
the following primers: forward, 5?-TTCTATCTGATTTGCTTTA-3?and
reverse, 3?-TGAAACATTCCATACATCC-5?. PCR was carried out in a
final volume of 50?L containing 1?g genomic DNA, 3?M of each
primer, 1.25U/?L of Taq DNA polymerase (Invitrogen Life Tech-
nologies), 5?L of supplied 10× buffer enzyme, 2.5mM MgCl2, and
0.1mM of each dNTP (Invitrogen Life Technologies). PCR was per-
formed by initial denaturation at 95◦C for 3min for the initial
denaturation followed by 30 cycles at 95◦C for 30s, 43◦C for 30s
and 72◦C for 1min, with a final extension at 72◦C for 7min. The
PCR resulted in a 233bp product. The amplified fragment of 3?UTR
1188 A/C polymorphism in the IL-12 p40 gene was digested for 1h
at 37◦C with 3U of TaqI (Invitrogen Life Technologies) restriction
enzyme. Restriction fragments were analyzed by electrophoresis
on 2% agarose gel (Invitrogen Life Technologies) and stained with
ethidium bromide. The C/C wild-type genotype was digested and
(TaqI site absent) migrated as a 233bp fragment.
2.3. IL-12 p40 mRNA expression
Total RNA was isolated from whole blood samples, as described
previously by Chomczynski and Sacchi  For each experiment,
equivalent amounts of intact RNA (2?g) were primed (70◦C for
5min) with oligo(dT) (0.5?g, Invitrogen Life Technologies). cDNA
was synthesized for 60min at 37◦C in a 20?L reaction mixture
containing 0.2mM deoxynucleoside triphosphates (Invitrogen Life
Technologies), 1× buffer, 200U of Moloney murine leukemia virus
reverse transcriptase (Invitrogen Life Technologies), 1.5mM MgCl2
(Invitrogen Life Technologies), and 1U of RNAasaOut (Invitrogen
Amp PCR System 9700; Applied Biosystems) in a 50?L reaction
mixture containing 3mM MgCl2, 1× buffer, 1.25U of Taq poly-
merase (Invitrogen Life Technologies), 3?M forward and 3?M
reverse primers (Invitrogen Life Technologies). All samples were
cycled with the following log-linear parameters: (i) for GAPDH
(glyceraldehyde 3-phosphate dehydrogenase), and 31 cycles of
denaturation (95◦C for 1min), annealing (60◦C for 1min), and
extension (72◦C for 1min 30s), and a final extension (72◦C for
2min), and (ii) for IL-12 p40, first denaturation at 95◦C for 30s and
32 cycles of denaturation (95◦C for 30s), annealing (60◦C for 40s),
and extension (72◦C for 2min 35s). A final extension of 10min at
72◦C was performed. The primer sequences were as follows: (i)
for GAPDH (GenBank accession number: BC013310), 5?-GCT CAG
ACA CCA TGG GGA AGG T-3?(forward) and 5?-GTG GTG CAG GAG
GCA TTG CTG A-3?(reverse) and (ii) IL-12 p40 (GenBank accession
5?-TGGGTCTATTCCGTTGTGTC-3?(reverse). PCR product mixtures
(10?L) were electrophoresed in a 2% agarose gel and stained with
ethidium bromide and an appropriate size ladder.
2.4. IL-12 p40 and p70 assay
Serum IL-12 p40 and p70 concentration in LL patients and
HS was measured using an ELISA R&D Systems. The minimum
detectable concentration is typically <15pg/mL for human IL-12
p40 and 0.5pg/mL for IL-12 p70. IL-12 p40 and p70 concentrations
were calculated from a standard curve using the corresponding
recombinant human IL-12 p40 and p70, respectively. All cytokine
measurements were done in duplicate.
2.5. Statistical analysis
The Hardy–Weinberg equilibrium and differences in the gene
Genotype and allele frequencies of IL-12 p40 3?UTR 1188 A/C polymorphism in LL patients and HS
GenotypeLL (n=51) % (n) HS (n=44) % (n) AlleleLL (n=88) % (n)HS (n=102) % (n)
‘A’ indicates the wild-type allele and ‘C’ indicates the presence of the TaqI polymorphic site. LL, lepromatous leprosy patients; HS, healthy subjects.
*p<0.05 vs. LL.
**p<0.05 vs. HS.
A. Alvarado-Navarro et al. / Immunology Letters 118 (2008) 148–151
differences in IL-12 p40 and p70 concentrations between groups
were analyzed using the Mann–Whitney U test. Spearman’s test
was used to evaluate correlations between variables where p<0.05
was considered significant. Statistical analysis was done by SPSS
3.1. Genotype and allele frequency of 3?UTR 1188 A/C
polymorphism in IL-12 p40 gene
The genotype and allele frequencies of the 3?UTR 1188 A/C poly-
morphism in the IL-12 p40 gene were evaluated in 44 LL patients
and 51 HS. The frequency of the CC genotype was higher in LL
patients than in HS. In contrast, the AC genotype frequency was
higher in HS (p<0.05) (Table 1). Allele frequency did not differ
significantly between LL patients and HS.
3.2. IL-12 p40 mRNA expression
The mRNA expression level of IL-12 p40 was higher in HS (5.76
RUA) than in LL patients (3.01 RUA) (p<0.05) (Fig. 1).
3.3. Serum IL-12 p40 and IL-12 p70 concentrations
The serum IL-12 p40 concentration was significantly higher in
serum IL-12 p70 concentration did not differ significantly between
LL patients and HS (Fig. 2).
The immune response in LL is characterized by defective cell-
mediated immunity, which affects the Th1/Th2 balance, possibly
because of altered IL-12 production. Several polymorphisms have
been described in the promoter and the 3?UTR region of the IL-12
A/C is associated with IL-12 production, which affects the delicate
We aimed to identify the genotype and allele frequencies of IL-
west of Mexico. The CC genotype carriers were significantly over-
represented among LL patients compared with HS (p<0.05). These
results indicate that the IL-12 p40 3?UTR 1188 A/C polymorphism
is associated with susceptibility to LL in patients from the west of
Fig. 1. The mRNA expression of IL-12 p40 was assayed by RT-PCR. Values are shown
as medians (horizontal lines) the 25th and 75th percentiles (boxes), and ranges
(whiskers). Values are reported in relative units of area (RUA). LL, lepromatous
leprosy patients; HS, healthy subjects.
These data concur with previous reports showing 10–50 times
lower p40 mRNA levels in lepromatous lesions than in tuberculoid
We also found higher serum concentration of IL-12 p40 in LL
patients but no significant differences in soluble IL-12 p70 concen-
p40 and low IL-12 p70 concentrations in PBMC cultures from LL
patients . These observations may be explained by the excess
secretion of IL-12 p40 as a free chain, which might associate to
priate signaling, in contrast to the p70 heterodimer [20,21]. These
data suggest that the defective cellular immune responses of the LL
patients might influence the disease evolution.
This unparalleled mRNA and protein production in both LL
is subject to posttranscriptional modifications. These modifica-
tion present significant difficulties because the mRNA is generally
plexes with diverse biophysical properties that could be involved
Fig. 2. Lepromatous leprosy (LL) and healthy subject (HS) IL-12 p40 and IL-12 p70 serum levels were assayed by ELISA. Values are shown as medians (horizontal lines) the
25th and 75th percentiles (boxes), and ranges (whiskers). (A) Distribution of serum IL-12 p40 values for LL patients and HS. (B) IL-12 p70 levels in LL patients and HS.
A. Alvarado-Navarro et al. / Immunology Letters 118 (2008) 148–151 Download full-text
in the translation of IL-12p40 and p70 in LL patients and HS. For
instance, Keene proposed an infrastructure termed the ribonome
between the genome and the proteome comprising the total col-
lection of mRNA and mRNA-associated proteins through which the
genetic information must flow and be processed. Upon reaching
the cytoplasm, most mature mRNAs are destined to be translated
into proteins. However, there is abundant regulation at the post-
transcriptional level to determine the fate of each mRNA, and this
fate may be linked to the function of the encoded protein .
In conclusion, we demonstrated that IL-12 p40 3?UTR 1188 A/C
polymorphism is associated with susceptibility to LL in patients
from the west of Mexico. These results highlight the potential
genetic influence on the development of the lepromatous pole in
this disease. However, other polymorphisms potentially involved
in cellular immune responses should be studied to understand this
This work was supported by Conacyt 31121-M.
 Araoz R, Honore N, Cho S, Kim JP, Cho SN, Monot M, et al. Antigen discovery: a
postgenomic approach to leprosy diagnosis. Infect Immun 2006;74:175–82.
 Lopez RR, Fafutis MM, Matsuoka M. Dapsone resistant Mycobacterium leprae
detected by DNA sequence analysis from a relapsed Mexican leprosy patient.
Revista Latinoamericana de Microbiologia 2006;48:256–9.
 Britton WJ, Lockwood DN. Leprosy. Lancet 2004;363:1209–19.
 Sieling PA, Modlin RL. Cytokine patterns at the site of mycobacterial infection.
 Wolf SF, Temple PA, Kobayashi M, Young D, Dicig M, Lowe L, et al. Cloning of
cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with
multiple biologic effects on T and natural killer cells. J Immunol 1991;146:
immunity. Nat Rev Immunol 2003;3:133–46.
leukin 12 receptor complex is composed of two beta-type cytokine receptor
subunits. Proc Natl Acad Sci USA 1996;93:14002–7.
 Scharton TM, Scott P. Natural killer cells are a source of interferon gamma that
drives differentiation of CD4+T cell subsets and induces early resistance to
Leishmania major in mice. J Exp Med 1993;178:567–77.
 Morahan G, Boutlis CS, Huang D, Pain A, Saunders JR, Hobbs MR, et al. A
promoter polymorphism in the gene encoding interleukin-12 p40 (IL12B) is
associated with mortality from cerebral malaria and with reduced nitric oxide
production. Genes Immun 2002;3:414–8.
 Morahan G, Huang D, Wu M, Holt BJ, White GP, Kendall GE, et al. Association of
IL12B promoter polymorphism with severity of atopic and non-atopic asthma
in children. Lancet 2002;360:455–9.
 Huang D, Cancilla MR, Morahan G. Complete primary structure, chromosomal
localization, and definition of polymorphisms of the gene encoding the human
interleukin-12 p40 subunit. Genes Immun 2000;1:515–20.
 Hall MA, McGlinn E, Coakley G, Fisher SA, Boki K, Middleton, et al. Genetic
polymorphism of IL-12 p40 gene in immune-mediated disease. Genes Immun
 Tsunemi Y, Saeki H, Nakamura K, Sekiya T, Hirai K, Fujita H, et al. Interleukin-
12 p40 gene (IL12B) 3?-untranslated region polymorphism is associated with
susceptibility to atopic dermatitis and psoriasis vulgaris. J Dermatol Sci
 Ridley DS, Jopling WH. Classification of leprosy according to immu-
nity. A five-group system. Int J Lepr Other Mycobact Dis 1966;34:255–
 Matsuoka M, Zhang L, Fafutis MM, Legua P, Wiens C. Polymorphism in the
rpoT gene in Mycobacterium leprae isolates obtained from Latin America coun-
tries and its possible correlation with spread of leprosy. FEMS Microbiol Lett
 Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for
extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:
 Chomczynski P,SacchiN. Single-step
acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem
 Modlin RL, Barnes PF. IL12 and the human immune response to mycobacteria.
Res Immunol 1995;146:526–31.
? differentially regulates interleukin-12 and interleukin-10 production in
leprosy. J Clin Invest 1997;99:336–41.
 Carra G, Gerosa F, Trinchieri G. Biosynthesis and posttranslational regulation of
human IL-12. J Immunol 2000;164:4752–61.
 Gillessen S, Carvajal D, Ling P, Podlaski FJ, Stremlo DL, Familletti PC, et al.
Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist. Eur J
 Keene JD. Ribonucleoprotein infrastructure regulating the flow of genetic
information between the genome and the proteome. Proc Natl Acad Sci USA
method of RNAisolationby